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claran

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modular-s2orc

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2018-10-14T07:37:27.722Z

2015-02-01T00:00:00.000Z

53749678

s2

Physicochemical Properties of Indonesian Pigmented Rice ( Oryza sativa Linn . ) Varieties from South Sulawesi This research was funded by Hibah Desentralisasi UPT Universitas Hasanuddin No. 4610/UN4.42/pm-17/2014. INTRODUCTION Rice is a strategic commodity in Indonesia, not only as a primary food crop cultivated by most of the Indonesian farmer but also as a staple food for more than 240 million of Indonesian peoples (Statistics Indonesia, 2012;Panuju et al., 2013). Indonesia is the third biggest rice producer in the world after China and India and South Sulawesi is the largest crop-producing regions in the eastern of Indonesia (FAOSTAT., 2012;Statistics Indonesia, 2012). There are many traditional cultivars of pigmented rice in South Sulawesi. The pigmented rice is varied in flavor, color, physicochemical character and nutrition composition depending on monsoon climate of rice crop area (Herniwati and Kadir, 2009). Yet, the experiments about pigmented rice is not as much as white rice in Indonesia (Shinta et al., 2014). Therefore, the aim of this study was to analyze the physicochemical properties of selected pigmented rice originating from western, eastern and transition zone of South Sulawesi climatic zones. Color, proximate content, total polyphenol, anthocyanin and antioxidant activity were parameters measured in this study. The results of this study can be useful in promoting pigmented rice varieties from South Sulawesi as a functional food. Preparation of samples: Thirteen of pigmented rice were collected from 2 districts in western zone, 2 districts in eastern zone and 1 district in transition zone of South Sulawesi. Rice seed was collected in the unhusked form and pounded to remove the husk before being analyzed. Grain Dimensions and Shape: Thirteen of rice seed samples were randomly collected. The dimensions were measured using dial micrometer to obtain the average length and width of rice seeds. Rice average length (mm) Length to width ratio (L/W) = Rice average width (mm) Based on the L value, the length of the husked rice was categorized by IRRI as follows: extra long = >7.50 mm, long = 6.61-7.50 mm, medium = 5.51-6.60 mm and short = 4.50 mm. Meanwhile, grain shape was characterized based on the L/W ratio: slender >3.0, medium 2.1-3.0, bold 1.1-2.0 and round 1.0 or less (Juliano, 1993;Cruz and Khush, 2000). Color analysis: About 100 g pigmented riced was placed on a flat container with size 10×10 cm 2 . Samples were set until the bottom of container covered. The color intensity was measured by attaching colorimeter TES 135 to the samples. It produced three color parameters i.e brightness (L*), redness (a*) and yellowness (b*). Prior to the assessment, calibration of the colorimeter was performed by using standardized white color in the instrument. Replication of examination were ten times for each samples (Yodmanee et al., 2011). Extraction: Pigmented rice was cleaned and crushed to a coarse powder. About 20 g pigmented rice were extracted with 50 mL HCl-ethanolic solution (HCl 0.01% in ethanol 96%). Maceration was done with modification using sonicator for 30 min, 40°C. The extract was collected and filtered. Filtration and re-extraction was conducted until a colorless solution obtained with total volume of the pigment about 800 mL. A part of rice pigment was used to analyze its anthocyanin content and the other was rotary evaporated at 50°C. The extract was subsequently placed in cool temperature in refrigerator. Yield: The extraction yield was calculated based on the dry weight of the sample used (the unit of g/100 g dry basis of the rice). Weight of rice extract after evaporation Yield (%, w/w) 100 Weight of rice powder before extraction   Determination of total phenolic contents: Total phenolic compounds of the HCl-ethanolic extracts were determined by the Folin-Ciocalteu method (Kumar et al., 2008;Males et al., 2006). A standard curve was constructed using various concentrations of gallic acid (Merck). The results were expressed as percentage of gallic acid. Determination of anthocyanin contents: Anthocyanin of the pigmented rice extracts were conducted by pH differential method (Wrolstad, 1993;Giusti and Worldstat, 2001;Rodriguez-Saona and Wroistad, 2001). Anthocyanin concentration was calculated as cyanidin-3-glycosides using a molar extinction coefficient of 26900 L cmG 1 mgG 1 and a molecular weight of 445.2. The absorbance value is calculated using the equation: The anthocyanin concentration expressed in mg CYE/100 g dry bases of samples. (CYE = cyanidin equivalent). Determination of DPPH scavenging capacity: Radical scavenging activity was performed by using DPPH method (Zhang and Lin, 2008). The percentage of DPPH binding capacity of each pigmented extracts was calculated based on the formula: Absorbance of control absorbance of sample Scavenging of free radicals (%) = 100 Absorbance of control   Each experiment was performed in triplicate, except proximate composition analysis. Grain dimension and shape: The grain shapes of the collected PR varieties are given below in Table 1. Thirteen of PR varieties had no significant differences in the length and L/W ratio (p<0.05). The PR length was in the range of 5.60-6.82 mm. There were two PR varieties longer than the others. The L/W ratio was in the range of 2.22-2.90. It means that all rice had medium shape (between slender and bold). Proximate analysis: In the development of a functional food product, it is important to understand the nature of each element in foodstuff. Although, the composition of foodstuffs is complex, the prominent elements in foodstuffs are water, minerals, carbohydrates, fats and proteins. The proximate analysis results of PR varieties are displayed in Table 2. Color analysis: The color of the rice was determined using colorimeter. Pigment parameters (Table 3) DISCUSSION In this study, 13 PR varieties were compared from South Sulawesi, Indonesia (Fig. 1), those have different grain dimension and shape (Table 1). Based on IRRI categorization, the length of PR varieties in this study ranges from extra long (H), long (A, C, D and I) and medium (B, E, F, G, J, K, L and M). For the grain shape, almost all of PR varieties are grouped as medium and only one PR variety that have slender grain shape (H). Grain dimension and shape indicate the quality and appearance of rice which are important considerations in rice preferences. In tropical area, such as in Indonesia, it is reported that the long to medium rice is generally preferred by the consumers (Unnevehr et al., 1992). Therefore, the PR varieties used in this study are mostly preferred by the consumers in Indonesia. A rice seed consists of several parts, i.e. the outer layer, pericarp and embryo and the endosperm. The endosperm comprises of aleurone layer, it is rich in many essential fatty acids, fiber, vitamins and minerals and endosperm proper containing starch. As the pigment is in the outer of pericarp layer (Juliano, 1993), rice was collected in the form of paddy and pounded manually prior to analysis. It prevented the loss of pigments during grinding and avoided the damage due to excessive exposure to the sunlight. Once the crop was harvested, the drying process should be conducted immediately to keep moisture level close to 14% (Dipti et al., 2003). The water content in pigmented rice (11.33-13.32%) is expected to maintain the stability of rice during storage. The ash content is the total amount of an inorganic residue from ignition process. It contains a wide variety of minerals derived from both of the rice tissue itself (physiologic-ash) and the residue of tracer elements (e.g. sand and soil) attached to the rice surface (un-physiologic-ash). Total ash contents in all rice varieties were between 1.42-1.62% (Table 2) but in the present research, the rice mineral contents have not been checked, so it could not claim whether the total ash is physiological ash or un-physiological ash. Lipid, protein and carbohydrate compositions in all rice varieties were between 1.06-2.78, 7.40-14.02 and 71.29-77.14%, respectively (Table 2). These levels were almost similar with pigmented rice from Southern Thailand (lipid and protein in the range of 1.44-1.93 and 6.63-8.46%, respectively) (Yodmanee et al., 2011) and Western Indonesia (lipid, protein and carbohydrate between 0.43-0.66, 8.68-10.55 and 87.46-89.28%, respectively) (Indrasari et al., 2010). The crude fiber contents of samples (0.66-0.99%) ( Table 2) were higher than crude fiber contents of pigmented rice grown in Southern Thailand (0.16-0.35%) (Yodmanee et al., 2011) and close to some rice varieties from Western Indonesia (0.57-1.14%) (Indrasari et al., 2010). The higher-fiber content, the better it is as a food supplement. Ambo awok variety was the black rice with the highest nutritionalvalue. The brightness (L*), redness (a*) and yellowness (b*) values of pigmented rice are indicators for its pigment component. These values were generally low in black rice and black glutinous rice depending on their anthocyanin content and genotype (Yodmanee et al., 2011). The darkest rice had the lowest L* as shown by black glutinous rice A, C and F varieties, except of J (black kobok) variety which was brighter. The highest and the lowesta* (redness) values were exhibited by D and G varieties, respectively (Table 3). Anthocyanins are abundant not only in violet and red fruits but also in colored grains. Abdel-Aal et al. (2006) reported that anthocyanin contents in black rice can reach as high as 25000 µg gG 1 , bigger than vegetables and fruits (200-10000 µg gG 1 ) depending on its planting condition. The amount of extracts produced by ethanolic-HCl extraction was varied. The highest and the lowest yields of extracts were A and B, consecutively (Table 4). These extraction yield were positively correlated with both of phenolic content (r = 0.868) and anthocyanin content (r = 0.849). It also suggested that ethanol solution can be used to extract the polar components of rice, especially anthocyanin. Methanol, ethanol and acetone are suitable extraction solution for anthocyanin and acidic condition needed because anthocyanin is more stable in acid (Giusti and Wroistad, 2001;Rodriguez-Saona and Wroistad, 2001). Anthocyanin contents were measured using pH differential method. At pH 1.0, anthocyanin is in colored oxonium or flavilium form. While at pH 4.5, anthocyanin is in uncolored carbinol form. Total anthocyanins were calculated by subtracting absorbance at a maximum wavelength of sample solution in two different pH of buffer (Wrolstad, 1993). Black gelatinous rice from western, eastern and transition zones showed higher anthocyanin and phenolic contents than black and red rice. Anthocyanin yields were 94.70-202.46 mg Cy-3-glc/100 g db for black glutinous, 66.08-113.83 mg Cy-3-glc/100 g db for black rice and 0-12.85 mg Cy-3-glc/100 g db for red rice. While phenolic contents of black glutinous, black and red rice were 292.74-746.25 mg GAE/100 g db, 119.74-230.10 mg GAE/100 g db and 12.52-64.52 mg GAE/100 g db, consecutively (Table 4). These data were similar with the results reported by Sangkitikomol et al. (2010a, b) and Abdel-Aal et al. (2006). Anthocyanins exert their antioxidant capacity due to their phenolic groups thereby can be used to treat a wide range of diseases caused by free radicals. Antioxidant activity test showed that black glutinous rice type had the highest DPPH free radical scavenging ability followed by this decreasing order A>I (black Lalodo) = J (black Kobok)>F. K (red Lea) harvested from transition zone had a higher antioxidant activity than the other red rice and not significantly different with black glutinous rice C, L (black Ambo Tanduk) and M (black AmbokAwok), although its polyphenols and anthocyanin levels were low (Table 4). These result exhibited that pigmented rice from transition zone (TanaToraja regency) was more valuable than rice from the other zones. TanaToraja is located in the higher altitude and have a colder climate compared to the other regions. This is consistent with research of Saenjum et al. (2012) which stated that the rice bran of rice varieties in the mountainous region have higher anti-free radical DPPH activity. It is presumably due to the presence of other antioxidant compounds extracted by acidic ethanol, among other vitamins and unsaturated oils, so it is necessary to analyze other antioxidant components in the extracts.

v3-fos

2017-04-30T21:28:32.838Z

2015-02-26T00:00:00.000Z

5020578

s2

Evaluation of Wheat Chromosome Translocation Lines for High Temperature Stress Tolerance at Grain Filling Stage High temperature (HT, heat) stress is detrimental to wheat (Triticum aestivum L.) production. Wild relatives of bread wheat may offer sources of HT stress tolerance genes because they grow in stressed habitats. Wheat chromosome translocation lines, produced by introgressing small segments of chromosome from wild relatives to bread wheat, were evaluated for tolerance to HT stress during the grain filling stage. Sixteen translocation lines and four wheat cultivars were grown at optimum temperature (OT) of 22/14°C (day/night). Ten days after anthesis, half of the plants were exposed to HT stress of 34/26°C for 16 d, and other half remained at OT. Results showed that HT stress decreased grain yield by 43% compared with OT. Decrease in individual grain weight (by 44%) was the main reason for yield decline at HT. High temperature stress had adverse effects on leaf chlorophyll content and Fv/Fm; and a significant decrease in Fv/Fm was associated with a decline in individual grain weight. Based on the heat response (heat susceptibility indices, HSIs) of physiological and yield traits to each other and to yield HSI, TA5594, TA5617, and TA5088 were highly tolerant and TA5637 and TA5640 were highly susceptible to HT stress. Our results suggest that change in Fv/Fm is a highly useful trait in screening genotypes for HT stress tolerance. This study showed that there is genetic variability among wheat chromosome translocation lines for HT stress tolerance at the grain filling stage and we suggest further screening of a larger set of translocation lines. Introduction High temperature (HT, heat) stress is one of the most important environmental stresses that adversely affects growth, development and yield of many field crops including wheat [1][2][3][4]. High temperature stress is a principal wheat yield-decreasing factor in Central Asia, North Africa, Europe, Australia, and the United States [5][6][7]. A simulation study in Australia showed wheat yield could decrease up to 50% when growing season temperature became 2°C warmer than the average [8]. Wheat is usually planted in late autumn to early winter and harvested to biotic stress such as powdery mildew, wheat curl mite, and leaf and strip rusts [29][30][31][32], but knowledge on performance of chromosome translocation lines under HT stress is limited. Because the translocation lines have chromosome segments from alien species native to stressed habitats, we hypothesize that genetic variability will exist among chromosome translocation lines for tolerance to HT stress. The objectives of this study were to evaluate wheat chromosome translocation lines for tolerance to HT stress at the grain filling stage and identify physiological and yield traits associated with the tolerance. Seeds were sown in 1.6-L plastic pots of dimensions 14 cm (height) × 50 cm (top perimeter) × 36 cm (bottom perimeter) filled with a potting mix (Metro Mix 360; Hummert International, Topeka, KS) and 8 g of Osmocote Plus (N:P 2 O 5 :K 2 O = 15:9:12; Scotts, Marysville, OH), a slowrelease fertilizer. Six pots of plants per genotype were randomly divided into three identical growth chambers (two pots per chamber) (Conviron, Winnipeg, MB, Canada) set at an optimum temperature (OT) of 22/14°C day/night,~85% humidity, 16 h photoperiod, and photosynthetically active radiation (PAR) of 650 μmol m -2 s -1 provided by cool white fluorescent lamps (Philips Lighting Co., Somerset, NJ). Thirty days after seeding, plants were thinned and staked, leaving four plants per pot. Granular Marathon 1% pesticide (a. i.: Imidacloprid, 1-((6-Chloro-3-pyridinyl) methyl)-N-nitro-2-imidazolidinimine) was applied to avoid sucking insect pest infestation, and plants were randomly relocated weekly within the growth chamber to avoid positional effect within the chamber. Throughout the growing season, growth chambers' air temperature and relative humidity were monitored every 20 min with HOBO U14-001 LCD Temperature/Relative Humidity (RH) Data Logger (Onset Computer Corporation, Bourne, MA). Thick paper caps were used on the top of the HOBO data loggers to shield against direct heat radiation from the lamps. The PAR was monitored once a month with a Field Scout Light Sensor (Spectrum Technologies, Inc., Plainfield, IL). At anthesis, two plants per pot were randomly selected and main stems were tagged. Ten days after anthesis, the temperature treatment was applied by moving one pot of each genotype from the OT regime (22/14°C day/night) to one of the growth chambers' set at HT of 34/26°C day/night,~85% humidity, 16 h photoperiod, and PAR of 650 μmole m -2 s -1 . The dates of movement (transfer) of pots to the HT chamber were different because of different anthesis dates. Another pot of each genotype remained at OT regime throughout the experiment and served as a control. The growth chambers' air temperature and relative humidity at HT were also constantly monitored with HOBO loggers with thick paper cap throughout the experiment period. The stress period was 16 d, after which pots were moved back to their original growth chambers. To avoid water stress, plants in both temperature regimes were fully irrigated by keeping pots in a tray filled with~2 cm water until physiological maturity. We recognized, however, that leaving plants sitting in water for so long may cause water-logging/anoxia; which should be documented or avoided in the future studies. Once a week until physiological maturity, plants were fertigated with Miracle-Gro water-soluble fertilizer (N:P 2 O 5 :K 2 O = 24:8:16; Scotts Miracle-Gro Products, Inc., Marysville, OH) per manufacturer's instruction. The daytime and nighttime temperature of all growth chambers were held for 12 and 8 h, respectively, with a 2-h transition period between them. During transition period, the rate of change in chamber temperature was set at 1°C/15 minutes. Data Collection The date of anthesis was recorded when 50% of plants in a pot reached Feekes growth stage 10.5.1; and the days to anthesis was calculated by subtracting planting date from anthesis date. Superoxide Dismutase and Superoxide Anions Leaf samples (flag leaf) collected from the secondary tiller of a tagged plant at 10 DAST were immediately frozen in liquid nitrogen and stored at-80°C until further analysis. Superoxide dismutase activity was assayed by measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT) at 560 nm following the protocol of [39] with slight modification. Frozen tissues were ground in a cold mortar and pestle using liquid nitrogen and suspended in 1.5 ml of ice-cold extraction buffer solution containing 50 mM Na-PO 4 buffer at pH 7.8, 1 mM EDTA.Na 2 and 2% (w/v) polyvinypyrrolidone. The suspension was centrifuged at 20000 × g at 4°C for 20 min. The supernatant was used to measure the enzyme activity. A reaction mixture of 3 ml containing 50 mM Na-PO 4 buffer at pH 7.8, 0.66 mM of EDTA.Na 2 , 10 mM of L-methionine, 33 μM of nitroblue tetrazolium and 0.0033 mM of riboflavin, and 50 μL of extraction buffer was added to the 150 μl of supernatant, and the tubes were illuminated with luminescent lamps for 10 min. The extinction was then read against a blank at 560 nm using a U-2000 double-beam UV/Vis spectrophotometer (Hitachi, Tokyo, Japan). One enzyme unit of SOD was defined as the amount required to inhibit photochemical reduction of NBT by 50% and was expressed as enzyme unit mg protein -1 . The concentration of superoxide anion was estimated according to Chaitanya and Naithani [40]. About 0.2 g of leaf samples were suspended in ice-cold 0.2 M sodium phosphate buffer at pH 7.2 containing diethyl dithiocarbamate (10 -3 M). The suspension was immediately centrifuged at 3000 × g at 4°C for 1 min. In 200 μl of supernatant, 2 mL of nitroblue tetrazolium (2.5 × 10 -4 M) was added and the ODA was recorded against a blank at 540 nm every 30 s for 150 s using a Hitachi U-2000 double-beam UV/Vis spectrophotometer. The superoxide anion was expressed as change in optical density min -1 g -1 fresh weight. Yield and Yield Components Plants were harvested by cutting at ground level. Spikes were counted, detached, and dried to constant weight in a 38°C incubator. The remaining aboveground biomass was oven-dried at 65°C to a constant weight. The dried spikes were hand-threshed. The spikes from primary tillers of two tagged plants were used to determine the number of grains per spike and grain weight per spike. Individual grain weight was estimated by dividing grain weight per spike by number of grains per spike and expressed in mg. Yield per plant was the mean grain weight in g from all the spikes of tagged plants. Heat Susceptibility Index (HSI) The heat susceptibility index (HSI) for grain yield was calculated according to Fischer and Maurer [41]: where Y is the average grain yield per plant of an accession at HT of 34/26°C, Yp is the average grain yield per plant of the same accessions at OT of 22/14°C, and D is the stress intensity, equal to 1-X/Xp, in which X is the mean Y of all accessions and Xp is the mean Yp of all accessions. The genotypes were classified as highly tolerant (HSI 0.5), tolerant (0.5 < HSI 0.75), moderately tolerant (0.75 < HSI 1.0), and highly susceptible. Similarly, HSIs for physiological and yield traits were also calculated and used in path coefficients to find relationships among heat tolerance of various traits including yield. They were also used to rank the genotypes for tolerance to HT stress. Statistical Analyses The experimental design was a split plot with three replications (three growth chambers under each temperature regime). The temperature treatment was randomly assigned to the growth chambers and considered as the main plot. The sub-plot comprised genotypes. SAS 9.2, PROC GLIMMIX (SAS Institute Inc., Cary, NC, 2003), was used to analyze the data with block, temperature, and genotypes as class variables; block and block × temperature as random effects; and all other variables as fixed effects [42]. The least square means were separated at p < 0.05 after Tukey-Kramer adjustment. The time series data on leaf chlorophyll, flag leaf temperature, and Fv/Fm were analyzed using the REPEATED statement and Type = CS, a covariance structure of compound-symmetry type. PROC REG in SAS was used to determine the relationship between two variables, and PROC CALIS in SAS was used to conduct path coefficient analysis. At the end, genotypes were ranked on the bases of heat susceptibility indices of physiological traits, yield, and yield components using the RANK function in Microsoft Excel (2010). Results The average day/night temperature recorded during the treatment period was 33.7±0.7/26.1±0.5°C in growth chambers set for the HT regime and 21.4±0.3/14.3±0.5°C in growth chambers set for the OT regime. As expected, there was no significant effect of temperature and interaction effect of temperature × genotype on days to anthesis; however, genotypes varied for this trait. The days to anthesis ranged from 88 in TA5599 to 114 in TA5088; and the mean, median, and mode were 98, 97, and 96 respectively. There were significant effects of temperature, genotype, and temperature × genotype interaction on physiological and yield traits, unless stated otherwise ( Table 2). Leaf temperature and leaf chlorophyll as a function of DAST are shown in Fig. 2. The relationship between leaf temperature and DAST under HT stress was significant and positive, with a slope of 0.2°C when averaged across the genotypes ( Fig. 2A), and the relationship between leaf chlorophyll and DAST under HT stress was significant and negative, with a slope of-2.03 SPAD unit (Fig. 2B). At OT, the relationship was either not significant (leaf temperature: slope = 0.02°C, R 2 = 0.007, and p = 0.46) or very weak (leaf chlorophyll: slope = -0.3 SPAD unit, R 2 = 0.10, p = 0.003); therefore, OT data are not presented or discussed further. An analysis of genotypic behavior at HT stress showed that the change in leaf temperature over the time period was not significant in TA4350-119, TA5594, or TA3008; however, TA5617, TA4350-118, and TA5639 had the highest rate of increase in leaf temperature (slope = 0.32 to 0.34°C; Table 3). Similarly, under HT stress, the rate of decrease in leaf chlorophyll content throughout the experiment was < 1 SPAD unit in TA4350-119, TA5594, and TA5617; between 1 and 2 in TA5088, TA5608, TA5636, TA4305-118, and TA5595; and >3 in TA5638 and TA5639. The difference between temperature regimes for maximum quantum yield of PSII (Fv/Fm, unitless) was significant at p < 0.001. High temperature stress at the grain filling stage decreased Fv/Fm by 38% (Fig. 1C) compared to OT when averaged across the genotypes and over the first 14 d of readings. Genotypes behaved differently according to the temperature treatment at p < 0.001 (Table 2). When averaged across the days, HT stress had no effect on TA4350-119, TA5594, TA5617, TA5088, and TA5599 for Fv/Fm compared with OT. However, Fv/Fm decreased by < 35% in TA5608, TA5595, TA5602, and TA4305-118 and by 60-64% in TA5640, TA5624-4, and TA5639. Fv/Fm as a function of DAST is shown in Fig. 2. A significant negative relationship was evident between Fv/Fm and DAST under HT stress when averaged across the genotypes, with a slope of-0.04 unit (Fig. 2C). At OT, the relationship of Fv/Fm with DAST was very weak (slope = -0.005 unit, R 2 = 0.08); therefore, data are not presented or discussed. Further analysis of genotypic behavior at HT stress showed that TA4350-119, TA5594, and TA5617 had the lowest rate of decrease in Fv/Fm (slope < -0.02 unit), and TA5638 and TA5601 had the highest rate of decrease (slope > -0.06 unit) during the time period (Table 3). Relationships among leaf temperature, leaf chlorophyll, and Fv/Fm are presented in Fig. 3. At OT, there was no relationship between leaf chlorophyll and leaf temperature (R 2 = 0.12, p > 0.05) (Fig. 3A), and a very weak positive relationship between Fv/Fm and leaf temperature (R 2 = 0.19, p = 0.05) (Fig. 3B). The relationship between Fv/Fm and leaf chlorophyll content was non-significant (R 2 = 0.0002, p > 0.05) (Fig. 3C). At HT, there was a highly negative relationship between leaf chlorophyll and leaf temperature (R 2 = 0.58, p < 0.001) (Fig. 3D); and Fv/Fm and leaf temperature (R 2 = 0.52, p < 0.001) (Fig. 3E). The relationship between Fv/Fm and leaf chlorophyll at HT stress was strongly positive (R 2 = 0.67, p < 0.001) (Fig. 3F). Superoxide Dismutase (SOD) and Superoxide Anion There was a significant difference between temperature regimes for SOD activities and concentration of the superoxide anion, a ROS. High temperature stress decreased SOD activity by 8% and increased superoxide anion concentration by 44% (0.12 changes in OD min -1 g -1 FW) compared to OT when averaged across the genotypes. Genotypes behaved differentially according to the temperature treatment for SOD at p < 0.001 (Table 4). The effect of HT stress was not evident in 17 genotypes for SOD, but SOD activity decreased by 29% in TA5638 and TA5600 and by 52% in TA4305-118. Genotype and temperature × genotype interaction was not significant for the superoxide anion, so data for this trait were not presented in further detail (p > 0.05) ( Table 2). Spike Number, Grain Number, Individual Grain Weight, and Grain Yield Effects of temperature and interaction of temperature × genotype were not significant for spike number per plant and grain number per spike; however, a significant variation among genotypes was evident at p < 0.001 ( Table 2). The average spike number per plant ranged from 1.2 in TA5088 to 2.9 in TA5608; and grain number per spike ranged from below 33 in TA5600, TA5601, TA5602, TA5624-4, TA4350-119, and TA4305-118 to more than 66 in TA5594, TA5595, TA5638, and TA5088. There was a significant difference between temperature regimes for IGW and grain yield per plant, and genotypes behaved differently according to temperature treatment for these traits at p 0.004 (Table 2). High temperature stress decreased IGW and grain yield by~43% compared with OT when averaged across the genotypes. As a result of HT, IGW decreased by 30% in TA5594, TA5088, and TA5608 and by 71% in TA5640 (Fig. 4A). High temperature stress significantly decreased grain yield in TA5595, TA5615, TA5636, TA5637, TA5638, TA5640, TA3008, and TA4350-118, and the decrease ranged from about 41% in TA5595 to 77% in TA5640. The decrease in grain yield because of HT stress was not statistically significant in other genotypes (Fig. 4B). Fig. 5. The genotypes TA5617 and TA4350-119 had 0.5 HSI for leaf chlorophyll; and TA5594, TA5599, TA5088, TA5608, TA4350-118 had HSI between 0.5 and 0.75. The genotypes TA5594, TA5617, TA5088, TA4350-119 had 0.5 HSI for Fv/Fm; and TA5599, TA4305-118 had HSI between 0.5 and 0.75. The genotypes TA5594, TA5088, TA5608 had 0.75 HSI for individual grain weight and all other genotypes had > 0.75 HSI. The genotypes TA5617 and TA5088 had < 0.5 HSI for grain yield, and TA5594, TA5639, TA5599, TA5608 had HSI between 0.5 and 0.75. The genotypes TTA5637, TA5638, TA5640, and TA3008 had > 1.0 HSI for all traits. Pearson's Correlation Coefficients and Path Coefficient Analysis At OT, there was no significant correlation between individual grain weight and grain yield per plant; and the maximum quantum yield of PSII (Fv/Fm) had no significant correlation with leaf chlorophyll and individual grain weight. A significant positive correlation was evident between Fv/Fm and grain yield per plant; and leaf chlorophyll had significant positive correlation with individual grain weight and grain yield per plant (Table 5). At HT, there was significant positive correlation among Fv/Fm, individual grain weight and grain yield per plant. Although leaf chlorophyll had strong positive correlation with Fv/Fm and individual grain weight, there was no correlation with grain yield per plant. A path coefficient analysis performed to identify correlation or direct and indirect effects of heat responses and relationships of heat susceptibility i.e. heat susceptibility indices (HSIs) of physiological and yield traits to each other and to HSI of yield are presented in Fig. 6. There was a strong positive correlation between SPAD HSI and Fv/Fm HSI . The SPAD HSI had neither direct nor indirect effect on IGW HSI and Yield HSI ; however, the Fv/Fm HSI had significant direct effect on IGW HSI and indirect effect on Yiled HSI . The IGW HSI had a strong direct effect on Yield HSI . Ranking of Genotypes Based on HSIs of Physiological, Yield Traits, and Yield The rankings of genotypes on the bases of their response to HT stress for traits i.e. Fv/Fm HSI , IGW HSI , and Yield HSI are presented in Table 6. There was evidence of direct and indirect effects of these traits on Yield HSI (Fig. 6). On the basis of final ranking estimated from a mean of all Evaluation of Wheat Translocation Lines under High Temperature Stress rankings, TA5594, TA5617, and TA5088 were the top three genotypes tolerant to HT stress; and TA5640 and TA5637 were highly susceptible genotypes with rankings of 19 and 20. Discussion We evaluated 16 wheat chromosome translocation lines that had a small chromosome segment from different wild wheat species introgressed into spring wheat cultivars for HT stress tolerance at the grain filling stage. The effect of HT stress was highly significant for all physiological and yield traits except grain number per spike, and we demonstrated genotypic variability in chromosome translocation lines for physiological traits, yield components, and grain yield. This information can be used in breeding programs to develop stress-tolerant cultivars. Canopy temperature shows the efficiency with which evaporative cooling is occurring from the plant surface, and it has been used to estimate heat stress in field crops under fully or limited irrigated conditions [16,[43][44][45]. Ayeneh et al. [46] showed a strong positive correlation between canopy temperature and flag leaf temperature (r = 0.91) in 13 wheat genotypes grown under HT stress. In this study, the HT stress at the grain filling stage increased flag leaf temperature by 6°C as compared to OT (Fig. 1A); and there was genotypic variation in flag leaf temperature. Genotypic variation in flag leaf temperature under HT stress was evident in wild wheat relatives [1]; and the variability in flag leaf temperature at HT stress might be attributed to differential decrease in vascular capacity and impairment of cooling mechanisms [47,48]. High temperature stress increased superoxide anions by 44% and decreased SOD activity by 8% (Table 4). Reactive oxygen species like superoxide anions are a product of regular subcellular processes and are strictly regulated by the antioxidant enzyme SOD to avoid their detrimental effects on cellular structures. High temperature stress increases the formation of ROS in chloroplast owing to the over excitation of chlorophyll molecules and decreases in antioxidant enzyme activity [13,17]. High temperature stress decreased leaf chlorophyll and maximum quantum yield of PSII (Fig. 1B, and 1C). Genotypic variation for these attributes was evident. High temperature stress is well known to decrease leaf chlorophyll [1,10,13], which could be the result of thylakoid membrane damage [11,12], lipid peroxidation of chloroplast membranes [13], and inhibition of chlorophyll biosynthesis [14]. The study showed that the longer the duration of HT stress, the higher the leaf temperature but the lower the leaf chlorophyll and the maximum quantum yield of PSII (Fv/Fm) (Fig. 2). It has been reported that light is one of the primary stress parameters for PSII performance, and significant differences in Fv/Fm can be found even among upper and lower leaves of a wheat plant due to shading [49]. In this study, as the fluorescence data was collected from the upper most flag leaf only; and the light level in both HT and OT chambers were about 650 μmol m -2 s -1 provided with the same make and model of white fluorescent lamps, we assumed that the influence of light source on Fv/Fm was negligible; and difference in Fv/Fm among genotypes was due to their differential tolerance to high temperature. Genotypic variation for the rate of increase or decrease of respective physiological traits at HT stress was evident (Table 3). In addition, under OT, there were no evidence of a relationship between leaf temperature and leaf chlorophyll or Fv/Fm, and leaf chlorophyll content and Fv/ Fm (Fig. 3), whereas under HT stress, the relationships were either highly negative (between leaf temperature and leaf chlorophyll or Fv/Fm) or highly positive (between leaf chlorophyll and Fv/ Fm). In this study, under HT stress, for a unit increase in leaf temperature, there was an evidence of decrease in leaf chlorophyll by 5.82 SPAD unit, and Fv/Fm by 0.105 unit; and for a decrease in one SPAD unit there was an evidence of decrease in Fv/Fm by 0.016 unit (Fig. 3). In this study, HT stress had no effect on grain number per spike ( Table 4). The absence of a treatment effect on grain number in this study contrasted with earlier findings from a controlled environment experiment, where winter wheat cultivar 'Karl 92' had a 12.5% reduction in grain number when temperature increased from 25/20°C to 35/20°C from 10 d after anthesis [18], and from a field experiment, where Zhong-hu and Rajaram [50] observed about a 13% decrease in grain number between late-and normal-planted wheat. Hays et al. [51] reported that HT stress at 10 d after anthesis induced ethylene production, resulting in kernel abortion in heat-susceptible winter wheat 'Karl 92' but not in heat-tolerant spring wheat cultivar 'Halberd'. This result showed that genotypes used in the current study were tolerant to HT stress for kernel abortion; however, we recommend further study of these genotypes to elucidate the mechanism of embryo tolerance to HT stress. High temperature stress decreased individual grain weight (Fig. 4A) by 43%, which is about 7% lower than the decline observed by Yang et al. [20] in 30 synthetic hexaploid wheat varieties and 13% higher than the decline observed by Gibson and Paulsen [18] in 'Karl 92' subjected to HT stress from 10 d after anthesis. The decrease in IGW under HT stress might be due to an increase in leaf senescence and decrease in grain-filling duration [8,10,20,21], and genotypic variation for this trait has been observed by other authors [15,[18][19][20]. High temperature stress decreased grain yield per plant by 44%. Yang et al. [20] and Gibson and Paulsen [18] observed about 54 and 78% decreases, respectively, in grain yield when HT stress was applied to wheat 10 d after anthesis. Grain number per spike and IGW are two important factors of grain yield per plant. The absence of treatment effect on grain number might be the reason for the smaller decrease in grain yield in this study compared with earlier studies. As in earlier work, genotypes differed significantly in their response to HT stress for grain yield. A correlation and path diagram analyses to investigate correlations between heat responses of physiological and yield traits (HSIs) to each other and to grain yield HSI showed that there was a strong correlation between SPAD HSI and Fv/Fm HSI (Fig. 6); however, SPAd HSI had no significant effect on IGW HSI and Yield HSI , whereas Fv/Fm HSI had a strong correlation with IGW HSI and Yield HSI . There was evidence that the nature of the effects of Fv/Fm HSI on IGW HSI and of IGW HSI on Yield HSI were direct but the effect of Fv/Fm HSI on Yield HSI was indirect. These results provide evidence that there might be strong associations (cause-effect relationships) among HSIs of Fv/Fm, IGW, and grain yield; this implies that the physiological trait such as Fv/Fm is useful in screening genotypes for HT stress tolerance. However, we are of view that gas exchange traits would have added further valuable information on the behavior of genotypes at HT; and therefore we recommend the collection of gas exchange data in future studies. On the bases of above result, we used Fv/Fm HSI , IGW HSI, and Yield HSI in ranking the genotypes for HT stress tolerance instead of using HSIs of Yield and/or IGW only. The ranking showed that TA5594, TA5617, and TA5088 were highly tolerant (ranked 1to 3), and TA5637 and TA5640 were highly susceptible (ranked 19 and 20) to HT stress compared with other lines and the check cultivars. Conclusions This study revealed genetic variability among wheat chromosome translocation lines for HT stress tolerance at the grain filling stage. It illustrated that a decrease in individual grain weight was the main reason for yield decline and quantified that an increase in HT stress (high leaf temperature) adversely affected leaf chlorophyll and Fv/Fm; and a decrease in Fv/Fm might be one of the physiological reasons for subsequent decline in individual grain weight. In this study, we identified TA5594, TA5617, and TA5088, having small segment of chromosome from Haynaldia villosa or Aegilops speltoides, as highly tolerant to HT stress at grain filling stage. These tolerant genotypes may be used to improve HT stress tolerance of wheat cultivars. We recommend further screening of a larger population of translocation lines. Because other translocation lines might have chromosome segments from different wild relatives or different genomes, there are good possibilities of discovering more HT stress tolerant genotypes.

v3-fos

2019-04-15T13:04:06.778Z

2015-02-09T00:00:00.000Z

59156248

s2

Effect of Different Regional Climates on Persimmon Quality Persimmon (Diospyros kaki) is grown in wide climate conditions, which may affect fruit biochemical characteristics such as vitamins, soluble solids and antioxidants. Therefore, the aim of this research is to evaluate the biochemical responses of fruit to these climate variables. For this purpose 5 districts of Kashan, Shahrud, Yazd, Kiasar and Sari were chosen to collect fruits. On November 2012, based on a complete block design ripen fruits were collected and quality factors were measured. The results of this research showed that local climate condition significantly (P < 0.01) influenced fruit biochemical characteristics. Fruit collected from Kashan had the highest vitamin C (1.74 mg /100 ml) acidity (pH = 6.6), while the samples from Shahrud had the highest soluble solids (22.13) and titratable acidity (0.04 mg/100 ml). Fruits collected from Yazd showed significant differences in Chland carotenoids contents, 0.07 and 0.42 mg/g, respectively, relative to other sites. These results show that arid and semi-arid districts enhance fruit quality. Introduction Persimmon (Diospyros kaki) belongs to the family Ebenaceae is widely grown in the climatic zones range in the world. This species is native to Japan and has wide leaves and delicious fruit. Planting this tree, because its fruits, first developed in Western countries and in the first decade of the 19 th century was introduced to California and Southern Europe [1], and later entered Iran. And has also in many parts of the country expanded. In color, the ripe fruit of the cultivated strains range from light yellow-orange to dark red-orange depending on the species and variety. The fruit has a high tannin content which makes the immature fruit astringent and bitter. It is edible in its crisp firm state, but has its best flavor when allowed to rest and soften slightly after harvest [2]. Fruit harvested at different times have a significant impact on fruit quality characteristics [3][4][5]. Harvest index, based on experience and ecological conditions of climate zones evaluated at the end of December is completed. Harvest index, based on experience and ecological conditions of climate zones is complete at the end of December. Fruit picked before ripening to increase during storage and maintaining quality characteristics it is very important. Fruits that are harvested too early, smaller and color, aroma and taste them down [6,7]. The plant growth is influenced by genetic and environmental characteristics, so it could be more due to the differences in a variety of environmental factors to genetic changes. Thus, the lack of environmental factors, plant growth will be disorder and will stop [8]. Temperature is one of the factors affecting photosynthesis and plant metabolism at the cellular level. Seasonal and diurnal temperature changes has a significant effect on plant growth [9]. The difference in the temperature range can be attributed to variations in latitude, topography, proximity to large bodies of water represented. This constructive climate, due to the high heat capacity of water, this constructive climate, due to the high heat capacity of Research Article Effect of Different Regional Climates on Persimmon Quality water, as evident in annual and the diurnal temperature range (DTR). Generally, higher temperatures lead to an increase in the rates of sugar accumulation in the fruit ripening and organic acids degardation, while low temperatures can lead to a reduction in the organic acids degradation and rates of sugar accumulation [10]. According to the daily temperature range, grape quality varies during the ripening period, because this parameter affects the sugar, anthocyanin compounds and as well as the aroma. During the day, photosynthesis occurs and at night photosynthesis product move from the source leaves to the fruit. Cool night temperature in during grape ripening favor the sugar accumulation and restrict the vegetative growth [11], Mori et al. [12], reported that at higher temperatures from 10˚C respiratory activity increases exponentially. Persimmon fruit is rich in the carbohydrates, organic acids, vitamins (especially A and C), minerals, phenolic compounds and carotenoids [13,14]. Phenolic compounds with ascorbic acid protects the body against oxidative stress [15,16]. The amount of ascorbic acid (vitamin C) is a basic indicator to define the marketability of fruit and vegetables [17]. It is believed that vitamin C acts as an intermediate in the biosynthesis and metabolism of substances that are involved in the immune system [18]. Ebrahimzadeh et al. [19], reported a significant difference among citrus species for vitamin C contents. Therefore, the aim of this study was to investigate the effects of different climate zones, as a manifestation of changes in temperature and humidity, is the persimmon fruit quality. Table 1. Meteorological data Daily data of maximum temperature, minimum temperature, average temperature and average relative humidity (During the growing season in 2014) of Iran Meteorological Organization (IMO) were received. Annual average calculated in Table 2 were reported. The difference between the dailymaximum and minimum temperature, the diurnal temperature range (DTR) was calculated. Fruits In this study, based on a randomized complete block design with three replications, from trees that have the same morphological characteristics, such as height, age and canopy, were sampled. To harvest fruit, the color index similar to citrus fruit (orange lightorange) was used. Measuring of biochemical characteristics of fruit To measure vitamin C from 2,6-Dichloroindophenoltitration method was used and the amount of vitamin C was calculated from the following equation [20]. Total soluble solid (TSS) in the juice was determined with a hand-refract meter (PR-32 palette, Atago Co., Japan) at room temperature and expressed as a Percentage [21]. Titratable acidity (TA) was determined in the presence of phenolphthalein (pH=8.2) and expressed as citric acid percent (Rabiei, 2006). pH of the juice was measured using a pH meter (Jenway,3020).To determine the amount of chlorophyll and carotenoid, first 0.5 g of sample was pulverized in the presence of 10 ml acetone 80%. The solution was centrifuged for 5 min at 5000rpm. Using a spectrophotometer (UV-1800 PC), the absorption rate in wavelength of 480, 510, 645 and 663 nm was recorded. Acetone 80% was used as a blank spectrophotometry [22]. The following formula for the amount of chlorophyll a, b, a * b and carotenoid were calculated: Fruit biochemical data were analyzed using ANOVA of SAS software (SAS 9.1), and the SNK post hoc test was employed to compare treatment means. Vitamin C (ascorbic acid) content Different climatic conditions in the regions studied, very significant effect on the amount of ascorbic acid (P ≤0.01). Based on research findings, fruits that were harvested from the areas of climate Yazd and Kashan, total vitamin C contentmore than the fruits that were harvested from other parts of the climate ( Table 2). According to Table 2, the highest vitamin C content of the fruit Table 1: Geographical location and climatic characteristics of the regions studied. *The term above mean sea level (AMSL) is the elevation or altitude of any object, relative to the average sea level. ** Average of annual temperature and precipitation. Table 2: Vitamin C, chlorophyll a, b and ab activity and carotenoid content of fruit harvested in different regional climate, during growth of persimmon fruit in 2012. harvested from Yazd and Kashan climate regions, respectively, with values of 74.1 and 63.1 (mg/100 ml of fruit juice) and the lowest level was in sari climate region (76.0 mg/100ml of fruit juice). Many actions and reactions that occur in plants, each to be affected by temperature fall. The role of temperature on photosynthesis and respiration reactions can be noted among. The net value of the daily work of photosynthesis by plants and accumulated difference between the amount of carbohydrate that is made during the day and the amount of carbohydrate per day (24 hours) consumed by respiration. Plant for biological activity and construction, only products that is added daily during the photosynthesis process, it can consume or store it in an organ or other plant organs accumulated. However, it was found that when the total daily respiration is more than the daily photosynthesis, the plant products that already had accumulated storage consumes, if the trend continues, with a lack of food plants will be lost. With increasing temperature, respiratory rate becomes faster than the rate of photosynthesis, and the pure substance that is made daily by the plant reduced. The process of photosynthesis is limited to circumstances where there is enough light during the day, while the respiration process continues over a day (every hour of the day) the night temperature?? Has an important influence ІІe on the pure substance that is made daily as a result of photosynthesis in plants. By breathing at 25°C, more food is consumed than 15°C [23]. Regional climate Thus, areas with relatively cool nights and warm days are the pure substance stored in the plant and the yield will increase. On the contrary, in areas that are relatively warm nights of the pure substance stored and the yield is generally low. Based on the findings of the report could be said that in areas where the temperature difference between day and night was higher than other regions, have increased the amount of material stored and also had a higher qualitative characteristics. Therefore, it is believed that the reason for the higher rate of vitamin C fruits harvested from climatic regions of Kashan, Yazd, and shahrud than the Kiasar and Sari climate is the temperature Hamedani et al. [24]. Temperature difference between day and night for them. Hamedani et al. [24], Showed that the amount of vitamin C content in the orange fruit is affected by the temperature and the amount varied at different temperatures. Chlorophyll and carotenoid contents of fruit Determining the chlorophyll a and b and ab activity fruits harvested from different climatic regions showed that the climatic conditions of the region had a significant effect on the chlorophyll a activity (P ≤ 0.01). While climatic conditions of the regions studied the activity of chlorophyll b and ab fruit had no significant effect ( Figure 1). Results showed that the chlorophyll a activity affected by regional climate. As fruits that were harvested from the region of Yazd, highest of the chlorophyll a activity, compared with other parts of the climate. While, the amount of activity chlorophylls b and ab in different climatic conditions studied did not change. Also, different climatic conditions have a significant effect on fruit carotenoids content (P ≤ 0.01). Based on the results, the carotenoid content of fruits that harvested from the region of Yazd, a higher rate than other parts of the climate (Figure 1). In the early season vegetative growth to reproductive parts (fruits) more. Therefore, many materials are synthesized by the leaves of the vegetative organs stores; but with the growth of fruit photosynthetic products in the fruit stored [25]. Therefore, the amount of chlorophyll in fruit during ripening is reduced (Fattahi moghaddam 2010). As the fruits exposed to higher and lower temperatures than optimal, the fruit quality decreases. Perveen et al. [26], showed at temperatures close to zero and less than zero, the amount of chlorophyll a, b and carotenoid content in wheat was zero temperature. Sharma et al. [27], reported that, at a temperature close to 30°C, chlorophyll and carotenoid content is highest than 45°C. Acidity (pH), Titratable Acidity (TA) and Total Soluble Solid (TSS) The results showed that the different climatic conditions had a significant effect on pH, TA and TSS of fruit (P ≤ 0.01). Thus, the fruits harvested in the Kashan region with value 6.6 had the highest pH while the highest TA and TSS (0.04 and 22.13 mg/100ml) was for the shahrud area, ( Table 3). The physiological activity of fruit in many species with exposure to high and low temperatures reduced [28]. Fruit soluble solids content more dependent on the synthesis and transport of assimilates from the leaves to fruits [29]. Role of the high temperatures in transfer of the fruit, during fruit ripening is known [30]. As low night temperatures reduce the soluble solids content [31]. Therefore, it is believed that high levels of soluble solids content in Kashan and Yazd climates than the Kiasar and Sari climates, related to the higher the day and night temperature in these areas. Based on the results of Javanmardi and Kubota [32], by studying changes in quality of tomatoes grown in greenhouse conditions showed that, Approaching the peak temperature and solar radiation Fruit titratable acid content decreased with the increase in sugar. The results of this study, the results Khanal et al. [33], to increase the soluble solids content and titratable acid reduced the fruit under different temperature conditions confirmed. Conclusion Based on the finding, the effect of different regional climate on persimmon fruit quality were significant (P ≤0.01).It is believed that, the day and night temperature changes on the amount of vitamin C, chlorophyll a, b and a, b activity and carotenoid content of harvested fruits was effective. Based on these findings, and considering the use of the properties of persimmon fruit in different sectors of agriculture, industry, medicine and nutrition, the importance of fruit quality should be taken into consideration more than ever.

v3-fos

2019-03-19T13:05:32.507Z

2015-01-01T00:00:00.000Z

55941268

s2

Isolation and Characterization of Psychrotolerant Serratia Quinivorans Strains Secreting β-D-galactosidase Cold adapted and extracellular β-D-galactosidase (EC 3.2.1.23) with high specific activity has potential in food industry. Two psychrophlic bacterial isolates (A5-2 and B8) were screened from soil collected from permanent glaciers of Himachal Pradesh, India. Both A5-2 and B8 isolates showed growth between 4-25°C, but not at temperature higher than 30°C, hence classified as psychrotolerants. Biochemical characteristics and 16S rDNA sequencing identified the isolates as Serratia quinivorans A5-2 and Serratia quinivorans B8, and deposited in NCBI GenBank under accesion numbers KJ 176660 and KJ 176661, respectively. The cold active, extracellular β-D-galactosidase activity of A5-2 isolate was three fold higher compared to its intracellular activity. Comparatively, the B8 bacterial showed negligible intracellular activity, and its extracellular activity was two folds higher as compared to that of A5-2 isolate. Interestingly, growth and β-D-galactosidase activity of A5-2 was enhanced in lactose supplemented medium;whereas, growth of B8 isolate was unaffected and its β-D-galactosidase activity was enhanced when grown in lactose supplemented medium. β-D-galactosidase activity was also increased, when the isolates were grown in galactose supplementation medium, but decreased when grown in the presence of glucose. The presence of milk sugars like lactose, glucose, or galactose, or the milk metal ions, namely Ca and Na ions did not inhibit the activity of β-D-galactosidase. Introduction A significant area of earth's biosphere is encompassed by cold regions with an average temperature of 4-5℃ [1]. These regions are occupied by diverse organisms like arctic fish, terrestrial invertebrates, amphibians, arctic birds and microorganisms such as eubacteria [2][3][4], archaea [5][6][7][8] and yeasts [9][10][11]. The cold adapted microbes are classified as psychrophiles, while cold-tolerant microbes as psychrotolerants [12][13]. Enzymes (example, proteases, lipases, amylases, cellulases, dehydrogenases, lactases/ β-D-galactosidases) produced by cold adapted microorganisms have been used in various industries such as detergents, food and dairy, cosmetics, textiles and biosensor applications [14]. Cold-active β-D-galactosidases have attracted the dairy industry for the production of lactose-free milk in cold storage to treat lactose intolerance. β-D-galactosidase hydrolyses lactose in milk into glucose and galactose, which increase the sweetness of milk and are also more fermentable than lactose. Hydrolysis of lactose from the whey generated in the cheese production process could reduce the pollution problem related to the dairy industry [15]. Cold-active β-D-galactosidases are useful for the synthesis of galacto-oligosaccharides (GO), which function as effective pre-biotics for humans. Although β-D-galactosidases from mesophilic Kluyveromyces lactis is commercially available as Lactozyme (Novo Nordisk Co.), cold adapted β-D-galactosidases have not been produced on industrial scale. The cold active β-D-galactosidase for such applications should be active in the presence of Na + and Ca 2+ ions/ lactose, galactose and glucose. In addition, intracellular enzymes pose additional cost for the recovery of enzymes, thereby increasing the demand for extracellular β-D-galactosidases. Cold-active β-D-galactosidases have been isolated from Arthrobacter sp. Collection of soil samples, isolation and screening of bacterial isolates The soil sample was collected from Kafnu (altitude, 4500 m) and Sangla valley (altitude 5500 m) of Kinnour district of Himachal Pradesh, India. The soil samples were collected by using sterile spatula in polyethylene bags and stored at -20℃. 5 g of each soil sample was enriched in 1% lactose solution at 4℃. After 21 days of enrichment, 100 µl of soil free supernatant was spread on LB agar plates and incubated at 4ºC till bacterial colonies were observed. The distinct colonies were purified by three successive streaking on LB agar medium and incubated at 25℃. The isolated colonies were subjected to Gram's staining and growth characterization at different temperatures i.e., 4℃, 10℃, 15℃, 30℃ and 37℃ by streaking on LB agar medium. Qualitative and quantitative β-D-galactosidase enzyme assays Both isolates were grown in LB broth medium for 24 h at 25℃. Cell cultures were centrifuged at 8000 rpm for 5 minutes. Cell lysate was prepared in Z-buffer (60 mM Na 2 HPO 4 , 40 mM NaH 2 PO 4 , 10 mM KCl and 1 mM MgSO 4 ,pH 7.0) by adding 10 µl of chloroform and 10 µl of 10% SDS and incubation at 37℃ for 15 minutes. The amount of total protein in whole cell lysate and cell free supernatant was estimated by Bradford method [28]. Two microgram of total proteins of whole cell extracts or cell free supernatant were spotted on LB agar medium supplemented with either X-gal (0.1 mM of X-gal in N, N dimethyl-Formamide), or X-gal and IPTG (25 mM) or X-gal and lactose (30 mM) and incubated at 25℃ for 24 h and analysed for the formation of blue colored product. For quantitative assay of β-D-galactosidase enzyme, bacterial cells were grown in LB broth medium for 96 h. The cell extracts and supernatant were assayed for β-D-galactosidase enzyme activity using 0.33 mM ONPG (ortho-nitrophenyl-β-D-galactopyranoside) as the substrate in Z-buffer [29]. The reaction was carried out at 30º C for 1 h and stopped by addition of 1 M Na 2 CO 3 . The release of ortho-nitrophenol from ONPG was measured at 420 nm and estimated using standard graph of o-nitrophenol. The specific activity of β-D-galactosidase enzyme was calculated as µmoles of o-nitrophenol released per minute per microgram (U/mg) of total protein. Effect of milk sugars and metal ions on growth and β-D-galactosidase activity of A5-2 and B8 isolates To study the effect of lactose on growth and β-D-galactosidase production, both A5-2 and B8 isolates were cultured either in LB broth or LB broth supplemented with 1% lactose and incubated at 25℃. Cell cultures were withdrawn at different time intervals (0 -160 h). At each time interval, cell density was measured at 600 nm and β-D-galactosidase activity in the cell-free supernatant (5 μg total protein) was determined using ONPG as substrate. Similarly, the effect of glucose and galactose on the growth of the isolates and β-D-galactosidase activity was studies at different time intervals of growth (0-100 h). To study the effect of milk sugars (glucose, galactose, and lactose) on β-D-galactosidase enzyme activity, A5-2 and B8 were grown in LB broth without supplementation of sugars and ONPG assays were performed using cell free supernatant (5 μg total protein) in the presence of different concentrations (2.5, 12.5 and 25 mM ) of glucose, galactose or lactose. To study the effect of metal ions, such as Ca 2+ and Na + on the β-D-galactosidase activity, cultures were grown without supplementation of sugars as described above. ONPG assays were performed using cell free supernatant (5 μg total proteins) in the presence of different concentrations (5,10, and 20 mM) of CaCl 2 and NaCl. Molecular identification of bacterial isolates A5-2 and B8 by 16S rDNA amplification and sequencing Total genomic DNA from A5-2 and B8 isolates was isolated using genomic DNA extraction (Fermentas Co., USA). Amplification of 16S rDNA was carried out using 100 ng of genomic DNA as template and the universal primers 27F-(5' GAGTTTGATCCTGGCTCAG-3') [31] and 1492R (5'-GGTTACCTTGTTACGACTT-3') [32]. The annealing temperature was 45˚ C. After PCR amplification, reaction products were separated on 1.0% agarose gel and visualized in a gel documentation system. DNA sequencing was done on both the strands using 27F and 1492R primers at Eurofins Genomics Pvt. Ltd., Bangalore, India. The 16S rDNA gene sequence was subjected to BLAST analysis in the NCBI database (http/www.ncbi.nlm/nih.gov) and aligned using the Clustal W program [33]. The phylogenetic tree for the data set was constructed using the Phylip computer programme package drawgram 3.66 [34]. The retrieved sequences were aligned using Phylip software and phylogenetic tree was constructed using Phylip computer programme package drawgram 3.66. PsychrotoLERANT SERRATIA QUINIVORANS STRains A5-2 and B8 Secrete Extracellular and Lactose Inducible β-D-galactosidase during the Decline Phase of growth In order to isolate psychrophilic bacteria, we collected soil samples from Kafnu and Sangla valleys of the Himalayan range located in Kinnour District, Himachal Pradesh. The valleys are covered with snow throughout the year. The soil microflora were enriched for lactose degrading bacteria in 1% lactose solution. The enriched microflora were plated on LB agar medium supplemented with lactose and incubated at 4℃. Distinct colonies were observed on 7 th day of plating (data not shown). 14 morphologically distinct bacterial colonies were picked and streaked by three successive streaking on LB agar medium and incubated at 4℃. To test the psychrophilic nature of bacterial isolates, all the isolates were streaked on LB agar medium and incubated at different tempertures ranging from 4 to 40℃ (Table 5.1). Only two isolates, namely A5-2 and B8 showed visible growth at 4℃ during the third day and no growth at temperature higher than 25℃. The optimum temperature for the growth of A5-2 was 20℃, and 25℃ for B8 isolate (Table 5.2). The optimal pH for both the isolates was 7 (Table 5.2). Thus, A5-2 and B8 were classified as psychrotolerants. A5-2 and B8 isolates were further pursued for the biochemical characterization of the isolates and extracellular nature of β-D-galactosidase. The Gram's staining revealed that both isolates were Gram's negative and uniform rods (Table 5.2). Both isolates were coagulase negative, catalase positive, oxidase negative, lactose fermenting, indole negative, MR-VP negative, lysine decarboxylase positive, motile and sugars fermenting (Table 5.2). Based on biochemical tests, both A5-2 and B8 are identified as Serratia sp. belonging to Enterobacteriaceae family (Table 5.2). To confirm the identity of Serratia sp. at the molecular level, and examine their phylogenetic position, total genomic DNA was subjected to PCR amplification of gene encoding 16S rRNA. A PCR product of approximately1500 bp was detected (data not shown). The 16S rDNA sequence thus obtained was subjected to homology search by BLAST searchof the NCBI database and both the sequences (A5-2 and B8) showed 98% similarities with Serratia quinivorans strain 4364. Phylogentic analysis revealed that A5-2 and B8 formed a distinct cluster from other Serratia spp. As well as from other members of Enterobacteriaceae family) (Figure 1 A & B). Based on biochemical properties, 16S rDNA sequence and phylogenetic analysis, we classified isolate A5-2 as Serratia quinivorans strain A5-2 and B8 as Serratia quinivorans strain B8. The multiple sequence alignment of 16S rDNA of both the isolates showed 99% similarities with substitution at positions G528A, T584C, G642A and addition of G575 in A5-2 isolate, and addition of T607 and C670 in the sequence of B8 isolate (data not shown). 16S rDNA sequences of Serratia quinivorans strain A5-2 and Serratia quinivorans strain B8 have been submitted to the NCBI GenBank database with accession number KJ 176660 and KJ 176661, respectively. We assumed that both the psychrotolerant strains are novel, and therefore characterized them for the extracellular production of β-D-galactosidase. Qualitative assays showed the presence of enzyme activity in the whole cell extracts and cell free spent medium of both A5-2 and B8 isolates, when tested by spotting assay on LB agar medium supplemented with X-gal as a substrate and lactose or IPTG as inducer (data not shown). This suggests an intracellular and extracellular nature of β-D-galactosidase enzyme produced by A5-2 and B8 isolates. Moreover, β-D-galactosidase activity was detected in the absence of IPTG or lactose, suggesting an auto-inducible nature of the enzyme. Quantitatively, we observed approximately 10 folds increase in the extracellular activity (1800 U/mg) as compared to intracellular β-D-galactosidase activity (186 U/mg) in Serratia quinivorans strain A5-2 strain ( Figure 2). Comparatively, very low amount of intracellular β-D-galactosidase activity (221 U/mg) was observed in Serratia quinivorans strain B8, and 14 folds more extracellular activity (2800 U/mg) (Figure 2). Due to the high levels of extracellular β-D-galactosidase production, A5-2 and B8 isolates were pursued for characterization of β-D-galactosidase. Figure 2. Estimation of β-D-galactosidase activity in A5-2 and B8 isolates. Cells were grown at 25℃ till A600 reached 1.0. Cell lysate (C) and supernatant (S) of A5-2 and B8 isolates were analyzed for β-D-galactosidase activity using ONPG as a substrate. The specific activity (U/mg) is plotted against the indicated samples. Effect of Substrate and Products on the Growth and Production of β-D-galactosidase To optimize the growth conditions for β-D-galactosidase production by A5-2 and B8 isolates, we studied the effect of lactose (substrate), glucose and galactose (products). Microbial growth of A5-2 and B8 isolates was markedly enhanced when cultured in the medium supplemented with lactose as compared to non-supplemented medium (Figure 3 A & B). In addition, the duration of stationary phase was prolonged in non-supplemented LB broth medium. In the case of A5-2 isolate, there was a marked increase in the β-D-galactosidase activity in the presence of lactose, with maximum activity (9000 U/mg) at 90 h of growth as compared medium without lactose supplementation, in which maximum activity (5500 U/mg) was achieved at 140 h of growth for ( Figure 3A). On the contrary, B8 isolate did not exhibit any significant difference in the growth pattern in the presence and absence of lactose, but showed a drastic increase in the β-D-galactosidase activity in lactose grown cultures ( Figure 3B). The maximum β-D-galactosidase activity (23000 U/mg) of B8 isolate was observed at 100 h in the lactose grown cultures as compared to 8000 U/mg at 130 h in the absence of lactose (Figure 2.4.3B). The correlation of enzyme activity with the growth kinetics indicated that maximum β-D-galactosidase activity was observed during the late stationary phase of growth of both the isolates (Figure 3 A&B). Figure 3. Effect of lactose on growth of A5-2 and B8 isolates and their β-D-galactosidase production. Both A5-2 and B8 isolates were inoculated in LB broth medium (Lac -) or LB broth supplemented with 1% lactose (Lac + ) and incubated at 25˚C. Samples were harvested at indicated time periods of incubation and β-D-galactosidase activity was measured in cell free spent medium. At each time point, cell density was measured by measuring absorbance at A600 nm. The β-D-galactosidase activity and the corresponding cell density (absorbance at 600 nm) for each time point are plotted against the time of incubation for A5-2 (panel A) and for B8 (panel B) isolates. Similarly, we studied the effect of glucose and galactose supplementation in the growth medium and its correlation with microbial growth and β-D-galactosidase activity. There was a marginal increase in the growth of A5-2 and B8 isolates in the presence of galactose and glucose (Figure 4 A & C). β-D-galactosidase activity of both A5-2 and B8 was enhanced when galactose was supplemented in the growth medium (Figure4 B & D). While glucose had no effect on the β-D-galactosidase activity of A5-2 ( Figure 4B), the activity was undetectable when B8 isolate was grown in the presence glucose (Figure 4 D). A B C D Figure 4. Effect of galactose and glucose on the growth and production of β-D-galactosidase of A5-2 and B8 isolates. A5-2 and B8 isolates were grown in LB broth supplemented with 100 mM of galactose or glucose. β-D-galactosidase activity and growth was measured at 24, 48, 72 and 96 h of incubation. The microbial growth and enzyme activity is plotted as indicated for A5-2 (A, microbial growth; B, β-D-galactosidase activity) and B8 (C, microbial growth; D, β-D-galactosidase activity) isolates. Metal Ions and Sugar Constituents of Milk do not Affect β-D-galactosidase Activity in vitro Since β-D-galactosidase has potential applications in dairy industries, it would be ideal if its activity is not inhibited by sugars (lactose, galactose and glucose) and metal ions such as Ca 2+ and Na + present in the milk and dairy products. To study the effect of sugars (galactose, glucose, and lactose) and metal ions on β-D-galactosidase activity, A5-2 and B8 isolates were grown in LB broth medium. The cell free spent medium was incubated with different concentrations of indicated sugars (2. 5, 12.5, and 25 mM) or metal ions (10-30 mM), followed by β-D-galactosidase assay. There was a mild reduction (10-30% inhibition) in the β-D-galactosidase activity of both A5-2 and B8 isolates in the presence of lactose, glucose or galactose ( Figure 5 A & B). More interestingly, the presence of metal ions such Ca 2+ and Na + at 10-30 mM did not affect the β-D-galactosidase enzyme activity of A5-2 and B8 isolates (Figure 6 A & B). (A) (B) Figure 5. Effect of sugars (lactose, galactose and glucose) on β-D-galactosidase activity. A5-2 and B8 isolates were grown in LB broth for 96 h at 25℃. The cell free spent medium was incubated with various concentrations of lactose, glucose or galactose (0.1, 0.5% and 1%) for 5 minutes, followed by β-D-galactosidase assay. The relative specific activity was calculated and plotted against the concentration of sugars for A5-2 (A) and B8 (B) isolates. These results clearly suggests that the presence of sugars in the production medium has greater role in either stimulating or inhibiting the β-D-galactosidase enzyme activity (3.2) as compared to the presence of sugars in the enzymatic assays (3.3). (A) (B) Figure 6. Effect of Ca 2+ and Na + on β-D-galactosidase activity. The cell free spent medium was incubated with indicated concentrations of either CaCl2 or NaCl for 5 minutes and β-D-galactosidase activity was measured by ONPG assay at 30℃. The relative β-D-galactosidase activity is plotted against the concentration of metal ions as indicated for A5-2 (A) and B8 (B) isolates. Discussion β-D-galactosidase enzyme is an essential tool for the commercial production of lactose-free milk and related dairy products. The cold storage of milk and milk products necessitates the use of cold active enzymes for their processing, which are not inhibited by constituents of milk like sugars and metal ions. Moreover, extracellular enzymes are commercially more viable than the intracellular ones. β-D-galactosidases naturally produced by psychrophilic microorganisms are either intracellular or expressed at low levels. Therefore, the present study was undertaken to isolate the psychrophilic bacteria producing cold active, extracellular β-D-galactosidases. We report the isolation of two new psychrotolerant bacterial isolates A5-2 and B8 that could grow optimally at 25℃ and exhibit no detectable growth at temperatures higher than 25℃, hence classified as psychrotolerant according to Morita [12]. Biochemical characterization of A5-2 and B8 isolates (Table 5.2) showed that both the isolates are Gram's negative rods and motile and belong to thegenus Serratia. In agreement with the biochemical characteristics, 16S rDNA sequence analysis revealed their close identity (98% homology) to Serratia quinivorans strain 4364. Both the isolates are closely related to Serratia quinivorans strain 4364, but differ from each other at nucleotide positions 528, 575,584, 607, 642, and 670 7 of the 16S rDNA. Thus the strains were named as Serratia quinivorans B8 and Serratia quinivorans A5-2. Isolates.. Phylogenetic analysis revealed that both the isolates form an independent cluster and are closely related to Yersinia sp., Hafni sp., Rahnella sp., and Obesumbacterium sp., as compared to the existing Serratia sp. Therefore, we conclude that S. quinivorans A5-2 and S. quinivorans B8 are novel members of the genus Serratia, which are also psychrotolerants. Very little is known about the existing S. quinivorans. It was originally classified as Serratia proteamaculans subsp. Quinivora, and later categorized as Serratia quinivorans [35,36]. To the best of our knowledge, there is no report of β-D-galactosidase from cold adapted Serratia quinivorans. Moreover, the extracellular nature of the enzyme makes it more important for industrial applications. Therefore, we further characterized and compared the β-D-galactosidase from Serratia quinivorans A5-2 and Serratia quinivorans B8 isolates. Microorganisms that produce β-D-galactosidase have been extensively studied. β-D-galactosidases naturally produced by psychrophilic microorganisms are either intracellular or expressed at low levels. In contrast, bacterial isolates S. quinivorans A5-2 and B8 secrete extracellular β-D-galactosidase (8000 and 23000 U/mg respectively), which is constitutively expressed. Conclusions In the present study, we isolated two novel species of psychrotolerant Serratia quinivorans which secrete cold active β-D-galactosidase. The β-D-galactosidase produced by Serratia quinivorans A-5-2 and B8 possesses two major advantages, 1) extracellular nature of the enzyme, and 2) enzyme activity not inhibited by the substrate (lactose), reaction products (glucose and galactose) and metal ions (calcium and sodium) present in the milk. Due to these unique features, Serratia quinivorans A5-2 and Serratia quinivorans B8 would be advantageous over the existing isolates for the production of β-D-galactosidase and its application in milk and dairy industry. The purification and characterization of the β-D-galactosidase enzyme from both the isolates would be the immediate need for its commercial exploitation.

v3-fos

2016-05-16T03:58:36.046Z

2015-09-01T00:00:00.000Z

9899638

s2

The effect of D123 wheat as a companion crop on soil enzyme activities, microbial biomass and microbial communities in the rhizosphere of watermelon The growth of watermelon is often threatened by Fusarium oxysporum f. sp. niveum (Fon) in successively monocultured soil, which results in economic loss. The objective of this study was to investigate the effect of D123 wheat as a companion crop on soil enzyme activities, microbial biomass and microbial communities in the rhizosphere of watermelon and to explore the relationship between the effect and the incidence of wilt caused by Fon. The results showed that the activities of soil polyphenol oxidase, urease and invertase were increased, the microbial biomass nitrogen (MBN) and microbial biomass phosphorus (MBP) were significantly increased, and the ratio of MBC/MBN was decreased (P < 0.05). Real-time PCR analysis showed that the Fon population declined significantly in the watermelon/wheat companion system compared with the monoculture system (P < 0.05). The analysis of microbial communities showed that the relative abundance of microbial communities was changed in the rhizosphere of watermelon. Compared with the monoculture system, the relative abundances of Alphaproteobacteria, Actinobacteria, Gemmatimonadetes and Sordariomycetes were increased, and the relative abundances of Gammaproteobacteria, Sphingobacteria, Cytophagia, Pezizomycetes, and Eurotiomycetes were decreased in the rhizosphere of watermelon in the watermelon/wheat companion system; importantly, the incidence of Fusarium wilt was also decreased in the watermelon/wheat companion system. In conclusion, this study indicated that D123 wheat as a companion crop increased soil enzyme activities and microbial biomass, decreased the Fon population, and changed the relative abundance of microbial communities in the rhizosphere of watermelon, which may be related to the reduction of Fusarium wilt in the watermelon/wheat companion system. Introduction Watermelon [Citrullus lanatus (Thunb.) Matsum and Nakai] is a widely cultivated fruit that is consumed globally. However, under continuous cropping patterns, the growth of watermelon is often threatened by the pathogen Fusarium oxysporum f. sp. niveum (Fon). The continuous cropping of the same crop in the same land can negatively affect the yield and quality of crops (Yu et al., 2000;Yao et al., 2006;Wang et al., 2014a) because it eliminates biological diversity (Blanco-Canqui and Lal, 2008). To overcome this problem, we proposed to increase the diversification of cultivated species and suggest that intercropping is the most efficient practice to reduce the incidence of soilborne diseases (Ren et al., 2008;Zhang et al., 2013). Soil enzyme activities are indicators of soil health and have been applied to analyze soil quality and ecosystems (Ndiaye et al., 2000). Most commonly, urease is used to monitor the soil nitrogen cycle and nitrogen utilization because it can hydrolyze urea to ammonia. Invertase is used to monitor the change of soluble nutrients in the soil (Li et al., 2012). Phenol oxidase may be involved in the decomposition of toxic compounds in soils (Sinsabaugh, 2010). These enzymes, as well as others, have been used to evaluate the effects generated by agricultural practice (Zhou et al., 2011;Wang et al., 2014a,b). The microbial biomass reflects the turnover of soil microorganisms and acts as both a source and a pool for nutrients (Irshad et al., 2012). Considering the species-specific effect of plants on the microbial community of a rhizosphere in an intercropping system, investigating the microbial biomass in the rhizosphere of intercrops compared with monocultures can provide meaningful data (Tang et al., 2014). The soil microbiome is thought to be responsible for biological processes that are necessary for maintaining a healthy soil and suppressing plant diseases (Garbeva et al., 2004;Pedersen and Mills, 2004;Andreote et al., 2014). Mazzola (2004) reported that a decrease in soil microbial diversity was related to the development of soil-borne plant diseases. Soils with a higher fungal diversity exhibited a higher potential of disease suppression (Penton et al., 2014). Intercropping can improve soil microbial diversity and change microbial communities; therefore, it plays an important role in controlling plant disease. For example, tomato blight disease can be controlled by using a tomato-marigold intercropping system (Gómez-Rodrígueza et al., 2003). Ren et al. (2008) demonstrated that intercropping with aerobic rice alleviated Fusarium wilt in watermelon by changing the microbial communities in the rhizosphere. In a previous study, D 123 wheat was used as a companion crop to reduce the incidence of watermelon Fusarium wilt (Xu et al., 2015). However, little information is known about how D 123 wheat, as a companion crop, exerts this effect. Therefore, the aim of this study was to evaluate the effect of D 123 wheat on soil enzyme activities, microbial biomass, and microbial communities in the rhizosphere of watermelon and explore the relationship between the effect and incidence of watermelon Fusarium wilt. Plant Material The D 123 wheat seeds were provided by the Vegetable Physiological Ecology Laboratory College of Horticulture at Northeast Agricultural University in Harbin, Heilongjiang Province. Seeds of the watermelon cultivar Jingxin No. 1, which is moderately susceptible to Fon, were bought from the Golden Seed Company, Beijing, China. Greenhouse Experiment This study was performed in a greenhouse located in the experimental center of Northeast Agricultural University in Harbin, China (45 • 41 ′ N, 126 • 37 ′ E). The soil used in pot experiments was collected from the surface of locations on Xiangfang farm in Harbin, China, where watermelon was cultivated continuously for 3 years and was infected by Fon. The soil contained 35.90 g·kg −1 of organic matter, 357.00 g·kg −1 of alkaline hydrolytic N, 378.80 g·kg −1 of available P, and 107.50 g·kg −1 of available K. The electrolytic conductivity was 1.27 ms·cm −1 , and the pH was 7.23 (1:5, soil:water). Two cropping systems were included in these experiments: (I) watermelon monoculture (CK2) and (II) watermelon/wheat companion system, in which D 123 wheat (D 123 ) was used as a companion crop because a previous study reported that the root exudates from D123 wheat can inhibit the mycelial growth of Fon (Xu et al., 2015). In addition, control pots were designed that did not contain any plants (CK1). All of the pots were arranged randomly with three repeats per treatment and 10 pots per repeat. Watermelon seedlings with five leaves were transplanted into plastic pots (20 cm in diameter and 17 cm in height). Each pot was filled with 3 kg fresh soil from the infected field mentioned previously. No fertilizers were added to the soil. In the watermelon/wheat companion system, D 123 wheat seeds were surface sterilized with 5% (v/v) H 2 O 2 for 30 min, rinsed four times with distilled water, and then directly sown on the side of the watermelon plant; each pot had 30 wheat seedlings and one watermelon seedling, and watermelon and wheat seedlings were kept apart 5-7 cm from each other. To ensure good aeration and avoid the shading of watermelon, the wheat seedlings were cut several times and kept to a 15 cm height during the experimental period. In the watermelon monoculture system, each pot contained one watermelon seedling only. Hand weeding was performed during the experiment. The water content of the soil was maintained by weight. No pesticides were sprayed. When the watermelon plants began to develop Fusarium wilt, the samples from the rhizosphere of the watermelon plant were collected from five plants in each repeat as described by Song et al. (2007). Briefly, five plants were excavated randomly, and the loosely adhered soil was shaken off; the tightly adhered soil was removed and mixed as the rhizosphere sample. Part of the sample was used to determine microbial biomass and soil enzyme activities, and the other part was stored at −70 • C for DNA extraction. Assessment of Disease Incidence The incidence of wilt was expressed as a percentage that was calculated by diseased plants over the total number of plants (Wu et al., 2009). Estimation of Microbial Biomass in the Rhizosphere Fumigation extraction methods were used to measure microbial biomass carbon (MBC), microbial biomass nitrogen (MBN), and microbial biomass phosphorus (MBP) in the watermelon rhizosphere as described by Brookes et al. (1985) and Vance et al. (1987). The 15 g fresh soil samples were placed in a 50 ml beaker, and another beaker was filled with 50 ml alcohol-free chloroform; both beakers were kept in a vacuum desiccator. As a control, soil that was not treated with chloroform was kept in another desiccator. Then, the two desiccators were kept in the dark for 24 h at room temperature. Subsequently, the two desiccators were evacuated using a vacuum pump, and the fumigated and nonfumigated samples were transferred to a 100 ml conical flask, respectively, and extracted with 0.5 M K 2 SO 4 by shaking for 30 min on a rotator at 300 rpm. The extracts were filtered through filter paper. The contents of organic carbon from fumigated and non-fumigated soil were measured using the dichromate digestion method. An extractability factor of 0.45 was used to calculate the MBC (Brookes et al., 1982). For MBN, the filtrates (20 ml) were digested with sulfuric acid (96%) in a 50 ml Kjeldahl bottle. The digested filtrates were team distilled using a semimicro Kjeldahl bottle and were titrated against hydrochloric acid (0.05 N). MBP was determined using the chloroform fumigationextraction method (Brookes et al., 1982). Soil DNA Extraction The genomic DNA of rhizosphere microorganisms was extracted with the PowerSoil R DNA Isolation Kit (MO BIO, USA) according to the manufacturer's instructions. Extracted DNA was stored at −20 • C until use. PCR Amplification and Miseq Sequencing The protocol used for PCR amplification was described by (Magoč and Salzberg, 2011). Each treatment (CK1, CK2, and D 123 ) was repeated three times, PCR reactions were performed in triplicate. The primer set of V338f (5 ′ -ACTCCTACGGGAGGCA GCA-3 ′ ) and V806R (5 ′ -ATGCAGGGACTACHVGGGTWTC TAAT-3 ′ ) was used to amplify the V 3 -V 4 region of the bacterial 16S rDNA, and the primer set of ITS1-737F (5 ′ -GGAAGTAAA AGTCGTAACAAGG-3 ′ ) /ITS2-2043R (5 ′ -ATGCAGGCTGCG TTCTTCATCGATGC-3 ′ ) was used to amplify the ITS2 (internal transcribed spacer 2) region of the fungi rDNA (White et al., 1990;Bazzicalupo et al., 2013). Because the entire ITS region is too long for high-throughput sequencing methods, recent highthroughput sequencing studies have selected either the ITS1 or ITS2 region (Mello et al., 2011;Orgiazzi et al., 2012). Due to systematic length differences in the ITS2 region as well as the entire ITS, Bellemain et al. (2010) found that ascomycetes will more easily amplify than basidiomycetes using ITS2 regions as targets. Ascomycota represents the largest phylum of fungi (Bellemain et al., 2010). Amplicon pyrosequencing was performed by Majorbio Bioinformatics Technology Co., Ltd. (Shanghai, China). The protocol used to determine the composition of the microbial communities in three treatments was described by Caporaso et al. (2010). All data sets were deposited into the NCBI Sequence Read Archive database (http:// www.ncbi.nlm.nih.gov/Traces/sra). Sequences were analyzed using the QIIME (Wang et al., 2007) software and UPARSE pipeline (Edgar, 2013). Pairs of reads from the original DNA fragments were merged using FLASH (Caporaso et al., 2010), which is a very fast and accurate software tool that was designed to merge pairs of reads when the original DNA fragments are shorter than twice the length of the reads. Sequencing reads were assigned to each sample according to the unique barcode of each sample. Briefly, the reads were filtered by QIIME (version 1.17) quality filters. Then, we used the UPARSE (version 7.1 http://drive5.com/uparse/) pipeline to pick operational taxonomic units (OTUs) to make an OTU table. Sequences were assigned to OTUs at 97% similarity. The chimeric sequences were identified and removed using UCHIME. We picked a representative sequences for each OTU and used the ribosomal database project (RDP) classifier (Caporaso et al., 2011) to assign taxonomic data to each representative sequence. The Analysis of Diversity The Shannon index (H shannon ) was determined as follows (Schloss et al., 2009): where S obs is the number of observed OTU S , n i is the number of individuals in the ith OTU, and N is the total number of individuals in the community. Additionally, the principal co-ordinate analysis (PCoA) was performed to estimate the community diversity between samples (Lozupone and Knight, 2005) based on the Illumina-MiSeq sequencing data with unweighted UniFrac distance matrix (Peiffer et al., 2013). Real-time PCR Assay The population of Fon in the soil samples was analyzed by real-time PCR. The primer pair of fn-1 (5 ′ -TACCACTTGTTG CCTCGGC-3 ′ ) and fn-2 (5 ′ -TTGAGGAACGCGAATTAAC-3 ′ ) was used to identify Fon (Zhang et al., 2005). The real-time PCR assays were performed with an IQ5 Real-Time PCR System (Bio-Rad Lab, LA, USA). The reaction volume was 20 µl, which contained 10 µl of 2 × Real SYBR Mixture (TIANGEN Biotech, China), 0.5 µl of each primer, and 2 µl of template DNA. The PCR process was 94 • C for 5 min; 95 • C for 30 s, 54 • C for 30 s, 72 • C for 30 s for 35 cycles in total; and a final elongation at 72 • C for 7 min. The signal threshold was set automatically by the system. To evaluate amplification specificity, melt curve analysis was performed at the end of the PCR run (see Supplementary Material). The plasmid standard for the quantification of Fon was generated from a cloned target gene from Fon genomic DNA. The amplicon was purified using the PCR Purification Kit (BioTeke Corporation, China) and ligated into a pMD18 T vector (Takara, Dalian) according to the manufacturer's instructions. Plasmid from the insert-positive clones was extracted with a Plasmid Extraction Kit (BioTeke Corporation, China). The concentration of plasmid DNA was measured and converted to copy concentration using the following equation as described by Whelan et al. (2003): DNA (copy) = 6.02 × 10 23 (copies mol −1 ) × DNA amount (g) DNA length (bp) × 660 (gmol −1 bp −1 ) Standard curves (see Supplementary Material) were performed with 10-fold dilution series of plasmids. Sterile water was used as a negative control to replace the template. All real-time PCR reactions were done in technical triplicates such that each treatment was analyzed nine times. Statistical Analyses Differences were calculated with One-Way analysis of variance (ANOVA) at the end of each assay. The data were analyzed by Duncan's multiple range test and expressed as the means ± standard error, and the incidence of watermelon Fusarium wilt was analyzed by independent samples T-test using the SAS statistical software. Incidence of Watermelon Fusarium Wilt was Affected by Companion with Wheat The incidence of watermelon Fusarium wilt was investigated. The rate was 63.3% (P < 0.05) in the monoculture system but was significantly lower at 21.1% (P < 0.05) in the watermelon/wheat companion system (Figure 1). Soil Enzyme Activities In this study, the activities of four soil enzymes in the watermelon rhizosphere (including control soils) were compared. The invertase activity increased significantly in the watermelon/wheat companion system compared with that in the monoculture system; however, the invertase activity in the control soil (without plants) was lower than that in the monoculture (P < 0.05; Figure 2A). The catalase activity in the control soil was lower than that in both the watermelon/wheat companion system and the monoculture system, but no significant difference was found between the watermelon/wheat companion system and the watermelon monoculture system (P < 0.05; Figure 2B). The polyphenol oxidase activity was higher in the watermelon/wheat companion system than that in the monoculture and control (without plants, P < 0.05; Figure 2C). The urease activity was highest in the watermelon/wheat companion system, lower in the monoculture system, and lowest in the control (without plants), and significant differences in urease activity existed between these treatments ( Figure 2D). Microbial Biomass No significant differences in MBC were detected among the three treatment soils (Table 1). However, the soil MBN and MBP were significantly higher in the watermelon/wheat companion system and the control (CK1) than in the monoculture system. Compared with the monoculture (CK2), the MBC/MBN ratio was significantly decreased in the watermelon/wheat companion system and control (CK1) ( Table 1, P < 0.05). Fon Population Real-time PCR analysis was performed to determine the copy numbers of the target DNA in the rhizosphere of watermelon collected from the watermelon/wheat companion system, monoculture system, and control soil, respectively. A significant difference was found between the watermelon/wheat companion system and the monoculture system (Figure 3). For the three treatment soils, the highest population of Fon was detected in the rhizosphere of watermelon in the monoculture system (up to 49.2 × 10 8 copies·g −1 soil). The population was lower in the control soil, at 31.1 × 10 8 copies·g −1 soil. However, the lowest Fon population was found in the rhizosphere of watermelon in the watermelon/wheat companion system, at 13.6 × 10 8 copies·g −1 soil. Pyrosequencing and Sequence Analysis The number of species detected in the samples, or the number of organisms at a given phylogenetic level, relies largely on the amount of analyzed sequences (Schloss and Handelsman, 2005). In the present study, the average sequence lengths for 16S rDNA and ITS2 were approximately 430 and 270 bp, respectively. After removing the low-quality sequence reads, a total of 174,251 sequences were obtained, in which 76,545 sequences were classified as bacterial and 97,706 were classified as fungal. They were used to evaluate the microbial richness and diversity and the differences among the treatments. Microbial Community Composition Analysis The maximum bacteria OTUs detected were 1854, 1763, and 1649 for the rhizosphere of the monoculture (CK2), the rhizosphere of the companion system (D 123 ), and the control soil (CK1), respectively ( Figure 4A). The cut-off for the analysis was at 97% sequence similarity. For fungi, 927 OTUs were found in the rhizosphere of the companion system (D 123 ), 829 OTUs were found in the rhizosphere of the monoculture watermelon (CK2), and 678 OTUs were found in the control soil (CK1) (Figure 4B). The rarefaction curves of both bacteria and fungi showed that the sequencing capability was not sufficiently large to capture the complete diversity of communities because the curves did not reach a plateau by increasing sample size. However, the data were sufficient to show differences among the treatments. All of the sequences were classified into 19 known classes by the mother program, "Others, " if labeled in data, meant that the sequences could not be classified into any known group. The results showed that the overall bacterial composition was similar for each treatment, whereas the relative abundance of each class varied in different treatments (Figures 5A-C). Alphaproteobacteria, Gammaproteobacteria, and Actinobacteria were the top three classes among all bacterial classes, which comprised 19. 06,18.39,and 13%,respectively,in CK1 soil;13.3,17.99,and 10.59%,respectively,in CK2 soil;and 16.35,16.2,and 10.83%,respectively,in D 123 soil. Sphingobacteria comprised 4.55,8.45,and 7.25% of the total bacterial communities in CK1, CK2, and D 123 soil, respectively. Gemmatimonadetes comprised 7.29, 6.23, and 7.92% of the total bacterial communities in CK1, CK2, and D 123 soil, respectively. Cytophagia comprised 3.37, 5.82, and 5.69% of the total bacterial communities in CK1, CK2, and D 123 soil, respectively. The overall fungal composition of the different soil samples was similar, whereas the distribution of each class was varied (Figures 6A-C). Compared with CK2, the relative abundance of Sordariomycetes was higher in the watermelon/wheat companion system, and Pezizomycetes was lower in the watermelon/wheat companion system. The PCoA analysis of the bacteria and fungi revealed three separated clusters (Figures 5D, 6D). Each cluster was distinguished from each other, which indicated that differences in the community diversity exist between the different cropping systems. The H shannon indices of soil bacterial and fungal community were also calculated. Compared with monoculture (CK2), the H shannon indices of soil bacterial and fungal community were increased in the watermelon/wheat companion system (Figure 7). Discussion This study showed that the incidence of watermelon Fusarium wilt was decreased in the watermelon/wheat companion system compared with a monoculture system (Figure 1). The result was consistent with the decline of the Fon population (Figure 3). These results suggest that the Fusarium wilt may be suppressed in the companion system, in accordance with the results reported by other studies (Larkin et al., 1993a,b). We also found that the activities of soil polyphenol oxidase, urease and invertase were increased significantly in the watermelon/wheat companion system compared with the monoculture system (Figure 2). Soil urease plays a vital role in utilization of soil nitrogen and the nitrogen cycle by decomposing urea into ammonia, carbon dioxide and water, which are beneficial for plant absorption (Wang et al., 2014a). The urease activity was sensitive to changes in cropping systems. The result is supported by other studies (Zhou et al., 2011;Xu et al., 2013). Extracellular phenol oxidases are deployed by both fungi and bacteria to mitigate the toxicity of phenolic molecules and aid in antimicrobial defense (Sinsabaugh, 2010). In our experiments, we observed that the polyphenol oxidase activity was higher in the watermelon/wheat companion system than in both the monoculture system and the control (without plants), which implied that the watermelon/wheat companion system was more beneficial in reducing the toxicity of phenolic acids in the rhizosphere of watermelon (Figure 2). Invertase widely exists in the soil and plays an important role in the transformation of carbon in the soil (Eivazi and Bayan, 1996). Dai et al. (2013) found that the intercropping of peanut with Atractylodes lancea effectively increased soil invertase activities. Additionally, Ahmad et al. (2013) found that pepper intercropping with green garlic significantly increased the activities of invertase in soil. The result obtained from this study is in agreement with their conclusion. In sum, the watermelon/wheat companion system (D 123 wheat as companion crop) changed the activities of soil enzymes, and these changes may reflect changes in the soil micro-environment (Iyyemperumal and Shi, 2008) and result in the inhibition of the growth of Fon, which exhibits a decreased incidence of the Fusarium wilt. Disease suppression is generally thought to be related to a global increase in soil microbial biomass (Janvier et al., 2007). In the present study, the watermelon/wheat companion system increased the total contents of MBN and MBP ( Table 1). The increase in MBN may be attributed to the increase in root exudates because the root exudates could come from both watermelon and D 123 wheat, which can lead to changes in the soil microbial community structure (Figures 5, 6). The MBP was also increased, which could be due to the watermelon/wheat companion system stimulating the transformation of P mediated by rhizosphere microorganisms. The ratio of MBC/MBN is often used to describe the structure and status of the microbial community. A high MBC/MBN ratio indicates that the microbial biomass contains a higher proportion of fungi, which means that the status of soil health is worse. In contrast, a lower ratio suggests that bacteria predominate in the microbial population, which represents good soil health (Campbell et al., 1992). In this study, the MBC/MBN ratio was decreased in the watermelon/wheat companion system compared with the monoculture system, which implied that the soil was healthier in the watermelon/wheat companion system. This may be another reason for the reduction of Fon wilt incidence. Sturz and Christie (2003) confirmed that the optimum microbial community can promote soil defense capability. In successively mono-cropped soil, specific microbial communities were formed due to the accumulation of specific exudates (Brussaard et al., 2007;Janvier et al., 2007), which could promote the increase of soil-borne pathogens. In this study, we found that the composition of microbial communities was changed (Figures 3, 5, 6), which suggests that Fusarium wilt may be suppressed in this system and is in concordance with previous studies (Larkin et al., 1993a,b). However, the mechanism by which the Fusarium pathogen is reduced remains unclear. We believe that the decline of the Fon population in the rhizosphere of watermelon in a wheat-based companion system is associated with the D 123 wheat root exudates because D 123 wheat root exudates may be inhibiting the mycelial growth of Fon (Xu et al., 2015). Furthermore, the MiSeq Illumina technology was applied to analyze the differences of bacterial and fungal communities. The different cropping system could affect the soil microbial diversity and composition (Larkin and Honeycutt, 2006;Acosta-Martínez et al., 2010). Our results indicated that, the relative abundance and diversity of the soil bacterial and fungal community were differed in different cropping system (Figures 5-7). The relative abundances of bacterial classes Alphaproteobacteria, Actinobacteria, and Gemmatimonadetes were increased, but the relative abundances of Gammaproteobacteria, Sphingobacteria, and Cytophagia were decreased in the watermelon/wheat companion system compared with the monoculture system (Figures 5B,C). The relative abundance of the fungal class Sordariomycetes was increased, but the relative abundances of Pezizomycetes and Eurotiomycetes were decreased in the watermelon/wheat companion system compared with the monoculture system (Figures 6B,C). In addition, the diversity of soil microbiota was increased in the watermelon/wheat companion system compared with the monoculture system (Figure 7). These differences may be due to the interactions between the soil microorganisms and different plant exudates (Singh et al., 2007;Song et al., 2007). Plant root exudates may play a strong role in shaping the rhizospheric community structure and function (Huang et al., 2014). The changes in the relative abundance and diversity of soil microbiota can also be used to explain the decrease in Fusarium wilt incidence. Conclusion In this study, the incidence of Fusarium wilt was decreased and the Fon population was reduced in a watermelon/wheat companion system. However, the soil polyphenol oxidase, urease and invertase activities were increased, MBN and MBP were increased, and the ratio of MBC/MBN was decreased in the rhizosphere of watermelon in the watermelon/wheat companion system compared with a monoculture system. These results suggest that the decrease in the incidence of Fusarium wilt may be related to changes in soil enzyme activities, microbial biomass, microbial relative abundance and diversity.

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Describing Paenibacillus mucilaginosus strain N3 as an efficient plant growth promoting rhizobacteria (PGPR) Abstract Bacterium Paenibacillus mucilaginosus strain N3 was isolated from agricultural farm soil (located at Boriavi village, Gujarat, India). Isolate showed an evidence of non-symbiotic nitrogen fixation, when grown in nitrogen-free bromothymol blue growth medium. It was tested positive for direct plant-growth-promoting traits like Indole-3-acetic acid production, solubilization of Tri-calcium-phosphate, and ammonia production. Further, N3 isolate was tested positive for siderophore production of catecholate type and catalase production as an indirect plant-growth-promoting trait. Biochemical tests along with 16s rRNA gene sequence analysis confirmed the strain N3 to be P. mucilaginosus. To determine its efficacy as a plant-growth-promoting rhizobacteria (PGPR), its talc-based biofertilizer was prepared and tested on the growth of green gram (Vigna radiata). Seeds treated with this biofertilizer showed an increase in overall dry biomass by 17% and sapling length by 28% (as compared to non-treated controls) after 10 days of sowing in pots. Thus, multiple plant-growth-promoting traits of P. mucilaginosus N3 determined in vitro along with its ability to promote growth in green gram in vivo we characterize this strain as an efficient PGPR. PUBLIC INTEREST STATEMENT Commercially available microbial biofertilizers contain strains of bacteria which associate with the roots of the plant and imparts beneficial effect on plant. Such microbial strains possess symbiotic relation with plants. Most widely available biofertilizers contain nitrogen-fixing microbes like Rhizobium, Azotobacter, Azospirillum, Blue green Algae, or contain phosphate solubilizing strains of Pseudomonas and Bacillus. To improvise microbial biofertilizers, there is a need to isolate and characterize new microbial strains which are more efficient or possess more than one trait which aids growth promotion in plants. Thus, with this article, we describe isolation and characterization of new microbial strain possessing ability to enhance growth of the plant, such research will introduce new and efficient strains which can be used to develop more efficient biofertilizers. Introduction Paenibacillus genus was separated from Bacillus before 20 years by Ash, Priest, and Collins (1993). They claimed that strains of Paenibacillus have dissimilarity in the consensus region of 16s rRNA as compared to strains of Bacillus. To our interest, species of Paenibacillus are similar to Bacillus in their action as plant-growth-promoting rhizobacteria (PGPR), but the nitrogen-fixing ability shown by some Paenibacillus strains provides them superiority. Characteristics that distinguish the genus Paenibacillus from other members of Bacillus includes, rod-shaped bacterial cells with a flagellum; producing ellipsoidal spores with swollen sporangia; positive for catalase; negative for H 2 S production; variable for oxidase; G + C content of their DNA ranging between 45% and 54 mol%, with anteiso-C15: 0 as the major cellular fatty acid, meso-diaminopimelic acid as the diagnostic diamino acid, and more than 89.6% intra-genus similarity in 16s rRNA gene sequences (Ash et al., 1993). Paenibacillus polymyxa (formerly Bacillus polymyxa) is most widely studied strain from this genus; it has ability to fix nitrogen and capable of producing antibiotic named polymyxin, and it is widely used as biocontrol agent for control of phytopathogens in agriculture (Eastman, Weselowski, Nathoo, & Yuan, 2014). Microbes are considered as a major resource for agricultural applications (Patel, Jha, Tank, & Saraf, 2011); however, strains other than P. polymyxa belonging to this genus are poorly studied for their role in agriculture. Importance of microbes in agriculture become popular since late 1970s and such plant beneficial bacteria are termed as PGPR (Kloepper & Schroth, 1978). But genus Paenibacillus was proposed much later and described in 2nd edition of Bergey's manual of systematic bacteriology which was published in 2004 (Naing et al., 2014). Since then there are limited reports describing strains of Paenibacillus as PGPR. Strains of Paenibacillus have not received comparable importance as efficient PGPR as strains of Acinetobacter, Agrobacterium, Arthobacter, Azotobacter, Sinorhizobium, Azospirillum, Burkholderia, Bradyrhizobium, Rhizobium, Frankia, Serratia, Thiobacillus, Pseudomonads, and Bacillus have received (Jacobson, Pasternak, & Glick, 1994;Sunar, Dey, Chakraborty, & Chakraborty, 2013;Vessey, 2003). However, over the period of last 10 years, few strains of Paenibacillus were rediscovered as nitrogen fixers which helped them to prove superiority over other extensively studied PGPR. Few nitrogen-fixing Paenibacillus strains include P. polymyxa, P. macerans, P. durus, P. peoriae, P. borealis, P. brasilensis, P. graminis, P. odorifer, P. wynnii, P. massiliensis, and P. sabinae (Beneduzi et al., 2010;Xie et al., 2014). Thus, there is a huge scope in isolating new stains of Paenibacillus and studying them for their plantgrowth-promoting traits which can help them to characterize as cogent PGPR. Such study will help to introduce new and efficient strains for agricultural applications. To portray any new strain as PGPR, it is necessary to know the mechanics employed by PGPR to enhance plant growth. The augmentative effect of PGPR occurs through various mechanisms which are primarily distinguished in the direct and indirect mechanisms. Direct mechanisms include production of plant hormones such as indole acetic acid (IAA), gibberellins, and cytokinins (Dey, Pal, Bhatt, & Chauhan, 2004;Patten & Glick, 1996) along with asymbiotic nitrogen fixation (Kennedy et al., 1997); and solubilization of phosphates (Banerjee & Yasmin, 2002;de Freitas, Banerjee, & Germida, 1997;Richardson, 2001). On the other hand, indirect mechanisms are the production of iron chelators, siderophores, and cyanides (Ahmad, Ahmad, & Khan, 2008;Cattelan, Hartel, & Fuhrmann, 1999; since they act as antagonists to plant pathogens. Under present study one such strain N3, capable of growing in nitrogen-free medium belonging to Paenibacillus genus was isolated from fertile agricultural soil. Strain was identified using 16s rRNA gene sequencing which showed high similarity with Paenibacillus mucilaginosus. The aim of the research was to portray this strain capable of growing in nitrogen-free growth medium, isolated from fertile rhizospheric soil as a PGPR. In-vitro biochemical analysis was performed to assess its multiple plant-growth-promoting traits which included ammonia production, phosphate solubilization, nitrogen fixation, IAA production, siderophore production, hydrogen cyanide (HCN) production, and catalase production. Plant-growth-promoting ability of strain N3 was in vivo determined by preparing talc-based biofertilizer and checking its effect on the seedling growth of green gram (Vigna radiata). Isolation of bacterial strain The rhizosperic soil samples were collected from the agricultural field 15 cm below the soil surface, located at Boriavi village, Anand, Gujarat, India (22°61′N, 72°93′E). Soil sample was aseptically suspended in sterile distilled water and after allowing it to stand for an hour, supernatant was spread on nitrogen-free medium (Glucose: 50 g l −1 , K 2 HPO 4 : 0.2 g l −1 , NaCl: 0.2 g l −1 , K 2 SO 4 : 0.1 g l −1 , CaCO 3 : 5.0 g l −1 , Agar: 25 g l −1 , pH 6.8, prepared in distilled water) with appropriate dilution and the plates were incubated at 27°C for 48 h. Identification of the organism Morphological analyses were performed by using a standardized method (Murray, Doetsch, & Robinow, 1994). Biochemical tests described in Bergy's Manual of Systematic Bacteriology were performed as per the methods described by Shi, Takano, and Liu (2012). All the media and reagents required to perform biochemical tests were obtained from HiMedia. For identification of bacterial isolate, 16s rRNA gene sequence was determined and analyzed. Genomic DNA from strain N3 was extracted and 16s rRNA gene was amplified using polymerase chain reaction using universal forward primer (5′-AGAGTTTGATCCTGGCTCAG-3′) and reverse primer (5′-AAGGAGGTGATCCAGCCGCA-3′) which were procured from 1st BASE (Agile Life Science Technologies India Pvt. Ltd) Goswami, Pithwa, Dhandhukia, & Thakker, 2014). The reaction was carried out in a 50 μl mixture containing 1.5 mM MgCl 2 , 0.2 mM each dNTP, 25 pmol of forward and reverse primers, 50 ng DNA template, and 5 U Taq DNA polymerase with its reaction buffer. Reaction was performed for 34 cycles using thermocycler at 94°C for 45 s, 58°C for 45 s, and 72°C for 105 s followed by a final extension of 10 min at 72°C. Amplified gene product of (1.6 Kb) was sequenced at 1st BASE (Agile Life Science Technologies India Pvt. Ltd). The BLASTn search program (http://www.ncbi.nlm.nih.gov) was used for sequence homology analysis. The gene sequences were also submitted to GenBank and accession numbers were assigned. The sequence obtained was then aligned by ClustalW using MEGA 4.0 software (Tamura, Dudley, Nei, & Kumar, 2007) and a neighborjoining (NJ) tree with bootstrap value 1,000 was generated using the software. Qualitative and quantitative estimation of phosphate solubilization Qualitative determination of phosphate solubilization was performed on Pikovskaya's agar plate. Isolate was spot inoculated and incubated at 27 ± 2°C, and the size of the halo corresponding to phosphate solubilization was measured after 7 days of incubation. Quantitative estimation of tri-calcium phosphate solubilization was performed by growing the strain in Pikovaskya's broth. The concentration of the soluble phosphate was determined from the culture supernatant at 10th, 15th, and 20th day after inoculation by stannous chloride method (King, 1932). Organic acid produced due to glucose metabolism (in Pikovaskya's broth) was determined by monitoring the change in the pH of the medium. IAA production Indole-3-acetic acid (IAA) production was estimated using the method described by Bric, Bostock, and Silverstone (1991). About 10% of exponentially grown culture of strain N3 was inoculated in 100 ml nitrogen-free medium with varying concentration of L-tryptophan which ranged from 0 to 500 μg ml −1 in different flasks. Bacterial cell-free supernatant was taken at 24, 48, and 72 h for performing quantative estimation of IAA. Briefly, 1 ml of cell-free supernatant was mixed vigorously with 1 ml of Salkowsky's reagent (1 ml of 0.5 M FeCl 3 in 50 ml of 35% HClO 4 ) followed by the addition of two drops of orthophosphoric acid and assay system was kept at 27 ± 2°C in dark for 20 min till pink color developed. Optical density of this colored solution was measured spectrophotometrically at 535 nm. The concentration of IAA in each sample was determined from the standard curve of IAA ranging from 10 to 100 μg ml −1 . Siderophore estimation Siderophore production was determined in defferated MM9 medium supplemented with 1% (w/v) Glucose, using CAS-shuttle assay (Payne, 1994). Strain N3 was grown in defferated MM9 medium with 1% (w/v) glucose. About 10 ml sample was withdrawn and centrifuged at 2,700 g for 15 min; 0.5 CAS assay solution was added to 0.5 ml of culture supernatant and mixed. This mixture was allowed to stand for 20 min. Siderophore (if present in the culture supernatant) will remove the iron from the dye complex, resulting in a loss of blue color of the solution. Decrease in the intensity of blue color was determined by measuring absorbance at 630 nm. Siderophore produced was calculated using the formula: where, A r is the absorbance of reference (minimal media + CAS assay solution) and A s is the absorbance of sample (culture supernatant + CAS assay solution). For qualitative estimation of catecholate type of siderophores, 1 ml of culture supernatant was mixed with 1 ml of 0.5 N HCl, followed by the addition of 1 ml of sodium molybdate reagent (10 g of sodium nitrite and 10 g of sodium molybdate in 100 ml of distilled water), and allowed to stand for 15 min. To this mixture, 1 ml of 1 N NaOH was added. Pink color, if develops in this solution, indicates the presence of catecholate type of siderophores (Carson, Holliday, Glenn, & Dilworth, 1992). Non-symbiotic nitrogen fixing ability and ammonia production Nitrogen-fixing ability was qualitatively studied by allowing the strain N3 to grow in nitrogen-free bromothymol blue (NfB) medium (Sucrose: 10 g l −1 , K 2 HPO 4 : 0.6 g l −1 , MgSO 4 : 0.20 g l −1 , NaCl: 0.2 g l −1 , K 2 SO 4 : 0.1 g l −1 , CaCO 3 : 2.0 g l −1 , pH 6.8, 2.0 ml of 0.5% Bromothymol blue solution). Here, the strain possessing the ability to fix nitrogen will increase the pH of the medium which is determined by the color change of the medium from green to blue (Jha et al., 2010). Ammonia production by the strain N3 was determined in peptone water broth. Briefly, strain N3 was inoculated into the medium and was allowed to grow at 27 ± 2°C, where the assay for ammonia production was performed for 4 days at an interval of every 24 h. Broth was collected, centrifuged, and the amount of ammonia in the supernatant was estimated by means of Nesslerization reaction where, 1 ml Nessler's reagent was added to 1 ml of supernatant and volume of this mixture was made up to 10 ml by addition of ammonia-free distilled water. Development of brown to yellow color indicated positive result for ammonia production and its optical density was measured by spectrophotometer at 450 nm (Cappucino & Sherman, 1992). The concentration of ammonia was estimated based on a standard curve of ammonium sulfate ranging from 0.1 to 1 μmol ml −1 . Catalase test Presence of catalase was checked qualitatively using the method described by . Solution of 6% H 2 O 2 was added on the colonies grown on nitrogen-free agar plates; effervescences of O 2 released from the bacterial colonies indicate the presence of catalase activity. Preparation of talc-based biofertilizer and seed bacterization The talc-based formulations of N3 isolate was prepared by the method described by Goswami, Vaghela, Parmar, Dhandhukia, and Thakker (2013). Briefly, talc powder was taken in a sterilized metal tray and its pH was adjusted to neutral by addition of 15 g of CaCO 3 per kg of talc. To this, 10 g of CMC was added and the mixture was autoclaved. About 400 ml of 48 h grown bacterial suspension was mixed with carrier-cellulose mixture under aseptic conditions. After drying (approximately 35% moisture content) overnight under sterile conditions, it was used for seed bacterization. Slurry of talc-based biofertilizer was prepared and surface-sterilized seeds (seeds dipped in 70% ethanol for 3 min and rinsed with sterile distilled water) were soaked overnight for the biofertilizer to get coated on seeds. Efficacy of biofertilizer was tested on wild-type seeds of green gram (V. radiata). These seeds treated with biofertilizer were sown in the pots sized (10 × 6 cm) and the growth parameters of seedlings were analyzed. All the pot experiments were carried out during the month of May where the average temperature was 37.0 ± 2.0°C and average humidity was 45%. Further, the soil used for the growth for chick pea and green gram was tested for its physical and chemical properties. Physical attributed Sieve analysis and Atterberg limit (Plastic limit, Liquid Limit and Plasticity Index; Dhandhukia, Goswami, Thakor, & Thakker, 2013). For chemical properties, total nitrogen, conductivity, TDS, pH, phosphates, sodium, potassium, calcium and magnesium were estimated from the soil sample (Ibekwe, Poss, Grattan, Grieve, & Suarez, 2010). Statistical analysis After the germination of test plants, the difference resulted by the biofertilizer treatment on the seedling growth was analyzed using statistical analysis. For this, analysis of variance (ANOVA) was carried out using triplicate value to identify significant difference in each vegetative parameter between treated and non-treated seeds. Mean values of triplicates were compared at significance levels of 5 and 1%. Isolation of bacterial strain From the soil sample used to isolate bacterial strains on nitrogen-free medium, total three types of colonies were obtained. The most dominant strain growing on the minimal medium was isolated and purified. Strain was designated as N3, where "N" stands for ability of the strain to grow in absence of nitrogen. Colonies were big, round, entire, transparent, colorless, sticky, and odorless. Image of colonies of strain N3 growing on nitrogen-free agar medium is shown in Figure 1. Identification of the organism Microscopic observation of strain N3 showed that it was gram negative and produced large capsules. Strain showed optimum growth at 30°C, with constant agitation at neutral pH of the growth medium. Biochemical tests showed that strain N3 was positive for oxidase, catalase, and nitrite reduction test; however, strain was found negative for amylase, gelatinase, urease, indole production, methyl-red test, and Vogus-Proskauer (VP) test. Strain showed acid production when grown with glucose, galactose, glycerol, sucrose, and mannitol; whereas, fructose, lactose, starch, sorbitol, maltose, citrate were not utilized by the strain (Table 1). Phylogenetic analysis was done by comparing 16s rRNA gene sequence of N3 and the sequences of other type strains of genus Paenibacillus from GenBank to identify the isolated strain. Accession numbers of Paenibacillus type strains were obtained from "Bergey's Manual of Systematic Bacteriology, Second Edition, Volume Three: The Firmicutes" to construct phylogenetic tree. Phylogenetic analysis showed that 16s rRNA gene sequence of strain N3 matched best with gene sequence of type strain P. mucilaginosus AS1.231 T under GenBank ID: DQ898308 (Figure 2). Based on biochemical characterization and phylogenetic analysis, strain N3 was designated as P. mucilaginosus strain N3and its 16s rRNA gene sequence was submitted to nucleotide sequence database under GenBank ID: JX154206. Estimation of phosphate solubilization It was observed that strain N3 could solubilize phosphate on Pikovaskya's agar where the zone diameter of phosphate solubilization was 20 mm and diameter of spot inoculant was 12 mm after its growth for 7 days. Quantitative estimation of phosphate solubilization was carried out in liquid Pikovaskya's medium which contained glucose as a carbon source. Here, quantative estimation of phosphate solubilized was determined at 10th, 15th, and 20th day of its growth. Strain N3 solubilized maximum of 11 μg ml −1 of tri calcium phosphate after 15 days of incubation, though on 20th day the determined phosphate solubilized was less than 11 μg ml −1 which may be due to utilization of solubilized phosphate by the strain. Analysis of pH altered in the growth medium by the bacterial metabolism suggested that the pH decreases from neutral to acidic due to organic acid production as a product of glucose metabolism (Figure 3). IAA production It was observed that strain N3 could produce IAA only when L-tryptophan was supplemented in the medium. IAA production by N3 isolate was determined at an interval of every 24 h after incubation (Figure 4). Maximum IAA produced was 13 μg ml −1 after 72 h of incubation when L-tryptophan concentration in the medium was maximum (500 μg ml −1 ). In the growth medium with absence of L-tryptophan, IAA was not detected even after 72 h. This shows that there is a direct correlation in IAA production and supplemented L-tryptophan in the medium. Note: Organic acids produced in the growth medium is attributed by drop in the pH of the medium. Goswami et al., Cogent Food & Agriculture (2015), 1: 1000714 http://dx.doi.org/10.1080/23311932.2014.1000714 Siderophore and catalase production Strain N3 showed production of siderophore in the iron-free MM9 minimal medium supplemented with 1% glucose which was detected by CAS-Shuttle assay. Maximum siderophore produced was 32% units ( Figure 5). The culture supernatant when mixed with CAS solution, its inherent blue color was gradually lost which indicated presence of siderophore (Figure 6(a)). Further, it was found that strain N3 could produce catecholate type of siderophores which was determined by the reaction of culture supernatant with sodium molybdate reagent under standard reaction condition produced pink color in the solution (Figure 6(b)). Strain N3 was also found to produce catalase as strong effervescences of O 2 evolved when 6% H 2 O 2 solution was flooded on the colonies grown on nitrogen-free agar. Non-symbiotic nitrogen fixation and ammonia production Ammonia production by strain N3 was determined in peptone water broth by Nesslerization reaction (Figure 6(d)). Maximum ammonia produced by the strain was 3.6 μmol ml −1 after 96 h of growth ( Figure 5). There are two evidences that prove strain N3 can fix nitrogen. First, the strain can easily grow in absence of any nitrogen source, indicating the nitrogen required for its growth must be available from the environment. Second, NfB medium which is nitrogen-free medium containing limiting amount of carbon source shows an increase in the pH when strain N3 is grown in it. The pH increase in the medium is visualized by the color change of the medium from green to blue, as the NfB contains pH sensitive dye bromothymol blue (Figure 6(c)). The increase in the pH caused by the metabolic activity of strain N3 in NfB medium is due to nitrogen fixation. Thus, these experiments are suggestive that the strain can fix atmospheric nitrogen. Effect of bio-fertilizer on seedling growth of green gram P. mucilaginosus strain N3 showed multiple traits of PGPR including production that included IAA, ammonia, siderophore, nitrogen fixation, and solubilization of phosphate; its efficacy as a PGPR was tested in vivo on the growth of green gram. Chemical properties of soil used for pot study are shown in Table 2. Green gram seeds were treated with biofertilizer prepared from P. mucilaginosus strain N3 and seeded in pots. After 15 days, the germinated seedlings were uprooted and their vegetative parameters including stem and root mass (Fresh mass and Dry mass), total length, root length, and stem length were measured. It was observed that biofertilizer-treated seedlings showed 29.0% increase in total length and 26.9% increase in the total fresh mass as compared to non-treated control (Figure 7). ANOVA analysis showed significant effect of biofertilizer treatment on vegetative parameters of the plant. Discussion Large soil microbial diversity is yet to be explored (Glick, 2014). Rhizosphere possess 100 folds higher bacterial density than in bulk soil as 5-21% of plant photosynthetic product is secreted by roots in form of different sugars which is in turn utilized by microbial populations (Govindasamy et al., 2011;Jha, Patel, Rajendran, & Saraf, 2010). Thus, the rhizospheric soil was selected for present study to isolate efficient strain (P. mucilaginosus strain N3) that can be categorized as efficient PGPR. Over the period of last 10 years, new Paenibacillus strains are identified as nitrogen fixers (Xie et al., 2014). Lu, Xue, Cao, Yang, and Hu (2014) reported various strains of P. mucilaginosus could fix nitrogen and suggested its potential application in agriculture. Under present study, nitrogen fixing ability detected by P. mucilaginosus strain N3 supports the findings of Lu et al. (2014). Despite nitrogen fixing ability possessed by Paenibacillus strains they are poorly characterized for other traits aiding plant growth promotion. This lack of research has opened a new horizon to portray on the Paenibacillus strains as PGPR for development of sustainable agriculture. To overcome this constrain, present study is focused to demonstrate P. mucilaginosus strain N3 as nitrogen fixing bacteria along with possessing other plant-growth-promoting traits like IAA production, phosphate solubilization, siderophore production, ammonia, and catalase production. Nitrogen and phosphorous are the most essential nutrient required for plant growth where, phosphorous is the second-most limiting nutrient for plants. Plants can absorb only mono and dibasic phosphate which are the soluble forms of phosphate (Jha, Patel, & Saraf, 2012). Phosphatesolubilizing microbes mineralize organic phosphorous in soil by solubilizing complex insoluble phosphates (Tri-calcium phosphate, Rock phosphate, Aluminum phosphate, etc.) and aid the phosphate availability to plants. Under present study, P. mucilaginosus strain N3 could efficiently solubilize tri-calcium phosphate in Pikovaskya's medium. It was also observed that strain could produce organic acids which increases acidity of the medium. Here, phosphate solubilized by the strain is ascribed by acid produced; as under acidic environment complex form of phosphates brakes in to simple form, which is considered primary mechanism for phosphate solubilization by microbes (Goswami, Pithwa, et al., 2014). Stains of Paenibacillus that are known to solubilize phosphate include P. telluris PS38, P. polymyxa B1-4, P. brasilensis PB177, and P. kribensis CX-7 (Ai-min, Gang-yong, Shuang-feng, Rui-ying, & Bao-cheng, 2013;Arthurson, Hjort, Muleta, Jäderlund, & Granhall, 2011;Lee, Kim & Yoon, 2011). PGPRs are known to produce several phytohormones such as gibberellins, cytokinins, auxins, etc. IAA-an auxin is an efficient molecule known to enhance plant growth by stimulating apical dominance and root growth (Bal, Das, Dangar, & Adhya, 2013). Majority of PGPR produce IAA form L-tryptophan as a primary precursor, whereas some does not require L-tryptophan (Idris, Iglesias, Talon, & Borriss, 2007). P. mucilaginosus strain N3 showed IAA production only when L-tryptophan was supplemented in the growth medium indicating the pathway used to produce IAA was L-tryptophan dependent. Notes: "ns" suggests (p value greater than 0.05) nonsignificant as compared to control (ANOVA); "*" suggests (p value between 0.05 and 0.01) significant at 5% (ANOVA); and "**" suggests (p value between 0.01 and 0.001) significant at 1% as compared to control (ANOVA). The most widely studied strain for IAA production among the genus Paenibacillus is P. polymyxa. Similar study was also performed by Lebuhn, Heulin, and Hartmann (1997) where they showed IAA production by P. polymyxa was directly dependent on supplementation of L-tryptophan. They also claimed that indole-3-ethanol, indole-3-lactic acid, and indole-3-carboxylic acid were also produced by P. polymyxa in addition to IAA on L-tryptophan supplementation. P. mucilaginosus strain N3 also showed production of siderophores. Siderophores are produced in the rhizosphere by PGPRs to quench iron. Iron-deficient condition in the rhizosphere caused by siderophore producing PGPR inhibits the growth of unwanted organisms. Thus, siderophoresproducing microbes antagonize the growth of plant pathogens in the rhizosphere eventually helps plant to remain healthy (Haas & Défago, 2005;Jan, Azam, Ali, & Haq, 2011). To portray any rhizobacterial strain as PGPR, it should be recognized to IAA production, phosphate solubilization, ammonia production, nitrogen fixation, and siderophore production, further, it is inevitable for strain to show plant growth promotion in vivo under pot trials (George, Gupta, Gopal, Thomas, & Thomas, 2013;Goswami et al., 2013). Our previous reports suggest that Pseudomonas spp. strain OG which showed several traits of PGPR also enhanced growth of green gram (V. radiata). Similarly for Bacillus licheniformis strain A2 and Kocuria turfanensis 2M4 isolated from saline soil, showed PGPR traits determined by in vitro biochemical tests could enhance growth of groundnut (Arachis hypogaea L.; Goswami, Pithwa, et al., 2014). Similarly, Mehta, Walia, Kulshrestha, Chauhan, and Shirkot (2014) described phosphate solubilizing, Bacillus circulans CB7 can enhance the growth of tomato (Solanum lycopersicum L.) and described the strain as an efficient PGPR. Ureaseproducing Pseudomonas aeruginosa strain BG isolated from marine water was reported to show several traits of PGPR-enhanced growth of chick pea (Cicer arietinum L.; . Under present study, we can categorize P. mucilaginosus strain N3 as an efficient PGPR as it possesses important PGPR traits and also shows growth promotion in green gram can be considered as an efficient PGPR.

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Extraction of Oil from Egyptian Oil Shale Oil shales are defined as fine grained sedimentary rocks containing abundant mainly sapropelic organic matter which produce oil on distillation. The present study highlights the characterization of Egyptian origin oil shale using various analytical techniques such as Elemental analysis, Infrared spectroscopy (IR), and the Morphology Study of oil shale (SEM). The study is focused on the characterization of shale oil samples obtained by solvent extraction method from oil shales. The paper studied the effect of amount and type of solvent, time of extraction, the temperature of retorting on the percentage yield of oil produced. The study is also focused on the analysis of shale oil samples by gas chromatography (G.C) and thermal analysis (T.G.A) which revealed that the oil has a high value of hydrocarbons. From Experimental studies it is found that the percentage yield of oil increased as the volume of solvent increased the best result is obtained by using methanol as a solvent. The yield also increased by increasing time of extraction and stirring rate. Extraction of Oil from Egyptian Oil Shale Introduction Oil shale is commonly defined as a fine-grained sedimentary rock containing organic matter that yields substantial amounts of oil and combustible gas upon destructive distillation. Most of the organic matter is insoluble in ordinary organic solvents; therefore, it must be decomposed by heating to release such materials. Underlying most definitions of oil shale is its potential for the economic recovery of energy, including shale oil and combustible gas, as well as a number of byproducts [1]. In terms of mineral and elemental content, oil shale differs from coal in several distinct ways. Oil shales typically contain much larger amounts of inert mineral matter (60-90 percent) than coals, which have been defined as containing less than 40 percent mineral matter. The organic matter of oil shale, which is the source of liquid and gaseous hydrocarbons, typically has a higher hydrogen and lower oxygen content than that of lignite and bituminous coal [2,3]. In general, the precursors of the organic matter in oil shale and coal also differ, much of the organic matter in oil shale is of algal origin, but may also include remains of vascular land plants that more commonly compose much of the organic matter in coal. The origin of some of the organic matter in oil shale is obscure because of the lack of recognizable biologic structures that would help to identify the precursor organisms. Such materials may be of bacterial origin or the product of bacterial degradation of algae or other organic matter [4,5]. The mineral component of some oil shales is composed of carbonates including calcite, dolomite, and siderite, with lesser amounts of alumino silicates. For other oil shales, the reverse is true-silicates including quartz, feldspar, and clay minerals are dominant and carbonates are a minor component. Many oil-shale deposits contain small, but ubiquitous, amounts of sulfides including pyrite and marcasite, indicating that the sediments probably accumulated in dysaerobic to anoxic waters that prevented the destruction of the organic matter by burrowing organisms and oxidation [5,6]. Oil shale differs from coal whereby the organic matter in coal has a lower atomic H: C ratio [6,7]. The heating value of the oil shale may be determined using a calorimeter. The heating value is useful for determining the quality of an oil shale that is burned directly in a power plant to produce electricity. The oil shale industry as represented by the six countries in Figure 1 Most of this decline is due to the gradual downsizing of the Estonian oil shale industry. This decline was not due to diminishing supplies of oil shale but to the fact that oil shale could not compete economically with petroleum as a fossil energy resource. On the contrary, the potential oil shale resources of the world have barely been touched [3]. Eastern Desert of Egypt ( Figure 2) is most notable for the presence of the phosphorites of Gebel Duwi Range discovered over 60 years ago. The shales of the Duwi Formation are interbedded with the phosphates of the overlying Dakhla Formation and are generally black to grey and rich enough in organic matter to warrant their description as black shales. The Duwi Formation of Campanian-Maastrichtian age [8,9]. Organic association in the shale includes the organic components C, H, N and S which seem to be responsible for accumulation of unusual amounts of some trace elements, especially V. The objective of the shown in Figure 5. The reaction vessel consists of a conical provided with a mechanical stirrer for mixing the reaction mixture, a hot plate adjusted at temperature reaction. A distillation unit was used to evaporate the solvent. For determining of oil produced weight a digital balance was used. Procedure of retorting: 100 gm of oil shale were put inside round metal dish (Dimension: 3×10 cm) then the metal dish was heated inside the oven at 200°C for 10 minutes (without cover) then put the cover of the dish and re-enter the oven and heat at 300°C for 120 minutes then the oven was turned off. Leave sample to cool down then weigh the sample. Procedure of solvent extraction: 100 gm of the crushed sample were stirred by magnetic stirrer in the flask as shown in Figure 5. A known volume of organic solvent was added and the stirring rate was adjusted at the required speed and the mixture was heated on the hot plate at the desired reaction temperature. After a known period the reaction mixture was filtered to separate the extracted oil with solvent then in the distillation unit the solvent can be recovered again. Analysis (Characterization) of oil shale Oil shale elemental analysis: The elemental analysis of oil shale sample was performed at Egyptian Petroleum Research institute in Cairo. The sample of the shale was analyzed on Vario El Elementar Germany instrument. A known weight of a sample about 3-5 mg was carefully weighed in tin boats and put in the auto sampler of the instrument. The sample was burned in oxygen gas at 1150ºC in the combustion tube. The product of combustion were then reduced by hot copper at 85ºC producing N 2 , CO 2 , H 2 O and SO 2 which were selectively adsorbed on columns. After heating of the columns they were determined using thermal conductivity detector with helium as carrier gas. The peak height was related to the gas and the percentage of element in the sample was determined. The elemental analysis data of oil shale sample studied is given in Table 1. work of this paper is divided into two parts: part one is concerned with investigations connected with study of composition and structure of oil shale from Al-Quseir area in Egypt which is the most perspective due to its high organic content while the other part is the characterization of shale oil obtained by solvent extraction using different organic solvent. Presented work is also focused on obtaining high percentage yield of oil from oil shale by studying variables like amount and type of solvent, time of reaction, the temperature of retorting. Materials and Methods Oil shale samples were obtained from Al-Quseir area, Egypt. All samples were dark in color. The oil shale samples used as shown in Figure 3 were crushed and sieved to a particle size range around 1-1.5 mm as shown in Figure 4. The elemental analysis of oil shale was performed at National Research Institute in Cairo, Egypt the data is given in Table 1. Experimental setup The experimental set up used in the present work is schematically The organic carbon content is a good indicator of the quality of organic matter in the oil shale. From the previous data it was observed that the Egyptian Oil shale contains much less percentage of S content than Indian shale and it gives the Egyptian shale a an advantage over to Indian shale because the presence of S content causes a lot of problems and has bad environmental impact specially in extraction of oil from oil shale which need special treatment to get rid of it and this costs a lot of money in industry. By comparing also the sulfur content of Egypt oil shale with The sulfur content of shale oil from the Jurfed Darawish and the Sultani deposits (Jordanian) is high (8-10%) [11] it gives an indication that Egypt oil shale is better than Jordanian oil shale in this percentage of S content another comparison of S and N content of china oil shale which contains 0.8% S and 0.57% N [12,13]. From Table 1 showing that H/C ratio is 0.3198 while the H/C ratio in Indian shale equal to 0.196 indicating that the Egyptian origin oil shale is carbon rich in nature and contains a significant amount of aromatics than Indian shale. The main characteristics of the organic matter of oil shale are high hydrogen and a low nitrogen percentage [12]. IR analysis: IR spectra of the oil shale sample is analyzed on Perkin-Elmer BX-II FT-IR instrument. By explaining the chart of IR spectrum of Egyptian origin oil shale as shown in Figure 6. It is observed that there was stretching peaks which indicate the aliphatic and aromatic bonds in the shale. The analysis of the sample shows the presence of various sharp carbon hydrogen bonds (C-H) in range 3000-4000 cm -1 (3975.42 cm -1 , 3796.04 cm -1 , 3618.58 cm -1 , 3427.62 cm -1 ). It was also observed that there is a large peak at 3427.62 cm -1 which indicates the O-H bond. Also it was observed that aliphatic hydrocarbon stretching bands are found at 2943.47 cm -1 . It was also observed that the peaks at 2519.12 cm -1 and 2324.30 cm -1 represent (C ≡ C) bonds. A large peaks are observed at 1437.02 cm -1 and 1033.88 cm -1 which indicate C-C bond and N-H group respectively. Whereas wave length 779.27 cm -1 indicates aromatic compounds specially benzene monosubstituted and di substituted, wave length 536.23 cm -1 indicates C-X bonds in which may be chloroalkanes, bromo alkanes or iodo alkanes, wave length 459.07 cm -1 indicates clay and mineral in oil shale [14][15][16][17][18]. Figure 7 (performed in City of scientific research & Technological applications -SRTA City). The purpose of this study is to show distribution of minerals in oil shale and to understand the microstructure relationships among the main constituent and the matrix of shale. The identification of different minerals through SEM was facilitated by comparing their characteristic morphologies with those shown in the SEM petrology Atlas of Welton [19]. SEM is a qualitative or semi quantitative technique useful for obtaining particle composition and morphology. It was found that is the major phases in oil shale are calcite, clay, and quartz [20,21]. Table 2 and Figure 8 show the effect of using different kinds of organic solvents and also different volumes of each solvent on the percentage yield of oil produced by solvent extraction. The results revealed that as the volume of solvent increased the percentage yield of oil produced increased. And this trend may be due to that the rate of extraction of bitumen depends on the type of organic solvent used so if the force of attraction between bitumen and solvent is greater than that between the solvent molecules of the dissolution of bitumen, then bitumen can be easily extracted. Results of experiments obviously showed that methanol gives the higher percentage yield of oil than other solvents. These results are in agreement with [21][22][23][24]. shows the comparison between different types of solvent used. From previous table and figure it is seen that the highest percentage yield of shale oil was obtained by using methanol as solvent, followed by kerosene. All other solvents used after that have approximately near percentage yield of oil produced. Figure 9 shows the G.C analysis of produced oil sample using Methanol as a solvent. From figure it was shown that the sample contains high hydrocarbon range from C12-C18 in high percentage. Characterization of shale oil by G.C chromatography From Figure 10 it is observed that as the time of extraction increased from 4 hr to 7 hr the percentage yield of oil produced increased. Beyond this time (7 hr) the effect of time is negligible. This time of 7 hours was an indication that the process has received almost completion. Tamimi and Uysal have shown that the effect of extraction time is more significant for larger particle diameter, greater than 2 mm. However in this study all experiments were conducted on particle size 1.5 mm. Effect of retorting on % yield of oil produced at 200-300°C From results and comparison of the percentage yield of oil produced in Table 3 and Figure 11 it was shown that the retorting process increases the yield produced of shale oil. Conclusions • The Elemental analyses of Egyptian Oil shale indicates that it contains lower percentage of S and N content and Higher H/C ratio than those from Jordanian and Indian oil shale. • The IR analysis of the shale indicates that contains high percentage of hydrocarbons. • The Morphology Study of oil Shale (SEM) indicates that the oil shale composed of the major phases present as calcite, clay, and quartz. • In the present study, the nature of solvent was found to have a very significant effect on the yield of recovery and the composition of the oils obtained. • The oil yield obtained by methanol extraction is significantly higher than those obtained by other solvents. Recommendations • Studying the effect of other organic solvents on the yield of oil produced. • Applying of other techniques to extract the oil from the shale (e.g. steam). Measuring the heat content of the oil shale.

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Correlations among Stress Parameters, Meat and Carcass Quality Parameters in Pigs Relationships among different stress parameters (lairage time and blood level of lactate and cortisol), meat quality parameters (initial and ultimate pH value, temperature, drip loss, sensory and instrumental colour, marbling) and carcass quality parameters (degree of rigor mortis and skin damages, hot carcass weight, carcass fat thickness, meatiness) were determined in pigs (n = 100) using Pearson correlations. After longer lairage, blood lactate (p<0.05) and degree of injuries (p<0.001) increased, meat became darker (p<0.001), while drip loss decreased (p<0.05). Higher lactate was associated with lower initial pH value (p<0.01), higher temperature (p<0.001) and skin blemishes score (p<0.05) and more developed rigor mortis (p<0.05), suggesting that lactate could be a predictor of both meat quality and the level of preslaughter stress. Cortisol affected carcass quality, so higher levels of cortisol were associated with increased hot carcass weight, carcass fat thickness on the back and at the sacrum and marbling, but also with decreased meatiness. The most important meat quality parameters (pH and temperature after 60 minutes) deteriorated when blood lactate concentration was above 12 mmol/L. INTRODUCTION The production of high quality pork has been a constant objective of the pig industry for many decades. The main goal is to obtain pigs with high lean percentage and good meat quality traits at the same time. However, this goal has proved difficult to achieve (Kusec et al., 2003). Meat and carcass quality are complex traits and are influenced by many antemortem and postmortem factors (Sellier and Monin, 1994;Huff-Lonergan et al., 2002). The prediction and evaluation of pork quality is of particular importance if these traits are to be improved. Many physical and biochemical factors have been evaluated in an attempt to assess meat quality (Huff-Lonergan et al., 2002). There are three important parameters to define pork quality: drip loss, ultimate pH and colour (Lee et al., 2000). Using those parameters, meat can be classified into the five quality categories; PSE (pale, soft, exudative), normal or RFN (red, firm, nonexudative), DFD (dark, firm, dry), RSE (reddish-pink, soft, exudative) and PFN (pale, firm, nonexudative) meat (Kauffman et al., 1992). It is well known that changes in some meat quality traits can affect many other meat quality attributes and pork quality overall (Huff- Lonergan et al., 2002). In addition, changes in physiological parameters affect meat quality. Increase in blood lactate concentration is associated with pre-slaughter stress, which has been shown to have a detrimental effect on pork quality (Hambrecht et al., 2005a, b;Edwards et al., 2010b). Hambrecht et al. (2004) determined that pigs exposed to aggressive handling just prior to stunning had a higher blood lactate concentration at slaughter and exhibited pork with higher drip loss and thus proposed that lactate was a potential indicator of both physical and psychological stress associated with the handling of pigs immediately before slaughter. Furthermore, the level of cortisol is an individual characteristic of each animal (Mormede, 2007) and it affects the amount of fat in the body, meatiness, and thus, carcass quality (Foury et al., 2005;2007;Skrlep et al., 2009). Higher cortisol levels were associated with higher initial and ultimate pH values, and consequently with lower Minolta L* and b* values and drip loss after 24 and 48 hours suggesting that cortisol level at slaughter reflects on meat quality (Skrlep et al., 2009). Considering relationships between biochemical traits and objective measures of quality, and complex biological mechanisms behind their development, the objective of this study was to determine to what extent different animal stress, meat quality and carcass quality traits were associated. Animals, housing and feeding The experiment was conducted on 100 commercial market pigs (31 gilts, 51 barrows and 18 boars), six months old and with live weight between 115 and 130 kg. All animals were of the same origin (cross between Naima sows and hybrids P-76 PenArLan boars) and came from the same farm. Pigs were housed in the finishing facility on partially slatted floors, in pens with 20 animals per pen (stocking density = 1 m 2 /pig). Pigs were provided with ad libitum feed and water. Pre-slaughter handling Before transport feed and water were not withdrawn. Pigs from the same pen were transported together, without mixing with other pigs, in batches of 20 animals in a transportation trailer (stocking density was 0.45 m 2 per pig). The distance between pens and vehicle at loading was about 20 m and the slope of the loading ramp was 15°. Transport from farm to the abattoir lasted 15 minutes. The surface of the vehicle and the unloading area were on the same level. After unloading, pigs entered a 10 m long corridor that led to the lairage pens. During loading and unloading sticks and electric prods were used to move pigs. During lairage, pigs were not mixed and stocking density was 0.70 m 2 per pig. Pigs were held in lairage less than 3 hours (slaughtered on the same day when they arrived, n = 28) or more than 14 hours (stayed overnight and slaughtered in the morning, n = 72). The exact length of lairage time was measured for each pig. Between lairage pens and stunning area was 5-m single file corridor. During pig handling from lairage pens to the stunning area stick and electric prod were used randomly. After lairage pigs were head-only electrically stunned (50 Hz, 2 A and 220 V) in batches of 6 animals without restraining. Immediately after stunning pigs were bled on the floor and then hoisted on a rail. Following bleeding carcasses were processed using conventional industry practice. Blood sampling and determination of blood lactate and cortisol content At slaughter blood samples were taken into plastic tubes and part of material was transferred to vacutainer tubes containing heparin (against blood coagulation). Thereupon blood lactate content was determined using a portable lactate analyzer (Lactate Scout, EKF Diagnostic, Magdeburg, Germany). The lactate analyzer was tested with a standard solution to ensure accuracy. After blood collection the vacutainer tubes were placed on ice and within 4 to 6 hours blood was centrifuged at 3,000 rpm for 3 minutes. Supernatants (plasma) were collected into microtubes and stored at -20°C until the determination of cortisol concentration by radioimmunoassay (RIA-CT Cortisol, INEP, Belgrade, Serbia). Meat and carcass quality analyses Meat quality measurements were carried out 60 minutes, 24 and 72 hours after slaughter on muscle Longissimus dorsi (LD), pars lumbalis. Values of pH 60 minutes and 24 hours postmortem (pH 60min , pH 24h ), and temperature 60 minutes postmortem (t 60min ) were measured using a pHmeter Testo 205 (Germany) calibrated with pH 4.00 and 7.00 phosphate buffer. Skin damages were assessed by three observers on three carcass regions (from head to back of shoulder, from back of shoulder to hind-quarters and region of hind-quarters) immediately after dressing using scores 1 (no damages), 2 (scratches or small wounds, less than 2 cm), 3 (bleeding wounds between 2 and 5 cm or a healed wounds of more than 5 cm) and 4 (deep and open wounds of more than 5 cm). The final skin damages score for each carcass was obtained by summing scores for the three regions, and ranged from 3 to 12. Degree of rigor mortis was estimated on right carcass side 3 hours post mortem by measuring degree of angle between body axis and foreleg (Davis et al., 1978). For that purpose photographic images were taken of carcasses, at distance of approximately 2 m and a height of 160 cm, parallel to the plane of the carcass and then the angle was calculated in AutoCAD. The size of the angle and intensity of rigor were inversely proportional, e.g. smaller angle meant a higher degree of rigor mortis. For determination of drip loss, colour and marbling, meat samples, 2.5 cm thick loin chops were taken 24 hours after slaughter from LD, between the 3rd and 4th lumbar vertebrae. Meat samples were weighed and stored for 48 hours at 4°C in a container (Honikel, 1998). After storage, meat samples were reweighed and drip loss (%) was calculated as the difference between sample weight before and after storage divided by the sample weight before storage. Sensory and instrumental colour (Commission Internationale de l'Eclairage [CIE] L*a*b*) (CIE, 1976) as well as marbling were determined at 24 hours postmortem, after approximatelly 60 minutes of blooming time (Honikel, 1998). L*, a*, and b* (CIE, 1976) values were determined using a Minolta Chroma Meter CR-400 (Minolta Co., Ltd., Osaka, Japan) utilizing a 65 light source and a 2° observer. Colour, marbling and drip loss were analyzed in duplicate. An analytical panel of three trained members assessed sensory colour and marbling of meat samples by using the scaling method (NPPC, 2000). Hot carcass weight and carcass fat thickness at two points (on the back between the 13th and 15th dorsal vertebrae and at the sacrum where is fat thickness thinnest) were measured. Meatiness (in percentages and kilograms) was determined according to regulation (Anon, 1985) on the basis of hot carcass weight and the sum of carcass fat thickness at two points (on the back and at the sacrum). Statistical analysis Statistical analysis of the results was elaborated using software GrapfPad Prism version 5.00 for Windows, GraphPad Software, San Diego CA, USA, www.graphpad.com. All parameters were described by descriptive statistics (mean, standard deviation, minimum and maximum value). Pearson's correlation was used to determine relationships among different stress, meat and carcass quality parameters. Student t-test was used for testing the effect of blood lactate concentration below and above 12 mmol/L on pH value and temperature measured 60 minutes postmortem. Characterization of the experimental population Characterization of the experimental population is presented in Table 1. In this study, blood lactate ranged from 1.3 to 24.6 mmol/L which is in accordance with results of other authors, who found values ranging from 1.1 to 20.6 mmol/L (Edwards et al., 2010a) and from 4.0 to 19.7 mmol/L (Edwards et al., 2010b). Blood cortisol content varied within studies (Hambrecht et al., 2004;Foury et al., 2005;Hambrecht et al., 2005b), which was also observed in this experiment (3 to 248 nmol/L). Those variations in cortisol content could be explained by influence of species, breed, time of day, stress, meal, physical and sexual activity and change of environment. Values of pH after 60 minutes ranged from 5.64 to 6.81, while after 24 hours were from 5.26 to 5.93. The highest carcass temperature was 40.3°C and the highest drip loss of meat samples was 9.50%. There were carcasses without skin blemishes (score 3) in the experimental group, as well as those with a maximum score for skin blemishes (score 12). The CIE parameters ranged from 35.11 to 60.22 (CIE L* value), from 4.15 to 11.83 (CIE a* value) and from 2.23 to 7.41 (CIE b* value), while sensory colour from 1.0 to 4.0. The average hot carcass weight in this study was 93.46 kg, which was similar to most EU countries (less than 95 kg) (Hansson, 2003). Carcass fat thickness ranged from 12 to 36 mm on the back, and from 5 to 34 mm at the sacrum. The reason for those variations, especially at the sacrum, was due to the presence of barrows, gilts and boars in the study, since boars have the lowest backfat thickness (Jaturasitha et al., 2006). The average meatiness was 43.79%, which was lower compared to data from 16 EU countries, where average meatiness ranged from 55 to 55.99% (Hansson, 2003). Unlike in the EU, in this study muscle tissue from the belly was not included in determination of meat yield, so differences in meatiness recorded by these different measuring practices could be up to 10%. The effect of lairage time Relationships among lairage time, blood lactate and cortisol concentration, and meat quality parameters in pigs are shown in Table 2. , pH values measured 60 minutes and 24 hours postmortem. 2 t60min, meat temperature measured 60 minutes postmortem; 3 L*, Lightness (a higher L* value indicates a lighter colour); a*, Redness (a higher a* value indicates a redder colour); b*, Yellowness (a higher b* value indicates a more yellow colour); 4 Sensory colour scale: 1 = pale pinkish-gray to white; 6 = dark purplishred. Although lairage time did not have a significant effect on cortisol concentration, other authors found the opposite (Perez et al., 2002;Warriss, 2003a), as cortisol decreased during lairage, particularly at night when pigs rested and became calm. On the other hand, in this study, lairage time was positively correlated with blood lactate, which, as a measure of stress, indicated that a longer lairage caused a higher level of stress. This could be explained by after longer lairage pigs were more susceptible to stress due to longer period of experiencing stressors in lairage (rough handling, fights, change of environment, food deprivation, etc.) (Stam et al., 2000;Bruijnzeel et al., 2001;Stam et al., 2002). Lairage time did not have a significant effect on initial or ultimate pH values, although higher pH values were found after longer lairage times in other studies (Perez et al., 2002;Hoffman and Fisher, 2010). This is explained by the fact that reserves of glycogen become depleted during lairage and after a longer lairage there is a lower muscle glycogen content and consequently higher pH value of meat. As in our study, Hoffman and Fisher (2010) determined lower drip loss after longer lairage. In addition, longer lairage increased the level of skin blemishes on the carcass. The percentage of skin blemishes is higher after a longer lairage time because it increases the aggressiveness of the pigs (Nanni Costa et al., 2002;Perez et al., 2002;Warriss, 2003a;Guardia et al., 2009). Since the rate of rigor mortis is positively correlated with the content of cortisol and lactate as indicators of stress (Warriss et al., 2003b), a greater degree of rigor mortis after longer lairage indicated a more stressful procedure. Lairage time affected meat colour, so after longer lairage meat became darker, less red and yellow which was also observed by Nanni Costa et al. (2002) and Hoffman and Fisher (2010). As in our study, Edwards et al. (2010a, b) found that the concentration of blood lactate was negatively correlated with the initial pH value (r = -0.25, p<0.001; r = -0.32, p<0.001). Higher drip loss of meat was expected in pigs with increased concentrations of lactate (Edwards et al., 2010a, b), but no significant correlation was determined between these two parameters in this study. Although correlation coefficients between blood lactate and meat quality parameters (pH 60min and t 60min ) were weak, they indicated that exsanguination blood lactate is a potential predictor of postmortem muscle metabolism and meat quality (Edwards et al., 2010b). As in our study, a higher degree of rigor mortis was found in pigs with higher levels of blood lactate (Warriss et al., 2003a). A positive correlation between blood lactate concentration and skin damages score indicated that lactate was not only associated with meat quality, but also with the level of stress and procedures prior to slaughter. Rabaste et al. (2007) found that skin blemishes score tended to be higher in pigs after rough preslaughter treatment (pigs moved quickly with an electric prod) compared to gentle (pigs moved slowly with a plastic board or whip), while Edwards et al. (2010c) observed higher concentrations of blood lactate in pigs that experienced more frequent use of electric prods. Higher levels of blood cortisol were associated with higher meat temperatures (r = 0.23, p<0.05) and marbling scores (r = 0.22, p<0.1). Warriss et al. (2003a) observed increased cortisol concentration in the blood of pigs having more developed rigor mortis. Initial pH value was not significantly correlated with most meat quality parameters, except with temperature (r = -0.16, p<0.1), L* (r = -0.19, p<0.1) and a* (r = -0.23, p<0.05) parameters of colour. In accordance with our results, Terlow and Rybarczyk (2008) found that a lower pH value after 40 minutes was associated with a higher temperature. Aaslyng and Barton Gade (2001) found that meat with lower pH value after 40 minutes had higher drip loss and became lighter. Other authors have found that drip loss increased when pH values after 45 minutes and 24 hours were lower (Hambrecht et al., 2003;Edwards et al., 2010a, b). Although a positive correlation can be found between initial and ultimate pH value, it is weak in most studies; p = 0.14 (Edwards et al., 2010b), 0.15 (Boler et al., 2008), 0.20 (Aaslyng et al., 2001), 0.33 (Borchers et al., 2007) and 0.44 (Klont et al., 2001). We found that relationship between initial and ultimate pH was not significant, probably due to the fact that these two parameters were under the influence of different factors. The extent of pH decline and the finally ultimate pH are primarily affected by amount of glycogen in muscles at the time of slaughter , while initial pH value depends on rate of postmortem glycolysis which is influenced by genetic (Barbut et al., 2007), pre-slaughter factors (Rosenvold and Andersen, 2003), postmortem carcass chilling (Tomovic et al., 2008) or combinations of all of these . Several meat quality characteristics are correlated with pH value after 24 hours. In pigs with lower ultimate pH values, higher drip loss (Huff- Lonergan et al., 2002;Hambrecht et al., 2003;Edwards et al., 2010b) and lighter colour (Huff-Lonergan et al., 2002;Hambrecht et al., 2003;Boler et al., 2008;Edwards et al., 2010b) were measured. In carcasses with rapid development of rigor mortis, ultimate pH values were significantly lower (Warriss et al., 2003a). Contrary to those findings, in this study, ultimate pH value was positively correlated only with marbling, as was also observed by other authors (Przybylski et al., 2007;Boler et al., 2008;Czarniecka-Skubiņa et al., 2010). This can be explained by the fact that energy reserves in muscle fibres are distributed among intramuscular fat and glycogen, and muscles with lower glycogen content have a higher intramuscular fat content. Therefore, higher marbling was accompanied by higher ultimate pH (r = 0.27, p<0.01), lower drip loss (r = -0.21, p<0.05) and darker colour expressed by L* value (r = -0.29, p<0.01) and sensory colour (r = 0.30, p<0.01), indicating lower glycogen content in muscles. Boler et al. (2010) also found a significant correlation between sensory colour and marbling (r = 0.29, p<0.05). In this study, there was no significant correlation between temperature and other meat quality parameters. Contrary to that, meat temperature after 40 minutes was positively correlated with L* value (Aaslyng et al., 2001) and drip loss (Aaslyng et al., 2001;Hambrecht et al., 2003), while Edwards et al. (2010b) found that increase in temperature reduced the amount of meat exudate. In addition, in the current study, pigs with rapid development of rigor mortis were observed to have a higher meat temperature, increased blood concentrations of cortisol and lactate, and lower ultimate pH value. Those changes in parameters indicated that stressful procedures prior to slaughter were accompanied by intensive metabolic processes in muscles and, consequently, more rapid development of rigor mortis (Warriss et al., 2003a). In this study, sensory colour was negatively correlated with L* (r = -0.60, p<0.001) and b* (r = -0.25, p<0.05) parameters, and positively with a* (r = 0.20, p<0.05) colour parameter. Higher L* values were correlated with higher b* (r = 0.60, p<0.001) values, and there was also a high correlation (r = 0.72, p<0.001) between a* and b* colour parameters, as found previously (Edwards et al., 2010b). Table 3 shows correlations between blood cortisol concentration and carcass quality parameters as well as correlations among carcass quality parameters. The relationship among blood cortisol concentration and carcass quality parameters Cortisol concentration was positively correlated with hot carcass weight (r = 0.26, p<0.1) and with the amount of fat in the body, expressed as carcass fat thickness on the back (r = 0.33, p<0.05) and at the sacrum (r = 0.25, p<0.1). Higher cortisol concentrations in the blood correlated with lower meatiness (r = -0.29, p<0.05). In accordance with this, Foury et al. (2005;2007) and Skrlep et al. (2009) have found that high concentrations of cortisol were associated with a greater thickness of subcutaneous fat tissue and lower meatiness. These results indicate that cortisol affects the metabolism of fats and proteins by stimulating the fat deposition at the expense of decreased synthesis and increased protein degradation (Mormede, 2007). In contrast to other authors (Foury et al., 2005;2007), we found a positive correlation (r = 0.22, p<0.1) between blood cortisol concentration and marbling, indicating that cortisol influenced deposition of adipose tissue not only under the skin and around organs, but also between and inside the muscle fibers. It is likely that the same mechanism controls deposition of fat regardless of location in the body, since Huff- Lonergan et al. (2002) have found a positive correlation (r = 0.38) between fat thickness on the back and marbling, which has been also observed in the present study (r = 0.19, p<0.1). Carcass quality parameters were inter-correlated, where hot carcass weight was associated with fat thickness on the back (r = 0.56, p<0.001) and at the sacrum (r = 0.40, p<0.001), as well as with marbling (r = 0.23, p<0.05). Fat thickness on the back and at the sacrum was negatively correlated with meat yield (r = -0.39, p<0.001, and r = -0.44, p<0.001). Similarly to our results, Huff- Lonergan et al. (2002) and Škrlep et al. (2009) found that increasing of hot carcass weight increased subcutaneous fat thickness and slightly reduced carcass meatiness. The effect of blood lactate concentration on pH and temperature at 60 minutes postmortem Figures 1 and 2 represent pH values and temperatures measured 60 minutes after slaughter in relation to the blood lactate concentration (below and above 12 mmol/L). In the group of pigs with the lower lactate concentration, a significantly higher (p<0.01) average pH value (6.37) was determined compared to the group of pigs with the higher lactate concentration (6.26). Furthermore, in the group of pigs with blood lactate concentration above 12 mmol/L, meat temperature (38.92°C) was, on average, higher (p<0.001) than in the group of pigs with lower blood lactate concentration (38.40°C). Drip loss did not differ between two groups (data not shown). Edwards et al. (2010b) compared the effect of low (up to 10 mmol/L) and high (more than 10 mmol/L) lactate concentration on drip loss and found a higher drip loss in the group with a higher lactate concentration. These results support the statement that lactate can be used as a predictor of meat quality (Edwards et al., 2010a) and the concentration of 12 mmol/L may be taken as a critical value above which the most important meat quality parameters deteriorate. Figure 1. In the group of pigs with the lower lactate concentration, a significantly higher (p<0.01) average pH value (6.37) was determined compared to the group of pigs with the higher lactate concentration (6.26). Figure 2. In the group of pigs with blood lactate concentration above 12 mmol/L, meat temperature (38.92°C) was, on average, higher (p<0.001) than in the group of pigs with lower blood lactate concentration (38.40°C). and after longer lairage, blood lactate and degree of injuries increased, meat became darker, while drip loss decreased. Higher lactate was associated with lower initial pH value, higher temperature and skin blemishes score and more developed rigor mortis, suggesting that lactate could be predictor of both meat quality and the level of preslaughter stress. Cortisol affected carcass quality, so higher levels of cortisol were associated with increased hot carcass weight, carcass fat thickness on the back and at the sacrum and marbling, but also with decreased meatiness. The most important meat quality parameters (pH and temperature after 60 minutes) deteriorated when blood lactate concentration was above 12 mmol/L.

v3-fos

2019-04-26T14:24:12.267Z

2015-04-01T00:00:00.000Z

132118568

s2

Effect of Land Use Types on Vulnerability Potential and Degradation Rate of Soils of Similar Lithology in a Tropical Soil of Owerri, Southeastern Nigeria A field study was conducted in 2014 to evaluate the effect of three land use types on the vulnerability potential and soil degradation rate of soils of similar lithology in a typic hapludult in Owerri, Southeastern Nigeria. Three land use types namely Fallow Land (FL), Cassava Cultivated Land (CCL) and maize and yam inter crop (MYC), located in three villages in Owerri North Local Government Area of Imo State Nigeria were studied. In each of the village and land use type, three soil samples were collected at the depths of 0-15 and 15-30 cm. Samples were prepared and analyzed using standard methods. Data generated from laboratory analysis were subjected to analysis of variance (ANOVA). Significant treatment means were separated using Least Significant Difference (LSD) while variations among soil properties and relation among soil properties was determined using coefficient of variation and linear correlation, respectively. Results obtained showed that irrespective of the land use, soils of the studied area were strongly acidic with high sand fraction (>70%) resulting to poor physical condition such as poor moisture retention and total porosity. The chemical properties showed moderate organic matter content, total nitrogen and available phosphorus. The exchangeable bases were low and below the critical limits with predominant exchangeable H and Al. Variations existed among soil chemical properties in the three land use types. Soils of the studied area have strong to moderate degradation rate and vulnerability potential. Relationship existed among soil physicochemical properties which positively or negatively interfere with nutrient availability. To reduce the high degradation rate and vulnerability potential of these soils and to improve the fertility status of the soils, it is recommended that farmers should be advised to plant acid tolerant plants. Organic fertilization and liming should be practiced. Cover cropping and conservational tillage should be practiced to reduce erosion and runoff. INTRODUCTION Decline in soil fertility and land degradation has been considered as some of the major constrains facing agricultural productivity in Southeastern Nigeria (Onwudike et al., 2015). Owerri in Imo State, Southeastern Nigeria, is a humid tropical rainforest characterized by high precipitation which causes runoff, leaching of nutrient elements and soil erosion (Onweremadu et al., 2011). Soils of these areas have been subjected to anthropogenic activities thereby resulting to variations in soil properties. Pando-Moreno et al. (2004) have shown that nutrient mining, absence of fallow periods, use of inappropriate farming practices and frequent changes in land uses (over-cultivation), variation in micro-climate, vegetation and parent material aggravate soil degradation, resulting to constant plummeting of soil fertility levels and Int. J. Soil Sci., 10 (4): 177-185, 2015 productivity. Research has shown that anthropogenic activities on the natural terrestrial ecosystem have resulted to variations in the physical, chemical and biological properties of soil (Conant et al., 2003). Some land use and cropping systems like cultivation, deforestation, overgrazing and mineral fertilization have been reported to cause significant variations in soil properties, terrestrial cycle and reduction of output (Conant et al., 2003;Saraswathy et al., 2007). Soil degradation is the lowering of soil physical and chemical fertility to a threshold that limits maximization of agricultural productivity (Ezeaku and Davidson, 2008). Soil degradation of similar lithology may be greatly affected by land use types and cropping systems and these play an important role in agriculture and the environmental management, especially with regard to soil fertility and soil quality (Ezeaku, 2013). There is dearth of information on the degradation rate and vulnerability potential of soil qualities in Owerri. Therefore, information on the rate of degradation, vulnerability potential as well as variation among soil physico-chemical properties due to land use types has become essential for practical and experimental agriculture. MATERIALS AND METHODS Study area: The study was carried out at Owerri North Local Government Area of Imo State, Southeastern Nigeria. The area lies between Latitude 5°17'N and 5°38' N and Longitude 7°11'E and 7°45'E. The area has an average annual rainfall range of 1949-2251 mm and annual temperature range of 27-30°C with average relative humidity of 78%. The geological material of soil in the study area is an ultisol and classified as Typic Hapludtult (FDALR., 1985), derived from Coastal Plain Sands (Benin formation) of the Oligocene-Miocene geological era and are characterized by low cation exchange capacity, low organic matter with high leaching of nutrient elements (Onweremadu et al., 2011). Tropical rainforest is the dominant vegetation of the area, though with remarkable ecological diversity caused by anthropogenic activities, especially farming and deforestation resulting into depleted vegetation as a result of demographic pressure. More than 50% of people in the area are subsistence farmers. Soil fertility restoration in the area is by bush fallow and application of inorganic and organic fertilizers. Soil sampling and experimental design: Soil samples were collected from three land use types in three villages in Owerri North local Government Area, Imo State, Southeastern Nigeria. A reconnaissance visit was made to the study locations to locate the sampling sites and to obtain information from the owners about the study sites. The villages were Ishiuzor village, Ofeuzor village and Umuayalu village. The three locations have similar lithological property (parent material) which is coastal plain sand and this guided the sampling locations. Fallow land (four years fallow) (FL), Cassava Cultivated Land (CCL) and maize and yam intercrop (MYC) were studied. Three soil samples were collected from each of the land use at each location which had similar history and agronomic practices. The three sampling points acted as replications while the land use types were used as treatments. Samples were collected at the root zones of 0-15 and 15-30 cm using soil auger and core samplers for bulk density determination. Each sample was air dried, passed through a 2 mm screen and the coarse fraction (>2 mm) separated. The <2 mm soil fractions were then subjected to laboratory analysis using standard procedures. Laboratory analysis: Particle size distribution was determined by hydrometer method according to the procedure of Gee and Or (2002). Bulk density was determined by core methods according to Grossmans and Reinsch (2002). Total Porosity (TP) was calculated from the result of bulk density as: Soil Sci., 10 (4): 177-185, 2015 (1) Silt/Clay ratio was calculated by dividing the value of the silt fractions by the clay fractions. Soil pH was determined in water and in KCl using pH meter in soil/liquid suspension of 1: 2.5 according to Herdershot et al. (1993). Organic carbon was determined using chromic wet oxidation method according to Nelson and Sommers (1982). Total Nitrogen was determined by kjeldahl digestion method using concentrated H 2 SO 4 and Sodium copper sulphate catalyst mixture according to Bremner and Yeomans (1988). The C/N ratio was determined by computation of organic carbon and total nitrogen values (Brady and Weil, 1999), while available phosphorus was determined using Bray II solution method according to Nelson and Sommers (1982). Exchangeable Mg and Ca was determined using Ethylene Diamine Tetra Acetic Acid (EDTA) (Thomas, 1982) while exchangeable K and Na was extracted using 1 N Neutral ammonium acetate (C 2 H 7 NO 2 ) and then determined using flame photometer (Thomas, 1982). Exchangeable Acidity was measured titrimetrically using 1 M KCl against 0.05 M sodium hydroxide (McLean, 1982), while, effective cation exchange capacity was calculated from the summation of all exchangeable bases and total exchangeable acidity. The Ca/Mg ratio was calculated by the value of exchangeable calcium with exchangeable Mg. Percentage Base Saturation (PBS) was calculated by the summation of the total exchangeable bases divided by effective cation exchange capacity and then multiplied by 100. Soil Vulnerability Potential (SVP) and Soil Degradation Rate (SDR) was estimated using the rating scheme for soil degradation according to Lal (1993). Soil texture, soil pH, soil organic carbon, soil total nitrogen, available phosphorus, effective cation exchange capacity and percentage base saturation was used in the assessment. The vulnerability potentials of these properties were also determined. For SDR, the weighting sequence was as follows: 1 = none, 2 = slight, 3 = moderate, 4 = severe and 5 = extreme. Good soils have the lowest SDR and poor soils have the highest value. For the SVP, the weighting sequence is the reverse of SDR such that: 5 = very low, 4 = low, 3 = medium, 2 = high and 1 = very high according to Lal (1993) and Akpan-Idiok (2012). Data were subjected to analysis of variance (ANOVA). Significant means among treatments were separated using Least Significant Difference (LSD) at 5% probability level. Variability among soil physical and chemical properties were determined using Coefficient of Variation (CV) and ranked according to Wilding et al. (1994) as % CV from 0-15 = low variation, 15-35 = medium variation and above 35 = high variation. RESULTS AND DISCUSSION Soil physical properties of the studied locations: Results of the physical properties of soil in the studied locations are presented in Table 1. Texturally, soils at the epipedon were sandy loam while the sub-soils were loamy sand irrespective of the land use. Sand fractions dominated the particle size distribution. The high sand fraction could be attributed to the parent material dominant in the area which is coastal plain sand since the texture of the soil is highly influenced by parent material over time (Oguike and Mbagwu, 2009). This result agreed with Onweremadu (2007) who observed similar textural characteristics on coastal plain soils in Owerri, Southeastern Nigeria. Also, the humid rainfall characteristics that promote illuviation or leaching of silt and clay particles below the epipedon could contribute to the texture of soils in the area. The bulk density of the soils increased down the depth in each land use. Fallow Land (FL) recorded the lowest bulk density of 1.05 g cmG 3 at the epipedon and 1.12 g cmG 3 at the sub soil. Higher bulk densities were recorded in Cassava Cultivated Land (CCL) and maize and yam inter crop (MYC). This could be attributed to tillage activities since tillage activities could reduce organic matter accumulation which reduces soil bulk density. However, the values of bulk densities were below the critical limit of 1.3 g cmG 3 recommended for tuber and cereal crops (Lal, 1986). Soil total porosity followed the same sequence with soil bulk density with FL recording the highest value of 60.6% at the epipedon and 57.3% at the sub soil. Increase in soil bulk density resulted to a decrease in soil total porosity which could be attributed to compaction of soil macro and micro pore spaces. The highest gravimetric moisture content was recorded in fallow land with the value of 527 g kgG 1 at the epipedon. The gravimetric moisture content of the soils was low and could be attributed to the high sand fraction and low porosity which hinders moisture retention. Soils with this property lack adsorption capacity for basic plant nutrient and water retention (Oguike and Mbagwu, 2009). Soil chemical properties of the studied locations: Results of soil chemical properties of the studied locations are presented in Table 2. Result showed that the soils had a mean pH value of 5.5 indicating strong acidity according to Esu (1991). The highest soil pH of 5.8 and 5.5 was recorded in the fallow land at the surface and sub-surface soils, respectively when compared to CCL and MYC. This could be attributed to litter falls which after decomposition increases soil organic matter and exchangeable bases thereby reducing the accumulation of H and Al ions on soil exchange complex (Onwudike, 2010). Soil organic carbon ranged from 10.9-18.8 g kgG 1 with mean of 13.0 g kgG 1 indicating medium organic matter content according to Esu (1991). The highest soil organic carbon was recorded in the fallow land 18.8 g kgG 1 at the surface and 13.6 g kgG 1 at the sub-surface. The highest organic carbon in the fallow land could be due to litter fall and expected increase in soil biodiversity (Miller and Gardiner, 2001). Bewket and Stroosnijder (2003) have observed that conversion of forest vegetation to agricultural land results in a decline of soil organic carbon content. The above trend was observed in soil total nitrogen and available phosphorus as shown in Table 2. Highest value of total nitrogen (1.6 g kgG 1 ) was recorded in the fallow land at the surface with mean value of 1.3 g kgG 1 . This value according to Enwenzor et al. (1989) is rated medium when compared with the range 2-5 g kgG 1 for productive soils. Similarly, the highest value (16.2 mg kgG 1 ) of available phosphorus was recorded in fallow land with mean value of 14.0 mg kgG 1 . The mean value indicates that the soil is rated medium according to Esu (1991) when compared with the values >20 mg kgG 1 for high available P. Highest value of total nitrogen and available phosphorus recorded in fallow land could be due to litter fall and higher soil organisms that help in organic matter decomposition since there is a positive correlation between organic matter and total nitrogen (Onwudike, 2010). The exchangeable cations were low in the three land use types with the highest values recorded in fallow lands. Exchangeable Ca, Mg, K and effective cation exchange capacity had the highest values of 3.1, 2.7, 0.8 and 7.8 cmol kgG 1 , respectively in fallow land at the epipedon. According to FAO (2006), the soils are rated low in exchangeable Ca (mean 2.2 cmol kgG 1 ), moderate in exchangeable Mg (mean 2.0 cmol kgG 1 ), low in exchangeable K (mean 0.3 cmol kgG 1 ) and very low in effective cation exchange capacity (mean 1.42 cmol kgG 1 ). Base saturation is ranked medium since the mean falls within 50-80% (Esu, 1991). The low exchangeable bases in these locations could be due to high rainfall which accelerates runoff and leaching of nutrient elements down the subsoil. Higher exchangeable bases in the fallow land could be due to the macro and micro climate that hinders the impact of rain drops on soil (Brady and Weil, 2002). Similarly, high ECEC and percentage base saturation in fallow land could be attributed to increase in exchangeable bases and organic carbon obtained from mineralization of litter falls in fallow land. Variability among soil physicochemical properties in the studied locations: Results of the relationship among soil physicochemical properties are shown in Table 3 and 4. Results showed that there was no variation among soil physical properties except in silt and silt/clay ratio that had low to moderate variation. Among soil chemical properties, there was variation in the three land use types. There was low to moderate variations in soil pH, high variation in soil organic carbon and total nitrogen as well as in exchangeable cations and ECEC. The parent material dominant in the locations could be responsible for no variations in the particle size fractions while different management practices existing in these three land use types such as tillage practices that alter the chemical equilibrium of the soil could attribute to variations among soil chemical properties. Degradation rate and vulnerability potentials of soils in the studied locations: Soil properties that were used to assess the degradation rate and vulnerability potentials of the studied Table 5 showed that the SDR for soil texture, soil pH, soil organic carbon, total nitrogen, available phosphorus, ECEC and base saturations was 4, 4, 3, 3, 4 and 3, respectively while their SVP was 2, 2, 3, 3, 2 and 3, respectively. The ratio of SDR and SVP for soil texture (4/2) showed that the studied soils have high susceptibility to degradation and vulnerability potential. This could be attributed to the parent material of the soil (coastal plain sand) that is characterized by high coursed sand fractions as well as high precipitation dominant in the region which enhances soil erosion. Similar observation was made by Akpan-Idiok and Ofem (2014) on Odudu cattle ranch soils of Southeastern Nigeria. The SDR/SVP for soil pH (4/2) showed severe acidity which could be attributed to high rainfall in the area, erosion and leaching of nutrient elements below the epipedon irrespective of the land use and crop management systems. The SDR/SVP for soil organic carbon, total nitrogen, available phosphorus, ECEC and base saturation indicate moderate degradation rate and vulnerability potential which showed that there are moderate availability of basic cations and organic matter in the studied soils which could support plant growth. Therefore, these results showed that soil organic carbon, total nitrogen, available P and base saturation are good soil quality indicators of soil in the studied location. These qualities together will soil pH and texture needs more improvement for maximum crop yield. Relationship mong soil physicochemical properties: Relationships between selected physical properties with soil chemical properties are presented in Table 6. Results showed that sand significantly correlated negatively with available P, base saturation, exchangeable Ca, ECEC, organic carbon, total N and soil pH. Clay correlated positively with the chemical properties except soil pH. There was positive correlation between total porosity and base saturation and negative with exchangeable H. Moisture content correlated positively with the chemical properties while bulk density correlated negatively with base saturation and positively with exchangeable H. High sand fractions in the studied location could reduce the nutrient concentration through runoff and (2014) and Lal (1993) leaching. Increasing the bulk density of the soil could reduce water infiltration, soil aeration and even soil biodiversity which will negatively affect the nutrient available to plants. CONCLUSION Soils of the studied locations are dominated by sand fraction, strongly acidic with low to moderate plant nutrient concentration. Variations existed among soil chemical properties in the three land use types. From the results obtained in this investigation, soils of Owerri Southeastern Nigeria have strong to moderate degradation rate and vulnerability potential. Relationship existed among soil physicochemical properties which positively or negatively interfere with nutrient availability. To reduce high degradation rate and vulnerability potential of these soil as well as to improve the fertility status of the soils, farmers should be advised to plant acid tolerant plants. Organic fertilization and liming should be practiced. Cover cropping and conservational tillage should be practiced to minimize erosion and runoff.

v3-fos

2018-04-03T00:15:32.017Z

2015-02-12T00:00:00.000Z

6260378

s2

Transcriptional response to petiole heat girdling in cassava To examine the interactions of starch and sugar metabolism on photosynthesis in cassava, a heat-girdling treatment was applied to petioles of cassava leaves at the end of the light cycle to inhibit starch remobilization during the night. The inhibition of starch remobilization caused significant starch accumulation at the beginning of the light cycle, inhibited photosynthesis, and affected intracellular sugar levels. RNA-seq analysis of heat-treated and control plants revealed significantly decreased expression of genes related to photosynthesis, as well as N-metabolism and chlorophyll biosynthesis. However, expression of genes encoding TCA cycle enzymes and mitochondria electron transport components, and flavonoid biosynthetic pathway enzymes were induced. These studies reveal a dynamic transcriptional response to perturbation of sink demand in a single leaf, and provide useful information for understanding the regulations of cassava under sink or source limitation. To examine the interactions of starch and sugar metabolism on photosynthesis in cassava, a heat-girdling treatment was applied to petioles of cassava leaves at the end of the light cycle to inhibit starch remobilization during the night. The inhibition of starch remobilization caused significant starch accumulation at the beginning of the light cycle, inhibited photosynthesis, and affected intracellular sugar levels. RNA-seq analysis of heat-treated and control plants revealed significantly decreased expression of genes related to photosynthesis, as well as N-metabolism and chlorophyll biosynthesis. However, expression of genes encoding TCA cycle enzymes and mitochondria electron transport components, and flavonoid biosynthetic pathway enzymes were induced. These studies reveal a dynamic transcriptional response to perturbation of sink demand in a single leaf, and provide useful information for understanding the regulations of cassava under sink or source limitation. C assava (Manihotesculenta Crantz), a tropical plant, is the most important root crop globally and provides food for over 800 million people. Since the storage roots of cassava are enriched in starch 1 and welladapted to barren soils under drought conditions that are common in the tropical world, cassava is considered an important source of carbohydrates to minimize global food scarcity 2 . Developing countries rely heavily on cassava for daily nutrition, comprising 51% (Africa) 29% (Asia) and 20% (South America) of the total diet on these continents 3 . Cassava tubers consist of 0.24% ash, 0.13% fat, 0.49% protein, 0.15% crude fiber and 98.4% starch on a dry mass basis 4 and therefore may also be well-suited to bioenergy and biomaterial production. One reason for cassava's elevated productivity relative to other crops is due to its unusually high rate of photosynthesis. Cassava has a carbon assimilation rate of approximately 43 mmol CO 2 /m 2 /s, that is optimal at high temperature (30-35uC) [5][6][7] and is similar or exceeds rates observed in highly productive C 4 crops including maize, sugarcane and sorghum 8 . As a C 3 plant, cassava lacks C 4 -Kranz anatomy, however it has a low CO 2 compensation point, low photorespiration, and highly active phosphoenolpyruvate carboxylase (PEPC, 15-35% of that in maize, a C 4 plant) [9][10] , characteristics that are consistent with efficient carbon assimilation. Field measurements of single-leaf photosynthesis among a wide range of cultivars collected in CIAT (International Center for Tropical Agriculture) have indicated significant positive correlations between leaf photosynthesis, total biomass and root yield [9][10] . Thus high-yield cultivars have been developed by exploiting genetic variations in leaf histology and biochemistry that enhanced photosynthesis efficiency and productivity 9 . Given the importance of leaf photosynthesis to starch production in cassava, a number of studies have focused on physiological measurements of photosynthetic parameters but few have assessed correlations with the transcriptome. As a result the molecular mechanisms that regulate photosynthesis and starch accumulation in the species remain unclear. The sequencing of the cassava genome (http://www.phytozome.net/), as well as the rapid improvement of second-generation sequencing techniques-such as Illumina sequencing-now provides an opportunity for detailed exploration of the complex regulatory networks that control photosynthesis in cassava. The phloem of higher plants is responsible for the transport of metabolic products and for the recycling of mineral nutrients from the shoot to the root or within the shoot from mature leaves to younger non-photosynthetic tissues 11 . Girdling, a technique using cold or ice treatment as well as the application of heat, vibration, electric or osmotic treatment [12][13][14][15][16][17][18] , can inhibit the transport of assimilates in the phloem, resulting in a decreased sink demand. This strategy is commonly used to study the mechanisms of source-sink balance. In a comprehensive study of multiple species, Goldschmidt and Huber 19 developed a hot-wax-girdling method to prevent the export of assimilates from leaves of soybean (Glycine max L. Merr. cv Ransom), cotton (Gossypiumhirsutum L.), cucumber (Cucumissativus L.), tomato (Lycopersiconesculentum Mill.), broad bean (Viciafaba L.), sunflower (Helianthus annuus L.), and bean (Phaseolus vulgaris L.). They reported different degrees of inhibition of photosynthesis that were species dependent and grouped results on the basis of leaf carbohydrate partitioning attributes (i.e. starch or sucrose stores). Using cold girdling, Krapp and Stitt 20 evaluated the direct and indirect mechanisms underlying the ''sink-regulation'' of photosynthesis in spinach (Spinaciaoleracea L.), and found that the inhibition of phloem export caused by cold girdling led to rapid changes in metabolism, as well as an almost simultaneous change in gene expression. Jeannette et al. 21 also confirmed that carbohydrate accumulation feedback inhibited photosynthesis on gene expression levels, though the signals evoking the inhibition response remain less clear. The ''end-product inhibition of photosynthesis'' hypothesis, as proposed ca. 150 years ago by Boussingault 22 stated that the accumulation of assimilates in an illuminated leaf may be responsible for the decrease in net photosynthesis rate. In most plant species, sucrose is the principal form of carbohydrate translocated through the veins from carbon-exporting source leaves to carbon-importing sink tissues 23 . To investigate transcriptional changes associated with photosynthesis and the genes affected by sucrose transport, and to identify genes that play a key role in balancing source supply and sink demand, we blocked phloem export by a modified heat treatment (first used in stems) for petioles on cassava leaves. RNA-Seq libraries were generated from phloem-blocked leaves and non-blocked controls to examine the transcriptional network response of photosynthesis in individual leaves. The data were assessed bioinformatically to identify transcriptional changes in genes that may play key roles in balancing source supply and sink demand in cassava. Other physiological and metabolic measurements were made to further link changes in the transcriptome with plant function. Results Effects of heat girdling on starch metabolism and photosynthesis. Heat-girdling was performed at the end of the day on the petiole of 120-day-old cassava plants that were developing storage roots (Supplemental Fig. 1, roots in pot). To minimize the effects of wounding and heat stress, a second girdling regime was utilized in which only a partial area of the petiole was heat girdled (partial girdling, PG). The fully girdled leaves (full girdling, FG) had significant amount of accumulated starch at the beginning of the light cycle; however, very little starch remained in the control. Since partially girdled leaves incompletely blocked the phloem transportation, starch accumulation was considerably lower relative to fully girdled plants after a night cycle, but remained 1.5to 2-fold higher than the non-treated control ( Fig. 1a and 1b). The sucrose content was modestly depressed in treated leaves and glucose and fructose contents did not differ significantly from the control (Supplemental Table S1). The development of storage roots may alter the source-sink balance of cassava; thus, we performed an identical treatment on the leaves of cassava seedlings (45 days old), and similar results were obtained (Fig. 1). Together the data indicate heat girdling is an effective way to alter carbon translocation from the leaf. An A/Ci curve was generated after heat girdling to examine the influence of treatments on the photosynthetic assimilation rate. As shown in Fig. 1c, heat girdling inhibited starch remobilization at night and significantly repressed CO 2 assimilation during the light cycle. This repression was not influenced by the development of the storage root. At the same CO 2 concentration, the assimilation rate was considerably lower in heat-girdled leaves than in the control, and this repression was more severe in FG than in PG. Net photosynthesis rates (Pn), stomata conductance (Gs), and intercellular CO 2 concentration (Ci) were all inhibited by the treatment (Supplemental Fig. 2). Mapping the RNA-Seq reads to the cassava reference genome. To identify the genes and pathways that responded to starch accumulation caused by heat girdling, we constructed RNA-Seq libraries from control and heat-girdled leaves from plants in the seedling and storage-root-development stages at 0, 2, and 4 h after light exposure. After examining the effectiveness of heat girdling on starch levels by IKI staining of individual leaf lobes, four leaves from each effective treatment were pooled for library construction and two biological replicates were performed (see Methods). A total of 36 libraries were sequenced using Hi-seq2500. Since the libraries from PG 4 h at the seedling stage were of low quality, we removed this time point from further analysis. A total of 272 million raw reads was generated from the leftover 30 samples. After trimming adapters and filtering out low-quality reads, ,238 million reads (,87.5%) were uniquely aligned to the cassava genome, and ,1.7% of reads aligned to multiple locations. For the unique reads, 74.7% were mapped to exon/protein-coding regions, 14.6% were mapped to the splicing junctions, and others were mapped to intron (3.1%) and intergenic (7.6%) regions. We defined 6,462 novel splicing junctions, which may be used to identify novel transcripts from the cassava genome. We identified a total of 22,284 genes expressed in the cassava leaves. Among these, 6,704 were considered significantly differentially expressed between treatment and control based on a cuffdiff pairwise analysis with the FDR controlled at 0.001. To confirm the accuracy and reproducibility of the Illumina RNA-Seq results, specific genes showing increased, decreased or unchanged expression after treatments were subjected to quantitative real-time (qRT)-PCR confirmation (Supplemental Table S2). As shown in Supplemental Fig. 3, the qRT-PCR expression trends of these genes showed significant similarities to the RNA-Seq data (r 2 5 0.85), which independently validated the gene expression analyses by RNA-seq. Functional category enrichment analysis reveals pathway changes after heat girdling. The log 2 fold changes of 6,704 significantly differentially expressed genes at each time point were entered in the Mapman and Pageman software for pathway analysis. Functional category enrichment was calculated using Fisher's exact test with FDR controlled by Benjamini and Hochberg's procedure at P 5 0.01. As presented in Fig. 2, blocking starch remobilization by heat girdling significantly influenced photosynthesis. The photosynthesis pathways were significantly down regulated compared to the control. This effect was more dramatic in fully girdled plants. Similar trends were observed for the N metabolism and tetrapyrrole synthetic (PG) and fully girdled (FG) cassava leaves. This analysis was performed using the Pageman software package http://mapman.gabipd.org/web 43 . Red boxes indicate that genes in a category were generally down-regulated in the corresponding set of samples relative to controls while blue boxes indicate that genes in a category were generally up-regulated in the corresponding set of samples. Fisher's exact test was applied to test for enrichment of functional category and FDR was controlled for by Benjamini and Hochberg's procedure at P 5 0.01. www.nature.com/scientificreports pathways. In contrast, the major CHO (carbohydrate) catabolic pathways including: glycolysis, TCA and mitochondrial electron transport pathways were significantly up-regulated in the heatgirdled leaves; and most dramatic in fully girdled leaves. The GO term enrichment assay gave similar results (Supplemental Table S3). To identify individual genes that responded to pathway changes, we used heat maps to quantify the expression of DE genes that were enriched in each functional category. Pathways repressed due to girdling. As shown in Fig. 3 (Supplemental Table S4), all photosynthesis-related pathways-including the light reactions, carbon assimilation and photorespiration were repressed significantly by the heat-girdling treatment. Since PG blocked starch remobilization only partially, the decrease in gene expression in PG was not as severe as in FG (Fig. 3, Supplemental Table S4). In addition, the gene expression patterns were similar at two different developmental stages of cassava plants. Girdling resulted in gene expression differences in the light harvesting components including PSI polypeptide subunits, such as PSA-D, PSA-F, PSA-L and PSA-N. PSII-related genes were also affected including chlorophyll a-b binding protein, PSII polypeptide subunits (e.g., PSBO2; PSBQ), NADH DH (NADH: plastoquinone dehydrogenase) complex, NDH-M and NDH-N, and ATP synthase complexes. Calvin-cycle-related gene expression was reduced after heat-girdling treatments including the expression of aldolase, FBPase (fructose-1,6-bisphosphatase), GAPA (glyceraldehyde-3-phosphate dehydrogenase A), phosphoglycerate kinase, PRK (phosphoribulokinase) and RPE (ribulose-phosphate 3epimerase). In addition, the expression of RCA (Rubiscoactivase), which activates Rubisco, was decreased 1.4-1.8-fold by PG treatment and 7.4-12.2-fold by FG treatment. Consistent with RCA, expression of the RuBisCO small subunit, which is involved in net photosynthetic CO 2 assimilation and photorespiratory carbon oxidation 24 , was decreased by 1.4-1.8-and 6.1-8.5-fold by the PG and FG treatments, respectively (Supplemental Table S4). The expression of genes encoding photorespiratory steps also decreased. Glycolate oxidase, alanine-glyoxylate transaminase, serine hydroxymethyltransferase, hydroxypyruvatereductase, and glycerate kinase were all expressed at lower levels in PG and FG samples (Supplemental Table S4). Likewise, Fd-GOGAT and glutamine synthetase 2 (GS2), the chloroplastic enzymes responsible for the reassimilation of photorespiratory ammonia, were also significantly reduced. Other genes that pertain to nitrogen metabolism were affected including NIR1 (nitrite reductase 1) and NIA1 (nitrate reductase 1), key enzymes in nitrate metabolism, and a putative glutamate receptor perceived to be involved in nitrogen transduction (Fig. 3, Supplemental Table S4). Inspection of tetrapyrrole biosynthesis genes (Fig. 3, Supplemental Table S4) that are tied to light harvesting through chlorophyll biosynthesis also produced a number changes in expression. Chlorophyll biosynthesis was repressed by PG and FG treatments, including magnesium chelatase GUN5; magnesium-protoporphyrin IX monomethyl ester (oxidative) cyclase required for the biosynthesis of chlorophyll; GSA2 (glutamate-1-semialdehyde 2,1-aminomutase), an upstream control point in chlorophyll biosynthesis; HO (hemeoxygenase), necessary for the biosynthesis of the phytochromechromophore; and GUN4, which functions as a regulator of chlorophyll synthesis and intracellular signaling. Pathways induced by girdling treatment. Genes involved in catabolic pathways including: glycolysis, the TCA cycle and mitochondrial electron transport changed due to girdling (Fig. 4A, Supplemental Table S5). The affected genes encoding enzymes related to glycolysis included glyceraldehyde-3-phosphate dehydrogenase, which catalyzes 3-glycerol phosphate aldehyde into 1,3 -2 glyceric acid phosphate and pyruvate kinase that produces pyruvate from phosphoenolpyruvate as well. Within the tricarboyxlic acid cycle (TCA) pyruvate dehydrogenase E1 and E2, citrate synthase, succinate dehydrogenase, succinyl-CoA ligase, and isocitrate dehydrogenase were all expressed at higher levels after heat girdling (Supplemental Table S5). The increase was more significant in FG compared with PG. Similar to TCA, most mitochondrial electron transport/ATP synthesis-related genes were induced after heat girdling (Supplemental Table S5), including cytochrome c, cytochrome c oxidase, cytochrome c reductase, and the genes encoding the NADH dehydrogenase complex and F-ATPase subunits. Likewise, metabolite transporters for the mitochondrial membrane, including ATP: ADP antiporters, and mitochondrial dicarboxylate carriers were also upregulated, possibly reflecting changes in the demand for ATP to maintain homeostasis. Girdling leaves exhibited a response to stress leading to enhanced gene expression in the flavonoid biosynthetic pathway that produces anthocyanins were induced. As shown in Fig. 4b (Supplemental Table S5), the genes encoding ACC1 and 4CL3, which act in the general phenylpropanoid pathways; CHS (TT4); CHI (TT5), F3H (TT6), and F3'H (TT7), the early steps upstream of the branch points of flavonoid biosynthesis pathways; DFR (TT3), which is important for anthocyanidin biosynthesis; and FLS, which is required for biosynthesis of flavonols, were significantly induced. As flavonoids play central roles in protection against harmful UV-light and excess visible light, the induction of this pathway may be indicative of imbalance of light energy in the starch accumulated leaves after treatment. This may also explain observed changes in cytochrome b5 family Table S5), which expression were increased by .100-fold after FG compared to the non-treatment control. Cb5 is an electron transfer protein localized mainly in the ER membrane, and may be involved in multiple biochemical activities in cells as an electron donor 25 . Change in starch and sucrose metabolism. Heat-girdling resulted in presence of starch during the light cycle the following day (Fig. 1) and influenced the expression of starch and sucrose metabolism-related genes (Fig. 5, Supplemental Table S6). Some starch biosynthetic genes, such as starch synthase, were expressed at lower levels after treatment; however, other genes, including APL3, which encodes the large subunit of ADP-glucose pyrophosphorylase and catalyzes the first and rate-limiting step in starch biosynthesis; and APS1, which is the major small subunit isoform required for large subunit stability and starch-branching enzymes, such as SBE2 were induced. Genes encoding enzymes involved in starch breakdown including starch phosphorylase (PHS2), and several sucrose-biosynthesis-related genes, including SPS (sucrose phosphate synthase) and SPP (sucrose-phosphatase), were significantly altered. These changes may indicate a reduction in sucrose production because of the imposed limitation on export and translocation and possibly some futile production and degrading of starch as a mechanism to initially cope metabolically with the unanticipated presence of starch at dawn. Generally, cell wall invertases (vacuolar invertases) were expressed at lower levels after heatgirdling treatment though one neutral invertase was less affected and SuSy, a key enzyme in sucrose breakdown in leaves, was induced by the treatments. Sugar sensors and transporters including: HXK1 and its homolog HKL1, SUT and STP13 were induced by girdling. STP13 is a hexose-specific/H 1 symporter that improves plant growth and nitrogen use and is involved in programmed cell death (PCD) 26 . Responses of transcription factors to the heat girdling treatment. After manual curation, we identified 431 putative TF genes that showed differential expression due to the girdling treatment. Hierarchical clustering segregated genes into four major groups according to their expression patterns (Fig. 6, Supplemental Table S7). The C1 and C2 clusters included genes expressed at higher levels by the treatment, and C3 included genes decreased in expression by the treatment. The 47 genes in C4 showed no clear expression trends. There were 87 genes in the C1 and 42 in the C2 clusters. Among them, several transcription factor family members that likely play a role in anthocyanin biosynthesis were identified, including those of the bHLH family TT8, MYB family (MYB111 and MYB4), and MADS family (AGL19 and AGL20) homologs. Apart from anthocyanins, a number of genes that respond to stress were noted. C3H zinc finger; C2H2 zinc finger (STZ), AP2/EREBP; and GRAP; and genes that respond to nutritional status, such as bHLH (cassava4.1_015506m.g), which responds to iron ion starvation; and MYB62, which responds to phosphate starvation, were included in the C1 and C2 clusters. A total of 255 genes were included in C3. Many TFs that are important to light signaling were included in this cluster, e.g. PAP2.4, CIB1, PIL1, PIL5 and ZFN1; as well as TFs that regulate photosynthesis activity, such as CIA2 (chloroplast import apparatus 2), which is important for protein chloroplast targeting; and GPRI1, which regulates photosystem II assembly and chlorophyll biosynthetic processes. Corresponding to the change in TFs associated with light signaling, we found down regulation of the expression of the blue-light photoreceptor CRY1 and PAS/LOV PROTEIN B, and genes involved in red/far-red signaling, such as FRS10 (FAR1-related sequence 10), and SPA family protein (SUPPRESSOR OF PHYA-105). However, the expression of phytochrome B (cassava4.1_000531m.g, Supplemental Table S7) was induced significantly by the treatments. Discussion Examining the coordination between source and sink demand in cassava is important to improve our understanding of regulatory aspects that govern photosynthesis and carbohydrate partitioning and facilitate breeding and genetic engineering efforts aimed at increasing yield. By overexpression of a modified bacterial AGPase in the storage roots of cassava, Ihemere 27 reported a significantly increased AGPase activities and biomass in both storage roots and above-ground tissues, which demonstrated that increasing source strength maybe an effective way to increase yield in sink tissues. However, the impact of reduced sink on source metabolism remains unclear. Heat-girdling of the petiole at the end of the light cycle during developing (120 days) and non-developing (45 days) storage root stages of cassava was used to perturb a single leaf sink demand. Others have used heat 18,20,28 or cold girdling [12][13][14] to explore source to sink relationships though the timing of the girdling process and length of studies varies extensively depending upon specific scientific questions. We were most interested in examining the photosynthetic response that occurs when girdling prevented the turnover of starch which would mimic a change in sink demand in a single leaf. The girdling process presumably inhibited starch remobilization at night based on the extensive starch accumulation and lower sucrose contents in the heat-girdled leaves at the beginning of the light cycle (Fig. 1). The extensive starch accumulation during the light cycle in leaves may also interference photosynthesis, as photosynthetic assimilation was significantly decreased (Fig. 1c). Understanding the direct cause of changes in photosynthetic metabolism has remained challenging. Though the correlation between starch accumulation and photosynthetic inhibition in our study are consistent with other girdling studies at various times 18,19,29,30 ; the direct linkage between the two remains questionable. Starch stores generally show an acute response to girdling that includes starch production and photosynthetic decline; however past studies have indicated starchless lines can also show a similar effect 18 . Using cold-girdling treatment of the low-starch-accumulating plant spinach, Krapp and Stitt 19 concluded that girdling inhibited photosynthesis only minimally; in contrast, in plants that accumulate greater amounts of starch such as maize, a significant decrease in photosynthesis occurred after heat girdling 20 . Thus the qualitative observation that the girdling process varies with species and starch accumulation also highlights the complexity of carbohydrate partitioning and photosynthesis. During the seedling stage, the majority of C 3 cassava plants accumulate starch in the leaves during the day, which is broken down at night to maintain growth 31 . Following their development, storage roots represent a strong sink that can drive assimilation; however, the phenotype resulting from inhibition of starch remobilization at night in a single leaf was similar in heatgirdled leaves irrespective of the presence of storage roots implying that the plant developmental state did not significantly impact the results. Though heat-girdling appeared to inhibit the remobilization of starch at night; it did not increase sucrose content indicating that sucrose production and starch breakdown were coordinated 31 . We observed a significant decrease in the expression of genes associated with the light reactions, the carbon assimilation, and photorespiration (Fig. 2, Supplemental Table S4) suggesting that the increased levels of starch may be inhibiting photosynthetic metabolism and resulting less starch and sucrose production. A 10-fold reduction in rubisco activase and rubisco small subunit expression in FG, and a 2-3-fold reduction in PG, implied a possible decrease in carbon fixation capacity, which may influence sugar metabolism in the cells, as reported previously in sugarcane 33 . Additionally, the expression of the major sugar sensors, HXK1 and HKL1 were induced and the cytochrome b5 family member was increased by .100-fold after FG treatment (Supplemental Table S5, S6), which has been linked to sugar starvation by interacting with sucrose and sorbitol transporters to regulate their affinity for substrate sugars 32 . Carbon and nitrogen metabolism are tied through amino acid metabolism biosynthesis and the production and consumption of energy and reduced nucleotide cofactors 34 . Transcripts including Fd-GOGAT, GS2, and NIR1, were reduced by the girdling treatment (Fig. 3) and may reflect the coordination with carbon and redox. Nitrate assimilation to ammonium and subsequent production of organic nitrogen in the leaf is an intensive process that requires a significant number of electrons supplied through photosynthetic metabolism. The altered capacity to carry out photosynthetic functions would result in an imbalance in carbon, energy, and redox and lead to diminished organic nitrogen biosynthesis 35 . A series of changes that are likely linked to stress metabolism were also observed including anthocyanin and flavonoid biosynthetic pathway genes. Similar to heat-girdled maize and cold-girdled sugarcane 20,33 , anthocyanin gene expression was elevated in girdled leaves. Arabidopsis pho3 mutants that accumulate soluble sugars and starch due to the lack of a phloem-loading Suc-proton symporter also exhibit increased anthocyanin accumulation 36 . Transcription factors including TT8, MYB111 and MYB4 ( Figure 6, Supplemental Table S7) that regulate anthocyanin production were induced as were genes, e.g. FLS, involved in other flavonoid biosynthetic pathways. Anthocyanins and other non-pigmented flavonoids serve to absorb UV light that protects plant DNA from UV damage 37 and their induced expression may suggest a stress response when photosynthesis was repressed. The accumulation of starch in leaves would presumably lead to an imbalance between light harvesting and carbon assimilating steps such that light stress occurred possibly resulting in a transcriptional response paralleling the exposure to UV. Nonetheless other antioxidants, such as catalase, SOD and peroxiredoxin, were principally not induced after girdling treatment (Supplemental table S8), suggesting that the leaves with extensive starch accumulation did not exhibit significant free radical genera- tion after 2 to 4 hrs light exposure. Furthermore, photorespiratory genes that could partially mitigate the generation of free radicals through redistribution of reducing equivalents were repressed. Our data suggest that heat girdling in a single leaf inhibits starch remobilization at night, which causes starch accumulation and inhibits photosynthesis during the light cycle. These changes strongly influence plant primary metabolism, including starch, sucrose metabolism, and nitrogen metabolism and secondary metabolism through the flavonoid and anthocyanin biosynthetic pathways. Changes at the transcriptional level were consistent with maintaining coordination between source and sink, and provide candidate genes to facilitate genetic engineering efforts that increase cassava yield. Methods Plant material and steam girdling of the petiole of leaves in intact cassava plants. The stem cutting of cassava (Manihotesculenta Crantz.), Nigerian TME 7 variety (also known as Oko-iyawo) collected from farms in Nigeria, Africa were established and maintained under tissue culture conditions at Donald Danforth Plant Science Center, USA 38 . The nodes containing an axillary bud were excised from the stems within about 5 mm of each node and collected in a sterile flask. The nodes were then surface sterilized with 15% (v/v) sodium hypochlorite solution containing 2-4 drops of Tween 20 (Sigma Aldrich) for 30 minutes with constant shaking at 150 rpm followed by rinsing with sterile water for 3-4 times. The nodes containing axillary buds cultured on MS (Murashige and Skoog 1962) medium containing 20 g/l sucrose and 2.5 g/l phytogel after trimming off the dead and bleached tissues. The cultures were incubated at 28uC for 3 weeks under 16 h photo period at 75 mmol/m 22 s 21 . The shoots emerging from the nodes were excised and maintained by sub culturing every 4 to 6 weeks on fresh MS basal media. About 1 to 1.5 cm of shoots from the in vitro cultures were transferred to MS media containing 20 g/l sucrose and 2.5 g/l phytogel for 3 to 4 weeks. The plantlets with well-developed roots were transplanted to Fafard 51 growing mixture, Professional Formula (60-65% composted bark, Canadian sphagnum peatmoss, perlite, and dolomitic limestone) under a 14-h light/10-h dark photoperiod (250 to 500 mmol s 21 m 22 irradiance), 29uC day/25uC night temperature cycles with a relative humidity of ,50% in the greenhouse at the Donald Danforth Plant Science Center, USA. The first fully expanded leaves of cassava plants at two developmental stages, 45 days (without storage roots) and 120 days (with storage roots), were subjected to steam-heating of the petioles at the end of the day. To control for heating stress, partial girdling (PG) by steam treatment on the upper side of the petiole, and full girdling (FG)-which involved treatment of the full circle of the petiole-were performed. The treated and non-treated controls of the first fully expanded leaves were collected at 0, 2, and 4 h after exposure to light on the morning of the following day. One leaf lobe from each treated leaf was cut for starch staining to examine treatment effectiveness; the others were immediately individually frozen in liquid nitrogen. A total of four leaves from each treatment were pooled, and two biological replicates were harvested. The same samples were also subjected to carbohydrate quantification and RNA-Seq sequencing. Net photosynthesis rate (Pn) and A-Ci curve determinations. Net photosynthesis (Pn) and CO 2 assimilation rates in steam-treated greenhouse plants were measured the following day at 0, 2, and 4 h after exposure to light, as described above, using the Li-6400 portable photosystem unit at light saturating conditions, irradiance of 1200 mmol m 22 s 21 (Li-Cor Biosciences Inc., Lincoln, NB, USA). The A-Ci Curve was determined accordingly. Starch stain and carbohydrate quantification. To determine the effectiveness of heat girdling on blocking sucrose transportation in phloem, leaf lobes were subjected to iodine potassium iodide (IKI) staining to visualize starch accumulation according to Ruzin 39 . Treated leaves were pooled and ground into a fine powder in liquid nitrogen. Methods of extraction and quantification of sugars were modified according to Lisec 40 . Samples were run on a Thermo Scientific ISQ single quadrupole GC-MS system coupled with a DB5 silica column (30 m length, 0.25 mm diameter, 0.25 m film thickness) for sucrose, glucose, and fructose quantification. After extraction for GC/MS analysis, the tissue residue was re-extracted three times with 80% (v/v) ethanol at 80uC to remove any soluble sugars, and the pellet was dried down. The residue was digested to glucose equivalents using the total starch assay kit (Megazyme, Wickow, Ireland) and spectrophotometrically quantified at 510 nm. The experiment was repeated three times and statistical analysis was performed using the SPSS software. Total RNA, poly(A) RNA isolation, and RNA-Seq library construction. Total RNA from the leaf tissues was extracted using RNA plant reagent kits (Tiangen Company) and purified using the TURBO DNA-free TM Kit (Ambion). The integrity and quality of the total RNA were examined using a NanoDrop 1000 spectrophotometer and formaldehyde-agarose gel electrophoresis. The poly(A) RNA was isolated from purified total RNA using poly(T) oligonucleotide-attached magnetic beads (Invitrogen). Following purification, the mRNA was fragmented into small pieces using divalent cations under elevated temperatures, and the cleaved RNA fragments were reverse-transcribed into first-strand cDNA using reverse transcriptase and random primers. Second-strand cDNA synthesis was performed using DNA polymerase I and RNaseH, and the cDNA fragments were processed for end repair, a single ''A'' base was added, and sequences were ligated to the adapters. These products were then purified and enriched by PCR to create the final cDNA libraries and sequenced on the Illumina Hi-Seq 2500 with 51-bp lengths according to the manufacturer's recommendations (Illumina). Pathway and clustering analysis. Gene expression changes (log 2 fold change of treatment versus non-treated control) were imported into the MapMan and Pageman software (http://mapman.gabipd.org/web/guest) for pathway and/or functional category analysis 43 , and FDR from Pageman enrichment assays were controlled using Benjamini and Hochberg's procedure 44 . HCL (hierarchical clustering) clustering embedded in the MEV software (http://www.tm4.org/mev.html) was used to perform clustering analysis. Quantitative RT-PCR analysis. To verify the RNA-Seq results, quantitative RT-PCR was conducted using SYBR-green (TaKaRa Biotechnology Co., Ltd, Dalian, China) and ABI PRISM/TaqMan 7900 Sequence Detection System (Applied Biosystems), as described previously. We used the Primer Premier v. 5.0 and Vector NIT v.11.0 software (Applied Biosystems) to design primers based on the sequences of key genes of interest identified in our library. The cassava actin gene was used as an internal control. The sequences of primers used in this study are provided in Supplemental Table S1. qRT-PCR reactions were repeated four times for each sample and the relative mRNA level was calculated as 2 2DDCT . Correlation and significance analyses were performed using Microsoft Office Excel 2007.

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In vitro Shoot Multiplication from Nodal Explants of Coccinia grandis (L.) Voigt. and It’s Antidiabetic and Antioxidant Activity Coccinia grandis (L.) Voigt. (Cucurbitaceae) is an important vegetable with high food and medicinal value. In this plant major problem is seed germination, seed setting and less number of fruit settings. Plant tissue culture technology may help to overcome these problems to a great extent. Therefore, the present study has been designed to develop an efficient protocol for in vitro shoot multiplication, as well as to check its antidiabetic and antioxidant activity. The nodal segments used as explants for cultured. Murashige and Skoog’s (MS) agar-gelled medium with different concentration and combination of BAP, KIN, NAA and NB6 for shoots multiplication and callus. Multiple shoots was seen after 10 days of sub culturing on MS medium. Maximum shoot formed (16±1.5) on MS medium containing 0.5 mg LG1 BAP combination with 0.2 mg LG1 NB6 after 15 days. Antidiabetic activity of C. grandis was also evaluated by using α-amylase inhibition assay and DNS assay. Methanolic extract of fruit, callus (BAP+NB6) and different combination mixture of callus at 1.0 mg LG1 concentration showed 54.29, 75.72 and 80.00% inhibition of α-amylase activity respectively. The result of agar diffusion amylase inhibition assay indicated that methanolic extract of callus show maximum inhibition (46.66%) compare to the fruit extract and fresh callus showed no inhibition. Antioxidant activity of C. grandis was evaluated by using DPPH assay method is based on the reduction of methanolic solution of coloured free radical DPPH by free radical scavenger. The DPPH radical scavenging activity and significantly decreased the formation of oxygen radicals generated in rat peritoneal macrophages plant extracts. The protocol described here is efficient with high number of shoot multiplication and can be employed for the large scale production and genetic manipulation for antidiabetic medicinal plant Coccinia grandis. INTRODUCTION In botanical, vegetables refers to "edible part of a plant" mostly collected or cultivated for their nutrition value for human (Hanif et al., 2006). In India, as a large section of people are vegetarian. So, for fulfilling the dietary requirements they depend on vegetables, approximately 400 vegetable crops grown commercially (Nandi and Bhattacharjee, 2005). India is the 2nd largest producer of vegetables in world. In India, 3% of total cropped area is used for crop cultivation. It does not fulfill the requirement of the everyday demand of day/person. This current status not only improves the nutritional requirement but also meet demand of food growing population of day/person is increased. In India, wide range of agro-climatic conditions present which is the maintaining Asian J. Biol. Sci.,8 (2): [57][58][59][60][61][62][63][64][65][66][67][68][69][70][71]2015 continues production of vegetable throughout the year. Vegetables gave more yield than any other crops like rice and wheat. They also provide higher quantity of food per unit area. They also give high farm income than any other crops and high export potential compare to any other field crops. Vegetables have given a boon to processing industry as they can be processed to form diverse varieties of food like sauces, chutney, pickles etc. The maintenance of good health is essential for balance diet including the vegetable. Vegetables provide the vitamin, fiber, protein, carbohydrate and minerals like iron, potassium and sodium. Vegetables have short production cycle which allow for multiple cropping and for that reason large volume of grow worldwide on small area in compare to other crops (Lampe, 1999). Majority of the plants are not amenable to vegetative propagation through cutting and grafting, thus limiting multiplication of desired cultivars. Moreover, many plants propagated by vegetative means contain systemic bacteria, fungi and viruses, which may affect the quality and appearance of selected items. During the last few years tissue culture technique has emerged as a promising technique for rapid and large scale propagation of various plants. Tissue culture is the in vitro aseptic culture of cells, tissues, organs or whole plant under controlled nutritional and environmental conditions, often to produce the clones of plants (Murashige and Skoog, 1962). The control condition provided to culture is required for their growth and multiplication of plant. It includes proper supply of nutrient, pH, temperature and proper gaseous. Research efforts in plant cell and tissue culture have increased dramatically worldwide in recent years including efforts in developing nations. Digestive system as a key enzyme use pancreatic alpha amylase. It catalyzes the starch hydrolysis and form mixture of oligosaccharides such as maltose. It is calved by glycosidase and degrade to glucose which is on absorption enters the blood stream. The disturbance in this pathway leads to hyperglycemia. When rapidly starch degradation occur, also glucose level increase rapidly in small intestine. The control of glucose level is important aspect for type 2 diabetes, hence control of starch degradation by enzyme such as α amylase and glycosidase play a key role in diabetes. This enzyme is major digestive enzyme and helps in intestinal absorption (Thakur et al., 2011). The treatment of diabetes is the potential targets in the development of lead compound are alpha amylase and glycosidase inhibitors (Nair et al., 2013). The lowering serum glucose level and reduction in glucose absorption because of inhibitors of this enzyme control starch degradation and also slow the process of carbohydrate digestion (Sivaraj et al., 2011;Loizzo et al., 2008). Antioxidants are substances which are present at low concentration, which search free radical and prevent damage caused by them, significantly delays or inhibits oxidation of that substrate. They can greatly reduce the damage due to oxidants by neutralizing the free radicals before they can attack the cells and prevent damage to lipids, proteins, enzymes, carbohydrates and DNA. Antioxidant can be classified into two types: enzymatic and non-enzymatic. The enzymatic antioxidants are produced endogenously and include superoxide dismutase, catalase and glutathione peroxidase. The non-enzymatic antioxidants include tocopherols, carotenoids, ascorbic acid, flavonoids and tannins which are obtained from natural plant sources. However, these compounds have some toxic effects like liver damage and mutagenesis. Nowadays, searching for natural antioxidant sources is more important (Munasinghe et al., 2011). Coccinia grandis, the ivy gourd, also known as baby watermelon and little gourd. Coccinia grandis belong to the Cucurbitaceae family, comprises 960 species. Most of the plants in Cucurbitaceae family are annual vines. It is aggressive wine, can form dense mats on lands that readily cover shrubs and small tree. Leaves are simple, arranged alternately along the stems, Asian J. Biol. Sci., 8 (2): 57-71, 2015 shapes are different from heart to pentagon shaped, up to 10 cm long, upper surface of the leaf is green colour and hairless while lower surface are pale-green colour with hair. Tendrils are simple. Flower is white, solitary, calyx five with recurved lobes, corolla lobe ovate and white, three stamens and ovary inferior. Fruit is slimy in touch, pulpy and ovoid to ellipsoid shaped or cylindrical. It possess about ten white stripes on posterior portion, It is green in color when young which turns to scarlet red when it ripes, 2.5-6.0 cm long, 1.5-3.5 in diameter, glabrous, fruit possesses numerous seeds which are oblong, 6-7 mm (Hussain et al., 2011;Starr et al., 2003;Pekamwar et al., 2013;Yadav et al., 2010). Coccinia grandis is mostly used due to its hypoglycemic and antidiabetic properties in India. It has been popularly used as an ayurvedic drug for the diabetes mellitus 2. Plant is roborant, emetic and used to treat inflammation, dyspnea, cough, emaciation, fever with burning sensation, convulsion, syphilis pulse and flower are used in jaundice. Fruit is applied to swelling and taken orally for disorder of blood, cure anemia, dried root powder is cathartic, the ash of root is applied for skin disease and also leaves and stems are antiplasmodic and expectorant, also useful in bronchitis. C. grandis fruit present the bioactive compound well known for its antimicrobial activity against pathogenic bacteria and inhibited the mycelium growth and sporulation (Satheesh and Murugan, 2011;Shaheen et al., 2009;Sivaraj et al., 2011;Farrukh et al., 2008). Also, it has anticancerous activity, antidyslipidemic, antipyretic (Bhattacharya et al., 2011), hepatoprotective activity (Moideen et al., 2011), antiulcerogenic effect (Mazumder et al., 2008) and anti-inflammatory (Ashwini et al., 2012). Coccinia grandis is seed germination, seed setting is low and long period of time dormant condition. This reason natural population day by day decline. It is urgent need to conservation for this species. The present study develop a large scale multiplication protocol for C. grandis using nodal culture and check their antidiabetic and antioxidant activity. Culture medium: The basal medium used for the culture was Murashige and Skoog medium (MS medium) with 3% sucrose and 0.8% agar and different growth hormones (Murashige and Skoog, 1962). Basically, a nutrient medium consists of the entire essential major and minor plant nutrient elements, vitamins, plant growth regulator and carbohydrate as carbon source with other organic substance as optimal additives. The concentrated stock solutions of all the ingredients were Likewise stock solutions of all other ingredients were also prepared and kept under refrigeration. Similarly, stock solutions of growth hormones were also prepared. The medium was prepared by adding required quantities of all the ingredients in the conical flask. After adding all the ingredients in required amounts, the final volume is making up with the help of distilled water. The pH of the medium is adjusted to 5.8 by using 1 M NaOH or 1 M HCl. After adjusting the pH, agar is added to the medium at the rate of 0.8% w/v for solidification of the medium. After pouring media (50 mL/300 mL bottle), bottles are tightly capped and labeled properly. After that media is autoclaved at 121°C for 20 min at 15 psi. These were then left to cool and solid. MATERIAL AND METHODS Explants sterilization: Nodal segments were excised from the grown plants of C. grandis. This segments placed in beaker and covered with net and washed for 30 min under running tap water to remove the all adhering dust particles and microbes from the surface, then sterilized with two to three drops of detergents in required quantity of water and kept for 7-8 min with frequent swirling and after again kept under running tap water until all detergents are removed from the water and followed by washed with distilled water. Surface sterilization treatment of explants: There were used many treatment for surface sterilization of C. grandis for standardization of treatment. For standardization use the antibiotic (Streptomycin), antifungal (Bavistin) and chemical (Hg Cl 2 ) of different combinations and time period. In this plant better result in the washing the explants in 1-2 drops of detergent in 100 mL water, followed by 1000 ppm bavistin solution for 10 min and 200 ppm streptomycin solution for 4 min. After the treatments of 0.1% HgCl 2 for 3 min as a surface sterilization. After all the treatments, explants were removed from the sterilizing solution and rinsed with sterile distilled water. All treatments were properly followed under laminar air flow unit. Inoculation of explants: All the experimental manipulations were carried out under strictly aseptic conditions in laminar air flow bench and follow the standard protocol for inoculation of explants (Thiripurasundari and Rao, 2012). Culture Condition: The tubes and bottles were shifted to culture room with controlled facility of diffused light (2000 lux) for 10 h daily at 28±2°C temperatures and 50-60% relative humidity. Establishment of culture: After approximately 7 days of inoculation, the axillary bud break was seen in the some explants. When the explants attain substantial bud proliferation, these cultures were then transferred to same media containing test tube after 21 days of incubation with a clean and sterilized forceps under laminar air flow unit. The initiated plants were taken out of the test tube, medium adhered to the plants were removed, undesirable/brownish leaves were also removed from the plants and were transferred to the culture test tube containing autoclaved semi-solid media having the same media combination as that for the culture initiation. Then, the all test tubes were placed in growth room under the standard condition. Different medium for establishment of culture are given in Table 1. Asian J. Biol. Sci., 8 (2): 57-71, 2015 Axillary shoots proliferation: Shoots multiplication were repeated subcultures on the multiplication medium. The preparation and sterilization steps for the medium, glassware and chamber were repeated as before. Multiple shoots were transferred from the cultured test tube to a sterile glass plate using sterilized forceps. The brown leaves were removed from the primary shoots and they were sectioned into one node piece after removing the leaves. These nodal segments were then transferred to the multiplication media. All this work was done with extreme care and inside the laminar unit to avoid any possible chance of contamination. This test tube was placed in growth room. Different medium for establishment of culture and multiplication of shoots are given in Table 1. Antidiabetic activity: The calluses (MS+BAP (0.2)+NB 6 (0.2)) were collected after 30-40 days. Calluses were washed with distilled water to remove all adhering particles. After that calluses were allowed to dry at room temperature. Similarly fruits were also allowed to dry in hot air oven. Extraction of callus: Dried sample of callus from in vitro grown nodal explants and fruits were grounded to powder with mortar and pestle. Five gram of samples was extracted using 20 mL of methanol in Erlenmeyer flask placed on shaker at 100 rpm for overnight at room temperature. The crude extract then filtered with filter paper (Whatman No. 1). The filtrate was collected and allowed to evaporate. After evaporation the remaining material was collected and different stocks were prepared by dissolving in methanol for antidiabetic study. Alpha-amylase inhibitory activity: The inhibition assay was performed using the chromogenic DNS method. One unit of enzyme activity is defined as the amount of enzyme required to release one micromole of maltose from starch per min under the assay conditions. The 5 mg of methanolic extract was first dissolved in10 mL of DMSO reagent. Then aliquots of that solution were taken as 0.1, 0.2, 0.3, up to 1.0 mL. Sodium phosphate buffer was added in all the tubes to make up the volume 1 mL. Then 0.5 mL of alpha amylase enzyme was added into each tube. All the tubes were incubated at 37°C for 10 min. After incubation, 0.5 mL of 1% starch was added. Again incubation was done at 37°C for 15 min. After that, 1 mL of 3, 5-dinitrosalisylic acid (DNS) was added in all the tubes. Then, all the tubes were placed in boiling water bath for 10 min and cooled to room temperature. Then, dilute by adding 9 mL distilled water to all the tubes. Control was prepared by taking distilled water instead of methanolic extract solution. The whole procedure was also done for the methanolic extract of callus powder. The 5 mg of extract was dissolved in 10 mL of DMSO reagent and then from that, aliquots were taken. The OD at 540 nm was taken. Percentage inhibition was calculated using following reaction: whereby, Reaction (%)= Enzyme activity in test/enzyme activity in control×100. Alpha amylase inhibition assay: This alpha amylase inhibition screening assay was performed using cup borer method. In this assay, 160 µL of alpha amylase enzyme and 120 µL of solution were mixed and incubated at 37ºC for 45 min. After incubation, the mixture was poured into the well made in the centre of petriplate containing sterile medium containing 1% (w/v) agar and 1% (w/v) starch. Plates were allowed to stand for 3 days at 25°C and then flooded with iodine solution and allowed to stand for 15 min. The diameter of clear zone of starch hydrolysis was measured. As a control, the enzyme was added into the well of the plate. The whole procedure was also done by using callus powder extract. The % inhibition was calculated by following equation: Diameter of test Amylase inhibition (%) Diameter of control 100 Diameter of control Antioxidant activity DPPH free radical scavenging activity: Required quantity of ascorbic acid was dissolved in methanol to give the concentration of 5, 10, 20, 30, 40, 50 µg mLG 1 . Stock solution of sample was prepared by dissolving 10 mg of dried methanolic extract in 10 mL of methanol to give concentration of 1 mg mLG 1 . The 1.5 mL DPPH solution was added to 3 mL methanol and absorbance was taken immediately at 517 nm for control reading. Different volume levels of test sample (0.1, 0.12, 0.14, 0.16, 0.18, 0.2) were added and made 2 mL of each dose level by dilution with methanol. Diluted with methanol with up to 3 and 1.5 mL DPPH solution was added to each test tube. Absorbance was taken at 517 nm in UV-visible spectrophotometer after 15 min using methanol as blank. The Free Radical Scavenging Activity (FRSA) (% antiradical activity) was calculated using the following equation: Control absorbance Sample absorbance Antiradical activity (%) 100 Control absorbance    Statistical analysis: All the experiments were repeated three times with 10 replicates per treatments. Data on the callus proliferation, percentage of regeneration, number of shoots per culture and shoot length was statistically analyzed using the procedure of SPSS package version X, correlation significance were assessed at 0.05 level. RESULTS AND DISCUSSION Coccinia grandis can be propagated through seeds and vegetative cuttings but this conventional propagation of C. grandis has limited potential for large scale cultivation. The application of tissue culture techniques might be of great value an alternative method to achieve large scale propagation independent of season and also used for secondary metabolite production. This study highlight the result of micropropagation of C. grandis and showed its antidiabetic and antioxidant activity. The result of the present investigation on shoot multiplication of Coccinia grandis (Tindora) are presented under the observation. Development of efficient and reproducible regeneration protocol from cell/tissue is a pre-requisite for the successfully application of recent cellular multiplication techniques for the improvement of crop plants. In this direction the choice of explants is of key role of importance and makes an absolute difference between success and failure in inducing regeneration in vitro condition. Effect of BAP on shoot proliferation from nodal explants of Coccinia grandis: Shoot initiation from nodal segments was mainly a cytokinin effect because the explants in cytokinin free medium did not respond. The role of BAP in bud breaking has been recorded in many medicinal plants such as Cucumis sativus (Ugandhar et al., 2011), Wadelia calendulacea (Emmanuel et al., Asian J. Biol. Sci., 8 (2): 57-71, 20152000, Vitex trifolia (Arulanandam and Ghanthikumar, 2011) and Wattakaka volubilis . Ghanthikumar et al. (2013) reported the MS augmented with 1.0 mg LG 1 of BAP in combination with 0.5 mg LG 1 of Kn showed maximum percentage shoot production (Ghanthikumar et al., 2013). Our results showed the best response on the medium containing 0.5 mg LG 1 BAP ( Table 1). Emergence of shoot bud was seen after 5 days of inoculation and after 10 days elongation of shoots was occurred ( Fig. 1c and d). Also, callus formed at the base of the nodal explants. Effect of KIN on shoot proliferation from nodal explants of Coccinia grandis: The MS medium supplemented with 0.2 mg LG 1 KIN show good response for shoot initiation (Table 1). After 5 days of inoculation shoots formation occurred. Secondary root formed after 20 days of inoculation. Similarly in 0.4 mg LG 1 KIN also shoot formation occurred (Fig. 1e). Maximum number of shoots regeneration was found at 4.0 mg LG 1 KIN. Whereas, the shoot buds induction was decreased at high level of KIN (Ugandhar et al., 2011). Effect of NB 6 on shoot proliferation from nodal explants of Coccinia grandis: The MS medium supplemented with various concentration of NB 6 (0.1-0.5 mg LG 1 ) produce shoot initiation after 5 days of inoculation. Shoot elongation was observed after 10 days of inoculation. Best response was seen on the MS medium supplemented with 0.5 mg LG 1 NB 6 (Table 1) (Fig. 1f). Also, root formed in the medium containing 0.3 mg LG 1 NB 6 growth hormones were observed. 0.1 and 0.4 g LG 1 concentration are nodal base callus production after 15th days (Table 1). The NB 6 growth hormone is very closed similar to BA. It is very important in the plant tissue culture for shoot initiations are used. Zhang (2009) reported the Phlox Paniculata, with axillary shoots as explants, basal MS media supplemented with 2.0 mg LG 1 6-benzyladenine and 0.1 mg LG 1 α-naphthalene acetic acid was the optimum media with 91% budding; MS plus 1.0 mg LG 1 6-benzyladenine and 0.1 mg LG 1 α-naphthalene acetic acid was highest shoot proliferation efficiency of 3.1 (Zhang, 2009). Effect of BAP+NAA on shoot proliferation from nodal explants of Coccinia grandis: Result indicated that best response for shoot formation was seen on MS medium containing 0.05 mg LG 1 BAP+0.01 mg LG 1 NAA. Shoot initiation after 7 days of inoculation and elongation of shoot was observed after 15 days (Table 1, Fig. 1f). Karim and Ahmed (2010) reported in teasle gourd, different concentrations and combinations of BAP and NAA had significant influence on shoot regeneration from shoot tips, inter-node, leaf and nodal segments. The explants was more suitable of producing multiple shoots compared to other explants,1.0 mg LG 1 BAP+0.1 mg LG 1 NAA produced shoots in shortest time (15 days) (Nabi et al., 2002). In our study, lower concentration better response for the shoot initiation and number of the shoot is increasing. After 25th day observe the culture root formation (Table 1). It is constant observed that the old culture shown more response towards root formation. It is advice that the after 20-25th day sub culturing is required. Effect of NB 6 +KIN on shoot proliferation from nodal explants of Coccinia grandis: The MS augmented with 1.0 mg LG 1 of BAP in combination with 0.5 mg LG 1 of KIN showed maximum shoot formation (Table 1). Callus induction was observed on the cut surface of nodal segments on MS augmented with BAP in combination with KIN. Literatures suggest that BAP is most active at combination of 1.0-2.0 mg LG 1 in many plant worked by the different authors Asian J. Biol. Sci., 8 (2): 57-71, 2015(Ghanthikumar et al., 2013. Results showing that NB 6 in combination with KIN showed more numbers of shoot formation. 0.4 mg LG 1 of NB 6 +KIN showed the best response for shoot formation (Table 1, Fig. 1g). Shoot initiated within 5 days of inoculation and elongation of shoot was observed after 10-12 days. Abu-Romman et al. (2013) reported in the plant Cucumis sativus L. cv. Sultan callusing ability of hypocotyl explants derived from 6-day-old in vitro seedlings of cv. Sultan was evaluated on MS medium supplemented with individual treatments of different auxins (2,4-D, IAA and NAA) or cytokinins (BA and Kn). Data was analyzed after four weeks of culture and the analysis showed that callus induction frequency, callus growth rate and nature of callus were affected by the type and concentration of the plant growth regulators (Abu-Romman et al., 2013). Effect of BAP+NB 6 on shoot multiplication from nodal explants of Coccinia grandis: The MS medium supplemented with various concentration of BAP combining with NB 6 gave best response than any other single or combine hormone. Shoot formation after 5 days of inoculation. After 10 days elongation of shoot was seen. Maximum shoot was also produced in BAP combining with NB 6 . Result showed that maximum shoot formation was seen on 0.5 mg LG 1 BAP with 0.5 NB 6 (Table 1) and also callus was seen at the base of the nodal explants. Mustafa et al. (2013) reported the MS medium supplemented with different concentration and combinations of BAP (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mg LG 1 ) and L-glutamic acid (0.5, 1.0, 1.5 and 2.0 mg LG 1 ). After two weeks of culture, explants induced little amount of callus and few shoots from the basal end of the explants. Each adventitious shoot was cut from the basal end and sub cultured again on the M S medium fortified with 2.0 mg LG 1 BAP and 0.5 mg LG 1 L-glutamic acid. The subcultures were maintained at interval of four weeks. The frequency of multiple shoots was enhanced on the same medium. The results indicated that the nodal explants were more capable of producing multiple shoots compared to internodes. Large numbers of shoots in short time were produced on MS medium. Effect of BAP+NB 6 on multiplication of shoot from nodal explants of Coccinia grandis: High frequencies of multiple shoot regeneration were achieved from the nodal explants. Explants were inoculated on MS basal medium with different concentration and combination of BAP and NB 6 for shoot multiplication. Nodal segments required 7-10 days to initiate shoots. Multiple shoots was seen after 10 days of subculture on MS medium. Maximum 16 numbers of shoot produce on MS medium containing 0.5 mg LG 1 BAP combination with 0.2 mg LG 1 NB 6 (Table 1, Fig. 1h). Also, after subculture elongation of shoot was observed. Mustafa et al. (2013) reported the bud explants of Momordica balsamina were cultured on MS media supplemented with different concentration of BAP. 1.0 mg LG 1 BAP stimulated the proliferation of bud meristems to form bud clusters and the co-efficient reached 6-8 (Mustafa et al., 2013;Sudha et al., 2011). It is clear that combination of BAP with NB 6 were found to be the best than single application. The MS medium supplemented with BAP with combination of NB 6 was most effective in multiplication of shoots. Also, callus was seen at the cut edges of nodal explants. In our study maximum numbers of the shoot produce in the combination of BAP and NB 6 combination (Table 1). This combination required the maximum 25th days maximum response in the nodal culture (Fig. 1i). Antidiabetic activity: Diabetes mellitus is a common endocrine system disease that causes metabolic disorders and which leads to multiple organ damage. Clinical admiral diabetes is divided into two types, with more than 90% of patients having type 2 diabetes. Type 2 diabetes is global health challenge and the WHO has recommended research and use of complementary medicines for the management of this disease. The number of diabetes cases was 171 million in 2000 and is expected to rise to 366 million in 2030 (Gomathi et al., 2012). The goal of treatment is to maintain normal levels of blood glucose and prevent the development of related disorders. Drugs that reduce post-prandial hyperglycaemia by suppressing hydrolysis of starch, such as PPA inhibitors have been found useful in the control of diabetes mellitus. The medicinal plants or natural products involve retarding the absorption of glucose by inhibiting the carbohydrate hydrolyzing enzymes. Several α-amylase inhibitors including acarbose, voglibose and miglitol are clinically used for treatment but their prices are high and clinical side effects occur (Sama et al., 2012). Many herbal extracts have been reported for their anti-diabetic activities and are currently being used in Ayurveda for the treatment of diabetes. However, such medicinal plants have not gained much importance as medicines due to the lack of sustained scientific evidence (Thakur et al., 2011). In the study, the α-amylase inhibition by DNS assay revealed that the methanolic extract of callus (BAP+NB 6 ) of C. grandis showed maximum inhibition of α-amylase enzyme activity compared with methanolic extract of fruits and different combine hormones used to produce callus (Fig. 2). Methanolic extract of fruit, callus (BAP+NB 6 ) and different combination mixture of callus at 1.0 mg LG 1 concentration showed 54.29, 75.72 and 80.00% inhibition of α-amylase activity respectively. Percentage of inhibition activity increased as the concentration of sample increased (Fig. 2). The plant has been used since ancient times as an antidiabetic drug by physicians who practice Ayurveda. A double-blind control trial conducted in India, demonstrated significant improvement in glycemic control following 6 weeks' use of powder from locally obtained crushed dried leaves of C. grandis in patients with poorly controlled or otherwise untreated type 2 diabetes (Kuriyan et al., 2008;Doss and Dhanabalan, 2008). Thus, data presented here indicate that methanolic extract of callus of C. grandis possesses significant antidiabetic activity. The mechanism by which callus of C. grandis exerted antidiabetic action may be due to its action on carbohydrate binding regions of α-1, 4 glucosidic linkage in starch and other related polysaccharides have also been targets for the suppression of postprandial hyperglycemia. This enzyme is responsible in hydrolyzing dietary starch into maltose which then break down to glucose prior to absorption. Since, α-amylase play as an important role in starch break down in human beings and animals, the presence of such inhibitors in food stuffs may be responsible for impaired starch digestion (Jiju et al., 2013). Food-grade phenolic α-amylase inhibitors from dietary plant extracts are potentially safer and therefore may be a preferred alternative for modulation of carbohydrate digestion and control of glycemia index of food products. This α-amylase inhibitor may be of value as novel therapeutic diabetic agents. Mode of inhibition of alpha amylase inhibitors: Nonetheless, it is important to mention here that α-amylase breaks down starch into disaccharides that are acted upon by isomaltases, especially α-glucosidase to release glucose. The presence of potent α-glucosidase inhibitory activity therefore, appears more important in controlling the release of glucose from disaccharides in the gut than α-amylase inhibition. However, moderate α-amylase inhibition with potent α-glucosidase inhibitory activity may offer better therapeutic strategy that could slowdown the availability of dietary carbohydrate substrate for glucose production in gut. Food-grade phenolic α-amylase inhibitors from dietary plant extracts are potentially safer and therefore may be a preferred alternative for modulation of carbohydrate digestion and control of glycemic index of food products. Furthermore, the present results demonstrate that the methanolic extract from C. grandis fruit, methanolic extracts of callus and methanolic extract of Callus-BAP+NB 6 contained potent α-glucosidase, α-amylase inhibitors and were effective for suppressing post-prandial hyperglycemia (Fig. 2). Amylase inhibition assay: The result of agar diffusion amylase inhibition assay indicated that methanolic extract of callus show maximum inhibition compare to the fruit extract and fresh callus showed no inhibition (Table 2). Gulati et al. (2012) reported the preliminary agar diffusion amylase inhibition assays indicated that all of the Australian aboriginal plant extracts showed complete inhibition of α-amylase enzyme such that no hydrolysis of starch was evident. Antioxidant activity Antioxidant activity of methanolic extract of Coccinia grandis DPPH Method (1, 1 diphenyl 2, picryl hydrazyl: DPPH assay method is based on the reduction of methanolic solution of color free radical DPPH by free radical scavenger. The procedure involves the measurement of decrease in absorbance of DPPH at its absorption maxima of 517 nm, which is proportional to concentration of free radical scavenger added to DPPH reagent solution. The activity is expressed as effective concentration EC 50 . Based on the data it can be concluded that methanolic extract of callus powder showed greater activity compared to the methanolic extract of fruits powder (Fig. 3). The activity of these two extracts is compared with standard ascorbic acids. The activity of methanolic extract is comparatively similar with standard ascorbic acid. The methanolic extract of callus showed more activity than the methanolic extract (Fig. 3). Rawri et al. (2013) reported the alcoholic leaves extract of C. grandis dose dependently demonstrated antioxidant potentials by DPPH scavenging activity. The DPPH scavenging potential of extract might be due to its reducing actions, which might donate hydrogen to a free radical, reducing it to non-reactive species. The presence of reeducates are responsible for reducing capacity, which involved in prevention of chain initiation, binding of metal ions, decomposition of peroxides and radical scavenging (Rawri et al., 2013). Coccinia grandis also showed the DPPH radical scavenging activity and significantly decreased the formation of oxygen radicals generated in rat peritoneal macrophages. The DPPH assay is one of the most widely used methods for screening antioxidant activity of plant extracts (Nanjo et al., 1996). The DPPH is a stable, nitrogencentered free radical which produces violet colour in ethanol solution. It was reduced to a yellow coloured product, diphenylpicryl hydrazine, with the addition of the fractions in a concentrationdependent manner. The reduction in the number of DPPH molecules can be correlated with the number of available hydroxyl groups. All the fractions showed significantly higher inhibition percentage (stronger hydrogen donating ability) and positively correlated with total phenolic content. CONCLUSION The result of the present investigation on Coccinia grandis is a perennial creeper, described as 'Indian substitute for Insulin' for in vitro shoot multiplication and assessment of antidiabetic and antioxidant activity. Maximum shoot formed on MS medium containing 0.5 mg LG 1 BAP combination with 0.2 mg LG 1 NB 6 . Also, after sub culturing elongation of shoot was observed. Thus, present protocol showed more efficient for more number of shoot produce with short time and more survival rate. Methanolic extract of callus of C. grandis possesses significant antidiabetic activity. Methanolic extract from C. grandis fruit, methanolic extracts of callus and methanolic extract of callus BAP+NB 6 contained potent α-glucosidase, α-amylase inhibitors and were effective for suppressing post-prandial hyperglycemia. The DPPH assay is one of the most widely used methods for screening antioxidant activity of C. grandis also showed the DPPH radical scavenging activity and

v3-fos

2018-04-03T05:01:40.844Z

2015-12-01T00:00:00.000Z

36901698

s2

Engineering soil organic matter quality: Biodiesel Co-Product (BCP) stimulates exudation of nitrogenous microbial biopolymers Biodiesel Co-Product (BCP) is a complex organic material formed during the transesterification of lipids. We investigated the effect of BCP on the extracellular microbial matrix or ‘extracellular polymeric substance’ (EPS) in soil which is suspected to be a highly influential fraction of soil organic matter (SOM). It was hypothesised that more N would be transferred to EPS in soil given BCP compared to soil given glycerol. An arable soil was amended with BCP produced from either 1) waste vegetable oils or 2) pure oilseed rape oil, and compared with soil amended with 99% pure glycerol; all were provided with 15N labelled KNO3. We compared transfer of microbially assimilated 15N into the extracellular amino acid pool, and measured concomitant production of exopolysaccharide. Following incubation, the 15N enrichment of total hydrolysable amino acids (THAAs) indicated that intracellular anabolic products had incorporated the labelled N primarily as glutamine and glutamate. A greater proportion of the amino acids in EPS were found to contain 15N than those in the THAA pool, indicating that the increase in EPS was comprised of bioproducts synthesised de novo. Moreover, BCP had increased the EPS production efficiency of the soil microbial community (μg EPS per unit ATP) up to approximately double that of glycerol, and caused transfer of 21% more 15N from soil solution into EPS-amino acids. Given the suspected value of EPS in agricultural soils, the use of BCP to stimulate exudation is an interesting tool to consider in the theme of delivering sustainable intensification. Introduction The greatest challenge for soil science over the next few decades is to help alleviate competing demands on soil for security of water, food, fuel, fibre, and ecosystem pressures without expanding the total area of soil under exploitation (Tilman et al., 2011). The need to engineer soils to deliver sustainable intensification objectives is therefore increasingly urgent. It is also increasingly achievable as the shortfalls between soil capability and actual condition become more apparent (McBratney et al., 2014). We propose that some alleviation to the pressure on soil functions may be found through employing native soil microbial populations to better utilise the waste streams from otherwise competing industries. If successful, this approach will challenge the simplistic perception that one agricultural output (e.g., food) necessarily conflicts with another (e.g., fuel). The global demand for biodiesel is driven by the need to reduce dependence on fossil fuels which exacerbate climate change. However, the energy embodied in fertiliser use for crop growth is significant, with fertiliser N production estimated to account for 2% of the total global energy budget (Smith et al., 2012). Moreover, the variable efficiencies of biodiesel production are strongly affected by subsequent loss of fertiliser N from soils, especially as N 2 O which has about 300 times the global warming potential of CO 2 on a mole for mole basis (Crutzen et al., 2008). Nitrogen dynamics (affected by soil management) are therefore central to the debate surrounding the efficiency of biofuel production and life cycle analysis (LCA) of the biodiesel industry. Biodiesel Co-Product (BCP) contains many biodiesel-processing residues, being a water soluble mixture of glycerol, salts of fatty acids, methyl esters, mono-and di-glycerides, potassium (or sodium) hydroxide, methanol and water. After production, BCP can be refined, for example to isolate glycerol for industrial/cosmetic uses. This was once an attractive option, but while it is possible to obtain glycerol (N 95% purity) from BCPthis requires financial outlay for refining facilities, is energy intensive, and so can be counter-productive (Raghareutai et al., 2010). More direct uses of BCP offer intriguing environmental prospects, e.g., as a component of soil treatments to engineer improved (or repair lost) soil functions. The application of BCP to soil as a substrate for the native soil microbial biomass was previously found to be N99% effective in causing nitrate (NO 3 ) capture from soil solution and thus preventing loss from surface horizon (23 cm) in winter (Redmile-Gordon et al., 2014a). BCP therefore has considerable potential for improving nitrogen use-efficiency and limiting the environmental damage caused by 'leaky' agriculture. While the previous study demonstrated that BCP prevented depletion of soil N, only about half of the N prevented from leaching could be accounted for by the microbial biomass N. The net gain in soil N observed suggested that a large alternative (extracellular) repository of N was created. Besides reducing losses of N, the agricultural advantages of increasing the mass of the extracellular matrix are suspected to include: (i) improving soil aggregate stability (Watts et al., 2005;Redmile-Gordon et al., 2013), (ii) increasing survivability of the soil microbial biomass under drought conditions (Or et al., 2007a) and (iii) improving overall water retention in sandy or dry soils (e.g., Rosenzweig et al., 2012). The recent development of methods to extract and quantify changes in the mass of the extracellular microbial matrix, or 'extracellular polymeric substances' (EPS; Redmile-Gordon et al., 2014b) mean that more descriptive investigations of soil organic matter (SOM) are now possible, and therefore of value in the investigation of 'organic engineering' methods which aim to cause the redirection of soil microbial resources into the extracellular matrix. An unpublished study of the soils treated by Redmile-Gordon et al. (2014a) indicated that BCP had indeed increased the production of EPS in soil. In the present study we hypothesise that, in a similar soil, increased allocation of the NO 3 -N pool to EPS-N would arise from BCP additions (in comparison to no amendment). Since the wide variety of labile carbon compounds in BCP is likely to support a diversity of microbial activity and niches, we also hypothesised that this soil amended with BCP would induce greater production of EPS-protein and EPS-amino acids (per unit of microbial biomass) than soil amended with a simple carbon source (glycerol). These hypotheses were investigated through 15 Nstable isotope probing of extracellular amino acids produced de novo, which were performed alongside more rapid colorimetric techniques. Increased abundance of heavy isotopes in the EPS was taken as evidence that the applied NO 3 -N had been immobilised from the soil solution as opposed to N derived from the atmosphere or soil organic matter. The content of extracts of EPS were also compared to the total hydrolysable amino acid pool (THAA; Knowles et al., 2010) to provide a contrast with the amino acid profile of the more general soil organic matter (SOM) pool. Since the purpose behind engineering an increased EPS production and reduced N loss in soil is towards delivering sustainable intensification, we also compared responses to BCP made from recycled vegetable oils (BCP R ; from a variety of restaurants) with BCP produced from foodgrade 'virgin' oilseed rape (BCP V ). The aim of this additional comparison was to contribute towards future development of more complete LCA's of biodiesel made from oil-crops grown for that sole purpose (BCP V ) and biodiesel made from recycled oils (BCP R ). Preparation of Biodiesel Co-Product (BCP) An alkali-catalysed transesterification of vegetable oils produced approximately 20 L of methanol rich BCP per 100 L biodiesel after separation. A catalytic quantity of 7 g pure KOH L −1 oil (corrected quantity for actual purity of 82%) plus a titrated quantity (2.5 g of 82% KOH to neutralise free fatty acids in the feedstock oil) was used for transesterification. Excess methanol was evaporated from 1 L of BCP in a 5 L beaker placed on a hotplate for 2 h (stirred at 70-80°C). The organic constituents of BCP were determined externally by Harvest Energy Ltd. (York House, London), and are provided in Table 1. Total C and N were determined by combustion analysis (LECO). Inorganic content was given by analysis of duplicates using ICP-MS after digestion of subsamples of BCP in a mixture of nitric and perchloric acid (Zarcinas et al., 1987). Of the 20 elements measured, BCP contained lower concentrations than material harvested from a non-contaminated grassland at Rothamsted (UK), with elements As, Cd, Cr, Ni and Pb always below 1 ppm (Supplementary Table 1). Importantly, the total N contents of both BCP's were negligible (b 0.0005%; below the limit of detection). Soil sampling Soil was sampled on 22 nd February 2012 from the Highfield Conversion Experiment at Rothamsted Research. This soil is a Batcombe Series (Soil Survey of England and Wales) fine silty loam, over clay drift with siliceous stones: roughly equivalent to a Chromic Luvisol as defined in the FAO soil classification system (Avery and Catt, 1995). Plot 12 (historically managed as grassland and switched to arable management in 2008) was sampled randomly with a 2.5 cm auger to a depth of 23 cm and sieved b 2 mm. Visible root material and organisms were removed. This specific soil management of grassland to arable was selected due to the intrinsic presence of a large pool of humified (and refractory) organic matter. The soil was pre-incubated at 25°C in the dark for one week to allow the mineralisation of substrates brought into fresh contact with microorganisms during sieving. Sand silt and clay proportions were 11, 66 and 23%, respectively (Watts and Dexter, 1997). The pH of soil in water was 6.50. The soil contained little easily available C (by design) due to the pre-incubation period and sampling time (late February 2012). Inorganic N availability was also likely to be low after exposure to 5-months rainfall and N leaching following the wheat harvest in August 2011 (Redmile-Gordon et al., 2014a). Treatments Glass equipment was used throughout. Treatments (Table 2) were prepared in water (18 MΩ resistivity). K 15 NO 3 (aq) was added to glycerol (39.13% C; Molecular biology grade, VWR Prolabo product# 444482 V), BCP R (51.52% C), or BCP V (39.17% C). BCP R was produced from reclaimed cooking oils and BCP V produced from the oil of pressed oilseed-rape. Since K + is a major component of BCP, and was always present in excess of biological requirement in treatments including BCP, we used it as the cation to balance counter-ion concentrations in amendments (OH − , Cl − ). BCP is variably alkaline and therefore the BCP treatments were adjusted to pH 8 using HCl (1 M). Further KCl (1 M) was added to BCP R , control and glycerol treatments to balance the concentration of Cl − . The glycerol and water treatments were brought to pH 8 using 0.1 M KOH. The remaining water was then added. Soil moisture was brought to 60% of its water holding capacity (WHC) by addition of each amendment. Each portion of moist soil was placed into 125 mL glass beakers thus forming 12 microcosms. The four aqueous solutions (control containing only water and N) were mixed immediately before drip-application to soil. All microcosms were thus provided with 150 μg 15 N g −1 soil as 99.7 Atom % Table 1 Composition of BCP's produced from i) waste cooking oil from a variety of restaurants (BCP R ), and ii) virgin oilseed rape (BCP V With the exception of the controls, all treatments gave 3 mg C g −1 soil, (either as BCP V , BCP R , or glycerol) with a substrate C/N of 20. Each microcosm was then placed in a desiccator (each desiccator forming 1 block of the treatment design) and experimental units randomly distributed within. A vial of soda lime was placed in the centre of each desiccator to prevent CO 2 accumulation. Silica gel in the base of each desiccator (380 g dry weight) was changed every 48 h in order to progressively dry the soil, which favours the production of EPS (drought was previously seen to maximise EPS production in sand; Roberson and Firestone, 1992). Soil was allowed to dry to 20% WHC before being remoistened to 40% WHC with deionised water: this was required twice over the experimental period of 10 days. Besides previously being observed to trigger an EPS response, moisture fluctuations ensured more complete circulation of solutes as would occur naturally in an exposed system. EPS extraction and colorimetric analyses EPS was extracted as described by Redmile-Gordon et al. (2014b). Briefly, after the removal of soluble microbial products from the soil with dilute CaCl 2 (0.01 M), EPS was extracted using cation exchange resin (CER) following separation by centrifugation. Three technical replicates of moist soil (2.5 g dry weight equivalent) were used to estimate microbial ATP before extraction and three were subjected to extraction of EPS. ATP concentrations of the extracted soils were compared with those of the non-extracted to estimate the extent of cell-lysis. ATP of the microbial biomass was quantified using the method described by Redmile-Gordon et al. (2011). EPS-polysaccharide content was estimated colorimetrically using the method of DuBois et al. (1956) and EPSprotein using a Lowry microplate assay modified for use with soil extracts (Redmile-Gordon et al., 2013). Polypeptide hydrolysis and purification 20 mL quantities were taken from each technical replicate of EPS extract, then combined and freeze-dried to provide lyophilate of 60 mL extract per biological replicate (or microcosm). Lyophilate was transferred to 10 mL thick-walled pyrex digestion tubes (threaded apertures with PTFE sealed caps) using 3 successive 1 mL aliquots of 0.1 M HCl. The HCl was then removed under a stream of N 2 at 60°C. Norleucine (20 μg) was added to each EPS extract and each soil (soils previously air-dried and milled) to serve as an internal standard (Jim et al., 2003). Amino acids in the soil (THAA) and EPS-AA were liberated by hydrolysis with 5 mL of 6 M HCl at 100°C for 18 h. Asparagine (Asn) and glutamine (Gln) could not be distinguished from their corresponding acids (Aspartate and Glutamate) as the amides were destroyed during hydrolysis (Roberts and Jones, 2008). The sums of original amides and deamidated amino acids are thus referred to as Glx, and Asx, respectively. All glassware had been washed with detergent (Decon 90; Decon Laboratories Ltd.), rinsed with double distilled water (DDW) then baked in a furnace at 450°C for ≥ 8 h. Double distilled water (DDW) was produced using a Bibby Aquatron still. Water was also extracted with dichloromethane (DCM) before use. Impurities were removed from the hydrolysate by washing through cation exchange resin (Dowex 50X-W8; Sigma Aldrich, Dorset, UK). Resin was pre-soaked for 12 h in 3 M NaOH to remove contaminants. NaOH was then removed and resin washed in DDW (× 6). To ensure all cation exchange sites were saturated with H + , resin was steeped in 6 M HCl for ≥24 h. Aliquots of resin (1 mL) were transferred to flash columns, and washed (6 × 2 mL DDW). Each soil hydrolysate was added to a dedicated column for the exchange of AAs. Non-affixed solutes were eluted to waste with 4 × 2 mL DDW before displacing the AAs with 6 × 1 mL 2 M NH 4 OH. The resulting solution was blown down to dryness under N 2 at 60°C. Derivatisation and GC-FID analyses A solution of AA standard was prepared containing 1 mg of each AA mL −1 0.1 M HCl (isotope ratios at natural abundance) firstly to validate retention times and secondly to confirm fragmentation weights and ratios given by mass-spectrometry (Section 2.7). All solvents were HPLC grade. N-acetyl, O-isopropyl derivatives of hydrolysed AAs were prepared firstly by conversion to their respective isopropyl esters followed by N-acetylation as described by Corr et al. (2007). Preparations were cleaned by phase extraction with saturated aqueous NaCl and remaining solvent was evaporated under a gentle stream of N 2 at 20°C. Potential residual water was removed by sequential blow down in DCM. Derivatised samples were capped, sealed with PTFE and stored in a freezer at −20°C for subsequent analysis. In preparation for GC-FID, samples were suspended in approximately 100 μL ethyl acetate and analysed using a Hewlett-Packard 5890 gas chromatograph fitted with a flame ionisation detector (FID) and VF-23MS capillary column (3-cyanopropylpolysiloxane stationary phase, 60 m × 0.32 mm internal diameter; 0.15 μm film thickness; Varian Ltd., Oxford, UK). About 0.5 μL of each sample was introduced using direct on-column injection. The carrier gas was H 2 with a flow rate of 3 mL min −1 ; head pressure 10 PSI. Initial oven temperature was 40°C and held for 1 min before heating at 15°C min −1 to 120°C (no hold), then 3°C min −1 to 190°C (no hold) and finally 5°C min −1 to 250°C and held for 20 min. The concentration of each AA was calculated by comparison of peak area with the internal standard (norleucine) and adjusted for flame response of each AA as given from the mixed standard. GC-combustion-isotope ratio MS (GC-C-IRMS) analyses and GC-MS Nitrogen stable isotope compositions were determined by GC-C-IRMS using a Thermoquest Trace GC connected to a DeltaPlus XP via a GC combustion III interface. A similar VF-23MS capillary column was used as for GC-FID (see above). Samples were introduced to a PTV 'splitless' injector via auto-sampler. δ 15 N values needed no adjustment for addition of the derivative (NAIP) as it contains no nitrogen (Corr et al., 2007). Compound identities were verified using a Trace GC/MS (Thermo Finnigan Ltd., Hemel Hempstead, UK) also equipped with a VF23-MS column, carrier gas was helium (2 mL min −1 ). Samples were introduced in splitless mode (inlet temp 250°C). Source held at 200°C, ionisation energy 70 eV with mass analyser scanning the range m/z 50-650. The GC oven was programmed as for GC-FID (described in Section 2.6). Mass spectra were consistent with those normally Statistical analyses Treatments were analysed for their contributions to variables of AA concentration and nitrogen isotope compositions. For direct comparison of nitrogen isotope compositions between the EPS and THAA extracts ANOVA was performed using Genstat software to analyse the experimental structure (blocks/treatment/extraction-method/AA). This enabled statistical distinction between the two pools of AAs to identify which pool contained proportionally greater 15 N (from KNO 3 ): THAA vs EPS-AA. Subsequently, THAA and EPS were analysed separately as different pools to quantify the likelihood of treatment effects and AA interactions (the 'interaction' being used to indicate AA specific changes in the AA profile or 15 N enrichments in response to treatment). Since residual variability increased with concentration, the data were transformed (log 10 ) to identify statistical significance of effects. 15 N % enrichment is presented on the natural scale as no systematic increase in residual variability was observed. All analyses were performed using Genstat software (15th edition). GC-FID quantitative analyses While glycerol and BCP R both appeared to potentially increase the concentrations of amino acids (AAs) in the total hydrolysable pool (THAA; Fig. 1A) ANOVA revealed no statistically significant effect of treatment (P = 0.363). The only statistically significant effect upon concentration was the identity of the AA being quantified (p b 0.001). In contrast, quantitative analysis of the EPS showed a statistically significant effect of treatment ( Fig. 1B; p = 0.02). An interaction effect was identified between treatments and AAs which was statistically significant (p = 0.001; Supplementary Table 3). This means that treatments exerted bias, augmenting some residues more than others (creating contrasting 'AA profiles'). Comparing EPS-AA concentrations per unit soil determined by GC-FID alone, shows no statistically significant differences in mean EPS-AA concentrations between treatments BCP V , BCP R , and glycerol (Supplementary Table 3)with all giving statistically significant increases in EPS-AA relative to 'N only' (l.s.d. = 0.095 μg g − 1 soil; data transformed log 10 ). However, Section 3.3 shows contrasting results when investigating EPS per unit microbial biomass ATP, i.e., as 'EPS production efficiency'. Treatment effects on nitrogen stable isotope compositions In the THAA pool 15 N-labelling was greatest following the treatment with glycerol and declined in the order glycerol N BCP V = BCP R N N only (l.s.d. 0.23%; Fig. 2A). This was true for every AA residue. Mean enrichments (by treatment) were 8.70, 7.57, 7.54 and 0.44%, respectively. However, in the EPS, mean AA 15 N enrichment was greatest for the BCP R treatment and declined in the order BCP R N BCP V N glycerol N N only. This was true for every AA, except hydroxyproline, which was most enriched by glycerol addition (Fig. 2B). Mean enrichments were 18.82, 16.74, 14.82 and 0.63%, for BCP R , BCP V , glycerol, and N, respectively (l.s.d. 0.43%). The 3 AAs most enriched with 15 N from addition of K 15 NO 3 (all treatments) were hydrophobic residues: leucine and isoleucine (branched chain amino acids), and phenylalanine. Phenylalanine is a precursor of tyrosine which was also the 4th most enriched AA. The 3 least enriched were glycine, aspartate/asparagine and hydroxyproline. With regard to overall enrichment, the mean AA 15 N enrichment was 6.06% in the soil total hydrolysable amino acid pool (THAA), and 12.75% in the EPS (with maxima of 13.51% and 29.24% 15 N, respectively). ANOVA confirmed there was far greater proportional enrichment of the EPS vs. THAA (p b 0.001; Fig. 2A vs. B). EPS colorimetric determinations and production efficiencies ANOVA of the quantitative colorimetric analysis of EPS-protein also showed statistically significant treatment effects ( Fig. 3; p = 0.025). BCP R caused the greatest increase of Lowry reactive EPS-protein of 61.9 μg g − 1 soil more than the 'N only' control (169.7 μg g −1 soil). Considering the BCP treatments alone, only BCP R resulted in a statistically significant increase in EPS-protein over 'N only' and glycerol (l.s.d. α = 0.05 = 36.70 μg EPS-protein g − 1 soil). EPS-polysaccharide (as glucose equivalents) showed a more uniform response to C addition with greater EPS-polysaccharide measured with all treatments inclusive of C (ANOVA treatment effect p = 0.002). There were no statistically significant differences in extracellular polysaccharide produced between the two BCP's and glycerol (l.s.d. α = 0.05 = 41.44 μg EPS-polysaccharide g −1 soil). The ATP concentration responses (Table 3) reflected the same pattern of observations as the THAA 15 N % response shown in Section 3.2 (glycerol N BCP V = BCP R N N only) except that BCP R caused a much smaller increase in ATP over the control (+1.8 nmol g −1 soil) than the large ATP increase in soil amended with glycerol (+ 6.71 nmol g −1 soil). Importantly this difference was statistically significant (Table 3; (Table 3; p = 0.024). p b 0.05) demonstrating that glycerol favours growth of the microbial biomass (intracellular biomolecules). The resulting EPS-production efficiencies showed greatest EPS-protein and EPS-polysaccharide production per unit ATP with BCP R . As with colorimetrically determined EPS-protein efficiency described above, the substrate-induced EPS-AA efficiency was greatest when no labile C was provided. However, among the treatments delivering Cincreasing both ATP and EPS -, BCP R resulted in the highest EPS-AA production efficiency (mean = 0.96 μg EPS-AA nmol ATP −1 ), followed by BCP V and then glycerol (Fig. 4). The production efficiencies induced by organic amendment reassuringly match the response pattern for 15 N enrichment (Fig. 2B), i.e., greatest for BCP R , followed by BCP V , then glycerol. For statistical comparison the data required transformation to stabilise residual variability (log 10 ). Efficiency differences between treatments were all statistically significant at the 5% confidence level, except for alanine (Fig. 4). Supplementary Table 2 compares the transformed mean AA data on a numerical basis. Extraction and analytical methods As with the more time-consuming analyses, the colorimetric protein assay showed that BCP R caused statistically significant increases in EPS-protein (Fig. 3). Considering that methods of hydrolysis can be incomplete for hydrophobic peptide residues (Roberts and Jones, 2008) colorimetric analysis of non-hydrolysed extracts remains a justifiable approach and low cost option for measuring EPS-protein in soil. The colorimetric results for polysaccharide (Fig. 3) show that all substrate additions (inclusive of C) caused increase in extracellular sugars, and indicate that polysaccharides form the bulk of the EPS matrix. This corroborates observations made by Schlecht-Pietsch et al. (1994) who also observed an increase in total soil polysaccharide in response to substrate addition, but were unable to distinguish between extra-and intracellular polysaccharide at that time. The amino acids measured in the total extractable pool (THAA) 10 days after treatment was applied accounted for more than half of the 15 N provided. Considering only 14 AAs were quantified, and based on a peptide N content of about 15.5% (Schindler et al., 2007) ⁎ Not directly comparable (not provided with carbon). reasonable evidence that most of the 15 N provided (150 μg N g −1 soil) had been biologically assimilated. This also shows that THAA extraction was highly efficient, which is in agreement with Roberts and Jones (2008) who stated that hydrolysis with HCl at around 100°C extracts most intracellular biomolecules. Considering this high extraction efficiency, the THAA pool is also likely to represent most AAs constituting the extracellular microbial peptides. Moreover, comparison of 15 N enrichment between hydrolysed EPS extracts (up to 29.2% 15 N) with soil THAA (maximum 13.5% 15 N) supports the premise that the THAA protocol extracts a larger proportion of pre-existing extracellular peptides with a natural abundance of 15 N/ 14 N (including pre-existing EPS and decaying organic matter). Conversely, the EPS extraction (using CER) removed a greater proportion of recently synthesised biomolecules. Extraction with CER was proposed to be selective for the extracellular polymers of biofilms (Frolund et al., 1996). The absence of extraction-induced cell lysis has been supported by extracellular ATP analyses in extracts of sediments (Takahashi et al., 2009) and intracellular ATP measurements in soil (Redmile-Gordon et al., 2014b). The present work shows that the EPS extraction procedure selects for microbial products produced de novo (enriched in 15 N) over the older pre-existing soil organic matter (SOM) which contained 15 N at natural abundance. This selectivity for EPS produced de novo was initially proposed to be important by Redmile-Gordon et al. (2014b) who used a colorimetrically determined estimate of co-extracted humified SOM as an indicator of (un)suitability for other EPS extraction techniques. Naturally, such an approach cannot be said to be conclusive due to the lack of a clear chemical definition for humified SOM (and the limited number of soils investigated) but the 15 N results discussed above provide much firmer support for this premise. The advantages of a technique which extracts newly synthesised EPS are that this facilitates scientific investigation by a) reducing the time required for experimental incubation, and b) minimising the risk of mis-allocating effects to EPS that may belong to some other coextracted pool of SOM. For example, Gillespie et al. (2011) demonstrated that functional impacts were likely to be mis-allocated to fungal glycoproteins by the application of over-inclusive extractions with hot aqueous citrate. Finally, c) the measurement of EPS recently exuded is likely to be of highest functional relevance to the contemporary microbes producing it. EPS is a more dynamic fraction of SOM Using highly inclusive extraction methods (Jim et al., 2003;Knowles et al., 2010) the quantitative extracts of total hydrolysable soil amino acids (THAAs) did not show any treatment induced change in soil AA concentration (Fig. 1A). Total soil peptide can be remarkably stable, persisting for years due to physical or chemical stabilisation (Schmidt et al., 2011). However, EPS concentrations were highly responsive (Fig. 1B). The results presented here demonstrate that 1) peptides in microbial EPS are a more dynamic pool of soil organic matter than total soil peptide, 2) the EPS extraction targets this specific pool, and 3) this pool is linked to immobilisation of NO 3 -N. The measurable increase of AAs in the EPS found using GC/FID, and the demonstration that the EPS was serving as an external repository for immobilised nitrate (GC-C-IRMS data) offer intriguing prospects: 1) for subsequent microbial uptake, and 2) direct plant uptake. While AA-N has been predicted to comprise up to 90% of total N flux into maize (Jones and Darrah, 1994), Jones et al. (2013) subsequently reported that 15 N from added glutamate was mostly taken up by plants after microbial mineralisation. Evidence concerning bioavailability is needed to test the assertion that EPS improves efficiencies of nutrient cycling (Flemming and Wingender, 2010) or conversely the possibility that this pool of organic N represents a route for C sequestration (e.g., Knicker, 2011;Cotrufo et al., 2013). In either case, longer term studies are required. The BCP effect Tracing the fate of 15 NO 3 improves our understanding of the mode of action underlying the biological retention of NO 3 -N in soil. The THAA 15 N enrichment firstly showed that microbial assimilation of NO 3 -N had occurred with provision of all labile C substrates. However, the mechanisms of glycerol-and BCP-induced retention were demonstrably different: the hypothesis that BCP would foster greater proportional investment into extracellular polymers was validated, and comparison of the increases in EPS due to substrate addition shows that EPS-AA production efficiency was much greater when provided with BCP rather than glycerol (Table 3). BCP thus promotes EPS exudation (both proteinaceous EPS and exopolysaccharide) with BCP produced from reclaimed oils (BCP R ) causing the greatest bias for exudation of AAs (Fig. 4). The BCP R contained a higher content of fatty acid methyl esters and organic salts of potassium than either BCP V or glycerol (Table 1) which may have contributed to the bias for EPS development and high production efficiencies per unit ATP ( Fig. 4; Table 3). Furthermore, due to the higher C content, less BCP R (total mass) was added than either BCP V or glycerol ( Table 1). Translation of these findings into agronomic practice may help to improve the overall efficiency of biodiesel production, with the greatest benefit arising when biodiesel is produced from reclaimed oils, with the BCP R subsequently being used directly to augment reserves of soil EPS. While this extracellular matrix is likely to be important for maintaining soil structure for agronomic purpose (Or et al., 2007b) field studies are still clearly needed. Addition of N alone caused only a very small increase in 15 N of THAA, indicating C limitation of the microbial biomass. The propensity for glycerol to favour growth of microbial biomass might be expected from its common use as a substrate in batch cultures. However, Hilliou et al. (2009) found that glycerol was also suitable as a sole carbon source for the production of EPS-polysaccharides in batch reactors containing Pseudomonas, and Shemesh and Chai (2013) found glycerol was one of the most effective of a range of substrates for promoting biofilm formation in Bacillus spp. (albeit when coupled with high Mn availability). While glycerol may be a useful substrate for developing biofilms, the concentration of microbial ATP presented in Table 3 suggests that bias for growth of the microbial biomass occurred in this soil. Furthermore, in the THAA pool which includes the bulk of intracellular material, 15 N was greatest in the AAs of soil given glycerol ( Fig. 2A). Among the AAs measured in the THAA fraction, Glx residues showed the greatest increase in 15 N, which is in accordance with Geisseler et al. (2010) who stated that glutamine/glutamate (collectively Glx) are the main intracellular products of inorganic-N assimilation in soil. In contrast to the sum of glutamate and glutamine (Glx) in THAA, the complimentary N carriers: aspartate and asparagine (Asx) contained little of the added 15 N (only Hyp residues showed less enrichment; Fig. 2A). Simultaneously, Asx was the most abundant AA residue in THAA (Fig. 1A). The low enrichment adds support to Payne's (1980) discussion that evolution has favoured the central role of Glx as a biological N carrier. Asx concentration in the EPS extracts was much lower (Fig. 1B) and featured even lower enrichment (Fig. 2B). The exact reasons for this are not clear and studies over time may be revealing. Amino acids in the EPS Leucine, phenylalanine, and tyrosine all show increased proportional abundance in EPS compared to THAA (Fig. 1A vs. B). Furthermore, all hydrophobic residues (Val, Leu, Ile, Phe, and Tyr) in EPS showed disproportionately high 15 N enrichments (GC-C-IRMS; Fig. 2B) compared to the concentration increases (GC/FID). This could be either due to a) increased turnover, or b) the incomplete hydrolysis of hydrophobic residues leading to underestimation by GC/FID (Roberts and Jones, 2008). Stable isotope probing (SIP) can thus give more robust indications of the relative differences between quantitative AA syntheses in soil. Nonetheless, GC/FID results show that BCP R caused the largest increases in concentrations of Leu, Phe, and Tyr in the EPS, with concentrations 70%, 61% and 153% higher than the control (N only), respectively. This more than doubling of EPS-tyrosine concentration through provision of BCP R is intriguing because EPS has long been postulated to serve important roles in colony adhesion of biofilms (e.g., Flemming and Wingender, 2001) and tyrosine has repeatedly been implicated in adhesion and structural roles in the extracellular space of eukaryotes (e.g., Stone et al., 2009), and was recently proposed to play a mechanistic role in the aggregate stability of aerobic sludges (Zhu et al., 2012). The propensity for aromatic residues to aggregate through non-covalent π-π stacking is well reported (e.g., Chourasia et al., 2011). In soil, aromaticity (e.g., phenylalanine and tyrosine) was found to be prevalent in the hydrophobic fractions of soil extracts (Nebbioso and Piccolo, 2011) which also contained more stable macrostructures in solution Piccolo, 2011, 2012). These BCPinduced EPS-tyrosine increases therefore deserve closer attention in the context of improving soil physical characteristics. BCP's potential to favour production of EPS over microbial growth is postulated to be of continued importance for the accumulation of highly water absorbent extracellular polymers in soil. This is because EPS exhibit both hydrophobic and hydrophilic properties (when soils are dry, and after a period of pre-exposure to water, respectively). Or et al. (2007b) describe this phenomenon and its potential significance for agriculture as it prevents osmotic stress and slaking of soil aggregates. Exopolysaccharide production was recently shown to be a process under positive feedback (Elsholz et al., 2014). In the aforementioned study, the EPS-polysaccharide exuded by Bacillus subtilis was found to also function as a signal to productionthus embodying a mechanism for positive feedback. This would be an evolutionarily favourable trait, as extracellular investment in EPS-polymers without a 'switch' to deactivate exudation when wasteful would be selected against (e.g., if extracellular biopolymers were being parasitised). Should this mechanism of positive feedback be ubiquitous in soils, maintenance of extracellular polysaccharide reserves may prove to be an important pre-requisite for the accumulation of organic matter in agricultural soils. Interestingly, hydroxyproline (Hyp) was an exception: becoming most enriched in the EPS through treatment with glycerol. Hyp has been suggested to be involved in metal nutrient acquisition and competitive microbial allelopathy (Vranova et al., 2011). In the current experiment, microbial biomass was found to be greatest following glycerol treatment, and thus probably also was interspecific microbial competition. The increase in 15 N enrichment was not coupled with an increase in the concentration of Hyp in EPS (from GC/FID). This could indicate increased turnover. Indeed, Hyp is hydrophilic which is a property said to contribute to higher bioavailability (Rothstein, 2010). Hyp was also found to be especially abundant in anti-listerial bacteriocins (Chakchouk-Mtibaa et al., 2014). If Hyp is considered an indicator of microbial allelopathy, then the present measurements lend support to the suggestion that EPS provides a direct competitive advantage to the organisms producing it (Flemming and Wingender, 2010). On a less conditional basis the present results concerning Hyp contradict the assertion of Philben and Benner (2013) that plants are the only major biochemical source of hydroxyproline in soil. Conclusions This work provides a clear demonstration using 15 N stable isotope probing with GC/C/IRMS that microbial allocation of nitrate-N to EPS-AA is stimulated through provision of BCP, and most notably by BCP R . Both BCP's from biodiesel manufactured with recycled oils and virgin oils caused greater incorporation of the isotopically labelled NO 3 into the EPS by a statistically significant margin compared to glycerol. The hypothesis that increased production of EPS-protein would occur in soil given BCP was also supported by simple colorimetric analysis of EPS-protein, with BCP R again causing the greatest increase. The BCP R treatment also resulted in the greatest EPS production efficiency per unit ATP (EPS-protein and EPS-polysaccharide). This measure was found to give greater sensitivity as an indicator of changing substrate quality as opposed to either total soil-EPS or THAA concentration. In all aspects, microbial EPS was shown to be more responsive to treatment than the more broadly defined and inclusive pool of SOM represented by THAA. The amino acids Ile, Leu, Phe and Tyr were more 15 Nenriched in EPS than in bulk SOM. The fact that EPS production efficiency was greatest using BCP R as a carbon source contributes to the already favourable LCA associated with the production of biodiesel from reclaimed oils over biodiesel crops grown only for fuel.

v3-fos

2018-04-03T00:11:54.123Z

2015-10-31T00:00:00.000Z

10730478

s2

Probiotic Characteristics of Lactobacillus plantarum FH185 Isolated from Human Feces Lactobacillus plantarum FH185 was isolated from the feces of healthy adults. In our previous study, L. plantarum FH185 was demonstrated that it has anti-obesity effect in the in vitro and in vivo test. In order to determine its potential for use as a probiotic, we investigated the physiological characteristics of L. plantarum FH185. The optimum growth temperature of L. plantarum FH185 was 40℃. L. plantarum FH185 showed higher sensitivity to novobiocin in a comparison of fifteen different antibiotics and showed higher resistance to polymyxin B and vancomycin. It also showed higher β-galactosidase and N-acetyl-β-glucosaminidase activities. Moreover, it was comparatively tolerant to bile juice and acid, and inhibited the growths of Salmonella Typhimurium and Staphylococcus aureus with rates of 44.76% and 53.88%, respectively. It also showed high adhesion activity to HT-29 cells compared to L. rhamnosus GG. Introduction The word 'probiotics,' which is derived from the Greek and means for life, was first used by Lilley and Stillwell (1965) to describe the substances secreted by one microorganism to stimulate the growth of another, as an antonym of 'antibiotic'. In 1974, Parker defined probiotic "as organisms and substances which contribute to intestinal microbial balance". Fuller (1989) redefined probiotics as "a live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance". Lactic acid bacteria (LAB) have complex nutritional requirements and are frequently used as probiotics or in the fermentation of food products. Probiotics consisting of one or more species of live bacteria, such as Lactobacillus and Bifidobacteria, not only affect the intestinal flora directly, but also affect other organs by modulating immunological parameters and intestinal permeability and producing bioactive or regulatory metabolites (de Vrese and Schrezenmeir, 2008; Delzenne et al., 2011;Gerritsen et al., 2011). Probiotics produce a health benefit when administered to animals, including humans. Several studies have reported the health-promoting effects of probiotics, including the maintenance of intestinal mucosal resistance to pathogenic microorganisms (Mennigen and Bruewer, 2009), prevention of diarrhea (Guandalini, 2008), stabilization of gut microflora (Gibson et al., 1997), alleviation of lactose intolerance (de Vrese et al., 2001), immunomodulation (Perdigón et al., 2001), reduced serum cholesterol levels (Nguyen et al., 2007), reduction of bodyweight and metabolic disorders , and the prevention of allergic diseases and cancers (Isolauri and Salminen, 2008;Kumar et al., 2010). Probiotics must be safe for their intended use. The 2002 FAO/WHO guidelines recommend that, though bacteria may be generally recognized as safe (GRAS), the safety of a potential probiotic should be assessed by the minimum required testing, i.e., determination of antibiotic resistance patterns, assessment of certain metabolic activities, acid and bile salt tolerance, ability to adhere to the intestinal epithelium of the hosts, antagonistic activity against pathogenic bacteria, and assessment of the ability to maintain their viability during processing and storage (Lin et al., 2006;Lonkar et al., 2005;Rial, 2000;Schlundt, 2002). In our previous study, L. plantarum FH185 was observed to exhibit lipase inhibitory activity of 70.09±2.04% and to inhibit the adipocyte differentiation of 3T3-L1 cells ( ARTICLE was also demonstrated that the strain has an effect on the reduction of adipocyte size and gut microbial changes in diet-induced obese mice. Thus, this study was performed to investigate the physiological characteristics of L. plantarum FH185 in order to determine its potential as a starter for functional food products. Materials and Methods Bacterial strains A LAB strain having an anti-obesity effect, namely, L. plantarum FH185, was isolated from the feces of healthy adults. In our previous study, L. plantarum FH185 was found to have lipase inhibitory activity of 70.09±2.04% and to inhibit the adipocyte differentiation of 3T3-L1 cells (18.63±0.98%) at a concentration of 100 µg/mL. It was also demonstrated that the strain has an effect on the reduction of adipocyte size and gut microbial changes in diet-induced obese mice (Park et al., 2015). The strain was incubated in a Lactobacilli MRS broth (Difco, USA) as the growth medium at 37 o C for 18 h. Growth of strain The number of viable L. plantarum FH185 was determined by serial ten-fold dilution in 0.1% peptone water. Ten microliter of L. plantarum FH185 was inoculated into 150 mL of 10% reconstituted skimmed milk (10 5 CFU/mL), and then the culture was incubated at 3 h intervals for 24 h at 34 o C, 37 o C and 40 o C. All of the pour plates were incubated aerobically at 37 o C for 48 h using a BCP plate count agar (Eiken, Japan). Antibiotic tolerance L. plantarum FH185 was grown at 37 o C for 18 h in MRS broth and inoculated (1%, v/v) into a MRS broth supplemented with antibiotics (amikacin, gentamicin, kanamycin, neomycin, streptomycin, penicillin-G, methicillin, oxacillin, ampicillin, bacitracin, rifampicin, novobiocin, lincomycin, polymyxin B and chloramphenicol; Sigma) at various concentrations in a two-fold dilution step. The minimal inhibitory concentration (MIC) was determined by checking the moment at which the strain stopped growing after incubation at 37 o C for 48 h. Enzyme activity An API ZYM kit (Apibio-Mérieux) was used to study enzyme activity. L. plantarum FH185 was grown at 37 o C for 18 h in MRS broth. Sediment from the centrifuged broth culture was used to prepare the suspension at 10 5 -10 6 CFU/mL. After inoculation, the cultures were incubated for 5 h at 37 o C. The addition of a surface active agent (ZYM A reagent) to the cupules facilitated the solubilization of the ZYM B reagent in the medium. Color was allowed to develop for at least 5 min, and values ranging from 0-5 (corresponding to the colors developed) were assigned. The approximate number for the free nmol hydrolyzed substrate was determined based on the color strength: 0, negative reaction; 1, 5 nmol; 2, 10 nmol; 3, 20 nmol; 4, 30 nmol; 5, 40 nmol or higher. Bile tolerance Bile tolerance was tested as described by Gilliland and Walker (1990). L. plantarum FH185 was grown at 37 o C for 18 h in MRS broth. Each 1% of the L. plantarum FH185 strain culture was inoculated into sterilized MRS broth containing 0.05% L-cysteine (Sigma) with or without 0.3% oxgall (Sigma), and then the growth potential was compared in the presence of the bile. Then, the cultures were incubated anaerobically at 1 h intervals for 7 h at 37 o C. All of the pour plates were incubated anaerobically at 37 o C for 48 h using a BCP plate count agar. Acid tolerance Acid tolerance was tested as described by Clark et al (1993). Solutions of 37% HCl in double-distilled water were adjusted to pH levels of 2.0, 3.0, and 4.0. Sterile double-distilled water (pH 6.4) served as the control. 10 mL of each pH solution was transferred into sterile test tubes. One milliliter of stock culture containing approximately 10 9 CFU/mL of L. plantarum FH185 using MRS agar containing 0.05% cysteine was then transferred into each of the four pH solutions. The pH solutions containing L. plantarum FH185 were then incubated anaerobically at 37 o C, followed by intermittent plating after 1, 2, and 3 h to stimulate the survival of L. plantarum FH185 under pH conditions common to the human stomach. Samples from the pH solution were re-suspended and subjected to serial dilutions. All of the pour plates were then incubated anaerobically at 37 o C for 48 h using a BCP plate count agar. Antimicrobial activity Antimicrobial activity was tested as described by Gilliland and Speck (1977). Escherichia coli KFRI 174, Salmonella Typhimurium KFRI 250, and Staphylococcus aureus KFRI 219 (obtained from the culture collection of the Korea Food Research Institute [Korea]) were enumerated on an EMB agar (Difco), on a Bismuth sulfite agar (Difco), and on a Baird Parker agar (Difco), respectively. All of the plates were incubated for 48 h at 37 o C. Both the control culture and the associative culture were incubated for 6 h at 37 o C. At the end of the incubation period, the samples were removed and placed in an ice bath until analysis. The number of CFU of pathogens per mL was determined using the appropriate selective medium. The percentages of inhibition were determined using the following formula: Adherence assay The adhesion of L. plantarum FH185 was studied using the HT-29 intestinal epithelial cell line (Kim et al., 2008). HT-29 cells were obtained from the Korea Cell Line Bank (Korea). The cells were cultured at 37 o C in a 5% CO 2 -95% air atmosphere in RPMI 1640 (GIBCO) supplemented with 10% FBS. The sub-cultured (3 times) L. plantarum FH185 was harvested by centrifugation at 12,000 rpm for 3 min, and then washed three times with PBS to remove any remaining MRS broth. The washed bacteria were then re-suspended in an RPMI 1640 medium to an optical density at 600 nm (OD 600) of 0.5 (approximately 10 7 CFU/mL). The re-suspended bacteria were appropriately diluted and plated on a BCP plate count agar. To investigate the adhesion activity, post-confluent HT-29 cells were washed twice with PBS. After washing, 1 mL of the bacteria in the RPMI 1640 medium was added to each well of the tissue-culture plate (12 wells), which was then incubated for 2 h. After incubation, the cells were washed five times with sterile PBS and harvested with a trypsin-EDTA (0.25% trypsin and 0.02% EDTA; GIBCO). It was appropriately diluted and plated on a BCP plate count agar to determine the number of viable cell-associated bacteria. Statistical analysis The results are expressed as the mean±standard deviation (SD). Statistical analysis was performed with a statistical analysis system (SAS, SAS Institute Inc., USA). The significance of the differences was analyzed by conducting a one-way analysis of variance (ANOVA) with Duncan's multiple range tests. Values of p<0.05 were considered statistically significant. Results and Discussion Growth of strain As the result of incubation of L. plantarum FH185 in 10% reconstituted skimmed milk at 34 o C, 37 o C and 40 o C for 24 h, the highest growth rate identified at 40 o C. The pH value was also the lowest at 40 o C. The optimum growth temperature of L. plantarum FH185 was found to be 40 o C (Fig. 1). Antibiotic tolerance Several studies on the antibiotic sensitivity and resistance of dairy starter bacteria were conducted over a period of many years . Although some resistance appeared to be a strain, a specific pattern for classification has not emerged (Reinbold and Reddy, 1974). Table 1 shows the tolerance of the L. plantarum FH185 strain to sixteen kinds of antibiotics. The results showed that L. plantarum FH185 showed itself to be more sensitive to novobiocin and penicillin-G in a comparison of fourteen different antibiotics, and exhibited the greatest resistance to polymyxin B and vancomycin. Vancomycin resistance is a matter of great importance in that vancomycin is one of the last antibiotics to remain widely efficacious against clinical infections caused by multidrug-resistant pathogens (Zhou et al., 2005). A few gram-positive bacteria, including Lactobacillus species, are essentially resistant to vancomycin (Swenson et al., 1990; Hamilton-Miller and Shah, 1998). Irreversible loss of antibiotic resistance from a strain as a result of a treatment known to eliminate plasmids is an indication that the resistance is plasmid-linked. However, in the case of the Lactobacillus strain, there has been no indication so far that vancomycin resistance would represent an inducible, transmissible genetic system . For confirmation of the safety, cloning and expression of the gene related to anti-obesity effect from L. plantarum FH185 could be an alternative way. Bile and acid tolerance Tolerance to gastric juice and bile salts is a crucial factor in the selection of probiotic strains (Caggia et al., 2015). In order to ensure their beneficial effects after consumption, probiotics must be viable in the food and survive the gastrointestinal ecosystem with a pH ranging from 1. *A value ranging from 0 to 5 is assigned to the standard color: zero represents a negative; 5 represents a reaction of maximum intensity. Values 1 through 4 represent intermediate reactions depending on the level of intensity. The approximate activity may be estimated from the color strength: 1 corresponds to the liberation of 5 nanomoles, 2 to 10 nanomoles, 3 to 20 nanomoles, 4 to 30 nanomoles, and 5 to 40 nanomoles or more. Fig. 2 shows the growth curves in MRS broth or MRS broth containing 0.3% bile. The log value of the population after incubation for 7 h without 0.3% oxgall was 8.9, but it was 8.6 with the addition of 0.3% bile. Therefore, the survival rate of L. plantarum FH185 in MRS broth containing 0.3% bile was 96.6%. Also, Fig. 3 shows the pH tolerance of L. plantarum FH185. It showed a 97.4% survival rate after incubation for 3 h in highly acidic conditions (pH 2.0). According to Guo et al. (2015), thirty kinds of Lactobacillus strains isolated from the suan-tsai and koumiss sample were tested with regard to their acid and bile tolerance. The acid resistance values of the lactobacilli ranged from 44.1 to 85.2%, while their bile tolerance values ranged from 4.6 to 34.2%. L. plantarum FH185 has probiotic potential because a comparatively high percentage of the strain survived in MRS broth containing 0.3% bile salt, under a highly acidic condition. Antimicrobial activity Foodborne diseases arising from the consumption of food contaminated with pathogenic bacteria such as Salmonella sp., Listeria monocytogenes, Staphylococcus sp., and E.coli is of vital concern to public health (Oussalah et al., 2007). As the number of multidrug-resistant pathogens expands, and recognition of the role that human microbiota play in health and disease increases, it is becoming increasingly interesting to use probiotics as a therapeutic agent (Britton and Versalovic, 2008). For instance, many researchers have demonstrated the anti-pathogenic effects of LAB (Casey et al., 2007). The antagonistic activities demonstrated by lactic acid bacteria may be due to the production of substances with antibacterial properties in particular: hydrogen peroxide, organic acid and bacteriocins (Tejero-Sarinena et al., 2012). Table 3 shows the antimicrobial activity of L. plantarum FH185 against various pathogenic strains. L. plantarum FH185 did not show any inhibition against E. coli, but it showed inhibition against S. Typhimurium and S. aureus at rates of 44.4% and 53.9%, respectively. The pH value of pathogens after incubation for 7 h was 6.4, but the pH value of a mixed culture with L. plantarum FH185 and pathogens was around 5.5-5.6. This means that even lactic acid produced during incubation affected the antimicrobial activity, it was not a large effect. Although L. plantarum FH185 did not show resistance against E. coli, the strain showed comparatively excellent inhibition against S. Typhimurium and S. aureus. Adhesion ability Adherence ability to the intestinal epithelium of the hosts is one of the main criteria for selecting probiotic strains. Attachment to mucosa prolongs the time probiotics can influence the gastrointestinal immune system and microbiota of the host. Thus, the ability to adhere to intestinal surfaces is thought to correspond to the efficacy of the probiotic strain (O'Halloran et al., 1997). As shown in Fig. 4, 19.62% of L. plantarum FH185 adhered to HT-29 cell and 17.02% of the L. rhamnosus GG strain adhered to the cell. L. rhamnosus GG was used as the positive control. In many studies, it was demonstrated that L. rhamnosus GG has a great ability to adhere to the epithelial cell line (Gopal et al., 2001;Tuomola and Salminen, 1998). Thus, we could define that L. plantarum FH185 exhibits great adherence to the epithelial surface. Conclusion In our previous study, Lactobacillus plantarum FH185 was isolated from the feces of healthy adults and demonstrated to have anti-obesity effects. We investigated the physiological characteristics of L. plantarum FH185 for potential use as probiotics. The essential and fundamental properties of probiotics -such as growth pattern, antibiotic tolerance, enzyme activity, bile tolerance, acid tolerance, antimicrobial activity and adhesion ability -were tested. The optimum growth temperature of L. plantarum FH185 was 40 o C. L. plantarum FH185 was able to survive in antibiotic conditions at a low concentration and did not produce carcinogenic enzymes such as β-glucuronidase. Moreover, it was found to be comparatively tolerant to bile juice and acid, and displayed inhibition against two kinds of pathogenic strains. It also showed high adhesion activity to HT-29 cells compared to L. rhamnosus GG. These results demonstrate that L. plantarum FH185 could be used as a probiotic.

v3-fos

2020-04-30T09:03:12.533Z

2016-01-01T00:00:00.000Z

251921538

s2

Expression of Aintegumenta-like Gene Related to Embryogenic Competence in Coconut Confirmed by 454-pyrosequencing Transcriptome Analysis A member of the Aintegumenta sub-family of Apetala gene family encoding two APETALA2 (AP2) domains was isolated and termed as Cocos nucifera Aintegumenta like gene (CnANT) . The deduced amino acid sequence of the conserved domains shared a high similarity with Aintegumenta-Like (ANT like) genes in Arabidopsis thaliana , Elaeis guineensis , Oryza sativa. Comparison of transcriptomes in different tissues revealed that CnANT transcripts were high in mature zygotic embryo (12 months after pollination; 12ME). Quantitative RT-PCR results confirmed the higher CnANT transcript accumulation in mature zygotic embryos while transcripts were rarely detected in vegetative tissues such as leaf. The expression data and global transcriptome data were therefore consistent across the embryo maturity stage and showed that CnANT could play a role in embryogenesis. Introduction Coconut (Cocos nucifera L.) is a perennial cross-pollinated plant that is cultivated mainly for edible oil in the tropical and sub-tropical regions. Despite its economic importance, coconut production has not been increased as we see in other oil crops. Production of high quality superior palms in large scale is a prerequisite for the development of coconut industry. Achieving this target using seeds as the planting material is impossible. Its propagation by tissue culture was first reported in 1983 (Branton and Blake 1983). Since then cloning of coconut via somatic embryogenesis has been addressed by several researchers in different research groups (Fernando and Gamage 2000;Hornung 1995;Karunaratne and Periyapperuma 1989;Perera et al. 2008;Perera et al. 2007;Verdeil et al. 1994). However, its highly recalcitrant behavior for in vitro conditions limits the success of coconut micropropagation (George and Sherrington 1984).Therefore, understanding of the molecular basis of coconut tissue culture would provide necessary information needed for the improvement of the in vitro propagation protocol. Members of the sub family Aintegumenta-Like (AIL) of the Apetala2/Ethylene-responsive element binding protein (AP2/EREBP) family play an important role during the transition from vegetative to embryogenic growth (Banno et al. 2001;Boutilier et al. 2002). BABY BOOM (BBM) is one of such genes known for its role in cell proliferation and morphogenesis during embryogenesis (Boutilier et al. 2002). These genes are expressed in dividing tissues where they have central roles in developmental processes such as embryogenesis. Overexpression of the AIL genes induces somatic embryogenesis and ectopic organ formation (Boutilier et al. 2002;Tsuwamoto et al. 2010). The characterization and functional analysis of markers such as AIL for somatic embryogenesis offer the possibility of determining the embryogenic potential of coconut in culture long before any morphological changes have taken place. Since zygotic embryo development always mimic the somatic embryogenesis, most studies on gene isolation have been carried out initially using zygotic embryo tissues (Cairney and Pullman 2007;Palovaara et al. 2010) . Next generation sequencing (NGS) technologies enable scientists to analyze the complete transcriptome at a minimal cost. Roche 454 Genome Sequencer (GS) FLX platform is widely used for de novo sequencing and EST analyses in non-model plants ( Barakat et al. 2009;Graham et al. 2010;Li et al. 2010;Wang et al. 2009;Novaes et al. 2008;Alagna et al. 2009) and is an ideal way to discover genes and markers, quantify transcripts and discover small RNA (Morozova et al. 2009;Brautigam and Gowik 2010). In the present study Aintegumeta like gene was identified in coconut zygotic embryos and expression was checked in four different plant tissues. The occurrence of this Aintegumenta like gene was further identified in a separate transcriptome analysis during AP2/EREBP family gene mining. Plant Material Seeds were from variety 'Sri Lanka Tall' and were harvested from bunches at 12-month maturity stage (given that 0 is the most mature unopened inflorescence embryos were used to extract RNA for gene isolation. Four different tissue types from the variety 'Sri Lanka Tall' namely, immature embryo at the age of nine months after pollination (9ME), mature embryo at the age of 12 months after pollination (12ME), a microspore derived embryo (MDE) (Perera et al. 2008) and developing leaves from an 8month old in vitro germinated coconut plantlet were used for the cDNA library construction for the 454 sequence analysis. RNA Extraction Total RNA from each tissue sample was extracted using the RNeasy® Plant Kit (Qiagen) according to the manufacturer's instructions. DNA contaminations were eliminated by treating with DNase I (Qiagen, UK) according to the manufacturer's instructions. The amount of RNA was quantified using a NanoDrop® ND-1000 Spectrophotometer and integrity was analyzed by 1% agarose gel electrophoresis. Cloning of coconut ANT-like cDNA Primers [2 F (5-TCT ATC TAC CGC GGC GTC-3′) and 4R (5-ACA AAC TCC TGT CGT GTC A-3′), 4 F (5-TGA CAC GAC AGG AGT TTG T-3′) and 5R2 (5-ATT CCA TTC CAA AGA TGG G-3′)] were designed to amplify the coconut ANT-like cDNA sequence on the basis of the most conserved nucleotide sequences of rice (Accession NM 001060643) and oil palm (Accession AY691196) Aintegumenta-like genes. Approximately 1 µg of total RNA was used to synthesize first strand cDNA using SensiMix 2 step kit (Quantace) according to the manufacturer's instructions. PCR was performed using 1 µL of cDNA in the presence of primers at 0.3 µM concentration. Each PCR reaction was carried out in a 25 µL reaction volume containing 12.5 µL 2X Biomix (Bioline). After an initial 2 min denaturation step at 94°C, 30 cycles were run, each with 30 s of denaturation at 94°C , followed by 30 s of annealing at 57°C and 45s of extension at 72°C. The final elongation cycle was at 72°C for 7 min. Purified PCR products were subjected to BigDye terminator cycle sequencing reaction and sequenced at the Bio-Centre, University of Reading. From these amplifications 1031 bp length fragment was obtained. Flanking sequence determination of 5' and 3' ends was carried out by the RACE method using the GeneRacer kit (Invitrogen) using the primers provided by the kit (GeneRacer 5 primer 5-CGA CTG GAG CAC GAG GAC ACT GA-3, GeneRacer 3′primer 5'-GCT GTC AAC GAT ACG CTA CGT AAC G-3, ′5′ nested primer 5′ GGA CAC TGA CAT GGA CTG AAG TAG AAA-3', 3′nested primer 5′-CGC TAC GTA ACG GCA TGA CAG TG-3′and gene specific primers 4R, 4F, 3R (5'-CGC CTT CTC CTC CTT ATC-3'), ANTF (5′-AAC TGG ATT ATG CAT GAT GA-3)′according to the manufacturer's protocol. Real-time RT-PCR expression analysis Two gene-specific primers; ANTF1: (5′-CGG TCT CTT CTC CTC TGG TG-3′) and ANTR: (5′-TCG TAA TTC CCT CCA AAT GC-3′) were designed based on the coding region of the CnANT gene to amplify a 180 bp region. The coconut elongation factor gene was used as the internal control gene (Morcillo et al. 2007;Olsvik et al. 2005). cDNA from the four tissue types 9ME, 12ME, MDE and LEAF were used for real-time RT-PCR analysis using SYBR premix Ex taqTM (Takara). Experiments were conducted with two biological samples, and the real-time qPCR reactions were performed in triplicate using the CAS-1200 liquid handling system, version 4.7.979 (Corbett Robotics). The real-time RT-PCR was performed on a Rotor-Gene 6000 real-time cycler (software 1.7, Corbett Research). The amplification parameters were one cycle at 95 °C for 1 min, 39 cycles of 95°C for 10s, 60°C for 20s, and 72°C for 8s. The relative expression level in different tissues was calculated by the standard curve method. cDNA synthesis and 454 pyrosequencing The first-strand cDNA was produced from 0.3µlg of total RNA. A modified SMART-Sfi1A oligonucleotide (5'-AAG CAG TGG TAT CAA CGC AGA GTG GCC ATT ACG GCCrGrGrG-3') was used in combination with the CDS-Sfi1B primer (5'-AAG CAG TGG TAT CAA CGC AGA GTG GCC GAG GCG GCC d (T) 20-3') to synthesize the first strand cDNA in the presence of PowerScript Reverse Trascriptase (BD Biosciences Clontech,UK). For doublestranded cDNA (ds cDNA) synthesis, the cDNA was diluted and amplified using PCR Advantage II polymerase (BD Biosciences Clontech,UK) in the presence of SMART PCR primer ( 5'-AAG CAG TGG TAT CAA CGC AGA GT-3'). The following the thermal profile: 1 min at 95ºC followed by 25 cycles of 95ºC for 7 s, 65ºC for 20 s, and 72ºC for 3 min was used for the amplification. Five micro liters of PCR product was electrophoresed in a 1% agarose gel to determine the amplification efficiency. The amplified cDNA PCR product was purified using QIAquick PCR Purification Kit (QIAGEN, CA), concentrated by ethanol precipitation and adjusted to a final concentration of 50 ngμ -1 . A total yield of 3 μg of cDNA was prepared for each tissue type by conducting several long distance PCR reactions. DNA sequencing of four libraries was performed at Centre for Genomic Research, University of Liverpool using a 454-GS FLX Genome Sequencer and the sequence data processing was performed with the GS FLX software v2.0.01. Sequences in each library were subjected to a BLAST search against the nonredundant protein database using BLASTX with an e value cut-off of 1E-6. AP2 family proteins were identified in each library based on the BLASTX search. Cloning of full length cDNA homologue of Aintegumenta-like gene A partial sequence (1029 bp) of Aintegumenta-like homologue gene was obtained by PCR using primer pairs 2F, 4R and 4F, 5R2. Amplification products of 5'-RACE and 3'-RACE generated the full length cDNA sequence that was 1782 bp in length. We named it as Cocos nucifera L. Aintegumenta (CnANT) gene. This contained a 1425 bp open reading frame (ORF), 62 nucleotides at the 5'untranslated region (UTR) and 305 nucleotides at the 3' UTR including 26 adenines from the polyA tail. The ORF encoded a putative peptide of a 474 amino acids (Figure 1). Sequence database searches revealed that the deduced polypeptide sequence of CnANT shows similarity to AP2/EREBP family proteins. This putative CnANT protein sequence contains two AP2 domains from protein residues 128 to 204 in repeat one and residues 230 to 298 in repeat two and a conserved linker region from residues 205 to 229 (Figure 1). Within the CnANT polypeptide sequence the highly conserved YRG elements encoded by amino acid residues from 128 to 149 and 230 to 251 were The amino acid sequences of two AP2 domains and the linker region connecting the two repeats of CnANT were aligned with the related AP2/EREB proteins. Black boxes indicate the amino acids that are identical in all members of the AP2 subfamily. Dark grey boxes shows the amino acids that are identical in all AIL sub group proteins. Amino acids that are identical in AP2 sub group are coloured in light grey. Note the 10 amino acid insertion in the AP2 repeat 1 (indicated by a red box) and one amino acid insertion in the AP2 repeat 2 (indicated by a blue box) which distinguish ANT genes from the rest of AP2 genes. observed for AP2 repeat 1 domain and repeat 2 domain, respectively. Two RAYD elements were identified from residues 161 to 204 in repeat 1 and residues from 255 to 298 in repeat 2. Furthermore, the specific central core of 18 amino acids within the RAYD element which has been predicted as forming an alpha helix were also detected (residues 169-186 in repeat 1 and 263-280 in repeat 2; Figure 1) (Okamuro et al. 1997). The 10 amino acid insertion in the AP2 repeat 1 and one amino acid insertion in the AP2 repeat 2 which distinguish ANT genes from the rest of AP2 genes were identified in the CnANT sequence (Figure 2). This clearly showed that CnANT belongs to the ANT sub group (Nole-Wilson et al. 2005). Furthermore, four conserved motifs identified (Kim et al. 2006) in the pre-domain region (EuANT 1, EuANT 2, EuANT 3 and EuANT 4) which are specified for the ANT sub group proteins were identified in the CnANT putative protein. Two AP2 domains and the linker region of the CnANT protein shows the highest similarity with EgAP2-1 (oil palm), ANT-like (rice), BBM, AIL5, AIL7, PLT1 and PLT2 (Arabidopsis), BBM (Brassica) and a number of recently identified hypothetical proteins available publicly. Amongst these genes, BBM (Boutilier et al. 2002), AIL5 (Tsuwamoto et al. 2010) Brassica BBM1 and BBM2 (Boutilier et al. 2002;Srinivasan et al. 2007), EgAP2-1 (Morcillo et al. 2007) have been characterized as embryogenesis related genes. Within the two AP2 repeat domains, these sequences share ~ 28% identity when consider both AP2 and ANT sub groups. However, the sequence identity is greatly increased (>75%) when only ANT group proteins are considered. When examined carefully it was noted that pairs of genes share similarity within the entire AP2 domain sequences. Coconut and oil palm ANT protein sequences show 98% identity with each other in the conserved domain regions while CnANT shares more than 80% identity in the same region with other ANT subfamily proteins when a pair wise comparison was conducted ( Figure 2). This observation emphasized that CnANT is strictly related with oil palm EgAP2-1 gene (Morcillo et al. 2007) and indicates its close relationship with the palm species sequence. In a recent study, Ouakfaoui et al. (2010) studied the conserved motifs of AP2 sub family outside of AP2 DNA binding domains and found ten sequence motifs of euANT lineage. Three of them identified at N terminal have been described by Kim et al (2006) previously. The euANT sub group was further categorized into BBM-like, PLT-like and AIL5like and oil palm EgAP2-1 was grouped as an AIL5-like gene (Ouakfaoui et al. 2010). Thus, similarity between oil palm EgAP2-1 and CnANT suggests CnANT to be grouped as AIL genes according to Ouakfaoui and colleagues classification and hence suggests that CnANT could play a role in embryogenesis as proposed for those orthologs. The four cDNA libraries were sequenced using a GS FLX sequencer (454/Roche), resulting in a total of 979428 reads. These reads were assembled into 32621 putative unigenes and 155017 singletons. ESTs had an average length of 460 bp, and represented 223.7 Mb. Assembled sequences were functionally annotated with blast2go. Homology-based functional assignment for putative sequences was accomplished through BLASTX queries against non-redundant protein database with an e value cut-off of 1E-6 to identify AP2 family proteins in each library. The highest number of APETALA family genes was found in the 9ME library which was represented by 16 contigs. The other three libraries 12ME, MDE and LEAF had 11, 12 and 10 contigs respectively which were encoded for AP2 family genes. As per the putative annotations made from BLASTX hits, most of them were of the ERF subfamily which has a single AP2 domain. Also there were contigs encoded for RAV subfamily genes (genes containing one AP2 domain and a second B3 domain) and AP2/ANT subfamily genes. Interestingly, CnANT gene which was described early in this paper could be identified in all embryo tissue libraries (Table 1; marked with an asterisk). These contigs returned the highest significant similarity [Evalue ranged from 1.04E-33 (9ME & MDE) to 1.66E -166 (12ME)] with the oil palm Aintegumenta-like gene; Figure 3. Relative expression levels of CnANT accumulation in the four different tissue types determined by RT-qPCR analysis. CnANT transcripts were compared relative to the elongation factor gene. LEAF: leaf, 9ME: embryo, 9 month after pollination, 12ME: embryo, 12 month after pollination MDE: microspore derived embryo EgAP2-1 (Morcillo et al. 2007) when subject to a BLASTX homology search. The EST abundance of this gene was high in the 12ME library. Even though the abundance was not as high in the 12ME library, a considerable number of ESTs was encoded by the contigs in 9ME and MDE. In 9ME, the contig was present 19 times while in MDE it appeared 34 times. However no transcripts encoding the CnANT gene were found in LEAF library. These observations are comparable with the results obtained from the quantitative real time PCR where the highest relative expression was observed in 12ME while LEAF tissues showed negligible relative expression ( Figure 3). Furthermore, the comparison of putative gene annotations, species and GI values of the contigs in different libraries revealed that a few more genes from the AP2 family are common to embryo tissue libraries ( Table 1). The present study provides evidence that CnANT is strongly induced in late embryogenesis and plays a role during zygotic embryo maturation phase. First, a qPCR experiment showed higher relative expression at the most mature stage of zygotic embryo (12 months after pollination). Secondly, global transcriptome analysis carried out using different stages of embryo maturity showed a higher accumulation of CnANT transcripts in the 12ME library compared to the 9ME library and the LEAF library based on the BLASTX analysis (sequence cut-off 1.0e -6 ). The expression data and global transcriptome data are therefore consistent across the embryo maturity stage. Higher expression of AIL5 and BBM genes in later stages of embryo development has been reported previously (Boutilier et al. 2002;Morcillo et al. 2007;Tsuwamoto et al. 2010). Unlike in BBM which was expressed in stages as early as the globular stage, AIL gene expression has been analysed after laser capture microdissection and in situ hybridization and shown to occur only in embryos older than the heart stage (Casson et al. 2005;Tsuwamoto et al. 2010). Based on the results, presence of AIL gene in coconut is reported and findings supports the fact that this gene is highly expressed in maturing stages of embryo. CnANT could be used as embryogenesis marker, since its expression changed with progression of embryo development. These findings may offer a valuable contribution to the evaluation of embryogenic culture responses in coconut.

v3-fos

2017-07-01T17:10:54.996Z

2015-03-11T00:00:00.000Z

10178648

s2

Comparison of Schmallenberg virus antibody levels detected in milk and serum from individual cows Background Schmallenberg virus (SBV) is a recently emerged virus of ruminants in Europe. Enzyme-linked immunosorbent assays (ELISA) are commonly used to detect SBV-specific antibodies in bulk tank milk samples to monitor herd exposure to infection. However, it has previously been shown that a bulk tank milk sample can test positive even though the majority of cows within the herd are seronegative for SBV antibodies. Development of a pen-side test to detect antibodies in individual milk samples would potentially provide a cheaper test (for which samples are obtained non-invasively) than testing individual serum samples by ELISA. Therefore, the aim of this study was to investigate the agreement between antibody levels measured in milk and serum. Results Corresponding milk and serum samples from 88 cows in two dairy herds in the UK were tested for presence of immunoglobulin G antibodies to SBV using a commercially-available indirect ELISA. A serum neutralisation test (NT) was also performed as a gold standard assay. The ELISA values obtained for the bulk tank milk samples corresponded with the mean values for individual milk samples from each herd (bulk tank milk values were 58% and 73% and mean individual milk values 50% and 63% for herds A and B, respectively). Of the 88 serum samples tested in the NT, 82 (93%) were positive. Although at higher antibody levels, the ELISA values tended to be higher for the individual milk samples than for the corresponding serum samples, the positive predictive value for milk samples was 98% and for serum samples 94%. The serum ELISA was more likely to give false positive results around the lower cut-off value of the assay. Conclusions The results indicate that testing of individual milk samples for antibodies against SBV by ELISA could be used to inform decisions in the management of dairy herds such as which, if any, animals to vaccinate. Background Schmallenberg virus (SBV), which emerged recently in Europe, causes subclinical or mild disease in adult ruminants with clinical signs including diarrhoea, fever and drop in milk yield in dairy cattle. However, infection of pregnant animals during a critical period of pregnancy can cause fetal deformities and may result in loss of the fetus or unviable offspring [1]. The first indirect enzyme-linked immunosorbent assay (ELISA) to detect SBV-specific antibodies in serum or milk samples became commercially available shortly after the emergence of SBV [2]. Testing of bulk tank milk samples by ELISA has been advocated as a convenient way to determine herd-level exposure to SBV [3]. With the availability of vaccines against SBV, it has become important to know the value of test results for informing herd management decisions; for example, whether a positive bulk tank milk sample result means that herd-level vaccination is not necessary as natural immunity is present. Since its emergence, SBV has spread rapidly across Europe and high levels of seroprevalence in cattle have been reported (reviewed in [4]). However, studies have also demonstrated that within-herd seroprevalence is variable. In addition to regional variation in seroprevalence, higher rates have been reported for herds that graze outdoors compared to herds that are housed indoors [5]. Furthermore, in one study, a bulk tank milk sample tested positive although only 25% of serum samples from individual animals within the herd were positive for antibodies to SBV [6]. The aim of this study was to examine the relationship between antibody levels detected in bulk tank milk and individual milk and serum samples from SBV-exposed cows in two herds using a commercially-available ELISA, with a serum neutralisation test as a reference. Methods Blood and milk samples were collected from Holstein-Friesian dairy cows in two herds (49 samples from herd A and 39 from herd B) on 2 nd October 2013. A bulk tank milk sample was also obtained from each herd. None of the cows had been vaccinated against SBV. All were clinically healthy at the time of sampling, but clinical signs suggestive of SBV infection (diarrhoea and drop in milk yield) had been observed around one month prior to sampling in herd B. All samples were stored at -20°C until tested. The study was approved by the School of Veterinary Medicine and Science's Ethical Review Committee. The presence of immunoglobulin G antibodies to SBV in milk and serum samples was determined using a commercially available indirect ELISA (SVANOVIR® SBV-Ab, Svanova) according to the manufacturer's instructions. As per the manufacturer's instructions, the percent positivity (PP) relative to the positive control serum supplied was calculated with a PP of ≥10% considered positive for serum samples and ≥8% for milk samples. Neutralization tests (NT) were performed on serum samples as previously described [7] using SBV strain BH80/11-4 (kindly provided by M. Beer, Friedrich-Loeffler Institute) with the minor modification that cells were fixed in 100% ethanol for 30 minutes then stained for 30 minutes with 0.1% v/v methylene blue in water. The cut-off value for a positive result was set at a titre of 1:8. Milk samples could only be tested by ELISA as milk is toxic to the cells used in the NT. Positive predictive (the probability that the disease is present when the test is positive) and negative predictive (the probability that the disease is absent when the test is negative) values were calculated for the ELISA using the serum NT as a reference. ELISA results with milk or serum were classified as true positive (TP) or true negative (TN) if in agreement with the serum NT. If results differed from the serum NT, they were classified as false positive (FP) or false negative (FN). Positive predictive value was calculated as TP/(TP + FP) and negative predictive value as TN/(TN + FN) and expressed as a percentage. Two-sample and paired t-tests as appropriate (with statistical significance set at p < 0.05) were performed using Minitab version 16. Bland-Altman analysis (to evaluate the variability between the serum and milk antibody levels measured by ELISA over the full range of results) was performed using GraphPad Prism v6. Results and discussion The bulk tank milk sample from herd A had an antibody level of 58% and from herd B 73%. Although the ELISA is only semi-quantitative, the mean of the individual milk sample values was consistent with the bulk tank milk sample values. A significantly lower (two-sample ttest, p = 0.037) mean antibody level was obtained for individual milk samples from herd A (50%) than for herd B (63%). Similarly, in a larger published study of bovine viral diarrhoea virus in which milk samples were tested for antibodies, individual milk and bulk tank milk results correlated well [8]. Thus, bulk tank milk testing might indicate the presence of individuals within a herd with lower antibody levels (and therefore at potentially greater risk of infection), but provides no information as to which (or how many) individuals are at potential risk of infection. In the analysis of samples from individual animals, six cows tested negative in the serum NT (four from herd A and two from herd B). Milk and serum samples from one of these cows also tested negative by ELISA. The other five animals all tested positive by serum ELISA (Table 1A) whereas only two of them tested positive by milk ELISA (Table 1B). Thus, the positive predictive values were 98% and 94% for the milk and serum ELISA, respectively and the negative predictive value for the milk ELISA was 100% but for the serum ELISA was 50%. Thus the serum ELISA was more likely to give both false positive and false negative results. The values obtained in the serum ELISA for the five 'false positive' samples were all at or just above the lower cut-off value of 10% (10-11%). Both the positive and the negative predictive values will be influenced by the high prevalence of SBV antibodies in the animals tested; in a high prevalence setting such as this, it is more likely that animals that test positive truly have antibodies to SBV and, conversely, that the negative predictive value is decreased [9]. The antibody levels measured in milk samples were significantly (paired t-test, p < 0.001) higher (mean PP 55%, standard error of the mean, SEM 3.13) than in serum samples (mean PP 42%, SEM 2.41). This is in contrast to other studies comparing antibody levels against bovine coronavirus and/or bovine respiratory syncytial virus in matched serum and milk samples, which found good agreement but generally lower antibody levels in milk compared to serum samples [10]. The distribution of the measured PP values is shown in Figure 1A. Bland-Altman analysis revealed a bias of -13.48. Differences between the milk and serum ELISA results were more apparent at mean PP values for the two tests of greater than 50% ( Figure 1B). Protective antibody levels have not been defined for SBV and the indirect ELISA is at best only semiquantitative. Therefore, if individual testing were conducted in order to inform management decisions such as whether to vaccinate potentially susceptible animals, the cut-off for deciding to vaccinate would be a discretionary one. A negative result in the serum or milk ELISA would clearly indicate a susceptible individual. However, PP values near the assay cut-off should be treated with caution, particularly for the serum ELISA. As testing a bulk tank milk sample may not provide an accurate reflection of the proportion of a herd that has antibodies, and milk samples can be obtained noninvasively, individual testing of a number of animals can provide an indication of the need to vaccinate the whole herd. However, testing using the currently available indirect ELISAs would, in most cases, be prohibitively expensive. Therefore, if informed decisions are to be made whether or not to vaccinate a dairy herd, a cheaper alternative pen-side test to detect antibodies in individual milk and/or serum samples is required. Conclusions The results from this study suggest that testing of either serum or milk samples from individuals rather than bulk tank milk testing is necessary to identify whether animals within a dairy herd are potentially susceptible to SBV infection.

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2015-07-23T00:00:00.000Z

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Dissection of drought response of modern and underutilized wheat varieties according to Passioura's yield-water framework Trait-based breeding is essential to improve wheat yield, particularly when stress adaptation is targeted. A set of modern and underutilized wheat genotypes was examined in a 2-year field experiment with distinct seasonal water supply. Yield formation and drought response strategies were analyzed in relation to components of Passioura's yield-water framework based on phenological, morphological, physiological, and root characteristics. Limited water supply resulted in 60% yield loss and substantially lower water use (37%), water use efficiency (32.6%), and harvest index (14%). Phenology and root length density were key determinants of water use. Late flowering underutilized wheat species with large root system and swift ground coverage showed greatest water use. Leaf chlorophyll concentration and stomata conductance were higher in modern cultivars, supporting their high biomass growth and superior water use efficiency. While, lower chlorophyll concentration and stomata conductance of underutilized wheats indicated a water saving strategy with an intrinsic limitation of potential growth. Harvest index was strongly dependent on phenology and yield components. Optimized flowering time, reduced tillering, and strong grain sink of modern cultivars explained higher harvest index compared to underutilized wheats. Cluster analysis revealed the consistent differentiation of underutilized and modern wheats based on traits underlying Passioura's yield-water framework. We identified physiological and root traits within modern cultivars to be targeted for trait-based crop improvement under water-limited conditions. High capacity of water use in underutilized genetic resources is related to yield-limiting phenological and morphological traits, constraining their potential role for better drought resistance. Still some genetic resources provide adaptive features for stress resistance compatible with high yield as revealed by high harvest index under drought of Khorasan wheat. Introduction Grain yield is the product of numerous developmental processes during crop growth. It is a trait governed by multiple genes and highly influenced by environmental conditions. Yield improvement in water-limited environments is complex and depends strongly on the drought regime, i.e., drought duration, intensity, and time of occurrence (van Ginkel et al., 1998;Blum, 2011). This complexity becomes evident when attributes contributing to yield loss mitigation in a given environment are not equally useful in other water-limited environments (Richards, 2006). Despite these difficulties, wheat yield was remarkably increased over the second half of the 20th century in all wheat growing environments (Calderini and Slafer, 1998;Fischer et al., 2014) as a result of genetic improvement, enhanced input of production factors, particularly water and nitrogen (Sinclair and Rufty, 2012), and a synergy between them (Richards et al., 2014). However, in the last decades, rates of yield improvement in wheat have declined to less than what would be required to meet projected demands for 2050 (Hall and Richards, 2013). Particularly in dry regions, yield increase was below breeding progress registered for high yielding environments (Trethowan et al., 2002;Graybosch and Peterson, 2010). Hitherto, only limited yield gains were realized using physiological traits for selection in drought prone environments (Richards, 2006;Reynolds et al., 2009). This is probably due to incomplete understanding of the physiological and genetic basis of drought resistance (Salekdeh et al., 2009) as well as insufficient consideration of drought environments when defining target traits for stress resistance (Rebetzke et al., 2013a). Also the upscaling of relevant drought defense mechanisms from the cellular level (e.g., dehydrins, Hassan et al., 2015;aquaporins, Maurel and Chrispeels, 2001) to the whole plant and stand level is challenging when searching for key traits in crop improvement. The conceptual framework of Passioura (1977Passioura ( , 2006 facilitates the dissection of drought-adaptive mechanisms for trait-based breeding under drought-prone environments (Richards, 2006;Salekdeh et al., 2009). The framework relates yield under water limited conditions to (i) crop water use (WU), (ii) water use efficiency (WUE), and (iii) harvest index (HI). In the past, wheat grain yield improvement has largely been driven by improvements in HI rather than biomass (BM). Thus, HI is already close to its theoretical limit (Perry and D'Antuono, 1989;Shearman et al., 2005;Sadras and Lawson, 2011). An option for yield improvement under drought stress is maximizing transpiration, i.e., better WU (Blum, 2009). This necessitates genotypes showing drought avoidance via uptake optimization, termed "water spenders" by Levitt (1980). In that respect, enhanced plant root systems are considered to be a promising approach (Wasson et al., 2012). WUE as target trait was critically discussed by Blum (2009) because (i) WUE defined as BM/WU is not independent of WU, and (ii) it might go along with reduced crop transpiration and hence yield under moderate stress conditions. Passioura (2006), however, pointed to the single leaf scale of gas exchange as a key for WUE. Thereby, WUE can be considered (scale) independent from the whole plant WU within the original yieldwater framework. High WUE of crops can be conferred by both stomata conductance and photosynthetic capacity. Udayakumar et al. (1998) suggested that only in those cases where high WUE is achieved via photosynthetic capacity, consistent yield increase could be expected. Condon et al. (2002) revealed that low stomata conductance as a reason for superior WUE generally expresses a conservative WU and leads to lower yields except for very dry environments where crop growth strongly relies on stored soil moisture. Underutilized wheat species are valuable genetic resources for secondary drought-adaptive traits (Reynolds et al., 2007;Trethowan and Mujeeb-Kazi, 2008). Nakhforoosh et al. (2014) revealed significant genotypic diversity for root traits as well as for root functionality in terms of soil water depletion. Khazaei et al. (2010) demonstrated an essential influence of ploidy level on stomata size in Iranian wheat landraces. Here we provide a comprehensive comparative analysis of modern and underutilized wheat germplasm based on their phenology, morphology, physiology, and root characteristics. The main objective is a trait based dissection of drought stress response strategies. We apply Passioura's yield-water framework and relate our investigated traits to the components of this analytical approach. Based on the identification of distinct strategies to cope with limited water supply, we will highlight strengths and weaknesses of underutilized wheat germplasm for trait-based breeding under water-limited conditions. Experimental Conditions Field experiments were carried out under rainfed conditions in Raasdorf (48 • 14 ′ N, 16 • 35 ′ E, 156 m) in the Pannonian plains of Austria. Long-term (1981Long-term ( -2010 annual precipitation and mean temperature are 516 mm and 10.3 • C, respectively. Daily weather data were obtained from a weather station located at the trial site. According to WRB (IUSS, 2014) soil is a chernozem with silt loam texture (sand 0.21 kg kg −1 ; silt 0.57 kg kg −1 ; clay 0.22 kg kg −1 ) with high water holding capacity (water content at field capacity: 0.286 cm 3 cm −3 , water content at wilting point: 0.118 cm 3 cm −3 ). Hydrological site conditions were characterized using the HYDRUS 1D software package (Šimůnek et al., 2013). The objectives of model based environmental characterization were (i) to define moisture conditions during the study years in relation to longtime site hydrology, and (ii) to provide a hydrological basis for the analysis of trait based stress response. The field experiments were sown on 8th March 2011 and 20th March 2012 in a four replicate randomized complete block design following a shallow seedbed preparation using a rotary harrow. Sowing was carried out by a plot seeder (Wintersteiger, Ried, Austria) with a seeding rate of 400 seeds per m 2 . Plot size was 7.5 m 2 with 10 rows spaced 12.5 cm apart. The site has high availability of P and K and was fertilized with 100 kg ha −1 N to exclude nutrient limitation. Yield and Yield Components After full ripening (BBCH 92), plants were hand harvested from a 0.25 m 2 area from the center of each plot. Total aboveground biomass, seed yield (oven dried at 60 • C for 48 h), number of fertile tillers, and number of seeds per ear were measured and expressed per unit area. Thousand grain weight was determined by weighing 400 seeds. Sensitivity of genotypes to water limitation was characterized by relative stress response (RSR) of traits between the two experimental years which differed strongly in seasonal water supply. RSR of yield and its components was calculated as: where Trait wet is the trait value under high water availability (i.e., 2011) and Trait dry is the value under low water availability conditions (i.e., 2012). Water use Traits Water use was calculated as a simplified water balance from soil water depletion ( S ) and cumulative rainfall. S was defined as the difference in soil water storage between sowing and harvest. Soil water content (θ) was measured weekly every 10 cm down to 90 cm soil depth by a capacitance probe (Diviner 2000 R , Sentek Pty Ltd., Stepney, Australia). Surface runoff can be neglected at the present experimental site. Deep drainage can't be quantified from soil water content measurements. From lysimeter studies at the site, however, it is known that due to low amount of rainfall and high soil water holding capacity, the amount of seepage water during the growing season is negligible (Nolz et al., 2014). The term WU ET is used to indicate that water use includes both plant transpiration as well as soil evaporation. In a water balance approach these two components can't be measured separately. Phenology was assessed using the BBCH decimal code (Lancashire et al., 1991). Time to any developmental stage was expressed in cumulative thermal time (CTT), measured in degree-days ( • C d) as described by Salazar-Gutierrez et al. (2013) and assuming a constant base temperature (T b ) of 0 • C as no information was available on possible T b differences among genotypes. Ground cover by leaf area was measured by digital imaging twice at early emergence and when canopy almost closed using a Canon EOS20D (Canon Inc., Tokyo) digital camera at 1.5 m height above the canopy. Digital images were analyzed individually by SigmaScan Pro Vers. 5.0 software (SystatSoftware Inc., Chicago) to identify green leaves and calculate the percentage of green ground cover as described by Richardson et al. (2001). Ground cover rate as an indicator of early vigor was calculated as the difference in ground cover between the two measurements divided by the CTT of the corresponding period. Ground cover rate could be hypothesized as a water use driver because of (i) higher early demand due to quicker leaf area development, (ii) possibly a related higher early rooting vigor, and (iii) higher allocation of available water to plant transpiration than soil evaporation. Root morphological traits were measured from soil cores and subsequent image analysis. In this study we will only refer to selected root data (i.e., root length density and root-to-shoot ratio). Details on root sampling and root system characterization are given in Nakhforoosh et al. (2014). Water Use Efficiency Traits Following Blum (2009), water use efficiency was dissected into biomass and water use, i.e., water use efficiency for biomass (WUE b ) equals BM/WU ET . Investigated traits related to WUE b were photosynthetic capacity and stomata conductance. Photosynthetic capacity was approximated by leaf chlorophyll content measured at heading (BBCH 50) using a SPAD-502 Plus chlorophyll meter (Konica Minolta Holdings, Inc., Tokyo). Ten plants were randomly selected in each plot and SPAD values of the flag and/or penultimate leaf of the main stem were recorded at 5 points along the proximal-distal axis of the leaf. Stomatal conductance was measured using an AP4 porometer (Delta-T Devices Ltd., Burwell, Cambridge, UK) in parallel with SPAD measurements at BBCH 50. Statistical Analysis Analyses of variance for single years and combined analyses across years were performed using the MIXED procedure of SAS 9.2 software (SAS Institute, Inc., Cary, NC). Genotypes were treated as fixed effects, block, block (year), year, and/or genotype by year interaction as random effects. The best linear mixed models were selected according to the corrected Akaike information criterion (AICC). To study direct and indirect relations of observed traits with the components of Passioura's framework regression analysis was applied via the REG procedure of SAS. Procedure CLUSTER was applied to determine similar groups of genotypes based on yield components, components of Passioura's framework and the underlying phenological, morphological, and physiological traits (Bodner et al., 2013). Rainfall Pattern and Soil Water Availability Hydrological conditions at the experimental site are displayed in Figure S1 (Supplementary Material). Longtime rainfall during the vegetation period of spring cereals is 237 mm, while plant available water (PAW) in the soil from stored winter moisture at time of sowing is 170 mm, i.e., 42% of total seasonal crop water supply ( Figure S1A). Monthly in-season rainfall increases toward summer, resulting in a favorable balance between climatic demand and supply. Therefore, the site can be described as predominantly supply driven. The two experimental years showed distinct hydrological conditions (Figures 1A,B). Although annual mean temperature and precipitation were similar (2011: 10.5 • C, 395 mm; 2012: 10.9 • C, 402 mm), in-season rainfall distribution and stored soil moisture at sowing differed strongly. During May and June, i.e., time of stem elongation, heading, anthesis, and early grain filling, rainfall was significantly lower in 2012 (77 mm Based on the simulated long-term site hydrology and the measured soil moisture pattern during the experiment, we determined the number of stress days using a threshold of ≤50% PAW and calculated the probability of occurrence of the two experimental years compared to the last 25 year average (1988-2013) ( Figure 1C). Seasonal water availability revealed that in 2011 hydrological conditions in May were among the wetter half of years, while June water availability was similar to 65% of years. Contrary, 2012 was a particularly dry year with a low probability of occurrence in 25 years. Due to low water storage over winter and reduced rainfall in spring, prolonged dry periods with water contents below 50% PAW were observed in May and June. The probability of occurrence of dry conditions of similar intensity as in 2012 is 8% for May and 32% for June, respectively. Thus, site hydrology revealed that only limited water stress occurred in June 2011, whereas 2012 was a particularly dry year with high stress incidence. Consequently, changes in crop performance between the 2 years can be interpreted in terms of drought response. Yield and Yield Components Significant (P < 0.001) genotypic variation was observed for grain yield and all other yield components. Combined ANOVA of the core set revealed also significant variation for year and genotype × year interaction (Supplementary Table S1). Grain yield varied from 209.2 (TRI5254) to 541.3 g m −2 (7060) and 37.9 (PI428154) to 237.7 g m −2 ("Floradur") in 2011 and 2012, respectively ( Table 2). Mean droughtinduced grain yield loss in 2012 was 60.6% for the core set. Adapted durum "Floradur" showed the highest grain yield among core set genotypes followed by early flowering "Matt" and Khorasan wheat "QK-77" (Kamut R ), whereas T. monococcum and T. timopheevi accessions were lowest yielding. Yield reduction in 2012 was lowest for Khorasan (20.5%), intermediate for "Matt" and "Floradur" (51.2 and 54.7%, respectively) and highest for the einkorn and Zanduri wheat (80.1-85.2%). Einkorn and Zanduri wheat showed a significantly higher number of fertile tillers, whereas Khorasan wheat had the lowest tillering capacity. Number of seeds per ear varied from 11.4 (PI428165) to 36.5 (7060) and 4.2 (PI428154) to 18.1 ("Floradur") in 2011 and 2012, respectively. Elite durum germplasm had a significantly higher number of seeds per ear compared to underutilized wheats, showing that seed number is a key component for high yielding cultivars. T. turanicum and T. monococcum showed the largest and smallest seed weight, respectively. Particularly for Khorasan wheat, seed weight was the component ensuring a relatively high yield. When analyzing the sensitivity of the core set genotypes for yield components in response to low water availability, it is evident that seed number per ear and tillering were highly sensitive, especially for the neglected species einkorn and Zanduri wheat with a RSR of 68.5 and 41.8%, respectively, while they had a relatively stable seed weight (11%). Durum varieties had high sensitivity for seeds per ear (40%) followed by seed weight (19.1%). Khorasan wheat responded to drought stress mainly with seed weight loss (26.5%) along with plant height reduction ( Table 2). Figure 2 shows the components of the yield-water framework according to Passioura (1977) and relations to traits that we hypothesized to constitute the crops' phenological, morphological and physiological drivers of WU, WUE, and HI. Beside direct relations of traits with Passioura's components, we also provide some secondary inter-trait relations suggesting hierarchical dependences among traits. Components of Passioura's Yield-water Framework and Related Traits Water use varied significantly between genotypes in each year and ranged from 223.2 (7063) to 277.8 mm (W9) and from 145.9 ("Matt") to 177 mm (PI428165) in 2011 and 2012, respectively ( Table 3). For all phenological and physiological traits ( Table 3) a significant (P < 0.05) difference between genotypes was observed. Water shortage in 2012 resulted in a 37% reduction in average WU ET among core set genotypes (i.e., from 256.9 in 2011 to 161.9 mm in 2012). Einkorn wheat PI428165 (226.3 mm) and durum cv. "Matt" (185.1 mm) showed the highest and lowest WU ET over the 2 years. With respect to flowering the genotypes can be classified into three groups (Figure 3, Table 3): (i) early flowering durum cv. "Matt, " (ii) intermediate flowering group incl. "Floradur" and other tetraploid and hexaploid wheat genotypes, and (iii) very late flowering underutilized wheats T. monococcum and T. timopheevi. In the core set, flowering hastened in 2012 (2011: 91.9 d; 1173.5 • Cd; 2012: 81.7 d, 1104.8 • Cd). This was more evident with respect to early growth stages, i.e., from emergence to stem elongation. Late flowering wheat relatives inevitably showed shorter grain filling periods than durum wheat over 2 years. The relation of time to flowering with WU ET was significant in 2011, when wetter soil profile alongside with in-season rainfalls provided appropriate conditions for longer root water uptake of late flowering genotypes. However, in 2012, low water availability in May and June obviously restricted prolonged water extraction by late flowering varieties and hence reduced variation in WU ET . Early vigor, as determined by ground cover rate, was significantly higher for the core set in 2012 than 2011 (0.171 vs. 0.156% • C d −1 , respectively) with a significant genotype by year interaction (Table S1). Despite an initial lag phase, which was especially evident for T. timopheevi W9, underutilized wheat relatives closed their canopy more swiftly than durum and Khorasan wheat, particularly in 2012. In both years a strong relationship between root length density and WU ET was observed. This indicates that roots are key determinant for the WU ET component in Passioura's framework. Differences in root length density and other root parameters are presented in detail in Nakhforoosh et al. (2014). Water use efficiency (WUE b ) showed significant differences among the germplasm in both years. "Floradur" along with durum lines 7060 and 7063 had highest WUE b in 2011. "Floradur" remained superior in WUE b also in 2012. Genotypes with lowest WUE b were Khorasan wheat TRI5254 in 2011 and einkorn wheat along with Iranian wheat "Tabasi" in 2012. Average WUE b of the core set dropped from 3.7 to 2.5 g m −2 mm −1 . "QK-77" was the most stable genotype of the core set in sustaining WUE b (15.4%) while "Floradur" (31.5%) and "Matt" (25.6%) had an intermediate response. Underutilized wheats were most susceptible to drought stress (41.4%). Stomatal conductance declined substantially for the core set in response to water scarcity in 2012 (i.e., from 457.0 to 166.6 mmol m −2 s −1 ). In 2011 Khorasan wheat (TRI5254, Kamut R ) along with durum cv. "Matt" showed highest stomatal conductance while einkorn and Zanduri wheat were characterized by the lowest stomatal conductance. In 2012, "Matt" and T. carthlicum W13 had highest stomatal conductance whereas, like in 2011, the underutilized species T. monococcum and T. timopheevii showed the lowest stomatal conductance. Chlorophyll concentration, as an indicator for photosynthetic capacity measured by SPAD, showed a significant decrease for the core set in 2012 which was more evident for underutilized wheat species. In 2011, durum wheats SZD3146 and "Clovis" were the genotypes with highest chlorophyll content followed by "QK-77" and "Floradur, " while in 2012 "Floradur" was the superior genotype. Accessions of einkorn and Zanduri wheat constantly had the lowest SPAD values in both years. Both WUE b components (biomass, water use) were influenced by the measured physiological leaf traits. Water use showed a negative association with both stomatal conductance and chlorophyll content, being significant in 2011 only. Water use of late-maturing einkorn and Zanduri wheat was higher, in spite of lower stomatal conductance. Biomass showed a significant relation with leaf chlorophyll content in the dry year 2012, suggesting this measurement as an appropriate indicator for WUE b under limited water condition. Similar to stomatal conductance, leaf chlorophyll content was positively related to earliness in both years. In 2011 the highest harvest index (HI) values were observed for durum wheat (mean 0.42), followed by Khorasan wheat (0.32) and the underutilized wheat species T. monococcum and T. timopheevii (0.26). In 2012 HI of the latter underutilized wheat species decreased significantly (0.13) in response to drought, while "Floradur" and "Matt" almost retained their HI. Interestingly, Khorasan wheat "QK-77" showed even an increase in HI. With respect to plant height T. turanicum was significantly taller than the other wheat species. Significant genotypic variation for root-to-shoot was observed only in the dry year 2012 (Nakhforoosh et al., 2014). HI was negatively associated with root-to-shoot ratio in this year. Trait Based Grouping of Genotypes Association between genotypes (and years) based on (i) yield components, (ii) Passioura components (WU ET , WUE b , HI), (iii) phenological, morphological, and physiological traits related to Passioura's components, and (iv) all traits was revealed by cluster analysis (Figure 4). Including all genotypes reveals the strength of group linkage driven by genotypic similarity (constitutive) and environmental influence (adaptive), respectively. Using different clustering variables shows which group of traits mainly expresses constitutive or adaptive linkage between genotypes. The distinction between underutilized einkorn and Zanduri wheat and the other genotypes appeared at the highest hierarchy with the exception of Passioura components. In this case the first grouping was according to years, which is explained by the strong water dependence of these traits. At a lower hierarchical level four clusters can be distinguished, subdividing the whole sample according to years and/or wheat species. For clustering based on yield components a differentiation at a lower level is even obvious between durum and Khorasan wheat. With respect to Passioura components only four main clusters can be distinguished. Interestingly, Khorasan wheat "QK-77" changes the group between years: in 2011 (high water availability) "QK-77" is grouped together with the other underutilized wheat species T. monococcum and T. timopheevii, while in 2012 (low water availability) it joins the group of modern durum cultivars. The most meaningful grouping at high and low distances is provided when considering all traits. Here, in 2011 T. durum and T. turanicum are grouped in different clusters. Among the 2012 clusters, hexaploid wheats are next to each other, while einkorn and Zanduri wheat form distinct groups. Discussion Drought Environment Characterization Understanding crop response to drought and relevant traits conferring better stress resistance requires a precise environmental characterization (Blum, 2011). Simulation models have been shown to be an appropriate tool for a proper description of the target environment for crop management and breeding activities (Chauhan et al., 2013). Continental climates as found in central-eastern Europe are distinguished by a higher proportion of in-season rainfall compared to stored soil moisture as source of crop water supply. Thereby, they differ essentially from storage driven Mediterranean winter rainfall FIGURE 4 | Hierarchical clustering of wheat genotypes based on yield components, components of Passioura's yield-water framework, phenological, morphological, physiological, and root traits related to components of Passioura's yield-water framework, and all traits. climates or subtropical sites where dry season crops grow on residual soil moisture. Still stored water can be essential to buffer temporary dry periods affecting crop yield particularly when their occurrence coincides with sensitive growth stages. The substantial change of crop performance due to low stored soil moisture together with low precipitation around flowering in our experiment clearly reveals that average climate variables (e.g., annual or seasonal rainfall sum) are insufficient to provide an appropriate picture on crop water stress. According to Blum (2009) an efficient use of available soil water should be targeted as selection criteria. In this regard site hydrology determines which plant traits support most effective water uptake. Generally rooting depth is considered the key trait for superior plant water supply (e.g., Wasson et al., 2012;Lynch, 2013). However, Nakhforoosh et al. (2014) demonstrated that dense root systems in the upper soil layers rather than deep rooting provide highest plant water uptake in an environment with high in-season rainfall and high storage soils. This is particularly valid for dry years with a lack of subsoil moisture from off-season winter rainfalls, when investing into deep rooting is of limited value to sustain high transpiration. Also Tron et al. (2015) in a modeling study could show that in strongly supply driven environments, rooting density can become more important for plant water acquisition compared to rooting depth. These findings are in agreement with results from an ecological study by Sperry and Hacke (2002) in a desert environment with soils of different storage capacity revealing that exploitative root traits (e.g., rooting density, root xylem cavitation resistance) allowed better adaptation than exploitative traits (e.g., deep rooting) when soil water availability was higher in the top soil compared to deep soil layers. There appear two keys to a water efficient root system: (i) spatio-temporal synchronization of root distribution with the distribution of available soil water (Schenk and Jackson, 2002) and (ii) high root hydraulic functionality to efficiently exploit available water in accordance with crop needs (Vadez, 2014). Plasticity of Yield Components Trait based strategies for better drought resistance in cereal crops require downscaling yield reduction under stress to the sensitivity of single yield components. In our study for example, "QK-77" (Kamut R ) stabilized its grain yield at the cost of shoot biomass via a significant decrease in plant height, resulting in an increased HI, suggesting a potential for partitioning of biomass to seeds as an important stress adaptive trait frequently found for cereals (Blum, 1998;Shearman et al., 2005;Dreccer et al., 2009). Reduction of competition from alternative sinks (stem and infertile tillers) is hypothesized as an opportunity to increase the partitioning to spikes and further increase HI beyond its current limit (Foulkes et al., 2011). Although "QK-77" can be considered a water stress tolerant genotype, it does not show high yield potential under favorable water condition. Contrary, T. monococcum and T. timopheevi significantly reduced the number of fertile tillers, their main yield component, in response to suboptimum water availability. Number of seeds per ear and seed weight, which are both related to grain sink strength (Miralles and Slafer, 2007;Acreche and Slafer, 2009), are very low in these species, resulting in significantly lower HI despite reasonable biomass production. Restricting tillering capacity is considered beneficial where water limitation requires a more conservative uptake strategy over the growing season to provide the crop with enough water during grain filling (Richards et al., 2010). The main yield component of durum varieties was number of seeds per ear followed by seed weight, whereas number of fertile tillers showed no plasticity. Slafer et al. (2014) recommended a balanced dependence of grain yield on single components to ensure both high yield potential and sufficient plasticity in response to water limitation. Shoot and Root Traits Underlying Passioura's Yield-water Framework Clustering genotypes based on Passioura's components revealed a clear distinction between tetraploid T. turgidum and underutilized T. monococcum and T. timopheevi, which was also demonstrated by their mean performances (Table S2). On the other hand, Khorasan wheat, a turgidum subspecies genetically similar to durum wheat but with lower breeding intensity, was more variable between and within clusters (Figure 4). Water use Traits Phenology was a major distinction among genotypes and a key driver of other morphological and physiological traits. The prolonged vegetative development of T. monococcum and T. timopheevi is obviously genetically determined. But also breeding history and origin can result in significant different phenology, e.g., "Matt" vs. "Floradur" (Figure 3). Beside the constitutive differences among genotypes, there is also phenological plasticity in response to water availability. In 2012 the transition from vegetative into reproductive phase was obviously stimulated by water stress. Plasticity of early growth stages until stem elongation is well-known in wheat while later growth stages are generally more stable (e.g., McMaster and Wilhelm, 2003). Flowering is the most sensitive stage to water shortage (Farooq et al., 2012). Progress has been achieved by breeding for earliness allowing crops to escape terminal drought stress and access enough soil water during flowering and grain filling (Salekdeh et al., 2009). However, vigorous growth and sufficient biomass prior to flowering is also critical for yield potential. In the present in-season rainfall environment yield limitation due to earliness was clearly demonstrated by low grain yields of early maturing cv. "Matt" compared to other advanced varieties and/or breeding lines. Grain yield of early maturing genotypes is largely limited by the potential number of grains per unit area which is determined between stem elongation and post-anthesis (Slafer et al., 2014). Among other factors, an overall lower water use seems to limit yield potential of very early cultivars, which can be attributed to a reduced rooting intensity (Mitchell et al., 1996). The dominant morphological difference within the investigated germplasm was the number of fertile tillers. T. monococcum and T. timopheevii exhibited a high number, T. turgidum subsp. durum an intermediate number and T. turgidum subsp. turanicum a low number. In 2012 water stress resulted in a reduced number of tillers. Highest plasticity with respect to number of tillers was found for genotypes of the "high tillering" group. In regard to water use, tillering is relevant due to the secondary nodal root system developing from tillers (Zobel and Waisel, 2010). Thus, a shortened period between emergence and stem elongation with limited tillering can also limit the development of nodal roots, resulting in lower water use. The consistent association between water use and root length density confirms this relationship (Figure 2). On the other hand, the high tiller number of underutilized wheats is evidently limiting yield as revealed by its negative correlation to harvest index. Therefore, optimization of tiller, related to nodal rooting, for high water uptake is constraint within tight limits. Other root system traits such as increased fine rooting in response to drought might provide alternatives to improve water use (Nakhforoosh et al., 2014). Such an adaptive response is more compatible to high yields than alteration of assimilate allocation between roots and shoots. An interesting trait promoting water use under conditions of limited availability was early vigor. Rapid ground cover can reduce evaporation losses by shading the soil (López-Castañeda and Richards, 1994), increase total photosynthesis by extending the duration of light capture (Parry et al., 2011) and enhance weed competitiveness of the crop (Bertholdsson, 2005). Our results showed a significant association of early vigor with water use only in the dry year 2012, suggesting secondary associations of this trait with phenology and root length density (Figure 2). With respect to inter-trait relations, however, results should be treated with caution if the data are concentrated at the two ends of the regression line. The associations might be a consequence of constitutive differences between underutilized wheat species and modern varieties rather than expressing causal inter-trait relations. Water Use Efficiency Traits Crop growth depends on acquiring CO 2 through open stomata, which in turn results in water loss through transpiration. Although upscaling from stomata gas exchange (intrinsic WUE) to whole plant WUE is complex (Hsiao et al., 2007), suitability of stomatal conductance as selection criterion has been demonstrated under both drought stress and well watered conditions (Rebetzke et al., 2013b). In both years the early maturing durum "Matt" was among the genotypes with highest stomatal conductance, suggesting an association between earliness and/or crop growth rate with stomatal conductance. Araus et al. (2002) pointed to higher stomatal opening as a consequence of crop earliness and lower leaf area index (LAI). Also in our study stomatal conductance was significantly and negatively correlated with LAI in the dry year 2012 (r = −0.75, p < 0.05; data not shown). Contrary, late maturing T. monococcum and T. timopheevii had the lowest values of stomatal conductance. An influence of ploidy level on stomata characteristics with diploid species, having the smallest stomata, was demonstrated by Khazaei et al. (2010). Low stomata conductance of einkorn and Zanduri wheat suggested a conservative gas exchange strategy. Their comparatively high water use is, therefore, explained rather by prolonged duration of transpiration than a high rate of water extraction due to conductive stomata. Stomatal conductance and photosynthetic capacity, traits underlying intrinsic WUE (Condon et al., 2002), seem to be strongly related to constitutive differences resulting from different breeding intensities. Similar to other studies, we found a significant association between stomata conductance and photosynthetic capacity (2011: r = 0.73, p < 0.01; 2012: r = 0.65, p = 0.058). This indicates a tight functional link between stomata opening ensuring high CO 2 inflow and photosynthetic capacity providing efficient fixation of available carbon in modern high yielding varieties. It also confirms the challenge of improving intrinsic WUE by lower stomata conductance without compromising crop productivity (Blum, 2005;Lawson et al., 2012). Fischer et al. (1998) demonstrated the association of leaf photosynthetic rate and stomatal conductance with yield progress in CIMMYT wheat genotypes. Also Reynolds et al. (1994) reported a significant association between photosynthetic rate and stomatal conductance with grain yield. Combining stomata conductance and leaf chlorophyll content measurements could allow the identification of germplasm combining improved WUE and productivity under both well watered or water limited conditions (Rebetzke et al., 2013b). Harvest Index Traits Genetic variation in harvest index within our germplasm was largely determined by distinct differences in yield components and phenology (Figure 2). Unlike modern cultivars, underutilized wheat species were more dependent on alteration of assimilate allocation between root and shoot in response to drought (Nakhforoosh et al., 2014). The observed association between harvest index and root to shoot in the second year most probably results from an intrinsic low harvest index of the underutilized wheat species resulting from their high allocation to roots under limited water availability. Dissecting the investigated germplasm according to Passioura's yield-water framework resulted in two contrasting patterns (Figure 5). Underutilized wheat species can be FIGURE 5 | Distinctive behavior of wheat genetic resources and/or underutilized wheat species vs. modern varieties within Passioura's yield-water framework. (Root trait differentiation is based on Nakhforoosh et al., 2014). considered as maximization types in terms of water use. Their phenology and morphology allows an intensive water extraction as a basis for pronounced vegetative growth. This seems to be a safety strategy based on a high number of tillers. Although the vegetative apparatus may suffer a high reduction of tillers in case of later water limitation, still the crop will avoid complete failure. Contrary, modern varieties are optimized with respect to effective water use which is well balanced between vegetative and generative demand. This strategy is most appropriate to sustainably supply less but still highly demanding generative sinks. In case of high water stress, this strategy may be risky and result in total crop failure if not sufficient water for their main yield components is available. In terms of WUE underutilized wheat species can be defined as conductance types and modern varieties as capacitance types. The high conductance, however, does not refer to the stomata scale but to the whole plant scale. The intense vegetative apparatus with high leaf area results in a high transpiring surface. This goes along with a low stomatal conductance and low photosynthetic capacity, both limiting assimilation potential. In modern varieties high stomatal conductance is linked to high photosynthetic capacity which ensures an efficient supply of assimilates. Water losses are controlled by an optimized total leaf area, ensuring sufficient light interception while avoiding unnecessarily high transpiring surface. Differences in harvest index between old and modern varieties are well documented. We characterized the distinctive pattern as source types for underutilized wheat with an extensive vegetative apparatus and as sink type for modern varieties where available resources are efficiently allocated to a strong generative sink. Conclusion Our study demonstrated that underutilized wheat species with low or no breeding intensity show serious limitations as source of novel traits of potential interest for wheat improvement. Their main strength is an efficient root water extraction linked to high assimilate translocation to roots, high tillering capacity, and long vegetative growth. In modern high yielding cultivars physiological traits such as stomata conductance combined with leaf chlorophyll concentration are responsible for their superior performance in well watered and stress conditions. The high yield stability of T. turanicum provides evidence that, despite limited yield potential, also some underutilized genetic material can be a source of interesting adaptive processes for future trait based breeding with respect to drought tolerance. Passioura's yield-water framework provides an appropriate conceptual model to guide such trait based analysis of breeding material. Our overall results suggest that crop improvement in water limited environments will likely profit more from making use of unexploited secondary traits in modern varieties than relying on wide crosses. Khorasan wheat, however, demonstrated that landraces or landrace selections of wheat subspecies of the same ploidy level may reveal promising drought stress response strategies that are currently not present in modern varieties.

v3-fos

2019-04-07T13:06:00.349Z

2015-01-10T00:00:00.000Z

101828008

s2

Comparison of the nutritional value of egg yolk and egg albumin from domestic chicken, guinea fowl and hybrid chicken The present study was conducted to compare the nutritional and physical quality of egg yolk and egg white of birds from three different ge notypes (domestic chicken, hybrid chicken and guinea fowl). The egg yolk and white f rom each of the bird were separated and analyzed for proximate, vitamins and minerals u sing standard analytical methods. The eggs of the 3 bird species showed similar conic al shape, however, weight of whole egg, egg white and yolk of hybrid chicken was much higher than that of domestic and guinea fowl. The moisture (60.45+ 0.14%) and vitamin C (121.50+.14mg/100g) contents of egg yolk were significantly higher in h ybrid chicken than in domestic chicken and guinea fowl while the protein (5.47+ 0.88%), ash (1.32+ 0.03%) and vitamin C (68.50+0.70mg/100g) contents of egg white was higher in hy brid chicken than domestic chicken and guinea fowl. However, moisture contents (87.45+ 0.71%) of egg white from guinea fowl was significantly (p<0.05) h ig er than hybrid chicken. All elements considered in this study had higher concen trations (mg/100g) in egg yolk than white except for Na whose concentrations were highe r in egg white than yolk. The concentration of K (321.50+7.62 and 119.50+ 2.6.2), Fe (12.45 +0.09 and 4.45+ 0.0.8) and Ca (26.60+0.63 and 9.23 + 0.22) for egg yolk and white respectively was significantly (p<0.05) higher in guinea fowl than d omestic and hybrid chicken. However, Na contents in hybrid chicken (850.00+ 22.40 and 975.00+ 09.00) for egg yolk and white respectively was significantly (p<0.05) h igher than that of guinea fowl and domestic chicken. It is concluded that egg yolk and white of hybrid chicken were riches in moisture, protein, ash , vitamin C and sodium than guinea fowl and domesti c ch cken. While egg yolk and white of guinea fowl were rich i n K, Fe and Ca than the eggs of domestic & hybrid chicken. INTRODUCTION A balanced diet is essential for normal growth, health and preservation of the human body. Eggs have constituted an important part of human diets for centuries because of its high quality protein [1]. They are known to supply the best proteins besides milk [2]. It is also rich in amino-acids, carbohydrates, easily digestible fats and minerals, as well as valuable vitamins [3]. The yolk and white components are all of high biological value and are readily digested [4]. Eggs play important culinary roles and are therefore prepared into different dishes. There are many types of poultry species' eggs consumable as a protein and amino acid supplement [5]. Nigeria has the highest number of poultry farm in Africa. Nigeria presently produces about 300,000 tons of poultry meat per annum officially and 650,000 tons of eggs [6]. A parallel record from Poultry Association of Nigeria (PAN), indicates that Nigeria produces presently above 1.25million tons of egg per year. South Africa is the second producer of eggs in Africa [7]. The question arises whether there are interspecies differences in poultry eggs quality which may affect the nutritive value and quality as human food. The guinea fowl is a bird native to the African continent [9]. It derives its name from the Coast of Guinea where it is believed to have originated. The indigenous guinea fowl (Numida meleagris) is widely distributed in Africa where it has distinct popularity among small holder farmers. It is believed that guinea fowls were taken to Europe and America by the Portuguese but in these regions the guinea fowls have been scientifically improved resulting in faster growth rate, bigger body size and enhanced egg laying capacity [10]. Guinea fowl breeding hens produce thicker shelled eggs in comparison to that of a regular chicken [11]. However there is paucity of information on the nutritional qualities of eggs from domestic fowl in comparison with eggs from other poultry species. In this report, we evaluated the nutritional levels of the proximate, vitamins and minerals composition in egg yolks and egg albumin collected from different bird's species. Source of Materials: Freshly-laid egg samples from birds of three (3) different genotypes (domestic chicken, guinea fowl and chicken breeders) were obtained between 21 and 22 September 2014 from a poultry keeper in Minna, Nigeria. The eggs were analyzed for nutritional compositions between 23 and 26 September 2014. At laying time the domestic fowl were approximately 28 weeks, guinea fowl were approximately 52 weeks and the hybrid chicken were approximately 26 weeks old. The birds' genotype was Hy-Line Brown (hybrid chicken), Pearl (guinea fowl) and normal feathered (domestic chicken). The domestic chicken and guinea fowl had outdoor access all the year round Fed with cereal, hay, clover, vegetables and green crop, according to the season of year. The hybrid chicken ate balanced biocomplete cereal-based mixed fodder with several additives daily. Sample Preparation The egg samples were thoroughly washed with distilled water in the laboratory federal university of technology, Minna. Nigeria. The yolk and albumin were separated by breaking a small part of the egg shell at one end and separating the egg albumin from the yolk. Evaluation of physical quality of egg Egg weight was measured with electronic weighing balance. Subsequently, yolk was separated from the white and weighed separately. The weight of shell was calculated by subtracting the weights of yolk and white from the weight of whole egg. Proximate Analysis Moisture and crude fat content were determined according to the standard methods of A.O.A.C [12]. Ash content was determined at 550 o C [13].Crude nitrogen was determined by Kjeldahl method [13] and crude protein determined by using the formula Crude protein = Crude nitrogen × 6.25 [14]. All the analysis was performed in triplicate. Mineral Analysis The method of A.O.A.C [12] was employed for the determination of mineral content. Vitamin Analysis Vitamin C and Vitamin A composition of each of the sample, were determined by the method of A.O.A.C [12]. Ethical Clearance Ethical clearance was given by Federal University of Technology, Minna/Nigeria ethical review board (CUERB) in accordance with international standard on the care and use of experimental animals. Statistical Analysis The data obtained were subjected to Analysis of Variance (ANOVA) using SAS statistical package. Means were separated using Duncan's Multiple Range Test (DMRT). Significance was accepted at P < 0.05. Physical Properties The eggs of domestic chicken, hybrid chicken and guinea fowl showed similar conical shape with blunt and pointed ends; however, weight of whole egg, egg white and yolk of hybrid chicken was much higher than that of guinea fowl and domestic chicken respectively (Table 1) Proximate The moisture and crude protein content was significantly higher in egg white than yolk while the crude fat and ash content was significantly higher in yolk than in egg white for the three (3) eggs sample. For the egg yolk the moisture and the ash contents was significantly higher in hybrid chicken than in domestic chicken and guinea fowl.The egg yolk from the 3 birds species show no significant difference in there protein and fat content while egg white of the hybrid chicken is significantly lowered in moisture (75.50+0.14%) but higher in ash (1.32+0.03) content as compared with the guinea fowl and domestic chicken. The protein content was significantly (p<0.05) lowered in domestic chicken (3.48+0.91) as compared with guinea fowl (5.81+0.62) and hybrid chicken (5.47+0.88) Table 3 presents the results of mineral analysis of domestic chicken (Gallus domesticus), guinea fowl (Numida meleagris) and hybrid chicken. All elements considered in this study had higher concentrations in egg yolk than in the white except for Na whose concentrations were higher in the egg white than in the yolk for all the species considered. Table 3 presents the results of vitamins A and C contents of egg yolk and white from domestic chicken (Gallus domesticus), Guinea fowl (Numida meleagris) and hybrid chicken. Vitamin C content of egg yolk is higher in hybrid chicken (121.50+0.14) and lowest in domestic chicken (97.50+0.71) For the egg albumin the highest concentration of vitamin C was recorded for hybrid chicken (68.50+0.70) and least was recorded for domestic chicken (47.00+2.11). However, no significant differences between the egg yolk and white of the 3 bird's species were found in the content of vitamin A. Physical properties Generally eggs of birds have oval shape with small differences among species. Despite its small differences, egg shape is considered as an important factor in characterizing bird species. In this study the eggs of domestic chicken, hybrid chicken and guinea fowl showed similar conical shape with blunt and pointed ends, however eggs of domestic chicken is more pointed than the two bird species. Similar findings have been reported for egg shapes of quail and guinea fowl [15]. The significantly higher weight of whole egg, egg white and yolk observed in hybrid chicken as compared to domestic and guinea fowl was obviously due to vast difference in the size of these three bird species. This difference could be attributed to the various feed additives, antibiotics or production stimulants fed to the hybrid chicken but deprived domestic and guinea fowl. The weight of hybrid chicken reported in this study (72.45+2.41) was higher compared to 56.41g reported for naked neck chicken and 40.5g for full feathered chicken [16] Proximate composition Protein is an essential component of human diet which is needed for the replacement of tissue and supply of energy. Protein deficiency cause growth retardation, muscle wasting, oedema, abnormal swelling of the body and collection of fluid in the body of children [17].This study revealed low protein contents in three poultry egg species and the little amount presents are more abundant in the egg white, however contrary findings have been reported by [15], who reported more protein contents in egg yolk than white from Quai and guinea fowl. Also in this study no significant difference in the protein content of egg yolk from the three poultry egg species, however the egg white from hybrid and guinea fowl chicken contain more protein than egg white of domestic chicken. Dietary fat functions in the increase of palatability of food by absorbing and retaining flavours. This study also revealed that the eggs yolk and white are good and poor source of lipids respectively. Although no significantly difference in fat contents of egg white from the 3 bird species, the low fat contents of egg white is an important consideration for people who suffer from elevated cholesterol level, and can also be recommend as part of weight reducing diets. The lipids contents of egg yolk for the three poultry egg species is high, a diet providing 1-2% of its caloric of energy as fat is said to be sufficient to human beings as excess fat consumption is implicated in certain cardiovascular disorders such as cancer and aging [18]. The fat contents of albumin from domestic chicken and guinea fowl in this study is comparable with that reported for chicken and guinea fowl egg [19]. The ash content gives a measure of total amount of inorganic compounds like minerals present in a food. This study revealed that the egg yolk and white of hybrid chicken contain more ash than domestic chicken and guinea fowl. This is an indication that the hybrid chicken will contain more minerals. This finding could be attributed to the variation in feed composition fed the birds. Similarly, low ash content has been previously reported for egg from Quai and guinea fowl [15], and chicken egg (0.91±0.03) [20]. This study revealed that egg white contain more moisture content than the egg yolk, also the egg yolk of hybrid chicken contain more water than those found in guinea fowl and domestic chicken. However high water contents of food have been implicated for low shelf life due to microbial attacked [21] Minerals Calcium helps in the regulation of muscle contraction required by children, infants and fetuses for bones and teeth development [22].The recommended dietary allowance value of calcium is 600-1400mg/kg [23]. The present study show that both egg yolk and white of guinea fowl contain high amount of calcium as compared to the hybrid and domestic chicken. Considering the importance of calcium, its concentration in egg yolk implies that this can contribute to the amount of dietary calcium. However the level of calcium observed in this study was lower than 38.2mg/100g reported for a whole chicken egg [20].The recommended daily value for sodium is 1100-3300mg/kg for adults [22]. Hybrid chicken contain high sodium concentration than the guinea fowl and domestic chicken. However, the concentration of sodium in egg yolk and albumin for all the three bird species observed in this study was lower than 134±20 reported for a whole chicken egg [20] The enrichment of iron in egg would provide improving the nutrition status of people especially in the risk of iron deficiency or anemia group especially infant, children, pregnant women and socioeconomic groups [24]. The recommended daily requirement of iron for man is 6-40 mg/kg [23]. The egg yolk and white was found to contain iron in concentration within the recommended daily requirement. However, the egg yolk contain more iron than the white, contrary findings have been reported by [25], who reported more iron contents in egg white than egg yolk from snail-eating turtle eggs. Also hybrid chicken was found to have the highest concentration of Iron, this poultry egg species from the result obtained can be used in improving the anaemic condition in iron deficient diabetic patients. Potassium is responsible for nerve action and is very important in the regulation of water, electrolyte and acid -base balance in the blood and tissues [26]. In this study, egg yolk of guinea fowl was found to contain the highest concentration of potassium. The level of potassium in this poultry eggs especially the egg yolks is a good indication that its consumption will enhance the maintenance of the osmotic pressure and acid-base equilibrium of the body [27] Vitamins The Recommended Dietary Allowances (RDAs) for vitamins reflect how much of each vitamin most people should get each day. Results of the present study revealed that the 3 poultry egg species studied contain considerable amount of vitamin A and C. However, vitamin C content of egg yolk and white is higher in hybrid chicken compare to domestic and guinea fowl. Vitamins A and C have been reported to have antioxidant properties and may protect body against some forms of cancer [28] The concentration of vitamins is influenced by genetics, rate of egg production and it varies with the composition of the hen's diet [29]. As the concentration of fat-soluble vitamins in the feed increases, so does the content of vitamins in the egg yolk [30]. However, according to [31] for some vitamins, such as vitamin A, the liver acts as a reservoir so that the concentration in the yolk is buffered against large changes in the diet. This finding is supported by the results of the present study as no significant differences between the egg yolk and white of the 3 bird's species were found in the content of vitamin A. CONCLUSIONS The findings of this study showed that eggs of guinea fowl, domestic and hybrid chicken are rich source of protein, vitamins and appreciable number of some essential minerals. However, from the tested parameters, egg yolk and white of hybrid chicken were rich in proximate, vitamin C and sodium than eggs of guinea fowl and domestic chicken. While egg yolk and white of guinea fowl were rich in K + , Fe 2+ and Ca 2+ than the other two studied birds

v3-fos

2016-05-04T20:20:58.661Z

2015-08-14T00:00:00.000Z

1300819

s2

2-DE proteomics analysis of drought treated seedlings of Quercus ilex supports a root active strategy for metabolic adaptation in response to water shortage Holm oak is a dominant tree in the western Mediterranean region. Despite being well adapted to dry hot climate, drought is the main cause of mortality post-transplanting in reforestation programs. An active response to drought is critical for tree establishment and survival. Applying a gel-based proteomic approach, dynamic changes in root proteins of drought treated Quercus ilex subsp. Ballota [Desf.] Samp. seedlings were followed. Water stress was applied on 20 day-old holm oak plantlets by water limitation for a period of 10 and 20 days, each followed by 10 days of recovery. Stress was monitored by changes in water status, plant growth, and electrolyte leakage. Contrary to leaves, holm oak roots responded readily to water shortage at physiological level by growth inhibition, changes in water status and membrane stability. Root proteins were extracted using trichloroacetate/acetone/phenol protocol and separated by two-dimensional electrophoresis. Coomassie colloidal stained gel images were analyzed and spot intensity data subjected to multivariate statistical analysis. Selected consistent spots in three biological replicas, presenting significant changes under stress, were subjected to MALDI-TOF mass spectrometry (peptide mass fingerprinting and MS/MS). For protein identification, combined search was performed with MASCOT search engine over NCBInr Viridiplantae and Uniprot databases. Data are available via ProteomeXchange with identifier PXD002484. Identified proteins were classified into functional groups: metabolism, protein biosynthesis and proteolysis, defense against biotic stress, cellular protection against abiotic stress, intracellular transport. Several enzymes of the carbohydrate metabolism decreased in abundance in roots under drought stress while some related to ATP synthesis and secondary metabolism increased. Results point at active metabolic adjustment and mobilization of the defense system in roots to actively counteract drought stress. Introduction Forest trees are of enormous ecological and economic value in global and local scale. They will be among the species most harmfully affected by the predicted climate changes with frequent temperature and precipitation extremes; however for a number of reasons our knowledge of the biochemical mechanisms to counteract inevitable environmental stresses is still very limited, especially concerning trees (Plomion et al., 2006;Abril et al., 2011). Quercus ilex is a dominant tree in western Mediterranean region, one of the main plant species of the so-called savannahtype woodland ecosystems (dehesas) which cover more than 4 million ha in western Mediterranean and northern African countries (David et al., 2007;Corcobado et al., 2013). Holm oak has double importance both from ecological and economic points of view, the latter coming from the fact that its acorns are major constituents in the diet of free range domestic animals; the rich nutrient composition and tannins of acorns give the original and specific taste of local meat products (David et al., 2007). According to climate model simulations, the Mediterranean region is expected to be a hot-spot which will be particularly affected by long term drought and warming episodes (Giorgi and Lionello, 2008). Holm oak is very well adapted to dry hot climate, due to morphological particularities like a deep and well-structured root system with relatively large surface area and rapid development, which allows efficient capture of water from deepest soil layers, and small evergreen sclerophylous leaves with minimal transpiration and economical water use efficiency (David et al., 2007;Tsakaldimi et al., 2009). More biomass in this species is allocated in roots, forming larger below-ground starch and lipid reserves (Sanz-Pérez et al., 2009). Physiologically, holm oak is considered to be a typical water-spender species which maintains low leaf water potential. Stomatal closure upon water stress does not inhibit carbon assimilation in holm oak contrary to plants with water-saving strategy like pines (Baquedano and Castillo, 2006). It is predicted that the natural habitats of Quercus ilex will not be greatly affected or will even be expanded in the context of future climate changes (David et al., 2007;Bussotti et al., 2014). The overall good adaptation potential and huge economic importance of holm oak make it principal and valuable species for Mediterranean reforestation programs, which fostered research on Quercus ilex variability and stress response, especially at the proteome level (Jorge et al., 2006;Echevarría-Zomeño et al., 2009;Valero Galván et al., 2011. Main limitation in reforestation practices is the very low field survival rate of planted seedlings. Early seedling establishment is an extremely vulnerable phase of plant development and drought is considered to be the main cause of mortality post-transplanting (Navarro Cerrillo et al., 2005;Tsakaldimi et al., 2009). Besides, prolonged drought weakens tree defense systems against fungal pathogens. The forest decline is attributed to fungal attacks (e.g., Hypoxylon mediterraneum (De. Not.) Mill., Biscogniauxia mediterranea (De Not.) (Kuntze), or Phytophthora cinnamomi Rands.) after severe drought combined with high temperatures (Corcobado et al., 2013;Sghaier-Hammami et al., 2013). An active response to drought is critical for tree establishment and survival, however, our knowledge concerning the biochemical mechanisms of trees to counteract drought stress is still very limited, especially at the protein level (Abril et al., 2011). Drought is one of the most deleterious abiotic stresses with respect to plant survival (Wang et al., 2003). Plants counteract drought using a combination of survival strategies like drought escape (through adjustment in the developmental program and ending reproductive cycle before severe drought development), drought avoidance (maintaining the internal water balance under drought conditions mainly through morphological and physiological adjustments), and drought tolerance (coping with water limitation mainly at cellular and biochemical level), which are subordinated to the natural climatic variations (Dolferus, 2014). In the genus Quercus for example, the rapid spring growth and allocation of high quantity of carbon reserves into roots could be regarded as elements of drought avoidance strategy-to develop robust root system and gain access to water in deeper soil layers (Sanz-Pérez et al., 2009). Survival under prolonged drought is more linked to tolerance strategies at the cellular level. Among them should be mentioned: synthesis of compatible solutes, enhanced protection against oxidative damage, protection of membranes, and proteins from denaturation through dehydrins and chaperones, degradation of unnecessary proteins and reusing the building blocks, and others (Wang et al., 2003;Dolferus, 2014). Roots are the plant part directly and primarily exposed to soil drought, so roots play a primordial role in water stress sensing and response. Besides the general anchoring and supportive function, root as a tissue provide water and mineral nutrients to all plant parts, and produce hormones for stress signaling (Ghosh and Xu, 2014). Root functions are indeed impeded upon severe water stress. Root tissue is very actively engaged in the adaptation to drought; for example water scarcity could stimulate root growth contrary to the aboveground plant part where growth is usually inhibited (Yamaguchi and Sharp, 2010). Contrasting metabolic changes have been reported in roots compared to shoots under drought stress-shoots have been metabolically inactivated and have had lower concentrations of sugars, amino acids, nucleosides, whereas roots have been metabolically activated, and more primary metabolites have been allocated to/synthesized in roots under water stress (Gargallo-Garriga et al., 2014). Increasing number of studies is accumulating now concerning root drought response at physiological, molecular biology, biochemical and proteomic levels (reviewed in Ghosh and Xu, 2014). As proteins are the main and direct executors of cellular functions, more pronounced impact of proteomic studies is recently observed in stress research on roots (Sengupta et al., 2011;Swigonska and Weidner, 2013;Oh and Komatsu, 2015). Proteomics along with metabolomics could be very useful tools in the so-called next-generation phenotyping methods of screening for stress tolerance (Dolferus, 2014). Proteomic studies on Quercus face the common problem with orphan species with non-sequenced genome which could be solved by homology-driven cross-species identifications based on high similarity to proteins from other plant species (Valero Galván et al., 2011). Preliminary studies of drought response in 1-year old plantlets of Quercus ilex using a proteomics approach have been performed in our group, analysing the changes in the leaf protein profile (Jorge et al., 2006;Echevarría-Zomeño et al., 2009). The aim of the present study was to follow the dynamic changes in root proteins of drought treated Quercus ilex seedlings at very early developmental stage, applying a gel-based proteomic approach, and to relate proteome changes to stress severity and to recovery from stress, thus, to build a picture on the functional meaning of the observed protein changes. Plant Material and Stress Treatment Holm oak (Quercus ilex subsp. Ballota [Desf.] Samp.) acorns were harvested on place in December 2011 from a region near Cerro Muriano (20 km apart from Cordoba). Mature fruits were dropped down directly from the trees, minimizing contact with soil. Acorns were immediately transported to the laboratory in tightly closed plastic bags, washed, surface-sterilized for 10 min in 10% sodium hypochlorite solution, extensively washed again, and inspected for worm damage. Healthy acorns were blotted dry, put in clean plastic bags and stored at 4-8 • C. Spontaneously germinated acorns after 3 months of storage in cold were used in this study. Plants were grown in individual containers (330 ml volume) in perlite at growth chamber conditions: 12/12 h photoperiod, fixed light (360 µE.m −2. s −1 ), 24/20 • C day/night temperature and 70% air humidity, at optimal water supply (120 ml tap water per 30 g dry perlite). Water stress was applied on 20 days-old holm oak seedlings with developed 9 ± 3 leaves on randomly selected sets of 12 plants, by carefully placing plants into new containers filled with perlite which was wetted with limited water quantity (40 ml water per 30 g dry perlite). Water limitation treatment was for a period of 10 and 20 days, followed by a 10 days recovery phase from stress by restoring the optimal water supply. Sufficient or limited water quantity was maintained by gravimetric measurements and daily compensation for the loss of water due to evapotranspiration. Sampling was performed at the appropriate time points-roots from drought treated plants for 10 (D10) and 20 (D20) days, and after 10 days recovery from 10 or 20-days water limitation period (R10, R20), with the respective age controls (C0-at the treatment beginning; C10for D10, C20-for R10 and D20, and C30-for R20). Water status and electrolyte leakage were monitored on fresh plant material. For proteomic analyses, roots were quickly but thoroughly rinsed with distilled water, blotted dry, quick-frozen in liquid nitrogen, ground to fine powder and stored at −80 • C. Stress Estimation Parameters-Plant Growth, Water Status, Electrolyte Leakage Stress was monitored by changes in water status, growth, and electrolyte leakage. These parameters were assessed as previously described (Simova-Stoilova et al., 2008). Time course changes were followed in the water content of roots and leaves along with observations for any visible changes in plants. Biomass reduction and recovery growth were registered gravimetrically. Root and shoot fresh weight was taken at sampling from all the sets of plants (n = 12). Water content was measured in 3 individual plants per treatment, calculated according to the formula (FW-DW)/FW where FW is fresh weight, DW-dry weight of the same sample by drying it at 70 • C to constant weight for 48 h, and was expressed in percentage. Water deficit in roots and leaves was estimated as (TW-FW)/TW where TW is the turgid weight after floating the tissues for 4 h at room temperature in deionized water, and expressed in percentage. Electrolyte leakage was estimated by measuring the conductivity of the effusate solution from intact tissue, kept for 4 h at room temperature in deionized water, relative to conductivity of the effusate from the same tissue after boiling it for 10 min and cooling down. Protein Extraction and 2-DE Separation Root samples after 10 and 20 days of drought (D10 and D20) with the respective age controls (C10, C20) and 10-days recovery after 10 days of drought (R10) were analyzed by gel-based proteomics. Proteins were extracted from 3 biological replicas (each replica from 3 individual plants) according to the protocol of Wang et al. (2006) using TCA/acetone-phenol-methanol. Protein content in samples was estimated by the method of Bradford (1976) with bovine serum albumin as a standard. Samples were isoelectrofocused in the range of pI 5-8 using a Protean IEF Cell system (Bio-Rad, Hercules, CA, USA), 17 cm IPG strips, at 400 µg protein load per strip, active rehydration for 16 h at 50 V, rapid voltage ramp to 10,000 V, 50,000 Volt-hours in total, 500 V maintaining focused state. The second dimension was run at 12% SDS (PROTEAN R Plus Dodeca Cell, Bio-Rad, Hercules, CA, USA) and gels were double stained with colloidal Coomassie. Broad range molecular weight standards (Bio-Rad, Hercules, CA, USA) run by side in the same gel were used for estimation of MW. Image Analysis and Selection of Spots of Interest Images of the gels were captured with a GS-800 densitometer (Bio-Rad, Hercules, CA, USA) and analyzed applying PDQuest software (Bio-Rad, Hercules, CA, USA). Ten-fold over background criterion was used to assess presence/absence of a spot. Data on normalized spot volumes were exported and subjected to multivariate statistical analysis including sample clustering, ANOVA and Principal component analysis using a free online-based software (NIA arrays analysis tools, http:// lgsun.grc.nia.nih.gov/ANOVA/index.html). Prior to statistical analysis the normalized values were log transformed to reduce dependence between abundance and standard deviation. Data were statistically analyzed by ANOVA using the following settings: error model max (average, actual), 0.01 proportions of highest variance values to be removed before variance averaging, 10 • of freedom for the Bayesian error model, 0.05 FDR threshold, and zero permutations. The principal component analysis (PCA) settings were: covariance matrix type, 4 principal components, 1.5-fold change threshold for clusters, and 0.5 correlation threshold for clusters. PCA results were represented as a biplot, with consistent proteins in those experimental situations located in the same area of the graph. Selected variable spots of interest (90 in total), well defined and presenting statistically significant changes (drought and/or recovery compared to the respective age controls), with at least 1.5-fold difference in abundance ratio, were manually cut for subsequent MS analysis. Mass Spectrometry Analysis, Protein Identification, and Functional Annotation The MALDI-TOF/TOF Mass Spectrometry and Protein identification analysis was carried out in the UCO-SCAI proteomics facility, a member of Carlos III Networked Proteomics Platform, ProteoRed-ISCIII. The excised spots of interest were digested with porcine trypsin (sequencing grade) and loaded onto a MALDI plate, by using a ProPrep II station (Digilab Genomic Solutions Inc., Cambridgeshire, UK). The gel specimens were destained twice over 30 min at 37 • C with 200 mM ammonium bicarbonate/40% acetonitrile. Gel pieces were then subjected to three consecutive dehydratation/rehydratation cycles with pure acetonitrile and 25 mM ammonium bicarbonate in 50% acetonitrile, respectively, and finally dehydrated for 5 min with pure acetonitrile and dried out over 4 h at room temperature. Then, 20 µl trypsin, at a concentration of 12.5 ng/µl in 25 mM ammonium bicarbonate was added to the dry gel pieces and the digestion proceeded at 37 • C for 12 h. Peptides were extracted from gel plugs by adding 1 µl of 10% (v/v) trifluoracetic acid (TFA) and incubating for 15 min. Then, extracted peptides were desalted and concentrated by using µC-18 ZipTip columns (Millipore, Billerica, MA, USA) and were directly loaded onto the MALDI plate using α-cyano hydroxycinnamic acid as a matrix. Mass analysis of peptides (MS) of each sample was performed with a MALDI-TOF/TOF 4800 Proteomics Analyzer (Applied Biosystems, Foster City, CA, USA) mass spectrometer in the m/z range 800-4000, with an accelerating voltage of 20 kV. Spectra were internally calibrated with peptides from trypsin autolysis (M + H + = 842.509, M + H + = 2211.104). The most abundant peptide ions were then subjected to fragmentation analysis (MS/MS), providing information that can be used to determine the peptide sequence. Proteins were assigned identification by peptide mass fingerprinting and confirmed by MS/MS analysis. Mascot 2.0 search engine (Matrix Science Ltd., London, UK; http://www.matrixscience.com) was used for protein identification running on GPS ExplorerTM software v3.5 (Applied Biosystems, Foster City, CA, USA) over non-redundant NCBI protein and Uniprot databases. The following parameters were allowed: taxonomy restrictions to Viridiplantae, one missed cleavage, 100 ppm mass tolerance in MS and 0.5 Da for MS/MS data, cysteine carbamidomethylation as a fixed modification, and methionine oxidation as a variable modification. The confidence in the peptide mass fingerprinting matches (p < 0.05) was based on the MOWSE score, and confirmed by the accurate overlapping of the matched peptides with the major peaks of the mass spectrum. Proteins with statistically significant (p < 0.05) hits were positively assigned identification after considering Mr and pI values. Annotation of their biological function was consistent with Bevan et al. (1998). To predict the most probable intracellular localization of proteins, results from different software were compared-WolfPSORT (Horton et al., 2007, http://www.genscript.com/ psort/wolf_psort.html), TargetP (Emanuelsson et al., 2000, http://www.cbs.dtu.dk/services/TargetP/), PredSL (http://aias. biol.uoa.gr/PredSL/input.html, Petsalaki et al., 2006), MultiLoc (http://abi.inf.uni-tuebingen.de/Services/MultiLoc/, Höglund et al., 2006). To construct a heat map the identified protein intensity, values were log10 transformed (base-10 logarithm of 1 + mean intensity values) and mean-centered to rescale them. Hierarchical clustering of samples and proteins were performed using MeV4.8 (http://www.tm4.org/mev.html) with agglomeration method set to average and the distances were calculated based on Pearson's correlation. Data Submission Starting from each individual search (Mascot.dat file), the identified spots were translated to PRIDE XML using the PRIDE Converter 2.0 software (Côté et al., 2012). A total of 90 PRIDE XML files together with the corresponding raw MS files, mzXML peak lists and the Mascot search results (.dat file) were submitted to the ProteomeXchange repository (Vizcaíno et al., 2014) following the ProteomeXchange submission guidelines. Data are available with identifier PXD002484. Holm Oak Morphological and Physiological Response to Drought In this study water stress was imposed on 20 days-old holm oak plantlets with well-developed root system consisting of small white tip part and larger brown lignified part, and well developed 9 ± 3 leaves. At this time the tissue linking cotyledon and plantlet was practically desiccated, and even lost somewhere, thus, cotyledons were not considered as influencing the plantlet. Water limitation was maintained for a period of 10 and 20 days, each followed by 10 days recovery. Appropriate age controls were used for comparison of stress treatments and recovery-C20 was for R10 and D20, C30 was for R20. The experimental design is presented schematically in Supplementary Material Figure S1. Leaf number was not significantly increased during the whole experimental period both in control and in treated plants. Stressed plants had longer and thinner roots with diminished white root part, which was more evident after 20 days of treatment and was not restored in recovery from the longer stress. Photos of control, stressed and recovered plants are presented in Supplementary Material Figure S2. Stress development was monitored by changes in plant growth parameters (Figure 1, dark columns 'part-root FW, white columns' part-shoot FW), in water status [relative water content Figure 2-separate columns for roots (dark) and leaves (white columns), and water deficit- Table 1 left part], and in electrolyte leakage ( Table 1 right part) as an indicator of membrane damage. Biomass was significantly reduced only after 20 days of stress, more in roots (by 37.5%) than in shoots (by 20.9%). Water content in control plants was in the limits: root white part-87-78% (slightly diminishing with plant age), leaves-38-45%. Drought stress induced change in relative water content only in roots (diminution by about 10-15%), not in leaves. In roots, this parameter did not decrease further with stress prolongation and was completely restored in recovery. Water deficit increased only in roots after drought treatment, along with increase in membrane instability. A 2-fold rising in relative electrolyte leakage (EL) was registered in roots under 10 FIGURE 1 | Plant growth parameters. Dark columns' part-root FW, white columns' part-shoot FW. Mean values (n = 12) are given. Vertical bars represent standard deviations. C-control plants, C0, C10, C20, C30-the respective age controls (of days of treatment). D-plants subjected to water limitation treatment for 10 days (D10) or 20 days (D20). R-recovery by resuming optimal water supply for 10 days after 10 days (R10) or 20 days (R20) of water stress. Different letters above columns denote statistically significant differences. days of drought stress. In leaves EL remained very low, possibly linked to establishment of xeromorphic leaf structure. Based on these data, an active strategy for metabolic adaptation to drought is expected to be found in root tissue at the proteome level. Quercus Ilex Root Proteome Changes after Drought and Recovery The protein extraction with modified TCA/acetone/phenol protocol resulted in protein yield of about 370-740 µg protein per g FW of root tissue ( Table 2). Protein extraction was also made of samples recovered from D20, but in R20 the quantity of extracted protein dropped substantially. Five variants of samples were analyzed applying gel based proteomics-C10, D10, R10, C20, D20, in biological triplicates. Due to the insufficient protein yield, R20 was not studied. The 2-DE gel images of each of the variants (pI 5-8, and 12% SDS PAGE), 400 µg protein load, Coomassie colloidal staining) are shown in Figure S3, Supplementary. Images were analyzed with PDQuest software. Approximately 359 ± 9 consistent protein spots were clearly resolved on the gels. Concerning variability in abundance on the basis of spot volume ratio (treated to age control variants), relatively more spots were found to be decreased in abundance than increased, and more variability was found in recovery compared to drought treatment ( Table 2). Sample clustering and PC analysis data (Figure 3) clearly separated the five sample variants -C10, D10, R10, C20, and D20. Identification of Differently Abundant Spots and Patterns of Protein Changes Selected well defined spots, presenting significant changes under stress (at least 1.5-fold change compared to controls) and consistent in the three biological replicas, were subjected to FIGURE 2 | Relative water content. Separate columns for roots (dark) and leaves (white). Mean values (n = 3) are given. Vertical bars-standard deviations. C-control plants, C0, C10, C20, C30-the respective age controls (of days of treatment). D-plants subjected to water limitation treatment for 10 days (D10) or 20 days (D20). R-recovery by resuming optimal water supply for 10 days after 10 days (R10) or 20 days (R20) of water stress. Different letters above columns denote statistically significant differences. 16.4 ± 9.5 9.9 ± 2.9 C10 8.6 ± 4.2 10.7 ± 1.1 20.9 ± 6.7 6.8 ± 1.7 D10 29.3 ± 6.9 11.5 ± 1.7 41.9 ± 4.9 7.0 ± 0.9 Remark 3 times increase n.s. change 2 times increase n.s. change Mean ± standard deviations from three independent replicates are shown; n.s.-nonsignificant changes. C0-control at the beginning of the treatment, D10-water limitation treatment for 10 days, C10-the respective age control. Tables S1, S2). There were five cases of low score unreliable identification and three cases of 2-3 different proteins identified in the same spot, which were excluded from these tables. In spite of the fact that Quercus ilex is an orphan species and identification was mainly based on homology, the majority of reliable hits for one given spot were for the same protein in different plant species. A few of the proteins were detected in more than one spot, like actin 2, enolase (EC 4.2.1.11), betaine aldehyde dehydrogenase (EC 1.2.1.8), cysteine synthase (EC 2.5.1.47), pyruvate decarboxylase (EC 4.1.1.1), but with some exceptions they presented similar trends of changes. Differences in protein abundance are clearly distinguished in the heat map built on the basis of normalized spot abundancy ratios of treatment to the respective age control (Figure 4). Different patterns of changes were found when looking at the identification Variable spots (>2-fold/>1.5-fold change) are related to the respective age controls. C-control plants, C10, C20-the respective age controls (of days of treatment). D-plants subjected to water limitation treatment for 10 days (D10) or 20 days (D20). R-recovery by resuming optimal water supply for 10 days after 10 days of drought (R10), the age control in this case is C20. results; however, the majority of spot abundance changes presented the same trend at drought stress irrespective treatment duration-D10 or D20. The following types of dynamic changes were observed-changes in abundance under stress unrelated to how long was the treatment (43 protein spots-24 up and 19 down), increase or decrease in abundance only at D10 (14 protein spots-8 up and 6 down in abundance), decrease only at D20 (2 protein spots), spot abundance changes in different directions comparing D10 and D20 (4 protein spots), prominent abundance changes in recovery, mainly diminution (8 spots), and changes beyond detection level, so-called qualitative changes (11 protein spots). Among the identified proteins with earlier and reversible increase in abundance under drought followed by recovery were found many enzymes related to secondary metabolism such as: (2 spots). The only protein spot with increase in recovery was proteasome subunit beta (EC 3.4.25.1). The most interesting qualitative differences were in one cysteine synthase isoform (EC 2.5.1.47), which spot was below detection under drought; however, there was another spot identified also as cysteine synthase with increased abundance under drought. Identified proteins were classified into functional groups according to Bevan et al. (1998): primary and secondary metabolism, protein biosynthesis and proteolysis, defense against biotic stress, cellular protection against abiotic stress, intracellular transport. In Table 3 are summarized the main functional groups and subgroups and the tendencies of changes in individual proteins within groups. It is seen that several enzymes of the carbohydrate metabolism were decreased in abundance in roots under drought stress while some related to ATP synthesis and secondary metabolism were increased. Results point at active metabolic adjustment and mobilization of the defense system in roots to actively counteract stress. A summary diagram of molecular mechanisms involved in Quercus ilex root response to water limitation is presented in Figure 5. Discussion Physiological Changes in Quercus ilex Roots Under Water Limitation Holm oak is regarded as relatively drought tolerant plant species, well adapted to Mediterranean type of climate. Its tolerance is due to the large well developed root system, the evergreen sclerophylous leaf anatomy, the very economic use of water resources, as well as some particularities in photosynthesis (David et al., 2007;Tsakaldimi et al., 2009). However, seedling establishment is one of the most stress vulnerable phases in plant development (Tsakaldimi et al., 2009). In our experimental system, much more plant biomass is allocated in roots than in shoots (2.5 to 3-fold more) at early seedling stage. Significant growth inhibition was detected after relatively long period of water limitation (20 days), both for roots and for shoots, which confirms the expected drought resilience of this tree species. Some reports emphasize the active root growth vs. shoot growth inhibition as an adaptive strategy for drought adaptation, which is reflected in changes in root to shoot biomass ratio (Yamaguchi and Sharp, 2010), while in other cases root growth is inhibited in response to progressive water stress (Sengupta et al., 2011), probably linked to stress duration and species peculiarities in stress tolerance. In the case of Quercus ilex seedlings, however, the total root biomass was more negatively affected by prolonged drought than the shoot biomass was, resulting in diminution in root to shoot ratio compared to the respective age controls. In the time course of the experiment, the root to shoot ratio in the age controls was constantly increasing, which supports the more active root growth compared to shoots. Besides root biomass diminution, prolonged drought leaded to changes in the root aspect-longer and thinner roots with less white tip part at D20, which may be linked to increased lignification as a response to water deficit. Increased degree of lignification in the basal part of root elongation zone has been reported in response to drought for other plant species . The difference in leaf and root response to water shortage was further supported by the observed changes in water status and membrane stability after drought stress, expressed mainly for the root system. Pronounced change in water status of roots subjected to drought stress, compared to leaves, is also documented for other plant species (Yoshimura et al., 2008; Wendelboe-Nelson and Morris, 2012). Secondary Metabolism is Activated in Roots Under Drought Treatment Drought adaptation of plants requires complex rearrangements of the metabolism with interactions between several metabolic pathways. One of the most striking observations in our comparative study was the relatively early increase of several cytoplasmic enzymes engaged in secondary metabolism in roots under water stress, like: shikimate dehydrogenase (EC 1.1.1.25), naringenin-chalcone synthase (EC 2.3.1.74), flavanone 3-hydroxylase (EC 1.14.11.9), dihydroflavonol 4-reductase (EC 1.1.1.219), (+)-neomenthol dehydrogenase (EC 1.1.1.208), and caffeoyl CoA 3-O-methyltransferase (EC 2.1.1.104). Shikimate dehydrogenase, an enzyme of the shikimic acid pathway leading to biosynthesis of aromatic amino acids and simple phenolics, catalyzes the reversible NADP + -dependent reaction of 3-dehydroshikimate to shikimate. Chalcone synthase (or naringenin-chalcone synthase) is a plant enzyme in the initial step and central hub for the pathway of flavonoid biosynthesis, leading to production of flavanoids, isoflavonoidtype phytoalexins, and other metabolites with stress protective functions for plants. Other enzymes of the flavonoid biosynthesis pathway which are found to be up-accumulated in concert with chalcone synthase under drought were: flavanone 3-hydroxylase which catalyzes the stereospecific conversion of flavanones to dihydroflavonols, and dihydroflavonol reductase, which catalyzes the reduction of dihydroflavonols to leucoanthocyanins (Dao et al., 2011). Chalcone synthase is induced under different abiotic and biotic stresses like UV, wounding, herbivory, and microbial pathogens, resulting in the production of compounds with antimicrobial, insecticidial, and antioxidant activity (Selmar and Kleinwächter, 2013). Flavonoids interfere with hormone signaling by inhibiting polar auxin transport (Dao et al., 2011). Increasing evidence suggests that plants exposed to drought accumulate secondary metabolites, and a plausible explanation could be to protect cells from oxidative stress by consuming NADPH + H + for the synthesis of highly reduced precursors Spot volume ratio changes of variants: D-plants subjected to water limitation treatment for 10 days (D10) or 20 days (D20). R-recovery for 10 days after 10 days of drought (R10); C10, C20-control plants (the respective age controls of days of treatment). Statistically significant differences between control and treated variants or between age controls (p < 0.05) are indicated by asterix. like aromatic amino acids, monoterpens, alkaloids (Selmar and Kleinwächter, 2013). Increased abundance under drought of the enzyme (+)-neomenthol dehydrogenase, which participates in monoterpenoid biosynthesis, observed in this study, could be linked to possible protective function of monoterpens. Caffeoyl-CoA 3-O-methyltransferase is engaged in the pathway of lignin biosynthesis. The accumulation of this enzyme under drought could be related to increased lignification of the cell wall-a modification in order to avoid water loss. Similar up regulation of Caffeoyl-CoA 3-O-methyltransferase in roots subjected to water stress is reported by several authors (Alam et al., 2010;Fulda et al., 2011). In concert with the changes in root growth (longer and thinner roots with less biomass) we observed an increase in abundance of actin 2 (component of microphilaments) as well as early decrease and late increase in peroxidase abundance-a cell wall cross-linking enzyme participating in cell wall lignification, defense against pathogen attack, and activated oxygen consumer. Increased content of peroxidase III in roots of wild watermelon under drought has been reported (Yoshimura et al., 2008). Carbon Metabolism and Energy Production-Glycolysis is Down-regulated in Roots in Water Deficit Conditions While ATP Synthesis is Stimulated In this study we observed concerted decrease in abundance of glycolytic enzymes-glucose-6-phosphate isomerase (EC 5.3.1.9), pyrophosphate-dependent phosphofructokinase (EC 2.7.1.90), 2,3-bisphospho glycerate-independent phosphoglycerate mutase (EC 5.4.2.12), as well as of the enzyme pyruvate dehydrogenase (EC 1.2.4.1)-mitochondrial enzyme which links the glycolysis metabolic pathway to the citric acid cycle. Decrease in the amount of the cytoplasmic aconitate hydratase (EC 4.2.1.3), an enzyme of the glyoxylate bypass in plants for utilization of fatty acids as a carbon source, was detected. As for enzymes of the tricarboxylic acid cycle, an increase in abundance of isocitrate dehydrogenase which catalyzes the rate-limiting step of the cycle, was detected (EC 1.1.1.42). Two protein spots related to ATP production were found to be increased in abundance under drought-ATP synthase (EC 3.6.3.14) subunit beta and NADH-ubiquinone oxidoreductase (EC 1.6.5.3). The general down-regulation of carbohydrate degrading glycolytic enzymes could be linked to reduced root biomass accumulation, and could be regarded as a mechanism to accumulate and store sugars for rapid growth in recovery. Similar decrease of glycolysis-related enzymes in roots under drought stress is reported for soybean (Alam et al., 2010). In the roots of other species-the xerophyte wild watermelon, an up-regulation of glycolysis and tricarboxylic acid cycle was found (Yoshimura et al., 2008). Tricarboxylic acid cycle is embedded into a larger metabolic network, constantly sharing substrates and products with other pathways (Sweetlove et al., 2010). Besides carbohydrates, the TCA cycle may be fuelled by products derived from protein and other macromolecules degradation, in order to produce sufficient ATP to meet the energetic needs under stress. The up-regulation of ATP-synthesis related enzymes could be explained by the need of energy for stress protection and maintaining tissue functional state under water limiting conditions. ATP energy is necessary for many cellular processes, including secondary metabolism and protein quality control. Dynamic Changes Are Observed in Enzymes Related to Amino Acid and One Carbon Metabolism We have found increased abundance under drought of some enzymes related to one-carbon and amino acid metabolism-S-adenosyl methionine synthase (EC 2.5.1.6) and formate dehydrogenase (EC 1.2.1.2); glutamine synthetase (EC 6.3.1.2) and cysteine synthase (E.C.2.5.1.47). Decrease in content was detected after prolonged drought for methylene tetrahydrofolate reductase (EC 1.5.1.20), a cytoplasmic enzyme member of one-carbon metabolism. The most interesting qualitative differences were in one cysteine synthase isoform which spot was below detection under drought; however, there was another spot identified also as cysteine synthase with increased abundance under drought. The amino acid cysteine is incorporated into proteins and glutathione (GSH); moreover, it is considered to be the bottleneck for GSH production. The cysteine synthase complex is considered to be the rate-limiting step of cysteine biosynthesis (Chan et al., 2013). Besides, cysteine acts as sulfur donor for methionine (Met) for S-adenosylmethionine and S-methylmethionine synthesis (Ravanel et al., 1998). Glutamine synthetase catalyzes the condensation of glutamate and ammonia to form glutamine, thus playing an essential role in nitrogen metabolism and ammonia assimilation. Ammonia is produced by nitrate reduction or amino acid degradation; on the other hand the amide group of glutamate serves as a readily mobilized nitrogen source for incorporation of amino group in various metabolites. Glutamine synthetase isoforms are reported to be highly responsive to drought (Yoshimura et al., 2008;Alam et al., 2010;Singh and Ghosh, 2013). S-adenosyl methionine synthase catalyzes the conversion, at the expense of ATP, of L-methionine into S-adenosylmethionine-AdoMet or SAM, the major methyl donor for proteins, nucleic acids, carbohydrates, lipids, and small molecules for lignin and many other biosynthesis, precursor for polyamine ant ethylene biosynthesis. Different trends of change in the enzymes related to one-carbon metabolism were reported in drought-stressed roots from different plant species and stress treatment (Yoshimura et al., 2008;Mohammadi et al., 2012;Grebosz et al., 2014). In our study, the accumulation of S-adenosyl methionine synthase and related enzymes in holm oak roots under drought could be linked to enhanced secondary metabolism and lignification which utilize activated one carbon particles. Protein Synthesis, Folding/processing and Degradation Processes Are Highly Responsive to the Applied Stress Changes in the protein complement of cells are indispensable for adaptation to stress. Within the large group of proteins related to protein synthesis, folding/processing and degradation, some spots with opposite trends of changes under stress were found, which may reflect the dynamic changes in cell protein profile. A DEAD box RNA helicase was detected among the identified proteins with earlier and reversible increase in abundance under drought. RNA helicases are involved in various aspects of RNA metabolism, including nuclear transcription, pre mRNA splicing, ribosome biogenesis, nucleocytoplasmic transport, translation, RNA decay, and organellar gene expression (Aubourg et al., 1999). Similar increase of DEAD box RNA helicase was reported in osmotically stressed triticosecale roots (Grebosz et al., 2014). On the other hand, glycyl-tRNA synthetase abundancy was found to decrease under drought and in recovery which is not in favor of up-regulation of translation under drought stress and may rather reflect changes in the composition of newly synthesized proteins. Chaperones and proteases are elements of the protein quality control machinery. Maintaining proteins in their functional conformation, preventing aggregation of nonnative proteins, refolding of denatured proteins and removal of non-functional and potentially harmful polypeptides are very important for cell survival under drought stress (Vaseva et al., 2012). Some molecular chaperones, which assist protein folding or renaturation, presented complex changes: chaperonin 60 kD, protein disulfide isomerase and chaperone of TCP-1 family decreased in abundance, while GroES chaperonin increased following water stress. TCP-1 family chaperones are related to cpn60/groEL chaperonin family and assist the folding of cytoskeletal proteins in the cytoplasm (Wang et al., 2004). Cpn60 is homologous to E. Coli GroEL found in chloroplasts and mitochondria. It acts in cooperation with GroES chaperonin in assisting proper folding of newly synthesized and membranetranslocated proteins into their native conformation. The observed different abundance changes in GroEL/GroES may reflect changes in composition within the chaperonin family. Under stress the folding capacity of cpn60 is suppressed while its binding affinity toward unfolded proteins is increased, thus protecting proteins from unfavorable conditions by sequestration (Vaseva et al., 2012). Protein disulfide isomerase is an enzyme located in the endoplasmic reticulum with the main function-correct arrangement of disulfide bonds in proteins. Decrease in abundance in this enzyme could reflect inhibition of protein synthesis at the endoplasmic reticulum. The proteasome plays a crucial role in the turnover of regulatory proteins, cellular house-keeping and stress tolerance (Kurepa and Smalle, 2008). Relatively early increase in abundance under stress was found for an ubiquitin activating enzyme E1 which may reflect the importance of stress signaling. The 6B regulatory subunit of 26S proteasome, involved in ATPdependent protein degradation in the cytosol and nucleus, decreased under drought, while the catalytic proteasome subunit beta type increased. Some evidence suggests that the control of proteasome function may be at the level of subunit composition rather than total increase in proteasome abundance (Kurepa and Smalle, 2008). The proteasome could function in two forms-26S and 20S, the latter containing only the catalytic core subunits and operating without ATP in degradation of oxidized proteins. Thus, 20S may play an important role in tolerance to the secondary oxidative stress developing under drought stress (Vaseva et al., 2012). Up-regulation of the 20S proteasome subunit was found in drought-treated poplar trees (Plomion et al., 2006), alfalfa plants (Aranjuelo et al., 2011), and rapeseed roots (Mohammadi et al., 2012). Opposite changes were observed for some aminopeptidases in roots under applied stress. Leucine aminopeptidase (EC 3.4.11.1) abundancy diminished under drought and in recovery, while Xaa-pro aminopeptidase (EC 3.4.11.9) increased. Aminopeptidases liberate free aminoacids from the N-terminus of the polypeptide chains. Amino acid metabolism was found to be among the top biological processes affected by drought (Kang et al., 2011). In conclusion, differently abundant identified protein species in holm oak roots subjected to water limitation treatment point at early activation of secondary metabolism, down-regulation of glycolysis and stimulation of ATP synthesis, accumulation of some enzymes related to aminoacid and one-carbon metabolism, and complex changes in protein synthesis, folding/processing and degradation processes, which emphasize the active metabolic adjustment and mobilization of the defense system in roots to actively counteract stress.

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THE EFFECT OF INJECTIVE APPLICATION OF SELENOPYRAN ON THE PROLONGED INCREASE OF THE SELENIUM CONTENT IN BLOOD AND SPERM OF RAMS 1 : Selenium is a microelement of big importance for male reproduction. As a part of the antioxidative enzyme gluthationperoxidase and structural Se proteins, it plays pivotal role in the defense of spermatozoa against generated ROS and in ensuring of its motility. During the last years the interests to the organic forms of selenium was enhanced because of its better biological utilization. The present work aimed to study the effect of injective application of organic compound selenopyran in rams on the distribution of selenium content in blood and sperm and on the changes in sperm quality. The experiment was conducted with 5 rams from the Synthetic Population Bulgarian Milk breed at the age between 3-7 years and live weight 85-90 kg. The animals were injected once with an oil solution of the selenopyran in dose of 0.1mg/kg live weight (selenium content 24%) 45 days before starting the breeding season. The blood was collected before treatment and 45 days thereafter. At the same time the first and second ejaculates of rams were collected using artificial vagina and analyzed by Sperm Class Analyzer. The selenium content was measured in plasma, blood cells and sperm by atomic absorption spectrometry using SpectrAA 55B double beam spectrometer (Varian, Inc.). The results showed that one injection of organic compound of selenium – selenopyran in dose of 0.1mg/kg live weight ensured the increase and support of high level of selenium in blood (plasma and blood cells) and in sperm during investigated period. That lead to the proper spermatogenesis in testis and allowed the production of qualitative ejaculates with higher number of total spermatozoa as well as of spermatozoa with progressive motility. Introduction During the last decades there are new evidences of selenium importance for the male reproduction, especially for the properly spermatogenesis. More than 25 selenoproteins were identified in the live organisms. Most part of them occurs in the male reproductive system at tissue (testis, epididymal epithelium), cellular (intracellular membranes) and subcellular level (sperm nucleus, mitochondrial capsule) (Ahsan et al., 2014). Se concentrations in rodent testes exceed that of other organs, except kidneys (Schriever et al., 2009). According to Kehr et al. (2009) the distribution of Se in midpiece and head of spermatozoa is 4:1. Spermatozoa may be more vulnerable to oxidative stress if the Se content in selenoproteins is low and likely decreases the possibility of fertilization (Beckett and Arthur, 2005). Also many investigations had underlined that selenium is essential for normal spermatozoa development and function in livestock animals (Shi et al., 2010;Kendall et al., 2000;Tareq et al., 2010). Se is actively incorporated into the developing spermatozoa of various mammalian species, including rats (Burk et al., 1972), bulls (Bartle et al., 1980) and rams (Pond et al., 1983). The polygamy of the rams and short breeding season requires good condition for semen production. For the achievement of the optimal reproductive performances during the breeding season, where rams are used intensively, their diets require an additional feed additives, including selenium at first. However, there is a lack of information for a definition of an optimal Se status in blood and sperm of rams with regard to fertility. Despite the many studies on the effects of selenium during spermatogenesis, more detailed investigations are required in order to provide a more clear understanding about relationship between selenium content in blood and in sperm. The aims of the present study were to examine: a) effect of injective application of organic compound selenopyran on the Se content in blood and sperm of rams; b) the correlation between the Se content in blood and sperm and sperm quality during the preparation of rams to breeding season. The effect of injective application of … 483 Material and methods The experiment was carried out with 5 rams from Synthetic Population Bulgarian Milk sheep breed, housed at Animal facility of the Institute of Animal Sciences -Kostinbrod. This breed is newly created (officially acknowledged during 2005) and it is the most spread breed in Bulgaria now. Rams were at the agebetween 3 and 7 years old with live weight of 80 to 95 kg. The animals were raised in pens and fed with meadow hay ad libitum and concentrated mix forage 500 g/head (250 g wheat and 250 g Dried Distillers Grains with Solubles). Salt and mineral licks were placed in pens (EuroLick MultiVit ® ), as the concentration of Se in licks was 10 mg per kg. 45 days before breeding season the experimental rams were injected once subcutaneously with oil solution of selenopyran (9-phenylymmetrical octahydroselenoxanthene) in dose of 0.1 mg /kg live weight. The Se content in this organic source was 24%. As mentioned in previous investigations (Abadjieva et al., 2014), the advantages of selenopyran are the lower toxicity in comparison with sodium selenite (LD 50 =1600 mg/kg against LD 50 =3.25 mg/kg) and ability to slowly liberate the selenium according to the needs of the organisms (Boryaev and Kravchenko, 2006). Blood collection The blood samples were collected before treatment and 45 days thereafter. The blood was collected from v. jagualris in vacutainers covered with EDTA. Plasma and blood cells were separated by centrifuge at 3000 rpm for 15 min and stored at -20°C until analysis. Semen collection and analysis From each ram the first and second ejaculates were collected by using artificial vagina in triplication (n=57 in total) before treatment and 45 days thereafter. At the time of sampling, the ejaculates were diluted (1:3, vol/vol) with 6A ram semen extender and transported to the IBIR-BAS laboratory within 1 hour. The estimation of semen quality parameters was done by Sperm Class Analyzer (SCA, Microptic, Spain) after appropriate additional dilution of samples. The total concentration, average percentage and number of motile sperm were measured by SCA software. Se measurement The content of selenium was analyzed in sperm, in blood plasma and blood cells by the atomic absorption spectroscopy method in the Central Laboratory for Chemical Testing and Control -Bulgarian Food Safety Agency. All samples were digested using microwave pressure digestion system MARSXpress (CEM) with IR temperature sensor control and XP-1500 Plus fluoropolymer closed vessels. 0.5g sample with 5ml concentrate HNO 3 was placed in vessels and heated to 185°C for 15min. The used reagents were of analytical reagent grade (Merck CGaA). For analysis stock solution of Se containing 1000 mg/ L (LGC Standards) was used for daily prepared analytical calibration standard with concentration 10µg/L by serial dilutions with 0.5% (v/v) HNO 3 . SpectrAA 55B double beam atomic absorption spectrometer (Varian, Inc.) was used for all determinations. Hallow cathode lamp from Varian operated at 10mA with spectral bandwidth of 1.0nm. The selected wavelength was 196.0 nm. Argon (99.996% purity) was used for carrier gas. The statistical processing of the data was done by the STATISTIC computer programme (Stat Soft Inc., Ver.10.0). The one-way and regression analysis were done. The mean differences considered statistically significant at P<0.05. Results and discussion The distribution of Se in ram blood plasma, blood cells and sperm are presented in the Table 1. The blood Se concentration measured before treatment showed that the animals should be considered as a Se deficiently because levels below 70 μg/L are subnormal (Pavlata et al., 2000). The injective application of selenopyran leads to significant increase of the Se level in both plasma and blood cells. Most literature data presents either serum or whole blood Se concentration analyses. Serum Se concentration reflects more acute or recent changes in Se nutrition or injective input of Se, whereas whole blood Se reflects more chronic or "historical" Se status (Stowe and Herdt, 1992). In our study we investigated blood plasma and blood cells separately and established that Se content in plasma was about 3 folders higher than in blood cells in both before treatment and 45 days thereafter. These results should be explained trough the injective application of selenopyran and its immediate introduction to blood plasma. The majority of the glutathione peroxidase contented Se is incorporated into the red blood cells at the The effect of injective application of … 485 time of erythropoiesis and the response to a Se treatment in blood cells requires a time. That corresponds with our results: the high level of Se appears in blood cells only 45 days after treatment. Compared to the reference range (Stowe and Herdt, 1992) of selenium in blood serum 120-150 μg/L for ewes, the average values in our rams, injected with 0.1mg/kg live weight selenopyran, were very high. Despite such high level of Se in blood we didn't observe any toxic effects. We suppose that the most part of Se in blood plasma is presented in the form of selenopyran and slowly released Se. Moreover, the quality of sperm after treatment with selenopyran was improved ( Table 2). The level of Se in sperm significantly increased and remained high till 45-th day after treatment. Also the close correlation between Se level in blood plasma and sperm was established after treatment with selenopyran ( Fig.1, r=0.84; p=0.049). These results confirmed low toxicity of preparation of organic compound selenopyran by its unique chemical structure where selenium atom is binding to the heterocyclic ring (Boryaev and Kravchenko, 2006). The similar results about low toxicity of organic selenium were reported in cows: the treatment with selenium yeast lead to high concentration of Se in blood (more than 1000 μg/L) without negative effects (Juniper et al, 2008). Whole blood Se is an indicator of circulating Se and it reflects Se status. After treatment we found the largest amount of Se in blood plasma, followed by blood cells and whole semen. Cheah and Yang (2011) underline that sperm count and concentration of selenium in semen are in direct ratio. The results of our experiment confirm this statement (Table 2): the concentration of spermatozoa increased with the increase of the Se content in semen. Also the number of spermatozoa with progressive motility was enhanced after selenopyran treatment. Figure 1. Correlation between selenium content in blood and sperm The mechanism of Se metabolism regulation is strongly dependet on species. In men, Iwanier and Zachara (1995) found similar to ours relations in Se content in blood and sperm. In bulls, the Se content was 10 times higher in seminal plasma than in blood serum (Saaranen et al., 1989). Our results demonstrate the important role of Se for ram reproduction showing significant increase of Se content in sperm before breeding season, when spermatogenesis initiates. The high Se concentration during spermatogenesis is related to its protective property and its associated enzymes, such as mitochondrial capsule protein in spermatozoa (Alabi et al., 2000;Kehr et al., 2009). The increase of Se content in sperm in our experiment probably ensures the sufficient amount of Se to be taken into the spermatozoa. Conclusion The obtained results showed that one injection of organic compound of selenium -selenopyran in dose of 0.1mg/kg live weight supports a high level of selenium in blood (plasma and blood cells) during the 45 days (spermatogenesis period in rams) and provides the sufficient amount of Se in sperm that ensures the quality ejaculates with higher number of total spermatozoa, as well as of spermatozoa with progressive motility. Acknowledgment The research and participation in the 4 International Congress "New perspectives and challenges of sustainablellivestock production" (Belgrade, Serbia, 7-9 October, 2015) is supported by grants №BG051PO001-3.3.06-0059 and № DKOF7RP02/17 under Ministry of Education and Science of Bulgaria and the European Social Fund and Operational Programme Human Resources Development.

v3-fos

2019-03-20T13:03:58.459Z

2015-07-03T00:00:00.000Z

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Assessment of miRNA expression profile and differential expression pattern of target genes in cold-tolerant and cold-sensitive tomato cultivars MircroRNAs (miRNAs) are small non-coding RNAs about 21 nt in length. These short transcripts regulate developmental and stress responses in plants. Cold stress is one of the most restraining abiotic factors adversely affecting the plant yield. In the present study, some cold stress-related miRNAs (miR167, miR169, miR172, miR393 and miR397) in tomato (Solanum lycopersicum) were assessed at early time points (0, 1, 4 and 16 h) of cold exposure. Relative expression of miRNAs was measured by stem–loop quantitative reverse transcription polymerase chain reaction. The results showed that miR167, miR169, miR172 and miR393 were activated in the early time points of cold treatment. Especially, miR172 was found to have highest expression level. Furthermore, target genes of selected miRNAs were identified and their expression profiles were assessed between cold-sensitive and cold-tolerant cultivars of tomato. It was found that inferred expression patterns of target genes were differentiated between the cultivars. Analysis of cis-acting elements showed that miRNAs had stress-responsive elements. Meanwhile, since no miR393 sequence is available, putative miR393 sequence and its secondary structure were predicted in tomato. These results may provide a framework for further analysis in terms of understanding the response of miRNAs against cold stress in tomato. Introduction Plant microRNAs (miRNAs), which are non-coding short transcripts, play a key role in development and stress response in plants. To date, hundreds of miRNAs have been found and characterized. miRNAs are often located in gene families and conserved between closely related species. [1À3] They repress gene expression by base-pairing with nearly perfect complementarity against coding sequences, leading to cleavage or translational repression of mRNAs.[4À6] Thus, miRNAs regulate the gene expression post-transcriptionally. [7] miRNAs have been shown to be involved in the response to various stress conditions. [8À10] In Arabidopsis, miR398 was identified to target two closely related Cu/Zn superoxide dismustase genes, and down-regulation of miR398 led to improved tolerance of transgenic lines under oxidative stress. [11] miR395 and miR399 were found to be involved in sulphate and phosphate starvation. [12,13] In another study, miR393 was strongly induced by cold stress. [8] In addition, miRNAs regulate many developmental processes, including leaf development, auxin signalling, phase transition, flowering and genome maintenance. [14À21] The knowledge about miRNAs mostly comes from Arabidopsis and accumulation of new data is still an ongoing process. To date, miRNAs have been identified and detected using high-throughput sequencing, cloning and bioinformatic approaches. [22À24] Conventional technologies such as northern hybridization and microarray analysis have been widely used for investigation of miRNAs but it was reported that it may not be sensitive enough to detect less abundant miRNAs. [25] Among the many techniques, stemÀloop reverse transcription polymerase chain reaction (stemÀloop RTÀPCR) provides more specificity and sensitivity since it uses linear primers due to basestacking and spatial constraint of the stemÀloop structure. [25,26] Furthermore, detection sensitivity is increased by a pulsed RT reaction. [27] Cold stresses, such as chilling and freezing, affect the agricultural productivity around the world. [28] Numerous physiological and molecular changes occur during cold acclimation. [29] Among them, repression of genes, mRNA export and mRNA degradation have been found to be of central importance for the cold-stress response. [29,30] However, knowledge on the role of miRNA in cold stress is still limited compared to other abiotic stresses. Therefore, elucidating the cold-stress-regulated miRNAs and determination of their expression profile could improve our understanding of miRNAs and their functions in biological systems. Recently, expression profiles of all known miRNAs under cold stress in Arabidopsis, poplar and rice were validated with microarray analyses. [19,24,28] Zhou et al. [7] established a machinelearning transcriptome-based approach for annotating cold-inducible miRNAs in Arabidopsis. They found that miR165/166, miR169, miR172, miR393, miR396 and miR397 were over expressed in cold stress. They also formed a sketch pathway, implying possible regulatory role of miR169. Tang et al. [31] found up-regulation of miR167 and role of trans-acting short-interfering RNA-Àauxin response factor (tasiRNAÀARF) in regulating the auxin-signalling pathway and possibly in the developmental response to cold stress in wheat. In rice, Jian et al. [32] found several cold-regulated miRNAs by direct cloning and sequencing. Zhang et al. [33] identified 28 cold responsive miRNAs in Brachypodium. Plants from temperate regions are tolerant to chilling but not to freezing; however, they can increase their freezing tolerance by being exposed to chilling temperatures, by a process known as cold acclimation. [34,35] On the contrary, tropical and subtropical plants, such as rice, maize and tomato are sensitive to cold stress and largely lack the capacity for cold acclimation. [35] Tomato suffers from chilling injuries at all stages of growth and development. [36] Prolonging cold temperature can further reduce the plant production and growth. It was predicted that there are many cold-induced miRNAs in plant species but knowledge about them is still limited. In this study, we investigated cold-induced miRNAs (miR167, miR169, miR172, miR397 and miR393) [7,8,31] by a sensitive method, stemÀloop qRTÀPCR, in cold-treated tomato plants. In order to understand the expression profiles of miRNA target genes, we also retrieved the microarray data of three tomato cultivars from a public database. Plant growth and cold treatment Tomato (Solanum lycopersicum var. H-2274) plants were grown in soil under greenhouse conditions with a 16:8 h (light:dark) photoperiod at 25 C § 2 C. After 4 weeks of growth, plants were subjected to cold treatment (4 C for 1, 4 and 16 h) with a light intensity of 20 mmol/(m 2 s). [37] Then plant leaves were quickly harvested and stored at À80 C. Control plants were not subjected to cold treatment. Preparation of RNA and cDNA Total RNA of the leaf samples were extracted using Trizol Ò (Invitrogen) reagent, according to the manufacturer's instructions. Extracted RNA was dissolved in Rnase-free water and stored at ¡80 C. RNA integrity was observed in a 2% agarose gel; three bands corresponding to ribosomal RNA (28S, 18S and 5S) were apparent. RNA concentration was calculated by using Cubit flour metric system (Invitrogen). An amount of 500 ng of total RNA was treated with 5 U of Rnase-free recombinant DNase I Ò (Roche) and incubated at 37 C for 15 min. Reaction was stopped by adding 2 mL of 0.2 mol/L ethylenediaminetetraacetic acid (pH 8.0) to a final concentration of 8 mmol/L and heating to 75 C for 10 min. StemÀloop RT primers were designed according to Varkonyi-Gasic et al. [25]. RT reaction contained 30 ng RNA, 1 mL stemÀloop RT primer (1 mmol/L) and 0.25 mmol/L deoxyribonucleoside triphosphates. The mixture was incubated at 65 C for 5 min and then 5£ reverse transcripatase buffer, 1 mL 20 units RiboLock RNase inhibitor, 1 mL RevertAid MÀMuLV RT (Thermo Scientific TM RevertAid TM First Strand Synthesis Kit) and Rnase-free water were added into the mixture up to 20 mL per sample. This mixture was incubated for 30 min at 16 C, followed by 60 cycles at 30 C for 30 s, 42 C for 30 s and 50 C for 1 s for pulsed RT. Finally, reverse transcriptase was inactivated by incubation at 70 C for 15 min. StemÀloop reverse transcriptionÀPCR Prior to quantification analysis, miRNAs of interest were amplified by conventional PCR (Techne Ò TC512 Gradient Termal Cycler) and checked in a 2% agarose gel (Figure 1(a)). For quantification analysis, primer-specific regions of targeted miRNAs were amplified using Luminaris TM Color HiGreen qPCR Master Mix kit (Thermo Scientific) and real-time PCR (qPCR) was performed in a RotorGene PCR machine (Qiagen). A miRNA-specific primer and a universal reverse primer were used for amplification of individual miRNAs (Table 1). Then, 0.5 mmol/L of each forward and reverse primer with 2 mL cDNA was mixed with 10 mL of qPCR Master Mix. The following program was set up: 95 C for 10 min, 40 cycles of 95 C for 15 s, 58 C for 30 s, 72 C for 30 s and melting analysis at a temperature gradient from 57 C to 95 C with an increment of 0.5 C/min. Relative quantity of individual transcripts was calculated by using a mathematical model, including an efficiency correction and crossing point (Cp) deviation of an unknown sample versus a control. [38] Tomato actin gene was used as internal control for qPCR analysis. The fold change (FC) in the expression of each miRNAs was computed according to Pfaffl [38]. Expression analysis of target gene under cold stress Mature miRNA sequences were obtained from miRBase (http://www.mirbase.org). [39] Target predictions were performed using the psRNATarget (http://plantgrn.noble. org/psRNATarget/) [40] with default settings. We also extracted the expression profile of five miRNAs' target genes from microarray data for three cold-treated tomato cultivars (LA1777, LA3969 and LA4024) in the Tomato Functional Genomics Database (http://ted.bti.cornell.edu/). The data was normalized using Print-tip lowess normalisation method. [36] We also compiled some target genes, which were reported from previous studies. However, expressions of some target genes were not available in databases. Thus, we excluded these targets. Probe sequences were further used as query sequences for the BLASTN search against SGN tomato whole-genome chromosome database (http://solgenomics.net/tools/blast/ index.pl) and NCBI (http://blast.ncbi.nlm.nih.gov/Blast. cgi#). Gene annotation analysis was performed using the Tomato Functional Genomics Database. The microarray data was deposited in the Tomato Functional Genomics Database with accession number E060, [36] for three tomato genotypes. The cold tolerance level of these genotypes was reported by Liu et al. [36]. In this manner, we were able to comparatively investigate how the expression profile of target genes differs among cold-tolerant and cold-sensitive tomato cultivars. Statistical analysis was carried out using the data obtained from three separate sets of biological samples. One-way analysis of variance with the post hoc test was performed using SPSS 18.0 for Windows. P < 0.05 was considered to be statistically significant. Isolation of sly-miR393 and cis-elements of miRNAs The pre-miRNA sequences for each of miRNA were obtained from miRBase and mapped to the sequences of tomato genome (http://www.phytozome.net/) using the BLAST search. Blast parameters were adjusted as follows: an expected value cutoff of 1; a low-complexity sequence filter; 1000 descriptions and alignments, and automatically adjusted parameters for short input sequences to improve the accuracy of outputs. For each miRNA, 1 kb of upstream putative promoter sequence was retrieved. [ Results and discussion Expression patterns of tomato miRNAs The obtained results showed that miR167 was induced by approximately four FC comparing to the control group at 4 h of cold treatment (Figure 1(b)). After 16 h of cold treatment, its transcript accumulation was decreased to control level. Transcript accumulation of miR169 was decreased at 1 h and then increased at 4 h of cold treatment. Expression of miR169 was not altered in comparison to control after 16 h of cold exposure. miR172 was induced to a highest extent among the tested miRNAs. Especially, its expression was significantly increased over six times at 4 h. Similar to miR167, its expression pattern showed a decrease in expression at 16 h of cold treatment as that in the control. The transcript abundance of miR393 was nearly 1.6 FC increased at 4 h. The expression level of miR393 was lower than that in the control at 1 h, whereas cold treatment did not significantly change the expression of miR397. When we compared the expression profile of miRNAs with that in the control group, it was miR172 that stood out as the most induced one (Figure 1(b)). Following miR172, the levels of expression of miR167 and miR169 were higher than that of other miRNAs. Interestingly, it was indicated that 4 h of cold treatment induced expression of miR172, miR167, miR169 and miR393. After 4 h, the expression of those miRNAs decreased but nevertheless they showed a nearly constant expression profile compared to the control. We suggest that 4 h of cold treatment was a suitable duration for up-regulation of miR167, miR169, miR172 and miR393 in tomato plants. Isolation of sly-miR393 and cis-element analysis of miRNAs We should note that miR393 expression in tomato was previously reported [42,43] but no sequence was deposited in any database. Using Solanum tuberosum (potato) and Arabidopsis mature miR393 sequences, which are well conserved, we identified putative mature sly-miR393 sequence location (100% identity with mature miRNA sequences of potato and Arabidopsis). Additionally, we tried to elucidate the sly-miR393 sequence by blasting stemÀloop miRNA sequences of potato and Arabidopsis against the tomato genome (http://www.phytozome.net/), and only potato miRNA sequences showed 93% identity (100% coverage; data not shown) with tomato sequence in the same location as those of mature sequences of both plants. The matched sequences were obtained and further screened for the potential hairpin structure by an RNAfold prediction program. The output of the program, which gave rise to the characteristic stemÀloop structure, is shown in Figure 2(a). The MFE of putative sly-miR393 was À39.30 kcal/mol and the MFEI was 1.22. This finding indicated that the miRNA precursor sequence had a significantly higher MFEI value than other non-coding or coding RNAs, e.g. tRNA, rRNA. [44] Furthermore, putative mature sly-miR393 sequences are in the stem portion of hairpin structures (Figure 2(a)). The gene expression level largely depends on cis-elements existing in the promoter region. [45] Cis-elements analysis could also be an effective approach for the miR-NAs as for protein-coding genes. [46,47] In the analysis, unknown cis-elements were not included. Cis-elements were heterogeneously distributed among miRNAs. The investigation revealed that abscisic acid (ABA)-response elements (ABRE, miR172 and miR393), ethylene-responsive element (ERE, miR172) and MYB-binding site involved in drought inducibility (miR167 and miR172), and defence-and stress-responsive elements (TC-rich repeats, miR167, miR172 and miR393), heat-stressresponsive elements (HSE, miR169, miR172 and miR397), light-responsive elements (G-Box, miR172, miR393 and miR397), and auxin-responsive elements AuxRR-core (miR167 and miR169) were present in miR-NAs (Supplementary Table S1). When comparing miRNA cis-elements, it was found that these cis-acting regulatory elements were mainly related to two physiological processes: light cycle and hormonal/environmental responses (Figure 2(b) and Supplementary Table S1). miR172 had 11 cis-acting elements related to hormonal/ environmental responses, while there were two cis-acting elements in miR169. On the other hand, miR393 had six cis-acting elements related to light cycle, whereas miR169 had two cis-acting elements. In the analysis, it the least number of cis-acting elements were found in miR397, which coincided with the low level of expression. In addition, some other cis-acting elements were also found, such as those possibly involved in regulation in response to methyl jasmonate, gibberellic acid, ethylene and salicylic acid. The transcription levels of miR172 and miR167 were higher than those of other miRNAs. Presumably, this could be related to higher number of hormonal/environmental responsive cis-acting elements contained in them. Predicted target genes We also identified potential target genes. Currently, bioinformatics tools facilitate revealing of target sequences based on the high degree of homology between miRNAs and target genes. [48] Most target genes are highly conserved across plants. The targets found in the analysis are shown in Table 2. Moreover, we investigated the target genes from which they had been previously identified in the literature. AFR6 targeted by miR167 [49] and TIR targeted by miR393 [31,50] were included in target gene expression analysis. The inferred expression of homeoboxÀleucine zipper protein 22 (HDÀZIP III; SGN-U216828) and annexin (SGN-U214256) targeted by miR167 were more induced in cold-sensitive (LA4024) than in coldtolerant tomato cultivars (LA1777 and LA3969) under cold treatment (Table 2). Similarly, HDÀZIP III expression was increased in cold-stored potato tubers. [35] SGN-U214038 and SGN-U214039 encoding multi-drug resistance protein mtdK showed higher FC in cold-tolerant cultivar LA1777 (4.9 and 5.5 FC, respectively). However, their expression in cv. LA3969 (1.6 FC) displayed lower pattern than cv. LA1777 but higher than cv. LA4024. SGN-U215435 (receptor-like protein kinase; RLK) was highly up-regulated in all cultivars. Similarly, in Arabidopsis, RLK1 was induced by several environmental stresses, such as dehydration, high salt and cold treatment. [51] Expression of AFR8 (SGN-U240026 and SGN-U227556) targeted by miR167 was repressed in cv. LA1777 and cv. LA4024. On the other hand, they were not significantly changed in cv. LA3969. Interestingly, the LA3969 cultivar showed higher expression profile of ARF6 (SGN-U230670) than others (1.7 FC). Following this observation, ARF6 transcript accumulation was decreased in LA1777, but it was not changed in LA4024. Although SGN-U214629 (ATP synthase IÀlike protein) was slightly up-regulated in cold-tolerant cultivars, it displayed constant expression level in the cold-sensitive cultivar. These findings suggest that gene expression profiles of cultivars may become differentiated in response to cold stress depending on the level of tolerance. Moreover, we retrieved two nuclear transcription factors Y subunit A-3 (NF-Y) targeted by miR169, which was repressed in all genotypes. Similarly, miR169 was induced in response to drought stress and led to repression of the expression of three nuclear factor Y subunit genes. [52] Contrary, multi-drug resistance protein ABC transporter was highly induced in all genotypes, especially in the most cold-tolerant tomato cultivar (LA1777; 5.8 FC). Multi-drug resistance-associated proteins (MRPs or ABCCs) are involved in cellular detoxification by transporting toxic compounds from cytosol into the vacuole. [53] Considering that, cold resistance of tomato genotypes may likely have similar mechanisms in the way MRPs are regulated. AP2-like transcription factor clade targeted by miR172 has many members. In the analysis, we found six AP2-like transcription factors in tomato as targets of miR172. Expression of AP2-like transcription factors differed based on the cultivars. AP2-like transcription factors such as SGN-U242104 and SGN-U218022 in cv. LA1777, SGN-U224195 and SGN-U226730 in cv. LA1777 and cv. LA4024 were down-regulated. However, SGN-U232455 in all cultivars, SGN-U218022 in cv. LA3969 and in cv. LA4024, and SGN-U214382 in cv. LA3969 showed over-expression. On the other hand, serine/threonine-protein kinase receptor (SGN-U219598) expression was highly induced in the LA3969 and LA4024 cultivars, but was not changed in cv. LA1777. TIR and TIR-like are involved in the auxin-signalling pathway, and are targeted by miR393. [32,52] SGN-U236908 (TIR) was repressed in all three tomato genotypes. The degradation of TIR mRNAs may subsequently prevent depression of auxin-signalling pathway. [7] Auxin F-box proteins 5 are another target of miR393. Auxin signalling primarily depends on F-box protein receptors and Arabidopsis has four redundant F-box proteins of the TIR1/ AFB2 auxin receptor clade. [54] Therefore, F-box proteins are closely related with TIR and TIR-like proteins. In addition to down-regulation of TIR, F-box protein (SGN-U213940 and SGN-U223813) accumulations were also decreased in all genotypes. As regards to SGN-U221001 (B3 domain-containing protein) and SGN-U231414 (U-box domain-containing protein) targeted by miR393, they showed slight changes in the expression levels. SGN-U216617 (laccase) targeted by miR397 exhibited low level of expression in cv. LA1777 and cv. LA4024, while SGN-U216616 (laccase 7) was overexpressed in cv. LA1777. On the other hand, SGN-U223145 (laccase 5) was repressed in all cultivars, while SGN-U226352 (laccase 3) was not significantly changed in cold-tolerant cultivars. Translocase of chloroplast 34 (Toc34) (SGN-U214046 and SGN-U214047) expression levels were significantly changed in cold-tolerant cv. LA1777. Toc34 is a member of the translocase of the outer membrane complex, which mediates the initial stage of protein import into chloroplasts. [55] Although studies on Toc34 are very limited, cold conditions did not seem to affect the function of Toc34. Interestingly, SGN-U224659 (1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase; PIP2) was repressed in all cultivars. Contrary to our finding, heat stress activated PIP2 accumulation in tobacco. [56] Comparative analysis Cold stress is one of the most severe abiotic stresses adversely affecting plants yields by restraining sowing time, causing tissue damage, and stunting growth. [28] Tomato is an important vegetable crop grown worldwide and prone to cold stress. Stress-related functions of miR-NAs may provide further insight in response to cold stress. However, little is known about the relation between the miRNAs and cold response. [19] In this study, miR167a, miR169a, miR172a and miR393 were up-regulated in response to initial phase of cold stress in tomato. Liu et al. [19] found that miR167a, miR393a and miR397a were up-regulated in cold-treated Arabidopsis based on the RTÀPCR analysis. Besides, they identified that miR169 and miR172 were induced as a stress response by microarray analysis. Contrary to our finding, miR169 was not detected in cold-stored potato tubers by deep sequencing and degradome analysis. [36] Meanwhile, miR397 was not induced in tomato under cold treatment. On the other hand, the expression levels of miR172, miR393 and miR397 were 1.88, 1.56 and 2.08 FC, respectively, in Arabidopsis. [19] In comparison to tomato, Arabidopsis miR397 was induced, whereas tomato miR397 showed nearly constant expression. In regard to miR393, it showed similar expression in both plants. The expression of ARF8, targeted by miR167, was higher in the cold-tolerant LA3969 cultivar, while it was at a low level in other cultivars. Although ARF6, targeted by miR167, was induced in cv. LA3969, it did not show significant expression level in cv. LA1777 and cv. LA4024. Therefore, up-and/or down-regulation of miR167 might play a potential role in cold resistance by affecting auxinsignalling pathways. [28] Also, miR393 targets TIR1 and other closely related F-box proteins in Arabidopsis. [12] These proteins are auxin receptors that target repressors of ARF. [31] The different expression profiles of ARF6 and ARF8, and down regulation of TIR1 and F-box protein in cold-tolerant tomato cultivars suggested that auxin perception and signalling may change in stress conditions based on tolerance. In addition to miR167, there could be a key regulatory role of miR393 in the auxin-signalling pathway under cold stress in tomato. NF-Y is post-transcriptionally regulated by miR169. [57] NF-Y is also known as heme-activated protein or CCAAT-binding factor. Moreover, CCAAT box is present in about 25% of eukaryotic gene promoters. [57,58] Over-expression of NFYB1 in Arabidopsis and maize conferred significantly increased drought resistance. [59] In drought stress, expression of NF-Y genes appears to be decreased in wheat leaves. [60] Similarly, NF-Y expression was down-regulated in response to cold stress in tomato genotypes. This result demonstrated that understanding the biological roles of NF-Y may have potential applications in enhancing stress tolerance in plants. Furthermore, laccase has been proposed as the target of miR397. [8,61] Expression analysis showed that accumulation of four laccase transcripts was differentiated in all cultivars. Laccase is involved in lignin metabolism and it has been reported to be induced under cold conditions in winter wheat. [8] However, it is not clear what the role of laccase is in response to cold. [62] StemÀloop qRTÀPCR analysis showed that miR172 was quickly activated by cold stress. Previous reports on miR172 and AP2 showed that miR172 act on both inhibition of the translation of AP2 and degradation of AP2 transcript. [4] An important role of AP2 is related to the flowering time and phase transition. [14] The expression profile of AP2-like fell within three categories in the analysis: up-regulated, down-regulated or slightly changed in the three tomato genotypes, which suggested the obscure behaviour of this transcription factor. AP2 transcript has been observed to be significantly reduced in tomato leaves agroinfected with tomato leaf curl New Delhi virus as miR172 transcript increased. [63] On the other hand, AP2 expression showed similar expression profile under cold and control conditions in wheat. [31] These data indicate that the detailed investigation on the role of AP2 under cold stress in tomato is needed. Analysis of the promoter elements of miRNAs revealed that they contain various hormone-regulated cisacting elements, such as ABRE, ERE, GARE and methyl responsive elements. miR172 and miR393 had GATAbox, which has been proposed to regulate light response [64,65] and to be related to cold stress. [8,66] In plants, most ABA-responsive genes have the conserved ABREs in their promoters, which are significant cis-acting elements for genes responsive to abiotic stress in Arabidopsis. [67] ABA plays an important role in the process of cold acclimation. Although miR393 has an ABRE motif, its expression was not as highly induced as miR172 in tomato. Nevertheless, expression of a number of genes induced by both dehydration and cold has revealed broad variation in the timing of their induction and differences in their responsiveness to ABA. [68] Another significant cold-related motif is the LTRE/DRE/C-repeats. [7] Interestingly, this motif could not be found in our miRNAs. CBFs (C-repeat binding factor) can bind many coldinducible genes, which have C-repeats/DRE regulatory element in promoters, and can activate their expression. [36] It is known that tomato also has a complete CBF cold-response pathway but its CBF regulon differs from that of Arabidopsis, [37] and the CBF cold-response pathway in tomato is not as important as in cold-acclimated plants. [36] At this point, the absence of LTRE/DRE/Crepeats in the promoter of five analysed miRNAs suggested that ABA-dependent signalling pathways and/or ABA-independent pathways may get involved in the regulation of cold tolerance of tomato. Despite the genome sequencing of tomato, low numbers of miRNAs (48) have been deposited from tomato. [69] In this study, we only analysed five miRNAs. It is also considered that the miRNA members in the same miRNA family often share the same cis-acting motifs in their promoter regions, suggesting that miRNA members of the same miRNA family may respond to the same biotic/abiotic or phytohormone stimuli. [70] Conclusions In this study, we analysed some cold-stress-related miR-NAs (miR167, miR169, miR172, miR393 and miR397) in tomato at early time points (0, 1, 4 and 16 h) of cold exposure by stemÀloop qRTÀPCR. In addition, we retrieved the expression profile of predicted target genes from three tomato genotypes, which were obtained from a public database. Different expression profiles of target genes were observed among cultivars. The result suggested that this differentiation among cultivars may highlight cold tolerance of tomato genotypes. Among the miRNAs assessed, miR167, miR169, miR172 and miR393 were up-regulated in early time points of cold stress in tomato. Moreover, the accumulation of the miR172 transcript was larger than that of others. Thus, it can be proposed that these miRNAs are activated in response to the initial phase of cold exposure in tomato. The level of miR397 was not significantly changed under cold conditions. Analysis of cis-elements showed that hormonal/environmental responses regulatory elements were predominant in the tested miRNAs. Furthermore, we proposed putative sly-miR393 with its secondary structure. Expression of predicted target genes showed different expression profile in three genotypes. Thus, we assumed that cold tolerance of tomato genotypes may affect the expression of target genes. However, further studies such as over-expression and/or RNAi strategies are needed to elucidate the precise role of these genes in tomato. Disclosure statement No potential conflict of interest was reported by the authors. Supplemental data Supplemental data for this article can be accessed at http://dx. doi.org/10.1080/13102818.2015.1061447.

v3-fos

2018-12-22T01:17:56.512Z

2015-01-22T00:00:00.000Z

91075559

s2

Seed yield and agronomic performance of seven improved cowpea (Vigna unguiculata L.) varieties in Ghana Cowpea is well adapted to environmental conditions that affect crop production such as drought, high temperatures and other biotic stresses compared with other crops. Notwithstanding, growth and development of many cowpea cultivars are affected by drought and high temperatures, especially during floral development. This is because cowpea cultivars tend to have narrow range of adaptation as cultivars developed for one zone usually are not very productive in other zones. A study on the yield and growth performance of seven cowpea varieties was conducted during the 2012 major and minor rainy seasons at the CSIR-Crops Research Institute, Kwadaso-Kumasi, Ghana to compare the performance of the seasonal variation on each variety. These improved varieties Nhyira, Tona, Asetenapa, Asomdwe, Hewale, Videza and IT 89KD374-57 were evaluated using a randomized complete block design and replicated three times. The results showed that varieties Hewale, Videza and Nhyira gave higher seed yields, whereas IT 89KD374-57 and Asetenapa had lower seed yields. Nhyira and Hewale gave comparatively better seed yields under both conditions. Hewale was the highest seedyielding genotype under both major and minor raining season. Cowpea production could be a profitable agribusiness for cowpea growers in Ghana considering the higher returns in terms of grain yield obtained in both seasons. INTRODUCTION Cowpea (Vigna unguiculata (L.) Walp) is one of the most ancient human food sources and has probably been used as a crop plant since Neolithic times (Summerfield et al., 1974). Cowpea is grown extensively in 16 African countries, with the continent producing two-thirds of the world total (Winrock, 1992). The crop is of major importance to the livelihoods of millions of people in the tropics. For resource-poor small-holder farmers, the crop serves as food, animal feed, cash and manure. Going beyond its importance for food and feed, cowpea can be regarded as a pivot of sustainable farming in regions characterized by systems of farming that make limited use of purchased inputs like inorganic fertilizer. The crop can fix about 240 kg ha -1 of atmospheric nitrogen and make available about 60 to 70 kg ha -1 nitrogen for succeeding crops grown in rotation with it (CRI, 2006;Aikins and Afuakwa, 2008). Cowpea is well adapted to environmental conditions that affect crop production such as drought, high temperatures and other biotic stresses compared with other crops (Martin et al., 1991). The aforesaid growth and development of many cowpea cultivars are affected by drought and high temperatures, especially during floral development (Dadson et al., 2005). In Ghana, cowpea covers 156,000 ha (IITA, 1993). The yields of the crop, however, are among the lowest in the world, averaging 310 kg/ha (Ofosu-Budu et al., 2007). Meanwhile, the crop is one of the widely cultivated legumes, mainly in the savannah and transition zones of Ghana (CRI, 2006). Hence, efforts have been made to improve cowpea production in all agro-ecological zones of Ghana through various means including the introduction of new varieties such as those used in this study. In recent years, several studies have evaluated the performance of cowpea genotypes in several ecological zones of Ghana. In selecting appropriate genotypes for different agro-ecological environments, it is important to know how various soils and climatic factors affect the growth and development of these new varieties in order to interpret the observed yields under these environments. Appropriate agronomic practices to improve the performance of new varieties of improved and dualpurpose cowpea under different agro-ecological zones are generally important for breeding and production purposes. Yield and growth performance could be increased through the evaluation of all these varieties under different agro-ecological zones for a better understanding of their morphological, physiological and biochemical response to the environment. This underscores the importance of evaluating the agronomic performance of cowpea varieties as a food security crop under the current and foreseeable future scenarios. The objective was to evaluate key yield related parameters among seven cowpea genotypes in the Forest zone of Ghana. Study site The study was carried out at the research field of the CSIR-Crops Research Institute, Kwadaso-Kumasi, Ghana. The area has a bimodal rainfall pattern, the major season (April to July) with maximum rainfall normally in June and the minor season (September to November) with the maximum normally in October. Figure 1 shows the rainfall amount and distribution during the study year of 2012. The area receives a mean annual rainfall of 1500 mm with an average monthly temperature range of 24 to 28°C. Soil analysis Soil samples were randomly collected before planting from four different cores at 0 to 15 cm and 15 to 30 cm for determination of soil physical and chemical properties using soil auger. The soil is a sandy loam classified as Ferric Acrisol (FAO, 1990), equivalent to Typic Haplustult in the USDA soil classification system. Table 1 gives the initial soil analysis. Experimental materials, design and treatments Cowpea varieties Tona, Nhyira, Asetenapa and newly developed genotypes; Asomdwe, Hewale, Videza and IT 89KD374-57 used in this study were collected from the CSIR-Crops Research Institute, Kwadaso-Kumasi, Ghana. Seeds of the varieties were planted during the major and minor seasons after the experimental sites have been disc-ploughed and discharrowed. The seeds were sown in March and August, 2012 for the major and minor seasons respectively. Each of the genotypes was grown into a six-row plot of 3.0 × 5.0 m with a spacing of 50 and 20 cm between and within rows respectively. The experimental design was a randomized complete block design with three replications. Two inner rows were harvested to determine the final seed yield. Other parameters such as plant height, root length, nodule count and number of branches were measured on 10 randomly selected plants from each plot. Data collection Using the stratified sampling method, plants from an area of 1 m 2 were carefully uprooted from each plot. In all, 21 plots were sampled and for each sample, the roots were cut using a pair of secateurs, placed in an envelope and labelled before they were sent to the laboratory for dry matter analysis. The roots were carefully washed to remove attached soil. Fresh weight, root length and dry matter were taken from the sampled plants. Plants were placed in an oven maintained at 80°C for 48 h. Samples from the 21 plots were sent to the laboratory for dry matter analysis (Table 2). Statistical analysis Data were subjected to analysis of variance (ANOVA) using the Genstat Discovery 4th Edition statistical package with subsequent mean separation using LSD at 5% level of significance. RESULTS Results of the seasonal variation studies on the agronomic and yield performance of the improved cowpea varieties are presented in Tables 3 and 4. Significant differences (p<0.05) were observed among the varieties for grain and fodder production ( Dry matter yields were highest in Hewale (30.6 g) and Asetenapa (32.3 g) compared to the other varieties for the major and minor seasons respectively (Table 3). Hundred seed weight (HSW) ranged from 14.1 to 19.2 g with a mean of 15.8 g for the minor season and 16.2 to 25.1 g for the major season. The highest 100 seed weight was obtained from Videza for both the major and minor growing season (Table 3). Plant height was higher during the long rainy season compared to the short rainy season (Table 3). Tap root length was significantly higher in Nhyira (21.8 cm) and least in IT89KD374-57 (15.3 cm) in the major season (Table 4). Nhyira again maintained the greatest root length in the minor season (19.0 cm) however this was only significantly different from IT89KD374-57 which had the least root length ((13.7 cm) (p<0.05) ( Table 4). Nodule count ranged from 15 to 20 and 12 to 20 for the major and minor seasons respectively. Videza recorded the highest nodule count for both the major and minor seasons (20.3 and 20.0 respectively), (p=0.05). Nhyira produced the highest stem diameter (0.86 and 0.88 mm) for the minor and major seasons respectively with Asomdwe recording the lowest (0.56 and 0.72 cm) for the minor and major seasons respectively (Table 4). Number of branches per plant (NBP) among the cowpea varieties ranged from 5.0 to 8.0 and 5.0 to 7.0 for the minor and major seasons respectively (Table 4). Videza attained the highest number of branches in both the major and minor season (Table 4). First pod height for the minor season was not significantly different among the seven cowpea varieties (Figure 2). The major season produced first pod heights which were higher than the minor season. IT 89KD374-57 produced the highest first pod height (64 cm) and Nhyira producing the lowest FPH (52 cm). On the other hand, final plant height at harvest was significantly different among the seven varieties for both seasons (Table 3). Videza recorded the highest height plant height of 82.3 cm whiles Tona recorded the lowest plant height of 54.2 cm for the major season (Figures 3 to 4). DISCUSSION The results of this study showed that rainfall is enough to support the growth and yield of cowpea in Kwadaso-Kumasi, Ghana, if other enabling factors are present in both the minor and major rainfall seasons. However, the cowpea varieties used in this study responded differently to the prevailing soil and climatic conditions. The highest yield of Videza was related to the continuous water supply. The results indicates that Nhyira and Videza will be more profitable than the other varieties in the minor and major seasons respectively and could serve as an alternative crop because of its desirable attributes and resistance to major biotic and abiotic constraints. Videza, Hewale and Asomdwe in this study gave lower seed yield under short raining season than the seed yield of the same variety grown under long raining season (Table 2). This is because cowpea cultivars tend to have a narrow range of adaptation, as cultivars developed for one zone with distinct climatic factors usually are not very productive in other zones with different climatic factors (Hall et al., 2003). Irrespective of the potentials of these varieties as a drought resistant crop, failure of rainfall or lack of irrigation is a frequent cause of shortfall in production especially in Ghana where cowpea production is primarily grown in dry areas. Drought could be considered as an important factor among several seed yield-reducing factors. Clearly, there is a potential for further increase in seed yield by planting high-yielding genotypes, providing optimum irrigation, adding fertilizers (Quin, 1997), planting early and spraying with suitable insecticides. Therefore, selection of cowpea genotypes that have higher tolerance to drought is needed to obtain higher and more stable seed yields and in this regard Nhyira, Tona and Hewale appear to have some drought tolerance potential due to their higher yield during the minor season. IT89KD-374-57 was observed to be low yielding due to its production of fewer nodules and dry matter. Low production of nodules means less nitrogen fixation by the variety. Production of relatively more leaves and branches with erect leave architecture in most cases reflect higher light interception and more photo-assimilate production that may result in increase yield. In the development and growth of most cowpea varieties in the Sub Saharan Africa, yield and seed development require the production of assimilates in leaves, translocation of these assimilates to the fruits, unloading of assimilates from phloem of the seed coat into cells of cotyledons and synthesis of the various seed storage compounds. Yield losses resulting from water stress are generally associated with decreases in the activity of these physiological factors and dry matter production. Among the varieties that provided the highest biological yield under short raining season conditions in 2012 were Asetenapa and Hewale, whereas under the long raining season conditions the highest biological yield was provided by Hewale and Tona. The growth habits of these genotypes were bushy, erect or semi-erect, characteristic which can be used as a cover crop as well as for grain. As observed in the grain yield, the biomass of the cowpea varied between the two seasons but the magnitude of the variation within the minor season was less than that observed in the major season. Differences in the seasonal fodder production of some varieties were not significantly different which showed that apart from rainfall some climatic factors like sun radiation might have influenced biomass production. Differences in day length between the major season and minor season in Ghana are that significant to affect crop production (Berchie et al., 2013). The significant differences observed with the dry matter showed that attainment of reproductive phase was a varietal characteristic related to the genetic constitution of the varieties. Dry matter production in the minor season was more affected by genetic composition of the variety than the seasonal variation. This perhaps may be due to the ability of cowpea to survive under extreme water limiting conditions and could respond against the later drought. This was mainly achieved by slowing growth and reducing transpiration, as reported by Vianello and Sobrado (1991) that drought stress during vegetative stage provides diminution of the growth in most crop leaves and stems. In this study, nodule numbers usually were lower at harvest than at earlier stages. The decline in number was especially noteworthy for nodules from taproots. Varietal differences account for nodule differences since the pattern of nodulation, most often, reflects the physical distribution of the root system in the soil. As reported by Hansen (1994), nodulation capacity is known to vary between and within legume species rather than rainfall variations as observed in this study. Varieties producing more nodules possess the capacity to fix nitrogen into the soil. However, genotypic effects on determinants of N 2 fixation resulting from nodulation are known to be complex. Lawn et al. (1974) suggested that the control of soybean nodule initiation occurs primarily in the root itself, but the control of nodule fresh weight occurs solely in the shoot and is related to the supply of assimilates. The result of this study has shown better crop performance in terms of vegetative and grain yield during the long rainfall season than the short rainfall season. The reason could be attributed to relatively higher rainfall and milder temperature experienced during the production season of major rainfall. According to the annual report of the Science Daily (2008), plants growing under water limiting conditions tend to grow taller in an effort to scramble for below nutrients around the growth environment. These present results are consistent with previous study on cowpea by Hayatu and Mukhtar (2010), who reported that the results for plant height at final harvest showed that, increases in plant height under both moderate and severe water stress were recorded at the expense of seed yield in IT00K-835-45 and IT98K-819-118. Plant population is reported to have effect on stem diameter, however, the results obtained from this study may be attributed to the better soil moisture availability, decreased plant competition and increased light penetration through plant canopy at such low plant population. The variation in stem diameter among cultivars might be due to genotypic differences. Conclusion From the results obtained in this study, it could be concluded that the performance of the three local and four improved varieties in terms of yield was higher in the major than minor season. Hewale and Videza are more suitable for high rainfall areas whereas Nhyira and Tona will be more productive and profitable in the drier areas. This study supports the clarion call that cowpea should become a successful legume crop for dry regions of Ghana.

v3-fos

2017-11-08T18:54:51.343Z

2015-11-25T00:00:00.000Z

5626029

s2

Developing single nucleotide polymorphism markers for the identification of pineapple (Ananas comosus) germplasm Pineapple (Ananas comosus [L.] Merr.) is the third most important tropical fruit in the world after banana and mango. As a crop with vegetative propagation, genetic redundancy is a major challenge for efficient genebank management and in breeding. Using expressed sequence tag and nucleotide sequences from public databases, we developed 213 single nucleotide polymorphism (SNP) markers and validated 96 SNPs by genotyping the United States Department of Agriculture - Agricultural Research Service pineapple germplasm collection, maintained in Hilo, Hawaii. The validation resulted in designation of a set of 57 polymorphic SNP markers that revealed a high rate of duplicates in this pineapple collection. Twenty-four groups of duplicates were detected, encompassing 130 of the total 170 A cosmos accessions. The results show that somatic mutation has been the main source of intra-cultivar variations in pineapple. Multivariate clustering and a model-based population stratification suggest that the modern pineapple cultivars are comprised of progenies that are derived from different wild Ananas botanical varieties. Parentage analysis further revealed that both A. comosus var. bracteatus and A. comosus var. ananassoides are likely progenitors of pineapple cultivars. However, the traditional classification of cultivated pineapple into horticultural groups (e.g. ‘Cayenne’, ‘Spanish’, ‘Queen’) was not well supported by the present study. These SNP markers provide robust and universally comparable DNA fingerprints; thus, they can serve as an efficient genotyping tool to assist pineapple germplasm management, propagation of planting material, and pineapple cultivar protection. The high rate of genetic redundancy detected in this pineapple collection suggests the potential impact of applying this technology on other clonally propagated perennial crops. INTRODUCTION Pineapple, Ananas comosus (L.) Merr., is a perennial herbaceous fruit crop belonging to the family Bromeliaceae. The crop is cultivated in all tropical and subtropical regions and ranks third in production among noncitrus tropical fruits, following banana (including plantain) and mango. The annual worldwide production reached 21.9 million metric tons in 2012 and the top seven producers (Brazil, Philippines, Thailand, Costa Rica, Indonesia, India, and China) jointly accounted for 90% of the global production (FAO, 2014). 1 The pineapple plant is indigenous to South America. 2,3 The putative center of origin is located in the Paraná-Paraguay River drainages between southern Brazil and Paraguay, based on the diversity distribution of related species and botanical varieties of pineapple in this region. [4][5][6] However, the eastern part of the Guiana shield has also been hypothesized as the center of domestication for pineapple, based on the variation of chloroplast and nuclear DNA markers, the high level of phenotypic diversity, and the large number of primitive cultigens in this area. 7,8 Pineapple was widely cultivated in tropical Americas before the arrival of Christopher Columbus, the first European to see this fruit, in 1493. 9 The introduction of pineapple into Asia and the Pacific began with the Spaniards in the early sixteenth century and pineapple reached Africa in mid-sixteenth century. 4 Since then, there have been multiple introductions and exchanges of germplasm among the pineapple producing countries. 4 Although many landraces and traditional cultivars exist in the Americas, only a few cultivars have been dispersed to Asia and Africa for use in commercial production. 4,10 About 70% of the world's production comes from a single cultivar, Smooth Cayenne, 10 which is a highly productive pineapple excellent for canning. 11 The current fresh fruit pineapple market is largely comprised of two cultivars bred by the Pineapple Research Institute, CO-2 and MD-2. 11 Developing new cultivars with desirable resistance and postharvest traits will depend on the available germplasm of this species. The United States Department of Agriculture (USDA) -Agricultural Research Service pineapple germplasm collection in Hilo, Hawaii, is one of the major collections in the world, along with the collections maintained by EMBRAPA/CNPMF in Cruz das Almas, Brazil, and by CIRAD-FLHOR in Martinique. As part of the ARS National Clonal Germplasm Repository for Tropical and Subtropical Fruit and Nut Crops, the collection at Hilo currently maintains over 180 accessions of pineapple cultivars and their wild relatives. As with many other tropical perennial crops, pineapple germplasm is almost exclusively maintained by vegetative propagation, by crowns, slips, suckers, or in vitro culture. Vegetative propagation has allowed the exchange of germplasm as clones among regions, countries, and continents. However, the exchange of vegetative planting materials has also resulted in problems for conservators of pineapple germplasm because records and labels of the cultivars have not always followed the same naming conventions, and accessions have limited information about their correct identity. Therefore, homonyms and synonyms are common among the names of pineapple cultivars and that restricts the sharing of information and materials among pineapple researchers and 1 hampers the use of pineapple germplasm in breeding. [12][13][14] Another major challenge for pineapple cultivar identification is that the protracted vegetative propagation has led to the accumulation of somatic mutations. Some mutations caused noticeable phenotypic effects and created intra-cultivar variation, which became the target of clonal selection. 10 While these selected mutants are important in horticultural production, it is necessary to identify them so that breeders and genebank curators can efficiently conserve and use these genetic materials. The utilization of biochemical and DNA molecular markers for pineapple germplasm management has been recently reviewed. 14 Using isozyme markers, Aradhya et al. studied pineapple germplasm and found considerable variation within and between species of Ananas. 15 In the Hawaii pineapple collection, they identified 66 distinct zymotypes that were able to differentiate all species and botanical varieties. Their results also suggested that, rather than genetic divergence due to reproductive isolating barriers, differentiation among the species of Ananas may be due to ecological isolation, and therefore may represent a species complex. Both dominant DNA markers (amplified fragment length polymorphism, AFLP) and co-dominant markers (restriction fragment length Polymorphism simple sequence repeat, SSR) have been used to assist pineapple cultivar identification and germplasm management. [16][17][18][19][20][21][22][23] In spite of the significant progress in marker-assisted germplasm management over the last 20 years, cultivar identification in pineapple remains a challenging task. Using AFLP markers, Kato et al. characterized 148 A. comosus accessions maintained in the USDA pineapple collection in Hilo, Hawaii. 20 They showed that a unique profile for major groups that had been classified by morphological traits, such as 'Cayenne', 'Spanish', and 'Queen', could not be established using AFLP-based DNA fingerprints. SSR markers likewise lacked congruence between phenotype and molecular marker-based classification in pineapple. 22,23 Moreover, neither AFLP nor SSR are the most suitable marker tool for detection of duplicates in the pineapple germplasm. Single nucleotide polymorphisms (SNPs) are the most abundant class of polymorphisms in plant genomes. Compared to SSR markers, SNP analysis can be done without requiring DNA separation by size and can, therefore, be automated in high-throughput assay formats. The diallelic nature of SNPs facilitates a much lower error rate in allele calling and promotes compatibility between laboratories. These advantages have resulted in the increasing use of SNPs as the markers of choice for accurate genotype identification and diversity analysis in perennial crops, as recently demonstrated in cacao (Theobroma cacao, 2013), 24 grapevine (Vitis vinifera, 2011), 25 pummelo (Citrus maxima, 2014), 26 strawberry (Fragaria spp, 2013), 27 tea (Camellia sinensis, 2014), and longan (Dimocarpus longan, 2015). Like other perennial horticulture crops, DNA fingerprinting that uses a small set of SNP markers is in great demand by the pineapple community for a broad range of research and field applications. These applications include, but are not limited to, identification of mislabeled accessions, parentage, and sibship analysis for quality control in breeding and seeds programs, and authentication and traceability to support the production of highvalue clones for premium markets. Nonetheless, this most powerful tool for germplasm management has not been applied to pineapple germplasm management. Ample genomic resources have been developed for pineapple. 14,28-31 The premier online database, 'PineappleDB' (http:// genet.imb.uq.edu.au/Pineapple/index.html), includes a more than 5,600 expressed sequence tags (ESTs) with 3,383 consensus sequences. The comprehensive sequence, bioinformatics, and functional classification of EST resources are available for text or sequence-based searches. A draft genome of A. comosus has been developed, which covers about 375 Mb (62%) of the estimated 526 Mb genome of this species. 14 These readily available genomic resources provide opportunities for mining new markers to use for pineapple germplasm management and breeding. The objectives of the present study were to develop SNP markers through the data mining of ESTs and transcriptome data and to assess their potential application for pineapple cultivar identification. The results reported herein represent the first validation study of SNPs in pineapple, demonstrating the utility of a transcriptome as an approach for rapid development of a high-quality genotyping tool. These SNP markers, as well as the genotyping method, will be particularly useful for intellectual property rights in varietal protection, germplasm management, and pineapple breeding programs. Mining of putative SNPs from EST and nucleotide sequences All available nucleotide sequences of Ananas spp. were downloaded from NCBI GenBank (http://www.ncbi.nlm.nih.gov, 4 October 2014). Redundant entries were examined and excluded using the CD-HIT program with a 95% sequence similarity threshold. The FASTA-formatted files of pineapple were merged into a single data set for further data mining. Putative EST-SNPs were detected using the QualitySNP program. 32 All of these selected clusters included a minimum of six EST sequences, whereas both the minimum redundancy threshold and minimal confidence score required by QualitySNP was set at three. In order to meet the requirements and constraints for primer design, all candidates for SNP markers with less than 50 nucleotides between two neighboring SNPs were removed. A subset of 96 identified SNP sequences was then chosen for design and manufacture of SNP assay. Validation of putative SNPs To evaluate the putative SNP markers for suitability of cultivar identification, we used a nanofluidic genotyping system and validated the SNPs for 170 pineapple accessions (Table 1; Supplementary Table S1). The pineapple germplasm samples were from the pineapple collection maintained by the USDA-ARS Tropical Plant Genetic Resources and Disease Unit, at the National Plant Germplasm Repository in Hilo, Hawaii (http://www.ars. usda.gov/main/site_main.htm?modecode=20-40-05-10) were harvested and dried in silica gel. DNA was extracted from dried pineapple leaves with the DNeasy Plant Mini kit (Qiagen Inc., Valencia, CA, USA), which is based on the use of silica as an affinity matrix. The dry leaf tissue was placed in a 2-mL microcentrifuge tube with one quarter-inch ceramic sphere and 0.15 g garnet matrix (Lysing Matrix A; MP Biomedicals. Solon, OH, USA). The leaf samples were disrupted by high-speed shaking in a TissueLyser II (Qiagen Inc.) at 30 Hz for 1 min. Lysis solution (DNeasy kit buffer AP1 containing 25 mg/mL polyvinylpolypyrrolidone), along with RNase A, was added to the powdered leaf samples and the mixture was incubated at 65 6 C, as specified in the kit instructions. The remainder of the extraction method followed the manufacturer's suggestions. DNA was eluted from the silica column with two washes of 50 mL buffer AE, which were pooled, resulting in 100 mL DNA solution. Using a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA), DNA concentration was determined by absorbance at 260 nm. DNA purity was estimated by the 260:280 ratio and the 260:230 ratio. Ninety-six putative SNP sequences were submitted to the Assay Design Group at Fluidigm Corp. (South San Francisco, CA, USA) for design and manufacture of primers for a SNPtype genotyping panel. The assays were based on competitive allele-specific PCR, and they enable bi-allelic scoring of SNPs at specific loci (KBioscience Ltd, Hoddesdon, UK). Data analysis Duplicate accessions were identified using pairwise multilocus matching among all individual samples. DNA samples that were fully matched at the genotyped SNP loci were declared the same cultivar or clones. The program GenAlEx 6.5 (2006, 2012) was used for computation. 37,38 After duplicate identification, the redundant samples were removed and descriptive statistics for measuring the informativeness of the SNP markers were calculated based on the remaining distinctive cultivars. The key descriptive statistics included minor allele frequency, observed heterozygosity, expected heterozygosity, Shannon's information index, and inbreeding coefficient. Computations were carried out using the same program. A cluster analysis using the neighbor-joining (NJ) method was used to further examine the genetic relationship among accessions. Kinship coefficient was chosen as genetic distance measurement of shared ancestry among the individual accessions. The computation was executed using MICROSATELLITE ANALYZER (MSA, 2003). 39 A dendrogram was generated from the resulting distance matrix using the NJ algorithm available in PHYLIP. 40,41 The unrooted tree was visualized using the web-based tool Interactive Tree of Life v2 (http://itol.embl.de/). 42 A model-based clustering algorithm implemented in the STRUCTURE software program was applied to the SNP data. 43 This algorithm attempted to identify genetically distinct subpopulations based on allele frequencies. The admixture model was applied and the number of clusters (K-value), indicating the number of subpopulations the program attempted to find, was set from 1 to 10. The analyses were carried out without assuming any prior information about the genetic group or geographic origin of the samples. Ten independent runs were assessed for each fixed number of clusters (K), each consisting of 1 3 10 6 iterations after a burn-in of 2 3 10 6 iterations. The DK value was used to detect the most probable number of clusters and the computation was performed using the online program STRUCTURE HARVESTER. 44 Of the 10 independent runs, the one with the highest Ln Pr (XjK) value (log probability or log likelihood) was chosen and represented as bar plots. To test the hypothesis that vars. ananassoides, bracteatus, and erectifolius are the putative progenitors for cultivated pineapples, we applied parentage analysis to verify the origin of the 53 accessions in A. comosus var. comosus (as labeled in Table 1). These cultivars or breeding lines were considered as 'offspring' for which parentage analyses were carried out. A. comosus vars. ananassoides, bracteatus, and erectifolius were used as candidate parents. A likelihood-based method implemented in the program CERVUS 3.0 was used for computation. 45,46 For each parent-offspring pair, the natural logarithm of the likelihood ratio (LOD score) was calculated. Critical LOD scores were determined for the assignment of parentage to a group of individuals without knowing the maternity or paternity. Simulations were run for 10 000 cycles, assuming that 10% of candidate parents were sampled, a total of 90% of loci was typed with a 1% typing error rate. The most probable single mother (or father) for each offspring was identified on the basis of the critical difference in LOD scores (D) between the most likely and the next most likely candidate parent at greater than 95 or 80% confidence. 45,46 RESULTS SNP discovery A total of 13 203 mRNA nucleotide and 5941 EST sequences from pineapple were gathered using methods previously described. After adapter removal, trimming, and quality control, 18 241 higher quality sequences were selected. The program CAP3, 47 using default parameters, was used to assemble sequences into 1793 contigs and 11 809 singlets with an average size of 3.59 sequences per contig, among which putative SNPs were detected in only 48 contigs using the QualitySNP program. Each of these selected clusters included a minimum of six EST sequences. In total, we obtained 213 putative SNPs, including 75C/T, 59A/G, 10A/T, 12A/C, 4T/G, 11C/G, 41 indel, and 1 high tri-allelic polymorphism. To select high-quality SNPs for validation, candidate SNP sites with at least 50 bp before and after the site were filtered. We calculated the number of all sequences in a cluster and the number containing the SNP type in this cluster. We then selected 96 SNPs for validation by genotyping the 170 pineapple accessions in the USDA-ARS pineapple collection. Screening for polymorphic SNP markers Out of the chosen 96 SNP markers, 80 were successful for genotyping. The failure of the remaining 16 SNPs was likely due to the sequence complexity or the presence of polymorphisms within the flanking sequences. However, among the 80 successful SNPs, 23 were monomorphic across the 170 pineapple samples (i.e. only one SNP variant was identified in all individuals). These monomorphic markers may have resulted from errors in transcriptome sequencing, which then led to the incorrect identification of SNP. It is also possible that some of these SNPs may correspond to rare alleles that were not present in the set of pineapple accessions we analyzed. A total of 57 polymorphic SNPs were retained for further analysis of this sample set. These 57 SNPs were reliably scored across the validation panel and thus were considered true SNPs. The flanking sequences of these 57 SNPs are listed in Table 2. Cultivar identification SNP profiles of the multiple accessions from the same pineapple cultivar showed that genotyping results were highly consistent (Table 3). Multilocus matching of SNP fingerprints revealed a high rate of duplicates in this pineapple collection. A total of 130 accessions could be classified into 24 synonymous groups ( Table 4). The largest synonymous group, which includes 36 accessions, was found in cultivar Cayenne. It is also noticeable that some accessions within the same synonymous group have apparent morphological differences, despite matching SNP profiles, indicting somaclonal mutation within the synonymous group. For example, Cayenne 7898 QC has atypical yellow flesh color, whereas Cayenne 7898 4N has a white color, but their SNP profiles are the same (Figure 1). Descriptive statistics and clustering analysis of 64 distinctive pineapple accessions From each of the synonymous groups, only one accession was retained and used for subsequent diversity analysis. Among the 170 genotyped accessions, there were 64 accessions with a unique SNP profile. Descriptive statistics were then computed for the 57 polymorphic SNPs across the 64 pineapple accessions with a unique SNP profile and the result is presented in Table 5. The minor allele frequencies of these 57 SNPs ranged from 0.090 to 0.495 with an average of 0.324. The mean information index was 0.601, ranging from 0.304 to 0.693. The observed heterozygosity ranged from 0.110 to 0.935 with an average of 0.520, whereas the mean expected heterozygosity was 0.414 ranging from 0.164 to 0.500 ( Table 5). The unrooted NJ tree grouped the 64 accessions into three main clusters ( Figure 2). The clustering patterns presented relationships among accessions based on the different botanical varieties or origins from different geographical regions. The first cluster includes all the accessions of A. comosus vars. ananassoides, bracteatus, and erectifolius, as well as the hybrids derived from these related botanical varieties. Within this cluster, vars. ananassoides bracteatus and erectifolius are clearly separated. This cluster also included several cultivated pineapple clones, such as Bogota, Pina Lisa, and Criolla from Colombia, Bermuda from Barbados, Cayenne Lot 520 from Hawaii, Cabezona from Puerto Rico, and Trinidad from Trinidad. The proximity between these cultivars and the two related botanical varieties indicates that these cultivars are either selected or derived from vars. ananassoides and bracteatus. The two Bolivian accessions (N94-92 Short Fruit#1 and N94-92 Long Fruit#2) were labeled as Ananas species in their passport record data. The cluster result showed that they should be A. comosus var. ananassoides or hybrids derived from A. comosus var. ananassoides. The second cluster comprised of exclusively A. comosus var. comosus, including several well-known cultivars such as Cayenne Hilo, Mauritius, and Antigua. Since these three cultivars represent the reference horticultural groups of 'Cayenne' (Cayenne Hilo) and 'Queen' (Mauritius and Antigua), respectively, their grouping here, Table 2. Flanking sequences and SNPs of the 57 polymorphic markers. SNP ID Flanking sequences and SNPs Ac4 (Figures 3 and 4) and this partitioning was largely compatible with the cluster analysis ( Figure 2 (Figures 3 and 4). The result of assignment by STRUCTURE is largely compatible with the result of clustering analysis (Figure 4). All the accessions assigned by STRUCTURE in the cluster of var. ananassoides or its hybrids were in the first cluster of the NJ tree. REZENDE Brazil A A A C C T A G C T A G C C C C C C C C C T C C A A G G A G G G A T G T T T A G A T A A G G C C G G A G C G C C A G C T C T G G C T C T Parentage analysis Among the 52 cultivars and hybrids derived from related botanical varieties, paternal or maternal parents were assigned (.80% confidence level) to 14 accessions (Table 6). A. comosus var. ananassoides was responsible for parentage of three accessions including Bogota, Bermuda, and Pina Lisa, whereas A. comosus var. bracteatus was assigned to parentage of 10 accessions. No parentage was assigned to A. comosus var. erectifolius. The result of parent-offspring assignment is largely compatible with the cluster analysis ( Figure 2). Accessions assigned as offspring from the same parent tended to be grouped together in the NJ tree ( Figure 2). For example, CB 17 was found to be the likely progenitor for Mauritius, Phu Qui, and Congo, all of which grouped together in the same subcluster in group 3 ( Figure 2). DISCUSSION Despite substantial progress in genomics research on pineapple, advanced molecular tools to support germplasm management are not available. Developing SNP markers from transcriptome sequences has been considered an efficient strategy for non-model species. In the present study, we validated 96 SNP markers based on the transcriptome sequences of pineapple at various development stages and used them to genotype a diverse panel of cultivated and wild germplasm. We obtained a success rate of approximately 60% for marker validation, which demonstrated that this approach can serve as a shortcut for SNP development. As shown in the present study, even a small set of SNP markers can significantly improve accuracy and efficiency in germplasm management. Pineapple cultivar identification Reliable identification of pineapple cultivars is invaluable for germplasm conservation and cultivar protection. In the present study, it has been demonstrated that the set of 57 SNP markers was effective for the assessment of genetic identity of pineapple germplasm. Results from multiple clones of the same cultivar showed 100% concordance, demonstrating that the nanofluidic system is a reliable platform for generating pineapple DNA fingerprints with high accuracy. The present result revealed a high rate of genetic redundancy in this pineapple collection. Some of the identified duplicates are well-documented synonymous cultivars. For example, the Cayenne cultivars are known to be derived from a few ancestral pineapple plants that originated from Cayenne, French Guiana. 10,48 But majority of the clones or synonymous groups have been less known to the pineapple community, such as Pernambuco vs. Sugar Loaf, Spanish Samoa vs. Natal, and Ruby vs. Los Banos. Identification of these clone groups will significantly facilitate the efficient exchange, conservation, and use of pineapple germplasm. However, caution is needed for the interpretation of genetic redundancy in pineapple. It is well known that somatic mutation is common in pineapple. Many phenotypic traits such as spiny leaves, fruit flesh color, acidity, and sugar content of fruit have been . 51 More comprehensive genomic approaches, such as nextgeneration sequencing, would be needed to detect which genes or alleles had been changed, thereby causing the phenotypic variation. For this reason, the reduction of identified duplicates in pineapple germplasm genebank needs to be considered on a case-bycase basis. Characterization of phenotypic traits among the synonymous group members is still essential to complement DNA fingerprinting for genotype identification. Classification of pineapple germplasm A. comosus is a mostly self-incompatible diploid with 2n52x550 chromosomes. 52 Table 1 and Supplemental Table 1. (Figures 2 and 3). This result indicates that the current system that classifies all cultivated pineapple into a single botanical variety (A. comosus var. comosus) may be questionable. It would be appropriate to consider cultivated pineapple as a complex of different botanical varieties, with possible significant gene flow among them. The third observation is about the validity of the horticultural classification of pineapple germplasm. Pineapple cultivars are classified into several horticultural groups. The commonly known groups include 'Abacaxi', 'Cayenne', 'Maipure', or 'Perolera', 'Queen', and 'Spanish'. 10,54,55 Despite these horticultural groups having been adopted by many users, little investigation has been done to show a genetic basis to reinforce this categorization. Kato et al. examined the efficacy of the horticultural groups and reported that the classifications of 'Cayenne', 'Spanish' and 'Queen' were not well supported by AFLP analysis. 20 Shoda et al. analyzed 31 pineapple accessions using SSR markers. 22 Their results also showed disagreement between the horticultural type and the results of the SSR analysis. The current study showed that the 'Cayenne' cultivars have a distinguishable genetic identity, and most of the affiliated accessions were grouped in a single cluster. However, accessions in the other groups did not appear well clustered. For example, cultivars Mauritius and Antigua are two well-known reference cultivars in the 'Queen' group, but in the NJ tree ( Figure 2) they were separated in different subclusters, where cv. Antigua showed higher proximity with the 'Cayenne' group than with Mauritius. Similar discordance was found between cultivars of the 'Spanish' group ( Figure 2). Therefore, our results support the previous conclusions of Kato et al. 20 and Shoda et al. 22 that the classification of pineapple cultivars into horticultural groups lacks consistency in terms of their genetic bases. Revision seems needed on this classification with the support of new evidence generated by DNA markers. Putative progenitors of pineapple Parentage analysis showed that both vars. bracteatus and ananassoides can be progenitors of pineapple cultivars ( Table 6). This result is in agreement with the fact that both var. bracteatus and var. ananassoides can intercross successfully with var. comosus to produce fertile offspring. 7,56 Coppens d'Eeckenbrugge and Leal hypothesized that var. ananassoides is the likely progenitor of cultivated pineapple, and it is likely that domestication happened in the Guiana shield. 7 One strong piece of evidence supporting this hypothesis is that all four chloroplast haplotypes that have been identified in cultivated materials are present in the wild var. ananassoides. 7 On the other hand, var. bracteatus was not considered as a progenitor in this hypothesis, mainly because var. bracteatus appeared to be a homogeneous variety with narrow genetic diversity, which is an unlikely basis for diverse domesticated cultigens of pineapple. 7 The present result, however, shows that 11 pineapple cultivars (Canterra, Papuri Vaupes Colombia, CB 30, Pina de Castilla, Rondon, Congo, Phu Qui, Mauritius, Cheese pine), which are dispersed across different clusters as shown in the NJ tree, Figure 2), could have their parentage (either male or female) traced back to var. bracteatus (Table 6). Ananas comosus var. bracteatus is native to Brazil, Bolivia, Argentina, Paraguay, and Ecuador but not to the Guiana shield. The present result thus indicates the possibility that pineapple could have been domesticated at multiple sites, involving both var. ananassoides and var. bracteatus. The Parana-Paraguay river drainage area could be one of the domestication sites, since both var. bracteatus and var. ananassoides are indigenously distributed in this area. 4,5 Geographically disparate origins of crop domestication are not uncommon in the Americas, as in the case of common bean (Phaseolus vulgaris), chili pepper (Capsicum spp.), potato (Solanum spp.), and cacao (T. cacao), as reviewed by Clement et al. 57 In conclusion, we conducted a study to develop a set of SNP markers for pineapple and employed them for fingerprinting the USDA's pineapple collection, using a nanofluidic array. This approach enabled us to generate high-quality SNP profiles for the purpose of pineapple cultivar identification. This is a highly useful tool for genebank management, which will also lead to more efficient crop improvement and, furthermore, has the potential to protect intellectual property rights of breeders. Our result also generated significant insight regarding the origin and domestication of pineapple. Efforts to sequence multiple cultivars from the same synonymous groups with somaclonal mutations are underway, in order to gain a comprehensive understanding about the genetic basis for mutation-based changes in important agronomic traits. This information will be highly useful for verification of pineapple cultivars and will improve the efficiency of pineapple genebank operation. The high rate of genetic redundancy detected in this Molecular characterization of pineapple germplasm using SNP1 L Zhou et al collection, also suggests the potential impact of applying this technology on other tropical perennial crops.

v3-fos

2016-05-12T22:15:10.714Z

2016-01-12T00:00:00.000Z

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Differential Toxicity of Bare and Hybrid ZnO Nanoparticles in Green Pea (Pisum sativum L.): A Life Cycle Study The effect of surface or lattice modification of nanoparticles (NPs) on terrestrial plants is poorly understood. We investigated the impact of different zinc oxide (ZnO) NPs on green pea (Pisum sativum L.), one of the highest consumed legumes globally. Pea plants were grown for 65 d in soil amended with commercially available bare ZnO NPs (10 nm), 2 wt% alumina doped (Al2O3@ZnO NPs, 15 nm), or 1 wt% aminopropyltriethoxysilane coated NPs (KH550@ZnO NP, 20 nm) at 250 and 1000 mg NP/kg soil inside a greenhouse. Bulk (ZnO) and ionic Zn (zinc chloride) were included as controls. Plant fresh and dry biomass, changes in leaf pigment concentrations, elements (Zn, Al, Si), and protein and carbohydrate profile of green pees were quantified upon harvest at 65 days. With the exception of the coated 1000 mg/kg NP treatment, fresh and dry weight were unaffected by Zn exposure. Although, all treated plants showed higher tissue Zn than controls, those exposed to Al2O3@ZnO NPs at 1000 mg/kg had greater Zn concentration in roots and seeds, compared to bulk Zn and the other NP treatments, keeping Al and Si uptake largely unaffected. Higher Zn accumulation in green pea seeds were resulted in coated ZnO at 250 mg/kg treatments. In leaves, Al2O3@ZnO NP at 250 mg/kg significantly increased Chl-a and carotenoid concentrations relative to the bulk, ionic, and the other NP treatments. The protein and carbohydrate profiles remained largely unaltered across all treatments with the exception of Al2O3@ZnO NPs at 1000 mg/kg where sucrose concentration of green peas increased significantly, which is likely a biomarker of stress. Importantly, these findings demonstrate that lattice and surface modification can significantly alter the fate and phytotoxic effects of ZnO NPs in food crops and seed nutritional quality. To the authors' knowledge, this is the first report of a life cycle study on comparative toxicity of bare, coated, and doped ZnO NPs on a soil-grown food crop. INTRODUCTION Engineered nanoparticles (ENPs), due to their high surface to volume ratio and greater numbers of atoms at the particle surface, have been widely used in the fields of medicine, agriculture (nano-fertilizers and nano-pesticides), manufacturing, electronics, and energy production (Ghormade et al., 2011;Roco, 2011;Bandyopadhyay et al., 2013;Gardea-Torresdey et al., 2014). It has been estimated that the global nanotechnology market will exceed to $3 trillion by the year 2020 (Venkatesan et al., 2004). In recent years, hybrid ENPs, e.g., doped and coated nanomaterials (NMs), have received increased attention due to their potential applications in microelectronics, semiconductors, optical device fabrication, and optics (Venkatesan et al., 2004;Ozgur et al., 2005;Dhiman et al., 2012). Commercially, available silane coupling agent (KH550) coated ZnO NPs and alumina doped (Al 2 O 3 ) ZnO NPs are two of the important hybrid NPs and are being used in the fabrication of detectors and optoelectronic devices (Zhang et al., 2010;Thandavan et al., 2015), preparation of novel polymerinorganic nanocomposites, among others (Abdolmaleki et al., 2012). Unique properties, such as, high reactivity and biocompatibility are two reasons for concern related to potential toxicity to biota. The rapidly increasing production and use have elevated the likelihood of ENP exposure in the environment (Mukherjee et al., 2014a,b). However, very little is known about the environmental health and safety of these newer hybridized materials. The literature has shown mixed effects of NP exposure on various animals, plants, and microorganisms; depending upon their species, growth conditions, NP type and exposure concentrations, among others. For example, Montalvo et al. (2015) reported improved phosphorus bioavailability through the application of hydroxyapatite nanoparticles to wheat (Triticum aestivum). Application of nanomaterials toward nanofertilizer development and plant disease suppression is described elsewhere (Liu and Lal, 2015;Servin et al., 2015). Conversely, ample evident of negative effects could also be found in the literature. For example, growth can be negatively affected by ENPs exposure (Lin and Xing, 2007;Sinha et al., 2011;Bandyopadhyay et al., 2012a,b;Gaiser et al., 2012;Hawthorne et al., 2012;Mukherjee et al., 2014b;Rico et al., 2014). There are several reports on the toxicity of different ENPs on food crops (Lin and Xing, 2007;Lee et al., 2008;Navarro et al., 2008;Sinha et al., 2011;Bandyopadhyay et al., 2012a,b;Gaiser et al., 2012;Hawthorne et al., 2012;Zhao et al., 2013aZhao et al., , 2014aRico et al., 2014;Mukherjee et al., 2014a,b). However, a mechanistic understanding of the impact of ENPs on edible/crop plants is needed for accurate exposure and risk assessment, but this knowledge remains elusive. "The Nanotechnology Consumers Products Inventory" identifies zinc oxide (ZnO) NP as the fifth most widely used material in terms of use in the consumer products (Maynard and Evan, 2006). ZnO NPs are commonly used in personal care products, anti-microbial agents, paints, and photovoltaics (Szabo et al., 2003;Hernandez-Viezcas et al., 2013). However, ZnO NPs have been shown to be potentially toxic in the environment (Kahru and Dubourguier, 2010). For instance, a 5-day exposure study with ZnO NP-DI water suspension in petri dishes showed root growth inhibition in ryegrass (Lolium perenne), radish (Raphanus sativus), and rape (Brassica napus) (Lin and Xing, 2007). NPs can also exert phytotoxicity by disrupting the water and nutrient pathways in plants (Szabo et al., 2003;Lin and Xing, 2008;Kahru and Dubourguier, 2010;Lopez-Moreno et al., 2010;De La Rosa et al., 2011). Lopez-Moreno et al. (2010) reported on the genotoxicity of ZnO NPs to soybean (Glycine max). A reduction in wheat (Triticum aestivum) biomass upon ZnO exposure, along with elevated reactive oxygen species (ROS) level, was reported by Dimkpa et al. (2012). Zhao et al. (2013a) observed reduction in chlorophyll production in corn (Zea mays) grown in soil amended with ZnO NPs at 800 mg/kg. Importantly, the toxicity of ZnO NPs may often be due to its greater dissolution or release of Zn 2+ ions into the growth media as a function of small particle size, opposed to the induction of oxidative stress by the parent ENPs (Hendry and Jones, 1980;Nel et al., 2006;Xia et al., 2006;Du et al., 2011;Kim et al., 2011;Priester et al., 2012). For example, released Zn 2+ ions from the dissolution of ZnO NPs can displace the central Mg 2+ of chlorophyll, effectively disabling the photosynthetic core, causing phytotoxicity (Rebeiz and Castelfranco, 1973;Hendry and Jones, 1980;Kupper et al., 1996;Oberdorster et al., 2005). There are very few reports on the effects of NPs on seed quantity, quality, or nutritional content. For instance, CeO 2 NPs change the nutritional quality of wheat (Triticum aestivum L.) . The fruit quality of soybean was impacted by ZnO and CeO 2 NPs (Priester et al., 2012). However, there appears to be no information available on the comparative toxicity of bare, doped, and coated ZnO NPs on green pea (Pisum sativum L.). The aim of this work was to evaluate the effect of surface coating and lattice doping on ZnO NP-plant interactions. Green pea was chosen because of its high global consumption and nutritional value (Iqbal et al., 2006). Pea plants were exposed to different concentrations of ENPs and bulk ZnO and zinc chloride. The accumulation/uptake of Zn, Al (present in doped NP), and Si (present in KH550 coating) in different plant tissues, as well as the mineral, carbohydrate, and protein content in seeds were also determined. Soil Sampling The soil was collected from the field at Texas AgriLife Research Center, El Paso, TX (31 • 41 ′ 44.98 ′′ N; 106 • 17 ′ 01.36 ′′ W, top 20 cm) and is a sandy loam with 3.73% clay, 12.15% silt, and 84.1% sand (Zhao et al., 2013a). The experiment was conducted in a 1:1 mixture of the native soil with high organic matter potting soil [Miracle-Gro Garden Soil for Flowers & Vegetables; N-P-K = 0.09-0.05-0.07] so as to improve the soil quality in terms of soil porosity, and water retention capacity, among others. Pot Preparation The bare ZnO NPs (10 nm commercial spheroid, Meliorum Technologies, New York) were obtained from the University of California Center for Environmental Implications of Nanotechnology (UC CEIN). Two percent wt Al 2 O 3 @ZnO (15 nm), and one percent wt KH550 coated ZnO NPs (20 nm) were obtained from US Research Nanomaterials (http://www. us-nano.com). ENPs and bulk ZnO were added as dry powder at 0 (control), 250 and 1000 mg NPs/kg of soil in black plastic containers (Ns-400; diameter: 20 cm; tall: 12.5 cm; volume: 3.925 L; Nursery Supplies). To achieve 1 wt% dissolved Zn, equivalent amount of 5 and 20 mg/kg zinc chloride was dissolved in 50 mL Millipore water (MPW) and added to the soil for ionic treatments. The soil was vigorously mixed with spatulas to maximize particle/ion homogeneity. Early rise variety of green pea (Seeds of Change, USDA organic, Home Depot, life cycle 65 days) were immersed in 4% bleach solution and rinsed three times with tap water. Seeds were soaked overnight in regular tap water and were sown in the test pots for a 65-day growth period. Two hundred milliliters of nutrient solution per day [0.72 g/L 15 N− 2.2 P− 12.5 K (Peters 15-5-15); EC = 1.80 dS/m; pH = 6.62] was added to each pot and the pots were maintained for 24 h in the green house for stabilization. The daily light integral (photosynthetically active radiation) was 15.3 ± 3.1 mol/m 2 /d. The greenhouse temperature was maintained at 26.9 ± 8.6 • C during the day and 13.7 ± 4.3 • C at night. The relative humidity was 41.6 ± 19.1%. Zeta Potential, Size, and pH of the NP Suspensions Particles were dispersed in 10 mL Millipore water (MPW) to achieve 250 and 1000 mg/L concentrations, sonicated for 10 min, and kept undisturbed for 1 h and the zeta potential and size were measured using a Zetasizer Nano-ZS 90, (Malvern Instruments Ltd., UK). The pH of the supernatants was measured. Each analysis was performed in triplicate. Dissolution of Different NPs in Soil Solution Release of Zn 2+ was measured by dispersing all the NPs and bulk ZnO in soil solution containing 5 g of soil mixture (1:1) in 20 mL DI water at a concentration of 1000 mg/kg soil. Zinc chloride was excluded due to its complete solubility in water. Each measurement was done in three replicates at three sampling intervals of 15, 30, and 45 days. These samples were used for the time-dependent dissolution study. Multiple serial centrifugations were used to remove suspended particles from the solution and to isolate the dissolved Zn ions. At the predetermined time (15, 30, and 45 days) intervals, samples were taken and centrifuged at 5000 rpm (Eppendorf AG bench centrifuge 5417R, Hamburg, Germany), and 2 mL aliquot of the supernatant was collected and centrifuged at 14000 rpm for 30 min. Subsequently, this supernatant was transferred and centrifuged again at 14000 rpm for 45 min. This process was repeated three times to remove particulate matters (Bandyopadhyay et al., 2015). The final supernatant was diluted to 15 mL with 2% HNO 3 and elemental concentrations were measured using ICP-OES/MS as described below. Elemental Analysis of Soil, Plant Tissues, and Seeds For each replicate, 1 g of native and 1:1 soil were collected separately from the stock pile and grounded in mortarpestle. Approximately, 200 mg of soil portions were digested in a microwave acceleration reaction system (CEM MARSx, Mathews, NC) with 1:4 plasma pure HNO3 (trace metals ≤ 1 ppb) and H 2 O 2 at 195 • C for 30 min (ramp 5 min; hold 25 min) in triplicate (Packer et al., 2007). Sixty five-day old pea plants were harvested and roots were washed with 0.01 M HNO3, with subsequent rinsing in DI water. The tissues were then oven dried at 70 • C for 2 days (Fisher Scientific Isotemp., Pittsburgh, PA; USA). The seeds were dried at room temperature for a week. Different tissues were weighed and digested similar to that described above. The digested samples were analyzed for elemental content using a Perkin Elmer optima 4300 DV inductively coupled plasma optical emission spectrometer (ICP-OES) or ICP-MS (ELAN DRC II; Perkin-Elmer) as required. Chlorophyll and Carotenoid Estimation in Leaf Approximately 0.5 gram fresh, razor blade chopped leaves were placed into 15 mL tubes. Five mL acetone was added and the samples were shaken overnight on a horizontal shaker (Revco Scientific DS1473AVA, 115 volts, 60 Hz, 7 amps). The supernatants were collected and absorbance was measured at 470, 645, and 662 nm using a Perkin Elmer Lambda 14 UV/Vis spectrometer (single-beam mode, Perkin-Elmer, Uberlinger, Germany). Concentrations of Chl-a, b, and total carotenoids were measured according to a previously described method (Wellburn, 1994). Determination of Starch, Total Soluble Sugars, and Reducing Sugars in Seeds The total soluble sugar extraction was performed following the method of Verma and Dubey (2001) with little modification. A sample of 100 mg of dried pea seed was ground in 2 mL of 80% ethanol and then heated (80 • C) in a water bath for 30 min. After cooling to room temperature, the extracts were centrifuged at 14000 rpm for 30 min (Thermo Scientific, Soruall T1, U.S.); a process that was repeated twice. All supernatants were combined and the total soluble sugar content was determined spectrophotometrically (λ = 490 nm, singlebeam mode, Perkin-Elmer, Uberlinger, Germany) following the method of Dubois et al. (1956). The reducing sugar content was measured spectrophotometrically (λ = 620 nm) by the procedure of Somogyi (1952). In both cases, sugar content was determined against a standard calibration curve of glucose. The amount of non-reducing sugar was determined by subtracting the value of reducing sugar from total sugar. Seed starch was also determined following the method of Verma and Dubey; the residue from total sugar extraction was used to measure the starch content (Verma and Dubey, 2001). The precipitate was dried at 70 • C for 24 h, 2 mL of MPW was added, and the mixture was heated in a water bath at 95 • C for 15 min. After cooling to ambient temperature, 1 mL of concentrated sulfuric acid was added. The suspension was stirred for 15 min, and the final volume was adjusted to 5 mL using MPW. The supernatant was centrifuged at 3000 rpm for 20 min, and the extraction was repeated once using 50% sulfuric acid. The supernatants were combined and diluted to 10 mL. The starch content was quantified following the method of Dubois et al. (1956) and expressed in mg/100 g dry weight. Protein Fractionation in Seeds Protein fractionation was performed according to Chen and Bushuk (1970). Dried pea seeds (100 mg) were extracted sequentially with 2 mL each of water, 0.5 mol/L NaCl, 70% ethanol, and 0.05 M acetic acid for 2 h. The extracted protein in each step was labeled as albumin (water soluble), globulin (saltsoluble), prolamin (alcohol-soluble), and glutelin (acid-soluble), respectively. Each fraction was centrifuged at 14,000 rpm; the supernatants were collected and analyzed using the methods of Bradford (1976). Statistical Analysis Unless otherwise noted, all the treatments were replicated four times. Data (means ± SE) were reported as averages of four replicates. We have run the Two-way ANOVA considering treatment type and concentration as variables with 6 and 2 levels respectively, followed by Tukey-HSD multiple comparison for the means to check the individual effects and their interactions at p ≤ 0.05 (R version 3.1.3). Pairwise comparison tests (with adjusted p-values) between concentration and nanoparticle type revealed high significance in some cases. However, we plotted only those interactions which resonate with the research goals, promote clarity and ease of comparison. RESULTS AND DISCUSSION Size, Zeta Potential, and pH of Different Particles in MPW In MPW, doped NPs had lower hydrodynamic diameter values than the other particles (Table S1). As expected, at 250 and 1000 mg/L, bulk ZnO possessed significantly higher diameter (1627 ± 198.9 and 9324 ± 236.8 nm) followed by coated (526.6 ± 14.2 and 608.5 ± 11.9 nm), bare-ZnO (397.5 ± 25.3 and 290.9 ± 20.2 nm), and doped NPs (362.2 ± 20.7 and 244.1 ± 25.6 nm). Interestingly, as the concentration increased, the size of aggregates of bare and doped nanoparticles decreased but that of the coated NP increased. This may be due to higher rate of aggregation and co-precipitation of bare and doped NPs with other suspended NPs and/or soil particles at the higher concentration, leaving behind smaller aggregates in the suspension. Conversely, the coated NP, due to its high negative surface charge (−26.4 ± 7.09 mV, Table S1), can form hydrogen bonding in MPW, leading to greater stability of the dispersion with larger NPs in diameter compared to bare and doped forms. With the exception of coated nanoparticles, all particles showed positive zeta potential. The order of magnitude was: coated < bare-ZnO < doped < bulk. The higher zeta potential for doped NPs compared to bare-ZnO NPs can be attributed to the fact that in doped NPs, Al 3+ replaced Zn 2+ in the ZnO lattice, which increases the surface potential. The negative zeta potential of the coated nanoparticles is understandable, given the nature of the surface coating; The ethoxy groups present in aminopropyltriethoxy-silane (KH550) can hydrolyze readily in water and generate hydroxyl silane (Wang et al., 2012). Thus, the attachment of KH550 onto the surface of ZnO NPs and corresponding hydrolysis could create a negative surface charge through the activity of the oxygen atoms, yielding a negative zeta potential. Although, there are differences in the numerical values of soil pH (7.7-8.5) among the treatments, these differences are not statistically significant (Table S1). This might be due to fewer number of replicates (three) and/or short exposure period. Particle Dissolution Dissolution data (mg/kg soil) of all the treatment is shown in Figure 1. We found no significant differences in dissolution across the three types of NPs in soil suspension (Figure 1). Similarly, the amount of released Si and Al remained unaffected at a given time, most probably, due to: (i) the very low amount of Al and Si in doped (2 wt%) and coated (1 wt%) NPs, respectively, with regard to the mass of ZnO NP and (ii) high background concentrations of Si and Al coming from the soil could make it difficult to quantify the source-specificity (NP vs. soil) of those two elements. On the other side, a variation in Zn dissolution FIGURE 1 | Zinc, silicon, and aluminum dissolution from all the particles after 15, 30, and 45 days at 1000 mg/kg soil concentration. Data points with same/no symbols (*) represent no statistical significance at p ≤ 0.05. Frontiers in Plant Science | www.frontiersin.org was observed with time. For instance, at 15th day, the amount of released Zn for all three NPs varied from 3.4 to 4.3 mg/kg soil but this difference was not enough to reach statistical significance. As expected, bulk ZnO particles released 1.5 mg Zn/kg soil, which was significantly less than all nano treatments (p ≤ 0.05). This could be attributed to the larger size of the bulk particles, which yields far less surface area and subsequently, less dissolution from the ZnO. The amount of dissolved zinc did not change between 30 and 45 days. Interestingly, the extent of dissolution after 30 and 45 days was lower than that of 15 days. This may be due to the production of zinc hydroxide that precipitates from solution and/or sorption of zinc ions to the different soil components, leaving behind fewer zinc ions upon reaching equilibrium (Wang et al., 2013;Zhao et al., 2013a). It is important to note that the dissolution study was neither intended to guide the toxicological experiments nor to elucidate the dissolution kinetics, but to check the trend of NP-dissolution in this particular soil type. It is noteworthy that we assumed 1 wt% zinc dissolution, although our dissolution study showed 0.06-0.43 wt% dissolution. The reasons for this difference are that the current study was performed under a closed system whereas the actual experiment was conducted under constant irrigation; this could significantly increase zinc dissolution. This is in part why we preferred to consider a "higher" amount reported in the literature, which was 1 wt% of ZnO NP (Bian et al., 2011). To achieve ∼1 wt% of zinc ion, we added 2 wt% of ZnCl 2 as the molar mass of Zn (65.4) is close to half the molar mass of ZnCl 2 (136.3). As such, 5 and 20 mg ZnCl 2 /kg soil approximate 1 wt% Zn 2+ dissolution from 250 and 1000 mg ZnO NPs /kg soil, respectively. Fresh/Dry Weights and Zinc/Aluminum/Silicon Bioaccumulation in Root/Stem/Leaf The elemental analysis of the native soil and 1:1 soil mix is shown in Table S2. Significant changes were observed in the elemental composition between the native and 1:1 soil (Table S2). The total amount of Zn, K, Mg, S, Mn, P, and Mo increased with the amendment of organic matter rich potting soil. However, the Fe concentration decreased and the Ca and Cu concentrations were unaltered. There was a slight numerical decrease in the pH values in the native (7.7 ± 0.18) and 1:1 soil (7.2 ± 0.08) but the difference was not of statistical significance. The biomass of plants exposed to Zn treatments is shown in Figure S1. At 250 mg/kg treatments, regardless of particle type, the fresh and dry weight were unaffected by Zn treatment. Similarly, at 1000 mg/kg, nano, doped and ionexposed plants had equivalent biomass, compared to the unexposed controls. However, the 1000 mg/kg bulk and coated NP treatments significantly increased the fresh weight, relative to the control plants; although the same trend was seen for dry weight, the differences were not statistically significant ( Figure S1). As expected, Zn treatments increased root Zn (Figure 2). At 250 mg/kg exposure, roots showed 5.8, 5.8, and 3 times more Zn for bulk, bare, and coated NPs, respectively, compared to controls. The doped NP exposure yielded a root Zn concentration 8 times higher than controls. Moreover, increases at 1000 mg/kg bare NP and doped treatments were 16-36 times higher than controls (Figure 2). The level of Zn in the 5 mg/kg ion exposed ("Ion-5") plants was equivalent to that of the control. The bulk, coated, and ion exposures did have nominal concentrations that were higher than the controls but large variability among these specific replicates resulted in statistical insignificance in these treatments. Concentration dependent increases in Zn content were evident for the nano, doped, and coated treatments; these trends were less clear for the bulk and ion exposures. Similar to the roots, green pea stems showed significant increase in Zn accumulation upon exposure, with the exception of the ionic zinc treatment (Figure 3). Increased Zn accumulation was in the following order: at 250 mg/kg, with the increases relative to control stems expressed parenthetically: bulk (5x), bare (7x), doped (4.7x), and coated (7x); at 1000 mg/kg, the values were as follows: bulk (9x), bare (11x), doped (20x), and coated (9x) (Figure 3). Unlike the roots, at 250 mg/kg there were no significance differences across the nanoparticle treatments. However, at 1000 mg/kg, similar to the roots, the accumulation of Zn from the doped nanoparticle treatment was significantly greater than the other nanoparticles. Similar to the roots, all ZnO treatments exhibited concentration dependent increases in zinc at the two exposure levels; this trend was not evident for the ion exposure. In leaves, all amendments except the ion treatment showed 4.6-5.3 fold increases in zinc uptake with exposure at 250 mg/kg but there were no differences among the particle types. At 1000 mg/kg, only the nano and doped treatments resulted in values significantly above the controls (5.5-11 times; Figure 4). No concentration dependent changes in Al and Si uptake were observed. Al and Si uptake by pea roots, stems, and leaves were largely unaffected across the different treatments. However, a few exceptions to this overall trend were noted. In stems, at the 1000 mg/kg treatments, doped and coated NPs accumulation showed 2.7 to 3.3 fold decreases in Al uptake compared to control ( Figure S2). Similarly, silicon uptake into pea roots was decreased significantly at 250 mg/kg bare (2.6x) and doped (2x) treatments compared to control ( Figure S3). In roots at 1000 mg/kg, bare nanoparticle exposure resulted in a 2.4 times decrease in Si content but no other differences were of statistical significance compared to control. Previously, Mukherjee et al. reported the differential effects of bare-ZnO NPs, bulk ZnO, and iron doped ZnO (Fe@ZnO) NPs on green peas cultivated in a growth chamber (Mukherjee et al., 2014a,b). At 250 mg/kg, in all the tissues, bulk and bare-ZnO NPs showed similar 3-6 fold increases in zinc uptake compared to control. However, in agreement with our current data, at higher concentrations (500 mg/kg) bare-ZnO NP showed 2.5 to 4 times higher Zn bioaccumulation compared to bulk treatment (Mukherjee et al., 2014a). Conversely, Mukherjee et al. (2014b) reported that roots of green pea exposed to 500 mg/kg Fe@ZnO showed lower Zn uptake (9x) compared to the NP treatment (12x). In addition, our current findings also indicate an opposite trend with a 36 fold increase in Zn uptake at higher concentrations of alumina doped, compared to bare-ZnO NPs. Therefore, changes in the doping agents (i.e., alumina or iron) can clearly change the uptake behavior of Zn in higher plants. Increases in element uptake from Al 2 O 3 @ZnO treatment compared to Fe@ZnO could be attributed to i) higher (more positive) surface charge due to alumina doping, which ensures greater adhesion/absorption to the root surface and ii) higher ion dissolution. At 1000 mg/kg, ZnO NPs@KH550 showed less (5x-9x) uptake across all tissues compared to all other particles. This might be attributed to larger size in soil and high negative surface change which exerts a repulsive force to the negatively charged root surface. Additionally, silicon has been proven to reduce the bioavailability of zinc ions in plants (Gu et al., 2011). Therefore, silicon released from the dissolution of coated NPs (KH550 or 3aminopropyltriethoxysilane) may be another cause for reduced Zn uptake compared to bare and doped NPs. In case of bare ZnO NP, intermediate size, and zeta potential could be two of the most important governing factors for keeping the extent of zinc uptake in-between doped and coated NPs. Although we do not know the reason for different Zn accumulation as a function of particle/exposure type, ion dissolution could be an important determinant too. Our data showed that ion release was greatest for doped, but the levels did not reach statistical significance (Figure 1). It has been reported that Si is not an essential element for plant growth (Epstein, 1999). However, the presence of Si in the coated NP makes it important to quantify the Si uptake in different plant tissues. The soil type was sandy-loam with ∼84% sand. The loading of the coating agent KH550 is only 1 wt% of NP. The Si content in the coated NP is negligibly small compared to that of soil. A similar scenario exists for Al content in soil (>6000 mg/kg soil), which was much higher than that in alumina doped NPs (2 wt% of NP). Consequently, due to very high background values, it is difficult to identify the effects of coating and doping on Si and Al uptake, respectively. There was a numerical decrease in Al and Si (except 1000 mg/kg doped) content in roots. Similar results were reported by Wang et al. (2013) where higher concentration of Zn (500 mg Zn/kg soil), lowered the bioavailability of Al "due to formation of ZnAl-layered double hydroxide (ZnAl-LDH)." Another, reason could be the coexistence of Si and Al with ZnO NPs, followed by adsorption onto the clay minerals (Zhao et al., 2013a). Silicon induced apoplastic binding of Al could also explain the lower translocation of Al through the shoot system of the plant (Wang et al., 2004). Moreover, alumina hydrolysis occurs in the acidic media (Balint et al., 2001) and the pH of the test media was in the basic range. That could be another reason for little or no dissolution of alumina in the soil. Nonetheless, synchrotron studies are essential to establish the relationship between NP composition and bioavailability. Ongoing speciation studies are focused on identifying the modes of interaction among bare, coated, and doped ZnO NPs with soil particles and higher plants. From the above results, it is clear that the phyto-toxicological response of green pea from exposure to these particles was very different. At the highest concentration, bare and doped NPs showed the greatest bioaccumulation in all the parts of the plant. However, no observable sign of toxicity was observed. Therefore, it is evident that the amount of zinc present in compound/particles is not the only determining factor for NP toxicity; the form (bare, coated, and doped) of ZnO NPs also plays a crucial role. Chlorophyll and Carotenoids in Leaf At 250 mg/kg, the amount of Chl-a increased with Zn exposure, although statistically significant increases were observed only with doped and ion treatments (3.2x-4.5x), compared to control (Figure 5). At 1000 mg/kg, all treatments resulted in 2.4-3.6 fold significant increases in Chla, compared to control, although there were no significant differences among the types of Zn amendments (Figure 5). Interestingly, there were no differences in the amount of chlorophyll-b (Chl-b) with Zn exposure ( Figure S4). Similar to the leaves at 250 mg/kg, the total carotenoid content trended upward with Zn exposure but only the doped and ion treatment enhancements (10x and 7x, respectively) were of statistical significance (Supporting Information Figure S5). The same trend was evident at 1000 mg/kg but only the bulk and doped particles resulted in statistically significant increases. Our findings are in good agreement with previous reports. For example, Prasad et al. (2012) reported higher chlorophyll content in peanut at 1000 mg/kg ZnO NP (25 nm) treatment. No effect on Chl-b in corn was observed at 400 mg/kg ZnO (Zhao et al., 2013a). Zhao et al. reported an increasing trend (but statistically insignificant) in total chlorophyll content in cucumber (Cucumis sativus) treated with 400 and 800 mg/kg bare-ZnO NP in soil (Zhao et al., 2013b). Zinc is an essential micronutrient in plants (Hansch and Mendel, 2009) but above a "threshold" concentration, the element can generate toxicity in different plant species (Broadley et al., 2007;Zhao et al., 2013b). For instance, Kupper et al. (1996) reported that zinc can substitute the central metal atom magnesium (Mg 2+ ) in chlorophyll, causing a breakdown of the photosynthetic process. It has been reported that above 200 mg/kg (threshold value) in leaf tissues, Bacopa monniera and Lolium perenne L. cv Apollo showed phytotoxicological responses (Ali et al., 2000;Bonnet, 2000). In our study, the maximum Zn concentration in leaf was <300 mg/kg DW. This value is likely less than the threshold Zn tolerance value (not determined here) for green pea leaves under our particular growth condition. Moreover, at 1000 mg/kg, carotenoid concentrations increased up to 9 fold, compared to control. Carotenoids are photo-absorbing pigments which might have protected Chl-a from photooxidation (Lichtenthaler, 1987). In leaf tissues, the unchanged (Chl-b) or increased (Chla, carotenoids) pigment content clearly suggests little or no toxicity to photosynthetic pigment production with Zn exposure. However, these findings may not exclude the possibility of damage to other components of the photosynthetic apparatus, e.g., electron transport chains and photosynthetic enzyme activities. Further biochemical investigations are warranted to evaluate the effects of ZnO NP exposure on other complex photosynthetic components. Effects of NPs on Green Pea Seed Quality Exposure to Zn, regardless of type, generally had little effect on the green pea pod characteristics. The pod length, pod weight, and number of seeds per pod did not change as a function of treatment, with the exception of doped 250 mg/kg nanoparticles (data not shown). Here, the number of seeds per pod decreased by 33% compared to that of bare ZnO NP treatment. Unlike bulk treatments, bare, doped, and coated NPs showed increase in Zn uptake at 250 mg/kg treatment, compared to control (Figure 6). At 1000 mg/kg, the Zn content increased by 2-2.5 times in all NP and bulk treatments as compared to control. The ionic treatments did not show any significant change in Zn uptake at 5 mg/kg or 20 mg/kg. Concentrations of Cu, Mg, and K in the seed did not change significantly with Zn exposure (data not shown). The Fe level was significantly elevated by the coated (250 mg/kg) and doped (1000 mg/kg) treatments. In addition, at 1000 mg/ kg coated treatment, P and Mn were significantly increased (Figures 6B-D). Overall, Zn exposure, regardless of type or concentration, had little impact on the protein or carbohydrate profile of the green pea seeds. The amount of acid-soluble (glutelin), salt-soluble (globulin), water-soluble (albumin), and alcoholsoluble (prolamin) protein fractions remained unaltered in all treatments ( Figure S6). There was a decrease in glutelin amount (50%) at 1000 mg/kg doped treatment, compared to control, but due to large variability and modest replicate numbers, the decrease was statistically insignificant. The amount of total sugar, starch, reducing sugars (glucose and fructose), and non-reducing sugar (sucrose) also remained largely unaltered. The exception was the 1000 mg/kg doped NP treatment where the sucrose content of pea seeds was significantly increased by 1.8 fold compared to all other treatments (Figure 7). Higher sucrose concentration in green pea at 1000 mg/kg doped treatment may be less of concern for seed quality but more problematic as an indicator of plant stress (Koch, 2004;Levitz, 2004;Zhao et al., 2014b). It has been reported that reducing and non-reducing sugars can contribute to the signaling pathways related to stress (Koch, 2004;Levitz, 2004;Zhao et al., 2014b). As mentioned earlier, green pea plants were chosen to evaluate the effects of NP exposure because of the crop worldwide production and consumption. Green pea seeds are rich in protein, certain minerals, and vitamins and have modest calorific content (Iqbal et al., 2006). Raw green peas are excellent source of vitamin K, C, B1, B9, A, B6, B3, and B2. The crop is also rich in Mn, P, Mg, Cu, Fe, Zn, and K (Iqbal et al., 2006). Among major legumes (i.e., lentil, green peas, and common bean, among others), green pea is the second best protein source (24.9/100 g raw green pea, Iqbal et al., 2006). It has been reported that a cup of raw green peas (=137.75 g) provides 30.3% fiber, 14.7% of protein, and only 6% calories as measured against typical daily nutritional values (Iqbal et al., 2006). There are very few reports available in the literature investigating the effect of nanoparticle exposure in soil under field-like conditions on pea seed quality. Several similar studies have been published focusing on bare-ZnO and CeO 2 NPs exposure. For instance, Rico et al. (2014) treated wheat plants at 0, 125, 250, and 500 mg/kg soil, and found changes in nutrient content (S and Mn), amino acid, and fatty acid profiles upon exposure to CeO 2 . Our findings agree well with Priester et al. (2012) where a 2.5 fold increase in zinc uptake by soybean pods was observed upon exposure to 500 mg/kg bare-ZnO NP as compared to controls. Peralta-Videa et al. (2014) found increased zinc concentration in soybean pods at 50, 100, and 500 mg/kg bare-ZnO treatments. Moreover, at "medium" concentration (100 mg/kg), significant bioaccumulation of Cu and Mn in soybean pods were also observed. Similarly, Zhao et al. (2014b) reported that treatment with 400 and 800 mg ZnO NP/kg soil resulted in changes of micronutrient and carbohydrate content without any alteration in protein profile of cucumber fruit. Elevated levels of Zn in the seeds was likely due to the enhanced mobility of Zn 2+ ions (Broadley et al., 2007;Wang et al., 2013) generated from the dissolution of NPs in soil. In terms of cellular uptake, there are different transporter genes and pathways present, which regulate the mobility of different metals across the plasma membrane. For example, Mn transport is regulated by natural resistance-associated macrophage protein (Nramp) transporters and zinc-regulated transporter/iron-regulated transporter (ZRT/IRT1)-related protein (ZIP) transporters, among others (Pittman, 2005). Currently, we have no information regarding the interaction among specific metal transporters and different NPs. As such, characterizing potential correlations between macro/macro nutrient uptake in seed with different NP exposure is too speculative with the current knowledge base. However, considering all the above data, it can be said that the mineral/nutrient concentration in the edible tissue was affected differentially by nanoparticle type, with the coated and doped ZnO exerting the greatest effects. Similarly, under high dose exposure (1000 mg/kg), doped NP altered (1.8 times higher) the carbohydrate profile (sucrose) of the seed. The implications of these NP-induced changes in fruit content/quality are currently unknown but are the subject of intense investigation. In summary, our study investigated the comparative phytotoxicity of bare-ZnO NPs, Al 2 O 2 @ZnO, and ZnO@KH550 NPs on green pea plants in terms of biomass, element bioaccumulation, changes in leaf photosynthetic pigment, along with the changes in seed quality. Our results confirmed that, in spite of possessing larger size in the commercial form, alumina doped ZnO NPs (15 nm) have greater effects on plant and seed quality, compared to bare-ZnO NPs (10 nm). The seed quality was affected most by the doped NPs at 1000 mg/kg where nutrient content and carbohydrate profile (sucrose) changed. It was suggested in the literature that doping (Fe doped ZnO NPs) could decrease the phytotoxicological effects of bare-ZnO NPs to higher plants (Mukherjee et al., 2014b). Nevertheless, our findings clearly demonstrate that Al 2 O 3 @ZnO NP treatments exerted more negative effects on green pea when compared to bare and coated ZnO NP. Therefore, the doping agents certainly play a crucial role in the phytotoxicological responses of NP exposure to the higher plants. Although, the mechanism is unknown, ion release and coating facilitated uptake of intact NPs are possible pathways of concern. Additional study into the broader implications of NP doping and coating type on food safety and on the fate and disposition of these materials in the environment is warranted. SUPPORTING INFORMATION Two tables listing details on particle characterization and elemental composition of native and 1:1 soil. Six figures describe different physiological and biochemical parameters of root, stem, leaf, and seeds.

v3-fos

2016-05-12T22:15:10.714Z

2015-12-01T00:00:00.000Z

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Detection of Mycobacterium avium subspecies paratuberculosis infection in two different camel species by conventional and molecular techniques Paratuberculosis (John’s disease) is infectious and chronically progressive granulomatous disease which affects domestic and wild ruminants. The causative agent is Mycobacterium avium paratuberculosis (MAP), a slow growing mycobactin dependent acid-fast bacillus. We investigated the detection and frequency of MAP in apparently healthy dromedary and Bactrian camels by insertion sequence 900 (IS900) polymerase chain reaction (PCR) and acid fast staining of fecal samples in Iran. Acid fast staining results showed that 6/50 (12.0%) samples of dromedary camels and 4/26 (15.3%) samples of Bactrian camels were suspected to MAP. Although the percentage of positivity for PCR assay of fecal dromedary camel was 8.0%, no bands corresponding to MAP detect in all samples of Bactrian camels. In conclusion, Although the incidence of MAP infection was low, further studies should be conducted to get more information on MAP infection in camel population, especially in areas where camels are close to other ruminants such as dairy cow, sheep and goat. Introduction Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease (JD), also called paratuberculosis, in domestic and wild ruminants throughout the world which estimated economic losses are over $250 per cow annually in highly infected dairy cattle herds, 1,2 because of severe loss of slaughter weight and reduced milk production. 3,4 Apart from its outbreak in cattle, sheep, goats and deer, paratuberculosis has also been diagnosed in a wide range of other free-ranging and domesticated ruminants including camels, llamas and alpacas. [5][6][7] Dromedary camel (Camelus dromedarius) and Bactrian camel (Camelus bactrianus) are valuable domestic animals in tropical and subtropical areas used for their meat, milk and wool. Camel milk and meat are considered as an important source of protein for wide range of population. 8 Paratuberculosis affects camels worldwide causing characteristic clinical manifestation of severe diarrhea resulting in death. 9 The disease in camels may have a more rapid course than in cattle, with death occurrence after 4 to 6 weeks of onset of illness. 10 In Bactrian camels, the disease was most severe in 3 to 5 year old animals. 7 Paratuberculosis has been reported widely in many populations of camels in Asia, Middle East, Africa and the former Soviet Union. 7,11,12 Paratuberculosis in Iranian camels is poorly documented in the literature even though the disease has been detected in some dairy cattle herds. [13][14][15][16][17] To the best of our knowledge there has not been any published report of JD in Iranian camels using molecular approaches. To reduce the infection rate in a herd, the test and culling strategy of JD control programs require sensitive and specific diagnostic techniques. Fecal culture is considered as the gold standard for the diagnosis of MAP infected animals but requires 12 to 16 weeks; 18 therefore the development of a rapid, sensitive and specific diagnostic method for the detection of MAP is essential in the control of Johne's disease in economically important animals. 19 The insertion sequence 900 (IS900) element is an insertion sequence considered to be a MAP-specific gene with 15 to 20 copies per genome and is a target for rapid detection of MAP by PCR. 20,21 The aim of this study was to detect MAP in apparently healthy dromedary and Bactrian camels by IS900 PCR and acid fast staining of fecal samples in Iran. Materials and Methods Collection of samples. A total of 76 fecal (50 dromedary camel and 26 Bactrian camel) samples from apparently healthy camels with different ages were obtained during six months from Semnan and Ardebil provinces (Iran). Approximately 5 g of feces were collected from each animal with a gloved hand directly from the rectum and placed in a sterile, leak proof container. The fecal samples were stored at 4 ˚C until they were transported to the laboratory. The unprocessed fecal samples were preserved at -20 ˚C for further studies. Ziehl-Neelsen acid fast staining. For Ziehl-Neelsen staining, fecal smears were stained with carbol fuchsin Ziehl-Neelsen acid-fast stain. The smears were washed for 2 min in tap water, decolorized in two brief washes of acid alcohol (1% hydrochloric acid in 70% ethanol), washed for 2 min in tap water, and briefly counterstained with methylene blue. 22 All smears were examined by three specialists, with multiple fields being evaluated. DNA extraction. According to Stabel et al., fecal samples (1 g) were diluted in 9 mL of 1× Tris-ethylenediamine tetra-acetic acid (EDTA) buffer (10 mM Tris-HCl, 1 mM EDTA; pH 7.6) in 15-mL polypropylene conical tubes. Samples were vortexed for 5 sec and allowed to settle for 2 min and then vortexed again. Samples were centrifuged at 200 g for 30 sec, and the supernatants from each sample were transferred to new 15-mL conical tubes. The supernatants were then diluted 1:10, 1:100, or 1: 1,000 in 1× TE (Tris EDTA) buffer. One milliliter of each dilution was placed into 1.5 mL sterile, DNase, RNase-free Eppendorf tubes (Eppendorf, Hamburg, Germany) and centrifuged at 13,000 g for 2 min. Supernatants were discarded and pellets washed 2 times with 1 mL of 1 × TE buffer. Pellets were resuspended in 500 μL of 1 × TE buffer and placed in a heating block at 100 ˚C for 10 min. A negative control containing 1 × TE buffer was included in the heating block as a sentinel for DNA crosscontamination during sample processing. After cooling to room temperature, 4 μL of RNAase (500 μg mL -1 ) was added to each sample. Samples were stored at -20 ˚C until PCR analyses were performed. 32 Polymerase chain reaction. IS900 PCR was performed as described by Corti and Stephan with minor modification. 24 Resuspended DNA (5 μL) was used as the template for IS900 PCR. The primers P90, 5 ' -GAA GGG TGT TCG GGG CCG TCG CTT AGG-3 ' and P91, 5 ' -GGC GTT GAG GTC GAT CGC CCA CGT GAC-3 ' were used for amplification cycles that generated a 413 bp product. The PCR mix consisted of 25 μL reaction volume containing 20 pmol of each primer, 5 mM MgCl2 (CinnaGen Co., Tehran, Iran), 40 μM 2 deoxyribonucleoside-5-triphosphate (dNTP; Cinna-Gen), 0.5 U Taq polymerase (CinnaGen) in 1× reaction buffer (CinnaGen). PCR program was as follows: 94˚C for 5 min (1 cycle); 94˚C for 1 min, 59˚C for 1 min, 72 ˚C for 2 min (30 cycles); 72 ˚C for 7 min (1 cycle). PCR products were visualized by gel electrophoresis on 1% agarose gel. Mycobacterium avium subspecies paratuberculosis ATTC 43105 was used as positive control for each PCR round. Statistical analysis. Prevalence of positive and negative results for Ziehl-Neelsen acid fast staining and IS900 PCR were primarily analyzed by contingency tables. All proportions were compared using the Fisher's exact test and a p-value less than 0.05 was considered significant by SPSS (version 16.0, SPSS Inc., Chicago, USA). Ziehl-Neelsen Staining analysis of the fecal smears. Acid fast staining results showed that 6/50 (12.0%) samples of dromedary camels and 4/26 (15.3%) samples of Bactrian camels were suspected to MAP. But there was no significant difference (p = 0.7) between the prevalence of positive acid fast staining results in two groups of dromedary and Bactrian camels. Polymerase chain reaction analysis. A total of four dromedary faecal samples out of 50 were positive by PCR assay for Map yielding an expected PCR product of size 413 bp (Fig. 1). The percentage of positivity for PCR assay of fecal dromedary camel was 8.0%. Although expected PCR bands size of MAP revealed in fecal samples of dromedary camels, there were not bands corresponding to MAP detection in all samples of Bactrian camels. No significant difference (p = 0.2) was found between the percentage of positivity for IS900 PCR assay in two groups of dromedary and Bactrian camels. Discussion Johne's disease is an infectious disease of cattle and other ruminants, caused by Mycobacterium avium subspecies paratuberculosis (MAP). 6,7 Diagnosis of paratuberculosis is difficult because of the fastidious growth pattern of the microorganism and the different host immune responses invoked during subclinical and clinical stages of infection. Traditionally, fecal culture for MAP is considered as the gold standard for diagnosis. However, fecal culture is time-consuming and detects only 38.0 to 50.0% of infected animals. 23 Serological tests such as enzyme-linked immunosorbent assay (ELISA) are even less sensitive than fecal culture, particularly in apparently healthy or sub-clinically infected animals. 8 Use of nucleic acid probes combined with the PCR for detection of MAP in fecal samples have vastly improved in recent years, leading to an increased sensitivity in detecting of low shedders, including a detection level of one colonyforming unit(s) per gram of feces. This is done by amplifying the IS900 gene sequence which is the most reliable way of detecting cows shedding low levels of MAP in their feces. 23,25,26 Use of PCR in contrast to culture and serological tests, allowed to detect nonviable as well as viable microorganisms and would be a more sensitive detection method. Therefore, in comparison with serological or culture methods, detection of MAP directly from bulk-tank milk or feces by IS900 PCR could be considered a valuable test for the estimation of herd-level prevalence. 27 The MAP has been detected in dairy herds throughout Iran. [13][14][15] A recent work in dairy cow herds in Fars province (southern Iran) showed a herd-level prevalence of 11.0% based on IS900 nested PCR on bulk-tank milk samples. 28 Another study done by nested PCR also showed that, 5 out of 90 samples (5.6%) were positive for MAP. However, the frequency of infection was diverse in different regions ranging from 4.2% to 7.7%. 26 Khaled et al., confirmed infection with MAP by PCR by targeting the IS900 gene. They extracted DNA from lymph node and liver samples of the five suspected camels resulted in amplification of a 229-bp PCR product which is the specific product of M. paratuberculosis-IS900. 25 Alhebabi and Alluwaimi reconfirmed the spread of MAP infection in the Saudi camel herds and concluded the ruminant ELISA was proved useful for the screening of the MAP infection in camel and could rule out the need for the species specific ELISA test. They have shown their feasibility as robust diagnostic tool for screening of John's disease in camel. They also proved that PCR was more practical than ELISA in detecting MAP. 8 In this study, according to previous study, 23 we used a rapid and simple DNA extraction method for the detection of MAP in fecal samples which were taken from two different camel species. Acid fast staining of suspected tissues is rapid and requires little optimization. However, Ziehl-Neelsen staining has been reported to falsely identify Nocardia and Corynebacteria and cannot differentiate among the various mycobacterial species. Previous reports have determined the sensitivity of Ziehl-Neelsen to be 36.4%. 29 A total of 4 out of 50 dromedary camel samples were positive in faecal samples by PCR. The percentage of positivity for PCR detection in this study was 8.0%. This result is in agreement with that of Alhebabi and Alluwaimi who reported only 97 positive samples out of 310 tested samples by PCR in dromedary camels. 8 Although the outbreak of MAP infection in Bactrian camels (Camelus bactrianus) was 15.3% based on acid fast staining, acid fast staining could not completely predicative. The results showed no bands on fecal samples by IS900 PCR, so it could be concluded that the genus Camelus bactrianus from our region are may be free of infection but further research is required. The study has confirmed the spread of MAP infection in the dromedary camel in Iran. But there is no significant association (p > 0.05) between the camel species and the prevalence of MAP infection by either acid fast staining or IS900 PCR methods. In the present study, IS900 PCR assay proved to be a more sensitive and reliable than acid fast staining for the detection of MAP in faecal samples of camels since the PCR assay was able to detect significantly more positive cases than acid fast staining. This study suggests that IS900-PCRbased detection of MAP could be used as a potential diagnostic tool for rapid and effective Johne's disease surveillance in camels. To the best of our knowledge, this study is the first description of a work of this kind performed in Iran. Although the incidence of MAP infection was low, further studies should be conducted to get more information on MAP infection in camel population, especially in areas where camels are close to other ruminants such as dairy cows, sheep and goats.

v3-fos

2019-04-01T13:16:04.698Z

2015-01-01T00:00:00.000Z

88605019

s2

THE POTENTIALS OF USING SELECTION INDEX IN THE ASSESSMENT OF BREEDING VALUES OF HOLSTEIN BREEDS IN SERBIA : The conducted research was aimed at constructing equations of selection index that would be used in the selection of the Holstein-Friesian breed animals in Serbia. The construction of the selection index includes the most important milk traits observed in standard lactation: milk yield (MY305), milk fat content (% MF305) and protein content (% MP305). The variance and covariance necessary for the construction of selection index are calculated using the mixed model by the method of least squares. The economic value of traits is expressed as a ratio of relative changes in costs per unit of traits included in the selection index. Livestock included in the research produced, in the first standard lactation, an average of 7681 kg of milk with 3.58% milk fat and 3.28% protein. The equation of the selection index presented in the paper is selected from the group of equations of selection index, as an equation with the highest correlation between the equation and the aggregate genotype, which amounted to 0.2156. Introduction Breeding domestic animals is a very complex zootechnical procedure both in terms of objectives to be achieved and in the methods used. A large number of participants are involved in this business, ranging from breeders, basic, regional and national breeding organizations, centres for artificial insemination, professional and scientific organizations and universities, who define breeding objectives in accordance with the breeding program (Stanojevic et al. 2015). In designing the breeding programs, selection objectives are defined which involve a larger number of traits that affect the economic efficiency of cattle production. One of the key issues when choosing parental pairs is the identification of genetic superior livestock that possess genes whose frequency we want to raise in the next generation. One of the most efficient ways for the assessment of breeding values for a larger number of traits is the use of the selection index (Sölkner et al., 2000). The selection index was used for the first time in the selection of plants, whereas Hazel and Lush (1942) were the first to use this method in the selection of domestic animals. The selection index combines production levels of two or more traits. The result of the selection index is the score whose basis serves for ranking and selecting livestock. In assessing the breeding value, selection index takes into account the economic value of all traits included in the index and their manifestation, heredity and connection and combines it all into one single value (selection score) that we use for ranking livestock when selecting (Hazel and Lush, 1942;Hazel 1943). Another advantage is its relatively simple application when the equation of selection index is determined (Radojkovic et al., 2010;Popovac et al., 2014). Thus obtained score is in maximum correlation with the genetic contribution of the individual unit. When assessing the breeding value by using the selection index, one can use the data on production results of the very individual unit and its relatives. In the past, the largest number of selection indices was designed in a way to include only productive traits such as milk yield, milk fat and proteins content (Miglior et al., 2005). Modern breeding programs have repositioned the focus from solely milk production traits to also include functional traits, longevity and traits of the type. Thus defined breeding programs are aimed at creating a healthier and economically efficient livestock units. The aim of this work is to design equations of selection index with differently expressed economic values of traits and their use in assessing the breeding value of cows and bulls of the Holstein breed. Materials and methods In the design of selection indices it is necessary to know the values of genetic and phenotypic variances and covariances, as well as the economic value for each trait involved in the design of selection index equation. For calculating the genetic and phenotypic variance and covariance, the research used production results that were achieved by 5123 primiparous in standard lactation. These livestock produced on 7 farms of the Agricultural Corporation Belgrade from 2006 to 2012. All the livestock were under milk yield control. The livestock were The potentials of using selection index in … 525 offspring to 53 bull-fathers, of which each bull had at least 5 daughters, while the average number of daughters per bull was 96.7. The breeding value rated by the selection index method can be represented by the following general equation: where: I -relative breeding value of the livestock unit estimated by the selection index, i.e. the value of selection index determined for the given livestock, b i -multiple regression coefficients for each trait included in the selection index, (X ii ) -the difference between the phenotypic value of the trait included in the selection index for a given individual and the population average for a given trait The construction of the selection index includes traits of primary importance regarding milk production, namely: milk yield (MY305), milk fat content (%MF305) and protein content (%MP305). All properties were observed in standard lactation. The values of genetic and phenotypic variances and covariances were calculated using the least squares method (Harvey, 1990) and by using the following mixed model: Where: Y ijklm -phenotypic manifestation of surveyed traits, µ-general population average, F j -fixed effect of the j farm, G k -fixed effect of the k year of calving, S l -fixed effect of the l calving season, U m -fixed effect of the m groups based on the age at first calving (I-age at first calving less than 24 months, II-age at first calving from 24 to 29 months, III-age at first calving from 29 to 35 months, IV-age at first calving from 35 to 41 months, V-age at first calving over 41 months), o i -random effect of the i sire, e ijklm -random error. In the absence of stable market relations over a longer period of milk production in our country, which are essential for determining the economic value of traits in terms of use of bio-economic model, this paper uses a methodology which is based on the use of the relationship between costs and expression of traits (Radojkovic 2000, Vukelic et al., 2004Popovac et al. 2014). The economic value expressed in this way represents a relative economic indicator. In calculating the economic value of traits included in the construction of the equation of selection index, the starting point was that all the traits included in the selection index are registered in standard lactation, and that all livestock achieved 305 feeding days during standard lactation. The main economic assumption is that the costs of one feeding day as economic size are the same throughout the surveyed period, which is not the case in practice, but higher costs at the initial phase of lactation compensate to some extent lower costs at later stages. The economic value is expressed as the difference in costs per trait unit that appeared as a consequence of the implementation of the breeding program. Milk yield in standard lactation (MY305) served as the primary trait in the research. The breeding objective here was set as milk yield of 9000 kg of milk with 3.70% milk fat and 3.40 protein. The economic value of traits included in the construction of the selection index is obtained by comparing the relative indicators of cost reduction between the primary trait and other two traits which appears after the implementation of the breeding program. The economic values of observed traits are given in Table 1 and Table 1a: The economic value of traits included in the construction of the selection index is also calculated according to the method used by Sharma and Basu (1986), Falconer and Mackay (1997) and Cameron (1997, where the relative economic value is expressed as 1/ σ p , where σ p is the phenotypic standard deviation of the observed trait. Results and Discussion Table 2 shows the average values and variability of milk traits in standard lactation achieved by the livestock included in the research: Table 2. Average values and variability of milk traits in standard lactation Livestock included in the research had, in standard lactation, an average production of 7681 kg of milk with 3.58% milk fat and 3.28% protein. The determined values are significantly higher than the values identified on the same population in the research by Đedović (2000) and Beskorovajni (2000), while the given results are in accordance with the results determined by Carlen et al. (2004) and Stanojević et al. (2012). Table 3 shows the results of examining the impact of factors on the traits included in the research. Table 4 shows the values of variance and heritability coefficients for observed traits. The values of heritability coefficients had their values from 0,065 in terms of protein content in milk to 0,165 in terms of milk yield. The determined values of heritability coefficients are significantly lower compared to the results gained by Carlen et al. (2004) andPham Manh Hung et al. (2008). Similar values were achieved in the research by Stanojevic et al. (2012) and Đedović et al. (2013).The determined values of heritability coefficients of milk production traits suggest the possibility of their improvement through selection, even though mainly external environmental factors influence their expression. Table 5 shows the values of coefficients of phenotypic (above the diagonal) and genetic (below the diagonal) correlations. After solving the system of normal equations and determining the coefficients of multiple regression, the equation of selection index was constructed with the following form: REV Selection indices equations r IAG REV 1 I=0,046 1 (X 1 -7681)+28,395 2 (X 2 -3,58)+157,44 3 (X 3 -3,28) 0,2156 REV 2 I=0,048 1 (X 1 -7681)+161,635 2 (X 2 -3,58)+281,772(X 3 -3,28) 0,1942 1 -the coefficient of multiple regression of coefficients for MY305, 2 -multiple regression coefficient for %MF305, 3 -multiple regression coefficient for %MP305, X1,2,3-phenotypic value of the livestock for traits included in the SI Presented equations of selection indices are selected from the group of equations of selection index as equations with the highest coefficient of correlation between the equation and aggregate genotype, which amounted to 0.2156 or 0.1942 for second equation of SI. Constructed equations combine, in an optimal way, phenotypic expression levels of the three traits that are included in it, where the resulting score is in maximum correlation with the breeding value of the livestock. Conclusion The research determined an average milk yield in the first standard lactation of 7681 kg of milk with 3.58% milk fat and 3.28% protein. The research identified a high variability of milk production traits, which is, on one hand, conditioned by hereditary factors and, on the other hand, environmental factors. The observed milk production traits were highly statistically influenced by the year of calving and calving season, as well as the age at first calving. The heritability coefficients calculated in the research indicate that the observed milk production traits in the studied population are hereditary low. Constructed equations of the selection index should serve as a simple and fast way to rank cows in their selection as parents of the next generation. Also, the constructed equations of selection index only include milk production traits. A selection conducted in this way would be one-sided and, in the future, there should be more work on the inclusion of a certain number of traits such as reproductive traits and fitness traits in the construction of selection index. Special attention should also be given to the introduction of more complex and precise methods for assessment of breeding values such as BLUP and BLUP AM method.

v3-fos

2017-08-03T00:42:34.571Z

2015-02-27T00:00:00.000Z

18895449

s2

Naturally p-Hydroxybenzoylated Lignins in Palms The industrial production of palm oil concurrently generates a substantial amount of empty fruit bunch (EFB) fibers that could be used as a feedstock in a lignocellulose-based biorefinery. Lignin byproducts generated by this process may offer opportunities for the isolation of value-added products, such as p-hydroxybenzoate (pBz), to help offset operating costs. Analysis of the EFB lignin by nuclear magnetic resonance (NMR) spectroscopy clearly revealed the presence of bound acetate and pBz, with saponification revealing that 1.1 wt% of the EFB was pBz; with a lignin content of 22.7 %, 4.8 % of the lignin is pBz that can be obtained as a pure component for use as a chemical feedstock. Analysis of EFB lignin by NMR and derivatization followed by reductive cleavage (DFRC) showed that pBz selectively acylates the γ-hydroxyl group of S units. This selectivity suggests that pBz, analogously with acetate in kenaf, p-coumarate in grasses, and ferulate in a transgenic poplar augmented with a feruloyl-CoA monolignol transferase (FMT), is incorporated into the growing lignin chain via its γ-p-hydroxybenzoylated monolignol conjugate. Involvement of such conjugates in palm lignification is proven by the observation of novel p-hydroxybenzoylated non-resinol β–β-coupled units in the lignins. Together, the data implicate the existence of p-hydroxybenzoyl-CoA:monolignol transferases that are involved in lignification in the various willows (Salix spp.), poplars and aspen (Populus spp., family Salicaceae), and palms (family Arecaceae) that have p-hydroxybenzoylated lignins. Even without enhancing the levels by breeding or genetic engineering, current palm oil EFB ‘wastes’ should be able to generate a sizeable stream of p-hydroxybenzoic acid that offers opportunities for the development of value-added products derived from the oil palm industry. Introduction In order for a lignocellulose biorefinery to function as an economically viable enterprise, it must be developed to yield value-added coproducts in the process [1,2]. Coupling the source of biomass to the waste stream of an associated commercial process is one way to create value in the overall system. The production of palm oil generates a substantial amount of empty fruit bunch (EFB) fibers that can be readily processed into sugars for biofuels production and a mixture of unfermentable components including lignin. The resulting lignin stream could then offer new opportunities for the development and isolation of value-added products derived from the oil palm industry [3]. For example, in 2013 Malaysia, the second largest oil palm producer after Indonesia, produced over 19 million tons of palm oil on a total plantation area of over 5 million ha, which also generated an estimated 70-80 million tons of biomass, mostly from oil palm EFB [4]. This biomass, which is a high-quality lignocellulosic fiber, has not been fully utilized commercially and is a largely untapped resource, although research in the biorefinery area has attempted to provide high value-added products via liquefaction [5], solvolysis [6] and pyrolysis [7]. Lignin, a stochastically generated biopolymer found in plants, forms by means of radical coupling reactions, primarily between a monolignol and the growing lignin polymer in an endwise process [8,9]. In angiosperms, coniferyl and sinapyl alcohols are the predominant monomers and, once incorporated into the lignin backbone, produce guaiacyl (G) and syringyl (S) units in the resulting polymer. A monolignol couples overwhelmingly at its β-position to the growing polymer at available 4-O-(G or S) or 5-(G only) positions. Branching is introduced by 5-O-4-or 5-5-coupling reactions between preformed oligomers, with the latter occurring only between G units and the former involving at least one G unit. Evidence continues to accumulate, particularly from studies of the lignin biosynthetic pathway using mutant and transgenic plants, that lignins can incorporate monomers beyond the traditional three monolignols. As reviewed previously [8][9][10][11], these include (but are not limited to) the novel caffeyl and 5-hydroxyconiferyl alcohols from incomplete methylation in monolignol biosynthesis (OMT-deficient plants); the immediate hydroxycinnamaldehyde monolignol precursors, coniferaldehyde and sinapaldehyde, and the hydroxy-benzaldehydes (vanillin and syringaldehyde) derived from them from incomplete reduction (CAD deficiency); and products such as dihydroconiferyl alcohol and its derived guaiacylpropane-1,3-diol in wild-type and mutant softwoods. Relatively little is known about the nature of p-hydroxybenzoylated lignins or how they arise. From oil palm trunk, nitrobenzene oxidation produced high levels of p-hydroxybenzoic acid (9.8-14.9 %) [37], which was also the main product of alkaline hydrolysis of parenchyma milled wood lignin (MWL) [31]. Small quantities of vanillic and syringic acids were also found. The authors concluded that the phenolic acids acylated the side-chain alcohol groups of lignin subunits. High levels of p-hydroxybenzoic acid were also released from oil palm lignins [42][43][44][45][46]34]. The 13 C-NMR chemical shifts in Salix (willow) MWL samples showed good concordance with expected shifts for free-phenolic γ-phydroxybenzoate esters based on model compound studies [28]. Rather than suspecting that the lignin side-chains were acylated after the polymer had already formed, p-hydroxybenzoyl monolignol conjugates were proposed as the precursors to these structures in early research [47]; monolignol acylation was further conjectured [25] and eventually proven by the observation of novel β-β-coupling products in lignins that could only have arisen from pre-acylated monolignols [29,14,48,32]. Recent studies have delivered the transferases p-coumaroyl-CoA:monolignol transferase (PMT) in grasses responsible for lignin p-coumarate esters via p-coumaroylation of monolignols [22,23,49,50] and now an exotic feruloyl-CoA:monolignol transferase (FMT) that feruloylates monolignols prior to lignification [27]; the putative p-hydroxybenzoyl-CoA and acetyl-CoA monolignol transferases have not yet been identified. The research presented in this paper seeks to answer several key questions regarding the intricacies of acylation of lignins by p-hydroxybenzoate, specifically: whether p-hydroxybenzoates exist as free-phenolic appendages on lignins, akin to the p-coumarates; whether they, like p-coumarates [51,52,23,49] and acetates [18] in most plants, primarily acylate S units; whether the various lignin substructures carry these units and thus implicate the incorporation of preformed monolignol γ-p-hydroxybenzoate conjugates into the lignification process; and whether diagnostic β-β-coupled units can be found to provide proof that lignification utilizes preformed monolignol p-hydroxybenzoate conjugates, thus, also establishing that monolignol p-hydroxybenzoate conjugates are lignin precursors. Although a survey across all palms is beyond the scope of this work, a sago palm lignin previously examined by analytical pyrolysis [35] and prior studies on coconut (Cocos nucifera) coir fibers [36] are used to suggest a degree of generality in palms. Together, such studies will help to elucidate the roles of putative genes and enzymes associated with the required transferase activity, knowledge that may clarify the poorly understood reasons for widespread lignin acylation in the plant kingdom. At the same time, it will also shed light on future paths that may be worth exploring for value-added products making lignocellulosic biofuels a more viable option; depending on the level of p-hydroxybenzoates found in a given biomass material, and the ease with which it can be cleaved and recovered from the polymer, p-hydroxybenzoic acid or its derivatives may be accessible value-added products [34,31]. A simple net search shows that p-hydroxybenzoic acid currently sells for~$US4,000/ton and has uses: as a source of esters that have antibacterial and antifungal properties, as an intermediate in pesticides and antiseptics, and for preparation of co-polyesters (including liquid crystal polymers). General Commercial chemicals, including solvents, were of reagent grade or better and used without further purification. Analytical thin-layer chromatography (TLC) was carried out on EM Science TLC plates pre-coated with silica gel 60F 254 ; TLC visualization was by UV. Purification by flash column chromatography was accomplished on silica gel (230-450 mesh). 4-Acetoxybenzoyl chloride pBz Cl was prepared according to a previous procedure [53]. Nuclear magnetic resonance (NMR) data and assignments for all model compounds are from welldescribed 1D and 2D NMR experiments (on a Bruker Biospin Avance 500 MHz spectrometer). High-resolution mass spectrometric (HRMS) data were acquired using electrospray ionization (ESI) on a Waters (Micromass) LCT ESI/TOF MS instrument. UV-vis analysis performed in 1-cm quartz cuvettes at λ=280 nm on a Shimadzu UV-1800. Plant Materials Palm Samples The African oil palm (Elaeis guineensis) fractions used in this study came from two sources. The first sample, consisting of oil palm fronds and empty fruit bunch (EFB) fibers, was supplied by the Forest Research Institute of Malaysia and is the same sample used in a previous study by Sun et al. [45] and will be denoted here as EFB-1. The second sample of oil palm EFB fibers was provided by the Ladang Tai Tak Sdn. Bhd. Palm oil mill in Kota Tinggi, Johor, Malaysia, and will be denoted here as EFB-2. These EFB fibers were steam-treated (45 psi, 130°C, 70 min) to remove the fruitlets and then shredded into fibers (4-6 in in length). Various data on both oil palm EFB samples are presented in Table 1. Isolated sago palm (Metroxylon sagu) lignin was the material previously described in a pyrolysis study [35]. Sample Preparation and Lignin Isolation All fibrous oil palm samples were dried at 50°C in an oven for at least 72 h before being ground. Two-step grinding was performed in a Wiley mill using a 2-mm followed by a 1mm mesh. Removal of extractives from the samples was carried out by successive extractions with water, ethanol, acetone, and chloroform. Extractions were accomplished by refluxing the material in Soxhlet equipment for at least 8 h with each solvent. Prior to analysis, extracted samples, frequently referred to as whole-cell-wall preparations, were dried for 2 weeks in a desiccator under vacuum using P 2 O 5 as drying agent, which was replaced regularly. The dried materials were ball-milled using a stainless steel vibratory ball mill for 1.5 h (with 0.5 h on, 0.5 h off, to minimize heating). The ballmilled materials (50 g) were submitted to enzymatic digestion with crude cellulases (Cellulysin, Calbiochem) for polysaccharide degradation [25,54,55]. As with aspen and poplar, the enzymatic treatment was particularly efficient at removing polysaccharides, leaving just 22.2 and 20.9 % of the cell wall from the EFB and frond samples, respectively. Samples of the enzyme lignins obtained (10 g) were then extracted with dioxane and water (96: 4) in complete darkness at room temperature for 2 days [56]. The soluble fractions were obtained by filtration and lyophilized overnight. The isolated lignins were washed extensively with water and 6 mM EDTA aqueous solution (pH 8). This removed as much of the remaining low-molecular-weight saccharides and metal ions as possible [25]. The washed Björkman lignins were lyophilized again overnight and stored. The yields of isolated EFB lignin used in a previous study by Sun et al. [45] were 19.9 % of the original cell wall material. As seen in Table 1, the AcBr lignin contents, measured with ε 280 =17.9, as previously described [57,58], of the EFB-1 fibers were 22.7±0.4 %, giving a MWL yield on a lignin basis of~88 %. The pBz content of the MWL was 4.2±0.1 %. For the EFB-2 sample, the AcBr lignin content was 18.3±0.2 %. Isolated lignins were acetylated using acetic anhydride and pyridine as previously described [59]. Solutions of the acetylated lignins in EtOAc were washed with 6 mM EDTA to more rigorously remove metal ion contaminants following methods described previously [25]. NMR of Lignins Spectra have been run over many years on a variety of Bruker instruments, including DP 360 and Avance 400, 500 and 600 MHz instruments, using standard experiments and conditions [13]. The 3D total correlation spectroscopy-heteronuclear single-quantum coherence (TOCSY-HSQC) experiment was run as described previously [60,55]. Derivatization Followed by Reductive Cleavage (DFRC) Method Adapted for the Analysis of Monolignol p-Hydroxybenzoate Conjugates The derivatization followed by reductive cleavage (DFRC) method [61,62] was adapted for HPLC analysis, as the monolignol p-hydroxybenzoate conjugates were found to be incompatible with GC-MS analysis. The modified procedure is detailed below. In a 2-dram vial equipped with a polytetrafluoroethylene (PTFE) pressure-release cap and containing a stir bar was suspended each extract-free EFB whole-cell-wall sample (50 mg) in a 20 % solution of acetyl bromide in acetic acid (5 mL). The suspension was heated at 50°C and allowed to stir for 3 h. The solvent was removed on a SpeedVac (Thermo Scientific SPD131DDA, 50°C, 35 min, 1.0 Torr, 40 Torr/min, RC lamp on). The crude film was suspended in absolute ethanol (1 mL), and the ethanol was then removed on the SpeedVac (50°C, 15 min, 6.0 Torr, 30 Torr/min, RC lamp on). To the residue was added a mixture of dioxane:acetic acid:water (5/4/1v/v, 5 mL) and nano-powder zinc (250 mg). The sample was sonicated to ensure that the biomass film was dissolved and the zinc was in suspension. The reaction was stirred in the sealed vial in the dark at room temperature for 16-20 h. Additional nano-powder zinc was added to the vial as required to maintain a fine suspension. The reaction was quenched with a mixture of dichloromethane (DCM, 6 mL), saturated ammonium chloride (15 mL), and internal standard (ISTD, diethyl 5-5′-diferulate diacetate, DEDF, 54.0 μg). After isolating the organic fraction using a separatory funnel, the aqueous phase was extracted with DCM (3×10 mL). The combined organic fractions were dried over anhydrous sodium sulfate, filtered, and the solvent evaporated in vacuo. The free hydroxyl groups were acetylated by adding pyridine/acetic anhydride (1/1v/v, 5 mL) and allowing the mixture to sit in the To remove most of the polysaccharide-derived products, the crude DFRC product was dissolved in ethyl acetate (EtOAc, 0.15 mL), diluted with hexanes (0.15 mL), and loaded onto an SPE cartridge (Supelco Supelclean LC-Si SPE tube, 3 mL, P/N: 505048). The products were eluted with hexanes:ethyl acetate (1:1, 8 mL), and the solvent was removed on a rotary evaporator. The product was dissolved in ethyl acetate and filtered through a 0.2-μm PTFE filter into a high-performance liquid chromatography (HPLC) vial. Analysis of the purified products was performed on an HPLC-PDA-ESI-MS (Shimadzu LC-MS 2020) with an amino stationary-phase column (Phemonenex Luna NH 2 , 100 Å, 250 mm×4.60 mm×5 μm). The mobile phase was hexanes and EtOAc with 0.1 % triethylamine, running at 0.7 mL/min, holding at 30 % EtOAc for 1 min then ramping at 1.2 %/min up to 95 % EtOAc. The column eluent was split 80:20 between a PDA detector (265-400 nm) and an ESI-MS detector operating in positive-ion mode. External synthetic standards of the predominantly trans-isomers were used for determination of the retention time, calibration of the PDA response, and authentication of each compound by MS, Table 2. The DFRC data is also summarized in Table 1. Syntheses of γ-p-Hydroxybenzoylated β-Ether Model Compounds As we determined by NMR and DFRC that p-hydroxybenzoates acylated essentially only S units, the prime model compounds of interest were the etherified and the free-phenolic S β-ether γ-phydroxybenzoate models 7 and their peracetates 8. These model compounds were prepared following the synthetic route shown in Scheme 1. Preparation of 4-Acetoxy-3,5-dimethoxyacetophenone 1 To a solution of the starting acetophenone (3,5-dimethoxy-4-hydroxy-acetophenone, 51.0 mmol, 10.0 g) in pyridine (30 mL) was added Ac 2 O (30 mL). The solution was allowed to stir overnight at room temperature. Upon confirmation by TLC that the starting material had been fully consumed, the reaction mixture was diluted with EtOAc (300 mL) and washed successively with 1 M HCl (3 × 150 mL), sat. NaHCO 3 (1×150 mL), and sat. NaCl (1×100 mL). The organic layer was dried over Na 2 SO 4 and concentrated in vacuo. Residual AcOH was removed by azeotropic distillation with toluene (3×50 mL), followed by removal of the toluene with MeOH co-distillation (2×50 mL). Recrystallization from 20 % EtOAc in Et 2 O afforded 1 (11.0 g, 91 %) as a pale yellow crystalline solid: 1 Bromination of compound 1 (preparation of compound 2) Compound 1 (46.0 mmol, 11.0 g) was dissolved in EtOAc (460 mL), and the reaction solution was cooled to 0°C. Pyridinium tribromide (55.4 mmol, 20.0 g) was charged into the reaction flask incrementally over 10 min, and the reaction was allowed to stir for 2 h, with the temperature gradually warming from 0°C to room temperature (23°C). If any starting material remained detectable by TLC at that point, additional portions of pyridinium tribromide (in increments of approximately 3 mmol, or 1 g) were added until TLC analysis showed full conversion. The reaction was then quenched with a solution of sat. NaHCO 3 (400 mL) and, after separation, the aqueous layer was extracted with DCM (1×100 mL). The combined organic layers were then washed with 1 M HCl Table 2 HPLC-PDI-ESI-MS elution parameters and observed mass-ion-ratios for parent or fragment ions from synthetic standards CA, SA, CA-pBz, SA-pBz, and internal recovery standard, DEDF (see Fig. 4 Procedure for 4-acetoxybenzoylation of the γ-OH in the β-O-4-linked dimers 4 (illustrated for preparation of compound 5 SG-pBz ) Alcohol 4 SG (1.3 mmol, 508 mg) was dissolved in dichloromethane (13 mL), and the resulting solution was cooled to 0°C. DMAP (0.33 mmol, 40 mg) and Et 3 N (3.9 mmol, 0.54 mL) were charged into the reaction mixture, followed by 4-acetoxybenzoyl chloride (pBzCl; 1.6 mmol, 318 mg). The reaction was allowed to warm to room temperature gradually with continued stirring for approximately 2 h. Once TLC showed no detectable starting material remaining, the mixture was poured into 1 M HCl (50 mL). After separation, the aqueous layer was extracted with DCM (1×30 mL), and the combined organics were washed with sat. NaCl (1× 50 mL). They were then dried over Na 2 SO 4 and concentrated in vacuo. Purification by flash column chromatography (50 g SiO 2 , gradient elution from 5 to 50 % EtOAc/hexane) gave ester 5 SG-pBz (547 mg, 76 %) as a white foam: 1 Typical procedure for reduction of α-ketones 5 (illustrated for preparation of the compound 6 SG-pBz ) Ester 5 SG-pBz (0.43 mmol, 250 mg) was dissolved in EtOAc (4.4 mL) and MeOH (0.4 mL), and the reaction mixture was cooled to 0°C. NaBH 4 (0.86 mmol, 33 mg) was charged to the flask and allowed to stir continuously for approximately 1 h. After confirming complete consumption of the starting material by TLC, the organics were diluted with EtOAc (25 mL), washed with 1 M HCl (1×30 mL) and sat. NaCl (1×30 mL), dried over Na 2 SO 4 , and concentrated in vacuo. The resulting clear, viscous oil 6 SG-pBz was used crude in the final deprotection, with an assumed 100 % yield; products 6 SG-pBz , 6 SS-pBz , 6 SG-OAc , and 6 SS-OAc were formed and used in the same way. Typical procedure for deprotection of phenolic acetates (illustrated for preparation of the compound 7 SG-pBz ) Acetates were removed essentially as in the recently described method [53]. Benzyl alcohol 6 SG-pBz (0.32 mmol, 175 mg) was dissolved in DMF (1.3 mL). Hydrazine acetate (1.1 mmol, 103 mg) was charged to the flask, and the reaction was allowed to stir at room temperature for 1 to 3 h. The solution was diluted with EtOAc (50 mL) and washed with H 2 O (5× 15 mL) and NaCl (1×10 mL). After drying over Na 2 SO 4 , the organics were evaporated under reduced pressure. The resulting crude oil was purified by flash column chromatography (10 g SiO 2 , gradient elution using either 10 to 70 % EtOAc/hexane or 1 to 10 % MeOH in DCM) to afford 7 SG- Typical procedure for peracetylation (illustrated for preparation of compound 8 SG-pBz ) Compound 7 SG-pBz (10.0 mg, 20 μmol) was charged into a small amber vial, followed by addition of a solution containing equal parts pyridine and acetic anhydride (500 μL each). The resulting solution was allowed to stir overnight, at which point, all organic solvents were removed in vacuo to yield 8 SG-pBz as a clear, colorless oil: 1 H NMR (CDCl 3 ) δ 7.98-7.94 (m, 2.7H, 2 C,e /6 C,e , 2 C,t /6 ,C, γOAc e ); 13 C NMR (acetone-d 6 ) Note that compounds 8 SG-OAc and 8 SS-OAc , if they are the desired final products, are obviously directly available simply by reducing and acetylating compounds 4 as previously reported [64]. Syntheses of model compounds 9-11 Model compounds for 9a, 10a, and 11a (Fig. 5) in which the 4-O-etherification is simply via a 4-O-methyl group were synthesized from sinapyl 4-acetoxybenzoate and sinapyl alcohol by peroxidase-H 2 O 2 oxidation, followed by separation of the individual products, methylation (MeI-K 2 CO 3 , acetone), and acetylation (Ac 2 O-Py). Analogous model compounds for 9b, 10b, and 11b were obtained in the similar way starting from sinapyl acetate and sinapyl alcohol as previously described [14]. Results and discussion The following sections address the various issues posed in the introduction regarding lignin p-hydroxybenzoylation in palms. The Nature of p-Hydroxybenzoates and Acylation Regiochemistry Determining whether p-hydroxybenzoates are in fact freephenolic entities acylating the γ-positions of lignin sidechains is most readily deduced from solution-state NMR of isolated lignins, analogously to similar determinations for pcoumarates [25]. Björkman milled wood lignins (MWLs) were prepared from the oil palm (Elaeis guineensis) EFB fibers, fronds, and trunk fractions; the EFBs had already been shown to be particularly rich in p-hydroxybenzoates [34]. Simple saponification of EFB whole-cell-wall (WCW) samples indicated that the EFB-1 and EFB-2 samples contained 1.09 and 1.05 wt% p-hydroxybenzoic acid or, based on AcBr lignin contents of 22.7 and 18.3 %,~4.8 and 5.8 % of the lignins ( Table 1). NMR of the underivatized (i.e., un-acetylated) isolated lignin was made difficult due to the high levels of iron in this sample, as previously reported [65]; unfortunately, we were not able to sufficiently effect its removal by complexation with EDTA. Nevertheless, 13 C NMR (Fig. 1) readily revealed the presence of p-hydroxybenzoates, assumed at this point to be on lignins. It is worth noting that these signals have sometimes been misinterpreted as arising from phydroxyphenyl lignin units or p-coumarates [43]. Acetylation of the isolated lignin facilitated removal of the metal contaminants. The observed changes in chemical shifts of the aromatic carbons following acetylation indicated that phydroxybenzoates were present overwhelmingly in their free-phenolic form on the lignin, Fig. 1c-d. The sago palm lignin, used in a previous study in one of our labs [35], had similar features, Fig. 1a; in fact the p-hydroxybenzoate level was highest on this particular lignin fraction. Also striking, and not observed in previous studies, was the high level of natural acetylation of the EFB lignin, Fig. 1b-c. The presence of natural acetates on lignins is frequently missed for a variety of reasons, including that lignins are routinely peracetylated to improve NMR properties, extraction conditions may hydrolyze the acetates [35], and lignin purification steps involving acetic acid can introduce artifactual acetates. Circumventing some of these issues, solid-state NMR spectra of various palm fractions indicate the presence of acetates, Fig. 1b. Furthermore, acetates are clearly observable in spectra from other published reports, where they have typically been attributed to polysaccharides [66,42]; historically, the contribution from lignin may have therefore been overlooked. The gradient-enhanced 2D HSQC NMR experiment provides a highly detailed view of the molecular structure of plant cell walls with minimal processing of the sample [67]. The fast relaxation caused by the metals lead to extremely poor spectra, Fig. 2a-b, but the chemical shift data are consistent with acylation of the lignin. The p-hydroxybenzoate (pBz) groups are revealed in the aromatic regions of Fig. 2b, along with the G and predominant S aromatics. The side-chain region, Fig. 2a, offers good evidence that lignin γ-hydroxyls, and not the alternative α-hydroxyls, are acylated; acylated β-ether α-H/C correlations, for example, would be seen at~6.2/76 ppm. After peracetylation of the lignins, followed by successive extraction into chloroform and washing with EDTA in order to remove metal contaminants, higher-resolution spectra, Fig. 2c-d, are obtained. At this point, the nature of the acylation (i.e., γvs. α-regiochemistry) is masked, and distinguishing acylation patterns indicative of p-hydroxybenzoates from those of acetates is less direct; this issue is somewhat ameliorated by the considerably higher resolution of the spectra. The shifts in the p-hydroxybenzoate HSQC correlations (especially pBz 3/5 ) following acetylation (Fig. 2d) concur with earlier observations regarding the 1D 13 C spectra, namely, that p-hydroxybenzoates on lignins exist in their free-phenolic form and are not etherified; were they etherified, the pBz group would not become acetylated and their correlations would be essentially unchanged from those in the unacetylated lignins (Fig. 2b). p-Hydroxybenzoates are therefore confirmed to be freephenolic pendant entities acylating the γ-hydroxyls of lignin side-chains. Additional proof is established below. Which Lignin Units are p-Hydroxybenzoylated? Next, we needed to establish which units in lignins were acylated by p-hydroxybenzoates. Consistent with observations on p-coumarates and acetates, were p-hydroxybenzoates largely on S units, and were they on a range of lignin unit types? In this case, NMR of the acetylated lignin derivatives could answer both of these questions as, fortuitously, the αprotons in acetylated γ-p-hydroxybenzoylated β-ether units B were rather well resolved from the normal acetylated β-ether units A (Fig. 2c). The 1D proton projection on the HSQC spectrum in Fig. 2c indicates how well the α-protons in the major β-ether units are resolved by proton NMR. The γ-p-hydroxybenzoylated βether units B have α-proton/α-carbon correlations at 6.20/ 76.8 ppm (threo) and 6.19/75.7 ppm (erythro), whereas those from the normal acetylated units A are at 6.06/76.8 ppm Fig. 1 1D 13 C NMR spectra of various palm lignins. a Solution-state spectrum from a sago palm MWL isolated as described previously [35]. b Solid-state (200 MHz) spectrum and c solution-state spectrum of MWL isolated from the oil palm empty fruit bunches (EFB) previously characterized [34,43]. d Solution-state spectrum of acetylated EFB isolated lignin (Ac-MWL). The solution-state spectra were run at 400 MHz (100 MHz 13 C); the acetylated MWL is in acetone-d 6 , underivatized MWLs in 9:1 acetone-d 6 :D 2 O. Red-colored peaks labeled pBz are for carbons in the p-hydroxybenzoyl moiety. The red lines between plot d and its axis denote the chemical shifts from model compound 8 SS-pBz . S syringyl unit. The side-chain regions are quite disperse but generally labeled α, β, and γ. γ-OpBz signifies p-hydroxybenzoylated γ-alcohols, γ-OAc is (naturally) acetylated γ-alcohols. Cyan-colored peaks in b-c indicate the natural γ-acetylation of these lignins; no acetates appear in the sago palm lignin in a presumably due to the isolation method [35]. Spectra of lignins isolated from other parts of the oil palm plant (trunk and fronds, not shown) are almost indistinguishable from those shown in c-d (threo) and 6.02/75.4 ppm (erythro). Initially, the α-1 H-13 C correlation contours were not sufficiently resolved, but running higher-resolution spectra restricted to acquiring just the side-chain region resulted in sufficient resolution to readily distinguish the various correlations (Fig. 2c). It is apparent from the aromatic region of HSQC spectra that the EFB lignin was S-rich, Fig. 2b, d. An S:G ratio of 83:17 can be derived from the data in Fig. 2d by volume integration of the S 2/6 and G 2 aromatic proton/carbon correlations. In support, the side-chain region (Fig. 2c) revealed very low levels of phenylcoumaran (β-5) and dibenzodioxocin (5-5/β-O-4) units that must involve G units-their contours appear below the levels plotted. Distinguishing whether p-hydroxybenzoates are attached to S or G units requires long-range 1 H-13 C correlation experiments [13]. The heteronuclear multiple bond correlation (360 MHz); a side-chain region, b aromatic region. The unacetylated material contained magnetic material, presumably iron compounds, that could not be effectively washed out; the spectra are therefore extremely broad, but a still clearly shows the degree to which γhydroxyls are acylated, either by acetate or p-hydroxybenzoate. c-d The EDTA-washed acetylated EFB MWL (500 MHz cryoprobe instrument); c side-chain region, d aromatic region. These much sharper spectra resolve the α-protons (and the corresponding correlations) of γ-phydroxybenzoylated B from γ-acetylated A units. e-g HSQC slices (F2-F3 dimension) from a 3D TOCSY-HSQC experiment on acetylated EFB lignin over a small proton chemical shift range in the F1 dimension: e 6.29-6.19 ppm (α-protons in γ-p-acetoxybenzoylated β-ether units B), f 6.13-6.07 ppm (α-protons in normal β-ether units A, both syn-and antiisomers), and g 6.04-5.98 (α-protons in normal β-ether units A, mainly anti-isomers). The colored symbols on the red contours in e are shifts from model compounds 8; clearly, only the SS-pBz moieties are present at significant levels in the lignin (with chemical shifts for the α-H/C correlations matching well with those from compounds syn-and anti-SS-pBz but not with those from the SG-pBz compounds; the β and γ data are not shown for the latter as they are less diagnostic). Units A are normal (acetylated) β-ether units, B are γ-p-acetoxybenzoylated β-ether units. G guaiacyl, S syringyl, pBz p-hydroxybenzoyl (HMBC) experiment can correlate a proton with carbons up to three bonds away. The simplest probe is via the resolved αprotons in β-ether units (in the acetylated lignins), which should correlate with carbons γ (3-bond), β (2-bond), 1 (2bond), and 2 and 6 (3-bond). The feature of this experiment that makes distinction of S-from G-units so elegant is that the identical 2/6 carbons in symmetrical S units resonate at about 105 ppm, whereas carbons 2 and 6 are distinctly different in the unsymmetrical G-units, resonating at 114 and 120 ppm. As shown in Fig. 3a, it is readily evident that the β-ethers in normal units A are both S (dark blue) and G (cyan), whereas it is only possible to detect γ-p-hydroxybenzoylated S units B (red). The prevalence of p-hydroxybenzoates on S over G units may be either a reflection of the control in the transferase-catalyzed acylation reaction or simply the timing of the conjugation and the levels of the two monolignols present at that time, i.e., that p-hydroxybenzoylation of sinapyl alcohol is either significantly favored over coniferyl alcohol, or that sinapyl alcohol is more prevalent at the time of acylation. The 3-bond correlations in the HMBC data between the γprotons and the carbonyl (Fig. 3b) also provide compelling proof that the p-hydroxybenzoates acylate lignin γ-positions. The HMBC experiment can also be used to identify the variety of units that are acylated. This is achieved here by examining the γ-protons correlating with the carbonyl carbon of the phydroxybenzoate moiety (Fig. 3b). However, determining the range of products is not as easy as it was for p-coumarates, as there are insufficient model compounds, and the dispersion between the unit types is limited. For example, it is currently difficult to prove that p-hydroxybenzoates acylate the phenylcoumaran (β-5) units or hydroxycinnamyl alcohol endgroups; each of those unit types is present only at low levels in this S-rich lignin. The dispersion that is afforded to the α-protons suggests that it should be possible for a 3D TOCSY-HSQC experiment to delineate whether p-hydroxybenzoates acylate both syn-(threo-) and anti-(erythro-) isomers of the β-ether units. This is because one can take 2D HSQC planes at given proton frequencies and exploit the α-proton differences between the γ-p-hydroxybenzoylated B and normal A units noted above. Similarly, one can take 2D HSQC-TOCSY planes through the α-proton region. Such 2D HSQC slices are shown in Fig. 2eg, and HSQC-TOCSY slices are shown in Fig. 3c-e, as described in the captions. It is revealed that there are syn-and anti-isomers of the normal β-ether units (Figs. 2f-g and 3de), with anti-isomers predominating because of the S-rich nature of the lignin, as already well established [68][69][70][71][72]. It is equally clear that p-hydroxybenzoates acylate both of the βether isomers (Figs. 2c and 3c). A comparison of the α-H/C correlation peak data for the lignin versus the overlaid data from the various model compounds (Fig. 2e) indicates that, in addition to the pBz's being almost entirely on S units as noted above, the data for the two SS-pBz isomers match very well, but those for the β-O-4-G analogs SG-pBz do not. Similarly, the data match well for the same SS compounds and not (especially for the β-H/C correlations) for the SG analogs (Fig. 3c). As in the case made early on for p-coumarates acylating grass lignins [25], the presence of p-hydroxybenzoates indiscriminately on both isomers of β-ether units suggests that they arise by lignification with preformed monolignol p-hydroxybenzoate conjugates. Determination of Regiochemistry and Quantification by DFRC Proving γ-regiochemistry (although not its exclusivity) and the S versus G nature of the units involved with p-hydroxybenzoates is elegantly made by the DFRC (derivatization followed by reductive cleavage) assay [61,62], Fig. 4a. This is possible as DFRC advantageously cleaves lignin βethers but leaves esters fully intact [62,51,18,23,27]. Unfortunately, the acetylated coniferyl and sinapyl phydroxybenzoate products anticipated from this method do not survive GC analysis conditions, so the products were analyzed by HPLC-ESI-MS, Fig. 4b. Quantification of the DFRC-released diacetates of coniferyl alcohol (CA), sinapyl alcohol (SA), coniferyl p-hydroxybenzoate (CA-pBz), and sinapyl p-hydroxybenzoate (SA-pBz) accounted for 0.9, 2.0, 0.1, and 1.3 wt% of the EFB-1 sample ( Table 1). The monolignol component, uncorrected for recovery and losses, accounted for some 16.7 % of the lignin. The DFRC-released total S to G monomer ratio was 73:27, whereas the ratio of SA-pBz to CA-pBz was 96:4, confirming the NMR observations that the p-hydroxybenzoates are primarily located on S units. Similar data were obtained for the EFB-2 sample ( Table 1). Proof that Lignin p-Hydroxybenzoates Arise via Lignification with Preformed Monolignol Conjugates All of the aforementioned data strongly suggest, but strictly do not prove, that the p-hydroxybenzoate groups inherent on these palm lignins arise from lignification incorporating preformed monolignol p-hydroxybenzoate conjugates. As we have described previously for acetates (e.g., on kenaf lignins), there is a way to provide compelling proof [62,14]. Given that post-coupling rearomatization of β-β-coupled quinone methide lignification intermediates is the one reaction that is perturbed by the presence of an acylated γ-OH, observing novel acylated β-β-coupling products in the lignin provides convincing evidence that acylated monolignols are involved in lignification [14]. As seen in Fig. 5, evidence for all three types of products 9a-11a indicating the involvement of sinapyl p-hydroxybenzoate monomers in lignification is provided by observing sets of signature H/C correlations in HSQC spectra, correlations that well match the data from synthesized model compounds for all of the structures 9a-11a. These units include both those arising from the cross-coupling of sinapyl phydroxybenzoate with sinapyl alcohol (9a, 10a) as well as the homo-dimerization of sinapyl p-hydroxybenzoate (11a). These spectra also provide evidence (with correlations for structures 9b-11b) that sinapyl acetate is a monomer conjugate in this palm EFB lignin, as was previously observed in kenaf [14]. Evidence from metabolite profiling of poplar wood for novel β-β-coupled heterodimers between sinapyl alcohol and sinapyl p-hydroxybenzoate [29,32] suggests that it is safe to contend that all lignin p-hydroxybenzoates, across the plant taxa that have them, derive from lignification using monolignol p-hydroxybenzoate conjugates. The evidence is therefore sufficiently compelling that currently unknown transferases are responsible for producing monolignol phydroxybenzoate conjugates that are used as 'monomers' for lignification in palms (as well as in willows, poplars, and aspen). The levels of p-hydroxybenzoates on lignins should be manipulable by normal methods of selection and breeding or by misregulating such genes, once found. If p-hydroxy benzoic acid or its derived products are sufficiently valuable as commodities and coproducts generated from a biomass conversion process, a long-term strategy might be to upregulate the p-hydroxybenzoate levels on lignin in target plants. As determined here, only some 23 % (Table 1) of lignin monomers are p-hydroxybenzoylated in palm EFBs, and the level is likely to be lower in poplar and willow, so there appears to be significant potential for its increase. Other factors being equal, for example, if all of the monolignols destined for lignification were p-hydroxybenzoylated, the amount of p-hydroxybenzoic acid available would be approximately 40 wt% of the lignin or roughly 8 wt% of the EFB fiber. Conclusions Our data provide compelling evidence that, as for acetates on kenaf (and many other plants) lignins and p-coumarates on grass lignins, p-hydroxybenzoates acylate the γhydroxyl of lignin side-chains and are found mainly on S units. Furthermore, like their p-coumarate analogs, the phydroxybenzoate moieties are not subjected to significant levels of radical coupling, or other etherification, during lignification and therefore remain overwhelmingly as freephenolic pendant units on lignin unit side-chains and can therefore be easily cleaved off to deliver a single compound in quite pure form. As has been demonstrated for p-coumarates, and as there is no a priori reason that p-hydroxy-benzoates can not undergo radical coupling, the logical reason is that such units are preferentially subject to radical transfer reactions to the G and S monomer and polymer phenolics present in the radical-limited lignification process [12,73]. The data presented here also provide evidence that, analogously to the p-coumarates and acetates found acylating lignin units, p-hydroxybenzoate units arise from lignification using the preformed monolignol p-hydroxybenzoate conjugates and, at least in these palm lignins, are almost entirely derived from sinapyl p-hydroxybenzoate. It is now logical to contend that any of the acylation products seen in any of the native lignins is via acylation at the monomer level and that post-polymerization (or postcoupling) acylation reactions are unlikely. We therefore Table 2 and elsewhere. The retention time, UV-vis spectrum, and mass spectrum of each compound were authenticated using external synthetic standards expect that p-hydroxybenzoyl-CoA:monolignol transferases are involved in lignification in the various willows (Salix spp.), poplars and aspen (Populus spp.), and palms (family Arecaceae) that have p-hydroxybenzoylated lignins, and that identification of the genes involved will allow p-hydroxybenzoate levels on lignin to be manipulated as recently demonstrated for p-coumarates [23]. To date, however, the in planta pathway to p-hydroxybenzoate (or its CoA derivative) remains ambiguous. If p-hydroxybenzoic acid becomes a viable coproduct from cellulosic bioenergy lignin streams, it could conceivably be upregulated or even introduced into other biomass plants. Right now, the current palm oil empty fruit bunch 'wastes' should be able to generate a sizeable stream of relatively clean p- Fig. 5c (γ-correlations) is simply the second (lowest field, highest ppm) γ-H/C correlation from the B unit of compound 9a hydroxybenzoic acid; when available at the multiton level, industrial applications are expected to increase beyond those already in use today.

v3-fos

2018-04-03T02:41:42.196Z

2015-04-30T00:00:00.000Z

3038449

s2

Structured Literature Review of Responses of Cattle to Viral and Bacterial Pathogens Causing Bovine Respiratory Disease Complex Bovine respiratory disease (BRD) is an economically important disease of cattle and continues to be an intensely studied topic. However, literature summarizing the time between pathogen exposure and clinical signs, shedding, and seroconversion is minimal. A structured literature review of the published literature was performed to determine cattle responses (time from pathogen exposure to clinical signs, shedding, and seroconversion) in challenge models using common BRD viral and bacterial pathogens. After review a descriptive analysis of published studies using common BRD pathogen challenge studies was performed. Inclusion criteria were single pathogen challenge studies with no treatment or vaccination evaluating outcomes of interest: clinical signs, shedding, and seroconversion. Pathogens of interest included: bovine viral diarrhea virus (BVDV), bovine herpesvirus type 1 (BHV‐1), parainfluenza‐3 virus, bovine respiratory syncytial virus, Mannheimia haemolytica, Mycoplasma bovis, Pastuerella multocida, and Histophilus somni. Thirty‐five studies and 64 trials were included for analysis. The median days to the resolution of clinical signs after BVDV challenge was 15 and shedding was not detected on day 12 postchallenge. Resolution of BHV‐1 shedding resolved on day 12 and clinical signs on day 12 postchallenge. Bovine respiratory syncytial virus ceased shedding on day 9 and median time to resolution of clinical signs was on day 12 postchallenge. M. haemolytica resolved clinical signs 8 days postchallenge. This literature review and descriptive analysis can serve as a resource to assist in designing challenge model studies and potentially aid in estimation of duration of clinical disease and shedding after natural pathogen exposure. B ovine respiratory disease (BRD) continues to be an economically important disease of cattle with losses estimated as $23.60 per treated calf. 1,2 Bovine respiratory disease is a multi-factorial disease involving infectious agents, compromised host immune system, and environmental factors ultimately resulting in bronchopneumonia. The viral pathogens associated with BRD include: bovine herpesvirus type 1 (BHV-1), parainfluenza-3virus (PI3), bovine viral diarrhea virus (BVDV), and bovine respiratory syncytial virus (BRSV). Bacterial pathogens associated with BRD include: Mannheimia haemolytica, Mycoplasma bovis, Pasteurella multocida, and Histophilus somni. Viral pathogens are capable of causing primary infection that is generally associated with mild clinical signs (CS) of BRD. [3][4][5][6][7][8] An important role for BRD viral pathogens is causing immune suppression which increases susceptibility to secondary bacterial infections. 6 Both BVDV and BHV-1 are spread via aerosolization with BHV-1 able to persist in a latent state in neural tissues and recrudesce during times of stress. [5][6][7]9 Parainfluenza-3 virus and BRSV are considered to be minor contributors to BRD and are spread via aerosolization. Similar to the viral pathogens, BRD bacterial pathogens are often present as co-infections. Mannheimia haemolytica is considered the most common bacterial pathogen in beef cattle BRD and is a normal inhabitant of the nasopharynx, becoming opportunistic during stress or viral infection. 5,6,10 Mycoplasma bovis can be a primary pathogen or co-infection, with some studies showing synergism with M. haemolytica. [11][12][13] Like M. haemolytica, Pastuerella multocida and Histophilus somni are also normal flora of the respiratory tract and become opportunistic colonizers of the lung after viral infection of the respiratory tract. 6 As BRD is a syndrome, the specific pathogens involved in individual cases or outbreaks are often unknown. Management and control of BRD outbreaks is influenced by disease risk factors as well as transmission dynamics of the pathogens involved. Understanding the cattle response and infectious period associated with each pathogen can lead to a better understanding of how to mitigate negative impacts of BRD in populations. While there are numerous challenge studies using the common BRD pathogens, a resource summarizing the time from exposure to a viral or bacterial BRD pathogen to exhibition of CS, pathogen shedding, and seroconversion does not exist. The objective for this study was to perform a structured literature review of the published literature and a descriptive analysis of cattle responses (the minimum time to onset of CS, time to peak outbreak, time to resolution of CS, minimum time to shedding, time to maximum shedding, time to resolution of shedding, time to seroconversion, and time to maximum seroconversion) to challenge with common viral and bacterial BRD pathogens. Materials and Methods A structured literature search was performed using PubMed, CAB, and Agricola databases to identify studies published in English that reported cattle BRD experimental challenge models for BHV-1, BVDV, PI-3 virus, BRSV, Histophilus, Pasteurella, Mycoplasma, and Mannheimia. The search strategies and keywords are listed in Table 1. Inclusion criteria for each study included: cattle confirmed pathogen-free before challenge, single pathogen exposure model, utilization a negative control group, and challenge animals receiving no other treatment or vaccination for BVDV, BHV-1, PI-3 virus, BRSV, Mannheimia haemolytica, Mycoplasma bovis, Histophilus somni, and Pasteurella multocida. Outcomes of interest included: minimum time to onset of CS, time to peak outbreak, time to resolution of CS, minimum time to rectal temperature exceeding 40°C, time to peak rectal temperature, return of rectal temperature to less than 40°C, minimum time to shedding, time to maximum shedding, time to resolution of shedding, time to seroconversion, and time to maximum antibody titers. Only challenge models were included as time to onset of CS, shedding, and resolution times were important outcomes and challenge models provide data with specific known time of pathogen exposure. The titles and abstracts from the combined search outcomes were evaluated for inclusion and exclusion criteria. Of the pertinent abstracts, the full text was reviewed to determine inclusion or exclusion from the structured literature review based on study criteria (attached Appendix S1 lists the references considered for inclusion). A hand search was performed of included studies to ensure no additional valid studies were omitted from the search results. A study could have multiple trials within the manuscript with multiple treatment group allocation. Therefore, a published manuscript was considered a study, whereas each individual challenge pathogen was considered a trial. Other data collected included: study length, challenge inoculum route, number of calves in the trial, frequency of sample collection, and blinding status. Bovine viral diarrhea virus type 1 and 2 were analyzed together and not separated into 2 separate categories. Trial day 0 was defined as the day the pathogen challenge was administered for all included studies. Trials were included for analysis regardless of completion of all outcomes of interest (eg, data only present for CS, or CS, fever, or shedding had not resolved before completion of the trial were still included for data analysis). Seroconversion data only included trials utilizing serum neutralization to test for antibody response. For the viral pathogens, shedding was determined by trials utilizing virus isolation from nasal swabs. For the bacterial pathogens, trials that utilized PCR for determination of shedding from nasal swabs were included in the structured literature review. Data points were collected for each outcome of interest from each trial. For CS: minimum time until CS was recorded as the day postchallenge calves began showing CS (eg, at least 1 animal) for each trial, time to peak outbreak recorded as the day the highest number of cattle were affected with CS for each trial, and the resolution of CS recorded as the day all cattle were asymptomatic for each trial (with the exception of outliers as reported and determined by the published trial when present). For rectal temperatures: minimum time until rectal temperature exceeded 40°C was recorded as the day postchallenge the mean rectal temperature of challenge calves was equal to or greater than 40°C for each trial, time to peak rectal temperature was the day the calves mean rectal temperature was the highest, time to resolution of rectal temperature less than 40°C was recorded as the day the calves mean rectal temperature was less than 40°C. For pathogen shedding: minimum time to shedding was recorded as the day calves began to shed the pathogen postchallenge (eg, at least 1 animal) for each trial, time to maximum shedding was recorded as the day the most calves with the highest titers obtained, time to resolution of shedding was recorded as the day all calves ceased pathogen shedding for each trial. Time to seroconversion was recorded for each trial as the day at least 1 calf has seroconverted, and time to maximum antibody titers was recorded as the trial day the challenge calves had the highest titer. A weighted mean accounting for the number of calves present in each study was utilized and descriptive statistics were performed to analyze the data. Box and whisker plots were produced summarizing the data points from each trial for each pathogen. Results After evaluation of article titles, abstracts, and then complete review of subsequent manuscripts, a total of 35 studies and 64 trials were included in the descriptive analysis. Table 2 shows the number of papers included for each pathogen during each stage of evaluation. No additional study was included after a hand search of references cited in included articles. All included studies were in the PubMed and CAB databases. No single trial contained all the desired areas of interest for structured literature review. Therefore, Table 3 demonstrates the number of trials that had data present and were analyzed for each area of interest. Bacterial shedding data were excluded from the analysis because of insufficient data present (only present for 1 study for M. bovis). Bovine Viral Diarrhea Virus We identified 12 BVDV trials from 8 studies for inclusion in the analysis. 3,[14][15][16][17][18][19] Table S1 summarizes the studies that were reviewed and analyzed. Blinding was reported for 9 of the trials. The mean trial duration was 15.5 days (range 9-27 days) with a mean of 10.2 calves (range 4-16 calves) included in each trial. Type 1 and type 2 BVDV were included in the same category for analysis. Type 1 BVDV was used as the challenge pathogen for 6 trials. Two trials utilized BVDV type 2 and 4 trials did not specify the BVDV type. Eight trials challenged the calves intranasal with the BVDV chal-lenge pathogen. Three trials challenged via aerosolized administration and 1 trial used a combination of intranasal and aerosolized for challenge exposure. The median for the minimum number of days until BVDV shedding was 2 days (range 1-5 days) for the 8 trials that reported time until CS started postchallenge (Fig 1). The median day for peak of BVDV shedding occurred at 7 days (range 4-9 days) postchallenge with resolution at 12 days (range 11-12 days) postchallenge. Median day that rectal temperatures began exceeding 40°C was 4 days (range 2-8 days) postchallenge. Rectal temperature peaks occurred with a median at 7 days (range 7-8 days) postchallenge with median time to resolution occurring 10 days (range 7-11 days) after challenge. Median time to onset of CS was 2 days (range 1-6 days) postchallenge. Median time to peak outbreak occurred 8 days (range 5-12 days) postchallenge with median days to resolution being 15 days Nine trials and 7 studies were included for BHV-1 analysis. 7,9,14,17,[20][21][22] Of the 9 trials, 3 reported using blinding with the other 6 trials either not being blinded or blinding status was not reported. The mean study duration was 33.1 days (range 14-55 days) with a mean of 6.2 study calves (range 3-14) for each trial. All trials utilized BHV-1 for the challenge model. Five trials utilized intranasal administration for the BHV-1 challenge and 4 trails challenged with aerosolization. Table S2 summarizes the studies that were reviewed and analyzed. The median time until BHV-1 began shedding was 2 days (range 1-3 days) (Fig 2). Median time for peak shedding occurred on day 4 (range 2-6 days) postchallenge for BHV-1. The median time until shedding of BHV-1 ceased was 14 days, but spanned a time frame of as early as 7 days and as long as 17 days. Median time to rectal temperatures exceeding 40°C occurred 2 days (range 1-10) postchallenge with median time to maximal rectal temperature on day 4 (range 3-10 days) postchallenge. Median time to rectal temperatures returning to less than 40°C occurred on day 8 (range 3-10 days) postchallenge. Median time to BHV-1 seroconversion occurred on day 17.5 (range 7-28 days) postchallenge with median time to peak antibody response on day 40 (range 28-56 days). The median time until CS began on day 2 after BHV-1 exposure with a range extending from 2 to 5 days after challenge. The median time to peak outbreak occurred on day 7 (range 2-8 days) with median time to resolution of CS on day 14 (range 10-15 days). Parainfluenza-3 Three trials from 3 studies investigating PI-3 virus were included for analysis. 3,14,17 Table S3 summarizes the studies that were reviewed and analyzed. The study length was 14 days for all trials with all 3 being blinded. The mean number of calves included for each trial was For virus isolation, minimum is defined as the day when shedding was first detected, peak is when shedding was at the maximum, and resolution when shedding ceased. For rectal temperature (Temp), minimum is defined as the day when rectal temperature first exceeded 40°C, peak when rectal temperature was highest, and resolution defined as when rectal temperature was less than 40°C. For serum neutralization (Serum), minimum is defined as the day when seroconversion was first detected and peak when serum neutralization was highest. For clinical signs, minimum is defined as the day when clinical signs were first detected, peak being when clinical signs were the most severe, and resolution when clinical signs resumed normal limits. 14.6 (range 13-16 calves). One trial had the PI-3 virus challenge administered via aerosolization and the other 2 trials had both intranasal and intratracheal administration. The median time shedding of PI-3 virus began 1 day (range 1-2 days) after challenge (Fig 3). Median time to peak nasal shedding occurred 4 days (range 3-5 days) after challenge and median time until shedding resolved was day 10 (range 10-13 days). Literature review data for CS onset were only available for the minimum (when CS first appeared after challenge) which the median time occurred on day 2 (range 2-3 days) postchallenge. Only 1 trial had resolution of CS by the end of the trial (day 14). The other 2 trials did not have resolution of CS by the end of the trial and the study length was 14 days after PI-3 virus challenge for both trials. Rectal temperature and serum neutralization data were only available for 1 trial; therefore, these outcomes were excluded for the structured literature review for PI-3 virus. Bovine Respiratory Syncytial Virus Investigations of BRSV for this review included 22 trials with 15 studies. 3,4,14,17,23-33 Table S4 summarizes the studies that were reviewed and analyzed. The mean study length was 15 days (range 6-42 days) with 12 trials being blinded and 10 trials either not blinded or blinding was not reported. The mean number of calves in each trial was 8 (range 4-15). Nine trials challenged with BRSV via aerosolization, 7 with intranasal challenge, and 6 with a combined intranasal and intratracheal method. Bovine respiratory syncytial virus median time to CS began on day 3 (range 1-6 days) postinoculation with time to peak median outbreak occurring on day 6 (range 2-11 days). Median time to resolution did not occur until day 12 (range 7-17 days) postinoculation. Median time rectal temperatures exceeded 40°C was day 5 (range 1-7 days) and median time to maximum rectal temperature occurred on day 6 (range 5-8 days). The median time rectal temperatures returned to less than 40°C was trial day 8 (range 7-10 days) postchallenge. Median time to seroconverison for BRSV was day 9 (range 5-21 days) postchallenge using serum neutralization. Time to maximum median antibody response occurred on postchallenge day 23 (range 9-32 days). Median time to BRSV shedding began 3 (range 1-5 days) days after challenge with median time Summary of IBR trials. For virus isolation (VI), minimum is defined as the day when shedding was first detected, peak is when shedding was at the maximum, and resolution when shedding ceased. For rectal temperature (Temp), minimum is defined as the day when rectal temperature first exceeded 40°C, peak when rectal temperature was highest, and resolution defined as when rectal temperature was less than 40°C. For serum neutralization (Serum), minimum is defined as the day when seroconversion was first detected and peak when serum neutralization was highest. For clinical signs (CS), minimum is defined as the day when clinical signs were first detected, peak being when clinical signs were the most severe, and resolution when clinical signs resumed normal limits. to peak shedding on day 5 (range 3-8 days) and median time to resolution on day 9 (range 7-14 days). Figure 4 summarizes this data. Mannheimia haemolytica Five trials from 5 studies investigating Mannheimia haemolytica met inclusion criteria for this structured literature review with the mean trial length being 23 days (3-84 days). [34][35][36][37][38] Table S5 summarizes the studies that were reviewed and analyzed. Of the trials, 4 used Mannheima haemolytica type A1 for challenge induction. One trial used Mannheimia haemolytica, the type was not reported. Two of the trials were blinded, 1 trial was not blinded and 1 trial did not report if the study was blinded. Four trials had the Mannheimia haemolytica challenge administered endoscopically and 1 trial administered the challenge intratracheally. The mean number of calves in each trial was 10.4 (range 3-19 calves). Area of interest data were only present for CS and rectal temperatures. Only 1 trial reported seroconversion. All trials reported the onset of CS occurred 1 day after challenge inoculation ( Fig 5). Median time to peak CS occurred 1 day (range 1-2 days) after challenge. All trials reported resolution 8 days after inoculation. Time until rectal temperatures exceeded 40°C was reported as 1 day after challenge by all trials included for the structured literature review. Peak rectal temperatures also occurred 1 day after challenge reported by all trial with the median time until rectal temperatures returned to less than 40°C on day 2 (2-6) postchallenge. Mycoplasma bovis Investigations of Mycoplasma bovis for this review included 8 trials and 4 studies. 12,[39][40][41] Table S6 summa-rizes the studies that were reviewed and analyzed. The mean number of calves included in each trial was 15.6 (range 8-20 calves) with a mean study length of 23.8 days (range 14-28 days). Two studies were blinded, 1 study was not blinded, and 5 did not state blinding status. Seven trials performed intratracheal inoculation and 1 trial challenged intranasally. The median time to onset of CS was 1 day (range 1--4 days) postchallenge with median time to peak CS occurring on day 2 (range 2-6 days). All trials either still had ongoing CS at the end of the trial or the time to resolution of CS was not reported (Fig 6). Median time to rectal temperatures exceeding 40°C occurred 1 day (range 1-8 days) after challenge with median time to peak CS on 4.5 days (range 1-8 days). Median time to rectal temperature resolving to less than 40°C occurred on day 8 (range 5-13 days). Median time to seroconversion was 21 days (range 14-28 days) postchallenge and median time to peak antibody titers on day 28 (range 21-28 days). Pasteurella multocida One study reporting 4 trials investigating Pasteurella multocida met inclusion criteria for the structured literature review. 42 Table S7 summarizes the study that was reviewed and analyzed. Each trial had 4 calves and all calves were challenged intratracheally. Blinding was not reported for the study. The study length was 4 days for all trials. Onset of CS for Pasteurella multocida occurred 1 day after challenge for all reported trials (Fig 7). Peak CS also occurred 1 day after challenge for all reported trials with median resolution on day 2 (range 2-4 days). Rectal temperatures also exceeded 40°C on day 1 postchallenge for all trials. Maximum rectal temperatures also occurred on day 1 postchallenge for all trials with resolution on day 2 postchallenge for all trials. For virus isolation (VI), minimum is defined as the day when shedding was first detected, peak is when shedding was at the maximum, and resolution when shedding ceased. For clinical signs (CS), minimum is defined as the day when clinical signs were first detected, peak being when clinical signs were the most severe, and resolution when clinical signs resumed normal limits. Summary of BRSV trials. For virus isolation (VI), minimum is defined as the day when shedding was first detected, peak is when shedding was at the maximum, and resolution when shedding ceased. For rectal temperature (Temp), minimum is defined as the day when rectal temperature first exceeded 40°C, peak when rectal temperature was highest, and resolution defined as when rectal temperature was less than 40°C. For serum neutralization (Serum), minimum is defined as the day when seroconversion was first detected and peak when serum neutralization was highest. For clinical signs (CS), minimum is defined as the day when clinical signs were first detected, peak being when clinical signs were the most severe, and resolution when clinical signs resumed normal limits. For rectal temperature (Temp), minimum is defined as the day when rectal temperature first exceeded 40°C, peak when rectal temperature was highest, and resolution defined as when rectal temperature was less than 40°C. For clinical signs (CS), minimum is defined as the day when clinical signs were first detected, peak being when clinical signs were the most severe, and resolution when clinical signs resumed normal limits. Histophilus somni was excluded from the structured literature review because no trials were identified that coincided with met our inclusion criteria. Discussion This structured literature review serves as a resource and summary for the common BRD pathogens with regard to expected times for CS, high rectal temperature, shedding, and seroconversion after pathogen exposure. For the viruses, the relationship between resolution of CS and shedding is interesting. For BVDV, median time for CS persisted 3 days after the resolution of shedding on day 15. However, 1 trial did not have resolution of CS until 23 days postchallenge. Unfortunately, this trial did not report virus isolation For rectal temperature (Temp), minimum is defined as the day when rectal temperature first exceeded 40°C, peak when rectal temperature was highest, and resolution defined as when rectal temperature was less than 40°C. For serum neutralization (Serum), minimum is defined as the day when seroconversion was first detected and peak when serum neutralization was highest. For clinical signs (CS), minimum is defined as the day when clinical signs were first detected, peak being when clinical signs were the most severe, and resolution when clinical signs resumed normal limits. Summary of Pasteurella multocida trials. For rectal temperature (Temp), minimum is defined as the day when rectal temperature first exceeded 40°C, peak when rectal temperature was highest, and resolution defined as when rectal temperature was less than 40°C. For clinical signs (CS), minimum is defined as the day when clinical signs were first detected, peak being when clinical signs were the most severe, and resolution when clinical signs resumed normal limits. data. Both median time to BHV-1 resolution of shedding and CS occurred on day 14 after challenge. These results correlate with other summaries reporting BHV-1 shedding resolution between 10 and 17 days and peak CS occurring between 4 and 6 days. 43 Our study found peak CS at 5 days postinoculation. For BRSV, median time CS resolved 3 days after shedding ceased on day 12. Sacco summarized that viral detection is expected until 7-10 days after infection with viral detection beginning at day 2-3 which correlates with our results of resolution at 9 days and shedding beginning at day 3. 44 Besides the outlier for BVDV, CS for the viral pathogens (BVDV, BHV-1, BRSV) resolved near the time of shedding cessation or up to 3 days after shedding ceased. This information could be vital to know in regard to instituting proper quarantine periods in association with onset of BRD CS. Unfortunately, investigations of PI-3 virus, M. haemolytica, M. bovis, and P. multocida did not report complete data sets to make comparisons between shedding and CS. Generally, most induced infections whether viral or bacterial in origin reported resolution of pyrexia before all CS resolve. For BVDV, high rectal temperature resolution occurred 6 days before the cessation of CS on day 15. Median time BHV-1 and BRSV resolved high rectal temperatures was 5 and 4 days before the resolution of CS. Median time M. haemolytica resolved high rectal temperature was day 2 which was 6 days before resolution of CS. M. bovis had median time to resolution of high rectal temperatures on day 8 postchallenge. However, we have no data regarding time to resolution of CS since all the trials included concluded before resolution of CS. This could be a result of short trial durations or it could be in conjunction with the known long, often chronic disease course associated with M. bovis. Parainfluenza-3 did not have data for time to resolution of CS and rectal temperatures. P. multocida was the only outlier of the common BRD pathogens with high rectal temperatures and CS resolving around the same time (high rectal temperature resolution on day 2, clinical sign resolution on day 2). Knowledge of high rectal temperature resolution with regard to time to resolution of CS could be an important disease progression indicator for producers, veterinarians, and researchers. For the most BRD pathogens, we can expect clinical sign resolution 4-6 days after rectal temperatures have returned to less than 40°C. Seroconversion is defined as the time at which antibodies are first detected in the serum. Median time to seroconversion occurred between 9 and 21 days for the pathogens in this study, with median time to seroconversion occurring on day 17 for BVDV, day 17.5 for BHV-1, day 9 for BRSV, and day 21 for M. bovis. Data were not available for PI-3 virus and M. haemolytica. Median time to peak seroconversion occurred on day 27 for BVDV, day 40 for IBR, day 23 for BRSV, and day 28 for M. bovis. For M. bovis, this partially concurs with 1 study evaluating response of na€ ıve calves being exposed to a herd endemically infected with M. bovis with antibodies first detected by day 29-35; however, peak antibody response did not occur until day 60 post-introduction. 45 The time to seroconversion or peak seroconversion could have been affected simply by the animals' ability to respond to the antigen and produce appropriate antibody or simply confounded by the sampling time selected by the researchers for each trial. For BVDV, BHV-1, BRSV, and M. bovis, seroconversion can be expected to occur in a range of 9 days to 21 with peaks between 23 days to 40. There are certainly limitations associated with this structured literature review and descriptive analysis. The biggest limitation would be the low number of trials for each pathogen. Unfortunately, the limited number of trials and the heterogeneity of the dataset limited any substantial statistics beyond descriptive. Thus, preventing any interpretation sample size has on outcome variables. For example, a larger study group might have an increase number of days to peak CS and resolution of CS over a smaller group. However, sample size may have no effect on time peak CS and resolution of CS since only challenge models were included. The answer to this question is beyond the ability of this manuscript. Ideally, the structured literature review would have been limited to studies that were blinded. Nonblinded studies were included because the number of trials included would have been severely limited. Additionally, nonblinding is not as likely to affect objective areas of interest such as: rectal temperature, seroconversion, and viral shedding. Another limitation is the lack of shedding data for the bacterial pathogens. However, interpreting shedding data (culture or PCR) for bacterial pathogens is difficult as bacterial pathogens are often normal flora of the nasopharynx of cattle. Ideally, a researcher would collect a deep nasopharyngeal swab for analysis before pathogen challenge ensuring the individual is negative for the pathogen challenge strain and thus correlating bacterial shedding postchallenge matches the appropriate strain. With this review, only 1 study looked at bacterial shedding through utilization of PCR without determining the resolution of shedding. Other factors to consider are the effects of pathogen strain, dose, and route of inoculation of the disease severity and disease course. Unfortunately, the limited dataset prevents utilization of any statistical analysis to determine if the study ranges are because of chance or trial variables (dose, strain, route of inoculation). One must be careful in extrapolating these data to clinical scenarios, as challenge studies may not represent a valid model for natural disease. However, the information in this structured literature review provides a resource when designing clinical trials for the specific pathogens of interest. This structured literature review serves as a valuable summary and resource for veterinary researchers, veterinarians, and producers interested in the duration of time between exposure to common BRD pathogens until expected time to resolution of CS, high rectal temperature, shedding, and seroconversion. Important conclusions are that CS resolved near the time of shedding cessation or up to 3 days after shedding ceased for BVDV, BHV-1, and BRSV; and high rectal temperatures resolved approximately 4-6 days before resolution of CS for BVDV, BHV-1, BRSV, and M. haemolytica. Supporting Information Additional Supporting Information may be found online in Supporting Information: Appendix S1. Articles reviewed for analysis inclusion.

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2015-02-15T00:00:00.000Z

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In vitro gastrointestinal digestion study of two wheat cultivars and evaluation of xylanase supplementation Background The filamentous fungus Talaromyces versatilis is known to improve the metabolizable energy of wheat-based poultry diets thanks to its ability to produce a pool of CAZymes and particularly endo-β(1,4)-xylanases. In order to appreciate their in vivo mode of action, the supplementation effect of two of its xylanases, XynD and XynB from families GH10 and GH11 respectively, have been evaluated on two different wheat cultivars Caphorn and Isengrain, which were chosen amongst 6 varieties for their difference in non starch polysaccharides content and arabinoxylan composition. Results Polysaccharides digestion was followed during 6 h along the digestive tract using the TNO gastrointestinal model-1, to mimic monogastric metabolism. Polysaccharide degradation appeared to occur mainly at the jejunal level and was higher with Isengrain than with Caphorn. For both cultivars, XynD and XynB supplementation increased notably the amount of reducing end sugars into the jejuno-ileal dialysates, which has been confirmed by a valuable increase of the soluble glucose into the jejunal dialysates. Conclusions The amounts of arabinose and xylose into the dialysates and ileal deliveries increased consequently mainly for Caphorn, suggesting that XynD and XynB supplementation in wheat-based diet could alleviate the anti-nutritional effects of arabinoxylans by limiting the physical entrapment of starch and could increase the available metabolizable energy. Background Due to its high content in storage polysaccharides (i.e. starch), wheat is a crucial source of energy in poultry diets contributing up to 650 g/kg diet for finishing broilers [1,2]. However, wheat grain contains 12-18% non-starch polysaccharides (NSP), which may result in low performance of growing broilers [3]. In wheat, as in other cereals, the physical entrapment of starch and protein by cell wall polysaccharides is one of the possible mechanisms by which NSP exert an anti-nutritional effect [4][5][6]. Anti-nutritional effects of NSP in many cereals are also due to the presence of high molecular weight soluble polysaccharides that enhance the viscosity in the digestive tract and reduce nutrient absorption. Enzymatic degradation of both soluble and insoluble NSP can also favour more effective hindgut fermentation and thereby improves overall energy utilization [7]. Arabinoxylans (AXs), the main cell wall polysaccharides in wheat, are composed predominantly of two pentoses, arabinose and xylose [8]. These polysaccharides are formed from a linear backbone of β(1,4)-linked D-xylose on which α-L-arabinofuranosyl units are attached as single side-chain units through O2 and/or O3 [9]. Other constituents such as galactose and glucuronic acid may also be present as side chains in addition to arabinose in heteroxylan isolated from the outer tissues of the grain. Low amounts of ferulic acid are esterified to arabinose side-chains [10]. Several studies have shown that supplementation of NSP-degrading enzymes could alleviate the antinutritional effect of cell wall polysaccharides, notably by partially breaking down the AX [9,[11][12][13][14]. As it is almost impossible to precisely assess the molecular effect of CAZymes along the animal's guts, the in vitro TNO gastrointestinal model-1 (TIM-1) represents an alternate choice to improve our knowledge of the enzyme action along the digestive tract [21]. Indeed, TIM-1 model simulates the successive dynamic conditions in the gastric/small intestinal tract such as body temperature, pH, concentrations of bile salts and gastroduodenal enzymes secreted in the successive subdivisions of the digestive tract. It also makes it possible to follow the kinetics of chyme transit through the stomach and proximal intestine and it simulates the absorption of low-molecular-weight molecules and water [22]. Previous studies on the evaluation of β-glucan and resistant starch digestibility using the TIM-1 method have shown similar results to that obtained in vivo (i.e. ileostomy patients) [23,24]. In addition, by using this in vitro model and AX extracted from different wheat cultivars, it has been shown that the AX structure might influence the improvement of digestibility related to supplementation with CAZymes [14,25]. However, the relationships between wheat characteristics, such as their NSP content and structure, and their responses to enzyme addition are not yet fully known and understood, especially in the gut. Moreover, the relationship between the in vitro versus in vivo modes of action of the enzymes is currently unknown. The aim of the present study was to evaluate the effects of XynD and XynB on wheat digestion, with emphasis on the carbohydrate soluble fractions that are associated with major detrimental effects of NSP on animal performances [26]. In order to understand the relationship between the wheat characteristics and the wheat response to supplementation by both xylanases, two wheat cultivars (Caphorn and Isengrain) with differing carbohydrate composition and viscosity [27][28][29] were chosen among 6 cultivars to be compared. The TIM-1 model allowed the study of the polysaccharide digestion kinetics in the different compartments of the gastro-intestinal artificial tract by following the formed products. Digestive solutions Three digestive solutions were used in the TIM-1. The first solution used in the gastric compartment, was the gastric salt solution, which contains 1 g/L of sodium chloride, 1.1 g/L of potassium chloride and 0.15 g/L of calcium chloride di-hydrate. The second solution used in the duodenal compartment, is the duodenal salt solution composed of 2% of trypsin solution (2 mg/mL), 25% of pancreatin solution (7% w/w), 49% of bile extract solution and 25% of small-intestinal salt solution. The third solution is the small-intestinal salt solution, used in the duodenal, the jejunal and the ileal compartments, and contains 5 g/L of sodium chloride, 0.6 g/L of potassium chloride and 0.3 g/L of calcium chloride di-hydrate. Digestion conditions The digestion medium for the TIM-1 trials was adapted from a previous study [14], and it was composed of 45 g (dry basis) ground wheat (grinding screen size 3 mm), 85 g of gastric salt solution, 5 g pepsin and lipase solution (90,000 and 11,200 U/mg, respectively). One unit of porcine pepsin corresponds to an increase of absorbance (at 280 nm) of 0.001 unit per min measured at 37°C and pH 2.0 using hemoglobin as substrate. One unit of pancreatic lipase is required to catalyze the formation of 1.0 μmol fatty acid from olive oil triacylglyceride in 1 h at 37°C and pH 7.7. Water was added to the gastric medium to adjust gastric content weight to 310 g. When added, XynD and XynB were used at 0.13 μL/g wheat, corresponding to 100.7 xylanase visco-unit/g wheat. One visco-unit of xylanase is defined as the amount of xylanase that hydrolyses low viscosity wheat AX (Megazyme International, Wicklow, Ireland), reducing its viscosity to change the relative fluidity of 1 (dimension less unit)/min under the assay conditions (pH 5.5 at 30°C). Digestion assays of wheat in TIM-1 The digestion assays were carried out on the TIM-1 developed at the TNO (Nederlandse Organisatie voor Toegepast Natuurwetenschappelijk Onderzoek) [21] (Figure 1). This in vitro model allows control of digestive secretions, pH, temperature and endogenous enzymes and mimics the gut peristalsis. The protocol of Minekus, adapted for pigs, was used along with some modifications to mimic poultry digestion [21]. The pH for the duodenal, jejunal and ileal compartments were adjusted at 6.5, 6.8 and 7.2, respectively. Before starting the incubation, the duodenal compartment was flushed with approximately 61 g of the duodenal salt solution described previously. The jejunal and ileal compartments were filled with 130 mL of the small-intestinal salt solution. The intestinal absorption was mimicked using haemodialyser HG-400 membranes with a molecular weight cut-off range of 5-10 kDa (Hospal Cobe, Lyon, France). The dialysis fluid was pumped at 10 mL/min and collected in the intervals 0-60, 60-120, 120-180, 180-240, and 240-360 min. The ileal effluents were collected between 0-120, 120-180, 180-240, and 240-360 min. The TIM-1 model was fed with 45 g of Caphorn or Isengrain ground (3 mm) wheat grains and the effects of XynD and XynB were evaluated in duplicate with a 2 × 2 factorial arrangement. Samples recovery Five types of sample were collected: ileal dialysate, jejunal dialysate, ileal deliveries, gastro-duodenal and jejuno-ileal residues. The ileal and jejunal dialysates were collected all along the digestion (see above) and were weighed and stored at −20°C until analysis. The ileal deliveries were also collected all along the digestion. All samples were centrifuged at 14,000 g for 10 min, supernatant and pellet were stored separately at −20°C. The gastro-duodenal residues and the jejuno-ileal residues correspond to the remaining fractions at the end of the digestion time-courses. Consequently, they were collected at the end of the run (i.e. 360 min) before to be centrifuged at 14,000 g for 10 min. The supernatants obtained, constituting the soluble fractions of the residues, were weighted and stored at −20°C until analysis whereas the pellets (insoluble part) were not taken into account for analysis. Sample analysis The supernatants collected in the different compartments were analyzed for their content of reducing ends and carbohydrate compositions (monosaccharides and oligosaccharides). The released reducing ends were determined according to the dinitrosalicylic acid (DNS) method [30], using 96-well microplates and a KRL microplate-spectrophotometer (Kirial International, Couternon, France). The blank contained water instead of the sample. The data were expressed as absorbance units, since the extinction coefficients varied greatly depending on the nature of the reducing residue (data not shown). The sugar quantifications were performed in triplicate. The monosaccharide composition of the wheat cultivars (Caphorn and Isengrain) and of the five samples recovered from the TIM-1 experiments (gastro-duodenal residues, jejuno-ileal residues, ileal deliveries, jejunal dialysis and ileal dialysis) was determined as alditol acetates by Gas Chromatography after acid hydrolysis [31]. The soluble extracts from wheat were prepared from 1 g of ground wheat grain in 4 mL of water [32]. The liquid fractions were hydrolyzed with H 2 SO 4 (4 mol/L) in the presence of inositol as the internal standard (5 g/L) for 2 h at 100°C, whereas the pellets (providing from the gastro-duodenal and jejuno-ileal residues) were first pre-hydrolyzed with H 2 SO 4 (13 mol/L) for 30 min at 25°C before inositol was added and hydrolysis was performed as for liquid fractions. After reduction sodium borohydride and acetylation, the samples were injected in the Gas-liquid Chromatography (GLC) system (Perkin-Elmer Autosystem, Courtaboeuf, France) using a 25 mm × 0.32 mm silica column (BP-225; J & W Scientific, Folsorn, CA, USA; temperature 205°C, carrier gas H 2 ) and a flame ionisation detector. The results are means of duplicate and the variation coefficient was always less than 4%. The AX content was calculated as the sum of arabinose and xylose. Statistical analysis Statistical analysis of repeated-measures data was performed using the mixed procedure of SAS 9.1.3 (SAS Institute, Inc., Cary, NC, USA). As the values were cumulated over time, the covariance structure was specified as ' Auto regressive type 1'. The restricted maximum-likehood method was used to estimate covariance parameters, and the tests of fixed effects (in model, contrasts and leastsquare means) were performed using the residual degrees of freedom (df; using the 'ddfm = RESIDUAL' option). Time was specified as the factor of repeated-measures ANOVA determinations. Focusing on each compartment (jejunum and ileum) and their sum, the following multifactor statistical model was used to the enzyme effects on the polysaccharide digestion parameters: β π α η δ γ. where α is the meaning effect; β, γ, π, η and μ are the adjusted coefficients of the fixed effects in the model; δ is the adjusted coefficient of the random effects in the model; ε is the random error associated with the jth treatment in experiment k assigned to the ith wheat at time l; subscripts i, j, k and l are the df of each factor: two wheat cultivars, three treatments, two experiments and five times. Time was considered as a repeated factor of the model. Regarding the effects of enzyme preparation on each cultivar, as the interaction 'treatment × wheat × cumulative time' was significant, a SLICE option was added to the LSMEANS statement in order to compute the treatment effect at each time for both cultivars separately. All statistical analyses were considered to be significant at P < 0.05. Carbohydrate compositions of different wheat cultivars Sugar composition of the whole grain (Table 1) and the soluble extracts (Table 2) were determined for Caphorn, Tapidor, Apache, Oratorio, Aztec and Isengrain wheat cultivars. The overall monosaccharide content in the whole grains was found to be globally similar for the six cultivars (between 78.10% and 83.15%) even if Caphorn and Isengrain present the higher amounts of total monosaccharide (83.15% and 82.72%, respectively). Moreover, the Tapidor, Apache, Oratorio and Aztec whole grains were composed of a very close rate of arabinose, xylose and glucose (Table 1). Mannose and galactose were present at low levels with little variations between the six cultivars and therefore were not discussed further. The proportions of polysaccharides in the soluble extracts ( Table 2) were however different between the six cultivars, with the highest levels of monosaccharides in the Caphorn and Isengrain extracts (9.95% and 4.09%, respectively). This was connected to high levels of glucose and total monosaccharide in the whole grain of both wheat cultivars. Furthermore, the Caphorn and Isengrain water extracts have shown the highest and the lowest AX contents (1.37% vs 0.54%) simultaneously with the lowest and the highest A:X ratio (0.68 vs 0.86). Finally, regarding these monosaccharide compositions, the AX amounts and the A:X ratio, Caphorn and Isengrain wheat cultivars were selected for the rest of the study for their contrasted soluble AX content and structure. Carbohydrate distribution into the different TIM-1 fractions after 360 minutes of digestion In order to evaluate the carbohydrates distribution into the soluble fractions from the different compartments of the TIM-1 throughout the digestion time, the amount of glucose, xylose and arabinose was measured without xylanase supplementations (Table 3). Both wheat cultivars presented a similar carbohydrate global repartition although the 3 main monosaccharides were differently distributed: 80% and 81% of glucose were found into the dialysates of Caphorn and Isengrain, respectively, while only 11% and 14% of arabinose plus xylose were found in the same compartments. Conversely, arabinose and xylose were mainly distributed in the soluble fractions of ileal deliveries and jejuno-ileal residues. Overall, no differences were observed for arabinose and xylose contents in the jejunal and ileal dialysates from Caphorn and Isengrain cultivars. With regard to the ileal deliveries and the soluble fractions of the gastro-duodenal residues, much higher levels of arabinose (+37.5 mg; P = 0.018 and + 12.0 mg; P = 0.020, respectively) and xylose (+65.7 mg; P = 0.009 and + 20.2 mg; P = 0.006, respectively) were measured for Caphorn than for Isengrain. The glucose content did not present any significant difference in either wheat cultivars regardless of the collected fractions in the TIM-1. These results indicated that AX were not degraded in the absence of xylanase along the digestive tract and were in accordance with the soluble monosaccharide compositions of the cultivars ( Table 2) where the AX content is higher in Caphorn than in Isengrain. Total reducing ends as a global digestibility marker of wheat in the TIM-1 compartmentseffects of xylanase supplementation The total reducing ends were measured, both with and without xylanases added in TIM-1, in order to evaluate the extent of polysaccharide hydrolysis ( Figure 2). As the reducing ends reflect both the hydrolysis of starch as well as NSP, these values can be considered as a marker of wheat digestion in the TIM-1. For both wheat cultivars, the total reducing ends in the jejunal and ileal dialysates represented the major part (70-80%) of the reducing ends measured in the TIM-1 after 360 min. Conversely, in the ileal deliveries and gastro-intestinal residues the total reducing ends represented only~10% in each (data not shown). In accordance with the results presented above, total reducing ends dialyzing at the jejunal and ileal levels in the absence of xylanase (control trial) were in the same range for both cultivars, with 48.4 ± 8.6% and 21.4 ± 2.3% for Caphorn vs 51.7 ± 3.5% and 20.7 ± 0.9% for Isengrain, respectively. The XynD and XynB supplementations increased total reducing ends for both wheat cultivars although the effect was slightly lower on Isengrain. However for Caphorn, XynD was effective all along the digestion tract (+5.3% and +8.1% relative to the control in the jejunal and ileal dialysates, respectively), whereas XynB was mostly effective at the beginning of the digestion (+12.3% and +1.0% vs the control in the jejunal and ileal dialysates, respectively). Conversely, XynD and XynB had a comparable effect on Isengrain with +5.0% and +5.6% in the jejunal dialysate and +2.1% and +2.6% in the ileal dialysate, respectively. Finally, these results suggested that the hydrolysis performed by XynD and XynB in the presence of salivary/pancreatic α(1,4)-amylases occurred mainly in Soluble extracts were prepared from 1 g of ground wheat grain in 4 mL of water [31]. Arabinose (Ara), xylose (Xyl), mannose (Man), galactose (Gal), glucose (Glc), arabinoxylan (AX), arabinose:xylose molar ratio (A:X). The results were expressed in g of component in water soluble extract/100 g of whole grain. The distribution (mg) of soluble arabinose (Ara), xylose (Xyl) and glucose (Glc) from 45 g (dry basis) of ground wheat, without xylanase supplementation, has been determined by gaz chromatography. The total expressed in percentage is calculated based on the initial content in mg. Mean values were based on three repetitions. SEM corresponds to the standard error of the mean estimated by the standard deviation (SD) divided by the square root of the sample size (n = 2), assuming statistical independence of the values in the sample. the proximal parts (from the gastric to the jejunal compartments) of the TIM-1. Kinetic effects on reducing ends release by the XynD and XynB supplementations on wheat digestion In order to follow the kinetic effect of a xylanase supplementation, the cumulative time course of reducing ends on the jejunal and the ileal dialysates is shown for XynD and XynB supplementation (Figure 3). For Caphorn cultivar, the XynB and XynD positive effect observed on Figure 2 appeared mainly at the end of the trials between 4 and 6 h. Indeed, the values of reducing ends measured at the previous times are not significantly different for the un-supplemented assay. However, the time course confirms that XynB was the most active at the jejunal level, whereas XynD was similarly active at both levels. When Isengrain was used, both enzymes tended to increase the contents in reducing ends in the jejunal dialysates during the last hours and gave a time course very similar to the control assay in the ileal dialysates. Effect of XynD and XynB supplementations on monosaccharide distribution on the TIM-1 compartments In order to get an overview of the effect of xylanase supplementation on monosaccharide content for each of the wheat cultivars, the values obtained for each monosaccharide in each compartment have been pooled together to define both the gastro-intestinal residues, and the jejuno-ileal dialysates (Figure 4). The enzyme effect was expressed as the percent increase or decrease of the monosaccharide release compared to the control without enzyme. XynD and XynB additions significantly affected the contents in monosaccharide in all the samples collected after 360 min of digestion. XynD increased the arabinose and xylose contents in the different fractions and for both wheat cultivars. Specifically, XynD supplementation increased the arabinose amount for Caphorn and Isengrain with +14.5% and +25.6% into the jejunoileal dialysates, +14.1% and +26.8% into the ileal deliveries and +28.9% and +30.0% into the gastro-intestinal residues, respectively. The xylose content in the presence of XynD followed the increase of the arabinose amount with for Caphorn and Isengrain, +78.7% and +115.0% into the jejuno-ileal dialysates, +13.9% and +23.2% into the ileal deliveries and +27.7% and +32.3% into the gastrointestinal residues, respectively. Finally, there was a trend for an increase of the glucose content for both wheat cultivars in the TIM-1 fractions collected in the presence of XynD. The largest increase of glucose content was observed on Isengrain cultivar into the jejuno-ileal dialysate (+39.7%). The XynB effect on the arabinose release was close to the XynD effect for the Caphorn cultivar, with +14.7%, +31.3% and +56.8% into the jejuno-ileal dialysates, the ileal deliveries and the gastro-intestinal residues respectively. For Isengrain the effect was controversial with −39.0%, +16.4% and −12.6% for the three compartments, respectively. The XynB effect on xylose contents for Caphorn and Isengrain was +58.3% and +89.5% into the jejuno-ileal dialysates, +27.1% and +25.2% into the ileal deliveries and +48.6% and −6.9% into the gastro-intestinal residues, respectively. Furthermore, it is interesting to note that the XynB effect on glucose appears later in the digestive tract for Isengrain than for Caphorn, with an increase of +15.5% in the corresponding ileal delivery. Kinetics of glucose appearance in jejuno-ileal dialysis in TIM-1 The kinetics of glucose appearance in dialysates was monitored in presence or absence of XynD and XynB in order to evaluate the impact of xylanase supplementation on the energy available for the animal ( Figure 5). For Caphorn and Isengrain cultivars, the glucose dialysis mainly took place into the jejunal compartment (between 2.11 and 2.53 times higher than into the ileal compartment for Caphorn and Isengrain, respectively). In the jejunal dialysate at 360 min, the content of glucose was higher for Isengrain than for Caphorn (+1,386 mg; P = 0.021). There were no significant differences between the two wheat cultivars in the ileal dialysates regardless of either digestion time or enzymatic treatment. However, XynD and XynB significantly increased the glucose content into the jejunal dialysates and consequently into the total dialysates for Caphorn cultivar throughout the digestion time. Finally at 360 min, the XynD supplementation on Caphorn cultivar increased the jejunal and total glucose contents by 17% (P = 0.014) and 9% (P = 0.060), respectively. The XynB supplementation increased the glucose contents in the same fractions by 19% (P = 0.006) and 10% (P = 0.035), respectively. The kinetics of appearance of arabinose and xylose in jejunal and ileal dialysates were almost identical to that of glucose, with the most evident effect of the enzymes during the last hours of transit. However, it should be noted that XynB showed a strong effect on xylose solubilisation from Isengrain cultivar in the jejunal compartment and no effect in the ileal dialysate confirming a proximal effect of XynB on AX hydrolysis (data not shown). Kinetics of arabinose, xylose and glucose appearance into the ileal delivery compartment The level of arabinose, xylose and glucose into the ileal deliveries was monitored along the digestion in order to evaluate the effect of xylanase supplementation on the monosaccharides and short oligosaccharides released which consequently transit to the colon compartment where they could play a prebiotic role ( Figure 6) [33]. Without the enzyme, ileal deliveries recovered from Caphorn feeding presented higher arabinose content than Isengrain all along the time course (8.3% vs 6.2% at 360 min, P = 0.005), and xylose contents (7.4% vs 5.2% at 360 min, P = 0.003). No significant differences were observed for glucose contents, with 5.3% for Caphorn vs 5.7% for Isengrain, at 360 min (P = 0.664). The XynD supplementation increased arabinose (at 360 min, +13.8% (P = 0.149) in Caphorn and +26.9% (P = 0.089) in Isengrain), xylose (at 360 min, +14.4% (P = 0.172) in Caphorn and +23.4% (P = 0.178) in Isengrain) and glucose (at 360 min, +3.8% (P = 0.833) in Caphorn and +7.1% (P = 0.669) in Isengrain) contents in the soluble fraction of ileal deliveries. Conversely, XynB showed globally a stronger effect than XynD on Caphorn cultivar with +31.7% (P = 0.009) for arabinose, +26.9% (P = 0.022) for xylose and +12.5% (P = 0.493) for glucose contents. With Isengrain the increases were +16.4% (P = 0.264), +27.0% (P = 0.148) and +15.5% (P = 0.367), for the 3 sugars, respectively. Finally, it is noteworthy that the effect of xylanase supplementation mainly took place during the three first hours of digestion especially concerning the glucose amount compared to arabinose and xylose. Indeed, the amount of arabinose and xylose increased progressively all along the digestion time whereas the glucose amount quickly increased in the two first hours, before leveling off. Moreover, Caphorn cultivar was more sensitive to XynB than to XynD supplementation, whereas Isengrain cultivar was less sensitive than Caphorn to xylanase supplementation, regardless of the xylanases used. Discussion In this study XynB (GH11) and XynD (GH10) from T. versatilis were evaluated for their ability to improve the digestibility of Caphorn and Isengrain wheat cultivars by using the in vitro gastro-intestinal model TIM-1. The sugar composition and AX ramification of both wheat cultivars studied here are in accordance with previous studies. Pritchard et al. determined the monosaccharide composition and the total A:X ratio of 211 wheat varieties and found that the total AX content ranges from 2.37% to 10.75% dry matter whereas the A:X ratio ranges from 0.4 to 1.3 [34]. Compared to Isengrain (0.54%) or to the literature values, the level of soluble AX measured in Caphorn was high (1.37%). Indeed, previous studies showed that soluble AX content in wheat whole grain ranges from 0.24% to 0.75% [35,36]. The viscosity of a polymer solution is dependent on different parameters: the primary structure of the polymer, its molecular weight and its concentration. Although AX structure and molecular weight can vary according to cultivars and environment, the amount of water-soluble AX is a good indicator of viscosity. In this study, Caphorn was found to contain water-soluble AX at three times higher levels than for Isengrain, which supports that Caphorn is a more viscous cultivar than Isengrain. As a matter of fact, previous authors reported that viscosity measurements, varied from 3.35 to 4.98 for Isengrain and from 7.86 to 10.2 for Caphorn [27][28][29]. Studies have demonstrated that chyme viscosity decreased by partial or complete hydrolysis of soluble NSP, supplementation in CAZymes (e.g. xylanases or β-glucanases) represents a good treatment to improve animal performance [7]. Based on this fact and previous work on xylanase supplementations, the preliminary step of this study required characterizing the behavior of both wheat cultivars along the in vitro gastro-intestinal model TIM-1. After 6 h digestion, it has been shown that the glucose levels in the dialysates were much higher than those of arabinose and xylose. This can be explained by a highest hydrolysis of glucose-containing polysaccharides (i.e. starch), compared to NSP (e.g. AX), releasing xylose and arabinose residues. In the TIM-1 model, the digestion process mimics the monogastric digestion, where only polysaccharides such as starch can be digested by salivary, gastric and intestinal enzymes. The soluble xylose and arabinose found in the different TIM-1 fractions provided from the hydrolysis of AX, originally located into the plant cell wall [37]. The gastro-duodenal residues and the ileal deliveries representative of the undigested fractions, present some differences in the soluble fractions with much more arabinose and xylose for Caphorn than for Isengrain. This difference is mainly linked to the fact that both wheat cultivars have different initial soluble polysaccharides contents, with especially a higher level of AX in Caphorn. The monitoring of time courses for sugar appearances in dialysates showed that the glucose dialysis mainly took place in the jejunal compartment. This observation was in accordance with the fact that α(1,4)-amylase and gluco-amylase quantities and activities were higher in this proximal part of the intestinal tract than in the distal parts [38]. In addition, there was slightly more dialyzed glucose for Isengrain than for Caphorn, suggesting that the starch digestion was more efficient in this cultivar. These results were in agreement with those obtained previously by Preynat et al. and Lafond et al. [39,25]. Hübener et al. and Torok et al. demonstrated that the CAZymes supplementation improves animal performance and nutrient utilization and also changes the composition and metabolic potential of bacterial populations in the gut microbiota [40,41]. Taking those different beneficial effects in consideration, the present work allowed estimation of the effects of XynD (GH10) and XynB (GH11) supplementations on Caphorn and Isengrain wheat cultivars. The addition of these enzymes in the TIM-1 induced an increase of the levels of xylose, arabinose and glucose into the dialysates fractions (corresponding to the physiological intestinal absorption), suggesting that the AX hydrolysis favored the starch degradation. In this regard, Classen et al. suggested that wheat NSP might act as a physical barrier preventing or slowing access of digestive enzymes to starch granules [42]. Romero et al. confirmed these observations in showing that a blend of β(1,4)-xylanase, α(1,4)-amylase and protease improved ileal digestibility of starch, fat and protein and thus significantly improved the poultry's ability to extract energy [43]. The microscopy study of Bedford and Autio reinforces the fact that there was indeed a considerable amount of starch surrounded by intact cell walls in the intestinal digesta of broilers fed wheat-based diets, which was mostly removed after NSP-degrading enzyme supplementation [5]. This hypothesis is strengthened by XynD and XynB supplementations that increased reducing ends content, with a direct effect on the soluble arabinose and xylose release and an indirect effect on glucose release (in both case mono-and/or oligosaccharides were produced), in the different TIM-1 collected fractions. Xylanase addition contributed to partial or complete degradation of soluble AX into arabinoxylooligosaccharides (AXOS) that were found primarily in the proximal part of the gut (i.e. jejunal dialysates) and also in a weak amount in the ileal deliveries. Consequently, the high amount of AXOS released from AX by both enzymes could: promotes a prebiotic effect on the colic microbiota, modulates the intestinal microbiota, and reduce the pathogens population in the gut and prevent some intestinal diseases [44,45]. Accordingly, Aulrich and Flachowsky found that the in vitro supplementation of the combination β(1,4)-xylanase plus β(1,4)-glucanase in wheat-based diet increased the amount of arabinose and xylose and reduced AX amount suggesting that solubilisation of insoluble cell wall polysaccharides by enzymatic hydrolysis and the degradation of solubilized material occurred simultaneously [46]. Isengrain cultivar showed a quite higher A:X ratio than Caphorn suggesting that xylan backbone of Isengrain AX is sensibly more substituted by arabinose than that of Caphorn AX. In our study, XynB had a greater global effect on Caphorn AX suggesting that it was much more efficient on low-substituted AX, whereas XynD had a greater global effect than its homologous from family GH11 on Isengrain which exhibits a higher substitution degree. These results are consistent with previous reports, where we observed that the XynD from T. versatilis was able to cleave the highly substituted xylan whereas XynB preferred less substituted xylan [19,20]. Moreover, it is known that xylanases from GH10 family are able to cleave the AX backbone in decorated regions and are less hampered by the presence of arabinose substitutions along the xylan backbone than GH11 members [47]. Although our experiments were carried out in a very complex system on solid matter and in non-optimal conditions for the enzymes, our results are directly linked to what was previously demonstrated in simple soluble media, containing only one substrate and one enzyme in optimal conditions. Consequently, it may be conceivable to observe a synergetic effect with supplementation of the two xylanases together. This assumption is based on the fact that XynD is active throughout the digestive tract while XynB shown a strong activity on the proximal part of the gut. These results were correlated to the membership of two different CAZy families and thus suggest a different mode of action in vivo. In this study we have shown globally a proximal effect of both enzymes, which decreased with digestion time. As we already described in 2010 for the Rovabio Excel TM the reduction in activity might be due either to enzyme degradation by the proteases available in TIM-1 or to a limitation in substrates [25]. However, based on previous studies [19,48], the recombinants XynB and XynD are both highly N-and O-glycosylated and it is well known that these kind of post-translational modifications induce a relative resistance to the protease [49]. We can thus suggest that N-and O-glycosylations of both enzymes confer a proteolysis resistance during the first times of digestion followed by a progressive degradation, inducing a loss of xylanolytic activity. Finally, XynD and XynB were both sensitive to the wheat proteinaceous inhibitors XIP-I (Xylanase-Inhibitor-Protein-I) with a K i of 10.2 and 89.7 nmol/L, respectively, whereas only XynB was found to be sensitive to TAXI (Triticum aestivum Xylanase Inhibitor) with a K i of 2.9 nmol/L (unpublished data from Lafond, [48,50]). Based on literature, Isengrain whole grain contains 560 μg/g of XIP-I and 190 μg/g of TAXI [51], which leads one to consider if these inhibitory levels are higher in Caphorn, and suggest a reduction of enzyme activities.

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2019-04-14T13:05:18.249Z

2015-07-13T00:00:00.000Z

54570639

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Obtained Yield and Financial Parameters of Organically Grown Mint in the Republic of Macedonia Aims: The objective of this paper is to present the soil and weather conditions, applied production technology and to demonstrate the economic viability of irrigated and organically grown Mint (Mentha piperita). Methodology: The obtained yield results are in reference to the open field growing conditions. In order to present the results in clear and understandable manner, standard methods for cost calculation were applied, usual for organic plant production. In order to obtain comparable results, the processed data in this paper are from the two-year production trials (2009 and 2010), of a newestablished mint plantation planted in October 2008, in the region of Ovche Pole central part of the Republic of Macedonia. Results: In the first year of utilization (2009), two cuttings were performed yielding 3500 kg/ha of Original Research Article Mihajlov et al.; BJAST, 10(4): 1-6, 2015; Article no.BJAST.17970 2 above-ground dry mass. In the second year of utilization (2010), there were three cuttings yielding 5155 kg/ha of above-ground plant dry mass. Economic analysis proves that profitability in organically produced Mint (Mentha piperita) is obtained in the second growing season year, recording profit of € 8125 per hectare. Conclusion: Based from the obtained results the benefits of growing Mint are: opportunities to optimize yield and achieve uniform high quality product. Organic way of growing Mint, can be a great business idea for the farm and family business. INTRODUCTION The origin of peppermint species cannot be determined. Dried leaves were found in ancient times, in the Egyptian pyramids. Two species of mint were used by the ancient Greek physicians. However, some writers debate whether one is the modern peppermint, though there is evidence that M. x piperita was cultivated by the Egyptians [1]. It is thought to be a natural hybrid between spearmint (M. spicata Linn. emend. Nathh.) and water mint (M. aquatic Linn), the latter itself being a hybrid of M. longifolia (Linn.) Huds, and M. rotundifolia (Linn.) Huds so M. piperita is a triple hybrid [2]. Mint is popular medicinal plant in several traditional systems of medicine. Medicinal, aromatic and the spice plants are widely used in pharmaceutical, food, cosmetics, alcoholic beverage industries and as well as in chemical (pesticide) industry. Up to the mid '50's of the last century, these plants were used as raw material from wild flora of these species, and some plants still are used how growing wild plants [3]. The WHO has estimated that more than 80% of the world's population in developing countries when satisfying basic health care needs depend primarily on herbal medicine. Approximately two thirds of the 50000 different medicinal plant species which are in use are collected out in the wilderness [4]. As far as production levels are concerned, the volume is estimated on more than 4000 t/year. Almost 80% of it is produced in USA. Mint production is also organized in Canada, UK, Australia and New Zealand, while in Asia this crop is mostly grown in India and China [5]. In South Africa under irrigation and good management, peppermint will yield 20 to 25 tons of plant material per hectare per year, at an oil recovery rate of 0,3% or 60 to 75 kg essential oil per ha. [5]. In Europe only 10% of medicinal species which are in commercial use are cultivated. Cultivation of some herbs has proved difficult because of low germination rate or specific ecological requirements [3]. Mints are widely cultivated on almost all types of soils and climates. The major commercially produced species are: Japanese or menthol mint (Mentha arvensis L. var. piperascens), peppermint (M. piperita L.), common or native spearmint (M. spicata L.), Scotch spearmint (M. cardiaca Baker), garden mint (M. viridis L.) and bergamot mint (M. citrata Ehrh). China, India, U.S.A, Japan, France, Italy, Russia and Bulgaria are some of the major producers of mint oils [6]. Peppermint Mentha piperita L., which is used in production of flavors, fragrances and pharmaceuticals, were investigated for their antimicrobial properties against 21 human and plant pathogenic microorganisms. Three of the largest producers of mint and mint related products, (such as essential oils) are the United States, India and China [7]. The U.S.А and India produce the largest supplies of mint oil used in chewing gum, toothpaste, mouthwash and several other products. Irrigation and nitrogen should be in adequate quantities for maximum growth; plant stress causes early bloom and production of menthofuran which reduces oil quality for some markets [8]. Republic of Macedonia has very favorable geographic position, diversity of soil types and low contamination of natural resources which provides conditions for cultivation of aromatic, spice and medical plants. The advantages of the Macedonian territory for organic cultivation of the medicinal aromatic and spice plants are: Areas with low degree of pollution or unpolluted resources (soil, water, air); Favorable geographical position, diverse landscape; fertile river valleys, flat lowlands and mountain slopes; diverse types of the soils. Naturally, there are weaknesses and these are: Unused agroenvironment potentials; Lack of Macedonian literature for growing technology of aromatic & medicinal herbs and spices; Lack of exploration experience in this area; Lack of skills on farmer level on growing methodology for certain aromatic spices and medicinal plants; Lack of devised strategy for cultivation medicinal plants that can penetrate on the market; Lack of information on potential yields, prices and markets for this group of the plants. Unfortunately, this is the first research attempts for growing Mint under the soil & weather conditions in the central part of Macedonia, hence we're unable to compare our results with any previously obtained, as they do not exist. Generally, the purpose of publishing this paper is to inform on the possibilities and the benefit of growing Mint under the organic principles in Macedonia. Difference in the values of the percentages of individual components in peppermint oil due to the different places of production of mint i.e. the different agro-ecological conditions [2]. Ovche Pole valley (GPS coordinates: N 41º49`, E 21º 59`), suffers negative effect by being overlapping area of two climate types. The Mediterranean and Continental climate, which are the main reason for inconsistent and uneven precipitation, especially in the summer when there are significant low precipitation values [4]. The soil on which Mint was cultivated according to the principles of organic farming belongs to vertisol type. Its characteristic and nutrient availability are presented in Table 1 (total area of 2 ha). The results from the agrochemical soil analysis presented in Table 1, performed on the soil from the trial fields on which the Mint was planted in Ovche Pole, indicates that the soil is with the following characteristics: good phosphorus availability (24.19 mg/100 g), rich in availability of potassium (74.1 mg/100 g), poor in humus content (1.94%) and slightly increased salinity [10]. The total nitrogen level is on satisfactory level (0.98 mg/g) while the pH value is on the alkaline side (7.65). Applied production technology of organically grown Mint, can be seen from data stated below, as well as from During the vegetation, in the stage of most intensive growth (up to the first cut) the prevention of the weeds was performed by 2 treatments between rows with cultivator. In-row weeds were reduced manually. Between each mowing, irrigation was applied using sprinklers providing 50 l/m 2 of water. Peppermint has a shallow root system and requires frequent irrigation with short sets, thus additional labor is required for moving irrigation equipment in peppermint compared to most field crops [7]. In the first year of exploitation (2009), due to youth fullness of the growing crop only two mowing were performed. The next year (2010), the number of mows increased by one. Practices described are based on production practices considered typical for the crop and area, but will not apply to every situation. Biomass Productivity Timing and types of establishment and cultural practices will vary among growers within the region and from season to season due to variables such as weather, soil, and insect and disease pressure [8]. Besides favorable agro climatic conditions for the production of high quality dried herb material, plants responds quite well to good irrigation and fertilizer management. As [11] indicates, the area of Ovche Pole is characterized by moderate low average amount of rainfall (640-750 l/m 2 ). Fig. 1 illustrates that ten years rainfall average (2001-2011), as well as average monthly precipitation in the years of study (2009)(2010), can be seen that in July, August and September, the average monthly precipitation values are lowest. From the abovementioned, it can be noted that for the cultivation of mint in this region, and to get more than two mowing, it is necessary to irrigate, especially in the months with low precipitation. Production of dried mint herb in these analyses ranged from 3500 kg/ha in first year of exploitations (2009), up to 5155 kg/ha in the following year (2010), as presented in Table 2 below, where it is presented and the ratio between dry leaves and stems. The analysis of organic mint production (baled dry mass), subject of this paper are grouped in accordance and mutual dependence of applied specific agricultural techniques and administrative expenses Financial Results In order to check the financial effect of the suggested crop, for which any crop would be interesting to the producers (in this case-the Mint), it was necessary to determine the production costs [12]. The determination of the economic effect of the production of organic Mint, gives opportunity to the producers for greater visibility, monitoring and planning of all activities in accordance with cost operations. Calculation of costs and the incomes in the production of organic mint in the year of establishment (2008) and in first year of utilizing (2009), includes many elements. These can be grouped depending on the price of certain agro-technical measures and certain administrative costs (Table 3). Table 4 presents that the overall cost structure, costs for propagating material, (underground stolons) are with biggest share (51.7 %). Due to the large volume of investments in the first year of establishment, the cost per unit is high (1.9 €/kg). For all the reasons mentioned previously, in the first year of utilizing the profit is significantly lower (1569 €/ha) compared to the second year (2010). Lower yield/profit in the first year of utilization is due to incompletely developed crop growing, and the lower number of mowing. Calculation of the Expenses in the Second Year of Utilizing the Crop (2010) In the second year of crop's utilization, the total costs (€/ha), and the costs per unit (0,15 €/kg), are significantly smaller. The total income is € 19050 ( Table 4). The income per unit area is 8125 €/ha. Calculation of costs and incomes in the second year of production of organic Mint is presented in Table 4. Data in this If of the total income (€ 19050), total costs are deducted (€ 2800), obtained value refer for second year profit (2010), which amounts to total of € 16250 or profit per unit area of 8125 €/ha. CONCLUSION Mint organic production has a lot of advantages because:  The characteristics of the obtained product is suitable for extended storage in controlled conditions;  Provides opportunities for monitoring and market analysis and possibly getting a higher price;  Excludes the application of synthesized chemicals for nutrition and protection of the growing crop;  Provides agro ecosystem sustainability through maintenance of biodiversity and antipollution impact on natural ecosystems for future generations.  It can to optimize yield and achieve uniform high quality product.  Relatively higher profit margin, through organic way of growing, can be a great business idea for the farm and family business.  At last, but no least important, following the profitability results of growing organic Mint, appears that this production orientation could be of great business opportunity for Macedonian farmers.

v3-fos

2016-03-01T03:19:46.873Z

2015-06-01T00:00:00.000Z

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Comparative Assessment of Phenolic Content and in Vitro Antioxidant Capacity in the Pulp and Peel of Mango Cultivars Mango (Mangifera indica L.), also called “the king of fruits”, is one of the most popular fruits in tropical regions. Pulp and peel samples of mango cultivars were analyzed to estimate total phenolic, total flavonoid and total anthocyanin contents. Phenolic acids, hydrophilic peroxyl radical scavenging capacity (hydro-PSC) and oxygen radical scavenging capacity (ORAC) in vitro were also determined. Total phenolics and flavonoid contents were found maximum in the peel of Xiao Tainang and Da Tainang cultivars, respectively, whereas Xiao Tainang also exhibited significant antioxidant capacity. Noteworthy, concentrations of gallic acid, protocatechuic acid, ferulic acid, chlorogenic acid and caffeic acids at 79.15, 64.33, 33.75, 27.19 and 13.62 mg/100 g fresh weight (FW) were quantified for Da Tainang, Xiao Tainang and of Jidan cultivars, respectively. Comparatively, a higher level of phenolics and significant antioxidant capacity in mango peel indicated that it might be useful as a functional food and value-added ingredient to promote human health. level of phenolics and significant antioxidant capacity in mango peel indicated that it might be useful as a functional food and value-added ingredient to promote human health. Introduction Humans have relied on nature throughout the ages for their basic needs of food and health. At present, there is an increasing interest in exploring new sources of plant bio-actives for applications in both the food and pharmaceutical industries [1]. Fruits and vegetables are the most important food sources, which supply essential nutrients and also contain an array of phytochemicals, such as phenolics and flavonoids, to maintain good health [2]. Fruits provide an opportunity for local growers to access the specialized markets where consumers show a preference for exotic characteristics and the presence of nutrients in food, capable of preventing degenerative diseases [3]. A number of reactive oxygen species (ROS), including superoxide anion, hydroxyl and hydrogen peroxide radicals, are produced in the human body by numerous enzymatic systems through oxygen consumption. These reactive oxygen species cause cancer, cardiovascular diseases, aging and neurodegenerative disorders [4]. The ingestion of fruits and vegetables has been connected with a distinguished health-protecting factor against diseases caused by oxidative stress [5,6]. Health benefits of fruits and vegetables have been attributed partly to the compounds having antioxidant capacity and an ability to overcome oxidative stress by neutralizing the overproduction of oxidant species [7,8]. It has been reported that the additive and synergistic effects provided by the complex mixture of phytochemicals present in fruits and vegetables cannot be achieved through micronutrient supplements [9]. Polyphenolic compounds, including phenolic acids, xanthones, gallotannins, carotenoids and vitamins (E and C), are important anti-radical, anti-mutagenic and anti-carcinogen agents [10,11]. They reduce the risk of chronic diseases, because of their safety, effectiveness and the presence of hydroxyl groups, which enable these compounds to have more diverse biological activities [12]. Phenolics antioxidants, such as hydroxyl benzoic acid, and their derivatives are potent free radical scavengers of singlet oxygen possibly concerning DNA damage and tumor promotion [13]. Phenolic acids are predominant compounds in the pulp of mango [14]. Consumption of ripened mango is better, as it contains a high content of phenolic acids, which play a significant role in quenching and neutralizing the free radicals to improve consumers' health [15,16]. Gallic acid is one the important anticancer agents, particularly against human prostate cancer cells in vitro and in vivo [17]. Ferulic acid is an important phenolic compound in fruits and vegetables, which is generated as a result of phenylalanine and tyrosine metabolism [18]. By virtue of effectively scavenging harmful radicals and suppressing radiation-induced oxidative reactions, ferulic acid serves as an important antioxidant, protects the body against different inflammatory diseases and is essential in preserving the physiological integrity of cells exposed to both air and impinging UV radiation [19]. In vitro and in vivo studies have revealed that chlorogenic acid is one of the most abundant polyphenols in the human diet, which exhibits significant anti-edematogenic, anti-nociceptive, antioxidant and anti-carcinogenic activities [20]. It has been reported that protocatechuic acid showed anti-proliferative activity against HL-60 cells by inducing apoptosis and is associated with the phosphorylation and suppression of Bcl-2 protein [21]. Caffeic acid has been proven as an inhibitor of hypertension and cardiotoxicity in rats by enhanced blood pressure, cardiac injury markers, restoration of the oxidant/antioxidant status, as well as decreasing histopathological changes [22]. Mangifera indica L. (mango), "the king of fruits" belonging to the family Anacardiaceae, is one of the most popular fruits in tropical regions. Mango has been cultivated for 4000 years and ranks only second to pineapple in quantity and value among internationally-traded tropical fruits. In Mainland China, mango was first introduced from India in 645 A.D. by Tang Xuangzang (Tang Dynasty), and its commercial cultivation was started in the 1980s. Now, China has become the seventh mango cultivation country in the world, with annual production of about 1,061,800 tones on 133,100 hectares [23]. Mango is considered as a good source of dietary compounds, such as ascorbic acid, phenolic compounds and carotenoids [15,24,25], which are beneficial to health due to their antioxidant capacity [26,27]. The pulp of mango is effective for leukemia, prostate, breast and colon cancers in vitro. Peels are the major by-products of different fruits and are good sources of phytochemicals and bioactive compounds [28][29][30]. Mango peel, which comprises 15%-20% of the fruit, is an edible tissue and a major by-product of the mango processing industry. Peel of unripe mango is used in making chutney and pickle, while that of the ripe fruit, due to its leathery nature, is not so satisfactory in taste, therefore being generally removed and discarded. In the food processing industry, mango peel is removed for technological and sensory advantages and usually ends up as a waste by-product [31]. Mango peel has been found to be a good source of polyphenols, carotenoids, dietary fiber, vitamin E and vitamin C [28,31], and it showed significant antioxidant properties [32,33]. Total phenolics, vitamin C and antioxidant activity have been reported in the fruit of mango varieties [15,24,25]. However, comparative assessment of total phenolic content, phenolic acids and in vitro antioxidant capacity in the pulp and peel of mango cultivars predominantly cultivated in China has rarely been reported before. In this context, the present study was designed to evaluate and correlate phenolic content and antioxidant activity and to assess the disparity in phenolic compounds and antioxidant capacity in the pulp and peel of nine cultivars of mango. Moisture Content Moisture levels as the percentage of moisture content determined in the pulp and peel of mango cultivars are given in Table 1. Overall, pulp samples contain higher moisture content than peel. Interestingly, maximum moisture content was determined in the pulp and peel of the Kaite cultivar at 89.47% and 87.04%, respectively. The Da Tainang cultivar showed the lowest level of moisture at 81.68 for pulp and 77.57 for peel sample. In the present study, the percentage of moisture content of the peel samples was higher than reported previously for Raspuri and Badami mango varieties (65%-75%) from India [32], which might be due to genetic variation and climatic conditions. Values are the means of three replicates ± SD. Different letters (a-f) within the columns indicate significant difference at p < 0.05. Total Phenolic Content Phenolics are among the major contributors that are accountable for antioxidant properties in fruits, vegetables, whole grains and other plant-based materials [34]. Although total phenolic compounds in the mango pulp have been reported before, to our knowledge, the phenolic composition in the peel of Chinese cultivars is estimated for the first time here. The measured levels of total phenolic content (TPC) in the pulp and peel samples of mango cultivars are presented in Table 2, which indicated that peel samples contained high phenolic content compared to pulp. Our results are consistent with a previous report [35] that peel always contains more phenolic contents than pulp at any stage of mango fruit. Peel is an important by-product of mango processing and is a good source of high-quality pectin and polyphenols [36]. On the whole, the peel and pulp of the Xiao Tainang cultivar exhibited a higher concentration of total phenolics among all of the studied samples. In peel samples, TPC ranged from 462.2-4071 mg gallic acid equivalent (GAE)/100 g fresh weight (FW). The Lvsong cultivar contained the lowest level of total phenolics, while in the peel of the Xiao Tainang variety, the TPC level was highest with a significant difference (p < 0.05). It was noted that the total phenolic contents reported in the peel of Pica mango from Chile [24] and the Ataulfo variety from Mexico [37] were slightly higher than the present results. Differences in the cultivars, their origins and genetic variation might result in the inconsistency among the findings [38]. In the pulp samples, the maximum concentration of TPC was estimated for Xiao Tainang at 97.47 mg GAE/100 g FW, followed by Aozhou, Da Tainang and Shuixian cultivars, while Lvsong showed the lowest content. The measured levels of total phenolic content in the pulp samples were in agreement, as reported earlier in different varieties of mango, such as Tommy Atkins and Pica mango from the USA and Chile [24][25][26][27], as well as in Brazil and Ecuador [37] and the Haden variety from Mexico [39]. Phenolic content expressed as mg of gallic acid equivalents per 100 g of fresh weight (FW); flavonoid content expressed as mg of catechin equivalents per 100 g of FW; anthocyanin content expressed as mg/100 mL of cyanidin 3-glucoside equivalents on a fresh weight basis; nd, not detected; different letters (a-i) within the columns indicate significant difference at p < 0.05; values are the means of three replicates ± SD. Total Flavonoid Content Estimated values of total flavonoid content (TFC) in the studied samples are given in Table 2, which revealed that peel samples exhibited a significant level of TFC compared to the pulp samples. These findings were in agreement that in mango, the peel contains more flavonoids than pulp [15]. In the peel samples, TFC ranged between 75. .90 mg catechin equivalent (CE)/100 g FW. The peel of the Da Tainang cultivar showed the highest content of total flavonoids at 75.35 mg CE/100 g FW, whereas the lowest values were calculated in the Kaite and Xiangya cultivars. These concentrations were statistically different (p < 0.05) among the studied samples. In the case of pulp samples, the concentration of total flavonoids was maximum in Aozhou (9.252 mg CE/100 g FW), whereas the minimum content was estimated for the Kaite variety at 0.904 mg CE/100 g FW. Our results demonstrated that the measured levels of total flavonoid content were comparatively higher than reported previous reports for the pulp and peel of Pica variety from Chile [24], for the pulp of Mallika variety from China [25] and Ataulfo mango [15,40]. Total Anthocyanin Contents Anthocyanins are well known because of their antioxidant properties and their pigmenting power that make them attractive to be used as food colorants [1,32]. It has been reported that anthocyanins are comparatively higher in ripe mango peel than raw peel [32]. Like phenolic and flavonoid contents, total anthocyanin content (TAC) was also estimated to be more in the peel samples than pulp ( Table 2). In the peel samples, the maximum contents of total anthocyanins were determined in the Guifei and Aozhou cultivars at 0.659 and 0.647 mg/100 mL of cyanidin 3-glucoside equivalents on a fresh weight basis, respectively. However, the contents of total anthocyanins in the pulp samples were very low. Anthocyanin contents were below the detection limit in the peel of Shuixian, Kaite and Xiangya and in the pulp of Lvsong, Shuixian, Da Tainang, Kaite, Xiao Tainang and Jidan. Compared to the literature, the present findings indicated that anthocyanin contents in Chinese cultivars were comparatively lower than reported in the peel of Indian mango [32], which might be attributed to genetic variation and their origin. Likewise, the reported level of TAC (2.1-26.8 mg of cyanidin 3-glucoside equivalent/100 g) in the peel of apple [41] was significantly higher than mango peel. In general, the present analysis revealed that mango peel contains more contents of total phenolics, flavonoids and anthocyanins. Therefore, it could be an excellent source of natural antioxidants and bioactive ingredients of functional food. Phenolic Acids Composition Measured levels of gallic acid, caffeic acid, chlorogenic acid, protocatechuic acid, vanillic acid and p-coumaric acid, which were identified and quantified for the first time by the HPLC method in the pulp and peel of different mango cultivars grow in China, are presented in Tables 3 and 4. Comparatively, elevated levels of phenolic acids were determined in the peel samples. Though similar types of phenolic acids have been reported before in different varieties of mango [10,15,36], in the case of pulp, concentrations of phenolic acids were different than reported for Ataulfo [15,40], Kent [14] and Tommy Atkins [34] varieties of mango. Gallic acid, caffeic acid, chlorogenic acid and protocatechuic acid were identified in all studied samples ( Figure 1A,B). Ferulic and vanillic acids were determined only in the pulp samples ( Figure 1A), while p-coumaric acid was estimated in the peel samples only ( Figure 1B). In the pulp samples, ferulic acid was predominant, followed by protocatechuic, chlorogenic, gallic, vanillic and caffeic acids. However, in the peel samples, gallic acid was predominant, followed by protocatechuic acid, chlorogenic acid, caffeic acid and p-coumaric acid. The Xiao Tainang cultivar exhibited the highest content of ferulic acid (33.75 mg/100 g) on a fresh weight basis, followed by Aozhou and Guifei. The lowest concentration of ferulic acid was present in the pulp of Lvsong, whereas in the Da Tainang and Kaite, cultivars ferulic acid contents were below the detection limit. In the peel samples, the gallic acid concentration varied from 79.15-1.450 mg/100 g FW. Gallic acid was maximum in the peel of the Da Tainang cultivar, whereas the minimum content was determined in Aozhou. In the pulp samples, the highest content of gallic acid was estimated for Aozhou, followed by the Lvsong and Da Tainang cultivars at 2.982, 2.492 and 2.369 mg/100 g FW, respectively. These values were significantly different at p < 0.05. In the peel samples, measured values of gallic acid were compatible with the reported levels in the Ataulfo variety, whereas pulp contained a lower concentration than the reported levels in previous studies [15]. It was noted that, in the peel of the Jidan cultivar, chlorogenic acid was maximum at 27.19 mg/100 g FW, whereas the lowest value was estimated in the peel of Shuixian. However, in the pulp samples, chlorogenic acid ranged from 0.957 mg/100 g FW in Xiangya to 6.147 mg/100 g FW in the Aozhou cultivar, and these values were considerably lower compared to Ataulfo mango [15]. A significant level of protocatechuic acid was quantified in the peel of Xiao Tainang at 64.33 mg/100 g FW, followed by the Aozhou and Jidan cultivars with a significant difference at p < 0.05. In the case of pulp samples, the maximum concentration of protocatechuic acid was estimated in the pulp of Aozhou, followed by the Jidan and Xiao Tainang cultivars. In the pulp samples, caffeic acid ranged between 1.117 and 0.250 mg/100 g FW. The highest concentration of caffeic acid was determined in the peel of Jidan at 13.62 mg/100 g FW, followed by Xiangya and Xiao Tainang at 7.070 and 2.989 mg/100 g FW, respectively, whereas it was undetectable in the peel of other cultivars. Antioxidant Capacity The results of in vitro antioxidant capacity determined PSC and ORAC assays are presented in Figures 2 and 3. In general, peel samples showed more antioxidant capacity compared to pulp. The hydro-PSC method was used for the first time to evaluate the antioxidant capacity of mango. In the peel samples, the PSC values ranged between 61.91 and 10.25 μM vitamin C equivalent/g FW. The highest peroxyl radical scavenging capacity was shown by the Aozhou cultivar, followed by Xiao Tainang and Da Tainang, whereas Lvsong showed the lowest level ( Figure 2B). In the pulp samples, the maximum PSC value was calculated for the Xiao Tainang cultivar at 8.713 μM vitamin C equivalent/g FW (Figure 2A). The results of oxygen radical absorbance capacity (ORAC) indicated that peel and pulp of the Xiao Tainang cultivar showed antioxidant capacity determined by the ORAC assay at 549.8 and 29.50 μM Trolox equivalent/g FW ( Figure 3A,B) among all of the studied samples. In the previous studies, the antioxidant activity of mango has been estimated by different methods [15,[24][25][26][27]32], which are incomparable to our findings. However, it was noted that measured values of oxygen radical absorbance capacity in mango pulp were compatible to [25,27]. To our knowledge, mango peel has never been analyzed to determine the oxygen radical absorbance capacity before. (A) (B) Figure 3. (A) ORAC value in pulp samples, the means of three replicates ± SD; different letters "a-c" indicate significant difference at p < 0.05; (B) ORAC value in peel samples; the means of three replicates ± SD; different letters "a-g" indicate significant difference at p < 0.05. Correlations In view of the fact that a large number of different antioxidants contribute to the total antioxidant capacity, it is not yet clear which components are more accountable for the observed antioxidant capacity [26]. Significant correlations between phenolic compounds and antioxidant activity in various kinds of fruits have been reported in previous studies [2,[42][43][44]. Tables 5 and 6 showed correlation coefficient matrices between phenolic content (i.e., TPC, TFC, TAC and phenolic acids) and antioxidant capacity in the pulp and peel samples. In the peel samples, highly significant coefficients of determination were calculated between TPC-ORAC, TPC-p-coumaric acid, ORAC-p-coumaric acid and ORAC-PSC (0.977, 0.962, 0.920 and 0.805, respectively). In the pulp samples, significant correlations were noted between TFC and chlorogenic acid (r = 0.858), TPC and vanillic acid (r = 0.822), protocatechuic acid-and chlorogenic acid (r = 0.807) and TFC-vanillic acid (r = 0.784), while total phenolic content exhibited a strong relationship with hydro-PSC (r = 0.675). These results were consistent with previous studies [45] and indicated that phenolic compounds contribute significantly to the antioxidant capacity. An inverse relationship between the consumption of foods rich in phenolic acids, such as chlorogenic acid and gallic acid, and the occurrence of different diseases has been suggested previously [25]. However, we observed significant correlations between phenolic acids and antioxidant activity in mango cultivars. Our data revealed that mango peel in particular is an excellent source of phenolic compounds, which are major source of natural antioxidants [25]. These findings are in agreement with the declaration that the antioxidant capacity of fruits and vegetables appears to be largely influenced by non-vitamin C phytochemicals [46]. Moisture Content The moisture content in the pulp and peel was determined by a modified oven-dried method [46]. Briefly, 10 g of pulp and 5 g of peel samples were dried in an oven at 105 °C until constant weight. Each drying test was performed in triplicate, and data were presented as the mean ± standard deviation (SD) of triplicates. Extraction Phenolics were extracted following the method as described by [47], with modifications [48]. Briefly, 5 g of fresh pulp and 3 g of fresh peel in triplicate were blended five times each with 25 mL of 80% chilled acetone for 5 min, followed by homogenization for 5 min in an electric homogenizer. Homogenates were centrifuged at 2500 rpm for 10 min; supernatants were pooled in rotary flasks and evaporated using a rotary evaporator at 45 °C until 10% of the filtrate was left behind. The filtrates were reconstituted with water to a final volume of 10 mL and stored at −40 °C for further analysis. Determination of Total Phenolic Content The Folin-Ciocalteu colorimetric method, as described earlier [49], with modifications [50], was used to determine the total phenolic content in the peel and pulp samples. All extracts were diluted with Milli-Q water to get readings falling within the range of the standard curve concentration: 0.0-600.0 µg gallic acid/mL. One hundred microliters of gallic acid solution or extracts were added to 0.4 mL of Milli-Q water in each test tube, followed by the addition of Folin-Ciocalteu reagent (0.1 mL). The solutions were allowed to react for 6 min to ensure the complete and speedy reaction of the Folin-Ciocalteu reagent with oxidizable phenolates in the sample. Then, 1 mL of 7% sodium carbonate solution was added to neutralize the mixture, followed by the addition of 0.8 mL Milli-Q water to adjust the final volume to 2.4 mL. The samples were mixed and allowed to stand for 90 min at room temperature. After color development, absorbance was measured at 760 nm on a DU 730 Nucleic Acid/Protein analyzer (BECKMAN, Inc., Fullerton, CA, USA). Total phenolic contents were calculated based on the standard curve of known gallic acid concentrations, and final values were expressed as milligrams of gallic acid equivalent per 100 grams on a fresh weight basis (mg GAE/100 g FW). Data were presented as the mean ± SD for triplicates analyses. Estimation of Total Flavonoid Content Total flavonoid content was estimated by the sodium borohydride/chloranil method (SBCM) as established in our laboratory [30]. Briefly, 1 mL of each extract was added into test tubes (15 × 150 mm), then kept under nitrogen gas until dried and reconstituted with 1 mL of tetrahydrofuran/ ethanol (THF/EtOH, 1:1, v/v). Freshly-prepared catechin hydrate (0.3-10.0 mM) in 1 mL of THF/EtOH (1:1, v/v) was used as the standard for analysis. Zero-point-five milliliters of each (NaBH4 (50 mM) and AlCl3 (74.6 mM)) solution were added into all test tubes with samples or standards and shaken on an orbital shaker at room temperature for 30 min. Additionally, 0.5 mL of NaBH4 (50.0 mM) solution were added into each test tube and shaken for another 30 min under the same condition. After shaking, 2.0 mL of chilled acetic acid (0.8 M) were thoroughly mixed, and the mixture was kept in the dark for 15 min. Then, 1 mL chloranil solution (20.0 mM) was added in each tube, and the mixture was heated at 95 °C in a shaking bath for 60 min. The reaction solutions were cooled with tap water, and the final volume was kept at 4 mL using methanol. One milliliter of 16% vanillin solution (w/v) was added into each tube, followed by the addition of 2 mL HCl (12 M), then mixed thoroughly and kept in the dark for 15 min. The reaction solutions were centrifuged at 2500 rpm for 10 min, and absorbance was immediately measured at 490 nm against a blank using a DU 730 Nucleic Acid/Protein analyzer (BECKMAN, Inc.). Total flavonoid content in each sample was calculated, using the standard curve of catechin hydrate concentration. The final value was expressed as milligrams of catechin equivalent per 100 gram of fresh weight (mg CE/100 g FW), and data were reported as the mean ± SD for triplicate analyses. Determination of Total Anthocyanin Content Total anthocyanin content was determined following the method as explained by [41]. Acetone extracts of pulp and peel samples in triplicate were mixed carefully with 0.025 M potassium chloride buffer (pH = 1) in 1:6 ratio. The absorbance was measured at 515 and 700 nm against distilled water blank (BECKMAN). Afterword, the extracts were mixed with sodium acetate buffer (pH = 4.5); absorbance was measured at the same wavelengths, and the total content of anthocyanins was calculated using the formula as follows: Total anthocyanins (mg/100 g of FW of samples) = A × MW × 1000/(ε × C) where A is absorbance = (A515 − A700) pH 1.0 − (A515 − A700) pH 4.5; MW is the molecular weight for cyanidin 3-glucoside = 449.2; ε is the molar absorptivity of cyanidin 3-glucoside = 26,900; and C is the concentration of the buffer in mg/mL. Anthocyanin content was expressed as milligrams of cyanidin 3-glucoside equivalent per 100 g on fresh weight basis (mg CGE/100 g FW), and data were reported as the mean ± SD for triplicates analyses. Identification and Quantification of Phenolic Acids Phenolic acids in the pulp and peel extracts of mango cultivars were determined by the method explained by [15]. Samples were injected automatically into an HPLC system (Waters Corp., Milford, MA, USA) equipped with a photodiode array detector. Absorption spectra for the main peaks were recorded at 280 and 320 nm. The HPLC system was equipped with a C18 reverse phase column (250 mm × 4.6 mm, 5 μm);the mobile phase was composed of 1% formic acid (A) and acetonitrile (B), and the isocratic elution gradient was 20% (B) in 40 min at a flow rate of 0.6 mL/min at 25 °C. The injection volume of the sample was 20 µL. Peaks were identified on the basis of retention time and chromatographs of the standards. Phenolic acids were identified and quantified on the basis of calibration curves and were expressed as mg phenolics per 100 g of FW. Data were reported as the mean ± SD for triplicate analyses. Antioxidant Capacity Assays Currently, researchers are paying more attention to natural antioxidants present in fruits, vegetables and whole foods because of their safety and potential nutritional and therapeutic effects [51]. The antioxidant potential of commonly-consumed tropical and subtropical fruit has been rated in the order of guava > mango > papaya > lemon [52]. Owing to the complex reactivity of phytochemicals, the antioxidant capacity of food and food extracts cannot be estimated by only a single method. However, at least two test systems have been recommended to establish legitimacy [53]. Consequently, the antioxidant capacity in the peel and pulp samples of mango cultivars was evaluated by the peroxyl scavenging capacity (PSC) and oxygen radical antioxidant capacity (ORAC) methods. Hydrophilic Peroxyl Radical Scavenging Capacity Assay The peroxyl scavenging capacity (PSC) assay is based on the oxidation of DCFH by peroxyl radicals and is used to determine the antioxidant capacity in hydrophilic and lipophilic extracts of fruits, vegetables, grains and whole food [47].The hydrophilic peroxyl radical scavenging capacity (hydro-PSC) assay, as explained by [47], with modifications [48,54], was used to assess antioxidant capacity in the pulp and peel of mango cultivars. Seventy five millimolar phosphate buffer (pH 7.4) was used to dilute samples in appropriate concentrations. Ascorbic acid and gallic acid were made fresh and diluted to (6.3, 4.8, 3.2, 2.4, 1.0) and (5, 3.5, 2.7, 1.4, 0.9) μg/mL concentrations, respectively, using phosphate buffer (75 mM, pH 7.4). The reaction mixture contained phosphate buffer (75 mM, pH 7.4), ABAP (40 mM), DCFH dye (13.26 μM) and the suitable amount of the pure antioxidant compound or sample extract. The dye was prehydrolyzed with 1 mM KOH to eradicate di-acetate before use and the reaction was carried out at 37 °C, in a total volume of 250 µL using a 96-well plate. Fluorescence generation was observed (excitation at 485 nm and emission at 538 nm) on a Fluoroskan Ascent fluorescent spectrophotometer (SoftMax systems, Molecular Devices, Sunnyvale, CA, USA). Data were analyzed using SoftMax Pro Software, Version 6.2 (SoftMax systems, Molecular Devices) running on a PC. The areas under the fluorescence reaction time kinetic curve (AUC) for both control and samples were included and used as the basis for the determination of peroxyl radical scavenging capacity (PSC) using equation: where SA is the AUC for the sample or standard dilution and CA is the AUC for the control reaction. Compounds or extracts inhibiting the oxidation of DCFH produced lesser SA and higher PSC values. EC50, the dose requisite to cause 50% inhibition (PSC unit = 0.5) for each pure compound or sample extract, was used to assess antioxidant activity of different compounds or samples. Final values of hydro-PSC were expressed as μmol of vitamin C equivalent per 100 g of FW (μM vCe/100 g of FW), and data were reported as the mean ± SD of each triplicate. Oxygen Radical Scavenging Capacity Assay The ORAC assay is a widely-used method to analyze the oxygen radical absorbance capacity of plant species extracts. This assay is based on free radical damage to a fluorescent probe through a change in its fluorescence intensity [55]. In the typical ORAC assay, the fluorescent loss of probes as phycoerythrin or fluorescein is followed over time in the absence and presence of antioxidant [41]. The oxygen radical absorbance capacity (ORAC) assay, as described by [55], with modifications [56], was conducted to measure the total antioxidant activity of the studied samples. Briefly, 20 μL of sample extracts in triplicate, diluted with 75 mM phosphate buffer (pH 7.4), were added in 96-well microplate, followed by the addition of 200 μL of fluorescein (0.96 μM), and incubated at 37 °C for 20 min. Outer wells were kept empty to avoid variation from inner wells. After incubation, 20 μL of freshly-prepared 119.4 mM AAPH in 75 mM phosphate buffer (pH 7.4) were added into each well, and the fluorescence intensity was measured immediately for 35 cycles every 4.5 min at an excitation of 485 nm and emission of 535 nm by the FilterMax F5 Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). Different concentrations of Trolox (range 6.25-50 μM) were used as a control. ORAC values were calculated by extrapolation on a calibration curve and expressed as the mean ± SD micromoles of Trolox equivalent per 100 g of fresh weigh (μM TE/100 g of FW) for three replicates. Statistical Analysis Statistical analyses were performed using SPSS software 13.0 (SPSS Inc., Chicago, IL, USA), and the dose effect was analyzed using Calcusyn software Version 2.0 (Biosoft, Cambridge, UK). Results were subjected to ANOVA, and differences among means were located using Tukey's multiple comparison test. A p-value less than 0.05 (p < 0.05) was regarded as statistically significant. Basic statistical parameters and correlation coefficients among the measured variables were also calculated. All data were reported as the mean ± SD for three replicates. Conclusions The present study was focused on comparative assessment of phenolic content and in vitro antioxidant capacity in the pulp and peel of mango cultivars. Though phenolic compounds showed a significant contribution in the inhibition of free radicals, the antioxidant capacity of mango and other fruits is not only due to the content of phenolic acids. It may also be due to the presence of various bioactive compounds, such as carotenoids, vitamins and other polyphenolics phytochemicals present in the pulp and peel of mango, which were not identified in the present study. Our results showed that the Xiao Tainang and Aozhou cultivars contained maximum phenolic content and exhibited remarkable antioxidant capacity. Gallic acid and protocatechuic acid were predominant in the peel and pulp of the studied samples. Highly significant correlations (r = 0.997, 0.962, 0.922, etc.) were calculated between phenolic and antioxidant properties, particularly in the peel samples. The present study revealed that the antioxidant capacity of mango peel is due to the synergistic actions of phenolics and other bioactive compounds present in it. Therefore, it is suggest that mango peel may contribute to promoting human health as a functional food or a value-added ingredient. To our knowledge, this is the first report on the comparative assessment of phenolic compounds and antioxidant capacity determined by ORAC and hydro-PSC assays in the peel and pulp of mango cultivars in China, particularly in peel. However, additional studies are desirable to assess the bio-absorption, mechanism of action and associations between these compounds after consumption.

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2018-01-01T10:59:15.547Z

2015-06-20T00:00:00.000Z

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Simultaneous extraction, optimization, and analysis of flavonoids and polyphenols from peach and pumpkin extracts using a TLC-densitometric method Background The use of medicinal plants has been reported throughout human history. In the fight against illnesses, medicinal plants represent the primary health care system for 60 % of the world’s population. Flavonoids are polyphenolic compounds with active anti-microbial properties; they are produced in plants as pigments. Quercetin, myricetin, and rutin are among the most well-known and prevalent flavonoids in plants, with an antioxidant activity capable of decreasing the oxidation of low density lipoproteins [LDLs]. To date, this research is the first of its kind to employ a coupled thin-layer chromatography (TLC) and a densitometric quantification method with a Box-Behnken design (BBD) response surface methodology (RSM) for optimization of ultrasonic-assisted extraction and determination of rutin and quercetin from peach and ellagic acid and myricetin from pumpkin fruits. Results The effect of process variables (extraction temperature (°C), extraction power (%) and extraction time (min)) on ultrasound-assisted extraction (UAE) were examined by using BBD and RSM. TLC followed by Quantity-One™ (BioRad) image analysis as a simple and rapid method was used for identification and quantification of the compounds in complex mixtures. The results were consistent under optimal conditions among the experimental values and their predicted values. A mass spectrometry (MALDI-TOF MS) technique was also used to confirm the identity of the natural products in the TLC spots resolved. Conclusion The results show that the coupled TLC-densitometric methods & BBD can be a very powerful approach to qualitative and quantitative analysis of; rutin and quercetin from peach extracts; and ellagic acid and myricetin contents from pumpkin extracts. Introduction The use of medicinal plants has been reported throughout human history [1]. In the fight against illnesses, medicinal plants represent the primary health care system for 60 % of the world's population. With the advent of chemistry, modern pharmacotherapy has depended more on synthetic drugs, however due to raised safety concerns and lower drug efficiency there is a growing interest to show direct evidence of the crucial role of natural secondary metabolites [2][3][4]. Flavonoids are polyphenolic compounds with active phytoalexins (anti-microbial) properties; they are produced in plants as pigments [5]. Quercetin, myricetin, and rutin are among the most well-known and prevalent flavonoids in plants, with an antioxidant activity capable of decreasing low density lipoproteins [LDL] oxidation [6]. Different types of hydro-alcoholic mixtures have been used to extract flavonoid from the plant material [7,8]. Methanol or ethanol was employed depending on the targeted compounds [9]. Extraction techniques for flavonoids are typically called traditional and modern methods [10]. Maceration, reflux, percolation, and Soxhlet extraction are within the lists of traditional methods, which have been significantly improved by automation [11]. Currently the focus has been on modern methods that were developed to be more efficient, faster, and with lower consumption of organic solvent [10,12,13]. Some of the modern methods are ultrasound assisted extraction (UAE), microwave assisted extraction (MAE), pressurized liquid extraction (PLE), and supercritical fluid extraction (SFE) [14,15]. Many methods have been used to isolate and measure the activity of antioxidant compounds [16]. Recently, thin layer chromatography (TLC) analysis of flavonoids in plant and animal samples was used for studying the application of scanning densitometry in quantification of flavonoids [17]. TLC is widely used because it is relatively simple, rapid, inexpensive, and accurate method for chemical identification when coupled with mass spectrometry (MS) [18]. TLC combined with densitometry and image analysis can have the ability to measure medicinal plant components [19]. Densitometry can be used to measure the differences among absorbance or fluorescence signals between a separated zone and the empty plate background across a range of wavelengths [20]. Image analysis methods are used to compare the spot color intensity with the plate color background. The peak area of the test spots are compared with data from calibration standards chromatographed on the same plate [21,22]. Several techniques have been used in the past for determining plant extracts, including TLC [23]; GC-MS [24]; and HPLC [25]. There are no known reports that described a coupled TLC-densitometry method for quantitative determination of; ellagic acid and myricetin from pumpkin; or quercetin and rutin from peach. This study employed a coupled TLC densitometric method and Box-Behnken design (BBD) with response surface methodology (RSM) for optimization of UAE. Isolated products identities were confirmed by using matrix-assisted laser desorption/ionization time-off-light MS (MALDI-TOF MS) analysis. Plant material All crops were grown on bare soil, a silt loam typical of southern Illinois. Fresh peaches (Red Haven) and pumpkins (Libbys Select) were harvested from several plants selected at random within a field at the Horticulture Research Center farm on Rowden Road near Southern Illinois University (Carbondale, IL). Peaches and pumpkins were grown according to conventional commercial methods for southern Illinois. Synthetic nutrients and pesticides were applied according to recommendations for peach and pumpkin production in southern Illinois. The samples were provided by Dr. Alan Walter (Department of Plant, Soil and Agricultural Systems, College of Agricultural Sciences, Southern Illinois University, USA). The fruits were cleaned and sliced into small pieces and crushed in a blender, and then sealed and stored in plastic bags in home refrigerator (−18°C) for five days before freeze-drying. Ultrasonic assisted extraction (UAE) An Elmasonic P30 (P30) ultrasonic device with heated water bath (Elma Hans Schmidbauer GMBH, Singen, Germany) set at 37 kHz was used for this study. User adjustable controls were heated bath temperature and power setting as a percentage of full power (30-100 %). The standard ultrasonic mode was used. The manufacturer rated the P30 with an effective power rating of 120 W. The P30 had a proprietary algorithm to adjust power based on the impedance of the system, resulting in the effective power rating. For a specific power setting, samples experienced the same degree of cavitation regardless of the load in the tank. For all treatments, the bath of the P30 contained 1.7 L of water before the treatment containers were added. Ultrasonic power was expressed as W/cm 2 , based on the power setting as a percentage of rated power and the volume of the bath solution. Although numerous variables my affect a process, identifying and controlling each variable with small contributions is practically impossible, therefore, variables were selected with known major effects [26]. The prior work of Altemimi et al. [27,28] with the same ultrasonic equipment was used as a guide and selected variables were bath temperatures of 30°C, 40°C, and 50°C; power level settings of 30 %, 50 %, and 70 %; and ultrasonic duration of 10 min, 20 min, and 30 min. The ultrasonic bath temperature was controlled by coupling with a cooling system using a cooling coil (Fisher Scientific Inc. St Louis USA) and water pump (model HJ-111, submersible pump, flow rate 250 L/h, Sunsun Inc., Zhejiang, China). Coupled heating and cooling helped to maintain temperatures that were evenly distributed across the ultrasonic water bath. Based on the manufacturer's effective power rating, the ultrasonic power for the three power settings inside the extract containers was 21 W/cm 2 , 35 W/cm 2 , and 49 W/cm 2 , respectively. A calorimetric method was used to independently verify the power settings [29]. Ten grams of the lyophilized samples were weighed and 100 mL of methanol were added to the samples in a 200 mL glass flask. Each flask was placed in the P30 and treated. After the samples had been exposed to ultrasound waves, the upper layer was filtered (Whatman no. 1) and placed in a rotary evaporator under vacuum at 40°C to remove solvent. Experimental design The effects of three independent variables of temperature, power, and time to optimize the extracted amount of compounds were investigated by using a BBD for RSM. The coded values of the experimental factors and settings for the experimental design were summarized in Table 1. The 17 ultrasonic treatments were completed in random order. The experimental data were analyzed with multiple regressions to fit the quadratic polynomial model in Eq. 1. Where Y is the predicted response; b 0 is the intercept; b 1 , b 2 and b 3 are the linear coefficients of temperature (X 1 ), power (X 2 ) and time (X 3 ), respectively; b 11 , b 22 and b 33 are the squared coefficient of temperature of sonication, power and time, respectively; b 12 , b 13 and b 23 are the interaction coefficients of temperature of sonication, power and time, respectively. The settings of the independent variables were represented as X i to X j . Thin layer chromatography chemical screening The glass TLC plates were 20 cm by 20 cm and pre-coated with silica gel 60 F254 (E. Merck/Millipore, Billerica, MA) (0.2 mm thickness). The following solvents were screened to determine the best separation compound for the TLC technique: 1) ethyl acetate 5: acetone 4 (v/v), 2) hexane 10: chloroform 10 (v/v), and 3) ethyl acetate10: formic acid 2: water 3 (v/v). The TLC plate was placed into oven at 110°C for 20-30 min to be completely dried. Each of the solvents was evaluated by mixing and placing 100 mL into a rectangular chromatography glass tank with ground edges. The glass tank was covered with a glass lid and solvents were allowed to saturate for 30-40 min before use. Two μL of each crude extract were added by syringe to a different TLC plate. The crude extracts were placed in a drop shape for identification and spread of the separated compounds according to Harbone [30]. Flavonoids have a weak natural fluorescence characterization and must be enhanced during separation on chromatography plates. The flavonoids fluorescence was enhanced by spraying the TLC plates with different complex agents. The most common complex agent used to increase the flavonoids fluorescence was the diphenyl-boric acid 2-amino ethyl ester (DPBA) [31]. Images of the TLC plates were analyzed using Quantity One™ densitometry software (Bio-Rad, Hercules, CA). The compounds in the samples were quantified by comparing density of the peaks and their areas (expressed as intensity per mm 2 ) from the samples with those from standard solutions of rutin, qurecetin, ellagic acid, and myricetin on the same plate. The best separation was obtained by ethyl acetate10: formic acid 2: water 3 (v/v) [32]. The software evaluated the area of separated spots by comparing the spot color intensity to the color of the TLC plate background. It was essential to chromatograph Two μL of each of the 17 plants extracts were applied on a TLC plate. The plate was developed and scanned as described in the TLC chemical screening process. The peak areas were recorded and the amounts of rutin and quercetin from peach extracts and ellagic acid and myricetin from pumpkin extracts were calculated using the respective calibration curves. Diagnostic checking of RSM model and validity testing Design-Expert™ software (version 9) was used to analyze the experimental results of the response surface design (State-Ease lnc. Minneapolis, MN, USA). P-values less than 0.05 were used to determine statistical significance of differences. Independent variables of extraction temperature, ultrasonic power, and extraction time were simultaneously optimized using RSM. Subsequently the output for each isolated compound was measured from peach and pumpkin extracts under the optimum ultrasonic conditions. The ultrasonic experiments using the optimum conditions were replicated three times and the results were compared with the predicted values for validation of the model. Mass spectrometric analysis The confirmation of each TLC spot identity was achieved using time-of-flight mass spectrometry. Each TLC spot of interest was excised and the compound was extracted into methanol. The freshly extracted compounds were then prepared for either matrixassisted laser desorption ionization (MALDI) or laser desorption ionization (LDI). Certified standards of quercetin, rutin, ellagic acid, and myricetin were analyzed in tandem to confirm the identity of each compound. A 1 − μL aliquot of TLC Spot Q, R, and M methanol extracts were spotted separately with 1 − μL of a MALDI matrix solution of α -cyano-4-hydroxycinnamic acid (αCHCA, 5 mg/mL αCHCA in 50:50 (v:v) acetonitrile: 0.1 % (v/v) trifluoroacetic acid in water). A 1 μL aliquot of TLC Spot E methanol extract was also spotted on the stainless steel sample plate with no MALDI matrix. The TLC Spots Q, R, M, and E were allowed to dry at room temperature. The stainless steel sample plate containing the dried MALDI and LDI samples was inserted into Bruker Daltonics (Billerica, MA, USA) MicroFlexLR time- Results and discussion Chromatographic separation and image analysis software TLC-densitometry coupled with image analysis detection was evaluated for the quantitative determination of induced flavonoids. According to the Figs. 1 and 2, the images allowed a visual evaluation of the flavonoids and polyphenolic acids (yellow-orange fluorescence) [33]. The method was suitable for rapid quantification of rutin and qurecetin in peach extracts and ellagic acid and myricetin in pumpkin extracts. It required less time for sample preparation and quantification compared to HPLC. These findings were in reasonable agreement with Nikolova et al. [22] and Naşcu-Briciu et al. [10] who found that TLC-densitometric analysis with image analysis software was complementary to the photodensitometric methods. Fitting the models The preliminary experiments were very advantageous in order to screen and choose the levels of independent variables for peach and pumpkin extracts. The experimental design for Box-Behnken and corresponding response data are presented in Table 1. According to the results in Table 1, the quadratic polynomial model was assigned for multiple regression analysis. The contribution of the quadratic model within regression coefficients analysis and the analysis of variance (ANOVA) are shown in Tables 2 and 3 [34]. In general, the variation in the data around the fitted model was examined using lack of fit test for the model [35]. Lack of fit must not be significant (p > 0.05) for an appropriate model. Effect of ultrasonic parameters on rutin and quercetin contents of peach and analysis of response surfaces ANOVA analysis and regression coefficients ( Table 2) were obtained in order to test the fitted quadratic surface models for rutin and quercetin content in peach extracts. For rutin content (μg/g of dry matter), the linear parameter (time) was significant and interaction parameters (temp*power, temp* time, time*power) were not significant (p > 0.05), whereas all quadratic parameters were significant (p < 0.05). In quercetin content (μg/g of dry matter), the interaction parameter (time* power) was significant at the level of p < 0.001 and the parameters (temp*power, temp*time) were not significant (p > 0.05) while all quadratic parameters were significant at the level of p < 0.05. F-values for lack-offit were 3.43 and 3.88 for rutin and quercetin, respectively. The lack-of-fit was not significant (p > 0.05). The R 2 of the models for rutin and quercetin content were 0.9572 and 0.9528, respectively. Moreover, the coefficients of variation (CV) were 1.43 and 1.57 for rutin and quercetin, respectively. Experimental results were predicted with good accuracy when a low coefficient of variation (CV) was obtained. Three-dimensional plots was used to better understanding the relationship between independent and dependent variables, then the following quadratic polynomial model equations (2, 3) were assigned to generate the contour plots: Rutin ¼ 2:89 þ 0:0325 Ã X 1 þ 0:02375 Ã X 2 þ0:06875 Ã X 3 −0:0025 Ã X 1 X 2 þ 0:0025 Ã X 1 X 3 þ 0:015 Ã X 2 X 3 −0:1525 Ã X 2 1 −0:09 Quercetin ¼ 2:77 þ 0:0325 Ã X 1 þ 0:02125 Ã X 2 þ0:06625 Ã X 3 −0:0024 Ã X 1 X 2 þ0:0025 Ã X 1 X 3 þ 0:015 Ã X 2 X 3 −0:1525 Ã X 2 The effects of parameter variables (ultrasonic temperature, power, and extraction time) and their interactions on rutin and quercetin contents in peach were studied. The third variable was assigned to be constant at the intermediate setting while surface plots of threedimensions were shown by two independent variables. As shown in Fig. 3a, with increase extraction temperature from 30°C to 41.08°C, the extraction amount of rutin quickly increased and reached the maximum value at 0 level of extraction time in the fixed extraction power of 53.24 %. However, with the increase of extraction temperature from 41.08°C to 50°C, the amount of rutin quickly decreased. This result confirmed that higher temperature can enhance the solubility of the solute thereby increases the yield of flavonoids. But, at the same time, increasing temperature can reduce the solvent density and consequently decreases the yield of total flavonoids. Therefore, the increase in temperature could have either a positive or a negative effect [36]. This finding was in agreement with Zhong [37] who reported that the thermal degradation of flavonoids and the decrease of number of acoustic cavitation bubbles were caused to decrease the amount of rutin. Figure 3b shows the effect of the interaction of extraction temperature and extraction time on the rutin content at a fixed extraction power of 0 level. Maximum rutin content was obtained at 41.08°C and then decreased slightly by increasing extraction temperature to 50°C in the fixed extraction time of 23.77 min. Figure 3c shows the effect of the interaction of extraction power and extraction time on the rutin content at a fixed extraction temperature of 0 level. Maximum rutin content was obtained at the highest extraction time in the fixed extraction power of 53.24 %. Moreover, the results found that extraction time (X 3 ) was the most significant factor affecting the responses at the level of p < 0.01. Figure 4a shows the effect of the interaction of extraction temperature and extraction power on the quercetin content at a fixed extraction time of 0 level. Maximum quercetin content was obtained at the lowest extraction temperature and reached the maximum value at 41.11°C of extraction temperature in the fixed extraction power of 52.92 %. Figure 4b shows the effect of the interaction of extraction temperature and extraction time on the quercetin content at a fixed extraction power of 0 level. Maximum quercetin content was also obtained at the lowest extraction temperature and then decreased slightly by increasing extraction temperature to 50°C in the fixed extraction time of 23.61 min. The decrease may be explained by oxidation and degradation of flavonoids due to sonication process with both highest extraction temperature and longest extraction time [38]. Figure 4c shows the effect of the interaction of extraction power and extraction time on the quercetin content at a fixed extraction temperature of 0 level. Maximum quercetin content was obtained at 52.92 % of extraction power in the fixed extraction time of 23.61 min. Table 3 lists the analysis of variance of the fitted quadratic polynomial model for ellagic acid and myricetin contents in pumpkin extracts. For ellagic acid content (μg/g of dry matter), the linear parameters (temp, time) were significant; interaction parameter (temp* power) and (temp*time) were significant (p < 0.05) while all quadratic parameters were significant at the level of p < 0.05. In myricetin content (μg/g of dry matter), the linear parameters (temp, time) were significant; the interaction parameters (temp*power, temp*time) were significant at the level of p < 0.001 and (power* time) was not significant (p > 0.05) while quadratic parameters X 2 1 ; X 2 3 À Á were significant at the level of p < 0.05. The F-value of 14.30, 15.30 of ellagic acid and myricetin contents respectively implied the model was significant. The lack-of-fit F-value of 0.7274 and 0.88 of ellagic acid and myricetin contents respectively reflects that the lack-of-fit was not significant. The R 2 of the models for ellagic acid and myricetin contents were 0.9484 and 0.9516, respectively. Moreover, the coefficients of variation (CV) were 1.10 and 1.06 for ellagic acid and myricetin contents, respectively. Effect of ultrasonic parameters on ellagic acid and myricetin contents of pumpkin and analysis of response surfaces Response surface models were used according to the following quadratic polynomial model equations (4,5) in order to study the effects of parameter variables (ultrasonic temperature, power, and extraction time) and their interactions on ellagic acid and myricetin contents of pumpkin extracts. The third variable was assigned to be constant at the intermediate point while surface plots of three-dimensions were made by two independent variables. Myricetin ¼ 2:96 þ 0:046 Ã X 1 −0:022 Ã X 2 −0:0445 Ã X 3 þ 0:060 Ã X 1 X 2 þ 0:043 Ã X 1 X 3 −0:025 Ã X 2 X 3 −0:11 Ã X 2 1 þ 0:00574 As shown in Fig. 5a, when extraction time was fixed at 0 level, ellagic acid contents were improved while the extraction temperature increased from 30°C to 38.81°C, and reached the maximum value in the fixed extraction power of 33.23 %, and then the amount of ellagic acid contents decreased when the extraction temperature reached 50°C due to the degradation of ellagic acid. The extraction amount of ellagic acid was affected by different ultrasonic extraction temperatures and ultrasonic extraction times as seen in Fig. 5b, when extraction power was fixed at 0 levels. It can be seen that the extraction amount of ellagic acid increased with the increasing ultrasonic extraction time and reached the maximum value at 18.51 min of extraction time. This finding was not in agreement with Zhang et al. [39] who found that the maximum value of ellagic acid from infructescence of P. latycarya strobilacea L. was at 40 min of extraction time. However, this result was concurred with Novak et al. [40] and Rostagno et al. [41] who confirmed that exposure to ultrasonic treatment for long time may cause loss to polyphenolic compounds due to denaturation, so it is very important to consider sonication time while processing. Figure 5c shows the effect of the interaction of extraction power and extraction time on the ellagic acid content at a fixed extraction temperature of 0 level. Maximum ellagic acid content was obtained at 18.51 min of extraction time in the fixed extraction power of 33.23 %. Moreover, the results were found that extraction temperature (X 1 ) and extraction time (X 3 ) were the most significant factor affecting the responses at the level of p < 0.01. Figure 6a shows the effect of the interaction of extraction temperature and extraction power on the myricetin contents at a fixed extraction time of 0 level. Maximum myricetin content was obtained at the highest extraction temperature and reached the maximum value at 38.98°C of extraction temperature in the fixed extraction power of 33.79 %. Figure 6b shows the effect of the interaction of extraction temperature and extraction time on the myricetin content at a fixed extraction power of 0 level. Maximum myricetin content was also obtained by increasing extraction temperature to 40°C in the fixed extraction time of 18.13 min. These results were in agreement with Shakthi Deve et al. [42] who found that the longest extraction time for flavonoids may result in loss to the polyphenols due to oxidation process. The oxidized products can convert to insoluble form compounds thereby diffusion of the polyphenols will be inhibited. Figure 6c shows the effect of the interaction of extraction power and extraction time on the myricetin content at a fixed extraction temperature of 0 level. Maximum myricetin content was obtained at the highest extraction power and reached the maximum value at 33.79 % of extraction power in the fixed extraction time of 18.13 min. Moreover, the results found that extraction temperature (X 1 ) and extraction time (X 3 ) were the most significant factor affecting the responses at the level of p < 0.01. Optimization and verification of the model for ultrasonic parameters In order to verify the rutin and quercetin contents simultaneously from peach extracts, there was one optimal extraction conditions, which was established to get the highest values: modifying the extraction temperature of 41.08°C to 40°C, extraction power of 53.09 % to 50 %, and extraction time of 23.68 min to 24 min. The results are shown in Table 4 and the amounts of rutin and quercetin contents respectively under the optimal predicted conditions and experimental conditions. There was significant difference (p > 0.05) between the experimental and predicted values. Thus, this modification was not appropriate to assign in order to optimize the process of rutin and quercetin contents from peach. It seems that it In order to facilitate the extraction process for pumpkin extracts, the optimal condition was modified as follows: the extraction temperature of 38.99°C to 40°C, and extraction power of 33.12 % to 33 %, and extraction time of 18.15 min to 18 min. The results are shown in Table 4 and the amounts of ellagic acid and myricetin contents respectively under the optimal predicted conditions and experimental conditions. There was no significant difference (p > 0.05) between the experimental and predicted values. Hence, the models can be used to optimize the process of ellagic acid and myricetin contents from pumpkin. Figure 7 shows the MALDI mass spectra obtained for TLC Spot Q-P (Fig. 7a) and TLC Spot R-P (Fig. 7c). Ion signals were observed at m/z 302.7 and 632.9. These ion signals were also observed in the MALDI mass spectra obtained from the certified quercetin (Q) (Fig. 7b) and rutin (R) (Fig. 7d) standards, respectively, and were assigned to the molecular radical cation (M +. ) of quercetin (Q) and the sodiated cation (M + Na + ) of rutin (R). Figure 8 shows the LDI mass spectrum obtained for the TLC Spot E-PP (Fig. 8a) and the MALDI mass spectrum obtained for the TLC Spot M-PP (Fig. 8c). Ion signals were observed at m/z 324.8 and 318.9, respectively, and were assigned to the sodiated cation (M + Na + ) of ellagic acid (E) and molecular radical cation (M +. ) of myricetin (M). Conclusion The results of this study indicated that the ultrasonic treatments had the ability to enhance and increase the amount of polyphenol extraction yields from plants extracts (peach and pumpkin). TLC-densitometric method and BBD can be a very powerful technique in quantitative analysis of rutin and quercetin from peach extracts and ellagic acid and myricetin contents from pumpkin extracts. A high correlation of the quadratic polynomial mathematical model was gained and could be employed to optimize rutin and quercetin from peach extracts and ellagic acid and myricetin contents from pumpkin extracts by ultrasonic-assisted assay. The modified optimal extraction conditions for measuring rutin and quercetin simultaneously from peach extracts were as follows: extraction temperature of 41°C, extraction power of 53 %, and extraction time of 24 min. Under these conditions, the experimental results of total rutin and quercetin contents were 2.816 ± 0.0305 μg/g of dry matter and 2.733 ± 0.0208 μg/g of dry matter respectively, which agreed closely with the predicted yield values. In contrast, the modified optimal extraction conditions for measuring ellagic acid and myricetin contents simultaneously from pumpkin extracts were as follows: extraction temperature of 40°C, extraction power of 33 %, and extraction time of 18 min. Under these conditions, the experimental results of total ellagic acid and myricetin contents were 2.96 ± 0.05 μg/g of dry matter and 2.953 ± 0.06 μg/g of dry matter respectively, which agreed closely with the predicted yield values.

v3-fos

2019-04-02T13:02:43.865Z

2015-05-28T00:00:00.000Z

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Growth and efficiency of water use of papaya cultivars (Carica papaya L.) under doses of bovine biofertilizer in hydroponics cultivation Among the fruit plants cultivated in Brazil, papaya (Carica papaya L.) stands out by having high productivity of fruit quality. The seedling production system of this culture needs a technology that promotes the production of plants with high physiological and sanitary quality. Thus, we aimed to evaluate the growth, dry matter accumulation and the efficiency of water use of papaya cultivars under doses of bovine biofertilizer in hydroponic culture. We used a completely randomized design with eight treatments in a factorial scheme 4 x 2, with six replications, and a useful plant per repetition totalizing 48 useful plants. Four doses of biofertilizers (D = 10, 20, 30 and 40% v/v) were tested and applied in two varieties of papaya (Sunrise Solo (C1) and Tainung-01 (C2)). During the first 60 days after sowing, the papaya cultivars were evaluated for growth, dry matter accumulation and water use efficiency in accordance to their doses of biofertilizers. The cultivar Tainung-01 has a higher potential for growth, biomass accumulation and efficient use of water in comparison with the Sunrise Solo cultivar. The doses estimated of 25 and 35% (v/v) of bovine biofertilizer promoted the greater growth and dry matter accumulation for the cultivars Sunrise Solo and Tainung-01, respectively. INTRODUCTION Among the fruit crops in Brazil, the papaya tree (Carica papaya L.) stands out for presenting high productivity of fruit quality. In the year of 2012, there was a national production of 1,517,696 tons, being the world's largest producer and the third largest exporter of papaya, with the Northeast Region (902,000 tons) being the largest producer of this fruit, followed by the Southeast (549,000 tons), North (42,000 tons), Midwest (6,000 tons) and the *Corresponding author. E-mail: vanies_agronomia@hotmail.com Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License South (4,000 tons), respectively (IBGE, 2014). The culture has shown great economic and social expression, mainly in the states of Bahia, Espírito Santo, Rio Grande do Norte and Ceará. Regarding the exports, the state of Espírito Santo accounts for 50% of the total (Serrano and Catteano, 2010). Due to the expanded cultivated areas and the need to increase productivity and final product quality, efforts are made to always improve productivity levels and reduce production costs (Guimarães et al., 2012). Thus, new technologies have been introduced in the papaya culture aiming to raise productivity levels. As such, the use of biofertilizers and seedling production systems through hydroponics emerge as a promising alternative, considering that the phase of seedlings and their initial development interfere directly in the orchard productivity (Trinidad et al., 2000). Among the papaya cultivars most commonly grown in Brazil are those of Solo and Formosa groups. Cultivars from the 'Solo' group are intended mainly for the export market, for having smaller fruits. The main cultivars of the 'Formosa' group are imported hybrids that produce larger fruits that are intended mainly for the domestic market, being used in these conventional, integrated and organic crop practices (Hafle et al, 2009;Serrano and Cattenao, 2010). The cultivation of papaya seedlings in a protected environment favors the production of high quality physiological and sanitary plants. According Fochesato et al. (2007), this needs to be done in containers where the seedlings produced alter their development complying with culture medium, when compared to the process in the field, with limited space for root growth. A good alternative for this is the optimization of propagation methods in hydroponics, which targets the time reduction to obtain seedlings, as well as a greater control of nutrition and phytosanitary conditions (Souza et al., 2013). In most cases of hydroponic cultivation, the nutrient solutions are produced from a mixture of different fertilizer salts of high solubility in water (Resh, 1997), but they can also be produced from organic biofertilizers, a system known as "organoponics", or as part of the solution, as it occurs in organic-inorganic hydroponics (Martins, 2000). Several studies have been reported in the literature with promising results of the use of biofertilizers in the seedling production from different cultures: Medeiros et al. (2008) with lettuce, Probst et al. (2008) in forage, Cocco et al. (2008) with tobacco and Dantas et al. (2014) with acerola. However, there are too few studies that enable the production of papaya seedlings using biofertilizers, especially when they are related to hydroponic production. Based on the above considerations, this study aimed to evaluate the growth, dry matter accumulation and efficiency of water use of papaya cultivars under doses of bovine biofertilizer in hydroponic cultivation. MATERIALS AND METHODS The experiment was carried out from February 3rd to April 3rd, 2012 in a seedling nursery at the Universidade Estadual of Paraíba (UEPB), Campus IV, Catolé do Rocha -PB, covered with a nylon shading screen for 50% brightness inside. We used a completely randomized design with 8 treatments in a factorial 4 x 2, with six replications, and a useful plant per repetition, totalizing 48 useful plants. Four doses of biofertilizers (D = 10, 20, 30 and 40% v/v) were tested and applied in two varieties of papaya (Sunrise Solo (C1) and Tainung-01 (C2)). The plants were grown in a hydroponic system using modified Leonard jars, made with pet bottles according to the methodology of Santos et al. (2009). The bottles were cut 14 to 15 cm from the base and together with the caps they underwent a sterilization process at a 250 L water tank with sodium hypochlorite (10%) for one hour. After this period, all parts of the bottles were rinsed in tap water to remove excess sodium. To each vessel, it was added one liter of washed sand, which was sterilized by autoclaving at a temperature of 121°C for two consecutive days number of hours. After being filled, the pots were seeded (three seeds per pot) and covered with paper bags, in order to prevent algae growth in the solution. The bovine biofertilizer was obtained by anaerobic fermentation, mixing equal parts of fresh cattle manure and slightly water win electrical conductivity -ECw = 0.8 dS m -1 , adding 2 kg of leaves and branches of the leguminous plant cowpea (Vigna unguiculata L.) (Table 1). For the preparation of the biofertilizer, plastics biodigesters with a capacity for 200 L were used, kept hermetically sealed for 45 days. To release the methane gas produced during fermentation, a thin hose was connected at the upper base and the other end was submerged in a water container to prevent the entrance of air and loss of quality of the organic feedstock (Santos, 1992). For being applied in liquid form, it was analyzed as if it were water for irrigation, as the data in Table 1, as a suggestion of Dantas et al. (2014). The total volume of the solution was 0.7 L, being replaced weekly based on culture evapotranspiration (ETc), as shown in Table 2. According to the methodology proposed by Benincasa (2003), relative growth rates in height (RGRH) were determined by equation 1 and in stem diameter (RGRSD) by equation 2. Based on the growth in stem diameter, and height the papaya seedlings reached in the end of the total emergency 15 after sowing in relation to the analyses performed at 30, 45 and 60 days after sowing (DAS). (1) In which: RGRH = Relative growth rate in height of plants (cm cm -1 Table 2. Water and biofertilizer consumption for papaya (Carica papaya L.) seedlings during 60 days in organic hydroponic cultivation. Total volume ml 10 1485 165 1650 20 1320 330 1650 30 1015 435 1450 40 870 (2) Water volume Biofertilizer volume In which: RGRSD = Relative growth rate stem diameter (mm mm -1 day -1 ); SD1 = plant stem diameter in the time t1 (mm); SD2 = plant stem diameter in the time t2 (mm), and ln = logaritmo natural. Also at 60 (DAS) the plants were collected to obtain the leaf dry matter (LDM) (g), stem dry matter (g) (SDM) (g) and root dry matter (RDM) (g), from the biomass partition of the collected material and packaging in an air circulating oven (DL-AF Dellta) at 65°C to dry the material for 72 h. After this period, the plants were weighed on an analytical balance (ABT 120-5DM Polimate). With the data of dry matter and water consumption by papaya, we determined the efficiency of water use (EWU) by the relationship between the produced dry matter and water consumed by the plant expressed in g L -1 . The results were submitted to analysis of variance (F test) and, when the parameters were significant, we used the Tukey mean comparison test (5%), for the cultivar factor and regression analysis, for the doses of biofertilizers with help from the SISVAR Software (Ferreira, 2011). RESULTS AND DISCUSSION To the relative growth in height of the cultivar C1 (Sunrise Solo), we verified a quadratic behavior at 30, 45 and 60 days after sowing, so that it reached the growth peak when cultivated under the doses of 24, 22 and 22% (v/v) of biofertilizer, respectively (Figure 1). Teixeira et al. (2009) also investigated reductions in height growth of papaya trees due to increasing doses of Lithothamnium. A fact confirmed by Dantas et al. (2014) in acerola seedlings, where the height of the seedlings responded in a quadratic way to bovine biofertilizer doses. The authors believe that these results were influenced by the increase in substrate fertility providing toxic effects. In order to cultivate C2 ('Tainung-01') a linear increase behavior was observed for the relative growth in height during the first 30 and 45 days after sowing due to the increase of biofertilizer doses up to the maximum level studied (40% v/v). It was also verified that at 60 days after sowing this behavior became quadratic, so that the higher relative growth rates in height were achieved at a 25% (v/v) dose of biofertilizer (Figure 1). Guimarães et al. (2012) also observed linear response of height growth in seedlings of Carica papaya. Tainung-01 in function of biofertilizer doses during the first 40 days after sowing. One can conclude from this that the need for larger doses of biofertilizer during the first 45 days after sowing may be related to lower efficiency of the plants' root system in this growth phase, so that at 60 days after sowing, when the seedlings had a more developed root system, they were able to meet their nutritional needs in hydroponic solution containing lower biofertilizer doses. For the growth in height, divergent behavior can be observed between the papaya (C. papaya) cultivars studied under biofertilizer doses at 30, 45 and 60 days after sowing (Figure 1). It was ascertained that the cultivar C1 ('Sunrise Solo') has lower nutritional requirements in relation to cultivar C2 ('Tainung-01'), thus demanding lower doses of biofertilizer to maximize its growth index. It was also observed that the cultivar C2 ('Tainung-01') holds the greatest potential for growth, especially under favorable nutritional conditions. For the growth of stem diameter, it is found that C2 ('Tainung-01') was similar to that observed in height, so that the papaya plants obtained linear relative growth rates of stem diameter according to biofertilizer doses during the first 30 and 45 days after sowing, denoting the initial growth potential of the cultivar and biofertilizer efficiency in papaya plant nutrition (Figure 2). However, at 60 days after sowing it was observed a quadratic behavior of the relative growth in stem diameter of C2 (Tainung-01), tending to reduce when cultivated in biofertilizer doses greater than 30% (v/v). Possibly after 45 days of sowing the papaya plants tend to reduce the growth in stem diameter due to the limitations of the container and a lower incidence of light in the nursery, reflecting the need for transplanting the seedlings. Thus, the reduction of growth limits nutrient and water absorption, such that larger doses of biofertilizer may have exerted a toxic effect on the papaya plants after this time. Sunrise Solo (C1) showed a quadratic behavior to the relative stem diameter growth at 30 days after sowing, reaching the maximum growth under 30% (v/v) dose of biofertilizer (Figure 2A and B). It was also observed that at 45 days after sowing, there was no significant influence of the doses in the relative growth in stem diameter of papaya plants ( Figure 2C and D). Such a fact may be related to the reduction of secondary growth activity of Sunrise Solo papaya plants, since at 45 days after sowing there was an increase in growth rates in height relative to the first 30 days after sowing, denoting the greater investment in primary growth. However, at 60 days after sowing, a quadratic behavior was once again verified in the relative growth of papaya cultivars, so that the seedlings produced at doses of 30% (v/v) of biofertilizer obtained the highest growth rates of 0.04 (mm day -1 ), similar to results obtained by cultivar Tainung-01, which also reached maximum growth at the respective dose. Lima et al. (2007) also found no differences in the relative growth of papaya plants Tainuing-01 and Golden due to the evaluation period. It is noteworthy that at 30 and 60 days after the sowing, both cultivars had similar growth rates, differing only in response to biofertilizer doses at 30 days, where cultivar Tainung-01 responds linearly to the doses, a fact that follows due to higher nutritional requirements of this cultivar in the first days after emergence, possibly due to having lower reserves from the seeds (cotyledons) compared to the cultivar Sunrise Solo who responded in a quadratic way to biofertilizer doses. For the leaf dry matter, a quadratic response was observed in both cultivars in relation to doses of biofertilizer ( Figure 3A), noting that the cultivar Tainung-01 had the highest leaf dry matter accumulation (0.71 g) under the dose of 34% (v/v) of biofertilizer. This value was 43.7% greater than the maximum leaf dry matter accumulation observed in cultivar Sunrise Solo (0.40 g), achieved at a dose of 21% (v/v) of biofertilizer. Based on these results, it is possible to explain the greater growth potential of cultivar Tainung-01 in relation to cultivar Sunrise Solo, given that the leaves are the organs responsible for the plant's photosynthetic activity and with it, the greater accumulation of leaf dry matter denotes the largest investment in active photosynthetic area, favoring the higher photosynthetic potential, de Paiva et al. 2319 encouraging further growth. This fact was observed in cultivar Tainung-01, that got high relative growth rates in stem diameter and height during the first 45 and 60 days after sowing respectively, in relation to cultivar Sunrise Solo (Figures 1 and 2). Similar behavior was ascertained by Diniz et al. (2011) in passion fruit plants, on which the supply of more than 50% (v/v) of biofertilizer caused decline in leaves dry matter accumulation. For stem dry matter, differing responses were verified between papaya cultivars depending on the increase of biofertilizer doses, and a quadratic response was found for cultivar Sunrise Solo with maximum accumulation of stem dry matter (0.27 g) in 26% (v/v) dose of biofertilizer ( Figure 3B). For that, a linear and increasing behavior of cultivar Tainung-01 was examined based on the biofertilizer doses reaching the maximum accumulation of 0.49 g in a dose of 40% (v/v) of biofertilizer, being this accumulation 45% higher than the cultivar Sunrise Solo ( Figure 3C). These results are possibly related to higher growth rates in height and stem diameter observed in cultivar Tainung-01 in relation to Sunrise Solo. In addition to that, the behavior observed for dry matter accumulation in papaya plants with biofertilizer doses was similar to that seen in the growth, so that the best responses from cultivar Sunrise Solo were at doses estimated close to 25% (v/v) of soil biofertilizer, while the best performance of cultivar Tainung-01 occurred at levels close to 40% (v/v) of biofertilizer. This denotes the genetic variability among papaya cultivars belonging to the Solo and Formosa groups regarding nutritional needs. As for the root dry matter, positive linear correlation was ascertained of cultivar Sunrise Solo to biofertilizer doses, obtaining increments of 0.007 g for each unit increase in the biofertilizer dose, reaching a maximum of 0.51 g in a dose of 40% (v/v) of biofertilizer ( Figure 3C). The stimulation of root growth may be related to the need for greater selectivity of nutrient in cultivation solution by the plant, promoting with this the exclusion of ions at high concentrations, considering a reduction of growth and leaf biomass accumulation of cultivar Sunrise Solo under higher doses of biofertilizer (Figures 1, 2 and 3A). For cultivar Tainung-01, a quadratic behavior was verified, a fact that confirms their stem and leaf dry matter accumulation. It was observed that in this cultivar the peak accumulation of root dry matter is reached in the dose of 34% (v/v) of biofertilizer, noticing a decrease thereafter ( Figure 3C). This behavior can be related to the toxic effect of some nutrients with an increasing dose of biofertilizer, making the plant reduce its root system due to the increase of nutrient concentration (salts) in solution. The divergence of the root system behavior of these cultivars due to the increase of biofertilizer doses may be related to its tolerance capacity to salt content in the solution. Sá et al. (2013) Sunrise Solo shows a higher potential of salt tolerance in relation to cultivar Tainung-01. This explains the root system growth capacity of cultivar Sunrise Solo even under the higher doses of biofertilizer, where there is a greater concentration of salts and nutrients in hydroponic solution. It was observed that efficiency water use of both papaya cultivars increased linearly with the increase of bovine biofertilizer doses in the cultivation solution ( Figure 3D). The efficiency water use is expressed by the relation between the biomass accumulation (CO 2 fixed during photosynthesis) and water consumption of the plant (sweating), so that the values denote the amount of carbon fixed by the plant by each unit of water lost (Taiz and Zeiger, 2013). It is believed that the higher doses of biofertilizer promoted an increase in the availability of nutrients in hydroponic solution, favoring root absorption, making it more efficient, and thereby promoting a reduction in the need of water uptake by the plants, since along with it, the essential nutrients for their growth are absorbed, favoring the increase in water use efficiency. It is noteworthy that cultivar Tainung-01 had the highest efficiency of water use in relation to cultivar Sunrise from doses greater than 15% (v/v) of biofertilizer, when compared under the same culture condition ( Figure 3D). It was also verified that the cultivar Taining-01 obtained unitary increments 75% higher than those observed for the cultivar Sunrise Solo with the increasing dose of biofertilizer. Thus, the greatest growth potential of cultivar Tainung-01 in relation to cultivar Sunrise Solo may be related to the first's higher efficiency in water use, denoting its greatest photosynthetic potential (CO 2 fixation) under increased nutrient availability. Conclusions The cultivar Tainung-01 has a higher growth potential, biomass accumulation and efficient use of water in comparison to the cultivar Sunrise Solo. The doses estimated of 25 and 35% (v/v) of bovine biofertilizer promoted the greater growth and dry matter accumulation for the cultivars Sunrise Solo and Tainung-01, respectively. The cultivar Sunrise Solo has lower nutritional requirements to achieve its maximum growth in relation to cultivar Taining-01.

v3-fos

2016-03-14T22:51:50.573Z

2015-06-30T00:00:00.000Z

5646359

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Identification of miRNAs and Their Targets in Cotton Inoculated with Verticillium dahliae by High-Throughput Sequencing and Degradome Analysis MicroRNAs (miRNAs) are a group of endogenous small non-coding RNAs that play important roles in plant growth, development, and stress response processes. Verticillium wilt is a vascular disease in plants mainly caused by Verticillium dahliae Kleb., the soil-borne fungal pathogen. However, the role of miRNAs in the regulation of Verticillium defense responses is mostly unknown. This study aimed to identify new miRNAs and their potential targets that are involved in the regulation of Verticillium defense responses. Four small RNA libraries and two degradome libraries from mock-infected and infected roots of cotton (both Gossypium hirsutum L. and Gossypium barbadense L.) were constructed for deep sequencing. A total of 140 known miRNAs and 58 novel miRNAs were identified. Among the identified miRNAs, many were differentially expressed between libraries. Degradome analysis showed that a total of 83 and 24 genes were the targets of 31 known and 14 novel miRNA families, respectively. Gene Ontology analysis indicated that many of the identified miRNA targets may function in controlling root development and the regulation of Verticillium defense responses in cotton. Our findings provide an overview of potential miRNAs involved in the regulation of Verticillium defense responses in cotton and the interactions between miRNAs and their corresponding targets. The profiling of these miRNAs lays the foundation for further understanding of the function of small RNAs in regulating plant response to fungal infection and Verticillium wilt in particular. Introduction MicroRNAs (miRNAs) are a class of endogenous non-coding small RNAs (sRNAs) that regulate gene expression at the transcriptional and post-transcriptional levels via mRNA cleavage or translational repression in plants and animals [1][2][3]. In higher plants, miRNAs play important roles in growth, development, stress responses, and many other biological processes [4][5][6]. In particular, there is increasing evidence that miRNAs are involved in regulating abiotic and biotic stress responses, including disease resistance [7][8][9][10]. Recently, sRNA-mediated gene silencing was found to play a significant role in plant defense against pathogens [8,[11][12][13]. In Arabidopsis thaliana, miR393 was found to contribute to basal defense against Pseudomonas syringae by regulating auxin signaling [8]. miR393 can be induced upon perception of flg22 (a 22-amino acid peptide), a PAMP (pathogen-associated molecular pattern) derived from bacterial flagellin, and negatively regulates transcripts of a number of F-box auxin receptors [8]. Moreover, miR160, miR167, and miR393 were identified as highly induced after infection using sRNA expression profiling on Arabidopsis leaves collected at 1 and 3 h post-inoculation of Pseudomonas syringae pv. tomato (DC3000hrcC). [12]. Interestingly, all three miRNAs negatively regulate auxin signaling by either targeting auxin receptor genes or auxin response factors [12]. Another recent study reported that miR162 and miR168 targeted Dicer-like1 and Argonaute proteins, which are likely up-regulated by infection and presumably positively regulated by plant defense responses, although their functions need to be confirmed by experimental data [13]. Cotton is one of the most important economic crops in the world. It is highly susceptible to cotton Verticillium wilt, a disease that significantly affects cotton yield and quality. Verticillium wilt is the primary disease attacking cotton crops and is mainly caused by a soil-borne fungal pathogen, Verticillium dahliae Kleb. (V. dahliae) [14]. The representative symptoms of susceptible cotton include leaf curl, necrosis and defoliation, and stem wilt [15]. It leads to discoloration of cotton leaves and stem vascular bundles, and it inhibits photosynthesis and increases respiration [16]. However, there is no proven control (chemical or cultural) for this disease as the mechanisms of Verticillium wilt remain poorly understood. Despite great efforts in producing wilt-resistant cotton cultivars by traditional breeding, very few Gossypium hirsutum varieties-the main species of cotton currently cultivated in the world-are resistant to the wilt [17]. Therefore, identification of new miRNAs and elucidation of their functions in response to V. dahliae infection will help us understand the regulation of pathogen defense responses. Recently, the Gossypium raimondii genome sequence was completed [18,19], and this will greatly advance biological research on cotton. Although many cotton miRNAs were identified in previous research, the role of miRNAs in the regulation of Verticillium defense responses is mostly unknown. To date, the majority of miRNA targets in cotton were predicted by bioinformatics approaches, and only a small portion were experimentally validated. Although no cotton cultivar is immune to Verticillium wilt, most cultivars of G. barbadense, such as Hai-7124, show significant advantages in Verticillium wilt resistance [17,20]. Therefore, to detect new miRNAs and their potential targets participating in the regulation of Verticillium defense responses, four sRNA libraries and two degradome libraries using RNAs from mock-and Verticillium-inoculated Gossypium hirsutum L. (G. Hirsutum) and Gossypium barbadense L. (G. Barbadense) roots were constructed and sequenced using a Solexa analyzer. A total of 140 known miRNAs and 58 novel miRNAs were identified and 107 genes sliced by 45 miRNA families were detected via degradome sequencing. The profiling of the miRNAs and their target genes provides novel information about the regulatory network of defense responses in cotton to V. dahliae. Overview of Small RNA (sRNA) Library Sequencing Four sRNA libraries were constructed and deep sequenced with total RNAs from G. hirsutum (Gh_CK: mock-inoculated; Gh_Ve: Verticillium-inoculated) and G. barbadense (Gb_CK: mock-inoculated; Gb_Ve: Verticillium-inoculated) roots. A total of about 26, 20, 21, and 20 million raw reads were obtained from Gh_CK, Gh_Ve, Gb_CK, and Gb_Ve, respectively (Table 1). After filtering out the reads of low quality and the adaptor sequences, there were approximately 26, 19, 20, and 20 million clean reads obtained in the Gh_CK, Gh_Ve, Gb_CK, and Gb_Ve libraries. To simplify the sequencing data, all identical sequence reads in each sRNA library were grouped and converted into unique sequence tags with associated counts of the individual sequence reads. There were 7,181,742; 6,033,991; 6,729,005; and 6,415,595 unique tags in the four sRNA libraries, respectively ( Table 1). The length distributions of sRNAs were very similar between the four libraries ( Figure 1). In these four libraries, the majority of sRNA sequences were 20-24 nt in size with 21 or 24 nt as the major size classes, which is typical for Dicer-derived products ( Figure 1). Identification of Known MicroRNAs (miRNAs) by sRNA Sequencing To identify known miRNAs in the four libraries, all mappable sRNA sequences were compared with the currently known plant miRNAs in the miRBase database (release 20.0), which contains 78 known cotton miRNAs. In total, approximately 77 known cotton miRNAs belonging to 52 families were identified in the four libraries. The numbers of reads of the 77 known cotton miRNAs in the four sRNA libraries are listed in Table S1. The miRNA families miR156 and miR166 were the most abundant in the four libraries. In addition, a total of the 63 known unique miRNAs with high sequence similarity to the other known plant miRNAs, representing 50 known miRNA families, were identified in the four libraries (Table S2). These known miRNAs, with a minimal folding free energy (MFE) of the predicted hairpins ranging from −21.34 to −130.6 kcal/mol (Table S2), covered almost all the plant-conserved miRNA families. Several known but non-conserved miRNA families that have previously been identified only from one or a few plant species were also found: e.g., miR477, miR530, miR827, miR1448, miR2111, miR2947, miR2950, miR3476, and miR5083. In the present study, most of the newly identified miRNA families, such as miR168, miR403, miR477, miR828, and miR1448, were from G. hirsutum, while most miRNAs from G. barbedense had already been identified previously (Table S2). Identification of Novel miRNAs in G. hirsutum and G. barbadense To predict novel miRNAs in G. hirsutum and G. barbadense, all mappable sRNAs were BLASTed to the G. raimondii genome sequence and known plant miRNAs in the miRBase database (release 20.0). The sRNAs that exactly mapped to the genome sequence and unknown plant miRNAs and their flanking sequences that could be folded into a secondary structure were considered as miRNA candidates. To increase predictive accuracy, five criteria described in the experimental section were mainly used to search for novel miRNAs. In total, 58 novel miRNAs were identified in the present study (Table 2). These new miRNAs were named temporarily in the form of novel_miR_number: e.g., novel_miR_1 and their lengths were 20, 21, 22, or 23 nt (Table 2). Among these miRNAs, 34 were detected in at least two of the four sRNA libraries, and 10 were detected in all four sRNA libraries. The predicted hairpins of their precursors had a MFE ranging from −21.1 to −143.2 kcal/mol with an average of −57.5 kcal/mol. All secondary hairpin structures are listed in Table S3. Expression Profiling of Differentially Expressed miRNAs in Response to V. dahliae Infection The expression profiles of miRNAs were analyzed and compared between the four libraries based on the number of clean reads generated from the high-throughput sequencing. After normalization, the reads of the tags of each miRNA family as "reads per million", p-value < 0.01, and the absolute value of |log2 Ratio | ≥ 1 were considered to indicate the statistical significance of miRNA expression. Interestingly, many miRNAs were differentially expressed between the libraries. miRNA expression between the mock-and Verticillium-inoculated treatments for the two cotton species was first analyzed. In G. hirsutum roots, a total of 19 miRNAs representing nine novel miRNAs were identified as V. dahliae-responsive miRNAs (Figure 2A). Among them, 11 miRNAs were preferentially expressed in the Gh_Ve treatment and eight were preferentially expressed in the Gh_CK treatment. In G. barbadense roots, a total of 26 miRNAs representing 13 novel miRNAs were identified as V. dahliae-responsive miRNAs ( Figure 2B). Among them, 20 were of higher abundance and only six were of lower abundance in the Gb_Ve treatment. Notably, novel_miR_13, novel_miR_21, novel_miR_25, novel_miR_27, and novel_miR_50 were of higher abundance in the roots of both cotton species after 24 h of treatment. The miRNA expressions between the two cotton species with mock-and Verticillium-inoculated treatments were also analyzed. We made a comparative analysis of miRNA expression between the two cotton species with mock-inoculated treatment ( Figure 2C). It was shown that a total of 35 miRNAs had a species-specific expression. Among them, 16 miRNAs were preferentially expressed in G. hirsutum roots and 19 were preferentially expressed in G. barbadense roots. Because of the genotype-specific expression of miRNAs under mock-inoculated treatment, it was plausible to assume that some miRNAs had preferential expression in one of the two species when both of them were inoculated by Verticillium. Thus, we compared the expression levels of miRNAs between Gb_Ve and Gh_Ve libraries. We found that 38 miRNAs had species-specific expression in Verticillium-inoculated treatments ( Figure 2D). Among them, 17 miRNAs were preferentially expressed in G. hirsutum roots and 21 were preferentially expressed in G. barbadense roots. Interestingly, most of the species-specific-expressed novel miRNAs (e.g., novel_miR_2, novel_miR_8, novel_miR_11, novel_miR_20, novel_miR_26, novel_miR_29, and novel_miR_37) were significantly preferentially expressed in Verticillium-inoculated G. hirsutum roots. To validate the existence and expression patterns of the predicted miRNAs in mock-and Verticillium-inoculated cotton roots, four novel miRNAs, as well as eight representative known miRNAs, were selected for qRT-PCR analysis. Although some non-conserved and novel miRNAs were identified in low read number or were undetectable in one or two sRNA libraries by Solexa sequencing, the 12 selected miRNAs were detected by qRT-PCR. The qRT-PCR results of those miRNAs were quite consistent with the results from the sequencing data, and confirmed the changes in miRNA expression in response to V. dahliae infection ( Figure 3A). Furthermore, to confirm the causality of the miRNA expression patterns and their target gene, we studied the expression of four miRNAs and their target genes by qRT-PCR in G. hirsutum and G. barbadense roots. Among the target mRNAs tested, four targets showed a significant inverse correlation in expression with their corresponding miRNAs ( Figure 3B). U6 snRNA and Ubiquitin1 were chosen as endogenous control genes. Error bars indicate standard deviation of three biological replicates.* and ** indicate significant differences relative to the Gh_CK at p < 0.05 and p < 0.001 by Student's t-test, respectively. Target Genes of miRNAs Identified by Degradome Analysis In the present study, degradome sequencing was used to search for the target genes of identified miRNAs in cotton. According to the relative abundance of tags at the predicted miRNA target sites, the identified targets were grouped into five categories (0-4) as described by Yang [21]. Category "0" was defined as >1 raw read at the position, with abundance at the position equal to the maximum on the transcript, and with only one maximum on the transcript. Category "1" was defined as >1 raw read at the position, with abundance at the position equal to the maximum on the transcript, and with more than one maximum position on the transcript. Category "2" included >1 raw read at the position, and abundance at the position less than the maximum but higher than the median for the transcript. Category "3" comprised transcripts with >1 raw read at the position, and abundance at the position equal to or less than the median for the transcript. Category "4" showed only one raw read at the position [21]. The representative miRNAs and corresponding targets are shown in Figure 4, in which the red arrows indicate the cleavage sites. A total of 83 and 24 targets were predicted to be cleaved by 31 known and 14 novel miRNA families, respectively (Tables 3 and S4). Among the identified targets of known miRNAs, squamosa-promoter binding protein (SBP) and squamosa-promoter binding protein-like (SPL) transcription factor genes, NAM, ATAF, and CUC (NAC) domain-containing protein genes, Class III homeodomain leucine-zipper (HD-Zip) protein genes, APETALA2 (AP2)-like factor genes, F-box/RNI-like superfamily protein (TIR1) genes, and growth-regulating factor (GRF) genes were targeted by several conserved miRNA families (including miR156, miR164, miR166, miR172, miR393, and miR396) that play significant roles in gene regulation (Table S4). ghr-miR7502, ghr-miR7505, ghr-miR7509, ghr-miR7510a, and ghr-miR7514, which were only reported in G. hirsutum, were predicted to cleave Nudix hydrolase, ATP binding and tetratricopeptide repeat-like superfamily protein, glutamate receptor 2 isoform 1, peroxidase superfamily protein, and RHO guanyl-nucleotide exchange factor 7 (Table S4). Among the identified targets of novel miRNAs, ubiquitin carboxyl-terminal hydrolase family protein gene was targeted by novel_miR_12; auxin response factor genes were targeted by novel_miR_16; purine permease 3 and ferrochelatase 2 genes were targeted by novel_miR_20; and TT12-2 (a T-DNA insertion line) MATE (multidrug and toxic efflux) transporter gene was targeted by novel_miR_24 (Table 3). Interestingly, we also found that leucine-rich repeat (LRR) containing protein genes and LRR and NB-ARC (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4) domains-containing disease resistance-like protein genes that play important roles in plant defense against pathogens were targeted by novel_miR_28 and novel_miR_56, respectively (Table 3). Unfortunately, target genes of some known miRNAs and novel miRNAs identified through deep sequencing were not detected in the present degradome analysis. Discussion Verticillium dahliae Kleb. is a soil-borne fungal pathogen that causes vascular wilt of more than 200 dicotyledonous plant species, including cotton crops [14]. Sea-island cotton (G. barbadense L.) exhibits relative resistance to Verticillium wilt, but upland cotton (G. hirsutum L.), the main species cultivated on a large scale, is sensitive to this disease [17,22]. Previous study has shown that a number of miRNAs and other small non-coding RNAs were involved in response to V. dahliae infection in G. hirsutum and G. barbadense roots [23]. However, most of these miRNA targets were previously predicted in cotton [23], and only a few miRNA targets have been identified experimentally [23,24]. The functions of most of these miRNAs in relation to the regulation of Verticillium defense responses remain unknown and further studies must be conducted. In the present study, the two cotton species G. hirsutum and G. barbadense were used as models to study the miRNA functions associated with the regulation of Verticillium defense responses. We constructed and sequenced four sRNA libraries and two degradome libraries from mock-and Verticillium-inoculated cotton roots. In total, 140 known miRNAs and 58 novel miRNAs were detected in G. hirsutum and G. barbadense by deep sequencing. In addition, 107 target genes of 45 miRNA families were identified by degradome library sequencing. The present study is the first to report comprehensive identification of miRNAs and their targets involved in cotton response to V. dahliae by using high-throughput sequencing and degradome analysis. This will provide useful information for improving the Verticillium wilt resistance of economically important crops. miRNAs have been identified experimentally in many plant species, especially in model plants. In plants, some miRNAs seem to be universally expressed, while others are present in only a few species. According to previous reports, conserved miRNAs are present throughout at least one major ancient clade of land plants, and non-conserved miRNAs, with a limited phylogenetic distribution, are characterized by primarily being single-copy genes [25]. In this study, most known miRNAs were conserved in other species and had been previously predicted. Many other studies have shown that a number of the most conserved miRNA targets common among the data sets include many transcription factors: SBP, SPL, auxin response factors (ARF), MYB (Myeloblastosis), NAC, TCP (Teosinte-like 1, Cycloidea, and Proliferating cell factor 1), NF-Y (Nuclear Factor Y), GRF, HD-ZIP, PPR (Pentatricopeptide Repeat), and AP2-like factors. It is possible that conserved miRNAs play a crucial role in universal mechanisms of regulation in different plant species and may help us understand the evolutionary relationships between cotton and other plants. Some known but non-conserved miRNAs (e. g., miR477, miR530, miR827, miR1448, miR2111, miR2947, miR2950, miR3476, and miR5083), which were also detected in the present study, have been only identified in one or few plant species so far. In addition, 58 novel miRNAs with a lower abundance than that of conserved miRNAs were identified by using universal rules for miRNA annotation [26]. They are likely to be cotton-specific miRNAs, which are classified into non-conserved miRNAs. It seems likely that these miRNAs evolved relatively recently [5], and may function only to regulate gene expression during Malvaceae-or cotton-specific biological processes. miRNAs regulate gene expression at the transcriptional and post-transcriptional levels via mRNA cleavage or translational repression. In higher plants, miRNAs mediate gene silencing mainly by slicing mRNAs [27]. miRNA-directed cleavage leaves a 5ʹ-uncapped 3ʹ-fraction of the sliced genes. Therefore, the powerful tool of degradome sequencing was applied to identify miRNA target genes in many species with greater throughput [28][29][30]. In our study, this experimental approach was performed to identify target genes for known and novel miRNAs in cotton. As expected, a number of target genes were predicted to be cleaved by known and novel miRNA families. Many of the identified target genes of known conserved miRNAs belong to diverse gene families of transcription factors (e.g., MYB, NAC, HD-ZIP and AP2-like factor) which are known to regulate diverse aspects of plant growth and development as well as the response to V. dahliae infection [31,32]. In addition, we found some novel miRNA targets (e.g., ubiquitin carboxyl-terminal hydrolase family protein, purine permease 3, TT12-2 MATE transporter, intracellular protein transport protein USO1, glutathione S-transferase 7, and bacterial-induced lipoxygenase), suggesting a new feature of miRNA regulation in cotton. The novel miRNAs and their targets might offer useful information in potential future studies on how miRNAs and their targets are involved in the response to V. dahliae infection, which should be further investigated. However, the target genes for some known miRNAs and more novel miRNAs were not detected in the present degradome analysis. It is possible that the levels of these sliced targets were too low to detect, or some miRNAs might inhibit target gene expression through translational repression [33,34]. In summary, degradome analysis has greatly accelerated the identification of miRNA targets and has sped up research on miRNA-target interactions. In our study, a number of the miRNAs exhibited altered expression in G. hirsutum and G. barbadense roots after infection with V. dahliae, indicating that V. dahliae infection could disrupt global gene regulatory networks during cotton development. These V. dahliae-responsive miRNAs might contribute to species-specific regulation, act as "early" regulators of signal transduction or be advantageous for adaptation to stressed environments [23,35]. In Populus, Ptc-miR482 was validated to cleave disease resistance protein genes, which were involved in the resistance of plants to biotic and abiotic stresses [36,37]. Previous studies have shown that the NBS (nucleotide-binding site)-LRR resistance gene might contribute to V. dahliae resistance in cotton [31,38]. In this study, ghr-miR482a, which targets LRR and NB-ARC domain-containing disease resistance-like proteins, was expressed at a very low level in G. barbadense mock-and Verticillium-inoculated roots compared with G. hirsutum roots, suggesting its possible role in relative resistance to Verticillium wilt of G. barbadense. The expression profiles of miR1886, miR3509, and miR3515 were down-regulated, while miR419 and miR2118 were up-regulated in G. barbadense roots after infection with V. dahliae. This was consistent with a previous study that showed that many miRNAs had a species-specific expression after infection of the fungal pathogen Verticillium in G. hirsutum and G. barbadense [23]. In plants, ARFs were involved in regulating the auxin signaling pathway, which plays an important role in growth, development, and environmental responses. In Arabidopsis, repression of auxin signaling could restrict P. syringae growth, implicating auxin in disease susceptibility and miRNA-mediated suppression of auxin signaling in disease resistance [8,39]. novel_miR_16, which targets ARF10, ARF17, and ARF19, was also induced by V. dahliae, indicating that it also played an important role in plant disease resistance, but this requires further experimental confirmation. There are still many other miRNAs involved in Verticillium-infection response; however, their target genes were not detected in the present degradome analysis and their functions in plants are unknown. Future analysis of target genes and molecular components downstream could help us to understand the significance of their roles in the process. Plant Material and Total RNA Isolation The G. barbadense L. variety Hai-7124 (resistant) and G. hirsutum L. variety Yi-11 (susceptible) seeds were grown in pasteurized sand which was placed in a greenhouse (day temperature 28 °C, night temperature 25 °C, and relative humidity 60%) with a photoperiod of 14/10 h of light/dark and watered with Hoagland culture liquid every 3 day. Verticillium dahliae isolates were provided by the College of Plant Protection, Shan Dong Agricultural University. After in-plate activation, V. dahliae was transferred to Czapek Broth liquid medium and cultured for 15 day (200 rpm, 25 °C). Then, after filtration through four layers of sterile gauze, V. dahliae was diluted to approximately 10 7 spores per ml of suspension with sterile water before inoculation. The cotton seedlings with two fully expanded leaves were inoculated with V. dahliae by root dip inoculation into a suspension of fungal spores for 5 min and were then returned to their original pots. Control plants were not inoculated but were otherwise treated and sampled with distilled water in the same way. After 24 h of inoculation, the roots of both pathogen-infected and control seedlings were harvested immediately, frozen in liquid nitrogen, and stored at −80 °C for RNA isolation. In each case, samples were harvested and pooled from 20 individual plants. Total RNA was isolated from each sample using a modified CTAB (cetyltrimethylammonium bromide) method [40]. sRNA Library and Degradome Library Construction and Sequencing sRNA library construction and deep sequencing were performed as described by Hafner [41]. A 20 µg aliquot of total RNA was sent to the Beijing Genomics Institute (Shenzhen, China) where the libraries were constructed and sequenced using an Illumina Genome Analyzer (Illumina, San Diego, CA, USA). Briefly, the sRNAs (~18-30 nt) were purified from 10 µg of total RNA by polyacrylamide gel electrophoresis, and ligated first to a 5ʹ-RNA adaptor and then to a 3ʹ-RNA adaptor. A reverse transcription reaction was followed by several cycles of PCR to obtain sufficient product for Sequencing by Synthesis (SBS) sequencing via Solexa technology. Two cotton degradome libraries (Gh and Gb) were constructed based on a method previously described [28]. Briefly, total RNAs were respectively extracted from Gh_CK and Gh_Ve, and mixed at an equal molar ratio as one sample for Gh degradome library construction. Total RNAs from Gb_CK and Gb_Ve were mixed for Gb degradome library construction in the same way. Approximately 200 µg of the mixed total RNA was used for polyadenylation using the Oligotex mRNA kit (Qiagen, Valencia, CA, USA), and then a 5ʹ-RNA oligonucleotide adaptor containing an MmeI recognition site was ligated to the 5ʹ-phosphate of the poly(A+) RNA by T4 RNA ligase. This was followed by purification of the ligated products using the Oligotex kit. Subsequently, five PCR cycles were performed on the products of a reverse transcription reaction which were then digested with MmeI and ligated to a 3ʹ-double DNA adaptor. Finally, the ligation products were amplified with 20 PCR cycles, gel-purified and subjected to SBS sequencing by the Illumina Genome Analyzer. Analysis of Sequencing Data Bioinformatic analysis of sRNA and degradome sequencing data was based on a method described previously [42]. For the sRNA sequencing data, the unique RNA sequences that perfectly matched the cotton genome were subjected to subsequent analysis. RNA reads showing sequences identical to known miRNAs from the miRBase 20.0 database were picked up as the miRNA dataset of cotton. Sequences matching non-coding rRNA, tRNA, snRNA, and snoRNA in the Rfam database were removed. Reads overlapping with exons of protein-coding genes were excluded to avoid mRNA contamination. The remaining sequences were used to predict their secondary structures by using the mfold web server [43,44]. A potential miRNA precursor must meet certain criteria: (1) Both a candidate miRNA and its corresponding reverse sequence (miRNA*) must be detected in the present high-throughput sequencing; (2) The candidate miRNA and miRNA* sequences must be found on the stem, and the number of mismatched bases between them must be less than four; (3) Within the miRNA/miRNA* duplex, the number of asymmetric bulges must be one or fewer, and the number of bases in the asymmetric bulges must fewer than two; (4) The miRNA and miRNA* should be located in opposite stem-arms and form a duplex with two nucleotide 3′overhangs; (5) The potential miRNA precursor must have higher negative minimal folding energy (MFE) with the MFE <−18 kcal/mol. To investigate the differentially expressed miRNAs between libraries, each identified miRNA read count was normalized to the total number of miRNA reads in each given sample and multiplied by a million. Then, the Bayesian method was applied to infer the statistical significance value [45]. After the Bayesian test, if the p-value <0.01 and the absolute value of |log2 Ratio | ≥ 1, a specific miRNA was considered to be differentially expressed. For the degradome sequencing data, 20-21 nt sequences of high quality were collected for subsequent analysis. The unique reads that perfectly matched cotton cDNA sequences were retained. The 15-nt of sequence upstream and downstream of the 5ʹ-end of matched reads was extracted to constitute 30-nt sequence tags for searching corresponding miRNA. The CleaveLand pipeline [27] was used to align the 30-nt sequence to cotton-known miRNAs from miRBase20.0 database and our newly identified miRNAs. All alignments with scores up to seven and no mismatches at the cleavage site (between nucleotides 10 and 11) were considered candidate targets. qRT-PCR To validate the presence and expression of the identified miRNAs and target genes, 16 miRNAs and four target genes were selected for qRT-PCR analysis. The expression profile of miRNAs and target genes were assayed in pathogen-and mock-infected roots of Hai-7124 and Yi-11 by SYBR ® Premix Ex TaqTM II (TAKARA, Dalian, China) on Bio-RAD iCycler iQ5 Machine. The primers used for qRT-PCR are listed in Table S5. qRT-PCR were performed using the One Step PrimeScript ® miRNA cDNA Synthesis Kit (TAKARA) and using 12.5 µL of SYBR ® Premix Ex TaqTM II (2×), 1 µL of PCR forward primer (10 µM), 1 µL of PCR reverse primer (10 µM), and 2 μL of fivefold diluted cDNA template in a 25-µL system with the following cycling profile: 95 °C at 30 s, followed by 40 cycles of 95 °C at 15 s and 60 °C at 30 s. All reactions of qRT-PCR were repeated three times for each sample. U6 snRNA and Ubiquitin1 gene were used as the internal control genes. All the gene expression data were obtained from three individual biological replicates and processed according to strict statistical methods [46]. Statistical significance was evaluated using a Student's t-test analysis. Conclusions In this study, four sRNA libraries and two degradome libraries were constructed from G. hirsutum and G. barbadense roots with and without V. dahliae infection for deep sequencing. A large number of miRNAs were identified in both species, including 58 novel and 140 known miRNAs. Meanwhile, 107 genes sliced by 45 miRNA families were detected via degradome sequencing. The differential patterns of miRNAs expression are a valuable resource for further studies on post-transcriptional gene regulation in the defense response of cotton to Verticillium wilt. Thus, the present study might provide valuable clues for exploring miRNA-mediated regulatory networks in Verticillium defense response. Author Contributions Yujuan Zhang and Fafu Shen designed the study. Yujuan Zhang performed the experiments, analyzed the data, and drafted the manuscript. Wei Wang and Jie Chen assisted with bioinformatic analysis and aided in writing the manuscript. Jubo Liu and Minxuan Xia aided in performing the experiments. All authors carefully checked and approved this version of the manuscript.

v3-fos

2019-03-20T13:05:03.667Z

2015-07-25T00:00:00.000Z

196585059

s2

Comparing the Effect of Gums on the Growth of Lactobacillus Species in Laboratory Medium and Fluid Milk This study compared the growth of three Lactobacillus strains in the presence of gums during a 12-hour incubation period at 37°C in order to determine which gum promoted the most growth. Our results showed that the populations of Lactobacillus rhamnosus GGB101 and Lactobacillus rhamnosus GGB103 were significantly higher in milk compared to growth in a laboratory medium, whereas Lactobacillus reuteri DSM20016 performed better in the medium. The recommended level of log 6CFU g-1 was exceeded for all tested trains during the incubation period. The addition of xanthan led to the highest growth of L. rhamnosus GGB101 (8.81±0.01logCFU/mL) and L. rhamnosus GGB103 (8.32±0.01 log CFU/mL) in milk. Carrageenan-maltodextrin promoted the highest growth (8.30±0.23log CFU/mL) of L. reuteri DSM 20016 in the medium and was found to support significant growth of Lactobacillus strains in both milk and medium. Our results showed that carrageenan-maltodextrin, xanthan and carrageenan could thus serve as functional ingredients for the enhanced growth and viability of Lactobacillus strains to promote quality probiotic dairy foods and thereby promote human health. Introduction Probiotics are living microorganisms (mainly Lactobacillus and Bifidobacteria), which confer health benefits to the host when taken in adequate amounts [1]. Most strains of Lactobacillus and Bifidobacteria are known for their probiotic qualities with associated health benefits [2][3]. Currently, Lactobacilli with probiotic functions are added to a variety of functional foods, and several studies have demonstrated their beneficial properties in human and animal health. These health benefits include alleviation of lactose intolerance, acceleration of intestinal mobility, reinforcement of gut mucosal immunity, decreased risk associated with mutagenicity and carcinogenicity, hypocholesterolemic effects, reduced duration of diarrhea, prevention of inflammatory bowel disease, prevention of colon cancer, prevention of allergies, and treatment and inhibition of intestinal pathogens [4]. However, such health benefits can only be attained when the probiotic Lactobacillus strains are viable and exceed a population of six million [4]. These health-promoting microorganisms are vulnerable to various environmental stressors to which they are subjected during fermentation, storage, and digestion. The survival of Lactobacillus microorganisms is also affected by food composition and the interaction between different bacterial strains [5]. To attain the target population level of six million, 100 g of food products containing 10 6 -10 7 live cells must be ingested daily [6]. Prebiotics are defined as non-digestible food substances that selectively stimulate the growth and activity of limited gut microorganisms and promote health benefits [7]. Some prebiotic carbohydrates such as fructooligosaccharides, lactulose, inulin and galactooligosaccharides from lactose (GOS-La) are currently available in the market [6,8]. However, there is also considerable interest in learning more about new carbohydrates with potential prebiotic qualities . Polysaccharide gums (such as, carrageenan, pectin, xanthan, alginate, gellan, zedo, konjac, starch, cellulose, and chitosan), proteins (casein, whey, gelatin, β-lacto globulin) and wax have been studied as prebiotics that could improve the viability of probiotic microorganisms [9][10][11][12]. Gums are complex polysaccharides extracted from plant, animal and microbial sources [5]. Gums impact sensory qualities, contribute fiber to foods [13] and promote growth in probiotics [12]. Gums are also used as thickeners and binders in cosmetics, medications, inks, paint, paper, and adhesives [13]. Gums are comprised of food ingredients such as carbohydrates, sugars, salts and minerals that could serve as additional carbon sources to enhance the growth of probiotic organisms in food and in the human gut. Thus, the aim of this study was to investigate the effect of different gums on the growth of three Lactobacillus strains in a laboratory medium and fluid milk as a practical means to enhance the viability of probiotics and ultimately improve the quality of dairy products. were obtained from the stock culture collection of the Food Microbiology and Biotechnology Laboratory at North Carolina Agricultural &Technical State University (Greensboro, NC, USA). Bacterial cultures were activated by transferring 100 µl of stock culture to 10 mL of deMan Rogosa Sharpe (MRS) broth (Neogen, Lansing, MI) and incubating at 37°C for 24h. Gum composition, preparation and inoculation Fluid milk (1% milk fat) and a modified basal medium were prepared by gradually dissolving 0.5% (w/v) of each of the following gums: pectin (PE), carrageenan (CA), carrageenanmaltodextrin (MC), pectin-carrageenan (PC), locust bean (LB), guar (GU), inulin (IN) and guar-locust bean-carrageenan (GL) and xanthan (XA) (Maryland, GA, USA) to a 100 mL batch of 1% fat liquid milk at 70°C. Samples without gums served as a negative control. Milk and media samples were then treated in a water bath at 65°C for 30 min and cooled to 42°C before use. Growth study Sterilized basal medium samples were inoculated with 1% of inoculum at a final inoculum level of 3 log CFU/ml. Bacterial inoculum was prepared by taking aliquots (1 mL) from appropriate serial dilutions from each active culture and aseptically adding the aliquots to each 100 mL batch of 1% fat milk and basal medium samples. Samples without gum served as the control. Inoculated samples were serially diluted at a final inoculum level of 3 logs CFU/ml, plated on MRS agar, and incubated at 37°C for 48h to determine initial bacterial counts. Inoculated samples were then incubated at 37°C for 12h. After incubation, samples were serially diluted and aliquots were placed on MRS agar to obtain final bacterial counts. Data and statistical analysis Each experiment was conducted three times in a randomized block design. The mean values and standard deviations were calculated from the duplicate tested samples. R-Project for Statistical Computingversion R-2.15.2 (http://www.r-project. org) was used to determine significant differences between the effect of different gums on the growth of Lactobacillus strains in milk and medium using one-way ANOVA (analysis of variance) at a significance level of p<0.05. Growth of Lactobacillus Strains A modified basal medium was prepared using basic components (Table 1) (Table 1) to study the effect of selected gums on the growth of Lactobacillus strains. The growth of Lactobacillus strains in the medium was compared to the growth in one percent fat fluid milk containing selected gums. Figures 1, 2 and 3 shows the effect of gums on three Lactobacillus strains in laboratory media and in milk during 12 hours of incubation at 37°C. The population of the L.rhamnosus GGB101 strain was significantly (p<0.05) higher in milk compared to medium in the presence of all tested gums ( Figure 1). However, the highest bacterial population was observed in milk containing xanthan at 8.81±0.01 log CFU/mL, which was followed by milk sample containing carrageenan (8.18±0.03 log CFU/mL). The addition of guar-locust bean-carrageenan resulted in the least growth of L. rhamnosus GG B101 (8.03±0.01log CFU/mL) in milk. Similarly, the addition of xanthan led to significantly (p<0.05) higher growth (7.65±0.00 0.04log CFU/mL) of L. rhamnosus GG B101 in the medium, with locust bean inducing the least growth (6.89±0.06log CFU/mL). However, the population of L. rhamnosus GG B101 was 1.16 logs CFU/mL higher in milk than in media in the presence of xanthan (Figure 1). Significantly (p<0.05) higher population of L. rhamnosus GG B103 was observed in milk compared to medium samples in the presence of all tested gums, except in the milk sample containing guar, pectin-carrageenan, and carrageenan where a slightly higher population of L. rhamnosus GG103 was found ( Figure 2). The addition of xanthan led to the highest population of L. rhamnosus GG B103 (8.32±0.01log CFU/mL) in milk during the incubation period. Compared to the control, the population of L. rhamnosus GG B103 was significantly (p<0.05) lower in the presence of pectin and locust beans in the medium, indicating an inhibiting effect of these gums on the growth of L. rhamnosus GG B103. The addition of tested gums led to a positive effect on the population of L. rhamnosus GG B103 in all milk samples compared to medium (Figure 2). The presence of carrageenan-maltodextrin resulted in the highest population of L. reuteri DSM20016 (8.3±0.23log CFU/mL) in modified basal media during the incubation period ( Figure 3). It was notable that carrageenan, carrageenan-maltodextrin, guar, and inulin promoted higher growth in the media compared to milk and also promoted higher growth in treatments than in the control. In contrast, gums (pectin-carrageenan, pectin, xanthan, guar-locust bean-carrageen and carrageenan) that led to a higher population of L. reuteri DSM20016 in treated samples than in control samples also enhanced bacterial growth in milk. This would indicate that the metabolic activity of Lactobacillus strains was affected directly by gums and indirectly by the medium of growth. This effect could be due to the interaction between the gums and the strains. L. rhamnosus GG that grew best in milk compared to the L. reuteri strain. Carrageenan, carrageenan-maltodextrin, guar, inulin and locust bean enhanced the growth L. reuteri DSM20016 in media. However, the addition of pectin-carrageenan, pectin, guar-locust bean-carrageenan and xanthan resulted in a slightly higher population of L. reuteri DSM20016 in milk ( Figure 3). The presence of carrageenan-maltodextrin led to the highest population of L. reuteri DSM20016 (8.3±0.23log CFU/mL) in media. It was also observed that carrageenan, carrageenan-maltodextrin, guar, inulin and locust bean which induced higher growth in the treatment group than in the control, also promoted higher growth in media compared to milk. Similarly, gums (pectin-carrageenan, pectin, xanthan, guar-locust bean-carrageen and carrageenan) that led to higher populations of strains in treated samples than in control samples also improved the growth of L. reuteri DSM20016 in media compared to milk. Xanthan significantly (p<0.05) stimulated the highest growth of L. rhamnosus GG strains in both milk and media whereas the addition of xanthan resulted in aslight inhibition in the L. reuteri strain in both milk and media. The presence of carrageenan-maltodextrin and carrageenan significantly (p<0.05) promoted the growth of all tested Lactobacillus strains in both media and milk. Discussion The results from this study demonstrated that gums could promote the growth of Lactobacillus strains. These results support previous findings [12,14] that showed carrageenan-maltodextrin could promote growth and viability of Lactobacillus strains. According to Su et al. [4], the addition of certain prebiotics such as fructose oligosaccharides (FOS) and soybean oligosaccharides (SOS) at 1.5% enhanced the growth of Lactobacillus in a basal medium at about 1 log CFU/mL. Buttermilk and whey supplemented with yeast extract also improved the growth of Lactobacillus strains, although the growth was lower compared to MRS [15]. The ability of gums to impact the growth of Lactobacillus could be due to the interaction between gums and the tested Lactobacillus strains as the metabolic activity of Lactobacillus strains tend to respond to the composition of the medium of growth [16]. Additionally, the structure and composition of gums could be a key factor in the enhancement of bacterial growth. Gums vary in chemical composition and carbon chains which impacts on their digestibility and availability to bacteria. According to Hernandez-Hernandez et al. [10] the length of carbon chains in carbohydrates such as galactooligosaccharides affects digestibility. The longer the carbon chains as in xanthan, the slower the digestibility, which subsequently impacts the growth of Lactobacilli. We would therefore expect to find the same trend in the digestibility of selected gums due to their composition and structure as well as carbon chain length with their consequent effect on growth and viability [10]. Additionally, gums added carbon and energy sources for the heterofermentative metabolic activity by L. reuteri and L. rhamnosus strains leading to enhance growth in the milk and media containing gums [16]. Conclusion Our results indicated that the tested gums could enhance the growth of Lactobacillus strains in milk compared to media as the addition of gums significantly improved the population of Lactobacillus strains in milk. In addition, carrageenanmaltodextrin and carrageenan showed enhanced growth of Lactobacillus strains in both milk and media. Xanthan stimulated the highest growth of L. rhamnosus GG strains but led to a slight inhibition in the growth L. reuteri strain in both milk and media.

v3-fos

2016-05-12T22:15:10.714Z

2015-04-05T00:00:00.000Z

11666699

s2

Aphidophagous Parasitoids can Forage Wheat Crops Before Aphid Infestation, Parana State, Brazil Aphid parasitoids are common in Brazilian wheat fields, and parasitize aphids at the wheat tillering stage. However, there is little information available about when this natural enemy occurs in wheat crops. This study investigated the initial occurrence of aphid parasitoids in four commercial wheat crops in northern Paraná during the 2009 crop season. We installed two Malaise traps at each wheat farm, and 400 tillers were assessed weekly in each field for aphid abundance. During this study, we captured 4,355 aphid parasitoids and 197 aphids. Three species of braconid parasitoids were identified, including Aphidius colemani (Viereck 1912), Lysiphlebus testaceipes (Cresson 1880), and Diaeretiella rapae (McIntosh 1855). The aphids species identified were Rhopalosiphum padi (Linnaeus 1758) and Sitobion avenae (Fabricius 1775). This study showed that aphid parasitoids are present in wheat crops even when aphid densities are low, and in one farm, occurred before the aphids colonization. These reports can justified the high efficiency of these natural enemies against aphids in wheat fields. Aphidophagous parasitoids, which are found in many regions of the world, help to regulate aphid populations in wheat crops (Adisu et al. 2002, Schmidt et al. 2003, Saethre et al. 2011, Plećaša et al. 2014, Elliot et al. 2014, Zhao et al. 2014. In temperate regions, is common aphids sexually reproduce during severe winters, and the female aphids usually lay their eggs on primary (harboreus) host plants (Starý and Havelka 2008). Sexual reproduction is a winter survival strategy because the eggs can endure the winter conditions. However, when the temperature begins to rise, the nymphs eclode and the young aphids migrate to the wheat, usually from the flowering stage onward (Schmidt et al. 2003, Thies et al. 2005, Caballero-López et al. 2012. In temperate regions, natural enemies such as aphidophagous parasitoids can survive winter in two ways: by starting diapause or by migrating to warmer regions (Jones et al. 2008). However, both these processes mean that these natural enemies need a long time to colonize wheat crops, which hinders initial aphid suppression. However, the warmer climate in tropical and subtropical regions is more favorable to aphid development, and aphid reproduction is often asexual (thelytokous parthenogenesis) (Simon et al. 2002). This implies that there is a different dynamic spatial and temporal relationship between aphids and their natural enemies in tropical and temperate zones. In Brazilian fields, most studies have investigated the occurrence of aphid parasitoids in a field by measuring the number of mummies (Ronquin et al. 2004, Alves et al. 2005, Macedo et al. 2010). However, this method severely restricts the understanding of the behavior of these natural enemies in the field. Many studies conducted in Brazil have shown that parasitized aphids (mummies) appeared during the tillering stage (Alves et al. 2005, Machado andSantos 2013), usually parasitized by polyphagous parasitoids (Starý et al. 2007). So, it is possible that this earlier occurrence and the broad spectrum of hosts, was the main reason that biological control was quite successful. Therefore, we believe that this natural enemy is present in the field immediately after aphid infestation. In this sense, to better understand it, this work aimed investigated when aphidophagous parasitoids colonizes wheat crops, in Parana State, Brazil. To confirm this hypothesis, we used traps that were designed to capture parasitoids, which allowed us to estimate the moment that aphid parasitoids appeared in wheat fields. This enabled us to evaluate the incidence and colonization of aphid parasitoids during the initial wheat development phase. In all the fields, wheat (Triticum aestivum L.) was sown in succession to soybean [Glycine max (Merrill) L.]. The crop was sown on April 29 (Rolândia), May 6 (Ibiporã-FBS), May 8 (Ibiporã-SAF), and May 11 (Londrina). The landscape complex around each farm is described in Table 1. To better support the work, the description was made considering the dispersal ratio of Aphidiinae parasitoids (2 km) (Thies et al. 2005). All fields wild radish (Raphanus raphanistrum L.) and black-jack (Bidens pilosa L.), however it was not quantified. Aphid infestation was assessed by demarcating two transects (90 m in length) per field, which were spaced 500 m apart. The transects were set up immediately after emergence (stage V1) of the wheat crops in all fields, except in Londrina, which was demarcated 1 day after sowing. Each transect contained 10 evaluation points, where 20 tillers per point were randomly evaluated (n ¼ 400 tillers/assessment in each field). All the aphids were quantified and identified to the species level. Aphidophagous parasitoid colonization was monitored using Malaise traps, which were placed in the center of each transect (n ¼ two traps/ field). The traps were made of a synthetic material and were shaped like a tent with an opening at the bottom that intercepted the insects during flight after they had collided with one of trap's septa. This type of trap is very efficient for capturing parasitoids, because after they collide with one of the septa, they go to the raised end where they are trapped in a collecting vessel (bottle). The traps allowed us to record the earliest occurrence of parasitoids in wheat fields. The traps were installed and positioned such that they faced north-where the most sunlight was received. Each trap was 1.80 m high by 1.80 m long. The collecting bottle contained 70% alcohol, which was changed weekly on the day on which the number of aphids was counted. Is important consider that this trap allow to capture parasitoids during dispersal flight among the patches. In the laboratory, the material collected was screened, and the aphidophagous parasitoids were identified using a stereoscope microscope according to the method described by Kavallieratos et al. (2006) and Pereira and Salvadori (2005). Infestation by aphids and colonization of their parasitoids began 7 days after the wheat was sown, except at Londrina where the assessment began 1 day after the wheat was sown. The assessments were carried out on a weekly basis, and aphid species were identified using a guide developed by Salvadori and Tonet (2001). To reduce interference, insecticides were not applied up to a distance of 5 m from the evaluation points. Finally, the data were descriptively analyzed and illustrated to depict the seasonal dynamics of aphids and aphid parasitoids. Discussion Our results showed that aphid parasitoids can forage wheat fields even in a low aphid infestation, and sometimes in absence of aphids. This observation helps us to understand the biodynamics of aphidophagous parasitoids and can be explained by two hypotheses. First, the tropical climate in northern Parana State probably means that the parasitoids can survive actively (parasitizing) for a longer period. For example, the temperature during the assessments (around 20.54 C 6 1.45 C) was optimal for the development of aphidophagous parasitoids (Jones et al. 2003, Rodrigues et al. 2004, Sampaio et al. 2005. Thus, the tropical climate in northern Parana allows aphidophagous parasitoids to appear in crops immediately after wheat emergence. Second, their polyphagous habit probably plays an important role in the maintenance of these parasitoids in fields because they can survive by attacking aphids from other plants such as weeds or other crops cultivated near wheat fields. The parasitoids captured in this study (L. testaceipes, A. colemani, and D. rapae) can parasitize a range of aphids, which occur in a several cultivated plants and weeds (Starý et al. 2007, Tepa-Yotto et al. 2013, Macedo et al. 2010, Hollingberya et al. 2012. Therefore, although aphid infestation in the fields was low, these parasitoids survived because of the presence of alternative aphid hosts. These included weeds such as wild radish (R. raphanistrum) present in all areas as well as maize fields (Table 1) cultivated around the fields studied. These plants can favor the presence of aphid parasitoids due the presence of aphids hosts, which facilitates the migration of aphid parasitoids to wheat fields. So, although the neighbor plants were not investigated, this hypothesis can explain the recordings at Londrina, where D. rapae and L. testaceipes were captured before wheat emergence. Other important factor is that the L. testaceipes parasitoid species, in particular, showed a low capacity to locate the host, since it has a limited response to volatiles released by the plants (Lo Pinto et al. 2004, Fauvergue et al., 2006. Therefore, this species in a plant with no wheat aphids may be captured owing to theirs disorientation or during transit, while they searched for hosts in other crops. In contrast, D. rapae responds strongly to volatiles emitted by brassica plants, even in the absence of hosts (Blande et al. 2007), which suggests that their natural occurrence is related to the presence of wild radish, inside the wheat fields. The 'early' reports of aphidophagous parasitoids in this study are very important for biological control, and it explains the high efficiency of these natural enemies in the regulation of aphid abundance. Finally, this exploratory investigation suggests that these natural enemies are well established in local agroecosystems owing to the large number of alternative hosts that are available during the growing season. Further studies into the behavior of these natural enemies in different climate and regions around the world are needed for efficient conservation of these agents of biological control.

v3-fos

2018-12-21T05:37:42.783Z

2015-09-01T00:00:00.000Z

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Effect of Enzyme Supplementation on Nutritive Values of Fermented Palm Kernel Cake Used to Substitute Soybean Meal in Broiler Diet Sinurat AP, Purwadaria T, Purba M. 2015. Effect of enzyme supplementation on nutritive values of fermented palm kernel cake used to substitute soybean meal in broiler diet. JITV 20(3): 184-192. DOI: http://dx.doi.org/10.14334/jitv.v20i3.1185 Two experiments were designed to improve nutritional values of palm kernel cake (PKC) by biofermentation process, followed by enzyme supplementation to substitute soybean meal (SBM) in broilers diet. A factorial of 2 x 2 design was applied in the first experiment, i.e. fermentation process (non fermented PKC and fermented PKC) and enzyme supplementation (no enzyme and +BS4 enzyme). Dry matter (DM) digestibility, AME and amino acids ileal digestibility (IAAD) of the treatment ingredients were measured in broiler chickens. Seven replicate were applied for the DM and AME assays and 3 replicate for IAAD assay. Second experiment was designed to study the effect of SBM substitution with enzyme supplemented FPKC (EFPKC). Four diets were formulated, i.e., control diet without EFPKC, 10%, 20 and 40% SBM substituted with EFPKC. All diets were formulated to meet the nutrient requirements of broilers. Each diet was fed to broilers from 1 to 35 d. Body weight, feed consumption, FCR and mortalities were measured. Carcass yield, abdominal fat and weight of liver and gizzard were measured at the end of experiment. Results showed that fermentation of PKC increased the DM digestibility, the AME was also increased but not significant. Enzyme supplementation did not affect the DM digestibility and AME of PKC. Fermentation process significantly (P<0.05) decreased IAAD of some indispensable amino acids. However, supplementation of enzyme did not affect the IAAD of indispensable amino acids. Substitution of soybean meal with EFPKC reduced the feed intake and growth rate of broilers. INTRODUCTION Modern chickens (broilers and layers) require high quality feed to support their rapid growth, high productivity and high efficiency in feed utilization. Soy bean meal (SBM) is one of good quality feedstuff that commonly used as a protein and or amino acids source in chickens diet. Its inclusion in broiler's diet varies between 15 to 30%, depends on the presence of other protein sources included in the diet. Even in countries with no SBM production such as Indonesia, SBM is commonly used in poultry diet. Increases of national broiler production causes increases in importation of SBM and price of feed. Indonesia imported 3.069 million ton SBM in 2010 and 4.250 million ton in 2014 (USDA 2015). In order to achieve self sufficiency in feed ingredients, it is important to seek alternatives to limit or replace SBM with local feed ingredients. As a world leading producer of palm oil, Indonesia produces palm kernel cake (PKC)-a by product of palm kernel oil production in a large quantity. The Indonesian PKC production in 2014 was estimated 4.55 million ton (USDA 2015). Its nutritive values including its digestible amino acids have been reported by some authors (Nwokolo et al. 1977;Onwudike 1986;Sue & Awaludin 2005;Sundu et al. 2006;Sinurat et al. 2014). The protein, amino acids and the digestible amino acids of the PKC are much lower than the SBM. Replacing the SBM with PKC as such for a protein source in poultry diet will deteriorate their performances. Sinurat et al. (2014) reported that the PKC could be fermented with Aspergillus niger to increase its crude protein from 14.76% to 20.04%. Some amino acids (except threonine and arginine) content increased varies between 5.2-117.2% and also its protein and ileal-amino acids digestibility. Although, the protein and amino acids contents and the ileal amino acids digestibility of the fermented PKC were higher than the PKC, it is still not comparable with those of the SBM. The feeding trial on laying hens showed that only 25-50% of the SBM could be substituted with enzyme supplemented fermented PKC without significantly affecting egg production and feed efficiency (Sinurat et al. 2014). In order to increase protein and amino acids content of PKC, Sinurat et al. (2015) have modified the fermentation method by adding cassava leaf meal as protein sources prior to PKC fermentation. This new method was able to increase protein content of fermented PKC from 21.91 to 28.97% as compared to the previous method (Sinurat et al. 2014). The methionine, lysine, tryptophan and threonine levels were also increased from 0.290 to 0.317%, 0.643 to 0.740, 0.150 to 0.273 and 0.31 to 0.273%, respectively. Enzymes have been widely used to increase nutrients availability of poultry feedstuff or feed. Enzyme complex (consist of xylanase, beta-glucanase and cellulose) have been reported to increase the crude protein digestibility and the AME of feed containing paddy and rice bran (Kang et al. 2013). Khan et al. (2006) also reported an increase on the dry matter-, organic matter-, crude protein and energydigestibilities of feed containing sunflower meal caused by supplementation of commercial enzymes. A multi enzyme produced by Eupenicillium javanicum has been developed in our laboratory (Purwadaria et al. 2003;Pasaribu et al. 2009). The enzyme, called BS4 has been reported effectively to improve nutrient digestibility of palm oil by products such as palm oil sludge (Pasaribu et al. 2009) and palm kernel cake (Sinurat et al. 2013). Sinurat et al. (2014) also showed that the enzyme improved the protein-and the amino acidsdigestibilities of fermented PKC. Therefore, an experiment was designed to test the biological values of the fermented PKC (produced with the new method) supplemented with the BS4 enzyme and the possibility of using the product to substitute SBM in broiler's diet as reported in this paper. Effect of enzyme supplementation on dry matter digestibility, AME and IAAD of PKC and fermented PKC (FPKC) Two steps of experiments were carried out in order to test biological values of fermented PKC. The first experiment was to determine AME and ileal amino acids digestibility of the FPKC produced with new fermentation method as described by Sinurat et al. (2015). The PKC was pretreated by autoclaving and supplemented with 10 % cassava leaf meal (9:1) before fermented with A. niger. For the digestibility (dry matter, protein, ileal amino acids digestibility and AME) study, a commercial broiler feed was used as basal diet (B). Basal diet was mixed with ingredients tested 50:50 and added 2% acid insoluble ash (celite) as an indicator as described in Ravindran et al. (2005) and Sinurat et al. (2014). The experiment was arranged in a 2 X 2 factorial design. The first factor was fermentation process, i.e., non fermented PKC and fermented PKC (FPKC) and the second factor was enzyme supplementation (no enzyme and with enzyme). The enzyme supplemented was BS4 enzyme (150 U/kg) as reported by Sinurat et al. (2014). Parameters measured were dry matter digestibility, apparent metabolisable energy (AME) and ileal amino acids digestibility (IAAD). One hundred (100) broiler chicks were reared on litter pens with standard rearing management from one day to 28 days old. At 28 d, 35 male chicks were selected and placed in individual wire cages. Each treatment diet was fed ad libitum to 7 (seven) chicks, and considered as replications. Treatment diets were fed for 5 days and samples of excreta was collected at the last 3 days for AME determination. After 5 days feeding the tested diet, 35 birds were sacrificed by CO2 asphyxiation and the digesta in the ileal was collected into plastic containers. Samples from 2 or 3 birds fed with the same diet were pooled to make 3 sample replications for each treatment. The digesta were immediately kept in the freezer for further chemical analyses. The feed and the tested ingredients were analysed for dry matter, nitrogen (protein), amino acids and acid insoluble ash (AIA) contents. The excreta collected was analysed for the dry matter, nitrogen (protein), gross energy and AIA contents while the ileal digesta were analysed for dry matter, amino acids and AIA contents. The dry matter and the nitrogen were analysed following procedures described by AOAC (2005), while the amino acids were analysed by HPLC method at Bogor Agricultural University laboratory. All data were subject to analyses of variance, followed by least significance difference when the ANOVA was significant (P<0.05) according to procedure described by Steel & Torrie (1997). Substitution of soybean meal with enzyme supplemented fermented palm kernel cake (EFPKC) in broilers diet The second experiment was designed to study the effect of substitution of SBM with the EFPKC on the performance of broilers. Four dietary treatments with graded levels of SBM substitution with EFPKC were formulated. All diets were formulated to meet the nutrient requirement of broilers according to Cobb (2012), i.e., crude protein 21.8%, metabolizable energy 3000 kcal/kg, digestible amino acids (lysine 1.185%, methionine + cystine 0.905%, tryptophan 0.202%, threonine 0.758), calcium 0.90% and available phosphorus 0.46%. The ingredients and nutrient composition of the experimental diets are shown in Table 1. The nutrient values of the EFPKC (ME and digestible amino acids) obtained from experiment 1, was used for the formulation of the treatment diets. The treatments consist of: 1. Standard diet (Control = C) with SBM level as in normal broiler diet 2. Diet with 10% of the SBM substituted with EFPKC 3. Diet with 20% of the SBM substituted with EFPKC 4. Diet with 40% of the SBM substituted with EFPKC Each diet was fed to 60 broilers (6 replicates and 10 birds per replicate) from 1 to 35 days old. The birds were reared in pens with rice hulls as litter. Feed and water were given ad libitum. Parameters observed were feed intake, body weight, mortalities, and feed convertion ratio. At the end of the feeding trial, 2 birds (1 male and 1 female) from each pen were slaughtered to determine carcass and abdominal fat. Data were analysed with analyses of variance (ANOVA) in a completely randomized design (4 treatments X 6 replicates). Duncan's multiple range test were applied to show difference between treatment means when the ANOVA was significant at P<0.05 (Steel & Torrie 1997). Nutrients digestibility of PKC and FPKC as affected by enzyme supplementation Effect of fermentation process and BS4-enzyme supplementation on dry matter digestibility and metabolisable energy (AME) of PKC and FPKC are presented in Table 2. The dry matter (DM) digestibility of PKC was only significantly (P<0.05) affected by fermentation (F) process, but not by enzyme supplementation (E) nor by the interaction of F x E. The DM digestibility of PKC in this study was 37.3% while previous study (Sinurat et al. 2013) showed a higher DM digestibility, i.e. 56.8%. The AME of the PKC was not significantly (P>0.05) affected by fermentation process (F), enzyme supplementation (E) nor by the interaction of F x E. The AME of the PKC obtained in this experiment was 2079 kcal/kg, almost similar to the previous study i.e., ME 2074 Kcal/kg (Sinurat et al. 2014) and 2091 kcal/kg (Sinurat et al. 2013). However, Saenphoom et al. (2013) reported a lower TME of PKC i.e., 4.71 MJ/kg (or 1126 kcal/kg), which may be due to different process applied in the production of the PKC. Some reports have shown different results on the effect of fermentation process on metabolisable energy of PKC. Sinurat et al. (2014) showed that fermentation of PKC with A. niger significantly decreased the AME (from 2074 to 1788 kcal/kg). Similar patterns were also reported by Muangkeow & Chinajariyawong (2009) after fermented with A. wentii. Dairo & Fasuyi (2008) also reported a decrease in the ME of PKC after fermentation without addition of a fungus as inoculum. In contrast, Bintang et al. (1999) and Iyayi & Aderolu (2004) showed an increase in the ME of PKC after fermented with A. niger and Trichoderma viride, respectively. In this study, PKC was mixed with 10% cassava leaf meal (CLM) prior to fermentation as described by Sinurat et al. (2015). Despite the ME of CLM, (i.e. 1160 kcal/kg according to Darma et al. 1989), was much lower than the ME of PKC, the fermentation process was still able to increase the ME of the PKC, although the differences were not significant (P>0.05). Enzyme supplementation did not significantly (P>0.05) affect the DM digestibility nor the AME of the PKC nor the FPKC, although it increased from 37.3% to 46.8% or 25.5% improvement. Previous study showed a similar but significant improvement (26.2%) in the DM digestibility of the PKC due to enzyme supplementation (Sinurat et al. 2014). The DM digestibility of the PKC was increased from 56.8% to 71.7% as affected by the enzyme supplementation. Although statistically not significant, the AME of the PKC was increased from 2079 to 2385 kcal/kg or 15% improvement, while previous study showed an increase from 2091 to 2317 kcal/kg or 11% improvement. The AME of the FPKC was only slightly increased as the effect of enzyme supplementation, i.e., from 2496 to 2554 kcal/kg or 2.3% improvement. Previous study (Sinurat et al. 2014) showed a similar trend, i.e., 4.4% improvement due to addition of similar enzymes. Saenphoom et al. (2013) showed a high (60.3%) improvement on the TME of PKC as effect of enzymes (cellulase and mannanase) supplementation. In their study, the PKC was soaked in water, added with the enzyme, and incubated for 18 hours before the digestibility study. Present results showed that the enzyme improved DM digestibility and AME less on fermented PKC as compared to the effect on non- fermented PKC. Less increase of AME due to the enzyme supplementation to FPKC compared to PKC might occur due to the saccharification activity of fibernolytic enzymes produced during fermentation. The FPKC may contain shorter fiber molecules than that of PKC, therefore they were less digested by the enzyme addition. Purwadaria et al. (1998) reported that fermented palm oil sludge with A. Niger produced mannanase and cellulase in the course of fermentation. Those enzymes might actively digest the fiber. Although the DM digestibility and the AME were increased due to fermentation process and enzyme supplementation, the effects were not significant (P>0.05) statistically. Perhaps, this is due to the high variability on the parameter observed in this study as shown by the coefficient of variation (CV), i.e., 22.14% and 14.8% for the DM digestibility and AME, respectively ( Table 2). The level of digestible amino acids in a feedstuff is calculated by multiplying the amino acids concentration in the feedstuff with the digestibility coefficient (percentage) of the amino acids. These values are commonly used for poultry ration formulation. The digestible amino acids of the PKC and FPKC as affected by enzyme supplementation are presented in Table 4. Effect of enzyme supplementation on ileal amino acids digestibility (IAAD) of PKC and FPKC is presented in Table 3. The IAAD of essential (or indispensable) amino acids were not significantly (P>0.05) affected by enzyme supplementation nor by interactions between fermentation process (F) and enzyme supplementation (E). Some indispensable amino acids, i.e., arginine, histidine, isoleucine, leucine, phenylalanine and valine were significantly (P<0.05) decreased by fermentation process. These results do not agree with the results of Muangkeow & Chinajariyawong (2009) which showed that true amino acids (except for arginine) digestibility of the PKC were increased after fermented with A. wentii. Sinurat et al. (2014) also reported an increase in IAAD of indispensable amino acids except the arginine, threonine, tryptophan and valine. Supplementation of cassava leaf meal in PKC fermentation process may have changed IAAD profiles of FPKC. The IAAD values of PKC obtained in this study were much higher than IAAD of the PKC used in the previous experiment (Sinurat et al. 2014) which indicate the difference in quality of raw material (PKC) used in fermentation. Among the indispensable amino acids, only digestible arginine, lysine and threonine were affected by treatments significantly (P<0.05). The digestible arginine level was significantly (P<0.05) reduced by the fermentation (F), but not significantly (P>0.05) affected by the enzyme (E) supplementation nor by their interaction (F x E). The digestible lysine was significantly (P<0.05) affected by the interaction (FxE), in which the enzyme supplementation increased the digestible lysine in PKC but not significantly (P>0.05) affected when applied in the FPKC. The digestible threonine was significantly (P<0.01) higher in the FPKC as compared to the PKC. In general, data on Table 4 showed that supplementation of enzyme did not increase the digestible amino acids in the FPKC. Solid substrate fermentation of palm kernel cake using A. niger USM F4 produced mannanase, a hemicellulytic enzyme for digesting NDF in PKM (Syarifah et al. 2012). Since the fermentation process of PKM in this experiment also produced enzymes, therefore supplementation of exogenous enzyme may have been in excess. As shown by Pourreza et al. (2007) supplementation of enzymes to a certain levels or activities improves the nutrients (dry matter, energy and protein) digestibility of feed, but supplementation of enzyme in a higher levels did not. Their data also showed that excess exogenous enzymes supplementation reduced nutrients digestibility of the feed. Effect of substitution of soybean meal (SBM) with the enzyme supplemented FPKC (EFPKC) on performance of broilers from day old to 35 days old is presented in Table 5. The feed intake of broilers at 1-21 d old was significantly (P<0.01) affected by treatments. The lowest feed intake was found when the SBM was substituted 10%, followed by 40% substitution. The feed intake was different significantly (P<0.05) between control (not substituted) with 10% and 40% substitution of SBM with EFPKC, however, substitution of 20% SBM did not show significant (P>0.050) difference with the control. Similar trend and significant (P<0.05) effect was also found on the feed intake during 1-35 d period. Since all dietary treatments were formulated with similar nutrient values, include the energy (ME) and the digestible amino acids, the difference on feed intake could not be due to the nutrient factors but may be there are some anti-nutrient substances in the EFPKC. If this assumption is true, then the more the SBM substituted, the more the EFPKC included in the diet. If the EFPKC contains some anti-nutrient substances, the lowest feed intake was expected to occur when 40% SBM was substituted with the EFPKC. This phenomenon has been reported by Sinurat et al. (2014) when the SBM was substituted with the fermented PKC (without enzyme supplementation) in laying hens diet. It could not be explained at this stage why this is not the case in this experiment. Body weight of broilers at 21 d was not significantly (P>0.05) affected by the substitution of SBM with EFPKC. However, the body weight at 35 d was significantly (P<0.05) affected by the treatments. Substitution of SBM with EFPKC produced lighter birds at 35 d, as compared with the control. The body weight is a reflection of feed or nutrients intake. The heavier birds were found on control diet (859.0 g at 21 d and 1929.6 g at 35 d) which consumed the highest feed and the body weight was lighter as the feed intake reduced. The feed convertion ratio (FCR) during 1 to 21 d was not significantly (P>0.05) affected by treatments, however the FCR during 1-35 d was significantly (P<0.05) affected. The FCR data showed that birds fed with the control diet were the most efficient in feed utilization. However, statistical analyses showed the FCR was only significantly different (P<0.05) between birds fed the control diet and those fed with 10% SBM substitution. The mortalities of birds during the experiment was not significantly (P>0.05) affected by treatments. The data, however, showed that the mortalities increased as the level of SBM substitution with EFPKC was increased. The effect of substitution of SBM with EFPKC on carcass percentage, weight abdominal fat, liver and gizzard of broilers as affected by treatments is presented in Table 6. Statistical analyses showed that there was no significant (P>0.05) effect of treatments on the dressed carcass percentage, abdominal fat-, liver-and gizzardweight of broilers. These results indicated that replacing the SBM with EFPKC did not change the metabolism of the birds. CONCLUSION This study concludes that fermentation process improved the nutrient values of the palm kernel cake. Supplementation of enzyme was not effective to improve AME and amino acids digestibility of fermented palm kernel cake. Supplementation of enzymes to fermented palm kernel cake was not recommended to substitute soybean meal in broiler diets, since it reduced feed intake and growth rate of broilers.

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Molecular characterization of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) gene family from Citrus and the effect of fruit load on their expression We recently identified a Citrus gene encoding SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factor that contained a sequence complementary to miR156. Genes of the SPL family are known to play a role in flowering regulation and phase transition. In Citrus, the mRNA levels of the gene were significantly altered by fruit load in buds; under heavy fruit load (ON-Crop trees), known to suppress next year flowering, the mRNA levels were down-regulated, while fruit removal (de-fruiting), inducing next-year flowering, resulted in its up-regulation. In the current work, we set on to study the function of the gene. We showed that the Citrus SPL was able promote flowering independently of photoperiod in Arabidopsis, while miR156 repressed its flowering-promoting activity. In order to find out if fruit load affected the expression of additional genes of the SPL family, we identified and classified all SPL members in the Citrus genome, and studied their seasonal expression patterns in buds and leaves, and in response to de-fruiting. Results showed that two additional SPL-like genes and miR172, known to be induced by SPLs in Arabidopsis, were altered by fruit load. The relationships between these factors in relation to the fruit-load effect on Citrus flowering are discussed. Introduction During their growth, plants undergo a series of developmental transitions, regulated by a complex network of molecular factors that are activated and interact in response to endogenous and environmental cues. Research of juvenile-to-adult and vegetative-to-reproductive phase transitions in model and crop plants has revealed the importance of two microRNAs, miR156 and miR172, which coordinate these processes in an opposite manner: miR156 is highly abundant during the juvenile phase and gradually decreases with age; its overexpression prolongs the expression of juvenile features and significantly delays flowering (Wu and Poethig, 2006;Chuck et al., 2007); in contrast, miR172 abundance increases as the plant ages and its overexpression accelerates flowering (Aukerman and Sakai, 2003;Chen, 2004;Lauter et al., 2005;Wu and Poethig, 2006;Jung et al., 2007). In Arabidopsis, miR156 targets 10 out of 16 members of the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) family of transcription factors (TFs), which are characterized by a 76-amino acid DNAbinding domain named SBP. SPLs influence flowering in a number of ways. In the leaf, they promote flowering upstream of FLOWERING LOCUS T (FT) by up-regulating miR172, a repressor of APETALA2 (AP2)-like TFs which inhibits FT transcription (Mathieu et al., 2009). In the apex, the expression of several SPLs increases during early stages of flowering transition ) and further activates important MADS-box genes by directly binding their promoters Yamaguchi et al., 2009). In addition, flowering via the gibberellin (GA) pathway is mediated by SPLs (Yu et al., 2012), whereas the miR156-SPL module directly regulates FT expression to control ambient temperature-responsive flowering (Kim et al., 2012). SPLs have also been identified as potential targets of the signaling molecule trehalose-6-phosphate, which positively regulates flowering in Arabidopsis (Wahl et al., 2013), suggesting a relationship between SPLs and the carbohydrate status of the plant. Aside from their regulatory role in floral transition, SPLs have been found to influence diverse physiological processes, such as sporogenesis (Unte et al., 2003), leaf development (Usami et al., 2009), copper homeostasis (Yamasaki et al., 2009), male fertility (Xing et al., 2010), gynoecium patterning (Xing et al., 2013), trichome development and anthocyanin biosynthesis (Gou et al., 2011). The involvement of miR156, miR172, and SPLs in the juvenile-to-adult phase transition has also been demonstrated in woody perennials, including English ivy, eucalyptus, poplar, acacia and oak, in a manner similar to that in annual plants . In addition to the age-dependent phase transition, perennials undergo seasonal phase transitions into flowering, which usually occur on an annual basis. However, in some tree species, flowering occurs biennially or even every few years. In biennial bearers, heavy fruit load 1 year (ON-Crop year) inhibits flowering the following year (OFF-Crop year), a phenomenon known as alternate bearing (AB) (Monselise and Goldschmidt, 1982). Complete fruit removal (de-fruiting) from ON-Crop trees induces next-year flowering (return bloom). In a search for regulatory and other processes which are altered in the buds as a result of contrasting fruit loads in Citrus, we previously identified an SPL-like gene whose mRNA level was relatively high during OFF-Crop years and low during ON-Crop years (Shalom et al., 2012). Moreover, de-fruiting significantly induced this gene's expression in the bud within a short time (Shalom et al., 2014). Although this SPL-like gene contained a miR156binding site, no changes in miR156 levels were detected between buds of ON-and OFF-Crop trees, suggesting its regulation by other factors as well. To the best of our knowledge, the above studies were the first to demonstrate a correlation between SPL-like gene expression and fruit load in fruit trees. However, further analyses are required to strength these relationships. In the current work, we performed a functional analysis of the Citrus SPL in Arabidopsis. The results showed it was able to promote flowering, while miR156 repressed its action. As in Arabidopsis, the Citrus genome contains additional SPL-like genes. Therefore, we also set out to identify all SPLs from Citrus and characterize their expression throughout the season and following de-fruiting. The responses of miR156 and miR172 to de-fruiting in Citrus buds are also characterized, and the relationships between them and SPLs with respect to AB are discussed. Identification and Phylogeny of Citrus SPLs Citrus SPL genes were identified using the Citrus clementina genome (http://www.phytozome.net). Arabidopsis SPL sequences were compiled from the TAIR database (https://www.arabidopsis.org/). Alignments were performed by MUSCLE program using default parameters (http://www.ebi.ac. uk/Tools/msa/muscle/ (Edgar, 2004). A phylogenetic tree was constructed based on the maximum likelihood (ML) framework using Phyml software (Guindon and Gascuel, 2003) by the JTT matrix-based model. The tree was graphically designed with the use of FigTree version 1.4 (http://tree.bio.ed.ac.uk/software/ figtree/). Full-length Sequencing of CiSPL5 mRNA An Expressed Sequence Tags (EST) based SPL consensus sequence was pooled from the HarvEST Citrus database (http:// harvest.ucr.edu/) according to the probe set ID of the Citrus GeneChip Microarray (Affymetrix, Inc., Santa Clara, CA). Total RNA was extracted from buds of 15-year-old Murcott mandarin (Citrus reticulata Blanco) trees grafted on sour orange (Citrus aurantium L.), using the CTAB extraction method (Chang et al., 1993). RNA was treated with RQ1 RNase-free DNase (Promega, Fitchburg, WI) according to the manufacturer's instructions. 5 ′ -RACE and 3 ′ -RACE for CiSPL5 were carried out using the FirstChoice RLM RACE Kit (Ambion, Austin, TX). For 5 ′ -RACE, 10 µg of total RNA was ligated to the RNA adapter after treatment with calf intestinal phosphatase and tobacco acid pyrophosphatase, followed by cDNA synthesis using random primers. For 3 ′ -RACE, cDNA was synthesized using the oligo d(T) adapter supplied by the manufacturer. Outer PCR and inner PCR were carried out using the adapter primers, and primers specific for Citrus SPL (Supplementary Table 4). RACE products were gel-purified and cloned into pGEM T easy vector (Promega) for sequencing. Plasmid Construction and Production of Transgenic Plants The CiSPL5 transcript sequence (open reading frame [ORF] + 3 ′ untranslated region [UTR]) and a sequence lacking the 3 ′ UTR (ORF no 3 ′ UTR) were PCR-amplified with Pfu DNA polymerase (Thermo Fisher Scientific, Waltham, MA) using cDNA as the template. CiSPL5 ORF + 3 ′ UTR was amplified using the following primers: forward CTAAAGGAAAAGAC TGTCAAGGATT, reverse GCGTAACGATTGATTCCTCAG. CiSPL5 ORF no 3 ′ UTR was amplified using the following primers: forward CTAAAGGAAAAGACTGTCAAGGAT T, reverse CTACTGAGGACCTACCCCTC. The sequence CiSPL5 ORF + mutated 3 ′ UTR was generated by introducing 10 mutations into the predicted miR156-binding site using recombinant PCR. Primers used for recombinant PCR were: forward TCGCATATTCACTACTCTCTTCCTTAGGCTCCT CCTCT, reverse AGAGAGTAGTGAATATGCGACCTGCAA TGCAGAAAGTT in combination with the primers used for amplification of CiSPL5 ORF + 3 ′ UTR. All three constructs were cloned downstream of the CaMV 35S promoter in pART27 using pART7 as an intermediate vector (Gleave, 1992). Arabidopsis plants (Columbia ecotype) were transformed using the floral dip method (Bechtold et al., 1993). Transformed plants were identified by kanamycin selection (50 µg/ml). Plants were grown under long-day (16 h light) or short-day (8 h light) conditions at 22 • C. Flowering time represents the appearance of the first open flower. Citrus SPLs Expression Analyses Complete fruit removal (de-fruiting) and sample collection were carried out as previously described (Shalom et al., 2014). For seasonal expression analyses, plant material was collected from a commercial orchard of 15-year-old Murcott mandarin (C. reticulata Blanco) trees grafted on sour orange (C. aurantium L.), located in the central coastal area of Israel, during the year 2014. Samples were collected from three biological replicates, each containing three OFF-Crop trees. Vegetative shoots, collected from the southeast side of the trees, were taken to the laboratory on ice. Buds and leaves were separated and immediately frozen in liquid nitrogen. Total RNA was extracted using the CTAB extraction method (Chang et al., 1993) and treated with RQ1 RNase-free DNase (Promega) according to the manufacturer's instructions. Primers for all of the identified Citrus SPLs were designed based on genomic sequences (Phytozome, http://www.phytozome.net/) using Primer3 software (Supplementary Table 1). To exclude putative antisense transcripts (ASTs) as templates in the realtime PCR, primers were designed to span exon-exon junctions or two different exons with a large intron between them. Real-time PCR was carried out as described (Shalom et al., 2012). miR156 and miR172 Analysis ctr-MIR156 and csi-MIR172a (one of several identified miR172 genes) were previously identified as Citrus microRNA genes transcribing precursors which generate mature miR156 and miR172 sequences, respectively (Song et al., 2009;Xu et al., 2010). Thus, measuring their expression may indicate the abundances of their compatible mature miRNAs. In the two available Citrus genome databases (Phytozome and http://citrus. hzau.edu.cn/orange/), both ctr-MIR156 and csi-MIR172a were not predicted as genes, probably duo to their relatively short lengths (<200 bp) and the lack of sufficient open reading frames, and are therefore represented here by their names given upon identification. For the analyses of ctr-MIR156 and csi-MIR172a expression, Citrus microRNA (miRNA) precursor sequences (Supplementary Table 2) were compiled from the miRBase database (http://www.mirbase.org/). Abundance estimates were calculated for each sequence and sample using the RSEM software package (Li and Dewey, 2011) and Bowtie alignment program (Langmead et al., 2009). TMM (trimmed mean of Mvalues-weighted trimmed mean of the log expression values) normalization was performed using code in edgeR, as described by Robinson and Oshlack (2010), and applied to scale the FPKMvalues provided by the abundance estimation software (RSEM) across all samples (Haas et al., 2013). The abundance of mature miR156 and miR172a sequences was determined using TaqMan R Small RNA Assay Kits (Thermo Fisher scientific, Walthem, MA, USA) according to manufacturer's instructions; 10 ng total RNA was used, and real-time PCR was run in a Rotor Gene Q instrument (Qiagen, Venlo, Netherlands). The results were normalized against the β-actin gene as described previously (Shalom et al., 2014). Statistical Analysis Real-Time PCR results were analyzed by One-Way analysis of variance (ANOVA) with Tukey-Kramer multiple comparison tests, as implemented in the software JMP version 10 (SAS Institute, Cary, NC, USA). Gene Structure and Phylogeny of SPL Family Members from Citrus To identify Citrus SPL members the following approaches have been taken: (1) all sequences containing an SBP domain were compiled from the Citrus clementina genome database (http:// www.phytozome.net/), (2) proper identification of all matching sequences was confirmed by performing BLAST against two different Citrus genome databases (Phytozome and http://citrus. hzau.edu.cn/orange/) using the 16 SBP-domain sequences from Arabidopsis as queries, and (3) all SPL-related unigenes were pooled from the Citrus EST database (http://harvest.ucr.edu/) and BLASTed against the C. clementina genome database. Following removal of redundant sequences and alternative transcripts, a total of 15 SPL members were determined in the C. clementina genome (Figure 1). To date, only one mature miR156 sequence from Citrus has been experimentally validated (Xu et al., 2010). Of the 15 putative Citrus SPL transcripts, 10 contained sequences complementary to the 20-nucleotide mature miR156 sequence, with one or two mismatches at the 1st, 7th, or 9th nucleotide (Figure 1). Analysis of the other five SPL transcripts resulted in no significant matches. Alignment of the full-length protein sequences of Arabidopsis and Citrus showed no consensus sequences other than the SBP domain (not shown). Therefore, only the SBP domains (Supplementary Table 3) were used for the phylogenetic analysis (Figure 2). The results of this analysis suggested that gene multiplication occurred before separation of Citrus and Arabidopsis, which are considered to be taxonomically related. Only Ciclev10020532 contained an SBP domain that was somewhat unique to Citrus (Figure 2). In general, these SPLs could be classified into three subgroups. The first one (green clade) consisted of SPLs characterized by relatively short protein sequences (<200 amino acids) and a miR156-binding site located within the 3 ′ UTR of the transcript. The Arabidopsis members of this group, AtSPL3, AtSPL4 and AtSPL5, showed the highest homology to three short Citrus SPL members: Ciclev10009879, Ciclev10017104 and Ciclev10016841 containing Table 3). SPLs were classified into three subgroups: <200 amino acids with miR156-binding site located within the 3 ′ UTR (green), >300 amino acids with miR156-binding site located within the ORF (blue), >300 amino acids without miR156 binding site (black). 130, 143 and 189 amino acids, respectively. The second group (blue clade) consisted of SPLs characterized by longer protein sequences (>300 amino acids) and a miR156-binding site located within the ORF. The seven Citrus SPL members within this clade were: Ciclev10020532, Ciclev10031391, Ciclev10032171, Ciclev10031834, Ciclev10031270, Ciclev10019546, and Ciclev10011938. The third group (black clades) consisted of SPLs which did not contain a miR156-binding site. The five Citrus SPL members within this group were: Ciclev10021106, Ciclev10004227, Ciclev10018697, Ciclev10000100, and Ciclev10004348. Molecular Characterization of miR156-Regulated CiSPL5 The roles of SPLs and miR156 as regulators of phase transitions have been extensively studied in Arabidopsis and other annual plant species; however, considerably less research has been done with trees. To gain insight into the roles of SPLs in Citrus and understand whether the presence of a miR156-binding site in an SPL transcript constitutes a real regulatory element, we performed a molecular and functional characterization of one Citrus SPL, Ciclev10009879, whose expression had been previously studied in relation to fruit-load effect on flowering induction of Citrus (Shalom et al., 2012(Shalom et al., , 2014. Full-length sequencing of Ciclev10009879 mRNA revealed that it is 843 bp long (with one alternative polyadenylation site at 750 bp) with a putative ORF encoding a 130-amino acid protein and a miR156binding site located in the 3 ′ UTR. An antisense transcript (AST) of about 2400 bp which encompasses the entire region of Ciclev10009879 was also identified (Figure 3). Surprisingly, RACE analyses indicated that the transcription start site of this AST is located in a neighboring upstream gene, Ciclev10009133, encoding a putative PROTEIN PHOSPHATASE 2C (PP2C). In fact, the full-length structure of the AST was similar to one of the predicted alternative transcripts of Ciclev10009133, only with a longer than predicted 3 ′ tail (long PP2C transcript, Figure 3). The RACE analyses identified four transcription start sites and four polyadenylation sites in the AST, suggesting a complex mode of transcription. In Citrus, fruit set takes place during May, whereas September-October are regarded as the last time points at which fruit removal during the ONyear reverses the AB trend (Martinez-Fuentes et al., 2010). The floral induction period starts in mid-November and lasts until approximately mid-January (Davenport, 1990). Expression analysis of the long AST in buds showed that it was expressed at higher levels from May to September and lower levels from November to January (Supplementary Figure 1). However, no significant differences were detected between buds of ON-and OFF-Crop trees, and no alterations were detected following fruit removal, putting its role in flowering control by fruit load into question. As mentioned above, Ciclev10009133 encoded a protein that is highly homologous to Arabidopsis PP2C (At3g15260; 79% identity). In the Arabidopsis genome, this gene is located jointly and in antisense orientation to AtSPL5, which belongs to the small SPLs subgroup (Figure 2). Therefore, based on SBPdomain sequence homology, protein length, miR156-binding site position and genomic coupling with PP2C, we determined that Citrus SPL Ciclev10009879 is the ortholog of Arabidopsis AtSPL5, and it is henceforth referred to as CiSPL5. Seasonal Expression Patterns of Some CiSPLs and Patterns Following Fruit Removal Coincide with Flowering Induction As woody perennials, Citrus trees undergo a series of developmental changes deriving from seasonal environmental and endogenous cues. Some of these changes, such as the shift to flowering and/or vegetation, might be regulated, at least in part, by SPLs. Therefore, we studied their expression patterns in leaves and buds of OFF-Crop trees, expected to flower the following spring, before and during the flowering induction period from November to January. Nine out of fifteen SPLs (Ciclev10020532, Ciclev10021106, Ciclev10031270, Ciclev10031391, Ciclev1000100, Ciclev10019546, Ciclev10032171, Ciclev10031834, and Ciclev10011938) were expressed at higher levels in buds than in leaves at most time points (Figure 5), whereas 4 out of 15 SPLs showed similar expression levels in buds and leaves at most time points. The mRNA levels of one SPL, Ciclev10017104, were similar in leaves and buds from May until September, but were higher in leaves from November to January. However, the mRNA of CiSPL5 was exceptional in that at all tested time points, it showed significantly higher levels in leaves than in buds. While CiSPL5 expression in leaves was relatively stable, with a transient threefold increase in November, its expression in buds gradually decreased from May to January (Figure 5, Supplementary Figure 3), consistent with our previous report (Shalom et al., 2012). Similarly, the mRNA levels of Ciclev10020532, Ciclev10021106, and Ciclev10031270 decreased moderately throughout the season in the buds. In contrast, the mRNA levels of Ciclev10019546 in buds showed a moderate increase from May to November followed by a slight decrease in January, similar to the trend in leaves. Another SPL whose expression is worth mentioning is Ciclev10016841, which showed a sharp increase in mRNA levels from November to January. As heavy fruit load inhibits flowering induction, the mRNA levels of SPLs were also investigated in buds of ON-and OFF-Crop trees and following fruit removal (de-fruiting). Most of the SPLs were of similar levels in ON-and OFF-Crop buds, and were not altered significantly by de-fruiting (Figure 6). However, the mRNA levels of Ciclev10020532 and Ciclev10021106 were higher in OFF-Crop vs. ON-Crop buds, and they were significantly upregulated (two-to threefold) 1 week after de-fruiting. In addition, the level of Ciclev10016841 mRNA was significantly higher (by about threefold) in OFF-Crop buds than in ON-Crop buds; however, its level was not altered by de-fruiting. Fruit Removal Alters the Expression of SPL-related miRNA Genes, but Not Necessarily Abundance of the Mature miRNA Primary miRNAs (pri-miRs) are capped and polyadenylated non-coding RNA transcripts containing mature miRNA sequences (Voinnet, 2009). Cleavage of the primary miRNA transcript eventually results in the release of biologically active mature miRNA and ultimately, the degradation or translational repression of mRNA targets. The miRBase database (http:// www.mirbase.org/) contains sequence data (precursor and mature) for 75 miRNAs from Citrus. We recently conducted an RNA deep-sequencing analysis of Citrus buds following fruit removal (Shalom et al., 2014). In the current work, we aligned the RNA deep-sequencing data to the Citrus precursor sequences from miRBase. Results of this analysis indicated that about two-thirds of them are expressed in buds; however, only a few were significantly affected by fruit load. Among these were ctr-MIR156 and csi-MIR172a, with the latter expressed to much higher levels ( Figure 7A). Expression levels of ctr-MIR156 were significantly higher in ON-Crop buds as compared to OFF-Crop buds and decreased following de-fruiting ( Figure 7B). In contrast, expression levels of csi-MIR172a showed the opposite trend, with OFF-Crop buds and buds after de-fruiting showing higher expression levels than ON-Crop buds. Alterations in both genes occurred as early as 1 week after de-fruiting ( Figure 7B). Abundance analysis of the mature miRNA sequences was performed next. While miR156 abundance was not affected by fruit presence, miR172 was more abundant in OFF-Crop buds than ON-Crop buds, and increased significantly (two to threefold) after de-fruiting ( Figure 7C). SPL Gene Family in Citrus Genes containing the SBP domain were originally identified in Antirrhinum majus and were named SQUAMOSA PROMOTER BINDING PROTEIN based on their ability to interact with the promoter of the floral meristem identity gene SQUAMOSA (Klein et al., 1996). Since then, SPLs have been identified and classified in a number of plant species, including Arabidopsis (Rhoades et al., 2002), rice (Xie et al., 2006), tomato (Salinas et al., 2012), and grape (Hou et al., 2013). Similar to other plants, about two-thirds of the Citrus SPLs contain sequences complementary to miR156. In Arabidopsis, 3 of the 10 miR156regulated SPLs, AtSPL3/4/5, differ from the others by the position of their miR156-binding site-it is located in the 3 ′ UTR and is believed to have moved there via exon degeneration (Guo et al., 2008)-and by their relatively short protein size, which mainly comprises the SBP domain (Wu and Poethig, 2006;Yamaguchi et al., 2009). Overexpression of each of these SPLs shortens the juvenile period and induces early flowering (Wu and Poethig, 2006). AtSPL3 activates LEAFY (LFY), FRUITFUL (FUL), and APETALA1 (AP1) by directly binding their promoter regions and evidence suggests that AtSPL4 and AtSPL5 have overlapping functions . Our phylogenetic analysis showed that AtSPL3/4/5 have three close orthologs in Citrus, with similar structural characteristics and therefore potentially similar functions. By its structure and genomic localization (i.e., proximity to a PP2C-like gene), Ciclev10009879 (CiSPL5) is suggested to be the ortholog of Arabidopsis AtSPL5. Antisense Transcription within CiSPL5 We identified an AST which encompasses the CiSPL5 genomic region. A large number of overlapping transcripts in antisense orientation with the potential to form double-stranded RNA structures have been identified in the Arabidopsis genome (Jen et al., 2005;Wang et al., 2005), for instance, in AtSPL3 (Wu and Poethig, 2006). Although the role of these ASTs remains unclear, they might function as an additional level of regulation. The identified AST is a long variant of a neighboring gene encoding PP2C-like protein homologs of At3g15260. Some PP2C proteins play a role in abscisic acid (ABA) signaling (Umezawa et al., 2009). ABA homeostasis is affected by fruit load (Shalom et al., 2014), and the decrease in the AST's expression during the winter months somewhat resembles the expression pattern of CiSPL5 (although it does not respond to fruit load). Therefore, exploration of ABA's possible signaling role in CiSPL5 regulation is warranted. Do SPLs and miR172 Play a Role in Fruit-load Effect on Flowering in Citrus? CiSPL5 was able to accelerate Arabidopsis flowering while miR156 repressed its action (Figure 4). Although no direct evidence was provided, it was likely that CiSPL5 regulated phase transition in Citrus, as well. A significant difference in its expression was detected between ON-and OFF-Crop buds, and it was induced by de-fruiting (Shalom et al., 2012(Shalom et al., , 2014, raising the posebility it also played a role in AB regulation. However, the findings that the levels of miR156 in Citrus buds were not affected by fruit load (Shalom et al., 2012, Figure 7C), questioned the possibility that fruit load regulated CiSPL5 expression through miR156. Previous investigations in various plant species demonstrated that miR156 was predominant in juvenile tissues, whereas miR172 was induced in adult tissues (Aukerman and Sakai, 2003;Chen, 2004;Lauter et al., 2005;Wu and Poethig, 2006;Chuck et al., 2007). Our transcriptomic data (Figure 7) showed low expression levels of ctr-MIR156 relative to csi-MIR172a, which is in agreement with the accepted dogma of their respective roles in juvenile and adult plants. This could explain why the levels of miR156 did not correlate with fruit load. Additional finding was the lack of correlation between the expression patterns of ctr-MIR156 and mature miR156 (Figure 7). It might be that the final level of this miRNA was ultimately determined by a combination of factors, including additional active miR156 genes or small interfering (si) RNAs. Unlike miR156, miR172a increased both at the precursor and mature levels following de-fruiting (Figure 7). In Arabidopsis, miR172 transcription is induced by AtSPL9 and AtSPL10 , and in turn promotes flowering by repressing AP2-like TFs which negatively regulate FT expression (Mathieu et al., 2009). Could this also be the case in Citrus fruit loadaffected flowering? In parallel to csi-MIR172a induction, three CiSPL genes were up-regulated following de-fruiting in the bud (Figure 6): CiSPL5 (Ciclev10009879), Ciclev10020532, and Ciclev10021106. While Ciclev10020532 has no close ortholog in Arabidopsis (Figure 2), Ciclev10021106 is the ortholog of AtSPL8, found to play important roles in the regulation of male fertility (Xing et al., 2010) and gynoecium patterning (Xing et al., 2013). Whether Ciclev10021106 has similar roles in Citrus is unclear; however, it is not targeted by miR156. Taken together, it is tempting to speculate that some of these SPLs regulate miR172a expression by a mechanism similar to that in Arabidopsis, as mentioned above. miR156's Negative Regulation of CiSPL5 in Arabidopsis Can Be Bypassed through the Photoperiod Pathway In addition to flowering promotion, CiSPL5 shortened the juvenile phase, as reflected by positive regulation of abaxial trichome production, which is characteristic of adult leaves. This has also been shown for other SPLs (Cardon et al., 1997;Wang et al., 2009;Wu et al., 2009). While long day promotes Arabidopsis flowering, under short day, flowering occurs after a long period of vegetative growth, as a result of miR156 downregulation accompanied by a gradual increase in AtSPL3 and AtSPL9 expression . Shifting plants from short days to long days leads to a rapid increase in AtSPL3 and AtSPL9 expression while miR156 levels remain unchanged. Our functional analysis of CiSPL5 in Arabidopsis supports the notion that the negative regulation by miR156 can be bypassed through the photoperiod pathway; when overexpressed in miR156insensitive forms, CiSPL5 accelerated flowering regardless of day length. The normal phenotype of lines overexpressing CiSPL5 in a miR156-sensitive form indicated that flowering in these plants is affected by endogenous factors, such as an age-dependent pathway (short day) or the photoperiod pathway (long day). Funding This work was supported by fund number 203-0870 of the Chief Scientist of the Ministry of Agriculture and Rural Development. Supplementary Material The

v3-fos

2019-03-12T13:05:44.524Z

2015-11-01T00:00:00.000Z

74219949

s2

Impact of the carrier state by Theileria annulata on milk yield in Tunisian crossbred ( Bos taurus ) cattle Asian Pacific Journal of Tropical Disease Objective: To estimate milk yield losses in Theileria annulata (T. annulata) carrier cows by a trial which was made in two cattle farms in the north of Tunisia. Methods: Eight non-infected cows were randomly and equally divided into two groups: the treated and control groups. Eight T. annulata carrier cows were also randomly and equally divided into two groups: the treated and control groups. Results: There was a negative correlation between milk yield and parasitemia in both treated ( r = -0.495, P = 0.037) and control carrier cows ( r = -0.662, P = 0.003). Average adjusted lactation curve showed that milk yield in treated carrier cows was higher than that in control carrier cows. Daily milk yield losses due to T. annulata infection have been estimated at 0.77 kg per of each T. annulata carrier cow. Conclusions: Since T. annulata infection induces a small but persistent decrease of milk yield in T. annulata carrier cows, further studies are needed in a larger animal group to confirm and improve this estimation. Introduction Tropical theileriosis [Theileria annulata (T. annulata) infection] is a protozoan disease transmitted by tick species belonging to the genus Hyalomma. In enzootic countries, T. annulata caused important losses, such as mortality, weight losses, abortions, milk yield losses, and control costs [1,2]. The impact of the carrier state of T. annulata infection on milk production was shown in a field study in 1991 by Singh [3]. It is estimated that the milk losses caused by the asymptomatic infection (carrier state) in zebu-taurine crossbred cows treated with buparvaquone at a daily average of 1.4 L, i.e. 126 L over a period of 90 days [3]. Minjauw and McLeod estimated these losses at 386 L/lactation period in carrier zebu-taurine crossbred cows [4]. Considering the prevalence of the carrier state in lactating cows from enzootic regions for tropical theileriosis [5], the estimation of the financial losses due to this infection state is an important step toward an accurate analysis of the financial impact of tropical theileriosis and to rank this infection among other animal health problems [6]. To our knowledge, the present study is the first in Africa to evaluate milk losses of T. annulata carrier cows (Bos taurus) by using a protocol based on injection of theilericidal drugs. [7]. DNA was extracted from blood samples and a PCR was run using the Tams primers [8]. After this time period, milk yield was recorded every two days during 30 days. The evolution of milk yield was illustrated by using observed and predicted yields before and after the buparvaquone injection. Predicted yields to a 305-day period were derived from fitting the Wood function to milk data points recorded during the trial period [9,10]. Adjusted lactation curves should show the evolution (increase or decrease) of milk production following the treatment. Materials and methods Milk yield losses were calculated by comparing yields recorded between treated and control animals during two periods. In order to decrease the heterogeneity, the milk yields were log transformed. The comparison of means of milk yield from Day 1 to Day 12 after buparvaquone injection was performed by using the student's t test with a cut-off value of 0.05 following One-way ANOVA where groups of cows (T0B0, T0B1, T1B0, and T1B1) were levels of the explanatory variable. Similar analysis was conducted for parasitemia. The correlation between parasitemia and milk yield was also computed (SPSS 13 for Windows). Results Before treatments, parasitemia was relatively high (maximum value: 20 parasites/100 fields in both control and treated animals). After buparvaquone injection, the parasitemia decreased in treated animals and remained fluctuant in control cows (Figure 1). were supposed to have their production declining (they were in the phase following the peak of lactation). Because of the short period of milk monitoring in the present study and since the recorded yields were carried out in the decreasing period of the lactation curve, the curves did not show the three phases of typical curves (increasing period, peak and decreasing lactation curve). Relative variation of milk yield in treated cows (11.63%) was higher than that in control cows (4.84%) (P < 0.05). The relative difference in milk yield was 6.79% with a mean daily loss per cow estimated at 0.77 kg. There was a significantly negative correlation between milk yield and parasitemia in both treated (r = -0.495, P = 0.037) and control carrier cows (r = -0.662, P = 0.003). Discussion In Tunisia, M'barek estimated milk yield losses in clinically infected cows during the month after treatment at 300 L per cow [11]. The present trial was carried out to estimate the milk yield losses due to the carrier state by T. annulata in the Tunisian context. To our knowledge, the only study that estimated such losses was carried out by Singh in crossbred taurine-zebu cattle [3], which was more resistant to T. annulata infection than pure taurines [12]. Another difference was that Tunisian cattle were exposed to the vector tick (Hyalomma scupense) from late June to early August [13], while in India the challenge occurs throughout the year. This difference had a high impact on the host-parasite relationship and the epidemiological features of the disease. The number of cows was low. That was due to the difficulties to find In India, Singh reported a more significant decrease of parasitemia passing from 0.0155% at Day 0 to 0.007 and 0.002% at Day 5 and 7, respectively [3]. This is due to a more innate resistance to T. annulata infection of these animals when compared of the present cow population [12]. In India, the animals are challenged throughout the year representing a fundamental difference when compared to the epidemiology of the disease in Tunisia, which occurs exclusively in the summer season (from June to August) [13]. Accordingly, the cows monitored in the present study were true carriers since they were not subject to any re-infection. This discrepancy with the milk yield level in the two countries should be taken into account when comparing the increase record in milk yields with the values reported in India [3]. The effect of buparvaquone injection is not comparable to its effect on clinically infected cows. In these animals, a dramatic and rapid decrease of parasitaemia is usually reported, and the disease can lead to a drying up of the cow [11]. In a study in Egypt, milk yield in T. annulata Friesian cows varied between 2.5 and 5.0 kg/day during the production peak in treated animals and between naught and 3 kg/day in non-treated animals [15]. Compared to our results, these losses are very high, which is due to the fact that these cows develope clinical episodes of tropical theileriosis while our animals were asymptomatic. During the 10-day post-injection, a progressive significant increase of milk yield was observed with a statistically significant negative correlation between milk yield and parasitemia. The results obtained herein were lower than those reported in India [3], where the mean daily losses were estimated to 1.4 L. This difference can be explained by the fact that these animals were challenged several times during the trial leading to higher parasitaemia levels. A significant effect of the carrier state on milk yield was identified in our study. However, it was carried out on a very low number of cows, and further trials are needed to confirm this decrease. Conflict of interest statement We declare that we have no conflict of interest.

v3-fos

2019-03-30T13:02:16.985Z

2015-03-04T00:00:00.000Z

56065299

s2

Response of Barley (Hordium vulgare L.) to Integrated Cattle Manureand Mineral Fertilizer Application in the Vertisol Areas of South Tigray, Ethiopia A study to investigate the effect of integrated mineral and cattle manure fertilizers on grain yield of Barley (Hordium Vulgar L.) was evaluated during 2013 and 2014 main cropping season on vertisols of southern Tigray Ethiopia. The treatment consists four level of N/P205 fertilizer combination (0/0, 23/23, 46/46, 69/69 kg ha -1 ) and five levels of farm yard manure (0, 4, 6,8,10 ton ha -1 ) and their interactions arranged in Randomized Complete Block Design (RCBD) in three replication. The combined statistical analysis over locations revealed significant main effects of FYM and NP fertilizers (p ≤ 0.05) and interactions effects on grain yield of barley. There were also highly significance variation among N/P fertilizer main effect for biomass, physiological maturity, plant height, seeds per spike and effective tiller per plant, however no significance variation between the FYM main effects and interaction effects of NP and FYM for biomass, physiological maturity, plant height seeds per spike and effective tiller per plant. Grain yield consistently respond to increasing level of fertilizations in the form of NP, FYM or their integration. The results of this finding showed that combined application of 69/69 N/ P205 kg ha -1 + 10 ton ha -1 FYM, 69/69 N/P205 kg ha -1 + 8 ton ha -1 FYM and 69/69 N/ P205 kg ha-1 + 6 ton ha -1 FYM significantly (P<0.05) increase the yield of barley than other treatments. Integrated application of 46/46 N/P205 kg ha-1 with 8 t ha -1 gave 18 % and 100% yield increment than current (46/46 N/P205 kg ha -1 ) blanket fertilizer recommendation in the area and the control. This may greatly benefits farmers in area where supply of mineral fertilizer is low or cases where farmers can’t afford the cost of high fertilizer input. Higher grain yield (2.9 ton ha -1 ) was obtained from residual effects of 8 ton ha -1 FYM applied in 2013 on barley grain yields in 2014 cropping season received 46/46 kg ha -1 N/P205. Introduction Barley (Hordeum vulgare) is one of the most important cereal crop, mainly grown by smallholder farmers at midand high altitudes Ethiopia, predominantly between 2200-3000 m a.s.l [1]. It is one of the most important small cereal crops, which ranks fourth after wheat, maize and rice in the world [2] and 5 th in Ethiopia both in terms of area and production after Teff, Maize, Sorghum and Wheat [3]. Food barley is commonly cultivated in stressed areas where soil erosion, occasional drought or frost limits the ability to grow other crops [4]. Low soil fertility has been recognized as one of the major biophysical constraints affecting agriculture in sub-Saharan Africa [5]. Soils in the highlands of Ethiopia exhibit low levels of essential plant nutrients and organic matter content [6 and 7]. This is largely consequence of the cerealdominated cropping history of most fields and continuous nutrient mining by crop removal [8 and 9], which eventually leads to depletion of soil nutrients [6 and 10]. Soil nutrient depletion has been exacerbated by low levels of chemical fertilizer usage [6] due to both high cost and constraints to timely availability of the fertilizer input [11]. The poor soil fertility in northern Ethiopia has been blamed for limiting the production and production stability of barley [12] and nitrogen and phosphorus are among the most productivity limiting nutrients [13]. In southern Tigray ,the current fertilizer blanket recommendation is 46/46 N/P 2 0 5 kgha -1 and is used by some farmers, but most of the resource poor farmers are using even below the recommendation due to increasing fertilize cost. Even if there is huge number of livestock in the study area the culture of using farm yard manure as fertilizer source is not practiced well and the rate of application and incubation method is not scientifically recommended. So reducing the amount of mineral nitrogen fertilizers applied to the field without a nitrogen deficiency is the main challenge in field management. Therefore, to maintain soil fertility and productivity, the use of other alternative option of soil fertility replenishment is indispensable. Farmyard manure (FYM) is one potential source of nutrients as a result of the high cattle production of the region where on average there are 14 livestock per family [14]. Application of organic materials alone or in combination with inorganic fertilizer helped in proper nutrition and maintenance of soil fertility [15].According to reference no [16] reported that organic manures increased the efficiency of chemical fertilizers. Beneficial effects of farm yard manure on crop production through improved fertility and physical properties of soil are an established fact [17] and providing greater stability in production, but also maintaining better soil fertility status [18]. The long term effects of the combined application of organic and inorganic fertilizers in improving soil fertility and crop yield have been demonstrated by many workers [19]. In reference no [20] reported that organic and inorganic fertilizers showed great benefits not only for the increase in the N uptake by the plant but also in the improvement of the fodder yield. Research efforts on how to use this resource and use of FYM together with low rates of mineral fertilizers could be one alternative solution for sustainable fertility management and alleviate food self sufficiency specially for resource poor farmers. More over there is no research recommendation on NP, FYM, FYM and NP integration in the study. Therefore the study is initiated to evaluate the effect of different integrated mineral fertilizer and FYM application rate grain yield of barley. [14]. Mixed livestock farming system is an agricultural production system practice in the area and livestock production is a major component of the livelihood system and provides draught power, food and income. Small ruminant production is one the component of the livestock production system. The rain fall pattern of the districts is presented in figure 1. The dominant soil type for the study area is vertisols with minimum and maximum air temperature of 8 and 22 0 c respectively. Treatments and Experimental Design` The FYM used for the experiment was well decomposed for three months under shade and applied all at planting with phosphorus fertilizer while N fertilizer was applied in split form with 1/3rd of the dose applied at planting and the remaining 2/3rd at tillering (40 days after sowing) stage of the crop. The source of phosphorus fertilizer was triple supper phosphate (TSP). Treatments were laid out in a randomized complete block design with three replications. The plot size was 3mx2.4m with 1.5 m between replication and 1m between plot alleys. The treatments consists four level of N/P205 combination (0/0, 23/23, 46/46, 69/69 kgha -1 ) and five levels of farm yard manure levels (0, 4, 6,8,10 ton ha -1 ) and their interactions. The variety used in the experiment was Shedho. Composite soil samples were collect from the plow layers (0-30cm) at each experimental site before applications of the treatments. A standard laboratory procedure for each parameter was followed in analyzing the composite surface soil samples. The results of the laboratory analysis of some physico-chemical properties of the soil used for the experiment are presented in Table 1. Accordingly, Soil samples were analyzed for texture, organic carbon, total nitrogen, Cation Exchange Capacity (CEC), available P, exchangeable Ca and Mg, PH, total nitrogen and available phosphorus. The methods used for physico-chemical analysis were: Organic matter content was determined by oxidation of organic carbon with acid potassium di-chromate (K2Cr2O7) by the Walkley and Black method [21]. Total nitrogen was analyzed by Micro-Kjeldhal method [22]. Soil pH was determined in 1:2.5 (weight/ volume) soil to water dilution ratio [21]. Cation exchange capacity was measured after saturating the soil with 1N ammonium acetate (NH4OAC) and displacing it with 1N NaOAC [23]. Available phosphorus was determined using Olsen method [24]. Agronomic Data • Days to maturity (DM): Physiological maturity was calculated by counting the number of days from 50 % emergence to the stage when 90% of the plant reaches physiological maturity. • Number of grains per spike (SPS -): Grains per spike were counted from ten randomly selected spikes of each plot, and the total grains number was divided by the sampled plants to get average number of grains per spike. • Grain yield (kg plot -1 ): The grain yield was taken from each plot by excluding the border rows and adjusted to 12.5% moisture level and then converted to hectare basis. Data Analysis The Analysis of Variance (ANOVA) on the relevant responsive variable was computed using the GLM procedure of SAS version 9.2 [25] following the standard procedures of ANOVA for RCB design [26]. The differences among locations and among treatments were considered significant if the P-values were ≤ 0.05. Least Significance Difference (LSD) was used to compare among varieties at 5% probability level. Result and Discussion Laboratory analytical results of selected physicochemical properties of the soil on which these on-farm experiments were conducted is presented in Tables 2. Soils in the study areas are dominantly clay in texture and vary from slightly acidic to neutral. The soil organic and total Nitrogen contents at all locations are very low and total indicating the low fertility status of the soils aggravated by continuous cultivation, and lack of incorporation of organic materials into the soils. The cation exchange capacity (CEC) of the experimental site was 44 and 46 Meq/100kg soil for ofla and Enda Mehoni respectively. The available phosphorus was also below critical. The combined statistical analysis over locations revealed significant (p ≤ 0.05) main effects of FYM, NP fertilizers and interactions effects on grain yield of barley. There were also highly significance variation among N/P fertilizer main effect for biomass, physiological maturity, plant height, seeds per spike and effective tiller per plant, however no significance variation between the FYM main effects and interaction effects of NP and FYM for biomass, physiological maturity, plant height seeds per spike and effective tiller per plant. Grain yield consistently respond to increasing level of fertilizations in the form of NP, FYM or their integration. Significantly more grain yields were obtained in treatments receiving combined application of 69/69 kg ha -1 N/P205 with 10 t ha -1 of manure and followed 69/69 kg ha -1 N/P205 with 8 t ha -1 and 69/69 kg ha -1 N/P205 with 6 t ha -1 respectively. Up to 18% and 100% grain yield increment was also recorded in integrated application of 46/46 N/P205 kg ha -1 + 6 tha -1 FYM than the present N/P205 fertilizer recommendation and control treatment respectively. This study, there for strongly confirms the role of manure and chemical fertilizer in increasing grain yield of barley but a combination of them has more effect on increasing in grain yield. Integrated soil fertility management involving the judicious use of combinations of organic and inorganic resources is a feasible approach too overcomes soil fertility constraints 27, 28 and 29] and contribute high crop productivity in agriculture [30]. In reference no [31, 32 and 33] reported also similar observations of getting higher yields of Barley grain with combined application of FYM and inorganic fertilizers. Significantly more grain yield was also obtained at 2014 from residual of farm yard manure in applied 2013 (Table4); this may due to the slow release of nutrients from FYM in the former cropping season. Mineral fertilizers in improving crop yields [34, 35 and 36]. Additional 0.4 ton per hectare of barley grain yield was obtained from residual interaction effect of 46/46 kg ha-1 N/P 2 0 5 + 8 t ha -1 FYM. Manure fertilizer treatments had beneficial residual effects on crop production and use from manure fertilizer for field fertilization and production of crops was better improved. Significantly high grain was obtained from residual application of 8 t ha -1 and is proportional with existing fertilizer recommendation. Therefore for resource poor farmers combined application of farm yard manure and mineral fertilizer is very economical than sole NP application. Conclusion From this finding the integrated use of farm yard manure, and N and P fertilizers are efficient than the use of either N/P or FYM alone. It can be concluded that use of farmyard manure and chemical fertilizer considerably improve grain yield of barley. The result in this investigation showed that use of 69/69 kg ha -1 N/P 2 0 5 chemical fertilizer integrated with 6 t ha -1 manure fertilizer could produce satisfactory yield of barley in the study area and farm yard manure treatments had beneficial residual effects on barley crop production.

v3-fos

2019-03-22T16:09:40.330Z

2015-04-14T00:00:00.000Z

85283639

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Toxicity of two plant powders as biopesticides in the management of Callosobruchus maculatus F. (Coleoptera: Chrysomelidae, Bruchinae) on two stored grain legumes Objective: The present study aimed to evaluate effects of leaf powder of Chenopodium ambrosioides (wormseed) and Adenia cissampeloides (snake climber) on insect populations and seeds weight loss percentage. Methodology and Results: Two leaf powders were applied at 2.5%, 5% and 7.5% (wt/wt). All bioassays were conducted at 27±2°C and 70±5%RH. Insect mortality was evaluated after 2, 4 and 6 days of exposure and the total progeny was assessed 34 days after. C. ambrosioides at 2.5% showed the best efficacy, recording 69.64% of mortality in Vigna subterranea groundnuts and 100% of mortality in Kerstingiella geocarpa one’s, 6 days after treatment. The lowest LC50 value after 6 days was obtained with A. cissampeloides applied at 2.37g/20g of V. subterranea groundnuts and with C. ambrosioides applied at 1.38 g/20g of K. geocarpa groundnuts. Conclusion and application of findings: Because of their effectiveness, the leaf powder of these plants could be recommended as grain protectant against C. maculatus. INTRODUCTION Pulses (grain legumes) are the second most important group of crops worldwide. About 870 million people are undernourished because of inadequate intake of proteins, vitamins and minerals in their diets (FAO, 2012). Pulses are excellent sources of proteins (20-40 %), carbohydrates (50-60%) and are good sources of thiamin, niacin, calcium and iron. As in many sub-tropical African countries, cereals and pulses are essential source of food for human consumption. In southern Benin, after harvest, the pulses are usually stored for long periods for seeds, trade or consumption. During storage period, they are seriously damaged by storage insect pests leading to severe losses Journal of Applied Biosciences 86:7900-7908 ISSN 1997-5902 Chougourou et al. J. Appl. Biosci. Toxicity of two plant powders as biopesticides in the management of Callosobruchus maculatus F. on two stored grain legumes. (Dinesh and Deepshikha, 2012). Among these pests, pulse beetle, Callosobruchus maculatus F. (Coleoptera: Chrysomelidae, Bruchinae) is a major pest that causes serious damage (Sharma, 1989) on stored pulse grains. In Nigeria for instance the damage due to C.maculatus on stored pulse reached 24 % of losses per year (Caswell, 1968). It is therefore necessary to reduce such losses by controlling pests on stored grains. When properly used, synthetic insecticides may play an important role in reducing storage losses due to insect pest (Menn, 1983;Redlinger et al., 1988). However, chemical pesticides have serious drawbacks such as development of pest resistance, toxic residue problems, toxicity risk on consumers and costs of application. Small-scale farmers generally use some traditional methods to protect stored foodstuffs from insect infestation in Eastern Africa (Hassanali et al., 1990;Poswall and Akpa, 1991). Plant materials have played an important part in those traditional methods in Africa where they have been mixed to stored grains. The mode of use and type of botanical material vary from place to place and appear to depend partly on the type and efficacy of suitable flora available in different locations. However, the number of plants that are known to possess insecticidal activity against storage insect pests is rather small. It appears necessary to develop alternative techniques to protect stored foodstuffs. Thus, this study aimed to evaluate effects of two ground leaf powder on population and damage of C. maculatus on stored pulse grains. C. ambrosioides (Chenopodiaceae) is a strongly aromatic, hairy, annual or perennial herb. It is abundant in the tropics and subtropics, especially in America and Africa (Rendle, 1983), and has been reported to have a wide variety of medicinal and insecticidal properties (Su, 1991;Quarles, 1992). A. cissampeloides (Passifloraceae) is a robust liana used in traditional medicine throughout tropical Africa. Most frequently recorded are the uses of an infusion or decoction of the root, stem or leaves for the treatment of gastrointestinal complaints, such as stomach-ache, constipation, diarrhea and dysentery (PROTA, 2010). MATERIALS AND METHODS Experiments were conducted in Laboratory of Applied Biology Research at University of Abomey-Calavi. C. maculatus was cultured in the laboratory at 27°C± 2%, 70%± 5% r. h. and 12h photoperiod. Collection and preparation of plant material: Leaves of C. ambrosioides and A. cissampeloides were collected at Godomey in southern Benin during November 2012. They were dried on laboratory benches at room temperature (26°C-28°C) for 5 days. The powder was obtained by grinding the dried leaves in a coffee grinder and was sieved through a mesh of 0.5 mm size. The obtained powder was mixed to pulse grains (Bambara and kersting's groundnuts) using various doses. Insects rearing: Adults of C. maculatus were reared in the laboratory under 27°C, 70% r. h. and 12/12 hours photoperiod. The original stock was obtained from stock cultures in Laboratory of Applied Biology Research of University of Abomey-Calavi. The food media used were Bambara and kersting's groundnuts. All grains used for this study were procured in Agbangnizoun, a southern Benin village. Treatments and experimental design : The powder obtained from dried leaves of C. ambrosioides and A. cissampeloides was mixed separately with 20 g of grains in 380 ml glass jars. For each product the three dosages (treatments) were considered as follows: 2.5% (0.5g/20g of seeds), 5% (1g/20g) and 7.5% (1.5g/20g).The plant materials were thoroughly mixed for 20 min with the grains using a rotary shaker (Multifix GmbH, Germany). For each set of treatments, a non-treated seeds was considered as control. Five pairs (5 males and 5 females) 1-3 day-old adults of C. Maculatus were introduced into the jars with treated or untreated grains. Each treatment was replicated three times. Each jar was covered with cotton cloths to allow air circulation. Data collection: The number of dead insects in each jar was counted 2, 4 and 6 days after treatment and the percentage mortality was corrected using the Abbott formula (Abbott, 1925): where PT= Corrected mortality (%), PO= Observed mortality (%) and PC = Control mortality (%). The number of damaged and undamaged grains was recorded in each treatment and weight loss due to insects was calculated using the formula of "Count and Weigh Method" (Adams and Schulten, 1978): where U = weight of undamaged grains, Nu = number of undamaged grains, D = weight of damaged grains and Nd = number of damaged grains. The F1 progeny population was assessed by keeping each sample in the laboratory until the emergence of new adults. Percentage reduction in adult emergence or inhibition rate (% IR) was calculated as follows: where Cn is the number of newly emerged adults in the untreated (control) jar and Tn is the number of insects in the treated jar. Statistical analysis: Data were analyzed using the SAS program version 9.2. Average means of parameters such as number of eggs laid, number of eggs hatched, dead insects, seed damage rates and progeny population were submitted to analysis of variance (ANOVA; Proc GLM; SAS Institute Inc. 2010) to compare the significance of various treatments. Means separation was performed using Student Newmann and Keuls test (SAS Institute Inc. 2010).For mortality tests, original data were corrected by Abbott's (1925) formula. Then mortality data were analyzed by probit analysis (Proc PROBIT, SAS 9.2, SAS Institute Inc. 2010) to determine LC50. RESULTS Oviposition and adult emergence: The effects of dried ground leaves on oviposition and F1 production are given in tables 1 for bambara groundnuts and table 2 for kersting's ones. The effects of powders were evaluated by comparing the total number of eggs laid, egg hatching percentage and inhibition rates in the treated and control jars. Results showed significant effect of leaf powders on the oviposition of the beetles (p < 0.05; Tables 1 and 2).The lowest number of eggs (12) was laid in samples treated with C. ambrosioides at 7.5% (Table 1).The highest oviposition inhibition rate (91.65%) was recorded in samples treated with C. ambrosioides at 7.5% (Table 2). Higher doses of vegetable powders severely reduced emergence, hatching and oviposition. Insect mortality and LC50: Insect mortality at 2, 4 and 6 days after treatment, was evaluated at three different rates 2.5%, 5% and 7.5% (Figure 1). Mortality rates increased proportionally to the increase of the dose of powders and to with the duration of exposure time. Mortality values of C. maculatus for all doses of plant powders were significantly greater than the control (p < 0.05). Exposure of adults showed that the different vegetable powders had a significant effect on the mortality of the beetles (p < 0.05; Figure 1). C. ambrosioides powder caused 100% mortality in adults fed on bambara groundnuts at 7.5%, 6 days after treatment (Figure 1b) and 69.64% mortality in adults fed on Kersting's groundnuts for the same duration (Figure 1a). The probit statistics, estimate of LC50 and their 95% fiducial limits for 2, 4 and 6 days after treatment are presented in tables 5 and 6. From this analysis, it was found that C. ambrosioides powder is the most toxic product (Tables 5 and 6). A. cissampeloides powder had the lowest toxic effect on C. maculatus (LC50= 24.10/20g of kersting's groundnuts 2 days after treatment). The C. ambrosioides powder had the highest toxic effect against pulse beetle and lowest LC50 values (3.57/20g of kersting's groundnuts 2 days after treatment) ( Table 6). Toxicity of two plant powders as biopesticides in the management of Callosobruchus maculatus F. on two stored grain legumes. Seed weight loss: After 45 days, low percentages weight losses were observed at 7.5% for both C. ambrosioides and A. cissampeloides powders (Figure 2). Plants powders ensured significant protection of seeds against C. maculatus. The lowest percentage weigh loss (0.84±0.38%) was obtained with C. ambrosioides at 7.5% ( Figure 2). Higher doses of vegetable powders generated high reduction of seed weight loss. DISCUSSION In this study, leaf powders of C. ambrosioides and A. cissampeloides, applied at different rates caused high mortality of C. maculatus compared to untreated controls but the C. ambrosioides leaf powder showed the best seed protection. Although the mode of action of C. ambrosioides powder is not clearly understood, its effectiveness was reported by Tapondjou et al. (2002) against C. maculatus. Moreover, Schoohoven (1978) demonstrated that insect death caused by C. ambrosioides leaves powder is due to anoxia or interference in normal respiration resulting in suffocation. This powder could also act as antifeedant or can modify the storage micro-environment thereby discouraging insect penetration and feeding (Obeng-ofori, 1995). In this study experiments, the C. ambrosioides powder was more effective (100% at 7.5%) than A. cissampeloides (26.78% at 7.5%) 6 days after treatment. Oviposition reduction reached 75% after applying each powder at the dose of 7.5% either in kersting's groundnut or in Bambara groundnut. The highest reduction rate (91.65%) was observed with C. ambrosiodes powder on kersting's groundnut. Malik and Mujtaba Naqvi (1984) reported that the powder of dried leaves of C. ambrosiodes had antifeedant effect on Rhyzopertha dominica, and that could be the case with C. maculatus. Similarly, Delobel and Malonga (1987) found that the dose from the dried powder of C. ambrosioides' leaves led to 90% mortality of Caryedon serratus adults 13 days after treatment. Moreover, insecticidal activity of botanical extracts of C. ambrosioides was reported on a big range of insect pests, especially those attacking stored products (Leach et Johnson, 1925;Hartzell and Wilcoxon, 1941;Su, 1991). According to Credland (1992), ovicidal effect of plant powders on bruchid is mainly through asphyxia because the powders obstruct the respiratory tract and prevent the normal exchange of gas between the chorion and the external environment. The effectiveness of these botanical insecticides could be due to the nature of their active components. As for P. guineene, for instance, it contains the piperine, the chavicine and the alkaloids (Lale, 1995). C. ambrosioides contains the ascaridiol known to have insecticidal activities on bruchids (Malloy, 1923;Pollack et al., 1990). CONCLUSION The presence of potential reduction of oviposition and insecticidal effects has been shown by the plant extracts in this study. Accurate identification and isolation of bioactive ingredients of these plant extracts should be explored as key issue for further study. In addition, the synergic effect from the mixture of these plant extracts should be tested for their effective use against field and storage insect pests.

v3-fos

2015-09-18T23:22:04.000Z

2015-04-21T00:00:00.000Z

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Incidence, Antimicrobial Susceptibility, and Toxin Genes Possession Screening of Staphylococcus aureus in Retail Chicken Livers and Gizzards Few recent outbreaks in Europe and the US involving Campylobacter and Salmonella were linked to the consumption of chicken livers. Studies investigating Staphylococcus aureus in chicken livers and gizzards are very limited. The objectives of this study were to determine the prevalence, antimicrobial resistance, and virulence of S. aureus and MRSA (Methicillin-Resistant Staphylococcus aureus) in retail chicken livers and gizzards in Tulsa, Oklahoma. In this study, 156 chicken livers and 39 chicken gizzards samples of two brands were collected. While one of the brands showed very low prevalence of 1% (1/100) for S. aureus in chicken livers and gizzards, the second brand showed prevalence of 37% (31/95). No MRSA was detected since none harbored the mecA or mecC gene. Eighty seven S. aureus isolates from livers and 28 from gizzards were screened for antimicrobial resistance to 16 antimicrobials and the possession of 18 toxin genes. Resistance to most of the antimicrobials screened including cefoxitin and oxacillin was higher in the chicken gizzards isolates. While the prevalence of enterotoxin genes seg and sei was higher in the gizzards isolates, the prevalence of hemolysin genes hla, hlb, and hld was higher in the livers ones. The lucocidin genes lukE-lukD was equally prevalent in chicken livers and gizzards isolates. Using spa typing, a subset of the recovered isolates showed that they are not known to be livestock associated and, hence, may be of a human origin. In conclusion, this study stresses the importance of thorough cooking of chicken livers and gizzards since it might contain multidrug resistant enterotoxigenic S. aureus. To our knowledge this is the first study to specifically investigate the prevalence of S. aureus in chicken livers and gizzards in the US. producing strains of S. aureus specially that most of these enterotoxins are heat stable. The objectives of this study were to determine the prevalence of S. aureus and MRSA in retail chicken livers and gizzards collected in Tulsa, Oklahoma and to characterize the recovered isolates for their antimicrobial susceptibility and possession of toxin genes. To our knowledge this study is the first to specifically investigate the prevalence of S. aureus in chicken livers and gizzards in the US. Isolation of Staphylococcus aureus from Retail Chicken Livers and Gizzards Chicken livers and gizzards samples were collected from several different grocery stores in the Tulsa, Oklahoma area for a period of six months starting January of 2010. A total of 195 chilled retail chicken liver and chicken gizzard samples were used in this study (156 chicken livers and 39 chicken Gizzards) ( Table 1). Meat samples were purchased from nine grocery stores that belong to six different franchises chains at variable locations in the city. The chicken livers and gizzards belonged to two major brands, which are designated brand A and brand B (Table 1). Samples were selected to be as variable as possible with different expiration and production dates. Chicken livers and gizzards samples were added to 10 mL of buffered peptone water (BPW) (BPW; EMD, Gibbstown, NJ, USA) in sterile plastic bags (VWR Scientific, Radnor, PA, USA) and massaged by hand for approximately 5 min. Ten milliliters was then transferred from the bag and added to 10 mL of enrichment broth of 2× Trypticase Soy Broth with 10% sodium chloride and 1% sodium pyruvate, then incubated at 37 °C for 24 h. A loopful was then streaked to Baird Parker (BP) selective media plates and incubated at 37 °C for 48 h [33]. Four suspected S. aureus colonies (those that have black colonies surrounded by 2 to 5 mm clear zones) were selected and streaked to Trypticase Soy Agar (TSA) plates and subcultured for confirmation on MSA (Mannitol Salt Agar) plates. Pure prospective S. aureus cultures were kept at −80 °C until PCR confirmation. DNA Extraction DNA was extracted from the prospective S. aureus strains using the single cell lysing buffer (SCLB) method [38]. One day old colonies were picked and suspended in 40 μL of single cell lysing buffer (SCLB) solution (1.0 mL of TE buffer (10 mM Tris-HCL and 1 mM EDTA) and 10 μl of 5 mg/mL proteinase K) in a 0.2 mL microtube. In a thermocycler, bacterial cells were lysed by initial incubation at 80 °C for 10 min, followed by 55 °C for 10 min, and then 95 °C for 10 min [38]. DNA extracted by the above mentioned method was stored at −20 °C until used as a DNA template for PCR. PCR Identification A multiplex PCR reaction was used to identify the isolated suspected S. aureus by using specific primers for S. aureus and MRSA to amplify a 108 bp and a 312 bp fragments respectively (Table 4). Twenty microliters PCR reactions, which included 10 µL of Qiagen Master Mix (Qiagen Inc., Valencia, CA, USA), 4 µL of sterile water (Qiagen), 1 µL of each forward and reverse primer (IDT, Coralville, IA, USA), and 2 µL of DNA template, were performed. The PCR protocol was as follows: initial denaturing at 95 °C for 5 min (followed by 35 cycles of denaturing at 95 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 1 min) and ending with extension at 72 °C for 10 min. PCR amplicons were subjected to agarose gel electrophoresis and DNA bands were visualized and recorded using a gel documentation system. Isolates showing resistance to cefoxitin and/oxacillin were subjected to PCR confirmation using a second set of MRSA primers that amplify a 533 bp mecA fragment and two other variant MRSA mecA primer sets (also known as mecC) that amplify 356 bp and 1800 bp fragments to confirm the MRSA phenotype (Table 1). Antimicrobial Susceptibility Testing A total of 115 S. aureus recovered isolates (87 chicken liver isolates and 28 chicken gizzard isolates) were subjected to antimicrobial resistance profiling against sixteen different antimicrobials that belong to ten different antibiotic classes ( Table 2). Isolates were grown on Mueller-Hinton (MH) agar (Difco) and incubated for 48 h at 37 °C . Cultures were then added to Mueller-Hinton broth (Difco), adjusted to turbidity equal to a 0.5 McFarland standard, and inoculated onto 6-inch MH agar plates supplemented with the appropriate antimicrobial at different concentrations (Table 2) including the breakpoint established for each antimicrobial according to the Clinical and Laboratory Standards Institute (CLSI) when available [39]. Plates were then incubated at 37 °C for 48 h and results were read for growth or no growth and denoted as resistant or susceptible, respectively according to the breakpoints for each of the tested antimicrobials ( Table 2). Detection of Toxin Genes A total of 115 Staphylococcus aureus isolates (87 chicken liver isolates and 28 chicken gizzard isolates) were screened for eighteen different toxin genes that belong to six different toxin gene groups (Table 3). Multiplex PCR was used to detect 18 different toxin genes of S. aureus isolates that include enterotoxins, toxic shock syndrome toxin 1, exfoliative toxins, leucocidins, Panton-Valentine leucocidin (PVL), and hemolysins (Table 3). Three multiplex reactions (A, B, and C), each of which included six toxin genes, were performed ( Table 3). The multiplex PCR targeting the toxin genes were performed in a 20 µL reaction solution that contained 10 µL of Green Master Mix (Promega), 2µL of sterile water, 2 µL of the DNA template and 0.5 µL of each of the toxin gene primers. The PCR protocol included an initial denaturation at 95 °C for 5 min, followed by 30 cycles of denaturation (94 °C for 1 min), annealing (57 °C for 1 min), and extension (72 °C for 1 min), ending with an extension at 72 °C for 7 min. PCR amplicons were subjected to agarose gel electrophoresis and DNA bands were visualized and recorded using a gel documentation system. The expected amplicon band sizes of S. aureus toxin genes are shown in Table 3. Several representative amplicons of each positive toxin were sequenced in house using the same amplifying primers to confirm PCR accuracy. Table 3. Multiplex PCR primers, reaction sets, references, and toxin groups for the screened toxin genes. Molecular Typing Using spa Genotyping A subset of the recovered Staphylococcus aureus isolates were subjected to molecular typing using spa typing. Molecular typing using spa was done according to published primers and protocols [43] and spa types were assigned using the BioNumerics Software (Applied Math, Austin, TX, USA) through the Ridom Spa Server. Prevalence of Staphylococcus aureus and MRSA in Chicken Livers and Gizzards A total of 195 chilled retail chicken liver and chicken gizzard samples were purchased from several Tulsa area grocery stores starting January 2010 for a period of 6 months. The number of chicken liver samples was 156 and the number of chicken gizzard samples was 39 ( Table 1). The chicken livers and gizzards collected in this study belonged to two major brands, which were designated brand A and brand B ( Table 4). As shown in Table 4, the overall prevalence of S. aureus in chicken livers and gizzards including the two brands together was 36/195 (18.5%). While 27/156 (17.3%) of chicken livers were contaminated with S. aureus, 9/39 (23.1%) of chicken gizzards were positive for the bacterium. The prevalence of S. aureus in brand A chicken livers was 26/71 (36.6%) while it was 9/24 (37.5%) in chicken gizzards of the same brand (Table 1). Only one out of 85 chicken liver samples (1.2%) of brand B was positive for S. aureus and none of the chicken gizzards of this brand was positive for S. aureus. No isolates of chicken livers and gizzards were positive for MRSA since none of them carried mecA or mecC genes. Even though the overall prevalence of S. aureus in chicken livers and gizzards was 36/195 (18.5%) in our study, the 36.6% and the 37.5% prevalence in brand A chicken livers and gizzards respectively is alarming (Table 4). While there were no available studies in the literature that specifically determined the prevalence of S. aureus in chicken livers and gizzards, a study in Turkey reported contamination in 9/30 (30%) of chicken giblets as a part of a larger study on chicken meat [10]. A second study in Japan reported a higher prevalence of S. aureus in chicken livers (63.8%) and chicken gizzards (58.1%) after enrichment while it was 47.9% and 22.6% respectively before enrichment [9]. The higher prevalence in the Japanese study might be due to differences between the US and the Japanese retail poultry markets. It can also be due to the methods used for identification since the Japanese study used only biochemical tests for identification of the S. aureus strains while molecular identification was used in our study. S. aureus was isolated from 56% of ground turkey collected from Maryland, USA [32] and was found in 25% of retail chicken in Detroit, Michigan where 3.9% were MRSA [31]. In another study in Iowa, 17.8% of retail chicken was contaminated with S. aureus [30], while in North Dakota a higher prevalence of S. aureus (67.6%) in retail chicken was reported [26]. The big difference between the overall prevalence of S. aureus in chicken livers and gizzards in brand A in our study (36.8%) and only 1% in brand B (Table 4) might be due to the difference in food safety and microbiology quality control handling protocols at the two production companies. While not conclusive, this data might suggest that chicken livers and gizzards contamination with S. aureus most probably occurs during handling at the slaughter house or at the retail packaging step. Antimicrobial Susceptibility of the Recovered Isolates A total of 115 Staphylococcus aureus isolates (87 chicken liver isolates and 28 chicken gizzard isolates) were subjected to antimicrobial resistance profiling against sixteen different antimicrobials that belong to ten different antibiotic classes (Tables 2 and 5). As shown in Table 5, the percentage of resistance of the 115 S. aureus isolates from chicken livers and gizzards to the sixteen tested antimicrobials were as follow: ampicillin (88.9%), tetracycline (71.3%), doxycycline (63.5%), penicillin (60.9%), erythromycin (45.2%), azithromycin (40,9%), vancomycin (39.1%), oxacillin with 2% NaCl (32.2%), ciprofloxacin (29.6%), trimethoprim/sulfamethazole (24.3%), rifampin (23.5%), cefoxitin with 2% NaCl (19.1%), clindamycin (12.2%), kanamycin (12.2%), chloramphenicol (10.4%), and gentamicin (10.4%). As shown in Table 5, the percentage of resistance to the sixteen tested antimicrobials varied between chicken livers and chicken gizzards isolates. The percentage of resistance found in the chicken gizzards was higher than chicken livers isolates for the following 10 antimicrobials: azithromycin, ciprofloxacin, oxacillin, cefoxitin, tetracycline, vancomycin, doxycycline, penicillin, kanamycin, and erythromycin (Table 5). On the other hand, for gentamycin, ampicillin, trimethoprim/sulfamrthoxazole, clindamycin, rifampin, chloramphenicol the chicken livers isolates showed a higher resistance (Table 5). This variability in antimicrobial resistance between isolates from chicken livers and gizzards might be attributed to the concentration of different antimicrobials in the liver and/or the fact that chicken gizzards often have more fats that would make some highly lipid soluble antimicrobials like azithromycin get to higher concentrations in the gizzards. Overall 35/115 (30%) of the screened isolates from chicken livers and gizzards were multidrug resistant to more than seven antimicrobials (data not shown) which is worrisome. Resistance to vancomycin was relatively high in isolates from chicken livers and gizzards in our study (Table 5).Vancomycin Resistant Enterococcus faecium (VRE) was previously reported in swine in Michigan, USA and was thought to be widespread despite the historical absence of the use of agricultural glycopeptides like avoparcin. Screening our phenotypically vancomycin strains for the presence of the vanA gene is currently underway as a part of a larger study focusing on vancomycin resistant S. aureus strains isolated from various US retail meats. Chicken livers and gizzards isolates in our study were highly resistant to ampicillin, tetracycline, doxycycline, penicillin, and erythromycin ( Table 5). The literature is lacking data about antimicrobial resistance of S. aureus strains isolated from chicken livers and gizzards. One study in Turkey [10] reported that S. aureus isolates from chicken giblets were resistant to penicillin G (22.2%) and erythromycin (33.3%). S. aureus recovered strains in our study that showed resistance to cefoxitin and/or oxacillin (highly prevalent in chicken gizzards as shown in Table 5) were subjected to additional PCR protocols to check for the presence of a mecA homologue ( Table 1). None of these isolates showed the presence of mecA gene or it homologues (mecC). Phenotypic MRSA isolates that do not harbor the mecA gene were reported [32,44]. This might be due to over production of Beta-lactamase enzymes or the presence of a variant mecA gene that does not amplify with the known PCR primers. The recent advancement in whole genome sequencing through next generation sequencing can help identifying such homologues. The high number of multidrug resistant S. aureus detected in our study is alarming. It raises concerns about inappropriate practices including the use of antimicrobials as growth promotors in food animal production and the frequent use of antimicrobials in poultry husbandry. Genes coding for antimicrobial resistance can move through horizontal gene transfer to clinical pathogenic strains and contribute to the creation of superbugs. Death in hospitals are often attributed to sepsis resulting from infections caused by multidrug resistant pathogens like MRSA, Pseudomonas aeruginosa or Candida albicans rather than the original cause of the hospitalization. Toxin Genes Possession Screening of the Recovered Isolates A total of 115 Staphylococcus aureus isolates (87 chicken liver isolates and 28 chicken gizzard isolates) were screened for eighteen different toxin genes that belong to six different toxin gene groups (Tables 3 and 6). As shown in Table 6, the prevalence of toxin genes in the 115 S. aureus isolates from chicken livers and gizzards to the eighteen tested toxin genes were as follow: hla (94.5%), hld (94.5%), hlb (48.7%), sei (42.6%), lukE-lukD (36.5%), seg (29.6%), seh (4.3%), sed (0.9%), sea (0%), seb-sec (0%), sec (0%), see (0%), sej (0%), tst (0%), eta (0%), etb (0%), lukM (0%), and lukS-lukF (0%). S. aureus hemolysin genes were found at a higher percentage in the chicken livers and gizzards than other groups of toxin genes screened. Also no isolates harbored enterotoxin genes sed, sea, sebsec, sec, see, or sej, the toxic shock syndrome toxin 1 gene tst, the exfoliative toxin genes eta, etb, or the Leucocidin gene lukM ( Table 6). The prevalence of enterotoxin genes seg (71.43%) and sei (92.9%) in chicken gizzards was higher than in chicken livers, where seg was 16.1% and sei was 26.4%. One isolate from chicken livers was positive for the entoretoxin gene sed (1.2%) and 23/87 of chicken livers isolates were positive for the entoretoxin gene sei (26.4%). The prevalence of hemolysin genes hla (97.5%), hld (97.5%) and hlb (64.4%) in chicken livers was higher than in chicken gizzards when it was 85.7%, 85.7% and 0% respectively. The Hemolysin gene hlb was present only in the chicken livers. Both chicken livers and gizzards isolates had similar prevalence for lukE-lukD. The literature is lacking data related to the prevalence of toxin genes in S. aureus isolated from chicken livers and gizzards. Even recent studies discussing S. aureus in US retail poultry in general lacks such toxin genes prevalence data. A study in Japan reported that 25.3% of their chicken liver isolates were enterotoxigenic while 36.4% of their chicken gizzards produced enterotoxins [9]. Even though they have not used PCR to detect enterotoxin genes in the Japanese study, their chicken gizzards strains showed a higher prevalence of enterotoxins that their chicken livers ones which is in agreement with our findings. The higher prevalence of hemolysin genes in chicken livers isolates than in the chicken gizzards ones might be due to the availability of blood in the liver which might select for S. aureus strains with blood lysing abilities. Chicken livers and gizzards should be cooked thoroughly since enterotoxins of S. aureus are known for their heat tolerance. So even if the cooking temperature was high enough to kill the pathogen, enterotoxins produced on the chicken livers or gizzards could tolerate such temperature increasing the risk of food poisoning. Genotyping Using spa Typing A subset of Staphylococcus aureus recovered strains (6 from chicken livers, and 5 from chicken gizzards) were subjected to molecular typing by spa typing (Figure 1). As it is shown in Figure 1, spa types were grouped into two major clusters with the majority of isolates in each cluster belonging to one source. As it is also shown in figure 1, the tested isolates showed higher diversity in regards to their spa types since 7 different spa types were detected among a subset of 11 isolates. The detected spa types (t1081, t064, t002, and t091) were not known to be livestock associated and hence, maybe of a human origin [45]. This is in agreement with what we discussed earlier in the introduction section about that the origin of S. aureus strains detected in US retail meats is mostly of a human origin rather than livestock associated as it is the case in Europe. Conclusions The prevalence of S. aureus in retail chicken livers and gizzards tested in this study varied between the two brands tested. While one of the brands showed very low prevalence of S. aureus, the second Chicken Liver brand showed prevalence close to 37%. The percentage of resistance to most of the antimicrobials screened was generally higher in isolates recovered from chicken gizzards. While no isolate harbored the mecA or mecC gene, a higher percentage of the chicken gizzards isolates were resistant to cefoxitin and/or oxacillin making them phenotypically similar to MRSA. A high percentage of S. aureus recovered strains particularly from chicken gizzards harbored enterotoxins seg and sei. The lucocidin genes lukE-lukD was equally prevalent in chicken livers and gizzards isolates. The hemolysin hlb gene was only prevalent in the chicken livers strains while hla and hld were prevalent in chicken livers and gizzards strains. Using spa typing, a subset of the recovered isolates showed that they are not known to be livestock associated and hence, maybe of a human origin. Data obtained from this study stress the importance of thorough cooking of chicken livers and gizzards since it might contain multidrug resistant enterotoxigenic S. aureus.

v3-fos

2018-04-03T03:42:30.271Z

2015-06-27T00:00:00.000Z

5600779

s2

Raw Milk Consumption There continues to be considerable public debate on the possible benefits regarding the growing popularity of the consumption of raw milk. However, there are significant concerns by regulatory, or public health, organizations like the Food and Drug Administration and the Centers for Disease Control and Prevention because of risk of contracting milkborne illnesses if the raw milk is contaminated with human pathogens. This review describes why pasteurization of milk was introduced more than 100 years ago, how pasteurization helped to reduce the incidence of illnesses associated with raw milk consumption, and the prevalence of pathogens in raw milk. In some studies, up to a third of all raw milk samples contained pathogens, even when sourced from clinically healthy animals or from milk that appeared to be of good quality. This review critically evaluates some of the popularly suggested benefits of raw milk. Claims related to improved nutrition, prevention of lactose intolerance, or provision of “good” bacteria from the consumption of raw milk have no scientific basis and are myths. There are some epidemiological data that indicate that children growing up in a farming environment are associated with a decreased risk of allergy and asthma; a variety of environmental factors may be involved and there is no direct evidence that raw milk consumption is involved in any “protective” effect. There continues to be considerable public debate on the possible benefits regarding the growing popularity of the consumption of raw milk. However, there are significant concerns by regulatory, or public health, organizations like the Food and Drug Administration and the Centers for Disease Control and Prevention because of risk of contracting milkborne illnesses if the raw milk is contaminated with human pathogens. This review describes why pasteurization of milk was introduced more than 100 years ago, how pasteurization helped to reduce the incidence of illnesses associated with raw milk consumption, and the prevalence of pathogens in raw milk. In some studies, up to a third of all raw milk samples contained pathogens, even when sourced from clinically healthy animals or from milk that appeared to be of good quality. This review critically evaluates some of the popularly suggested benefits of raw milk. Claims related to improved nutrition, prevention of lactose intolerance, or provision of ''good'' bacteria from the consumption of raw milk have no scientific basis and are myths. There are some epidemiological data that indicate that children growing up in a farming environment are associated with a decreased risk of allergy and asthma; a variety of environmental factors may be involved and there is no direct evidence that raw milk consumption is involved in any ''protective'' effect. Nutr Today. 2015;50(4):189Y193 BACKGROUND In 1908, Chicago became the first US city to introduce cow's milk pasteurization into municipal law (except for cows that were certified tuberculosis-free). However, it took another 8 years before it was fully adopted in Chicago owing to political wrangling and a debate over ''pure milk'' (raw milk) versus ''purified milk'' (pasteurized milk). 1 Around that time, public health officials became greatly worried about the transmission of bovine tuberculosis from cow's milk to humans. By 1900, it was estimated that as many as 10% of all tuberculosis cases in humans were caused by infection via milk consumption, and in 1910, a tuberculosis epidemic spread through Illinois, infecting over 300 000 cattle. 1 Certification of herds as tuberculosisfree became very difficult to manage/administer, and pasteurization became increasingly popular because of its ability to process large quantities of milk in a cost-effective approach. Tuberculosis was 1 of the major human health concerns of the early part of the last century; for example, between 1912 and 1937, it is estimated that about 65 000 persons in England and Wales died of tuberculosis that originated from bovine sources. 2 In 1924, the US Public Health Service developed a regulation known as the Standard Milk Ordinance for voluntary adoption by state and local agencies; this is now called the Grade ''A'' Pasteurized Milk Ordinance (PMO). 3 Pasteurization is defined as ''the process of heating every particle of milk or milk product, in properly designed and operated equipment, to any 1 of the specified pasteurization time/ temperature combinations.'' 3 These time-temperature combinations are designed to destroy all human pathogens, and the most common pasteurization treatment is rapidly heating milk to not less than 72-C and holding that temperature for at least 15 seconds (Table 1). 3 In some countries, milks are subjected to higher (ultra) heat treatments (eg, 138-C for 2Y4 seconds); if the product is packaged normally, then this milk is called ultrapasteurized; if the process is done aseptically, then the milk can be stored at ambient temperature, this product is called ultra-high temperature (UHT). In the subsequent decades, more states started to use the PMO approach. If we look back to just before World War II, in 1938, it was estimated that milkborne outbreaks constituted 25% of all disease outbreaks (related to food/water) in the United States. Today, with the widespread use of pasteurization and other sanitation procedures outlined in the PMO, milk and fluid milk products account for less than 1% of reported outbreaks caused by food/water consumption. 3 Today, tuberculosis is a forgotten disease in the United States because of the success of eradication programs and the implementation of milk pasteurization. There is an ongoing popular debate about the risks and potential benefits from the consumption of raw milk. A significant number (3.4%) of US consumers were recently reported to consume raw milk. 4 The objective of this review is to discuss what are the scientifically demonstrated microbiological (health) risks and to determine if there are any proven health/nutritional benefits to the consumption of raw milk. RISK: PRESENCE OF HUMAN PATHOGENS Surveys from various countries have monitored the presence of different types of pathogens in raw milk, with prevalence levels as high as 13% for bacteria like Campylobacter jejuni and Listeria monocytogenes ( Table 2). In some studies, almost a third of all milk samples contained at least 1 type of pathogen. 5 Thus, we must assume that raw milk is likely to contain pathogens. The prevalence of pathogens in milk is influenced by numerous factors, including farm size, number of animals on the farm, hygiene, farm management practices, milking facilities, season, and others. 5 Raw milk can be contaminated with pathogens even when sourced from clinically healthy animals. 6 Even milk that appears to be of good quality (ie, low total bacteria count) may contain pathogens. 5,7 There are at least 4 different mechanisms by which raw milk becomes contaminated by pathogens: direct passage from the blood (of the cow) into milk (systemic infection), mastitis (udder infection), fecal contamination (external contamination of milk from the environment during or after milking), or contamination from human skin. Dairy farms are an important reservoir of various foodborne pathogens. 8 The relative importance of the various sources of contamination depends on the farming practices and may be different for each pathogen. 6 Pathogens are not visible to the naked eye, and measurements of their numbers can take several days to complete, so it can be extremely difficult to determine the safety of raw milk before that milk has been consumed. Occasional testing of raw milk does not guarantee that pathogens are absent from the milk supply on days when no testing is done (eg, because of possible contamination during a single milking occasion). Ensuring the safety of raw milk by occasional testing is difficult because of the following 5 : & Difficulties in having sufficient sampling since contamination of milk may be sporadic, and bacterial loads can vary from day to day (ie, sampling and testing every day provides more confidence to a claim of safety). & Bacteria/spores are often associated with the fat phase and are not evenly distributed in milk. & It is possible that the number of organisms (pathogens) present is too low to be detected by the test method but the numbers may be sufficient to cause illness if the effective dose is low (which is the case for several key pathogens; see Table 1). & There might have been very low initial numbers of a pathogen, which were below the limits of the test method at the time of sampling, but the pathogen might grow if milk was stored improperly. & It is impossible to test for every single different type of human pathogen. Raw milk has frequently been identified as the source of foodborne illness outbreaks. US statistics for dairy-associated outbreaks of human disease during the period 1993Y2006 9 There were 121 dairy product outbreaks where the pasteurization status was known; among these, 73 (60%) involved raw milk products and resulted in 1571 reported cases, 202 hospitalizations, and 2 deaths. A total of 55 (75%) outbreaks occurred in the 21 states that permitted the sale of raw milk. States that restricted the sale of raw milk had fewer outbreaks and illnesses. In an updated report covering the 6-year period from 2007 to 2012, the average number of outbreaks associated with nonpasteurized milk was 4-fold higher during this 6-year period (average 13.5 outbreaks/year) than that reported in the previous review of outbreaks during 1993Y2006. 10 Even in states where raw milk sales are illegal, outbreaks due to the consumption of raw milk have been tracked; for example, between 1998 and 2009, in Wisconsin, there were 6 outbreaks resulting in 261 reported cases and 27 hospitalizations. 11 However, the number of illnesses investigated as part of well-documented outbreaks likely only represents a small proportion (tip of the iceberg) of the actual number of illnesses associated with raw milk consumption. For example, analysis of routine surveillance data in Minnesota during 2001Y2010 revealed that 3.7% of patients with sporadic, domestically acquired enteric infections had reported raw milk consumption during their exposure period. 12 Children were disproportionately affected, and 76% of those younger than 5 years were served raw milk from their own or a relative's farm. 12 During the study period, the number of patients with sporadic laboratory-confirmed infections who reported raw milk consumption was 25 times greater than the number of raw milkYassociated outbreak cases among Minnesota residents. Furthermore, they estimated that up to 20 500 Minnesotans, or 17% of raw milk consumers, may have become ill with enteric pathogens during the study period after they consumed raw milk. 12 SUGGESTED HEALTH BENEFITS A number of different claims have been made about the possible health benefits that could hypothetically be derived from the consumption of raw milk. Recent scientific reviews by various international groups have concluded that there was no reliable scientific evidence to support any of these suggested health benefits. 13Y15 Nutritional During pasteurization, there is no significant change in the nutritional quality of milk. 16 Pasteurization does not cause any change in protein quality; minor levels (G7%) of denaturation of whey proteins have been reported due to pasteurization, but protein denaturation has no impact on protein nutritional quality. Pasteurization does not cause any change in the concentrations of minerals; minerals are very heat stable. Pasteurization may cause very minor losses (G10%) of vitamin C, folate (vitamin B 9 ), vitamin B 12 , vitamin B 6 , and thiamine (vitamin B 1 ). Of these vitamins, milk is an excellent source of only vitamin B 12 ; milk has only low concentrations of most of the vitamins listed previously, which might show some minor losses on pasteurization. Pasteurization does not change the concentration of riboflavin (B 2 ) (which is very heat stable) or fat-soluble vitamins like vitamin A or E. 15 Other factors like type of packaging material, light exposure, and storage time/temperature have much larger impacts on vitamin losses in milk. Feed (like pasture grazing) can greatly influence milk composition, and sometimes proponents of raw milk confuse feed-related changes in milk composition with those caused directly by pasteurization. Other milkprocessing approaches, like ultra-pasteurization and ultrahigh temperature, have only a minor impact on the nutritional quality of milk. 13 Allergy Food allergy is an abnormal immunological response due to sensitization to a particular food (usually a protein). Cow's milk proteins can trigger an immunoglobulin EYmediated reaction in patients called cow's milk protein allergy (CMA). Young infants usually outgrow this allergy within the first year of life. Young infants may be more susceptible to CMA owing to their milder digestive systems (weaker pepsin/enzyme activity, higher stomach pH), which exposes them to more allergic responses from ''intact'' proteins or larger peptide sequences. 17 In children with CMA, neither raw milk, unhomogenized and pasteurized milk, or homogenized and pasteurized milk was tolerated by CMA patients. 18 It can also be mentioned that epidemiological data indicate that the dietary intake of pasteurized milk is not correlated with any increased risk of the development of respiratory allergies or atopic dermatitis. 19 Several epidemiological studies have shown that growing up in a farming environment is associated with a decreased risk of allergy and asthma. 20Y22 A possible factor that has been hypothesized as being involved in this effect is the early ingestion of raw cow's milk. One issue is that at the farm level, milk is either consumed raw or boiled (heated in a pot or container until the milk boils); boiling is a much more severe heat treatment than the mild pasteurization process used commercially. It is not clear why there would be much difference in the allergenicity of cow's milk proteins from raw milk or from a mild heating process like pasteurization, which causes only minimal modification/ denaturation of (the allergic) milk proteins. 17 Loss et al 22 hypothesized that any possible protective effect of raw or mildly heated (eg, pasteurized) milk on asthma might be associated with the whey protein fraction of milk, which would be impaired when farm milk was severely boiled (heating to 985-C). The intestinal microbiome is getting significant attention for its potential impact on human health. This complex Volume 50, Number 4, July/August 2015 microflora is initially developed during infancy, and many factors, including the type of milk consumed (breast, raw, pasteurized), could influence this system, which in turn could impact the sensitivity of an infant to the development of allergy. 23 It is important to note that most health organizations recommend that babies be exclusively breastfed for about the first 6 months of life. It is known that farm kids also come into contact with a wider range of bacteria/allergens compared with children who live in modern cities. It is possible that early exposure to these farm allergens could help some infants develop a more robust immune system. Microorganisms have been identified from the farming environment (eg, barns and milk houses) that have been reported to have an allergyprotective effect. 24 It should be noted that there would be considerable ethical concerns with intentionally exposing infants to raw milk in an attempt to try to boost their immune function because of the fact that human pathogens are routinely found in raw milk. 20 Recently, using a murine model of gastrointestinal allergy, Hodgkinson et al 25 demonstrated that drinking milks exposed to different treatments (raw, gamma-sterilized or heat treated) changed the allergic responses to a nonrelated dietary antigen. However, they found that the group fed raw milk had a greater allergic response than did those fed heated milk. Thus, at present, there is no direct evidence of any beneficial impact of raw milk consumption for CMA, but this topic needs further study. Lactose Intolerance (Raw Milk Enzymes) All types of milks (including human or breast milk) contain the sugar lactose, and when we consume milk, the lactase enzyme (A-galactosidase) hydrolyzes it into glucose and galactose, which are then absorbed by the body. Many individuals lose the ability to digest lactose as they age, and they can develop a condition known as lactose intolerance, in which people have digestive symptomsVsuch as bloating, diarrhea, and gasVafter eating or drinking milk or milk products. One of the claims made about raw milk is that it alleviates lactose intolerance. A recent randomized controlled study found that raw milk failed to reduce lactose malabsorption or lactose intolerance symptoms compared with pasteurized milk among adults positive for lactose malabsorption. 26 Because there is no A-galactosidase enzyme present in raw milk, there is no obvious reason why raw milk could assist with lactose intolerance. Yogurts, which contain high levels of bacteria that have this A-galactosidase enzyme, are tolerated better by individuals with lactose intolerance. Although raw milk contains low levels of some proteases and lipases, no physiological role in human digestion has been demonstrated for these enzymes. Both the indigenous milk proteinase (plasmin) and lipase (lipoprotein lipase) are relatively heat stable, so there would be little loss of activity in pasteurized milk relative to raw milk. Anyway, raw milk enzymes are likely degraded/hydrolyzed in the human digestion system (due to the stomach acid, pepsin, etc). Beneficial Microflora and Antibacterial Systems Some media reports claim that raw milk is healthy because of the presence of ''good bacteria.'' Probiotics are defined as ''live microorganisms that, when administered in adequate amounts, confer a health benefit on the host.'' 27 Some lactic acid bacteria are considered probiotics. However, key probiotic bacteria like Bifidobacteria or Lactobacillus acidophilus should be present only at quite low levels in raw bovine milk, as they do not compete well with the more common types of lactic acid bacteria. Instead Bifidobacteria are found at high numbers in the gastrointestinal tract of cows and humans, and the presence of Bifidobacteria in raw milk has been used as possible indicator of fecal contamination. 28 When probiotic cultures are used in commercial products like yogurt, it is considered highly desirable that (a) the specific probiotic strain used was originally isolated from a human source (not from animals like cows), and (b) the specific strain conveys proven health benefits when used at high levels (ie, millions of colony forming units per milliliter). None of these conditions are met with any fecal contamination of raw milk by probiotic bacteria. There are a number of potential antimicrobial systems in milk, including lactoferrin, lactoperoxidase, lysozyme, bovine immunoglobulin, bacteriocins, oligosaccharides, and xanthine oxidase. Lactoperoxidase and lysozyme retain 70% or more of their activity in pasteurized milk, whereas the other components listed above retain all their activity in pasteurized milk. 5 Collectively, these antimicrobial systems are unable to prevent pathogen growth in raw milk. Mastitic milk often contains elevated levels of lactoferrin and immunoglobulins, which would indicate that the milk is infected and these antibacterial systems are elevated to help fight this bacterial infection. At the farm level, prudent steps that can be taken by the farmer to reduce pathogen numbers in their raw milk include minimizing fecal/pathogen contamination and maintaining low storage temperatures to reduce growth of pathogens. In conclusion, raw milk is not inherently safe and carries a significant food poisoning risk with its consumption. 5,6 There is no evidence that raw milk has any inherent health or nutritional benefits those media claims were shown to be myths. Pasteurized milk has an excellent food safety record and remains an important dietary source for many important nutrients (Table 3), especially for children and young adults. 29

v3-fos

2015-09-18T23:22:04.000Z

2015-07-31T00:00:00.000Z

8562369

s2

Caesalpinia decapetala Extracts as Inhibitors of Lipid Oxidation in Beef Patties In this study we investigated the effects of Caesalpinia decapetala (CD) extracts on lipid oxidation in ground beef patties. Plant extracts and butylated hydroxytoluene (BHT) were individually added to patties at both 0.1% and 0.5% (w/w) concentrations. We assessed the antioxidant efficacy of CD by the ferric reducing antioxidant power (FRAP) assay and evaluated their potential as natural antioxidants for meat preservation by thiobarbituric acid reactive substance (TBARS) values, hexanal content, fatty acid composition and color parameters. These were tested periodically during 11 days of refrigerated storage. TBARS levels were significantly lower (p ≤ 0.05) in the samples containing plant extracts or BHT than in the non-treated control. In addition, the beef patties formulated with the selected plant extracts showed significantly (p ≤ 0.05) better color stability than those without antioxidants. These results indicate that edible plant extracts are promising sources of natural antioxidants and can potentially be used as functional preservatives in meat products. Introduction Lipid oxidation, one of the major causes of quality deterioration, is also important because it can negatively affect sensory attributes such as color, texture, odor, and flavor as well as the nutritional quality of the product. Meat mincing, cooking and other processing prior to refrigerated storage disrupt muscle cell membranes facilitating the interaction of unsaturated lipids with pro-oxidant substances such as non-hemeiron, accelerating lipid oxidation leading to rapid quality deterioration and development of rancidity. Initially lipid oxidation in meat products results in a cardboard flavor and progresses with the development of painty, rancid and oxidized flavors [1]. Antioxidants are substances that at low concentrations retard the oxidation of easily oxidizable biomolecules, such as lipids and proteins in meat products, thus improving the shelf life of products by protecting them from deterioration caused by oxidation [2]. Natural extracts have been developed in response the recent demand for natural products and consumers' willingness to pay significant premiums for natural foods. Many plants have been recognized as possessing antioxidant activity, including barks of cinnamon (Cinnamomum iners), buds of clove (Syzygium aromaticum Linn), rhizomes of ginger (Zingiber officinale Rosc.), leaves of green tea (Camellia sinensis) and leaves of thyme (Thymus vulgaris Linn.) [3]. C. decapetala (Roth) Alston is a climbing shrub that belongs to the genus Caesalpinia of the Fabaceae family. C. decapetala (Roth) Alston is widely distributed around the world, but mainly distributed in the southern regions of the Yangtze River in China. The plant is locally known as "Yan wang ci" in Guizhou Province, China. The roots of C. decapetala (Roth) Alston are used in folk medicine to treat bronchitis, prevent colds, and as an antimalarial agent. Previous chemical investigations on C. decapetala (Roth) Alston revealed that the main chemical components were terpenoids and flavonoids [4]. Recently, the chemical constituents of C. decapetala (Roth) Alston have been systematically investigated and the antitumor activities of the compounds have been tested to validate the medicinal use of C. decapetala (Roth) Alston. C. decapetala has been shown to contain antioxidants. The leaves of C. decapetala contain cassane diterpenoid, caesaldecan, spathulenol, 4,5-epoxy-8(14)-caryophyllene, squalene, lupeol, resveratrol, quercetin, astragalin and stigmasterol [5]. Our objective in this study was therefore to evaluate the effectiveness of C. decapetala extract in preventing or reducing lipid oxidation as well as color changes in ground beef patties during storage at a chilled temperature (4 °C). Antioxidant Capacity Assays (AOC) AOC determined by the ferric reducing antioxidant power (FRAP) assay at 24 h and after 11 days are presented in Figure 1. In order to obtain an accurate value for the total antioxidant activity (Table 1), both the hydrophylic and lipophilic antioxidant activity analyses were done on the same samples. and FRAP lipid; assay for each treatment: Control (C), CD1 (0.1%), CD2 (0.5%) and BHT after 11 days of storage. The values represent mean ± standard error; Treatment means that do not share a common letter are different (p < 0.05). The hydrophylic and lipophilic antioxidant activity values were higher in the sample containing C. decapetala leaf extract (0.5%). The hydrophylic antioxidant activity (0.20 ± 0.003 mol Trolox equivalent/mL sample) had a higher value than the lipophilic FRAP value with no significant difference (p ˂ 0.05) from the sample of BHT (0.21 ± 0.01 mol Trolox equivalent/mL sample). The sample with the lowest antioxidant activity as expected was the control. The FRAP value is a measure of the capacity of the antioxidant to reduce Ferric (III) ions to Ferrous (II) ions [6]. In our study the final hydrophylic and lipophilic values of antioxidant activity of the sample containing C. decapetala (0.5%) were higher than those reported by Topuz et al. [7]. They studied the effect of addition of sauces containing olive oil and pomegranate juice into marinated anchovy to retain the initial quality, by preventing undesired chemical and oxidative alterations during storage at 4 °C. The total antioxidant activity value of CD2 (0.39 ± 0.03) was similar to those (0.31 ± 0.05) reported by Bubonja-Sonje et al. [8]. The antioxidant activity of the hydrophylic and lipophilic extracts can be attributed to different antioxidants. The hydrophylic extract contains antioxidants such as phenolic derivatives of benzoic acid (gallic acid) and cinnamic acid or flavonoids [9]. In the lipophilic extract the major contributors to the antioxidant activity are hydrophobic compounds such as carotenoids, tocopherols, polymeric proanthocyanidins and high molecular weight tannins [10]. The extracts showed a higher ability to reduce Fe 3+ . The assay showed higher AOC values in the assay carried out with the lipophilic extract compared to the values for the hydrophylic extract. Effects on Metmyoglobin Formation The effect of C. decapetala and BHT on MetMB percentage in beef patties is presented in Figure 2. The relative MetMb percentage increased with time for the 11 days of refrigerated storage. The samples treated with leaf extract and BHT had a lower (p < 0.05) concentration of MetMb compared to the control, thus demonstrating some ability to inhibit formation of MetMb. After 10 days, the control sample exhibited higher MetMb concentration (73.48 ± 0.20). No significant difference was found between the control and sample CD 1 0.1%. Antioxidant effect was best in samples containing leaf herb extract (66.57% ± 0.3% for C. decapetala at 0.5%). The sample with BHT had a very similar behavior to the CD2 extract, with no significant difference between these samples at the end of the study. Although many factors can influence the color stability of meat and meat products, the susceptibility of myoglobin to autoxidation is a predominant factor. The discoloration of meat from red to brown during storage results from the oxidation of OxyMb to MetMb [11]. The radical species produced during muscle phospholipid oxidation may act to promote OxyMb autoxidation. Conversely, superoxide anion released from oxidized OxyMb can dismutate to hydrogen peroxide and hydroxyl radical, which are potent lipid pro-oxidants [11]. The free radical scavenging effects of phenolic compounds occurring in C. decapetala leaf extract are the most likely reason for the retardation of MetMb formation. In a previous study, Sánchez et al. [12] reported that beef patties treated with rosemary did not exceed 40% of metmyoglobin after day 8 of storage. Significant correlations (95%) were observed between metmyoglobin formation and values from the thiobarbituric acid reactive substance (TBARS) assay. This confirms that both parameters reflect the oxidation rate for the samples during the study period, showing the control as the most oxidized sample. The addition of 0.5% C. decapetala was effective in inhibiting myoglobin oxidation and maintained the redness of the beef patties due to its ability to maintain oxymyoglobin stability, and to reduce the formation of metmyoglobin. Figure 2. Effects of C. decapetala extract added at 0.1% and 0.5% (w/w) and BHT on metmyoglobin changes in beef patties during 11 days of refrigerated storage at 4 °C. Results are given as mean ± standard error. Different letters in the same day (a-c) indicate significant differences between samples. Volatile Compounds The hexanal content increased together with the TBARS values, thereby suggesting lipid oxidation development ( Figure 3). The hexanal content of meat stored at 4 °C increased rapidly over the first four days of storage. The trend observed for hexanal values was as follows (p ˂ 0.05): control ˃ CD1 ˃ CD2 = BHT. The antioxidant herb extract added to beef patties reduced the amounts of volatile compounds formed. After eight days, the control and CD1 sample showed the highest hexanal concentration throughout the storage period. CD2 (0.5%) extract and BHT samples, which also had the lowest TBARS values, formed the least volatiles with 7.16 ± 0.1 and 6.89 ± 0.1 ppm hexanal, respectively. Flavor and aroma compounds found in meat include a broad array of compounds, including hydrocarbons, aldehydes, ketones, alcohols, furans, thiophenes, pyrroles, pyrazines, oxazoles, thiazoles, and sulfurous compounds. Also, flavor and aroma are attributes most easily detected and assessed by consumers as either acceptable or not [13]. Aldehydes are the most prominent volatiles produced during lipid oxidation and have been used to successfully follow lipid oxidation in meat or meat products where they are reported to contribute to the overall off-flavor of oxidized meat. Hexanal is reported to be the most sensitive indicator for lipid oxidation [14]. Hexanal and heptanal are degradation products from the oxidation of long chain polyunsaturated fatty acids n − 6, mainly linoleic acid [15]. Long chain polyunsaturated fatty acids are known to be less stable towards oxidation than monounsaturated fatty acids, and the high hexanal values observed in this study can be attributed to degradation of linoleic acid. Similar observations have been also made by Juntachote et al. [16] in cooked ground pork sausages with various added antioxidants. Sampaio et al. [17] indicated that natural antioxidants including honey, oregano and sage exhibited greater antioxidant efficacy than that shown by BHT, when assessed by hexanal formation. Thiobarbituric Acid Reactive Substance (TBARS) Value The antioxidant effects of C. decapetala leaf extracts and the synthetic antioxidant BHT in ground beef patties (0.1% and 0.5%, w/w) are shown in Figure 4. The extracts showed effective antioxidant activity against lipid oxidation, although the TBARS content of the patties treated with edible plant extract (0.5%) was lower than that of the patties treated with BHT. As expected, the TBARS values of the control sample increased most by 5.6 mg malondialdehyde/kg sample after 11 days, whereas the TBARS values of patties containing 0.1% and 0.5% C. decapetala extract increased by 2.9 and 1.7 mg malondialdehyde/kg sample, respectively, after 11 days-significantly less than the control (p < 0.05). The ethanolic extract of C. decapetala was moderately antioxidant at both 0.1% and 0.5% in beef patties, with significantly lower (p < 0.05) TBARS values than the control, and the concentration 0.5% was more effective as an antioxidant than BHT treatment. We concluded that a 0.5% C. decapetala leaf extract is more capable than BHT of maintaining lipid stability and efficiently delaying lipid oxidation in refrigerated beef patties. Our results are consistent with various other studies, all of which reported that natural antioxidants from culinary herbs and edible plants were effective at controlling lipid oxidation and extending the shelf life of meat products. Fasseas et al. [18] reported that both oregano essential oil (3%) and sage essential oil (3%) significantly reduced oxidation. Mitsumoto et al. [19] reported that adding tea catechins (200 or 400 mg/kg) to minced meat inhibited lipid oxidation in both raw and cooked beef. Similar results to ours were reported by McCarthy et al. [20], where an addition of rosemary extracts (0.2%) to beef patties stored in refrigeration had antioxidant activities similar to BHA/BHT (0.01%/0.1%). Formanek et al. [21] noted that rosemary extracts worked synergistically with vitamin E to inhibit the formation of malondialdehyde (TBARS). Han and Rhee [22] showed that 0.25% (w/w) extracts of rosemary, sappanwood, and red or white peony almost completely inhibited lipid oxidation in raw beef patties. In general, the effectiveness of these natural antioxidants is proportional to the number of -OH groups present on the aromatic rings. If their solubility is compatible with a particular meat system, the fact that they are natural and have antioxidant activity that is as good as or better than the synthetics makes them particularly attractive for meat products. Figure 5 shows the effects of C. decapetala added at two concentrations (0.1% and 0.5%) w/w and BHT (0.1%) on the pH values in raw beef patties during cold storage for 11 days. The control sample had the highest pH value (5.50), and the pH values of the other treatments decreased with storage time. Samples treated with C. decapetala (0.5%) had the lower pH value after storage (5.39). The changes in pH value during storage might be due to acidity produced by bacterial action on the muscle glucose and accumulation of the microbial metabolites due to bacterial spoilage in pork meat patties [22]. Figure 5. Effects of Caesalpinia Decapetala added at 0.1% and 0.5% (w/w) and BHT added at 0.01% (w/w) on the changes in pH values of raw beef patties. Data is presented as mean ± standard deviation. Different letters at the same time (a-d) indicate significant differences between samples. Color Changes The CIE color values in raw ground beef samples with/without spice extracts are shown in Table 2. The color of meat and meat products after slaughter and manufacturing is altered by increased metmyoglobin. The formation of metmyoglobin is associated with the oxidation of oxymyoglobin (light pink color) during storage. L* values showed a small difference for all samples throughout the storage period. Different authors refer to these slight changes in the values of L* in meat through the storage time [23,24]. The a* value (redness) is the most important color parameter in evaluating meat oxidation, as a decrease in redness makes the meat product unacceptable to consumers. In all samples, the redness (a* value) decreased as storage time progressed. At the end of the study period (Day 11), the intensity of each color parameter was lower than the value measured at Day 0 as a result of the oxidation process, leading in this way to a change in color. It is clear that the protective effects of the test extracts against the color loss (a* value decrease) in stored beef patties were not as pronounced as their effects against lipid oxidation. At the end of storage, the a* values of the CD1 and CD2 samples (−1.02 ± 0.33) were significantly lower (p < 0.05) than those of others samples. BHT displayed the highest value of a* at the end of the experiment. Therefore, the natural plant extracts affected meat color, specifically redness, and are therefore potentially useful in prolonging the shelf life of the meat product. Several authors have reported an a* value decrease in different meat and meat products stored under a modified atmosphere [24,25]. The samples had an initial yellowness (b*) value of 3.33 ± 0.82. Significant differences (p < 0.05) were observed in b* values in all samples throughout storage. Results of color changes are expressed as mean (SD). Means with different letters (a-d) in the same day are significantly different at p < 0.05. Plant Material C. decapetala was collected from Perú in the spring of 2012. This was crushed and stored (inside a desiccator) in the dark, at room temperature until use. Sample Extraction The powder (1.5 g) of the dried leaves of C. decapetala was stirred using a magnetic stirrer with 25 mL mixture of ethanol/water (5:5) for 24 h at 4 °C. The extraction was done in triplicate. Then the mixture was centrifuged at 2500 rpm and the separated liquid was collected. A portion of the separated liquid was stored at −80 °C until use to determine the antiradical capacity, and the remaining part was concentrated in a rotary evaporator and lyophilized. The lyophilized extract was stored in a desiccator until use. Preparation of Beef Patties Fresh beef samples were purchased from a local fresh food market and minced three times. The meat was mixed with salt (1.5%). Four formulations were prepared: negative control (without antioxidant), CD1 (C. decapetala 0.1% w/w), CD2 (C. decapetala 0.5%) and BHT (0.01%). The mixing process for each formulation was replicated. The ingredients were mixed manually in a steel bowl for about 1 min to obtain a homogeneous mixture, and then burgers were formed manually. The beef patties were randomly selected and packed in plastic trays which were filled with a gas mixture of 70% O2 + 20% CO2 + 10% N2 before sealing and they were kept refrigerated at 4 °C for 11 days. Antioxidant Capacity Assay (AOC) AOC was determined by the ferric reducing antioxidant power (FRAP) assay. The sample preparation for AOC studies involved extraction of C. decapetala with distilled water or a lipophilic extraction solvent system (acetone/ethanol/distilled water; 5:4:1, v/v/v, according to Linden [26].). These solvent systems were used to extract the hydrophylic and lipophilic antioxidants. The determination of the antioxidant capacity of antioxidants extracted was performed by the FRAP assay (ferric reducing antioxidant power). Based on this, the methods are called FRAPwater, or FRAPlipid assays. Prior to extraction, deep frozen muscle samples were minced by a disintegrator. Muscle homogenate (5 g) was weighed into a glass tube and mixed with 5 mL of extraction solvent for 30 s using an Ultra-Turrax. Two different extractions were performed. The first was made with distilled water to dissolve the hydrophylic antioxidants and the second was carried with an acetone/ethanol/water mixture (5:4:1, v/v/v) to dissolve the lipophilic antioxidants in the muscle. Then the samples were centrifuged at 4 °C for 30 min. Supernatants were filtered using a folded filter and stored in the dark and on ice until immediate analysis. Once the supernatant was obtained, the FRAP assay was performed. Measurements were carried out in triplicate. FRAP Assay The FRAP assay was carried out according to the procedure described by Benzie and Strain [6] with minor modification The determination of reducing capacity was performed with microplates, mixing the FRAP reagent incubated at 37 °C with the samples (in an appropriate dilution to cause the absorbance to fall in the range 0.1-1.0). The FRAP reagent was prepared from sodium acetate buffer (300 mM, pH 3.6), 10 mM TPTZ solution (40 mM HCl as solvent) and 20 mM iron (III) chloride solution in a volume ratio 10:1:1, respectively. The samples were measured in triplicate. The absorbance of the reaction mix was then detected at 593 nm. The results were expressed as milimoles of Trolox equivalents/g of dry plant. Headspace Volatile Analysis Hexanal was measured using a TRACE GC gas chromatograph equipped with a mass spectrometer DSQII (Thermo Fisher Scientific) with automatic injector TRIPLUS with a Head Space module. One gram of homogenized patties was placed in a 10 mL glass vial capped with an aluminum cap with PTFE/silicone septum. The sample was shaken and heated at 60 °C for 30 min in an autosampler heating block before measurement. Vapor phase (1 mL) was injected using a special gas syringe maintained at 65 °C. Hexanal concentrations were determined from peak areas using a standard curve prepared from authentic hexanal. Determination of Secondary Oxidation by TBARS The TBARS method was used to measure the lipid oxidation over the storage period as described by Grau et al. [28].The TBARS reagent was prepared from 15% trichloroacetic acid, 0.375% thiobarbituric acid and 2.1% hydrochloric acid. Briefly, 1 g of sample was weighed and protected with 1 mL aqueous EDTA, and was mixed with 5 mL of thiobarbituric acid reagent using an Ultra-Turrax (IKA, Staufen, Germany) at 32,000 rpm speed for 1 min. It was then filtered with a Whatman filter (0.45 μm) to obtain the part soluble in the solvent. All procedures were carried out in the dark and all samples were kept on ice. Immediately, the filtered samples were immersed in a water bath preheated to 95 °C ± 1 °C for 10 min. Samples were cooled at room temperature for 10 min and the absorbance was measured at λ = 531 nm. TBARS value was calculated from the calibration curve of malondialdehyde (MDA). The results are expressed as mg malondialdehyde (MDA)/kg of meat. pH Measurement The pH of 5 g samples was determined with a pH meter (Mettler-Toledo GLP 21, Schwerzenbach, Switzerland). Color Measurements Color measurements were performed at four points on the surface of the patties using a Minolta Chromameter CR-300. Color measurements were observed at 11 days of chilled storage. The L*, a* and b* values (CIE L*, a*, b* color system) were assessed as a measure of lightness, redness and yellowness, respectively. The instrument was calibrated using a black-and-white glass tile, provided with the instrument. The meat samples were kept inside the plastic dishes in triplicate, and then the instrument was directly placed on the surface of the meat at three different points for each plastic dish. For every point result was a mean of nine measurements. The mean and standard error for each parameter were calculated. Statistical Analysis The data were reported as means ± standard deviation (SD). Statistical analyses were conducted using the Minitab software program. The significance of differences was determined by the one-way analysis of variance (ANOVA) with Duncan's pairwise comparison (p < 0.05). Conclusions Our experiments with raw beef patties indicated that the C. decapetala extract may be promising as a source of natural antioxidants for meat products. The analysis of TBARS, fatty acid degradation, antioxidant activity and concentration of volatile compounds provides a complete assessment of the consequences of lipid oxidation in burger patties. The addition of this extract to the beef patties at 0.5% was the most effective antioxidant. This concentration inhibited formation of TBARS and volatile compounds more effectively than the synthetic antioxidant BHT over the course of 11 days. Using this herb extract as an ingredient in burger patties may be an efficient strategy to enhance the nutritional value and safety of these meat products.

v3-fos

2018-04-03T04:09:01.702Z

2015-10-26T00:00:00.000Z

15612459

s2

Data in support of the proteomic analysis of plasma membrane and tonoplast from the leaves of mangrove plant Avicennia officinalis The data provides information in support of the research article, Proteomics 2014, 14, 2545–2557 [1]. Raw data is available from the ProteomeXchange Consortium via the PRIDE partnerRepository [2] with the dataset identifier PXD000837. Plasma membrane and tonoplast proteins from the leaves of Avicennia officinalis were identified using gel electrophoresis (one and two dimensional) combined with LC–MS analysis. Based on GO annotation, identified proteins were predicted to be involved in various biological processes. a b s t r a c t The data provides information in support of the research article, Proteomics 2014, 14, 2545-2557 [1]. Raw data is available from the ProteomeXchange Consortium via the PRIDE partnerRepository [2] with the dataset identifier PXD000837. Plasma membrane and tonoplast proteins from the leaves of Avicennia officinalis were identified using gel electrophoresis (one and two dimensional) combined with LC-MS analysis. Based on GO annotation, identified proteins were predicted to be involved in various biological processes. & 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Subject area Biology More specific subject area Membrane proteomics and mass spectrometry (MS) Type of data MS data and annotations How data was acquired Mass spectrometry: Data was acquired on the instrument TripleTOF 5600 (SCIEX, Foster City, CA, USA). Data format Raw and processed data Experimental factors Plasma membrane and tonoplast membrane protein fractions were isolated from the leaves of field-growing Avicennia officinalis trees using two-aqueous-phase partitioning and density gradient centrifugation, respectively. Experimental features The plasma membrane and tonoplast membrane proteins were fractionated by 1DE and 2DE. The peptides resulting from in-gel tryptic digestion were fractionated and analyzed using LC-MS/MS. Value of the data Avicennia officinalis is a salt secreting mangrove plant that exhibits salt tolerance to high salinities. To our knowledge, this represents the first dataset on the membrane proteomics of a mangrove plant. A total of 254 plasma membrane and 165 tonoplast proteins were identified. This data would be valuable in understanding the mechanism of salt secretion in mangroves and salt tolerance in plants. partitioning method while the tonoplast fraction was isolated by density gradient centrifugation. The membrane proteins were fractionated by one-and two-dimensional gel electrophoresis. The trypsin digested fragments from the gels were used for LC-MS/MS analysis. MS data was acquired using a TripleTOF 5600 system and the peptide identification was carried out using the ProteinPilot 4.5 software. Membrane preparation Leaves were collected from the Avicennia officinalis trees growing in the equatorial mangrove swamps in Singapore. Plasma membrane and tonoplast fractions were prepared from freshly harvested leaves ( $ 100 g) using two-phase partitioning [3] and density gradient centrifugation [4] methods, respectively. Briefly, to isolate PM, freshly harvested leaves were rinsed with ice-cold distilled water and homogenized in a blender using pre-chilled homogenization medium consisting of 50 mM Tris, 500 mM sucrose, 10% glycerol, 20 mM EGTA, 20 mM EDTA, 5 mM β-glycerophosphate, 1 mM phenantroline, 0.6% PVP, 10 mM ascorbic acid, 1 mM PMSF, protease inhibitor tablets (Roche), 1 mM leupeptin, 5 mM DTT and 1 mM Na-orthovanadate after adjusting to pH 8.0 with MES. The resulting homogenate was filtered through a nylon cloth (100 mm) and centrifuged at 26,000 g for 25 min at 4°C. The resulting supernatant was centrifuged at 84,000 g for 30 min at 4°C to obtain a microsomal pellet. The pellet was resuspended in microsomal buffer consisting of 5 mM phosphate buffer pH 7.8, 330 mM sucrose and 2 mM DTT. The microsomal fraction was further purified using a MATRYWVASLPIGEGSSASSLWGRLQESVSKQSFDTSLYRFNIPNLRVGTLDSLLALSDDLLKANSFIEGVSHKI RRQIEELERVSGVVASSLTVDGVPVDSYLTRFVWDEAKYPTMSPLREIVDGIHVQIAKIEDDLKVRVAEYNNVRS LLNAINRKQAGSLAVRDLSNLVKPQDIVSSEHLTTLLAIVPKYSQKDWLSSYETLTTYVVPRSSKMLHEDNEYVL YTVTLFSRDADNFRTKARERNFQIRDFEYNPETQESHKQELEKLNQDQETLRSSLLQWCYTSYGEVFSSWMHFCA VRVFSESILRYGLPPSFL dextranpolyethylene glycol (PEG) two-phase system consisting of 20% dextran T -500, 6.4% PEG, 5 mM phosphate buffer pH 7.8, 5 mM KCl and 300 mM sucrose. The upper phase was recovered and partitioned with fresh lower phase twice. The final upper phase was diluted at least two-fold with plasma membrane washing buffer consisting of 10 mM Tris, 10 mM boric acid, 300 mM sucrose, 9 mM KCl, 5 mM EDTA, and 5 mM EGTA and was centrifuged at 176,000 g for 30 min at 4°C to obtain a PMenriched pellet. For the isolation of tonoplast, freshly harvested leaves were rinsed with ice-cold distilled water and homogenized in a pre-chilled grinding medium consisting of 0.25 M sorbitol, 5 mM EGTA, 1 mM PMSF, 2.5 mM potassium metabisulfite, 1.5% (w/v) PVP, 50 mM MOPS-KOH pH 7.6, 10 mM β-glycerophosphate, 0.45 mM butylated hydroxytoluene, protease inhibitor tablets (Roche), 5 mM DTT and 1 mM Na-orthovanadate. The tissue homogenate was filtered through 4 layers of gauze. The filtrate was centrifuged at 3600 g for 15 min at 4°C. The resulting supernatant was centrifuged at 150,000 g for 40 min at 4°C. The pellet was re-suspended in a buffer containing 15% sucrose (w/v), 10 mM potassium phosphate pH 7.8, 1 mM EGTA and 2 mM DTT. This suspension was overlaid with a buffer containing 0.25 M sorbitol, 5 mM MOPS-KOH pH 7.3, 1 mM EGTA and 1 mM DTT and centrifuged at 120,000 g for 1 h at 4°C. The tonoplast membranes at the interface between sucrose and sorbitol layers were collected and diluted $ 5-fold with a buffer containing 5 mM MOPS-KOH pH 7.3, 0.25 M sorbitol, 1 mM EGTA and 1 mM DTT. The suspension was centrifuged at 150,000 g for 30 min at 4°C to obtain a tonoplast enriched pellet. All membrane fractions were carbonate washed, following the method described by [5] to remove the soluble proteins and were stored at À 80°C. One-and two-dimensional polyacrylamide gel electrophoresis (1DE and 2DE) For 1DE, samples containing 5-10 mg of purified PM and tonoplast proteins in gel loading buffer (62.5 mM Tris adjusted to pH 6.8 with HCl, 2% (m/v) SDS, 0.1 M DTT, 10% (v/v) glycerol, 0.1% (m/v) bromophenol blue) were loaded on to precast, 4-12% gradient gels (Nu PAGE, Invitrogen). The SDS-PAGE was carried out at a constant voltage of 200 V for $ 1 h. After the completion of separation, the protein bands were visualized by staining with coomassie brilliant blue and/or silver. Three independent experiments were carried out for both PM and tonoplast fractions from three biological replicates. The bands that appeared consistently in all biological replicates were selected for MS/MS analysis. For 2DE, the PM and tonoplast pellets were resuspended in rehydration solution consisting of 7 M urea, 2 M thiourea, 4% CHAPS, 40 mM DTT, and 0.002% w/v bromophenol blue, vortexed followed by a 1 h incubation at room temperature. The supernatant consisting of solubilized PM and tonoplast proteins was then collected by centrifugation at 14,000 g for 30 min. For the first-dimension electrophoresis, 17-cm long pH 4-7 ReadyStrip IPG strips (Bio-Rad, Hercules, CA) were passively rehydrated overnight at room temperature with 340 mL of rehydration buffer containing 50 mg protein and 0.5% v/v pH 4-7 carrier ampholytes. IEF was carried out in a PROTEAN IEF cell (Bio-Rad) at a current limit of 50 mA per IPG strip at 20°C for a total of 37.55 kV h. Each focused IPG strip was equilibrated by soaking, with mild stirring for 15 min in 10 mL of equilibration buffer 1 consisting of 6 M urea, 0.05 M Tris pH 8.8, 2% w/v SDS, 20% v/v glycerol, 2% w/v DTT followed by soaking in 10 mL of equilibration buffer 2 (same content as equilibration buffer 1 except DTT was replaced with iodoacetamide) for 15 min. The second dimension of 2DE was carried out by placing the IPG strips on to a separating gel (12% polyacrylamide, w/v). Gel electrophoresis was performed at 30 mA per gel with circulating cooling and was completed in 5 h. Protein spots were visualized by staining with silver and gel image was captured. All 2DE analyses for both PM and tonoplast fractions were carried out in three biological replicates, and spots that appeared consistently in all biological replicates were selected for MS/MS analysis. In-gel digestion The proteins were digested in-gel using MS-grade Trypsin Gold (Promega) according to the manufacturer's instructions. The selected bands from 1DE and spots from 2DE were carefully excised from the gels using a clean razor blade and incubated at 4°C for 24 h in a washing buffer containing 2.5 mM NH 4 HCO 3 and 50% ACN followed by incubation of the samples in fresh washing buffer at 37°C for 10 min with constant shaking. Later, the samples were dried in a vacuum centrifuge (SpeedVac). Reduction of samples were carried out using 10 mM DTT followed by alkylation with 55 mM iodoacetamide. Later, alternative washing with 100 mM NH 4 HCO 3 and dehydration using ACN was carried out. Finally, the samples were vacuum dried and trypsin digested by preincubating in 10-20 mL trypsin (0.01 mg/mL) solution at 4°C for 30 min followed by an incubation at 37°C for 16 h. LC-MS/MS analysis The analysis was carried out as described earlier [1]. Eksigent nanoLC Ultra and ChiPLC nanoflex (Eksigent, Dublin, CA, USA) in trap-elute configuration were used to separate the digested peptides. Sep-Pak C18 Elution Plate (Waters, Milford, MA, USA) was used to desalt the digested samples. The desalted samples were reconstituted with 15 mL of diluent (2% ACN, 0.1% formic acid (FA)). A total of 10 mL of the sample was loaded onto a 200 m  0.5 mm ChromXP C18-CL trap column and eluted onto a 75 mm  150 mm ChromXP C18-CL analytical column. Peptides were separated by a gradient formed by 2% ACN, 0.1% FA (mobile phase A) and 98% ACN, 0.1% FA (mobile phase B): In 5-7% of mobile phase B for 0.1 min, in 7-30% of mobile phase B for 10 min, in 30-60% of mobile phase B for 4 min, in 60-90% of mobile phase B for 1 min, kept at 90% of mobile phase B for 5 min, 90-5% of mobile phase B for 0.1 min, and maintained at 5% mobile phase B for 10 min, at a flow rate of 300 nL/min. TripleTOF 5600 system (SCIEX, Foster City, CA, USA) in information-dependent mode was used to perform MS analysis. MS spectra were acquired across the mass range of 350-1250 m/z in high-resolution mode (4 30,000) using 250 ms accumulation time per spectrum. A maximum of 20 precursors per cycle were chosen for fragmentation from each MS spectrum with 100 ms minimum accumulation time set for each precursor and dynamic exclusion for 8 s. Tandem mass spectra were recorded in highsensitivity mode (resolution 415,000) with rolling collision energy on. ProteinPilot 4.5 software Revision 1656 (SCIEX) was used for peptide using the Paragon database search algorithm (4.5.0.0.). The data with all the MS/MS spectra were searched against several databases, including databases of Swiss-Prot, Plant Reference Sequences (total 248,485 entries) and a database consisting of all the available mangrove plant protein sequences in NCBI. Only the proteins with highest coverage were retained while the redundant proteins identified from each band/spot were filtered. The search parameters were as follows: Sample Type-Identification; Cys Alkylation-Iodoacetamide; Digestiontrypsin; Special Factors-Phosphorylation emphasis; Species-None. The processing was specified as follows: ID Focus-Amino Acid Substitutions; Search Effort-Thorough; Detected Protein Threshold-0.05. No FDR analysis was performed on the dataset. Instead, peptides identified with confidence interval Z95% (green color coded peptides as shown in Fig. 2) were taken into account. In addition, peptides identified with confidence level o95% but Z 50% (yellow color coded peptides as shown in Fig. 3) were manually inspected and only those were included in which all the Y ions and majority of the intense peaks were assigned. The plasma membrane and tonoplast proteins identified in the study are listed in Supplementary Tables 1 and 2 and the index to Spot/band numbering and PRIDE numbering is given in Supplementary Table 3.

v3-fos

2019-03-20T13:07:04.216Z

2015-04-01T00:00:00.000Z

83986837

s2

Utilization of Leucaena Leucocephala in Traditional Fattening Program of Bali Cattle in Amarasi This field study was aimed to evaluate the availability and the utility of forage on Bali cattle in Amarasi system in dry land area such as Kupang. Amarasi system is a fattening production system (paronisasi), which traditionally utilizes Leucaena leucocephala forage in dry season of Amarasi region. This field study was conducted during the period of July to December 2013 in Oesena and Merbaun Villages. The method used in this research was field survey. This type of survey was used to identify the forage availability and utility for Bali cattle; including determining the level of feed intake, measuring carrying capacity of both observe villages, and recording body weight of cattle. Cluster random sampling was applied in this study to determine sample used in the research, those were two villages with different altitudes, namely Oesena Village and Merbaun Village. Data were analyzed descriptively to describe the related existing conditions in the field. T-test was conducted to determine leucaena consumption and Bali cattle body weight in both villages. The results showed that during observation period, availability of leucaena forage for cattle was low based on dry matter intake 3.60 and 3.58 kg/head/day, which led to low consumption of cattle. Nevertheless, the use of leucaena in this system increased average body weight gain of Bali cattle up to 0.77 kg/day, due to high crude protein consumption. The conclusion of this study was the availability of leucaena as Bali cattle feed did not meet the cattle needs, but the use of leucaena increased the average of Bali cattle body weight gain. INTRODUCTION The forage portion in ruminant ration reaches approximately 3% of the body weight. Nutritionally leucaena, is source of fiber, mineral and protein, which traditionally fulfill animal requirements for growth. The difficulties in providing sustainable amount of forage often occur in Kupang due to long dry season. It causes low productivity of ruminants. Amarasi region is located in Kupang Regency in East Nusa Tenggara province and consists of four sub districts namely Amarasi, West Amarasi, East Amarasi, and South Amarasi. The climates in these areas are influenced by the monsoon winds from Australia. Monthly rainfall ranges between 4 mm (in October) to 316 mm (in February) with humidity ranging from 64% to 84% (BPS, 2013). In Amarasi, farmers applied a traditional fattening system called "paronisasi" by using only leucaena as one of the most important forage on dry season. Leucaena is daily feed on dry season in this system by cut and curry method. Farmers grow leucaena in their garden and harvest the forage regularly. This system was initially applied in 1930 under the rule of the Dutch government in Baun Village of Amarasi region (Metzner, 1981) which then being reinforced by the local customs authorities in 1932. Regulations made by the local authority (king) have significantly supported this system to a success story compared to breeder-farmers in other regions of the province. Since this system was introduced, great numbers of farmers are interested in implementing this system and are entirely relying on L. leucocephala as the main feed source. Forage L. leucocephala can be used as a protein, minerals, and vitamin sources (Haque et al., 2008;Kang et al., 2012). Dahlanuddin et al. (2013) reported that Bali cattle fed leucaena increase average daily gain by 0.47 kg/head/d. Furthermore, addition of S. cerevisiae and L. leucocephala on low-quality feed improved nutrient digestibility and cattle performance (Herawaty et al., 2013). Nevertheless, the flea's attack in the early 1986 had tremendously destroyed leucaena stands and directly influenced the declining of livestock productivity in Amarasi. Leucaena shortage in beef cattle affected the fattening program period is longer and the number of cattle are fattened be less. In such condition, the Amarasi system became better and are flea's tolerant leucaena being re-cultivated. Based on the above information this study was undertaken to evaluate the availability and the use of leucaena (L. leucocephala) on Amarasi system in Kupang. Leucaena Intake Measurement Edible part of leucaena intake was recorded by weighing the forage before feeding and the residue of leucaena on the next day. Forage intake was observed for seven consecutive days. The number of respondents were 15 farmers for each villages and coded as F1, F2, until F15 to collect primary data including leucaena consumption, chest circumference to estimate body weight, and nutrient quality of leucaena. These farmers were part of total respondent (75) that were interviewed. Research Accomplishment The survey was conducted to investigate information regarding cattle condition by farmers, field, and farmer's profiles. Along with this, questionnaires were used to obtain information on general condition of production system and feed resources in farm level. Data used in the study were primary and secondary data. Primary data were obtained from the results of leucaena intake, chemical composition of forage and cattle body weight. Secondary data contained climatic data, village resources agriculture. Cluster random sampling was applied in this study to determine sample used in the research, those were two villages with different altitudes, namely Oesena Village located in the hills at 500 m above sea surface in Amarasi sub district and Merbaun Village which located near the shore areas at 30 m above sea surface in West Amarasi sub district (BPS, 2013). The number of samples was calculated using the following formula (Som, 1996): n= N/(1 + Ne 2 ) Notes: N : total farmers e : error (10%) n : total sample The number of respondents was 75 farmer families from both villages. The information regarding farmer, number of cattle, and forage supply patterns were obtained by using questionnaires. T-test was used to determine Bali cattle body weight and leucaena consumption value in both villages. Carrying Capacity Measurement Carrying capacity is a number of cattle that can graze on a pasture without destroying the land, crops, and livestock. The carrying capacity was calculated based on the space of plant with assuming the population of leucaena was 2500 trees/ha and plant spacing was 2 x 2 m and cutting frequency was 3 times in dry season (8 months). Carrying capacity was calculated using a modified method of Halls et al. (1964). Body Weight Gain Estimation Body weight gains were estimated from chest circumference using the pattern Zurahmah (2011). Data Analysis The data were analyzed descriptively to describe the general state of the research sites. Those were temperatures, humidity, rainfall and livestock rearing systems and patterns of green fodder supply that can support the efforts of ruminant livestock in East Nusa Tenggara. Analysis of Chemical Composition of Leucaena The chemical composition of leucaena forage was analyzed using proximate analysis method (AOAC, 2005) and Van soest (Tillman, 1998). Forage feed samples were taken from the both Oesena and Merbaun vilages. Total digestible nutrient (TDN) from leucaena forage was calculated using the following formula as described by Hartadi et al. (1980) General Condition of the Research Area Farmer in Oesena Village and the Merbaun Village spread evenly because of the existence land in the villages. The lands were used not only for L. leucocephala but also to graze livestock traditionally in both extensive and semi-intensive system. In addition the cattle were kept in cage during the day and some of the Bali cattle kept freely around the house. L. leucocephala was planted as fence around the house in some farmers, but some other farmers planted leucaena as garden to fed Bali cattle. The average of farmer ages in both villages were 33-48 years old. Farming has become an integral part of the culture and is existed as a complementary income generator that is inherited from each generation. The farmers came from different formal education background such as elementary, junior high school, senior high school and bachelor degree (S1), and also farmers who had not a formal education. The main occupations of the farmers in both villages were varied, they were mostly working as a farmer, gardener, and motorcycle taxi drivers. Hence, it could be assumed the average income of farmers without fattening program in economic standard of farmers were low. As it was considered as another source of income, the farmers took care of their cattle once the main occupation had finished. The profit from derived fattening was budgeted for paying children school fee, marriage, and other traditional events. On average, each farmer in the two villages had their own 2-6 cattle as the heritance from the parents as well as purchasing their own cattle. Bali cattle production system used in the two villages was semi-intensive, cattle are tied up and then being fed with leucaena twice a day in the morning and afternoon. This system is also called "paronisasi". Defoliation systems of leucaena forage conducted in the village is cut and carry. Residue of leucaena forage in the form of twigs and stems are collected by the farmers and are used as firewood and poles for construction. Another function of L. leucocephala was reforestation, poles for construction, firewood, shade in permanent plantations, traditional medicine (Nehdy et al., 2014), pulp production (Lopez et al., 2008), green manuring (Sharma & Behera, 2010), and in wood production (Prasad et al., 2011). Forages given to cattle were collected from their own garden or around the yard. There is a law that is regulated by the government since ancient time that is when farmers stole forage from other breeders, they will have to pay approximately 1 million IDR. The law enforces the farmers to grow leucaena in their garden. In accordance to the results of our interview, each farmer in average possessed 2.5 ha of leucaena field in Oesena, while in Merbaun Village they had 6.25 ha. Based on the field observation, Psyllid was found in some stands of leucaena in both villages. Integrated pest management applied by the community and village government to eliminate the fleas was entirely successful. Since leucaena was still considered as a major feed in Amarasi, new tolerant fleas varieties of Lamtoro cv. Tarrambah, was extensively cultivated in the area (Nulik et al., 2013). Leucaena has been chosen due to its ability to adapt to a longer dry season and palatable. Besides, support from the government to apply the forestation program, pave the way to make leucaena as the major feed crop in Amarasi until now. Nutrient Quality of Leucaena The chemical compositions of leucaena forage in both study areas were not significantly different. Crude protein contain in leucaena forage grown in Oesena (mountainous) 27.35% was higher than Merbaun which was located nearby the beach 19.88% (Table 1). Protein content in Oesena village was higher than Merbaun village, this could be due to the fertility of the soil and climate. Merbaun village is located near the beach so it has a higher salinity levels and lower rainfall than Oesena village which stated in the hills. Leucaena Consumption The average of dry matter intake of leucaena (DMIL) in Oesena was 3.60±0.83 kg/head/d, while in Merbaun Village DMIL was average of 3.59±0.72 kg/head/d ( Table 2). The DM consumption on rates of leucaena in both village were still low about 1.59% of body life weight and 2.05% of body life weight or approximately 53% and 72% of total leucaena feed in Oesena and Merbaun, respectively. According to Kearl (1982), daily DMI for cattle as about 2.8% body weight. Based on the calculation of the consumption, the weight of the cattle in the two villages were livestock feed shortage. Kearl (1982) states that the average cow body weight between 150-200 kg with an increase in body weight 0.50 kg/head/d of dry matter intake 4.2 kg/h/day or 2.8%. The shortages occurred due to long period of drought that lead to the declining of feed availability. Prolonged drought and high temperatures resulted in lost of leucaena leaves. Then, the flea invasions also caused the two villages to the lack of forage availability. Nevertheless cattle in these two villages were able to utilize nutrients leucaena well; this was followed by an increase in body weight of Bali cattle in wet season. DMIL in Merbaun is significantly higher P<0.05 (T-test) than those in Oesena. This was perhaps due to greater area of leucaena possessed by farmers in Merbaun than in Oesena. Crude Protein and TDN Consumptions Consumption of crude protein in Oesena Village was higher 0.98±0.21 kg/head/d than Merbaun Village 0.71±0.08 kg/head/d (Table 2), it was because of the high crude protein content in leucaena forage from Oesena (27.35%). Crude protein consumption in the cattle was higher than crude protein of requirement 0.57-0.67 kg/d as recommended by Kearl (1982). TDN consumption of two villages did not much different, and it fulfilled the TDN need in cattle. TDN consumption in Oesena and Merbaun village was 2.61±0.33 kg/head/d and 3.87±0.46 kg/head/d (Table 2). Based on the calculations of TDN requirement Kearl (1982) standard for 150-200 kg cattle body weight was 2.75-3.55 kg/d. Bali Cattle Body Weight Gain Average daily gain (ADG) of cattle in two villages were varied. It can be grouped into two ADG level ie: ADG ≤ 0.50 kg/head/d as group A and ADG ≥ 0.50 kg/ head/d as group B. The lowest daily gain average of Bali cattle of group A in the Oesena was 0.17 kg/head/d and Merbaun Village was 0.32 kg/head/d (Table 3). Cattle in group A was growing cattle, so feed consumed was (Table 3). Kearl (1982) stated that the standard of protein consumption for cattle with ADG 0.50 kg/head/d was 0.60-0.70 kg/head/d. The excess of the protein was converted into energy. Carrying Capacity The average of leucaena leaf dry matter production in both villages Oesena and Merbaun were 3.2 ton/ha and 2.4 ton/ha, respectively (Table 4). Leucaena production and carrying capacity were calculated based on population leucaena per hectares with a spacing 2 x 2 m. Therefore, in one hectare of leucaena farm were 2500 trees by three times cutting during the dry season. Season has an important role on the productivity of the leucaena farm, so that the rest leucaena period used was 70 days. In the dry season, the result of carrying ca-pacity at Oesena and Merbaun village was 1.94 AU and 1.43 AU, respectively (Table 4). Rainfall is a major factor that affecting the growth of the plant. The low and spread of intensity of rainfall can bring a difficulties for farmers to get the forage for cattle, particularly during the dry season. In addition, topography also influences the availability of forage. The land slope is relating to land management and erosion. The land elevation is relating to temperature and solar radiation. CONCLUSION The availability of leucaena as a fodder for Bali cattle in two villages did not meet the cattle need as seen from the dry matter intake and low carrying capacity. Nevertheless the use of leucaena as Bali cattle feed was able to meet the nutrient requirement of crude protein, thereby increasing daily body weight gain of Bali cattle.

v3-fos

2016-05-04T20:20:58.661Z

2015-06-25T00:00:00.000Z

11598367

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Association of Agronomic Traits with SNP Markers in Durum Wheat (Triticum turgidum L. durum (Desf.)) Association mapping is a powerful approach to detect associations between traits of interest and genetic markers based on linkage disequilibrium (LD) in molecular plant breeding. In this study, 150 accessions of worldwide originated durum wheat germplasm (Triticum turgidum spp. durum) were genotyped using 1,366 SNP markers. The extent of LD on each chromosome was evaluated. Association of single nucleotide polymorphisms (SNP) markers with ten agronomic traits measured in four consecutive years was analyzed under a mix linear model (MLM). Two hundred and one significant association pairs were detected in the four years. Several markers were associated with one trait, and also some markers were associated with multiple traits. Some of the associated markers were in agreement with previous quantitative trait loci (QTL) analyses. The function and homology analyses of the corresponding ESTs of some SNP markers could explain many of the associations for plant height, length of main spike, number of spikelets on main spike, grain number per plant, and 1000-grain weight, etc. The SNP associations for the observed traits are generally clustered in specific chromosome regions of the wheat genome, mainly in 2A, 5A, 6A, 7A, 1B, and 6B chromosomes. This study demonstrates that association mapping can complement and enhance previous QTL analyses and provide additional information for marker-assisted selection. Introduction Durum wheat (Triticum durum Desf.) is a tetraploid species consisting of A and B genomes (AABB). It was resulted from domestication of wild emmer wheat (T. dicoccoides) derived from a spontaneous cross between T. urartu (AA genome, 2n = 14) and an ancient relative of diversity pattern and population structure in durum wheat germplasm [41][42][43]. However, there are few reports published on trait-marker associations in durum wheat. Therefore, the major objective of this study is to reveal associations between quantitative traits and the SNP markers in durum wheat. Plant materials and field trials One hundred and fifty durum wheat accessions of worldwide origin were investigated in this study. The collection of the durum wheat germplasm was classified into seven groups based on their geographic origins. Of the accessions, 24 originated from West Asia (WA), 25 from East Asia (EA), 33 from North America (NA), 33 from different parts of Europe (EU), 12 from South America (SA), 16 from North Africa (AF), and 7 from Australia (AU). The name, place of origin and identifier number for each accession is listed in S1 Table. In order to obtain reliable phenotypic data, field trials of all the accessions with replications were conducted in four consecutive years. The field trials got the approval of Huazhong Agricultural University, and were performed on the experimental farm of Huazhong Agricultural University, Wuhan, China. The land accessed is not privately owned nor protected, which is belong to Huazhong Agricultural University. All of the materials used in this study were acquired by Dr. Junhua Peng from USDA (United States Department of Agriculture), and no any protected species were sampled in the field trials. The trials with three replications were planted around the end of October in 2009, 2010, 2011 and 2012, respectively, in two rows with 1 m in length and 20 cm between rows, 6 plants in each row. Because some of the accessions were very tall and easy-lodging, we installed frames made of bamboo sticks in each plot before heading to prevent lodging, or reduce lodging impact on the traits. Phenotyping of the key traits Measurement of key traits. After full maturity, we randomly harvested four individual plants from each plot. The following 10 traits were measured. The mean value of a trait in each replication was calculated. Kolmogorov-Smirnov test was performed to test for normal distribution. Data transformation is performed for the traits that did not fit the normal distribution. Calculations of the descriptive statistics, analysis of variance (ANOVA) and broad-sense heritability (H 2 ), and correlation analysis were performed using SPSS programs (IBM SPSS Statistics, Chicago, IL, USA). DNA extraction, SNP genotyping and marker data analyses Before the elongation stage of wheat plants, approximately 1.0 g of young leaf tissue was collected from each of the accessions. The tissue was placed in a 1.5 ml Eppendorf tube, immediately frozen in liquid N, and stored in a -80°C freezer [43]. The cetyltrimethyl ammonium bromide (CTAB) method was used to extract the total genomic DNA [44]. The DNA samples were shipped to University of California at Davis, USA for genotyping. A set of 1,536 genome-specific SNP markers were applied to genotype the germplasm. These SNP markers were discovered in a panel of 32 lines of tetraploid and hexaploid wheat (http:// avena.pw.usda.gov/SNP/internal/protocol/id.htm), and downloaded from the Wheat SNP Database (http://probes.pw.usda.gov:8080/snpworld/Search). The SNP-genotyping was performed using the Illumina Bead Array platform and Golden Gate Assay (Illumina, San Diego, CA) at the UC Davis Genome Center (http://www.genomecenter.ucdavis.edu/dna_ technologies). The SNP markers were treated as co-dominant markers. The details of genotyping and genetic analyses were described in Ren et al. [43]. Linkage disequilibrium It is essential for association mapping to examine the degree of LD in the genome and chromosome [45,46]. The fraction of locus pairs indicating significant LD increases with decreasing significance level. A high significance level of p<0.001 was chosen for comparative purposes. If all pairs of adjacent loci within a chromosomal region were in significant LD, this region was treated as a LD block [47]. LD between markers was measured using R 2 , square of correlation between the markers [48]. The values of R 2 and P were calculated using the software TASSEL 3.0.124 (http://www.maizegenetics.net/). Association analysis Association mapping analysis between SNP markers and the 10 quantitative traits (PH, ES, LMS, SMS, RLMS, LFPMS, NSPP, GNP, GWP, and KGW) was performed based on the general linear model (GLM) and the mixed linear model (MLM) using software TASSEL 3.0.124 (http://www.maizegenetics.net/tassel). The population structure was estimated using STRUCTURE 2.3.4 software [49] as in Ren et al. [43]. The pair-wise kinship coefficients were estimated according to the method of Lynch and Ritland [50], performed in the program SPA-GeDi [51] (http://ebe.ulb.ac.be/ebe/SPAGeDi.html). The number of permutation runs was set as 10,000 to obtain the permutation-based significance in GLM analysis. MLM was fitted for each marker and phenotype, accounting for Q-Matrix of the population structure as a covariate and pair-wise kinship coefficients (K matrix) as random effects [34]. Significance of associations between marker loci and traits was tested at a corresponding level of the experiment-wise P-value. Significance of associations between loci and traits was described as P-value and the QTL effects were evaluated by marker-R 2 [52]. SNP markers and population structure Multiplexed 1,536 Illumina Golden Gate SNP assay involving in 150 durum wheat accessions generated 230,400 data points. Out of the examined SNPs, 1,366 (89%) were successfully amplified, and other 10% were missing. The detailed analyses on the SNP markers were reported in Ren et al. [43]. The SNP loci were well distributed across the seven homoeologous chromosome groups. The total marker number ranged from 161 in group 5 to 236 in group 7 chromosomes. The number of polymorphic markers ranged from 108 in group 5 to 161 loci in group 6 chromosomes [43]. The structure analysis was performed in Ren et al. [43], and the result suggested that the observed durum wheat germplasm can be divided into two genetically distinct groups (Group I and Group II). The cluster analysis showed that the group II can be further divided into four subgroups, IIa, IIb, IIc, and IId. The dendrogram of 150 durum wheat landraces based on the shared-allele genetic distance calculated from 1,366 SNP markers was showed in Ren et al. [43]. Linkage disequilibrium among intra-chromosome SNP loci A total of 1,338 SNP markers with a mean marker density of 95-96 markers per chromosome, ranging from 66 (3B) to 130 (7A) for all the 14 chromosomes, were used to calculated the extent of LD. The pattern of LD was measured using R 2 of allele pairs between 2 loci according to Weir and Cockerham [53] on both chromosome and genome levels (Tables 1 and 2). There were 894,453 possible pair-wise loci in the matrix of 150 genotypes and 1,338 SNP markers. Of these locus-pairs, 5.43% showed significant LD (p<0.001) ( Table 2). There were 2,145 (3B) to 8,385 (7A) possible locus pairs in the 14 chromosomes. The percentage of locus pairs showing significant LD (p<0.001) ranged from 3.76% (1A) to 8.01% (6B), respectively. The average R 2 values varied from 0.038 (1A) to 0.081 (4A) among the 14 chromosomes (Table 1). A small percentage of significant locus pairs had R 2 value >0.1 (p<0.001). On the average, the highly significant pairs (R 2 >0.1; p<0.001) were 251 per chromosome, ranging from 97 (4B) to 550 (7A). The percentage of all possible locus pairs showing highly significant LD (R 2 >0.1; p<0.001) ranged from 3.03% (1A) to 6.59% (6B) ( Table 1). The extent of LD was varying with chromosomes. Table 2 showed LD value versus genetic distance in the locus pairs on genome level. There were 749 and 589 loci available for LD evaluations in the A and B genome, respectively. Across all 1,338 loci, 65,328 possible pairs of linked loci (in the same linkage groups) and 829,125 pairs of unlinked loci (from different linkage groups) were detected. The observed locus pairs of linked and unlinked loci were 30,868 and 390,935, respectively. Among the linked locus pairs, 2,357 (5.86%) possessed significant LD (P<0.001) in genome A, whereas, 1,709 (6.82%) had significant LD in genome B. As to the unlinked locus pairs, 1,236 (5.01%) had significant LD (p<0.001) in genome A, whereas 8,483 (5.73%) in the B genome. The mean R 2 values for all the linked pairs in genome A and B were 0.062 and 0.056, respectively. Therefore, the number of possible pairs, number of significant pairs, and mean R 2 of the genome A were larger than the genome B except for the percentage of significant pairs ( Table 2). The extent of LD was varying with chromosomes. The percentage of significant LD (R 2 >0.1; p<0.001) pairs in the A chromosomes generally was higher than the corresponding B (Tables 1 and 2). Thus the extent of LD of A genome was larger than the B genome on both the chromosome and genome levels in general. Variation of the key traits Features of the examined traits. All the durum accessions were observed for 10 agronomic and morphological traits in replicated field trials for four consecutive years (Table 3). Distribution histograms of the 10 traits were showed in Fig 1. In general, Kolmogorov-Smirnov test showed that most of the observed traits fitted the normal distribution except for PH, ES and LMS. PH significantly deviated from the normal distribution (P<0.05 in all the 4 years) and showed the feature of binomial distribution. ES significantly deviated from the normal distribution in 2010 and 2013 (P<0.05), and nearly significant in 2011 (P = 0.062). LMS showed significant deviation (P<0.05) in 2011-2013 and nearly significant deviation in 2010 (P = 0.059) (Fig 1). Therefore, most of the observed traits are quantitatively inherited. But PH seems controlled by a single gene together with polygene of minor effects in the population, and distribution of ES and LMS seems varying with the environment. Trait variation with year and genotype. The trait distribution pattern was similar over the four years, and most of the traits generally showed normal distribution. The year effect was highly significant for most of the observed traits as revealed by the analysis of variance (ANOVA). The genotypic variation was highly significant for all the 10 traits. The genotype × year (G × E) interaction effect was also highly significant for all the examined traits. Estimation of broad-sense heritability (H 2 ) showed that most of the traits (6/10) have high heritability (H 2 >65%) ( Table 4). Therefore it is meaningful to conduct association analyses between the traits and SNP markers. Correlation among the observed traits. Table 5 showed correlation coefficients among the 10 observed traits. Out of the 45 possible correlation pairs, more than 75% (34) were significant or highly significant. LMS, RLMS, LFPMS and SMS showed highly significant positive correlations with PH. NSPP, GNP and GWP showed highly significant positive correlations with ES, while SMS and KGW showed significant and highly significant negative correlations with ES. LMS showed significant positive correlations with RLMS and NSPP. The correlations between LFPMS and GNP, GWP were positive and highly significant. SMS was highly and positively correlated with NSPP, while negatively correlated with KGW. This indicated that the more SMS, the more NSPP correspondingly. In another word, the growth condition of main spike reflected the growth condition of the other spikes to some extent. And the more SMS and NSPP mean lighter and smaller grains. As a result, KGW was negatively correlated with SMS (Table 5). Association analysis Association analyses between SNP markers and the 10 quantitative traits (PH, ES, LMS, SMS, RLMS, LFPMS, NSPP, GNP, GWP, and KGW) were conducted preliminarily under the GLM and MLM models by using the computer software TASSEL 3.0.124. Comparison between these two models showed that MLM decreased the total number of significant associations (p<0.01) (data not shown), and most of the significant associations were consistent between the two models. Yu and Buckler [34] suggested incorporating the pair-wise kinship (K matrix) as random effects into a mixed model to correct relatedness and reduce the number of false positives in association analysis. In addition, association analyses in Yang et al [38] and Zhu and Yu [54] indicated that MLM (K+Q) model was better for correcting false positives associations than GLM. Therefore, the results under the MLM model that accounted for both Q and K matrixes were presented in this paper. Some imperfect markers were excluded out of the 1,536 SNP markers. Thus 1,366 SNPs were used for association analysis in this study. Table 6 and S2 Table showed In 2010, sixty markers were significantly associated with the ten observed traits. The distributions of the association pairs were uneven among the traits. Most of the associations were detected between markers and the yield traits. More than half of the markers were associated with GNP, and the number of associated markers for other traits range from 1 (ES, LFPMS and RLMS) to 10 (LMS). The percentage of the variation explained by marker ranged from 5.4% (CD454448_6_A_84 associated with KGW) to 18.2% (BG605368_2_A_Y_310 associated with LMS). In 2011, we detected 26 marker-trait association pairs. The number of the associated markers ranged from 1 (ES and NSPP) to 6 (LMS and GWP) ( Table 6). The percentage of the variation explained by marker was in a range between 5.4% (BG274294_1_B_382 associated with SMS) and 13.1% (BG605368_2_A_Y_310 associated with LMS). In 2012, 45 marker-trait associations were detected. The number of the associated markers ranged from 2 (NSPP) to 10 (GNP). The percentage of the variation explained by marker varied from 5.2% (BG312827_6_A_Y_305 associated with PH) to 11.6% (BM134437_3_A_Y_233 associated with LMS). For the year 2013, 70 associations were detected. The percentage of the total variation explained by marker varied from 5.0% (BE444144_2_B_N_138 associated with SMS) to 26.1% (BF474284_1_B_Y_357 associated with LMS) ( Table 6, S2 Table). Moreover, taking consideration of all the four years, the number of markers associated with each trait ranged from 1 (LFPMS) to 54 (GNP), and the percentage of the total variation explained by marker ranged from 5.0% (BE444144_2_B_N_138 associated with SMS) to 26.1% (BF474284_1_B_Y_357 associated with LMS) (S2 Table). We found that one trait associated with many markers (e.g., GNP with 54 markers), and single markers were associated with multiple traits (BE590553_7_A_190 associated with GNP, NSPP and SMS, and BE443538_5_A_ 1436, BE590521_6_B_N_331 associated with GNP, GWP and RLMS, etc.). This may indicate that quantitative traits are always conferred by multiple loci, and QTLs conferring multiple agronomic traits may cluster around the single regions/markers due to pleiotropic effects of genes [55]. Seven associations (4 for LMS, 3 for PH) were detected in all the four years. Two associations (1 for PH, and 1 for SMS) were detected in three of the four years. Eleven associations were detected in two of the four years (S2 Table). These reproducible associations were significant and more reliable. Associations for morphological traits. Plant height (PH): six significantly associated SNPs were detected in four years of 2010-2013 (Table 6). Three SNP markers, BE405269_4_ B_84, BF475120_6_B_67, and BF475120_6_B_Y_75 were detected to be significantly associated with PH in all the four years. Other three SNPs, BG312827_6_A_Y_305, BE443948_2_A_ Y_345 and BE490041_1_A_371 were significantly associated with PH in three or two of the four years (S2 Table). Furthermore, PH showed feature of the binomial distribution (Fig 1) and thus may be controlled by the polygene including a single major gene and some minor genes in the populations. These PH-associated SNP markers were mainly located in chromosome 1A, Association of Agronomic Traits with SNP Markers in Durum Wheat 2A, 4B, 6A and 6B. Several marker loci, significantly associated with PH, were previously detected on chromosomes 4B, 5A, 5B, 6B, 7A and 7B [56]. RLMS and LFPMS: A total of 5 and 1 SNP markers were detected in the four years for RLMS and LFPMS, respectively (Table 6, S2 Table). Markers significantly associated with the traits were present on chromosome 1B, 5A, 6A and 6B. BE443538_5_A_1436, BE590521_6_ B_N_331 and BG314205_1_B_33 were associated with RLMS, GNP and GWP. Correlation analysis indicated significant positive correlations of RLMS with GNP and GWP (Table 5). Flag leaf and rachis internode were related to photosynthesis and photosynthetic product accumulation and transfer, and thus played important roles in grain filling process [57]. Therefore, it is understandable that SNP markers associated with RLMS and LFPMS are also related with GNP and GWP. LMS: Six to fifteen associations were detected between LMS and SNP markers in the four years ( Table 6, S2 Table). The SNP markers associated with LMS were located on chromosome 1B, 2A, 3A, 4A 5A, 6A, 7A and 6B. Four SNP markers BE445667_6_B_Y_285, BF474284_ 1_B_Y_357, BG605368_2_A_Y_310 and BM134437_3_A_Y_233, were significantly associated with LMS in all the four years. Five SNPs showed significant associations with LMS in two of the four years (S2 Table). The marker BF484028_5_A_Y_97 corresponding to the Vrn-A1 region in the interval of 5AL10-0.57-0.78 was significantly associated with LMS. Some associations were founded to be located in the same regions for LMS-related traits (GNP and GWP etc.) ( Table 5, Fig 2). Associations for yield traits. ES and NSPP: A total of 13 and 16 SNP markers were associated with ES and NSPP in the four years, respectively ( Table 6, S2 Table). Some SNP markers were associated with both ES and NSPP. Highly significant positive correlation was detected between ES and NSPP (Table 5). SMS, GNP and GWP: A total of 22, 54 and 18 significant associations with SNP markers were detected for SMS, GNP and GWP in the four years, respectively ( Table 6, S2 Table). BG314551_3_A_Y_162 was significantly associated with SMS in three of the four years. This SNP explained over 8.1% of the variation ( Table 6, S2 Table). The EST represented by BG314551_3_A_Y_162 was located in the same region as Eps gene (earliness per se). GWP showed positive correlation with GNP. Several SNP markers are thus associated with both GNP and GWP. Linkage disequilibrium in durum wheat The variation patterns of LD at both the chromosome and genome levels reflect the complicated evolutionary and breeding history in wheat [58]. In the present study, we demonstrated an extensive amount of LD in durum wheat using 1,338 SNP markers (Tables 1 and 2). The extent of LD in A genome is higher than in B genome in general. The similar result was reported in previous study [59]. In their study based on SSR markers, the highest extent of significant LD was observed in D genome, followed by the A and B genomes of the bread wheat [59]. The genomic locations of genes controlling important adaptive traits were different. These can have a differential influence on LD in different genomes. Vrn-A1 gene on chromosome 5A has higher number of widely distributed haplotypes than the Vrn-B1 gene on chromosome 5B and thus more likely to have a stronger effect on LD [60]. In our study, chromosome 4B had the lowest percentage of significant LD pairs and mean R 2 value, and thus possessed relatively low LD extent in chromosome 4B (Table 1). Akhunov et al. [61] also reported that chromosome 4B had the lowest number of haplotypes per locus and lowest haplotype diversity. This may indicate that the haplotype diversity and genes controlling important adaptive traits have a differential influence on LD in chromosome 4B. Therefore, the divergence in the extent of LD is probably related to breeding history and selection pressure applied to genes located in the different chromosomes and genomes during the process of cultivation [62]. The genetic diversity of genome A is lower than genome B [43,55]. The extant LD in genome A is higher than in genome B, on the contrary. On chromosome level, some chromosomes have the similar extant LD (like 2A and 2B, 3A and 3B, 4A and 4B etc.) ( Table 1). Chao et al [62] reported similar result. The extant LD was related to genetic diversity in the individual breeding program. The domestication history of genome A is longer than genome B in wheat [55,63]. Genome A thus probably has more genes controlling important adaptive traits. Under the natural and artificial selections in the breeding programs, the genome A of cultivars captured comparable number of adaptive traits/genes, and widely distributed haplotypes resulting from the high extant LD [62,63]. As mentioned above, breeding/domestication history and selection specific to each breeding program have influence on LD to some extent. Candidate QTLs revealed by association analysis In the present study we performed association analysis using big number of SNP markers in durum wheat consisting of worldwide accessions. A total of 201 association pairs between SNP markers and 10 quantitative traits were detected in the four years (S2 Table). Fifty-two known regions were marked on the 14 chromosomes (Fig 2), which may represent the candidate QTLs. Four credible SNP associations for PH were reproducible at least in three of the four consecutive years. These associations were located on 4B, 6A and 6B. Two markers (BF475120_6_B_67 and BF475120_6_B_Y_75) located on the same position in the region 6BL5-0.40-1.00 of the long arm of chromosome 6B, were associated with PH in all of the four years, and these two associations possibly represent a single credible QTL explaining over 7.2% of the variation in the four years (S2 Table). Several QTLs were reported in the similar region of 6BL by Börner et al. [64] and Cadalen et al. [56]. Four credible associations for LMS were reproducible in the four consecutive years. These associations were located on 1B, 2A, 3A and 6B, respectively, and thus might represent 4 QTLs. BG605368_2_A_Y_310, located on 2AL, was associated with LMS and explained 10.8% of the variation in the four years (S2 Table). Similar QTL for LMS was detected in the region of 2AL using SSR and EST-SSR markers in Yao et al [52], and Peng et al. [55] mapped over ten QTLs involving similar traits (PH, GNP, KGW and LMS) and defined two domestication factors in this chromosome arm. BE445667_6_B_Y_285, located on 6BL, was associated with LMS in the four years (S2 Table). QTLs involving similar traits (PH, GNP, KGW and LMS) were detected also in this region by Börner et al. [64]. The credible candidate QTLs may reside in a region containing several candidate genes conferring the examined traits. The candidate genes may have pleiotropic effects or several genes are clustered in the same region and acting on different traits [55]. Therefore, the candidate QTLs or the QTL-carried regions are potential reference regions for gene cluster. These QTLs and the clustering regions are worthy of further precisely QTL locating and gene detecting and cloning. QTL clusters in the genome As shown in Fig 2, most of the SNP associations were located on chromosomes 2A, 5A, 1B and 6B. The number of association effects in the A genome was larger than that in the B genome (Table 1, Fig 2). The genome A has longer domestication evolution history than the genome B in wheat, and thus probably has more genes controlling important adaptive traits [1,55]. Chao et al. [62] demonstrated that the genome A of wheat cultivars captured comparable number of adaptive trait genes under the natural and artificial selection and in the breeding programs. It is noteworthy that several associations co-locate in the same chromosome regions, even for the unrelated traits. There are several regions with association clusters especially on chromosomes 2A, 5A, 6A, 7A, 1B and 6B. For example, seven associations for PH, GNP, KGW and LMS are located on the proximal region C-2AL1-0.85 of chromosome 2 (S2 Table, Fig 2). Peng et al. [55] mapped over ten QTLs involving similar traits (PH, GNP, KGW and LMS) and defined two domestication factors in this chromosome arm. Yao et al. [52] detected similar QTLs for spike length, thousand kernel weight and spike number per plant in the same region. This region may be a convincible region for cluster of QTLs. On the chromosome 5A, we detected association clusters for LMS, GNP, GWP and SMS mainly in the short arm (5AS1-0.40-0.75) and the long arm (5AL12-0.35-0.78) (Fig 2). Kato et al. [65] and Gadaleta et al. [66] reported QTL clusters for yield components (thousand kernel weight, grain yield per spike and kernel number per spike) in similar region 5AL15-0.67-0.78. Peng et al. [55] mapped 19 QTLs involving 11 traits including LMS, GNP, GWP and SMS and also defined two domestication factors in this chromosome 5AL arm. In Gadaleta et al. [66], many SNPs mapped in the bin 5AS1-0.40-0.75 on the short arm have duplicated loci in bin 5AL5-0.46-0.55 on the long arm. The bin on 5AS may have undergone a duplication followed by an insertion into the 5AL of the same chromosome 5A. This may explain the similar associations mapped in the regions of 5AS1-0.40-0.75 and 5AL12-0.35-0.78 (Fig 2). Another significant cluster of associations for PH, GNP, KGW and LMS was detected on the long arm of chromosome 1B (1BL1-0.47-1.00) (Fig 2). Similarly, Börner et al. [64] detected QTLs for spike length and grain weight in this region. Similar result was reported by Cadalen et al. [56]. Peng et al. [55] mapped 8 QTLs involving 8 traits including LMS, GNP, GWP and SMS and defined one domestication factor in this 1BL chromosome arm. Phenomenon of QTL clustering was formally reported by Peng et al. [55] for domestication-related traits in wild emmer wheat. They defined a cluster of QTLs co-located in the same chromosome region as domestication syndrome factor [55]. Actually this phenomenon of QTL clustering was repeatedly observed, although not verbally using the term of 'QTL cluster', in wheat [52,56,[64][65][66][67][68]. In the present study, we demonstrated obvious QTL clusters represented by SNP-based associations in durum wheat (Fig 2). More and more studies tend to show that genes often reside in the genome in clusters. This seems especially true for resistance genes and QTLs for quantitatively inherited traits. The genetic mechanism for this universal phenomenon is the pleiotropic effect of genes [55]. Nevertheless, the genomic regions of QTL clusters need further validation by fine mapping and cloning of QTLs or genes. Genes for plant height Plant height (PH) is the key agronomic trait in wheat. We found six marker-trait associations for PH located on chromosomes 1A, 2A, 4B, 6A and 6B in four years. Each of the two markers, BF475120_6_B_67 and BF475120_6_B_Y_75, associated with pH explained >7.0% of variation in four years (S2 Table). In the chromosome region 6BL5-0.40-1.00 of BF475120 (http://wheat. pw.usda.gov/GG2/index.shtml), the SSR marker Xfbb250-6B was founded to be significantly associated with PH [56]. As shown in NCBI database (http://www.ncbi.nlm.nih.gov/), BF475120 is an EST sequence fragment derived from wheat salt-stressed crown cDNA library. The encoded protein of BF475120 has very high homology (E = 1e -53 ) with the protein GDSL esterase/lipase from Aegilops tauschii. One member of rice GDSL esterase family might be involved in lipid yield [69]. Esterase/lipase is involved in the entire process of plant growth and development. Furthermore, Börner et al [64] detected two QTLs for PH on the similar region 6BL5-0.40-1.00 of 6BL. Thus it is reasonable that BF475120 is associated with PH. The SNP marker BG312827_6_A_Y_305 associated with PH explained >5.2% of variation in the four consecutive years. The EST BG312827 was derived from T. monococcum early reproductive apex cDNA library (http://www.ncbi.nlm.nih.gov/). The encoded protein has very high homology (E = 1e -63 ) with the DNA replication licensing factor, a mcm5-A-like enzyme from Brachypodium distachyon (http://www.ncbi.nlm.nih.gov/). DNA replication licensing factor expressed in shoot apex and flower buds is essential to undergo a single round of replication initiation and elongation per cell cycle [70]. Arabidopsis MCM2 to MCM5 and MCM7 genes contain E2F consensus sites in their promoters. Their transcripts are elevated in plants expressing E2FA/DPA which not only regulates the mitotic cell cycle progression but also plays a role in the endocycle. It is a prerequisite for normal plant development [70][71][72]. Therefore BG312827 closely relates with apex cell division and growth, and thus undoubtedly associate with PH. Additionally, the marker BE405269_4_B_84 without exact site, located on chromosome 4B, was associated with PH in all the four years. This reproducible significant association is reliable. Rht-B1, located on chromosomes 4BS, is known to have major effect on PH [73]. The marker BE405269_4_B_84 was located in the same chromosome with Rht-B1, while the exacted region and relations need to be further explored. Genes for length of main spike For length of main spike (LMS), we found a total of 23 SNP associations located on chromosomes 1B, 2A, 3A, 4A 5A, 6A, 7A and 6B in the four years. These reproducible associations are significant and reliable. BF484028_5_A_Y_97 associated with LMS (S2 Table), and was mapped in the interval of 5AL10-0.57-0.78 (http://wheat.pw.usda.gov/GG2/index.shtml). Two genes Vrn-A1 and Fr1, are located in the same chromosome interval as BF484028_5_A_Y_97 [74]. Vrn-A1, a member of Vrn-1 genes, regulates flowering-time, an important criterion for regional adaptation and yield in all the cereal crops [75]. Vrn-1 gene is associated with heading date, spike length and grain yield. Vrn-A1 had a greater effect on spike length [75][76][77]. Furthermore, Vrn-1 completely links to MADS-box gene AP1 [78] which defines the pattern of where floral organs arise, as well as determines development of the floral meristem [79,80]. Therefore, the gene marked by BF484028_5_A_Y_97 may affect LMS through Vrn-A1 gene regulating vernalization. The marker BF474284_1_B_Y_357 associated with LMS explained >8.6% of the variation in the four consecutive years. BF474284 is an EST derived from wheat vernalized crown cDNA library. It has complete homology (E = 0.0) with TAVDAC2 gene located on the long arm of chromosome 1B in wheat (http://www.ncbi.nlm.nih.gov/). The Tavdac cDNAs express in meristematic tissues (floral tissues and embryos), regulate the mitochondrial functions during the period of floral development to embryo formation [81]. Therefore, Tavdac is indirectly related to floral development and embryo formation in some ways, e.g., regulating the mitochondrial functions. This explained why BF474284 was associated with LMS to some extent. Gene for number of spikelets on main spike For number of spikelets on main spike (SMS), we found a total of 22 significant associations in the four years. One reliable SNP marker BG314551_3_A_Y_162, significantly associated with SMS in three years, explained over 8.1% of the variation (S2 Table). This SNP was located in the bin 3AS4-0.45-1.00 on chromosome arm 3AS in the same region as Eps gene (earliness per se). This gene is usually responsible for the fine-tuning of wheat flowering time. RFLP markers linked with Eps explained significant variation of plant height, thousand kernel weight, kernel number per spike, and grain yield [82,83]. Thus BG314551_3_A_Y_162 represent a significant factor from early reproductive apex greatly impacting SMS. Candidate gene for grain number per plant Grain number per plant (GNP) is a key yield component factor in wheat. A total of 54 significant SNP associations were detected for GNP in the four years. Several reliable QTLs could be suggested for this trait ( Table 6, S2 Table). BF293541_4_A_Y_88 is located in the bin 4AL5-0.66-0.80 on chromosome arm 4AL (http://wheat.pw.usda.gov/GG2/index.shtml). This region was associated with spike length, spikelets density, grain number per spike [84]. The EST of BE498418_7_A_148 was also derived from pre-anthesis spike cDNA library and mapped on 7AL (C-7AL1-0.39). This EST has very high homology (E = 1e -104 ) with UDP-Dxylose epimerase 3 coded by UXE3 gene from UXE gene family in Hordeum vulgare (http:// www.ncbi.nlm.nih.gov/). The abundant transcript of HvUXE was possibly correlated to arabinoxylan deposition in cell walls in the starchy endosperm during grain development. There was a substantial increase in HvUXE1 and HvUXE3 mRNA levels at the differentiation stage of endosperm development [86,87]. The chromosome region of BE498418 was also proved to carry the QTL for grain weight [64]. This further confirms the association of BE498418_7_ A_148 with GNP. The EST of BG263521_2_A_61 mapped in chromosome bin C-2AS5-0.78 (http://wheat.pw. usda.gov/GG2/index.shtml), was also derived from wheat pre-anthesis spike cDNA library, and has very high homology (E = 2e -126 ) with putative serine/threonine-protein kinase WNK1 (http://www.ncbi.nlm.nih.gov/). WNK1 gene is member of WNK gene family, which involved in the regulation of flowering time in Arabidopsis [88]. Several QTLs for grain yield and kernel number per spike were detected within this region [89]. Therefore, the associations between BG263521_2_A_61 and GNP may be true. Gene marked by SNP BG263521_2_A_61 affects GNP by regulating flowering time just as WNK does. Candidate gene for the 1000-grain weight The 1000-grain weight (KGW) is another key yield component factor. A total of 7 significant associations between KGW and SNP markers mainly located in chromosomes 2A, 5B, 6A, 7A and 7B with R 2 >5.4%, were detected in all the four consecutive years (S2 Table). The SNP marker AY244508_5_B_Y_26, significantly associated with KGW and GNP and explained over 11% of variation, was located in the same region as AP1 and Vrn-B1. AP1 defines the genesis pattern of floral organs, as well as determines development of the floral meristem [79,80]. WAP1, a wheat APETALA1 homolog, plays a core role in the phase transition from vegetative to reproductive growth [90,91]. Therefore, associations of AY244508_5_B_Y_26 with KGW and GNP may be attributed to the role of AP1 and VRN1. BG605368_2_A_Y_310 was associated with KGW, and explained 9.71% of variation (S2 Table). As discussed above, BG605368_2_A_Y_310 was also associated with LMS in all the four years. The EST BG605368 was derived from wheat pre-anthesis spike cDNA library. It is highly homologous (E = 1e -127 ) with Exopolygalacturonase from T. urartu. Exopolygalacturonase expressed in pollen and young developing tissues, suggesting that they could be implicated in the cell wall modifications and related to cell elongation and/or expansion in these tissues [94]. BG605368 may be related to flower development. Several QTLs for grain weight and yield in the region (C-2AL1-0.85) of the EST were detected in previous study [64,95,96]. Therefore, the association between BG605368_2_A_Y_310 and KGW and LMS should be credible. Conclusions The previous studies indicated that both QTL analysis and association mapping are suitable and effective tools for mapping quantitative loci in wheat and barley [7,9,55,[97][98][99]. We detected 201 significant associations in total between SNP markers and 10 quantitative traits in durum wheat in four years. Some of the associations are corroborated by the previous QTL analyses, and further supported by the functions of the deriving ESTs and the homologous genes. The plausible QTLs represented by the associated SNP markers are generally clustered in specific chromosome regions of the wheat genome, especially 2A, 5A, 6A, 7A, 1B, and 6B chromosomes. Nevertheless, the associated SNP markers need to be further confirmed before they can be utilized in marker-assisted selection breeding programs [7,9,100].

v3-fos

2019-03-30T13:05:56.945Z

2015-04-23T00:00:00.000Z

196609377

s2

Response of rooted simmondsia chinensis cuttings to foliar fertilizers at different times of year under green-and-shade house conditions Jojoba (Simmondsia chinensis (Link) Schneider) is a new industrial crop being grown commercially in hot arid and semiarid regions of the southwestern United States.1 The primary product of this evergreen shrub is a unique liquid wax contained in the seed. This wax is used as a natural base for a wide range of cosmetic products, it has heat-resistant lubricating properties, and is potentially useful in the chemical industry. Nutrient requirements are important for good growth of rooted jojoba cuttings particularly when plants rooted under intermittent mist irrigation under greenhouse conditions. These nutritious elements could be secured for jojoba plants easily in the controlled conditions such as green house or shade house using foliar fertilization, especially when plants lose their nutritious contents from leaves due to regulated mist irrigation. Further, treating jojoba plants with different kinds of foliar fertilization in those controlled conditions makes plants grow well and reach a suitable size for planting early. Feldman2 reported that leaf contents of N, P and K were reduced after 12 weeks under intermittent mist and approached deficiency levels for jojoba plants. Therefore early growth following rooting was directly related to leaf concentration of the nutrients either macronutrients or micronutrients. Nursery fertilization with Osmocote (macronutrient fertilizers containing NPK @ 19-6-12 respectively) at either 1.48 or 2.97kg/m3 enhanced nursery growth over that of control liners.2 Macronutrients are essential for different activities in the plant such as N, which is one of the essential nutrients needed by plants mainly for buildup associated with high photosynthetic activity.3,4 Phosphorous (P) also is an important structural component essential for energy storage and transfer (ADP and ATP) for subsequent use in growth and reproductive processes.5 Potassium (K) has important role in increasing water uptake and consequently in cell expansion.3 Growth of young olive trees improved with NPK fertilization according to Bouranis et al.,4 Micronutrient fertilizers such as Zn, Mn and B were also related to early growth of rooted cuttings particularly during the spring. Deficiencies can be overcome if proper nutrition is applied to the liners in the nursery stage. However, time required to reach plantable size may then be increased.2 Generally, there is lack of information about the potential of rooted jojoba cuttings and its response to different kinds of foliar fertilization. Therefore, the target of these investigations was to study the effect of foliar fertilization of macronutrients (MA), micronutrients (MI) and sea algae extract (SAE) individually or in combinations at different times of year on the growth of rooted Simmondsia chinensis cuttings under green-andshade-house conditions. Introduction Jojoba (Simmondsia chinensis (Link) Schneider) is a new industrial crop being grown commercially in hot arid and semiarid regions of the southwestern United States. 1 The primary product of this evergreen shrub is a unique liquid wax contained in the seed. This wax is used as a natural base for a wide range of cosmetic products, it has heat-resistant lubricating properties, and is potentially useful in the chemical industry. Nutrient requirements are important for good growth of rooted jojoba cuttings particularly when plants rooted under intermittent mist irrigation under greenhouse conditions. These nutritious elements could be secured for jojoba plants easily in the controlled conditions such as green house or shade house using foliar fertilization, especially when plants lose their nutritious contents from leaves due to regulated mist irrigation. Further, treating jojoba plants with different kinds of foliar fertilization in those controlled conditions makes plants grow well and reach a suitable size for planting early. Feldman 2 reported that leaf contents of N, P and K were reduced after 12 weeks under intermittent mist and approached deficiency levels for jojoba plants. Therefore early growth following rooting was directly related to leaf concentration of the nutrients either macronutrients or micronutrients. Nursery fertilization with Osmocote (macronutrient fertilizers containing NPK @ 19-6-12 respectively) at either 1.48 or 2.97kg/m 3 enhanced nursery growth over that of control liners. 2 Macronutrients are essential for different activities in the plant such as N, which is one of the essential nutrients needed by plants mainly for buildup associated with high photosynthetic activity. 3,4 Phosphorous (P) also is an important structural component essential for energy storage and transfer (ADP and ATP) for subsequent use in growth and reproductive processes. 5 Potassium (K) has important role in increasing water uptake and consequently in cell expansion. 3 Growth of young olive trees improved with NPK fertilization according to Bouranis et al.,4 Micronutrient fertilizers such as Zn, Mn and B were also related to early growth of rooted cuttings particularly during the spring. Deficiencies can be overcome if proper nutrition is applied to the liners in the nursery stage. However, time required to reach plantable size may then be increased. 2 Generally, there is lack of information about the potential of rooted jojoba cuttings and its response to different kinds of foliar fertilization. Therefore, the target of these investigations was to study the effect of foliar fertilization of macronutrients (MA), micronutrients (MI) and sea algae extract (SAE) individually or in combinations at different times of year on the growth of rooted Simmondsia chinensis cuttings under green-andshade-house conditions. Plant material The investigations were carried out at Jojoba Naturals Company greenhouse and shade house, Sana'a, Yemen during the years 2013 and 2014. The plant material for these two experiments was the rooted cuttings of jojoba plants (10-12cm height), which had been rooted previously by the application of a suitable auxin (IBA) to semi-hardwood cuttings. These rooted cuttings were taken out from rectangular flat trays, which had been kept under intermittent mist propagation conditions for rooting duration, and were transferred to pots The importance of jojoba plants comes due to its multi uses and unique features. It is a desert plant, drought tolerant, salty resistant and adapts well in different types of soil. Jojoba plants produce liquid wax which meets global demand owing to its use in cosmetics and other important aspects of life. Production of high quantities of the jojoba liquid wax needs elite and active plants in the growth. Thus, the objective of these present investigations was to study the effect of fertilizers and time of spray on the vegetative growth of rooted jojoba cuttings. Two experiments were conducted in the greenhouse and shade house using rooted jojoba cuttings and different types of fertilizers such as macronutrients and micronutrients additional to some of sea algae extract either individually or in combinations at four times of year with 30 day intervals under greenhouse and shade house conditions. The results pointed out significant effect by the spray of micronutrient fertilizers compared to other type of fertilizers in both greenhouse and shade house in terms of height of plant and number of shoots and leaves per plant. The fourth spray of fertilizers gave the highest values of the different parameters studied. The interaction between time of spray and type of fertilizers did not differ significantly. Greenhouse condition was more favorable for growth of jojoba rooted cuttings than shade house. This study created an importance of foliar fertilization of jojoba plants by micronutrient fertilizers. Experiment 1 Rooted cuttings of jojoba plants were sprayed with different types of liquid fertilizers i.e., macronutrients (MA) (Bio 20; Omex, Agrifluids, Lmt., England), micronutrients (MI) (Omex; micromax, Agrifluids, Lmt., England), and sea algae extract (SAE) (Gaefol AL-Khair, Chema Industries, Egypt) (use to strengthen root system and increase nutrient uptake) individually and in combinations viz, MA+MI, MA+SAE, MI+SAE, MA+MI+SAE additional to the control (untreated plants), each combination had the same ratio in volume. Plants were kept under greenhouse conditions under temperature range less than 35 ͦ C by using Pad Cooling System and the humidity range was not more than 60%. Both of them were measured with Hygro-Thermometer device (Jumbo Display Hygro-Thermometer, USA). Treatments were achieved in four periods, starting time was the mid-Dec then mid-Jan, mid-Feb and the mid-March with 30 day intervals during the years 2013 and 2014. Evaluation of obtained data of jojoba plants for different parameters such as height of plant and number of shoots and leaves per plant were calculated for four times, but starting time was from the mid-Jan. to the mid-Apr. Studying only of these three parameters is owing to that these characters give fast reading and perfect view regarding important aspect of plant i.e., growth of plant. Experiment 2 Rooted cuttings of jojoba plants were sprayed with the same fertilizers and its combinations in different times of the year as indicated in Experiment 1 but under shad house conditions (55% shade). The temperature range in this period was between 11-18⁰C whereas humidity range was between 40-45%. Evaluated parameters were as in Experiment 1. Experimental design and data analysis Experiments were conducted in a factorial completely randomized block design (FCRBD) with three replicates, each with 10 rooted cuttings per replicate. Obtained data of different parameters of rooted jojoba cuttings were subjected to statistical analysis according to Jomez et al. 6 and Sastry. 7 ANOVA values were obtained with Opstat1 software (O.P Sheron, Programmer, Computer Section, CCS HAU, Hisar, India) and means were separated with least significant difference (LSD) at P = 0.05. Further, comparison of greenhouse and shade house effect on the all parameters studied was done independently according to Systat software, version 10, SPSS, Ine. 2000, by calculating T-test at P=0.01%. Results and discussion Growth of rooted jojoba cuttings as affected with different types of fertilizers and spraying times under green house conditions (Table 1). Significantly, the height of plant increased after the first spray by the use of fertilizers in the mid-Dec. to the last spray in the mid-March (13.58 cm to 19.04cm respectively) irrespective of the type of fertilizers. This increase in height of plant is a normal result because of the consumed time between first and last spray which took around 120days in which the growth of plant was significantly increased. Similarly, the growth of other parameters studied such as number of shoots and leaves per plant took place. This reason is suitable to interpret the result of all parameters studied regarding why the last spray of fertilizers gave the highest value for all characters in both conditions of greenhouse and shade house. Among various types of fertilizers and their combinations, micronutrients yielded significantly the highest height of rooted jojoba cuttings (18.58cm) compared to other type of fertilizers except with macronutrient fertilizers, there was no significant difference (18.50cm). The least height of rooted jojoba cuttings(13.29cm) was recorded with the spray comprising MA+MI+SAE. Number of shoots per rooted jojoba cutting did not differ significantly at all time of sprays regardless of the type of fertilizers used. The greatest number of shoots per plant (4.66) was observed with the spray of micronutrient fertilizers in comparison with other type of fertilizers whereas the least number of this character (2.50) was recorded with spray containing MA+MI+SAE. Regarding number of leaves per plant, it was similar to those mentioned in the first and second parameters in which the highest number of leaves per plant was seen after spray of fertilizers in the mid-March whereas the lowest number of leaves per plant was noticed after the first spray of fertilizers in the mid-Dec. irrespective of the time of spray of fertilizers. Significantly, the greatest number of leaves (27.75) was recorded by the spray with micronutrient fertilizers but the least number of leaves (11.25) was gained by the spray with treatment containing MA+MI+SAE. The ideal type of foliar spray fertilizer was the micronutrient fertilizer in majority of parameters studied, this might be due to bleaching of some nutritional elements from plant leaves either macro or micro elements. Therefore, the plant element's requirement was higher from the micronutrient fertilizers over other type of fertilizers and this result appeared clearly almost in all parameters investigated. Regarding why the spray containing MA+MI+SAE yielded the lowest value almost in all parameters studied, this could be due to weakness uptake of elements by plant foliage when added at one time, perhaps some clashing was created among cations and anions of different elements either macro or micro elements. The data of interaction between time of sprays and type of fertilizers regarding height of plant and number of shoots and leaves per plant was illustrated (Figure 1-3). The results indicated differences among different values of the various parameters but with no significant difference. Table 2 shows the effect of time of spray and type of fertilizers in terms of height of plant, number of shoots and leaves per plant on the growth of rooted jojoba cuttings under shade house conditions. The greatest height of plant (22.91cm) was obtained markedly after the fourth spray of fertilizers in the mid-March irrespective of the type of spray of fertilizers, this was followed by the third spray of fertilizers (21.50cm) which was in the mid-Feb. and then the second spray of fertilizers (18.29cm) which was in the mid-Jan. whereas the lowest value of this character was recorded after the first spray of fertilizers (13.91cm) which was in the mid-Dec. The fertilizer factor affected significantly the height of jojoba rooted cuttings. The spray of rooted jojoba cuttings by the use of micronutrient fertilizers yielded higher height of plant than the plant sprayed with SAE and MA+MI+SAE The data of interaction between time of sprays and type of fertilizers regarding height of plant and number of shoots and leaves per plant was illustrated (Figure 4-6). The results pointed out differences among different values of the various parameters but with no significant effect. A comparison was made between greenhouse and shade house conditions independently on the all parameters studied after treated jojoba rooted cuttings with different fertilizers used (Table 3). It is clearly seen that green house condition was more favorable for the growth of plant particularly for the height of plant and no. of shoots and leaves per plant of jojoba rooted cuttings than shade house condition. On the other hand, the difference in means between greenhouse and shade house conditions for all parameters investigated pointed out that the green house condition was better with significant effect for height of plant or high significant effect for number of shoots and leaves per plant at P=0.01% in comparison with shade house conditions. This superiority of greenhouse condition versus shade house condition might be attributed to the nature of conditions inside greenhouse; where optimal temperature and relative humidity are supplied to plants and there was no loss in the fertilizers' quantities when plants sprayed due to controlled condition. In general, foliar fertilization using micronutrients fertilization was the best compared to other types of fertilizations with its combinations in the greenhouse over shade house conditions. Comparisons were made between G & S in each column, *means presence of significance between difference in means, and **means presence of high significance between difference in means according to T-test at P=0.01%, SD: Standard Deviation. Conclusion Jojoba plants seem to have better growth after the forth spray in the mid-March by the use of micronutrient fertilizers in both green-andshade house conditions in comparisons with other time of sprays and type of foliar fertilizations. Fertilization of plants in the greenhouse was better than in the shade house. The findings could be of great value to the nursery men to spray their rooted jojoba cuttings by foliar application of micronutrient fertilizers especially when plants rooted under mist irrigation system.

v3-fos

2017-10-21T03:17:30.366Z

2015-08-01T00:00:00.000Z

14613197

s2

Effect of replacing oat fodder with fresh and chopped oak leaves on in vitro rumen fermentation, digestibility and metabolizable energy Aim: A study was conducted to evaluate the effect of replacing oat fodder (OF) with fresh oak leaves (FOL) or chopped oak leaves (COL) on rumen fermentation and digestibility through in vitro gas production technique (IVGPT). Materials and Methods: Nine different diets were prepared by mixing OF with oak leaves (either FOL or COL) in different ratios (100:0, 75:25, 50:50, 25:75, and 0:100). The rations were evaluated through Hohenheim IVGPT with 200 mg substrate and 30 ml of buffered rumen liquor. All the syringes were incubated at 39°C for 24 h in buffered rumen liquor of cattle. After 24 h, the total gas production was recorded, and the contents were analyzed for in vitro methane production, protozoa no. and ammonia-N. Results: Chopping (p<0.01) reduced the tannin fractions as well as non-tannin phenol. Increase in levels of oak decreased total gas production, methane, organic matter (OM) digestibility, and metabolizable energy (ME) values. The polyphenol content of the substrate did not show any significant difference on the protozoal count. Conclusion: In vitro studies revealed that the addition of oak leaves reduced the methane production and ammonia nitrogen levels; however, it also decreased the OM digestibility and ME values linearly as the level of the oak leaves increased in the diet. Chopping was effective only at lower inclusion levels. Further studies, especially in vivo studies, are needed to explore the safe inclusion levels of oak leaves in the diet of ruminants. Introduction Availability of fodder among Asian countries particularly with countries like India is not adequate to meet the ever growing livestock population. There is a great need to explore new feed resources to meet this deficit without competing with food chain [1]. Tree fodders are the alternate source of small ruminant feeds to that of conventional green fodders which have the potential to mitigate the gap between demand and supply of feeds [2]. Tree fodders have similar nutritive value as that of leguminous fodders [3], which plays an important role in the nutrition of grazing animals where there is less scope of conventional fodders. Oaks (Quercus spp.) are one such tree fodder which is the dominant, climax tree species of the moist temperate forests of the North Western Himalayan region (NWHR). During extreme climatic condition in this agro-climatic zone, when ruminants cannot go out to graze oak leaves take cares the nutritive requirement of such animals. Even though the oak leaves are abundantly available in the NWHR, toxicity problems exist due to sole feeding of oak leaves in the diet of ruminants [4]. Moreover, previous workers reported oak toxicity even on feeding oak leaves partially in the diets of the ruminants [5]. Quercus species are reported to contain the high levels of hydrolysable tannins (HT) which are the main reason for the toxicity in the livestock. HT undergo acid and microbial hydrolysis to release simple phenolics which there by cause toxicity [6,7]. Chopping of the oak leaves is the simple procedure by which the polyphenol content of the oak leaves can be reduced. Chopping helps the phenolic oxidases to get exposed with tannins which results in tannin reduction. So, the present study was undertaken with two objectives: (i) To identify the safe inclusion level of oak leaves in replacing the conventional high quality oat fodder (OF) for feeding ruminants through Hohenheim in vitro gas production technique (IVGPT) (ii) To explore the additional benefit of chopping oak leaves on reducing the polyphenol content and on rumen metabolism through IVGPT. Ethical approval The study was undertaken after taking necessary approvals from the Institutional Animal Ethical Committee of the University. Sampling of the oak leaves The fresh mature oak (Quercus leucotrichophora) leaves were manually lopped from the nearby forest area of Palampur, Kangra District, Himachal Pradesh, India. A part of the lot was chopped by a Chemical analysis The chemical composition of the FOL and COL, and OF were determined by the method of AOAC (2000) while fiber fractions were estimated as per the methods suggested by Van Soest et al. [8]. Polyphenol profile of oak leaves was estimated by the method of Makkar [9]. Total phenols (TP) and non-tannin phenols (NTP) were estimated by Folin-Ciocalteau method in combination with polyvinylpolypyrrolidone, with tannic acid as a reference standard [9]. The condensed tannins (CT) were estimated by using butanol-HCl method. Rumen liquor sampling Rumen liquor was collected from two rumen cannulated cattle (body weight = 220 kg), strained through a four-layered muslin cloth and pooled together which was used as an inoculum source for in vitro studies. The donor animals were fed 60% wheat straw and 40% concentrate. Five different diets were prepared mixing OF with oak leaves (FOL and COL) in the ratios of 100:0, 75:25, 50:50, 25:75, and 0:100 and evaluated through Hohenheim IVGPT suggested by Menke et al. [10] with 200 mg substrate and 30 ml of buffered rumen liquor. All the syringes were incubated at 39°C for 24 h in buffered rumen liquor of cattle. After 24 h, the total gas production was recorded, and the contents were analyzed for in vitro methane production, protozoal count, and ammonia-N. In vitro methane production was estimated in gas-liquid chromatography (gas chromatography [GC], Nucon 5765, Nucon Engineers, New Delhi, India) equipped with a flame ionization detector. The column was of stainless steel packed with a propak-q (length 1.8 m; o.d 0.3 mm; i.d 2 mm; mesh 80-100). The analytical condition of GC was carrier gas N 2 flow 40 ml/min, H 2 30 ml/min, air 300 ml/min, and temperature range at injection port was 150°C, column 60°C, and at detector was 130°C. The peak was compared with the standard (50% CH 4 and 50% CO 2 from SPANCAN calibration gas, Spantech, Surrey, UK) and the analysis and calculation used the Aimil chromatography data system (WINACDS, New Delhi, India). Metabolizable energy (ME) values of samples were calculated by a formula derived by Menke and Steingass [11]. The microbial protein and digestibility were calculated with the 400 mg substrate incubated in 40 ml of buffered rumen liquor. ME (MJ) = 2.20 + 0.136* gas (ml/200 mg DM) + 0.0057 * CP + 0.0029*EE Microbial protein was estimated by the following formula: Microbial protein (mg) = TD (mg) -(2.25 × net gas volume) Whereas, TD = True digestible matter (substrate incubated−NDF) The in vitro gas production was completed in three runs (statistical replicates) with each sample incubated in triplicate (analytical replicates). Statistical analysis The analytical replicates were averaged prior to statistical analysis with each run being the statistical replicate. The data were analyzed using one-way analysis of variance procedures (SPSS base 7.5 for windows [1997]) and the difference between the treatments means were compared by Duncan's multiple range tests. Results are presented as means and standard error of means. Treatment effects or differences were considered significant if p<0.05. Chemical composition The chemical composition of the oat fodder (OF) and oak leaves are presented in the Table-1. The chemical composition of OF at the early maturity was comparable to that reported by earlier workers [12][13][14][15][16]. The organic matter (OM), crude protein (CP), ether extracts (EE), nitrogen free extract (NFE), and Crude fibre (CF) content of the OF was 88.4, 14.7, 3.66, 46.47, and 23.57%, respectively. There was no significant difference between the values in the OM, CP, EE, NFE, and CF content between FOL and COL. The chemical composition of the oak leaves was comparable to that reported by earlier workers [17][18][19][20][21]. Polyphenol content Polyphenol content (on DM basis) of the OF, and the oak leaves are presented in the Table-2. There was 10.1, 9.43, 10.3, 7.0, 11.1% reduction in the TP, NTP, TT, CT and HT values due to chopping of the oak leaves. There was a significant difference in the TP, NTP, TT, and HT content between the FOL and COL. The reduction of the polyphenol content of the oak leaves is due to the higher susceptibility of COL to oxidative enzymes and conversion of higher polymerization leading to inert phenols. The degree of susceptibility of HT to the oxidative enzyme is relatively more than the other polyphenol [22,23]. Total gas production The total gas production per 200 mg substrate was higher at the 100% oat concentration (41.17 ml). Increase in levels of oak decreased total gas production (ml/200 mg DM) (Graph-1) at a decreasing rate. Gas production was more in the COL group than the FOL at the same ratio. There was a significant difference (p<0.01) between the values of 25% oak leaves in COL and FOL. The gas produced in the syringes is largely due to acetate and butyrate, and lower gas production is associated with propionate production. Easily fermentable carbohydrates yield higher propionate therefore leading to less gas. The gas production is negatively related with the neutral detergent fiber (NDF) content and positively with the starch content. Tannins especially HT at higher levels are toxic to the rumen microbes, therefore, leading to less gas production [24]. Methane The methane production was maximum in the 100% OF level (16.9 ml/200 mg). As the concentration of oak leaves increased in the substrate, the methane production decreased (Graph-2). The effect on methane production was parallel to decreased total gas production. Even at the 25% inclusion level of oak, there were 5.2% and 21.24% reduction of methane in fresh and chopped oak, respectively. There was significant (p<0.01) difference between the methane produced in COL and FOL group (except at 25% oak). There was less production of methane in COL than the FOL of the same ratio. Studies using CT-containing forages such as big trefoil (Lotus peduncalatus, 53 g/kg CT), sulla (Hedysarum coronarium, 27-68 g/kg CT), red clover (Trifolium pretense, 3 g/kg CT), and Sericea lespedeza (Lespedeza cuneata, 177 g/kg CT) reported reductions in CH 4 emissions [25][26][27][28]. Tannins present in different plants such as Calliandra calothyrsus [29] and Onobrychis viciifolia [30] and Populus deltoides [31] reduced methane production under in vitro conditions. Similar results were reported by Woodward et al., [26] that CT containing H. coronarium forage reduced methane production per kg DM intake (19.5 vs. 24.6 g) in grazing cows. Similarly, sheep fed L. corniculatus silage reduced methane production [32]. Waghorn et al. [25] reported 16% reduction in methane production in lambs fed on CT containing Lotus pedunculatus (lotus). Microbial protein synthesis Microbial protein synthesis was estimated through equations with the help of total gas production. Microbial protein was higher at the 100% OF (164.69 mg), however, as the oak leaves percentage increased in the substrate incubated the production of microbial protein decreased linearly. There was no significant difference between the values in COL and FOL. The linear reduction in the microbial protein was due to the toxic effect of the polyphenols to the rumen microbes [33]. For microbial protein synthesis synchronization of the rate of degradation of N and carbohydrate components in the rumen is important Graph-1: Total gas (ml/200 mg dry matter). Graph-2: Methane production (ml/200 mg dry matter). for efficient utilization of rumen ammonia nitrogen. Therefore, there is a reduction of microbial protein synthesis with respect to ammonia nitrogen. True dry matter (DM) and OM digestibility Both the DM and the OM digestibility were higher in the 100% OF (81.14 and 81.73%, respectively). Both DM and OM digestibility followed the same trend, i.e. when the percentage oak leaves increased the digestibility decreased (Graph-3). There was a significant difference between the values at 50% oak leaves in COL and FOL. The reduction in the digestibility is attributed to the high tannin and lignin content in the diet [34,35]. Tannins reduce digestibility by reducing the activity of rumen microbes, by binding with rumen enzymes, or by binding with feed components [36]. Particularly in case of tree leaves, tannins are present in NDF and acid detergent fiber (ADF) fractions in certain amounts which are tightly bound to the cell wall and cell proteins and it is believed to be involved in decreasing digestibility [37]. Ammonia nitrogen The ammonia nitrogen (mg/30 ml) in the different dietary combination is presented in Table-3. The ammonia nitrogen (mg/30 ml) produced was maximum in the 100% OF (6.6). There was a significant difference (p<0.01) in the ammonia nitrogen production in all the ratios between COL and FOL groups (except at 25%). Tannins are known to bind with proteins, which is the key reason for the reduction of rumen ammonia concentration [38]. Many authors have indicated that the principal effects of tannins in ruminal fermentation include a reduction in proteolysis of dietary protein and subsequently lower concentrations of ammonia in rumen fluid [39,40]. All ammonia concentrations were higher than the 100 mg/L reported by Van Soest et al. [8] as optimal for the efficiency of amino acid synthesis and microbial growth. Although that value might depend on a number of factors, such as the amount of available fermentable energy [41], ammonia concentrations were probably adequate for optimal rumen fermentation in all cases. Protozoal count The number of protozoa in the different ratio of oat: Oak is represented in the Table-3. The polyphenol content of the substrate did not show any significant difference on the protozoal count. Similar results were reported with Q. leucotrichophora [20]. Tavendale et al. [42] suggested that inhibition of growth of methanogens is due to the bacteriostatic and bactericidal effects of CT. Since some of the methanogens are ecto-and endo-symbiotically associated with protozoa, a reduction in methanogens would probably affect the protozoal population [43]. The effects of tannin on the protozoal number are conflicting, some authors claim in the reduction of protozoal number with tannin supplementation, but others claim no effect. Monforte-Briceno et al. [44] studied the defaunating properties of 15 tree fodders containing tannins, but the inhibitory effect on protozoa was observed in Acacia farnesiana, C. calothyrsus and Lysiloma latisiliquum. Tannins present in tanniferous plants are not equally efficient in reducing the protozoal count. ME values The calculated ME (MJ/kg DM) values were higher in the 100% OF group (7.9). A similar trend Graph-3: Effect of replacing oat fodder with fresh or chopped oak leaves on in vitro digestibility. was seen as that of the gas production in oak groups, i.e. as the % oak increased in the substrate, the ME values decreased. The ME values of the fresh oak group was 7, 6.1, 5.07 and 4.09 MJ/kg DM and in chopped oak group was 7.34, 6.41, 5.09 and 4 MJ/kg DM at 25, 50, 75 and 100% respectively. The ME values of 100% FOL and COL were also estimated by Ajith [20], ME value of FOL and COL reported by him were 5.76 and 5.52 MJ/kg DM. It is well-known fact that NDF, ADF and CT are negatively associated with ME of feedstuffs [45]. Conclusion The current in vitro study revealed that the addition of oak leaves reduced the methane production and ammonia nitrogen levels; however, it also decreased the OM digestibility, microbial protein synthesis and ME values linearly. Oak tannins didn't have any effect on the protozoal number. Even though, there was a reduction in polyphenol content due to chopping, it was effective only at lower inclusion levels (i.e. 25% and 50%). However, comprehensive in vivo studies with animal hosts need to be undertaken to evaluate the sustainability of oak leaves supplementation to mitigate rumen methanogenesis without detrimental effects on the animal as a whole. Authors' Contributions RB and AK planned and supervised the entire research work. KR and RJ carried out the experimental work and laboratory analysis. BS and GM prepared the manuscript along with data analysis. All authors read and approved the final manuscript.

v3-fos

2019-04-04T13:06:13.403Z

2015-03-01T00:00:00.000Z

93147627

s2

Using electrical signals of microbial fuel cells to detect copper stress on soil microorganisms A method based on microbial fuel cells (MFCs) was used to evaluate the effects of copper (Cu2+) on soil microorganisms. Soil spiked with 50–400 mg kg−1 of Cu2+ as CuCl2 was incubated for 24 hours before being packed into the MFC anode chambers and assayed for dehydrogenase activity (DHA), substrate‐induced respiration (SIR) and microbial biomass carbon (Cmic). Soil was amended with 5% (w/w) glucose to accelerate ‘start‐up’ and improve power generation, followed by 150 hours of operation. Anode biofilm and soil was extracted to recover total nucleic acids and the 16S rRNA gene was subjected to PCR‐DGGE, sequencing and phylogenetic analysis. Results showed that increases in soil Cu2+ concentrations reduced voltage and postponed start‐up. The quantity of generated electrons within 48 hours was 32.5 coulomb (C) in the without‐Cu control and decreased with increasing Cu2+ concentrations (11.7, 7.7, 2.0 and 1.3 C under 50, 100, 200 and 400 mg kg−1 Cu2+, respectively). Cyclic voltammetry identified decreased soil electrochemical activity with increasing Cu2+ concentrations. The results indicate that Cu2+ reduced electrical signals by inhibiting the electrochemical activity, metabolic activity and biomass of microorganisms. The 16S sequences of recovered anodic bacteria were assigned to Firmicutes, including Bacillaceae, Acetobacteraceae, Clostridium, Bacillus and Sporolactobacillus. In general, the DGGE band intensity of anodic bacteria decreased with increasing Cu2+ concentrations, except for bands assigned to Firmicutes and Bacillus, which increased with increasing Cu2+ concentrations. We suggest that the short‐term electrical signals generated from MFCs with contaminated soil can be used to assess the toxic effect of heavy metal pollutants on soil microorganisms. Introduction A microbial fuel cell (MFC) is a device that converts chemical energy from organic compounds into electricity by using electrogenic bacteria as biocatalysts (Bond & Lovley, 2003). A dual chamber MFC usually consists of an anode chamber and a cathode chamber separated by a proton exchange membrane. In the anode chamber, electrogenic bacteria degrade organic compounds and transfer electrons to the anode. The electrons then flow through a conductor to the cathode, where they bind with an acceptor such as oxygen or ferricyanide and thus current is generated. Because the current is closely correlated with the metabolic activity of electrogenic bacteria, MFC-based biosensors have been developed to monitor the biological oxygen demand in wastewater (Di Lorenzo et al., 2009), heavy metal toxicity (Stein et al., 2010(Stein et al., , 2011 and acid toxicity (Shen et al., 2012). In MFC-based biosensors, a culture of mixed electrogenic bacteria from other active MFCs (Stein et al., 2010) or a pure culture of Geobacter or Shewanella have been used to generate a current with growth medium in the anode chamber (Dávila et al., 2010). During the sensing process, wastewater or chemicals were added to the anode chamber after electrogenic bacteria enriched on anode and generated stable current (Shen et al., 2013). The current decreased with increasing pollutant toxicity and increased with increasing BOD concentrations (Kim et al., 2008). It is known that soil can be used to generate electrical power in MFCs and a diversity of electrogenic bacteria have been detected. Ringelberg et al. (2011) found that a variety of soils had electrogenic activity and bacteria belonging to -and -Proteobacteria and Firmicutes dominated anodic biofilms in soilbased bio-electrochemical systems. Ishii et al. (2008) found that Clostridiales, Chloroflexi, Rhizobiales and Methanobacterium dominated the anode biofilm in MFCs inoculated with soil from a rice field. Kaku et al. (2008) operated plant-MFCs in which soil organic carbon and rice exudates served as energy resources, and found that Natronocella acetinitrilica, Beijerinckiaceae bacterium and Rhizobiales bacterium were dominant on the anode. In recent studies, more than 30 isolates belonging to Firmicutes and -, -, -and -Proteobacteria were verified as electrogenic bacteria (da Rosa, 2011) and many more microorganisms detected on the anode by molecular methods were thought to have electrogenic activity. As electrogenic bacteria are widely distributed in soils and soil bacteria are sensitive to environmental changes, stress on soil microbial communities may inhibit electrogenic activity. For example, Deng et al. (2014) found that the voltage generated by a soil varied synchronously with incubation temperature. We hypothesize that the electrical signals produced by electrogenic bacteria in soil can be used to assess heavy metal toxicity in soil. In the present study, we used soil with different amounts of Cu 2+ added and subjected it to a series of measurements that included glucose dehydrogenase activity (DHA), substrate-induced respiration (SIR), microbial biomass carbon (C mic ) and the electrical signals of MFCs, including voltage and the quantity of electrons. The DHA, SIR and C mic characteristics are sensitive to environmental changes and are conventional bioindicators of soil pollution (Wang et al., 2007). In our study the monitoring process is different from that for wastewater. The pure culture electrogenic bacteria and medium are unnecessary. They increase cost and the medium, if it were to be discharged, may lead to pollution. Instead, we used the endogenous soil electrogenic bacteria and, because soil usually contains adequate nutrients, we only amended soil with glucose to minimize the 'start-up' time for power generation and to improve the voltage output (Kim et al., 2000). A regression analysis was conducted to compare responses between the electrical signals, DHA, SIR and C mic to Cu 2+ toxicity. The effect of Cu 2+ exposure on microbial community structure on the anode and in the soil was assessed by using DGGE, sequencing and phylogenetic analysis based on the 16S rRNA gene. The intent of this study was to develop a MFC-based method to detect heavy metal toxicity in soil microorganisms. Soil sampling and chemical analysis Soil samples were collected in October 2012 at a depth of 0-20 cm from a broad-leaf forest at Nanjing Normal University, Nanjing City, China. The climate is sub-tropical and wet with an average annual precipitation of 1100 mm and a mean annual temperature of 15 ∘ C. After sampling, the soil was gently separated by hand to preserve soil structure, passed through a 2-mm mesh and thoroughly mixed. Part of the sieved soil was air-dried for physiochemical analysis. Soil texture was determined with the pipette method (Gee & Bauder, 1986). Total carbon and total nitrogen were measured with an elemental analyser (Vario EL III, Elementar, Hanau, Germany). Soil pH was measured at a soil:water ratio of 1:2.5 (w/v). Total Cu and total iron (Fe) in soil were determined using inductively coupled plasma (Prodigy, Leeman Labs Inc., Hudson, NY, USA). Soil electrical conductibility (EC) was determined with an electrical conductibility meter (DDSJ-308F, INESA Scientific Instrument Co., Ltd, Shanghai, China). The soil is categorized as an Alfisol (IUSS Working Group WRB, 2007). The soil physiochemical properties were as follows: soil texture, clay loam; total carbon, 18.1 mg g −1 ; total nitrogen, 0.62 mg g −1 ; soil pH, 7.57; total Cu, 41.1 μg g −1 ; total Fe, 29.4 mg g −1 ; EC, 76.6 S cm −1 . Copper stress experiment The soil was divided into five aliquots, each with 600 g soil (dry mass). Thirty millilitres of CuCl 2 solution was added to each aliquot to yield 50, 100, 200 and 400 mg Cu 2+ kg −1 dry mass soil at a final soil moisture content of 18%. The remaining aliquot received 30 ml distilled water to serve as a control. The soil of each aliquot (600 g) was placed into a vial (1 litre) and incubated in the dark at 26 ∘ C for 24 hours. To help maintain the moisture content, a lid was placed loosely on each vial. After incubation, each soil aliquot was thoroughly mixed and equally divided into three sub-aliquots (200 g for each) as three replicates before the measurements of electrical signals, SIR, DHA and C mic . MFC set up Fifteen dual chamber MFC reactors were constructed in oroglas ® . An MFC reactor consisted of an anode chamber and a cathode chamber (each chamber with a dimension of 6 × 6 × 6 cm 3 and a working volume of 170 ml) separated by a Nafion ® cation exchange membrane ( Figure 1). The anode and cathode were made of carbon felt with the same area of 16 cm 2 (4 × 4 cm 2 ), fixed in parallel and separated by the cation exchange membrane. The distance between electrodes was 6 cm. The cathode chambers of the 15 MFC reactors were filled with 50 mm potassium ferricyanide (in 50 mm phosphate buffer solution (PBS)). The 15 reactors were randomly divided into five groups (three MFCs per group) according to Cu 2+ concentration (control, 50, 100, 200 and 400 mg kg −1 ). One hundred and fifty grams (dry mass) of contaminated or control soil was amended with 5% (w/w) glucose and packed into one anode chamber. Distilled water was added to the anode chamber to keep soil saturated. The anode and cathode were connected to a 1000 Ω external load by titanium wire. The 15 MFCs were operated at 26 ∘ C in a constant temperature incubator. The voltage was recorded with a data acquisition module every 10 minutes for 150 hours. To determine whether power originated from the microbial process or chemical processes, the voltage of an MFC reactor with uncontaminated soil sterilized by the chloroform fumigation method (Wolf et al., 1989) was recorded. Electrochemical activity The electrochemical activity of soil from the anode chambers was examined by cyclic voltammetry (CV) following 150 hours of operation. Briefly, 50 g of Cu 2+ -spiked soil from the MFC anode chamber and soil sterilized by CHCl 3 fumigation were collected and centrifuged at 1400 g for 10 minutes. The extracted soil solution was filtered through 0.45-μm pore size membrane and aerated with nitrogen gas for 10 minutes. Cyclic voltammetry measurement of the extracted soil solution was conducted with a potentiostat (CHI 1040 C, Shanghai Chenhua, Shanghai, China) with a three-electrode configuration consisting of the working electrode (glasses carbon), a counter electrode (platinum wire) and an Ag/AgCl reference electrode. The scanned potential between −1 and +1 V (against standard hydrogen electrode (SHE)) was performed at a scan rate of 50 mV s −1 under quiescent conditions with a working volume of 5 ml. DHA, SIR and C mic Values of DHA were determined according to Casida et al. (1964). Briefly, 1 g soil (dry mass equivalent) was combined with 2 ml 1% (w/v) 2, 3, 5-triphenyltetrazolium chloride (TTC) as a substrate. The triphenylformazane (TPF) produced was determined by spectrophotometry at 485 nm. Measurement of SIR was according to Lipson et al. (1999). Glucose was added to 10 g of soil (dry mass equivalent) in a 250-ml vial to give a final concentration of 30 mg g −1 dry soil. The vials were sealed and incubated for 12 hours at a constant 26 ∘ C. The CO 2 was trapped in 0.2 m NaOH solution in a 15-ml flask placed inside the 250-ml vial and determined by titration with 0.05 m HCl following the addition of 1 m BaCl 2 . C mic was determined using the protocol described by Vance et al. (1987). Ten grams of soil (dry mass equivalent) were fumigated with ethanol-free CHCl 3 and extracted in 0.5 m K 2 SO 4 . The organic carbon in the soil extract was measured with an automated TOC analyser (TOC-VCSN, Shimadzu, Kyoto, Japan). DNA extraction and PCR After the 150-hour operation of MFCs, a piece (1.5 × 1.5 cm 2 ) of the anode and 0.5 g of soil from the anode chamber were collected. The anode was rinsed with sterilized deionized water to remove soil before DNA extraction (He et al., 2009). The genomic DNA of the collected anode and soil was immediately extracted by using the Fast DNA SPIN kit for soil (BIO101, MP Biomedicals, Carlsbad, CA, USA) and following the manufacturer's instructions. The purity and the quantity of the DNA were determined by a nanodrop ND-1000 UV-Vis spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) at 230, 260 and 280 nm. DGGE, sequencing and phylogenetic analysis The DGGE was performed with 8% polyacrylamide gel with a denaturing gradient of 40-80% (100% denaturant = 7 m urea, 40% (v/v) formamide) using the Bio-Rad Protean II System for 15 hours at a constant voltage of 100 V and a constant temperature of 60 ∘ C in 1 × TAE buffer. After electrophoresis, the gel was stained for 30 minutes with 1:10 000 dilution SYBR™ Green I nucleic acid gel stain (Invitrogen, Shanghai, China). The stained gel was immediately photographed under UV light with the Gel Doc XR + gel imaging system and digitized with Quantity One (version 4.4.0) software (Bio-Rad Laboratories, Hercules, CA, USA). Eleven dominant bands were carefully excised from the DGGE gel and DNA was eluted by incubating each band in 40 μl sterilized distilled MilliQ water overnight at 4 ∘ C. Five microlitres of eluted DNA was used as a template for PCR amplification, using the same primers (341f/907r) and thermocycling programmes as described above. PCR products were cloned using the pEASY™-T3 Cloning Kit (TransGen Biotech, Beijing, China) according to the manufacturer's recommendations, and transformed into trans1-T1 phage-resistant chemically competent E. coli cells (TransGen). Transformed colonies were screened for inserts of the correct size by PCR amplification with the specific primers M13F (TGT AAA ACG ACG GCC AGT) and M13R (TCA CAC AGG AA ACA GCT ATG AC). Sequencing of correct colonies (five replicate colonies for each band) was carried out with vector primer set M13F/M13R (Invitrogen, Shanghai, China). Vector sequences were removed by using DNASTAR Lasergene 7.1. The gene sequences of the DGGE bands were subjected to taxonomic assignments with reference sequences using Blastn (http://ncbi.nlm.nih.gov/blast). Phylogenetic analysis was performed using a neighbour-joining algorithm and distance calculation by MEGA4. Sequences that were 97% or more identical were considered as a unique operational taxonomic unit (OTU). All the sequences determined in this study were deposited in the GenBank database and have been assigned accession numbers KF186478 to KF186493. Statistical analysis All statistical tests were performed using SPSS software (version 14.0). Significant differences between means (n = 3) were determined using one-way anova followed by the least significant difference test (LSD) at a level of P < 0.05. Exponential regression analysis was conducted to relate DHA, SIR and C mic to the quantity of electrons present within 48 hours. The quantity of electrons, Q (coulomb, C), produced was evaluated with the following formula: where U is the voltage (V), I is the current (A), R is the external loading 1000 Ω, n is the number of data recorded by a data acquisition module with a time interval of 600 s, and m is the total number of voltage data within 48 hours. to reach 'start-up' and another 3 days to reach a peak voltage of 52 mV (data not shown). Glucose addition accelerated the start-up and increased the peak voltage. For the control MFC with 5% glucose, start-up occurred after 15 hours of operation and reached a peak voltage (354 mV) after 25 hours of operation (Table 1). The copper additions at 50 and 100 mg kg −1 delayed the start-up, reduced the peak voltage and decreased the quantity of electrons produced significantly (P < 0.05). The MFC amended with 200 and 400 mg kg −1 Cu 2+ did not show a peak voltage; instead, the voltage of these treatments increased throughout the operation time. The MFC with fumigated soil had voltage below 1 mV during the 150 hours of operation, indicating that the electrical power was produced by soil microorganisms. The response of MFC voltage to Cu exposure Values of DHA, SIR and C mic decreased significantly with increasing Cu 2+ concentrations (Table 1) and were significantly correlated with the quantity of electrons produced within 48 hours, as indicated by an exponential regression (Figure 3). The transition from a steep to a flat slope of the exponential curve occurred with the decreasing Cu 2+ concentration and greater numbers of electrons produced. Electrochemical activity The electrochemical activity of the soil solution recovered from anode chambers was determined with cyclic voltammetry (Figure 4). The fumigation treatment did not show any electrochemical activity. With the increase of Cu 2+ concentration, the reduction peaks at around −550 mV became less negative. A pair of symmetric oxidation and reduction peaks at around −50 and 50 mV were from Cu 2+ . Sequencing and phylogenetic analysis of the DGGE pattern The DGGE profiles of the bacterial 16S rRNA gene showed three distinct bands 4, 10 and 11, which were dominant in the anode samples rather than in the soil samples ( Figure 5). Bands 4 and 11 were mainly detected in the 400 mg kg −1 Cu 2+ treatment while band 10 was mainly in the control treatment. The other bands were largely shared between the anode and soil. Unlike many other bands, the intensity of band 6 did not decrease with increasing Cu 2+ amount. The phylogenetic tree for 16S rRNA gene sequences ( Figure 6) from the anode samples only clustered bands 4 and 11 with Firmicutes and Bacillus, respectively. Band 10 was clustered with Acetobacteraceae and Bacillaceae. Bands 1, 2, 3, 5 and 6 were assigned to Clostridium. Bands 7, 8 and 9 were assigned to Sporolactobacillus. The electrical signals from soil MFCs In MFCs, the electrogenic bacteria require time to enrich the anode to start-up and to produce power (Borole et al., 2010). The addition of easy-to-degrade carbon sources, such as glucose and acetate, accelerates the start-up of MFCs and improves power output (Deng et al., 2012). In our study, soil was mixed with 5% glucose (w/w) to accelerate start-up effectively and improve voltage. We noted that peak voltage did not increase and start-up was not accelerated with the addition of more glucose (8%, w/w) (data not shown). This could be because the current was saturated with 5% added glucose (Lee et al., 2008), although we cannot discard the possibility that the organic acids produced from the fermentation of excess glucose inhibited microbial activity (Sundberg & Jonsson, 2005). Cyclic voltammetry was used to identify the electrochemical activity of soil in the anode chamber in response to Cu 2+ addition. The electrochemical activity decreased with increasing soil copper concentrations and was absent in the sterilized soil. This result indicated that increasing soil Cu 2+ concentrations had a negative effect on the redox activity of the anode system, and that the measured electrochemical activity was related to soil microbial activity, either by electrogenic bacterial growth or by the presence of electrochemically active metabolic products. The reliability of the MFC-based method The quantity of electrons produced within 48 hours decreased significantly with increasing soil Cu 2+ concentrations. This result demonstrates the potential use for MFCs in assessing heavy metal (a,f) The without-Cu control and fumigation treatment were set as references. toxicity in soil microorganisms. To confirm the agreement of the MFC-based method with traditional methods, SIR, DHA and C mic were also studied. Values from DHA provide one of the most sensitive indicators for soil microbial activity (Goyal et al., 2008). The value of SIR largely depends on the metabolically active component of microbial biomass and is thus an indicator of the viable microbial community activity (Deng et al., 2009). The C mic is a measurement of the abundance of living microorganisms. Results of our study indicated that SIR, DHA and C mic decreased with increasing amount of Cu 2+ , which is consistent with a large number of previous studies. Large concentrations of heavy metals can adversely affect soil microorganisms, inhibiting enzyme activities and thereby reducing overall microbial activity. However, C mic measurements include active, inhibited and dormant cells: therefore it could be less sensitive to stress than DHA and SIR (Guo et al., 2013). The exponential regressions used to fit DHA, C mic and SIR in relation to coulombs measured suggest that the quantity of electrons produced decreased to a greater extent than did SIR and C mic in control and 50 mg kg −1 Cu 2+ . Exocellular electron transfer by electrogenic bacteria is carried out by membrane-bound electron transport proteins, such as c-type cytochromes (Logan, 2009). In contrast, bacterial metabolic activities are controlled largely by endoenzymes. Copper toxicity is mitigated by a series of internal cellular mechanisms (Silver & Phung, 1996). In addition, as a specific function, electrogenic activity is guaranteed by less redundant metabolic pathways or species than general functions of SIR or C mic . Therefore, the measure of power generation was more sensitive to the smaller Cu 2+ concentrations than either SIR or C mic . These results indicate that electrical signals may be useful in identifying the presence of small amounts of pollution in soil. We also suggest that soil be tested under controlled conditions with constant temperature to prevent power generation fluctuations (Deng et al., 2014). The DNA-based analysis of anodic bacteria Phylogenetic analysis of the 16S rRNA gene from the anode demonstrated the composition of anodic bacteria. Three bands distinct to the anode samples were assigned to Firmicutes, Bacillus, Acetobacteraceae and Bacillacea. Previous research has assigned electrogenic bacteria to the Proteobacteria and Firmicutes phyla (Kim et al., 2008) and within the Firmicutes, Acetobacterium, Bacillus, Clostridium, Corynebacterium, Lactobacillus, Lactococcus and Streptococcus were identified. A number of anodic bacteria have been isolated and their electrogenic activities confirmed (Park et al., 2001;Chung & Okabe, 2009;Xing et al., 2010;Wu et al., 2013). Bacillus sp. transfer electrons to the anode mainly through soluble redox-active mediators (Nimje et al., 2009). In a study of forest soil, Kappler et al. (2004) found that iron (Fe(III)/Fe(II)) and humic acids acted as electron mediators. Acetobacterium plays a role in current generation by producing acetate, one of the most favourable substrate for electrogenic bacteria, through glucose fermentation or homo-acetogenic processes (Ziv-El et al., 2012). In addition to the three distinct bands from the anode samples, bands shared by both anode and soil were identified and assigned to Clostridium and Sporolactobacillus. Sporolactobacillus ferment glucose and produce D lactate (Wang et al., 2011), but an electrogenic activity has not been reported. Clostridium organisms are electrogenically active. The Firmicutes, Clostridium sp. and Bacillus sp. represented by bands 4, 6 and 11, respectively, are possibly Cu-resistant. The Bacillus sp. dominate bacterial communities in heavy metal-polluted soils (Ellis et al., 2003). However, in spite of the presence of Firmicutes, Bacillus and Clostridium in 200 and 400 mg kg −1 Cu 2+ -amended soil, the measured voltage was very small with a slow start-up. The Cu 2+ exposure severely inhibited the electrogenic activity and the start-up of voltage probably resulted from adsorption of Cu 2+ on soil, and resilience of microbial activity (Deng et al., 2009). Conclusions Our study is the first to use the electrical signals of soil-based MFCs to monitor the effects of Cu 2+ on soil microorganisms as far as we are aware. We conclude that the electrical signals of MFCs with glucose-amended soil can be used to evaluate the ecotoxicity of soil pollutants. The electrical signals produced are very sensitive to small stresses. Nevertheless, there are several limitations that should be overcome to facilitate application, including (i) minimizing the volume of MFC reactors and the amount of soil for a test, (ii) using oxygen as a sustainable electron acceptor rather than ferricyanide, and (iii) reducing the cost and improving the performance of electrodes and the cation exchange membrane. Further research is needed to validate the technique and to assess the reliability of the method with different stressors and with different soil types.

v3-fos

2019-08-19T07:46:58.993Z

2015-04-01T00:00:00.000Z

208002377

s2

Biochar’s effect on soil nitrous oxide emissions from a maize field with lime-adjusted pH treatment Introduction Conclusions References Tables Figures Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | time, is highly vulnerable to the consequences of a changing climate (IPCC, 2014). With its 300 fold warming potential compared to CO 2 , nitrous oxide (N 2 O) from soil is a downside of the large productivity increase in agriculture, due to synthetic nitrogen fertiliser application. Reducing agricultural N 2 O emissions would reduce the GHG induced radiative forcing (IPCC, 2014), improve the stability of the stratospheric ozone layer 5 (Ravishankara et al., 2009) and reduce agriculture's energy intensity when achieved with a lower nitrogen fertiliser use (IAASTD, 2009). Biochar is produced by thermal decomposition of organic material in a low-oxygen environment, called pyrolysis. This stable charcoal-like material has the potential to contribute to the mitigation of climate change by increasing soil carbon (C) (Lehmann, 10 2007; Woolf et al., 2010;Lal et al., 2011). In addition, biochar can increase crop yields (Jeffery et al., 2011;Biederman and Harpole, 2013;Crane-Droesch et al., 2013) and reduce water stress, which helps to adapt to climate change (Mulcahy et al., 2013). Its application to soils that have a small cation exchange capacity and low organic carbon content is associated with higher crop yields (Crane-Droesch et al., 2013) with 15 an overall mean response of 10 % (Jeffery et al., 2011). Biochar also controls nitrogen (N) cycling (Clough et al., 2013). Biochar can reduce N leaching (Steiner et al., 2008;Güereña et al., 2013) and soil-borne N-containing GHG (van Zwieten et al., 2015). Especially nitrous oxide (N 2 O) emissions from soil are reduced on average by 54 % in lab studies and 28 % in field measurements (Cayuela 20 et al., 2015). In field situations, N 2 O reduction effects are typically difficult to verify because of less uniform conditions and a large spatial and temporal variability of fluxes (Felber et al., 2013;Schimmelpfennig et al., 2014). A few field experiments indicated an increase in N 2 O (e.g., Verhoeven and Six, 2014;Liu et al., 2014), many showed no significant effects (Angst et al., 2014;Karhu et al., 2011;Scheer et al., 2011;Suddick 25 and Six, 2013; Anderson et al., 2014) while other studies indicated decreasing N 2 O emissions (e.g., Felber et al., 2013;van Zwieten et al., 2010;Taghizadeh-Toosi et al., 2011;Zhang et al., 2010;Case et al., 2014). Only few studies with biochar have looked (Verhoeven and Six, 2014), hence there is a large uncertainty about longer term effects of biochar addition. Biochars are often alkaline and therefore increase soil pH after application (Joseph et al., 2010). Denitrifying bacterial communities have the potential to increase their N 2 O-reducing activity with increasing pH, which may reduce N 2 O emissions from soils 5 (Cavigelli and Robertson, 2001;Simek and Cooper, 2002;Čuhel et al., 2010). Some authors suggest that the elevated soil pH is responsible for reduced N 2 O emissions following biochar application through increased activity of N 2 O reducing bacteria (van Zwieten et al., 2010;Zheng et al., 2012). In contrast, Yanai et al. (2007) argue that the suppression of N 2 O emissions by biochar is not through increased N 2 O reduction ac-10 tivity because biochar ash also increases soil pH but does not reduce N 2 O emissions. Cayuela et al. (2013) showed that biochar's acid buffer capacity was a more important factor in denitrification than the pH shift in soil. There are indications that biochar enhances nosZ expression, the gene responsible for the transcription of the N 2 O reductase in denitrifying microorganisms (Harter et al., 2014;Van Zwieten et al., 2014). 15 This could be a mechanistic link to the observed reduction in N 2 O emissions through biochar increasing soil pH and microbial activity. In contrast, under conditions favouring nitrification and not being as sensitive to pH as total denitrification, biochar addition increased N 2 O emissions in the lab (Sánchez-García et al., 2014) and possibly in the field (Verhoeven and Six, 2014). 20 In this study, we test (i) whether N 2 O emissions are reduced following the application of biochar to soil of a temperate maize cropping system and (ii) whether this possible reduction in N 2 O emissions is due to an increase in pH. The latter was tested by a treatment where limestone was added to increase soil pH to the same level as that from the addition of 20 t ha −1 biochar. N 2 O emissions and maize yield were quantified 25 during one growing season in the field. Group WRB, 2006) it is a Eutric Mollic Gleysol (Drainic). The untreated soil has a pH of 6.3 in water (1 : 2.5 w/v), total organic carbon content of 26.2 g kg −1 , total N of 0.29 g kg −1 and bulk density of 1.3 g cm −3 . Biochar Several biochars were screened in advance to pick one with a high liming capacity and 15 with properties in agreement to the guidelines for polycyclic aromatic hydrocarbons (PAHs), C-and N-content of the European Biochar Certificate (EBC, 2012). The chosen biochar was produced in a Pyreg reactor (Pyreg GmbH, Dörth, Germany) by Verora in Edlibach ZG, Switzerland in late 2013 (see chapter 30, case study 2 in Lehmann and Joseph, 2015). Pyreg reactors use slow pyrolysis in a continuous system with 20 an average residence time of circa 25 min and a peak temperature of approximately 650 • C. The feedstock was green waste mainly from tree pruning. The biochar has the following properties: 64.9 % total C; 62.1 % Corg, pH 9. Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | the time of application was 12 %. Biochar was sieved < 3 mm shortly before it was spread on the field. Experimental setup Three different treatments were introduced; 20 t ha −1 biochar, control without additions and a limestone treatment to increase the soil pH to the same level as with biochar. The 5 field was split into 3 × 3 plots with a size of 2 by 3 m (6 m 2 per plot and 3 replicates for each treatment). One meter buffer zones were established between plots on all sides. The 3 different treatments were arranged in a randomized complete block design with the 3 × 3 grid accounting for spatial variability. The whole field, including the buffer zones, were planted with maize (zea mays). Initial pH values were not different among 10 treatment plots (see pH measurement in January on Fig. 2). Field management The field was ploughed in autumn 2013 after the maize harvest. In January 2014, 20 t ha −1 biochar and 2 t ha −1 limestone were spread on the wet, ploughed field surface. Freshly applied biochar was gently mixed with the first 1-3 cm of soil by hand at the 15 same time. In mid-February 2014, the automated GHG chamber system was installed and in March the field was harrowed by a rototiller to a depth of circa 15 cm. The chamber frames were reset into the soil again and Decagon TE5 temperature and humidity sensors (Decagon Devices Inc., Pullman Wa, USA) were placed at a depth of 8 cm in the centre of each plot. 20 In May, potassium (K) and phosphorus (P) fertiliser was applied at a rate of 41.4 and 132 kg K ha was not in the same range as the biochar plots. Maize (Padrino from KWS SAAT AG, Einbeck, Germany) was sown on the 8 May with 0.14 m distance within rows that were 0.6 m apart from each other. For plant protection only one herbicide application was conducted on the 19 June with 1 L ha −1 Dasul (Syngenta, Basel, Switzerland) 1 L ha −1 Mikado (Bayer CropScience, Germany) and 1 kg ha −1 Andil (Omya AG, Switzerland). 5 Despite manual weeding and herbicides a considerable amount of weeds emerged. Plots were harvested on the 13th of October. Nitrous oxide measurement N 2 O and CO 2 emissions were measured with static chambers of a fully automated measurement system (Flechard et al., 2005;Felber et al., 2013) consisting of nine stainless steel chambers (30 × 30 × 25 cm). These chambers were placed on PVC frames inserted 3 cm deep into soil. Two frames were placed on each plot at a similar distance to the plot borders. These positions were moved three times during the growing season to obtain a better spatial representation of each plot. After maize had been sown, the chamber positions were between rows and no vegetation was grown 15 within the chamber frame. Each of the 9 chamber lids were automatically closed and opened sequentially (over a period of 3.5 h) allowing N 2 O and CO 2 to accumulate in the chamber headspace for 15 min. Chamber headspace air was circulated (1 L min −1 air flow) through an inlet and outlet line from each chamber through polyamide tubes (4 mm I.D.) to the analytical system and back to the chamber headspace continuously 20 after sample analysis. The analytical and chamber control instruments were installed in a nearby field cabin under temperature controlled air conditioning. N 2 O concentrations were continuously measured and stored every minute using a gas filter correlation technique (TEI Model 46C, Thermo Environmental Instruments Inc., Sunnyvale, CA, USA Hence a temperature correction factor was applied to the raw data from a regression of the device temperature with data during calibrations in May. N 2 O and CO 2 fluxes from soil were calculated from the continuous concentration measurement (resolution 1 per min) when chamber lids were closed. Data from the first 3 min of the total 15 min closure time were omitted from the flux calculation to re-5 move signal noise due to gas exchange from the system during chamber switching and closing (Felber et al., 2013). The same flux estimation procedure (R-script by R. Fuss on bitbucket.org, see Fuss, 2015) was used as in Leiber-Sauheitl et al. (2014). It is a modification of the HMR package (Pedersen et al., 2010) that chooses between exponential curvature for non-linear chamber behaviour (Hutchinson-Mosier regres-10 sion) and robust linear regression (Huber and Ronchetti, 1981). The exponential HMR scheme considers non-linear concentration increase in the chamber due to a possibly decreasing concentration gradient, chamber leakage and lateral gas transport. Robust linear regressions provide a more reliable flux estimate for low fluxes when there is a lot of variation due to limited measurement precision and outliers. The resulting flux 15 estimates from this procedure were then filtered for implausible large N 2 O uptake by soil. N 2 O fluxes smaller than −50 ng-N 2 O m −2 s −1 (Neftel et al., 2010) were removed as well as data associated with a likely invalid chamber functioning (i.e. frozen lids) when CO 2 flux < −0.5 µmol m −2 s −1 (Felber et al., 2013). In total 302 CO 2 and 351 N 2 O data points from the entire dataset (14 068 points) were rejected. 20 Yield The yield was separated into grain (kernels) and plant material. Cobs were threshed and dried whereas the plants were weighed freshly on the field, chaffed and a subsample was then dried to measure water content and for further plant nutrient analysis. From both plant and grain, dry matter total N and P were measured (FAL, 1996). Soil sampling and analysis Soil samples for pH, ammonium (NH + 4 ) and nitrate (NO − 3 ) measurements were taken on the 31 January, 31 March, 26 May, 16 June and 4 September 2014. At each sampling, five randomly distributed soil cores per plot were taken (0-10 cm) and pooled. Soil pH was determined in moist soil samples using water at a ratio of 1 : 2.5 w/v and 5 measured with a PH100 ExStik pH meter (Extech Instruments Corp., Nashua, NH, USA). Soil bulk density was measured on the 27 June at a depth of 3-8 cm using 100 cm 3 steel cores, 3 per plot. For soil NO − 3 and NH + 4 concentrations, 20 g of moist soil were mixed with 100 mL 0.01 M CaCl 2 solution. The suspension was shaken for 30 min, filtered and then anal-10 ysed by segmented flow injection analysis on a SKALAR SANplus analyser (Skalar Analytical B.V., Breda, the Netherlands). Statistical analysis The obtained fluxes from the automated chamber system were aggregated to 8 h means producing a regular, smoothed dataset. The system was able to measure each 15 chamber three times for every 11 h calibration cycle during regular operations, hence on average 2.2 measurements for each chamber were included in each a 8 h mean. Still missing values after this aggregation step were linearly interpolated for each chamber. Treatment averages and standard deviations were calculated from the 3 chambers on the replicated plots. 20 Statistical analyses were performed with R (version 3.0.1, The R Project, 2014). Significance level was chosen at p < 0.05 for all procedures, unless indicated otherwise. Significant treatment effects for cumulated fluxes were determined using ANOVA from rbase package (treatments: control, biochar and lime; n = 3). Bartlett test of homogeneity of variances showed conflicting ANOVA assumptions for the cumulative fluxes. 25 This could be solved by log transformation of the flux data. In addition, a generalized least squares model (GLS) was constructed with weekly cumulated N 2 O emissions as dependent variable, and weekly averages of soil volumetric water content (VWC) and the treatments (control, biochar, lime) as explanatory variables. A restricted maximum likelihood generalised linear model from nlme R package was used to calculate the GLS. Meteorological data on the field The year started with above average temperatures and low rainfall (Fig. 1). End of May to June was dry with high temperatures being on average for Switzerland 1.5 • C above the 1981-2010 norm (Meteoswiss, 2015). The soil's volumetric water content fell to 10 circa 20 %, inducing high water stress on the young maize seedlings. The lack of soil moisture presumably hampered the dilution of the first application of 40 kg ha −1 N in the soil solution. Along with the 2nd N fertilisation the field was therefore irrigated with 33 mm water (shown as green bar in the precipitation dataset). The summer months following (July and August) were rather cold and wet with daily mean air temperatures 15 below 20 • C (Meteoswiss, 2015). The GLS model indicated a significant, treatment specific (p = 0.0202) effect of weekly mean soil VWC on weekly cumulated N 2 O fluxes (p = 0.0034). Biochar plots had significantly higher soil water content than lime and control plots (p < 0.001). However, there is no interaction between treatment and VWC on a weekly basis (p = 0.542). Soil pH and nitrogen Soil pH increased with limestone and biochar addition in medium terms by circa 0.4 pH units (Fig. 2). The initial soil pH was on average 6.3 and not different among treatments. Following biochar application soil pH increased to up to 7.4 whereas with addition of limestone soil pH increased to up to 6.9 (averages across replicates). The pH sharply Introduction Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | decreased after the initial peak, especially in those two liming plots, which were treated with another 1 t ha −1 in May. Soil pH of biochar and lime treatments were not significantly different at any sampling time, whereas soil pH of the control treatment was systematically below that of the amended soils. Mean soil bulk density was not statistically different between treatments (1.31 g cm −3 5 in the control, 1.29 g cm −3 in biochar and 1.36 g cm −3 in the liming treatment). Soil mineral N was not statistically different between treatments (Tables 1 and 2). N 2 O fluxes Emissions were characterized by peak events, particularly in summer, and by background emissions in spring and autumn (Fig. 3). Main emissions occurred after the second fertilisation event of 80 kg-N ha −1 around early August. Afterwards, there were only emissions from one of the lime plots but almost none until the end of October from all the other plots. This also corresponds to the low amounts of available soil N, indicating that the plants had taken up most of it. All treatments revealed similar temporal N 2 O emission dynamics but the height of the peaks differed. During peak events 15 emissions from the biochar treatment were often lower than those from the other treatments, especially compared to the control. This resulted in an increasing difference in cumulative fluxes (Fig. 4) that were directly influenced by the N-fertiliser applied (between 26 May and 13 August = approx. 3 months) and subtract half of the cumulative emissions from the residual period measured (approx. 6 months). This resulted in IPCC emission factors of 0.58 % for biochar, 1.28 % for control and 1.25 % for the lime treatment. 10 Maize yields were not significantly different between treatments, for both grain and plant dry matter (Fig. 5). Nitrogen and P uptake did not differ among treatments (Figs. 6 and 7). same authors under field conditions (28 ± 16 %). In our temperate maize field, N 2 O emissions thus decreased with biochar addition as much as they have been shown to be reduced under controlled lab conditions. Our results show no a decrease in N 2 O emissions when limestone is used to increase the soil pH to the same level as that with biochar. This finding does not support 5 the hypothesis that biochar's N 2 O reduction effect is solely due to a geochemical manipulation of soil pH. However, it must be considered that the large variability among the three replicates hampers the power of this conclusion. The high variability solely in the liming treatment might be due to additional lime application to the field in May 2014 and the high spatial-temporal variability of that soil property in general. The two replicates that received additional limestone were the ones that emitted more N 2 O than the other plot. Hence, instead of reducing emissions by increasing the pH, the additional limestone application could have provoked local arbitrary disturbance to soil chemistry leading to emission hotspots. To determine the biochar effect on N 2 O emissions, we therefore also compared only the biochar and control treatments; the cumulative emis-15 sions in the biochar amended plots are significantly lower (by 53 %) than in the control treatment. Maize yields and plant growth The GLS model shows that not only treatment but also water content affects soil N 2 O emissions. However, the mechanism behind the overall negative feedback of VWC on N 2 O emissions (i.e. higher VWC leads to lower emissions) can not be derived from our 20 data. Biochar effects on soil physical properties have been shown to increase waterholding capacity, reduce bulk density and increase soil sub-nanopore surface together with a 92 % decrease in N 2 O emissions (Peake et al., 2014;Mukherjee et al., 2014). This suggests that increased soil aeration by biochar dominates the effect of increased water content and hence does not favour denitrification (van Zwieten et al., 2010). -Toosi et al., 2011;Liu et al., 2012). A number of studies found no significant effect of biochar addition in the field (Schimmelpfennig et al., 2014;Angst et al., 2014;Scheer et al., 2011;Karhu et al., 2011;Anderson et al., 2014). Often the much higher variability in the field and the low number of replications make it difficult to reproduce reduction effects observed in laboratory studies. In particular, Angst et al. (2014) found 5 no significant difference but there was a tendency for lower emissions with biochar addition. However there are also studies that showed increased emissions from biochar application in the field (Verhoeven and Six, 2014;Shen et al., 2014). Sánchez-García et al. (2014) found that biochar increases soil N 2 O emissions produced by nitrification-mediated pathways. In our study, the water content ( Fig. 1) was 10 high during periods of high emissions and suggesting that during periods of high water content denitrification dominates the N 2 O production in soil. The high emissions were thus often triggered by large precipitation events. There are many indications from lab experiments that biochar can reduce N 2 O emissions in denitrifying conditions at high water content (Felber et al., 2013;Harter et al., 2014;Singh et al., 2010;Yanai et al., 15 2007). Under denitrification conditions, the pH exerts control over the N 2 O : N 2 ratio (Simek and Cooper, 2002). Various studies have suggested that an elevated soil pH is responsible for reduced N 2 O emissions following biochar application through increased activity of N 2 O reducing bacteria (van Zwieten et al., 2010;Zheng et al., 2012). In contrast, Yanai et al. (2007) argued that the suppression of N 2 O emissions by charcoal is 20 not due to increased N 2 O reduction activity because biochar ash increased pH to the same degree as biochar but did not reduce N 2 O emissions. Also Cayuela et al. (2013) found no N 2 O mitigation when soil pH was increased to the same level as biochar did but with CaCO 3 addition. They also showed that biochar's buffer capacity but not biochar pH was highly correlated with lower N 2 O emissions compared to pH-adjusted 25 biochars (Cayuela et al., 2013). In our case, we used a biochar with rather high liming capacity (17.2 % CaCO 3 ) and pH (9.8). We can confirm that with this kind of biochar N 2 O emissions can effectively be reduced also in real field conditions, although the high variability in the pH adjusted control does not allow us to reject the hypothesis of Van Zwieten et al., 2014). Some authors relate this enhancement of N 2 O reducing bacteria to biochar's redox activity that facilitates electron shuttling for the sensitive process of N 2 O reduction (Kappler et al., 2014;Cayuela et al., 2013). This shuttling might be the connection between reduced N 2 O emissions and low H : Corg ratios (Cayuela et al., 2015) in biochar that 10 refers to condensed aromatic structures and its quinone/hydroquinone moieties being electro-active by allowing electron transfer across conjugated pi-electron systems (Klüpfel et al., 2014). Such high electro-catalytic activity has also been shown in Ndoped C nanotube arrays (Gong et al., 2009). Hence, in contrast to a promotion of microbial N 2 O reduction, there is also the possibility that biochar abiotically reduces 15 N 2 O through its electrocatalytic abilities represented by a high aromaticity with low H : Corg ratios. Indeed, this is one of the various abiotic mechanisms that reduce N 2 O emissions suggested by Van Zwieten et al. (2015). Yield and nutrients In our experiment, grain yield and plant biomass production were not increased by show clear effects within the first year of application yet. Our data is also in agreement with Jay et al. (2015) who showed that biochar had no effect on harvest yield of different crops after a single rotational application (20 and 50 t ha −1 ) in a sandy loam under intensive management. Nitrogen uptake was not changed by biochar or liming. Although there was no signif-5 icant difference in P uptake between the treatments, green plant material from biochartreated plots tended to have higher uptake then the control (+100 % increase). Vanek and Lehmann (2014) showed significant increase in P availability through enhanced interactions between biochar and arbuscular mycorrhizas.

v3-fos

2018-12-15T11:40:53.702Z

2015-03-01T00:00:00.000Z

56565867

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Effect of Crude Palm Oil (Cpo) Protected by Formaldehyde on Physical and Chemical Quality of Lamb This study was conducted to determine the effect of crude palm oil (CPO) protected byformaldehyde on chemical and physical quality of lamb. The research design applied was completelyrandomized design with 3 treatments and 5 replications Fifteen local male sheeps aged 9-12 monthsweighing 14-17 kg were divided into 3 groups for different ration treatments. The first group receivedonly basal ration (R0), the 2nd group received basal ration and 3% of CPO (R1), while the 3rd groupreceived basal ration and 3% of CPO protected by 2% of formaldehyde (R2). The data were analyzed byanalysis of variance. The differences among treatments were tested by Duncan's New Multiple RangeTest. The results showed that the addition of CPO protected by formaldehyde (R2) in the sheep dietincreased lamb protein and fat content (P<0.05), produced tenderer lamb (P<0.01) with lower cookingloss (P<0.01). The diet with CPO protected by formaldehyde can improve the chemical and physicalquality of meat sheeps. INTRODUCTION Fatty acids in ruminant meat were dominated by saturated fatty acids. This is becaused unsaturated fatty acids (linoleic-C18:2 and linolenic-C18:3) in the diet are hydrogenated by rumen microbes become saturated fatty acids, especially stearic acid (C18:0) (Jenkins et al., 2008;Wang et al., 2010), so that only about 10% joined the lipid tissues (Wood et al., 2008), whereas 90% were hydrogenated into saturated fatty acids. Based on the final product, hydrogenation of unsaturated fatty acids are completely done by two groups of bacteria, namely (1) group A, which hydrogenate C18:2 and C18:3 with the final product of trans-C18: 1; and (2) group B, which hydrogenate trans-C18:1 with the final product of C18:0 (Bauman et al., 2003). In addition, unsaturated fatty acids in ruminants diet would disturb rumen fermentation and reduce the utilization of fiber (Hristov et al., 2009;Vafa et al., 2009). This is caused fat can wrap feed particles and close access of microbial cell membrane to contact feed, thereby disrupting the enzymes production to degrade feed, so that decrease feed digestibility and meat quality. The in vitro study has been done using crude palm oil (CPO) as a source of unsaturated fatty acids as much as 0%; 1.5%; 3%; 4.5%; 5% and 6% of the dry matter that is mixed by expired milk powder (1:2) and protected with technical formaldehyde as 0%; 1%; 2% and 3% of the mixture. The result showed that CPO as much as 3% of the dry matter protected with 2% of formaldehyde can protect unsaturated fatty acids from rumen microbial hydrogenation and no negative effect on fermentation parameters and rumen microbial activity (Tiven et al., 2011). This is in line with Kitessa et al. (2001) andde Veth et al. (2005) research, that protection of fat with formaldehyde can reduce the hydrogenation of unsaturated fatty acids in feed by rumen microbial. Results of in vitro studies need to be applied in vivo to determine the effect of CPO protected with formaldehyde in the diet on the chemical and physical quality of lamb. The success of this study is expected to be a reference using this feed that is high in saturated fatty acids to improve chemical and physical quality of lamb in order to reduce consumers susceptibility to cardiovascular disease. Animals and Feed Fifteen of local male sheep aged 9-12 months with a body weight of about 14-17 kg were raised in individual equipped with places to eat and drink. Sheeps were randomly divided into 3 groups according to the treatment of feed; each group consisted of 5 animals. Completely randomized design was used in this study. Basal diet was consisted of forage and concentrate with a ratio of 60:40. Forage used was elephant grass, while the concentrates were consisted of 30% rice bran and 10% soybean meal. Nutrient contents of basal ration were 62.98% of total digestible nutrients, 45.5% of dry matter, 14.48% of crude protein, 4.70% of crude fat and 21.93% of crude fiber. The first group received only the basal diet (R0), the 2 nd group received the basal diet and 3% of CPO (R1), while the 3 rd group received the basal diet and 3% of CPO protected with 2% formaldehyde (R2). Chemical and Physical Meat Properties After feed treatment for 3 months, sheep were slaughtered. Halal slaughtering is done, starting with the neck cut to the jugular vein severed, esophagus, and trachea (near the lower jaw bone). The Longissimus dorsi (LD) muscle on the back of the carcass were taken for analysis of chemical meat composition (AOAC, 2005) and meat physical properties, consisting of pH (AOAC, 2005), water holding capacity using Hamm's method (Soeparno, 2005), tenderness with Warner Bratzler tools and cooking loss (Suryati et al., 2008). Data Analysis The data were analyzed by analysis of variance. The differences between treatments were tested further by Duncan's New Multiple Range Test. Data processing was done by using the SPSS program 17.0 for Windows Evaluation Version (Oramahi, 2008). Chemical Composition of Meat Water Content The effect of CPO protected by formaldehyde on water content of lamb is presented in Table 1. The results showed that the addition of CPO in the basal diet (R1) caused a decrease in the water content of the meat (P<0.05) about 1.76% compared to sheep that were given basal diet (R0). The decrease in water content was much higher (P<0.05), i.e. 2.83%, in sheep fed CPO protected with formaldehyde in the basal diet (R2), but it was not significantly different than that of R1. The decrease in water content was due to an increase in fat content that was negatively correlated to the water content. The water content in this study ranged from 73.95 to 76.78%. Water is the largest component of meat, namely 75% (Lawrie, 2003) with a range of 60-80% (Forrest et al., 1975). Manso et al. (2009) reported that the addition of sunflower oil (SFO) in Merino sheep diet obtained 75.20% of water content. Protein Content The effect of CPO protected by formaldehyde on protein content in lamb is presented in Table 1. The results showed that the addition of CPO in the basal diet (R1) causes an increase in protein levels (P<0.05) by 0.82% compared to sheep that were given basal diet (R0). The increase of protein content was much higher (P<0.05), i.e. 1.41%, in sheep fed CPO protected by formaldehyde in the basal diet (R2), but it was not significantly different than that of R1. The increase is due to increase of rumen microbial protein that might accumulate in the meat, it was caused by the availability of N, the source of energy and carbon skeleton as a precursor for the synthesis of microbial protein (Tiven et al., 2011). The increase of this protein content in line with Kitessa et al. (2003) who reported that protected tuna oil (PTO) with formaldehyde can increase the protein content of milk in lactating sheep from 54 g/kg to 56 g/kg. The protein content in this study ranged from 18.21 to 19.62%. Meat contains protein by 19% with a range between 16-22% (Forrest et al., 1975). The results of this study is higher than the research of Manso et al. (2009) which added hydrogenated palm oil (HPO) and sunflower oil (SFO) in Merino sheep diet, obtained the protein content of 17.63% and 19.27%. Fat Content The effect of CPO protected by formaldehyde on fat content in lamb is presented in Table 1. The results showed that the addition of CPO in the basal ration (R1) increased the fat content (P<0.05) by 0.71 % compared to sheep that were given only the basal ration (R0). This increase is due to the extra fat from palm oil in the diet. Increase of fat content was much higher (P<0.05), i.e. 1.27%, in sheep fed CPO protected with formaldehyde in the basal ration (R2). The increase is due to formaldehyde can protect CPO fat; especially unsaturated fatty acids and reduce microbial degradation in the rumen, so that it can accumulate in the meat. The protection of CPO with formaldehyde can increase unsaturated fatty acids, i.e. oleic, linoleic and linolenic acid (P<0.01) (Tiven et al, 2011). The increase of fat content is in line with the research of Kitessa et al. (2003) which protected tuna oil (PTO) with formaldehyde, can increase the fat content of milk in lactating sheep from 74 g/kg to 77 g/kg. Fat content in this study ranged from 3.12 to 4.39%. According to Forrest et al. (1975), meat fat content is about 2.5% with a range between 1.5 to 13%. The results of this study is higher than the research of Manso et al. (2009) which reported that the addition of hydrogenated palm oil (HPO) and sunflower oil (SFO) in Merino sheep rations, gained fat content of 2.46% and 3.20%. According to Savell and Cross reported by Soeparno (2005), the fat content of beef accepted by the consumer is about 3-7%. Referring to the statement, the lamb produced in this study might be accepted by consumers, because the fat content is within that range. Ash Content The effect of CPO protected by formaldehyde on ash content in lamb is presented in Table 1. The results showed that the addition of CPO in the basal ration (R1) and CPO with formaldehyde in the basal ration (R2) were not significantly affect on ash content of lamb. The average ash content in this study was 1.35%. The ash content is also influenced by the fat content, which is negatively correlated to the ash content. According to Forrest et al. (1975), ash content was influenced by the fat content, the higher the fat content of meat, the lower the ash content. The addition of sunflower oil (SFO) in Merino sheep diet, gained 3.20% of fat content and 1.64% of ash content, whereas the diet without the addition of oil (control) gained 2.73% of fat content and 1.79% of ash content (Manso et al., 2009). Most of the minerals relatively contained in lean meat because mineral components were primarily associated with water and meat protein (Soeparno, 2005). Meat Physical Properties Meat pH The results showed that the addition of CPO in the basal diet (R1) and CPO protected with formaldehyde in the basal diet (R2) were not significantly affect on meat pH ( Table 2). The average value of meat pH was 6.22, that was higher than the ultimate meat pH, with a range of 5.8 (Soeparno, 2005). The high value of ultimate pH was due to low muscle glycogen reserves when it was slaughtered so that the accumulation of lactic acid stopped, because muscle glycogen reserves was exhausted before the meat ultimate pH was reached. Stress before slaughtered, such as: weighing livestock, can reduce muscle glycogen. According to Lawrie (2003), glycogen can reduce because the livestock are tired, hungry or scared before slaughtered. This pH value is lower than the study of Cooper et al. (2004), which used protected linseed and soya bean (protected linseed and soybean -PLS) with formaldehyde in sheep diet obtained meat pH value of 6.52 at 45 minutes after slaughtered, whereas after 24 hours the pH value became 5.68. Water Holding Capacity (WHC) The effect of CPO protected by formaldehyde on lamb WHC is presented in Table 2. The results showed that the addition of CPO in the basal diet (R1) and CPO protected with formaldehyde in the basal diet (R2) were not significantly affect on meat WHC. The average value of meat WHC was 25.47%. The value of WHC was influenced by the meat pH. Large drop of postmortem pH will affect on WHC, i.e. the higher of ultimate pH, less of WHC (Lawrie, 2003). Tenderness The effect of CPO protected by formaldehyde on lamb tenderness is presented in Table 2. The results showed that the addition of CPO in the basal ration (R1) was not significantly affect on the tenderness of lamb compared to those that were given only the basal diet (R0). However, the addition of CPO protected with formaldehyde in the basal ration (R2) made the lamb become tenderer (P<0.01) compared to lamb from sheep given only the basal diet (R0) and sheep given the basal diet with the addition of CPO (R1), each at 3.93 kg/cm 2 and 2.69 kg/cm 2 . Better meat tenderness in treatment R2 was affected by the higher of saturated fatty acids content, compared to R1 and R0. Mauger et al. (2003) stated that the high saturated fatty acids caused ruminant fat becomes harder and can lead to cardiovascular disease in consumers. Cooking Loss The effect of CPO protected by formaldehyde on lamb cooking loss can be seen in Table 2. The addition of CPO in the basal diet (R1) was not significantly affect on meat cooking loss, compared to meat from sheep given only the basal diet (R0). The addition of CPO protected with formaldehyde (R2) in the diet made cooking loss that was lower (P<0.01) than lamb from sheep given only the basal diet (R0) and lam from sheep given the basal diet with the addition of CPO (R1), each at by 7.63% and 7.98%. The low meat cooking loss on R2 treatment was influenced by the higher fat content of meat compared to R0 and R1 (Table 2). According to Forrest et al. (1975), that the cooking loss was influenced by the fat content in meat and fat translocation. During cooking, the fat will melt and distributed in the meat so the meat that has marbling will have smaller cooking losses. According to Soeparno (2005), the equitable distribution of fat throughout the meat can act as a barrier liquid to escape during cooking. Generally, cooking loss varies with the range of 15-40% (Soeparno, 2005). The meat with low cooking loss relatively has a better quality than the meat with high cooking loss, because of little nutrients loss during cooking. CONCLUSION The CPO protected by formaldehyde in the diet can increase meat protein and fat content, and produce tenderer lamb with lower cooking loss.

v3-fos

2016-05-12T22:15:10.714Z

2015-10-20T00:00:00.000Z

18740387

s2

Genotype by environment interaction and breeding for robustness in livestock The increasing size of the human population is projected to result in an increase in meat consumption. However, at the same time, the dominant position of meat as the center of meals is on the decline. Modern objections to the consumption of meat include public concerns with animal welfare in livestock production systems. Animal breeding practices have become part of the debate since it became recognized that animals in a population that have been selected for high production efficiency are more at risk for behavioral, physiological and immunological problems. As a solution, animal breeding practices need to include selection for robustness traits, which can be implemented through the use of reaction norms analysis, or though the direct inclusion of robustness traits in the breeding objective and in the selection index. This review gives an overview of genotype × environment interactions (the influence of the environment, reaction norms, phenotypic plasticity, canalization, and genetic homeostasis), reaction norms analysis in livestock production, options for selection for increased levels of production and against environmental sensitivity, and direct inclusion of robustness traits in the selection index. Ethical considerations of breeding for improved animal welfare are discussed. The discussion on animal breeding practices has been initiated and is very alive today. This positive trend is part of the sustainable food production movement that aims at feeding 9.15 billion people not just in the near future but also beyond. The increasing size of the human population is projected to result in an increase in meat consumption. However, at the same time, the dominant position of meat as the center of meals is on the decline. Modern objections to the consumption of meat include public concerns with animal welfare in livestock production systems. Animal breeding practices have become part of the debate since it became recognized that animals in a population that have been selected for high production efficiency are more at risk for behavioral, physiological and immunological problems. As a solution, animal breeding practices need to include selection for robustness traits, which can be implemented through the use of reaction norms analysis, or though the direct inclusion of robustness traits in the breeding objective and in the selection index. This review gives an overview of genotype × environment interactions (the influence of the environment, reaction norms, phenotypic plasticity, canalization, and genetic homeostasis), reaction norms analysis in livestock production, options for selection for increased levels of production and against environmental sensitivity, and direct inclusion of robustness traits in the selection index. Ethical considerations of breeding for improved animal welfare are discussed. The discussion on animal breeding practices has been initiated and is very alive today. This positive trend is part of the sustainable food production movement that aims at feeding 9.15 billion people not just in the near future but also beyond. ANIMAL BREEDING AND ANIMAL WELFARE Although an increase in overall meat consumption is expected in the coming decades resulting from the ever growing human population, the dominant position of meat as the center of meals is on the decline. This is motivated by religious, health, moral, and environmental considerations. Rauw (2015) reviewed the history of ethics of animal use and consumption from Pythagoras to Bentham (c 500 BC to the end of the 18th century), which describes the origins of health and moral objections to the consumption of meat. Of a much more modern origin are environmental considerations, and public concerns with animal welfare in livestock production systems; the latter particularly came about in response to the publication of Harrison's (1964) book "Animal Machines: the New Factory Farming Industry". Rapid turnover, high-density stocking, and a high degree of mechanization resulted in a public awareness of the results of intensification of livestock production practices and "factory farming" in the 60s and resulted in an increasing number of philosophical writings on animal rights from the 70s on (Singer, 2005;Stamp Dawkins, 2013). Factory farming is characterized by overcrowding, restricted movement, unnatural diets and unanesthetized surgical procedures resulting in physical pain and necessarily in reduced animal welfare (Frank, 1979). Frank (1979) suggested that intensive farming differs from factory farming in that it involves increasing productivity through better management and breeding techniques but without necessarily involving crowding and thus significantly altering the pattern of life the animal leads. However, this situation no longer applies since it became recognized that animals in a population that have been selected for high production efficiency are more at risk for behavioral, physiological and immunological problems (Rauw et al., 1998). Examples are most pronounced in populations that are selected for narrow yield goals at high intensity of selection, such as broiler chickens selected for increased body weight at a certain age (Rauw et al., 1998;Rauw, 2009). As Oltenacu and Algers (2005) write regarding dairy cattle: "[Improved production efficiency] should optimize the use of resources, increase farm profit, and reduce cost for consumers. In many European countries, yield per cow has more than doubled in the last 40 years. The dramatic increase in yield per cow is due to rapid progress in genetics, nutrition and management, " however, due to the resulting fertility problems, increasing incidence of health problems, and declining longevity in modern dairy cows, "genetic selection for increased milk yield increasingly is viewed as increasing profit at the expense of reducing animal welfare. " As a result, animal breeding practices have become part of the debate that deals with issues of animal welfare and animal production ethics and at a wider scope with sustainable agriculture and livestock production. Frank's (1979) definition of intensive farming practices which do not negatively affect the pattern of life of the animals involved is now newly captured under the banner of "sustainable intensification" of livestock production, i.e., improving productive output while maintaining animal health and welfare (Gamborg and Sandøe, 2005;Charles et al., 2014). The Farm Animal Welfare Council has emphasized welfare concerns in relation to animal breeding strategies since 1992 in their reports (FAWC, 2004(FAWC, , 2012MacArthur Clark et al., 2006). For example, the 1992 report on the welfare of broiler chickens reads: "Genetic selection has the potential for positive as well as negative effects on welfare. However, the selection of stock for liveweight gain and food conversion efficiency in preference to, and to the detriment of, factors necessary for the welfare of the birds should be discouraged" (FAWC, 2004). The 1998 Council Directive 98/58/EC concerning the protection of animals kept for farming purposes reads: "Natural or artificial breeding or breeding procedures which cause or are likely to cause suffering or injury to any of the animals concerned must not be practiced" (EUR-Lex, 2015). In 2000, the Sustainable European Farm Animal Breeding and Reproduction project was initiated by the Farm Animal Industrial Platform (currently the European Forum of Farm Animal Breeders); one of the aims was an agreement by breeding organizations to develop Codes of Practice (MacArthur Clark et al., 2006;Neetesonvan Nieuwenhoven et al., 2006). The main objectives of the resulting Code-EFABAR launched in 2006, a voluntary "Code of Good Practice, " are to be the standard instrument for defining and maintaining good practices for farm animal breeding, and to create transparency for society (Code-EFABAR, 2006). As MacArthur Clark et al. (2006) conclude, failure to address the issues arising from bad breeding practices presents a significant risk to Governments, to the livestock industry, and to animal welfare. HOW SHOULD WE BREED? Animal production is basically an input-output system to which the first law of thermodynamics, or the law of conservation of energy, applies, in the same way as it does for any other energetic system: energy cannot be created nor destroyed, but can only be changed from one form to another. Energy in output (production, losses) requires an equal amount of energy input (eventually this comes down to food intake). In other words: an animal from a population genetically selected for increased production will only be able to realize this potential in an environment in which resources are adequately supplied (Beilharz et al., 1993;Rauw, 2009). However, while this holds even intuitively, in practice, livestock animals are often genetically selected for increased levels of production (output) at the same time that they are selected for decreased levels of energetic input (improved feed efficiency, decreased levels of fatness; Rauw, 2012). A clear example of selection practices that have resulted in a mismatch between input and output is the voluntary feed intake capacity of young sows which has been reduced as a consequence of selection for high lean growth, resulting in animals that are constrained by limited body reserves and/or limited feed intake capacity at the time of lactation when they have to support a genetically increased litter size and growth rate. As Knap (2005) writes regarding pig production: "Increasing genetic potential requires advances in animal nutrition and animal management to support its expression, but these advances have often been poorly addressed or overlooked. " This results in the inability to maintain a successful balance of biological needs and consequently, inadvertently, in animals that are less robust, showing undesirable side effects of genetically improved levels of production (Siegel and Dunnington, 1997;Rauw et al., 1998;Knap, 2005). In addition, livestock animals are required to perform in a wide variety of environmental conditions, regarding climate, housing facilities, social environment, disease pressure, and differences in feed quality and composition (Knap and Wang, 2006;Star et al., 2008;Mormède et al., 2011). The farm animal of the future is thus described as robust, adapted, and healthy (Mormède et al., 2011), i.e., having "the ability to combine a high production potential (growing or reproductive) with resilience to stressors, allowing for unproblematic expression of a high production potential in a wide variety of environmental conditions" (Knap, 2005). After Knap (2005), the literature on selection for robustness traits has increased considerably, becoming a rapidly developing key area in farm animal breeding (Knap, 2009). Knap (2009) indicates that there are two options for breeding for animal robustness, which can be implemented simultaneously in an evaluation system for performance-relevant robustness: through the use of reaction norms analysis by estimating breeding values for the environmental sensitivity of the genetic potential for production performance (indirect approach), or through the inclusion of directly measurable robustness traits in the breeding objective and in the selection index (direct approach). This review presents a historic overview of gene by environment interactions (including the concepts of reaction norms, phenotypic plasticity, canalization, and genetic homeostasis), the applicability of reaction norms analysis in livestock production, and the feasibility of selecting for the different reaction norm parameters (the level vs the slope). The review ends with a discussion of the feasibility of directly including robustness traits in the breeding objective and selection index, a discussion of the ethical consideration of selection for robustness, and with a short synthesis of all the material discussed in this paper. The Influence of the Environment The influence of the environment on the phenotype and on evolution was of course most famously recognized by Jean Baptiste de Lamarck in his book "Philosophie Zoologique" in his chapter (translated) "Of the influence of the environment on the activities and habits of animals, and the influence of the activities and habits of these living bodies in modifying their organisation and structure" published in 1809. Indeed his statement that "the environment affects the shape and organisation of animals, that is to say that when the environment becomes very different, it produces in course of time corresponding modifications in the shape and organisation of animals (. . .) [because] great alterations in the environment of animals lead to great alterations in their needs" has become a "truth, which, once recognized, cannot be disputed" (Lamarck, 1914). He thus recognized the continuous dynamic geological, climate, and geographic changes in the environment as opposed to a static world, and in order to adjust to these changes, organisms had to evolve (Mayr, 1972). According to Lamarck, because "nature is forced to submit her works to the influence of their environment, (. . .) this environment everywhere produces variations in them" (Lamarck in Shaner, 1927). Resulting from this, "Nature has produced all the species of animals in succession, beginning with the most imperfect or simplest, and ending her work with the most perfect, so as to create a gradually increasing complexity in their organisation, (. . .) [forming] a branching series, irregularly graded and free from discontinuity, or at least once free from it. . ." (Lamarck in Shaner, 1927). As Shaner (1927) notes, it was Lamarck who first thought of the animal kingdom as a great family tree, initiating the modern theory of evolution. However, to his disfavor, Lamarck is mostly known for his concept of inheritance of acquired characteristics formulated in his second law: "All the acquisitions or losses wrought by nature on individuals, through the influence of the environment in which their race has long been placed (. . .) are preserved by reproduction to the new individuals which arise" (Lamarck, 1914). This was similar to that proposed by Erasmus Darwin in his work "Zoonomia" published earlier in 1794: "[F]rom the first rudiment, or primordium, to the termination of their lives, all animals undergo perpetual transformations; which are in part produced by their own exertions in consequence of their desires and aversions, of their pleasures and pains, or of irritations, or of associations; and many of these acquired forms, or propensities, are transmitted to their posterity" (Darwin in Harrison, 1971). Darwin and Lamarck had failed to distinguish between the influence of the environment on individual animals (resulting in non-heritable modifications) vs. the influence of the environment on animal populations (resulting in evolution). It was Erasmus' grandchild Charles who successfully challenged the inheritance of acquired characters in individuals when he recognized the influence of the environment on evolution of animal populations, resulting from natural selection in the struggle for existence. But as to how variations were produced on which natural selection could act, he wrote: "I have hitherto sometimes spoken as if the variations-so common and multiform in organic beings under domestication, and in a lesser degree in those in a state of nature-had been due to chance. This, of course, is a wholly incorrect expression, but it serves to acknowledge plainly our ignorance of the cause of each particular variation" (Darwin, 1869). In an aim at answering this question of the origin of variation, he developed the hypothesis of pangenesis based on modifications and amplifications of earlier existing theories. Each unit of living tissue continually produced minute particles or "gemmules" at each stage of its development which would multiply and develop themselves into new cells and which were transmitted from parents to offspring via the reproductive organs (Geisen, 1969). This idea is similar to that proposed far back in antiquity by Hippocrates: "For the seed comes from all parts of the body, healthy seed from healthy parts, diseased seed from diseased parts" (Hippocrates in: Zirkle, 1946). However, not different from Lamarck, it was still a naïve conception of transmission of personal qualities as the heritable elements to the progeny. Reaction Norms and Phenotypic Plasticity This approach to heredity was very different from the first controversial but accurate model by Mendel, first published in 1865 but not seriously considered until 1900, introducing "elements" of inheritance. These elements were later coined "genes" by Johannsen in 1909 and recognized as a segment of a chromosome after the discovery of the structure of DNA by Watson and Crick in 1953 (Portin, 2002). The discovery of Mendelian inheritance resulted in a temporary popularity of discontinuous "saltations" by mutations as the primary mechanism of evolutionary change as opposed to Darwin's concept of evolution through natural selection acting on small continuous variations (Sarkar, 1999). Woltereck (1909), in order to prove Darwin right, studied phenotypic variation of continuous traits in morphologically distinct pure-line strains of Daphnia species subjected to variations in environmental factors. Plotting the response curves of the phenotypes (relative head height) of the different strains to the environmental variation (nutrient level) showed that the resulting reaktionsnorm (reaction norm, or standard pattern of the response curve) was different in the different strains (Woltereck, 1909;Sarkar, 1999). In his understanding, the genotype of an animal was synonymous to the shape of this curve, i.e., the reaction norm, and thus constituted the unit that was inherited, resulting in hereditary change. Johannsen, who had proposed the term "genotype" as the "sum total of all the "genes" in a gamete or in a zygote" agreed that "[t]he very appropriate German term "Reaktionsnorm" used by Woltereck is, as may be seen, nearly synonymous with "genotype, " in so far as the "Reaktionsnorm" is the sum total of the potentialities of the zygotes in question. (. . .) [It] emphasizes the diversity and still the unity in the behaviour of the individual organism; certainly, the particular organism is a whole, and its multiple varying reactions are determined by its "genotype" interfering with the totality of all incident factors, may it be external or internal. Thence the notion "Reaktionsnorm" is fully compatible with the genotype-conception" (Johannsen, 1911). However, he did contest that Woltereck's observations disproved evolutionary saltations since he held that continuous transitions exhibited by phenotypes, as expressed in the reaction norm, result from discontinuous saltations in the genotype, i.e., through mutations. Three years later, Nilsson-Ehle, discussed the "acclimatization or adjustment" to the climate by plants, i.e., "the plant's ability to change their characteristics in one way or another such that it thrives in a new environment" (Nilsson-Ehle, 1914, quote translated from Swedish). Referring to a particular example of a 10-year study by Bonnier (1894), who described the adaptation of individual plants of the same genotype (cuttings of the same seedlings) to the climate at different altitudes in the Alpes and the Pyrenees with respect to their size, color, and shape, he concluded (translated from Swedish): "Summarizing all experience in this area, then you can also say that the climate's influence can hardly be explained in a purely causal-mechanical way. One has to, as (. . .) even Johanssen explicitly holds, count with the organisms' ability of self-adjustment or self-regulation, the appropriate reaction norm. This plasticity, depending on various external conditions, is in fact neither easier nor more difficult to interpret then the organism's appropriate characteristics at all. " Nilsson-Ehle is by many recognized as being the first scientist to use the word "plasticity" ("plasticitet, " Nilsson-Ehle, 1914, p. 549) to describe the effect of the environment on the phenotype of an organism (Fuller, 2003), however, it was Bonnier himself who proposed it ("plasticité") some 10 years earlier based on his own work that Nilsson-Ehle had referred to (translated from French): "The influence of the climate of the Alpine region is not only visible in the modification of the diverse exterior characteristics; it also has a profound effects on the development and the nature of the different tissues of the organism, each affected to a more or lesser extent. (. . .) Among the plants that support the climate change, from the plain to high altitudes or vice versa, some show almost complete modifications the first year, whereas others only show the beginning of transformation after 10 years. Therefore, all the degrees of plasticity are possible, depending on the species considered" (Bonnier, 1895). By 1918, Fisher had introduced a method that allowed for the separation of different causes of variability: "It is therefore desirable in analyzing the causes of variability to deal with the square of the standard deviation as the measure of variability. We shall term this quantity the Variance of the normal population to which it refers, and we may now ascribe to the constituent causes fractions or percentages of the total variance which they together produce" (Fisher, 1918). At the time he considered that the variation due to environment was nihil (probably less than five percent) and that most of the variation instead was due to ancestry, Mendelian segregation and dominance. Although later he did reconsider the environment as a possible source of variation and with it the relationship between environmental and heritable variation when he first presented the "analysis of variance" table (Fisher and Mackenzie, 1923;Tabery, 2008), the effect of the environment really created a potential complication for assessing the relative importance of heredity and so it was to be considered and then either dismissed or eliminated or at least minimized by experimental design (Tabery, 2008;Strandberg, 2009). Not for Lancelot Hogben, however, who further developed his thoughts on the relationship between differences of genetic constitution and the external environment in the process of development. He thus recognized three different sources of variability: genetic, environmental, and that which "arises from the combination of a particular hereditary constitution with a particular kind of environment, " or Genotype × Environment interaction (Hogben, 1932;Tabery, 2008). Canalization Meanwhile, in the Soviet Union, the concept of the reaction norm was further developed in the 1920s, such as resulting from the work of Dobzhansky on the "abnormal abdomen" mutation of Drosophila funebris (Sarkar, 1999). Much in line with Johannsen, he held that it was the entire reaction norm that was inherited and that mutation resulted in a change in this norm of reaction (Nicoglou, 2014). Subsequently, Schmalhausen (1949; originally published in Russian in 1938) clearly recognized the influence of the environment on the evolution of the reaction norms: different environments will expose different portions of the reaction norm that will be subjected to natural selection, whereas the portions not exposed, or no longer exposed when the environment changes, will be subjected to drift. Changes in the environment eventually result in adaptive modifications that will again "stabilize" into new adaptive phenotypic response curves. The reactivity of the reaction norms, stabilized by means of processes of autoregulation through underlying reactions, would thus be buffered or "canalized" into a more specific optimal norm (Schmalhausen, 1949;Pigliucci, 2001). This idea is similar to that proposed (independently) by Waddington a few years later (1942): "The main thesis is that developmental reactions, as they occur in organisms submitted to natural selection, are in general canalized. That is to say, they are adjusted as to bring about one definite end-result regardless of minor variations in conditions during the course of the reaction. (. . .). The canalization, or perhaps it would be better to call it the buffering, of the genotype is evidenced most clearly by constancy of the wild type. " The constancy of the wild type was recognized earlier by Darwin (1869) when he wrote observing a "much greater variability, as well as the greater frequency of monstrosities, under domestication or cultivation, than under nature. " Since canalization thus reduces the phenotypic expression of variation, it can actually result in the undetected accumulation of selectively neutral underlying genetic variation and mutation accumulation, a concept that is extensively discussed by Schlichting (2008). In other words, the genotype "absorbs" a certain amount of its own variation such as that resulting from new mutations ("genetic canalization") or that resulting from environmental perturbations ("environmental canalization"; Waddington, 1942;Pigliucci, 2001). Lerner (1954) coined this ability of a Mendelian population of organisms to equilibrate its genetic composition and to resist sudden changes "genetic homeostasis", as grounded in the concept of physiological homeostasis proposed earlier by Cannon (1932) (Hall, 2005). Thus, canalization of a character can be equated with homeostasis of that character. In effect, "[b]y insensible gradations this functional homeostasis merges with physiological reactions which result in developmental homeostasis. (. . .) A given repertory of functional and developmental homeostatic mechanisms is, of course, determined by the norm of reaction of each genotype" (Dobzhansky and Levene, 1955). And, similar to physiological homeostasis, straying away from the limited variety of possible reaction norms established in evolution under the control of natural selection would result in death (Dobzhansky and Levene, 1955). Although Lerner's genetic homeostasis was described for Mendelian populations and not for individuals, he argued that it was brought about by the same mechanisms as those which underlie the other forms of homeostasis (Dobzhansky and Levene, 1955). It was implied that Darwinian fitness, resulting from homeostatic adjustment through self-regulation to environmental or genetic disturbances, was manifested by true heterosis or hybrid vigor (Woolf and Markow, 2003). And hybrid vigor, in turn, was considered to be a consequence of heterozygosity, as first proposed independently by Shull and East in 1908, and after by Dobzhansky in 1950. Dobzhansky proposed that it was particularly coadapted heterozygosity that was a component of Darwinian fitness, referring to polygene complexes which have become mutually adapted by natural selection in the course of evolution; however, some years later he concluded that heterozygosity may produce higher fitness even without prior coadaptation (Woolf and Markow, 2003). Lerner (1954) also emphasized the heterozygote buffering advantage associated with coadapted polygenic systems resulting from evolutionary history, especially in natural populations, although he also indicated that heterozygosity at a single locus (or coadapted homozygosity in self-fertilizing plants) and epistasis may play a role in determining adaptation (Woolf and Markow, 2003). In addition, he held that no population can afford to maintain too many heterotic loci or blocks simultaneously (Lerner, 1961). Phenotypic Plasticity vs Canalization According to Lerner (1954), the superior buffering ability of heterozygotes at complex multigenic systems would serve two important functions: it would allow for individuals with combinations of phenotypic properties that are expressed near the optimum (canalization), while at the same time it would result in genetic variation, although "hidden" in the phenotypes, and potential plasticity (Woolf and Markow, 2003;Hall, 2005). As Dobzhansky and Levene (1955) note, homeostasis does not prevent the development from switching from one of the historically established paths to other established paths, as long as they remain within the canalized norm. The ability of the organism to follow any of these paths (or to change paths) is, in fact, highly adaptive. This emphasizes the complementary relationship between the processes of canalization and plasticity. Indeed, as given by Waddington (1953) and Dobzhansky and Levene (1955), homeostasis does not imply a stationary state but a dynamic (plastic) stability (canalization); "homeostasis is brought about by changes in some processes which result in stability of other processes. " And following Cannon (1932): "Constancy is in itself evidence that agencies are acting, or ready to act, to maintain this constancy. " Schmalhausen (1949) considered that those animals that are best in responding adaptively to changes in the environment (i.e., those with highest plasticity) while simultaneously best withstood environmental perturbations (i.e., those with highest canalization) would be favored by natural selection (Willmore et al., 2007). Also Bradshaw (1965), in a key contribution to the field, emphasized the adaptive value and evolutionary significance of plasticity, in particular in plants since they are not able, as animals are, to evade adverse conditions: plasticity of certain characters may lead to homeostasis (canalization) of others (Bradshaw, 1965). An example of a plastic mechanism in animals that results in overall robustness (phenotypic stability) is protein turnover, which is responsive to various physiological and developmental scenarios, and provides the flux that is necessary for metabolic regulation and adaptation. Because it is involved in maintenance of homeothermy, reproduction, development, the repair of damaged tissue, maintenance of the immune system, combating infection, and the nutritional/physiological status, a high turnover rate may improve robustness by improving the ability of an animal to adapt to new dietary and physiological conditions (Baldwin et al., 1980;Rauw, 2012). Also plasticity in the functioning of the hypothalamic-pituitary-adrenal axis, which is the most important stress-responsive neuroendocrine system and shows large differences across species, breeds and individuals, has been found to improve robustness through its effects on metabolism, the immune system, inflammatory processes and brain function (Mormède et al., 2011). Bradshaw (1965) proposed that plasticity of a character can be (a) specific to that character, (b) specific in relation to particular environmental influences, (c) specific in direction, (d) under genetic control, and (e) radically altered by genetic selection. According to Via (1993) and De Jong (1995), "plasticity can be produced either by environment-specific gene expression or by allelic effects that vary across environments. " De Jong (1995) defined the reaction norm as the total pattern of expression of a character along a continuous gradient, and plasticity as the difference in character value between environments, i.e., the first derivative of the function in that environment. When the environment cannot be described along a continuous gradient, than it will be mandatory to describe the phenotypic expression as a series of character states, i.e., values as points on the curve. However, when the environment can be described by a continuous variable, it is possible to describe a character by a function (the reaction norm) and use the function values, coefficients and derivatives for traits (De Jong, 1995). Although reaction norms are mostly described as linear relationships, they can take any shape. Figure 1 presents phenotypic character states of three different (imaginary) genotypes as a function of an environmental gradient at values −1 (an unfavorable environment), 0 (a "neutral" environment) and 1 (a favorable environment). Animal 1 shows a steady increase in phenotypic performance when the environment improves. Animal 2 increases its phenotypic performance slightly when the environment becomes more favorable, however, it is particularly negatively affected when the environment becomes more challenging. Animal 3, as animal 1, shows a steady increase in phenotypic performance when the environment improves, but at a slower rate. REACTION NORMS ANALYSIS IN LIVESTOCK PRODUCTION Reaction norms analysis in animal breeding involves quantification of resilience of phenotypic values of production performance expressed by a genotype or by various genotypes across a gradient of a descriptor of the environment (Knap, 2005). Intuitively it holds that an abundant environment will result in a better production performance whereas a restricted environment will depress production. It is proposed that an animal (a genotype) that is best at maintaining its production across this gradient is more robust (i.e., less sensitive) because it has a greater ability to adapt to environmental fluctuations. It is clear from the description that this method is not much more specific than the trait it aims to measure, but it does visualize what robustness represents: a combined production ability (y-axis, the level) and environmental adaptability (x-axis, the slope) trait that can be described in different ways depending on how the variables along the axes are quantified. For example, along the x-axis, environmental factors affecting animal production can be thought to include disease exposure, social stress, stocking density, temperature, nutrient quality, feeding regime, etc. In addition to the ability to maintain production performance, the animal in question will need to be healthy with a sufficient welfare, as it can only be considered robust when its production is qualified as "unproblematic. " In order to include this last part in the analysis, a multi-dimensional representation could be imagined, not only including production traits measured across a gradient of a descriptor of the environment, but also health and welfare traits measured across a gradient of the environment or of the production response. In plants, a particular individual genotype can be represented by identical clones, however, in animal breeding, the reaction norm of an individual "genotype" (often the sire) can be approximated by its offspring which is spread across a wide environmental range, usually through AI (Knap, 2009). The following three sections give an overview of the use of reaction norms analyses today in dairy cattle, beef cattle, and pigs. The aim of these sections is to review how x-and y-axis traits are formulated in these different livestock species, and to indicate some of the results that followed from the analyses. Reaction Norms in Dairy Cattle The reaction norms method has been mostly applied to dairy cattle, which can count on large numbers of daughters for each sire that are producing at a wide variety of herd environments at which a wide variety of characteristics are recorded. This wide range of available production characteristics facilitates investigation of a descriptor of the environment as a continuous variable instead of being limited to describing the environment as discrete classes, i.e., as a series of character states. Zwald et al. (2001), Fikse et al. (2003), and Calus and Veerkamp (2003) describe several continuous climate and herd management characteristics that can be used as descriptors of the environment, such as "mean peak yield" and "persistency" as an overall measure of the quality and intensity of the management system, "days to peak yield" reflecting differences in dry cow management and health and nutrition programs, "herd size" as an indirect measure of differences in facilities and treatment of cattle, "day of calving" as a variable that could separate rotational grazing herds with seasonal calving from other types of herds that feature year-round calving, "percentage of animals with completed lactations" as a measure to explain differences in culling strategies between farms, "fat:protein ratio" as a measure of the feeding system, "body condition score" as a measure of the ability of management to tune the feed intake to the energy requirements of the animal, and a temperature and humidity indicator as a measure of the heat stress on cows. As Calus and Veerkamp (2003) indicate: "Potentially a large number of environmental parameters could be defined, but parameters used (. . .) were chosen because they: (1) reflect management and environment, (2) are obtainable from the available data, (3) are continuous rather than categorical (. . .), and (4) are not too strongly correlated with each other. " Strandberg et al. (2000) used the herd-year effect as a general measure of a complex of environmental values to which they linearly regressed 305-d protein yield and days open to estimate breeding values in Nordic dairy cattle (Finnish Ayrshire, Norwegian Dairy Cattle, and the Swedish Red and White Breed). Crossing of reaction norms indicated reranking in the presence of genotype × environment interactions for both traits. Calus et al. (2002) performed a random linear regression of 305-d heifer protein production on herd-year-season in Dutch Holstein Friesian dairy cattle. The level of the reaction norm had such a great impact that the slope had very little influence on the total breeding value, and no genotype × environment interaction was observed. They suggested that another more environmentalspecific parameter or defining another scale for the environmental parameter might contribute to increase the influence of the slope. In addition, they suggest that non-linear reaction norms might explain sire variance better. Ravagnolo and Misztal (2002) estimated the genetic component in heat tolerance for nonreturn rate in Holstein cows using an animal linear model augmented by a random regression on a temperature-humidity index. They observed a negative, unfavorable genetic correlation between merit for milk yield and non-return rate at 90 days after first insemination but indicated that simultaneous selection for improving both traits is feasible. Kolmodin et al. (2002) regressed first lactation 305-d protein production and days open on the herd-year average in Nordic dairy cattle (Danish Red Dairy Breed, Finnish Ayrshire, Norwegian Dairy Cattle, and the Swedish Red and White Breed). They evaluated three different reaction norm models: (1) a random regression on an environmental variable, (2) a regression model including the level and the slope of the reaction norm of the sire, and (3) an extension of model (2) to include a set of regressions on a second environmental variable. The models were similar in both the level and the slope. Results showed that the genetic parameters changed over environments, and that a significant variation for the slope of the norm resulted in little reranking of sires, except between extreme environments. Fikse et al. (2003) regressed 305-d milk yield on fifteen environmental parameters in Guernsey-sired cows (from Australia, Canada, United States, and the Republic of South Africa). Nine parameters had a significant effect and results indicated that reranking of animals may occur in extreme environments. Calus and Veerkamp (2003) estimated breeding values for milk, fat, and protein yield and percentage, of daughters by applying a random regression on various values of environmental parameters for each sire in Dutch dairy cattle (mostly Holstein-Friesian and Meuse-Rhine-Yssel). Twelve of fourteen environmental parameters gave significant reaction norms, but reranking hardly occurred across environments. Hayes et al. (2003) investigated the magnitude of genotype × environment interactions of milk, protein, and fat yield from a random regression on four different environmental descriptors in Australian Holstein-Friesian dairy cattle. Interactions were observed for average herd protein yield and temperature humidity index. Bryant et al. (2006) investigated the environmental sensitivity of Holstein Friesian and Jersey dairy cattle and their crosses for 2-year milk, fat and protein yields in relation to the range of herd milksolid yields (as a proxy for feeding level) in New Zealand using first and second degree polynomial regression functions. Their results indicated that Holstein Friesians originating from overseas (mostly from North America), exhibited higher levels of production (level) but also higher environmental sensitivity (slope) than Holstein Friesians from New Zealand and Jerseys. The overseas Holstein Friesians, which are selected in an environment where high levels of concentrate are offered and high levels of production are achieved, improved their ranking in a high production level environment, whereas New Zealand Jerseys, which are selected in pasture-based, low production level environments with high levels of environmental heterogeneity due to the variable nature of pasture supply, improved their ranking in a low production level, grassland-type environment. Haile-Mariam et al. (2008) regressed not only milk production traits (milk, fat, and protein yield and percentage) but also fertility traits (calving interval, calving to first service interval, 25-d nonreturn rate at first service, and pregnancy rate) and survival to the next lactation on the environmental descriptors "level of herd milk production, " temperature-humidity index, and herd size in Australian Holstein-Friesian dairy cattle. There was no evidence for the presence of a large genotype × environment interaction that resulted in economically significant reranking of bulls. Shariati et al. (2007), fitting a reaction norms model to first test-day records for first lactation milk, protein, and fat of Danish Holstein cows, reported the presence of genotype × environment interaction, but with a small effect on reranking of candidates for selection. Streit et al. (2012) applied reaction norm random regression sire models to corrected test day records for milk, protein, and fat yield and somatic cell score as a function of herd test day solutions as environmental descriptors in German Holstein dairy cattle. Results indicated the presence of minor genotype × environment interactions which did not result in reranking of sires. Corrêa et al. (2010) evaluated differences in sire genetic values by a reaction norms hierarchical model for post weaning gain in response to estimates of contemporary group effects in Brazilian Devon cattle. They reported the existence of genotype × environment interaction. Most reranking of sires happened in restrictive environments, indicating that importing genetic material should be carefully assessed when the selection conditions of the animals in the exporting countries are greatly superior to local production environmental conditions. Pégolo et al. (2009) assessed genotype × environment interaction for 450-day adjusted weight and body weight gain in Brazilian Nelore cattle using a random regression reaction norms model on heard-year and herd-year-season-management groups, and heard-year-season-management group solution estimates. The models generated consistent parameter estimates. Important genotype × environment interactions were found with low genetic correlations among extreme environments, indicating a significant reranking of sires in different environments. Mattar et al. (2011) investigated the presence of genotype × environment interactions for long-yearling weights of Brazilian Canchim cattle using reaction norms of the trait as a response to a "contemporary group" effect that combined year and season of birth, sex, genetic group of dam, herd at weaning and long-yearling, and feeding regimen from birth to weaning and from weaning to long-yearling. Their results showed that all animals increased their performance with the environmental improvement, that there was some reordering of genotype ranks, and that there existed variability in phenotypic plasticity. Cardoso and Tempelman (2012) investigated alternative linear reaction norms models for post-weaning body weight gain to a "contemporary group" effect of herd-year-season-sexmanagement subclasses in Brazilian Angus cattle. They observed genotype × environment interactions and possible reranking, and furthermore concluded that environmental sensitivity of imported North American Angus bulls was significantly larger than that of local Brazilian Angus sires which tended to be more robust to environmental changes. Santana et al. (2013) determined the presence of genotype × environment interaction for birth weight, weaning weight, postweaning weight gain and yearling scrotal circumference in Brazilian composite beef cattle from reaction norms taking the environmental covariate of the reaction norms (the contemporary group) as the environmental descriptor. A genotype × environment interaction was observed and reranking of animals and it was concluded that it can be important to include phenotypic plasticity in the breeding goal. Reaction Norms in Pigs Reaction norms in pig production are scarcely described. Knap and Su (2008) estimated linear reaction norms of total litter size at birth as a function of routine herd-year-season effects in two PIC lines of pigs and their cross. Daughters of sires were spread over North and Latin America, Europe, Asia and Australia, providing for a wide range of environmental effects of a climatic, nutritious, management-related and infectious nature. Environmental sensitivity showed a progressively lower genetic component with increasing data volume, and progressively less frequent reranking of genotypes across the environmental range. Consequently it was recognized that reaction norms analysis is indeed a demanding process, requiring large data volume and a wide environmental range in order to produce meaningful results (Knap and Su, 2008). Reaction Norms for Behavior and Welfare So far, a behavioral reaction norm as suggested here previously has not been applied in livestock production, however, Sih et al. (2004) proposed that behavior can be included in phenotypic plasticity and reaction norms models. Similar, Dingemanse et al. (2009) describe that animal responsiveness (behavior) can be described as a function of environmental variation (context), and that this can be considered a complementary aspect of the individual phenotype. Examples given are the relationships between parental provisioning rate and offspring begging intensity, between dispersal behavior and wind velocity, or between anti-predator behavior and predation risk (Dingemanse et al., 2009). In addition, animal personality is suggested to express itself as a coping strategy that is consistent across contexts (Koolhaas et al., 1999); Sih et al. (2004) refer to such suites of correlated behaviors in an individual as "behavioral types, " which show consistency in behavior across multiple situations. This behavioral consistency may be represented by the individual behavioral response as a function of a stimulus that can vary across a gradient, as an index of its behavioral stability (Sih et al., 2004;Dingemanse et al., 2009). Personality does not imply that each individual is necessarily completely consistent in behavior, such that variation in plasticity may be observed between individuals and populations (Dingemanse et al., 2009). Coping styles are important in livestock production as they form general adaptive response patterns that have genetically evolved in reaction to everyday challenges and are thus closely related to individual adaptive capacity and vulnerability to stress-related disease (Koolhaas et al., 1999). Dingemanse et al. (2012) used the reaction norms approach to estimate the quantitative genetics parameters of the exploration behavior of an open-field of over 1000 offspring of two populations of wild-caught three-spined stickleback fish. They found heritable variation and population differences in both the average level of exploration and behavioral plasticity. Examples in livestock production of environmental gradients can be thought to include group size and composition, temperature, photoperiod, environmental enrichment, but might also include production parameters such as growth rate or milk production. Smiseth et al. (2008) described behavioral reaction norms to investigate parent-offspring conflict and co-adaptation. They indicate that behavioral interactions can include other questions where the expression of traits depends upon the behavior of other individuals, "encompassing the whole field of animal communication, " such as aggression related to competition for resources. A similar analysis may be applicable to social interactions in livestock production systems. Selection for Increased Production, Against Environmental Sensitivity The breeding value as estimated from reaction norms analysis is built up of two parts: the environment-independent part (the level), and the environment-dependent part (the slope; Calus et al., 2002). Thus, the ideal reaction norm in animal production has a high level and a flat slope (Strandberg et al., 2000). According to De Jong (1995), the level and the slope are genetically correlated; however, this does not necessarily mean that separate genes for plasticity and trait mean exist. Su et al. (2006) indicate that in reaction norms analysis a linear relationship between the phenotypic expression of a given genotype and the covariate representing a particular environmental effect is assumed, which is approximated by using the mean phenotypic performance in the appropriate environment, without the need to know the actual covariate. However, the variance among phenotypic means of production environments includes a genetic component, resulting in overestimation of the variation of environmental values, even in a random mating population. In addition, computer simulation indicated that it results in an underestimation of variance components associated with the slope, and an overestimation of the variance components associated with the level. Instead, they suggest a more satisfactory alternative by inferring environmental values simultaneously with the other parameters in the model using a Bayesian Markov Chain Monte Carlo approach, which was shown to lead to estimates of parameters with no detectable bias and with smaller mean squared errors. To account for a scale effect on residual variances in reaction norms models such that larger environmental effects are associated with larger residual variances, Cardoso and Tempelman (2012) proposed two alternative extensions to the model to allow for heteroskedastic residuals: an exponential function and a best fitting environmental classification model; the latter seemed to provide a better fit than the exponential function. Lillehammer et al. (2007Lillehammer et al. ( , 2009) described a different approach by investigating not the effects of genotypes but the effects of single genes in response to environmental variation using quantitative trait loci (Lillehammer et al., 2007) and single nucleotide polymorphisms (Lillehammer et al., 2009). This is important since QTLs and SNPs with an environmental interaction can be hard to detect even though they have a large average effect. In the SNP analysis they report a genetic correlation between general production and environmental sensitivity from 0.55 to 0.88, indicating that most genes should affect the level and the slope in the same direction. This supports earlier work by Kolmodin et al. (2002) who observed that animals with genetically high production tended to be more sensitive to changes in the production and fertility environment, and by Kolmodin et al. (2003), who studied the effect on environmental sensitivity (the slope) of selection for high phenotypic value (the level) in combination with a continuously improving environment in a simulation study. They detected a significant selection response, suggesting that environmental sensitivity will increase with selection for high phenotypic values. These observations were also supported by later work, for example by Knap and Su (2008), who indicated that the very precisely estimated correlation between the intercept and the slope was extremely high: "Hence, irrespective of genetic effects, the performance of sows with a high reproductive capacity is practically always highly sensitive to environmental disturbance. [The same pattern applies to] the genetic level; [it is clear] that for litter size, the performance of high-potential genotypes (and of high-capacity sows) will likely come down strongly when environmental conditions become unfavourable. " However, because of the low heritability of the slopes, environmental sensitivity would be increasing at a slow rate. The negative correlation between high levels of production and increased environmental sensitivity can result from resource allocation patterns described by Beilharz et al. (1993). Resource demanding physiological processes show trade-offs resulting from limits in the resource availability, food intake and digestive capacity and/or limiting resource allocation patterns which typically result in a genotype × environment interaction. Animals that are genetically driven to produce at high levels may need to reallocate resources away from other process, leaving the animal lacking in ability to respond to other demands, such as coping with disease and stress. This will consequently result in an animal that is more sensitive to environmental fluctuations (Rauw, 2009). Indeed, Friggens and Van der Waaij (2009) indicate the single-trait limitation of the reaction norms approach and developed resource allocation models, based on the model of Van der Waaij (2004), providing a framework for a multitrait definition of robustness. This model explicitly examines the partition of resources between different life functions and provides a framework for exploring trade-offs. The equations allow for relating total fitness to environmental variation and resource availability, defining plasticity in terms of more than one trait. This is more biologically meaningful since adaptation to environmental change is essentially a process that results from a combination of physiological mechanisms (Friggens and Newbold, 2007). However, as reviewed by Friggens et al. (2013), the challenge of linking prediction of nutrient partitioning to its consequences on health, reproduction, and longevity is only recently being addressed, and so far the models developed, for the most part, remain research models that need to be further developed to be applied in the field. As Kolmodin et al. (2003) notes, high sensitivity may be beneficial when the environment is highly controllable and predictable, since the benefit from improvements of, for example, management and feeding would be substantial, while the risk of environmental deterioration, causing drastic reductions in levels of production, would be relatively low. However, since populations of animals with high production potential will be more dependent on highly controlled environments this may be of ethical concern. Lillehammer et al. (2009) indicate that their results show that a small fraction of the genes affect only production (the level) or only environmental sensitivity (the slope). In addition, even a category of possible selection gene candidates was found that affects production and environmental sensitivity in opposite directions. Such genes would facilitate selection for increased production and robustness at the same time. DIRECT INCLUSION OF ROBUSTNESS TRAITS IN THE BREEDING OBJECTIVE The second option for breeding for animal robustness is the direct approach, which encompasses the inclusion of directly measurable robustness traits in the breeding objective and in the selection index. These robustness traits can include the same physiological, immunological and reproduction traits that are affected as a result of selection for high production efficiency (Rauw et al., 1998). They are often referred to as "functional traits, " i.e., traits that are closely related to biological functional ability or fitness, such as longevity, health and fertility. Although these traits are important to all livestock animals, the term is mostly used in dairy cattle production, where they can include structural soundness, udder and teat conformation, frame score, disposition/temperament, body condition score, fertility, calving ease and mothering ability, and adaptability to the environment (Peck, 2006;Egger-Danner et al., 2015). Similar fitness traits related to longevity, health and fertility are described for other livestock species. The Nordic countries (Sweden, Norway, Denmark) in particular have broadened breeding goals to also include fertility and health, which became possible since these countries implemented well-established, national recording systems for health data (Herringstad et al., 2000). Since the mid-1990s also several European and North-American breeding organizations have included fertility and health in their breeding objectives (Oltenacu and Broom, 2010). The International Committee for Animal Recording (ICAR) promotes since 1951 the development and improvement of activities of performance recording and the evaluation of dairy cattle and its Functional Traits Workgroup is in particular involved with recommendations regarding functional traits in dairy cattle. Heritabilities of functional traits and feasibility of inclusion of these traits in the breeding objective has been described in a number of works and several reviews (e.g., Groen et al., 1997;Essl, 1998;Herringstad et al., 2000;Lawrence et al., 2004;Egger-Danner et al., 2015). According to Knap (2009), genetic improvement of robustness traits can improve profitability of production at a similar rate as by improvement of a conventional production trait. In spite of antagonisms between robustness and production performance, a positive genetic trend in both traits can be achieved at the same time when robustness traits are properly included in the breeding goal and selection criteria (Knap, 2009). In addition, several authors discussed the feasibility of including behavioral traits that are related to animal welfare in the selection criterion. These traits will improve animal welfare and can be expected to lead to improvements in mortality, disease resistance, efficiency, longevity, reproductive performance and carcass wastage as a correlated effect (Turner, 2011). For example, Jones and Hocking (1999) extensively reviewed the feasibility of using selective breeding to improve welfare, describing results of selective breeding studies in which fear, adrenocortical stress responses, social motivation, feather pecking, and growth rate were manipulated in quail and chickens. Star et al. (2008) described including, besides immunological and physiological traits, also behavioral traits in laying hens. Rydhmer and Lundeheim (2008) proposed to include improved piglet survival, stronger legs, a better constitution, improved disease resistance, less aggressive behavior, reduced fear of humans and a great appetite in the breeding programs of pigs. D'Eath et al. (2010) discussed the possibilities of selection for farm animal behavior in livestock species in general, indicating that in many cases, estimated heritabilities are of comparable magnitude to traits already included in the breeding program (0.1 to 0.4) which suggests that selection for behavior would result in a positive selection response. Turner (2011) explored the genetic contribution to harmful social behavior traits using as examples regrouping and poor maternal care in pigs, and oral manipulation of penmates in pigs and laying hens, and concluded that for most traits, improvements in harmful behavior can be made by careful breed choices and selective breeding. Dawkins and Layton (2012) describe the feasibility of breeding for better welfare in broiler chickens, noting that "Broiler chicken welfare is most likely to be improved in practice if animal welfare traits such as good walking ability, good feathering and healthy legs and feet are seen as compatible, rather than in conflict, with other goals such as commercial production. " Canario et al. (2013) reviewed the feasibility of including behavioral traits in the selection criteria of cattle, pigs, poultry and fish. They note that animal behavior is a welfare indicator since it relates both to the existence of stressors and to the animal's ability for behavioral adaptation to physical and social environmental stressors. Mormède et al. (2011) proposed to select animals for a higher activity of the stress-related hypothalamic-pituitary-adrenal axis (which releases cortisol or corticosterone) to improve animal robustness and welfare. And finally, Oliveira et al. (2010) proposed assessment of play behavior as a new and promising potential indicator of animal welfare. According to Allen and Bekoff (2005), there are evident emotions associated with play-joy and happiness-that drive animals into it. Indeed, animal play only if they are healthy, safe, well-fed and in a relaxed state, but not if they are under a stressful condition (Burghardt, 2005). According to Held and Špinka (2011), play may signal both the absence of bad welfare and the presence of good welfare, however, it does not consistently reflect favorable environmental conditions. Rauw (2013) investigated the consistency of a behavioral play marker in piglets and proposed investigating the feasibility of using play markers in the selection criterion of livestock species. The challenge to including behavioral traits in the selection criteria is to define quantifiable traits or proxy measures thereof that can be recorded cost-effectively and reliably on the large number of animals that are necessary for a breeding program (D'Eath et al., 2010;Turner, 2011). In addition, which trait(s) to select for in order to truly improve animal welfare is complicated by the many different conceptions and definitions of animal welfare proposed, defined in terms of, e.g., animal function, the balance of enjoyment or pleasure vs. suffering or pain, preference satisfaction, or natural living (Duncan and Fraser, 1997;Lassen et al., 2006). As Turner (2011) notes, it may be difficult to identify behavior in the recipients vs. the donors (for example of aggression), and it may be challenging to attribute an accurate economic value to behavioral traits. In addition, D'Eath et al. (2010) warn for selecting animals that do no longer show outside signs of negative welfare, but still experience the negative feelings associated with the unwanted behavior, for example in the case of docile animals that are too frightened to move. It may thus be necessary to first further investigate the cognitive processes and emotional experiences underlying the phenotypes (Turner, 2011). Finally, in addition to production traits, functional traits and behavioral traits, Olesen et al. (2000) discussed the need to define animal breeding goals as an integrated part of sustainable production systems, i.e., based on a holistic, longterm perspective. They stress that higher productivity should not only be balanced with (short-term) improved health, fertility, and feed intake capacity, but also with (long-term) important non-market values of animal traits, such as ethical values of improved animal welfare and possibly also with natural capital and ecosystem services (depletion of fossil energy, degradation of the atmosphere) and social issues. Also Kanis et al. (2005) proposed including "societally important" traits, such as product safety, welfare, and environmental impact, which do not have a clear economic value. They present a retrospective selection-index method to obtain the proper weights for those traits. Olesen et al. (2000) emphasize that animal breeding practices must become part of the pluri-and interdisciplinary, philosophical and ethical debate. Code-EFABAR also follows the principles of sustainable breeding in their Code of Good Practice; the general definition of sustainable farm animal breeding is defined as: "the extent to which animal breeding and reproduction, as managed by professional organizations, contribute to maintenance and good care of animal genetic resources for future generations" (Gamborg and Sandøe, 2005;Code-EFABAR, 2006). ETHICAL CONSIDERATIONS Artificial selection was already described by Mago from Carthage in his work "Treatise on Agriculture" several centuries BC in which he recommended choosing oxes that were "young, stocky, sturdy of limb with long horns, darkish and healthy, with a wide and wrinkled forehead, hairy ears and black eyes and chops, the nostrils well-opened and turned back, the neck long and muscular, and dewlap full and descending to the knees, the chest welldeveloped, broad shoulders, the belly big like that of a cow in calf, the flanks long, the loins broad, the back straight and flat or a little depressed in the middle, the buttocks rounded, the legs thick and straight, the hooves large, the tail long and hairy and the hair on the body thick and short, red-brown in color and very soft to the touch" (Koster, 2015). Selective breeding has been responsible for the domestication of 14 animal species and about 100 plants yielding valuable domesticates (Diamond, 2002). Before the 1940s, breeding objectives were mostly visual with the expectation that form determines performance (Darlow, 1958). Subsequently, breeding industries evolved toward objectives involving performance, such as rapid growth and high milk yield (Harris and Newman, 1994). Breeding value estimation was limited to the data that was available for evaluation. This first included single traits, until models were developed for combining several traits into a selection index by Hazel and Lush (1943), and methods were developed such as those for the estimation of variances and covariances for unbalanced animal data by Henderson (1953) (Philipsson et al., 1994). In dairy cattle, as reviewed by VanRaden (2004), the national index of Swedish dairy cattle included 12 traits in the selection index as early as 1975, including milk production, growth rate, female fertility, stillbirths, ease of milking, temperament, and six conformation traits (Philipsson et al., 1994), but the USDA introduced its first net merit index in 1994, which combined productive life, and somatic cell score with yield traits (VanRaden, 2004). The USDA selection index subsequently included conformation traits in 2000 and cow fertility and calving ease in 2003. Only recently is selection for production traits under scrutiny for the consequential undesirable side effects that this may produce affecting animal welfare (Rollin, 1986), thus leading the British Farm Animal Welfare Council to recommend that new and existing breeding technologies and breeding programs should be evaluated for welfare and ethical issues that may arise as a result (FAWC, 2004). Broadening the breeding objective and including more traits in the selection criteria may alleviate and possibly even prevent such negative side effects, with as the only negative consequence a reduced selection response of production traits. However, genetic modification may also result in an intrinsic ethical concern when breeding affects animal integrity. Rollin (1986Rollin ( , 1995 used the Aristotelian concept of the telos of an animal to describe animal nature, i.e., the differences "rooted in biological, genetically based, empirically ascertainable, environmentally expressed "blueprints"" giving rise to "the pigness of a pig, the dogness of a dog. " Bovenkerk et al. (2002) write: "It implies that the animal is intact or whole, which is an attribute of the animal itself, not just some value we have placed on it. " Any artificial genetic modification may be seen as changing the telos, however, D'Eath et al. (2010) suggest that animal behavior is much more easily considered to be part of the animal's nature than any other production trait. As to the question of changing the telos by means of changing the genetic make-up, Rollin (1986) writes: " [O]ne cannot argue that because it is wrong to violate the various aspects of a certain animal's telos given the telos, it is therefore wrong to change the telos. This is true only if the change in the telos is likely to engender more unhappiness in the animals, given the environment in which they live, than would have accrued to them before" (Rollin, 1986). Indeed, Rollin believes that there is no moral problem if welfare could be improved by changing animal natures, even altering animals such that they can be made happier in questionable environments (Rollin, 1995;Bovenkerk et al., 2002). For example, animals bred to have fewer desires or animals with a reduced sentience will be more easily satisfied and consequently have a higher welfare than the population before such selection (D'Eath et al., 2010). In the same way, blind chickens do not show feather pecking or cannibalism, therefore, blind hens may not suffer (Sandøe et al., 1999). Strains that are improved to disguise welfare threatening conditions may discourage the development of higher standards of environmental provisioning (MacArthur Clark et al., 2006). As a consequence, in extreme cases, genetic modification of animals into senseless, emotionless machines that have no desires could be considered a solution to the animal welfare problem. However, intuitively, a large amount of the human population believes that genetic modification of animals is troubling and morally problematic; as such the public opinion can be expected to influence breeding decisions made by producers that would eventually prevent producing animal machines (Rollin, 1998;Thompson, 2010). As Bovenkerk et al. (2002) note, animal integrity is an intuitive concept, and because it lacks objectivity it is therefore not of practical use since that would entail objective criteria to measure it. However, not different from ethical considerations in humans, the concept of integrity can be used in the ethical discussion on livestock breeding, and in the same way that concepts of human rights based on integrity are formulated into laws, from discussions regarding the ethics of livestock breeding can follow similar agreements and regulations (Bovenkerk et al., 2002). Rauw (2015) suggests that although consumer demand may influence decision making and although consumers may be willing to pay more for products that are produced in more welfare friendly production systems, legislation should really be based on ethics independent of consumer demand and willingness to pay. Similar to the option to buy clothes cheap that are produced unethically versus paying more for clothes that are produced under humane circumstances, we as consumers should not be able to have that option (Rauw, 2015). The Farm Animal Welfare Council, in its 2012 report, writes: " we were concerned about general trends in breeding, given the commercial pressures on breeders and farmers alike. Today matters are improving: we still have concerns but we are encouraged that many breeding goals now include aspects of animal welfare, e.g., disease resistance. " Conclusion 105 of the report reads: "Farm animal breeding companies should be congratulated for the progress made on breeding goals aimed at improving robustness and health and welfare traits. However, there are still some issues associated with high production levels resulting in poor animal welfare. " The discussion on animal breeding practices has been initiated and is very alive today. This positive trend is part of the sustainable food production movement that aims at feeding 9.15 billion people not just in the near future but also beyond. However, the discussion is taking place in Europe and North America which are home to the largest livestock breeding companies that hold most of the market share (Gura, 2007). These developed countries are projected to account for only part of the increase in meat consumption, whereas more than half of the increase is projected to be accounted for by developing countries in Asia, Latin America, and Africa, countries that still depend heavily on agriculture for their livelihoods (Borlaug and Dowswell, 2005;Thornton, 2010;Appleby and Fuentesfina, 2015). Although the technology and genetic resources are available, this may be of limited use to local farmers when they are threatened by poverty, governmental regulation and intellectual property rights (Borlaug and Dowswell, 2005). In addition, concern for animal welfare and rights is generally stronger in Europe than in Asia (Phillips et al., 2012) and it remains to be seen if European (breeding) companies will apply their animal welfare standards on a global basis, as suggested by Fraser (2008), or whether this may eventually influence breeding decisions in the future when such standards are not required by international food companies and their customers. SYNTHESIS Since environmental resources (land, water, and energy) are limited, a 70-100% increase in the projected need for food by 2050 must necessarily come from what is called "sustainable intensification. " As Godfray et al. (2010) write: "A threefold challenge now faces the world: Match the rapidly changing demand for food from a larger and more affluent population to its supply; do so in ways that are environmentally and socially sustainable; and ensure that the world's poorest people are no longer hungry. " Increasing production limits both in crops and in livestock are inevitably part of satisfying the global food demand in the future. A further increase in livestock yields with continued selection will be facilitated by superior selection methods including genome-wide selection, more sophisticated progeny testing and tracking methods, and a greater predictive power of total genetic merit indices that integrate genomic markers with multiple traits (Hume et al., 2011). However, at the same time, animals in populations that have been selected for high production efficiency are found to be more at risk for behavioral, physiological and immunological problems (Rauw et al., 1998). As a result, in the last few decades, breeding practices have become of ethical concern and consideration of the possible effects on animal welfare are called for (e.g., FAWC, 2012). The farm animal of the future is described as robust, adapted, and healthy (Mormède et al., 2011). Options for breeding for improved robustness include: (1) estimating breeding values for the environmental sensitivity of the genetic potential for production performance through the use of reaction norms analysis, and (2) direct inclusion of measureable robustness traits in the breeding objective and in the selection index (Knap, 2009). Theories on reaction norms analysis have their basis in genotype by environment interactions that have been described since Lamarck and Darwin. Reaction norms describe phenotypic production values as a function of a gradient of a descriptor of the environment (Knap, 2005). They were first applied in plants, whereas application of reaction norms analysis in livestock production (mostly dairy and beef cattle) is of a much more recent origin. Linear reaction norms are built up of two parts: the level and the slope. A generally observed negative correlation between these parameters suggests that improvement in production yield will result in animals that become more sensitive to changes in the production environment (Kolmodin et al., 2002). Although livestock selection indexes include multiple, mostly yield-related, traits for several decades, direct inclusion of functional, robustness, traits became more seriously applied since the 90s (Oltenacu and Broom, 2010). Of more recent origin is the consideration of inclusion of behavioral traits (Turner, 2011) and even important non-market values of animal traits, such as ethical values or environmental impact (Olesen et al., 2000). Despite an often antagonistic relationship between robustness and production performance, a positive genetic trend in both traits can be achieved when both are properly included in the breeding goal and selection criteria (Knap, 2009). According to the Farm Animal Welfare Council, farm animal breeding companies may be congratulated for the progress made so far toward breeding more robust and healthy animals. The discussion and efforts on animal breeding practices is very alive today and will remain to be an important part of the sustainable intensification debate in the future. ACKNOWLEDGMENTS This work was financed by a Marie Curie Reintegration Grant from the European Union, project no. PIRG08-GA-2010-277031 "Selection For Welfare, " and by a grant from the Ministerio de Economía y Competitividad del Gobierno de España, project no. AGL2012-39137 "Group competition, feed efficiency and welfare in traditional and genomic selection programs in aquaculture. " The reviewers are gratefully acknowledged for their suggestions on improving this manuscript.

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A Century of Gibberellin Research Gibberellin research has its origins in Japan in the 19th century, when a disease of rice was shown to be due to a fungal infection. The symptoms of the disease including overgrowth of the seedling and sterility were later shown to be due to secretions of the fungus Gibberella fujikuroi (now reclassified as Fusarium fujikuroi), from which the name gibberellin was derived for the active component. The profound effect of gibberellins on plant growth and development, particularly growth recovery in dwarf mutants and induction of bolting and flowering in some rosette species, prompted speculation that these fungal metabolites were endogenous plant growth regulators and this was confirmed by chemical characterisation in the late 1950s. Gibberellins are now known to be present in vascular plants, and some fungal and bacterial species. The biosynthesis of gibberellins in plants and the fungus has been largely resolved in terms of the pathways, enzymes, genes and their regulation. The proposal that gibberellins act in plants by removing growth limitation was confirmed by the demonstration that they induce the degradation of the growth-inhibiting DELLA proteins. The mechanism by which this is achieved was clarified by the identification of the gibberellin receptor from rice in 2005. Current research on gibberellin action is focussed particularly on the function of DELLA proteins as regulators of gene expression. This review traces the history of gibberellin research with emphasis on the early discoveries that enabled the more recent advances in this field. Introduction The origins of gibberellin research can be traced to the late 19th century in Japan with the demonstration that a disease of rice that produced symptoms of excessive seedling elongation and infertility, among others, was the result of fungal infection (Hori 1898). Culture filtrates of the fungal pathogen were later shown to reproduce the symptoms in rice, and the active growth-promoting principle was named gibberellin after the perfect (reproductive) form of the fungus, Gibberella fujikuroi. Various names for the disease were used by Japanese farmers depending on location, the most well-known being ''bakanae'', translated as silly seedling. The early research leading to the discovery, isolation and structural determination of gibberellins and the realisation that these compounds may be endogenous growth regulators in plants has been reviewed in detail by Phinney (1983). His review contains photographs of the principal scientists involved in this research. Phinney points out that while work on gibberellins before 1945 was restricted to Japan, some coverage of this research was available to the West in the 1930s through Chemical Abstracts, but did not inspire interest. However, following the 2nd World War, with freer communication with Japan, scientists in the USA and UK realised the importance of these compounds and active research programs were initiated in the 1950s. These and continuing work in Japan resulted in the isolation and structural determination of the main active compound from the fungus, named gibberellic acid in the UK and gibberellin-X in the USA, with the name gibberellic acid being agreed between them. The same compound was known as gibberellin A 3 (GA 3 ) in Japan. Gibberellic acid was found to have profound effects on plant growth, with the ability to rescue dwarf mutants of maize and pea, and induce bolting and flowering in rosette species. These effects could also be obtained with plant extracts, providing a strong indication that gibberellins were endogenous plant metabolites. This was confirmed by the isolation of gibberellin A 1 (GA 1 ) from immature seeds of runner bean, Phaseolus coccineus, in 1958 (MacMillan andSuter 1958). Since this time there was steady progress in understanding gibberellin biosynthesis, the pathways being delineated in G. fujikuroi and higher plants by the 1970s, with the nature of the participating enzymes characterised for plants by the 1980s. With the cloning of the transcripts encoding these enzymes in the 1990s, the way was open to investigate how gibberellin metabolism is regulated, a topic of research that is still very active. Progress in understanding gibberellin action was initially slow, with much of the early work focused on the cereal aleurone, which responds to gibberellin by synthesising and secreting hydrolytic enzymes such as a-amylase. However, major breakthroughs in the 1990s and 2000s transformed our understanding of gibberellin function at the molecular level. With the cloning of the GAI cDNA in Arabidopsis thaliana (Arabidopsis) and its mutant allele gai, which produces GA-insensitivity, Peng and others (1997) suggested that gibberellins act to relieve growth repression by GAI (a member of the DELLA subgroup of the GRAS family of transcriptional regulators). The demonstration that gibberellin induces DELLA protein degradation via the ubiquitination-proteasome pathway, and the isolation of the GID1 GA receptor have enabled a detailed understanding of the early events in gibberellin perception and action. DELLA proteins are now known to act in partnership with transcription factors to regulate gene expression, and their function is currently an active area of research. This article traces the major events in the gibberellin research timeline focussing particularly on the earlier work, which tends to become lost in the mists of time. Gibberellins as Fungal Metabolites: Early Research The plant pathologist Kenkichi Sawada, working at the Imperial Research Institute at the Department of Agriculture in Taipei, Taiwan, was the first to suggest that the bakanae fungus provided the stimulus that caused the overgrowth symptoms in rice (Sawada 1912). This was later confirmed by his colleague Eiichi Kurosawa, who published a paper in 1926 showing that the symptoms of the disease could be reproduced by application of sterile fungal cultures (Kurosawa 1926). He found that the secreted ''toxin'' stimulated the growth of seedlings of several species besides rice. This landmark publication was followed by numerous reports on the properties of the secreted substances, and in 1935, the chemist Teijiro Yabuta, who was Professor of Agricultural Chemistry at the University of Tokyo, obtained a purified sample with high biological activity, which was called gibberellin after its fungal source (Yabuta 1935). Subsequently, the sample yielded two crystalline substances, which were named gibberellin A and gibberellin B (Yabuta and Sumiki 1938), with the names apparently being reversed in later publications. Although there were a number of reports on the chemical properties of these substances, they were later shown to be impure so these studies were inconclusive. It was not until the 1950s that chemists at Tokyo University, including Nobutaka Takahashi and Saburo Tamura, returned to the chemical nature of gibberellin A, and showed that it was a mixture of at least three compounds, which were isolated as their methyl esters and named gibberellin A 1 , gibberellin A 2 and gibberellin A 3 (Takahashi and others 1955). This system of nomenclature was later adopted for all gibberellins that were subsequently isolated (see below). The nature of gibberellin B is still unclear. Research on gibberellins outside of Japan began in the 1950s when the Japanese work and its significance were finally appreciated in the West. It started at about the same time in the UK and USA. The British group led by Percy Brian at the ICI Akers Laboratories in Welwyn, north of London, was alerted to the early Japanese work by reports in Chemical Abstracts and began screening the ICI Fusarium collection for gibberellin production. Crystalline active preparations were passed for structural studies to the chemistry group, which was led by John Grove and included Jake MacMillan, Brian Cross, Philip Curtis and Paddy Mulholland. They were able to obtain a pure crystalline compound, which they called gibberellic acid (Curtis and Cross 1954). A structure for gibberellic acid was proposed in 1956, the evidence appearing in a series of papers, and reviewed by Grove (1961). An X-ray crystal structure for gibberellic acid as its di-p-bromobenzoate methyl ester was published in 1963 by Hartsuck and Lipscomb (1963). Studies on gibberellin production in the USA were initiated by John Mitchell, a mycologist working at Camp Dietrick, Maryland. He procured a gibberellin-producing strain from Japan from which he was able to obtain growthpromoting extracts (Mitchell and Angel 1951). On Mitchell's recommendation, a unit headed by Kenneth Raper was set up at the USDA laboratories in Peoria, Illinois, to produce gibberellin for agricultural trials. Further strains of the fungus were provided by Yusuki Sumiki, who had taken over as Professor of Agriculture at Tokyo University after the retirement of Yabuta in 1950. Sumiki presented the results of the Japanese research on gibberellins to the West, visiting the Peoria laboratories in 1951 and the Akers Laboratories in 1953. After initial difficulties, the Peoria group was able to produce good yields of gibberellin, and by 1953 under the leadership of the chemist Frank Stodola had obtained a pure crystalline product, gibberellin-X (Stodola and others 1955). As described above, gibberellin-X and gibberellic acid were found to be identical, and the name gibberellic acid was agreed on. This compound also proved to be identical with the Japanese gibberellin A 3 . Its availability opened the way for detailed studies on the effects of this fungal metabolite on plant growth and development. Gibberellins in Higher Plants In the mid to late 1950s, numerous reports on the effects of gibberellin on plants appeared in the literature. Of particular note was the ability of gibberellic acid to rescue the growth defect in dwarf mutants of pea (Brian and others 1954;Brian and Hemming 1955) and maize (Phinney 1956) and to induce bolting and flowering in a number of biennial rosette species (Lang 1956;Wittwer and others 1957). At this time, auxin was the only known endogenous plant growth regulator, but the remarkable properties of gibberellins prompted the suggestion that they may also be naturally occurring in plants. The idea that dwarf peas may lack gibberellin prompted Margaret Radley, working with Percy Brian at the Akers laboratory, to apply extracts of tall peas to dwarf peas and demonstrate that they produced a similar growth response as gibberellic acid (Radley 1956). In similar experiments, Bernard Phinney and colleagues at UCLA used dwarf maize in bioassays to show that extracts from a number of plant species contained gibberellin-like substances (Phinney and others 1957). The first definitive evidence for the occurrence of gibberellins in plants was provided by Jake MacMillan and P.J. Suter, who isolated 2 mg of gibberellin A 1 from 87.3 kg of immature seeds of runner bean (Phaseolus multiflorus, later reclassified as Phaseolus coccineus) (MacMillan and Suter 1958). They later identified gibberellins A 5 (MacMillan and others 1959), A 6 and A 8 (MacMillan and others 1962) from the same source. Following the first characterisation of gibberellins from runner bean, new gibberellins were isolated from different plant sources and given names in a rather haphazard fashion based on their plant origin, as, for example, bamboo gibberellin (Murofushi and others 1966) or Lupinus gibberellin-I (Koshimizu and others 1966). However, in the naming of new gibberellins such as A 4 and A 7 isolated from Gibberella fujikuroi, the numerical system that had been employed in Japan was continued. Furthermore, MacMillan and colleagues adopted this system in naming the gibberellins from P. coccineus seed. To put gibberellin nomenclature on a more systematic basis, it was agreed at the International Conference on Plant Growth Substances held in Ottawa, Canada, in 1967 that this numbering system would be used for all gibberellins, with Jake MacMillan and Nobutaka Takahashi assigning numbers to new gibberellins as they are identified (MacMillan and Takahashi 1968). After their retirement, this task was taken over by Yuji Kamiya and Peter Hedden. It is now common practice to abbreviate gibberellin A x as GA x , with the generic abbreviation GA commonly used for gibberellin. It has led to the misconception that GA is an abbreviation of gibberellic acid, but as will be clear from the above discussion, gibberellic acid is a specific compound and is synonymous with GA 3 . As it turns out, GA 3 is a minor gibberellin in higher plants. Initially, structural characterisation of novel GAs required the isolation of large quantities of pure material, with structures based on chemical degradation to simpler compounds of known structure. As more chemically characterised GAs and related compounds became available, it was often possible to use conversion to known compounds in relatively few steps to confirm novel structures. Furthermore, the use of nuclear magnetic resonance reduced the required amounts of material to mg, and more recently lg quantities. However, these methods required the isolation of pure material, which, with concentrations of GAs in plant tissues often at levels of ng.g -1 fresh weight, is rarely feasible. The development of combined gas chromatography-mass spectrometry (GC-MS) for the analysis of GAs (and other plant metabolites) in MacMillan's laboratory in the late 1960s offered new opportunities (Binks and others 1969). GC-MS was much more sensitive than other analytical methods available at the time and could be used with impure extracts. It was ideal for identifying known compounds for which mass spectra were available, although it could not be used to determine the structures of novel compounds directly. However, in many cases, characteristic fragmentation patterns allowed structures to be predicted, and the assumed structures could then be synthesised for GC-MS comparison with the native compound. By this means, the numerous novel GA-related structures synthesised in several chemistry labs, including those of Lewis Mander at the Australian National University, Canberra, Australia and Jake MacMillan at the University of Bristol, UK, have enabled the number of naturally occurring GAs to expand to 136. The source of the first 126 GAs in plants, fungi and bacteria was catalogued by MacMillan (2002). Many of these occur in developing seed at often high concentration, but their function is unknown. It is noteworthy that no new GA has been characterised in over 10 years, although further natural GAs must exist. This may be due in part to their structures not being easily synthesised, but also reflects the current lack of chemistry laboratories with an active GA program, with only Lewis Mander active in the field in recent years. His provision of isotopically labelled GAs for analytical and metabolism studies has been vitally important for GA research. Gibberellin Metabolism The Biosynthetic Pathways Following the structural determination of gibberellic acid (GA 3 ), experiments to determine its biosynthetic origin in G. fujikuroi began in the late 1950s. Incorporation of 14 Clabelled substrates, including acetate and mevalonate (MVA), into GA 3 in fungal cultures followed by degradation confirmed its diterpenoid nature (Birch and others 1958). Later Cross and others (1964) demonstrated that the tetracyclic diterpene hydrocarbon, (-)-kaurene, now more commonly referred to as ent-kaurene, was incorporated into GA 3 , establishing it as an intermediate. At about this time, Jan Graebe, who was a graduate student working with Bernard Phinney and Charles West at UCLA, attempted to prepare cell-free preparations from the fungal mycelia, but achieving no success turned instead to the endosperm-nucellus of the California wild cucumber (Marah macrocarpus, formerly Echinocystis macrocarpa), which Phinney and others (1957) had shown to be a rich source of gibberellin-like substances. Jan Graebe's endosperm system was extremely active and he could demonstrate conversion of MVA into ent-kaurene and ent-kaurenol (Graebe and others 1965). Later, on establishing his own laboratory at the University of Göttingen, Germany, Jan Graebe continued to work on GA biosynthesis in endosperm of another member of the Cucurbitaceae, pumpkin (Cucurbita species), with considerable success (see below). Following this first demonstration of ent-kaurene synthesis, cell-free systems from a number of other plant sources, mainly developing seeds, were shown to convert MVA into ent-kaurene, but the Marah and Cucurbita systems, in contrast to the others, produced ent-kaurene as the major product in high yield (reviewed in Hedden and others 1978). The main pathways for GA biosynthesis in G. fujikuroi, pumpkin endosperm and vegetative organs of higher plants are shown in Figs. 1 and 2. The pathways in the fungus and plants differ in the order of the 3b-hydroxylation and 13-hydroxylation steps, the former occurring early in the fungus (Fig. 1), whereas it is the last step in plants (Fig. 2). In contrast, 13-hydroxylation commonly occurs before loss of C-20 in plants and is the last step in GA 3 biosynthesis in G. fujikuroi. Work on GA biosynthesis in G. fujikuroi and higher plants, mainly in cell-free systems from developing seeds, continued in parallel in the 1960s and 1970s, resulting in the main features of the pathways being established. Many of the initial experiments were conducted in Charles West's laboratory at UCLA. A cell-free system from G. fujikuroi mycelia established trans-geranylgeranyl diphosphate as a precursor of ent-kaurene, to which it is converted in two steps via ent-copalyl diphosphate (Fall and West 1971;Shechter and West 1969). The sequence of steps from ent-kaurene to ent-kaurenoic acid and then to ent-7a-hydroxykaurenoic acid was shown in the Marah and pumpkin cell-free systems (Dennis and West 1967;Graebe 1972;Lew and West 1971). These steps were subsequently confirmed in a number of other cell-free and intact systems (reviewed in Hedden and others 1978). The next intermediate, GA 12 -aldehyde, the first with the ent-gibberellane carbon skeleton, was shown to be formed from MVA in the pumpkin cell-free system, which also produced GA 12 (Graebe and others 1972). No intermediates beyond GA 12 were obtained until Mn 2? , which was included to enhance ent-kaurene formation, was omitted from the cell-free system. In the absence of Mn 2? , GA 12 was converted to a number of products, included GA 43 , which is a major endogenous GA in pumpkin endosperm, and to the C 19 -GA, GA 4 (Graebe and Hedden 1974;Graebe and others 1974a, b). Refeeding these products established the pathway shown in Fig. 2 (blue arrows). The reason for the inhibition of these later reactions by Mn 2? became apparent once the nature of the enzymes catalysing these steps was established (see below). In the 1970s, considerable progress was made in determining the GA-biosynthetic pathways in G. fujikuroi. This included particularly work conducted in MacMillan's laboratory in Bristol using liquid cultures of a GA-deficient mutant, B1-41a, provided by Phinney (Bearder and others 1975). B1-41a contains a lesion in ent-kaurene oxidase and allowed unlabelled substrates to be used without dilution by endogenous metabolites. These experiments and those in James Hanson's laboratory at the University of Sussex, UK, showed that 3b-hydroxylation occurred on GA 12aldehyde and that GA 14 , but not GA 12, was on the pathway to GA 3 (Bearder and others 1975;Evans and Hanson 1975). Hanson and colleagues determined the stereochemistry of many of the steps, showing, for example, that the 1a, 2a-H atoms are lost in the dehydrogenation of GA 4 to form GA 7 (Evans and others 1970). However, it was not possible to determine the immediate precursor from which C-20 was lost in the formation of C 19 -GAs since the potential C 20 intermediates in the oxidation of C-20, GA 37 and GA 36 did not accumulate and were not metabolised by the fungal cultures. In contrast, since the oxidised C 20 precursors accumulate in plants and are readily metabolised, it was possible to show using cell-free systems from pumpkin endosperm and pea seeds that C-20 was lost from the aldehyde (Graebe and others 1980;Kamiya and Graebe Fig. 1 Early and intermediate steps of GA biosynthesis in higher plants (green arrows) and the fungus Fusarium fujikuroi (red arrows). In plants, ent-kaurene is synthesised in plastids, predominately via the methylerythritol phosphate pathway, while in fungi, it is biosynthesised from mevalonic acid. Conversion of ent-kaurene to GA 12 and GA 53 (plants) and GA 14 (fungi) is catalysed by membrane-associated cytochrome P450 monooxygenases. Arrows running through structures indicate multiple steps catalysed by single enzymes 1983). This confirmed earlier suggestions by Hanson and White (1969) and Durley and others (1974). The difference between the fungus and higher plants could be later explained by the nature of the GA 20-oxidase enzymes that catalyse the sequential oxidation of C-20, the fungus utilising a cytochrome P450 monooxygenase for these reactions as opposed to a 2-oxoglutarate-dependent dioxygenase in plants (Hedden and others 2002). Although it has not been demonstrated, it is likely that the oxidised C-20 intermediates in the fungus are not released by the enzyme during this multi-step reaction. The biosynthetic pathways in plants were also being studied in intact organs at this time. Developing seeds of legumes are particularly rich in GAs and have been used both as intact and cell-free systems to study the later stages of biosynthesis. Notably, experiments by Sponsel and MacMillan (1977) in which substrates were injected into cotyledons of immature pea seeds provided evidence of two parallel pathways leading to 13-hydroxylated and 13-deoxy GAs, respectively, with 13-hydroxylation occurring early in the pathway. This system also demonstrated high levels of 2b-hydroxylation, particularly in the Fig. 2 Late steps of GA biosynthesis in vegetative plant tissues (green and brown arrows), pumpkin endosperm (blue arrows) and the fungus Fusarium fujikuroi (red arrows). The main bioactive GAs in plants, GA 1 and GA 4 , are boxed in green, while the product of the fungal pathway, GA 3 , which is also active and produced as a minor product in some plants, is boxed in red. Brown arrows indicate inactivation of C 19 -GAs by 2bhydroxylation and further C-2 oxidation to catabolites (shown for GA 29 and GA 51 , but can also occur for GA 8 and GA 34 ). The reactions are catalysed by soluble 2-oxoglutaratedependent dioxygenases in plants and cytochrome P450 monooxygenases in the fungus, except for the fungal desaturase that converts GA 4 to GA 7 , which is a 2-oxoglutaratedependent dioxygenase. Arrows running through structures indicate multiple steps catalysed by single enzymes J Plant Growth Regul (2015) 34:740-760 745 testa, with the 2b-hydroxylated C 19 -GA products being further oxidised on C-2 to form the GA catabolites (Sponsel 1983). Because 2b-hydroxy GAs have low biological activity, their formation was recognised as an inactivation process, which is important in the regulation of bioactive GA concentrations. It does not occur in G. fujikuroi, in which oxidation at C-2 occurs on the a face. Other inactivation mechanisms known to occur in plants are conjugation, primarily with glucose (Schneider and Schliemann 1994) and the recently described epoxidation of the C-16,17 double bond (Zhu and others 2006). The epoxides are hydrated to the 16,17-dihydrodiols, which have been known for some years to be endogenous GA metabolites (for example, Hedden and others 1993). Several of the biosynthetic steps were of particular interest from a mechanistic standpoint. The formation of ent-kaurene from ent-copalyl diphosphate involves a complex rearrangement proposed to arise from a carbonium ion formed by heterolytic cleavage of the diphosphate (evidence reviewed in MacMillan and Beale 1999). Contraction of ring C from six to five carbons in the formation of GA 12 -aldehyde from ent-7a-hydroxykaurenoic acid occurs with extrusion of C-7. It was proposed (Evans and others 1970) and subsequently confirmed (Castellaro and others 1990;Graebe 1980;Graebe and others 1975) that ring contraction is initiated by stereospecific removal of the C-6b H atom. In pumpkin endosperm and the fungus G. fujikuroi, a by-product, ent-6a, 7a-dihydroxykaurenoic acid, accompanies GA 12 -aldehyde formation. Thus, the intermediate formed after the removal of the 6b-H, assumed to be a free radical, undergoes either rearrangement and further loss of H • to give GA 12 -aldehyde or recombines with HO • to form the dihydroxykaurenoic acid. This latter product is further oxidised, but is not converted to GAs. Other by-products of GA biosynthesis are formed in pumpkin endosperm and G. fujikuroi, in which entkaurenoic acid is converted to ent-kaur-6,16-dienoic acid (and then to the kaurenolides), by stereospecific removal of the 6a, 7a-H atoms (Beale and others 1982;Castellaro and others 1990;Hedden and Graebe 1981). It has been shown for the fungus that all of these by-products are produced by the highly multifunctional enzyme that converts ent-kaurenoic acid to GA 14 (Rojas and others 2001). The equivalent enzyme, ent-kaurenoic acid oxidase, in pumpkin endosperm can be assumed to have similar catalytic properties, although it lacks 3b-hydroxylase activity, forming GA 12 rather than GA 14 . However, there is no evidence for these by-products being formed in vegetative plant tissues, which presumably possess ent-kaurenoic acid oxidases with tighter specificity. The mechanism for the loss of C-20 from the aldehyde is still unclear. Bearder and others (1976) showed that in the fungus both oxygen atoms in the c-lactone of C 19 -GAs were derived from the carboxylic acid on C-4 (C-19). Later Yuji Kamiya demonstrated in a cell-free system from developing pea cotyledons that C-20 is lost from the aldehyde as CO 2 (Kamiya and others 1986). This would require two oxidation steps, although no intermediate between the aldehyde and C 19 product has been identified. The GA 20-oxidase (GA20ox) enzyme responsible for removing C-20 also catalyses the oxidation of C-20 from a methyl to the aldehyde via an alcohol. More recently, on the basis of experiments with a recombinant GA20ox from Arabidopsis, it was proposed that an initially formed free radical on C-20 decomposes by an unknown oxidative mechanism to produce a C-10 radical, which captures the C-4 carboxyl group (Ward and others 2002). The Enzymes The properties of the diterpene cyclases that convert GGDP to ent-copalyl diphosphate and then to ent-kaurene were first studied in West's laboratory at UCLA. The activities were originally named ent-kaurene synthetase A and B (erroneously as they do not require ATP), but the names ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS) were proposed by MacMillan (1997) and have been universally adopted. In the fungus, the two activities reside on a single polypeptide, which was purified by Fall and West in 1971. The activities were partially purified from M. macrocarpus and could also not be separated (Frost and West 1977), although subsequent work showed them to be separate enzymes, which may act in association (Duncan and West 1981). Early indications that ent-kaurene synthesis occurred in plastids (for example, Simcox and others 1975) were later confirmed by Aach and others (1995), who showed conclusively that GGDP was converted to ent-kaurene in plastids from pea shoot tips and pumpkin endosperm. Furthermore, following the cloning of their cDNAs (see below), both CPS and KS were found to contain transit sequences for plastid targeting. It is notable that despite the many demonstrations of ent-kaurene synthesis from MVA in cell-free systems, ent-kaurene was later shown to be produced mainly from pyruvate and glyceraldehyde phosphate via the methylerythritol phosphate (MEP) pathway in plants ( Fig. 1; Kasahara and others 2002). Work with cell-free preparations from Marah, pumpkin, pea and Gibberella showed that the oxidative activities for the conversion of ent-kaurene to GA 12 -aldehyde were present in microsomes and were stimulated by NADPH. West and colleagues demonstrated that the enzymes catalysing the conversion of ent-kaurene to ent-7a-hydroxykaurenoic acid had the properties of cytochrome P450dependent monooxygenases (Hasson and West 1976a;Murphy and West 1969). In the pumpkin cell-free system, GA 12 -aldehyde is oxidised to GA 12 by both microsomal and soluble enzymes (reviewed in Hedden 1983), whereas a microsomal preparation from pea cotyledons converted GA 12 -aldehyde to GA 12 and thence to GA 53 by 13-hydroxylation (Ropers and others 1978). Thus, it was demonstrated that in higher plants, the middle section of the pathway from ent-kaurene to GA 12 and GA 53 was catalysed by monooxygenases. After the cloning of cDNAs encoding these enzymes (see below), it was found that just two enzymes, ent-kaurene oxidase (KO) and ent-kaurenoic acid oxidase (KAO), were required for GA 12 formation from ent-kaurene, with a third enzyme responsible for 13-hydroxylation. The demonstration in Graebe's laboratory that these enzymes were present in the endoplasmic reticulum (Graebe 1980) was later confirmed using GFP fusions by Helliwell and others (2001a, b), who showed that KO was also present in the plastid envelope. The fungal cell-free system being investigated in West's laboratory was capable of forming GA 14 , but no activity could be obtained for the further steps (West 1973). There is still no explanation for this conundrum. After the early 1980s, there was a hiatus in research on fungal GA biosynthesis, but the topic was reactivated by Bettina Tudzynski and her collaborators in the late 1990s through the identification and characterisation of the biosynthetic genes, which are present as a cluster. Through targeted gene knock-out and expression of individual genes in a mutant strain lacking the gene cluster, they could demonstrate the function of each of the seven enzymes responsible for GA 3 biosynthesis (Linnemannstöns and others 1999;Tudzynski and others 2003). With the exception of a 2-oxoglutarate-dependent dioxygenase that converts GA 4 to GA 7 (Bhattacharya and others 2012), the steps from ent-kaurene, including the 20-oxidation of GA 14 to GA 4 , are catalysed by cytochrome P450 monooxygenases. When conversion of GA 12 -aldehyde and GA 12 to endogenous GAs was achieved with the pumpkin endosperm system, there was considerable interest in discovering the nature of the enzymes, which were found to be soluble and therefore different from the monooxygenases responsible for the earlier steps (Graebe and Hedden 1974). Experiments with this system and with others, such as those from Phaseolus seeds (Patterson and others 1975), indicated that the enzymes required Fe 2? , which could be removed by Fe chelators such as EDTA. This explained the inhibition by Mn 2? and other heavy metal ions which could displace Fe at the enzyme active site. Enzyme activity was lost after gel filtration, indicating the requirement for a small molecule cofactor. The demonstration that activity could be restored by 2-oxoglutaric acid and stimulated by ascorbic acid established the enzymes to be 2-oxoglutaratedependent dioxygenases (ODDs) (Hedden and Graebe 1982). Four potential ODD activities were present in the pumpkin system: 20-oxidation, 3b-hydroxylation and 2bhydroxylation, which are universal in higher plants, and the 7-oxidation of GA 12 -aldehyde to GA 12 . This last enzyme appears to have a restricted distribution, being so far identified in members of the Cucurbitaceae (Pimenta Lange and others 2013). After the identification of the enzymes, the next step was to purify them, and this was undertaken in several laboratories, particularly in order to facilitate their cloning (Griggs and others 1991;Kwak and others 1988;Lange and others 1994b;Smith and MacMillan 1984). In fact, cloning was enabled both by enzyme purification and the use of mutants. Mutants and Genes The importance of GA-deficient mutants of pea and maize in establishing GAs as plant hormones has already been described. These and mutants in other species, most notably Arabidopsis, were to prove extremely valuable for studies on GA biosynthesis and in identifying transcripts and genes encoding the enzymes. Bernard Phinney at UCLA and Ian Murfet in Hobart, Tasmania assembled a series of single gene mutants of maize and pea, respectively, for which, through a combination of substrate feeding and product identification by GC-MS, the sites of the lesions in the biosynthetic pathway were identified. For example, the dwarf-1 and le mutants of maize and pea, respectively, were shown to be defective in the 3b-hydroxylation of GA 20 to GA 1 (Ingram and others 1984;Spray and others 1984). There was particular excitement in defining the le lesion because it was responsible for one of the traits (difference in stem height) used in Mendel's classic experiments on the nature of inheritance. Later, the cloning of the LE cDNA allowed the amino acid substitution, causing impairment of enzyme function in the le mutant to be defined (Lester and others 1997;Martin and others 1997). The first characterisation of a GA-biosynthetic mutation was reported for maize, in which it was demonstrated using cell-free systems from shoots that the dwarf-5 mutant was defective in KS activity, producing ent-isokaurene rather than ent-kaurene (Hedden and Phinney 1979). Also in maize, Phinney and Spray (1982) demonstrated in bioassays with dwarf-1 that GA 1 , but none of its precursors, possessed biological activity, so confirming the structural requirements for activity, which were later substantiated when the GA receptor was identified (see below). In 1980, Maartin Koornneef at Wageningen, The Netherlands, produced a number of GA-sensitive mutants in Arabidopsis, naming them ga1 to ga5 on the basis of epistasis (Koornneef and van der Veen 1980). The ga1, ga2 and ga3 mutants were extreme dwarfs, and were sterile with non-germinating seeds, whereas the ga4 and ga5 phenotypes were much less severe. Analysis of the GAs in ga4 and ga5 by Talon and others (1990b) indicated that they were defective in 3b-hydroxylation and 20-oxidation, respectively. Redundancy for the GA3ox and GA20ox enzymes catalysing these reactions was later to explain the relatively mild phenotype, whereas the GA1, GA2 and GA3 genes are present as single copies. The ga1-3 mutant, which was produced by neutron bombardment and contained a large deletion, was utilised in the first cloning of a GA-biosynthetic gene using genomic subtraction (Sun and others 1992). GA1 could then be shown by expression in E. coli to encode CPS (Sun and Kamiya 1994). Soon after, the Anther ear1 (An1) gene of maize, predicted to encode CPS, was cloned by transposon tagging (Bensen and others 1995). Both GA1 and AN1 contained chloroplast-targeting leader sequences. The cloning of the Arabidopsis CPS was quickly followed by the identification of cDNAs for the other biosynthetic enzymes. Theo Lange working with Jan Graebe and Peter Hedden purified a GA20ox from pumpkin endosperm and obtained partial sequences (Lange 1994), allowing the production of peptide antibodies that were used to isolate the cDNA from an expression library (Lange and others 1994a). The identity of the clone was confirmed by functional expression in E. coli. The nucleotide sequence of the pumpkin clone allowed the isolation of three GA20ox cDNAs from Arabidopsis through PCR by Andy Phillips at Long Ashton Research Station, UK (Phillips and others 1995). The three GA20ox enzymes were functionally similar, oxidising GA 12 to the C 19 -GA, GA 9 , in contrast to the pumpkin GA20ox that produced the tricarboxylic acid GA 25 as the major product. Expression of the genes showed different tissue specificity and was down-regulated by application of GA, confirming feedback regulation (see later). A similar strategy was used in Jan Zeevaart's laboratory to clone one of the Arabidopsis GA20ox cDNAs, which they showed to correspond to GA5 (Xu and others 1995). T-DNA tagging enabled Chiang and others (1995) to clone the Arabidopsis GA4 gene, which was later confirmed to encode a GA3ox by expression in E. coli (Williams and others 1998). Shinjiro Yamaguchi, working with Yuji Kamiya at the RIKEN in Wako, Japan, cloned KS from pumpkin cotyledons after purifying the enzyme (Yamaguchi and others 1996), allowing him to isolate the homologous cDNA from Arabidopsis and, through mutant complementation, demonstrate its identity with GA2 (Yamaguchi and others 1998). GA3 was cloned by Helliwell and others (1998) at the CSIRO laboratory in Canberra, Australia, by map-based cloning and random sequencing. The same group cloned KAO from barley, where it is defined by the grd5 mutation, and then from Arabidopsis, which contains two fully redundant copies (Helliwell and others 2001a). They demonstrated that the enzymes carry out the threestep conversion of ent-kaurenoic acid to GA 12 by heterologous expression in yeast. The availability of these genes provided a means to modify GA content through ectopic expression in transgenic plants. Such studies showed that in Arabidopsis, GA biosynthesis is limited particularly by GA20ox activity (Coles and others 1999;Fleet and others 2003;Huang and others 1998). The potential benefits of modifying GA metabolism in crop species were a powerful driver for such experiments, particularly with the aim of reducing GA content to control growth. Chemical growth retardants had been available since 1949 (Mitchell and others 1949), with notable early examples being 2 0 -isopropyl-4 0 -(trimethylammonium chloride) -5 0 -methylphenyl piperidine-1-carboxylate (AMO-1618; Wirwille and Mitchell 1950) and chlormequat chloride (CCC; Tolbert 1960), the latter still in use primarily as an anti-lodging agent. As growth inhibition by these chemicals could be reversed by application of GAs, they were thought to function as anti-gibberellins, and they were found to inhibit GA biosynthesis in the fungus (Kende and others 1963). Subsequently, AMO-1618 and other quaternary ammonium-type inhibitors were shown to inhibit ent-kaurene synthesis (Dennis and others 1965). Further growth retardants acting on different stages of the biosynthetic pathway have been developed, with KS, KO and GA3ox, the principal sites of action (reviewed by Rademacher 2000). As an alternative to growth retardants, expression of GA deactivating genes, such as GA2ox (encoding 2b-hydroxylases), was an attractive option. To isolate GA2ox clones, Steve Thomas, working with Peter Hedden and Andy Phillips, returned to the material from which GAs were first identified, immature P. coccineus seeds, a known rich source of 2b-hydroxylase activity (Durley and others 1971). The simple successful strategy involved screening a cDNA expression library for clones that released 3 H from [1,2-3 H 2 ]GA 9 (Thomas and others 1999). The bean enzyme and three GA2ox enzymes identified by homology from Arabidopsis accepted C 19 -GA substrates, oxidising them to 2b-hydroxy products, with some also producing GA catabolites. GA2ox cDNAs were subsequently cloned from immature pea cotyledons by Dave Martin working with William Proebsting in Corvallis, Oregon, and Diane Lester in James Reid's group in Hobart (Lester and others 1999;Martin and others 1999). Later, a new class of GA2ox which hydroxylates C 20 -GAs was identified in Arabidopsis by activation tagging (Schomburg and others 2003). Both classes of GA2ox are ubiquitous in higher plants and have important roles in regulating GA content. Their overexpression has proved to be a very effective method for producing dwarfism (Phillips 2004). The recent cloning of GA 12 13-hydroxylases from rice (Magome and others 2013) means that genes have now been identified for all the enzymes in the pathway. The two rice cDNAs encode cytochrome P450 monooxygenases that are closely related to the inactivating 16, 17-epoxidase (EUI). Indeed, Magome and others (2013) suggested that 13-hydroxylation may be a form of mild deactivation because overexpression of these cDNAs caused reduced growth. This is an interesting and unexpected conclusion since in most plant species, Arabidopsis being a notable exception, the 13-hydroxylation pathway predominates. The increasing number of plant genome sequences now available has simplified the identification of GA-biosynthetic genes. However, the genes are often incorrectly annotated, and in only a few cases, their functions are demonstrated biochemically, being assigned on the basis of sequence homology. The focus of research on GA metabolism has now moved to its regulation by developmental and environmental factors and the determination of the underlying mechanisms. This topic has been covered in a recent review . Gibberellin Action Investigations into the physiological responses of higher plants to GA were advanced even before the active compounds had been isolated and structurally characterised. The early work was reviewed by Stowe and Yamaki (1957), who listed the numerous effects of GAs on plant development. Some of these are illustrated in Fig. 3, which compares a wild-type Arabidopsis plant with a GA-deficient mutant. With remarkable foresight, they noted that ''there is little doubt that the gibberellins must correspond in their action to naturally-occurring compounds in higher plants'' and suggested that GA acts by removing a limitation to growth. Promotion of elongation in young (still growing) stems is one of the most obvious effects of GA and it occurs without a change in the number of nodes. Internode growth is promoted through enhanced cell elongation, shown later to be due to relaxation of the cell wall rather than increased cell turgor (Cosgrove and Sovonick-Dunford 1989). However, GAs also promote cell division in some circumstances, notably in the induction of bolting in rosette species (Sachs 1965). Stowe and Yamaki noted that GA promotes leaf expansion, but inhibits root growth, from which they concluded that GA changes the root-shoot ratio. It is now known that GA action is essential for root elongation, but high GA concentrations are inhibitory and in most cases roots contain close to saturating GA levels (Tanimoto 2012). Another notable action of GA is the promotion of seed germination: of particular note was the observation that GA substituted for the light requirement for germination of photoblastic seeds, whereas it reversed the light inhibition of stem elongation. These contrasting effects could be later explained by the opposite responses of GA metabolism to red light in these tissues (reviewed by Kamiya and Garcia-Martinez 1999). The effect of GAs on flowering is complex and can be promotive, inhibitory or neutral depending on the species (Pharis and King 1985;Zeevaart 1976). Some long-day plants growing under non-inductive conditions can be induced to bolt and flower by GA application, while others will bolt without flowering. The ability of GA to substitute for long-days prompted speculation that it was the long sought-after leaf-derived signal, florigen, and, although this is now generally recognised to be flowering locus T (FT) or related peptides, there is no doubt that GA can act as a mobile inductive signal, if not the major one (King 2012). Until the discovery of GAs, elongation growth was thought to be regulated exclusively by auxin, and many of the early experiments tested the hypothesis that GA acted by stimulating auxin levels (reviewed in Paleg 1965). However, the reverse scenario is now known to occur with auxin promoting stem elongation by increasing GA biosynthesis (Ross and others 2001), although in a recent report, it was shown that GA is required for auxin transport (Willige and others 2011). The molecular mechanisms of GA action has in recent years become intensively researched, but for practical reasons, much of the early research on GA function was conducted with germinating cereal grain. Germination in cereals is associated with the production and secretion of hydrolytic enzymes, including a-amylase, in the aleurone layer for the breakdown of macromolecules in the endosperm as a source of nutrient for the growing embryo. Research on this topic was stimulated by the importance of the process for malt production in brewing. In 1940, Takeshi Hayashi, working at the Imperial Agricultural Station, Hongo, Tokyo showed that barley grain germination and amylase activity were stimulated by GA (Hayashi 1940). The topic was reactivated in 1960 when Leslie Paleg, at the Waite Institute in Adelaide, Australia and Harugoro Yomo, working at the Takara Shuzo Company in Kyoto, Japan, reported independently that GA stimulated amylase production in embryo-less barley grain (Paleg 1960a;Yomo 1960). Margaret Radley (1959) had shown earlier that barley grain contained GA-like substances which increased during germination, prompting the suggestion that the embryo was the source of GA that stimulated amylase production in the endosperm (Paleg 1960b). This proposal has been substantiated many times since (reviewed in Bethke and others 1997). It was later shown by Chrispeels and Varner (1967) that the source of a-amylase was the aleurone, a layer of living cells surrounding the dead starchy endosperm. The cereal aleurone proved an ideal experimental system to study GA action, since it was dependent on an external source of GA and gave a well-defined biochemical response. It could be easily isolated to produce a uniform population of cells and was amenable to the production of protoplasts which retain their GA response (with some changes), allowing experiments on membrane properties, such as patch clamping, unencumbered by the cell wall. Gibberellin was shown to promote a-amylase mRNA production in the barley aleurone (Higgins and others 1976), but the response occurs relatively late and is preceded by increases in cytosolic free Ca 2? , changes in cytosolic pH, and in the concentrations of calmodulin and cyclic GMP (reviewed in Bethke and others 1997). The role of these factors in the GA response is still not well understood. It has, however, been established that GA promotes expression of a MYB transcription factor (termed GAMYB), which binds to the promoters of a-amylase genes and activates their expression (Gubler and others 1995). GAMYB mRNA production following GA treatment is not affected by the translation inhibitor cycloheximide, indicating that GAMYB may be a primary response gene. GA was shown also to promote programmed cell death of aleurone cells (Bethke and others 1999), a process that also occurs in the tapetum via a GA-regulated mechanism involving GAMYB (reviewed in Plackett and others 2011). A number of lines of evidence indicated that the GA receptor in aleurone cells was present on the plasma membrane. Although membrane-impermeable GA induced a-amylase production in oat aleurone protoplasts (Hooley and others 1991), GA injected into barley aleurone protoplasts was ineffective (Gilroy and Jones 1994). Furthermore, experiments with an agonist and inhibitor of heterotrimeric G proteins suggested their involvement in the response of the oat aleurone to GA (Jones and others 1998). However, a membrane GA receptor has not been identified, and the discovery of a soluble, nuclear-localised GA receptor (GID1) in rice (Ueguchi-Tanaka and others 2005) has placed some doubt on its existence, particularly with the recent report that GID1 was the only GA receptor in rice (Yano and others 2015). Indeed there is some debate as to whether plants actually contain G-protein coupled receptors (Taddese and others 2014). Nevertheless, the demonstration that the rice GA-insensitive dwarf1 mutant is defective in the Ga subunit of a heterotrimeric G protein suggests that these proteins may play some role in GA signalling (Ueguchi-Tanaka and others 2000). The mechanism by which GAs promote growth, summarised in Fig. 4, has been formulated over the last 20 years, with particular progress following the discovery of the GID1 receptor in 2005. The basic concept that GAs act by suppressing a growth inhibitor was proposed from studies with GA-insensitive mutants (Harberd and others 1998). The characteristics of such mutants had been known for many years. In 1970, Margaret Radley showed that the Japanese dwarf wheat cultivar Norin-10 and related dwarf lines did not respond to applied GA, unlike tall lines, and that they accumulated much higher levels of GA-like substances than the tall cultivars (Radley 1970). She suggested that in these lines, a ''block to the utilisation of GA causes an accumulation of the hormone''. This proposal proved correct, although the link between GA action and Fig. 4 Representation of GA perception and signal transduction. Binding of a bioactive GA results in a conformational change in the GID1 receptor that promotes interaction with DELLA proteins. Recruitment of an F-box protein initiates ubiquitination of DELLA by an SCF E3 ubiquitin ligase targeting the DELLA for proteasomal degradation. Loss of DELLA relieves growth repression and suppresses other DELLA-mediated responses metabolism was not as direct as Radley may have envisaged. Norin-10 is the source of the Reduced height (Rht) genes that were introduced by Norman Borlaug into high yielding wheat varieties in the Green Revolution to stabilise the stem and increase harvest index (Hedden 2003). The two homoeologous semi-dwarfing genes present in Norin-10, Rht1 (renamed RhtB1b to indicate its genome location and allele) and Rht2 (RhtD1b), are still used widely in modern wheat cultivars. Appleford and Lenton (1991) showed that near isogenic lines containing Rht-B1b or the more severe Rht-B1c (Rht3) dwarfing allele accumulate C 19 -GAs, but have reduced levels of C 20 -GAs compared with the tall (Rht-B1a) line. Similar results had been obtained for the GA-insensitive dwarf-8 mutant of maize (Fujioka and others 1988) and GA-insensitive (gai) (Talon and others 1990a), an Arabidopsis deletion mutant obtained by Koornneef and others (1985). In contrast, Potts and others (1985) reported that slender pea mutants containing the la cry s gene combination grew independently of GA status and possessed abnormally low levels of GA-like substances. Similarly, slender, an overgrowth mutant of barley with a constitutive GA response was shown to contain lower levels of C 19 -GAs, but elevated C 20 -GA levels relative to its wild type (Croker and others 1990). On the basis of these observations and the ability to normalise GA precursor levels in the maize dwarf1 (3b-hydroxylase) mutant by treating with GA, Hedden and Croker (1992) proposed that GA action resulted in reduced GA20ox activity, that is, GA20ox was under feedback regulation. When GA20ox cDNAs were cloned from Arabidopsis, transcript abundance for these genes was shown to be regulated by GA (Phillips and others 1995). The demonstration by Cowling and others (1998) that the transcript level for GA4, which encodes a GA3ox enzyme (AtGA3ox1), was similarly regulated by GA signalling extended the number of genes under feedback control. Subsequently, it was reported that some GA2ox genes are up-regulated by GA (Thomas and others 1999), whereas the GID1 receptor genes are down-regulated (Griffiths and others 2006), indicating the existence of a complex system of homeostatic regulation in GA signalling. A breakthrough in GA signalling was achieved with the cloning from Arabidopsis of the genes responsible for GAinsensitivity. Nicholas Harberd and colleagues at the John Innes Centre, Norwich, UK, cloned GAI and a related gene GRS and demonstrated that the gai mutant contained a 17-amino acid deletion in the N-terminal region (Peng and others 1997). On the basis of genetic evidence, they proposed that GAI, which had the characteristics of a transcriptional co-activator, is a growth repressor, that the repression is relieved by GA signalling, and that the gai mutant form is resistant to GA, that is, gai is a gain-offunction mutation. Tai-ping Sun and colleagues at Duke University, USA, substantiated this scenario when they characterised a loss of function mutation that partially rescued the semi-dwarf phenotype of the GA-deficient mutant ga1-3 (Silverstone and others 1997). They showed that the gene, called REPRESSOR of ga1-3 (RGA), was identical to GRS and that the encoded protein, which was 82 % similar to GAI, was degraded by GA signalling (Silverstone and others 1998). Furthermore, a mutant form of RGA with the same deletion as in gai was resistant to GA-induced degradation. Thus, the N-terminal region is required for degradation in the presence of GA, but not for growth repression. GAI and RGA belong to a plant-specific family of transcriptional regulators, named GRAS after its first three members, GAI, RGA and SCARECROW (Pysh and others 1999), but GAI and RGA form a subgroup of GRAS proteins with conserved DELLA and VHYNP motifs at the N-terminus not present in SCARECROW and related proteins. These motifs are essential for GA-regulated degradation of this subgroup (Dill and others 2001), known as DELLA proteins (Wen and Chang 2002). After the identification of GAI and RGA, other DELLA genes were cloned: Arabidopsis was found to contain three further DELLA proteins, RGA-like1, -2 and -3 (Hussain and Peng 2003), while, of particular significance, the Harberd group showed that wheat Rht and maize Dwarf-8 encode DELLA proteins and that the gain-of-function mutations that produce GA-insensitivity are due to disruption to the N-terminus (Peng and others 1999). As the Rht-B1b and Rht-D1b mutations create stop codons in the DELLA region, it was assumed, although it has still not been demonstrated, that re-initiation of translation produces a truncated product lacking the DELLA motif. As for wheat and maize, barley and rice contain a single DELLA protein, SLN1 and SLR1, respectively (Chandler and others 2002;Ikeda and others 2001). Strikingly, missense mutations in their N-terminus produce dwarfism (gain of function), while loss of function mutations result in an overgrowth (slender) phenotype. Further progress was made in 2003, when the Arabidopsis SLY1 and rice GID2 genes were cloned and shown to encode the F-box components of SCF ubiquitin ligases (McGinnis and others 2003;Sasaki and others 2003). Mutations in these genes caused an accumulation of DELLA protein and GA-insensitive dwarfism suggesting that DELLA degradation involved ubiquitination, which targeted the protein for proteasome-mediated proteolysis. The involvement of GA in this process became clear when Ueguchi-Tanaka and others (2005) demonstrated that GID1, loss of which also caused GA-insensitivity and DELLA accumulation in rice, encoded a soluble, nuclearlocalised GA receptor with similarity to hormone-sensitive lipases. They showed that association of GA with GID1 promoted interaction with SLR1, the rice DELLA protein. On the basis of domain analysis and mutagenesis experiments, Ueguchi-Tanaka and others (2007) proposed a molecular model, later confirmed by the X-ray crystal structure of GID1 (Shimada and others 2008) and of an Arabidopsis ortholog AtGID1a (Murase and others 2008), whereby binding of GA (GA 4 was the most effective GA) in a pocket allowed the flexible N-terminal strand of GID1 to associate with the top of the pocket, acting as a lid. This conformational change is necessary for interaction with the DELLA protein, which occurs through this protein's DELLA and VHYNP motifs. The interaction with GID1-GA promotes DELLA's association with the F-box protein and hence its degradation (Griffiths and others 2006), although the details of this process at the molecular level are still unclear. Rice contains a single GID1 receptor, whereas Arabidopsis has three paralogs (Nakajima and others 2006), with considerable redundancy such that loss of a single paralog has no effect on the phenotype, while the two double knockouts produce different phenotypes and loss of all three receptors results in a very extreme GAinsensitive dwarf (Griffiths and others 2006). This redundancy may explain why GID1 was discovered in rice rather than Arabidopsis, in which mutant screens for the receptor were unsuccessful. The establishment of DELLA proteins as key components of GA signalling has focused research on DELLA function and down-stream events. It is known that they regulate gene expression with as many genes activated as suppressed (Zentella and others 2007). They do not contain a recognisable DNA-binding domain, but act in association with transcription factors. The first reported examples of a direct association with transcription factors were the independent demonstrations by two groups that DELLAs interact with PHTOCHROME INTERACTING FACTORs (PIFs) in the Arabidopsis hypocotyl and thereby prevent their activation of gene expression (de Lucas and others 2008; Feng and others 2008). However, apart from this sequestration of transcription factors, DELLAs have also been shown to act as co-activators of gene expression through interaction with INDETERMINATE-type transcription factors (Yoshida and others 2014). A recent example of this is the interaction of GAI with GAI-ASSOCIATED FACTOR1 (GAF1) (Fukazawa and others 2014). Intriguingly in association with DELLA protein, GAF1 promotes expression of GA-biosynthetic genes that are subject to feedback regulation so providing the molecular basis for this regulation and the accumulation of GAs in DELLA gain-of-function mutants, as observed by Radley 45 years ago. A number of DELLA partners are components of signalling pathways for other hormone classes, as, for example, the transcription factors BZR1, involved in brassinosteroid signalling (Gallego-Bartolome and others 2012), and JAZ in jasmonate signalling (Hou and others 2010), indicating the high degree of cross-talk between GA signalling and these pathways. Gibberellin Transport The early application experiments indicated that GA 3 was mobile in plants and the first studies to investigate GA transport, such as that by Kato (1958) in which he measured movement through pea stems between agar blocks, established that GA transport, unlike that of auxin, was non-polar, with equal movement in acropetal and basipetal directions. To enable detection, Kato used very high amounts of GA, but subsequent experiments by several groups with radiolabelled GAs at physiological concentrations confirmed the non-polar nature of GA transport in shoot tissue sections, although there was evidence for polar, basipetal movement from root tips (reviewed in Jacobs and Jacobs 2001). The rate of movement was much less than for polar auxin transport. Based on the observed inhibition of [ 3 H]GA 1 movement through oat coleoptiles by sodium azide, Drake and Carr (1979) concluded that GA transport is symplastic, occurring via plasmodesmata. This accorded with the ''ion trap'' model in which the weakly acidic GAs are ionised in the alkaline environment of the cytosol and unable to diffuse through the plasma membrane, whereas in the more acidic apoplast, they would be protonated and rapidly taken up into cells. Kramer (2006) estimated that the decay length of GAs in the apoplast and xylem would be measured in micrometers, with 13-hydroxylated GAs surviving slightly longer in this environment. O'Neill and others (1986) had reached a similar conclusion based on the high permeability of GA 1 in cowpea membrane vesicles, and predicted it would be translocated efficiently in the weakly alkaline phloem. They suggested also that accumulation of GA 1 in the cytosol would disrupt the membrane pH gradient and stressed the importance of metabolism to more polar metabolites that could be stored in the vacuole. This group had previously suggested from work with leaves and protoplasts from cowpea and barley that GA 1 is converted by 2b-hydroxylation to GA 8 , which was compartmentalised in the vacuole, mainly as the glucoside (Garcia-Martinez and others 1981). Indeed, Musgrave and others (1971) had suggested earlier that accumulation of [ 3 H]GA 1 in barley aleurones was associated with metabolism to more polar products. What is the physiological relevance of GA transport? On the basis of the co-location of genes encoding GA-biosynthetic enzymes and signalling components, Kaneko and others (2003) concluded that GAs are synthesised at their site of action in shoot apices and stamens of rice. However, some organs are dependent on an external source of GAs, notable examples being the cereal aleurone, which receives GA from the embryo scutellum (Lenton and others 1994), and petals, which are dependent on the anthers as their GA source (Weiss and Halevy 1989). Long distance transport of GAs from leaves has been implicated in floral initiation at the shoot apex in a number of species (King 2012), and in the promotion of elongation and secondary growth of the stem (Dayan and others 2012;Garcia-Martinez and Rappaport 1982). Rescue of GA-deficient mutants in grafting experiments has also demonstrated long distance movement of GAs, and while grafting between wild-type and mutant maize seedlings implied movement of bioactive GA (Katsumi and others 1983), experiments with pea and potato indicated that the precursor GA 20 rather than GA 1 was the mobile form (reviewed in Ross and others 2006). Recently, grafting experiments with Arabidopsis mutants provided clear evidence that GA 12 is the main mobile form in this species in both the xylem and phloem (Regnault and others 2015). These grafting experiments demonstrate that leaves and roots are capable of providing GAs and/or precursors to support the growth of shoots. However, as shoots would normally be autonomous for GA, the physiological relevance of these observations needs clarification. The identification of GA-like substances in phloem and xylem exudates (Hoad and Bowen 1968;Reid and others 1969) is consistent with GAs being transported by both these routes. However, as discussed above, while phloem transport of GAs would be predicted, transport in the xylem is not consistent with the ion trap model based on passive diffusion of the neutral molecules through membranes. Furthermore, on the basis of scanning colorimetry and electron spin resonance experiments with artificial phospholipid membranes, Pauls and others (1982) concluded that GA 4 and GA 7 associate with the membrane surface, but do not penetrate. Transport of GAs would therefore appear to require transmembrane transporters, particularly efflux transporters, which would also fit with the apparent high structural specificity of the GAs that are transported (Regnault and others 2015). The recent report that GA-fluorescein conjugates accumulated in the endodermis of Arabidopsis roots is evidence also of cellular specificity (Shani and others 2013). GA transporters are now being identified, although they lack specificity and are capable of transporting other hormones as well as unrelated molecules (Chiba and others 2015;Saito and others 2015). It is anticipated that further GA transporters will be found in the near future. Evolution of Gibberellin Biosynthesis and Signal Transduction The availability of genome sequences for numerous organisms has prompted interest in the evolution of GA production and signalling. The lycophyte Selaginella moellendorffii, but not the bryophyte Physcomitrella patens, contains functional GA-biosynthesis and signalling pathways, indicating that they evolved in vascular plants (Hirano and others 2007;Vandenbussche and others 2007;Yasumura and others 2007). In Selaginella, GA signalling regulates sporulation, but not growth, and it is suggested that the pathway evolved to regulate GAMYB, which is involved in reproductive development even in less advanced plants such as Physcomitrella (Aya and others 2011). The development of a role for GA in growth responses in higher plants may have occurred through modifications to DELLA that extended the range of transcription factors with which it can interact. Gibberellin production has evolved in some fungal and bacterial species and, at least in fungi, this seems to have occurred independently of that in plants (Hedden and others 2002). G. fujikuroi is now known to consist of a number of mating populations, the rice pathogen being reclassified as Fusarium fujikuroi (Leslie 1999). Members of this species complex have distinct plant hosts, and many have lost the capability to produce GAs through mutation and/or loss of parts of the GA-biosynthesis gene cluster (Malonek and others 2005), perhaps indicating that GA production is no longer beneficial to the fungus. On the other hand, GA production is present in a number of distantly related species (Kawaide and Sassa 1993;Rademacher and Graebe 1979), and may have been passed between fungal species by gene transfer. Gibberellins have no known physiological function in fungi, which secrete GAs to modify their host plants, with evidence that they may compromise the plant's defence mechanism by interfering with jasmonate signal transduction (Hou and others 2010;Navarro and others 2008). Some bacteria also produce GAs, the nitrogen-fixing endophyte Bradyrhizobium japonica, for example, is capable of producing GA 9 (Mendez and others 2014), although there is as yet no indication of function. Present and Future Since the first experiments in the late 1950s, the chemistry, biochemistry and genetics of GA biosynthesis have been resolved to a considerable extent. Nevertheless, a few unsolved questions remain. For example, an alternative GA 20-oxidase that converts the lactone form of the C-20 alcohol to the aldehyde (Ward and others 1997) is likely to make a major contribution to GA biosynthesis, but has not been characterised. Furthermore, the precise mechanism by which C-20 is lost is still unresolved. Although GA 13-hydroxylases have been identified in rice as cytochrome P450s, other enzymes with this activity must be present, as mutants lacking both GA13ox paralogs are not completely deficient in 13-hydroxy GAs (Magome and others 2013). The regulation of GA biosynthesis by developmental and environmental factors is an area of considerable current interest, and the recent progress in understanding the molecular mechanism for GA homeostasis at the transcriptional level is an important advance. However, work suggesting that GA feedback regulation may also operate at the level of protein stability (Lee and Zeevaart 2007) needs to be followed up. Research on GA signalling is focussed on identifying the transcription factors with which DELLA proteins associate to activate or suppress gene expression, as well as their gene targets. A non-transcriptional mechanism for DELLA was reported in the regulation of microtubule assembly, through nuclear sequestration by DELLA of the chaperone component Prefoldin5 (Locascio and others 2013). By enabling microtubule assembly and orientation in the cytosol, GA promotes the transverse orientation of microfibrils, producing the anisotropic cell growth characteristic of GA action (Shibaoka 1993). As well as alternative DELLA functions, there remains the question of whether GA signalling can occur independently of DELLA, as has been suggested for GA-mediated fruit growth in Arabidopsis (Fuentes and others 2012). Mapping precisely the sites of GA biosynthesis and action in plants is an essential prerequisite for understanding how GA signalling is regulated. The sensitivity of physicochemical methods for analysing GA concentrations, GC-MS and more recently liquid chromatography-mass spectrometry has improved enormously, but is still not sufficient for measuring the concentrations of GAs and precursors at the cellular level. The development of in situ methods for identifying the cells that produce, accumulate and respond to bioactive GAs is an important objective as is the further characterisation of GA transporters. Although the GA field has developed immeasurably in the last 100 years, there is still considerable scope for further advances.

v3-fos

2018-04-03T06:12:41.493Z

2015-09-15T00:00:00.000Z

14511947

s2

Soil N retention and nitrate leaching in three types of dunes in the Mu Us desert of China A large reservoir of soil nitrate in desert subsoil zones has been demonstrated in previous studies; however, information on the subsoil nitrate reservoir and its distribution characteristics in the deserts of China is still limited. This study investigated the distribution patterns of soil total nitrogen (N), nitrate, ammonium, and stable isotopic ratios of 15N (δ15N) in shallow (1 m) and subsoil (5 m) profiles in three types of dunes in the Mu Us desert of China. We found that soil N retention of the fixed and semi-fixed dunes followed a progressive nutrient depletion pattern in shallow soil profiles, whereas the subsoil nitrate of the fixed, semi-fixed and mobile dunes maintained a conservative accumulation pattern. The results indicate that the subsoil of the Mu Us desert may act as a reservoir of available nitrate. Furthermore, a soil δ15N analysis indicate that the nitrate content of the fixed dune is likely derived from soil nitrification, whereas the nitrate content in the mobile dune is derived from atmospheric nitrate deposition. Within the context of looming climate change and intensifying human activities, the subsoil nitrate content in the deserts of northern China could become mobilized and increase environmental risks to groundwater. Deserts cover one-third of the land surface worldwide and play an important role in the function of global ecosystems and biogeochemical cycling [1][2][3][4] . Desert soils are generally thought to be nutrient poor and low in total nitrogen (N) 1,5 . However, recent studies have demonstrated that desert subsoil represents a large reservoir of bioavailable N in the form of nitrate, suggesting that this N pool has been previously overlooked [6][7][8][9] . Investigations of this subsoil N storage could increase estimates of vadose-zone N content by 14 to 71% for warm deserts and arid shrublands worldwide 6 . Moreover, subsoil nitrate may contaminate groundwater and exert further negative effects after land-use or climate change in deserts 6 . Therefore, additional investigations of subsoil nitrate reservoirs are required in the field of desert environmental research. China contains several of the largest areas of desert and desertified land in the world. The total area of desert in China is estimated at approximately 1.53 × 10 6 km 2 , and deserts occupy approximately 15.9% of the total national land area 10,11 . Numerous studies have suggested that desert subsoil in China could accumulate a large amount of bioavailable N from massive atmospheric nitrate depositions and active N fixation by biological soil crusts 8,[12][13][14] . However, to our knowledge, few studies have been conducted to characterize the subsoil nitrate distribution and dynamics in Chinese deserts. Moreover, numerous studies have demonstrated that the climate of northwest China has experienced an increasing warming and wetting trend over the past several decades and suggest that this trend will continue throughout the 21 st century [15][16][17] . Increases in precipitation would lead to additional leaching and mobilization of subsoil nitrate in deserts and heighten the environmental risk to groundwater. Therefore, investigations of the distribution characteristics of subsoil nitrate reservoirs in Chinese deserts are urgently required, and the resulting information will broaden our understanding of desert N cycling. The Mu Us desert is located in the southeastern region of the Ordos Plateau in northern China and covers an area of 40000 km 2 18 . Over the past decade, the region has been the most active area of economic growth in China because of its rich coal, oil and natural gas resources. Groundwater is the main water source in the Mu Us desert, and soil nitrate is readily mobilized to the groundwater because of the shallow groundwater table 19 . Moreover, the rapid development of petroleum and coal industries in the region could significantly increase the atmospheric deposition of nitrate and pose an additional potential threat to the groundwater 20 . However, little is known of the distribution patterns of subsoil nitrate in the region, which limits our understanding of whether a subsoil nitrate reservoir occurs in the area and how it might be characterized. In this study, three typical landforms in the Mu Us desert -fixed dune, semi-fixed dune and mobile dune -were selected, and soil samples from shallow profiles (1 m) and subsoil drilling cores (5 m) were analysed to determine the total N, nitrate, ammonium, and δ 15 N (Fig. 1). The objectives of this study were to (1) investigate the characteristics of soil N retention in shallow soil profiles and (2) characterize the distribution patterns of soil nitrate in the subsoil horizon. Table 1). The fixed dune presented the highest vegetation cover, plant species composition, silt and clay content, and bulk density, and Artemisia ordosica and Hedysarum fruticosum were the dominant species. The semi-fixed dune presented lower vegetation cover, plant species composition, silt and clay content, and bulk density compared with that of the fixed dune, and A. ordosica was the dominant species. The mobile dune was barren and presented the highest sand content and lowest soil bulk density. Characteristics of soil N retention in the upper 1 m of the soil. In the upper 1 m of the soil, the content of soil total N, nitrate and ammonium presented minimal changes with increasing soil depth for the mobile dune, whereas for the fixed and semi-fixed dunes, the soil N content decreased with increasing depth and showed a progressive nutrient depletion pattern (Fig. 2a-c). A statistical analysis demonstrated that the fixed dune has the highest content of soil total N and ammonium among the three types of dunes (total N: P = 0.015; ammonium: P = 0.012), whereas soil nitrate does not show significant differences (nitrate: P = 0.151). Ammonium was the dominant form of soil inorganic N preserved in the upper 1 m of the soil, and the ratios of soil nitrate to ammonium averaged 0.24 for the three types of dunes (Fig. 2d). Distribution patterns of soil nitrate in the 5 m soil layer. In the 5 m soil layer, soil nitrate exhibited obvious pattern of leaching for all of the three types of dunes (Fig. 3). In the mobile dune, the soil nitrate content presented minimal changes within the depth range of 0-3.5 m, whereas below 3.5 m, the soil nitrate content significantly increased and showed an obvious pattern of leaching. In the fixed and semi-fixed dunes, nitrate in the shallow soil profiles showed a progressive nutrient depletion pattern, whereas nitrate in the deep soil horizon exhibited an increasing pattern, although the distribution patterns varied. Soil δ 15 N values among the three types of dunes. The values of δ 15 N in the upper 1 m of the soil exhibited considerable variation among the three types of dunes, averaging 1.74%, 0.80% and − 0.15% for the fixed, semi-fixed and mobile dunes, respectively (Fig. 4). A statistical analysis demonstrated that the fixed dune had the highest values of δ 15 N, whereas the mobile dune had the lowest (P < 0.001). Discussion The three types of Chinese desert dunes evaluated in this study were found to have large differences in their levels of vegetative cover, which is the primary causal factor for the corresponding differences in the soil N retention and nitrate leaching. In the fixed dune, the vegetation cover was approximately 85%, with a large number of biological soil crusts observed to cover the soil surface 21,22 . Dense vegetation cover constitutes a powerful system for biological cycling of soil N and usually leads to progressive nutrient depletion patterns in shallow soil profiles 23,24 . Moreover, the dominant species of the fixed dune were A. ordosica and H. fruticosum. H. fruticosum is a species of the legume family, and biological N fixation by H. fruticosum and soil crusts also promotes soil N increases at the soil surface 25,26 . Moreover, a high degree of vegetation cover and biological soil crust formation can interfere with the atmospheric deposition of N 27,28 . These factors contributed to the current situation in which the fixed dunes showed Previous studies have demonstrated that the high soil-surface temperatures of deserts (greater than 50 °C), which are driven by solar radiation, can cause abiotic losses of N in the form of NO y (all forms of oxidized gaseous N) and NH 3 29 . Moreover, the soil pH tends to be high in extremely dry areas, which is also a key driver of ammonium loss associated with volatilization 30 . Therefore, ammonium is generally considered difficult to preserve in the desert soil. In this study, we found that ammonium was the dominant form of inorganic N preserved in the upper 1 m of the soil and that the level of soil ammonium was nearly three times higher than that of soil nitrate (Fig. 2d). We conclude that the relatively low temperature and soil pH played a critical role in regulating the soil ammonium retention at the study site. Hu et al. (2008) reported that soil ammonium retention is both negatively and significantly correlated with the air temperature and soil pH in the drylands of central East Asia, where low temperature and soil pH are found to correspond to high soil ammonium content 31 . In this study, the annual mean temperature of the area was 6.7 °C and the monthly mean temperatures from April to October range from 7.4-21.9 °C 32 . Moreover, the soil pH of the study area is not too high due to a relatively high rate of precipitation (with an annual average of 345 mm) ( Table 1). Numerous studies have reported that low temperature and soil pH could inhibit the ammonium oxidation rate and suppress ammonia volatilization 30,[33][34][35] , which are both beneficial to the soil ammonium storage 31 . Moreover, the fixed dune had the highest content of soil ammonium measured among the three types of dunes (P = 0.012), indicating that the net soil N accumulation dominates the ecosystem N cycling process as the biological N fixation of the legume species and soil crusts. In deserts, the storage of available soil N is low [36][37][38] ; as such, the resulting nitrate leaching from desert soils is also expected to be low. We found, however, that the soil nitrate content increased significantly in the subsoil horizon, which indicates that high levels of available soil N in the presence of nitrate have leached into the deeper soil. This result is similar to the findings obtained by Walvoord et al. (2003) 6 reported that large near-surface nitrate pools were found in the soils capped by desert pavement in the Mojave Desert 7 . Therefore, we speculate that subsoil nitrate reservoirs may exist in deserts. In this study, the increase seen in the nitrate content of subsoil indicates the likely presence of a nitrate reservoir in the subsoil zones of the Mu Us desert. However, because of the large differences in the vegetation cover, the three types of dunes showed different patterns of subsoil nitrate distribution. The mobile dune is barren and does not provide physical buffering and biological regulation of vegetation. Consequently, the mobile dune showed a very clear process of nitrate leaching (Fig. 3). In the fixed and semi-fixed dunes, the presence of vegetation was seen to affect the soil N cycling (Figs 2 and 3). As a result, the fixed and semi-fixed dunes had a fluctuating pattern of nitrate leaching (Fig. 3). Walvoord et al. (2003) explained that available soil nitrate is not completely consumed by plants or returned to the atmosphere 6 , which is a possible cause of nitrate leaching in deserts. Moreover, Gebauer and Ehleringer (2000) discovered that desert plants do not necessarily consume water and nutrients simultaneously 40 , which could also contribute to nitrate loss. In this study, the relatively high precipitation rate was found to also promote nitrate leaching, while the soil δ 15 the mobile dune had the lowest values of δ 15 N (− 0.15%). Nitrate is known to have a more negative δ 15 N value than ammonium [41][42][43] , indicating a source with increased nitrate content, whereas a more positive value of δ 15 N indicates a source with increased ammonium content 43 . In this study, the highest value of soil δ 15 N was observed in the fixed dune, thus indicating that the biological N fixation of legume species and soil crusts dominates the surface soil N process, producing ammonium and maintaining the nutrient demands of the desert ecosystem. Therefore, nitrate in the fixed dune was most likely to have been derived from soil nitrification. Correspondingly, the lowest soil δ 15 N content was found in the mobile dune, indicating that the soil N in this dune was primarily derived from the atmospheric nitrate deposition. A number of studies have indicated that changes in climate or land use could mobilize the subsoil nitrate reservoirs that have accumulated over thousands of years in deserts 6,7 . The activated subsoil nitrate can contaminate groundwater and adversely affect public water supplies 44 . In the Mu Us desert, the risk of nitrate pollution to the groundwater is high because of the large-scale coal mining operations in the area. Moreover, Hong et al. (2014) demonstrated that the rates of summer precipitation in arid eastern Central Asia (to include northwestern China) have increased steadily over the past 8,500 years 45 and suggested that the trend of a wetter climate would continue in the future 15,16,45 . Increasing precipitation in the arid regions of China has the potential to mobilize the subsoil nitrate in deserts and increase the environmental risks to groundwater. Therefore, the adverse effects of subsoil nitrate on the groundwater quality should be considered when managing water resources in the arid and semiarid regions of China. Study area. This study was conducted at the Chinese Academy of Sciences' Ordos Sandy Grassland Research Station, which is located in the Mu Us desert of Inner Mongolia, China (see Supplementary Fig. S1 online). The area has a typically semiarid climate with marked seasonal and diurnal temperature variations and low precipitation. The annual mean temperature is 6.7 °C, with monthly mean temperatures falling below 5 °C from November to March and ranging from 7.4 °C to 21.9 °C from April to October. The annual mean precipitation is 345 mm, with an annual mean evaporation of 2535 mm. From April to October, the mean precipitation is 322 mm, which accounts for approximately 93% of the annual precipitation 32 . The topography of the area is characterized by sand dunes and desert shrub vegetation, and only a small area of grasslands is distributed in the lowland and upland areas 18 . In the vicinity of the Ordos Sandy Grassland Research Station, three typical landforms -fixed, semi-fixed, and mobile dunes -were selected as sampling sites. The fixed dune sampling site lies at 111° 11′ 39″ E and 39° 29′ 43″ N and is 1313 m above sea level. Vegetation cover accounts for approximately 85% of the area, and the main plant species are A. ordosica, H. fruticosum, Hedysarum scoparium and Stipa bungeana 21,46,47 48 . The semi-fixed dune sampling site lies at 111° 8′ 8″ E and 39° 28′ 51″ N and is 1289 m above sea level. Vegetation cover accounts for approximately 25% of the area, and the main plant species are A. ordosica, P. villosa and C. komarovii 21,46,47 , with A. ordosica as the dominant species. The percentages of sand, silt and clay measured at depths of 0− 20 cm in the soil profile are 80.54%, 16.94% and 2.52%, respectively 48 . The mobile dune sampling site lies at 111° 11′ 50″ E and 39° 28′ 39″ N and is 1317 m above sea level. The mobile dune is barren, and the percentages of sand, silt and clay measured at depths of 0-20 cm in the soil profile are 94.50%, 4.53% and 0.97%, respectively 48 . Soil sampling and laboratory analysis. Soil sampling was performed in July, 2013. In the study area, three soil profiles with depths of 0-100 cm were established in each of the fixed, semi-fixed, and mobile dune sampling sites. The distance among the three soil profiles of the fixed dune was approximately 150 m. At each fixed-dune profile, we used a soil corer with a diameter of 5 cm to collect the soil samples. After removing the biological soil crust and litterfall, soil samples were collected at 10-cm intervals to a depth of 100 cm below the soil surface. Soil samples were stored using polyethelene bags and brought to laboratory for further analysis. In the sampling sites of the semi-fixed and mobile dunes, the distance between the soil profiles was observed to be approximately 200 m. Soils were sampled by the same method as used for the fixed dune. In total, 30 soil samples were collected from each sampling site. Moreover, three replicate samples were collected from each profile at intervals of 10 cm to conduct a soil bulk density analysis using a 100 cm 3 stainless steel cylinder. These shallow soil samples were analysed for soil total N, nitrate, ammonium, δ 15 N and pH. To obtain the subsoil samples, a hand-held auger 6 cm in diameter and 500 cm in length was used to collect soil cores from a depth of 0-500 cm. Soil samples were collected at 20-cm intervals, and 25 soil samples were obtained at each drilling core. Each sampling site contained one core, and 25 soil samples were collected from each sampling site. These subsoil samples were then analysed to measure soil nitrate content. All of the collected soil samples were air-dried in the laboratory, and any remaining roots were carefully removed. The soil pH was measured in a deionized water suspension (soil:water, 1:2.5) using a DMP-2 mV/pH detector (Quark Ltd., Nanjing, China). For the soil N analysis, the air-dried soil samples were ground in an agate mortar and passed through a 0.15-mm sieve. The soil total N content Scientific RepoRts | 5:14222 | DOi: 10.1038/srep14222 was measured through micro-Kjeldahl digestion, which was followed by distillation and titration 49 . Soil nitrate and ammonium were extracted with a 2 M KCl solution (soil:solution, 1:5) and then filtered through a 0.45-μ m filter 50 . The extracted solutions were analysed to determine their nitrate and ammonium concentrations using a Continuous Flow Analyser. A soil stable N isotopic analysis was performed in the Environmental Stable Isotope Laboratory of the Chinese Academy of Agricultural Sciences. These measurements were performed using an EA-IsoPrime100 Stable Isotope Ratio Mass Spectrometer (Isoprime Ltd, U.K). The 15 N/ 14 N ratio was expressed in δ notation as parts per thousand deviations (%) from the Pee Dee Belemnite (PDB) standard: where R represents the isotope ratio ( 15 N/ 14 N) and R standard is the 15 N/ 14 N ratio for atmospheric N 2 . The analytical precision for δ 15 N was 0.2%. Data analysis. Statistically significant differences in the soil total N, nitrate, ammonium and δ 15 N among the three sampling sites were identified using a one-factor analysis of variance (ANOVA) and least significant difference calculations at an alpha level of 0.05 (a < 0.05). All statistical analyses were performed with the Statistical Program for Social Sciences (SPSS 11.0; SPSS Inc., Chicago, IL, USA).

v3-fos

2016-05-04T20:20:58.661Z

2016-01-19T00:00:00.000Z

10211757

s2

A De novo Transcriptomic Approach to Identify Flavonoids and Anthocyanins “Switch-Off” in Olive (Olea europaea L.) Drupes at Different Stages of Maturation Highlights A de novo transcriptome reconstruction of olive drupes was performed in two genotypes Gene expression was monitored during drupe development in two olive cultivars Transcripts involved in flavonoid and anthocyanin pathways were analyzed in Cassanese and Leucocarpa cultivars Both cultivar and developmental stage impact gene expression in Olea europaea fruits. During ripening, the fruits of the olive tree (Olea europaea L.) undergo a progressive chromatic change characterized by the formation of a red-brown “spot” which gradually extends on the epidermis and in the innermost part of the mesocarp. This event finds an exception in the Leucocarpa cultivar, in which we observe a destabilized equilibrium between the metabolisms of chlorophyll and other pigments, particularly the anthocyanins whose switch-off during maturation promotes the white coloration of fruits. Despite its importance, genomic information on the olive tree is still lacking. Different RNA-seq libraries were generated from drupes of “Leucocarpa” and “Cassanese” olive genotypes, sampled at 100 and 130 days after flowering (DAF), and were used in order to identify transcripts involved in the main phenotypic changes of fruits during maturation and their corresponding expression patterns. A total of 103,359 transcripts were obtained and 3792 and 3064 were differentially expressed in “Leucocarpa” and “Cassanese” genotypes, respectively, during 100–130 DAF transition. Among them flavonoid and anthocyanin related transcripts such as phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonol 3′-hydrogenase (F3′H), flavonol 3′5 ′-hydrogenase (F3′5′H), flavonol synthase (FLS), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), UDP-glucose:anthocianidin: flavonoid glucosyltransferase (UFGT) were identified. These results contribute to reducing the current gap in information regarding metabolic processes, including those linked to fruit pigmentation in the olive. INTRODUCTION The olive tree (Olea europaea L. subsp. europaea var. europaea) is one of the most important and widespread fruit trees in the Mediterranean area. It belongs to the Oleaceae family, which includes 600 species within 25 genera. It is widely distributed on all continents, from temperate areas in the north to sub-tropical regions and from low to high altitudes. Native to Mediterranean regions, Olea europaea is the only species within the genus Olea that produces edible fruits (Green and Wickens, 1989;Wallander and Albert, 2000;Green, 2002;FAOSTAT, 2008 1 ). The quality of its products, olive oil and table olives, is highly dependent on the agronomic and organoleptic characteristics of its drupes. These characteristics vary in relation to the genetic traits, varieties, the stage of ripeness, as well as in relation to the different susceptibility to environmental growth conditions (Loumou and Giourga, 2003;Conde et al., 2008). The genuineness of olive oil is important within the "Mediterranean diet." Several research and epidemiological studies link healthy aspects of its components; in particular, olive oil is known to exert protective effects against vascular disease and the onset of cancer (Vauzour et al., 2010). These features are correlated to the high percentage of monounsaturated fats as well as to the high content of antioxidant compounds such as phenols and tocopherols, which, together with other components, characterize the nutraceutical profile of olive products (Pérez-Jiménez et al., 2007;Bruno et al., 2009;Muzzalupo et al., 2011). Phenolic compounds represent a complex mixture in olive derived products responsible for the anti-atherogenic and anti-cancerogenic effects, and for antioxidant properties (Hashim et al., 2008;Llorente-Cortes et al., 2010;Martinelli and Tonutti, 2012). Despite the importance and uniqueness of olive products, the long juvenile developmental phase and its intrinsic self-incompatibility mechanisms slow down current olive breeding programs, which are still very long. Although the current breeding strategies can now benefit from the availability of new polymorphic genetic markers, characterization of the olive germplasm is still far from complete Muzzalupo, 2012;Muzzalupo et al., 2014). Therefore, it is of prime importance to focus research programs toward innovative improvement strategies to support conventional programs. In particular, a wider characterization of genes related to plant product quality and to adaptive mechanisms, could provide new information and tools to support both Marker Aided Selection (MAS) strategies and biotechnological approaches. This would aid the development of new growing techniques to increase productivity and quality of this unique species. Anthocyanins are the most widely distributed group of pigments in plants. They are synthesized via the phenylpropanoid pathway and are mainly responsible for the mauve, red, blue, and purple colors in flowers, fruits, leaves, seeds, and other organs in most flowering plants. As one of the most ubiquitous class of flavonoids, anthocyanins possess a multitude of biological roles, including protection against solar exposure and ultraviolet radiation, free radical scavenging and antioxidative capacity, defense against many different pathogens, and attraction of predators for seed dispersal. Anthocyanins also play a role in consumer preference for flower and fruit quality, potential food health properties, and related horticultural attributes. As a result, classical breeding, as well as transgene technologies, have been used to enhance or create novel colors in ornamental and food crops (Chalker-Scott, 1999;Schaefer et al., 2004;Takahama, 2004;Stommel et al., 2009). The enzymes involved in the anthocyanin biosynthetic pathway are well characterized. Many of the genes encoding these enzymes have been cloned and share high sequence similarity across species and exhibit tissue-or development-specific expression. Chalcone synthase (CHS) is the first enzymatic step of the biosynthetic pathway (Coe et al., 1981;Dooner, 1983;Koes et al., 1989Koes et al., , 2005. Subsequently chalcone isomerase (CHI) catalyzes the isomerization of chalcone to naringenin (van Tunen et al., 1988(van Tunen et al., , 1989Grotewold and Peterson, 1994;Griesbach and Beck, 2005). Flavanone 3-hydroxylase (F3H) converts naringen into dihydrokaempferol, which is converted to anthocyanins by the action of three enzymes. Dihydroflavonol is first converted to a colorless leucoanthocyanidin by dihydroflavonol 4-reductase (DFR). Leucoanthocyanidins are subsequently converted to colored anthocyanidins by anthocyanidin synthase (ANS) finally, the UDP-glucose-flavonoid 3-O-glucosyltransferase (UFGT) creates the anthocyanin-3-glucoside. Within the path, the CHS is the first and key regulatory enzyme of flavonoid biosynthesis and the DFR is the first committed enzyme of anthocyanin biosynthesis in the flavonoid pathway (Holton and Cornish, 1995;Ramsay and Glover, 2005;Martinelli and Tonutti, 2012). Despite having been recently studied in different olive cultivars Galla et al., 2009;Martinelli and Tonutti, 2012) the molecular mechanisms involved in the regulation of biosynthesis are still unknown. It has been suggested that a functional MYB-MYC-WD complex directly binds the cis-element of structural gene through MYB transcription factor, while R-like MYC might bind indirectly via a hypothetical R interaction protein (RIP) (Ramsay and Glover, 2005). R-like MYC is centered in the complex that interacts with a MYB factor with WD proteins on its sides. Together, they activate the entire set of anthocyanin biosynthesis genes (Stommel et al., 2009). The aim of this work was to define the main transcriptomic profile differences during olive drupe development and to identify the transcripts involved in flavonoid and anthocyanin metabolism. We have chosen to analyze the transcriptome profile at 100 and 130 days after flowering (DAF), through an Illumina RNA-seq approach, to identify the transcripts along flavonoids and anthocyanins biosynthetic pathways and to monitor their expression levels during ripening. A de novo transcriptome reconstruction of olive fruits was performed together with a full expression analysis between samples from "Leucocarpa, " an olive variety characterized by a switch-off in skin color at full ripeness, and "Cassanese, " used as control plant. Significant differences in flavonoid and anthocyanin transcript expression profiles emerged, both during fruit maturation and in relation to genotypes. Consequently, from the wide array of information obtained, our attention was focused on the identified candidate genes set, the expression of which was confirmed by quantitative PCR. In addition, the expression patterns of different MYB, MYC, and WDR transcriptional activators was compared to CHS, DFR, and ANS genes during fruit ripening (Matus et al., 2009;Ravaglia et al., 2013). Plant Materials Olive drupes, of Olea europaea L. Leucocarpa and Cassanese cv were used. Drupes were collected from 20-year-old plants, clonally propagated and belonging to the olive germplasm collection of the Agricultural Research Council-Olive Growing and Oil Industry Research Centre, CREA-OLI in Mirto-Crosia (Cosenza, Calabria, Italy). Olive trees were grown using the same field conditions and were located at latitude 39 • 37 ′ 04.57 ′′ N, longitude 16 • 45 ′ 42.00 ′′ E and altitude 8 m asl). Fruit sampling was performed as previously described (Matus et al., 2009): for each cultivar, drupes (n = 30,) were randomly collected at 100 and 130 DAF ( Figure S1). In order to minimize the effects related to asynchronous fruits maturation within the same tree, drupes with similar pigmentation were picked from all around the external parts of the tree canopy. Concerning drupe pigmentation, the epimesocarp tissues, was totally green in color at 100 DAF whereas at 130 DAF the pulp pigmentation was 50% brown in "Cassanese" and totally unpigmented in "Leucocarpa" drupes ( Figure S1). All samples were fixed in liquid nitrogen and stored at −80 • C for both RNA-seq and qRT-PCR experiments. RNA-Seq Library Preparation and Sequencing In order to obtain a general overview of the transcripts and metabolic pathways involved in fruit maturation and to avoid cross contamination from non-homogeneous tissue separation, sample pooling strategy has been here used (Peng et al., 2003). Pooling reduces variability by minimizing individual variation and represents an alternative approach to biological replicates in experiments where the interest is not on the individual but rather on characteristics of the population (e.g., common changes in expression patterns; Lilley, 2007, 2009). Total RNA was extracted from the epi-mesocarp tissues of drupes collected together, using the RNeasy Plant Mini kit (Qiagen) according to the manufacturer's instructions. Each RNA sample was subjected to DNase digestion (DNase I, Roche) to remove any DNA contamination and pooled equally, as previously described (Muzzalupo et al., 2012). RNA was quantified by the NanoDrop Spectrophotometer ND-2000 and quality was checked by electrophoresis (28S rRNA/18S rRNA ratios). Samples with a concentration of ≥400 ng/µl, OD260/280=1.8∼2.2, RNA 28S:18S ≥ 1.0, and RNA Integrity Number (RIN) ≥ 7.0 were used for cDNA library preparation. Standard RNA-seq library preparation and sequencing via Illumina HiSeq TM 2000 was carried out by Technology Services of the Institute of Applied Genomics (IGA, Udine, Italy). For each sample a single-end (SE) sequencing cDNA library was constructed with a fragment length range of 50 bp. Each library was created using two replicates, consisting of a separate pool of 30 homogeneous fruits. RNA-Seq Data Filter and De novo Assembly by Trinity The raw Fastq "reads" (NCBI PDA/SRAaccession numbers: SRR1574719, SRR1574772, SRR1573503, SRR1574328, Table 1) were analyzed and filtered, respectively with FastQC and Fastx Toolkit softwares to obtain high quality de novo transcriptome sequence data. Each sequence set was filtered using the following criteria: (i) reads containing the sequencing adaptor were removed; (ii) reads with unknown nucleotides comprising more than 5% were removed; (iii) low-quality reads with ambiguous sequence "N" were trimmed and discarded. Since the olive tree does not have a reference genome, the de novo assembly of the clean reads into transcripts was performed using the Trinity program (Grabherr et al., 2011;Haas et al., 2013), a useful method for the efficient and robust de novo reconstruction of transcriptomes from RNA-seq data (Ward et al., 2012;Gutierrez-Gonzalez et al., 2013;Liang et al., 2013;Liu et al., 2013;Pallavicini et al., 2013;Tulin et al., 2013). Trinity was run via script using 128 GB of ram, 12 cpu thread and a minimum assembled contig length to report set to 300 bp. Trinity sequentially combines Inchworm, Chrysalis and Butterfly modules to process large RNA-seq reads data, partitioning the sequence data into many individual de Bruijn graphs, representing transcriptional complexity at a given gene or locus (Grabherr et al., 2011;Haas et al., 2013). Analysis of Transcript Assembly For non-model organisms, one metric for evaluating the transcript assembly quality is to examine the number of transcripts that appear to be full-length or nearly full-length if compared to a closely related organism to examine fulllength coverage. In this context, a more general analysis was performed aligning the assembled transcripts against all known plant proteins determining the number of unique top matching proteins that are aligned in 70-100% range of its length by full-length transcript analysis (Haas et al., 2013). Therefore, a blastable database has been created to perform a local blastx search where only the single best matching Trinity transcript was outputted for each top matching entry. To validate our de novo assembly read remapping has been realized using bowtie2 (Langmead and Salzberg, 2012); for each data set a bowtie2 index was created, and then the number of reads that map to our transcriptome have been counted. Abundance Estimation and Differentially Expressed Trinity Transcripts For abundance estimation of transcriptome assemblies RSEM software was used (Li and Dewey, 2011). RSEM is a package for estimating gene and isoform expression levels from RNA-seq data. The current version of RSEM, was bundled with the Trinity software package. Moreover, Trinity currently supports the use of Bioconductor tools (edgeR and DESeq) to compute differential expression analysis in the assembled transcriptome (Anders and Huber, 2010;Grabherr et al., 2011;Haas et al., 2013). In order to identify statistically significant differences in transcript expression between samples, the number of reads/transcripts, the depth of sequencing, the transcripts length (longer transcripts generate more fragment reads) and the expression level of the transcripts were considered. Expression values, normalized for each of these factors were measured in FPKM (fragments per feature kilo base per million reads mapped) (Trapnell et al., 2010;Robinson and Oshlack, 2010) and used to make a comparison across multiple samples and replicates. Trinity supports the use of TMM (trimmed mean of M-values) normalization (Lekanne Deprez et al., 2002;Dillies et al., 2012), to account for differences in the mass composition of the RNA-seq samples, which does not change the fragment count data, but provides a scaling parameter that yields an effective library size (total map able reads) for each sample. This effective library size is then used in the FPKM calculations. Quantitative PCR Gene expression analysis was performed by quantitative realtime PCR on a 7500 fast real time PCR system (Applied Biosystems) with SYBR R Select Master Mix. The oligonucleotide primer sets (Table 1) used for qRT-PCR analysis were designed using Primer3 (http://primer3.ut.ee/). Each primer pair (Supplementary data, Table S1) generated a single specific amplicon on the 3 ′ -end of target sequence. PCR products were about 150-200 bp long and primer pair average efficiency ranged between 0.95 and 1.0. The housekeeping olive ELONGATION FACTOR 1 (EF1) gene (CAQ17046.1) was used to normalize the expression levels (Galla et al., 2009;Trapnell et al., 2010). Amplification reactions were prepared in a final volume of 20 µl according to the manufacturer's instructions. All reactions were run in triplicate in 96-well reaction plates, and negative controls were set. The cycling parameters were as follows: one cycle at 95 • C for 3 min to activate the Taq enzyme, followed by 40 cycles of denaturation at 95 • C for 10 s and annealing-extension at 58 • C for 30 s. To confirm the occurrence of a unique PCR product, the "melting curve" (Lekanne Deprez et al., 2002) was evaluated by an increase of 0.5 • C every 10 s within a 60-95 • C range and a unique "melting peak" in every reaction was observed. The comparisons of cycle threshold (CT) values were obtained analysing data with the 2 − CT method (Livak and Schmittgen, 2001). The means of gene expression levels were calculated from two biological repeats, obtained from two independent experiments. Blast2GO To assign gene ontology (GO) terms in our DE data sets, we used BLASTx 2.2.26+, BLOSUM62 similarity matrix, and Blast2GO database version August 2011 programs (Conesa et al., 2005;Morgulis, 2008). The definition of each GO term was determined by the GO Consortium: http://www.geneontology. org and can be found using the EMBL European Bioinformatics Institute QuickGO: http://www.ebi.ac.uk/QuickGO or the Gene Ontology Normal Usage Tracking System, GONUTS: http://gowiki.tamu.edu/wiki/index.php/Main_Page. Pathway assignments were determined following the Kyoto Encyclopedia of Genes and Genomes pathway database (Kanehisa et al., 2008) using BLASTX with an E-value threshold of 1.0E-5. MapMan (http://mapman.gabipd.org/) analysis was done using our DE transcripts rearranged as input experimental dataset. Using the Mercator web application we can assign MapMan "Bins" to DNA sequences (Thimm et al., 2004;Lohse et al., 2014). The output was used as a mapping file for data visualization in MapMan. The Mercator tool generates functional predictions by searching a variety of reference Frontiers in Plant Science | www.frontiersin.org FIGURE 1 | Whole transcripts expression during fruit ripening. In (A,B), MA plot for differential expression analysis generated by EdgeR: for each gene, the log 2 (fold change) (log 2 (100DAF/130DAF)) between the 100 and 130 DAF samples is plotted (A, y axis) against the gene's log 2 (average expression) (M, x axis). In (C,D), the Volcano plot reports a FDR (−log 10 FDR, y axis) as a function of log 2 (fold change) between the 100 and 130 DAF samples (logFC, x axis). Transcripts that are identified as significantly differentially expressed at most 0.1% FDR are colored in red. In (E), the heat map show the relative transcript/sample expression levels. Green and red colors are used to indicate the transcripts up to four-fold up-and down-regulated, respectively. Expression values (FPKM) are log 2 transformed and then median-centered by transcript. The dendrogram, on the left, orders whole transcripts set in relation to their level of expression. databases (BLAST-based, RPSBLAST based and InterProScan) and subsequently evaluating and compiling the search results for each input gene to propose a functional Bin. RNA-Seq Library Sequencing and De novo Transcriptome Assembly by Trinity Starting from four RNA-seq libraries, corresponding to two fruit developmental stages (100 and 130 DAF) of Olea europaea "Leucocarpa" and "Cassanese, " 147,789,544 raw reads were generated from 50 bp insert library. A total of 142,479,595 highquality SE reads were identified and used for Olea europaea trascriptome assembly, through the Trinity software. Using the 25-mer in Trinity and a minimum assembled contig length set to 300 bp, we found 103,359 transcripts. The total used reads, the total assembled transcripts, N50 statistics for each sample and remapping results are indicated in Table 1. A total of 93,623 likely coding sequences were extracted with the Transdecoder utility, to identify the longest ORF (Open Reading Frame) from the transcript assembly, reporting that ORF scored according to the Markov model. In all, 9597 of the olive transcripts had a BLAST hit with an E-value of less than 1e-20, and 19,708 of the extracted reference coding sequences are considered to be approximately "full length, " with the Trinity contigs aligning the matching UniProt reference transcript's length by more than 70%. Differential Expression Analysis To estimate the differential gene expression between fruits of both considered cultivars at each developmental stage, a single assembly, based on combining all reads across all samples as inputs was generated. A single assembly was chosen to avoid difficulty in comparing the results across the different samples, due to differences in assembled transcript lengths and contiguity. Then, reads were aligned separately back to the single assembly, in order to identify the number of DE transcripts with a False Discovery Rate (FDR) value of at most 0.001 and at least four-fold difference in expression values according to the Trinity protocol. For this purpose, it was possible to identify the DE transcripts sets of each cultivar, during the 100-130 DAF transition from Trinity scripts that leverage the R software. In this context, 3792 and 3064 DE transcripts (of 49,162 and 54,194 total transcripts, respectively) were identified in "Leucocarpa" and "Cassanese." The fold change and the statistical significance values between different developmental stage and cultivar were also estimated. Trinity facilitates analysis of RNA-seq data, including scripts for extracting transcripts that are above some statistical significance (FDR threshold) and fold-change in expression. To examine expression across multiple samples, the FPKM expression values across samples have been normalize, which will account for differences in RNA composition, afterwards TMM normalization generate a matrix of normalized FPKM values across all samples. These adjusted library sizes are used to recompute the FPKM expression values. Although the raw fragment counts are used for differential expression analysis, the normalized FPKM values are used below in examining profiles of expression across different samples, each DE set of transcripts was displayed as MA plots (where M = log ratios and A = mean values) (Figures 1A,B), volcano plots (Figures 1C,D) and clustered heat maps (Figure 1E). A correlation matrix (Figure S2) for the different developmental stages across cultivars, reveals that samples are more highly correlated within cultivar than between cultivar. Functional Annotation of Differentially Expressed Transcript Sets The in silico analysis of the entire sets of DE transcripts, performed by querying databases of genes and proteins (NCBI, ExPASy, InterProScan) and the functional annotation software Blast2GO, have allowed for each sequence to be traced back to the gene family and to be annotated according to the terms of the three main Gene Ontology vocabularies (Figure 2). Since analyses were conducted on the same organ and developmental stages, in both analyzed cultivars a fairly overlapped distribution of GO terms was observed during the developmental transition. In particular, the most represented ontological categories were membrane (GO:0016020), cell (GO:0005623) and organelle (GO:0043226). Molecular functional categories were strongly represented by terms related to catalytic activity (GO:0003824) with 47 and 46% in Leucocarpa and Cassanese cvs, respectively, followed by binding (GO:0005488) and transporter activity (GO:0005215). Finally, more than 10 categories were identified at the biological process level with metabolic and cellular processes (GO:0008152, GO:0009987), among the groups most represented, highlighting the intense and complex metabolic and regulatory activities during fruit maturation. In order to trace back to the pathways, such as flavonoids and anthocyanin, (map 00941 and 00942, Figures S3A,B, respectively), which were more closely involved in the transition FIGURE 4 | Relative transcripts expression during fruit ripening in Leucocarpa (dark gray) and Cassanese (light gray) cvs. The qRT-PCR results (log fold change) are presented as a proportion of the highest value after normalization with the EF1 house-keeping gene; for each cv 100 DAF samples are used as calibrator. The means ± s.e. of two independent biological replicates are reported. between 100 and 130 DAF, the whole DE transcripts set was examined through the Kyoto Encyclopedia of Genes and Genomes (KEGG). Functional analysis was implemented in Mapman, to focus gene expression changes via Image Annotator. All obtained results are consistent with a down regulation of flavonoid and anthocyanins metabolism in Leucocarpa cv, while an opposite trend was observed in "Cassanese" (Figure S4). Gene Expression during Olive Fruits Ripening We performed a quantitative RNA-seq analisys in a cultivar of Olea europaea species, whose fruits are characterized by a switchoff in skin color at full ripeness, to identify the genes involved in flavonoid andanthocyanin biosynthesis. The Table S1). Moreover, it was possible to identify different member of MYB, MYC and WD transcription factors related to the regulatory complex that controls anthocyanin structural genes at the transcriptional level (Takahama, 2004). Interestingly, the quantitative gene expression analysis does not seem to show significant differences during olive fruit development in Leucocarpa and Cassanese cvs ( Table 2). Indeed, focusing attention on the paths that control the biosynthesis of pigments and the natural reduction of photosynthetic pigments during the veraison stage (Pua and Davey, 2010), the "Leucocarpa" was characterized by a broad down-regulation of CHS, DFR, and ANS transcripts (Figure 3), during the 100-130 DAF transition compared to Cassanese cv. The estimated fold change of the selected genes was also confirmed by quantitative PCR experiments (Figure 4). In particular, the expression of transcripts putatively involved in the selected pathway were more highly expressed in "Cassanese" genotype than in "Leucocarpa." This genome-wide overview on flavonoid and antocyanidin genes also allowed us to select different members of MYB, MYC, and WD transcription factors (TF), within the differentially expressed gene set, linkable to anthocyanins regulatory circuit (Dixon et al., 2005;He et al., 2008;Tian et al., 2008;Stommel et al., 2009;Jaakola, 2013). The abundance estimation analysis made it possible to compare the identified TFs in all analyzed samples. In the "Cassanese" plant, despite a slight decline, the amount of transcripts during 100-130 DAF transition was consistent with the increased anthocyanin structural gene expressions and metabolite accumulation during growth of fruits; whereas in the Leucocarpa cv the identified TFs are primarily characterized by lower expression levels and a general reduction in expression abundance during ripening transition. The differences were most evident when the comparison was carried out at the same stage FIGURE 5 | Comparisons in MYB, MYC, and WDR transcripts abundance between samples. Each data is displayed as a stacked bar. Transcripts expression levels were taken from the complete FPKM normalized matrix that were identified as differentially expressed. (100 or 130 DAF) of maturation. 9 MYB, 5 MYC, and 7 WD TF undergo a decrease in expression during transition, in contrast to Cassanese cv where they appear to participate in the activation pathway ( Figure 5). DISCUSSIONS In the present work we used the Illumina RNA-seq technology to identify the transcripts along flavonoids and anthocyanins biosynthetic pathways and to monitor their expression levels during ripening, by comparing two olive cultivars characterized by different phenological behavior at ripening in terms of anthocyanin accumulation and general pigmentation. We also used a de novo transcriptome assembly strategies performed in many plants, including rice, maize, sesame, bamboo, poplar, sweet potato, Eucalyptus tree, chickpea, and orchid (Mizrachi et al., 2010;Wang et al., 2010;Fu et al., 2011;Garg et al., 2011;Wei et al., 2011;Zhang et al., 2011a). The characterization of the genetic entity of olive cultivars has benefited from new molecular biology and high-throughput sequencing methods Galla et al., 2009;Bazakos et al., 2012;Muñoz-Mérida et al., 2013). Through the analysis of massive data it is possible to identify/investigate the genetic pathways that underlie specific, or more general, agronomic traits in the physiological performance of the plants belonging to the Olea europaea species. Between different high-throughput methods, the Illumina sequencing is the best next generation technology, both less costly and more efficient, for transcriptome analysis, if compared with 454 platform, in particular when used in non-model organisms, where genomic sequences are unknown. Even though this technology has been previously restricted to the re-sequencing of organisms with available reference genomes (Nagalakshmi et al., 2008), its recent improvement has enabled the development of de novo strategies for robust trascriptome reconstruction for non-model plants from short reads and their assembly into unigenes. In addition, different transcription factor members with similarity to MYB, MYC, and WD40 family and involved in anthocyanin biosynthesis were also found. Furthermore, the transcripts abundance of identified genes was correlated to the accumulation rate of anthocyanin metabolites. The anthocyanin biosynthesis pathway has been extensively studied in several plant species, such as petunia, pears, goji berry, bilberry and black raspberry (Jaakola et al., 2002;Zeng et al., 2014). During the ripening progression, many species including the olive tree accumulate anthocyanin in their fruits (Jaakola et al., 2002;Sweetman et al., 2009;Zhang et al., 2011b). In this context, anthocyanins are considered potent marker to monitor ripening stages and organoleptic quality of fruits. In apple, the regulatory circuit in anthocyanin biosynthesis is tuned by the MYB-MYC-WD40 protein complexes (Ramsay and Glover, 2005;Schaart et al., 2012). Moreover the R2R3-MYB and bHLH TFs are able to activate structural genes, including CHS, DFR and ANS, and ultimately promote anthocyanin accumulation in fruits (Chagné et al., 2013;Umemura et al., 2013;Zeng et al., 2014). In our case the transcripts abundance of MYB, MYC, and WD40-type TFs was higher in Cassanese cultivar than in Leucocarpa and was also directly related to anthocyanin accumulation. In conclusion, the comparative approach performed provide an invaluable resource to identify genes involved in fruit maturation and to define the metabolic pathway and tissue specific functional genomics in non-model plant species. The characterization of transcripts from flavonoid and anthocyanin biosynthetic pathways and the analysis of their expression level in olive fruits is an important goal to understand the veraison event of fruits and to increase the knowledge on these antioxidant molecules, important for human health. AUTHOR CONTRIBUTIONS DI performed research and discussed results. AC designed research analyzed data and discussed result. IM designed research analyzed data and discussed results. All authors contributed to improving the papers and approved the final manuscript. ACKNOWLEDGMENTS The authors are very grateful to Dr. Sabrina Micali (CREA-FRU of Roma, Italy) for excellent technical and scientific assistance. This research was supported by the "Certificazione della composizione varietale, dell'origine geografica e dell'assenza di prodotti di sintesi negli oli extravergini di oliva-CERTOLIO 2012-2014" projects and by grant from University of Calabria (MIUR ex 60) to Prof. AC.

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Effects of Sequence and Expression of Eight Anthocyanin Biosynthesis Genes on Floral Coloration in Four Dendrobium Hybrids 1Special Research Unit in Microalgal Molecular Genetics and Functional Genomics, Department of Genetics, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand 2Center of Advanced Studies for Tropical Natural Resources, Kasetsart University, Bangkok 10900, Thailand 3Department of Biology, Faculty of Science, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand 4Center for Agricultural Biotechnology, Kasetsart University, Kamphaeng Sean Campus, Nakhon Pathom 73140, Thailand 5National Center for Genetics and Biotechnology (BIOTEC), Pathum Thani 12120, Thailand Introduction Anthocyanins are a group of flavonoid glycosides constituting the major color pigments in flowers and fruit. Anthocyanins are synthesized along with flavonoid biosynthesis through a series of enzymatic reactions that convert chalcone into three major anthocyanidin types: cyanidin (red to magenta), pelargonidin (brick red to scarlet) and delphinidin (purple to for three branches of subsequent biosynthesis cascades. This leads to variations in pigment production via the three dihydroflavonols, namely, dihydroquercetin, dihydrokaempferol, and dihydromyricetin, which are then catalyzed by DFR and subsequently ANS, producing cyanidins, pelargonidins and delphinidins, respectively (Tanaka et al., 2008). Additionally, along with anthocyanin biosynthesis, flavone and flavonol are synthesized through the activity of flavone synthase (FNS) and flavonol synthase (FLS), respectively (Holton and Cornish, 1995;Martens and Mithöfer, 2005). Many studies have demonstrated the effects of mutations in anthocyanin structural or regulatory genes on anthocyanin pigmentation. Loss-of-function mutations in CHS, CHI, F3H, DFR, and ANS normally cause a block in the biosynthesis, and plants harboring these mutations often produce white flowers or are colorless in tissues that usually contain color pigments (Britsch et al., 1992;Franken et al., 1991;Inagaki et al., 1996;Nakatsuka et al., 2005;Napoli et al., 1999). Studies of several yellow-flowered varieties of ornamental plants including Dianthus and Cyclamen showed that recessive mutations in CHI caused the accumulation of naringenin chalcone, resulting in yellow flowers (Forkmann and Dangelmayr, 1980;Miyajima et al., 1991). However, in some cases, CHI mutations did not result in complete disruption of anthocyanin production because some portion of the CHI substrate, naringenin chalcone, could be spontaneously catalyzed and proceed into the pathway (Forkmann and Dangelmayr, 1980;Miles and Main, 1985). Mutations in either F3'H or F3'5'H in many cases caused color alterations. Studies in roses, Dianthus and Chrysanthemum demonstrated that the lack of blue-purple in these plants was due to the loss of F3'5'H, which encodes the key enzyme responsible for delphinidin synthesis (Tanaka and Brugliera, 2006). F3'H switches anthocyanin biosynthesis to red-colored cyanidins and, in some varieties of Chrysanthemum in which F3'H is mutated, anthocyanin biosynthesis proceeds towards pelargonidin production resulting in orange flowers . Furthermore, certain types of mutation in DFR led to alterations in enzyme specificity towards its substrates. For example, while DFRs from many species such as Dianthus caryophyllus and Gerbera hybrida have broad specificity to the three types of dihydroflavonol, DFRs from petunia and Cymbidium cannot reduce dihydrokaempferol efficiently and, therefore, cannot produce pelargonidin-based color pigments (Forkmann and Rahnau, 1987;Helariutta et al., 1993;Johnson et al., 1999;Stich et al., 1992). Dendrobium hybrids have been commercially distributed throughout the globe due to their elegant, colorful flowers with diverse shapes. Research has been conducted to understand the control of coloration in Dendrobium for improving flower production. However, knowledge regarding this issue is currently limited. Analysis of pigment compositions in 28 commercial Dendrobium species and hybrids has shown that cyanidins are the major pigment and pelargonidins are found in a few hybrids with peach or red flowers (Kuehnle et al., 1997). Previously, CHS and DFR genes were isolated and characterized to verify the difference of floral coloration controls in two Dendrobium hybrids producing purple and peach flowers. The analysis suggested that the substrate specificity of DFR was a possible cause of the color difference (Mudalige-Jayawickrama et al., 2005). Furthermore, CHS, DFR, and F3'5'H were recently isolated from D. moniliforme, and the analysis of F3'5'H expression suggested its role in floral coloration (Whang et al., 2011). In this report, we aim to determine the coloration controls of four Dendrobium hybrids producing different flower colors including purple, peach, and two types of white. Full-length cDNA of CHS, CHI1, CHI2, F3H, DFR, ANS, F3'5'H, and FLS was isolated. Sequence and expression analyses revealed variations of deduced amino acid sequences and expression patterns of the eight genes in the four hybrids. Data obtained from this study were discussed on the basis of the different coloration in each hybrid. Furthermore, the analyses were extended to two selected Dendrobium mutants producing paler-and darker-colored flowers to address the possible causes of their color alterations. Plant materials and RNA isolation Four Dendrobium hybrids were obtained from local orchid farms in Thailand. These included a hybrid producing purple flowers named 'Sonia Earsakul' (SE), a peach-flowered hybrid called 'Sirin classic' (SC), and two white-flowered hybrids named 'Suree white' (RW) and 'Jasmine white' (JW). Two SE mutants that produce a paler color (L, ER6-329) and a deeper purple color (D, ER5-1129) on both petal and sepal were selected from the SE mutagenized population generated by acute gamma-irradiation of protocorm-like bodies at 30 Gy. The mutants were monitored for their consistency in color production in at least four consecutive flower generations. Four floral development stages, namely, early young bud (~0.5 × 1.5-2 cm: width × height), young bud (~1 × 2.5-3 cm), mature bud (~1.5 × 3.5-4 cm) and fully open flower stages, were collected for RNA isolation. Total RNA was isolated from whole buds and flowers by a method described by Yu and Goh (2000). Briefly, buds or flowers were ground in liquid N 2 , suspended in the extraction buffer [50 mM CTAB, 40 mM Tris-HCl pH 7.5, 20 mM EDTA, 2 M NaCl, 1% (w/v) PVP-90 and 2% (v/v) β-mercaptoethanol] and incubated at 60°C for 15 min. The mixture was centrifuged at 7000 × g and 4°C for 15 min. Supernatant was mixed with an equal volume of chloroform:isoamylalcohol (24:1) and centrifuged at 7000 × g at 4°C for 15 min. This step was repeated twice. Supernatant was mixed with one-third volume of 10 M LiCl and kept at −20°C overnight for RNA precipitation. RNA was collected by centrifugation at 12000 × g at 4°C for 20 min and then washed with 2.5 M LiCl and 70% (v/v) ethanol in DEPC-treated water. RNA was dried and dissolved in DEPC-treated water. RNA was then subjected to DNase treatment (Promega, Medison, WI, USA), phenol: chloroform extraction and RNA precipitation. RNA was analyzed by agarose gel electrophoresis and quantified using Nanodrop (Thermo Scientific, Waltham, MA, USA). 5'-and 3'-RACEs and full-length cDNA cloning Initially, partial sequences of CHS, CHI1, CHI2, F3H, DFR, ANS, F3'5'H, and FLS were amplified from cDNA of the SE hybrid, and these were used for priming sequences in 5'-and 3'-RACE reactions. Total RNA from buds and flowers of the SE hybrid was used for isolation of the 5' and 3' ends of the transcript of the eight anthocyanin biosynthesis genes using GeneRacer ® Kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer's protocol. 5' and 3' amplification reactions included 1 μL of GeneRacer-cDNA of the SE hybrid, 0.5 μM for either 5' or 3' RACE primers and gene-specific primers, 100 μM dNTPs, 1× Taq polymerase buffer and 0.5 units of Taq polymerase (NEB, Ipswich, MA, USA) in a total volume of 20 μL. PCR conditions were as follows: 2 min at 94°C; 35 cycles of 30 s at 94°C, 30 s at 50-60°C and 1 min at 72°C; and finally 72°C for 10 min. PCR products were resolved by electrophoresis in 1% (w/v) agarose gel, stained with ethidium bromide and visualized under UV light. DNA fragments were purified and cloned into the pGEM-T Easy Vector (Promega) for subsequent sequence analysis. 5' and 3' end sequences of each gene were used for primer design for full-length cDNA cloning. For full-length cDNA cloning, cDNA of SE, SC, RW, JW, ER6-329, and ER5-1129 was synthesized using SuperScript TM III Reverse Transcriptase (Invitrogen). Briefly, a reaction including 1 μg of total RNA, 2.5 μM oligo(dT) 15-18 , 0.5 mM dNTP and DEPC-treated water in a total volume of 13 μL was incubated at 65°C for 5 min before being cooled down on ice. Four microliters of 5× First-Strand buffer, 1 μL of DTT, 1 μL of RNaseOUT TM (Invitrogen) and 1 of μL SuperScript TM III Reverse Transcriptase were added into the reaction and incubated at 50°C for 60 min and then at 70°C for 15 min. Full-length cDNA sequences of CHS, CHI, F3H, DFR, ANS, F3'5'H, and FLS were amplified from Table 1. Primer sequences for full-length cDNA cloning, quantitative real-time PCR, and semi-quantitative RT-PCR analysis. Gene Forward primer (5′-3′) Reverse primer (5′-3′) z These were used for both quantitative real-time PCR and semi-quantitative RT-PCR. the cDNA with the primers listed in Table 1 using Easy-A ® High-Fidelity PCR Cloning Enzyme (Stratagene, La Jolla, CA, USA) and subsequently sequenced. Nucleotide and deduced amino acid sequences were aligned using ClustalW version 3. Quantitative real-time PCR analysis cDNA was synthesized from total RNA obtained from young buds, mature buds and fully open flowers of SE, SC, RW and JW hybrids using SuperScript TM III Reverse Transcriptase (Invitrogen). cDNA synthesis reaction was performed in the same manner as described in the full-length cDNA cloning section with the exception that 50 ng of random hexamers (Invitrogen) were used as replacement for 2.5 μM oligo(dT) 15-18 primer. Quantitative real-time PCR was performed using 25fold diluted cDNA samples. Each quantitative PCR reaction contained 7.5 μL of 2× SsoFast TM EvaGreen ® Supermix (BioRad, Hercules, CA, USA), 2 μL of cDNA and 0.2 μM forward and reverse primers in a total volume of 15 μL (see Table 1 for primer sequences). Thermal cycling was performed on Eppendorf Mastercycler ® ep Realplex Real-Time PCR Systems (Eppendorf, Hamburg, Germany) using a preheating step at 98°C for 30 s followed by a two-step cycle: 5 s at 98°C and 30 s at 60°C. 18S rRNA amplification was used as an internal standard. Data were analyzed using the ΔΔCt method with default parameters. Error bars represent the SD of three biological replicates and each was conducted in triplicate. Semi-quantitative RT-PCR analysis cDNA was synthesized from total RNA of the four flowering stages of SE, ER6-329 and ER5-1129 in the same manner as described for quantitative real-time PCR analysis. cDNA products were diluted 10-fold with dH 2 O, and semi-quantitative RT-PCR was performed in reactions containing 1 μL of cDNA, 0.5 μM for each primer, 100 μM dNTPs, 1× Taq polymerase buffer and 0.5 units of Taq polymerase (NEB) in a total volume of 20 μL. The reaction mixture was incubated in conditions of 2 min at 95°C; followed by 10-29 cycles of 30 s at 95°C, 30 s at the annealing temperature specific for each primer pair and 1 min at 72°C; and finally 10 min at 72°C (see Table 1 for primer sequences). 18S rRNA amplification was used as a reference. The analysis was performed in triplicate. Isolation and sequence analysis of full-length cDNA Full-length cDNA of putative CHS, CHI, F3H, DFR, ANS, F3'5'H, and FLS was isolated from buds or flowers of Dendrobium SE hybrid. Two distinct putative CHI sequences, designated as CHI1 and CHI2, were isolated, whereas single amplicons were obtained from the other genes. This indicated that at least two CHI homologues are present in the SE hybrid genome. Alignments of deduced amino acid sequences of the isolated cDNAs with previously reported anthocyanin biosynthesis genes from other plant species showed high sequence similarity within each gene group (data not shown). The sequences were deposited in the NCBI database. Details of the eight full-length cDNA sequences regarding accession numbers, sequence lengths, deduced amino acids, the highest sequence identity match and their conserved domains are presented in Table 2. Sequence analysis of the eight anthocyanin biosynthesis genes among four Dendrobium hybrids To gain insight into the basis of color variations in flowers of Dendrobium hybrids, we examined nucleotide and deduced amino acid sequences of the eight genes from four hybrids that produce different flower colors. These included hybrids with flower colors ranging from dark purple (SE), peach (SC) and white (RW) to greenish white (JW) (Fig. 1). Nucleotide and amino acid sequence alignments showed a number of differences in nucleotide sequences that caused amino acid changes, a gene deletion and an immature translation termination in the eight genes from the four hybrids (Table 3). Open reading frames of the eight genes from SC, RW and JW hybrids were intact and similar to 86 those from the SE hybrid in terms of both size and amino acid contents, except for CHI2 from RW and F3'5'H from JW. A single adenosine deletion at position 498 of the CHI2 coding sequence from RW was observed. This caused frame-shift mutation and consequently premature translational termination, which resulted in a shorter polypeptide product containing 156 rather than 209 amino acids. A 117-nucleotide deletion observed in F3'5'H from the JW hybrid resulted in a loss of 39 amino acids at position 7-45 compared with that of the SE hybrid. Furthermore, amino acid alterations located in the conserved region of each gene were also noted. Expression analysis of anthocyanin biosynthesis genes in four Dendrobium hybrids To examine the correlation between gene expression and flower colors, temporal expression of the eight genes in the four hybrids was analyzed using quantitative real-time PCR. The expression levels were monitored at three stages: young buds, mature buds, and anthesis. Variations in the expression levels of the eight genes were observed (Fig. 2). Generally, in all four hybrids, the expression of the anthocyanin biosynthesis genes was observed in young bud and gradually increased, reaching the highest levels in mature bud, before declining to very low levels at the anthesis stage, with the exception of F3'5'H, of which the expression remained high through to the anthesis stage. In the purple-flowered SE hybrid, the expression levels of CHS, CHI1, CHI2, F3H, DFR, ANS, and FLS were very high in young and mature buds compared with those from the other hybrids, before decreasing to very low or no expression in fully open flowers. In contrast, the expression levels of the eight genes in the SC hybrid were about one-third to one-half of those in the SE hybrid. The overall low levels of gene expression somehow reflected the fact that the color intensity of the SC hybrid flowers is far lower than that of the SE hybrid, regardless to the color. In white-flowered hybrids, distinct expression patterns were observed. We noticed that, in the JW hybrid, the expression of F3H, DFR, and ANS was almost undetectable and the expression of CHI1 was dramatically high compared with that of the other hybrids, whereas, in the RW hybrid, the expression level of FLS was distinctively high, about 5-fold higher than in the other hybrids, and CHI1 was expressed at a high level at the anthesis stage. Notably, the expression of CHI1 and that of CHI2 somehow compensated for each other. The expression of CHI2 was markedly high in young bud and mature bud stages but was reduced at the anthesis stage, whereas the expression of CHI1 was very low at the two early stages but abruptly increased in the anthesis stage. Table 3. Amino acid changes in the anthocyanin biosynthesis genes of SE hybrid compared with those of SC, RW, and JW hybrids. Premature translation termination and deletion events were observed, and the numbers in brackets indicate the predicted size of the translated protein and the position of the deleted amino acid, respectively. Expression analysis of anthocyanin biosynthesis genes in irradiated Dendrobium mutants We explored further the causes of the color changes in Dendrobium SE by examining two SE mutants that exhibit flowers with a paler color (L, ER6-329) and a darker color (D, ER5-1129) using sequence and expression analyses. Analysis of nucleotide sequences from eight anthocyanin biosynthesis genes showed no point mutation in both L and D mutants (data not shown). Semi-quantitative F3H,F3'5'H,DFR,and FLS showed that, while the expression levels of the five genes in the wild type and the D mutant were similar, the expression levels in the L mutant were generally lower than those in both the wild type and the D mutant (Fig. 3). This indicated that the paler-colored flowers in the L mutant were the result of simultaneous reductions of gene expression in at least five genes involved in anthocyanin biosynthesis. Discussion The results from sequence alignments strongly suggest that the full-length cDNAs cloned from Dendrobium SE hybrid are indeed their corresponding anthocyanin biosynthesis genes. The alignment of CHS, CHI1, CHI2, DFR, and F3'5'H showed that sequences with the highest percent identity (74-100%) were from either Dendrobium or other orchid species. We noted that our CHS and DFR sequences were exact matches (100% identity) to previously described Den-CHS-4 and Den-DFR-1 isolated from Dendrobium Sw. (UH503) (Mudalige-Jayawickrama et al., 2005) and CHS from Dendrobium SE (Pitakdantham et al., 2010). Although there were no F3H, ANS, and FLS sequences available from orchids, our sequences were grouped very closely with those from monocots, with 71-95% identity. Considering sequence alterations of the eight genes among the four hybrids, although some of the alterations could be anticipated, it was difficult to determine which specific amino acid changes would affect the protein functions and, therefore, alter the flower colors. According to the CHS alignment result, the alterations of CHS amino acid sequences observed in SC and JW hybrids were in variable regions and, thus, this is unlikely to affect the function of CHS. This is supported by the fact that the SC hybrid produced colored flowers. However, this does not preclude the possibility that this is the cause of white flowers in the JW hybrid. On the basis of a crystallography study of CHI protein structure (Jez et al., 2000), premature termination of the CHI2 in the RW hybrid, with more than 25% Cterminus polypeptide loss, is likely to result in functional disruption. Nevertheless, the presence of the CHI1 homolog is likely to compensate for this loss. We compared our deduced sequences to specific amino acid changes that have been previously reported to alter anthocyanin production, but no match was found. These changes included amino acid sequences of DFR from petunia, potato and Caryophyllales (De Jong et al., 2003;Johnson et al., 2001;Shimada et al., 2004), ANS from Caryophyllales, gentian and onions (Kim et al., 2005;Nakatsuka et al., 2005;Shimada et al., 2005), and F3'5'H and FLS from soybean (Takahashi et al., 2007(Takahashi et al., , 2010. Nevertheless, it is worth noting that the amino acid alterations might potentially affect the flower colors. On the basis of the amino acid changes observed in the conserved regions of the coding proteins, these include changes in F3'5'H from the SC hybrid, F3H from the RW hybrid, and DFR and ANS from the JW hybrid. The expression analysis showed that, in coloredflower hybrids including SE and SC, CHS, CHI, F3H, DFR, and ANS were generally expressed from young bud to mature bud stages. These expression data match with the flower colors as these five genes have been reported to be the key genes responsible for anthocyanin production (Mol et al., 1998). In all four hybrids, while the expression of the eight genes mostly occurred at the young and mature bud stages and dramatically declined to very low levels at the anthesis stage, the expression of F3'5'H was observed at elevated levels in the progression towards the anthesis stage. Despite the presence and expression of the F3'5'H gene, delphinidins have never been found to be the color pigments of Dendrobium species, and myricetin and syringetin 3',5'hydroxylated flavonols were shown to represent F3'5'H enzymatic activity in Dendrobium (Kuehnle et al., 1997). Thus, our results suggest that the function of F3'5'H mostly occurs at late stages of flower development and this could contribute to the production of 3', 5'-hydroxylated flavonols, which act as co-pigments for coloration in Dendrobium flowers. Previous assessments determining the color pigments of the flowers of Dendrobium hybrids proposed a plausible cause of color changes from cyanidin type in lavender flowers of Dendrobium Sw. (UH503) to pelargonidin type in peach flowers of Dendrobium hybrid (K1224), which could be due to a mutation in F3'H, rather than in DFR (Mudalige-Jayawickrama et al., 2005). Such mutation resulted in the inefficient production of dihydroquercitin and led to more abundant dihydrokaempferol and, therefore, pelargonidins. This could be similar to our case in purple-flowered SE and peach-flowered SC hybrids. However, F3'H was not included in our study. Nonetheless, because the alterations of DFR sequences between SC and SE hybrids were neither in conserved regions nor in other positions that have been shown to affect its function, it could be anticipated that a mutation(s) in F3'H could be the cause of peach flowers in the SC hybrid. Previous studies of anthocyanin contents suggested that the color intensity of Dendrobium flowers corresponded directly to pigment contents within the colored cells (Kuehnle et al., 1997). Considering the gene expression results, it is possible that the low color intensity in the SC hybrid could be because of the very low expression levels of CHI1 and CHI2. The reduction in CHI expression could, therefore, result in the low production of naringenin, downstream pigment production and color intensity, as a consequence. Despite its white flowers, the expression profiles of the eight genes in the RW hybrid were similar and even higher for CHS, CHI1, and CHI2 than those in the SC hybrid. Two notable features were that the expression level of FLS was about 5-fold higher than those from the other hybrids and there was likely to be an interplay between the expression of CHI1 and CHI2. On the basis of our results, the production of white flowers in the RW hybrid could possibly be explained by either or both of the following causes. First, 6 amino acid alterations in the conserved regions of F3H of the RW hybrid compared with those of the SE hybrid suggest a high potential for F3H functional alteration. This could result in the blockage of subsequent anthocyanin biosynthesis and the loss of color pigments (Nakatsuka et al., 2005). Second, FLS catalyzes the production of colorless flavonols and competes with DFR in terms of anthocyanin production. Previous studies in white-flowered petunia containing high levels of flavonols demonstrated that the flower color could change from white to pink when either FLS was down-regulated or DFR was overexpressed (Davies et al., 2003). Giving that the CHI2 function was disrupted by premature termination and CHI1 expression was very low at early floral developmental stages, the level of the catalytic product of CHI could be very low in RW floral tissues. Thus, the white flowers of the RW hybrid could be the result of low expression levels of CHI1 and high expression levels of FLS, which cause the conversion of color flavonoid intermediates into colorless flavonols. To rule out these possibilities, further study of anthocyanin and flavonol compositions in RW flowers is required. If the RW hybrid exhibited white flowers because of CHI and/or F3H deficiencies, the accumulation of both anthocyanins and flavonols should be greatly reduced, whereas if this white flower phenotype is caused by the upregulation of FLS, the accumulation of flavonols should be increased and that of anthocyanins should be reduced. In the JW hybrid, F3H was hardly expressed throughout the flower stages, being about 30-fold less than those from the other hybrids. No expression was detected for DFR and ANS. These three genes are the key for anthocyanin pigment production (Martin and Gerats, 1993;Mol et al., 1998). A number of studies have shown that their expression had dramatic effects on color production. For example, loss or very low expression of either F3H or ANS was shown to be responsible for white flowers in Vanda hybrids and a gentian hybrid (Homoi), respectively (Junka et al., 2011;Nakatsuka et al., 2005). Simultaneous loss of expression in F3H, DFR, and ANS was also shown to be responsible for white coloration in both a gentian hybrid (Polano white) and a group of white-flowered Sim carnations (Mato et al., 2000;Nakatsuka et al., 2005). This simultaneous loss of expression is similar to our observation and we suggest that this would be the case for the white coloration in JW hybrid flowers. Common regulatory factors controlling the expression of genes in the anthocyanin biosynthesis pathway are the key for colorations in flowers and other specific tissues (Davies et al., 1993). Analysis of white flowers in Sim carnation and gentians in comparison to their colored-flower counterparts suggested that mutations in regulatory proteins could result in the failure to induce a set of anthocyanin production genes during flower development (Mato et al., 2000;Nakatsuka et al., 2005). Thus, our results imply that, in the JW hybrid, F3H, DFR, and ANS were coordinately controlled, and the loss of expression of these genes was potentially due to a change in anthocyanin regulatory elements. In maize, CHS, CHI, F3H, DFR, and ANS are transcriptionally regulated by three regulatory protein families known as MYB, bHLH, and WD40 (Petroni and Tonelli, 2011). In particular, in maize seeds, mutations of MYB-C1 or bHLH-R1 have been shown to cause a colorless phenotype, and a mutation in WD40-PAC1 90 was associated with reduced pigmentation (Dooner et al., 1991;Selinger and Chandler, 1999). Similarly, a recent report on Phalaenopsis showed that white coloration in the petals of P. amabillis resulted from the loss of anthocyanin-specific MYB transcripts, which subsequently caused the loss of DFR expression (Ma et al., 2009). From this, we suggest that the simultaneous reductions of gene expression of the five genes observed in L mutants was likely due to a mutation(s) in one of the regulatory genes in anthocyanin biosynthesis. Furthermore, a novel bHLH gene identified in maize (referred to as IN1) was shown to function as an inhibitor in anthocyanin biosynthesis, and in1 mutants exhibited very intense pigmentation (Burr et al., 1996). This may coincide with our observations and we suggest that the increase of color intensity in the flowers of D mutants might be because of a mutation in one of the bHLH inhibitors. Data obtained from our study allowed postulations of factors affecting color variation in Dendrobium hybrids. Peach coloration in the SC hybrid is likely derived from the combination of changes in pigment production from cyanidin to pelargonidin through a mutation in F3'H and low levels of CHI1 and CHI2 expression. White coloration in the RW hybrid likely results from mutations in specific genes responsible for pigment production or increase in the expression of FLS that converts color pigments into colorless co-pigment molecules. Alternatively, in the JW hybrid, it is likely a result of the simultaneous loss of gene expression of a number of genes involved in the biosynthesis process. Furthermore, analysis of SE hybrid mutants bearing pale and dark flowers demonstrated that the expression levels of anthocyanin biosynthesis genes influence the intensity of color pigments in the flowers. Information obtained from these hybrids implying different approaches in flower coloration could benefit flower improvements by providing various strategies for genetic manipulations in Dendrobium and related orchids.

v3-fos

2017-09-07T05:56:47.500Z

2015-01-01T00:00:00.000Z

198430637

s2

Grain Sorghum Yield Response to Water Availability Yield effects of irrigation on sorghum and corn were compared, focusing only on the grain sorghum phase. Average water use for irrigation was 22 in., and dryland sorghum used 17 in. Average yields based on 12.5% grain moisture for dryland and irrigated sorghum were similar, with 138 bu/a for the irrigated and 142 bu/a for the dryland environment. Irrigated sorghum yields were similar, but in dryland, the Pioneer 84G62 hybrid yielded 149 bu/a, a 10 bu/a increase over Pioneer 84Y50 and DKS 53-67 hybrids, which yielded 139 bu/a and 138 bu/a, respectively. Although there was a difference in the yield between the hybrids on the dryland block, there were no significant differences between water use and water use efficiency (WUE). Introduction Decreases in available irrigation water and increased water restrictions necessitate exploration of more economical ways to use available irrigation water. Under low-yielding environments (<80 bu/a grain sorghum), sorghum has a yield advantage over corn because of its lower input costs and superior WUE and heat tolerance. Sorghum's yield potential is not as high as corn's, however, so the goal of this study is to determine at what point in available water, both under dryland and irrigation management scenarios, it is better to plant sorghum rather than corn. Procedures In a randomized complete block design, grain sorghum was planted in a dryland and a fully irrigated block at the Scandia Unit of the North Central Kansas Experiment Field. Within each block, three treatments of different grain sorghum hybrids were planted (Pioneer 84G62, Pioneer 85Y40, and DKS 53-67) with four replications. The plot size was 10 ft × 45 ft (length), and sorghum was planted in 30-in. rows (four rows per plot). The center two rows were harvested for final grain yield and its components. Plant populations and fertility were based on yield goals of 160 bu/a for the fully irrigated sorghum and 125 bu/a for the dryland. Grain sorghum was planted on May 22 with seeding rates based on a goal of 90,000 plants/a in the irrigated block and 50,000 plants/a in the dryland block. Fertilizer was applied based on recommendations for corn because sorghum fertilizer recommendations for the target yield were lower, and we wanted to eliminate variables that would cause different yields for corn vs. sorghum. Nitrogen (N) was applied preplant at 100 lb/a on both dryland and irrigated sorghum and was supplemented with 130 lb/a N on the irrigated block (applied June 11, 2014). Based on soil tests, phosphorus (P) was also applied on June 11 at 30 lb/a on dryland and 35 lb/a on irrigated treatments. Potassium (K) was not applied because of high soiltest potassium levels. Because this study evaluates crop production under limited irrigation, water usage was measured at diverse growth stages. After emergence, 6-ft aluminum tubes were installed in the center of each plot halfway between the center two rows. These tubes were used to take water content measurements throughout the growing season using a neutron probe at depths of 0.5, 1.5, 2.5, 3.5, and 4.5 ft. The moisture readings were taken at emergence, mid-vegetation, flowering, mid-reproductive, harvest, and 55 days after seed maturity. Results Yields for both irrigated and dryland blocks were similar (Tables 1 and 2). This could be owing to several factors, including time of harvest or lodging. A glitch in the new program on the irrigation system also caused dryland sorghum to receive 1.2 in. of water near flowering, which is the most sensitive time for sorghum. The irrigated sorghum had an average yield of 138 bu/a, and dryland had an average yield of 142 bu/a. Although yields were similar, water use and WUE differed between irrigated and dryland treatments. The mean water use for the irrigated and dryland were 21.9 in. and 17.0 in., respectively. The dryland was more efficient with the water that it used, with WUE of 8.3 bu/in. compared with 6.3 bu/in. for irrigated sorghum. Irrigated sorghum hybrids did not differ in water use, yield, or WUE, but a significant difference was detected among hybrids in the dryland environment, although not for water use and WUE (using a 95% confidence interval). The Pioneer 84G62 hybrid yielded significantly higher at 149 bu/a, whereas 85Y40 and DKS 53-67 had similar yields of 139 and 138 bu/a, respectively.

v3-fos

2019-04-01T13:14:45.267Z

2015-03-08T00:00:00.000Z

55017817

s2

Molecular and Biochemical Characterization of Some Egyptian Genotypes Rhizobium (Vicia Faba) Isolates Thirteen Rhizobial isolates were recovered from the root nodule of Faba bean (Viciae faba L.) grown in different geographic locations and soil properties in Egypt. The tested isolates were identified as R. leguminosarum sv. Viciae on the bases of morphological, biochemical characteristics and sequences of the gene encoding 16s rRNA. Rhizobium isolates were tested for their ability to utilize different carbon sources. Mannitol and Glucose were the best source of carbon. All tested isolates from Vicia faba differ in IAA production. The maximum amount of IAA production was in the range of (2.04 μg/ml) for Al Arish isolate to (7.5 μg/ml) for Ismailia isolate among the studied isolates. A great ability to degrade Roundup herbicide among the tested isolates was observed. Sues City isolate was the best active degrading Roundup herbicide on plates. All of the tested isolates showed resistances to (25 and 50 mg/ml) expect isolate RL7 was sensitive at high Roundup herbicide concentration, while isolates RL5, RL6, RL8, RL10 and RL13 were the most tolerance at 50 mg/ml herbicide. South Sinai, Zefta, Rafah, El-Menia, Cairo and Ismailia isolates shared a common band with mol. wt. of 70 KDa. New protein types were detected due to the differential response of the five isolates to the effects of the environment stress. Molecular and Biochemical Characterization of Some Egyptian Genotypes Rhizobium (Vicia Faba) Isolates Introduction Bacteria belonging to the genera Allorhizobium, Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium [1] are generally called Rhizobia. They are best known for their ability to establish symbiotic relationships with leguminous plants of agricultural and environmental importance, a process that results in biological nitrogen fixation [2]. Nitrogen-fixing leguminous increase soil fertility and quality by enhancing soil N content, organic matter and soil aggregates [3]. Fundamentally, nitrogen (N) is one of the most vital nutrients required for the synthesis of the vast array of metabolites such as proteins, enzymes, chlorophyll, and nucleic acids and consequently, determines critically the health of all living organisms including microbes and plants [4,5]. Despite of the presence of this element in the atmosphere in huge amounts (78%), the growing plants cannot utilize it in the present gaseous form (N2). For this, the nitrogen fixing microorganisms residing in the specific organs of root systems of leguminous plants have devised a novel strategy to convert the plant-unavailable atmospheric N2 into plant-utilizable forms (ammonia) through biological N2 fixation (BNF) using enzymatic machinery known as nitrogenase [6]. BNF represents an economically beneficial and environmentally sound alternative to chemical fertilizers [7]. More than 50% of the applied of nitrogen fertilizers are somehow lost through different processes which not only represent a cash loss to the farmers but also consequently polluted the environment. Scientists all over the world are facing this alarming situation and they are trying to overcome this condition by exploring alternative sources which are cost effective and save the environment. Biofertilizer, an alternative source of N, especially rhizobia in legume symbiosis, is a promising technology. The United Nations Food and Agriculture Organization (FAO) estimates that the total demands for agricultural products will be 60% higher in 2030 than present time and more than 85% of this additional demand will come from developing countries. For over half a century, the world has relied on increasing crop yields to supply an ever increasing demand for food. Pesticides accumulation into soils at an abnormal level causes spectacular changes in composition, diversity and functional activities of important soil microflora [8]. Round up causes decreased nodule numbers and their dry weight in cowpea roots with no nodule development on side [9]. Generally Pesticides also interact with rhizosphere microorganisms including rhizobia and restrict the root growth, in that way lead to the reduction in the number of the root sites available for the rhizobial infection [10]. Also decreases the total microbial biomass not only adversely affect the growth of bacteria, fungi and actinomycetes [11] by inhibiting protein synthesis, the metabolic enzymes including dehydrogenase and phsphatase. In addition to damaging structural proteins through biochemical alterations in membrane composition and geno-toxicity [12]. Pesticides are divided into categories according to the target organisms they are designed to control e.g., herbicides are used to kill unwanted weeds; insecticides control insects and fungicides eradicate phyto-pathogenic fungi. Pesticides are also classified according to their chemical structures or on the basis of their modes of action ( Figure 1). Pesticides inhibit symbiotic signaling between host plants and rhizobia by interfering in the interaction between plant-secreted flavonoids and the rhizobial NodD proteins; thus they effectively block molecular communication and disrupt the initiation of symbiosis [13]. In addition, pesticides not only inhibit the biochemical signaling between the hosts and cognate rhizobia but also block the initial attachment of complementary rhizobia to lectins present on root hairs as the recognition sites by protecting them [14]. Herbicides may have negative effects on growth of rhizobia [15]. Protein profiles [16] have been studied in rhizobia. Proteins induced in response to stress may suggest that they have an important role in homeostasis and maintenance of vital cellular functions [17]. The SDS-PAGE analysis of whole cell proteins not only helps in identifying of the Rhizobial strains [18,19] but also useful in the differentiation among the isolates within the same group [20]. Also molecular tools for the identification of bacteria are now available and are used routinely in laboratories. The electrophoretic screening of large plasmids, although limiting the analysis to the extra chromosomal elements, has a particular importance in the screening of Rhizobium. These bacteria have large plasmids (> 50 kb in size) that account for a substantial portion of their genome and contain several genes coding for nodulation and nitrogen fixation [21]. These plasmids are often present in variable numbers and sizes; traits that provide the basis for an accurate strain characterization when the extracted plasmids are separated by gel electrophoresis [22]. The principle objectives of this study are isolation and evaluation the genetic diversity of some root-nodule bacteria isolates from Faba bean plants grown in different geographic locations in Egypt based on morphological, biochemical and molecular characteristics to determine the biodiversity among tested isolates. In addition, plasmid profile and herbicide tolerance. Collection and isolation of Rhizobium isolates Thirteen representative sites were chosen from ten Egyptian governorates as presented in Table 1 and Figure 2. Rhizobial isolates collected from the root nodules of Faba bean (Vicia faba L.) plants. Plant roots containing several nodules from each plant and transported in plastic bags (each plant in a separate bag). Bacteria were generally isolated in the same day as plant harvesting. Isolation of rhizobium isolates All nodules were cut off in a laminar flow cabinet with small pieces of root and washed thoroughly with 2.5% NaOCl and sterile water. Nodules were surface sterilized with 70% ethanol alcohol for 5 min and exhaustively washed in sterile distilled water. After washing the root nodules were taken from the roots with care not to damage the surface, washed thoroughly in distilled water. Nodules were then transferred to 0.2% (W/V) solution of mercuric chloride for 3-5 minutes. Each nodule was crushed under aseptic conditions and streaked onto a Yeast-Mannitol Agar plate (YMA) using a sterile loop and incubated at 28°C. Single colonies were picked up from the original streaked plates. Pure cultures of Rhizobium leguminosarum were isolated according to [23] methods and restreaked on YMA containing Congo red to ensure purity before storage in 20% glycerol at -20°C. Stock cultures of the Rhizobium isolates were maintained on slants of YEM at 4°C and refreshed periodically. Purification and storage of rhizobium isolates Agar plates were incubated at 28 ± 1°C for 3 days. Individual colonies appearing over this period were re-streaked onto YEMA plates, and stored at 4°C until the time of processing. Phenotypic characteristics Antibiotic resistance pattern: All isolates were evaluated for their responses as resistant or sensitive against 24 different antibiotic disks [24]. Growth rate at different carbon source: All of the Rhizobia isolates were tested for utilization of different carbon sources by replacing mannitol in YEM broth medium with equal amounts of different carbon sources including glucose, fructose, mannose, and maltose to study the effect of different carbon sources on growth of isolates. The growth was measured after 24h and 48h of incubation at 28 ± 1°C. Roundup herbicide resistance test by agar plate and broth medium Laboratory experiment: The sensitivity or resistance of herbicide on growth of Rhizobium was determined by two methods. YEMA: The effect of Roundup on R. leguminosarum isolates under laboratory conditions were studied on Petri dishes containing YEM agar. In the first method bacterial sensitivity streak plate method, the Rh13 Ismailia City, Al Ismailia Governorate. calculated amount of herbicide was mixed with YEMA medium and poured into sterile Petri plates. Plates without herbicide were prepared to use as control. Plates were streaked with broth suspension 107cells ml −1 of test organism. The plates for Rhizobium were incubated at 28°C for 4 days, after which growth was examined. Growth was visually grouped into 4 categories: +++ (good), ++ (moderate), + (poor) and−(no growth) at different concentration of 0, 25, 50,100 mg/ml respectively. Three replicates of Petri dishes were used to each isolate as well as three Petri dishes were used without Roundup as control. The Petri dishes were incubated at 28ºC. After 4 days, were inspected to estimate the efficacy of Roundup against Rhizobium isolates. YEM broth: The second method was bacterial sensitivity broth culture method in 150 ml flasks containing 50 ml YEM, pH of medium was adjusted to 7, and the control cultures were grown at 28ºC. The flasks were incubated on rotary shaker for 4 days at 180 rpm. The growth of rate in Rhizobial isolates was measured by recording optical density (OD). The experiment was conducted in three replicates. The turbidity was measured using Jenway UV-VIS. spectrophotometer model UV-6305 at 600 nm against the blank (sterilized uninoculated YEM broth) and the mean generation time was calculated for each isolates. Biochemical identification of rhizobia IAA production: All the isolates were tested to IAA production in Yeast Extract Mannitol (YEM) broth [23] supplemented with 100 µg/ ml L-tryptophan and added to YEM medium without L-tryptophan. The test tubes were covered with brown paper and incubated at 28ºC for 5 days on a rotary shaker. The broth was centrifuged at 3,000 rpm for 30 minutes. 2 ml of supernatant was collected and 2-3 drops of o-phosphoric acid were added. The aliquots were shaken and 4 ml of Salkowski reagent (1 ml of 0.5) M FeCl 3 in 50 ml of 35% perchloric acid (HClO 4 ) was added then vortexes thoroughly. The samples were incubated at room temperature for 25 minutes. Development of a pink color indicates IAA production. IAA concentrations were quantified by Spectrophotometer, the developed pink color from previous reagent was read at 530 nm using spectrophotometer, and concentration of produced IAA was determined from a prepared standard curve of pure IAA (10-100 μgml -1 ) [25]. Uses different IAA concentrations are prepared as aqueous solution of IAA ranging from 10 microgram/ml to 100 micrograms/ml, each 1ml of the standard, 2 ml of 2% 0.5 M FeCl 3 in 35% perchloric acid (Salkowaski) reagent is added and readings are taken after 25 minutes at 530 nm by UV-Visible spectrophotometer. Catalase test: The catalase enzyme decomposes hydrogen peroxide (H 2 O 2 ) to water and O 2 . Hydrogenperoxide is very toxic for bacteria. Bacteria are smeared in a drop of hydrogen peroxide and checked for formation of oxygen gas bubbles. Protein profile: Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) Glass slabs and spacers were washed with water and wiped with tissue paper. Rizobia were grown on yeast extract mannitol broth [23] at 28°C for three days. According to [16] protein was extracted, Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) as described previously [26]. DNA extraction and amplification of 16S rRNA Gene of rhizobia DNA was extracted from bacterial cultures using SDS/CTAB Lysis and phenol/chloroform extraction method [27]. Sequence analysis of 16S rDNA and subsequent BlastN analyses indicated that the majority of isolated strains were Rlv [28]. Plasmid isolation from Rhizobium: Plasmid DNA was isolated by the method of [29]. Restriction fragment analysis: The extracted plasmids were digested with EcoRI and MSPI enzymes according to (Thermo scientific fermentase) as following: Combine the following reaction components at room temperature in order indicated: Water, nuclease-free 15 µl, 10x FastDigest or 10x FastDigest Green Buffer 2 µl, DNA 2 µl, FastDigest enzyme 1 µl, Total volume 20 µl, Mix gently and spin down, incubate at 37°C in a heat block or water thermostat for (5-15) min and load an aliquot of the reaction mixture in 2% agarose with TBE (Tris -Boric-EDTA) buffer. Electrophoreses were carried out at 100 V for 1 h. Gels were stained with ethidium bromid"EtBr" (2.5 mg/10 ml) and photographed under UV illumination. Statistical analysis The data were subjected to one-way ANOVA using SPSS (2002) analysis program (version 11.5). The significant differences among individual means were analyzed by Duncan's multiple range tests and statistical significance was determined at 5% level. Phenotypic characteristics On the basis of morphological parameters, we have confirmed differences between the isolates (Table 1) represent thirteen isolates of Rhizobium leguminosarum symbiovar Vicia isolated from nodules of Egyptian Faba beans (Vicia faba L.) collected from different geographic areas. This isolates were tested by Congo red technique according to [30]. To ensure that all isolates were rhizobia and did not contaminated. Rhizobial isolates were found to be microscopically similar, isolates identified as Rhizobium leguminosarium symbiovar. Vicia. According to the negative reaction to gram stain. Strains were found to be motile. On YEM agar, the growth is generally moist, whitish, smooth and gummy. Antibiotic resistance pattern: All isolates were evaluated for their responses as resistant or sensitive against 24 different antibiotic disks [24]. Table 2 and Figure 3. All the thirteen Rhizobium isolates were tested to utilize a wide range of carbohydrates as a sole carbon source. The Rhizobium isolates in this present study also preferred monosaccharides as well as mannitol for proper growth [31]. In the present study, the Rhizobium isolates from V. faba showed better growth with mannitol then glucose, fructose, mannose and maltose when mannitol was replaced with four other carbohydrates in equal quantities. Our results showed that Rhizobia prefer mannitol and glucose as best carbon sources for growth [32]. Furthermore all the tested isolates were able to utilize almost all tested carbon sources in this investigate. All of the isolates obtained from Vicia faba nodules were able to utilize mannitol, glucose, Fructose, Mannose and Maltose as carbon sources. These carbon sources are generally utilized by bacteria of the genus Rhizobium [33]. The ability of the isolates to utilize a broad range of carbon substrates is also related to the survival of the isolates under acidic environments. Generally, Mannitol and Glucose were the best source of carbon. Mannitol measurements were ranged from (O. Roundup herbicide resistance test by agar plate and broth medium It is clear that the higher dose of Roundup herbicide inhibited the growth of Rhizobium and we observed varied growth in vitro under different herbicide treatments between the tested isolates. In broth culture method, untreated rhizobia (control) had the highest OD ( Figure 5). The OD (240) Compared with control (without Roundup Herbicide), whereas growth was visually grouped into 4 categories: +++ (good), ++ (moderate), + (poor) and − (no growth) at different concentration of 0, 25, 50,100 mg/ ml respectively in solid media (Tables 3 and 4). Growth inhibition was shown at highest concentration. Generally, the higher concentration of herbicide has higher toxicity to the bacteria. Therefore, the number of colonies tolerant to Roundup decreased when concentration increased. About 38% of isolates were tolerant to the higher concentration. This experiment resulted in selected Rhizobium strains tolerant to environment stress i.e., high concentration of Roundup herbicide. Isolates from different locations in this study showed a great ability to degrade Roundup herbicide. They differed in their ability to Roundup degradation. It is clear that Ismailia City isolate was the most active degrading Roundup in this investigates as shown in Table 3. All isolates are tolerant to 25 mg/ml concentration of herbicide. All used isolates are very sensitive to 100 mg/ml concentration of herbicide and intermediate for 50 mg/ml concentration of herbicide. All Isolates showed inhibition of growth by increasing concentration of herbicide. All isolates grew on YEMA media [23]. The results of different concentrations of herbicide effects on Rhizobial growth showed that 25 mg/ml concentration was the best concentration for all isolates growth; there was no growth at all at 100mg/ml for all isolates. As consequently different concentrations of herbicide decrease in the growth of rhizobia untreated rhizobia had the highest O.D. and each successive increase in the concentration of herbicide decreased the O.D. These results are in agreement with those obtained by [9] who studied the effect of different herbicides on the nodulation and growth of Rhizobial isolates. Also some studies have evaluated the effect of different herbicides on Rhizobium growth and nitrogen fixation activity. The effect depends on the herbicide, its concentration, and different weather conditions. Applied research methodology also may depend on the Rhizobium or Bradyrhizobium species and even the strain used [34]. Biochemical identification of rhizobia IAA production: All isolated Rhizobia showed variations in red colour reaction with salkowaski reagent indicating their variations ability to produce IAA. Comparison between the isolated Rhizobia showed variation for their ability to produce IAA. Rhizobium isolates from Vicia faba differ in IAA production. In majority of isolates; the amount of IAA produced was more than that in the control. The maximum amount of IAA production was in the range of 2.04 for isolate No.8 from Al Arish City to 7.5 μg/ml for isolate No.13 from Ismailia City in all the isolates studied. Comparison of means shows that there is a difference between isolates. Indole -3-acetic acid (IAA) is the common natural auxin, to produce auxin. This substance can be converted to IAA by soil beneficial bacterial activities. Tryptophan (L-trp) is physiologically a precursor of auxin biosynthesis in higher plants and microorganism [35]. Various reports showed Tryptophan derived microbial auxins present in the rhizosphere area play a very important role in growth and development of plant rooting system and crop yield [36]. Many of rhizobial species enable to produce IAA. Some studies shows that auxins play a key role in creating nodule in leguminous plants and generally, in establishing a symbiotic association with rhizobia. Also has been proved that flavonoids, promoting nodulation gene, increase the production of IAA by rhizobial bacteria [37]. Rhizobia are known to produce significant levels of IAA both in free living conditions and also symbiotically in nodules [38], (Table 5 and Figure 6). Table 6 and Figure 7 revealed that the biochemical characteristics of Rhizobium isolates showed positive production of Catalase enzyme, by all of the tested isolates with exception of isolate RL 3 and RL 12. The genetic diversity among thirteen isolates representing the indigenous population of rhizobia was investigated using SDS-PAGE pattern. SDS-PAGE profile of protein extracted from thirteen R. leguminosarum isolates ( Table 7, Figures 8 and 9). A dendrogram developed from similarity between the different isolates based on Protein banding pattern formed two groups of proteins were identified in Rhizobium leguminosarum sv viciae isolates ( Table 8). The first group contained eleven isolates, whereas the second group contained remains isolates RL8 and RL9 located in South and North Sinai respectively. It showed a total of 78 bands with different molecular weight values ranged from 130 to 15 KDa. Catalase test: Results present in The protein bands of the tested isolates varied in numbers of bands. The electrophoresis banding pattern of thirteen R. leguminosarum isolates showed either absence or presence of different polypeptides in this study. One band was for only one isolate RL4 (130 KDa), some of these bands appeared in each of the studied isolates such as 70, 40, 35, 25, 20 and 15 KDa which were considered monomorphic bands. Whereas each isolate contained at protein banding range 5-7 bands. Our results were agreement with [39] who mentioned that protein patterns, peroxides isozymes and DNA fingerprints were used for analysis of somaclonal variations among three R. leguminosarum isolates to determine Rhizobium genetic diversity under environment stress. Preparation of total protein from all rhizobial isolates showed seventy eight bands ranged from (15 to 130 KD), these bands were not necessarily present in all isolates. Among tested Rhizobium isolates there were some variations in protein banding pattern. As presented in Figure 10. There were sixty five bands common in all Rhizobial isolates and thirteen bands showed variability among them. This is may be due to the differences of their genetic background according to the different locations; similar conclusion was also obtained by [40] who stated that Rhizobial isolates could be distinguished by protein banding pattern. Our results indicate that under salt stress, SDS-PAGE revealed a powerful characterization of method Rhizobium leguminosarium genotypes. On the other hand, phenotypic and genotypic diversity among tested strains play important role for their tolerance or sensitive stress. The results finding is in agreement with [41] that used protein pattern to analyze the genetic variations among three Rhizobium leguminosarum and observed variation in protein banding pattern (10, 12 and 11 polypeptide bands). This study was conducted to employ SDS-PAGE as simple tool to determine the genetic variability among R. leguminosarum isolates ( Figure 11). Plasmid isolation from Rhizobium Plasmid profile of Rhizobial isolates were presented in Figure 12. Results showed variation in number of bands based on variation in size of plasmids. Plasmid genotypes of rhizobia nodulating Faba bean from various Egyptian locations were extracted and characterized. Plasmid profile analysis revealed different plasmid types having differ of size ranging from 10 to 0.5 Kb pairs. A variation in number of symbiotic (Sym) plasmid was observed. The size of these plasmids showed marked diversity among the isolates and varied in size from 10 to 0.5 Kbp. Isolates were characterized according to their plasmid profiles. All studied isolates contained a 10 Kbp plasmid and four isolates RL4, RL9, RL12 and RL13 contained additional small plasmid with molecular weight about 2 Kbp, 2 Kbp, 7 Kbp and 5 Kbp respectively as shown in Figure 12. These data are parallel to that achieved by [42][43][44] who found that R. leguminosarum sv. Viciae strain T83K3 which had six plasmids of 480, 440, 300, 255, 210 and 155 kb.

v3-fos

2018-04-03T01:01:56.101Z

2015-12-01T00:00:00.000Z

4855057

s2

Nano-TiO2 Is Not Phytotoxic As Revealed by the Oilseed Rape Growth and Photosynthetic Apparatus Ultra-Structural Response Recently nano-materials are widely used but they have shown contrasting effects on human and plant life. Keeping in view the contrasting results, the present study has evaluated plant growth response, antioxidant system activity and photosynthetic apparatus physiological and ultrastructural changes in Brassica napus L. plants grown under a wide range (0, 500, 2500, 4000 mg/l) of nano-TiO2 in a pot experiment. Nano-TiO2 has significantly improved the morphological and physiological indices of oilseed rape plants under our experimental conditions. All the parameters i-e morphological (root length, plant height, fresh biomass), physiological (photosynthetic gas exchange, chlorophyll content, nitrate reductase activity) and antioxidant system (Superoxide dismutase, SOD; Guaiacol peroxidase, POD; Catalase, CAT) recorded have shown improvement in their performance by following nano-TiO2 dose-dependent manner. No significant chloroplast ultra-structural changes were observed. Transmission electron microscopic images have shown that intact & typical grana and stroma thylakoid membranes were in the chloroplast, which suggest that nano-TiO2 has not induced the stressful environment within chloroplast. Finally, it is suggested that, nano-TiO2 have growth promoting effect on oilseed rape plants. Introduction Use of nanomaterials is one of the rapidly growing research areas during the last decade [1]. Nanomaterials are being applied in almost every field like cosmetics, medicine and agriculture etc. [2]. Nanomaterial's (NMs) have tremendous potential to generate new ways to manipulate genome, DNA delivery and growth regulation in plants [3,4]. There is an extensive interest to investigate applying NMs to plants for agricultural use. Nano-TiO 2 is diversely used nanoparticles. Nano-TiO 2 materials are being utilized as a disinfectant, antibiotic, biological sensor, tumor killing agent and antibacterial products [5]. Contrasting effects of nano-TiO 2 on plant growth have been reported. Some studies have reported that nano-TiO 2 is cytotoxic [6] but others are showing opposite results [7]. One study has shown that nano-TiO 2 application may induce aged seeds vigor and chlorophyll content in spinach [8] and other has shown that nano-TiO 2 is toxic for seed germination and root growth [9,10] which are considered as the most important basic toxicity research tools for plants. Overall, few systematic studies have been conducted to determine the effects of nano-TiO 2 on plant physiology and plant development at the organism level. Information available about nano-TiO 2 effect on chloroplast ultra-structural changes is scarce. However, literature has suggested that any abiotic change in the plant environment induce oxidative stress which in turn may damage the membrane system especially mitochondria and chloroplast ultra-structures [11] and ultimately hampers physiological performance of photosynthetic apparatus in plants. Limited literature is available on the plant biological and physiological effects of nano-TiO 2 for its practical application in agriculture. Therefore, it is imperative to continue such studies to understand the effects of nano-TiO 2 on plant growth and physiology. Oilseed rape is considered as one of the main source of edible oil not only in China but all over the world. [12]. Therefore, oilseed rape potential must be exploited against various environmental stresses like nano-TiO 2 . Keeping in mind the importance of oilseed rape and contrasting biological responses of plants to nano-TiO 2 toxicity, the present study was planned. The study was executed with a wide range of nano-TiO 2 toxicity on plant growth, antioxidant system and photosynthetic apparatus especially the chloroplast ultra-structures. Results from this research may help to understand the effects of nano-TiO 2 in oilseed rape plants and further their application in the laboratory or field. Plant material and treatment conditions Seeds of Brassica napus L. (cv. Zhongshuang No. 11) were purchased during August, 2014 from a local seed company-Wuhan Zhongnongyou Seeds Technologies Co., Ltd., Wuhan, Hubei Province, China (30°69N, 114°19E). The seeds were vernalized for 2 weeks and were sterilized for 10 min in 10% sodium hypochlorite solution before use. Healthy seeds of Brassica napus L. (cv. Zhongshuang No. 11) were sown in plastic pots (30 cm diameter) having commercial soil (Sunshine Mix #5, Sun Gro, Canada). Five uniform plants per pot were allowed to grow after three weeks and there were four replications for each treatment. Forty four days old seedlings were sprayed with water or different concentrations of nano-TiO 2 suspensions (500, 2500 and 4000 mg/l). Data for different parameters were recorded from the next day for four times with an interval of a week. Left over plants for each treatment were allowed to grow till maturity and then harvested to record the data for yield and yield components. Morphological parameters Five plants for each treatment were randomly sampled next day after nano-TiO 2 treatment to record the data for taproot length, plant height and biomass of the seedlings and this action was repeated four times with an interval of a week. Chlorophyll was extracted from the leaves by soaking in acetone and alcohol (1:1) mixture solution. Total chlorophyll contents were spectrophotometrically recorded [18]. Transmission electron microscopy Completely unfolded leaves at the top of plants were used to obtain the control and treated samples excluding veins. Samples were washed thrice with glutaraldehyde-4% in phosphate buffer-0.1M after treatment with the same buffer for more than 12 hours. After incubation for 1 h in OsO 4 -1%, samples washing were repeated thrice after every ten minutes. In the next step, samples were dehydrated using gradually increased concentration of ethanol from 50-100% and ultimately for twenty minutes with acetone. After infiltration and embedding with Spurr's resin, samples were heated at 70°C for nine hours. Finally, transmission electron microscope (JEOL TEM-1230EX) was used to observe the ultra-structures of the samples on copper grids [13]. Photosynthetic gas exchange Photosynthetic gas exchange characteristics of healthy leaves were recorded at 10: 00 am in the morning with a CIRAS-1 portable photosynthesis system (PP-Systems, UK). Randomly three healthy and functional leaves were selected for each measurement. Photosynthetic parameters like net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO 2 concentration (Ci) and transpiration rate (Tr) were recorded and replicated at least eight times [19]. Statistical analysis Analysis of variance was performed with statistical package prism 5.0. Data means were subjected to Student 0 s t test for comparison at p<0.05. Plant growth response Phytotoxic effects of nano-TiO 2 were recorded on plant growth in terms of taproot length, plant height and fresh biomass (Fig 1). A positive but not significant change was recorded for taproot length of oilseed rape plants ( Fig 1A). However, oilseed rape plants height was increased after treated with different concentrations of nano-TiO 2 (Fig 1B). Maximum height was recorded with 4000 mg/l nano-TiO 2 . The total biomass of plant vegetation (leaves, stems, and roots) of nano-TiO 2 -exposed seedlings increased approximately by 30-40% compared with control seedlings (Fig 1C). Increase in the biomass followed the dose dependent pattern of nano-TiO 2 . Minimum biomass was recorded in control plants and maximum at 4000 mg/l nano-TiO 2 treated plants. Exogenous Nano-TiO 2 Improves Oilseed Rape Growth and Photosynthesis Antioxidant enzymes activity The effect of nano-TiO 2 on the protective antioxidant enzymes activity such as SOD, CAT and POD of oilseed rape is shown in Fig 2. Results have shown that immediately after exposure to nano-TiO 2 at 45 days, the SOD, CAT and POD activity of oilseed rape depicted no significant change with the increase of nano-TiO 2 as compared to control plants. However, during next three weeks (52, 59 and 66 th day) SOD, CAT and POD activity of B. napus was significantly increased compared to the respective controls and followed the dose dependent pattern of nano-TiO 2 (Fig 2A-2C). Maximum activities for all the enzymes tested were recorded in 66 days old plants treated with 4000 mg/l nano-TiO 2 . Overall, enzymes results have shown that the effect of nano-TiO 2 on the activity of protective enzymes follow the same trend. Nitrate reductase activity and chlorophyll content Effect of nano-TiO 2 on nitrate reductase activity and chlorophyll content is shown in the Fig 3. Results have shown that increasing dosage of nano-TiO 2 has not induced significant change in the NR at 45 th & 52 nd day old seedlings. However, later on the activity of NR increased significantly and followed the concentration dependent pattern. Maximum values of NR were recorded at 66 th day (Fig 3A). Chlorophyll content data recorded for 45 days old seedlings showed no significant change with the increase of nano-TiO 2 dosage (Fig 3B). However, the chlorophyll content of oilseed rape seedlings during the next weeks were dramatically higher and followed the nano-TiO 2 concentration dependent pattern with maximum chlorophyll content in 66 days old plants treated with 4000 mg/l nano-TiO 2 . These results have shown that nano-TiO 2 can significantly increase chlorophyll content of oilseed rape. Fig 4 shows the photosynthetic apparatus physiological changes induced by nano-TiO 2 in oilseed rape plants. Photosynthetic parameters, i.e. net photosynthetic rate, stomatal conductance, internal CO 2 concentration and transpiration rate showed no significant change just after the exposure to nano-TiO 2 at 45 days old seedlings. However, a significant increase was recorded with the increase in TiO 2 concentration during the coming weeks and generally this increase was gradual. Maximum performance for all these parameters was recorded at 66 th day of seedling age with 4000mg/l nano-TiO 2 concentration except intercellular CO 2 concentration whose results were indifferent. Ultra-structural changes in chloroplasts Thylakoid membrane system was observed typical with no drastic changes in nano-TiO 2 treated chloroplasts compared to the control. Grana and stroma membrane stacks were intact and there was no swelling in the stroma when treated with nano-TiO 2 . Starch grains were present in the chloroplast and more importantly plastoglobuli were lesser in number especially in 4000 mg/l nano-TiO 2 treated plants compared to the control plants. Generally speaking, an increase in the dosage of nano-TiO 2 didn't induce any negative change in chloroplast ultrastructures (Fig 5A-5D). Effect on yield and yield components At maturity, effect of nano-TiO 2 on yield and yield components was monitored in terms of pods per plant, seeds per pod, 1000-seed weight and seed yield as shown in Fig 6. There was no statistical significant change for number of seeds per pod (Fig 6A). However, 1000-seed weight, seed yield per plant and number of seeds per pod was improved by increasing dose of nano-TiO 2 especially at higher dose of 4000 mg/l (Fig 6B-6D). Generally speaking, results are showing that nano-TiO 2 have no toxic effect on the ultimate seed yield of oilseed rape plants rather improves it. Discussion Nano-toxicity to plant life is an emerging arena and is being focused in the recent years [20]. Contrasting effects of nano-materials on plant growth and development is a feature of the previous studies. The present study is an effort to understand how diverse range of nano-TiO 2 impacts the plant growth and development in oilseed rape. Enhanced root length, plant height, biomass in the present study indicates that nano-TiO 2 might have induced the absorption of water and fertilizer [21]. Increase in growth parameters also demonstrates that nano-TiO 2 has catalyzed the photosynthetic process as shown in Fig 4. This promotion in plant growth may also be due to the increased inorganic nitrogen (such as NO 3 --N and NH + -N) conversion into organic nitrogen i-e protein and chlorophyll, which ultimately improve the plant growth [22,23]. It could be speculated that relatively higher nitrate reductase activity (Fig 3A) in our present study might have provided the pool of NH 4 + nitrogen by transforming the NO 3 to NH 4 + in vivo during nitrogen metabolism and then accelerate the formation of chlorophyll (Fig 3B) which ultimately has induced the plant growth [24]. Nitrate reductase activity enhanced in the present study might be linked with nitrate absorption as it has been reported that nitrate reductase activity induced under nitrate [25]. Results of this study are consistent with the findings of all the tested plant species i-e Brassica compestris L., Lactuca sativa L. and Phaseolus vulgaris L. for root length [26] and spinach for biomass [27]. Although nano-TiO 2 is not phytotoxic as revealed by the performance of plant growth indices but it has activated the antioxidant system (Fig 2) of oilseed rape plants as a matter of defense in response to oxidative stress [28,25]. This means that abiotic stress was induced by nano-TiO 2 . Dose dependent increase in enzymes activities suggest us that increasing dose of nano-TiO 2 would have increased the production of reactive oxygen species (ROS) and in turn antioxidant system enzymes activities were activated in the same fashion [29,30]. Enhanced antioxidants activities suggest that nano-TiO 2 induced stress was not severe to destroy the antioxidant system apparatus in the plants, rather activated it as a matter of defense and ultimately overall growth of plants. Plant growth response and grain yield is ultimately controlled by the production of photosynthates. Induced photosynthetic gas exchange capacity by nano-TiO 2 by following the dose dependent manner suggest that nano-TiO 2 might has increased the absorption of nitrogen and magnesium minerals to promote the chlorophyllase activity and hence the chlorophyll synthesis which in turn might have increased light absorbance, improved light energy traffic and ultimately has avoided the chloroplasts damage ( Fig 5) and prolonged the photosynthesis time of chloroplasts [31]. To analyze the chloroplast damage, transmission electron microscopic chloroplast ultrastructural images were executed which have revealed intact grana-stacks and no swelling in stroma (Fig 5) which means that either there was no over-production of ROS in the chloroplast or scavenged by the antioxidant system activated (Fig 2) by nano-TiO 2. Chloroplast serves as an apparatus for photosynthesis reactions [32]. Reports have shown that chloroplast ultrastructures are affected by toxicity and in turn hamper the photosynthesis [33]. Under stress conditions, transpiration is hampered due to stomata closure, which declines the CO 2 concentration within chloroplasts and consequently affects NADPH + production and let the ferredoxin electrons reduce O 2 , ultimately induces the formation of reactive oxygen species like H 2 O 2 , OHetc. [34]; these species may deteriorate the membrane system of the plant such as chloroplast. Few plastoglobuli observed in the chloroplast (Fig 5) is an indication of no lipid peroxidation of thylakoid or cell membrane. Presence of starch granules in the chloroplast also reveals that there was no stressful environment induced by the TiO 2 in chloroplast, as complex sugars have not transformed into simple soluble sugars which are supposed to be the major compatible solutes for osmotic adjustment [35]. Improved yield and yield components data for our study is the ultimate response of nano-TiO 2 on oilseed rape plants which confirms that nano-TiO 2 is not toxic rather improves plant performance. In summary, this article suggests that nano-TiO 2 is non-phytotoxic as revealed by the improved photosynthetic apparatus physiological performance, no drastic changes in the thylakoid membranes, improvement in the plant growth and ultimately better yield of the oilseed rape plants. However, further studies are required to establish it, keeping in mind dose, exposure time, plant species and growth stage variability.

v3-fos

2018-04-03T03:06:25.888Z

2015-10-13T00:00:00.000Z

168797

s2

In Vitro Seeds Germination and Seedling Growth of Bambara Groundnut (Vigna subterranea (L.) Verdc. (Fabaceae)) Bambara groundnut (Vigna subterranea (L.) Verdc.) is an indigenous grain legume. It occupies a prominent place in the strategies to ensure food security in sub-Saharan Africa. Development of an efficient in vitro regeneration system, a prerequisite for genetic transformation application, requires the establishment of optimal conditions for seeds germination and plantlets development. Three types of seeds were inoculated on different basal media devoid of growth regulators. Various strengths of the medium of choice and the type and concentration of carbon source were also investigated. Responses to germination varied with the type of seed. Embryonic axis (EA) followed by seeds without coat (SWtC) germinated rapidly and expressed a high rate of germination. The growth performances of plantlets varied with the basal medium composition and the seeds type. The optimal growth performances of plants were displayed on half strength MS basal medium with SWtC and EA as source of seeds. Addition of 3% sucrose in the culture medium was more suitable for a maximum growth of plantlets derived from EA. Introduction Bambara groundnut (Vigna subterranea (L.) Verdc.) plays an important role in reducing food insecurity and child malnutrition and improving the economic power of households. Ranked among the minor species, the plant has risen in recent decades, a renewed interest among research funders due to its ability to contribute to the diversification of food crops in Africa [1,2]. As a leguminous plant, Bambara groundnut is useful in crop rotations as a source of residue nitrogen for the subsequent crop through nitrogen fixation [3]. Despite its importance, Bambara groundnut cultivation is limited by its sensitivity to plant pathogens and low and unpredictable yields (650-850 kg⋅ha −1 ) for the majority of semi-Arid Tropics [4]. Furthermore, the presence of antinutritional factors in the seeds, such as tannins and trypsin inhibitors [5], limits digestion and availability of nutrients from the seed. The popularization of Bambara necessarily involves intensification of its production and in particular the creation of improved varieties to overcome the numerous cultural constraints of the plant. Improvement of Bambara groundnut can be achieved by genetic recombination and selection, but reports in this domain are limited [6]. Artificial hybridization is extremely difficult and very low success rates have been reported (<2% harvested hybrid seeds) [2]. Moreover, conventional breeding methods are time consuming and laborious. Recent advances in biotechnology including the direct integration of genes into plants have offered the opportunity to develop improved germplasm by overcoming the difficulties to cross distant taxa. The development of such technologies largely depends on the development of efficient plant regeneration protocols. Before achieving this goal, the availability of healthy explants derived from in vitro seedling is a prerequisite. Therefore, in vitro germination of seeds 2 The Scientific World Journal was attempted in order to obtain young and surface sterile plant material for the subsequent establishment of in vitro propagation protocols, necessary for the intended exploitation of this indigenous plant through biotechnological means. Seedlings obtained in vitro are of great importance since they are directly used for other tissue culture experiments without any sterilization. In vitro tissue culture is important to offer high rates of multiplication from segments of tissue; however, it is also an efficient tool for obtaining large numbers of individuals free of contaminating sources [7][8][9]. In Bambara groundnut, seeds germination and the development of in vitro plantlets using only one basal medium (BM) have been reported [16,17]. Similarly, in vitro germination of seed for obtaining in vitro plantlets of Bambara was carried out in our previous works [18][19][20]. However, assessment of this process has never been undertaken. Herein, the purpose of this research was to establish optimal conditions for the seeds germination and in vitro seedling growth of Bambara groundnut based on the responses to the culture media composition and the seeds types used as explants. The results from this study can be used for plant regeneration of different Bambara groundnut landraces. Seed Sources and Surface Sterilization. The mature seeds of Bambara groundnut (cv. Ci2) were collected from the plants grown on experimental plots in University Nangui Abrogoua. The seeds were stored in air tight plastic bottles at room temperature. Under a laminar airflow cabinet, the seeds were surface-sterilized sequentially with 70% (v/v) ethyl alcohol (30 sec.) and 7% (w/v) calcium hypochlorite solution (30 min) and finally rinsed thoroughly three times. The seeds were soaked overnight in 100 mL beaker containing 50 mL of sterile distilled water (SDW). After soaking overnight, the water was discarded, the seeds were rinsed 3-4 times with SDW, and then three (03) types of seeds were used for in vitro germination trials. (i) Seeds with seed coat (SWC) (Figure 1(a)), (ii) seeds without seed coats (SWtC) (Figure 1(b)), and (iii) the embryonic axis carefully isolated after the cotyledons have been separated (Figure 1(c)). Effect of Different Basal Media. The three types of seeds were cultured on five (5) basal media, namely, MS [21], B5 [22], SH [23], MC [24], and Chu N 6 (CHU) [25]. 3% sucrose was added to these basal media containing no growth regulators. The pH of the culture media was adjusted to 5.8 before adding 0.6% (w/v) agar. Culture medium without any fortification in nutrients served as control. Effect of Medium Strength. To optimize germination and the in vitro development of plantlets, the seeds types were inoculated on three strengths of MS medium, namely, full strength MS salts (MS), half strength MS salts (1/2 MS), and quarter strength MS salts (1/4 MS). 2.4. Effect of Carbohydrate Sources. The best culture medium from the previous experiment was selected for testing different sources of carbohydrate. Thus, in this germination medium, 3% of sucrose, glucose, and fructose have been added, respectively, for their effectiveness in promoting the germination and the seedling subsequent growth. Effect of Sugar Concentrations. Different concentrations (1, 2, 3, 4, 5, and 6%) of the best carbon source defined in the previous experiment were tested. Seeds were cultured on these culture media with a composition varying in sugar concentration. 2.6. Culture Conditions. All the culture media were autoclaved at 120 ∘ C for 20 min. For all the experiments described above, the cultures were transferred into a culture room, where they were maintained at 25 ± 2 ∘ C under 16/8 hours' (light/dark) photoperiod with light intensity of 3000 lux provided through white fluorescent tubes. Data Collection on Germination and Growth Parameters. Germination was defined as the appearance of a 2 mm radicle and is referred to as physiological germination throughout this paper. Germination was monitored daily for each seed type until no further germination was recorded and the mean germination time (MGT) was calculated using the formula cited by [26] given below: where is the number of seeds newly germinated at time at 25 ∘ C; represents hours from the beginning of the germination test; ∑ is the final germination. The total numbers of the germinated seeds for each treatment were summed up to determine the cumulative germination and the rate of germination was calculated following the procedure of [27]. Four (4) weeks after sowing, seedlings height, epicotyl and primary root length, and the number of leaves, branches, and secondary roots were recorded. The dry weights of aerial and root systems were obtained by drying them in an oven at 65 ∘ C for 72 hours until a constant dry weight. Statistical Analysis of Data The experiment was carried out in a completely randomized design with ten replicates and each individual treatment was repeated three times. Data were submitted to analysis of variance (ANOVA) to detect significant differences between means. Means differing significantly were compared using Newman-Keuls multiple range test at the 5% probability level using statistical software program Statistica version 7.0. Effect of Basal Medium on In Vitro Germination of Seeds and the Plantlets Development. After incubation on the culture media, seeds became swollen quickly and germination occurred within the first two weeks of culture (Figures 1(d)-1(i)). The mean germination time (MGT) and the mean germination rate (MGR) of the three seed types on different culture media after a 3-week incubation period are presented in Figures 2 and 3, respectively. Regardless of the seed type, the MGT was not influenced by the different culture media tested. However, an effect of seed type was observed with all culture media. The shortest time for germination (4-5 days) was observed with the embryo axis (EA) followed by the seeds without seed coat (SWtC) which germinated in 8-9 days while the seeds with coat (SWC) have taken between 10 and 14 days to germinate ( Figure 2). Compared to the embryonic axis, the time taken by the water to cross the barrier of the integument and to hydrate the cotyledons to initiate the physiological process of germination could explain the delay in germination observed with seeds with or without seed coat. Similarly, [28] demonstrated in a study involving Uapaca kirkiana Müll. Arg. that the presence of hard outer seed coat layers delays seed germination due to impermeability and restriction of radical emergence. Similar periods required to achieve in vitro germination by EA, SWtC, and SWC, respectively, have already been reported in Bambara groundnut by previous authors [16,17]. After three weeks of cultivation, no significant difference in the germination rate was observed among the culture media including the control regardless of the type of seed. This lack of significant difference between basal media and the control containing only agar suggested that macroand microelements were not necessary for germination in Bambara groundnut and, thus, the success of seed germination was mainly related to water availability. This finding is consistent with the observations reported on the seed germination of Withania coagulans Dunal by [29]. Moreover, on the same medium, the germination rate largely varied among the seed types ( Figure 3). Indeed, the germination rates recorded with EA and the seeds without seed coat (SWtC) were similar but significantly higher than the rate obtained with SWC. Obviously, because of the removal of integument, the access to water in SWtC and the EA was more important than in SWC. The mechanical restriction by the seed coat is the reason for the lowest rate of germination obtained with SWC in this species. On the other hand, continuous supply of water is needed to start and complete germination [30]. Studies have also shown that the hard seed coat renders the seeds impermeable to water and oxygen needed for germination process [31]. Higher percentage seed germination was achieved when outer and inner seed coat layers were removed completely [32]. Unlike our results, [17] reported that EA explant gave the highest germination rate among the three explant types throughout the period of culturing. The mean values of growth parameters of plantlets obtained from the three explants types on different basal media after culturing for four weeks are presented in Table 1. Significant differences in performance/development were observed among plantlets derived from embryo axis explants and those from seeds either with or without coat. The lowest growth performances were observed with the plants grown on the control medium containing no inorganic salts. This result shows that the mineral salts are essential for plant growth after the germination phase and, thus, it is apparent that the nutritional requirements for initiation of germination in Bambara groundnut are considerably different from those required for optimum growth of plantlets. On the control medium, no significant difference was observed in the development of plants grown from seeds with or without coat. However, the growth of these plants was higher than that of developed plants from the embryonic axis. This relation was also expressed on basal media containing macro-and microelements. This might be due to adequate nutrient reserves stored in the cotyledon of the SWC and the SWtC with the embryonic axis explants having little stored nutrient reserves. The authors of [17] made The Scientific World Journal 5 similar observations in their studies on Bambara groundnut seeds types. Furthermore, among the five culture media tested, the highest size of the seedling (10.48 cm) and the most important root length (21.51 cm) were observed with the plants developed from seed without coat on MS basal medium. All tested media contain mineral salts that vary not only in their concentrations but also in their available forms. The media used in this present study were different from one another in their chemical composition. The distinguishing feature of the MS inorganic salts is their high content of nitrate, potassium, and ammonium in comparison to other salt formulations. MS medium is highly enriched with macro-and microelements and the inorganic salts in this medium were enough to support the maximum growth of the plant. The concentration and the quality of nitrogen in MS medium may be the reason of prolific growth obtained with plant derived seed types incubated on this medium. Indeed, nitrogen is supplied to medium in inorganic form as the anion NO 3 − or the cation NH 4 + . The author of [33] found that the ammoniated form of nitrogen is more appropriate than the nitrate form and reported the fastest growth of Dactylorhiza species seedlings at 50-100 mg⋅L −1 NH 4 NO 3 . It was also earlier reported by [34] that an efficient concentration of organic and inorganic nitrogen sources can promote the growth of explants. In addition, better plant growth from embryonic axis was observed on MS medium compared to other basal media in Juglans regia L. [35]. Effect of MS Medium Strengths on the Growth of Seedlings Developed from the Embryonic Axis and the Seeds without Seed Coat. On the different strengths of MS basal medium used, a significant difference was recorded for growth parameters when comparing the plantlets derived from EA and SWtC ( Table 2). Regardless of the strengths of MS medium, plantlet height, root length, and the biomass were highest with the plants developed from SWtC. No significant difference was noticed between the three strengths of MS basal medium in terms of number of leaves, root length, and biomass of plantlets derived from both EA and SWtC. But a significant reduction in plantlet height was observed on 1/4 MS. This result shows that a very low amount of macro-and microelements is not effective for plantlet growth. Half strength of MS gave satisfactory results for Bambara groundnut seedling development suggesting that an adjustment can be performed from the full composition of MS basal medium without any significant reduction in plantlet growth. Thus, half MS was selected as the culture medium for the subsequent assessment of carbohydrate treatments. Effect of Carbohydrate Sources. Plants growing under tissue culture conditions are semiautotrophic [36] and leaves formed during in vitro growth may never attain photosynthetic competence [37]. Moreover, plantlets growing under in vitro conditions have limited accessibility to CO 2 inside the vessel [36]. Therefore, sugar is supplemented as a carbon source to maintain an adequate supply of carbon source for in vitro multiplication and growth of plant cell, tissue, and organs or whole plantlets. Continuous supply of carbohydrates to plants cultured in vitro is essential because the photosynthetic activity of in vitro-grown tissues is usually low. These compounds are also necessary as osmotic agents In the same column, the numbers followed by the same letter are statistically identical to the 5% threshold (Newman-Keuls test) (average ± standard error). In the same column, the numbers followed by the same letter are statistically identical to the 5% threshold (Newman-Keuls test) (average ± standard error). in the culture media. Hence, sugars have a potential effect on the physiology, growth, and differentiation of cells [38]. Therefore, the optimal carbon source needs to be considered. Thus, different sugars such as glucose, fructose, and sucrose at 3% (w/v) were incorporated into the basal medium 1/2 MS. After four weeks of cultivation, the growth of the plants developed from EA was recorded and presented in Table 3. Plants grew in the presence of sugar in the medium. The kind of sugar (sucrose, glucose, or fructose) did not seem to have a significant effect on the number of leaves, the root length, and the plant biomass. Similar findings have also been reported in Arabidopsis thaliana (L.) Heynh. by [39]. However, among the three types of sugars, the highest plant height (7.94 cm) was observed on medium containing 3% sucrose. The positive effects of sucrose on growth of explants under in vitro condition are linked with its high solubility in water, its electrical neutrality, and its lack of inhibitory effect on the majority of biochemical processes [40]. This positive effect of sucrose resulted in its wide use in tissue culture as a carbon source [41,42]. Supplementation of sucrose in growth medium meets the energy demands for growth and physiological function [36]. Effect of Different Concentrations of Sucrose. Development of in vitro plantlets derived from EA was further investigated to study the effect of different concentrations of sucrose, 1, 2, 3, 4, 5, and 6% (w/v), on plant growth. The results obtained after four weeks of culture are recorded in Table 4. Increasing the sugar concentration from 1 to 3% has a visibly stronger influence on plant height and biomass production. But, above the concentration 3% sucrose, no significant difference was observed. Moreover, among the different concentrations of sucrose tested, a nonsignificant difference was recorded for the number of leaves and the root length. From these results, a 3% sucrose concentration in the basal medium seems to be sufficient for normal plant growth. Sucrose is the most widely used carbon source in most of the plant species, as it is the main sugar translocated in the phloem [19]. As a carbon source, sucrose supports growth of plant cells in culture [43]. A sucrose concentration of 1-5% is generally used for in vitro tissue culture, since it is also synthesized naturally by the tissue [44]. For tissue culture, workers generally use 3% sucrose in the medium as per recommendation of [21]. Besides serving as energy source, it also provides the carbon precursors for structural and functional components [45]. The Scientific World Journal 7 Conclusion The overall objective of this investigation was to define the optimal conditions for in vitro seed germination and plant growth of Bambara groundnut. The main results showed that the composition of the germination medium did not influence the germination capacity of different types of seeds used in Bambara. Germination occurs faster when the embryonic axis is used as a seed source. The best seedling growth is observed with the seeds without coat followed by the embryonic axis on half MS medium containing 3% sucrose. This established protocol would provide sufficient materials as source of explants for initiating in Bambara groundnut different types of tissue culture.

v3-fos

2019-04-02T13:05:52.263Z

2015-06-30T00:00:00.000Z

55021707

s2

Role of Pellets and Capsules of Acacia nilotica and Sapindus mukorossi in Combination of Seed Bio-Priming with Microbial Antagonists in the Supression of Root Infecting Pathogenic Fungi and Promotion of Crop Plants Acacia nilotica (L.) Willd. ex Del. is also known as Gum Arabic tree, Babul, Egyptian thorn, or Prickly. Acacia is a multipurpose tree and play a great role in fixation of nitrogen [1]. Species of Acacia contains secondary metabolites including amines and alkaloids, cyanogenic glycosides, cyclitols, fatty acids and seed oils, fluoroacetate, gums, nonprotein amino acids, terpenes (including essential oils, diterpenes, phytosterol and triterpene genins and saponins), hydrolyzable tannins, flavonoids and condensed tannins [2]. A. nilotica also possess antimicrobial activity against some micro-organisms [3]. Mashram has observed the antimicrobial activity of Acacia nilotica, against microorganisms like, B. subtilis, S. aureus and E. coli in vitro and found that the extracts of bark and leaf showed inhibition zone between 7.5-16 and 8-15.5 mm respectively and most effective against E. coli. and B. subtilis. recorded antifungal activity of methanolic extracts and aqueous extract of A. nilotica with percentage inhibition ranging from 34.27 ± 1.45 to 93.35 ± 1.99 [4]. Similarly Sapindus mukorossi (L.) is well known for its folk medicinal values. Recently many of the pharmacological actions of this plant have been explored which includes the antimicrobial cytoxoic molluscicidal insecticidal and fungicidal [5-9].The leaves are used in the baths to relieve joint pain and the roots are used in the treatment of gout and rheumatism. Since ancient times S. mukorossi has been used as a detergent for shawls and silks. The fruit of S. mukorossi was utilised by Indian jewellers for restoring the brightness of tarnished ornaments made of gold, silver and other precious metals [10]. Dawar et al. [11] studied that leaves, stem, bark and fruit powder of Eucalyptus sp., has ability to reduce the infection of root infecting fungi viz., Fusarium sp., R.solani and M. phaseolina. Introduction Acacia nilotica (L.) Willd. ex Del. is also known as Gum Arabic tree, Babul, Egyptian thorn, or Prickly. Acacia is a multipurpose tree and play a great role in fixation of nitrogen [1]. Species of Acacia contains secondary metabolites including amines and alkaloids, cyanogenic glycosides, cyclitols, fatty acids and seed oils, fluoroacetate, gums, nonprotein amino acids, terpenes (including essential oils, diterpenes, phytosterol and triterpene genins and saponins), hydrolyzable tannins, flavonoids and condensed tannins [2]. A. nilotica also possess antimicrobial activity against some micro-organisms [3]. Mashram has observed the antimicrobial activity of Acacia nilotica, against microorganisms like, B. subtilis, S. aureus and E. coli in vitro and found that the extracts of bark and leaf showed inhibition zone between 7.5-16 and 8-15.5 mm respectively and most effective against E. coli. and B. subtilis. recorded antifungal activity of methanolic extracts and aqueous extract of A. nilotica with percentage inhibition ranging from 34.27 ± 1.45 to 93.35 ± 1.99 [4]. Similarly Sapindus mukorossi (L.) is well known for its folk medicinal values. Recently many of the pharmacological actions of this plant have been explored which includes the antimicrobial cytoxoic molluscicidal insecticidal and fungicidal [5][6][7][8][9].The leaves are used in the baths to relieve joint pain and the roots are used in the treatment of gout and rheumatism. Since ancient times S. mukorossi has been used as a detergent for shawls and silks. The fruit of S. mukorossi was utilised by Indian jewellers for restoring the brightness of tarnished ornaments made of gold, silver and other precious metals [10]. Dawar et al. [11] studied that leaves, stem, bark and fruit powder of Eucalyptus sp., has ability to reduce the infection of root infecting fungi viz., Fusarium sp., R.solani and M. phaseolina. Pelleting and encapsulation of plant parts gaining importance in recent times stated that pyrophyllite mixed Avecenia marina plant parts pellets (leaves and stem powder mixed separately with pyrophillite) had played a marked role in the elevation of growth parameters as well as in the reduction of soil borne root rot fungi like F. oxysporum, M. phaseolina and R. solani on cowpea and bringal plants [12]. Ghaffar, 1995 observed prominenet reduction in M. phaseolina infection on chickpea and mungbean plants when pellets of sodium alginate were mixed in the soil @ 1 and 10 pellets inside the plastic pots. Walker and Connick [13] used alginate type pellets in formulations of microbiological and chemical herbicides. According to, Tariq and Dawar [14] encapsulation of halophytic plant parts powder such as stem and leaves powder @ five capsules per pot prominently decreased the incidence of root-infecting pathogenic fungi on okra and mungbean plants. Apart from pelleting and encapsulation techniques, bio-priming of seeds with beneficial micro-organisms is also gaining importance in the control of many plant pathogens as another alternative to synthetic fungicides in present times. Bio-priming of seeds with antagonistic microbes are capable of colonizing the rhizosphere by potentially providing the advantages to the plant beyond the seedling emergence stage. Using antagonistic microorganisms was such an approach which was used increasingly on a commercial scale both in field and green house crops. Antagonistic micro-organisms like Trichoderma species are important biological control agents (BCAs) of many soil borne plant pathogens [15]. Different mechanisms have been used by Trichoderma in order to control plant pathogens which include competition for nutrients and habitat, mycoparasitism, release of antibiotics and fungal cell wall breaking enzymes [15][16][17]. Besides fungal antagonistic microbes, beneficial bacterial isolates have also been used for seed biopriming. These beneficial bacterial micro-organisms contain antifungal and good plant growth promoting attributes. Bacterial strains in soil that have tremendous effects on plant growth and health are commonly known as plant growth promoting rhizobacteria (PGPR). PGPR enhance plant growth indirectly or directly with the biocontrol of pathogens, production of plant hormones or improvement of plant nutritional status [18]. Present research work was therefore carried out on role of pellets and capsules of A. nilotica and S. mukorossi in combination of seed bio-priming with microbial antagonists in the suppression of root infecting pathogenic fungi and promotion of crop plants. Collection of plants and antagonistic agents A. nilotica and S. mukorossi leaves parts were collected from Campus of University of Karachi, air dried separately and finely powdered in an electric grinder. Cultures of Rhizobium meliloti and Trichoderma harzianum were obtained from the Karachi University Culture Collection (KUCC). Preparation of pellets and filling of capsules A. nilotica and S. mukorossi pellets were prepared with the help of research method of Tariq and Dawar [12]. Similarly, A. nilotica and S. mukorossi pellets were prepared with the help of multiple pellet sampler of equal size and weight (1 g pellet containing 0.5 g pyrophyllite and 0.5 g leaves powder). These pellets were air dried under laminar air flow chamber. Leaves powder of A. nilotica and S. mukorossi were filled in empty capsules (0.5 g in each capsule). Preparation of spore/cell suspension and bio-priming of seeds Spore/cell suspension of T. harzainum and R. melilotii were prepared in distilled sterilized water and seeds were bio-primed in these suspensions separately for about 10 minutes and then these seeds were air dried for sowing. Pots setup under screen house Plastic pots containing 300 g of soil were placed under screen house in Department of Botany, University of Karachi under randomized block design. These experimental pots containing (a) amendment of A. nilotica and S. mukorossi pellets in the soil separately in which non treated seeds were sown (b) T. harzianum and R. melilotii primed seeds were sown in the soil separately in non-amended soil (c) combined effect of both bioprimed seeds and pellets amended soil (d) only pyrophyllite pellets were mixed in the soil which served as control no. 1 (e) 0.5 g of A. nilotica and S. mukorossi leaves powder were filled in empty capsules separately and mixed in the soil containing non treated seeds (f) combined effect of both capsules and bioprimed seeds (g) empty capsules mixed in the soil which regarded as control no. 2. These experimental pots were kept under screen house for about 30 days and for maintaining the moisture content watered regularly. After 30 days the plants were uprooted for the observation of growth parameters and their roots were washed in running tap water for estimation of roots colonization by root infecting fungi. Statistical Analysis Data obtained from the experiment were analysed with the help of ANOVA (analysis of variance) and LSD (least significant difference) test at P=0.05 and DMRT (Duncan's multiple range test) in order to compare treatment means, using STATISCA computer software. Results There was significant (p<0.001) enhancement in growth parameters of peanut when A. nilotica pellets (pyrophyllite and leaves powder @ 50:50 ratio) in combination with bio-priming of seeds with T. harzianum spore suspension was used as compared to the control in which only pyrophyllite pellets was amended in the soil (Figure 1). Similarly combine effect of A. nilotica capsules (filled with 0.5 g leaves powder) and bio-priming of peanut seeds with T. harzianum spore suspension gave significant increase in root length, shoot length, root weight and shoot weight and prominent decrease was also noticed in the colonization of Fusarium sp (p<0.001), R. solani (p<0.01) and M. phaseolina (p<0.5) as compared to the control (empty shells of capsules) ( Figure 1). In chickpea, root length, shoot length, root weight and shoot weight increased significantly (p<0.001) when A. nilotica pellets and capsules were amended in the soil and seeds were bio-primed with T. harzianum, whereas, M. phaseolina reduced significantly (p<0.5) in comparision with both the controls ( Figure 2). Combine impact of A. nilotica (pellets) and S. mukorossi (capsules) along with T. harzianum bio-primed seeds significantly (p<0.001) elevated the growth parameters of okra and significant suppression in root infecting fungal pathogens like R. solani (p<0.001) and Fusarium sp (p<0.001) was also observed ( Figure 3). In sunflower, combined application of (A. nilotica pellets and biopriming of seeds with T. harzianum) and (encapsulation of S. mukorossi and T. harzianum primed seeds) gave significant (p<0.001) health and vigour to growth parameters such as root length, shoot length, root weight and shoot weight and significant decrease in root infecting fungi such as Fusarium sp (p<0.001), R. solani (p<0.001) and M. phaseolina (p<0.5) was also recorded ( Figure 4). Of all the treatments and amendments, it was observed that pellets and capsules of A. nilotica, and S. mukorossi in combination with biopriming of seeds with T. harzianum spore suspension was found to be most effective for the promotion of growth and suppression of root infecting pathogenic fungi like Macrophomina phaseolina, Rhizoctonia solani and Fusarium sp on leguminous and non-leguminous crops. Discussion In the present research, encapsulation and pyrophyllite mixed pellets of A. nilotica and S. mukorossi alongwith T. harzainum primed seeds played a marked role in the enhancement of growth parameters as well as in the reduction of fungal pathogens like M. phaseolina, R. solani and Fusarium sp of leguminous and non-leguminous crops. Significant (p<0.001) enhancement in growth parameters of peanut was noticed when A. nilotica pellets (pyrophyllite and leaves powder @ 50:50 ratio) in combination with bio-priming of seeds with T. harzianum spore suspension was used. Tariq and Dawar [12] used formulations of A. marina plant parts pellets with pyrophyllite @ 50:50 ratio and observed that there was prominent increase in growth parameters of leguminous and non-leguminous plants and root infecting fungi like M. phaseolina, R. solani and Fusarium sp was also reduced significantly .Our results showed that in chickpea, root length, shoot length, root weight and shoot weight increased significantly with the combined effect of A. nilotica pellets, capsules and T. harzianum primed seeds whereas, M. phaseolina reduced significantly. Similarly, when mixed alginate pellets in the soil, there was significant suppression in the colonization of M. phaseolina on mung bean and chickpea plants [19]. According to our results, combined application of A. nilotica (pellets) and S. mukorossi (capsules) along with T. harzianum bio-primed seeds significantly elevated the growth parameters of okra and significant suppression in root infecting fungal pathogens like R. solani and Fusarium sp was also observed. Tariq and Dawar [20] investigate the effect of mangrove plant parts filled capsules and pellets and recorded that formulations of mangrove combined parts powder and pellets when amended in the soil found to releases compounds which are nematicidal in nature and reduced the activity of Meloidogyne javanica on okra and mungbean plants and thus increase plant growth and crop yield. Besides pelleting and encapsulation reports, several studies on biopriming of seeds with beneficial micro-organisms have shown that seed bio-priming with microbial antagonists played a prominent role in the health and vigour of seedlings as well as in the reduction of many soil borne diseases. Rafi and Dawar [21] studied the effects of bio-priming on leguminous and non-leguminous crops at different time intervals and concluded that growth parameters markedly improved when seeds were bio-primed with microbial antagonists like Trichoderma harzianum and Rhizobium meliloti for 10 minutes whereas, root infecting fungal pathogens reduced significantly when seeds were bio-primed with T. harzianum, Bacillus sp and R. meliloti conidial/cell suspensions for 5, 10 and 20 minutes time interval .Present investigation found that in sunflower, combined application of (A. nilotica pellets and biopriming of seeds with T. harzianum) and (encapsulation of S. mukorossi and T. harzianum primed seeds) gave maximum growth parameters such as root length, shoot length, root weight and shoot weight and significant decrease in root infecting fungi was also recorded. Harman [22] observed that strain T-22 of Trichoderma harzianum causes plant to have more extensive root systems and not only suppressed the diseases but also improve the plant metabolism. Present research investigation clearly suggests that pelleting and capsulation of A. nilotica, and S. mukorossi in combination with biopriming of seeds with T. harzianum spore suspension was found to be most effective for the promotion of growth and suppression of root infecting pathogenic fungi like Macrophomina phaseolina, Rhizoctonia solani and Fusarium sp on all the tested plants.

v3-fos

2018-12-10T23:50:58.687Z

2015-09-29T00:00:00.000Z

55848567

s2

RETRACTED ARTICLE: Nutritional, technological, and medical approach of finger millet (Eleusine coracana) AbstractFinger millet (Eleusine coracana L.) is also known as African millet and is commonly called “ragi” in India. It has excellent nutritional value and is even superior to other common cereals. It is a richest source of calcium (344 mg) and magnesium (408 mg) than other millets. Predominant fatty acids of this millet are oleic (49%), linoleic (25%), and palmitic acids (25%). Finger millet contains both water-soluble and lipo-soluble vitamins. Emerging bakery products prepared from this millet are pasta, noodles, vermicelli, and bread. Being gluten free, it is suitable for individuals suffering from celiac disease. Finger millet grain is a rich source of several phytochemicals. Finger millet possesses blood glucose lowering, cholesterol lowering, and antiulcerative, wound healing properties as indicated by in vitro and in vivo studies. Commonly used processing techniques for this millet are milling, malting, popping, and decortications. Introduction Finger millet (Eleusine coracana L.) is also known as African millet and is commonly called "ragi" in India. Being one of the ancient grains, it is believed to be originated in East Africa and introduced PUBLIC INTEREST STATEMENT Finger millet (Eleusine coracana L.) has excellent nutritional value. It is a richest source of calcium (344 mg) and magnesium (408 mg) than other millets. This minor millet is known for having several health benefits such as blood glucose lowering, cholesterol lowering, antiulcerative, and wound healing properties which are attributed to its polyphenol and dietary fiber contents. Being gluten free, it is suitable for individuals suffering from celiac disease. Therefore, the aim of the current study was to review the nutritional, technological, and medical approach of finger millet (E. coracana). into India by sea traders around 3000 B.C. The word ragi originated from a Sanskrit word "raga" meaning red. In India, it is commonly grown for human consumption and also in many other arid and semi-arid areas of the world. This millet can grow in almost all types of soils and climatic conditions including alkaline soils with pH as high as 11 and at an altitude of 2,500 m from sea level, with average annual rainfall ranging from 800 to 1,200 mm. It is known as the poor man's food because of its long sustenance as it can be stored safely for many years without infestation by insects and pests. This property makes it a very necessary famine reserve food. The world production of finger millet is about 4.5 million tons per annum, out of which India produces nearly 55% of the total production. Finger millet is commonly cultivated in India, Nepal, Sri Lanka, East China and Bangladesh, Kenya, Tanzania, Rwanda, Uganda, Burundi, Ethiopia, Sudan, Zimbabwe, Malawi, and Madagascar. E. coracana is the most common species of this millet cultivated for food use. Other wild and semi-wild species of this millet are Eleusine indica and Eleusine africana. Its grain has distinct morphological features. It is a small seeded grain and its kernel is not having a true caryopsis but a utricle. Its pericarp (glumes) in the utricles is not fused with the seed coat or testa, thus its pericarp can be easily removed by rubbing or by soaking in water or sometimes it gets detached from the seed during threshing. Large variations in color, appearance, and size of this millet kernel have been observed among varieties. Its grain color varies from white to orange, deep brown, and purple to almost red. But the most common color of the seed is brick red. Its endosperm is white in color, similar to rice. The kernel shape may be spherical, globular, or oval, and varies in size from 1 to 1.8 mm. Its seed coat contains five layers and these are attached tightly to the endosperm (McDonough, Rooney, & Earp, 1986). Embryo, endosperm, and seed coat of this millet account for about 15% of the kernel mass. The endosperm of this millet is soft and fragile and is divided into three parts namely, peripheral, corneous, and floury. Floury endosperm comprises about 83% of whole grain. The endosperm of finger millet is mostly filled with starch granules. The shape of starch granules may be spherical, polygonal, or lenticular in floury area corneous and at peripheral regions. According to the data of United States Department of Agriculture (USDA), world millet production, which was 32.5 million tons in 2010/11 season, declined to 27.4 million tons in 2011/12 season and reached 30.4 million tons with an increase in approximately 3 million tons in 2012/13 season. USDA projects that world millet production decreased to 29 million tons in 2013/14 season, which will reach 30.3 million tons level again in 2014/15 season. Millet is intensely produced in India, Nigeria, and Niger. These three countries realize 63.7% of world millet production. Solely India realized 10.6 million tons of 30 million ton world millet production in 2013/14 season. Ranking second after India, Nigeria realized 5 million tons of millet production in 2013/14 season, while Niger realized 2.9 million tons of production. Lipids Finger millet is having a lipid content of 1.5%. Predominant fatty acids of this millet are oleic (49%), linoleic (25%) and palmitic acids (25%). About 72% of total lipids are present as neutral lipids, 13% as glycolipids, and 6% as phospholipids (Mahadevappa & Raina, 1978). Lipids of finger millet are mostly triglycerides and these are known to reduce the incidence of duodenal ulcer. Dietary fiber Total dietary fiber content of finger millet grain is 22.0% and is relatively higher than most of other cereal grains, e.g. 12.6 wheat, 4.6 rice, 13.4 maize, and 12.8% sorghum, respectively. Dietary fibers are categorized as water soluble or insoluble. Chethan and Malleshi (2007) reported that finger millet grain contained 15.7% insoluble dietary fiber and 1.4% soluble dietary fiber, while (Shobana & Malleshi, 2007) reported 22.0% total dietary fiber, 19.7% insoluble dietary fiber, and 2.5% soluble dietary fiber in finger millet. Vitamins Finger millet contains both water-soluble and lipo-soluble vitamins viz., thiamin, riboflavin, niacin, and apparently vitamin C plus the (tocopherols) vitamin E Obilana and Manyasa (2002). Watersoluble B-vitamins of finger millet are concentrated in the aleurone layer and germ, while lipo-soluble vitamins are mainly located in the germ. Post harvesting operations of finger millet The harvesting of finger millet crop takes place mainly during October through November. There are two methods of harvesting. Harvesting of only panicles After crop maturity, the panicles (ear heads) of finger millet are collected by cutting with the help of a sickle, leaving the plant stalks as such in the field. The operation is being carried out at one time or at intervals depending on the uniformity of maturity. The harvested panicles are gathered in a container such as bamboo baskets (tokri) before heaping them in a convenient place. The panicles staked in heaps are left for sun drying for a period ranging from one week to more than a month. The heat generated within the heap will help in easy separation of grains during threshing. Harvesting of stalks along with panicles This is the most commonly used method. In this method, harvested stalks are spread in rows in the field for sun drying for a couple of days depending on the weather conditions. After sun drying, the harvested stalks are bundled and staked near the threshing yard. During rainy days, a stacking practice involving arranging the bundles in the field in closed lines in slanting position and covering with dried straw to prevent dampening is practiced. After few days, the cover is removed and allowed to dry for one to two days before staking at the yard. Threshing of finger millet grains Separation of grains from panicles (ear heads) is done by spreading panicles or stalk in the morning and threshing starts from 10 o'clock. Threshing of panicles or stalks is usually done using bullocks (4-5 in number) for trampling or by a stone roller drawn by a pair of bullocks. For large-scale operation, in some places, tractors are used by farmers for grain separation. Farmers also use paddy threshers. Bamboo sticks are also used for threshing in small-scale operations. Storage Before storage, grains of finger millet are sun dried. Various types of structures (Bhakari, Kalanjiam, Semiliguda, turjhulla, Dumbriguda, and Chatka) are used by farmers for storage of this millet. Closed structures are commonly used for storage of seeds. In present days, gunny bags or nylon-woven sacs are used by farmers for grain storage. However, storage period for this millet varies from region to region. Traditional food products from finger millet Finger millet is usually pulverized and wholemeal is utilized for the preparation of traditional foods. In addition to traditional foods, it is also processed to prepare popped, malted, and fermented products. Noodles for diabetic patients were successfully developed from finger millet by Shukla and Srivastava (2011). Thirty-fifty percent finger millet proportion was blended with refined wheat flour for the preparation of noodles. On the basis of sensory evaluation, 30% finger millet-incorporated noodles were selected and evaluated for glycemic response compared to control. Results revealed that 30% finger millet-incorporated noodles have low glycemic index as compared to control. Finger millet seed coat is an edible material and contains a good proportion of dietary fiber, minerals, and phytochemicals. Seed coat matter (SCM) forms a byproduct of the millet milling, malting, and decortication; this can be utilized as composite flour in biscuit making. Krishnan, Dharmaraj, Sai Manohar, and Malleshi (2011) developed biscuit from finger millet seed coat. On the basis of sensory evaluation, they found that 10% of SCM from native and hydrothermally processed millet and 20% from malted millet could be used in composite biscuit flour. Saha et al. (2011) prepared biscuit from composite flours containing 60:40 and 70:30 (w/w) finger millet:wheat flour and these were evaluated for dough characteristics and biscuit quality. These reported that a composite flour of finger millet:wheat flour (60:40) was best, particularly regarding biscuit quality. Muffins were also prepared by replacing wheat flour with 0, 20, 40, 60, 80, and 100% finger millet flour (FMF), emulsifiers, and hydrocolloids (Rajiv, Soumya, Indrani, & Rao, 2011). Effect of finger millet, emulsifiers, and hydrocolloids on the batter microscopy, rheology, and quality characteristics of muffins was also studied. They found that a combination of additives with 60% FMF significantly improved the volume and quality characteristics of muffins. New food products made from finger millet which are currently being explored are noodles, vermicilli, prepared either from finger millet alone or in combination with refined wheat flour (Shukla & Srivastava, 2011;Sudha, Vetrimani, & Rahim, 1998), pasta products (Gull, Prasad, & Kumar, 2015;Krishnan & Prabhasankar, 2010), halwa mixes (a sweet dish prepared with flour, sugar, and clarified butter) and composite mixes (Itagi, Singh, Indiramma, & Prakash, 2013), papads (flattened and dried dough products which are toasted or deep fried and used as adjuncts with a meal) (Kamat, 2008;Vidyavati, Mushtari, Vijayakumari, Gokavi, & Begum, 2004), roller-dried finger millet-based soup mixes (Guha & Malleshi, 2006), bakery products such as muffins (Jyotsna, Soumya, Indrani, & Venkateswara, 2011), bread, and biscuits (Krishnan et al., 2011;Saha et al., 2011;Singh, Mishra, & Mishra, 2012), and complementary foods (Stephen et al., 2002) are also being prepared and marketed in selected markets. Breads from millet-based composite flours, wheat in combination with finger millet, barnyard millet, and proso millet were also prepared. Ready-to-eat nutritious supplementary food from popped FMF was also prepared by . This is a whole-grain product rich in macronutrients, micronutrients, dietary fiber, and usually mixed with vegetable or milk protein sources such as popped bengal gram, milk powder, and oil seeds sweetened with jaggery or sugar. Expanded finger millet has also been recently developed (Ushakumari, Rastogi, & Malleshi, 2007) from the decorticated finger millet. Its dietary fiber content is lower than that of popped finger millet, as it is prepared from decorticated finger millet which is devoid of seed coat. Ragi soup is prepared by mixing ragi flour in water. Then, this mixture is heated for 15 min and stirred frequently to avoid lump formation. After that the mixture is removed from heat; curd and salt are then added to it. Finally, it is served either warm or cold. In addition to the above-mentioned products, many other local preparations are in practice making use of finger millet depending upon the local habits. Few modern products incorporating finger millet are now available in the market such as, ragi health drink (baby vita), foodles, multigrain noodle, and ragi biscuit. Medicinal properties of finger millet Chronic metabolic disorder "Diabetes mellitus" is characterized by hyperglycemia with alterations in carbohydrate, protein, and lipid metabolism. It is a most common endocrine disorder and results in deficient insulin production (Type 1) or combined resistance to insulin action and the insulin secretory response (Type 2). Whole-grain foods are suggested to be beneficial for the prevention and management of diabetes mellitus and epidemiologically lower incidence of diabetes has been reported in millet-consuming populations (American Diabetes Association, 2005;Kim, Hyun, & Kim, 2011;Shobana et al., 2009). Consumption of finger millet-based diets resulted in significantly lower plasma glucose levels, mean peak rise, and area under curve that might be due to higher fiber content of finger millet than rice and wheat. This lower glycemic response of whole finger millet-based diets may have been due to the presence of antinutritional factors in whole FMF which are known to reduce starch digestibility and absorption (Lakshmi Kumari & Sumathi, 2002). Role of finger millet feeding on skin antioxidant status, nerve growth factor, and wound healing parameters in healing impaired early diabetic rats has been reported. Increased levels of oxidative stress markers accompanied by decreased levels of antioxidants play a vital role in delaying wound healing in diabetic rats. However, finger millet feeding to the diabetic animals for 4 weeks controlled the glucose levels and improved the antioxidant status which hastened the dermal wound healing process (Rajasekaran, Nithya, Rose, & Chandra, 2004). Almost all countries of the world are facing increase in rates of cardiovascular disease. It has been demonstrated that rats fed with a diet of native and treated starch from barnyard millet had the lowest blood glucose, serum cholesterol, and triglycerides compared with rice and other minor millets (Kumari & Thayumanavan, 1997). Serum triglycerides concentrations in finger and proso millet groups of hyperlipidemic rats were significantly lower than those of white rice and sorghum groups. In addition, serum concentrations of high density lipoprotein (HDL) and low density lipoprotein (LDL) cholesterols were significantly higher in the sorghum group than in the white rice, finger millet, and proso millet groups. Thus, finger millet and proso millet may prevent cardiovascular disease by reducing plasma triglycerides in hyperlipidemic rats (Lee, Chung, Cha, & Park, 2010). Phenolic extracts from kodo, finger, proso, foxtail, little, and pearl millets were evaluated for their inhibitory effects on lipid peroxidation in vitro, copper-mediated human LDL cholesterol oxidation, and several food model systems namely cooked comminuted pork, stripped corn oil, and a linoleic acid emulsion. At a final concentration of 0.05 mg/ml, millet extracts inhibited LDL cholesterol oxidation by 1-41%. All varieties exhibited effective inhibition of lipid oxidation in food systems used in this study and kodo millet exhibited superior inhibition of lipid peroxidation, similar to butylated hydroxyanisole at 200 ppm (Chandrasekara & Shahidi, 2011b). Millet grains are known to be rich in phenolic acids, tannins, and phytate that act as "antinutrients" (Thompson, 1993). It has been established that these antinutrients reduce the risk for colon and breast cancer in animals (Graf & Eaton, 1990). Van Rensburg (1981) reported that populations consuming sorghum and millet have lower incidences of esophageal cancer than those consuming wheat or maize. There is a growing demand for novel, tasty, and healthy foods. People suffering from celiac disease have given birth to a new market consisting of cereal products made from grains other than wheat and rye. For this reason, oat, sorghum, and millet have gained a special position (Angioloni & Collar, 2012). Celiac disease is an immune-mediated enteropathy, triggered by the ingestion of gluten in genetically susceptible individuals. It is a lifelong disorder worldwide. Since millets are gluten free, these are suitable for individuals suffering from celiac disease (Chandrasekara & Shahidi, 2011a, 2011bTaylor & Emmambux, 2008;Taylor, Schober, & Bean, 2006). Therefore, millet grains and their fractions have a potential to be useful for producing food products for celiac people. Millet grains are rich in antioxidants and phenolics; however, it has been established that phytates, phenols, and tannins can contribute to antioxidant activity important in health, aging, and metabolic syndromes (Bravo, 1998). Methanolic extracts from finger millet and kodo millet have been found to inhibit glycation and cross-linking of collagen (Hegde, Chandrakasan, & Chandra, 2002). Therefore, there is a potential usefulness of millets in protection against aging. Extracts and fractions of millet grain were found to have antimicrobial activity. In one study, phenolic acids from finger millet-milled fractions (whole flour, seed coat, 3, 5, and 7%) were isolated. Seed coat extracts of finger millet showed higher antimicrobial activity against Bacillus cereus and Aspergillus flavus than whole flour extract. Therefore, the results indicated that potential exists to utilize finger millet seed coat as an alternative natural antioxidant and food preservative (Viswanath, Urooj, & Malleshi, 2009). Milling Generally, finger millet is pulverized to flour for preparation of food products. First, it is cleaned to remove foreign materials such as stones, chaffs, stalks, etc., then passed through abrasive or friction mills to separate out glumes (non-edible cellulosic tissue), and then pulverized. Normally, it is pulverized in stone mill or iron disk or emery-coated disk mills. Sometimes, pearling or decortications is used to dehusk the finger millet grain; it results in pulverization of both the seed coat and endosperm. Hence, finger millet is invariably pulverized along with the seed to prepare wholemeal. Centrifugal sheller can also be used to dehull/decorticate the small millets. Decortication This is a very recent process developed for finger millet (Malleshi, 2006). It is also known as debranning. This method is used for debranning all cereals, but it is not effective for finger millet, owing to its seed coat being intactly attached to the fragile endosperm. However, hydrothermal processing is used to decorticate finger millet; this involves hydration, steaming, and drying which harden the endosperm of grain and enable it to withstand mechanical impact. The decorticated finger millet could be cooked as such, as rice is cooked. Popping Popping is one of the traditional methods to prepare popped FMF. In this process, millet is mixed with 3-5% additional water to raise the moisture content, tempered for 2-4 h, and then popped by high temperature and short time treatment by agitation in sand to about 230°C. This process results in the development of highly desirable aroma because of the Maillard reaction between sugars and amino acids. Popped finger millet is a precooked ready to eat product. Also, it can be pulverized and mixed with protein-rich sources to prepare ready nutritious supplementary food (Premavalli, Majumdar, Madhura, & Bawa, 2003). However, popping contaminates the product with particles of sand, which is used as a heat transfer media, and thus affects its eating quality. To overcome this drawback, air popping in a suitable mechanical process that has been successfully explored. But this method lacks the characteristics aroma compared to that using sand (Malleshi & Desikachar, 1981). Popped finger millet can be prepared at household community or at industrial level. Malting Malting of finger millet is commonly practiced for specialty foods. During this process, bioavailability of proteins, carbohydrates, and minerals are enhanced. Some B-group vitamins are synthesized and concentration of antinutritional factors is also reduced. Malting involves soaking of viable seeds in water to hydrate and to facilitate sprouting. These sprouts are then kiln dried. Finally, the rootlets are separated from the grain manually by rubbing with hand. All these operations influence the quality of malt. Seed germination is the most important step because during this process, the hydrolytic enzymes are developed which cause endosperm modification and increase nutritional properties. Malting of finger millet has been successfully utilized for developing various health foods such as infant food, weaning food, milk-based beverages, and confectionary products . Economic status of finger millet Millets are generally consumed in the region where they are produced. Thus, the millet amount subjected to the world trade is significantly low. USDA doesn't have open data for world millet trade. World millet trade announced by United Nations Food and Agriculture Organization (FAO) with formal, semi-formal, and forecast data and import and export amounts are not equivalent. FAO's current data belong to the year 2011. While the export was 385,000 tons, the import was 416,000 tons. An important factor in this difference is that the data cannot be obtained in a healthy way. According to the data of FAO, India ranks first in world millet export, the USA with 60,000 tons, Russia with 54,000 tons, following India, which exported 132,000 tons of millet in 2011. Sudan ranks first in imports, Pakistan with 33,000 tons, Belgium with 32,000 tons, and Germany with 19,000 tons, following Sudan which imported 67,000 tons. Conclusion Potential health benefits and nutritive value of millet grains were found comparable to major cereals. Several processing technologies were found to improve nutritional characteristics of millets. Utilization of millet grains as food is still limited to populations in rural areas. This is due to the lack of innovative millet processing technologies. This review provides a scientific rationale use of finger millet as a therapeutic and health-promoting food. In addition, to promote the utilization of millet grains in urban areas to open new markets for farmers to improve their income, developing highly improved products from millets is needed. Finger millet can be used in different food formulations for making value-added products due to its well-balanced protein profile and gluten-free properties. Although the consumption pattern of this millet is specific and continues to remain as such; therefore, its popularization in the broader range is essential and specific design of foods acceptable to the population can help in promoting the consumption of this millet.

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2016-05-12T22:15:10.714Z

2015-09-22T00:00:00.000Z

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Genetic Divergence and Heritability of 42 Coloured Upland Rice Genotypes (Oryzasativa) as Revealed by Microsatellites Marker and Agro-Morphological Traits Coloured rice genotypes have greater nutritious value and consumer demand for these varieties is now greater than ever. The documentation of these genotypes is important for the improvement of the rice plant. In this study, 42 coloured rice genotypes were selected for determination of their genetic divergence using 25 simple sequence repeat (SSR) primers and 15 agro-morphological traits. Twenty-one out of the 25 SSR primers showed distinct, reproducible polymorphism. A dendrogram constructed using the SSR primers clustered the 42 coloured rice genotypes into 7 groups. Further, principle component analysis showed 75.28% of total variations were explained by the first—three components. All agro-morphological traits showed significant difference at the (p≤0.05) and (p≤0.01) levels. From the dendrogram constructed using the agro-morphological traits, all the genotypes were clustered into four distinct groups. Pearson’s correlation coefficient showed that among the 15 agro-morphological traits, the yield contributing factor had positive correlation with the number of tillers, number of panicles, and panicle length. The heritability of the 15 traits ranged from 17.68 to 99.69%. Yield per plant and harvest index showed the highest value for both heritability and genetic advance. The information on the molecular and agro-morphological traits can be used in rice breeding programmes to improve nutritional value and produce higher yields. Introduction Rice (Oryza sativa L.) is the staple food and most important crop in most Asian countries. It belongs to the Poaceae family and is the main source of carbohydrate in these countries. Additionally, rice comprises about 20% of calories consumed worldwide [1]. About 90% of both rice Experimental design and layout The selected forty two coloured rice genotypes were germinated in a petri dish after which they were transferred into growing buckets (23× 21 cm) in the glasshouse at the rate of 5 plants/ bucket. The experimental design was randomized complete block design (RCBD) with three replications. Agronomic practice such as weed control was done manually while disease control was through the application of 5g per bucket Furadan (PT Bina Guna Kimia, Indonesia) and 5 ml per 1 L Malathion (Hextar chemicals Sdn. Bhd., Malaysia). The fertilisers urea, muriate of potash (MOP), and triple super phosphate (TSP) were applied 3 times at 5, 25, and 55 days after planting, to provide N, K and P nutrition, at the rate of 160 kg N/ha, 80 kgP 2 O 5 /ha, and 60 kgK 2 O/ha. Data collection Fifteen agro-morphological traits were identified by measuring five plants per genotype in each replicate and their means were used for further analysis. These traits include: (i) plant height; (ii) number of tillers per plant; (iii) number of panicles per plant; (iv) percentage of filled grain; (v) 100 grain weight; (vi) harvest index; (vii) days to first flowering; (viii) days to maturity; (ix) grain dimension and shape; (iix) length of flag leaf; (xi) panicle length; (xii) kernel length; (xiii) length breadth ratio;and (xiv) chlorophyll SPAD reading at 40 days and (xv) 60 days. DNA extraction protocol DNA was extracted from the seed samples using the modified conventional method [17]. About 100 mg of each seed sample was ground in the mortar using a pestle, and 400 μL extraction buffer (200 mMTris-HCL, 200 mMNacl, 25mM EDTA, 0.5% SDS). Then, the solution was transferred into a 2 ml microcentrifuge tube and 400 μl of CTAB solution (2% CTAB, 100 mMTris-HCL, 20 mM EDTA, 1.4 M NaCl, 1% PVP) was added. Next, 400 μl mixture, ratio of chloroform: isoamyl alcohol: phenol (24:1:5%), was added in the same tube. The mixtures were then well mixed by vortex and centrifuge (14,000 rpm, 5 minutes) at room temperature. The supernatant was then transferred into a new 2 mL microcentrifuge tube and 2/3 volume of isopropanol added. The mixtures were gently mixed by inverting the microcentrifuge tube and then incubated at room temperature for 10 minutes. Following this the mixture was centrifuged again (14,000 rpm, 5 minutes) at room temperature. The supernatant was then discarded and the pellets rinsed with 70% alcohol for a few minutes. Subsequently, pellets were air dried and re-suspended in 50 μL of TE buffer. The quality and quantity of the DNA was measured by NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, USA). Data analysis The presence and absence of alleles were scored using the binary system '1' and '0' respectively. The observed number of alleles, effective number of alleles and Shannon's Information index were determined using the Popgen software. Polymorphism information content (PIC) and expected heterozygosity (He) were also calculated using the PIC calculator (http://www.liv.ac. uk/~kempsj/pic.html). Genetic similarity was calculated using the dice coefficient [18]; the sequential agglomerative hierarchal and nested (SAHN) clustering was performed based on genetic similarity and unweighted paired group method with arithmetic averages (UPGMA); the principal component analysis (PCA) was performed on the matrix of the genetic similarity coefficients; and data analysis was carried out using the NTSYS version 2.1. The agro-morphological traits collected were subjected to Analysis of Variance (ANOVA) using SAS version 9.2. The Pearson correlation coefficient among all agro-morphological traits calculated using SAS version 9.2. A dendrogram was then constructed based on UPGMA and PCA based on agro-morphological data using NTSYS version 2.1. The broad sense heritability, genetic advance and other variance estimates were calculated using the method of Allard [19]. The following formulas were used to calculate the genetic parameters: Genotypic variance (σ² g ) = (MS 2 -MS 3 )/b Error variance (σ² e ) = MS 3 Phenotypic variance (σ² p ) = σ² g + σ² e Genotypic coefficient of variation ðGCVÞ ¼ ffiffiffiffiffiffiffi Phenotypic coefficient of variation ðPCVÞ ¼ ffiffiffiffiffiffiffi s 2 p p = X Â 100 MS 2 = mean square of populations MS 3 = mean square of error b = number of blocks X = mean of the trait Heritability (h 2 B ) = σ² g/ σ² p Expected genetic advance (GA) and genetic gain (GG) (as percentage of the mean) were calculated using the method of Allard [15] where selection intensity (K) was assumed to be 5% Expected genetic advance ffiffiffiffiffiffiffiffi ffi K is a constant which represents the selection intensity, when K is 5% the value is 2.06, ffiffiffiffiffiffiffi s 2 p p represents the phenotypic standard deviation, h B 2 is the heritability while X represents the mean of the characteristic being evaluated, using the formulae suggested by Burton [20]. (Table 3). Cluster analysis from SSR markers The dendrogram based on the UPGMA method grouped the 42 selected coloured upland genotypes into seven groups (Fig 2) at the coefficient of 0.62. Dice coefficient ranged from 0.50 to 1.00. Group 1 comprised the landrace cultivars from Thailand, India and Vietnam. Group 2 comprised all breeding and inbred line genotypes from Philippines and one from Myanmar whereas Group 5 comprised just one genotype from the Ivory Coast. Principal component analysis from SSR markers The first three components from the PCA analysis explained about 75.28% of the total variation present in these genotypes. About 5 distinct groups were obtained from the three dimensional PCA (Fig 3). Group 1 comprised landraces and traditional cultivars from India, Thailand and Vietnam. The clustering patterns found in these genotypes group via PCA was similar to that found in the dendrogram where all the breeding and inbred lines clustered to form Groups II and group IV, respectively. Genetic divergence and morphometric variability in the genotypes All the genotypes showed higher significant differences at p 0.05 and p 0.01 based on the quantitative traits, as revealed by ANOVA (Table 4). Genotype (V22) IR 5533-15-1-1 showed the highest number of tillers and panicles. Plant height ranged from 82.14 to 170.20 cm for all the genotypes with 124.72 cm being the average (Table 5). Genotype V5(C) recorded the highest panicle length at 30.35 cm. while genotype V33 (chirikata 2) showed the highest percentage of filled grain. Additionally, wide variability was observed in grain yield per plant among all the genotypes ranging from 0.57 V13 (Bibili al) to 6.62 g V5 (C) and showed the lowest and highest grain yield per plant. Genotype V7 (Chokoto 14) showed the highest rice kernel length. Length breadth ratio for all genotypes ranged from 2.77 to 4.85, while genotype V4 (Bi-e-gaw) recorded the earliest days for 50% flowering and days to maturity, which was 49 and 114 days, respectively. Clustering analysis from agro-morphological traits data The similarity coefficient as shown in the dendrogram varied from 0.15 to 1.44 (Fig 4). All the genotypes were grouped into 4 groups (Fig 4) at 0.79 coefficient. Group1 comprises of red rice genotypes from several geographical origins. This group had the highest mean value of agromorphological traits, such as plant height (148.78 cm), length of flag leaf (53.18 cm), percentage of filled grain (66.45%), panicle length (25.46%), kernel length (6.05 mm), and harvest index (0.77). Three genotypes were found in group 4, and this cluster had the lowest mean for traits such as number of tillers (2.04), number of panicles (1.55), percentage filled grain (31.51%), harvest index (0.09), grain yield/plant (0.65g), kernel length (5.59 mm), and kernel length/breadth ratio (3.19). Principal component analysis from agro-morphological traits data Using the PCA all the rice genotypes were grouped into 4 distinct groups which were similar to the cluster analysis grouping (Fig 5). All agro-morphological traits showed the highest cumulative percentage (!70%) except for length breath ratio (69%), chlorophyll SPAD value at 40 days (58%) and 60 days (56%) ( Table 6). Correlation among the agro-morphological traits data Yield per plant had positive correlation with number of tillers, number of panicles, percentage filled and unfilled grain, harvest index, panicle length, and chlorophyll SPAD reading at 60 days (Table 7). Chlorophyll SPAD reading at 40 days showed significant negative assosication with plant height, flag leaf and panicle lenght. Genetic parameters The genetic parameters calculated, such as genotypic variance (σ 2 g), phenotypic variance (σ 2 p), heritability (h 2 B ), and genetic advance (GA), are presented in Table 8. The broad sense heritability for 15 agro-morphological traits ranged from 17.68% (chlorophyll SPAD value at 40 days) to 99.69% (kernel length), respectively. Yield per plant showed the highest value of genetic advance (126%) among all the traits. Broad sense heritability was high for virtually all the yield component traits with the lowest (percentage filled grain) being approximately 62%. Discussion Genetic divergence as revealed by the molecular markers and agro-morphological traits is important for breeding and improvement of existing rice genotypes to suit consumer demands. Genetic divergence helps in breeding resistance and tolerance to various biotic and environmental stresses and also as a tool to investigate the effects of climate change, in order to select the genotypes with higher potential for use in breeding programmes. Among the 25 SSR primers used in this experiment, only 21 show polymorphism among genotypes. The marker used must be informative in order to reveal the genetic divergence among the genotypes. The mean polymorphic information content (PIC) recorded was 0.41 for all the SSR markers tested. The marker is informative if the PIC value is higher than 0.5 [21]. For instance, Ravi et al. [22] reported the mean PIC value of 0.578 for genetic diversity analysis of rice cultivars using SSR data. Cluster analysis of SSR primers data grouped all the genotypes based on geographical origin and status of the genotypes. Additionally, only one genotype was placed in Group 5. This genotype was an advanced cultivar from the Ivory Coast. The cophenatic correlation value (r) calculated from the dendrogram was 0.83 which shows the good fit of the data for diversity analysis if (r>0.8). Based on the grouping in the PCA, some groups could not be separated using clustering analysis. Groups 3 and 5 (Fig 2), for example, consisted of mixed groupings of genotypes and this cannot be resolved by PCA. Thus, this analysis can be informative for differentiation among the major groups and it will help the breeder to select from diverse breeding lines. All agro-morphological traits differed significantly. Three genotypes showed the lowest values for yield per plant and percentage of filled grain. The breeding line showed good agro-morphological traits, such as lower plant height, high filled grain, and higher grain yield per plant compared with the landrace or traditional cultivars. Thus, cross breeding between traditional cultivars and breeding lines can be done to produce plants with good yield characteristics. The dendrogram for agro-morphological traits was constructed based on the matrix of average of taxonomic distance using the UPGMA method. The dendrogram showed that Group 1 consisted of all the landrace and traditional cultivars. These genotypes displayed similar agromorphological characteristics, such as plant height, panicle length and length of flag leaf. Group 3 showed higher mean value of grain yield per plant (3.9g) as it was from the breeding lines from Philippines. From the dendrogram constructed based on SSR marker and agro-morphological traits, there are differences in grouping of the genotypes. Agro-morphological traits were influenced Genetic Divergence and Heritability of Coloured Rice Genotypes by environmental factors such as light intensity, disease and humidity. Additionally, clustering based on SSR markers was more accurate. This is because their usage is not influenced by environmental factor thus it will reflect the actual level of genetic difference existing among the genotypes [23]. In addition, SSR markers can detect slight differences of DNA structure. From PCA analysis, the first four components explained about 83.76% of variation [24]. Since the variation is high (! 25%), this analysis can be used along with cluster analysis to show the relatedness among the genotypes [25]. From the results of the first three components, it was seen that days to first flowering, number of panicles, number of tillers and percentage of filled grain played an important role in explaining the variation. This is because positive eigenvalue is shown for each of the 3 components. Plant height showed a negative correlation with number of tillers and number of panicles. Thus, lower plant height is a good characteristic because it results to higher tiller and panicle numbers. From the result (Table 5), traditional varieties showed the highest plant height when compared with breeding line. In modern rice breeding, gene sd-1 is one of the most important genes controlling dwarfisim in rice plant. It ressesive character helps to improve lodging resistance as a result of shortened culm [26]. Lodging is the most common problem affecting upland rice in the field. Thus reducing the plant height is one of the means of resolving the lodging problem. Most of the modern rice varieties have short plant height and this is the focus of selection by breeders for improvement of rice plant. Thus, plant height is one of the most important traits which needs to be considered in production of high yield variety. In addition, yield per plant was positively correlated with number of tillers, number of panicles, percentage of filled grain, harvest index and panicle length. This information is valuable to know the agromorphological traits that contribute to the to yield of the plant. From the present study, higher phenotypic variance was recorded than genotypic variance for all traits evaluated. The difference in value of genotypic variance and phenotypic variance was due to environmental factor. Our data shows that environmental factors have pronounced effect on each of those traits compared to the genotypic factors. Broad sense heritability was found to be high for plant height, panicle length, 100 grain weight, harvest index, yield per plant, days to flowering, days to maturity, kernel length and length breadth ratio. The heritability results must be combined with expected genetic advance to achieve reliable results [27]. Yield per plant and harvest index showed the highest value for both heritability and genetic advance. These traits need to be considered for yield improvement of rice plant genotypes through breeding programmes. High heritability and genetic advance for grain yield per plant and harvest index has also been reported by Bisne et al. [28]. From the analysis it is shown that all the 42 coloured upland rice genotypes can be selected for breeding programmes based on the SSR primers and agro-morphological traits evaluation. Genotypes such as C, Chirikata 2, Ble Chu Cau, and IR 9669-PP 836-1 have high potentials for selection. This is because, these genotypes have good agro-morphological traits such as, high percentage of filled grain, high harvest index and high grain yield per plant. Furthermore, the selected genotypes are clustered in different groups based on the SSR marker analysis which indicates that they are genetically divergent and thus breeding from these genotypes can produce good progenies with superior traits. Conclusions Cluster analysis from the SSR markers grouped all the genotypes into 7 groups according to geographical origin and status of the cultivar. All agro-morphological data showed significant differences at p 0.05 and p 0.01 for all traits which shows the presence of diversification among the 42 coloured upland rice genotypes. Four groups were constructed using agro-morphological data clustering analysis. Yield contribution factors traits, which are yield per plant and harvest index, showed the highest value of heritability and genetic advance. Selection based on these traits can be done for further breeding programmes. Potential genotypes such as C, Chirikata 2, Ble Chu Cau, and IR 9669-PP 836-1, are recommended for selection and further evaluation in future breeding programmes. This is based on their molecular and agromorphological information. These genotypes, selected from groups 2, 3, 4 and 5 from the SSR primer grouping, also have high grain per yield and harvest index values. For further studies, the evaluation of nutritional value of coloured upland rice can be done since red and purple bran may contain many phytochemical and neutraceutical functional foods. Thus, it will give high impact on rice breeding programme for development high yield and functional rice.

v3-fos

2019-04-07T13:05:49.333Z

2015-09-14T00:00:00.000Z

101118017

s2

Physicochemical characteristics of groundwater from Kumbakonam Taluk of Thanjavur District - Tamilnadu (India) The aim of present study was to assess the quality of ground water from Kumbakonam region in Thanjavur district, and check its fitness for drinking and other purpose. An eight ground water samples were collected from various parts of Kumbakonam region. The physicochemical parameters such as calcium, magnesium, chloride was determined by titration method, sulphate, nitrate, nitrite were analyzed by spectrophotometric method, pH was determined by pH metric method and other parameters were analyzed by Indian standard method. Physicochemical parameter of ground water samples were compared with standard limits recommended by BIS. The comparative study of ground water to this region, all the collected water samples are not suitable for drinking purpose, because in this region ground water samples had excess of manganese, calcium, magnesium and iron content. INTRODUCTION Water is extremely indispensable for survival of all living organism. The quality of water is vital concern for mankind since it is directly linked with human safety. In India, most of the population is dependent on groundwater as the only source of drinking water supply. Groundwater is believed to be comparatively much clean and free from pollution than surface water [1]. But expanded discharge of industrial effluents, domestic sewage use of fertilizers and pesticides, waste deposit causes the groundwater to become polluted and creates health problems [10]. In developing countries contamination of water supplies by organic chemicals is lesser concern, because most of the health problems are found to be associated with the presence of inorganic chemicals and pathogenic organisms in drinking water. In Thanjavur region some of ground water samples was contaminated, because in the presence of excess of calcium hardness, magnesium hardness, TDS and total alkalinity [8]. The hard water is said to cause serious health problems such as urolithosis, cardiovascular disorder, kidney problems and cancer [7]. Additionally, WHO reports that excess intake of calcium is associate with kidney stones and that of magnesium leads to diarrhea and laxative effect due to change in bowel habit. Water quality of Thiruvarur region most of the areas groundwater is not suitable for drinking purpose, because in the presence of excess hardness [12; 3]. In view of the above aspects of groundwater contamination, the present study was undertaken to investigate the possible impact on the groundwater quality of some bore wells of Kumbakonam region in Thanjavur district. Thus, in this paper an attempt has been made to assess the physical and chemical properties of groundwater. Study area: Kumbakonam is located in Thanjavur district. The studies area lies at 11 0 38 ' N and 75 0 45 ' E latitude. Fig -1 showed the location of ground water samples was collected from Kumbakonam region and table -1 are given about sampling points of Kumbakonam region. Methodology Water samples were collected in previously sterilized poly ethylene plastic bottles with cap. The sampling has been carried out in month of MAY -2015. The samples in the canes (Bottle) were kept in the refrigerator. The physicochemical parameters were determined by IS analytical method. Table-2 is given about methods used for estimation of variation physicochemical parameters. RESULTS AND DISCUSSION The water samples were collected from various parts of Kumbakonam region, and were analyzed for their physicochemical characteristics. The physicochemical parameters like TDS of water samples was analyzed by gravimetric method, turbidity of the water samples was analyzed by nephelo metric method, alkalinity, calcium, magnesium, chloride was estimated by titrimetric method, and other parameters were analyzed by Indian standard methods. The results were compared with BIS drinking water quality guideline. The estimated physicochemical parameters are reported in the table-3 and table-4. Appearance: Colour of water may be due to the presence of fine particles in suspension (or) due to certain mineral matter in solution. The entire collected sample had slightly yellow colour, (Table-3). Odour: Organic and inorganic chemicals originating from domestic wastes and by decomposition of vegetables matter contributes odour to the water. Entire collected ground water samples had odour less nature, (Table-3). Turbitity: Turbitity in natural water is caused by clay, organic matter, phytoplankton etc., and the turbidity of ground water samples from Kumbakonam region ranged from 10 NTU to 55 NTU, the data are shown in table-3. Electrical Conductivity: Electrical conductivity is the capacity of water to convey current and this may be due to the presence of soluble salts and ionic species which act as conducting medium. Conductivity of the samples ranged between 550 µS/cm to 1790 µS/cm. The data are given in table-3, (Fig-2). Total Dissolved Solids: Many dissolved substance are undesirable in water. Dissolved minerals, gases and organic constituents may produce aesthetically displeasing colour, taste and odour. The total dissolved solids of Kumbakonam region ground water range between 385 mg/l to 1253 mg/l. The TDS of ground water sample number S1, S3, S5, S6, S7 and S8 is high in Kumbakonam region, (Table -3), (Fig -3). If the TDS of drinking water is more than 2000 mg/l, would result to affect gastro intestinal irritation to human beings. Hydrogen ion concentration (pH): pH is the measure of capacity (or) alkalinity natural water is alkaline due to the presence of carbonates. The desirable pH range for drinking water is 6.5 to 8.5. The water samples had pH ranged from 6.8 to 7.2. All the samples pH lying with in BIS permissible limit, showing that all the samples were almost neutral and harmless, (Table-4), (Fig-4). International Letters of Chemistry, Physics and Astronomy Vol. 59 3 Alkalinity: Alkalinity in water is due to the presence of carbonates, bicarbonates and hydroxides. Bicarbonates are the major contributes since they are included from the basic materials in the soil. Alkalinity is also a measure of water to absorb H + ions. Total alkalinity of the samples was found to range from 212 mg/l to 460 mg/l, (Table-4), (Fig-5). The entire collected ground water samples had total alkalinity lying above the BIS desirable limit. Calcium: The calcium hardness was recorded in 32 mg/l to 128 mg/l. Ground water sample number S7 and S8 were exceeding the BIS limit. Excess calcium hardness of ground water samples causing kidney problem, urolithosis, to human beings. The data are given in table-4 and fig-6. 10. Iron: Irons usually exist in ferrous and ferric forms. Generally, the ferric form is predominant in natural water. Excess iron causes stripping of clothes. The samples had iron levels ranged between 0.30 mg/l to 1.47 mg/l, (Table-4), (Fig-8). The entire ground water samples of Kumbakonam region iron content is above the BIS desirable limit. Manganese; Manganese was recorded in 0.33 mg/l to 3.36 mg/l. All the ground water samples of Kumbakonam region the manganese content level is high compare than BIS desirable limit, (Table-4), (Fig -9). Generally the manganese is non -toxic to animals. However, when exceed 100 ppm it cause toxicity to human body and produces fever, muscular fatigue. Nitrite: Nitrite in water is due to incomplete oxidation of organic matter containing nitrogen. Nitrites should never be present in drinking water. Nitrite of the samples was found to range from 0.09 mg/l to 1.65 mg/l. The nitrite content of ground water sample number S1, S3 and S4 had high concentration compare than BIS desirable limit. High concentration of nitrites may cause blue-baby syndrome in children, (Table-4), (Fig-10) Nitrate: This is the highest oxidized form of Nitrogen. Biological oxidation of nitrogenous substance from sewage is the main source of nitrate. All the samples were found to have nitrate concentration ranging from 1mg/l to 8 mg/l. All the water samples had nitrate content lying within the BIS limit, (Table-4), (Fig-11). Chlorides: Discharge of domestic sewage is the main source of chloride in water. The chloride content estimated in the samples ranged between 44 mg/l -284 mg/l, (Table-4), (Fig-12). The ground water sample number S1, S7 and S8 have high concentration of chloride ions. An excess of chloride ion react with organic matter in water and produces cancer -causing compounds. Fluoride: Industrial waste is the main source of fluoride. The fluoride content was recorded in 0.1 mg/l to 0.2 mg/l. An entire ground water samples had fluoride content lying well below the detectable limit, (Table-4), (Fig-13). Sulphate: Sulphate occurs naturally in all kinds of water. Drainage wastes are the main source of high sulphate concentration. Excess sodium and magnesium sulphate may cause cathartic action. The samples had sulphate levels ranged between 3 mg/l to 75 mg/l. Entire collected ground water samples lying within BIS limit, (Table-4), (Fig-14). Phosphate: Generally phosphate occurs in natural water as inorganic (or) organic phosphates. Domestic sewage, agricultural effluents and detergents are the main source of phosphate in water. Excess phosphate may lead to growth of unwanted algae. The sample had phosphate content ranged from 0.03 mg/l to 0.86 mg/l, (Table-4), (Fig-15). CONCLUSION The physicochemical analysis of water samples concluded that the water quality of Kumbakonam region most of the areas groundwater is not suitable for drinking purpose, because in the region ground water had excess of TDS, manganese, chloride and iron. So human being of that region is suffering with various diseases such as gastro intestinal irritations and skin irritation. Rainwater harvesting is one of the solution to minimize the heavy metals concentration in drinking water.

v3-fos

2017-07-06T10:26:52.324Z

2015-03-01T00:00:00.000Z

23606107

s2

Phytochemicals and Other Characteristics of Croatian Monovarietal Extra Virgin Olive Oils from Oblica, Lastovka and Levantinka Varieties Virgin olive oils from the fruits of Croatian autochthonous varieties Oblica, Lastovka and Levantinka were characterized for the first time. Headspace volatiles were analyzed by HS-SPME/GC-FID/MS. The main volatiles were C6 compounds. The most abundant was (E)-hex-2-enal (62.60%–69.20%). (Z)-Hex-3-enal was not found in Lastovka oil, while Levantinka oil did not contain hexanal. Tocopherols, chlorophylls and carotenoids were determined by HPLC-FL. Levantinka oil was characterized by the highest α-tocopherol level (222.00 mg/kg). Total phenolic contents (TPs), as well as antioxidant activity (DPPH assay) of the oils hydrophilic fractions (HFs) were assessed by spectroscopic methods. The antioxidant activity of Oblica oil HF was the most pronounced (0.91 mmol TEAC/kg) and the HF contained the highest TPs amount (212.21 mg/kg). HFs phenolic composition was determined by HPLC-DAD. The main identified phenols were secoiridoids dominated in Oblica oil: decarboxymethyl ligstroside aglycone (p-HPEA-EDA up to 158.5 mg/kg), oleuropein aglycone (3,4-HPEA-EA up to 96.4 mg/kg) and decarboxymethyl oleuropein aglycon (3,4-DHPEA-EDA up to 93.5 mg/kg). Introduction Virgin olive oil (VOO) has been a valuable vegetable oil extracted from fresh and healthy olive fruits (Olea europeae L.) by mechanical and other physical methods (washing, decantation, centrifugation or filtration). Nowadays, it is well-known that regular dietary consumption of VOO manifests in health benefits associated with Mediterranean diet [1]. The nutritional value of VOO arises from high level of oleic acid and from minor components such as phytosterols, carotenoids, tocopherols and hydrophilic phenols. The major phenolic compounds are oleuropein derivatives, based on hydroxytyrosol which are strong antioxidants and radical scavengers [2]. The content of phenolic compounds is an important factor to be considered when evaluating the quality of VOO, since these compounds exhibit potent antioxidant activity and contribute significantly to the extraordinary oxidation stability of VOO [3]. Chain-breaking antioxidants, such as phenolic compounds, react with lipid radicals to form nonreactive radicals, interrupting the propagation chain. In fact, these compounds are able to donate an electron or a hydrogen atom to the lipid radical formed during the propagation phase of lipid oxidation [2]. Phenolic compounds are intimately associated with the positive attributes of bitterness and pungency which are typical sensory notes of VOO obtained from the olives that are green and turning color [4]. The polar phenolic compounds of VOO belong to different classes: phenolic acids, phenylethyl alcohols, hydroxy-isochromans, flavonoids, lignans and secoiridoids [2]. The amount of polar phenols and volatile compounds in the olive oil depend on many factors such as cultivar, agronomic practices, ripeness index, fruit pre-storage, extraction, procedure, storage conditions, etc. [4]. Color is one of the major attributes that affects consumer perception of VOO quality. The lipophilic nature of chloroplast pigments (chlorophyll and carotenoids) determines their affinity for the oily phase and the pigments are mainly responsible for the color of VOO ranging from yellow-green to greenish gold [5]. The presence of chlorophylls and carotenoids in olive oil depends on the fruits genetic factors (olive variety), the stage of fruits ripeness, environmental conditions, production year, extraction process and storage conditions. Carotenoids, together with polyphenols and tocopherols provide oxidative stability to the olive oils and exhibit synergistic antioxidant and anticarcinogenic action at physiological concentration [6]. In addition, extra VOO is appreciated worldwide for its taste and flavor that is colored by various volatile compounds: aldehydes, alcohols, esters, hydrocarbons, ketones, furans and others [7]. It has been stated [8] that C6 compounds, the major components of VOO headspace, mainly contribute to green odor notes. These compounds are produced by lipoxygenase-mediated oxidation of polyunsaturated fatty acids containing cis-cis-penta-1,4-diene structure during the crushing and malaxation steps of the oil production [9]. Variable amounts of hexanal, hexanol and hexyl acetate derive from degradation of linoleic acid, while (Z)-hex-3-enal, (E)-hex-2-enal, (E)-hex-2-enol, (Z)-hex-3-enol and (Z)-hex-3-enyl acetate result from the enzymatic degradation of linolenic acid [10]. VOOs exhibit positive effect on human health as well as specific and desirable sensory properties, which is why the demand for these oils is constantly growing with request to mark their geographical and varietal origin [4]. There are about 30 autochthonous cultivars of olives in Croatia. Oblica, Lastovka and Levantinka are the main autochthonous varieties in Dalmatia region (south Croatia) and Oblica is the most abundant [11]. The aim of present research is to perform detail chemical characterization of Oblica, Lastovka and Levantinka VOOs (first report including statistical analysis) by: (1) determination of basic characteristics (acidity, peroxide value, K232 and K270) according to EU regulations; (2) total phenols, chlorophylls and carotenoids evaluation by UV/VIS including targeted tocopherols analysis by HPLC-FL; (3) the headspace volatiles profiling (HS-SPME/GC-FID/MS); (4) HPLC-DAD targeted phenolics analysis of the hydrophilic oil fractions (HFs) and assessing the HFs antioxidant activity (DPPH assay). Results and Discussion To avoid the influence of other factors, olive trees were cultivated in the same orchard under identical agronomic (i.e., fertilization or irrigation) and pedoclimatic conditions and the olive fruits were picked at the same stage of ripeness and the oils were extracted with the same processing system (Section 3.1.). Therefore, it was possible to attribute the observed results exclusively to different cultivars. Basic Characteristics of the Samples The acidity, peroxide value (PV), K232 and K270 of investigated VOOs were assessed by EU method (Table 1). [12]). From Table 1 it can be seen that Oblica variety exhibited the lowest acidity (0.12%), while the highest value was found in Lastovka oil (0.17%). Statistically significant differences were obtained in free acidity and peroxide value among the oils from the studied cultivars. Although significant differences were determined by Tukey's test, the absolute values of measured acidity of all oils are very similar. Peroxide value should amount less than 20 mEq O2/kg for extra VOO. Determined PVs ranged from 2.96 to 4.00 mEq O2/kg. The lowest PV showed Oblica oil (2.96 mEq O2/kg) and the highest Lastovka oil (5.32 mEq O2/kg). Determined PVs are significantly different in all tested oils, but in absolute values they are very close. K232 value demonstrates conjugated dienes and their oxidation products (absorbtion at λ = 232 nm) and K270 value indicates conjugated trienes and secondary oxidation products (carbonyl compounds; absorbtion at λ = 270 nm). K232 and K270 values ranged from 1.70 to 2.11 and from 0.12 to 0.19 (Table 1). Oblica oil showed the lowest K232 and K270 values while Lastovka oil showed the highest values. The investigated parameters (Table 1) were within the limits of EC Reg. 1989Reg. /2003Reg. (2003 [13] indicating the category of extra VOOs. Headspace Composition Olive oil, compared to other vegetable oils, is distinguished by a characteristic aroma. The sensory characteristics, together with high nutritional value are the main features that have resulted in the increase of VOO consumption in recent years [14]. Aroma is an important criterion for VOOs. Consequently, the identification of the compounds contributing to the aroma is considered as a key for quality and authentication control [15]. The headspace profile of tested monovarietal oils was dominated by C6 volatile organic compounds, mostly aldehydes ( Table 2). The C6 compounds responsible for green and fruity VOO perception are produced through lipoxygenase pathway during the olive fruit crushing and malaxation and incorporated into resulting oil. The most abundant was (E)-hex-2-enal (up to 63.8%, 69.8% and 63.6% respectively). (E)-Hex-2-enal was the most important positive contributor of lawn perception [9]. Considering other C6 compounds it can be seen that (Z)-hex-3-enal was not found in Lastovka oil, while Levantinka oil did not contain hexanal. Different values of identified C6 aldehydes in the samples could be due to different acyl hydrolase activity and consequently good or poor availability of free polyunsaturated fatty acids [16]. The results are in accordance with previous research [17] confirming that the cleavage by heterolytic hydroxydeperoxide lyase was the most important process (higher abundance of C6 compounds in comparison with C5 metabolites). The representatives of C5 compounds (Table 2) were pent-1-en-3-one, pentan-3-one and (E)-pent-2-enal. The percentage of pent-1-en-3-one is positively correlated with bitter taste of VOO [18]. In Lastovka oil the level of this compound was the highest (3.57%), while Oblica oil exhibited the lowest value (1.67%). In contrast to Oblica and Lastovka oils, only Levantinka oil contained (E)-pent-2-enal which can be useful potential biodiversity marker. Several hydrocarbons (such as α-copaene, trans-β-ocimene and two isomers of 3-ethyloct-1,5-diene) were found ( Table 2). trans-β-Ocimene was not identified only in Levantinka oil, whereas, α-copaene was identified in Oblica and Levantinka oils. 3-Ethyloct-1,5-diene isomers were detected in all the oils. Statistically significant differences were found almost among all identified compounds of Oblica, Lastovka and Levantika VOOs. According to [19] the most common parameters that influence the composition of VOO volatiles are: variety, growing area, the degree of maturity of fruits, harvesting method, storage conditions, storage of olive fruits after harvest, processing of fruits into oil, others. The olive fruits of different varieties grown in the same area give VOOs of different volatiles composition, but similar results were found for VOOs of the same varieties grown in different geographic areas [19] Brkić Bubola et al. [20] investigated volatiles and sensory characteristics of the oils from three Istrian (Croatia) olive varieties (Buza, Črna and Rosinjola) and found that the volatiles generated depending on the varieties which indicated a close relation with the activity of a genetically determined enzyme. Targeted Tocopherols Analysis and Total Chlorophylls and Carotenoids Tocopherols are considered as the most important lipid soluble natural antioxidants and they increase oxidation stability of the oils during storage [14]. The amount of their main component, α-tocopherol, varies by up to 300 mg/kg [21]. The concentration of β-, γ-and δ-tocopherols range from traces up to 25 mg/kg. A synergistic relationship between the antioxidant actions of some phenolics and tocopherols was demonstrated [14]. It is well accepted that tocopherols content seem to be reduced during the fruit's ripening, refining and hydrogenation process [22]. The levels of tocopherols, chlorophylls and carotenoids determined in the oils from all varieties are shown in Table 3. The results showed the dominance of α-tocopherol in all studied oils, followed by γ-tocopherol as expected for a typical VOO. Levantinka oil was characterized by the highest level of α-tocopherol (222.0 mg/kg), while significantly the lowest value was found in Lastovka oil (177.82 mg/kg). Considering γ-tocopherol, significantly the lowest value was measured in Lastovka oil, whereas Oblica and Levantinka oils contained higher values (33.19 and 31.84 mg/kg, respectively). The obtained results are in accordance with our previous paper [23] on Croatian varieties Krvavica and Mašnjača as well as by Ranalli et al. [24] on Italian varieties Leccino, Frantoio and Moraiolo and Douzane et al. [25] on several Algerian varieties. The results agree with Perrin [26] findings that tocopherols concentration is generally greater than 100 ppm in good quality oils with α-tocopherol representing about 95% of the total fraction. The color intensity of the oil is determined by changes in the source fruits' pigment content during ripening. Although the pigment concentration in the fruit differs greatly with the variety, it decreases with ripening when chlorophylls disappear faster than carotenoids [27]. Chlorophyll and carotenoid pigments greatly influences the color of VOOs ranging from green-yellow to golden, depending on the variety and the stage of maturity [28]. As shown in Table 3, significant differences among cultivars (p < 0.05) were observed in α-tocopherol and γ-tocopherol among Lastovka VOO in comparison with Oblica and Levantika VOOs. The results show significantly higher total pigment content (chlorophylls + carotenoids) in Lastovka oils, with mean value of 6.81 mg/kg. Oblica and Levantinka oils contained total pigments in the amount of 5.96 and 5.86 mg/kg respectively. Chlorophylls and carotenoids ranged, respectively from 3.86 to 4.75 mg/kg and from 1.89 to 2.06 mg/kg. These findings are in agreement with previous results [29]. Moreover, Giufrida et al. [30] reported that the presence of the pigment in the oil depends on several factors, such as the cultivar, soil and climatic conditions, fruit ripeness and the processing procedures. Data on the pigments composition of those Croatian olive oils could be used, along with other parameters, to guarantee the genuineness and authenticity of the products. These compounds also exhibit biological and health properties and occur in the oils at concentrations which usually correlate with those of phenols and volatiles [31]. Total Phenolic Amounts and Targeted Phenolic Analysis The phenolic composition of olive oil is very complex and the average concentration of these compounds depends on several factors including maturation stage, part of the fruit, variety, season, packaging, storage, climatologic conditions and the production technology [14]. VOO is well known for its high content of phenolics along with oleic acid and tocopherols that exhibit health-promoting properties [32]. Phenolic compounds influence the sensory properties (flavor, bitterness, etc.) of olives and the oil, and they protect against oxidative rancidity by acting as antioxidants, which are increasingly being recognized by playing a beneficial role in the diet [33]. Significant differences in total phenols (TPs) content among several HFs of the samples were observed (Table 4). Oblica oil exhibited the highest TPs amount (212.21 mg/kg), while Levantinka oil showed the lowest TPs content (144.60 mg/kg). Lastovka oil was quite similar to Oblica oil considering TPs amount (206.09 mg/kg). It is generally accepted that the TP's level varies in the oils obtained from different cultivars and areas. Discrimination among the olive oil samples with the same geographical origin and different cultivars was possible by comparing TPs [32]. Another authors [34] claim that phenolic composition was found to be not useful in discriminating the olive oil samples due to the fact that TPs content of the oils was affected not only by the olive cultivars, but also by the climatic and environmental conditions, agronomic practice and the technological process. TPs content ranged from 50 to 1.000 mg/kg, but the values usually amount from 100 to 300 mg/kg [35] that is similar to the values found in the investigated samples. TPs content of the samples in this study could be considered as medium-high levels in accordance with previous reports [23,32,34,36]. Olive oil hydrophilic extracts contain a large number of phenolic compounds including simple phenols, lignans, and secoiridoids, which exhibit antioxidant properties [37]. Among the phenolic compounds found in extra-virgin olive oils, o-dihydroxyphenolics are very potent antioxidants [38]. Free radicals are generated in the human body through aerobic respiration and exist in different forms, including superoxide, hydroxyl, hydroperoxyl, peroxyl and alkoxyl radicals. Natural antioxidant enzymes in healthy individuals remove these free radicals while dietary antioxidants assist the body in neutralizing free radicals. Therefore, it is important to consume foods with high contents of antioxidants, such as virgin olive oil, to reduce the harmful effects of oxidative stress [39]. Antioxidant activity measured by DPPH assay showed the highest value in hydrophilic fraction of Oblica oil (0.91 mmol TEAC/kg) and statistically significant lowest value in Levantinka oil (0.55 mmol TEAC/kg). The difference in antioxidant activity of tested oils may depend on the total phenol content in the varieties. Most of the previous studies reported strong correlation between total phenols and antioxidant capacity [23,37,39,40]. HPLC-DAD was used for targeted analysis of the polar compounds in Oblica, Lastovka and Levantinka VOOs. The most abundant secoiridoids of the samples (Table 5) were dialdehydic form of elenolic acid linked to hydroxytyrosol or tyrosol (p-HPEA) respectively assigned as 3,4-DHPEA-EDA, p-HPEA-EDA and 3,4-HPEA-EA. Statistically significant differences among phenolic levels were observed for the oils. Levantinka and Oblica VOOs were characterized statistically by highest amount of p-HPEA-EDA. Lastovka VOOs contained the highest amount of 3,4-HPEA-EA. 3,4-DHPEA-EDA, the dialdehydic form of decarboxymethylelenolic acid linked to hydroxytyrosol, was statistically different in all tested oils with the highest value in Oblica oil (85.0 mg/kg) and lowest value in Lastovka oil (34.4 mg/kg). Flavones, such as apigenin and luteolin, showed in total the highest value in Oblica oil, while the lowest value was measured in Levantinka oil. Lignans, such as pinoresinol, were estimated only in Oblica oil. These results are similar to those reported by several authors for other monovarietal VOOs [31,40,41]. The Samples and Preparing of the Hydrophilic Fractions All the olive oils were produced from the fruits from the Dalmatia region (Zadar hinterland, Croatia) in 2014. The olive trees were cultivated under same agronomic and agrotechnical conditions. 12-year-old olive trees were planted in squares (7 × 5 m spacing) in the same orchard (ca. 2 ha). There was no irrigation. Five batches of the healthy olive fruits (replicates; each with 200 kg of the fruits) were handpicked from each olive variety at the same maturity index (MI = 4.3). MI was calculated as a subjective evaluation of the skin color and flesh as proposed by Uceda and Frias [42]. The fruits of each variety were separately processed in the extraction plant Molinova TG (Gruppo Pieralisi, Pieralisi S.p.A. Jesi, Italy) within 24 h of collection. Before the extraction of each batch, the plant was cleaned. The fruits were crushed with a hammer crusher and olive paste was malaxed for 35 min at 26 ± 1 °C in the mixer. The olive oil was separated by centrifugation through two phase decanter (without addition of warm water). All oils were filtered through 25 mm GD/X 0.45 μm cellulose acetate filters (Whatman, Milan, Italy) and thereafter stored in dark glass bottles at 4 °C until the analyses. Hydrophilic fractions (HFs) of the oils were prepared by adding 5 mL of CH3OH-H2O (80:20 v/v) mixture in 3 g of the oil placed in 20 mL screw cap test-tube. The mixture was blended in an ultrasonic bath for 15 min at 30 °C (the emulsion was allowed to separate). The hydrophilic layer was placed in a round flask. The oil extraction was repeated two times; the hydrophilic extracts were combined and evaporated on a rotary vacuum evaporator at 30 °C. The residue was dissolved up to 5 mL with the 80:20 CH3OH-H2O solution and filtered through a Whatman 13 mm GD/X 0.2 μm cellulose acetate syringe filter (Whatman, Milan, Italy). Determination of the Oils Basic Characteristics Free acidity (% of oleic acid (%18:1)), peroxide value (mEq O2/kg of the oil) and UV absorption characteristics (K232 and K270) were determined according to the European Union Commission Regulations EC 1989 as in our previous paper [23]. K232 and K270 were determined with an UV spectrophotometer (Specord 200, Analytik Jena AG, Jena, Germany) at 232 and 270 nm using 1% solution of the oil in cyclohexane and a path length of 1 cm. All parameters were determined in triplicate for each sample. Headspace Solid-Phase Microextraction (HS-SPME) HS-SPME was performed on SPME fiber (divinylbenzene/carboxen/polydimethylsiloxane; DVB/CAR/PDMS) purchased from Supelco Co (Bellefonte, PA, USA). The fiber was conditioned according to Supelco Co instructions before the extraction. VOO (5 g) was placed in 15 mL glass vial and sealed with PTFE/silicone septa. The vial was placed in a water bath at 40 °C for equilibration (15 min) and extraction (40 min) under constant stirring with a magnetic stirrer (1000 rpm). After sampling, the fiber was inserted into the injector (250 °C) of GC-FID/MS for 6 min for thermal desorption of the volatiles into the GC column. Gas Chromatography and Mass Spectrometry (GC-FID/MS) An Agilent Technologies (Palo Alto, CA, USA) gas chromatograph (7890A) with flame ionization detector, mass selective detector (5975C) and HP-5MS capillary column (5%-phenyl)-methylpolysiloxane Agilent J & W GC column; 30 m, 0.25 mm i.d., coating 0.25 μm) was utilized. Helium was applied as carrier (1.5 mL/min). The injector operated in split mode (2:1 split ratio) at 260 °C. The column was heated at 40 °C for 3 min, thereafter to 100 °C (5 °C/min) and later to 260 °C (3 °C/min) and then held to 260 °C for 3 min. The MS conditions were: source temperature 230 °C; quadrupole at 150 °C; transfer line at 270 °C; EI 70 eV and m/z 29-350. The peaks were identified by comparison of the retention indices with authentic samples (relative to C9-C25 n-alkanes) and literature as well as by comparing their mass spectra with Wiley 9 MS library (Wiley, New York, NY, USA) and NIST08 (Gaithersburg, MD, USA) database. The percentage composition was computed from the peak areas using the normalization method (without correction factors). The component percentages ( Table 2) were calculated as mean values from duplicate GC-FID analyses of each sample. Liquid Chromatography with Diode Array Detector (HPLC-DAD) HF phenolic compounds were detected and quantified with the HPLC-DAD method described by Tuberoso et al. [43]. A ProStar HPLC system (Varian Inc., Walnut Creek, CA, USA) was employed equipped with a pump module 230, an autosampler module 410, a ThermoSeparation diode array detector SpectroSystem UV 6000lp (Thermo Separation, San Jose, CA, USA) and a Gemini C18 column (150 × 4.60 mm, 3 μm, Phenomenex, Casalecchio di Reno, BO, Italy). The mobile phase was 0.2 M H3PO4 (solvent A) and CH3CN (solvent B) at a constant flow rate (1.0 mL/min), mixed (linear gradients) as follows: at 0 min A:B ratio 85:15 (v/v), reaching 60:40 (v/v) in 30 min, then 40:60 (v/v) in 10 min and finally at 100% B until 50 min. Prior to each injection the system was 10 min stabilized with A:B ratio 85:15 (v/v). The injection volume was 10 μL. The phenols analysis was carried out at 280 nm (hydroxytyrosol, tyrosol, vanillic acid, pinoresinol, oleuropein, and ligstroside derivatives) and 360 nm (luteolin and apigenin). Oleuropein and ligstroside derivatives were tentatively identified according to the literature data [44,45]. The obtained chromatograms and spectra were elaborated with a ChromQuest V. 2.51 data system (ThermoQuest, Rodano, Milan, Italy). Stock standard solutions were prepared in CH3OH and working solutions in CH3OH-H2O (80:20, v/v). The method was validated according to the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidance note [46] as previously reported [23]. All compounds were dosed using the calibration curve constructed with corresponding standard, except oleuropein and ligstroside derivatives that were dosed using the oleuropein calibration curve. The correlation values were comprised between 0.9992 and 0.9999. Determination of Total Chlorophylls and Carotenoids The oil solutions of 5% (w/w) in CH3COCH3 were prepared and absorbances were measured (at 464 nm for carotenoids and at 669 nm for chlorophylls) on a 10 mm quartz cuvette utilizing a Varian Cary 50 UV-Visible spectrophotometer (Varian, Leini, TO, Italy). Chlorophyll a and β-carotene stock standard solutions were prepared in CH3COCH3, as well as working solutions that were prepared with proper dilutions (0.1-2.0 mg/kg, r = 0.9997 and 0.02-0.50 mg/kg, r = 0.9994 for chlorophyll a and β-carotene, respectively). Folin-Ciocalteu Assay HF total phenolic content was determined spectrophotometrically with a modified Folin-Ciocalteu method [47]. Shortly, HF (100 μL) was added to Folin-Ciocalteu phenol reagent (500 μL). After 5 min, 3 mL of 10% Na2CO3 (w/v) was added, the mixture was shaken, and thereafter diluted with H2O to a final volume of 10 mL. After a 90 min incubation period at room temperature, the absorbance was read at 725 nm (against a blank) on a 10 mm optical polystyrene cuvette (Kartell 01937, Kartell Spa Noviglio, Mi, Italy) utilizing a Varian Cary 50 spectrophotometer (Varian, Leini, TO, Italy). The total polyphenol content expressed as mg/kg of gallic acid equivalent (GAE) were obtained using a calibration curve of a freshly prepared gallic acid standard solution (5-100 mg/kg, r = 0.9999). DPPH Assay HF antiradical activity was assessed with the DPPH spectrophotometric method and the obtained data were expressed as Trolox equivalent antioxidant capacity (TEAC) [47]. HF (50 μL) was dissolved in 2 mL of 0.04 mmol/L DPPH in CH3OH. Spectrophotometric readings were carried out at 517 nm with a Varian Cary 50 spectrophotometer using 10 mm optical polystyrene cuvette after an incubation period of 60 min in dark at room temperature. A Trolox calibration curve (0.02-1.00 mM) was prepared (r = 0.9997) and data were expressed in TEAC (mmol/kg). Statistical Analysis Statistical analysis by SPSS 17.0 statistical software (SPSS Inc., Chicago, IL, USA) was applied to datasets to perform descriptive multivariate statistical study. The mean values were compared using the Tukey's honestly significant difference test (p < 0.05). Conclusions In continuation of our research on monovarietal extra VOOs from Croatian autochthonous olive varieties, it was established that Oblica, Lastovka and Levantika VOOs exhibited all characteristics within the limits for the extra VOO category. Among the headspace volatiles, the most abundant was (E)-hex-2-enal. (Z)-hex-3-enal was not found in Lastovka oil and was different abundant in Levantika and Oblica oils and therefore useful for their differentiation. Levantinka oil did not contain hexanal. The results showed α-tocopherol dominance in all samples followed by γ-tocopherol. The lowest value of γ-tocopherol was measured in Lastovka oil. Higher total pigment content (clorophylls + carotenoids) was found in Lastovka oil. The most abundant secoiridoids were the dialdehydic forms of elenolic acid linked to hydroxytyrosol or tyrosol, particularly 3,4-DHPEA-EDA, p-HPEA-EDA and 3,4-HPEA-EA, which dominated Oblica oil, and 3,4-HPEA-EA in Lastovka oil. Different abundance of these compounds could be useful to distinguish olive oils from different cultivars.

v3-fos

2019-02-13T14:07:19.655Z

2015-08-30T00:00:00.000Z

60900217

s2

Effect of Magnesium on Biomineralization of Bone Zhaolong Huang a , Shaoping Feng , Ying He , Mengshu Lai , Yan Jiang, Ruiming Xiao, Lida Sun School of Science, Honghe University, Mengzi, Yunnan 661100, P. R. China Key Loboratory of Natural Pharmaceutical & Chemical Biology of Yunnan Province, Yunnan 661100, P. R. China The State Administration for Entry-Exit Inspection and Quarantine of Hekou, Hekou 661300,China zlhuang0132@126.com, shaopingfeng@126.com, heyeng@126.com, Introduction Biomineralization is an important process in the formation of hard tissues in human and other vertebrates. Not only amounts of calcium, phosphorus and collagen are required, but also regulatory protein factors, enzymes and other biological activation component have play an important role on regulating and inducting the process of biomineralization of bone [1,2].About 74% of dry bone is inorganic constituent (principal components, hydroxyapatite, HA) and rest of dry bone is organic matter (principal components, collagen). It is known that there is a close relationship between magnesium ion and many proteins, nucleic acid, enzyme structure, metabolism, but the particular effects which magnesium ion played on bone tissue growth and natural environment biological mineralization of collagen in bone wasn't reported frequently. In literature review,C.M. Serre pointed out that magnesium ion could slow down the degradation of calcium phosphate and high concentration of magnesium ion be poisonous to bone cells [3]. L.P. Zhao reported that magnesium avail fracture healing [4,5]. It was found that magnesium has an effect on the process of collagen self-assembly mineralization and the process was slow down on the condition that the magnesium existed in bone in our study [6]. In this paper, the influences of magnesium ion on calcium, phosphorus levels and inorganic components in chicken bones were studied and some rules were found during chicken growth. Experiment Materials and Methods. Live chicken 27 bought from the local markets, which had grown for one month from hatch, and they were similar each other in age and weight. Other inorganic reagent were analytical reagent. Experiment. 27 chicken was grouped into 3 groups, the first group was normal group (marked NG), the second group was fed solid MgSO 4 (marked SG) and the third group was fed aqueous solution of MgSO 4 (marked LG). SG and LG ate 0.43 gram of magnesium every day. Nine chicken 3rd International Conference on Material, Mechanical and Manufacturing Engineering (IC3ME 2015) which every three chicken come from the same group (NG, SG and LG ) was killed and removed out the bones to test elements every month. Elements analysis provided by Honghe agricultural comprehensive testing center. Result and discussion Variation of calcium content with magnesium in chicken bones. We analyzed the calcium content of chicken bones at 2 months, 3 months and 4 months respectively, and the results were shown in Table 1. As it can be seen from Table 1, the content of bone calcium of the liquid group(LG) fed with the magnesium salt solution is higher than the normal group(NG), while the calcium content of the solid magnesium group(SG) and the normal group(NG) isn't relatively obvious. It showed that the content of bone calcium has raised with the increasing of the content of magnesium, this is being that it is the magnesium salt solution that the chicken can be easy to absorb, while the solid magnesium salt is not obvious, as the overwhelming reason being that absorption of solid magnesium salt might be difficult to the chicken. A possible reason why the calcium content could be increase when chicken have ate additional magnesium salt is that there was an antagonism between serum calcium and serum magnesium(both of them could be form complex compound). The antagonism could accelerate free calcium ion into the bone cell to form HA when the concentration of serum magnesium increased. Another reason was that activity of some coenzyme increased by magnesium, accelerating the transference of calcium. Calcium content in SG was high slightly compared with NG,but it was lower than that in NG when chicken were fed to 4 month, as the reason was not known. The calcium content in LG was always higher compared with SG and the reason explained above. It should be indicated that bone mineral density, bone strength will raise with the increasing of calcium content of bone, this is being that magnesium ion could accelerate the increase of bone mineral density. Variation of phosphorus content with magnesium in chicken bones. The effect of phosphorus content in chicken bones with different magnesium salt was showed in Table 2. 2 showed that the phosphorus content of three groups in the bone raised with the increase of feeding time. In addition, the phosphorus content of the LG was slightly higher than the NG in February and April, the phosphorus content of the SG was significantly more than that the NG at the beginning of feeding time, but was unnatural in the coming time, especially in April,as the reason may be related to poor solid magnesium absorption efficiency. The change of phosphorus content with time for LG showed that feeding magnesium salt was helpful to increase phosphorus content in chicken bones. Variation of molar ratio of calcium to phosphorus with magnesium in chicken bones. The effect of molar radio of Ca and P in chicken bones with magnesium salt was showed in Table 3. As seen from table 4, the molar ratio of Ca to P (MRCP) in the bone decreased with the feeding time in three groups and the decline for LG was slower than other both groups. In the same growth period, the MRCP of two groups of LG and SG were higher than the control group(NG) except the second month, the group of LG was close to the NG and the SG was lower than the NG. It was known that normal inorganic composition in the bones is hydroxyapatite (Ca 10 (OH) 2 (PO 4 ) 6 , marked HA), molar ratio n (Ca)/n (P) is equal to 1.67 in HA. Analysis of growth rule in chicken bones, it was found that MRCP in three groups exceeded 1.67 when feeding for 2 months in three groups, regardless of feeding or not. Generally believed that the new bone may partially exist (CaHPO 4 , the MRCP was 1) in the form of calcium hydrogen phosphate, and then gradually transformed into hydroxyapatite (HA, the MRCP was 1.67), but the old chicken bones calcium phosphorus ratio is higher than that of 1.67, the possibility reason being that calcium compounds of chicken may also exist in other [7]. From the beginning of the third month, MRCP in three groups was below normal value 1.67 and the LG was the maximum value near 1.67. MRCP of LG was reduced to 1.24 which was still higher than the NG (1.19) at the fourth month, while the SG was 1.49, as might be related with feeding solid salt and absorption abnormally. Analysis of the change of MRCP during feeding from 2 to 4 month, variation of MRCP for LG was 1.23 and for NG was 1.33, indicating that bone mineral composition may tend to be more the ACH of the smaller ratio.The constituent in bone is HA when MRCP was 1.67:1, while is ACH when MRCP was 1:1, as is indicating that more HA existed in chicken bones with feeding magnesium salt. Therefore, we conclude that magnesium element could promote the stability of chicken bones and decrease the ratio of HA decomposition. Variation of magnesium content in chicken bones. The variation of magnesium content in the bone was showed in Table 4. As seen from Table 4, magnesium content in the bone for LG and SG was more than NG, and LG was high compared with the SG. It is generally believed that magnesium ion could replace calcium ion at the same position in the bone and cause the change of bone property, but the calcium content in bone also increased, this is being that the increase of magnesium can not enough influences chicken growth of bone calcium content with the the growth of chicken. The reason of the increase of calcium content in chicken bones might be that magnesium can accelerate the activity of some coenzyme in bone and promote the calcium ions migrate into the interior of bone cells, except replacing of calcium ion. It is known that the more of serum magnesium concentration, the higher of carboxyl that was bonded at serum, which weakens the complex of calcium and serum carboxyl, accelerating the migration of calcium ion to bone cells and increasing the content of bone calcium. Summary Magnesium played a role on increasing calcium and phosphorus content in chicken bones. Soluble magnesium salt is better than solid salt to rise calcium and phosphorus content in the bone and the effect on the calcium content is more than phosphorus content. MRCP of chicken bones in three groups decreased with the extension of feeding time and the decline for LG was slower than other both groups. It was speculated that some change process had happened, as CaCO 3 change into HA and HA+ACH, which may be related to the process of growth and faded about the chicken. Although magnesium content in the bone increased with the extension of feeding time after feeding magnesium salt, the effects of magnesium on chicken bones composition were mainly derived from the factors on the role of coenzyme and other biochemical factors.

v3-fos

2017-05-29T11:13:51.281Z

2015-09-07T00:00:00.000Z

691847

s2

Identification and classification of silks using infrared spectroscopy ABSTRACT Lepidopteran silks number in the thousands and display a vast diversity of structures, properties and industrial potential. To map this remarkable biochemical diversity, we present an identification and screening method based on the infrared spectra of native silk feedstock and cocoons. Multivariate analysis of over 1214 infrared spectra obtained from 35 species allowed us to group silks into distinct hierarchies and a classification that agrees well with current phylogenetic data and taxonomies. This approach also provides information on the relative content of sericin, calcium oxalate, phenolic compounds, poly-alanine and poly(alanine-glycine) β-sheets. It emerged that the domesticated mulberry silkmoth Bombyx mori represents an outlier compared with other silkmoth taxa in terms of spectral properties. Interestingly, Epiphora bauhiniae was found to contain the highest amount of β-sheets reported to date for any wild silkmoth. We conclude that our approach provides a new route to determine cocoon chemical composition and in turn a novel, biological as well as material, classification of silks. Summary: FTIR analysis performed on unspun silks and cocoons from 35 different species provides information regarding the biochemical diversity and evolution of this group of materials. INTRODUCTION Silkworm silk is a high-value agricultural product offering sustainable harvesting that directly contributes to poverty alleviation in rural communities (Astudillo et al., 2014;Dooley, 2004). Yet, it also has growing technical applications (Borkner et al., 2014;Omenetto and Kaplan, 2010). Developments in mulberry sericulture and the increasing use of fibres from 'wild' silkworms provide the backdrop for increased interest in understanding the diversity of all silks. Not surprisingly, millions of years of divergent evolution have resulted in a rich biodiversity of silks (Scoble, 1999). Typically used in cocoons, this class of materials consists of a silk fibroin protein thread of up to 1 km long coated with sericin proteins acting as a resin/matrix glue (Chen et al., 2010b). This non-woven composite structure (Chen et al., 2010a) regulates gas flow and humidity (Danks, 2004;Horrocks et al., 2013;Roy et al., 2012), as well as protecting the encased pupae from predation (Ishii et al., 1984), micro-organisms (Franceschi and Nakata, 2005) and the environment (Chen et al., 2012b). Silkworms produce cocoons with a broad variety of morphologies and architectures, ranging in porosity from loose meshes to full shells, with or without an exit opening (Chen et al., 2012c). Cocoons may also incorporate extraneous materials as well, such as integrated leaves for camouflage or an internally applied calcium oxalate solution that hardens the cocoon and may impart toxicity (Arnott and Webb, 2000;Chen et al., 2012c;Franceschi and Nakata, 2005;Gheysens et al., 2011;Takahashi et al., 1969;Teigler and Arnott, 1972b). The diversity of lepidopteran silk materials includes a molecular dimension, with amino acid analysis showing widely varying chemical compositions of silkworm silk (Hwang et al., 2001). However, while more advanced biochemical methods can inform on protein size (Hwang et al., 2001;Inoue et al., 2000;Mita et al., 1994), amino acid residue patterns (Navarro et al., 2008) and propensity to fold (Dicko et al., 2008), they are often labour intensive and expensive. Hence, only a handful of fibroin proteins have been sequenced to date (Tanaka and Mizuno, 2001). Furthermore, these methods are often focused on one specific molecular component of the cocoon and are unable to account for the other compounds present. An alternative approach to achieve a broader assessment of chemical diversity is to employ complementary spectroscopic and scattering techniques (Gheysens et al., 2011;Warwicker, 1954). For example, the use of attenuated total reflection infrared spectroscopy (ATR-IR) is particularly well suited to studying silks in all forms as it is capable of measuring rough and deformable solids (Chen et al., 2012a;Gheysens et al., 2011), as well as turbid and concentrated protein solutions (Boulet-Audet et al., 2011). Requiring only minimal sample preparation, ATR-IR can selectively probe the inside or outside surface of silk cocoons, providing information on the local chemical composition (Boulet-Audet et al., 2014;Chen et al., 2012aChen et al., , 2007. This spectroscopic method can determine (i) the level of protein crystallinity (Boulet-Audet et al., 2008), (ii) secondary structure (Goormaghtigh et al., 2006), and (iii) specific protein components such as sericin (Barth, 2000;Teramoto and Miyazawa, 2003). Infrared spectra are also indicative of (iv) non-protein molecules present in silk, such as the amount of water (Boulet-Audet et al., 2011), calcium oxalate (Chen et al., 2012b;Gheysens et al., 2011) and carbohydrate (Lu et al., 2011;Schulz and Baranska, 2007). In addition, the multivariate analysis of infrared spectra can (v) discriminate and classify samples based on their degree of relatedness. This infrared-based classification approach can even discriminate bacterial species (Kansiz et al., 1999;Preisner et al., 2007), types of human hairs (Panayiotou and Kokot, 1999), and coffee bean varieties (Briandet et al., 1996), as well as providing information for the construction of taxonomic trees (Zhao et al., 2006). In this study, we analysed unspun native silk feedstock from six species across the Saturniini and Attacini tribes, and spun silks from 35 species across the Lepidoptera and Arachnida. Multivariate and hierarchical clustering analysis performed on over 1000 individual spectra allowed us to build taxonomic trees and compare them with trees based on protein-coding nuclear genes (Chen et al., 2012c;Regier et al., 2005Regier et al., , 2008aRegier et al., ,b, 1998Regier et al., , 2002. As we demonstrate below, we identified several interesting outlier species that produce silk with very different chemical compositions and provide a hypothesis as to their origin. These newly characterised silks could even have greater potential for use in industrial and biomedical applications than those currently employed today. Native feedstock spectral features To evaluate the chemical variability of unspun silk without exogenous material, we used infrared spectroscopy to compare the native feedstocks of key species from the Lepidoptera genera Actias, Attacus, Bombyx and Saturnia, with the spider Nephila edulis as the outgroup. Bombycidae feedstocks such as Bombyx mori silk comprise heavy and light chain fibroins as well as P25 linked with disulphide bonds (Chevillard et al., 1986;Mita et al., 1994) in a 6:6:1 ratio (Inoue et al., 2000). In contrast, feedstocks of the Saturniidae such as Antheraea yamamai, Actias luna, Attacus atlas and Saturnia pavonia (Tanaka and Mizuno, 2001) comprise a homodimer (double heavy chain, H-H) protein mixture. As arthropods, spiders are very distantly related to the silkworms, yet by all accounts evolved silk production independently around 400 million years ago (Craig, 1997). Yet, the similar flow properties of their feedstocks thus represent an excellent example of convergent evolution (Craig, 1997;Holland et al., 2006). Fig. 1 illustrates the distinctive features of native silk feedstock infrared spectra between 900 and 1500 cm −1 for silk from a variety of species. Table 1 indexes band assignments. Peaks between 1340 and 1456 cm −1 are commonly assigned to the vibration mode of residues (Barth, 2000). The strong 1383 cm −1 band associated with CH 2 bending for wild silks (the top four curves in Fig. 1) suggests a higher proportion of long-chain residues in feedstocks from B. mori and N. edulis major ampullate silk glands. Another important distinction for wild silk feedstocks is the presence of the well-resolved 1308 cm −1 peak in the amide III region. Monitored by Rheo-IR (Boulet-Audet et al., 2014), this band vanishes under shear-induced denaturation (see supplementary material Fig. S1) and is absent from cocoon spectra. We have assigned this component to β-turns that are precursors to β-sheets formed after spinning (Bandekar and Krimm, 1980;Cai and Singh, 2004;Rousseau et al., 2006). The arginineglycine-aspartic acid (RGD) residue pattern (Sukopp et al., 2002) is believed to procure a greater fibroblast proliferation rate to the wild silkworm Antheraea mylitta compared with domesticated B. mori silk (Minoura et al., 1995;Navarro et al., 2008). Hence, we hypothesized that RDG patterns might contribute to the wild silkspecific vibration mode at 1308 cm −1 . The amide III shoulder at 1270 cm −1 results from α-helices (Cai and Singh, 2004;Krimm and Bandekar, 1986), and also appears stronger in wild silkworm silk feedstock. The neighbouring peak at 1245 cm −1 is commonly assigned to random coil secondary structures (Cai and Singh, 2004;Shao et al., 2005;Taddei and Monti, 2005;Yoshimizu and Asakura, 1990), and is strongest in the non-wild silks of B. mori and N. edulis. While present for all silk feedstocks, the peak at 1165 cm −1 , associated with the stretching of the N-C α bond, is clearly broader for non-wild species, suggesting a wider distribution of conformations. Actias luna is the only species probed that shows a well-resolved peak at 1144 cm −1 . We speculatively assigned this distinct band to the C-O stretching of sericin-like components used as a binding resin/matrix, as this species produces a cocoon with low porosity and high density (Chen et al., 2012c). While this study focused on unprocessed silk, extracting the sericin for further analysis could help to clarify this speculative assignment. The 1103 cm −1 band appeared on all spectra collected, although it was stronger in wild silkworm feedstocks. In the skeletal vibration region, this peak is likely to be caused by the C-C stretching of tyrosine aromatic rings, tryptophan or phenolic compounds (Andrus, 2006;Barth, 2000;Taddei and Monti, 2005). The adjacent component at 1075 cm −1 is present in all silk feedstocks (except sericin-free spider silk) and is also observed in pure sericin spectra, but is strongest in A. luna, thus reinforcing our previous assignment of the 1144 cm −1 peak for this species (Anghileri et al., 2007;Barth, 2000;Gupta et al., 1997). We also assigned the band at 1052 cm −1 to sericin C-O stretching, which is well resolved in most silkworm silks (Gupta et al., 1997;Taddei and Monti, 2005;Teramoto and Miyazawa, 2003). Cocoon silk spectral features Our findings (above) show that silk feedstocks have clear spectral differences between species. Therefore, we must assume that the cocoons produced from these feedstocks would also show variability. Moreover, we would also expect this diversity to increase as construction introduces variables such as the larva's spinning behaviour, other silkworm secretions such as faeces, and exogenous materials such as tannins diffusing from leaves. Previous work has shown that the properties of silk cocoons vary between the innermost and outermost layers (Chen et al., 2012a). Thus, to examine these sources of chemical diversity, we compared the infrared spectra of the Data are for domesticated silkworm silk (Bombyx mori), wild silkworm silk (Attacus atlas, Antheraea yamamai, Actias luna, Saturnia pavonia) and spider silk feedstock (Nephila edulis major ampullate). The 1700-1500 cm −1 region is not shown as little difference between species was observed. Infrared spectra were collected from feedstocks extracted directly from the animal and kept at a native concentration (∼22% dry weight). innermost and outermost layers of cocoons from 34 species of silkworm alongside the spectra of N. edulis dragline silk. Because of silk's molecular alignment, spectra will vary depending on the orientation of the fibres relative to the beam path (Boulet-Audet et al., 2008;Papadopoulos et al., 2007). For a fair comparison against cocoons without preferential orientation (Chen et al., 2012b), spider silk filaments were arranged in a similar random orientation order. Fig. 2A shows spectra acquired from the innermost part of the cocoons from selected distinctive species. While the primary constituent of these cocoons is still silk proteins, the cocoons' infrared signature differed substantially from that of their respective feedstocks. We assigned these differences to a number of causes: the water content is lower in cocoons, reducing the ratio of amide I to amide II height (1642/1508 cm −1 ); and precursor helical structures and random coils present in the feedstocks are converted via spinning into β-sheets, resulting in decreasing absorbance at 1642, 1547, 1308 and 1245 cm −1 and rising absorbance at 1699, 1620, 1508, 998 and 961 cm −1 (see Tables 1, 2). The relative absorbance of these β-sheet peaks can serve as an indicator of protein crystallinity. The peaks at 1699 and 1620 cm −1 in the amide I region are commonly used to determine the anti-parallel β-sheet content, but this overlaps with adjacent components from the fibroin and other compounds present in cocoons. In contrast, the lowfrequency component at 961 cm −1 assigned to poly-alanine (A) n is much better resolved (Moore and Krimm, 1976;Papadopoulos et al., 2007;Taddei and Monti, 2005). This band also appears in the N. edulis dragline and most spectra of wild silk cocoons, particularly that of Epiphora bauhiniae. In contrast, some species like B. mori and Anaphe panda have two weaker peaks at 998 and 975 cm −1 as their β-sheets are constituted instead of poly(alanine-glycine) segments (Moore and Krimm, 1976;Taddei and Monti, 2005). Fig. 2B demonstrates that the distinctive spectral features observed in the innermost layer of the cocoons are even more prominent in the outermost layer. For example, there is a higher relative absorbance of bands between 1395 and 1058 cm −1 , which is consistent with the greater amount of sericin in the outermost layer for species like B. mori. For comparison, a pure spectrum of sericin is included in Fig. 2A (Chen et al., 2012a). Another clear difference between the two layers is the amount of calcium oxalate [Ca (COO) 2 ] crystals found embedded in the outermost layer of some species (Freddi et al., 1994(Freddi et al., , 1993Gheysens et al., 2011;Takahashi et al., 1969;Teigler and Arnott, 1972b). Calcium oxalate vibration modes at 1315 and 779 cm −1 dominate the outermost layer of Gonometa postica cocoons yet are much weaker in the innermost layer. Another spectral contrast between layers was found in Opodipthera eucalypti, where the shoulder at 1733 cm −1 can be assigned to the carboxylic acid and the polyphenol hydroxyls around 1000 cm −1 (Andrus, 2006;Silverstein et al., 1981). Bandekar and Krimm, 1979;Garside et al., 2005;Miyazawa and Blout, 1961;Moore and Krimm, 1976;Miyazawa, 2005 1642 Amide I, unordered All silks Boulet-Audet et al., 2008;Goormaghtigh et al., 2006;Jeong et al., 2006;Venyaminov andKalnin, 1990 1620 Amide I, β-sheets All spun silks *E. bauhiniae -Audet et al., 2008;Moore and Krimm, 1976;Sonoyama andNakano, 2000 1547 Amide II, unordered All silks Boulet-Audet et al., 2008;Goormaghtigh et al., 2006;Jeong et al., 2006;Venyaminov andKalnin, 1990 1516 Tyr Anghileri et al., 2007;Barth, 2000;Teramoto and Miyazawa, 2003 1383 δ(CH 2 ), (AG) n All unspun silks *A. attacus *S. pavonia Barth, 2000;Moore and Krimm, 1976 1370 δ(CH 2 ), (AG) n All spun silks *E. bauhiniae Barth, 2000;Moore and Krimm, 1976 1340 Thus, in summary, we established that it is possible to use structural and chemical markers to determine the type of crystallinity, the presence of sericin and calcium oxalate, and the polyphenol content in the measured cocoons. Calcium oxalate mineral crystals Calcium oxalate, also called raphide, forms highly toxic needle-like crystals, which can tear soft tissues and are thought to represent a plant defence mechanism (Arnott and Webb, 2000). Because no known metabolic pathways process calcium oxalate in silkworms, we assume that calcium oxalate presence in the cocoon is a result of the ingestion of leaves containing the compound and resultant excretion by the silkworm. While this may be the case for wild silkworms, it appears that artificial selection has changed the behaviour of the B. mori silkworm to prevent this excretion into the cocoon. Fig. 3A shows the relative intensity of the band at 779 cm −1 achieved by integrating the absorbance between 740 and 800 cm −1 . This well-resolved band was used as a relative indicator of the amount of microscopic calcium oxalate monohydrate [Ca(COO) 2 ] mineral crystals present in the cocoons (Chen et al., 2012b;Gheysens et al., 2011). Our ATR-IR results identified cocoons from G. postica as having the highest calcium oxalate content. The host plant of G. postica, Acacia, is also rich in calcium oxalate as a means to detoxify calcium ions (Franceschi and Nakata, 2005;Martin et al., 2012;Teigler and Arnott, 1972a). The high calcium oxalate content of G. postica and Antheraea genera cocoons measured is in agreement with previous reports and electron microscopy observations on these cocoons (Chen et al., 2012b;Gheysens et al., 2011). Cocoons from Samia, Hylophora and Attacus species also indicate the presence of calcium oxalate, but in lower proportions while other species measured have only minute amounts in their cocoons. The presence of calcium oxalate in the cocoon is known to complicate the industrial reeling as it prevents the extraction of long lengths of fibre (Gheysens et al., 2011). Calcium oxalate is notoriously toxic to humans and responsible for kidney stone formation (Evan et al., 2007). The commonplace edetic acid (EDTA) treatment for dissolving kidney stones was found to be equally effective at demineralizing wild silk cocoons containing calcium oxalate crystals and enabling industrial processing (Gheysens et al., 2011). Thus, the ability to detect and quantify the amount of calcium oxalate present in a cocoon prior to processing may have industrial advantages in minimizing reagent use or in selecting low mineral content cocoons in the first place. β-Sheet crystallinity X-ray scattering initially showed the presence of β-sheet nanocrystals inside silk fibres (Warwicker, 1954). Conveniently, polyalanine (A) n and polyalanine glycine (AG) n β-sheet structures also give distinctive peaks in silk infrared spectra, indicative of the degree of crystallinity present, and by extension may relate to mechanical properties (Boulet-Audet et al., 2008;Moore and Krimm, 1976;Porter et al., 2005;Sonoyama and Nakano, 2000). Using the integrated absorbance of (A) n antiparallel β-sheets peaking at 931-983 cm −1 , Fig. 3B shows the relative (A) n β-sheet content across the species tested. Our results suggest that E. bauhiniae has the highest degree of crystallinity amongst all the (A) n -containing silks measured, followed by species from the Samia, Antheraea and Attacus genera. For most species, the (A) n β-sheet content appears greater in the innermost layer, probably due to non-fibroin compounds contributing to the infrared signal more on the outermost layer. Spider silk dragline from N. edulis appears to have a comparable (A) n β-sheet crystallinity to that of most silkworm silks measured. Gonometa, Argema and Caligula genera seem to have the lowest (A) n β-sheet content amongst all the species studied. Integrating the region between 984 and 1006 cm −1 quantified the contribution of the (AG) n peaks at 975 and 998 cm −1 while excluding the (A) n β-sheet peak at 961 cm −1 . Only three species appear to have (AG) n repetitive segments, B. mori, Bombyx mandarina and A. panda (see Fig. 3C). Unlike Bombycidae and Noctuidae families, none of the Saturniidae cocoons displayed peaks associated with the (AG) n structure (Moore and Krimm, 1976;Taddei and Monti, 2005). This fundamental distinction could be related to their appurtenance to different taxonomic families (see below). Tannins and phenolic compounds Wild silkworms naturally secrete some phenolic compounds in their silk (Brunet and Coles, 1974), but our results suggest that additional hydroxyl-containing compounds, such as polyphenols, could come from exogenous sources. By integrating the absorbance between 1035 and 1094 cm −1 , the relative amount of these molecules can be estimated. Fig. 3D shows that a few species had phenolic compounds, located mainly on the outside of the cocoon, including O. eucalypti, Saturnia pyri, Hyalophora gloveri, Attacus edwardsii, Antheraea polyphemus and A. luna. This finding agrees with the hypothesis that leaves incorporated by the silkworm into the cocoon structure leech water-soluble plant polyphenols when wet. In contrast, species that do not integrate leaves into their cocoons, such as A. mylitta and A. atlas, showed low phenolic compound parameter scores. Sericin protein gum Sericin proteins are essential to cocoon construction as they are used to bond fibres together (Chen et al., 2012a). The amount of sericin present in a cocoon can be inferred from the absorption bands between 1384 and 1403 cm −1 associated with the amino acid serine, which is present in high quantities in sericin but not in fibroin (Teramoto and Miyazawa, 2003). Fig. 3E suggests that Bombyx genus silks have the most sericin along with Actias, Antheraea, Saturnia and Samia genera silks. Our results indicate that there is less sericin in the coats of the high-porosity cocoons of species such as Cricula trifenestrata, Graellsia isabellae and Loepa katinka (Chen et al., 2012b). Differences in sericin abundance between the innermost and outermost layers of the cocoons tested indicate that B. mori cocoons have more sericin in the outermost layer, consistent with previous findings (Chen et al., 2012a). However, because of the additional mineral and phenolic components of the wild silks, it is challenging to interpret the distribution reliably in the other silks tested. Classification of silk species Our results show that the integration of infrared spectra bands assigned to individual compounds can provide select windows into a silk cocoon's chemical composition. However, single variable analysis exploits only a small fraction of the information contained within the spectra with thousands of data points. In contrast, multivariable analysis is far more powerful for classifying and discriminating samples. Hence, we first performed a principal Bandekar and Krimm, 1980;Krimm and Bandekar, 1986;Rousseau et al., 2006Rousseau et al., 1270 Amide III, α-helices All silks Cai and Singh, 2004;Bandekar, 1986 1237-45 Amide III, random coil All silks Cai and Singh, 2004;Shao et al., 2005;Taddei and Monti, 2005;Yoshimizu andAsakura, 1990 1217 Amide III, β-sheets All spun silks *E. bauhiniae component analysis (PCA) (Pearson, 1901) to reduce the number of variables while retaining most of the variability. The first principal component (PC) expresses the largest variance between samples. The PC scores, indicating the relative importance of these PC for each spectrum, were subsequently used for the linear discrimination analysis (LDA) to model the differences between species with a set of factor coefficients and scores. The LDA scores were able to discriminate broadly between wild and domesticated silks as well as spider silk. Of the 25 measurements selected randomly for validation, the method identified the correct species for 100% of the 'unknown spectra' (see Materials and methods). Supplementary material Fig. S2 shows the tree generated from the LDA scores using hierarchical clustering analysis (HCA). However, as previously noted, once the silk has been spun into the cocoon structure, even more variables are introduced, and thus our multivariate approach becomes even more powerful. The multivariate analysis had an identification hit rate of 70% for species and 75% for genus, tested using the randomly selected validation group of 200 'unknown spectra' (see Materials and methods). Our initial multivariate analysis of cocoon diversity is summarized in Fig. 4, which highlights the values of the first and second factor scores calculated from the cocoon spectra. The primary cluster encompasses most silks from wild silkworm species with Antheraea silks near its centroid (green markers). Antherina suraka, L. katinka, E. bauhiniae and Samia cynthia silks appear in the periphery of the cluster, suggesting a greater dissimilarity with the average of the measured silks. Clearly discriminated species outside this cluster such as A. panda, B. mori and B. mandarina appear as outliers. The N. edulis spider dragline silk is also outside the primary cluster, and easily discriminated from silkworm cocoons with the second factor scores. Notably, our LDA implies that E. bauhiniae is the silkworm species producing the closest silk to the N. edulis dragline. However, more species from other families would need to be studied to identify which of the thousands of silkworm species spins 'spider silk'. To develop this analysis further and begin to draw quantitative links between species, our HCA used the scores of the 10 most important factors to group these species according to their similarity. 1135 cm −1 (Fig. 3D), this result suggests that these species were grouped together partly based on their high phenolic content. Except for C. trifenestrata, these species' cocoons appeared substantially tanned with a dark brown coloration (Chen et al., 2012c). Also, these species do not present calcium oxalate crystals on their surface (Chen et al., 2012c), as confirmed in Fig. 3A. Group 1 also appears to have a lower β-sheet content than the other groups. Group 2: Argema Group 2 contains the Argema genus. Unlike cocoons from group 1, they do not appear to have a high phenolic content or calcium oxalate, and although largely comparable to neighbouring groups these two factors could explain the large Euclidean distance from group 1. Group 3: Antheraea From our classification, it appears that Antheraea silks all have small Euclidean distances relative to one another and as such they were all grouped together with A. suraka in group 3. Previous studies based on morphological feature classification argued that A. suraka could be more closely related to the African Bunaeini tribe than other species of the Saturniini (Oberprieler, 1997). The comparable Euclidean distance between A. suraka and Antheraea frithi weakens this hypothesis. As Antheraea is the genus with the most calcium oxalate (Fig. 3A), the LDA method could have regrouped these silks mainly on mineral content. However, A. suraka and A. frithi show less absorption between 740 and 800 cm −1 (calcium oxalate) and are more distant from the other species of this group. In addition, group 3 has weaker phenolic compound bands than group 1 and yet has an average amount of sericin. The next closest species to these groups are L. katinka and G. isabellae, which both present a low sericin content and high porosity according to other studies (Chen et al., 2012b,c). Group 4: Attacus and Samia Much more distant are the species classified in group 4, including Samia, Hyalophora and Attacus genera along with Callosamia promethea. When compared with previous reports, the morphology of the cocoons classified together in group 4 appears characteristic as the innermost layers are much more compact than their outer layers (Chen et al., 2012c). This morphological difference could explain the higher amount of sericin measured in the innermost layer (Fig. 3E). Adding to the composition variation between the innermost and outermost layers, these cocoons have an intermediate content of (A) n β-sheets, sericin and tannin when compared with those from groups 1 and 3. Branching from group 4, E. bauhiniae has the largest amount of (A) n β-sheets, little phenolic compounds, no calcium oxalate and little sericin. Group 5: Bombyx The LDA placed B. mori and B. mandarina silks into a distant group. Even though B. mandarina appears to have more (AG) n β-sheets than B. mori (Fig. 3C), the difference between their spectra is subtle in comparison with the other species presented. This result suggests that the artificial selection of B. mori might have played a lesser role than natural selection in differentiating this species from other Lepidoptera families. Silk from other superfamilies While still very distant, A. panda had the smallest Euclidean distance to Bombyx. Our results suggest that A. panda also has (AG) n β-sheets, sericin and no calcium oxalate. Testing more silks from these two families would confirm whether these silks share the same spectral features. Social spinning behaviour is another interesting characteristic of A. panda, which partners with many other worms to build a communal cocoon nest (Mbahin et al., 2007). The cocoon's structure does not depend on the silk quality of a single individual, resulting in different natural selection constraints. Also from another superfamily (Lasocampiadae), G. postica silk is very distinct from all other species studied. The innermost layer appears closer to Saturniidae silks (Fig. 2), whereas the major difference between their outer layer is likely to come from the large amount of calcium oxalate present. Comparison of ATR-IR and phylogenetic trees The ultrametric tree generated from the infrared spectra (see Fig. 5A) was compared with the phylogenetic tree built from the sequencing of a few protein-coding nuclear genes by Regier et al. (2008aRegier et al. ( ,b, 2002; see also Chen et al., 2012c). The genes selected to construct this phylogeny produce proteins other than silk, with various enzymatic functions such as carbamoylphosphate synthetase, aspartate transcarbamylase, dihydroorotase (Moulton and Wiegmann, 2004), dopa decarboxylase (Fang et al., 1997), enolase (Farrell et al., 2001) and wingless (Brower and DeSalle, 1998). Although a quantitative comparison between an ultrametric and a unitless tree is not possible, they are strikingly similar, except for few species. DISCUSSION By assessing the diversity of wild silks, this study compared the biochemical composition of native silk feedstock from six species and silk cocoons from 34 species using infrared spectroscopy and 4. Factor scores of the cocoon spectra. The first and second factor scores contribute to 62% of species discrimination of the linear discriminant analysis. multivariate analysis. For unspun native silk feedstocks, we identified new spectral markers unique to wild silkworm silks, which we assigned to β-turn secondary structures. The hierarchical clustering of the feedstocks also profiled the dissimilarity of Saturniidae silks to the silks of Bombycidae and spiders. Collecting spectra from silkworm cocoons provided information not only on the spun fibre but also on the non-protein chemical content and distribution across the layers. The specific infrared bands revealed the relative content of sericin, calcium oxalate, phenolic compounds, (A) n and (AG) n β-sheets. The multivariate analysis also permitted the hierarchical classification of 35 species (including one spider silk) into groups based on their chemical composition. This analysis revealed the presence of interesting outlier species with very dissimilar spectra, which could manifest as distinctive mechanical or chemical properties. Amongst these outliers were G. postica cocoons, which had the highest calcium oxalate of all species measured. Furthermore, the species with the most β-sheets, E. bauhiniae, also appeared to have the closest chemical composition to N. edulis spider silk dragline. The Bombyx genus stood out from all other species measured, representing an outlier group. Consequently, using B. mori as the model species for silk studies could lead to conclusions that are not applicable to all types of silks. Although our sampling had a bias towards Saturniidae silk, Antheraea silks were found to have median PC scores, suggesting that Antheraea silks are more representative of silk biodiversity. Not only did the multivariate analysis have a species identification hit rate of 70% but also the ultrametric trees were created from the infrared spectra. Our analysis thus suggests a relationship between non-silk coding nuclear genes selected by Regier et al. and the silkworm cocoon's overall biochemical composition (Regier et al., 2005(Regier et al., , 2008a(Regier et al., ,b, 2002. Such a link implies that infrared spectra could be used as a proxy for the phylogenetic classification of species. Despite huge similarities between these trees, a few silk species were classified differently under these two approaches. This difference could be the result of non-protein-based variation such as temperature or humidity or the incorporation of exogenous material into the cocoons. For instance, C. trifenestrata was expected to be closer to the Antheraea silks rather than classified into group 1. The difference could be due to the fact that C. trifenestrata lives in an environment with a warm climate, requiring more ventilation than Antheraea silk cocoons found in colder regions (Kakati and Chutia, 2009). Interestingly, G. isabellae silk should have been very similar to Actias silks, but was classified by our analysis outside group 1 along with L. katinka. Their separate classification could result from the high concentration of tannins measured in the cocoons of these species. Oberprieler and Nassig (1994) suggested that these species might have been misclassified, and our study strengthens the hypothesis that Graellsia and Actias are two distinct genera. As expected, E. bauhiniae was classified in the Attacini tribe but is rather distant from the other species of group 4, most likely because of its higher (A) n β-sheet crystallinity content. In summary, despite minor differences in the classifications, our method represents a powerful but straightforward hierarchical classification tool to help resolve some of the ambiguity in the relationships of Lepidoptera species. 0 400 800 1200 1600 Dissimilarity (Euclidean distance) B A Because of the intense selection pressure on this vital biological structure, we believe silk cocoons represent a model for the phylogenetic analysis of all silkmoth species. This untapped proxy method not only adds to more traditional gene and protein sequencing but is also less time consuming and cheaper than, for example, protein sequencing. As silk cocoons are commonly part of entomology collections spanning hundreds of years of sampling, they can be readily sourced and rapidly tested in a non-destructive manner. Such powerful longitudinal studies could shed light on silkmoth evolution and ecology by helping to resolve some of the relationship ambiguities of Lepidoptera species. Furthermore, with the advent of affordable handheld IR instruments, our approach could also allow such analysis to take place in the field. Thus, combining ATR-IR with multivariate analysis could aid in unravelling the evolution and biodiversity of silk-producing species as well as inform us regarding which species is best suited to a particular industrial application. Native silk feedstock preparation All wild silkworm eggs were purchased from Worldwide Butterflies (WWB, Dorset, UK). Actias luna, A. yamamai, A. atlas and S. pavionia were fed with walnut (Juglans regia), hawthorn (Crataegus monogyna), privet (Ligustrum vulgare) and hawthorn (C. monogyna), respectively. When larvae started spinning their cocoon, native silk feedstocks were extracted from the silk glands of last instar silkworms. Final instar B. mori worms were fed with white mulberry leaves (Morus alba). Nephila edulis major ampullate glands and dragline were extracted from mature female spiders fed with Drosophila spp. and Caliphora spp. and reared in-house under controlled temperature and humidity as described elsewhere (Holland et al., 2006). Silk cocoon preparation We analysed cocoons from 34 species across the Lepidoptera. The superfamilies Saturniini and Attacini are highlighted in Fig. 6. The International Centre of Insect Physiology and Ecology (icipe; African Insect Science for Food and Health) in Kenya provided G. postica silkworm cocoons. The other cocoon species were purchased from WWB; the species chosen cover four Lepidopteran families. At least four, 3.5 mm round cocoon discs were cut from each cocoon using a plier punch for analysis by infrared spectroscopy. Spectral acquisition and treatment A Golden Gate single bounce diamond ATR accessory (Specac Ltd, London, UK) coupled to a Nicolet 6700 FTIR spectrometer equipped with a MCT nitrogen cooled detector (Thermo Scientific, Madison, WI, USA) was used for spectra collection. Spectra acquisition was performed at a 4 cm −1 resolution from 500 to 6000 cm −1 , averaging 32-64 scans at a 5.06 cm s −1 mirror speed. Although the fibres in a cocoon sample are randomly oriented, spectra were collected with the infrared beam polarized perpendicular to the plane of incidence (s) with a zinc selenide holographic wire grid polarizer (Thermo Scientific). The ATR diamond's internal reflection element (IRE) had a refractive index of 2.417 and an angle of incidence of 45 deg. For this configuration, the evanescent wave emerging out of the IRE could probe around 1.2 μm deep into the sample, the penetration varying with the wavelength (Harrick, 1967). The liquid state of native feedstock spectra ensured a good contact with the IRE for data collection. As the cocoons have an inherent roughness greater than tens of micrometres, an anvil was used to press on the cocoon discs to ensure a good contact with the IRE. For acquisition consistency, the pressure applied on cocoon discs was kept to the minimum necessary to obtain an absorbance of 0.1 for the amide II band. By aiming to keep the absolute absorbance consistent, the anomalous dispersion of the refractive index was therefore comparable for each spectrum collected (Boulet-Audet et al., 2010). Before each measurement, the crystal was cleaned with tissue and demineralized water before a new background was acquired. This method helped to compensate for the detector's signal fluctuations as well as preventing contamination between measurements. The innermost and outermost layers of these discs were measured by collecting at least 18 distinct spectra from each species for a total of 1185 spectra across the 35 species studied. This spectroscopic study thus encompasses the largest number of wild silk types to date. Data pre-processing Spectral operations were performed using OMNIC 7.3 (Thermo Scientific) using a custom-written VBA code. An offset was first subtracted from all spectra as calculated from the average of the region from 1950 to 1900 cm −1 . Spectra were then normalized using the integrated absorbance from 1900 to 800 cm −1 to compensate for absolute signal variations incurred by differing cocoon contact with the IRE. For the single component analysis, the relative area of each peak integrated was calculated by subtracting a linear baseline between the interval limits from the integrated absorbance. Multivariate analysis and dendrogram generation Despite high spectral reproducibility, the absolute absorbance values vary between measurements depending on the contact between the porous Bombycoid complex Bombycoidea Bombycidae (2) Sphingidae Saturniidae Attacini (9) Saturniini (21) Regier et al. (2008a). Numbers in parentheses represent the number of species measured in the subfamilies. The images on the right are the cocoons of the species measured. cocoon and the IRE. To discriminate silks based on their spectral line shape and peak position rather than absolute absorbance values, the multivariate analysis was performed on the first derivative of the spectra. The second derivative is as effective, but enhances the noise further (Kansiz et al., 1999). Mid-infrared spectra contained 2853 variables, but were not all interdependent as a single compound spectral line often contributed in different regions simultaneously. To reduce the number of variables while preserving most of the dataset variability, a Pearson PCA (Pearson, 1901) was performed using XLSTAT (Addinsoft, Paris, France). Keeping only 10 PCs still preserved 90% of native silk feedstock spectral variability, while selecting the first 40 PCs still accounted for 86% of cocoon spectral variability. A LDA (Fisher, 1936;Yang et al., 2005) was performed on the PC scores to find a linear combination of features that separate infrared spectra from silks of different species; 27 of 52 native silk spectra and 962 of the 1162 cocoon spectra were randomly selected for the training (estimation) group to construct the discrimination function. The remaining 25 native silk and 200 cocoon spectra were used to validate the discrimination function. The LDA achieved a hit rate of 100% for native silk and 70% for cocoon spectra while assigning 75% to the correct genus. The LDA factor centroid scores were subsequently used to calculate the Euclidean distance between each species for Ward's HCA (Mariey et al., 2001;Ward, 1963). This method minimizes the total variance within clusters starting from singleton clusters (one species per cluster) in a topdown approach. The resulting HCA dendrograms were subsequently compared with the phylogenic tree dendrogram built from genetic data (Chen et al., 2012c;Regier et al., 2005Regier et al., , 2008aRegier et al., ,b, 2002. Supplementary material Fig. S1 shows the infrared spectra before and after shear-induced denaturation of B. mori and A. atlas along with their corresponding difference spectrum. Supplementary material Fig. S2 shows the ultrametric tree generated from the infrared spectra of native feedstock primary canonical functions. Supplementary material Fig. S2 shows the tree generated from the infrared spectra of native feedstock main canonical functions using HCA. Sharing common spectral features such as the 961, 1103 and 1308 cm -1 , feedstocks from A. luna, S. pavonia and A. atlas are more closely related. This result corroborates the fact that these four species are from the same Saturniidae arthropod superfamily. Although much more distant, the closest to these species is the silkworm silk B. mori feedstock as it is also a silkworm silk feedstock containing sericin proteins. With fewer types of fibroins and no sericin, spider silk feedstock infrared spectra are very distinct. Relative to the silkworm feedstock's dissimilarity, the spider silk feedstock tested has a much greater Euclidean distance.

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Promoting flowering, lateral shoot outgrowth, leaf development, and flower abscission in tobacco plants overexpressing cotton FLOWERING LOCUS T (FT)-like gene GhFT1 FLOWERING LOCUS T (FT) encodes a mobile signal protein, recognized as major component of florigen, which has a central position in regulating flowering, and also plays important roles in various physiological aspects. A mode is recently emerging for the balance of indeterminate and determinate growth, which is controlled by the ratio of FT-like and TERMINAL FLOWER 1 (TFL1)-like gene activities, and has a strong influence on the floral transition and plant architecture. Orthologs of GhFT1 was previously isolated and characterized from Gossypium hirsutum. We demonstrated that ectopic overexpression of GhFT1 in tobacco, other than promoting flowering, promoted lateral shoot outgrowth at the base, induced more axillary bud at the axillae of rosette leaves, altered leaf morphology, increased chlorophyll content, had higher rate of photosynthesis and caused flowers abscission. Analysis of gene expression suggested that flower identity genes were significantly upregulated in transgenic plants. Further analysis of tobacco FT paralogs indicated that NtFT4, acting as flower inducer, was upregulated, whereas NtFT2 and NtFT3 as flower inhibitors were upregulated in transgenic plants under long-day conditions, but downregulated under short-day conditions. Our data suggests that sufficient level of transgenic cotton FT might disturb the balance of the endogenous tobacco FT paralogs of inducers and repressors and resulted in altered phenotype in transgenic tobacco, emphasizing the expanding roles of FT in regulating shoot architecture by advancing determine growth. Manipulating the ratio for indeterminate and determinate growth factors throughout FT-like and TFL1-like gene activity holds promise to improve plant architecture and enhance crop yield. Introduction Plants sense multiple environmental cues and endogenous signals to determine the appropriate timing of flowering, which is an orchestrated process through the integration of multiple environmental cues and endogenous signals. Genetic and molecular analyses of flowering-time mutants in Arabidopsis have established the current model, in which five major pathways mainly control the transition from the vegetative to reproductive phase. The photoperiodic and vernalization pathways are responsive to the appropriate environmental conditions, whereas the autonomous, gibberellin, and age pathways reflect the internal status of plants (Srikanth and Schmid, 2011;Yamaguchi and Abe, 2012), which all converge on the 'hubs' known as the integrator genes. Among them, FLOWERING LOCUS T (FT) and its paralog TWIN SISTER OF FT (TSF), encodes a ∼20 kDa globular proteins of the phosphatidylethanolamine-binding protein (PEBP) family, which has a central position in mediating the onset of flowering (Kardailsky et al., 1999;Kobayashi et al., 1999;Yamaguchi and Abe, 2012;Hiraoka et al., 2013). FT as well as TSF proteins including tomato SINGLE FLOWER TRUSS (SFT) and rice HEADING DATE 3a (Hd3a; Lifschitz et al., 2006;Corbesier et al., 2007;Mathieu et al., 2007;Tamaki et al., 2007;Notaguchi et al., 2008), nicknamed florigen, were produced in the phloem companion cells. They are subsequently transported to the shoot apical meristem (SAM), where they form a complex involving a bZIP transcription factor FLOWERING LOCUS D (FD) to activate the expression of floral meristem identity genes, including SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1), APETALA1 (AP1), and LEAFY (LFY; Abe et al., 2005;Wigge et al., 2005;Yoo et al., 2005;Kaufmann et al., 2010), which are important regulatory of hubs in the control of flowering time. Phylogenetic studies of PEBP-like genes in angiosperms revealed that they fall into three subfamilies: the FT-like, the TEMINAL FLOWER1 (TFL1)-like and the MOTHER OF FT AND TFL1 (MFT)-like (Chardon and Damerval, 2005;Hedman et al., 2009). FT-like and TFL1-like genes modulate flowering transition and inflorescence architecture (Kobayashi et al., 1999;Hanzawa et al., 2005;Ahn et al., 2006), but their functions in flowering control are opposite. FT promotes the transition to reproductive development and flowering, whereas TFL1 represses this transition. Numerous studies have concluded, FT orthologs possessing floral inductive function in woody perennials (Hisada et al., 1997;Endo et al., 2005;Böhlenius et al., 2006;Hsu et al., 2006;Carmona et al., 2007;Kotoda et al., 2010;Song et al., 2013); grasses (Yan et al., 2006;Tamaki et al., 2007;Kikuchi et al., 2009;Meng et al., 2011;Wu et al., 2013;Coelho et al., 2014); legumes (Kong et al., 2010;Ono et al., 2010;Hecht et al., 2011;Laurie et al., 2011); ornamental (Hayama et al., 2007;Hou and Yang, 2009;Imamura et al., 2011), CsFTL3 from chrysanthemum (Chrysanthemum seticuspe; Oda et al., 2012;Xiang et al., 2012;Li et al., 2013); and others such as BvFT2 from sugar beet (Beta vulgaris; Pin et al., 2010), NtFT4 from tobacco (Nicotiana tabacum; Harig et al., 2012), StSP3D form potato (Solanum tuberosum; Navarro et al., 2011), AcFT2 from onion (Allium cepa; Lee et al., 2013), PaFT from avocado (Persa americana; Ziv et al., 2014), LsFT from lettuce (Lactuca sativa; Fukuda et al., 2011; Above information was listed in Supplementary Table S1). Previously study suggests a conserved ancestral function of FT-like proteins in transmitting inductive signals in plants. However, recent studies showed that FT-like genes in numerous species play important roles in various physiological aspects other than flowering (Pin and Nilsson, 2012). In Arabidopsis, FT and TSF regulates stomatal guard cells opening by activating H + -ATPase (Kinoshita et al., 2011), meristem maintenance in cooperation with SHOOT MERISTEMLESS (STM) and FD during inflorescence development (Smith et al., 2011), and prevention of indeterminate growth, floral reversion and aerial rosette (Melzer et al., 2008). FT and TSF modulate lateral shoot outgrowth in Arabidopsis, and link the floral transition and lateral shoot development to maximize the reproductive success of a plant (Hiraoka et al., 2013). FT has also been demonstrated to be involved in multiple steps of axillary bud development, likely to coordinate axillary shoot development with flowering (Niwa et al., 2013). Ectopic overexpression of FT in cotton through virus-induced flowering uncouples flowering from photoperiodic regulation and promotes determinate growth habit in all aerial organs (McGarry and Ayre, 2012). In tomato, SINGLE FLOWER TRUSS (SFT) regulates reiterative growth and termination of shoots, influences leaf maturation, compound leaf architecture, stem growth, and abscission zone formation (Shalit et al., 2009). Florigen is thus established as a plant protein functioning as a general growth hormone. Also, allelic variation at the SFT locus is implicated in heterosis of yield (Krieger et al., 2010), suggesting a single overdominant gene may improve productivity in other agricultural organisms, which supports the overdominance model for heterosis. PtFT1 controls short-day (SD) induced growth cessation and bud set in autumn (Böhlenius et al., 2006). Some members of FT-like gene family modulate growth of underground storage organs. StSP6A functions as a mobile 'tuberigen' that induces the photoperiod-sensitive process of tuberization in potato (Navarro et al., 2011), and AcFT1 and AcFT4 play role in bulb formation in onion (Lee et al., 2013). The Gossypium (Cotton) is one of the most important cash crops worldwide, having a large impact on our economy and everyday life. Gossypium species are naturally a photoperiodic that does not flower until the shorter days of late summer or fall. Domestication of the two allotetraploid that comprise the majority of world-wide cultivations, Gossypium hirsutum and G. barbadens gradually lose their photoperiod sensitivity (McGarry and Ayre, 2012). Cotton originated from a tropical region, and its growth is very sensitive to low temperature and soil conditions in temperate cultivation regions. Flowering earliness is an important objective in most cotton breeding programs. However, the molecular mechanisms regulating the transition from vegetative to reproductive growth in cotton are less well characterized than in other plant species, mostly due to the complexity of cotton genome and scarcity of cotton flowering time mutants. In previous study, we isolated and characterized an FT-like gene GhFT1 from G. hirsutum, and we investigated its temporal and spatial expression profile during cotton multiple develop stages (Guo et al., 2015). Overexpression of GhFT1 in Arabidopsis obviously generated early flowering phenotypes in both LD and SD conditions, showing that GhFT1 is a putative FT ortholog in G. hirsutum that regulates floral transition, similar to Arabidopsis (Guo et al., 2015). In this study, we further dissected its roles by ectopic expression of GhFT1 in wildtype (WT) tobacco. As expected, GhFT1 obviously promotes the floral transition in transgenic tobacco plants by producing terminal flower. However, boosting flowering is just one of the pleiotropic functions of GhFT1. In addition to precocious flowering, we observed that tobacco plants carrying 35S::GhFT1 had more lateral shoots outgrowth at the base, axillary buds at rosette axil, altering leaves morphology and causing flower abscission. Our data suggests that sufficient level of transgenic cotton FT homolog might disturb the balance of endogenous FTlike proteins and disorder the ratio of inducer and repressors, resulting in inflorescence and plant architecture change. Plant Materials and Growth Conditions The seeds of N. tabacum cv. NC89 and N. benthamiana preserved in our lab were surface-sterilized for 20 min with 2.8% sodium hypochlorite solution containing 0.1% surfactant (Triton X-100, Sigma-Aldrich, Munich, Germany), and rinsed several times with sterile water. Then seeds were stratified for 3 days at 4 • C in darkness and then plated on the Petri dishes with half-strength Murashige and Skoog (MS) medium containing MS salt (pH 5.7; Duchefa, Haarlem, the Netherlands) mixture, 1% (w/v) sucrose and 0.8% (w/v) agar. Petri dishes were then placed in light growth incubator at 28 • C for 15 days under SD conditions (8 h light/16 h dark). The aseptic seedlings of N. tabacum for transformation were then transferred into a sterile flask containing half-strength MS medium at 28 • C for another 30 days. The N. benthamiana seedlings for transient expression assay were transplanted into soil after germination and grown in phytotron under long-day (LD) conditions (16 h light/8 h dark), and the light intensity for tobacco growth is 200 µmol m −2 s −1 . Constructions of Overexpression Vectors 35S::GhFT1, 35S::GFP, and 35S::GhFT1-GFP constructs were the same vectors that were used in Guo's study (Guo et al., 2015). We first replaced the GUS fragment in the binary vector pCAMBIA1301 (CAMBIA, Canberra, ACT, Australia) by 525 bp of GhFT1 encoding sequence (digested with Nco I and Bst EII restriction, respectively) to construct pCAMBIA1301-GhFT1. The 5.7 kb upstream sequence of Arabidopsis thaliana FT was amplified by polymerase chain reaction (PCR) using pDONR207-8.1kbAtFTpro plasmid as template and next cloned into the Pst I and Nco I restriction site of the pCAMBIA1301-GhFT1 vector to construct the 5.7kbAtFTpro::GhFT1 plasmid. 35S::GhFT1, 35S::GhFT1-GFP and 5.7kbAtFTpro::GhFT1 were all transfected into Agrobacterium tumefaciens GV3101(pMP90RK) by electroporation. The transient expression assays in tobacco were performed according to the method described by Voinnet et al. (2003). The A. tumefaciens strain GV3101 (pMP90RK) carrying 35S::GhFT1-GFP was grown at 28 • C in LB medium with kanamycin and rifampicin to OD 600 = 0.5−0.6. The agrobacteria cells were centrifuged and re-suspended in 10 mmol L −1 MgCl 2 , 10 mmol L −1 MES-KOH (pH 5.7) and 150 µmol L −1 acetosyringone to OD 600 = 0.5. The agrobacteria cells were left to standing for 3 h at room temperature and then infiltrated into the abaxial side of leaves of 4-weeks-old N. benthamiana plants. After 3-5 days the infiltrated leaves were selected to detect GFP fluorescent by CLSM. Gene Expression Analysis Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacture's protocol. The cDNA synthesis reactions were performed using the Superscript R First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions with 1 µg of total RNA per reaction used as template. qRT-PCR was carried out using Applied Biosystems 7500 Fast Real-Time PCR System and Fast SYBR R Green Master Mix (Life Technologies, Foster City, CA, USA) to detect the expression of GhFT1 and endogenous genes in transgenic tobacco lines and WT plants. Primers information used in this research are listed in Supplementary Table S5. NtActin-F and NtActin-R were used to amplify the NtActin gene (GenBank accession no. U60495), which was used as an internal control. At least three replicate assays were performed with independently isolated RNA for all experiments. Each RT reaction was loaded in triplicate for qRT-PCR analysis. qRT-PCR data were analyzed using the PCR analysis program 7500 software v2.0.6 (Life Technologies, Foster City, CA, USA). Semi-quantitative RT-PCR was performed as described by Xu et al. (2013). Gene-specific primers GhFT1-F2 and GhFT1-R2 were used to analyze the expression of GhFT1 in 35S::GhFT1-GFP transgenic tobaccos, and NtActin was used as an internal control. Amplification was performed for 28 cycles at 94 • C for 30 s, 58 • C for 30 s, and 72 • C for 30 s. PCR products were subsequently separated on a 1.2% (w/v) agarose gel, then stained with ethidium bromide and photographed under UV light. Chlorophyll Content Determination Determination of chlorophyll content in transgenic plants grown under LD and SD conditions were estimated according to the method described by Xu et al. (2013). 0.1 g plant tissue was homogenized in 80% acetone and incubated in dark for 6 h. The homogenate was centrifuged at 10,000 rpm for 10 min. Supernatant obtained was read at 649, 665 nm in Spectra Max plus-384 (Molecular device, USA). Leaf Mass Per Area (LMA) Measurements Leaf area was measured using paper-cutting method described by Hattersley (1984) with some modification. We digitally photographed leaves of different transgenic tobacco lines and WT plants. Photos of different lines were printed randomly with A4 paper (No. 3954,Deli,Ningbo,China). Then the printed photos were cut off carefully. Weight of leaves (W1) and their corresponding cut-off papers (W2) were weighed. Then LMA was calculated using the following formula: LMA = (7 × W1)/(1000 × W2) cm −2 . At least three replicate assays were performed independently in this experiment. Photosynthetic Rate Curve The photosynthetic rate of tobacco plants under LD and SD conditions were measured by LI-6400 (LI-COR Inc., Lincolin, NE, USA) with auto-measure program. The CO 2 concentrations in sample phytotron were controlled at 400 µmol CO 2 mol −1 . And different red-blue light intensity 2000, 1800, 1500, 1200, 1000, 800, 500, 300, 200, 100, 50, and 0 µmol m −2 s −1 were applied to measure the net CO 2 uptake rate. Light curve data were analyzed using the built-in program in LI-6400 system. Cytoplasm and Nucleus Location of GhFT1 Protein We previously confirmed that GhFT1 located in the cytoplasm and nucleus by detecting the fused green fluorescence protein (GFP) in the Arabidopsis root cells carrying 35S::GhFT1-GFP (Guo et al., 2015). In this study, we initially generated up to 12 independent 35S::GhFT1-GFP transgenic tobacco plants by transformation with A. tumefaciens. We next to observed the green fluorescence using CLSM. As expected, root tip cells expressing GhFT1-GFP fusion protein revealed strong fluorescence in the nucleus, and GFP signal was also obvious in the cell membrane (Supplementary Figure S2), which was similar to that of the 35S::GhFT1-GFP transgenic Arabidopsis seedling (Guo et al., 2015). To exclude the possibility of cell wall association of GhFT1-GFP, we performed the plasmolysis assay by sucrose treatment. However, we were not able to observe an ideal picture of plasmolysis due to the very thick cell wall of tobacco root tips. Furthermore, the resulting construct of 35S::GhFT1-GFP (Supplementary Figure S1A) was transformed transiently into N. benthamiana. We next monitored the subcellular location of the GhFT1-GFP fusion protein by CLSM in the leaf epidermal cells of N. benthamiana. Green fluorescence was detected in the peripheral cytoplasm (surrounding the vacuole) as well as in the nucleus, which was similar to the cells expressing GFP alone ( Figure 1). As our previous report (Guo et al., 2015), we further confirmed that GhFT1 localized in both the cytoplasm and nucleus in plant cells. We next transferred all the 35S:GhFT1-GFP transgenic tobacco plants into pots containing soil. Under SD conditions, these transgenic plants flowered at 63 ± 6.6 days after sowing with 14.5 ± 0.6 leaves (Supplementary Figure S3A), compared with 94.5 ± 3.3 days in the WT tobacco plants with 15.7 ± 0.5 leaves (Supplementary Table S2). To investigate whether GhFT1 was highly expressed in the 35S::GhFT1-GFP transgenic lines, semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed. As shown in Supplementary Figure S3B, GhFT1 was expressed in all the selected transgenic lines, and Ectopic Expression of GhFT1 Promoted Flowering in N. tabacum Our previous research showed that overexpression of cotton GhFT1 in Arabidopsis caused early flowering both LD and SD conditions (Guo et al., 2015). To explore the potential of GhFT1 in the regulation of flowering in tobacco, this gene was overexpressed in N. tabacum under the control of the strong and constitutive cauliflower mosaic virus (CaMV) 35S promoter by transformation with 35S::GhFT1 construct (Supplementary Figure S1B). We obtained numerous transgenic lines from two times of independent transform assays, and all of them were confirmed by PCR (data was not provided). The majority of 35S::GhFT1 primary transformants flowered much early than the WT, both in terms of time and the number of leaves before flowering (Supplementary Table S2). In the homozygous T3 plants, 14 showed significantly early flowering phenotype under LD (line 1 and line 2 are shown as an example in Figure 2A), and 12 also showed precocious flowering compared with the WT plants under SD conditions (line 15 and line 16 are shown as an example in Figure 2B). In LD conditions, the flowering time in the 35S::GhFT1 transgenic lines was 45.8 ± 4.8 days after sowing by producing 8 ± 0.7 leaves, compared with 106.3 ± 4.8 days by producing 16 ± 0.8 leaves in the WT (Supplementary Table S2). Likewise, under SD conditions, the flowering time of the 35S::GhFT1 transgenic lines was about 57.2 ± 4.9 days by producing 7 ± 0.9 leaves, compared with 94.5 ± 3.3 days by producing 15.7 ± 0.5 leaves in the WT siblings (Supplementary Table S2). In addition, transgenic lines had rapidly elongated internodes and reduced internodal length, and thereby developed dwarf stature when flowering than the controls under both conditions. Previously, it has been shown that a transgene consisting of 5.7-kb sequence upstream of the Arabidopsis FT translation start site fused to the FT cDNA was sufficient to rescue the late flowering phenotype of ft-10 plants grown under inductive Frontiers in Plant Science | www.frontiersin.org extended SD conditions (Adrian et al., 2010). To further analyze the function of GhFT1 in promoting flowers, we designed the 5.7kbAtFTpro::GhFT1 construction by using 5.7-kb Arabidopsis FT gene promoter fused to the GhFT1 cDNA (Supplementary Figure S1C). We next generated up to 14 independent transgenic lines expressing GhFT1 cDNA by transformation with A. tumefaciens. All of them flowered earlier than the WT plants under non-inductive SD conditions (Figure 2E). The average flowering time for these 5.7kbFTpro::GhFT1 transgenic lines was approximately 50.5 ± 2.1 days average by producing 13.5 ± 1.0 leaves, whereas the flowering time in WT sibling was 94.5 ± 3.3 days by producing 15.7 ± 0.5 leaves (Supplementary Table S2). These data combined with previous report by Guo et al. (2015) further supported that the gene product of GhFT1 function as a floral activator to promote flowering in cotton. To explore whether the early flowering phenotype correlated with GhFT1 expression in the transgenic tobacco lines, we used quantitative Real-time PCR (qRT-PCR) methods to analyze gene expression level. As shown in Figures 2B,D,F, higher GhFT1 expression was observed in early flowering transgenic lines more than in those with a less phenotype, whereas no GhFT1 expression was seen in WT. Ectopic Expression of GhFT1 Caused Lateral Shoot Outgrowth in Tobacco Nicotiana tabacum is an annually grown herbaceous plant with little branches. Many flowered inflorescences arise at the terminal after floral transition of the plant (Amaya et al., 1999). Axillary buds are formed at the axillae of foliage leaves, and will further develop into an inflorescence shoot ( Figure 3A). Surprisingly, we also observed that the 35S::GhFT1 transgenic plants produced more axillary buds after floral transition under LD conditions, but these axillary buds could not further develop into lateral shoots (Figures 3B,D), whereas the WT tobacco plants could. Conversely, more lateral shoots were generated at the base of transgenic plants, which do not usually appear in the WT plants of laboratory accession, N. tabacum cv. NC89 (Figures 3C,E; Supplementary Figures S4A,B). Under SD conditions, the formation of axillary buds in all the 35S::GhFT1 transgenic plants were not as obvious as in the LD conditions. They also could not develop into lateral shoots finally, and more lateral shoots were generated from stem base at early buds stage (Figures 3F,G; Supplementary Figure S4C). These observations suggested that ectopic expression of GhFT1 in tobacco could modulate lateral shoot outgrowth and axillary bud set in additional to floral transition. Overexpression of GhFT1 Influenced Leaf Morphology in Tobacco Surprisingly, we also noted that change of leaf morphology appeared in all the 35S::GhFT1 transgenic plants. To decipher the function of GhFT1 further, the 35S::GhFT1 transgenic line 5 and line 6 were used to observe their leaves phenotype under LD and SD conditions. We compared leaves morphology from apical to basal position among the transgenic lines and the WT tobacco siblings. The leave area in the transgenic lines was significantly smaller than in the WT plants. Under LD conditions, the leaves in line 5 appeared to be much longer and narrower than that in the WT plants, but leaves in line 6 appeared to be much shorter and wider ( Figure 4A). We next measured the leaf length to width (L/W) ratio in the transgenic lines and WT tobacco plants, respectively. Accordingly, line 5 had the largest L/W ratio value, followed by the WT plant and Line 6, respectively ( Figure 4B). Strikingly, the leaves of all the 35S::GhFT1 transgenic lines looked much more green and fleshy than the WT plants. We then measured chlorophyll content, suggesting both line 5 and line 6 had higher total chlorophyll content than the WT plants ( Figure 4C). Similar phenotype was also observed in the 35S::GhFT1 transgenic lines in SD conditions (Supplementary Figure S5A). Leaf mass per area (LMA) is a key trait in plant growth and an important indicator of plant strategies, which is most closely correlated with a relative growth rate, and has been used wildly in plant ecology, agronomy, and forestry (Poorter et al., 2009). The transgenic lines showed higher LMA values than the WT plants (Figure 4D), contributing to more fleshy Figure S5). We next set out to explore whether the change of leaf morphology and increasing of chlorophyll content in transgenic plants could enhance photosynthesis. The efficiency of photosynthesis was determined by LI-6400 portable photosynthesis system in different light intensity. As shown in Figure 5, the transgenic tobacco lines showed higher photosynthetic efficiency than the WT plant in LD as well as SD conditions. This suggests, GhFT1 might play important roles in the modulation of leaf development in cotton by increasing photosynthesis and chlorophyll content other than floral transition. Ectopic Overexpression of GhFT1 Caused Flowers Abscission Nicotiana tabacum cv. NC89 is a typical cymose inflorescence in which the first-formed flower develops from the growing region at the top of the flower stalk, and the development of the flower at the apex is followed by two new flower axes developing from buds opposite on another (Amaya et al., 1999; Figure 6A). Both LD and SD conditions, strikingly, overexpression of GhFT1 in tobacco caused extremely early flowering (Figure 2). In addition, 86% tobacco plants overexpressing GhFT1 showed obvious premature flowering abscission in early flower developmental stages. For example, in the 35S:GhFT1 line 18, after the first flower opened, it abscised form the stalk ( Figure 6B) under SD conditions, so it produced very few flowers and few mature seeds. In transgenic line 17, it could not present the regular flowers, due to their abscission before opening ( Figure 6C). One of the reasons may be their flowers failed to enter meiosis, and eventually the plants did not produce any seed capsules. No cymose inflorescences similar to the WT plants ( Figure 6A) were formed in all these transgenic lines. Similar to SD conditions, we also observed that these transgenic lines showed buds abscission at the initiation of early bud set under LD conditions (Figures 6D,E). However, the extent of floral bud abscission was alleviated, and flowers could normally open and seed set at later developmental stage (Figures 2A,C). Viewed form outside, the transgenic plants showed normal flower development, produced fertile flowers and normal seeds (Supplementary Figures S6A,E). To investigate whether overexpression of GhFT1 affected flower organs development in tobacco, we dissected the flowers of −1 days of anthesis (DOA) and 0 DOA in the WT and 35S::GhFT1 transgenic FIGURE 5 | Overexpression of cotton GhFT1 increased photosynthetic efficiency in tobacco. Photosynthetic efficiency among the WT and the 35S::GhFT1 transgenic tobacco lines was determined of using LI-6400 portable photosynthesis system at different light intensity under LD conditions (A) and SD conditions (B), respectively. The WT plant and transgenic plants were 7 weeks-old after transfer to the phytotron. Data represent the mean ± SE of three independent experiments (n = 4). Asterisk denotes significant difference compared with WT plants at P < 0.05, according to the Student's t-test, respectively. lines, respectively. For example, line 5 and line 6 showed a smaller flower than the WT control, but no difference of phenotype in stamen, stigma, petal, ovary, and sepal were observed (Supplementary Figures S6B-D), suggesting that the product of GhFT1 had no influence on the development of flower organs. Influence of GhFT1 Overexpression on the Expression Level of Other Genes in Tobacco In the present model, FT protein, is now widely established as a major component of florigen, a systemic signal that induces flowering in responsible to daylength, which is translocated through the phloem to the SAM (Corbesier et al., 2007;Mathieu et al., 2007;Notaguchi et al., 2008), where they form a complex involving a bZIP transcription factor FD to promote the transition to flowering by activating the expression of multiple flower meristem identity genes, such as SOC1 and AP1 (Abe et al., 2005;Fornara et al., 2010). The MADSdomain transcription factor AP1 is a key regulator of Arabidopsis flower development, controlling the onset of flower development (Wigge et al., 2005;Kaufmann et al., 2010). SOC1 integrates multiple flowering signals including photoperiod, temperature, hormone, and age-related signals, involving in the process of floral organ formation, meristem determinacy, and prevention of secondary growth and shoot longevity (Lee and Lee, 2010;Hiraoka et al., 2013). LFY, which encodes a plant specific transcription factor, plays dual roles in determining floral meristem identity and floral organ patterning via AP1 and other floral homeotic genes (Moyroud et al., 2010). We next detected the expression profiles of the tobacco flower meristem identity genes among the transgenic and the WT siblings by qRT-PCR. The results indicated that NtAP1, NFL (the likely Nicotiana FLO/LFY homolog; Amaya et al., 1999) and NtSOC1 were obviously upregulated in the transgenic lines under LD conditions (Figures 7A-C). Under SD conditions, the three genes were also obviously upregulated in 35S::GhFT1 transgenic tobacco plants (Figures 7D-F). Four FT-like genes have been identified in N. tabacum genome, NtFT1, NtFT2, NtFT3 and NtFT4, which acts antagonistically to regulate floral initiation (Harig et al., 2012). The NtT1, NtFT2, and NtFT3 proteins are floral inhibitors, whereas NtFT4 is a floral inducer (Harig et al., 2012). To explore whether the early flowering phenotype was correlated with the endogenous NtFTs expression in 35S::GhFT1 transgenic lines, we next further detected the expressions profile of the four FT paralogs. As is shown in Figure 8A, higher NtFT4 expression was observed in line 5 and line 6 than the WT plants under LD conditions. NtFT4 was also observed highly expressed in transgenic tobacco plants under SD conditions ( Figure 8B). Surprisingly, the expression of NtFT2 and NtFT3 were also upregulated in the 35S::GhFT1 transgenic line 5 and line 6 under LD condition (Figures 8C,E). However, both NtFT2 and NtFT3 were downregulated expressed in line 5 and line 6 in SD conditions (Figures 8D,F). It was previously reported that overexpression of NtFT2 and NtFT3 showed a delayed flower phenotype, but the exact biological functions of NtFT2 and NtFT3 remain unclear. We were unable to detect NtFT1 expression under both conditions, while Harig et al. (2012) were unable to detect the expression of these genes under LD condition, which may be associated with the different tobacco varieties. Conserved Fuctions of FT-Like Proteins as Floral Promotes A wide spectrum of research of FT orthologs from angiosperms has been demonstrated their conserved function in the regulation of flowering time (Pin and Nilsson, 2012). However, the developmental mechanisms targeted by FT orthologs to transform vegetative meristems into reproductive organs remain unclear. We previously identified a FT-like gene GhFT1 from cotton (G. hirsutum), which was highly expressed in all the tissues except in root, and the strong sequence identity and critical amino acids residues Tyr88 (Y) and Gln144 (Q) to FT-orthologous genes of other species indicates that the GhFT1 might be also involved in the control of flowering. Ectopic expression of GhFT1 promoted precocious flowering under both LD and SD conditions in Arabidopsis (Guo et al., 2015), suggesting that GhFT1 is a potential FT ortholog that regulates floral transition in cotton. To investigate the developmental mechanism targeted by GhFT1 protein, we further unveiled its overall growth effects by overexpressing GhFT1 in tobacco. The transgenic tobacco plants carrying the 35S::GhFT1 construct flowered earlier and had fewer leaves at flowering than the WT plants in both LD and SD condition. Furthermore, the 5.7kbAtFTpro::GhFT1 construction by using 5.7-kb Arabidopsis FT gene promoter fused to the GhFT1 cDNA could also accelerate flowering in transgenic tobacco ( Figure 2E).The precocious flowering phenotype regardless of photoperiod indicated its conserved roles in floral induction. Although FT orthologs have been identified and characterized from numerous plant species, the subcellular distributions of many of them have not been clearly studied. Here, we examined the distribution of functional GhFT1-GFP fusion protein expressed in leaf epidermal cells of N. benthamiana or in hypocotyl of tobacco by transform with 35S::GhFT1-GFP. In both cases, GhFT1-GFP was observed in the nucleus and cytoplasm (Figure 1; Supplementary Figure S2), which was consistent with Guo et al. (2015) results. Similar results using a GFP or YFPtagged FT were also reported in Arabidopsis (Abe et al., 2005), rice (Taoka et al., 2011), tomato (Lifschitz et al., 2006), and tobacco (Harig et al., 2012). FT-GFP fusion protein in transgenic plant has been detected to move through the phloem from the leaves as the place of light perception to the shoot apex as the position of flower formation only in limited plant species (Supplementary Table S3). FT-GFP fusion proteins induced early flowering were previously reported in Arabidopsis (Corbesier et al., 2007), rice (Tamaki et al., 2007), and tomato (Shalit et al., 2009). However, the size of the fusion protein restricted the long-range function of FT. The larger FT-GFP protein may move less effectively to the SAM from the minor veins than from the larger veins (Supplementary Table S3). The 35S::GhFT1 with C-terminal translational fusion of GFP induced precocious flowering in tobacco, indicating GhFT1-GFP protein has similar activity like the WT FT protein. Gene expression using qRT-PCR analysis revealed that the expression of NAP1, NtSOC1, and NFL1 were significantly more upregulated in transgenic lines than in the WT plants under LD or SD conditions (Figure 7). These results were consistent with the finding that NAP1 and NFL1 were highly expressed in the flower buds of 35S::CgFT tobacco plants (Xiang et al., 2012). These data indicates that the expression of NAP1, NtSOC1, and NFL1 may be regulated by FT. Therefore, GhFT1 might have upregulated them to regulate flowering in transgenic tobacco plants. Beyond Flowering Promotion: Pleiotropic Functions of GhFT1 Tobacco is a determinate species in which main shoot terminates by converting into a flower, with subsequent growth occurring only from lower meristems ( Figure 3A). A number of axillary meristems generated below the apex also develop into terminal flowers in a cymose pattern (Amaya et al., 1999). Although florigen was originally proposed as a flowering hormone, it is now apparent that FT is a universal growth factor affecting several aspects of plant architecture. In addition to promoting flowering, we observed that the transgenic tobacco plants showed pleiotropic phenotype different from the WT control, suggesting that GhFT1 played multifaceted roles during plant development. That Arabidopsis FT is involved in the promotion of lateral shoot outgrowth and axillary bud initiation were previously proposed (Hiraoka et al., 2013;Niwa et al., 2013), but the single overexpression of Arabidopsis FT gene is insufficient to promote initiation or early development of axillary buds, and must combined with LFY (Niwa et al., 2013). However, ectopic overexpression of GhFT1 in tobacco resulted in more axillary buds transition (Figures 3B,D) and more lateral shoots generation at the base of main shoot (Figures 3C,E- Figure S4). It has been previously reported that elevation of FT concentration promotes more determinate habit, and influences leaf development (Shalit et al., 2009;McGarry and Ayre, 2012). Here, we also observed that leaf morphology of the 35S::GhFT1 transgenic tobacco lines was very different from that of WT. Firstly, the leaves of transgenic lines appeared much more dark green and fleshy compared with WT plants. Accordingly, the chlorophyll content in transgenic lines was higher than that in the control plants ( Figure 4C; Supplementary Figure S5C). Secondly, compared with other WT plants, leaves were shorter and wider in some transgenic lines, whereas leaves were longer and narrower in other transgenic lines. However, both had bigger value of L/W ratio and LMA under LD and SD conditions than the WT plants (Figures 4B,D; Supplementary Figures S5B,D). Likewise, transgenic lines showed higher photosynthetic efficiency than the WT plants ( Figure 5). The results suggested that high GhFT1 level could function to modulate leaf development by increasing L/W ratio, LMA and photosynthesis, and developing into smaller leaf. We surmised that GhFT1 could link the transition to floral with leaf development. The leaf morphological change in the 35S::GhFT1 transgenic tobacco lines is reminiscent of resent reports on overexpression FT orthologs in different plant species. It was previously reported that the florigen-dependent SFT/SP regulatory hierarchy could determine leaf architecture in tomato and overexpression of SFT induced simple lanceolate-like leaves (Shalit et al., 2009). When Arabidopsis FT is ectopically overexpressed in ancestral cotton accession TX701 through virus-induced flowering, it also generated the lanceolate leaf shape (McGarry and Ayre, 2012). Endo et al. (2005) reported that constitutive expression of CiFT in trifoliate orange altered the leaf shape and color; the leaf in the transgenic plants containing 35S:CiFT was small, lacked color on the margin, and had a leaflet at the center of trifoliate leaf that was smaller than other leaflets. Xiang et al. (2012) reported that transgenic tobacco lines expressing CgFT showed the early release of axillary buds, and the rapid elongation of internodes enabled the formation of thinner stems and reduced leaf sizes. Teper-Bamnolker and Samach (2005) reported that overexpression of Arabidopsis FT induced the high level expression of FUL and SEPTAL (SEP3) in Arabidopsis and leaded to small-sized leaves. Overexpression of tobacco NFL1 in tobacco results in dwarf stature, reduced internode length, and thickened leather-like leaves (Ahearn et al., 2001). Furthermore, Flachowsky et al. (2010) reported that ectopic expression of Arabidopsis LFY in apple showed an altered phenotype, which is similar to the columnar phenotype, and leads to shortened internodes and a significantly reduced length of the regrowing shoot. The high expression of NFL1 (Figure 7) in tobacco driven by the overexpression of GhFT1 might contribute to the leaf shape and plant architecture. Ectopically overexpressed transgenic plants containing FT orthologous genes exhibited similar phenotype in leaf shape and plant architecture, suggesting that the function of FT-like gene family is highly conserved during evolution. Surprisingly, we also observed obvious premature flowering abscission in early developmental stages in the extremely early flowering transgenic lines carrying 35S::GhFT1, resulting in fewer mature seeds (Figure 6). In tomato, SFT was also reported to accelerate mature and promote abscission zone formation (Shalit et al., 2009). Overexpression of Arabidopsis FT in the ancestral cotton accession TX701 delivered by virus-induced flowering caused many of flowers abscission before producing mature bolls (McGarry and Ayre, 2012). The abscission trait is considered as an innovation in angiosperms, and is regulated by multifactor, including auxin, ethylene, and jasmonic acid even day length (Shalit et al., 2009). Further study would clarify the possible mechanism for the precocious floral organ abscission in tobacco plants overexpressing cotton GhFT1. Overexpression of GhFT1 Might Disturb the Balance between Inducer and Repressor of FT Parologs in Tobacco It is now apparent that the relative ratios of FT to other members of the PEBP gene family have influenced the balance of indeterminate and determinate growth in many plant species, and play important role in the floral transition and architecture formation. For example, the tomato SFT/SP ratio regulates the reiterative growth and termination cycles typical of perennial plants, accelerates leaf maturation, influences the complexity of compound leaves, the growth of stems and the formation of abscission (Shalit et al., 2009). The recent report showed N. tabacum possesses four FT-like proteins (Harig et al., 2012), suggesting that the balance of FT-clade in tobacco plays important roles in the floral transition and plant architecture. However, the exact mechanism in the control of floral transition remains very unclear. As expected, NtFT4 showed significantly upregulated expression in all the transgenic tobacco plants under both light conditions (Figures 8A,B). Surprisingly, the expression of NtFT2 and NtFT3 were upregulated in transgenic plants under LD light conditions, but their expression levels were downregulated in SD conditions (Figures 8C-F). The contrast expression profiles of NtFT2 and NtFT3 under LD and SD conditions leads us to have a profound consideration of the florigen paradigm for influencing plant architecture. The balance model predicts that FT and TFL1 concentration fluctuate, and balance are re-fined in local tissues to give rise to different architecture (McGarry and Ayre, 2012). The expression levels of GhFT1 in LD condition tobaccos were much higher than that in SD condition (Figure 2), suggesting that in LD and SD conditions, tobacco might have different concentration of florigen for mediating floral transition. High expression of cotton GhFT1 completely influenced the expression level of endogenous FT genes in tobacco. As a floral inducer, NtFT4 expression was highly upregulated under both LD and SD conditions, whereas NtFT2 and NtFT3 expression, acting as a floral repressor, were upregulated in LD condition and downregulated in SD, resulting in disorders of the balance between inducers and repressors in transgenic tobacco plants, therefore influences the FT/TFL1 genes expression, and further changes FT/TFL1 proteins concentration in the transgenic tobacco. The problem balance will further influence the expression levels of flowering meristem identity genes, such as NAP1, NFL1, and NtSOC1 (Figure 7), resulting in developing multifaceted phenotypes: early flowering, axillary buds set, lateral shoot outgrowth, leaf development change and flower abscission. However, further extensive research is needed to clarify these scenarios. Taken together, overexpression of cotton GhFT1 in tobacco promotes precocious flowering uncouple from photoperiod, showing that FT paralog evolves a conserved function of floral promoter in fiber plants. Introducing of transgenic cotton FT disturbs the balance of endogenous FT paralogs including inducers and repressors, and further disturbs other PEBP family members balance through antagonistic functions. We here present evidences that sufficient levels of FT activity might modulate axillary and lateral shoot outgrowth, influence leaf development and promote flower abscission, supporting the view that florigen functions as general growth hormone mediating growth and termination. These finding further extends the knowledge for plant florigen. Judicious manipulation of the ratio for indeterminate and determinate growth factors, mediated by a balance of FT-like and TFL1-like gene activities by transgenic technology, holds promise for improved plant architecture optimized for region-specific environment and enhanced crop yield in order to meet the agricultural demand of the rapidly expanding global population. Author Contributions XH and XW designed the experiments and organized the manuscript. CL, YZ, KZ, and DG performed the experiments. XW and BC edited the manuscript. All the authors discussed the results and contributed to the manuscripts.

v3-fos

2018-12-07T17:42:47.226Z

2015-01-28T00:00:00.000Z

55926747

s2

Influence of psychrotolerant plant growth-promoting rhizobacteria (PGPR) as coinoculants with Rhizobium on growth parameters and yield of lentil (Lens culinaris Medikus) Cold-tolerant plant growth-promoting rhizobacteria (PGPR) are of immense agronomic importance due to the fact that the winter season crop growing cycle is subject to varying spells of low temperatures and such PGPRs are metabolically functional at cold conditions. Thirty-five (35) rhizobacterial isolates were isolated from lentil rhizospheric soil, six isolates showing maximum exponential growth at both 10 and 15°C after 24 h were selected and characterized belonging to genera Bacillus and Pseudomonas. Production of indole acetic acid (IAA) ranged from 12.7-17.5 μgml at 10°C to 18.4-20.4 μg/ml at 15°C. At 15°C, an isolate J-17 was found to be strong HCN producer whereas at 10°C, only two isolates: J-26 and J-30 were moderate producers. P-solubilizion at 15°C, ranged from 62.5 77.2 μgml. However, at 10°C none of the isolates solubilized P. At 15°C, catechol type siderophore production ranged from 523.4 606.1 μgml. On the basis of the PGP traits, four isolates J-3, J-17, J-18 and J-30 were selected for evaluation under field conditions of lentil at research farm of PAU, Ludhiana, Punjab, India. Coinoculation exhibited a significant increase in nodule number, nodule dry weight, plant and root dry weight, chlorophyll and leghaemoglobin content over Rhizobium alone. Application of J-3, J-17, J-18 and J-30 along with Rhizobium further enhanced the grain yield (1.8, 4.4, 3 and 1.4%). INTRODUCTION Lentil is the world's fifth largest pulse crop with annual production of 3-4 Mt (Sharpe et al., 2013). The important lentil-growing countries of the world are India, Canada, Turkey, Bangladesh, Iran, China, Nepal and Syria (Ahlawat, 2012). In Punjab, lentil occupied an area of 1.0 thousand hectares with a production of 0.73 thousand tones during the year 2011-2012. The average yield was 725 kg/ha (Anonymous, 2013). Lentil seeds contain 1-2% fat, 24-32% proteins and minerals (iron, cobalt and iodine) and vitamins (lysine and arginine). Being a legume crop, it can fix its own nitrogen (N) from the atmosphere and help in restoring the soil fertility. Presently, agricultural research has shifted to sustainable agriculture by supplementing chemical fertilizers with organic amendments. Lentil is constrained by extremities of temperature which are detrimental to the survival and functioning of most introduced mesophilic microorganisms. Such climatic conditions warrant the use of highly adapted microbial strains that retain their biological functions (Mishra et al., 2008). The seed inoculation with the appropriate rhizobia at sowing is a recommended practice, but in recent years, the potential of combined inoculation of N 2 -fixers and plant growth-promoting rhizobacteria (PGPR) is more effective than single organism (Khanna and Sharma, 2011). Sindhu et al. (2002) reported that coinoculation of some Bacillus strains with effective Bradyrhizobium resulted in enhanced nodulation and plant growth of green gram (Vigna radiata L.). The present investigation deals with the isolation, characterization, determination of the growth promotion of cold tolerant PGPR and their coinoculation effects with Rhizobium leguminosarum in lentil (Lens culinaris L. Medikus) under field conditions. Isolation of rhizobacteria Rhizobacteria were isolated from ten different soil samples collected from different lentil growing fields in Punjab. Pour plating was done on nutrient agar (NA) for Bacillus and on King's B for Pseudomonas. The isolates were grown at 10 and 15°C in respective media. Growth in terms of optical density was recorded at 10 and 15°C. The cultures showing maximum growth were selected for further analysis. Biochemical characterization of rhizobacteria Biochemical characterization of bacterial isolates was done on the basis of Gram reaction, catalase production, nitrate reduction, starch hydrolysis and methyl red test were conducted as per the standard methods (Cappuccino and Sherman, 1992;Holt et al., 1994). Indole acetic acid production Characterization of isolates for the production of IAA was done by method of Gordon and Weber (1951). HCN production HCN production was inferred by the qualitative method of Bakker Kaur et al. 259 and Schipper (1987). Phosphate solubilization Phosphate solubilizing isolates were screened by spotting on Pikovskaya's medium (Pikovskaya, 1948). Inoculated plates were incubated at 10 and 15°C and the diameter of clear zone (halo) surrounding the bacterial growth as well as the diameter of colony was measured after four weeks and P-solubilization index was calculated. Phosphate solubilization index = colony of Diameter zone) halo (colony diameter Total  The colonies forming clear halos were considered as phosphate solubilizers. Microbial solubilization of insoluble phosphates in liquid media was detected by the method of Jackson (1973). The total phosphorus was estimated with the help of standard curve using different concentrations of phosphorus. Production of siderophore Siderophore production by the isolates was estimated qualitatively at two different incubation temperatures, that is, 10 and 15°C. Siderophore production was detected by the Chrome Azurol-S (CAS) assay given by Schwyn and Neilands (1987) in 100 mm Petri dishes. Catechol-type siderophores were estimated using the method given by Arnow (1937). Field study Field experiments were conducted during Rabi season of 2013-2014 on lentil at the research farms of Punjab Agricultural University, Ludhiana, Punjab, India ( 30°54'5"N 75°47'53"E). Lentil seeds of variety LL 931 were inoculated with recommended Rhizobium strain and PGPR, as per treatments. In single inoculation of Rhizobium (1x10 8 cell/g of carrier), inoculant was applied to seeds. In dual inoculation (Rhizobium + PGPR), 500 mg each of the inoculant was used. In uninoculated treatment (absolute control), seeds were treated with sterilized charcoal only. Before inoculation, equal volume of different organisms were mixed and allowed to stand for 30 min at room temperature. Inoculated seeds were dried at room temperature under shade before sowing. Crop was sown on 14 th November, 2013 following the recommended agronomic practice and harvested on 11 th April, 2014. The experimental design used was randomised block design and five treatments were used having three replications each. Each plot size was 12 m 2 . The experiment was performed as per the recommended practices. Symbiotic and plant growth parameters such as nodule number and nodule dry weight, root dry weight, shoot dry weight, chlorophyll and leghaemoglobin contents were recorded both at vegetative and flowering stages, and grain yield was recorded at harvest. Five randomly selected plants were carefully uprooted from each plot at 60 and 90 days after sowing (DAS) with root system intact. The roots were washed in running tap water and nodules were detached carefully with forceps, and number of nodules per plant was counted as average. The detached nodules were dried in oven at 60°C for two days and their dry weight per plant was recorded in mg. Dry weights of shoots and roots of five randomly uprooted plants from each plot were taken after drying at 60°C for 2 days. (1971) and leghaemoglobin content by the method of Wilson and Reisenauer (1963). Grain yield from each plot (g/plot) was recorded at final harvest and was expressed in kg/ha. Data was analysed using an analysis of variance appropriate for completely randomized block design (RBD). Statistical analysis was done using CPCS1 software developed by the Department of Mathematics, Statistics and Physics, PAU, Ludhiana. Isolation and characterization Thirty-five rhizobacterial isolates were isolated from lentil rhizospheric soil sample collected from 10 different locations of Punjab and six isolates showing maximum growth in terms of optical density at both 10 and 15°C when incubated for 24 h were selected (Figures 1 and 2). Two isolates from Kings B medium showed the characteristic yellowish-green pigmentation, whereas the other 4 isolates from NA showed off-white to creamish colour. The isolates were evaluated for cultural, morphological and biochemical characteristics as per Bergey's manual of systematic bacteriology. On the basis of these tests, the isolates were tentatively assigned to genera Bacillus and Pseudomonas (Table 1). Indole acetic acid production The auxin IAA is an important phytohormone produced by PGPR, and reports have shown that treatment with IAA producing rhizobacteria increase the plant growth (Hayat et al., 2010). Indole acetic acid production ranged from 12.7 -17.5 µgml -1 in the presence of tryptophan after 5 days of incubation at 10°C, whereas at 15°C, IAA production ranged from 18.4 -20.4 µgml -1 in the presence of tryptophan after 5 days of incubation (Table 2). Isolate J-17 produced IAA at the same rate at 10 and 15°C. Similar results were reported by Selvakumar et al. (2008) in a cold tolerant Serratia marcescens strain SRM (MTCC 8708) which produced 11.1 µg ml -1 IAA at 15°C. HCN production At 15°C, isolate J-17 was found to be strong producer of HCN (colour changed from yellow to reddish-brown), whereas J-18 was moderate producer (orange brown colour) as compared to yellow coloured control (Table 2). However, at 10°C, isolates J-26 and J-30 were found to be moderate producers. Pseudomonas sp. strain PGERs17 was reported to exhibit the HCN (cyanogenic compound) production at 15 and 4°C (Mishra et al., 2008). Phosphate solubilization At 15°C, P-solubilisation on solid media started on the 5th day and increased till the 15th day. P-solubilizing index ranged from 2.9 to 5.4, highest being recorded with isolate J-17 followed by J-18. The quantitative estimation showed that P-solubilization ranged from 62.5 -77.2 µgml -1 (Table 3). The bacterial isolate J-17 was found to be the most potent P-solubilizer. Selvakumar et al. (2008) reported that at 15°C, the isolate S. marcescens strain Siderophore production The six isolates produced distinct halo (golden yellow colour) after three days of incubation, reaching a maximum after seven days. Diameter of halo varied from 1.5 -3.0 cm on CAS agar plates, maximum being produced by fluoresecent Pseudomonas isolate J-18 (3 cm) (Table 3). Arnow's test gave positive reaction, indicating the presence of the catecholate group of siderophores. At 15°C, isolate J-17 was the highest catechol producer. However, at 10°C no zone was formed. The isolates J-3, J-17, J-18 and J-30 were selected for coinoculation with Rhizobium in lentil under field conditions. Effect of co-inoculation of Rhizobium and PGPR on symbiotic parameters and grain yield of lentil Isolate J-17 showed highest nodule number (27.1 and 52.8 nn/plant) followed by J-18 (26.8 and 52.3 nn/plant) as compared to Rhizobium (23.2 and 30 nn/plant) at 60 and 90 DAS, respectively (Table 4). This may be credited to the abilities of PGPRs to produce IAA. An increase of 50 to 98% was reported in nodulation through dual inoculation of Pseudomonas and Serratia with Rhizobium leguminosarum, when compared with control in lentil (Zahir et al., 2011). Table 3. P-solubilisation and siderophore production by rhizobacterial isolates at 15°C. Application of J-3, J-17, J-18 and J-30 alongwith Rhizobium further increased nodule dry weight (28.7, 34.6, 34.5 and 26.6 mg/plant) as compared to Rhizobium alone. Seed inoculation of lentil with effective strain of rhizobia ensures enhanced nodulation and N 2 fixation and combined use of Rhizobium with PGPR being more beneficial is well documented (Khanna et al., 2006;Siddiqui et al., 2007). Coinoculation of R. leguminosarum-PR1 with PGERs17 culture has also been reported to improve the symbiotic effectiveness in terms of nodule number (15.2%) and nodule dry weight (4.1%) in comparison with inoculation with R. leguminosarum-PR1 alone (Mishra et al., 2012). At 60 DAS, maximum shoot dry weight was recorded (Table 4) in Rhizobium along with J-17(1.2 g/plant) followed by Rhizobium along with J-18 (1.03 g/plant) as compared to Rhizobium (0.71 g/plant). The findings are in agreement with the studies of Ogutcu et al. (2008) and Elkoca et al. (2008). Coinoculation of non rhizobial Bacillus thuringiensis-KR1 with Bradyrhizobium japonicum-SB1 was reported to increase nodule number, root and shoot biomass, root length of soybean in comparison with B. japonicum-SB1 alone (Mishra et al., 2009). At 90 DAS, analogous results (Table 4). Highest chlorophyll content was recorded by Rhizobium+J-17 (1.07 and 2.10 mg/g) and Rhizobium+J-18 (0.919 and 2.02 mg/g) which was at par with Rhizobium (0.649 and 1.64 mg/g), respectively at 60 and 90 DAS (Table 5). A study showed that treatment with Pseudomonas sp. strain PGERs17 supernatant was also effective in reducing chlorosis and increased total chlorophyll (6.9%) over non-inoculated control plants in field pea as reported by Mishra et al. (2012). Rhizobium inoculation along with PGPR application showed significantly higher leghaemoglobin content, that is, 4.5, 4.9, 4.6 and 4.4 mg/g of nodule with J-3, J-17, J-18 and J-30, respectively, in comparison with Rhizobium (3.2 mg/g) alone (Table 5). Coinoculation of Pseudomonas sp. strain PGERs17 with R. leguminosarum-PR1 resulted in 17.4 and 4.76-fold increase in leghaemoglobin content over control and R. leguminosarum-PR1 treated plants, respectively (Mishra et al., 2012). Grain yield is the most important economic trait. Improvement in grain yield due to Rhizobium inoculation has been reported in legume crops Togay et al., 2008). Application of PGPRs J-3, J-17, J-18 and J-30 along with Rhizobium enhanced the grain yield (1.8, 4.4, 3.1 and 1.4%) over Rhizobium inoculation alone. The results are in corroboration with Saini and Khanna (2012) who reported that dual inoculation enhanced grain yield of lentil by about 13-15% over control. Kumar and Chandra (2008) reported that dual inoculation of Rhizobium sp. + PGPR showed significant increases in grain yields (20.8 and 23.5%) over Rhizobium sp. and PSB alone inoculation, respectively. The increase in yield can be attributed to complementation of the functionality traits of the co-inoculants, resulting in improved symbiosis and nutrient uptake. Conclusion The present study reveals that a vast diversity of PGPRs possessing an array of functionality traits is available for exploiting as bio-inoculants for sustainable crop production. In the present study, native PGPR isolate Pseudomonas species (J-17) alongwith recommended Rhizobium inoculant improved plant growth, symbiotic parameters and enhanced grain yield by 4.43% over Rhizobium alone.

v3-fos

2018-04-03T04:17:02.868Z

2015-11-25T00:00:00.000Z

16470247

s2

Pseudomonas aeruginosa in Dairy Goats: Genotypic and Phenotypic Comparison of Intramammary and Environmental Isolates Following the identification of a case of severe clinical mastitis in a Saanen dairy goat (goat A), an average of 26 lactating goats in the herd was monitored over a period of 11 months. Milk microbiological analysis revealed the presence of Pseudomonas aeruginosa in 7 of the goats. Among these 7 does, only goat A showed clinical signs of mastitis. The 7 P. aeruginosa isolates from the goat milk and 26 P. aeruginosa isolates from environmental samples were clustered by RAPD-PCR and PFGE analyses in 3 genotypes (G1, G2, G3) and 4 clusters (A, B, C, D), respectively. PFGE clusters A and B correlated with the G1 genotype and included the 7 milk isolates. Although it was not possible to identify the infection source, these results strongly suggest a spreading of the infection from goat A. Clusters C and D overlapped with genotypes G2 and G3, respectively, and included only environmental isolates. The outcome of the antimicrobial susceptibility test performed on the isolates revealed 2 main patterns of multiple resistance to beta-lactam antibiotics and macrolides. Virulence related phenotypes were analyzed, such as swarming and swimming motility, production of biofilm and production of secreted virulence factors. The isolates had distinct phenotypic profiles, corresponding to genotypes G1, G2 and G3. Overall, correlation analysis showed a strong correlation between sampling source, RAPD genotype, PFGE clusters, and phenotypic clusters. The comparison of the levels of virulence related phenotypes did not indicate a higher pathogenic potential in the milk isolates as compared to the environmental isolates. Introduction Pseudomonas aeruginosa (family Pseudomonadaceae) is an aerobic, motile, Gram-negative rod, widely present in the environment, e.g. in water and in humid settings [1], but it is also an important opportunistic pathogen for humans, plants and animals. P. aeruginosa can cause acute and chronic infections in different mammalian hosts and organs, due to the production of a wide arsenal of virulence factors. Virulence factors associated with P. aeruginosa include flagella, adhesion proteins and extracellular proteins, or secondary metabolites, with proteolytic and/or cytotoxic activity (e.g. exotoxin A, elastase, proteases, pyocyanin, hemolysins) [2]. In humans, chronic infections caused by P. aeruginosa can persist for months or even years, resisting host defense mechanisms and repeated antibiotic treatments [3]. In animals, P. aeruginosa is occasionally involved in enzootic or epizootic outbreaks of mastitis in bovine species [4] but also in small ruminants [1,5]. Small ruminant mastitis is an important issue for veterinarians and animal scientists. Indeed, the number of small ruminant dairies and the consumption of small ruminant milk is increasing worldwide, and the need for producers to provide people with animal proteins in the form of milk and meat will continue to increase [6]. Economic issues derive from production losses, treatment costs, and milk payment policies depending on cellular quality in certain areas [7][8][9]. In addition, mastitis caused by P. aeruginosa represents a serious health risk for the human consumer due to possible infections or intoxications through milk, cheese and yogurt [7][8][9]. P. aeruginosa infections generally occur in the post-partum period and sometimes during drying-off [1], with either clinical or subclinical intramammary infections (IMI) [5]. Studies performed on P. aeruginosa strains isolated from clinical and subclinical mastitis in small ruminants and cows analyzed colony morphology, biochemical traits and biofilm production [5]. Recent research regarding phenotypic behavior of small ruminants P. aeruginosa strains is reported by Wright et al [10]. In this study the authors found that a clone isolated from ovine mastitis showed high expression of genes associated with biofilm and twitching motility [10]. While a high number of studies have been carried out on P. aeruginosa strains isolated from human infections, few data are available on strains isolated from animal infections. The aim of this retrospective study was to investigate the epidemiology and pathogenicity of P. aeruginosa isolated from milk and environmental samples in a Saanen dairy goat herd located in Northern Italy, and to analyze their genotypic and phenotypic traits. Case Report An unusual increase in clinical and subclinical IMI caused by P. aeruginosa in a dairy goat herd owned by the Department of Animal Science, Veterinary College of Milan, was recorded from September 2007 to October 2008; the herd is located in Northern Italy (coordinates 45°30' 21.85" N -9°01' 17.01" E). Herd production data were measured monthly by the regional breeding association (Associazione Provinciale Allevatori Milano, Italy) and were referred to the whole period of lactation. These data were made available by Prof. Rapetti, who was responsible of the goats management of the farm. During this period, the herd had an average of 26 Saanen lactating goats, with an average milk production of 58.84 kg per day, meaning an average production per doe from a minimum of 1.14 kg per day to a maximum of 3.38 kg per day; with 3.67% and 3.19% protein and fat content, respectively, and a bulk tank milk somatic cell count of 2.457 x 10 6 cells/ml. The goats were divided into 4 pens, 3 for lactating animals and 1 as a sick pen. All pens were bedded with straw renewed 1-2 times a week. In September 2007, 1 goat (goat A) was affected by a severe case of clinical mastitis, characterized by high fever, rapid decrease of milk and atypically associated with diarrheal symptoms. Milk bacteriological analysis showed the presence of P. aeruginosa. The goat was treated with intramammary tube of nafcillin/benzylpenicillin/streptomycin (Nafpenzal lattazione, Intervet S.r.l., Italy) every 24 h for 2 times and intramuscular treatment with 0.4ml/20kg of tylosin (Tylan 200, Eli Lilly Divisione Elanco, Italy) every 24 h for 3 times and with tolfenamic acid (Tolfedine CS, Azienda Terapeutica, Italy) 1 ml/20kg every 48 h for 2 times. Because these products were not approved by authorities for use in goats in Italy, each drug was administered under Italian regulations (Legislative Decree n. 193 of April 6, 2006) for extra-label drug use (ELDU). After the treatment, a reduction in the clinical symptoms was observed but, at the same time, the monthly Dairy Herd Improvement (DHI) data of this goat revealed a remarkable increase in milk SCC, from an average of 4.150 x 10 5 cells/ml from April to August 2007, to 4.787 x 10 6 cells/ml in September 2007. After this single outbreak, all the lactating goats were monitored for 1 year, and milk samples were collected before dry off (November 2007 and October 2008) and after kidding (April 2008). Additional sampling was performed after intramammary antibiotic treatment of the infected animals with cefoperazone in May 2008. In November 2007, bacteriological analysis of composite milk samples was performed on 22 lactating goats before dry off. Two goats with subclinical IMI (asymptomatic, with high SCC, with no evident macroscopic milk changes and with positive bacteriological analysis) were found positive for P. aeruginosa, goat A (the goat with previous clinical mastitis) and goat B. The farm manager decided to dry off all the lactating goats with ELDU of 1 tube of nafcillin/ benzylpenicillin/streptomycin (Nafpenzal asciutta, Intervet Italy S.r.l) and collect their half udder milk samples immediately after parturition. In April 2008, post-partum half udder milk sampling performed on 29 lactating goats indicated that 4 other goats with subclinical IMI (goats C, D, E and F) were infected by P. aeruginosa. Regarding the 2 previously positive goats, only goat A remained infected, still showing high milk SCC (1.316 x 10 6 cells/ml). Post-partum milk samples from goat B were culture-negative, but with very high milk SCC (3.491 x 10 6 cells/ml) and low milk production (0.7 kg/day), thus the farm manager decided to sell this animal and treat the 5 remaining infected animals. Antimicrobial susceptibility towards 12 antibiotics (i.e. amoxicillin/clavulanic acid, cefalonium, cefapirin, cefoperazone, cefquinome, ceftiofur, danofloxacin, marbofloxacin, nafcillin/benzylpenicillin/streptomycin, neomycin/bacitracin/tetracycline, penethamate and rifaximin) was tested on all isolates, by means of the disk diffusion method. Overall, samples were found sensitive towards only 3 of the tested antibiotics (i.e. cefoperazone, danofloxacin and marbofloxacin). The positive animals were segregated, treated with ELDU of 1 intramammary tube of cefoperazone (Pathozone, Zoetis, Italy) and monitored. In May 2008, bacteriological analysis confirmed that goats A, C and F remained infected, whereas goats D and E were culture-negative. Since the infected animals showed no clinical signs, only goat C was culled due to its low milk production. In October 2008, only goat A and another goat (goat G) out of 26 lactating goats were bacteriologically positive, although without clinical signs of infection. The 2 goats were segregated and culled in November 2008 before dry off, in order to avoid further spread of the infection. Table 1 summarizes P. aeruginosa detection in goat milk during the study period. Bacteriological analysis Bacteriological analyses were performed on 142 milk samples ( The 12 milk samples positive for P. aeruginosa had pure cultures (mean 3 x 10 3 CFU/ml) and a high SCC (mean 5.13 x 10 6 cells/ml). Statistical analysis performed on the somatic cell score (SCS) revealed a significant difference (p<0.05) between culture-negative and P. aeruginosa-positive samples. Conversely, no statistical difference was observed between culture-negative and other microorganism-positive samples or between P. aeruginosa-positive and other microorganism-positive samples. Environmental samples were collected for microbiological analysis in October 2008, after the last 2 infected does were segregated from the herd. This sampling was performed in order to ensure that eliminating all positive animals and maintaining good hygiene practices and biosecurity would reduce the risk of infection from the environment. Environmental samples analysis was also carried out in order to define the sources of the P. aeruginosa outbreak, and to investigate the interrelationships among milk and environmental isolate genotypic and phenotypic traits. Out of 41 samples collected from the farm environment, P. aeruginosa was isolated only from 2 watering trough samples derived from pen number 3 and pen number 4 (sick pen), with 60 CFU/l and 800 CFU/l, respectively. Twenty-six P. aeruginosa isolates from the 2 watering troughs (disclosing different colony morphotypes) were selected for further analyses, plus 7 P. aeruginosa milk isolates (1 per infected goat) ( Table 2). Isolates genotyping A positive PCR amplification of the eta gene from all 33 isolates, followed by sequencing, confirmed the isolates to be P. aeruginosa. The isolates selected for rpo gene sequencing (Table 2), were submitted to BLAST analysis (http://blast.ncbi.nlm.nih.gov) which confirmed a 100% identity of P. aeruginosa. The RAPD-PCR assay showed the presence of 3 different DNA-banding patterns (Fig 1). All the milk isolates showed an identical DNA-banding pattern, named G1 (n = 7), while the environmental isolates showed 2 different patterns, G2 (n = 2) and G3 (n = 24). In addition to the RAPD-PCR assay, a PFGE assay was performed (Fig 2). By considering a value of 85% as the correlation threshold, in accordance with previous studies [11], 4 major clusters were defined. Cluster A (isolates 2, 3 and 7) and B (isolates 1, 4 and 6) shared a high degree of homology (81.45%) and contained milk isolates, while clusters C (isolates 8 and 9) and D (isolates from 10 to 33) contained only environmental isolates. The environmental cluster C showed 56.84% homology with the milk clusters A and B, while the environmental cluster D disclosed a reduced homology (42.15%) with the other 3 clusters. A high correspondence was found between the RAPD-PCR and PFGE results. Indeed, G1 corresponded to clusters A and B (milk isolates), G2 corresponded to cluster C, and G3 to cluster D. Phenotypic characterization of P. aeruginosa isolates In order to assess possible correlations between the P. aeruginosa sampling source, phylogenesis and pathogenic potential, the 33 P. aeruginosa isolates were tested for the expression of virulence-related phenotypes. The phenotypes included motility (swimming and swarming), hemolytic activity, and the production of biofilm and secreted virulence factors (proteases, pyocyanin, elastase, gelatinase; S1 and S2 Figs). Overall, each isolate showed a unique pattern of production/expression of these virulence-related phenotypes, making it difficult to infer correlations between the pathogenic potential and the phylogenetic cluster and/or with the source of isolation. Phenotypic clustering of P. aeruginosa isolates and correlations within variables tested in this study The phenotypic analysis described above suggested a relevant degree of variance within each genotypic cluster. Hence, clustering aggregation analysis of virulence factor production was performed in order to reveal possible correlations between phenotypes and genotypes. Results showed that the P. aeruginosa isolates can be divided into 5 major phenotypic clusters (named PC 1, PC 2, PC 3, PC 4 and PC 5, Fig 3), considering a value of 22.34% as the correlation threshold (software automatic truncation). Cluster PC 1 included only milk isolates, i.e. isolates 2 and 3 (PFGE cluster A) and isolates 1 and 4 (PFGE cluster B), and showed a great phenotypic dissimilarity (58.72%) with the remaining 3 clusters. Cluster PC 2 included the remaining milk isolates, i.e. isolate 7 (PFGE cluster A) and isolates 5 and 6 (PFGE cluster B), and showed 28.48% of phenotypic dissimilarity with cluster PC 4, the latter including only environmental isolates 10-19 and 21-25 (all belonging to PFGE cluster D). Cluster PC 3 included the environmental isolates 8 and 9 (PFGE cluster C), and showed a high degree of phenotypic dissimilarity (95.92%) with respect to the other clusters. Cluster PC 5 included the environmental isolates 20 and 26-33 (PFGE cluster D) and showed a considerable phenotypic dissimilarity (39.93%) with clusters PC 2 and PC 4. Correlations within variables are shown in the correlation matrix, reporting a high positive association between phenotypic clusters, sampling source and genotypic clusters (Table 3). These high correlations (r>0.7) suggest that genotypic isolate grouping corresponds to a phenotypic one and that this clustering is also in accordance with the sampling source. Further correlation analyses were performed in order to identify every possible association between the different variables recorded in the study, to find other statistically significant correlations (p<0.05), both positive and negative (Table 3). Variables relating to the sampling source, PFGE genotype and phenotypic behavior have certain virulence factors in common, i.e. hemolysis and swimming are virulence factors that are positively correlated (Table 3). Also other virulence factors are correlated (Table 3), indicating possible relationships that would promote specific phenotypic behavior. Principal Component Analysis (PCA) was performed in order to infer the distribution of the 33 P. aeruginosa isolates in relation to genotypic and phenotypic clusters. Overall, genotypic clustering and phenotypic behavior were significantly correlated. The first two dimensions, plotted on X and Y axes (Fig 4) captured 98.46% of the total variability. The Pearson's correlation matrix (data not shown) highlighted a consistent association between the analyzed factors (isolates vs PFGE clusters: r = 0.746; isolates vs phenotypic clusters: r = 0.885; PFGE clusters vs phenotypic clusters r = 0.914) (Fig 4). Statistical analyses results relating to the production of virulence factors Clustering investigations have highlighted correlations that allowed us to study the average production of each virulence factor for each different genetic cluster (Fig 5). A preliminary analysis showed that the average production of each virulence factor was not significantly different in PFGE clusters A and B (data not shown). This finding is consistent with the high genetic similarity of these 2 clusters, which together include exclusively all the milk isolates. Hence, for the sake of simplicity, the average level of each virulence factor was calculated by considering the milk isolates as a unique group, corresponding to RAPD cluster G1 and to PFGE clusters A and B together ( Fig 5). Overall, the comparison of the levels of virulence related phenotypes, produced on average by each cluster, did not indicate a higher overall pathogenic potential in the milk isolates with respect to the other strains ( Fig 5). Indeed, only the average gelatinase production was significantly higher in the milk isolate cluster, while the average levels of other virulence related phenotypes (i.e. biofilm production, hemolytic activity and swimming motility) were lower, with respect to both clusters of the environmental isolates ( Fig 5). Antimicrobial susceptibility testing and carbapenemase confirmation tests Minimum Inhibitory Concentration (MIC) of the 33 P. aeruginosa isolates was analyzed in order to better understand their antimicrobial resistance patterns (Table 4). All the isolates displayed resistance to 3 or more classes of antibiotics. The main pattern of antimicrobial multiresistance found in 22 of the isolates was penicillin G, oxacillin, ampicillin, amoxicillin/clavulanic acid, cephalosporins I, II, and III generation, macrolides, lincosamides, trimethoprim/sulfamethoxazole, tetracyclines, chloramphenicol. The remaining 11 isolates showed an overall similar pattern of antimicrobial multi-resistance but were susceptible or intermediate to doxycycline. The highest level of resistance was found against the most widely used beta-lactam antibiotics approved in Europe for food animals (ampicillin, amoxicillin/clavulanic acid, oxacillin), and to all the cephalosporins tested. Three isolates from milk (goats A, C and G) were found resistant to imipenem and therefore were tested for the carbapenemase production. All the strains were susceptible to imipenem and meropenem (with an imipenem inhibition zone that ranged from 23 to 25mm and from 30 to 36mm for the meropenem) according to the KPC/ MBL and OXA-48 Confirm Kit and to the Combined-disk method with imipenem and cloxacillin. All the combined-disk tests using the carbapenemase/AmpC inhibitors yielded negative results and Carba NP test did not detect the presence of carbapenemase production. In addition the PCRs for the bla VIM , bla IMP , bla GES and bla OXA-48 genes were also negative. None of the P. aeruginosa isolates met the criteria of multidrug-resistant P. aeruginosa, which is defined as resistance to both imipenem and gentamicin [12], nor displayed resistance to both of the fluoroquinolones tested (enrofloxacin and marbofloxacin). The resistance to ticarcillin was the only difference found by comparing milk and environment isolates. Overall resistance was higher in milk isolates than in environmental isolates (14% vs 4%). Discussion Outbreaks of P. aeruginosa mastitis in small ruminants [13][14][15] and cows [16][17][18] have occasionally been described. The adaptive behavior of P. aeruginosa is possible thanks to its genetic flexibility which is supported by an extended genome, containing a high number of accessory genes [3]. Genome sequencing of the extensively studied P. aeruginosa strain PAO1 revealed the presence of a great number of genes involved in metabolic adjustment, transport, and release of organic elements, as well as numerous chemotaxis systems [19,20]. These features virtually concur to the notable capability of this bacterium to fit into a wide range of ecological niches [20]. During mastitis outbreaks, P. aeruginosa has been isolated in contaminated wash hoses in milking parlors, in water and spray nozzles and in contaminated antibiotic preparations [21]. This microorganism is often found in fresh water such as lakes and rivers in concentrations of 100 CFU/l to >10,000 CFU/l [22]. In small ruminants, P. aeruginosa and other opportunistic pathogens are able to cause enzootic or epizootic outbreaks that mostly appear during the post-partum period and sometimes during drying-off [1]. Additional information regarding udder health is provided by the milk SCC, a valuable tool for the prevalence assessment and screening of IMI in cows and ewes. In goats, the correlation between milk SCC and IMI is difficult to assess, and infection-independent factors play an important role in defining milk SCC values [23]. Hence, in goat herds, it is usually difficult to distinguish infected from healthy udder halves based on milk SCC values only [1]. Nevertheless, P. aeruginosa positive samples revealed a significantly higher milk SCC when compared to the culture-negative samples, while no difference was found between the negative and positive samples for other microorganisms. This result confirms previous studies showing that infections caused by P. aeruginosa trigger a greater inflammatory response than other pathogens [24]. After the first outbreak, the milk of goat A, showing clinical signs only at the beginning of the case study, remained positive for P. aeruginosa throughout the entire period of investigation, indicating the establishment of a chronic infection. In addition, the subclinical goats C and F were positive in 2 consecutive samplings. This confirms previous reports showing that the persistence of subclinical IMI, depending on the causative pathogen and the interaction with the host, is generally high since it is poorly eliminated, at least during lactation, with an average shedding duration of 3-4 months [1]. The persistence of IMI during the dry period must also be taken into account, although an overall self-cure rate of 20% to 60% of halves is estimated in goats [23]. The results reported in this study confirm those recorded in literature [1]. As reported in Table 1, the dry period is focused from November 2007 to April 2008; 2 goats (goat A and B) resulted positive to P. aeruginosa before dry off (November 2007), while only 1 goat (goat A) still resulted positive after kidding (April 2008), implying that the infection was able to persist during the dry period and Ampicillin ( through to the subsequent lactation without any clinical manifestation, showing a cure rate of 50%. A previous study by Contreras et al [8] reported that an infection can be considered a true and persistent IMI when the same pathogen is isolated twice or more consecutively from the same half udder. Accordingly, isolates deriving from goats which were positive in only 1 sample (isolates 2, 4, 5 and 7; Table 1) can be categorized as transient; while those from goats that were positive for at least 2 consecutive samples (isolates 1, 3, 6; Table 1) can be defined as persistent. Statistical analyses were carried out to compare virulence factor production within transient and persistent isolates, finding no significant difference (data not shown). By examining the phenotypic data, the huge amount of variation existing among each one of the analyzed virulence factors was evident. Each isolate revealed a high degree of dissimilarity from the other isolates, even if belonging to the same genotype. P. aeruginosa phenotypic changeability is documented in literature, from studies on chronic cystic fibrosis isolates [25]. These isolates, including clones deriving from the same sample, revealed great differences in motility, excreted virulence factors and biofilm formation [25]. Despite this high variation, it was possible to define important phenotypic correlations among the isolates. The degree of genotypic homology confirms the phenotypic profiles of the clusters. Indeed, no significant virulence profile difference was observed between clusters A and B. Therefore, given their common sampling source (goat udder), high genotypic homology and phenotypic similarity, the 2 PFGE clusters were considered to contain isolates belonging to a single ecological niche. This subset corresponds to the G1 genotype detected by RAPD-PCR. The same comparison was made for the environmental isolates, revealing how the lower genotypic homology was related to differences in phenotypic traits. Therefore, it was possible to define the isolates analyzed in the study as having 3 distinct phenotypic features, corresponding to G1, G2 and G3 genotypes (Fig 5). In accordance to a previous study comparing P. aeruginosa isolates from human (clinical) sources and from the environment [26], no correlation was found between the pathogenic potential of milk isolates (both clinical and subclinical) and environmental isolates. Altogether, the results obtained by PFGE and RAPD-PCR, well correlated with the phenotypic analysis, revealed that P. aeruginosa milk isolates belonged to the same clonal line, shared distinctive phenotypic traits and were not retrieved from the environment. These results clearly indicate transmission of the infection within the herd. Although it was not possible to determine the environmental origin of the P. aeruginosa isolate which caused the outbreak, the observation that all goats were infected by 2 closely related clades of P. aeruginosa suggests that these 2 clades share a common ancestor particularly well adapted to the udder environment. Since the first goat to show clinical symptoms (goat A) remained chronically infected throughout the entire sampling period, it is not unlikely that the outbreak originated from this animal. In accordance with data from literature, it is tempting to speculate that such spread occurred during the milking routine, promoted by insufficient cleaning procedures such as teat cleaning with a paper towel and without post-milking teat disinfection [21]. Strikingly, the specific antibiotic therapy with cefoperazone (administered in April 2008) was effective in curing only goats D and E, while goats A, C and F remained infected. The lack of response in the antibiotic therapy of P. aeruginosa infections is not a rare event, being P. aeruginosa lung infection a paradigmatic example in human cystic fibrosis [27]. The subsequent analysis of the antimicrobial resistance patterns using the MIC methods showed two main patterns of antimicrobial multi-resistance that included beta-lactam antibiotics and macrolides. These multi-resistance patterns are due to an intrinsic resistance mechanism linked to a reduction in the outer membrane permeability of P. aeruginosa [28]. The level of resistances found are consistent with the literature data in dairy cattle mastitis that reported a high number of strains resistant to ampicillin, ceftiofur, and 1 st generation cephalosporins [29,30], while no data are available for small ruminants mastitis. Three strains of P. aeruginosa were found resistant to imipenem with a MIC of 16 μg/ml for two of them, and of 8μg/ml for the third, but the following carbapenemase phenotypic screening and confirmation tests showed a susceptibility to carbapenems and the absence of carbapenemase production. A possible explanation for this disagreement between the MIC and the carbapenemase confirmation test is that, in the absence of carbapenem-hydrolyzing enzymes, the mechanism leading to carbapenem resistance or reduced susceptibility to carbapenems is usually multifactorial in P. aeruginosa. Several antibiotic efflux systems, together with AmpC hyperproduction and/or porin loss, contribute to multidrug resistance in this species [31,32]. Additionally, an important feature that emerged on analyzing the correlation matrix (Table 3) is the positive correlation among the sampling source, RAPD-PCR and PFGE clustering, hemolysins production and swimming motility. In conclusion, this study shows how caprine P. aeruginosa isolates could be compared to human isolates, being phenotypically diverse, able to cause both transient and chronic infections, with clinical and subclinical manifestations, and triggering a greater inflammatory response with respect to other pathogens. The study demonstrates that P. aeruginosa is also able to persist during the dry period through the subsequent lactation, demonstrating the difficult clearance of this pathogen from the mammary gland. Multidrug resistance patterns shown by all the isolates tested assists this resistance to cure IMI. Indeed, the highest levels of resistance were found against the most commonly used beta-lactam antibiotics. Phenotypic assays showed unique but distinguishable patterns of virulence-related phenotypes. In addition, the combined analysis of data, both as discrete values or as clusters, led to the detection of correlations that would allow the definition of certain isolates as better adaptable to the udder environment. Sampling source, genotypic and phenotypic clustering are highly positively correlated. Association between hemolysins production and swimming motility might suggest that some virulence factors would be capable of making an isolate more adaptable to the goat udder. One relevant aspect, which broadens the field of interest of this study to beyond veterinary microbiology, emerges when it considering that chronic infections of P. aeruginosa, in contrast to acute infections, have received much less attention, likely due to the lack of suitable animal models and the difficulty of recreating these long-lasting infections in vitro [33]. Up to date, animal models used to study P. aeruginosa pathogenesis have been mainly based on induced infections in rodents, for example through burns, wounds or direct inoculum of high microbial counts in lungs [34]. This study highlights the fact that increasing knowledge on the pathogenic aspects involved in P. aeruginosa mastitis in small ruminants could be useful to better understand the behavior of P. aeruginosa in an animal models where the chronic infection can occur naturally. Before milking the animals, teat ends were swabbed with chlorhexidine, dried with a disposable paper towel and, after discarding the first 3 streams, milk samples were collected in sterile tubes. Samples were kept at 4°C and immediately transported to the laboratory for microbiological analysis. Milk and environmental samples A total of 41 environmental samples were collected from the following sites: goat drinking water (n = 4; 4 from the watering troughs, 1 from each of the 4 pens); goat feed (n = 6; 4 from troughs, 1 from haystack, 1 from pelleted feed); goat resting areas (n = 4; 4 from bedding material, 1 from each pen); goat feces from animals with diarrheal symptoms (n = 10; rectal swab samples); straw used for goat bedding (n = 1); mammary skin from animals with diarrheal symptoms (n = 10); milking apparatus (n = 6; 4 swabs from teat cup liners after milking, 2 from washing water before and after milking). Samples were collected in sterile tubes and immediately refrigerated. Grab samples from bedding and feed were collected in sterile sample bags. Samples were stored in a cooler with ice packs and transported to the laboratory, where processing was initiated within a few hours after sample collection. Bacteriological analysis The milk samples (n = 142) were cultured as recommended by National Mastitis Council (NMC) [35]. A 0.01 ml aliquot of each sample was plated onto 5% sheep blood agar plates (each sample from a goat udder half was placed on a ¼ plate). Plates were incubated aerobically at 37°C and examined at 24 h and 48 h. Single isolated colonies were picked up, subcultured and presumptive identification was performed based on colonies appearance, Gram-staining, catalase and coagulase-testing. Hemolytic staphylococci suspected to be S. aureus but yielding negative results in the coagulase-test were frozen in nutrient broth with 15% glycerol added before further speciation. For each milk sample, the somatic cell count (SCC) was determined by an automated fluorescent microscopic somatic cell counter (Bentley Somacount 150, Bentley Instrument, Chaska, MN, USA). All bacteriological procedures on the environmental samples were performed on Pseudomonas Selective Agar (PSA; Microbiol Diagnostics, Cagliari, Italy). Pseudomonas spp. cells from swabs from goat rectums, mammary skin and teat cup liners were directly isolated by streaking on PSA plates. Pseudomonas spp. cells were isolated from 100 ml water samples by filtration through 0.22 μm pore size sterile cellulose acetate membrane filters (International PBI, Milan, Italy) and growth on PSA plates. Isolation from bedding and feed was made by adding 10 g of each sample to 90 ml of sterile saline and manually mixing the solution for 10 min. The liquid phase was withdrawn by an automatic pipette, serial 10-fold dilutions were made in sterile saline (final dilution, 1:1,000) and 0.1 ml of each dilution sample was plated on the same medium. Multiple isolated colonies were collected from each plate. The API 20E Diagnostic Kit (BioMerieux, Marcy l'Etoile, France) was used to identify biochemically each isolate to the genus or species level. Bacterial isolates P. aeruginosa PAO1 was used as a control strain. All bacterial isolates characterized in this study are listed in Table 2. Genomic DNA extraction Genomic DNA extraction from P. aeruginosa isolates was based on a standard phenol-chloroform extraction. Briefly, all bacterial isolates were grown in Luria-Bertani (LB) broth overnight at 37°C and 1 ml of the bacterial suspension was pelleted at 3,000 g for 10 mins. After supernatant removal, 400 μl of TE Buffer 1X (1.2% Triton), 50 μl of 10% (w/v) SDS, and 50 μl of 20 mg/ml Proteinase K (Novagen, Merck Millipore Italy, Milan, Italy) were used to digest cell pellets at 37°C for 1 h. The lysate was extracted twice with 1 ml phenol:chloroform (1:1, v/ v). The upper aqueous phase was added to 1/10 v of 5M sodium acetate and 0.6 v of isopropanol, mixed gently and incubated at room temperature for 5 mins, until the DNA precipitated. The DNA was recovered after centrifugation at 12,000 g for 10 mins, the supernatant was discarded and the DNA pellet was washed with 70% ethanol. After a final centrifugation at 7,000 g for 5 mins, the DNA pellet was dried and finally re-suspended in 100 μl elution Tris-EDTA (TE). DNA concentration was calculated by measuring the absorbance at 260 nm; then 200 ng/ μl DNA stock solutions were prepared for each sample. PCR for detection of eta gene P. aeruginosa isolates were identified via PCR using exotoxin A-specific primers, ETA1 (5'gacaacgccctcagcatcaccagc-3') and ETA2 (5'-cgctggcccattcgctccagcgct-3') [36]. The PCR assay was performed in a final volume of 25 μl containing 1x PCR buffer, 2 mM MgCl 2 , 240 μM of each nucleotide, 0.24 μM of each primer (Invitrogen, Life Technologies, Monza Brianza, Italy), 1U Taq polymerase (Invitrogen, Life Technologies, Monza Brianza, Italy) and 2 μl of bacterial DNA template. The chosen amplification protocol was 95°C for 4 mins, followed by 35 cycles of 94°C for 45 secs, 54°C for 45 secs, 72°C for 1 min, and a final extension at 72°C for 5 mins. Ten microlitres of the amplification products were analyzed on 2% agarose gel stained with ethidium bromide in order to evaluate the banding patterns at 367 bp. Bacterial typing Representative isolates selected by PFGE analysis (1 isolate per cluster, Table 2), were submitted to BMR Genomics (Biotechnology Center, Padova, Italy) for targeted sequencing, using PCR primers specific to a region of rpo gene [37]. RAPD-PCR analysis For typing P. aeruginosa isolates, primer D-10514 (5'-tggtggcctcgagcaagagaacaaag-3') was used [38]. RAPD-PCR was performed in a final volume of 25 μl containing 1X PCR buffer, 4 mM MgCl 2 , 200 μM of each nucleotide, 0.5 μM primer (Invitrogen, Life Technologies, Monza, Italy), 1U Taq polymerase (Invitrogen, Life Technologies, Monza, Italy) and 2 μl of bacterial DNA template. Parameters for the amplification were 95°C for 3 mins, followed by 45 cycles of 94°C for 60 secs, 38°C for 60 secs, 72°C for 60 secs, and a final extension at 72°C for 5 mins. Ten microliters of the amplification product was analyzed on 2% agarose gel stained with ethidium bromide in order to evaluate the binding patterns. PFGE typing P. aeruginosa isolates were also typed by pulsed-field gel electrophoresis (PFGE), as previously described by van Mansfeld et al. [39], with the following modifications. Bacterial isolates were grown in LB broth at 37°C overnight. For each bacterial strain, 3.5x10 8 cells were mixed with an equal volume of 2% low melting agarose. The resulting plugs were incubated for 5 hours in G-positive lysis buffer (6mM Tris pH8, 1M NaCl, 100mM EDTA-Na 2 pH 8, 0.5% (w/v) Brij58, 0.2% (w/v) sodium-deoxycholate, 0.5% (w/v) lauroyl sarcosine pH 7.5) with the addition of lysozyme (1 mg/ml final concentration). Plugs were further incubated overnight at 55°C in Gnegative buffer (500mM EDTA-Na2 pH9.5, 1% (w/v) lauroyl sarcosine) with the addition of proteinase K (500ug/ml final concentration). DNA restriction was performed for a 2mm plug slice with 20U SpeI (Invitrogen, Life Technologies, Monza, Italy), incubating overnight at 37°C. Lambda ladder PFGE marker was loaded in the first lane of each run. Electrophoresis was carried out in 1.2% (w/v) agarose gel in 0.5X Tris-borate-EDTA (TBE) at 12°C on a CHEF DRIII PFGE system (BioRad, Hertsfordshire, United Kingdom). PFGE run setting were: initial switching time 5 s; final switching time 45 s; gradient 6Vcm 2 ; 120°C angle; run time 21 h. Samples 6, 7, 12, 34 gave a resolved fingerprint only with electrophoresis in TBE0.5x plus 50uM thiourea. Grouping of the PFGE profiles was obtained with the BioNumerics 5.0 software package (Applied Maths, Kortrjik, Belgium) by using the Pearson product moment correlation coefficient and the UPGMA (unweighted pair group method using arithmetic averages) cluster analysis. Isolates with correlation coefficient > 85% were defined as genetically identical [11]. Phenotypic assays The crystal violet binding assay was used to investigate the ability of P. aeruginosa to produce biofilm and was basically performed as described in [40]. Isolates were grown for 24 h in 5 mL of TSB at 37°C. The absorbance at 600 nm (A 600 ) of each sample was normalized to 1.65±0.1. Samples were diluted 1:1 in TSB with 0.25% glucose and 200 μl of this solution was incubated in 96-well plates overnight at 37°C without shaking. Media with suspended bacteria was then removed; the plates were carefully washed four times with sterile water and air-dried before 125 μl of 0.99% crystal violet solution (Merk, Darmstadt, Germany) was added for 15 mins at 30°C. After removing the dye solution and washing four times with sterile water, the attached dye was solubilized with 95% ethanol for 10 mins at room temperature. Absorbance at 570 nm of the solubilized sample was determined by a microtiter plate reader (MULTISCAN EX, Labsystems, Helsinki, Finland). The elastolytic activity in the culture supernatant of the isolates was determined by the Elastin Congo Red (ECR; Sigma-Aldrich, Milano, Italy) assay, as previously described [41]. Isolates were grown in LB at 37°C for 24 h. The absorbance at 600 nm (A 600 ) of each sample was normalized to 1.65±0.1. The reaction mixture was centrifuged at 3,000 g for 10 mins and the absorbance at 495 nm of the supernatant was determined. A negative control tube containing 0.75 ml buffered ECR and 0.25 ml LB broth and a positive control tube containing 1U Elastase (Elastase, Sigma-Aldrich, Milano, Italy) were used. For measuring gelatinase production, isolates were grown in LB at 37°C for 48 h. The A 600 of each culture was adjusted to 2.00±0.1. Then, 2 μl of each culture was spotted on LB agar plates containing 5% of gelatins and incubated overnight at 37°C. 600 μl of 15% MgCl 2 and 20% HCl 1N reagent was added pouring it on plates and turning them to distribute it with homogeneity and, after 3 minutes of incubation, gelatinase production was determined by measuring the area of the circular clear zone around the colonies. Hemolysis was measured by the blood agar assay. Isolates were grown in LB at 37°C for 24 h. The A 600 of each culture was adjusted to 1.65±0.1. Then, 2 μl of each culture was spotted onto 5% sheep blood agar plates and incubated at 37°C for 48 h. Haemolytic activity was determined by measuring the area of the circular clear zone around the colonies. Protease production was determined by the milk plate assay. Isolates were grown in LB at 37°C for 24 h. The A 600 of each culture was adjusted to 1.65±0.1. Then, 1 μl of each culture was spotted on Litmus Milk (Oxoid, Milano, Italy) agar plates and incubated overnight at 37°C. The alkaline protease production was determined by measuring the area of the circular clear zone around the colonies. Pyocyanin levels were measured in P. aeruginosa supernatants as previously described [42]. Isolates were grown in LB broth at 37°C for 48 h. The A 600 of each culture was adjusted to 2.00±0.1. Samples were centrifuged at 9,000g for 10 mins and pyocyanin from 5 ml of the supernatant was extracted with 3 ml of chloroform, centrifuging at 3,000g for 5 min. Pyocyanin was then re-extracted into 1 ml of 0.2 N HCl, to give a pink to deep red solution, and centrifuged at 3,000g for 5 mins. The absorbance of this solution at 520 nm was measured. Concentrations, expressed as micrograms of pyocyanin produced per milliliter of culture supernatant, were determined by multiplying the OD at 520nm by 17.072 [43,44]. Swarming motility assays were conducted as previously described [45]. Isolates were grown for 24 h in LB at 37°C. The A 600 of each culture was adjusted to 2.10±0.1. Swarming plates contained 25 ml of the following medium: 0.8% nutrient broth, 0.5% agar, 0.5% glucose. 2 μl of each culture was spotted on the swarming plates and incubated for 24 h at 37°C. Determination of the percentage of swarm plate surface coverage was performed as previously described [45]. Swimming motility assays were conducted as previously described [46]. Isolates were grown for 24 h in LB at 37°C. The A 600 of each culture was adjusted to 1.65±0. Swimming plates (1% tryptone, 0.05% yeast extract, 0.5% NaCl, 0.3% agar) were inoculated with a sterile toothpick and incubated for 14 h at 37°C. Swimming motility was determined by measuring the areas of the circular turbid zone formed by the bacterial cells migrating away from the inoculation spot. Each sample was tested at least in duplicate. MIC and carbapenemase confirmation tests MIC of 18 antimicrobials (Table 4) were determined for all the 33 P. aeruginosa isolates using broth dilution test, according to the procedure described in CLSI guidelines VET01-A4 [47]. A commercially available microdilution MIC system, the Sensititre Compan1F Plate Format veterinary panel and Sensititre ARIS system (Trek Diagnostics Systems, East Grinstead, UK) was used. This panel includes antimicrobials and respective MIC dilution ranges listed in Table 4. Results were interpreted using CLSI resistance breakpoints according to M100-S24 guideline [48] for amikacin, doxycycline, imipenem, and VET01-S2 guideline [49] for all the other antimicrobials. For ticarcillin, ticarcillin/clavulanic acid, gentamicin, amikacin and imipenem specific resistance breakpoints for P. aeruginosa were available, while for doxycycline ampicillin, cefpodoxime, trimethoprim/sulfamethoxazole, enrofloxacin and marbofloxacin were used the resistance breakpoints for Enterobacteriaceae. For amoxicillin/clavulanic acid the breakpoint for "other organisms" was used; while for chloramphenicol the breakpoint select was for "organisms other than streptococci". For cefazolin the only breakpoint available was used, that is recommended in CLSI VET01-S2 for all the microorganisms tested. For oxacillin and cefoxitin the only cited resistance breakpoints for Staphylococcus spp. were used; for the same reason, for clindamycin the resistance breakpoints for Streptococcus spp. was used; for penicillin and erythromycin the resistance breakpoints for Enterococcus spp. were used. Finally, the resistance breakpoint for cattle mastitis was used for ceftiofur. The Sensititre plate reading was performed manually recording the last concentration of antimicrobial without turbidity or deposit of cells at the bottom of the well. Considering the great concern for public health induced by the presence of P. aeruginosa strains producing metallo-beta-lactamases (MBLs) that inactivate carbapenems, the strains that displayed resistance to imipenem, and therefore with a suspected carbapenemase production, were tested by using agar tablet/disc diffusion method by the KPC/ MBL and OXA-48 Confirm Kit (ROSCO Diagnostica A/S, Taastrup, Denmark). A combineddisk method using imipenem and cloxacillin (4000μg), as inhibitor of intrinsic cephalosporinase AmpC, was also evaluated to discriminate between carbapenemase-producing and nonproducing P. aeruginosa. [50]. Carba NP test was performed as previously described [51] in order to detect the presence of carbapenemase. The presence of the most frequent carbapenemase genes found in Pseudomonas (bla VIM , bla IMP ) [52][53][54] and the bla GES and bla OXA-48 genes were tested by Polymerase-chain-reaction (PCR) [55,56]. Statistical analyses SCCs were transformed to SCS, in order to normalize their distribution using the formula log 2 (SCC/100,000) + 3 [57]. Statistical analyses were computed using the IBM SPSS 21.0 software for Windows (SPSS Inc., Chicago, IL, USA). SCS, production of biofilm, elastase, gelatinase, protease, hemolysins and pyocyanin, swarming and swimming motility were the response variables. The explanatory variable was the strain relation to a genotype. Data distribution was tested with Shapiro-Wilk test, which found crystal violet biofilm, elastase, gelatinase, protease, hemolysisns, pyocyanin, swimming and swarming data not normally distributed, therefore a non-parametric Mann-Whitney test was used to asses significant differences. On the other hand, Shapiro-Wilk test revealed a normal distribution of data regarding SCS, therefore a oneway ANOVA was used. Levene's test was used to assess the variance homogeneity [58]. This assumption was satisfied, therefore Bonferroni test was performed. The statistical significance threshold chosen was p<0.05. Further analyses were computed with the XLSTAT 2014.6.01 statistical software for Excel (Addinsoft, New York, NY, USA) in order to identify clusters of complex trait phenotypes exhibiting different patterns and discern correlations between different strain sources, genotypes and phenotypic behavior. Phenotypic clustering was estimate through Agglomerative Hierarchical Clustering (AHC) calculating the Euclidean distance and using Ward's agglomeration method [59]. Correlations within variables tested in the study were obtained through Pearson's correlation matrix with a significant correlation level chosen at p<0.05. Data were also analyzed through PCA in order to visualize significant factors' correlations (p<0.05) on a dimensional map. The analyzed factors were represented by isolates, PFGE and phenotypic clusters.

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Influence of Multi-Gene Allele Combinations on Grain Size of Rice and Development of a Regression Equation Model to Predict Grain Parameters Background Grain size is one of the key factors determining yield and quality in rice. A large number of genes are involved in the regulation of grain size parameters such as grain length and grain width. Different alleles of these genes have different impacts on the grain size traits under their control. However, the combined influence of multiple alleles of different genes on grain size remains to be investigated. Six key genes known to influence grain size were investigated in this study: GS3, GS5, GS6, GW2, qSW5/GW5, and GW8/OsSPL16. Allele and grain measurement data were used to develop a regression equation model that can be used for molecular breeding of rice with desired grain characteristics. Results A total of 215 diverse rice germplasms, which originated from or were developed in 28 rice-consuming countries, were used in this study. Genotyping analysis demonstrated that a relatively small number of allele combinations were preserved in the diverse population and that these allele combinations were significantly associated with differences in grain size. Furthermore, in several cases, variation at a single gene was sufficient to influence grain size, even when the alleles of other genes remained constant. The data were used to develop a regression equation model for prediction of rice grain size, and this was tested using data from a further 34 germplasms. The model was significantly correlated with three of the four grain size-related traits examined in this study. Conclusion Rice grain size is strongly influenced by specific combinations of alleles from six different genes. A regression equation model developed from allele and grain measurement data can be used in rice breeding programs for the development of new rice varieties with desired grain size and shape. Electronic supplementary material The online version of this article (doi:10.1186/s12284-015-0066-1) contains supplementary material, which is available to authorized users. Background Rice is one of the most important crops in the world alongside wheat and maize. The top five countries for rice production and consumption are China, India, Indonesia, Bangladesh, and Vietnam. According to a 2009 survey, the combined population of these countries represented nearly half of the total world population (http://data.worldbank.org/). Numerous additional countries are also engaged in rice production and consumption, as well. This indicates the importance of rice as a staple food crop worldwide, and particularly in Asia. The world population continues to increase rapidly and this increase has led to a growing demand for rice. Enhancement of grain yield as well as grain quality is therefore of key agricultural importance. Rice yield potential is affected by at least four characteristics: panicles per unit area of land, number of spikelets per panicle, percentage of filled grains, and 1000-grain weight (KGW). Of these, KGW is a particularly complex trait that is determined by a combination of grain size-related traits such as grain length (GL), grain width (GW), and grain length to width ratio (LWR) (Tan et al. 2000). Grain size is a further quality trait used by the global market. Grain size preference varies depending on the geographical location. For instance, rice consumers in Thailand, Lao PDR, Cambodia, Malaysia, and the Philippines prefer long and slender grains, while those in Korea, Japan, Northern China and Taiwan prefer shorter and plumper grains (Calingacion et al. 2014). Grain size therefore influences the market available for any given crop (Redoña and Mackill 1998). A combination of different factors influence the grain size of rice. These include GL, GW, LWR, and grain thickness, all of which significantly correlate with grain weight (Tan et al. 2000). In summary, rice grain size and yield are closely related to one another and can be explained by a combination of grain size-related traits. A large number of genes are associated with rice grain size, and many of their functional roles have been elucidated in a range of studies (Zuo and Li 2014). In most cases, Quantitative trait loci (QTLs) linked to grain size parameters, such as length and width, allowed genes involved in grain size regulation to be fine mapped and characterized. For instance, GRAIN WIDTH and WEIGHT2 (GW2) gene, which encodes a RING-type E3 ubiquitin ligase, was fine mapped from a major QTL responsible for rice grain width and weight (Song et al. 2007). From two rice varieties showing contrast grain width, Fengaizhan 1 (FAZ1; small-grain indica-type variety) and Wuyujing 3 (WY3; large-grain japonica-type variety), two alleles of GW2 gene were distinguished by a single nucleotide polymorphism (SNP) in exon 4. The SNP resulted in a premature translational termination of GW2 protein, and was thought to be responsible for the enhanced grain width and weight in WY3 compared to FAZ1. GRAIN SIZE 3 (GS3) gene, which encodes a putative transmembrane protein, was fine mapped from a major QTL responsible for grain length and weight (Fan et al. 2006). The functional role of GS3 has been suggested as a negative regulator to prevent the growth of the grain size. Two alleles of GS3 gene, A and C, were distinguished by a SNP in exon 2 (Takano-Kai et al. 2009). Similarly, this SNP also resulted in a premature translational termination of GS3 protein. It was reported that the allelic variation of GS3 gene was significantly associated with the different grain length (Fan et al. 2009). In addition, an association study revealed that the Aallele (C165A mutation) of GS3 gene was significantly linked to enhanced rice grain length (Takano-Kai et al. 2009). GRAIN SIZE 5 (GS5) gene, which encodes a putative serine carboxypeptidase, was fine mapped from a QTL responsible for grain size . It was also suggested that higher expression of GS5 was significantly correlated with larger grain size. Three alleles of GS5 gene were distinguished by natural variations in the promoter region, and the rice varieties carrying these alleles also showed significant differences in grain width: H94-(narrow grain), Zhenshan97-(medium grain), and Zhonghua11-allele (wide grain). Therefore, these natural variations in the promoter region were thought to be responsible for the different grain width. qSW5/GW5 gene was fine mapped from a major QTL responsible for grain width, QTL for rice seed width on chromosome 5 (qSW5) (Shomura et al. 2008). Compared to the Kasalath (aus-type variety), the qSW5/GW5 gene region in Nipponbare (japonica-type variety) and in several indica-type varieties contained a large deletion and number of SNPs. Three alleles of qSW5/GW5 gene were distinguished by length of deletion at the qSW5/GW5 locus (Yan et al. 2011): Kasalathallele (no deletion), indica II-allele (950 bp deletion), and Nipponbare-allele (1,212 bp deletion). Analysis of variance (ANOVA) showed that germplasms carrying the Nipponbare-allele had wider grains than germplasms with the other two alleles (Shomura et al. 2008). GRAIN WIDTH 8/SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 16 (GW8/OsSPL16) gene, which encodes a positive regulator of cell proliferation, was fine mapped from a major QTL responsible for grain width, qGW8 (Wang et al. 2012). It was also demonstrated that higher expression of GW8/OsSPL16 resulted in enhanced rice grain width and yield due to promoted cell division and grain filling. In Basmati 385 (indica-type traditional Basmati variety), 10-bp deletion was found in the GW8/OsSPL16 promoter region compared to other two indica-type varieties (HJX 74 and TN1), and named Basmati-allele. This deletion was thought to be responsible for the slender grain in Basmati 385 compared to HJX 74. In addition, two alleles of GW8/OsSPL16 gene were also distinguished by a SNP in exon 1: HJX 74and TN1-alleles. Beside these, several genes involved in regulation of rice grain size were also identified by other approaches. For instance, GRAIN SIZE 6 (GS6) gene was isolated from an ethylmethanesulfonate (EMS)-induced mutant, gs6 (Sun et al. 2013). GS6 encodes a member of GRAS family proteins, which plays an important role in reducing rice grain size. Three alleles of the GS6 gene, type I to III, were distinguished by nucleotide variants in the promoter region. Of these, the type I-allele is found predominantly in japonica-type varieties, which have wider and heavier grains than varieties with the other two alleles. Despite recent advances in our understanding of grain size regulation in rice, the influence of combinations of alleles from multiple different genes remains largely unknown. It is therefore important to understand the influence of allele combinations on grain size when considering breeding for preferred grain shape and subsequently enhanced rice grain yield. In this study, we examined allele combinations of above-mentioned six key genes that were previously shown to influence grain size. A total of 215 rice germplasms from diverse countries were examined. The data were used to develop a regression equation model with potential for use in molecular breeding of rice. These results will facilitate prediction of grain size and facilitate efficient breeding of rice varieties with desirable grain yield and shape characteristics. Diverse Grain Size in the Rice Germplasm Collection A total of 215 rice germplasms collected from 28 riceconsuming countries were used to investigate the influence of allele combinations of six key genes on rice grain size (Additional file 1). This collection contained diverse rice germplasms from wild species to modern cultivars: tropical japonica (24), temperate japonica (63), indica (85), aromatic (9), aus (15), wild species (1), traditional (1), and ungrouped (17). The average grain size differed substantially between the different germplasms with respect to four grain size-related traits (GL, GW, LWR, and KGW). The GL range was 6.09-10.81 mm, GW range was 2.21-4.32 mm, LWR range was 1.79-4.34, and KGW range was 14.49-51.91 g (Additional file 1). Significant correlations between these four traits were observed in the germplasm collection (Additional file 2). This suggests that the four traits determining grain size are tightly related to one another and play important roles in regulating rice grain size. Allele Distributions of Six Genes Involved in Determining Grain Size Numerous genes are associated with rice grain size, and the functional roles of many of their protein products have been elucidated in a range of studies (Zuo and Li 2014). However, the allelic contributions of multiple genes to grain size and shape have not yet been explored. In this study, allelic variation in several genes was used to develop a regression equation model that could be used for molecular breeding in rice. Six key genes were investigated: GS3 (Os03g0407400), GS5 (Os05g0158500), GS6 (Os06g0127800), GW2 (Os02g0244100), GW8/OsSPL16 (Os08g0531600), and qSW5/GW5 (Os05g0187500). The genes each had two or three functional alleles in the diverse germplasm collection, and grain size parameters such as length and width varied considerably with these alleles. The allele distributions of the six genes were determined in the germplasm collection, then considered in the context of grain measurements. Two alleles of GS3 gene, A and C, were distinguished by a SNP in exon 2 (Takano-Kai et al. 2009). A functional nucleotide polymorphism (FNP) marker, GS3-PstI, which was developed by Yan et al. (2011) was used to determine the allele distribution of GS3 gene in the germplasm collection. A-and C-alleles were found in 27 and 83 germplasms, respectively. Interestingly, an additional allele was also uncovered (named the B-allele) that contained a 45-base pair insertion in the first intron. The B-allele was found in the remainder of the collected germplasms. The additional B-allele necessitated use of a higher percentage agarose gel (2.5 %) than was used previously. The different GS3 allelic groups exhibited differed grain parameters: (1) germplasms with the A-allele had significantly higher GL and LWR values, and lower GW values, than the other allelic groups; (2) germplasms with the B-allele had intermediate GL and LWR values;and (3) germplasms with the C-allele had significantly lower GL and LWR values, and significantly higher GW values, than the other germplasms (Table 1). In summary, the three alleles of GS3 gene were significantly associated with differences in grain size-related traits (except for KGW) in the diverse germplasms used in this study. Three alleles of GS5 gene, Zhenshan97, H94, and Zhonghua11, were distinguished by natural variations in the promoter region . Two derived cleaved amplified polymorphic sequence (dCAPS) markers were used to investigate the allelic distribution of GS5 gene in the germplasm collection: (1) GS5-TaqI for classification of the H94-and Zhenshan97-alleles, and (2) GS5-SalI to distinguish the Zhonghua11-allele. The Zhonghua11-allele was most abundant (135 out of 215 germplasms) and the Zhenshan97-and H94-alleles were found in 17 and 63 germplasms, respectively. Consistent with the previously published results , the 135 germplasms carrying the Zhonghua11allele had significantly higher GW values than the other germplasms. In addition to the GW, we also observed significant differences between the allelic germplasm groups for GL and LWR (Table 1). Three alleles of GS6 gene, type I to III, were distinguished by nucleotide variants in the promoter region (Sun et al. 2013). For this study, as only the type I-allele was associated with enhanced grain parameters, the type II-and type III-alleles were considered together (i.e., two categories were used: type I, and type II/III). An insertion-deletion (InDel) polymorphism marker, indel-GS6, was designed in the promoter region of GS6 gene to distinguish the different alleles. Type I and II/III-alleles were found in 104 and 114 germplasms, respectively. As noted previously (Sun et al. 2013), germplasms carrying the type I-allele had significantly higher GW and KGW values than the other germplasms (Table 1). In addition to GW and KGW, we also observed significant differences between the allelic germplasm groups for GL and LWR. Two alleles of GW2 gene, FAZ1 and WY3, were distinguished by a SNP in exon 4 (Song et al. 2007). A dCAPS marker, GW2-ScaI, was designed to examine allele distribution. The FAZ1-allele was found in almost all the germplasms, with only three carrying the WY3-allele (Hokuriku 130, Dearybbyeo 1, and Oochikara). As noted previously (Song et al. 2007), the WY3-allele germplasms had significantly higher GW and KGW values (Table 1). This suggests that the WY3-allele of GW2 gene is valuable for grain size enhancement, but is relatively rare. Three alleles of qSW5/GW5 gene, Kasalath, indica II, and Nipponbare, were distinguished by length of deletion at the qSW5/GW5 locus (Yan et al. 2011). A previously designed InDel marker, N1212del (Shomura et al. 2008), was used to investigate the allele distribution of qSW5/GW5 gene in the germplasm collection. All three qSW5/GW5 alleles were found in the collection. The Kasalath-allele was found in 90 germplasms, Indica IIallele in 41 germplasms, and Nipponbare-allele in 84 germplasms. Consistent with the previous study (Shomura et al. 2008), the 84 germplasms carrying the Nipponbare-allele of qSW5/GW5 gene had significantly higher GW values than the germplasms with the other two alleles (Table 1). In addition, we also observed significant differences between the allelic germplasm groups for GL, LWR, and KGW. Here, an InDel marker, indel-GW8, was designed to identify germplasms carrying the Basmati-allele. In addition, a specific genomic region containing the 5′ untranslated region (UTR) and first exon was sequenced to distinguish between the HJX74-and TN1-alleles. The Basmati-allele of GW8/OsSPL16 was found in 111 germplasms, and 66 and 38 germplasms contained the HJX74-and TN1-alleles, respectively. In contrast with the previous study (Wang et al. 2012), germplasms with the Basmati-allele had wider grains than germplasms carrying the other two alleles. The three alleles of GW8/ OsSPL16 gene were significantly associated with differences in GL, GW, LWR, and KGW (Table 1). Taken together, our data show that allelic variations of the six genes are widely distributed in our germplasm collection. In most cases, allelic variants are significantly associated with differences in three or more of the major traits (GL, GW, LWR, and KGW) for grain size. Influence of Allele Combinations on Grain Size-related Traits The genotyping results showed that several germplasms had a similar grain size and also shared a particular allele combination of the six key genes examined in this study. For example, 33 germplasms had a GL of 6.16-8.15 mm and had the same allele combination (B-allele of GS3, Zhonghua11-allele of GS5, Nipponbare-allele of qSW5/ Data represent mean±standard deviation; ANOVA test, *P < 0.05 a, b, and c were ranked by Duncan's test GW5, type I-allele of GS6, and Basmati-allele of GW8/ OsSPL16). This indicated that similarity in rice GL could be attributed to certain allele combinations even in the presence of different genetic backgrounds. We therefore grouped our germplasm collection according to allele combinations that were significantly associated with four major traits (GL, GW, LWR, and KGW) for grain size and yield (Additional file 3). With respect to GL, significant differences were observed between alleles of five genes (GS3, GS5, qSW5/ GW5, GS6, and GW8/OsSPL16) (Table 1). Twenty-six of the 162 possible allele combinations for these five genes Table 2 Allele combinations including a single gene-specific allelic variation Grain size-related traits Gene name Group name Germplasms were found in 166 of the 215 germplasms. These combinations were termed group LA to LZ. Three or more germplasms constituted a group. Several combination groups showed a single gene-specific allelic variation. For instance, group LA, LB, and LC all had the same allele combinations for four genes (GS5, qSW5/GW5, GS6, and GW8/OsSPL16), but only differed at GS3. Other groups (LD-LF and LJ-LL) also showed single genespecific allelic variation at GS3. Despite the similar allele combination patterns in these groups, average GL was significantly different only for group LA-LC (Additional file 4). These results indicate that allelic variation at GS3 plays an important role in regulation of rice GL in the presence of a certain allele combination of four other genes (Zhonghua11-allele of GS5, Nipponbare-type of qSW5/GW5, type I-allele of GS6, and Basmati-allele of GW8/OsSPL16). Although several other groups also varied at only one gene (GS5, qSW5/GW5, GS6, or GW8/ OsSPL16), the average GL was not significantly different between these groups. As for GW, significant differences were observed between alleles of all six genes (GW2, GS3, GS5, qSW5/ GW5, GS6, and GW8/OsSPL16) (Table 1). Of the 324 possible allele combinations, 26 were present in a total of 164 germplasms and were named group WA-WZ (Additional file 3). Of these, average GW was significantly different in groups containing single gene-specific allelic variation at one of three genes (GS3, GS6, or qSW5/GW5), particularly when certain allele combinations of the other five genes were present (Table 2). For instance, average GW was significantly different between WN and WV, which different only at GS6 gene. For LWR, significant differences were observed between alleles of five genes (GS3, GS5, qSW5/GW5, GS6, and GW8/OsSPL16) (Table 1). Of the 162 possible allele combinations, 22 were observed in a total of 189 germplasms and were named group RA-RV (Additional file 3). Of these, the average LWR was significantly different in groups containing single gene-specific allelic variation at one of two genes (GS3 or qSW5/GW5), particularly when certain allele combinations of the other three genes were present (Table 2). For instance, the average LWR was significantly different between groups RN and RP, which varied only at qSW5/GW5 gene. Significant allelic differences for KGW were observed in four genes (GW2, qSW5/GW5, GS6, and GW8/ OsSPL16) (Table 1). Of the 36 possible allele combinations, 14 were found in a total of 206 germplasms and were termed group KA-KN (Additional file 3). Of these, KGW was significantly different in groups containing single gene-specific allelic variation only at GW8/OsSPL16 gene, particularly when certain allele combinations of the other three genes were present (Table 2). For instance, KGW was significantly different between KM and KN, which varied only at GW8/ OsSPL16 gene. Our results, taken together, indicate that a certain type of allele combination plays an important role in regulation of rice grain size and yield, even in the presence of difference genetic backgrounds. The results also suggest that particular allele combinations have a strong influence on single gene-specific allelic variation for grain size and yield. Development of a Regression Equation Model Allelic distribution data for the six genes examined in this study were used to develop a regression equation model. Allele data were first converted into dummy variables. Genes with significant allelic associations with grain size-related traits (GL, GW, LRW, and KGW) were used as independent variables (Table 1). Regression analysis was performed using these data. Parameter estimates, standard deviations, and, t values were calculated accordingly (Tables 3 and 4). These values indicated the contribution levels of each molecular marker to differences in grain size-related traits. For instance, the influence of allelic variation at GW2 gene in GW and KGW was significantly analyzed by use of a dCAPS marker, GW2-ScaI. These values were used to develop a regression equation model for prediction of rice grain size. Validation of a Regression Model To evaluate the regression equation model, allele and grain size-related trait data were gathered from an additional 34 germplasms of diverse origin and grain size (Additional file 5). The estimated values of each grain size-related trait (GL, GW, LRW, or KGW) were calculated using the regression equation model, and these values were then compared with actual measurements. The model had substantial predictive power for three traits (GL, R 2 = 0.640; GW, R 2 = 0.542; and LWR, R 2 = 0.735) (Fig. 1). However, predictive power for KGW was low (R 2 = 0.260), suggesting that grain weight is possibly regulated in a complex manner compared to grain length and width. Discovery of an Additional Allele of GS3 Gene A causal C to A mutation in the second exon of the GS3 gene is highly associated with GL in rice (Fan et al. 2009). This mutation, which creates a prematurely truncated GS3 protein, results in enhanced GL. Here, 110 of the 215 germplasms tested had A-(C165A mutation) or C-alleles (Table 1). These germplasms differed in GL according to their GS3 allelic variation. The remaining 104 germplasms contained a novel allele (termed the Ballele) (Additional file 6), which had a 45-bp insertion in the first intron and did not exhibit the C165A mutation (Takano-Kai et al. 2013). Germplasms carrying the Ballele of GS3 gene had an intermediate GL compared to the germplasms carrying the A-and C-alleles (Table 1). As noted above, enhanced GL in C165A varieties was attributed to the truncated GS3 protein (Mao et al. 2010). It has been reported that aberrant splicing can be generated by insertions in intron regions (Sironen et al. 2006). We propose that such an aberrant splicing event could have been generated by the 45-bp intronic insertion in the B-allele, and that this led to modified translation of GS3 protein. Comparisons of the B-and C-allele transcripts would verify such aberrant splicing. The presence of the novel GS3 allele in a large proportion of the tested germplasms highlights its importance. The B-allele will enhance our understanding of the influence of GS3 on grain size-related traits. The Influence of Allele Combinations from Six Genes on Rice Grain Size A large number of genes are involved in regulation of rice grain size, and there are consequently thousands of possible allele combinations governing grain traits. Here, we examined the influence of allelic variation in six genes on grain size-related traits: GS3, GS5, GS6, GW2, GW8/OsSPL16, and qSW5/GW5. Allele data and grain parameters were used to develop and test a regression equation model for prediction of grain size. Our results showed that a relatively small number of allele combinations persisted in the diverse rice germplasm collection, and that these combinations were significantly associated with differences in grain size. We also noted that single gene-specific allelic variation played an important role in regulation of grain size in the presence of certain allele combinations. For example, GL was significantly different between allelic groups LA, LB, and LC, which had the same alleles for four of the five genes under consideration, but not between LD-LF or LJ-LL, which also differed at only one of the five genes (Additional file 4). These results suggest that particular allele combinations have substantial influences on rice grain size. Application of a Regression Equation Model for Prediction of Rice Grain Size A regression equation model was generated using allele and trait data from 215 germplasms. The model was then tested against data from a further 34 diverse germplasms. R-square values indicative of correlation were obtained for three grain size-related traits (GL,0.640;GW,0.542;and LWR,0.735), but correlation was low for KGW (R 2 = 0.260) (Fig. 1). These values indicate the utility of our regression equation model for prediction of grain size in rice. Our data show that grain size is likely to be strongly influenced by combinations of certain alleles, and the regression equation model therefore provides a useful tool for rice molecular breeding. For example, rice grain size preferences vary between countries, and the regression equation model could be used to develop novel rice varieties with grain length and width within a desirable range. However, it is important to note that, although the R-square values seen in this study indicate some correlation, the current model is limited in its predictive ability as only six genes were examined. The model will be improved by inclusion of novel alleles as they are discovered (such as the B-allele of GS3 discovered in this study) and by addition of data from more genes. In addition, the model developed here can be used alongside other models to refine breeding for other yield potential and grain quality traits. For example, several regression equation models have been developed to estimate rice eating quality (Lestari et al. 2009;Lestari et al. 2015). Conclusion Allelic variation of six key genes involved in regulation of rice grain size was widely distributed in a collection of 215 germplasms, indicating that the collection was representative of genetic diversity in rice (Oryza sativa L.). Several germplasms in our collection that had similar grain traits (such as length and width) also shared allele combinations. These results suggest that rice grain size is likely determined by particular allele combinations of several genes involved in regulation of grain size. The results were used to develop a regression equation model that can be used for rice molecular breeding programs. Our data and regression model will be valuable for Plant Materials and Grain Size Measurements A total of 215 rice germplasms that were developed in, or originated from, 28 rice-consuming countries were used in this study (Additional file 1). Rice plants were examined under natural field conditions in the experimental farm of Seoul National University, Suwon, Korea. Normal agricultural practice was followed for field management. Harvested rice grains were air-dried for 1 month prior to measurement. A total of ten fully filled grains were randomly chosen for each rice germplasm. The size of selected grains (GL, GW, and LWR) was measured using SmartGrain Version 1.2 software (Tanabata et al. 2012). The KGW of each germplasm was determined by measuring 200 fully filled grains and multiplying by five. Genomic DNA Extraction, PCR, and Sequencing Genomic DNAs were extracted from young rice seedlings using a modified CTAB DNA extraction procedure (Murray and Thompson 1980). PCR was performed in a reaction volume of 20 μL containing 40 ng of genomic DNA, 0.2 μM of each primer, 200 μM of each dNTP, 10 mM Tris-Cl (pH 8.3), 50 mM KCl, 1.5 mM MgCl 2 , 0.01 % gelatin, and 0.5 U of Taq DNA polymerase. PCR amplifications were carried in a DNA Engine Tetrad 2 Peltier Thermal Cycler (Bio-Rad) using the following program: 5 min at 94°C; 32 cycles of 45 s at 94°C, 30 s at 47-65°C, 20 s at 72°C; and 10 min final extension at 72°C (Additional file 7). Amplified PCR products were separated on a 1-3 % agarose gel to validate the expected fragment size.

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Efficacy of sub lethal concentration of entomopathogenic fungi on the feeding and reproduction of Spodoptera litura In the present investigation, impact of sub lethal concentrations of entomopathogenic fungi, namely Isaria fumosorosea, Beauveria bassiana and Paecilomyces variotii, secondary metabolite on feeding, growth, fecundity and hatchability of Spodoptera litura was performed. The S. litura treated with I. fumosorosea and B. bassiana metabolites exhibited renounced food consumption. The growth rate of treated S. litura with metabolite of I. fumosorosea had drastic reduction. In the case of approximate digestibility (AD), maximum impact was established by the I. fumosorosea isolate, which significantly reduced the approximate digestibility of the IV and V instar larvae. The III instar larvae of S. litura treated with I. fumosorosea metabolite showed significantly lower efficiency of conversion of digested food (ECD) and efficiency of conversion of ingested food (ECI) values than IV and V instars. However the performance of metabolites on fecundity and hatchability of S. litura was immense. Therefore, metabolites of I. fumosorosea could be reliable biocontrol agent, which has been highly recommended for S. litura management in commercial crops. . Robert (1981) provided a complete overview on these fungal toxins. According to Wang et al. (2007) studies, it has been observed that, the fungal metabolites are potential insecticide against insect pest. Thomsen and Eilenberg (2000) stated that the lepidopteran insects are vulnerable to the destruxins. In support of this, Hu et al. (2007) stated that, the secondary metabolites produced by the entomopathogenic fungi particularly M. anisopliae, were toxic to Spodoptera litura. Therefore, the present study, aims to study the role of secondary metabolite of entomopathogenic fungi on the feeding, growth and development of S. litura. Screening of secondary metabolites The selected entomopathogenic fungi were subjected to solvent extraction (Ethyl acetate) for the isolation of active secondary metabolites. The extracts of the entomopathogenic fungi were subjected to study the Rf value, by separating them by using TLC and were observed under UV trans-illuminator and ninhydrin spray. Feeding experiment Different sub lethal concentrations were used for the feeding experiment study against S. litura in in vitro. The feeding experiment was performed on different parameters such as consumption index (CI), growth rate (GR), approximate digestibility (AD), conversion of digested food (ECD) and conversion of Ingested food (ECI) (Tables 1, 2, 3). The one tailed t test analysis report was presented in Table 4. Feeding experiment The S. litura treated with I. fumosorosea and B. bassiana fractions exhibited reduced food consumption. I. fumosorosea fraction diminished the consumption efficiency at the mean difference of 0.035, 0.582 and 0.692 with respect to III, IV and V instar larvae of S. litura (Table 1) with respect from control to 1, 2, 3, 4 ppm accordingly. The calculated CD (0.05 %) (0.002, 0.002 and 0.002) was comparatively lower than that of the mean difference (0.004, 0.699 and 0.765) which implies the significant difference between control and treatment. The GR of treated S. litura with fractions of I. fumosorosea had drastic reduction in III, IV and V instar larvae ranged from 0.193 (control) to 0.180 (4 ppm) mg dry wt −1 live larvae −1 ; 0.198 (control) to 0.185 (4 ppm) mg dry wt −1 live larva −1 and 0.201 (control) to 0.181 (4 ppm) mg dry wt −1 live larva −1 . The mean difference of the growth rate of the control and treatment was 0.004, 0.013 and 0.006, which was higher than the calculated CD value (0.05 % level) (0.002, 0.002 and 0.003). Similarly, in the P. varioti isolate, there was a sharp decrease in the growth rate observed ( Table 2). The reduction in terms of weight was 0.07, 0.010 and 0.20 mg dry wt −1 live larva −1 . The mean difference was significantly differed from control. In the case of B. bassiana, the reduction of growth rate was found higher in 3rd instar larvae, whereas it become remain the same in the 4th and 5th instar larvae (0.020 mg dry wt −1 live larva −1 ) ( Table 2). The mean difference of the growth rate between the control and treatment was 0.004, 0.009 and 0.011. It was higher than the calculated CD (0.05 % level) value (0.002, 0.003 and 0.001), which implies the significant difference in the growth rate of control and experiment. The AD sharply decreased with increasing the concentration. The maximum impact was established by the I. fumosorosea isolate, which significantly reduced the AD of the IV and V instar larvae. The III instar exhibited over 90 % AD which declined sharply in the IV and V instars to a maximum of 88.05 and 83.33 %. The mean difference of the control and treatment with respect to III, IV and V instar larvae was 1.16, 2.85 and 0.006. Similar reduction was not observed in P. variotii, while B. bassiana isolates exhibited the similar pattern of reduction from III to V instar larvae. The AD of the P. variotii and B. bassiana isolate from III to V instar at 4 ppm ranged from 83.33 (Table 2) and B. bassiana stated that, the AD of the tested instars was significantly differed from control group. The III instar larvae of S. litura treated with I. fumosorosea toxin showed significantly lower ECD and ECI values than IV and V instars ( Table 1). The similar impact was also observed in the III instar larvae (Table 2) treated with fraction of P. variotii (ECD and ECI) and B. bassiana (Table 3) (ECD and ECI). Exceptionally, V instar larvae of S. litura treated with B. bassiana had outrageous impact and have least conversion efficiency of 23.01 and 22.90 % with respect to ECD and ECI. There was a significant difference in the control and treatment was observed for ECD and ECI values of S. litura treated with P. variotii and B. bassiana. Effect of ethyl acetate fraction on the fecundity and hatchability The fractions of EPF had adverse effect on egg laying as well as growth stages of S. litura. The fecundity of S. litura inversely proportional to the concentration of the fraction of I. fumosorosea used (Table 5; Fig. 1). The fecundity of the S. litura treated with I. fumosorosea fraction drastically reduced since lowest concentrations recorded 167 eggs −1 batch −1 insect −1 , while at 5 µl showed 67 eggs −1 batch −1 insect −1 . The mean difference between the control and experiment was 8.07, which was higher than the calculated CD value (0.049 and 0.034 at 0.01 and 0.05 % respectively). The difference in fecundity found between the lower to higher concentration was 100 eggs batch −1 insect −1 . No such reduction in the egg laying was observed in the case of P. variotii (Table 5) (Fig. 1). However the performance of fraction on fecundity and hatchability of S. litura was immense. In addition to that, they shortened and mutated the each stage up to adult emergence in a panic and adverse way. Hence the fractions were not only pathogenic but also mutagenic to the S. litura. However, the instars treated with selected EPF showed significant difference in the treatment with respective fractions of EPF. Discussion In this study, the tested fractions showed good sign of infection during the life cycle of S. litura. Vey et al. (2001) reported the in vivo effects of fungal metabolites in insects, measured as growth depression and changes in mortality, fertility, egg viability and metamorphosis. In the present study, a drastic decline in food consumption was observed with fraction of I. fumosorosea compared to B. bassiana and P. variotii. It was well supported by Tefera and Pringle (2003) who observed that, the significant reduction in consumption has been attributed to the production of toxic substances by the entomopathogenic fungi inside the host that lead to mechanical disruption in the insect structural integrity. The drastic reduction in food consumption in the present study was supported by Assaf et al. (2005) who stated that the reduction in feeding associated with the production of toxins by the fungus I. fumosorosea. Sahayaraj and Tomson (2010) observed 33.34 % reduction in bodyweight of Dysdercus cingulatus treated with metabolites of B. bassiana fraction 2 (BBF2). It supported the present study that, the reduction in the range of 0.02-0.010 mg in larval weight of S. litura. Similar observation was also attained by Malarvannan et al. (2008) who found The early instar larvae of S. litura had shown normal digestion compared to control while proceeding the larvae failed to show a good sign of digestion process in terms of reduced AD. The higher AD values in the early instars of infected larvae may be due to the little consumption of the treated part. Likewise, the lower AD values in the late instars were because these caterpillars consumed food indiscriminately to meet the demand for energy and nitrogen (Hussain et al. 2009). In support of the present investigation, Hussain et al. (2009) observed higher AD values in Ocinara varians larvae infected with entomopathogenic fungi compared to the healthy control. The present investigation recorded reduced conversion potential (digested as well ingested food) of the treated larvae. Slansky and Scriber (1982) found that the utilization efficiencies (ECI) of 11 predaceous insects were between 4 and 75 %, while in the present investigation, the isolate I. fumosorosea fraction treated larvae had shown comparatively higher reduction in ECD and ECI which ranged from 75 to 46 % with respect to III to V instar larvae. In support of the present study, Sintim et al. (2009) observed 45.8 % ECI value in S. litura when fed with artificial diet. Fraenkel (1981) states that, under suitable conditions, a growing insect could convert maximum of 2/3 of its ingested food to body materials remaining will be utilized for metabolic processes. Abnormal reduction in hatchability of eggs in I. fumosorosea treatment was in accompany with Leckie et al. (2008) observed delayed development, lower weights and high mortality of larvae of Heliothis zea when fed on diets containing mycelia of B. bassiana. Malarvannan et al. (2010) observed the complete arrest of fecundity by 2.4 × 10 7 spore mL −1 concentration of B. bassiana. Similarly, Gindin et al. (2006) reported reduction of 80-82 % in the hatchability of red palm weevil adults, Rhynchophorus ferrugineus treated with B. bassiana. From this it was confirmed that, the deleterious impact of this insecticidal toxin or fraction on the larval anatomy has been noticed. Conclusions Surveillance of impact of fungal fraction on growth and development of S. litura was performed in the present study. Sub lethal dose of I. fumosorosea fraction had significant impact on the consumption and digestion rate of S. litura. There was severe damage in the midgut regions were noticed. In addition to that, the next generation seeds, the eggs, were heavily suffocated in development and thereby I. fumosorosea fraction severely reduced the hatching and thus leads to significant production of malformed pupae for the next generation. Therefore, I. fumosorosea fraction could be reliable biocontrol agent that can highly recommend for S. litura management in cotton as well as sunflower field. Isolation of entomopathogenic fungi One gram of soil was diluted with 10 ml of distilled water and was serially diluted. From each dilution, 100 µL was plated on Potato Dextrose Agar (PDA) medium and it was fortified with streptomycin (10 mg/100 ml). It was allowed to grow for 7 days at 27 ± 2 °C in the respective media. After 7 days of incubation, the fungal colony was identified and was sub-cultured in Sabaroud Dextrose Agar (SDA). In the case of cadavers, each cadaver is carefully held with a light forceps and the conidia drawn slowly into a vial using a 00 camlin brush. Five milli-gram of dry spore was taken and added with 10 ml of sterile 0.02 % tween 80 solution. The sterilized SDA medium was transferred into sterile petridishes and test tubes that were then inoculated with pure conidia of entomopathogenic fungi by streak plate method (Haraprasad et al. 2001) The isolated fungi were transferred on to Potato Dextrose Agar (Hi-Media, India) petri dishes (9 cm in diameter) (Borosil ® ) and incubated at 25 °C for 1-2 week to produce conidia which is used as inoculums for the further study. Screening of secondary metabolites After 2 weeks of incubation, dense sporulated PDA plate was used for harvesting the inoculum preparation. The plate was flooded with 20 mL of sterile distilled water supplemented with 0.02 % tween 80 (Hi-Media, India) and scraped with stainless steel spatula (Hi -Media, India). It was then filtered through muslin cloth and the resulted spore solution was subjected for spore count using Haemocytometer. The spore concentration was then adjusted to 1 × 10 8 and 1 mL from this stock was poured into the Potato dextrose broth (PDB) (Hi-Media, India) for secondary metabolite production. After 14 days of incubation, crude extracts of the cultured broth were obtained following the method reported by Hu et al. (2007) with minor modification. The thick mycelia mat was removed and culture medium was harvested and centrifuged (Remi, India) at 8000 rpm for 10 min. The supernatant was extracted with ethyl acetate (Sample: Ethyl acetate = 5:2, v/v) and the organic phase was evaporated by placing it in incubator at 40 °C. The concentrate was diluted with 4 times volume of water and incubated at 4 °C overnight until the precipitation was observed. Finally, dried precipitate was used for bioassay as secondary metabolite. The precipitate was dissolved in ethyl acetate and used for feeding experiment. Feeding experiment The first instar larva of S. litura was separated from stock culture and fed with Ricinus communis leaves ad libitum. After the larvae reached the III instar, the initial weight of the larvae and leaves provided to them were calculated using digital balance. The leaf consumption rate was calculated from III to V instar along with the growth rate of the larvae. The faecal matter of these larvae was also collected at each stages were dried and weighed from which the a P. variotii milation rate of the larvae was calculated. It was adopted to larvae of S. litura to calculate its consumption index (CI), growth rate (GR), approximate digestibility (AD), efficiency of conversion of digested food (ECD) and efficiency of conversion of ingested food (ECI) by gravimetric analysis (Waldbauer 1968) by using the following formula. Consumption index (CI) Consumption index (CI) or the rate of feeding relative to the weight of the insect in a definite time can be expressed as: where, F is the weight of food eaten; A is the mean weight of insect during the feeding period, T is the duration of the feeding period (Days) Growth rate (GR) This measurement explains the rate at which the digested matter is available to the insect during the experimental period. Approximate digestibility (AD) Earlier workers have used to call approximate digestibility as "co-efficient of utilization", "coefficient of digestibility" and "degree of absorption" for express P. variotiing the digestibility of food material. Waldbauer (1968) pointed out that this measure is misleading and should be referred to as "approximate digestibility". Efficiency of conversion of digested food (ECD) The amount of food digested can be calculated by subtracting the weight of faeces from the weight of food ingested. This index has also been termed by some workers as "coefficient of growth". Efficiency of conversion of ingested food (ECI) Efficiency of conversion of ingested food measurement indicate the overall efficiency of the insect to utilize the food for growth. Statistical analysis Data of fecundity and hatchability both control and treatment was subjected to one tailed t test by using Statistical Packages for Social Sciences version 17. The critical difference (CD) value was calculated by using the software WASP 1.0. Authors' contributions PVM and CB designed this study and PVM executed the study. CB, SS and AR collected the reference data and corrected the manuscript. PVM wrote the manuscript. All authors read and approved the final manuscript. Authors information Author PVM, did his research in the field of biological control of insect pest using entomopathogenic fungi. He did his doctoral thesis in the same field at Thiagarajar College affiliated to Madurai Kamaraj University. GR = Weight gained by the insect Duration of feeding period Days × Mean weight of insect during the feeding period AD = Weight of food ingested − weight of faeces Weight of food ingested × 100 ECD = Weight gained by the insect Weight of food digested × 100 ECI = Weight gained by the insect Weight of food ingested × 100

v3-fos

2019-03-20T13:05:43.332Z

2015-06-24T00:00:00.000Z

84032577

s2

Growth and Economic Performance of Clarias gariepinus Fry Reared at Various Stocking Densities The biological and economic performance of Clarias gariepinus fry was investigated at various stocking densities. Five aquaria tanks were stocked at 100 (STK1), 150 (STK2), 200 (STK3), 250 (STK4) and 300 (STK5) fry/L. Triplicate groups of experimental fish were fed for 28 days with commercial diets. Water quality parameters were monitored throughout the experimental period, with pH, dissolved oxygen and temperature within the recommended range for the culture of C. gariepinus . Mean weight gain was significantly higher (P<0.05) in STK1 and STK 2 groups. Specific growth rate also followed the same trend, with the highest (8.47) recorded in STK 1, while the least (6.55) was recorded in STK 5. Performance indicator reduced with increasing stocking density. Profitability index were high in STK1 (3.21) and STK2 (3.30) while Incidence of costs were significantly lower in these two groups. This study reveals that stocking density significantly affects the performance and survival of fry of the African catfish, therefore stocking density of 100- 150 fry/L is recommended for C. gariepinus fry. Introduction The ultimate goal of aquaculture is the production of high quality fish in the highest quantity, within the shortest possible time at the least cost. This is achievable with careful selection of species, appropriate feeding, and good water quality and to a great extent, the density to which the fish are stocked [1]. The determination of the optimum carrying capacity of an aquatic environment is therefore very important. The number of organism that can be stocked in a culture medium per unit area or volume is referred to as stocking density and described optimum stocking density as the density at which yield is maximized without negatively affecting growth rate. Studies concerning the relationship between stocking density and growth in fish have shown that optimal stocking density for obtaining the highest possible fish yields depend upon the amount and the quality of food available [2]. In Nigeria, Clarias gariepinus is widely cultured because they tolerate many extremes in water quality and generally adapt to culture environment, although intensive culture, where fish are kept crowded under high stocking density, fish natural abilities to repel diseases are weakened and thus more susceptible to disease attacks [3]. Schreck and Essa [4][5] noted that high stocking density as a technique to maximize water usage and thus increase stock production has an adverse effect on growth on Oncorhynchus kisutch and the hybrid of Oreochromis niloticus X O. aureus. Studies on the stocking densities of C. gariepinus fingerlings and other bigger sizes abound, however, information on the optimal stocking density of fry to actualize the farmers' objective of economic growth and profit maximization is inadequate. This study investigates the growth, survival and economic performance of C. gariepinus fry at different stocking density, to establish the optimum stocking density for this species. Experimental fish and rearing units The experiment was conducted using fifteen aquaria (105 Litre capacity) filled with 80 L of aerated-water. Fry of C. gariepinus (initial mean weight 0.021 g) were obtained from the University of Ibadan Fish hatchery. At the start of the experiment, a total of 50 fry were taken in duplicate from the common stock in order to determine initial weights. Wet weight was recorded using an electronic weighing balance (accuracy of ± 0.01 mg). Fish were counted and stocked at 100, 150, 200, 250 and 300 fry/L to give STK1 (8000 larvae), STK2 (12000 larvae), STK3 (16000 larvae), STK4 (20000 larvae), and STK5 (24000 larvae) respectively, and each treatment replicated thrice. Daily water exchange in the tanks was 1.5 L/min. Fish were fed encapsulated brine shrimp (Artemia salina) four times daily to satiation for the first 3-days, after which they were fed at 10% of total biomass three times daily (At 7:00, 12:00 and 17:00 hrs.) with commercial diet (45% Crude protein). Excess or uneaten feed and faecal wastes were siphoned using 2 mm hose with a screen once daily. Dead fish in each tank were removed and recorded. Tanks were uniformly aerated. Water temperature and dissolved oxygen were measured using a Combined Digital Probe (YSI Model 57, VWR Company, New Jersey), weekly pH were measured using pH meter (Metler Toledo-320 model, U.K), while ammonia and nitrite were measured using API test kits. Fifty fish were weighed per tank biweekly, using electronic toploading balance (OHAUS corporation model: V21PW15) and the average weight calculated, this was used to adjust for feed requirement. Economic analysis The cost of production of experimental fish was calculated as described by [6] Statistical analysis The data obtained were subjected to a one way analysis of variance and the difference between means were separated using Duncan Multiple Range Test at P>0.05, using SPSS version 17. Results and Discussion The growth performances of experimental fish are shown in Table 1. The results showed that mean weight gain were significantly higher (p<0.05) in STK1 and STK2 groups. Highest weight gain (0.21 g) was recorded in STK1, while the least value (0.11 g) was recorded in STK5. This same trend was observed in the SGR. Availability of space and reduced competition and stress may have contributed to the result obtained in this present study. Higher stocking density was observed to increase stress, leading to higher energy requirements, causing a reduction in growth rate and food utilization [7]. This result is in agreement with [8] who reported better growth in Heteropnestes fossilis at lower stocking density. Significant decrease in weight gain was also reported in the works of [9,10] for C. gariepinus and the hybrid as a result of high stocking density. Superior SGR in fish stocked at lower stocking density was observed in [11]. Significant variation (p<0.05) is observed in the feed intake of experimental fish. Higher stocking density resulted in reduced feed intake per fish. Reduction in growth could directly be attributed to this, as growth in fish has been shown to depend on food intake and a host of intrinsic factors [12]. The result of this present study is corroborated by [2]. The AFCR values of 1.04 and 1.01 recorded in STK1 and STK2 respectively, were superior to values recorded in other groups. This shows that feed conversion may be more efficient when competition is less. Feed utilization efficiency has also been reported to decrease with increased stocking density [13,14]. The highest performance indicator (0.72) was recorded in STK1 and this continuously reduced with increase in stocking density. Survival rate ranged from 81.0% in STK5 to 96.0% in STK1, indicating that stocking density significantly affected survival of C. gariepinus fry. This negative influence of high stocking density on survival rate has earlier been reported in the work of [1] on Clarias batrachus. However, harvested biomass was higher at higher stocking density, as shown in Table 2. For profitability of aquaculture investment, it is important for stocking density to be economically viable [15]. Investment cost analysis (ICA) was significantly increased (P<0.05) with increase in stocking densities. The highest Net Present Value (NPV), Profit Index (PI) were recorded in STK2, while the Incidence of cost (IC) was significantly lower (P<0.05) in STK1 and STK2. From the result of this study, an economic stocking density is evident in STK2 group. Hydrogen ion concentration (pH) ranged from 6.91 and 7.18; water temperature ranged from 28.46 to 29.56; and dissolved oxygen from 4.95 to 5.40. All these parameters as shown in Table 3 fall within the recommended range [16]. Nitrate levels in experimental units were significantly higher (p<0.05) in STK4 and STK5, while nitrite levels marginally increased with increase in stocking density. This may be as a result of the increase in waste generation associated with increasing biomass. The result obtained here is in agreement with [17]. In conclusion, the results of the present study have shown that higher stocking density affected the feed utilization, growth performance and survival of C. gariepinus fry. Therefore, a stocking density of 150 fry/L is recommended.

v3-fos

2017-07-07T06:50:00.060Z

2015-10-28T00:00:00.000Z

10141951

s2

Cromolyn-mediated improvement of intestinal barrier function is associated with enhanced piglet performance after weaning Background Previous work showed that weaning stress causes gut barrier dysfunction partly by triggering the release of corticotropin releasing factor (CRF) and thereby inducing the degranulation of intestinal mast cell (MC). This study investigated the hypothesis that attenuating the weaning-induced activation of the CRF-MC axis via administration of a MC stabilizing agent (cromolyn) may improve gut permeability and piglet performance after weaning. Results To test the hypothesis twenty piglets were weaned (20 ± 1.0 d of age; 6.4 ± 0.4 kg of BW) and injected intraperitoneally with saline (control, n = 10) or 20 mg/kg BW of sodium cromolyn (cromolyn, n = 10) at – 0.5, 8 and 16 h relative to weaning. Piglets were housed individually and fed ad libitum a pre-starter diet from one to 15 d post-weaning followed by a starter diet until the end of the study on d 36. Cromolyn improved intestinal permeability as indicated by the reduced recovery of cobalt and mannitol in plasma samples. Cromolyn treated pigs consumed more feed (369 vs. 313 g/d; P < 0.009), gained more BW (283 vs. 238 g/d; P < 0.006), and grew more efficiently (0.60 vs. 0.40; P < 0.042) than their control counterparts. As a result, cromolyn treated pigs were 1.4 kg heavier than those in the control group by d 36 after weaning (16.5 vs. 17.9 kg; P < 0.002). Conclusions In agreement with our hypothesis, present data indicate that the cromolyn-mediated improvement of intestinal permeability is associated with enhanced pig performance after weaning. Background Mammals must constantly cope with the external environment to maintain homeostasis. To this end, they are provided with extended and complex selective surfaces like the gut mucosa. This epithelium, which is the largest mucosal surface in the body, plays the critical function of ensuring the absorption of nutrients while limiting the entrance of detrimental compounds such as toxins and bacteria that inhabit the luminal environment [1]. Over the last decade stress has been identified as a trigger of gastrointestinal dysfunctions through activation of the brain-gut axis. This mechanism entails the release of corticotropin-releasing factor (CRF), the main neuro-mediator of stress and a key effector of stress-related intestinal disturbances [2,3]. In the gut, CRF binds to CRF 1 and CRF 2 receptors which are expressed in multiple effector cells in the gut including neurons, epithelial cells, and immune cells such as the mast cells (MC) serving thereby as end-effector of the brain-gut axis. Activation of MC by CRF induces their degranulation and release of several cytokines and proteases causing increased intestinal permeability through a mechanism that involves disruption of tight junctions [2,[4][5][6]. In most settings of pig production piglets are weaned at an early age (19 -28 days of life), which expose them to several stressors like mother and littermate separation, transportation, dietary changes, and commingling with unfamiliar mates. This combination of stressful events compromises piglet welfare and health by resulting in transient anorexia, increased susceptibility to diarrhea and enteric infections, and reduced growth [7,8]. Furthermore, neonatal maternal separation induces longterm impairment of the intestinal barrier function [2]. In turn, compromised gut permeability facilitates transmigration of endotoxins and other harmful compounds from the lumen into the lamina propria and from there into systemic circulation. Increased levels of circulating endotoxins trigger the release of pro-inflammatory cytokines causing a shift in the partitioning of energy from growth towards maintenance of the immune response, finally resulting in decreased animal performance [9,10]. In line with this notion, it has been demonstrated that early weaning induces long term intestinal hyperpermeability in pigs [11] and that stabilization of MC with cromolyn, a drug that prevents MC degranulation trough a mechanism that appears to involve inhibition of the opening of Ca 2+ channels [12,13], can attenuate weaning-induced mucosal dysfunction [6]. Although compelling evidence indicates that treating pigs with cromolyn around weaning can improve intestinal barrier function, the impact of this treatment on animal performance after weaning has not been systematically investigated. The current study is based on the hypothesis that lessening the weaning-induced increase in gut permeability with cromolyn may improve piglet performance during the post-weaning phase. Therefore the aim of the present study was to investigate the effect of treating piglets with cromolyn immediately before and after weaning on gut barrier function and animal performance during the nursing phase. Animals and housing All experimental procedures were approved by the Laboratory Animal Care Advisory Committee of the Faculty of Veterinary Sciences of the Universitat Autónoma de Barcelona, Spain. Two days before weaning, twenty piglets (50:50 male:female ratio; Large White x Landrace × Piétrain) belonging to 12 different litters were selected and identified by ear-tag at the farm of origin (Cassá de la Selva, Spain). Piglets were weaned at 20 ± 1.0 d of age and 6.4 ± 0.42 kg of BW and immediately transported (50 km) to the nursery facility located at the Swine Experimental Unit of Lucta S.A. (Girona, Spain). At arrival, piglets were distributed into 20 individual pens (0.35 m 2 /pen) equipped with fully slatted plastic floors plus a nipple drinker and a feeder, offered ad libitum access to water and a non-medicated pre-starter diet from d one (weaning) to d 15 and a nonmedicated starter diet from d 16 to d 36 of experiment (Table 1). Individual BW was measured at weaning (initial) and then weekly until d 36 after weaning. Feed consumption was measured weekly from weaning until d 36 after weaning. However, feed consumption on week 5 (d 30 to 36) was excluded from statistical analysis because of technical problems that did not allow a proper weighing of feed orts on d 36. The health status of piglets was evaluated and registered daily by trained personnel. Experimental design and treatments On d one, 30 min before weaning, animals were randomly assigned to two groups paired by body weight and sex (n = 10) that were control (injected i.p. with 4.5 mL of saline solution) and cromolyn (injected i.p. with sodium cromolyn at 20 mg/kg BW; Sigma-Aldrich). The same procedure was repeated at 8 and 16 h relative to weaning time. The dose of sodium cromolyn was established based on a previous study [6]. Sample collection and analysis for gastrointestinal permeability Intestinal permeability was assessed in vivo on d 36 after weaning. Eight pigs per treatment were fasted for two h and subsequently sedated with a mixture of xilazine (1.5 mg/kg BW im) and ketamine (11 mg/kg BW im) in order to minimize handling stress. Ten minutes after sedation, animals were intragastrically dosed (gastroduodenal feeding tube, Levin type; VEC Medical) with a marker solution containing 0.5 g mannitol (Sigma-Aldrich, Madrid, Spain) and 0.6 g Co-EDTA [14] dissolved in 15 mL deionized water for a total dose of 1. [14]. Tissue collection On d 36, eight pigs per treatment were sacrificed with captive bolt and exsanguinated. The abdomen was opened, the intestines were removed and the ileum (from the first Peyer's patch to the ileocecal valve) was dissected. A 10 cm segment was removed from the midsection of the ileum, divided into 5-cm halves, opened longitudinally and flushed with saline. One of these samples was fixed in JB-fix [15] for later MC count. The remaining sample was fixed in 10 % buffered formalin for later histologic determination. A 5-cm sample was collected from the ascendant colon, opened longitudinally and flushed with saline. The mucosa was scraped, stored at −80°C, and subsequently analyzed by ELISA for cortisol, tumor necrosis factor alpha (TNF-α) (Cusabio Biotech, Hubei, China) and MC tryptase (MCT) (Elabscience, WuHan, China). Histological analyses Samples of ileum were dehydrated and embedded in paraffin, sectioned (~4 μm), and stained with hematoxylin and eosin. Villus height, crypt depth and number of goblet cells in crypts were measured in 10 well-oriented villi and crypts by using a light microscope (Olympus) and a linear ocular micrometer (Olympus). All measurements were performed by the same person who was blinded to the treatments, as described previously [16,17]. For quantification of MC, ileum samples were fixed in zinc-based fixative [15] and sectioned for immunohistochemistry. Tissue sections were then processed for immunohistochemistry and stained for C-Kit (YR145) (Cell marquee, Rocklin, California). Mast cells were counted at a 40 × magnification using a micrometer grid fitted within an eyepiece. At this magnification, the grid covered a 0.5 mm 2 area. For each tissue slide, six non-overlapping areas above the muscularis mucosae were counted for the estimation of mucosal MC numbers, which were expressed as cells per mm 2 . Statistical analysis In view of the objectives of the study, data for body weight and average daily gain were analyzed from weaning until the end of nursing phase (d 36). Average daily feed intake (ADFI) and growth efficiency expressed as the ratio between mean ADG and mean ADFI were analyzed from weaning until d 29. For these analyses, the pig was the experimental unit. A mixed-effect model with repeated measures was used in which the effect of pig nested within treatment entered the model as random and the effect of treatment, time (week after weaning) and their two-way interaction were considered fixed. Results for the recovery of mannitol and Co in plasma samples were analyzed with a mixed-effects model in which the pig nested within the treatment was considered a random variable and treatment as fixed effect. The same model was used to analyze data for ileum histology and concentration of inflammation markers in colon. Least squares means were separated into significant effects using Tukey's adjustment. In all cases, the smallest value for the Akaike's information criterion was used to identify the most appropriate covariance structure and ANOVA was performed using the mixed-model procedure of SAS (release 9.2; SAS Institute). Differences were considered significant when P < 0.05, whereas when P < 0.05 but ≤ 0.10, differences were considered to indicate a trend toward a significant effect. Overall, the amount of feed consumed, growth rate and BW that were achieved throughout the study resemble values observed under current setting of pig production [18]. Remarkably, cromolyn-treated pigs had a significantly higher (P < 0.009) feed intake (Table 2), gained more BW (P < 0.006; Fig. 2) and grew more efficiently (P < 0.042) than pigs in the control group (Table 2). Discussion In line with published results [6], data reported herein indicate that the administration of a MC-stabilizing agent (cromolyn) to piglets around weaning had beneficial and long-lasting consequences for the integrity of the intestinal mucosa. Specifically, treating piglets with a drug that blocks the weaning-induced activation of the intestinal CRF-MC axis [5,6] reduced the recovery in plasma of molecules (i.e., Co and mannitol) that enter the bloodstream mainly via paracellular leakage through mucosal tight junctions [8,14,19]. These findings demonstrate that cromolyn was effective at preserving intestinal integrity in early-weaned pigs. Noteworthy, the fact that intestinal permeability was assessed 36 d after cromolyn administration suggests that counteracting the intestinal effects of weaning stress with an inhibitor of MC degranulation improved the long-term function of the intestine. Previous work provides compelling evidence indicating that exposure to stress early in life results in sustained dysfunction of the intestinal barrier function through a complex mechanism in which the interaction between neuroimmune factors and intestinal MC plays a fundamental role [20][21][22]. Barreau and co-workers [23] showed that maternal deprivation during the neonatal phase increased gut mucosal permeability, the number of MC and the expression of cytokines in the colon of rats 12 weeks after exposure to stress. More recently, Smith et al. [11], reported that weaning pigs at 15 to 21 d of age resulted in various signs of paracellular leakage that were still evident at 63 d of age. These effects were attributed to chronic activation of the HPA-axis with a concomitant increase in the lysis of intestinal MC, release of MCT, and alteration of mucosal immune homeostasis. Based on these observations, we speculated that the cromolynmediated improvement in intestinal permeability would be paralleled by reduced degranulation of ileal MC and concentration of colonic MCT on d 36 after early weaning. Although changes in these variables were as expected, their magnitude was not large enough to become significant. We cannot rule out, therefore, that in our study cromolyn improved the intestinal integrity of pigs through actions that are unrelated to the inhibition of MC degranulation in the intestine. Indeed, although cromolyn is commonly defined as a "mast cell-stabilizer" that suppresses MC degranulation, previous findings [24,25] support the idea that the targets of this molecule are not restricted to MC. In order to assess if changes in gut permeability caused by cromolyn treatment were associated with alterations in the architecture of the mucosa we measured the morphology of the ileal epithelium on d 36 after weaning. The dimensions of ileal villi and crypts as well as the number of goblet cells did not differ between control and cromolyn-treated pigs. Even though other intestinal sections were not examined in our study, these results are not surprising because the morphology of the intestinal mucosa appears to recover much faster than its barrier function after weaning-induced damage. Indeed, Hu et al. [26], found that in pigs the architecture of the intestinal epithelium returned to pre-weaning levels 14 d after weaning whereas the gut barrier function was still impaired. Considering that activation of intestinal MC dysregulates the expression and function of tight and adherence junction proteins [27], it is reasonable to suggest that the cromolyn-mediated improvement in gut permeability might have involved functional changes at the level of tight junctional complexes between adjacent mucosal cells. Further mechanistic studies are needed to investigate this possibility at a molecular level. We hypothesized that reducing the negative impact of weaning stress on gut permeability can have long-lasting effects on the mucosal barrier function and thereby enhance animal growth long after weaning. The association between gut hyperpermeability in response to maternal separation and long-term reduction in BW has been already demonstrated in rats [23]. To the best of our knowledge, however, this is the first study to show that reducing the impact of weaning stress on intestinal permeability via treatment with cromolyn is associated with improved performance of pigs fed cereal-based diets. Remarkably, cromolyn treated pigs had a 20 % faster rate of growth and were 1.4 kg heavier than control animals on d 36 after weaning. Provided the magnitude of these effects, confirmation of results under commercial settings of pig production is warranted. Because cromolyn administration also improved the efficiency of feed conversion, the enhancement in BW gain is attributable to factors other than solely increased feed consumption. Noteworthy, enhanced intestinal permeability facilitates the translocation of bacteria from the gut lumen to systemic organs thereby triggering inflammatory and immunological responses [28]. These findings along with data reported herein support the suggestion that reduced gut permeability in the cromolyn-treated pigs might have resulted in a diminished stimulation of the proinflammatory immune response when compared with control animals. If this is true, then it is possible that cromolyn-treated animals grew more efficiently partly by using a larger proportion of absorbed nutrients for growth rather than to support the immune system [10]. Overall, this proof-of-concept study clearly demonstrates that using a MC-stabilizing agent to limit the impact of weaning stress on intestinal permeability resulted in significant improvements in pig performance long after weaning. Interestingly, recent cellculture studies have shown that various plant bioactives, including some polyphenols, can also inhibit MC degranulation [29]. Therefore, available data provide a rationale for exploring the value of such compounds to mimic the impact of cromolyn on gut permeability and performance of weanling pigs. Conclusions In summary, treating piglets at weaning with the MCstabilizing agent cromolyn has long-term effects in reducing intestinal permeability deterioration of the gut and enhancing feed consumption, growth, and efficiency of growth long after weaning. Taken together, our results suggest that targeting intestinal MC at weaning may illuminate ways to reduce the magnitude and persistency of the negative impact that weaning stress has on the intestinal integrity, growth, and welfare of pigs.

v3-fos

2019-05-16T13:03:46.011Z

2015-05-01T00:00:00.000Z

154697251

s2

Effects of different drip irrigation regimes on saline–sodic soil nutrients and cotton yield in an arid region of Northwest China A field experiment was conducted on a saline wasteland in Xinjiang, Northwest China, during 2008–2010 to evaluate the nutrient behavior and cotton yield during reclamation, applied by different drip irrigation regimes. The experiment included five treatments in which the soil matric potential (SMP) thresholds at 20 cm depth were controlled at −5, −10, −15, −20 and −25 kPa. The results indicated that both soil salinity and sodicity declined significantly at 0–40 cm depth and greater reductions were achieved at higher SMP thresholds (−5 and −10 kPa) than in other treatments. The distributions in soil inorganic nitrogen (N) and available phosphorus (P) and potassium (K) in the soil profile were mainly influenced by the point-source characteristic of drip irrigation, drip irrigation regime and fertilization mode. With the reclamation of both soil chemical and physical properties, there were dramatic increases in soil N, P and K concentration by the end of 2010. The soil nutrient concentrations in N, P and K were all proportional to the SMP thresholds, as higher SMP could result in greater reductions in soil salinity and sodicity. Since crop growth became more vigorous during reclamation, there was also a considerable increase (9.7–31.9%) in soil organic carbon by the end of 2010, and the concentrations were also proportional to SMP thresholds. −1 The highest cotton yield was obtained in S1 (−5 kPa) treatment for both 2009 (2.87 Mg ha ) and 2010 (3.60 Mg ha−1). Additionally, the soil C:N ratios were inversely proportional to the SMP thresholds in 2009 and 2010. Considering the soil reclamation efficiency, soil nutrient stocks and cotton yield, SMP thresholds of −5 and −10 kPa could be used as effective measures to trigger irrigation in the first 3 years of saline–sodic soil reclamation in Xinjiang, Northwest China. © 2015 Elsevier B.V. All rights reserved. Introduction Soil salinity and sodicity, and their combination, are worldwide problems posing significant threats to the sustainable development of agriculture, especially in arid and semiarid regions (Oster et al., 1996;Qadir et al., 1997;Ma et al., 2012). In Xinjiang, a typical arid region of Northwest China, there are approximately 1.1 × 10 7 ha of saline wasteland, of which 7.27 × 10 6 ha are overly saline-sodic soils (Xi et al., 2005). Salinization and sodification of soils are serious land degradation issues in Xinjiang, where it is estimated that onethird of the arable land is affected by salinity and sodicity, which greatly reduces agricultural output in the area (Chen et al., 2010). Under saline conditions, the reduced growth of crops is mainly attributed to the osmotic effect, which can reduce soil water potential and nutrient availability and uptake by plants (Al-Karaki, 1997;Elgharably, 2011). Sodicity increases clay dispersion and reduces aggregate stability, which results in declining air permeability, infiltration and hydraulic conductivity (Wong et al., 2010). This can seriously hinder root respiration, hence reducing plant growth and activity of soil organisms. However, the typical inland arid conditions and water resource shortages in Xinjiang increase the difficulty of reclaiming saline-sodic soil. Drip irrigation with its characteristic of applying water at low discharge rate and high frequency over a long period of time can maintain constant and high soil water potential in the root zone and reduce salinity levels in soil water by leaching, particularly near the drip emitters (Keller and Bliesner, 1990;Wang et al., 2011). Among all the factors, drip irrigation scheduling is the most important in salt leaching efficiency. Previous studies have evaluated the impact of different levels of soil matric potential (SMP) to trigger drip irrigation in arid and semiarid regions (Dou et al., 2011;Sun et al., 2012;Wang et al., 2012). These studies have http://dx.doi.org/10.1016/j.agwat.2015.01.025 0378-3774/© 2015 Elsevier B.V. All rights reserved. mainly focussed on soil salinity, sodicity and soil hydraulic properties, particularly with regard to soil salt movement and plant health. However, very few studies have explored soil nutrients during the reclamation process under drip irrigation, and hence understanding of nutrients in saline-sodic soil under drip irrigation is limited. Soil structure and root activity decline with increasing sodicity in a saline-sodic soil, thus reducing nutrient mobilities and leading to nutrient deficiencies (Wong et al., 2010). Therefore, organic matter and mineral nutrients in saline-sodic soils are generally at low levels. Nutrient behavior in saline-sodic soils during the reclamation process needs to be evaluated because of the changes in soil chemical composition during and after reclamation. Especially under conditions of drip irrigation with mineral fertilizer input, the variations of soil nutrients during reclamation remain largely unknown. The objectives of this study were (1) to investigate the effects of drip irrigation triggered by different SMP thresholds on distribution of soil mineral nutrients (inorganic N, available P and K); (2) to ascertain variation in soil organic carbon (SOC) and (3) to measure the effects of different SMP thresholds on soil carbon-to-nitrogen (C:N) ratio and seed cotton yield during 3 years of reclamation under drip irrigation. Experimental site The field experiments were conducted during 2008-2010 on a saline wasteland at Karamay farm (latitude: 45 • 22 N and longitude: 84 • 50 E, 350 m a.s.l.), which is located in the middle of the Jungger Basin in the Xinjiang Province, Northwest China. The area has a typical inland arid climate with annual precipitation of about 105 mm, mainly concentrated in June-August, and average annual evaporation capacity and temperature of about 3545 mm and 8.0 • C, respectively (Wang et al., 2007). The average depth to groundwater is about 2.5-3.0 m, and the electrical conductivity of the groundwater (EC gw ) ranged between 30 and 52 dS m −1 . Irrigation water is pumped from the reservoir in the west suburbs of Karamay, with EC iw of 0.3 dS m −1 . The soils in the area are chloride-sulfatetype saline-sodic soils, which are typically in Xinjiang (Wang et al., 1993;Xi et al., 2005). The climate and special geographical conditions make this region liable to accumulate salt on the soil surface. The EC e (electrical conductivity of saturated paste) and the nutrient concentration of soil samples at different soil depths are reported in Tables 1 and 2. Plot layout and irrigation water management The experiment included five water treatments (S1-S5) based on the SMP, measured with tensiometers located at a depth of 20 cm beneath a dripper near the center of the plot for each treatment (Fig. 1), that determined when to irrigate or to trigger irrigation. The SMP thresholds that triggered irrigation were −5 (S1), −10 (S2), −15 (S3), −20 (S4) and −25 kPa (S5). These treatments were replicated three times in a completely randomized block design. Plots consisted of 20 rows of cotton planted on 10 raised (15 cm) beds during 2008-2010, spaced at 0.8 m. The beds were mulched with white polyethylene sheets after sowing. Each bed was 0.4 m wide and 3.8 m long (Fig. 1). The size of plots was 8.0 m × 3.8 m. The location of water treatments was the same during the 3 years of the experiments. Each treatment was irrigated with an independent irrigation system. The system consisted of a water tank (1000 L) and 30 drip tubes (10 tubes per plot). A tank filled with irrigation water was placed at 1 m above the ground to maintain water pressure in the irrigation system within the range of 10-20 kPa. The drip tubes with 20 cm emitter intervals were placed at the center of each raised bed. To enable seedlings to emerge, 40 mm of water was applied to all treatments immediately after seeding. Cotton seedlings were thinned to the spacing described below, and irrigation treatments were initiated on 15 June (16 days after seeding), 2 June (23 days after seeding) and 5 June (26 days after seeding) in 2008, 2009 and 2010, respectively. Thereafter, 9.8 mm of water was applied when SMP reached the target values. The tensiometers were observed three times daily: 8:00, 12:00 and 18:00 h. Plant and fertilizer management Seeds of cotton were sown on 30 May 2008, 10 May 2009 and 8 May 2010 in double rows. The rows were 30 cm apart; within a row, the seeds were sown 10 cm apart. Soon after emergence, the plants were thinned to a spacing of 30 cm. Since the seeding date was relatively late in 2008, the emergence rate was low and the bolls did not grow well enough for harvest due to the low temperature in the late growing stages. Accordingly, there were no yield data for 2008. In 2009 and 2010, harvest was started on 2 October and 30 September and finished on 20 and 25 November, and the total harvest period lasted 49 and 57 days, respectively. The seed cotton was picked by hand at 4-7-day intervals, and the total weight per plot was checked at each harvest time. Basal dose of 450 kg ha −1 of a compound fertilizer (16% N, 35% P 2 O 5 and 8% K 2 O) was uniformly applied to the plots at the time of plowing before seeding in 2008-2010. This fertilizer dose was supplemented with urea (46% N), applied with the irrigation water; 0.15 L of an 11 mg kg −1 urea solution was added to the irrigation tank every time irrigation was applied. Soil sampling and chemistry analyses In a saline soil, the soil salinity distributions clearly showed leaching near the drip lines, an area where the root density has been found to be maximum (Hanson et al., 2006;Hu et al., 2009). Since the low EC e zones we found were usually within 40 cm (Wang et al., 2011), a soil depth interval of 0-40 cm was analyzed. Soil samples were obtained on soil cores from each plot with an auger (2.0 cm in diameter and 15 cm long) on 12 May 2008 (before seeding), 13 September 2008, 14 September 2009 and 13 September 2010 (after irrigation ended). The distances of sampling points to drip emitters were 0, 5, 10, 15, 20, 25, 30, 35 and 40 cm, and all sample depths were the same: 0-5, 5-10, 10-20, 20-30 and 30-40 cm (Fig. 1). All soil samples were air-dried and sieved through a 1 mm sieve. The soluble salt estimates were based on extracts of saturated soil. The EC values were determined with a conductivity meter (DDS-11A, REX, Shanghai). Sodium adsorption ratio (SAR) was Table 1 Soil texture, bulk density, electrical conductivity of saturated paste extracts (ECe) and SAR of the initial soil profile at Karamay, Xinjiang, Northwest China. Table 2 The concentration of total N, soil organic carbon (SOC), available P and K and inorganic N (NO3 − -N and NH4 + -N) in the initial soil profile at Karamay, Xinjiang, Northwest China. (1) Concentrations of Na + were determined by flame photometer method and those of Ca 2+ and Mg 2+ by EDTA titration method (Bao, 2005). Total nitrogen (TN) was determined using the micro-Kjeldahl method, with 2.0 g of soil being digested in H 2 SO 4 and H 2 O 2 solution before measurement (Stuart, 1936). The inorganic N (ammonium and nitrate: NH 4 + and NO 3 − ) was determined in 2 mol L −1 KCl extract on a Skalar automatic analyzer (Bundy and Meisinger, 1994). Olsen P (available P) was extracted with 0.5 mol L −1 NaHCO 3 at pH 8.5 and determined according to Olsen and Sommers (1982). Available potassium (K) was determined by flame photometer method using 1.0 mol L −1 CH 3 COONH 4 adjusted to pH 7.0 as the extractant. Soil organic carbon (SOC) was determined by high temperature combustion on a LECO CNS-2000 analyzer (Wong et al., 2008). The values for soil organic carbon (SOC) and total nitrogen (TN) were used to calculate the carbon-to-nitrogen ratio, C:N. Statistical analyses Tukey's significant difference (TSD) was used to compare and rank the treatment means at p < 0.05 for the EC e , SAR, TN, C:N ratio and cotton yield. Two factors, sampling date (n = 6) and treatment (n = 5), were included in the ANOVA for soil EC e , SAR and nutrients. Statistical analyses were performed using SPSS (version 15.0) and Excel. Soil nutrient spatial distributions were mapped with Surfer 7.0. Weather and irrigation The summer temperatures (July and August) in 2008 were higher than those in 2009 and 2010, while in other months, the mean temperatures were very similar among the 3 years (Fig. 2). Total rainfall during the experimental period was 70.8, 81.2 and 114.6 mm in 2008, 2009 and 2010, respectively, with correspondingly seven, six and nine effective rainfall (>5 mm) events. There were four heavy rainfall events (rainfall intensity > 16 mm h −1 ) in early July 2008, which was twice as much as observed in 2009 and 2010. Comparatively, the temporal distribution of rainfall was more uniform in 2010 than in the other years (Fig. 2). Before cotton emerged, all treatment plots were irrigated with the same amount of water to ensure uniform germination. After that, irrigations were triggered by different SMP thresholds. Higher SMP threshold resulted in more irrigation times and hence greater amounts of irrigation water (Table 3). The most irrigation water was applied in S1 (−5 kPa) each year, while the maximum (665.6 mm) was in 2008. Temporal changes of soil EC e and SAR The variation of soil EC e and SAR after irrigation terminated in each year is shown in Fig. 3. During the irrigation season, salts moved downward with water, thus after the irrigation ceased in fall (September), the EC e value significantly declined for each treatment compared with the initial values in 2008 before seeding (13 May) (Fig. 3a). Compared with the initial EC e in 2008, the reduction in EC e after 3 years at 0-40 cm depth varied from 56% (S5) to 89% (S1) for the five treatments. The reduction of EC e decreased with the decreasing SMP threshold for each year. Thus more reclamation occurred in S1 (−5 kPa) and S2 (−10 kPa) than for the other treatments. By the end of the third cropping season, the EC e for S1and S2 treatments had reduced from 47.0 to 5.3 and 8.1 dS m −1 , respectively, which are in the range of threshold salinity of 7.7 dS m −1 for cotton (Maas and Hoffman, 1977). This could only occur if more water was applied to the soil than was used by the crop within this depth, thus causing leaching. The changes in SAR with time ( Fig. 3b) were similar to those of EC e . There were considerable reductions of SAR at 0-40 cm depth. After the third irrigation season, the declines in SAR, relative to the initial level, were 76, 60, 52, 46 and 41% for S1-S5 treatments, respectively (Fig. 3b). In the fall, the reduction of SAR at 0-40 cm soil depth also decreased with the decreasing SMP threshold. The reductions in SAR could be enhanced in calcareous soil under cropped conditions, because plant roots can affect the soil chemical environment by increasing the partial pressure of carbon dioxide (CO 2 ), which will facilitate the dissolution of calcite, thereby providing a source of Ca to replace exchangeable Na (Qadir et al., 2002). This process is more efficient in cropped than in uncropped soils as plant roots can exert higher CO 2 partial pressure (Robbins, 1986a,b;Qadir et al., 2002Qadir et al., , 2007. Apart from root action, the decrease in SAR can also be attributed to Ca 2+ supplied in irrigation water and lime dissolution through soil microbial activity (Qadir et al., 1997). Soil N In this study, soil inorganic N (NO 3 − -N and NH 4 + -N) distribution within 40 cm depth was analyzed after irrigation ceased for each treatment in 2010. The two types of inorganic N accumulated mainly in the upper soil layer (0-20 cm) (Fig. 4). The NO 3 − -N and NH 4 + -N formed a concentration gradient with soil depth, with the highest values concentrated around the drip emitter. This may be due to the point-source characteristic of drip irrigation and the fertilization mode. Under point-source infiltration, water can form a wetting area in close proximity to a dripper ); hence, the nutrient dissolved in and moving with irrigation water can form a similar shape to the wetting area in the soil profile. Compared with the initial value (Table 2), there were dramatic rises in both the NO 3 − -N and NH 4 + -N in the soil profile for each treatment after 3 years of the experiment. There are two main reasons for this: applied fertilizer and crop roots. Applying N fertilizer, on the whole, can increase the inorganic N concentration and therefore the NO 3 − -N and NH 4 + -N stocks in the soil profile (Yin et al., 2007). Additionally, soil reclamation was more efficient at higher SMP thresholds (Fig. 3), and thus more roots distributed in soil for S1 (−5 kPa) and S2 (−10 kPa) treatments could promote adsorption, transportation and biometabolic action of ionic species in soil, which results in accumulation of nutrients (Ma et al., 2012). This can also explain why more NO 3 − -N and NH 4 + -N accumulated at higher SMP thresholds after the experiment ended in 2010 (Fig. 4). After the first growing season, there was a significant increase in total N in the upper 40 cm of soil in all treatments, compared with those before seeding (Fig. 5); the increase rates in the range of 8.4-38.9% were proportional to the SMP thresholds. In the following 2 years, the total N for each treatment steadily increased. Compared with the initial value, the total N increased by 79.5, 69.4, 43.3, 52.8 and 35.6% for S1-S5 treatments, respectively, after the experiment ended in 2010. The considerable increase in soil N was mainly due to the soil reclamation and fertilizer application. During reclamation, both the aboveground and underground parts of the crop can flourish yearly, and large amounts of tiny holes and channels observable in the soil with growing crops indicate a fibrous root system, creating micropores in the soil (Hayashi et al., 2006;Tejada et al., 2006). This can improve hydraulic conductivity in the soil structure and provide available water and nutrients for crops to grow. However, the biologically fixed N through the crop roots, the shedding of older leaves and the dead plants may also have increased the organic matter and N content in the upper 40 cm of soil (Qadir et al., 1997). Soil available P The soil available P distributions in the soil profile (Fig. 6) were similar to those for soil inorganic N (Fig. 4). By the end of 2010, the average value of soil available P in 0-40 cm depth had increased to 11.1-6.8 mg kg −1 for S1-S5 treatment. Most soil available P accumulated in the upper soil layer (0-20 cm), which might be mainly due to the point-source characteristic of drip irrigation and Pcontaining fertilizer. In addition, more CaCO 3 present at shallower soil depths may have absorbed more P than in deeper soil layers (Wang et al., 2011. Soil moisture content influences the effectiveness and availability of applied P (Campo et al., 1998); moreover, the higher water availability in the topsoil under drip irrigation could be related to a higher transformation from organic to available P (Yang et al., 2011). This can explain the phenomenon of available P content in the soil profile being higher in S1 and S2 than in the other treatments (Fig. 6). For S1 and S2 treatments, there was more available P concentrated in deeper soil layers (30-40 cm) under drip emitters than in other treatments. This might be caused by the large amount of applied water, which could leach soil P to deeper layers. In the cropped treatments, the mineral weathering of apatite, dissolution of slightly soluble P compounds and mineralization of some organic P are believed to be the factors responsible for increased soil P content (Qadir et al., 1997;Havlin et al., 2005;Chai et al., 2014). Irrigation method can alter soil properties, thus affecting the transformation and movement of soil nutrients and so affecting soil P content (Yang et al., 2011), which can also explain the changes of soil P content under drip irrigation in the present study. Soil available K After 3 years of the experiment, the soil had available K in the range of 155-179 mg kg −1 in 0-40 cm depth of soil for the five treatments (Fig. 7). By the end of the experiment, the soil available K had increased by 55.1, 36.5, 32.5, 34.8 and 45.4% for S1-S5 treatments, compared with the initial value, respectively. The increase in soil available K was mainly due to the release of K from illite and applied fertilizer. Like many soils in the area, the soil in this study had illite as the dominant clay mineral (Cheng and Fukuhara, 1994; Guan et al., 2011), which serves as a good reservoir of plant-available K. The shallower samples had slightly lower concentrations of K, which was similar to those for soil N and P (Fig. 7). SOC In saline-sodic soil, the effects of salinity and sodicity on plant health adversely impact on SOC stocks, generally leading to less SOC (Wong et al., 2010). Comparing with the initial value, there were significant increases in SOC for S1-S3 treatments after the first irrigation season (13 September 2008), and the increase rates from S3 to S1 were in the range of 5.3-17.4% (Fig. 8). For S4 Tukey's significant differences (TSDs) (p≤ 0.05) for SOC were 0.03 and 0.05% among the treatments and dates, respectively. and S5 treatments, the increase rates were not statistically significant. This was mainly due to the more vigorous cotton growth in the S1-S3 than in the other treatments, as soil reclamation was more efficient at higher SMP thresholds (Fig. 3). In the second irrigation season, there was a constant increase in SOC for all treatments, and the highest increase rate of 8.3% was for the S1 treatment compared with that in the first year (13 September 2008). The presence of plants can increase the SOC in several ways (Jinbo et al., 2006). In the present study, the plant residue after harvest was incorporated into the soil before plowing in the next spring, which was an important factor leading to the consistent increase in SOC each year. Comparing with the initial value, SOC increased by 31.9, 24.2, 18.5, 14.0 and 9.7% for S1-S5 treatments, respectively, when the experiment ended in 2010. In highly saline-sodic soils, enhanced SOC loss can occur through dispersion of aggregates on wetting, which increases substrate accessibility and availability to the soil microbial biomass (Oades, 1984). At higher SMP thresholds, soil reclamation was more efficient both for nutrients and for structure, which could explain the differences among the five treatments in SOC each year (Fig. 8). Cotton yield and soil carbon-to-nitrogen (C:N) ratio As shown in Table 4, the highest seed cotton yields for both 2009 (2.87 Mg ha −1 ) and 2010 (3.60 Mg ha −1 ) were obtained in S1 treatment, for which the yields were 67 and 84% of the average level for non-saline soil in this region, respectively. Statistical analysis showed that maintaining different SMP values during the growing season had a significant effect on seed cotton yield in both 2009 and 2010. The yields of the five treatments in 2010 were 8-42% greater than those in 2009. Moreover, the seed cotton yield increased as the SMP thresholds increased, which is in agreement with the results Values for each separate year in a column followed by the same letter are not significantly different at p≤ 0.05. of a study conducted by Jiao et al. (2006) and in Ningxia, Northwest China. Soil carbon-to-nitrogen (C:N) ratio is one of the key factors influencing soil organic matter (SOM) decomposition (Cong et al., 2012). Relatively high C:N ratio (>30) can result in the competition for nitrogen and adverse impact on crop yields (We et al., 2011). In this study, the ratios were significantly influenced by SMP thresholds and were inversely proportional to the SMP thresholds both in 2009 and in 2010 (Table 4). Compared with those in 2009, the ratios in 2010 decreased by 7.4, 7.7, 6.9, 9.4 and 13.4% for S1, S2, S3, S4 and S5 treatments, respectively. In crop land, the SOC pool is dependent on inputs from vegetation (Wong et al., 2010). Moreover, soil TN change is generally assumed to follow SOC dynamics because both elements are bound into organic compounds (Cong et al., 2012). In saline-sodic soil, the sum of the osmotic and matric stresses limited crop yields, the amount of applied N, P and K taken up by the crop and the amount of C produced by the crop in the form of roots and foliage. Apparently, in regard to the C:N ratio, the limitation on N uptake was greater than that on the production of C (Table 4). In high SMP threshold (−5 kPa) treatment, more soil reclamation occurred, which resulted in vigorous plant growth and more soil microorganism quantity (Batra and Manna, 1997). This can lead to the increasing rates of SOC consumption as well as the soil TN accumulation. Therefore, of all treatments, TN was the highest and SOC was the lowest for the S1 treatment. Consequently, for the combined data of 2009 and 2010, C:N ratio decreased with increasing cotton yield, and a logarithmic function relationship was found between them (Fig. 9). Conclusions The soil salinity, sodicity and nutrients were significantly affected by the SMP thresholds under drip irrigation during the 3 years of reclamation. There were larger reductions of EC e and SAR in S1 (−5 kPa) and S2 (−10 kPa) than in the other treatments at 0-40 cm soil depth. The distributions of soil inorganic N and available P and K in the soil profile were mainly influenced by the point-source characteristic of drip irrigation, drip irrigation regime and fertilization mode. With the reclamation in both soil chemical and physical properties, there were dramatic increases in soil N, P and K concentration by the end of 3 years of the experiment. The soil concentration of inorganic and total N, available P and K were proportional to the SMP thresholds, as higher SMP resulted in more efficient soil reclamation. Since cotton growth became more vigorous during reclamation, there was also a considerable increase (9.7-31.9%) in SOC by the end of 2010, and the increased rates were proportional to SMP thresholds. The highest cotton yield was obtained in S1 (−5 kPa) treatment for both 2009 (2.87 Mg ha −1 ) and 2010 (3.60 Mg ha −1 ). The soil C:N ratios were inversely proportional to the SMP thresholds in 2009 and 2010. Overall, considering the soil reclamation efficiency, soil nutrient stocks and cotton yield, SMP thresholds of −5 and −10 kPa could be used as effective measures to trigger irrigation in the first 3 years of saline-sodic soil reclamation in Xinjiang, Northwest China.

v3-fos

2016-03-01T03:19:46.873Z

2015-05-01T00:00:00.000Z

15148991

s2

Polymorphisms of the Ovine BMPR-IB, BMP-15 and FSHR and Their Associations with Litter Size in Two Chinese Indigenous Sheep Breeds The Small Tailed Han sheep and Hu sheep are two prolific local sheep in China. In this study, the polymorphisms of BMPR-IB (Bone morphogenetic protein receptor IB), BMP-15 (Bone morphogenetic protein 15) and FSHR (follicle stimulating hormone receptor) were investigated to check whether they are associated with litter size in Small Tailed Han sheep and Hu sheep. Consequently, three polymorphisms, FecB mutation in BMPR-IB (c.746A>G), FecG mutation in BMP-15 (c.718C>T) and the mutation (g. 47C>T) in FSHR were found in the above two sheep breeds with a total number of 1630 individuals. The single marker association analysis showed that the three mutations were significantly associated with litter size. The ewes with genotype FecBB/FecBB and FecBB/FecB+ had 0.78 and 0.58 more lambs (p < 0.01) than those with genotype FecB+/FecB+, respectively. The heterozygous Han and Hu ewes with FecXG/FecX+ genotype showed 0.30 (p = 0.05) more lambs than those with the FecX+/FecX+ genotype. For FSHR gene, the ewes with genotype CC had 0.52 (p < 0.01) and 0.75 (p < 0.01) more lambs than those with genotypes TC and TT, respectively. Combined effect analyses indicated an extremely significant interaction (p < 0.01) between the random combinations of BMPR-IB, BMP-15 and FSHR genes on litter size. In addition, the Han and Hu ewes with BB/G+/CC genotype harbor the highest litter size among ewes analyzed in current study. In conclusion, BMPR-IB, BMP-15 and FSHR polymorphisms could be used as genetic markers in multi-gene pyramiding for improving litter size in sheep husbandry. Introduction The FecB (Fec = Fecundity, B = Booroola) mutation plays a vital role in increasing ovulation rate and prolificacy in ewes. This mutation (c.746A>G) was in BMPR-IB (Bone morphogenetic protein receptor IB) gene that located on chromosome 6,which was first found to be significantly associated with litter size in Booroola Merino ewes [1][2][3]. BMP-15 (Bone morphogenetic protein 15) gene belongs to the TGFβ (Transforming growth factor-β) family, which acts as a key regulator of granulosa cell (GC) processes in ovarian follicular development [4,5]. The sheep BMP-15 gene is located on the X chromosome [4]. The c.718C>T mutation (named FecX G ; Galway mutation) in BMP-15 gene was first identified in Cambridge and Belclare sheep, which increased ovulation rate and infertility [6]. FSHR (Follicle stimulating hormone receptor) gene was first identified in rat Sertoli cells and may have an influence on the FSH (follicle stimulating hormone) signal transduction [7]. Additionally, FSH has been reported to play an important role in the development of antral follicles [8,9]. A variety of mutations were found in the 5' flanking region of ovine FSHR gene, which were significantly associated with litter size in Australian sheep, Hu sheep and Small Tailed Han sheep [10][11][12]. The Small Tailed Han sheep and Hu sheep were originally raised in Shandong Province and Jiangsu Province, China [13]. They quickly gained the attention of Chinese sheep breeders and were largely used in the modern hybridization system as female parents because of their reputation for high fertility. To date, there are no reports about the combined effect of BMPR-IB, BMP-15 and FSHR genes on litter size of Small Tailed Han sheep and Hu sheep. Therefore, the objectives of this study were to investigate the relationships of single nucleotide polymorphisms (SNPs) in BMPR-IB, BMP-15 and FSHR, and their combined effect with litter size, which may serve as valuable markers for female fertility selection at the early stage in Small Tailed Han sheep and Hu sheep. Genotyping and Allele Frequency Analysis A 140 bp PCR product containing the c.746A>G polymorphism in the coding region of BMPR-IB gene was digested using the Ava II restriction enzyme. The digestion generated three fragments, and the 140 bp, 110 bp + 30 bp and 140 bp + 110 bp +30 bp bands represented ++, BB and B+ genotypes, respectively ( Figure 1). Additionally, the 141 bp PCR product of the c.718C>T polymorphism of the BMP-15 gene was digested with Hinf I, generating two fragments: the 141 bp + 111 bp + 30 bp and 111 bp + 30 bp bands represented G+ and ++ genotypes, respectively ( Figure 2). In ewes studied, none of them carried homozygous genotype (GG). The 244 bp PCR product of the g.47C>T of the FSHR gene was digested by BsiE I and the result is shown in Figure 3. The fragment length of the TT genotype was 244 bp, while CC and CT genotypes were 154 bp + 90 bp and 244 bp + 154 bp + 90 bp, respectively. The allele and genotype frequency were analyzed in the experimental populations ( Table 1). All these three mutations were detected in both Small Tailed Han sheep and Hu sheep. The c.746A>G polymorphism of the BMPR-IB gene showed a higher frequency of allele G (B) than allele A (+), and the GG (BB) genotype was predominant in total population. The CC (++) genotype of the c.718C>T polymorphism of the BMP-15 gene was higher in all population. Whereas, TT genotype of the g.47C>T polymorphism of FSHR gene was higher than the CC and TC genotypes in the population. Single Marker-Trait Association The least squares means and SE (standard error) for litter size of individuals with different genotypes in Small Tailed Han sheep and Hu sheep are shown in Table 2. The association analysis revealed that the three polymorphisms found in this study were significant, or tend to be associated with (BMPR-IB, p < 0.01; BMP-15, p = 0.05; and FSHR, p < 0.01) litter size in Hu and Han sheep. Association analysis results indicated that the ewes with genotypes BB and B+ had 0.78 (p < 0.01) and 0.58 (p < 0.01) gave birth to more lambs than those with genotype ++ in the experimental population, respectively, while the ewes with genotype G+ had 0.30 (p = 0.05) had more lambs than the ++ genotype ones. The ewes carrying genotype CC had 0.52 (p < 0.01) and 0.75 (p < 0.01) had more lambs when compared with the ewes carrying genotypes TC and TT, respectively. More specifically, the ewes with genotypes BB and B+ had 0.85 (p < 0.01) and 0.57 (p < 0.01) more lambs than those with genotype ++ in Small Tailed Han sheep, respectively. The c.718C>T polymorphism of the BMP-15 gene was not significantly associated with litter size in Small Tailed Han sheep (p > 0.05). The ewes carrying genotype CC and TC of the FSHR gene had 0.63 (p < 0.01) and 0.18 (p < 0.01) more lambs than the ewes carrying genotype TT, respectively. In Hu sheep, the ewes with genotypes BB and B+ had 0.74 and 0.59 had more lambs than those with genotype ++, respectively. While the ewes carrying genotype G+ had 0.52 (p < 0.05) more lambs than the ++ genotype ones. The ewes carrying genotype CC had 0.80 (p < 0.01) and 0.54 (p < 0.01) had more lambs when compared to the ewes carrying genotypes TT and TC, respectively. The different superscript letters in the same column for each gene in lowercase represent significant level at p < 0.05, which letters in uppercase represent significant level at p < 0.01, and the same letter represents no significant difference (p > 0.05). B means FecB mutation; G means FecX G mutation; + means wild-type; C means FSHR g.47C>T mutation; T means wild-type. Combined Effect Analysis of BMPR-IB, BMP-15 and FSHR Genes on Litter Size Highly significant interactions were observed if we randomly combined two of three genes studied and all the three genes. The combined effect of two genes (BMPR-IB/BMP-15, BMPR-IB/FSHR and BMP-15/FSHR) on litter size is presented in Table 3. For BMPR-IB/BMP-15, the ewes with BB/G+ genotype had the largest litter size and with the ++/++ genotype having the lowest litter size among all the six genotypes. The effect of the BMPR-IB gene mutation was greater than that of the BMP-15 gene mutation on litter size in this population. For BMPR-IB/FSHR, the ewes with BB/CC genotype had greater litter size than those with other genotypes. The effect of the BMPR-IB gene mutation was greater than that of the FSHR gene mutation on litter size. For BMP-15/FSHR, the ewes with G+/CC genotype had greater litter size than those with other genotypes. The effect of the FSHR gene mutation was greater than that of the BMP-15 gene mutation on litter size. Therefore, the ewes carrying mutations in both the BMPR-IB and FSHR genes had greater litter size than the other two genes combinations, and the effect of the BMPR-IB mutation was the greatest among these three genes. The combined effect analysis of three genes (BMPR-IB/BMP-15/FSHR) on litter size is presented in Table 4. The BB/G+/CC genotype had significantly greater contribution on litter size than any other genotypes. Discussion In the present study, we selected the ovine BMPR-IB, BMP-15 and FSHR as candidate genes to analyze the effect of single-marker and multi-marker on litter size. The FecB gene is crucial in the regulation of prolificacy phenotype in sheep [1,2]. Several studies indicated that ewes carrying FecB-mutation have significantly higher ovulation rates if compared with their wild-type contemporaries [1,2,14]. In this study, the FecB-mutation was found in Small Tailed Han sheep and Hu sheep, and was significantly associated with litter size, which is consistent with previous reports [15][16][17][18]. Ovine BMP-15 gene plays a vital role in growth and differentiation of early ovarian follicles [19][20][21][22]. In Inverdale and Hanna sheep, the c.718C>T mutation of the BMP-15 gene has been reported to show an increased ovulation rate under heterozygous conditions, and homozygotes are otherwise infertile [17]. Chu et al. (2005), Wang et al. (2005) and Davis et al. (2006) failed to detect the BMP-15 (FecXI) mutation in Hu sheep [17,23,24], but a BMP-15 (FecXG) was identified in the Small Tailed Han Sheep by Chu et al. [15]. In the present study, the BMP-15 (FecXG) mutation was detected in both Small Tailed Han and Hu sheep breeds. We also found that the c.718C>T mutation of the BMP-15 gene was significantly association with litter size, similar with previous studies in the Inverdale and Hanna sheep [17]. Interestingly, there were no homozygotes (GG genotype) detected in Small Tailed Han sheep (n = 869) and Hu sheep (n = 761). Chu et al. also reported the absence of GG genotype in Small Tailed Han sheep [15]. There are two potential reasons for the lack of GG genotype ewes in the population, one simple explanation is that GG ewes did not exist in our population, another reason is that the GG ewes may have existed in our population, but we selected the ewes with litter size records, and the infertile GG ewes were excluded in this study. About 29% of ewes in the population are G+ genotype and mating occurred under a random model; we believe GG genotype ewes should be generated under this model and therefore the GG genotype ewes should be detected in the infertile group. Mating of the G+ genotype rams and G+ genotype ewes can help verify this speculation. Numerous reports have shown that the FSHR gene plays a key role in animal reproduction [25][26][27]. Chu et al. found two mutation (g.681T>C and g.629C>T) in the 5' flanking region of the FSHR gene in Hu sheep and three novel mutations (g.200G>A, g.197G>A and g.98T>C) in Small Tail Han Sheep [12]. In our previous study, a novel SNP (g.47C>T) was found in the 5' flanking region of the FSHR gene in the Small Tailed Han sheep and Hu sheep [14]. This SNP was significantly associated with litter size. Therefore, the ovine FSHR gene could be selected as a candidate gene for improving litter size traits in sheep husbandry. Interestingly, several groups have reported the multi-marker combination effect on litter size in sheep. Chu et al. reported that the Small Tailed Han ewes carried BB/G+ genotype (BMPR-IB and BMP-15) showed more litter size than those with either mutation alone [15]. Individuals in Cambridge and Belclare breeds with mutations in both the GDF9 and BMP15 genes were found to be associated with greater ovulation rate than those with either single mutation [6]. In the present study, mutations in both the BMPR-IB and BMP-15 genes were also detected in Hu sheep and Small Tailed Han sheep and we also found a third mutations (FSHR g.47C>T) in these two breeds. The single marker-trait association analysis revealed that each mutation in ovine BMPR-IB, BMP-15 and FSHR genes was significantly associated with litter size in this population, and multi-marker analysis showed that individuals with the BB/G+/CC genotype had more lambs than those with only one predominant genotype, indicating that multiple markers may have a greater effect on contributing to the litter size in sheep and that the BB/G+/CC genotype combinations of BMPR-IB, BMP-15 and FSHR genes was considered as the superior genotype. Ethics Statement The experimental procedures were performed according to protocols approved by the Biological Studies Animal Care and Use Committee of Gansu Province, China. All efforts were made to minimize any discomfort during blood collection. Experimental Population A total of 1630 ewes aged from 12 to 30 months were collected from Gansu Zhongtian Sheep Ltd., including 869 Small Tail Han Sheep and 761 Hu sheep. All the sheep were in the artificial insemination system and raised in the same managed conditions. The litter size data for ewes was from the first or second parity (Table 5). DNA Extraction and Genotyping Genomic DNA was extracted from the venous jugular blood samples (5 mL per ewes) by the phenol-chloroform method, then dissolved in TE buffer solution (10 mM Tris-HCl and 1 mM EDTA, pH 8.0), and kept at −20 °C. The polymorphisms were genotyped by PCR-RFLP. The primers and restriction enzymes used in the genotyping analysis are listed in Table 6. The information of the primers of BMPR-IB, BMP-15 and FSHR are shown elsewhere [1,6,13]. The PCR was performed in a volume of 10 μL, containing 10× PCR buffer, 0.15 μM primer, 35 μM of dNTP, and 20 ng of genomic DNA, 0.5 U Taq DNA Polymerase (TransGen Biotech, Beijing, China). The PCR was performed as below: 5 min at 94 °C, followed by 35 cycles for 30 s at 94 °C, 30 s at 58~63 °C, 25 s at 72 °C and a final extension of 5 min at 72 °C. Five μL of each PCR product was digested with 3 U restriction endonuclease overnight at 37 °C, then the different sizes were separated on a 3% agarose gel, subsequently stained by GelRed. PCR fragments from different genotypes were cloned and sequenced for validation. Statistical Analysis The association analysis between single marker and litter size was performed by GLM (General liner model) procedure in the SAS software package (SAS Inst. Inc., Cary, NC, USA). The linear model was as follows: Yijl = μ + Gi + Bj + Sl + εijl where Yijl was the ijl traits' observation value; μ was the mean; Gi was the effect of the ith genotypes; Bj was the effect of jth breeding; Sl was the effect within season and εijl was residual corresponding to the traits observation value with var (ε) = Iσe 2 . Conclusions In summary, our present study indicated that the Small Tailed Han sheep and Hu sheep carried three polymorphisms (FecB, FecG and FSHR g.47C>T) associated with litter size. The ovine BMPR-IB, BMP-15 and FSHR genes have a combined effect on litter size in Small Tailed Han sheep and Hu sheep. Using BMPR-IB, BMP-15 and FSHR genes as genetic markers for multi-gene pyramiding can provide a way to improve litter size and shorten the breeding process of highly prolific sheep.

v3-fos

2017-04-03T10:14:37.817Z

2015-03-04T00:00:00.000Z

5045351

s2

Fate of Antibiotic Resistant Bacteria and Genes during Wastewater Chlorination: Implication for Antibiotic Resistance Control This study investigated fates of nine antibiotic-resistant bacteria as well as two series of antibiotic resistance genes in wastewater treated by various doses of chlorine (0, 15, 30, 60, 150 and 300 mg Cl2 min/L). The results indicated that chlorination was effective in inactivating antibiotic-resistant bacteria. Most bacteria were inactivated completely at the lowest dose (15 mg Cl2 min/L). By comparison, sulfadiazine- and erythromycin-resistant bacteria exhibited tolerance to low chlorine dose (up to 60 mg Cl2 min/L). However, quantitative real-time PCRs revealed that chlorination decreased limited erythromycin or tetracycline resistance genes, with the removal levels of overall erythromycin and tetracycline resistance genes at 0.42 ± 0.12 log and 0.10 ± 0.02 log, respectively. About 40% of erythromycin-resistance genes and 80% of tetracycline resistance genes could not be removed by chlorination. Chlorination was considered not effective in controlling antimicrobial resistance. More concern needs to be paid to the potential risk of antibiotic resistance genes in the wastewater after chlorination. Introduction Excessive use of antimicrobial drugs for human and veterinary infection results in the wide dissemination of bacterial resistance in the community and the environment. Occurrence of many antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) have been frequently reported in sewage, treated drinking water, river water, soil, and even air [1][2][3][4][5]. ARGs are widely considered as emerging contaminants for their potential threats on public health [6][7][8]. The need for "a global strategy to contain resistance challenges" has been strongly proposed [9]. Because of variable mixtures of bacteria, abundant nutrients and antimicrobial agents, municipal wastewater is considered favorable for both the survival and the transfer of bacterial resistance [10]. Microbial safety is one of the concerns during wastewater reclamation, and disinfection is normally applied for microbial control in wastewater treatment plants (WWTPs). However, antimicrobial resistance has just attracted attention, and limited studies have investigated the influence of disinfection techniques on the control of ARB and ARGs. Chlorination has a long history of application, and some previous studies have investigated the removal of various ARB by wastewater chlorination. Most researchers reported effective removal of ARB number by chlorination [11,12]; whereas there are also inconsistent results indicating that chlorination did not contribute to significant reduction of ARB even for the same kind [13,14]. Some antibiotic-resistant organisms, such as chloramphenicol-, trimethoprimand cephalothin-resistant bacteria, are reported to be tolerant to chlorine, weakening the effect of chlorination [12,[15][16][17]. However, conflicting results still exist concerning the removal of the ARB percentage by chlorination. Grabow et al. reported that the percentage of ampicillinresistant bacteria in sewage after chlorination changed slightly or even decreased [18]. Another study by Iwane and colleagues also found that chlorination treatment did not significantly affect the percentage of resistance in E. coli to one or more antibiotics (from 14.7 to 14.0%), or specifically to ampicillin (constant at 7.3%) and tetracycline (from 8.0 to 6.7%) [19.20]. The lack of data makes the question still unclear, and the effectiveness of chlorination in ARB reduction needs further exploration. On the other hand, the effective inactivation of ARB may not indicate elimination of antibiotic resistance in wastewater. The fate of ARGs needs to be considered as well. ARGs released to the environment have been observed to persist for a long time and then eventually transfer into new hosts [6]. Besides, ARGs have been discovered to occur as part of multidrug-resistant (MDR) superintegrons, which may result in multiple drug resistance in microorganisms. Chlorination, therefore, needs to be evaluated concerning its effectiveness on ARGs reduction. A study of Shi et al. conducted on drinking water chlorination reported that chlorination promoted most ARGs' abundance, although the gene of sul(I) was significantly removed [5]. Another study by Luo et al. suggested that significant levels of NDM-1 gene, the New Delhi metallo-β-lactamase, were present in WWTP effluents treated by chlorination (from 1316± 232 to 1431 ± 247 copies/mL) [21]. Karumathil et al. investigated the effect of chlorine on the survival of A. baumannii (a multidrug resistant pathogen) in water and transcription of genes conferring antibiotic resistance; results revealed that all A. baumannii isolates survived the tested chlorine doses. Additionally, there was an up-regulation of all or some of the antibiotic resistance genes in A. baumannii [22]. So far, limited attempts have been made concerning the characteristics of ARGs in wastewater chlorination based on molecular biological methods. In this study, the traditional cultivation method, as well as a culture independent method (quantitative real-time PCR), were applied for comprehensive assessment of chlorination effects on both ARB and ARGs reduction in the secondary effluents of a WWTP. The effects of chlorination on heterotrophic bacterial resistance to nine antibiotics (cephalexin, ciprofloxacin, chloramphenicol, erythromycin, gentamicin, rifampicin, sulfadiazine, tetracycline and vancomycin) were studied. The inactivation of MDR bacteria (erythromycin &tetracycline resistant) by chlorination was explored. Secondly, four erythromycin resistance genes [ere(A), ere(B), erm(A) and erm(B)] and four tetracycline resistance genes [tet(A), tet(B), tet(M) and tet(O)] in treated wastewater were investigated to evaluate the effectiveness of chlorination on ARGs reduction. The tested ARGs represent various mechanisms of antibiotic resistance. The ere(A) and ere(B) genes encode enzymes that hydrolyze the lactone ring of the macrocyclic nucleus [23,24], while the erm(A) and erm(B) genes encode rRNA methylase of target modification [25]. Similarly, the tet(A) and tet (B) genes encode efflux pumps, and the tet(M) and tet(O) genes encode the ribosomes protection protein [26]. The aim of our study was to evaluate the effect of chlorination on ARB and relevant ARGs simultaneously in wastewater. Treated wastewater sampling Treated wastewater samples were obtained from the effluent of a biological aerated filter (BAF) process in Q WWTP of Shanghai, China. The plant treatment process is shown in Fig. 1. The biological process of the plant was the anaerobic-anoxic-oxic (A 2 /O) process with a total hydraulic retention time (HRT) of 7.5 h. The load for the BAF was 0.35 kg NH 3 -N/(m 3 Ád) with HRT of 1 h. Wastewater quality indexes of the treated wastewater are given in S1 Table. The samples were collected in 500 mL clean Polyethylene bottles. The samples was stored at 4°C during the transportation time within 2 h, until subsequent processing in the lab, within 12 h. Chlorination procedures The chlorination of the treated wastewater sample (300 mL) was carried out in 500-mL Erlenmeyer flasks with magnetic stir bars to mix samples under room temperature (25°C). Referring to the study of Huang et al. [12], sodium hypochlorite was added to the samples to achieve various doses of chlorination (by DPD method), (0, 0.5, 1.0, 2.0, 5.0 and 10.0 mg Cl 2 /L) for a reaction time of 30 min. The pH was monitored during the process for all samples to keep constant at 7.0. 20 mL sample of each dose was applied for the determination of residual chlorine, while the remainder was added with sodium thiosulfate solution (1.5%) to terminate chlorination process. The CT value (the production of initial chlorine concentration and contact time) was used to express the dose of chlorination. The CT values in chlorination process were 0, 15, 30, 60, 150 and 300 mg Cl 2 min/L, respectively. The chlorine disinfection process at each concentration was conducted in duplicate. 30 mL of the disinfected sample at each dose was applied for microbial analysis. The remainder was saved at 4°C for the subsequent DNA extraction. Microbial resistance analysis To determine the bacterial resistance to nine types of antibiotics after chlorination, a certain amount of each antibiotic was added into the nutrient agar. In details, 1 mL of the chlorinated samples were spiked, serially diluted, and plated on nutrient agar (beef extract 3 g/L, peptone 10 g/L, NaCl 5 g/L and agar 15 g/L, pH: 7.2) containing antibiotics in duplicate. The plates were incubated for 24 h at 37°C to determine the number of antibiotic resistant heterotrophic bacteria. The plates with no antibiotic added were also conducted in the process to determine the total heterotrophic bacteria number. All samples were conducted according to the standard count technique. According to our previous study, the antibiotic concentrations added were defined as the maximum value of the Minimum Inhibition Concentration (MICs) of bacteria listed in CLSI [27,28]. The concentrations of the nine types of antibiotics, and a combination of two antibiotics that were used, are given as follows: cephalexin (CEP), 16 DNA extraction and quantification Each 100 mL chlorinated sample was filtered through a 0.22 μm micropore filter (Millipore, USA). The filters were then cut into small pieces and added to the DNA extraction tubes as described by Pruden et al [6]. The extractions were carried out in duplicate according to the Fas-tDNA Spin Kit for Soil (MP Biomedicals, USA). The extracted products were then checked for the yield and the quality using agarose gel electrophoresis and spectrophotometry (NanoDrop 8000, NanoDrop Technologies, USA). Quantitative real-time PCRs Four ERY resistance genes [ere(A), ere(B), erm(A) and erm(B)] and four TC resistance genes [tet(A), tet(B), tet(M) and tet(O)] were qualified by SYBR Green II q-PCR, as described by our previous study [28]. In brief, primers of four ERY resistance genes were developed in our previous study [28]. The development of four TC resistance gene primers have been prepared by Aminov et al. [29] and Zhang and Zhang [30]. The information of the primers is presented in Table 1. The target ARGs were amplified and then cloned to PMD-18T (Takara, Japan) to establish q-PCR standard curves. The Ct value of each sample was measured to calculate ARGs abundance according to the standard curves. Table 1) and 72°C for 30 s. Each sample was conducted for Q-PCR reaction in triplicate. The Q-PCR efficiency of the eight ARGs was 88.6%~110.3%, and R 2 values were always > 0.99 for all the standard curves. In addition, serial dilutions of extracted DNA were added to the plasmids containing each ARG (1 × 10 6 copies) to check for Q-PCR inhibition. The concentration of the template DNA was kept below 0.25 ng/ μL to avoid the suppression. The q-PCR amplified fragments specificity was then confirmed according to agarose gel electrophoresis and melt curves. Quantitatively evaluation of antibiotic resistance The proportion of each ARB was determined as follows: Proportion (%) = count number of each ARB (CFU/mL)/ total heterotrophic bacterial count (CFU/mL) × 100% Here, total heterotrophic bacteria count includes both resistant and non resistant bacteria. SPSS (Ver.19 ) was applied to perform all statistical tests in the study. The t-test was used to evaluate the differences between ARB or ARGs concentrations at a p level of 0.05. Ethics Statement Sample collection in the study was approved by Shanghai Chengtou wastewater Treatment CO.LTD in Shanghai, China. Effect of chlorination on the reduction of antibiotic resistant heterotrophic bacteria Heterotrophic bacteria resistant to nine types of antibiotics were all detected in the treated wastewater (Fig. 2). VAN-, SD-, CEP-and ERY-resistant bacteria were the four prevalent ARB with each proportion more than 40%. 4.4×10 4 and 3.1×10 4 CFU/mL of RIF-and GEN-resistant bacteria were found, with prevalence of 10% and 7%, respectively. In addition, TC-, CIP-and CHL-resistant bacteria were detected with rather low proportions, less than 3%, yet still with the absolute concentrations of more than 10 2 CFU/mL. In the chlorination process, all ARB were effectively inactivated with increased chlorine dose (Fig. 2). VAN-, CEP-, RIF-, GEN-, TC-, CIP-, and CHL-resistant bacteria did not exhibit tolerance to chlorination. They were eliminated down to the detection limit (1 CFU/mL) at 15 mg Cl 2 min/L (Fig. 2). The significant effectiveness of chlorination was consistent with previous reports of Murray et al. [16] and Macauley et al. [11]. It is worth noting that SD-and ERY-resistant bacteria shared similar initial concentrations with the VAN-and CEP-resistant bacteria, but showed tolerance to low chlorination dose. They were not eliminated until the dose was up to 60 and 150 mg Cl 2 min/L, respectively (Fig. 2). The tolerance of the two ARB was also observed previously, especially in actual WWTPs [14], where low effective dose of chlorine was probably conducted and thus weaken the chlorination effect. In addition, the existence of ARB tolerant to chlorination was frequently detected. A study of Rizzo et al. reported an antibiotic-resistant E.coli (MDR2) exhibiting resistance to high initial chlorine dose of 240 mg Cl 2 min/L [31]. In a recent study, Oh et al. revealed that 10% of E. coli DH5α (containing a multi-resistance gene) in a synthetic wastewater survived more than 30 mg/L of chlorine with 15 min exposure [32]. The kind of ARB tolerance to chlorination might be connected to the similar mechanism of gene resistance to antibiotic and chlorine (e.g., gene resistance to chlorination and the antibiotic might be both mediated by multidrug efflux pumps). This kind of 'co-resistance' might result in the ineffectiveness of chlorination on bacteria containing some ARGs. In general, chlorination was observed to greatly influence the ARB resistance. The MDR bacteria (ERY & TC-resistant) were observed in the treated wastewater with a proportion of only 2%. However, the data implied that there were 48% of TC-resistant bacteria exhibiting resistance to erythromycin, even without consideration of other antibiotics. This result emphasizes the wide abundance of MDR bacteria in WWTPs, as previous researches had observed [33,34]. ERY & TC-resistant bacteria did not exhibit tolerance to chlorination, which were inactivated to the detection limit at the lowest chlorination dose (15 mg Cl 2 min/L). Chlorination was considered effective in reducing ARB as well as MDR bacteria in the treated wastewater. It was observed that chlorination contributed to the effective inactivation of all the tested ARB; however, antimicrobial resistance might still not be eliminated completely. It was reported that genes encoding microbial resistance were likely to survive and keep active for a long time, even with the absence of their hosts [35]. Therefore, the effect of chlorination on ARGs was also evaluated in the following study. Effect of chlorination on the reduction of ERY and TC resistance genes The fates of four ERY resistance genes [ere(A), ere(B), erm(A) and erm(B)] and four TC resistance genes [tet(A, tet(B), tet(M) and tet(O)] during the chlorination process were investigated. Their abundances in samples treated by various chlorine doses are presented in Fig. 3. Among the four ERY resistance genes, ere(A) and erm(B) were observed to be dominant in the treated wastewater before chlorination, with concentrations of (3.2 ± 0.1) × 10 7 copies/L and (2.1 ± 0.2) × 10 7 copies/L, respectively (Fig. 3a). By comparison, less than 10 5 copies/L of ere(B) and erm(A) were detected. Chlorination exhibited significant reduction of the ere(A) or erm(B) genes, with 87% of ere(A) and 40% of erm(B) removed at 15 mg Cl 2 min/L. However, further increases of the chlorine dose did not result in higher reduction of these two genes. (6.6 ± 0.1) × 10 6 copies/L of ere(A) and (1.3 ± 0.1) × 10 7 copies/L of erm(B) always persisted in chlorinated samples. By comparison, no significant changes of the ere(B) and erm(A) abundances were observed during chlorination (p > 0.05). Tet(A) was the dominant gene in treated wastewater prior to chlorination among the four TC resistance genes, with a concentration of (1.3 ± 0.1) × 10 7 copies/L (Fig. 3b). The tet(A) concentration decreased significantly (p < 0.05) after wastewater chlorination. However, 76% of tet(A) still persisted in wastewater after chlorination, which could not be further eliminated with increasing chlorine doses. The concentrations of tet(B) and tet(O) in wastewater prior to chlorination were (4.1 ± 0.1) × 10 3 copies/L and (1.1 ± 0.1) × 10 6 copies/L respectively, and their concentrations changed only slightly with increased chlorine doses. The tet(M) gene was non-detectable in all samples. Apparently, chlorination could not eliminate the ARGs. To further explore the fates of ERY-and TC-resistance genes during the process, the total abundances of four genes carrying resistance to each antibiotic were summed respectively (S1 Fig.). Chlorination significantly decreased the total concentrations of ERY-or TC-resistance genes (p < 0.05). However, increasing the chlorine dose did not contribute to further reduction of the two genes abundances. Significant levels of ERY [(2.0 ± 0.4) × 10 7 copies/L] and TC resistance genes [(1.0 ± 0.1) × 10 7 copies/L] always persisted in samples treated by chlorination. Reduction levels of the total ERY-and TC-resistance genes by chlorination were calculated to be 0.42 ± 0.12 log and 0.10 ± 0.02 log, respectively. The results indicated that the reduction of ARGs abundance by chlorination was not as effective as ARB. Indeed, large amounts of ERY or TC resistance genes existed after chlorination when no ERY-or TC-resistant bacteria survived. The poor effect of chlorination on the ARGs removal was also confirmed by Shi et al., who investigated the effect of drinking water chlorination and found that chlorination even caused enrichment of ARGs abundance, such as the amp (C), aph(A2), bla TEM-1 , tet(A), tet(G), erm(A) and erm(B) genes [5]. Results of Dodd and Rizzo et al. also implied that the effective reduction of chlorine on bacterial DNA may be occurred only for extremely high dose [20,36]. This phenomenon might be related to the mechanism of chlorination. Chlorine inactivates bacteria by the strong oxidability of HOCl, which can easily enter bacterial cells, destroying the enzyme system inside and at last inactivating the bacteria. However, ARGs might not be destroyed in the process, and they might survive as dissociative DNA even without the presence of their hosts, causing large amounts of ARGs not detected after chlorination. Therefore, it is hypothesized that the decrease of ARGs concentration after chlorination probably results from the non-detection of the dissociative DNA, rather than the damage of ARGs. To evaluate the effect of chlorination on antibiotic resistance control in the treated wastewater, the normalized ratios of ARB and ARGs resistant to ERY or TC after chlorination are presented in Fig. 4. It was found that about 40% of ERY resistance genes and 80% of TC resistance genes could not be removed by any chlorine dose, while by comparison all ARB were below detection limit. The persistent ARGs represent the great potential risk of gene transfer to new hosts. Oncu et al. reported that chlorination could not affect plasmid structure at all studied doses, also did not change its transformability to competent cells [37]. In our recent study, we found that chlorination could not inhibit the ARGs transfer in wastewater; low dose of chlorine had slight effect on the ampicillin resistance gene transduction, or even promoted the frequency of tetracycline resistance gene conjugative transfer. Therefore, chlorination was considered not so effective in eliminating antibiotic resistance in treated sewage. By comparison, other disinfection techniques might be more conducive in controlling antibiotic resistance. Previous studies indicated that ultraviolet (UV) disinfection could effectively reduce the abundance of both ARB and ARGs given sufficient fluence [11,28]. Ozone and photocatalytic treatment were also reported to result in conformational changes of ARGs and the damage increased with doses [37]. Further evaluation of other wastewater treatment processes on antibiotic resistance is proposed, to explore more effective techniques in minimizing their potential risks. Furthermore, the results implied that only the detection of ARB may not be able to reflect accurately antibiotic resistance in wastewater, especially in those WWTP effluents treated by chlorination. On the other hand, only the detection of ARGs might also be difficult to reflect the general resistance to a specific antibiotic. Combinations of both ARB and some relevant ARGs might be more effective in determining antibiotic resistance in wastewater. Conclusions Chlorination exhibited effective inactivation of all ARB. SD-and ERY-resistant bacteria exhibited tolerance to low doses of chlorination, while other ARB species were inactivated down to detection limit at 15 mg Cl 2 min/L. Chlorination decreased limited ERY-or TC-resistance genes significantly, with the removal levels of overall ERY-and TC-resistance genes at 0.42 ± 0.12 log and 0.10 ± 0.02 log, respectively. However, 40% of ERY-resistance genes [(2.0 ± 0.4)×10 7 copies/L] and 80% of TC-resistance genes [(1.0 ± 0.1)×10 7 copies/L] still persisted in the wastewater after chlorination. The results indicated that chlorination could not eliminate the potential risk of antibiotic resistance in the treated wastewater. However, the fate of persisted extracellular ARGs in the wastewater after chlorination is not well examined yet. Further studies on their opportunities of repair, reactivation and transfer in subsequent aquatic environment are proposed. Table. Characteristics of the wastewater used in this study. (PDF)

v3-fos

2018-12-15T16:46:41.144Z

2015-10-23T00:00:00.000Z

84104071

s2

EFFECT OF PLANTING DATES ON THE YIELD OF BROCCOLI GENOTYPES This experiment was conducted during September, 2011 to March, 2012 in the experimental field of Department of Horticulture, Bangabandhu Sheikh Mujibur Rahman Agricultural University (BSMRAU), Gazipur to find out the effect of planting date on the yield of broccoli genotypes. There were five genotypes viz. Early green, Forest green, Green calabrese, Premium crop and Green king and four planting dates viz. 2 October, 27 October, 21 November and 16 December. The treatment effects were statistically analyzed and found significant in most of the characters studied. Genotype Green calabrese was the highest in average plant height (53.70 cm). Green king produced the maximum spread diameter (69.23 cm), stem diameter (30.35 mm) and early initiation of floral head. Genotype Early green performed the best regarding head weight (343.87 g), yield per plant (477.4 g) and yield (19.10 t/ha). Broccoli planted on 21 November initiated early flower head, maximum head diameter (16.99 cm), head weight (314.49 g), yield per plant (453.64 g) and total yield (18.15 t/ha). The genotype Early green planted on 21 November showed the best performance in yield per plant (580.17 g) and yield hectare (23.21 t/ha). Introduction Broccoli (Brassica oleracea var. italica L.), is an important member of "Cole" crops, belongs to the family Brassicaceae. Broccoli originated from west Europe (Prasad and Kumer, 1999). The word "Cole" means a group of highly differentiated plants originated from a single wild Brassica oleracea var. oleracea (Sylvestris L.) commonly known as wild cabbage (Bose and Som, 1986). Cole crops are the most widely grown vegetables in the temperate zones. After the Second World War these have spread rapidly to both tropical and subtropical areas and cabbage has increased in Africa by 31.5% and in Asia by 8.9% compared to the total increase in the world by 5.8% from 1970 to 1980. In Bangladesh, broccoli was introduced about two decades ago. Broccoli is grown in winter season in Bangladesh as an annual crop. It is environmentally better adapted and can withstand comparatively high temperature than cauliflower (Rashid et al., 1976). Its wider environmental adaptability, higher nutritive value, good taste and less risk to crop failure due to various biotic and abiotic factors indicate that there is enough scope for its promotional efforts. Its popularity to the consumers of urban area is increasing day by day in our country. But its cultivation has not spread much beyond the farms of different agricultural organizations. This is mainly due to the lack of awareness among the people about its importance and lack of available information production technology about it. Cultivation of broccoli in our country are confined into a very limited area with a minimum production and its average yield is only about 10.5 metric tons per hectare (Anon., 2004) which is very low compared to other broccoli growing countries like 24 t/ha in Italy, 20 t/ha in Japan and 18 t/ha in Turkey (Ahmed et al., 2004). The planting dates have significant effect on yield and other yield contributing characters of broccoli. The yield decreased with delay planting. Head yield is higher when crops are planted earlier and show a linear decreasing trend with delay in planting dates (Bianco et al., 1996). Early planted crops resulted in longer duration and produced taller plants with more number of leaves, higher plant spread and more leaf size index as well as the lowest percentage of abnormal curds than late planted crops and finally attributed to higher curd yield (Gautam et al., 1998). So there is enough scope to identify the optimum planting date to maximize the broccoli yield. Broccoli genotypes have also significant effect on yield of broccoli. Cultivar "Captain" produced the highest total yield as well as top and lateral head yields, the largest top head weight and marked earliness which was followed by cultivars Lucky, General, Griffen, Liberty and Milady (Toth et al., 2007). Several broccoli genotypes are cultivated in our country those differ in yield. So it is essential to identify high yielding genotypes to maximize broccoli yield. Therefore, the present experiment was undertaken to find out optimum planting date and appropriate genotype for maximum yield of Broccoli. Materials and Method The experiment was conducted at the Horticultural Research Farm, BSMRAU, Gazipur during September, 2011to March, 2012. The location of the experimental site was at the centre of Madhupur Tract (24.09 0 N latitude and 90.26 0 E longitudes) at 8.5m above the sea level (Anon., 1995). The soil of the experimental field was silty clay of shallow Red Brown Terrace type under Salna Series of Madhupur Tract in agroecological zone (AEZ) 28 (Brammer,1971 andSaheed,1984). The soil contained pH of 6.4 (Anon., 1998 andHaider et al., 1991). There were four planting dates viz. 2 October, 27 October, 21 November and 16 December and five genotypes viz. Early green, Forest green, Green calabrase, Premium crop and Green king. The field experiment was laid out in a factorial Randomized Complete Block Design with three replications. The unit plot size was 4m x 1.5m. The genotypes of broccoli were collected from the market of Siddique bazaar of Dhaka. The land was manured and fertilized with N, P , K and Molybdenum as Cowdung, Urea, TSP, MoP and Molybdenum @ 1500, 210, 120, 100 and 1kg/ha, respectively. The entire amount of Cow dung, TSP, MoP and Molybdenum were applied at the time of final land preparation and the entire urea was applied as top dressing in two equal split at 15 and 30 days after transplanting. Healthy and uniform 30 days old seedlings were transplanted in the main field at each date of planting maintaining a spacing of 50 cm x 50 cm. Weeding, irrigation and other cultural practices were done as and when necessary. Data were recorded from randomly selected plants for individual plant performance and from total plot for per hectare yield. Collected data were statistically analyzed by MSTATC. Plant height Significant variation in plant height was observed due to different planting dates ( Table 1). The highest plant height was obtained from 2 October planting at 15, 25 and 35 DAT (30.10, 40.75 and 54.83 cm, respectively) while it was the minimum with delayed planting i.e. 16 December). This result was in agreement with the result obtained by Ahmed and Abdullah (1986) The interaction effect between genotypes and planting dates varied significantly at 15 DAT, but it did not vary significantly at 25 and 35 DAT ( Table 2). The highest plant height (34.50 cm) was recorded from the genotype Premium planted on 27 October which was similar to G 4 P 3 (32.50 cm) and the lowest Means bearing same letter(s) in a column do not differ significantly at 1% level of probability. The combined effect of genotypes and planting dates was found significant in respect of spread diameter ( Table 2). The maximum spread diameter was obtained in Forest green planted on 2 October (44.47 and 64.60 cm) at 15 and 25 DAT, respectively followed by G 5 ×P 1 (42.87 and 63.57cm). At 35DAT, the plants of G 3 ×P 1 produced the maximum spread diameter (80.93 cm), which was statistically identical with G 5 ×P 3 (79.00cm) closely followed by G 2 ×P 3 (77.87 cm) and G 4 ×P 3 (76.57 cm) while it was minimum (57.57 cm) in G 1 ×P 1 . Diameter of stem Significant variation in stem diameter was observed due to the influence of planting dates ( There were significant differences in stem diameter due to interaction of genotypes and planting dates ( Head initiation Variation in days to head initiation due to planting date was observed (Fig. 1). It was revealed that maximum number of days (75.3) for first head initiation was required in 2 October planting which showed a decreasing trend with the later planting. Similar trend were also observed in 50% head initiation and total head initiation. In both cases maximum number of days 84.7 and 89.3 were required for 50% and 100% head initiation, respectively in 2 October planting, which decreased to 70.3 and 75.3 days in most delayed planting i.e. 16 December planting. Head formation in broccoli was quite similar to the curd formation of cauliflower which was primarily influenced by temperature. When broccoli sown late exhibited premature head initiation i.e., head initiation started before completion of vegetative growth. Similar results were obtained in cauliflower by Salter and Ward (1972) who reported that cauliflower plants grown in late planting at low temperature passed from vegetative phase to reproductive phase rapidly. Fig. 1. Effect of planting dates on days to head initiation. Genotypic differences were observed on days to head initiation ( Figure 2). Green king required minimum number of days 67.33, 71.5 and 76.66, respectively for first head initiation, 50% head initiation and total head initiation while these were maximum in Green calabrese, which were 86.33, 93.5 and 103.66 days for first head, 50% and 100% head initiation, respectively. date 472 HAFIZ et al. Head diameter Broccoli planted on 21 November produced the highest diameter of head (16.99 cm) and it was the lowest (13.64 cm) in 2 October planting ( Table 3). The result was in agreement with the findings of Anon. (1980) who reported that transplanting of broccoli in November produced the largest sized main head. Diameter of head significantly varied due to genotypes, planting dates and the interaction between them (Table 3).The largest head diameter obtained from in the genotype Premium (17.34 cm), which was statistically identical with Early green (16.89 cm) and Forest green (16.36 cm) while the smallest head diameter (12.92cm) was found in genotype Green calabrese (Table 3). Head weight Head weight varied due to planting dates (Table 3). The maximum head weight (314.44 g) was found from 21 November planting, which was statistically identical with 16 December planting and the minimum head weight (154.12 g) Means bearing same letter(s) or without letter in a column do not differ significantly at 1% level of probability. (1977) who reported that planting of broccoli on 28 November gave the maximum weight of head. Central head weight was significantly influenced by the genotypes, planting dates and their interaction effects ( Table 3). The maximum weight was recorded from genotype Early green (343.87 g) followed by Premium (264.07 g) which was statistically identical with Green king (239.11 g) and Forest green (236.22 g) while it was minimum (147.39 g) in Green calabrese (Table 3).Genotype Early green produced the highest head weight might be due to producing higher number of leaves and head diameter. Srivastava (1960) reported that good head depends on the number of leaves, their size (length and breadth) and ability to store carbohydrates and other nutrients within a particular temperature range. Due to combined effect of genotype and planting dates head weight differed significantly ranging from 76.79 to 437.42 g ( Table 4). The maximum head weight (437.42 g) was recorded from G 1 ×P 2 (Early green planted on 27 October) that was statistically identical with G 1 ×P 3 (406.69 g). The minimum head weight (76.79 g) was obtained from G 3 ×P 4 (Green calabrese planted on 16 December). The plants of G 1 P 2 and G 1 P 3 performed better might be due to prevailing suitable temperature for vigorous vegetative growth resulting in higher head weight. Similar results were obtained by Bianco et al., (1996) who reported that central head yield was higher when crop planted earlier. Number of secondary heads per plant Number of secondary heads per plant also varied significantly due to different planting dates ( The secondary head was those, which develop after harvest of main head. The results revealed that there was a variation in number of secondary heads among the genotypes (Table 3). Maximum number of secondary heads per plant (3.83) was recorded in genotype Early green. The minimum number of secondary heads per plant (1.92) was found in Green calabrese. A significant variation in number of secondary heads per plant was observed due to interaction of genotypes and planting dates ( Table 4). The maximum number of secondary heads per plant (4.67) was obtained from G 1 ×P 3 (Early green planted on 21 November) which was statistically identical to G 1 ×P 2 (4.00), G 2 ×P 3 (4.00), G 4 ×P 3 (4.00), G 1 ×P 1 (3.67) and G 5 ×P 3 (3.67).The minimum number of secondary heads per plant (1.67) was observed in treatment G 3 ×P 1 and G 3 ×P 4 . 476 HAFIZ et al. Secondary head weight Secondary head weight differed significantly among planting dates ranging from 139.16 g to 81.65 g ( Table 3). The maximum weight of secondary head (139.16 g) was obtained from 21 November planting which was statistically different to others, while the minimum (81.65 g) was found from 16 December planting. There was significant variation on weight of secondary head per plant among the genotypes ( Table 3). The highest weight (133.54 g) of secondary head per plant was found in Early green and the minimum was in Green calabrese (66.53 g) while rest of the genotypes produced statistically identical weight of secondary heads per plant. The combined effect of genotypes and planting dates was found significant on secondary head weight (Table 4). The secondary head weight varied from 173.48 to 52.38 g. The maximum head weight (173.48 g) was obtained from treatment combination G 1 ×P 3 (Early green planted on 21 November) which was statistically identical to G 2 ×P 3 (149.04 g), G 4 ×P 3 (148.82 g), G 1 ×P 2 (140.91 g) and G 5 ×P 3 (137.34 g) while it was the minimum (52.38g) in G 3 ×P 4 (Green calabrese planted at 16 December). Yield per plant Yield per plant varied significantly with planting dates (Table 3). The maximum yield per plant (453.64 g) was found in 21 November planting followed by 16 December (376.46 g) and 27 October planting (334.07 g) whereas it was the minimum (242.46 g) in 2 October planting. Genotypes of broccoli differed significantly regarding yield per plant (Table 3). The genotype Early green produced the maximum yield per plant (477.41 g) followed by Premium (372.35 g), which was statistically similar to Green king (350.13 g) and Forest green (344.48 g). The minimum yield per plant (213.92 g) was found in Green calabrese. Significant variation was observed in yield per plant due to the interaction of genotypes and planting dates ( Table 4). The highest yield per plant (580.17 g) was obtained from G 1 ×P 3, which was statistically identical to G 1 ×P 4 (538.94 g) followed by G 4 ×P 3 (500.93 g) and G 2 ×P 3 (452.53 g) while it was minimum (129.17 g) in G 3 P 1 . The treatment combination G 1 P 3 performed the best might be due to the production of higher number of leaves, head diameter, head weight and secondary head weight by the genotype Early green in presence of good environmental condition on 21 November planting. Total yield Planting dates also had significant influence on the yield of broccoli (Table 3). Broccoli of 21 November planting produced the highest yield (18.15 t/ha) followed by the 16 December planting (15.06 t/ha). It might be due to favorable low temperature for the head setting and development. On the other hand, minimum yield (9.70 t/ha) was obtained from 2 October planting. It might be due to comparatively higher temperature than the optimum at that time. The total yield of broccoli consisted of the main head and the secondary head those develop after the removal of the main one. Although the core of stem is also edible, it is usually not included as part of the yield. Significant variation in yield (t/ha) was observed among the genotypes (Table 3).The maximum yield (19.10 t/ha) was obtained from genotype Early green. This might be due to best performance of this genotype in head diameter, head weight and secondary head weight. The lowest yield (8.56 t/ha) was recorded from Green calabrese. Interaction effect of genotypes and planting dates on yield per hectare was found significant (Table 4). The plants of G 1 ×P 3 produced the maximum yield (23.21 t/ha) which was statistically identical to G 1 ×P 4 (21.56 t/ha) followed by G 4 ×P 3 (20.04 t/ha) while the lowest yield was obtained from G 3 ×P 1 (5.17 t/ha). Conclusion Considering yield contributing characters and yield potential of Broccoli, genotype Early green was found the best among the studied genotypes. 21 November was found to be the optimum date of planting for Broccoli. So the genotypes Early green should be planted on 21 November for maximum yield of Broccoli.

v3-fos

2019-04-01T13:16:20.626Z

2015-06-10T00:00:00.000Z

88664731

s2

A Milky Way to Healthy Gut: The Probiotic of All Ages This article innovatively discusses functional benefits of milk for optimal gut physiology and health. This involves optimizing the gut microbial ecology. Milk with its diverse nature of nutrients and functional items stimulates gastrointestinal digestive and assimilative functions. Milk’s special proteins and lactose help gut microbes retain adequate diversity to allow beneficial strains reproduce and inhibit harmful strains from generating toxins. Milk is an overlooked probiotic of all ages that must be highly included in healthy regimens. Science and Logics Humans worldwide continue to suffer from a multitude of gastrointestinal diseases and cancers that claim many lives and largely depress life quality. This usually happens in the darkness of insufficient dairy products consumption of mainly fresh milk [1,2]. The lack of serious exercise and suboptimal food intake rhythms and times are exacerbated by greatly inadequate milk consumption in many parts of the world [3][4][5][6]. This public policy article establishes a global pragmatic science for serious practice to accommodate sufficient milk in daily diets to help gut's internal and microbial actions remain most beneficial towards improved gut integrity and intermediary metabolism. Discussion of Innovations Milk contains numerous functional items that work beyond their merely nutritional value [1]. Specialized proteins, peptides, amino acids, vitamins, calcium, and lactose all independently and integratedly stimulate host and microbial enzymes production. Milk lactose can induce an active gut environment for both digestion and fermentation [7,8]. Despite the common wisdom that lactose intolerance is undesirable, certainly it could be favourable from a microbial and gut health perspective. Bloat and colic may in some degree occur in cases of lactose overintake, but prolonged milk consumption has the great potential to reduce the signs of intolerance. Lactose and milk driven gut fermentation is considered quite healthful for keeping the gut from passivity and inadequate movement. Due to its diverse and inter-fitting collection of nutrients and functional components, milk is pragmatically irreplaceable as far as body fitness and overall health are concerned. Milk is an optimal food pre-and post-exercise. Iron, copper and vitamins enriched milk is greatly exclusive in having essential nutrients in a watery environment fitting that of the body. That is the main reason that mammals' most crucial stage of life during infancy -and in humans during brain development -relies solely and entirely but indeed outstandingly on milk [8][9][10]. From the age of two years on, when breast milk is no longer fed, milk consumption must not be overlooked in daily human diets and must regularly continue through youthhood, adulthood and advanced aging. This is very determining when aging brings more susceptibility to and risks from a multitude of morbidities including stomach and gut related diseases of both short-and long-term types. The rising global concerns on the outbreak of obesity and related cardiovascular problems can be significantly helped to be overcome through prolonged milk consumption. Such a healthy milk intake does not mean drinking less than 0.5 L of fresh milk a day. Complementary dairy intake will come from yogurt, cheese, and other healthy products. Implications Human daily consumption of milk must not be overlooked after two years of age when breast feeding does no longer occur. Milk possesses an irreplaceable exclusive collection of functional items that are considered crucial for normal and healthy gut and liver function. Milk healthfully stimulates host and microbial food digestion and substrate assimilation that are required for normal gut physiology and dynamics. Milk-driven temporary watery defecation must not be considered unhealthy, as it helps the gut steadily but effectively accommodate milk towards body's natural and functional longevity. This is what the aging human populations strive for the most.

v3-fos

2017-08-03T02:46:18.829Z

2015-08-01T00:00:00.000Z

18352891

s2

Utilization of information from gene networks towards a better understanding of functional similarities between complex traits: a dairy cattle model Our study focused on quantifying functional similarities between complex traits recorded in dairy cattle: milk yield, fat yield, protein yield, somatic cell score and stature. Similarities were calculated based on gene sets forming gene networks and on gene ontology term sets underlying genes estimated as significant for the analysed traits. Gene networks were obtained by the Bisogenet and Gene Set Linkage Analysis (GSLA) software. The highest similarity was observed between milk yield and fat yield. A very low degree of similarity was attributed to protein yield and stature when using gene sets as a similarity criterion, as well as to protein yield and fat yield when using sets of gene ontology terms. Pearson correlation coefficients between gene effect estimates, representing additive polygenic similarities, were highest for protein yield and milk yield, and the lowest in case of protein yield and somatic cell score. Using the 50 K Illumina SNP chip from the national genomic selection data set only the most significant gene-trait associations can be retrieved, while enhancing it by the functional information contained in interaction data stored in public data bases and by metabolic pathways information facilitates a better characterization of the functional background of the traits and furthermore — trait comparison. The most interesting result of our study was that the functional similarity observed between protein yield and milk-/fat yields contradicted moderate genetic correlations estimated earlier for the same population based on a multivariate mixed model. The discrepancy indicates that an infinitesimal model assumed in that study reflects an averaged correlation due to polygenes, but fails to reveal the functional background underlying the traits, which is due to the cumulative composition of many genes involved in metabolic pathways, which appears to differ between protein-fat yield and protein-milk yield pairs. Electronic supplementary material The online version of this article (doi:10.1007/s13353-015-0306-5) contains supplementary material, which is available to authorized users. Introduction Recently in genetic analysis of complex traits the focus has been shifted from single genes identified via genome-wide association studies (GWAS) to genes identified via a functional analysis (Evangelou et al. 2014;Visscher et al. 2012). While genes selected by GWAS represent a selection of variants with (very) high effects on disease risk or on trait genetic variation, sets of genes selected by the functional approach are likely to also contain variants with moderate to small effects manifested through participation in important functional processes (Eleftherohorinou et al. 2009;Wang et al. 2010). In our study we were interested in the incorporation of functional information from gene network analysis into the assessment of similarity between selected quantitative traits. This idea was first introduced by McGary et al. (2010), but later Woods et al. (2013) developed this concept to derive phenologs, i.e. phenotypes orthologous between species, e.g. by showing that mouse phenotypesclonic seizures and abnormal brain wave patternare genomically similar to Communicated by: Maciej Szydlowski Electronic supplementary material The online version of this article (doi:10.1007/s13353-015-0306-5) contains supplementary material, which is available to authorized users. human epilepsy. Our study focussed on a within-species phenotype comparison by quantifying functional similarities between traits routinely recorded in dairy cattle. Recently, Pszczola et al. (2013) used the so-called predictor traits with widely available records in cattle populations, e.g. fat-proteincorrected milk, to enhance the accuracy of genomic prediction for other traits with less phenotypic information available such as, e.g. dry matter intake. Conceptually this can be considered as a within-species phenolog approach on an additive polygenic basis, i.e. with the underlying assumption of an infinitesimal mode of inheritance of phenotypes, with identification of neither particular genes nor the pathways. Our goal was to compare similarities between traits based on the functional information gathered through gene networks and thus assuming an underlying complex mode of inheritance. Material Deregressed estimated breeding values predicted in the national routine evaluation of 2601 bulls from the Polish Holstein-Friesian dairy cattle breed were used in this analysis. Breeding values comprised production traits: milk-, fat-and protein yields (MKG, FKG, and PKG), somatic cell score (SCS), two type traits: stature (STA) and body size score (SIZ), as well as two fertility traits: non-return rate for heifers (NRH) and non-return rate for cows (NRC). All those traits undergo a complex mode of inheritance determined by major genes, as well as a large number of genes with moderate and low effects with heritabilities in the Polish population estimated at 0.33 for MKG, 0.29 for FKG and 0.29 for PKG, 0.32 for SCS, 0.54 for STA, 0.50 for SIZ, as well as 0.02 for NRH and NRC. Genotypes comprise SNPs from the Illumina BovineSNP50 Genotyping BeadChip, which consists of 54, 001 SNPs (version 1) and 54,609 SNPs (version 2). Genetic samples were provided within the frame of the MASinBULL project and comprised semen probes acquired via a routine semen collection procedure. Genotype preprocessing comprised elimination of SNPs with minor allele frequency below 0.01 and call rate under 90 % and resulted in 46,267 SNPs selected for the analysis. GWAS Effects of the 46,267 SNPs were estimated using a SNP-BLUP model as described in Szyda et al. (2011). Statistically, this is a mixed model with random effects of SNPs described by a diagonal covariance matrix and bulls' pseudophenotypes as dependent variables. Based on the estimated SNP effects, information of SNP genomic location and the pairwise linkage disequilibrium between SNPs, underlying gene effects were calculated and tested for significance using a normal approximation of the t-test, as described in detail by Szyda et al. (2012). Genomic and functional information Genes showing effects significant with a maximum 20 % type I error rate were selected, separately for each trait, as scaffolds for the network construction. For better result validation two software packages were used to generate networks, i.e. the Bisogenet plugin (Martin et al. 2010) to the Cytoscape software (Shannon et al. 2003) and the stand alone Gene Set Linkage Analysis (GSLA) programme (Zhou et al. 2013). Both approaches construct networks of genes based on retrieving biological relations stored in multiple public data bases. For gene network generation Bisogenet utilizes data on protein-protein and protein-DNA interactions stored in publicly available data sets, as well as information from KEGG and signalling pathways. GSLA utilizes data on protein interactions predicted by the HIR V1 prediction model and 69,586 experimentally reported interactions. In both programmes the human data base was utilized, since interaction information for cattle available to date is very limited. The functional information was expressed either by sets of genes in generated networks or by the sets of gene ontology (GO) terms associated with the genes which were significant in GWAS. Genomic similarities The sets of genes composing each network and the sets of GO terms associated with significant genes were summarized in a design matrix (Supporting information Table S1), which was then used to calculate similarity scores between traits. Two measures were used to quantify similarities between pairs of traits by comparing the sets of genes underlying networks for each trait and by comparing sets of GO terms related to genes, which effects were estimated as significant in GWAS analysis. The cosine similarity between traits i and j is given by: where N ij represents the number of times a feature (i.e. gene or GO term) was significant for both traits, N i (N j ) is the number of times a feature was significant for trait i(j). Spatially, the metric represents an angle between two vectors of features. The Jaccard similarity coefficient, defined as the quotient between the intersection and the union of the pairwise compared variables: J ac ¼ N i j N i þN j þN i j . In addition, Pearson correlation coefficients were calculated between SNP and gene effect estimates for each pair of traits. Genes For size and non-return rate for cows and heifers no gene effect exceeded the 20 % significance threshold and thus the traits were not used for further analysis. For milk yield seven genes located on BTA14 were selected as significant, with effects ranging between 2.79 kg milk and 7.52 kg milk. For fat yield nine genes were selected, all located on BTA14, with effects between 0.11 kg fat and 0.39 kg fat. For protein yield six genes located on BTA03, BTA08, BTA17, BTA18, BTA19 and BTA29 were selected, with effects of 0.08 and 0.09 kg protein. Most genes (29), all with moderate standardized effects varying between 1.29 and 1.79, were selected for somatic cell score and were located on BTA01, BTA07, BTA09, BTA10, BTA12, BTA13, BTA17-20, BTA22-24 and BTA29. For stature two genes with standardized effects of 1.29 and 1.66 were selected on BTA5. Gene networks The networks obtained by Bisogenet and GSLA for production traits consisted of 98 and 34 genes for MKG with 17.4 % of genes overlapping between both programmes, 97 and 64 genes for FKG (23.6 % overlap), as well as 44 and 87 genes for PKG (24.43 % overlap). The largest network consisting of 1255 and 1437 genes with a 32.4 % overlap between programmes was obtained for SCS and the smallest network was observed for STA with 26 and 59 genes (10.6 % overlap). The list of genes selected for the analysed traits, representing vectors used for the calculation of genomic similarities, is given in the Supporting information Table S1. Similarities between traits Similarities between traits based on gene and GO term sets underlying the gene networks, calculated using two different measures, i.e. the cosine and the Jaccard coefficients, were very consistent. While comparing sets of genes constituting a gene network for each trait the highest similarity of 0.455 was observed between MKG and FKG, while no similarity, expressed by metrics equal to 0, was observed between PKG and STA. Considering sets of GO terms characterizing the significant genes the highest similarity score of 0.622 was also calculated for MKG and FKG, while the lowest score of 0.049 was attributed to PKG and FKG (Fig. 1). Pearson correlation coefficients calculated between 4345 estimates of gene effects were highest for PKG and MKG (0.762) and lowest (−0.011) for PKG and SCS, whereas correlations between 46,267 SNP estimates ranged from 0.779 for PKG-MKG to 0.025 for PKG-STA (Fig. 2). When comparing the results it is noteworthy that for many trait-pair combinations polygenic based information expressed by Pearson correlation coefficients is not consistent with the functional similarity measures, particularly all of the trait pair comparisons involving PKG indicated high polygenic similarity, but low functional similarity. Discussion The approach to derive functional information on complex phenotypes from GWAS, applied in this study, has already been postulated by Eleftherohorinou et al. (2009). Those authors stressed that using GWAS only the most significant gene-trait associations can be retrieved, which merely Brepresent the tip of the iceberg^of all potential genes involved in the determination of a quantitative phenotype, most of which have small individual effects. The impact of such genes on the determination of a complex trait is then manifested through their cumulative effect within functional pathways. This reasoning is exactly in line with our understanding of the genetic determination of complex traits and the corresponding methodology applied in our study attempts to extract most of the genomic background. A potential drawback of the experimental design of our study is connected with a relatively low coverage of the bovine genome by the 46,267 SNPs available for the analysis. The average intermarker distance was 51,728 bp, indicating some long gaps of the genomic sequence without SNP information. Therefore, out of over approximately 30,000 genes identified for dairy cattle we were able to pinpoint only 4345 with direct or closely located SNPs. Another aspect often neglected in association studies is that not all estimated significant associations may really represent physical linkage between a SNP and the genomic region. Some of the associations may arise through selection and the associated nonrandom mating in the population (Falconer and Mackay 1996). A technical limitation of the proposed approach results from the fact that it is based on GWAS results to select genes used as a scaffold for gene networks or GO terms. Since GWAS is only able to pinpoint genes of moderate to high effects on a quantitative trait variation, no scaffold can be created for traits with a pure polygenic (i.e. without major genes) mode of inheritance, in our case SIZ, NRH and NRC. On the other hand, due to a very small effective population size in dairy cattle, linkage disequilibrium is very strong within 1000 bp of physical distance (Qanbari et al. 2010), assumed as a threshold distance between a SNP and a gene in our study, the 4345 gene effects are expected to be accurate even if most of the polymorphisms are not located within a gene and thus do not represent causal mutations. Moreover, a large sample size and a very low level of residual noise thanks to the bull pseudophenotypes used being a function of thousands of records further contribute to the accuracy of the results. The apparently surprising result of our study is that the functional similarity observed between protein yield and milk/fat yield contradicts moderate genetic correlations estimated earlier for the same population based on a multivariate mixed model (Jesiołkiewicz et al. 2011). The discrepancy indicates that an infinitesimal model assumed in that study reflects an averaged correlation due to polygenes, but fails to reveal the functional background underlying the traits. As discussed by Shipley (2000) genes that are statistically the most significant are not necessarily direct, physiological causes of a complex phenotype. Consequently it appears that metabolic pathways underlying PKG and FKG/MKG appear to be different to a large extent. Such an outcome could have been expected when considering experimental resultse.g. Bauman and Griinari (2010) reported that the diet-induced low-fat milk syndrome in dairy cows does not affect protein yield, while food supplementation with biotin increased milk yield, but showed only a limited effect on milk composition (Girard and Matte 1988). On the other hand, similarities between PKG and FKG/MKG were reported in studies where an averaged genetic effect was considered, e.g. in a selection experiment reported by Kay et al. (2005) where selection on increased milk yield also resulted in an increased protein yield, or in studies focused on major genes with pleiotropic effects, e.g. DGAT1 reported to jointly influence MKG, PKG and FKG (Grisart et al. 2002). Polygenic based correlations between milk production traits (MKG, FKG, PKG) and SCS reported in the literature are very low, practically equal to zero (Miglior et al. 2007), which is in agreement with results estimated based on gene set similarity. The two similarity measures provided very concordant results. However, the Jaccard metric based on gene sets was consistently lower than values based on GO term set based similarity. It should be noted here that GO terms were related to genes, which were significant in GWAS and thus represent only the most significant associations, whereas gene sets include information on interactions and thus provide a broader insight into the functional background of traits. The major idea behind our study was to show that one does not need to rely only on Braw^information from gene effects estimated in GWAS or polygenic effects estimated in conventional mixed models, since they do not take into account other sources of biological information other than phenotypegenotype correlations. The mixture of the two sources of information, i.e. results of GWAS and functional information contained in public interaction data bases and metabolic pathways better characterizes the functional background of quantitative traits and furthermore facilitates their comparison.

v3-fos

2017-09-07T05:49:28.889Z

2015-01-01T00:00:00.000Z

128224181

s2

Response of Wheat to Residual Fertilizer Nitrogen Applied to Previously Failed Corn When drought conditions result in poor corn growth and yield, the potential exists for carryover of fertilizer nitrogen (N) to wheat. Soil sampling at the wheat jointing stage showed that NO3-N levels increased slightly as previous N rate increased up to 240 lb/a N, but did not appear sufficient for the wheat yield increase to previous N rate. The relationship between wheat normalized difference vegetative index (NDVI) measurements at jointing and wheat yield was linear. The use of crop active sensors such as the GreenSeeker (Trimble Navigation Ltd., Sunnyvale, CA) may provide plant response data to supplement soil sampling to more adequately determine residual effects on a following wheat crop. Introduction In 2012, extreme hot and dry conditions reduced corn crop yields. These drought-induced, low-yielding conditions likely resulted in low N uptake by corn. As a result, the potential exists for unused fertilizer N left in the soil, but the potential carryover of unused N fertilizer is uncertain because of the dynamics of N cycling. The objective of this study was to determine the effect of residual N that had been applied to a previous, drought-failed corn on the following wheat crop. Experimental Procedures A study was started in 2012 to determine the effect of N rates and nitrification inhibitors on short-season corn grown with no tillage. The experimental design was a splitplot arrangement of a randomized complete block with four replications. Nitrogen fertilizer rates were the whole plots and nitrification inhibitors were the subplots. An untreated control was included in each replication. Because of replanting and hot, dry weather, corn yields were less than 30 bu/a with no response to nitrification inhibitors and a slight decline in yields as N rate increased (data not shown). Because many farmers rotate winter wheat after corn and the 2012 experiment would not be repeated, 'Everest' wheat was drilled on October 12, 2012, with no added fertilizer and no tillage. The same plots with the same experimental design were used to study the residual effect of the N treatments. Wheat was harvested on June 25, 2013. In early April when the wheat was beginning to joint (Feekes 6), soil samples were taken from each plot to a 12-in. depth and analyzed for NH 4 -N and NO 3 -N. At the same time, a GreenSeeker handheld crop sensor was used to take NDVI readings. Results and Discussion The use of nitrification inhibitors on the previous corn crop had no residual effect on soil inorganic N levels and wheat NDVI readings taken in early April or wheat yield in June (data not shown), but residual from the previous N rate treatments did show carryover effects on soil NO 3 -N and NDVI readings at jointing and on wheat yields (Table 1). However, previous N rate treatments had no effect on soil NH 4 -N levels in the top 12 in., which were less than 20 lb/a N. The residual soil NO 3 -N levels in the top 12 in. increased from 5 to 20 lb/a N as the previous N rate increased from 0 (control) to 240 lb/a N. This small increase found at jointing from the control to the highest previous N rate was consequently expected to have minimal effect on wheat yield. Even though NDVI values were less than 0.70, the NDVI values increased with initial increments in previous N rate, but little change was measured at previous N rates above 120 lb/a N. Wheat yield increased more than 17 bu/a as N rate increased from the control to the previous 120 lb/a N fertilizer rate, but with no statistical increase with greater previous N rates. To assess fields, producers should first sample for available N in the soil. In this situation, because NH 4 -N levels were constant, a change in soil NO 3 -N of only 5 lb/a appeared to result in improving yield from 60% of the maximum to more than 90%, but there was little change as soil NO 3 -N increased another 10 lb/a ( Figure 1A). In contrast, the relationship between NDVI at jointing and relative wheat yield was linear ( Figure 1B). The potential for carryover of fertilizer N when the corn crop fails because of drought exists for a following wheat crop. A producer's first step to determine potential fertilizer N residual is to soil sample; however, with the dynamics of N processes, those results may not always be a reliable indicator of the residual effect of previous N fertilization. The use of crop active sensors, such as the GreenSeeker, may provide plant response data to supplement soil sampling to more adequately determine residual effects on a following wheat crop. Figure 1. Effect of soil NO 3 -N levels in the 0-12-in. depth and wheat normalized difference vegetative index (NDVI) readings taken at jointing (Feekes 6) on relative wheat yield in 2013.

v3-fos

2015-03-21T17:44:09.000Z

2015-02-05T00:00:00.000Z

651151

s2

Selection of a protein solubilization method suitable for phytopathogenic bacteria: a proteomics approach Background Finding the best extraction method of proteins from lysed cells is the key step for detection and identification in all proteomics applications. These are important to complement the knowledge about the mechanisms of interaction between plants and phytopathogens causing major economic losses. To develop an optimized extraction protocol, strains of Acidovorax citrulli, Pectobacterium carotovorum subsp. carotovorum and Ralstonia solanacearum were used as representative cells in the study of phytopathogenic bacteria. This study aims to compare four different protein extraction methods, including: Trizol, Phenol, Centrifugation and Lysis in order to determine which are more suitable for proteomic studies using as parameters the quantity and quality of extracted proteins observed in two-dimensional gels. Results The bacteria studied showed different results among the tested methods. The Lysis method was more efficient for P. carotovorum subsp. carotovorum and R. solanacearum phytobacteria, as well as simple and fast, while for A. citrulli, the Centrifugation method was the best. This evaluation is based on results obtained in polyacrylamide gels that presented a greater abundance of spots and clearer and more consistent strips as detected by two-dimensional gels. Conclusions These results attest to the adequacy of these proteins extraction methods for proteomic studies. Background The practice of agriculture brings as consequence the occurrence of plant diseases in levels that require their control. The most recommended method for this control is the use of genetic resistance [1]. However, not all plants are resistant to pathogens, and not every resistant variety is adapted to different regions of cultivation [2]. The bacteria Acidovorax citrulli (Ac), Pectobacterium carotovorum subsp. carotovorum (Pcc) and Ralstonia solanacearum (Rs), respectively cause bacterial fruit blotch, which is the main bacterial disease of melon culture, being responsible for heavy losses in production and depreciation of fruits [3]; soft rot in several hosts, among which lettuce, potatoes and tomatoes [4]; and bacterial wilt, which is the main worldwide vascular disease and attacks more than 50 botanical families, mostly the Solanaceae family, causing great economic losses [5,6]. The proteome is defined as the set of proteins expressed in a cell, tissue or any biological sample at a given time or under specific conditions [7]. The identification and characterization of these microorganisms using proteomic technologies can integrate the knowledge base necessary for the understanding of the mechanisms that phytobacteria use to cause diseases in their host [8]. In comparison with genomic studies, investigations at the proteome level provide detailed information, such as the abundance of proteins and post-transcriptional modifications [9,10]. The extraction of proteins is challenging, inconsistent and has been a problem for scientists [11]. Many techniques, including physical methods and those based on detergents, are available for protein extraction and are used for various purposes [12]. In proteomic studies, the development of an extraction method that can produce high yields and result in the complete dissolution, breakdown, denaturing and reduction of the greatest possible number of proteins present in the sample is an absolutely essential step for good results in two-dimensional gel electrophoresis (2D-PAGE) and mass spectrometry [13]. However, there are few studies that have compared the efficiency of these methods [14,15]. In this study, we compared four extraction methods: Trizol, Phenol, Centrifugation and Lysis, to determine their effectiveness in the separation of proteins by 2D-PAGE of three important phytobacteria: Acidovorax citrulli, Pectobacterium carotovorum subsp. carotovorum and Ralstonia solanacearum. Results In this study, four different extraction methods were compared to determine which of them increase the protein solubilization of phytobacteria for subsequent analysis by 2D-PAGE. Considering that non-protein impurities can severely affect the quality of 2D-PAGE separation, this study was critical to evaluate, standardize and select efficient methods for protein analysis of Acidovorax citrulli, Pectobacterium carotovorum subsp. carotovorum and Ralstonia solanacearum. The four extraction methods tested were effective in obtaining and concentrating proteins and the results are presented in Table 1. Although all methods presented appropriate yields for bacteria Ac and Pcc, the largest amount of proteins was obtained by the Centrifugation method. However, for Rs the best result was observed with the Lysis method, where there was a significant difference compared to the other methods tested. The SDS-PAGE analysis showed that the extractions presented good quality proteins, with well-defined bands without signs of degradation ( Figure 1). For bacteria Rs and Pcc it was noted that all methods seem well suited. However, for Ac the Lysis method presented a loss of proteins with a molecular weight above 38KDa in addition to little definition of the bands, which from this analysis suggest that this method is less efficient compared to the other methods tested for this specie. In this gel, it is possible to observe that there is a difference in the patterns and intensity of the bands observed amongst the methods in each of the phytobacteria. Thus, for the study in question, the best extraction method was considered the one that is most comprehensive, namely, the method that presents the greatest possible number of proteins with the best definition in 2D-PAGE gels. The results of the two-dimensional gels ( Figure 2 and Table 2) showed clarity and resolution, but were different for each of the bacteria. To define which method is best suited for the organism under study, one should consider the relative quality of the sample for analysis in 2D-PAGE and the number of protein spots obtained. Ac presented the best results with the Centrifugation method, showing 224 spot with a pH distribution between 4 to 7 and a molecular weight of 10 to 80 KDa; the bacteria Pcc and Rs presented respectively 212 spots, pH of 4 to 7 and molecular weight between 10 and 70 KDa and 369 spots, pH of 4 to 9 and a molecular weight of 20 to 70 KDa. These results showed a good range and are recommended for use in proteomic studies. Discussion Proteomic studies of high resolution depend mainly on a sample of good quality, so the method applied in the extraction of proteins is a key step to that end [11]. There is a great diversity of types of samples, therefore, an efficient protein isolation process for each one of them must be assumed empirically and tested in order to determine its real efficiency for the sample used, in order to obtain reproducible results in addition to the greatest possible representativeness of proteins in 2D gels [10]. During sample preparation in bacteria cells, problems may arise in cellular rupture due to the presence of thick cell walls and polysaccharide capsule in certain bacterial groups. Some bacteria can be lysed just by lysis buffer constituents, while others must be mechanically broken; in some cases it is necessary to use enzymes for the digestion of the cell wall [16]. Although many methods have been developed and reported, there is no single method for efficient isolation of all proteins of interest (cytosolic soluble or of a highly insoluble membrane) of an organism. Therefore, protein extraction methods continue to be a challenge for scientists in the accurate analysis of proteins [11]. In this regard, the chosen method should be simple and quick, with low cost and toxicity. These are important aspects in the selection of the method to be used, without selectively losing proteins while removing contaminants to the maximum extent possible [10]. The Lysis method has been applied in others organisms, such as sugarcane [17], soybean [11] and also in Xanthomonas campestris pv. viticola [18] with similar efficiency to that observed in this work. The lysis buffer composition allows quick access to the proteins, promoting denaturation, keeping them in the primary structure and thus protecting them against degradation agents. The preparing of protein samples consists in three fundamental steps, present in all methods: cell disruption, inactivation or removal of interfering and proteins solubilization [19]. The solubilization of proteins is considered the most problematic step in preparing protein samples for proteomic studies. The better solution is the buffer with a combination of urea and thiourea, associated with appropriate detergents, as tested by Chan and collaborators [20] for Prorocentrum triestinum. They observed an increase in the number of spots in electrophoresis gels when using urea and an even greater increase when using the combination of urea and thiourea. This is due to the fact that urea is a chaotropic agent, efficient in the rupture of hydrogen bonds, denaturing proteins by breaking the non-covalent and ionic links between aminoacid residues [19], leading to the split and denaturation of proteins. In turn, thiourea is very suitable for breaking hydrophobic interactions, increasing the solubilization of membrane proteins [21,22]. CHAPS and DTT are two important components in the proteins solubilization because they prevent hydrophobic interactions and promote the re-oxidation of disulfide bonds, respectively, avoiding the loss of proteins by aggregation or precipitation. There are several advantages to the use of the Lysis method in protein extraction: it is a method that is simple, fast (about 1 h), most interfering materials (nonprotein components) are effectively removed, the proteins are protected against degradation by proteases, thus not requiring the addition of protease inhibitors, in addition to having low toxicity. Furthermore, the composition of the extraction buffer ensures that proteins are under the same conditions as 2D-PAGE. The Centrifugation method was very efficient for Acidovorax citrulli, result that suggests its utilization for specific studies with this specie. The presence of SDS in the extraction buffer used in this method allows access to the proteins by breaking the membrane and, associated with heating at 100°C, inactivating proteases. The use of DNAse I and RNAse A enzymes, with subsequent precipitation with acetone, guarantees the elimination of contaminating in the final sample [23]. Some studies have focused on comparing protocols for protein extraction from a wide variety of organisms. For example, a study of lactic acid bacteria, which presented the comparison of three extraction methods for sonication, Centrifugation and FastPrep, found the best results with the latter [24]. In aphids, the TCA/acetonebased method was the more efficient in comparisons for 2DE than detergent and phenol based methods [13]. Unlike the results obtained in this work, in dinoflagellates the Trizol method presented better results when compared to the Lysis method [25]. Proteomic studies conducted with the bacterial phytopathogen Xanthomonas axonopodis pv. citri showed that the Phenol method was employed with success [26]. In some cases, it is necessary to develop a new method due to peculiarities of the sample in question as noted by Barbarino and Lourenço in 2005 [27] due to high concentrations of salts present in the sample. Conclusions For new proteomic studies with organisms that have not been registered in the literature, a pre-test of different methods for the preparation of the sample is strongly recommended in order to determine which is best suited for this type of analysis. In the case of the phytobacteria used in this study, the recommended methods are Centrifugation for Acidovorax citrulli and Lysis for Pectobacterium carotovorum subsp. carotovorum and Ralstonia solanacearum. Growing conditions Bacterial isolates were Acidovorax citrulli (Aac Were grown in 20 ml NYD medium (dextrose 10 g/l; meat extract 3 g/l; yeast extract 5 g/l; peptone 5 g/l) during 24 h at 28°C under constant agitation of 150 rpm for the formation of the pre-inoculate. Following this, 180 ml of the same media was added and the culture maintained under the same growth conditions for 24 h. Consequently, the bacterial growth was collected by centrifugation at 10.000 × g for 5 min, to obtain the cell mass for the extraction of total protein. Three biological replicas were made (independent cultures) and the samples were collected at an optical density (OD 600 = 0.5 ± 0.05) corresponding to the exponential phase 1 x 10 7 CFU/ml of each of the strains. Extraction of proteins Four different protein extraction methods were tested including modified Trizol, Phenol, Centrifugation and Lysis. After extraction, the supernatants containing the proteins of each of the methods were stored at -20°C until later analysis. Trizol method Protein extraction followed the instructions set out by the manufacturer of Trizol (Invitrogen ®) with some modifications. Briefly, 500 μl Trizol reagent were added to the cell pellet and lyse cells in sample by pipetting up and down several times. Subsequently, 200 μl of chloroform were added to the cell lysate before shaking vigorously for 15 s. The mixture was allowed to stand for 5 min at 25°C before being centrifuged at 12000 × g for 15 min at 4°C. The aqueous phase was removed. 300 μl of ethanol were added in order to resuspend the reddish bottom layer and the mixture centrifuged at 8000 × g for 5 min at 4°C. Supernatant was transferred to a new tube and 1.5 ml of isopropanol were added. The mixture was allowed to stand for at least 20 min for protein precipitation at 25°C. It was then centrifuged at 12000 × g for 10 min at 4°C. The pellet obtained was briefly washed with 95% ethanol before allowed to air dry. Finally, the proteins were ressolubilized in 500 μl of sample preparation solution (7 M Urea; 2 M thiourea; 4% CHAPS). Phenol method Total protein extraction was done as described by Metha and Rosato in 2001 [28]. The cell pellets were washed in phosphate buffer (7 mM K 2 HPO 4 ; 3 mM KH 2 PO 4 ; 0.15 mM NaCl; pH 7.2) and 750 μl of extraction buffer were added (0.7 M sucrose; 0.5 M Tris-HCl; 30 mM HCl; 50 mM EDTA; 0.1 M KCl and 40 mM DTT; pH 8.5), followed by incubation for 15 min (25°C). The same volume of phenol was added, and after 15 min of agitation in a vortex, the suspension was centrifuged at 14.000 x g for 3 min at 4°C and the phenolic phase was recovered. This procedure was repeated two more times. Proteins were precipitated with the addition of 5 volumes of 0.1 M ammonium acetate in methanol. The precipitate was washed with 1 ml of 80% acetone and solubilized as previously described. Centrifugation method The pellets were resuspended in 500 μl of extraction buffer (0.3% SDS; 200 mM DTT; 48 mM Tris; 28 mM HCl; pH 8.8). The microcentrifuge tubes containing the cell suspension were agitated gently for 10 min at 4°C, followed by removal of the cells by centrifugation at 14.000 x g for 10 min at 4°C. The extraction was incubated at 100°C for 5 min and then cooled on ice. Subsequently, 24 μl of assay buffer (0.5 M Tris; 476 mM HCl; 50 mM MgCl 2 pH 8.5; 1 mg/ml DNAse I; 0.25 mg/ml RNAse A) were added and the extraction incubated again for 15 min on ice. The reaction was stopped by the addition of four volumes of acetone cooled on ice and precipitation of proteins was left to occur for 20 min on ice. The cellular debris were removed by centrifugation at 14.000 × g for 10 min at 4°C [29]. The proteins were resolubilized in in 500 μl of sample preparation solution (7 M Urea; 2 M thiourea; 4% CHAPS) Lysis method The centrifuged pellets of bacteria were resuspended in 500 μl of lysis buffer (7 M urea; 2 M thiourea; 4% CHAPS) and homogenized in a vortex for 5 min at 25°C. The homogenized sample was centrifuged at 10.000 x g for 30 min at 4°C. The supernatant was transferred to a new 1.5 ml tube [17]. Quantification of proteins Total cellular protein concentrations were determined using a commercial protein colorimetric assay kit, 2D Quant Kit, according to the manufacturer's protocol (GE Life Sciences®) with bovine serum albumin (BSA) as a standard of measurement and absorbance at 480 nm. The kit is reported to not interfere with any chemicals used during extraction protocols and is therefore compatible with isoelectric focusing (IEF). The samples and the standard were read in triplicate. 2D-PAGE Two-dimensional electrophoresis (2-DE) was carried out according to the method of Görg and collaborators [31]. In the first dimension isoelectric focusing, 100 μg proteins were added to a rehydration buffer (7 M urea; 2 M thiourea; 2% CHAPS; 2 mM DTT; 1% IPG buffer pH 3-10 and 0.2% bromophenol blue) for a final volume of 250 μl. The sample was loaded onto 13 cm pH 3-10NL immobiline DryStrips (GE Life Sciences) with overnight rehydration, followed by isoelectric focusing for a total of 15.500 V/hrs. Strips were equilibrated in SDS equilibration buffer (6 M urea, 30% glycerol, 2% SDS) for 15 min with 10 mg/ml DTT, then 15 min in fresh buffer with 25 mg/ml 15 min. The second dimension was performed in homogeneous vertical acrylamide gel 15%. The equilibrated strips were applied onto the gel and sealed with agarose 0.5% and bromophenol blue 0.01%. The proteins electrophoretic separation was performed at 15°C in two stages: the first at 15 mA per fixed gel for 20 min and the second at 45 mA per gel for approximately 2 hours. Rainbow was used the molecular weight standard (GE Life Sciences). After the second dimension electrophoresis, the proteins were stained as in the SDS-PAGE. Rainbow was used as the molecular weight standard (GE Life Sciences). After the second dimension electrophoresis, the proteins were stained as in the SDS-PAGE. Analysis of two-dimensional gels After stained, the gels were scanned using an ImageScanner (Amershan Biosciences) scanner in transparency mode with a resolution of 300 dpi and images were saved in .mel format. These were analyzed using the ImageMaster Platinum v. 7.0 (Amershan Biosciences) computer program. The detection of each spot of protein was validated by manual inspection. The program provided the number of spots of each of the gels. Statistical analysis Data were analyzed by oneway analysis of variance (ANOVA) followed by Tukey's posthoc test and shown as mean and standard deviation. In all statistical analyses, p < 0.05 as taken as the level of significance.

v3-fos

2019-03-30T13:12:41.731Z

2015-02-12T00:00:00.000Z

85830321

s2

University of São Paulo “Luiz de Queiroz” College of Agriculture Exogenous hormonal manipulation to increase reproductive efficiency in dairy cows ............................................................................. 47 3. INTRODUCTION In dairy cattle, fertility is critical to improve the efficiency of dairy production. It was observed that, in high producing herds (12,500 kg in 305 days of lactation), by reducing days open from 161 to 98, there was an increase in milk production of 1.51 kg/day/cow (RIBEIRO et al., 2012). On the last 50 years, it was observed a marked increase in the average of milk production associated to a decrease of fertility in dairy cattle (LUCY, 2001;WASHBURN et al., 2002). In the United States, in the late 70's, when average milk production was ~17 kg/day/cow, there were approximately two services per conception, whereas in 1999, almost three services were needed per conception, and milk production was greater than 25 kg/day/cow (WASHBURN et al., 2002). Increase of incidence of clinical and subclinical diseases , increased steroid metabolism associated with increase of dry matter intake (DMI) (PARR et al., 2002) and heat stress affect reproductive efficiency (SARTORI; BASTOS; WILTBANK, 2010). High milk production is associated with increase of DMI (HARRISON et al., 1990) and liver blood flow, consequently, to the elevated steroid hormone metabolism SANGSRITAVONG et al., 2002). Lactating dairy cows had bigger ovulatory follicles and corpora lutea (CL), nevertheless, they had lower plasma concentration of estradiol-17β (E2) and progesterone (P4) than dairy heifers . Cows with higher milk production have lower circulating E2 at estrus, lesser duration of estrus, less events and standing times of estrus, despite having greater ovulatory follicles (LOPEZ et al., 2004) and multiple ovulation rate (WILTBANK et al., 2006). It has been shown also that cows ovulating larger follicle had lower pregnancy per artificial insemination (P/AI) (PEREIRA et al., 2014). For improvement of reproductive performance of dairy cows, by using two GnRH doses and a single Prostagladin F2α (PGF) treatment, Pursley et al. (1995) created the first fixed time artificial insemination (FTAI) protocol known as Ovsynch (GnRH -7 d -PGF -2 d -GnRH -1 d -AI) . Nevertheless, to obtain higher P/AI at the Ovsynch, cows should be treated with the first GnRH between d 5 and d 13 of the estrous cycle (VASCONCELOS et al., 1999). This strategy resulted in greater synchronization of wave emergence, presence of CL during the protocol and greater ovulation rate. Although E2/P4-based protocols (BO et al., 1993) have limitations due to prohibition in some countries, they have been effectively used for FTAI in other countries ( SOUZA et al., 2009;VASCONCELOS et al., 2011;PEREIRA et al., 2013PEREIRA et al., , 2014. Despite been widely used, P4/E2-based FTAI protocols also present a relatively high incidence of ovulation failure, and not optimized P/AI (around 30%) (SOUZA et al., 2009), requiring more studies to understand the cause of these failures in dairy cows. Currently, a 2.0 mg dose of estradiol benzoate (EB) at the onset of the protocol has been used in Nelore (Bos indicus) cattle with good results (MENEGHETTI et al., 2009;PERES et al., 2009;SA FILHO et al., 2009). Nevertheless, it is has been shown that after treatment with EB, nonlactating Holstein cows presented about half the circulating E2 in relation to nonlactating Nelore cows independent of EB dose (1.0, 2.0 or 4.0 mg) (BASTOS et al., 2011). Therefore, it is likely that the 2.0 mg dose of EB may be insufficient to provide an optimized wave emergence synchronization, especially for lactating dairy cows, which have even higher E2 clearance than nonlactating Holsteins (WILTBANK et al., 2006). For improving the response to the FTAI protocols a presynchronization strategy has been proposed (MOREIRA et al., 2001;. Although this concept is well defined in the GnRH-based protocols, few studies have evaluated the use of presynchronization protocols in dairy cows when using E2/P4-based FTAI protocols. Presynchronization-based protocols were used to increase the synchronization rate of wave emergence to the Ovsynch protocol ensuring greater P/AI (MOREIRA et al., 2001). Not only the phase of the estrous cycle is important for obtaining higher fertility (VASCONCELOS et al., 1999), but it is also necessary to consider that high concentration of P4 during emergence and development of the future ovulatory follicle increases the fertility of dairy cows (WILTBANK et al., 2006;. Another presynchronization protocol, called Double-Ovsynch, was developed with positive and consistent results . It has been shown that high circulating P4 both before and after AI is associated with increased fertility (WILTBANK et al., 2006). Thus, supplemental P4 has been used to increase fertility in dairy cows before and after AI (WILTBANK et al., 1956;ROSS;FOURT, 1958;LARSON;BUTLER;CURRIE, 2007). Moreover, LH receptors presence in the CL (FITZ et al., 1982) suggests LH involvement on CL developmental and function during metestrus and diestrus. In vitro, luteal cells increased P4 production, when challenged with human chorionic gonadotropin (hCG; similar molecule to the LH; FITZ et al., 1982). Studies have shown that P4 supplementation after ovulation has benefits for embryo development O'HARA et al., 2014). However, fertility data are scarce on the effects of P4 supplementation in recipient cows prior to a single embryo transfer. High concentration of circulating P4 on metestrus or early diestrus has been associated with advancement of conceptus elongation O'HARA et al., 2014), an associated increase in interferon-τ (IFN) FRAY;LAMMING, 2006). The IFN is the signal for maternal recognition of pregnancy in ruminants associated with prevention of luteolysis. This signal ensures continued production of P4 by the CL (PLANTE; HANSEN; THATCHER, 1988). During early pregnancy, the presence of the conceptus in the uterus suppressed the expression of both oxytocin and E2 receptors in the uterus (ROBINSON et al., 2001). The absence of oxytocin receptor in the endometrium prevents the release of luteolytic pulses of PGF, thereby sustaining lifespan of the CL and P4 production (DORNIAK; SPENCER, 2013). In addition to preventing luteolysis, IFN also induces expression of a large number of IFN stimulated genes (ISGs) in the endometrium (HICKS et al., 2003;GIFFORD et al., 2008). Not only endometrial cells express ISGs, but also other tissues, such as peripheral blood mononuclear cells (PBMC). Pregnant cows have shown increased ISG expression than nonpregnant cows (MONTEIRO et al., 2014;. ISGs, such as 2',5'oligoadenylate synthetase, myxovirus resistance 1/interferon-inducible p78 (MX1), myxovirus resistance 2 (MX2), interferon-stimulated gene 15 (ISG15), receptor transporter protein 4 (RTP4) were found expressed in PBMC showing difference on the expression between pregnant and nonpregnant ewes (YANKEY et al., 2001;OLIVEIRA et al., 2007;GIFFORD et al., 2008;) and cows (MONTEIRO et al., 2014;. 1.1 Hypothesis 1.1.1 The increase in the dose of EB from 2.0 mg for 3.0 mg would increase synchronization of follicular wave emergence and thereby increase synchronization during the protocol; 1.1.2 Presynchronization with a single GnRH treatment either 3 or 7 days before EB, would synchronize the stage of the follicular wave at the start of the E2/P4 protocol and produce a greater synchronization rate to the E2/P4 protocol; Champaign, v. 17, p. 386-390, 1958. Stoneham, v. 52, p. 1067Stoneham, v. 52, p. -1078Stoneham, v. 52, p. , 1999. Introduction Reproductive efficiency in dairy herds around the world has declined for a number of decades (LUCY, 2001;MEE, 2012), although recent data on daughter pregnancy rate in the USA has indicated a stabilization in genotypic values and a dramatic increase in phenotypic values for reproduction (NORMAN et al., 2009;PURSLEY, 2014). The improvement in reproductive performance can be partially explained by the use of systematic reproductive management programs in the USA including programs for synchronization of ovulation and fixed time AI (FTAI). These programs allow cows to be inseminated at a predesignated time without the need for detection of estrus and thus increase the AI submission rate (WILTBANK et al., 2014). Two types of hormonal programs have been utilized for synchronization of ovulation. Programs such as Ovsynch, use GnRH and prostaglandin F2α (PGF) to synchronize ovulation (PURSLEY; MEE; WILTBANK; 1995; PURSLEY; KOSOROK; WILTBANK, 1997), whereas programs in many parts of the world combine estradiol (E2), progesterone (P4), and PGF to synchronize ovulation (BO et al., 1995b;SOUZA et al., 2009;BARUSELLI et al., 2012). Both types of FTAI programs attempt to synchronize follicular waves, corpus luteum (CL) function, and circulating reproductive hormones in order to allow ovulation of an optimal-sized follicle in an optimal hormonal environment at a preappointed time. In GnRH-based programs it is well known that many cows are not correctly synchronized by the program. For example, Giordano et al. (2012) reported that only about 50% of cows treated with Ovsynch were correctly synchronized, whereas Double-Ovsynch resulted in about 70% synchronization. To date, synchronization rates during E2/P4-based programs in lactating dairy cows have not been adequately evaluated. Early studies that established the physiological basis for these programs utilized beef heifers and found that treatment with various forms of E2 led to suppression of gonadotropins, regression of growing follicles, and emergence of a new follicular wave about 4 d after E2 treatment (BO et al., 1993(BO et al., , 1994(BO et al., , 1995. Souza et al. (2009) evaluated 45 lactating dairy cows using daily ultrasound evaluations and reported that 84.4% (38/45) had synchronized emergence of a follicular wave after treatment with 2 mg E2-benzoate (EB) combined with insertion of controlled internal drug release containing P4. In addition, they found that 83.3% of cows had synchronized ovulation at the end of the protocol (Souza et al., 2009). Thus, similar to GnRH-based programs, protocols using E2/P4 may have problems with correct synchronization of the follicular wave at the beginning of the program and synchronized ovulation at the end of the program. Critical features of FTAI protocols that need to be optimized include: follicular wave emergence and growth (WILTBANK et al., 2011), concentration of P4 during follicular growth WILTBANK et al, 2014), complete lysis of the CL at the time of AI PEREIRA et al., 2013b;WILTBANK et al., 2014), and size of the ovulatory follicle (WILTBANK et al., 2011;PEREIRA et al., 2013aPEREIRA et al., , 2014. Programs that use E2/P4 to synchronize ovulation have generally used 2 mg of EB at the start to synchronize the follicular wave, although the early studies used 5 mg of various E2 derivatives, sometimes combined with P4 (BO et al., 1993P4 (BO et al., , 1994P4 (BO et al., , 1995a. The studies of Souza et al. (2009) reported that higher-producing lactating dairy cows had earlier follicular wave emergence than lower producing dairy cows after treatment with 2 mg EB. They postulated that the high E2 metabolism that has been found in lactating dairy cows (SANGSRITAVONG et al., 2002;WILTBANK et al., 2006) may be responsible for a more rapid decrease in circulating E2 concentrations after EB treatment. Thus, high-producing lactating dairy cows may require a greater dose of EB to achieve follicular wave regulation than is required in lower-producing cows or beef cattle. The GnRH-based FTAI programs have better fertility when initiated on certain days of the estrous cycle ( VASCONCELOS et al., 1999;MOREIRA et al., 2000), such as d 6 or 7 when there is a high ovulation rate in response to the first GnRH treatment and high circulating P4 during the protocol. Presynchronization programs have been developed to increase the percentage of cows at these optimal stages of the estrous cycle such as Presynch-12-Ovsynch Thus, these studies were based on three hypotheses related to E2/P4 protocols. First, we hypothesized that an increased dose of EB would increase synchronization of follicular wave emergence and thereby increase synchronization during the protocol. Second, we hypothesized that presynchronization with a single GnRH treatment would synchronize the stage of the follicular wave at the start of the E2/P4 protocol and produce a greater synchronization rate to the E2/P4 protocol. Finally, we hypothesized that complete characterization of the physiological response to E2/P4 protocols would demonstrate that synchronization rate following the protocol was not optimal and was due to specific physiological responses to the protocol. Two experiments were performed with the following objectives. The first experiment evaluated whether 3.0 mg of EB produced better synchronization of follicular wave emergence than 2.0 mg. The second experiment evaluated whether pre-synchronization with GnRH, 3 or 7 d before starting the protocol, would improve the synchronization rate of the cows. Finally, the results from both experiments were combined to evaluate specific physiological reasons for lack of synchronization to the protocol. Materials and methods The Animal Research Ethics Committee of "Luiz de Queiroz" College of Agriculture [ELANCO, 2009]) and age of 3.9 ± 0.1 yr (range, 3.0 to 4.8). During the experimental period, cows were housed in a tie stall barn equipped with sprinklers and fans. Cows were milked two times daily, and milk yield for each cow was recorded. Feed was provided two times daily, concurrent with milking. Cows were fed ad libitum a TMR-based diet of corn silage, with corn-soybean meal-based concentrate and minerals and vitamins, which was balanced to meet or exceed the nutritional requirements of lactating dairy cows (NATIONAL RESEARCH COUNCIL -NRC, 2001). Protocols and treatments The cows were randomized to receive either 2.0 mg (EB2; n = 23) or 3.0 mg (EB3; n = 21) of EB (Sincrodiol, Ourofino, Ribeirão Preto, SP, Brazil) at the start of a FTAI protocol Two d after ECP treatment (d 10), all of the cows were inseminated with commercial semen from bulls with proven fertility (Figure 2.1). Ultrasonography evaluation. During the protocol, ovaries of all cows were examined by transrectal ultrasonography (DP-2200, Mindray, Huntingdon, UK, PE29 6SZ) using a 7. cows were housed in a free stall barn equipped with sprinklers and fans. Cows were milked three times daily, and milk yield for each cow was recorded automatically. Feed was provided three times daily, concurrent with milking. Cows were fed ad libitum a TMR-based diet of corn silage, barley, and corn and soybean meal-based concentrate with minerals and vitamins, which was balanced to meet or exceed the nutritional requirements of lactating dairy cows (NRC, 2001). Protocols and treatments. The cows were subjected to the same FTAI protocol as used in experiment 1, except that all cows received 2 mg of EB on d 0 (Figure 2.2). Prior to the FTAI protocol cows were randomly assigned to receive GnRH (10.5 µg buserelin acetate; Sincroforte, Ourofino) either: 7 d before the start of the FTAI protocol (n = 40) or 3 d before the start of the FTAI protocol (n = 42). Three groups were designated following determination of ovulatory response to the GnRH treatment: Cows that ovulated to the d 7 GnRH treatment (G7), cows that ovulated to the d 3 GnRH treatment (G3), and cows that did not ovulate following GnRH treatment (NoOv). Cows were considered positive for emergence of a new wave when emergence occurred between d 1 and d 6. Cows that had the ovulatory follicular wave emerge before d 1 were classified as lacking emergence of a new wave and if ovulation occurred from this follicular wave were classified as ovulating a persistent follicle. Blood sampling and analysis of progesterone in plasma. Blood was sampled from all 82 cows every 24 h from d 0 until d 5, and on d 7 and d 10, by puncture of the coccygeal vein or artery into evacuated tubes containing Sodium Heparin (Vacutainer, Becton Dickinson, Franklin Lakes, NJ, USA). Immediately after collection, tubes with blood were placed on ice and kept refrigerated until transported to the laboratory within 4 to 5 h after collection. Blood tubes were centrifuged at 1,700 x g for 15 min at 4ºC for plasma separation. Aliquots of plasma were frozen at -20ºC until assayed. Concentrations of P4 were analyzed by RIA using a commercial kit (Coat-a-Count, Siemens Healthcare Diagnostics, Los Angeles, CA). The sensitivity of the kit was 0.02 ng/mL. The intra-assay CVs were 5.4% in assay 1 and 4.3% in assay 2. The inter-assay CV was 4.1%. Analysis of synchronization using cows from both experiments Cows in both studies were treated with a similar EB/P4-based FTAI protocol and had daily ultrasound evaluations during the protocol. Therefore, an analysis was performed to determine the reasons that cows failed to synchronize during the protocol. In this analysis, data from all 126 cows from both experiments were evaluated (n = 44 from Experiment 1; n = 82 from Experiment 2). Cows that had emergence of a new follicular wave after EB treatment and ovulated at the end of the protocol were considered synchronized. Statistical Analysis Both experiments the sample size was calculated using the POWER procedure of SAS Continuous data with repeated measures over time were analyzed by ANOVA using the MIXED procedure of SAS version 9.3 with models fitting a Gaussian distribution. Data were tested for normality of residuals, and data with residuals not normally distributed were transformed before analysis. The models included the fixed effects of treatment, day of measurement, parity, breed, BCS, milk yield categorized within parity during the week of d 0 as above or below the mean value, interactions between treatment and day and treatment and parity, and the random effects of cows nested within treatment. The covariance structure that resulted in the smallest Akaike's information criterion was selected for the model. Model fitting was evaluated using the fit statistics. Categorical data were analyzed by logistic regression using the GLIMMIX procedure of SAS fitting a binary distribution. The models included the fixed effects of treatment, parity, breed, BCS category, and milk yield categorized within parity on the week of d 0 as above or below the mean value. The Kenward-Roger method was used to calculate the denominator degrees of freedom to approximate the F tests in the mixed models. Model fitting was evaluated using the fit statistics. The estimates were back-transformed using the ILINK function of SAS to generate the adjusted percentages. The FREQ procedure of SAS was used for the analyses of the combined data from experiment 1 and 2 or for analyses in experiment 2 when a treatment outcome was 0 or 100%. In all analyses, differences were considered significant when P < 0.05, whereas tendencies were considered when 0.15 > P ≥ 0.05. Experiment 1 Day of emergence, day of deviation, and size of future ovulatory follicle at time of deviation were not affected by EB dose (Table 2.1). The percentage of cows with a CL on d 7 was reduced (P < 0.05) for cows receiving 3 mg of EB compared to 2 mg. The percentage of cows ovulating at the end of the protocol was high (~90%) and not altered by EB treatment. However, time of ovulation was later (P < 0.01) for cows that received 2 mg EB compared to 3 mg EB. In addition, multiple ovulation was surprisingly high (33%) but not affected by treatment. There was also no effect of treatment on P/AI ( This may have influenced size of the ovulatory follicle which was greater for cows in the NoOv group compared to G3 and G7 groups (Table 2.2). Independent of treatment group, cows that had a CL on d 7 ovulated a smaller (P = 0.05) follicle (14.9 ± 0.3 mm) than cows that did not have a CL on d 7 (16.5 ± 0.5 mm). A comparison was made between milk production and various measures of timing and size of the future ovulatory follicles, which includes only those cows with emergence of a new follicular wave (d 1 to d 6) and single ovulation (data not shown). Day of follicle wave emergence was delayed (P = 0.03) in cows with lower milk yield (3.4 ± 0.20 d) compared to cows with higher milk yield (2.8 ± 0.20 d). However, milk yield did not affect the day of deviation, day of ovulation, size of future ovulatory follicle at deviation, or maximum size of ovulatory follicle. In addition, the percentage of cows that had emergence of a new follicular wave (d 1 to d 6) or percentage of cows with multiple ovulation was not affected by milk yield. In Figure 2.3 is shown the dynamics of ovulatory follicle growth for cows that ovulated a persistent follicle (ovulated a single follicle that was present on d 0; n = 17) compared to cows that ovulated a single follicle that emerged from a new follicular wave (d 1 to d 6 of protocol; n = 42). On all days during the protocol, the future ovulatory follicle was larger for cows that ovulated a persistent follicle than cows that ovulated a follicle that emerged during the protocol (P < 0.05). Cows ovulating a new follicle tended (P ≤ 0.07) to have higher P/AI (Figure 2.3). Concentration of progesterone. At the time of protocol initiation (d 0) circulating P4 concentrations were greater in G7 and NoOv cows compared to G3 cows (Figure 2.4). As the protocol progressed, the P4 concentrations progressively decreased in NoOv, as CL continued to regress, and increased in G3, with growth of the new CL. Therefore, concentrations of P4 were greater in G7 and G3 than in NoOv cows on d 4, d 5, and d 7 of the protocol. By d 7, circulating P4 concentrations had decreased (P < 0.05) in G7 and NoOv cows compared to d 0 values but had not changed in G3 cows. A comparison of overall circulating P4 concentrations during the entire period indicated that there was a tendency for a treatment effect (P = 0.06), an effect of day (P < 0.001), and a treatment by day interaction (P = 0.01). Concentrations of P4 were 0.5 ng/mL greater in G7 (3.2 ± 0.3) compared to NoOv (2.7 ± 0.2) cows, with G3 (2.7 ± 0.3) cows not significantly different from the other two groups. Independent of treatment group, cows that were pregnant on d 31 or d 59 after FTAI had greater circulating P4 concentrations during the protocol than cows that were not pregnant (P = 0.01; Figure 2.5). There was also an effect of day (P < 0.001), but no day by pregnancy status interaction. For all days that were analyzed during the protocol, except d 7, cows that were later found to be pregnant had greater circulating P4 compared to cows that were subsequently found to be non-pregnant. On the day of FTAI, the circulating P4 concentrations were categorized in order to evaluate whether individual cows had undergone complete luteolysis (P4 < 0.10 ng/mL) or partial luteolysis (P4 = 0.10 to 0.22 ng/mL). The percentage of cows that ovulated at the end of protocol was greater (P = 0.04) for cows with complete compared to partial luteolysis ( Figure 2.6). In addition, there was an effect of complete luteolysis on P/AI at either d 31 (P = 0.03) and d 59 (P = 0.05; Figure 2.6). When only cows that ovulated were considered in the analysis, luteolysis status tended (P = 0.08) to influence P/AI on d 31 and d 59 (P = 0.14) after FTAI ( Figure 2.6). Ovulation rate and pregnancy per AI. Day of ovulation and multiple ovulation rate were not different among groups, but ovulation rate tended to be higher on NoOv than G7, and G3 did not differ from the other treatments ( x,y P value > 0.05 and ≤ 0.10 1 Only cows that had emergence of a new wave between d 1 and d 6 were included 2 Number of cows that had emergence of a new wave between d 1 and d 6 divided by the total of cows treated 3 Only cows that had emergence of a new wave (d 1 to d 6) and single ovulation were included 4 Only cows that had a CL on d 0 were included in the analysis 5 Number of cows that ovulated divided by the number of cows treated Cows were considered to have ovulated a persistent follicle (n = 17) when they ovulated a follicle that was present on d 0. Cows ovulating a new follicle (n = 42) were considered when they had emergence of a new follicular wave between d 1 and d 6. There was an interaction between follicle type and day (P < 0.001). Cows that ovulated a persistent follicle tended to have a lower P/AI on d 31 (P = 0.06) and d 59 (P = 0.07) than those that ovulated a new follicle. * Within day, effect of follicle age (P < 0.05) Days after the onset of the protocol Cows received FTAI on d 10 of the protocol. From d 0 to 5 and on d 7, concentrations of P4 averaged 2.5 ± 0.23 and 3.5 ± 0.29 ng/mL for nonpregnant and pregnant cows, respectively. There were effects of pregnancy status (P = 0.01) and day (P < 0.001), but there was no interaction between pregnancy status and day Figure 2.6 -Ovulation rate (percentage of cows that ovulated at the end of the protocol) and Pregnancy/AI (P/AI) at d 31 and d 59 after fixed time AI (FTAI) for cows that had complete luteolysis (n = 49; P4 < 0.10 ng/mL) or partial luteolysis (n = 33; P4 ≥ 0.10 and < 0.22 ng/mL) on day of FTAI. * Effect of complete vs. partial luteolysis (P ≤ 0.05) Finally, independent of treatment group, ovulatory follicle size had no effect on P/AI that did not have ovulation at the end of the protocol (0/28). Therefore, overall synchronization rate, based on follicle emergence and ovulation, was 59.5% (75/126) with much greater (P < 0.01) P/AI in synchronized compared to non-synchronized cows (Table 2.3). Table 2.3 -Results of the analysis of synchronization to the protocol using all cows from both experiments. Cows were classified by whether they had emergence of the new follicular wave during the protocol (d 1 to 6) and whether they had ovulation at the end of the protocol in order to determine the percentage of cows that were synchronized to the FTAI protocol and the Pregnancies/AI (P/AI) for synchronized (Yes) and non-synchronized ( Cows that ovulated between d 9.5 and 11.5 of the protocol 3 Cows were considered synchronized when they had new wave emergence and ovulated Discussion Reproductive management in many parts of the world use hormonal programs that synchronize ovulation in order to allow for FTAI of all eligible cows. This research evaluated the dynamic changes in ovarian structures and reproductive hormones during modifications of an E2/P4 FTAI synchronization protocol. Our first hypothesis, that increasing the dose of EB would increase synchronization, was rejected as the higher dose of EB (3 mg vs. 2 mg) did not improve follicular synchronization but instead led to earlier regression of the CL and earlier ovulation at the end of the protocol. Although we have been unable to find other studies that have tested the specific hypothesis of dose of EB compared to synchronization, our results on increased CL regression are consistent with other research results, as discussed below. Our second hypothesis, that synchronization could be improved by initiating the E2/P4 protocol at a specific stage of a follicular wave, was proposed due to the success of presynchronization programs on fertility in GnRH-based protocols (MOREIRA et al., 2001;HERLIHY et al., 2012; WILTBANK; PURSLEY, 2014). However, we also rejected this second hypothesis, as we observed that irrespective of the stage of the follicular wave at protocol initiation, many cows did not ovulate at the end of the protocol or ovulated a persistent follicle, due to lack of emergence of a new follicular wave following EB treatment. Probably, the most interesting results from this study were found when we combined the results of both experiments to evaluate the specific physiological abnormalities that led to lack of synchronization during the protocols. Only about 60% of cows were synchronized by the E2/P4 protocol, based on emergence of a new follicular wave and ovulation at the end of the protocol. Cows that were synchronized had excellent fertility to the FTAI (~ 60%) but cows that were not synchronized had low fertility (15.7%). Although the results of these experiments are consistent with previous research, as discussed below, they provide novel physiological insights into problems and possible resolutions associated with E2/P4-based protocols for synchronizing ovulation. Treatments with P4 and various types of E2 have been shown to result in the emergence of a new follicular wave (BO et al., 1995a(BO et al., , 1995b, however, consistent with the results of a previous study in lactating dairy cows (SOUZA et al., 2009), more than 25% (33/126) of dairy cows in our study did not demonstrate emergence of a new follicular wave after EB treatment. Insufficient EB does not seem to explain this problem, since increasing the dose of EB from 2.0 to 3.0 mg did not reduce the percentage of cows that ovulated a persistent follicle (29% persistent follicles after 3 mg EB; 6/21). It is unclear why some cows did not have new wave emergence after EB treatment but one possibility, that was not tested in our study, is that there was insufficient suppression of circulating FSH/LH concentrations in response to EB and P4 treatment at protocol initiation and therefore follicular wave turnover did not occur. Lactating dairy cows have increased E2 and P4 metabolism due to elevated liver blood flow (SANGSRITAVONG et al., 2002;WILTBANK et al., 2006) and therefore the P4 implant and EB may have produced an insufficient increase in P4 and E2 to suppress gonadotropins. In addition, similar circulating P4 concentrations seem to be less inhibitory to LH pulses in lactating compared to non-lactating cows (VASCONCELOS et al., 2003). Another important consideration is that treatment with EB, particularly the 3 mg dose of EB, was associated with regression of the CL. Increasing the EB dose from 2 mg to 3 mg clearly produced greater CL regression during the protocol as the number of cows with CL at the time of PGF treatment (d 7) decreased from about 60% with 2 mg to less than 20% after 3 mg EB treatment. We did not measure circulating P4 in this first study, however CL regression would be followed by a substantial decrease in circulating P4 and probably increased frequency et al., 1975;THATCHER et al., 1986). It is unclear if 2 mg EB caused CL regression, since we did not have a control group that received no EB treatment. However, it is clear that most CL regress in response to 3 mg EB. Treatment with E2 can increase PGF production by the uterus (FORD et al., 1975;THATCHER et al., 1986). This effect appears to be mediated by binding of E2 to estrogen receptor α, subsequent upregulation of oxytocin receptor expression, binding of oxytocin to the oxytocin receptor, and subsequent production of PGF pulses due to the pulsatile pattern of oxytocin pulses (McCRACKEN; CUSTER; LAMSA, 1999;FLEMING et al., 2006;SPENCER et al., 2007). Thus, although greater doses of EB might be expected to produce greater suppression of gonadotropins, it appears this effect may be more than conteracted by the decrease in circulating P4 due to increased CL regression with a greater dose of EB. It seems likely that a better way to produce a more consistent regression of all follicles is to increase the amount of P4 simultaneously to EB treatment, such as by using high doses of injectable P4 or by including two P4 implants during the protocol, but these approaches also need to be tested. Our observations of low fertility in cows that ovulated a persistent follicle are similar to results from numerous other studies (AHMAD et al., 1996;BLEACH;GLENCROSS;KNIGHT, 2004;CERRI et al., 2009;SANTOS et al., 2010) and are consistent with the idea that increasing the dominance period of follicles reduces fertility in the oocyte that is ovulated from the persistent follicle. This effect may be due to premature germinal vesicle breakdown due to excessive LH stimulation of the dominant follicle (AHMAD et al., 1994;REVAH;BUTLER, 1996). Oocytes from persistent follicles are fertilized but generally undergo cessation of cellular division and embryo degeneration during the first few day of embryo development (AHMAD et al., 1994;CERRI et al., 2009). For example, ovulatory follicles that were 12 d old compared to 9 d of age had a lower percentage of freezable embryos and a greater percentage of degenerate embryos (CERRI et al., 2009). In addition, lower circulating P4 concentrations during follicle growth can result in reduced fertility of the follicle, independent of follicle age (RIVERA et al., 2011;WILTBANK et al., 2014b). This idea was supported by our data as circulating P4 concentrations were consistently greater in cows that became pregnant compared to cows that became non-pregnant, independent of treatment group ( Another important hormonal problem that we observed during the protocol was that some cows had a slight elevation in circulating P4 near the time of AI (> 0.1 ng/mL). This elevation was associated with a decrease in P/AI at the d 31 and the d 59 pregnancy diagnoses. One important reason for the decrease in fertility was a decrease in ovulation at the end of the protocol in cows with small P4 elevations. Another study also reported that an elevation in circulating P4 to more than 0.1 ng/mL resulted in a reduced P/AI in lactating dairy cows treated with an E2/P4-based FTAI protocol (PEREIRA et al., 2013b). This appears to be a lower threshold for P4 than has been reported in studies using GnRH-based protocols in which the threshold for reduced fertility was between 0.3 to 0.5 ng/mL for P4 BRUSVEEN;WILTBANK, 2009;BISINOTTO et al., 2010;GIORDANO et al., 2012GIORDANO et al., , 2013. It seems likely that any difference in P4 threshold could relate to inhibition of an ECP-induced LH surge by low concentrations of P4, since E2 action is blocked by P4 at the hypothalamic GnRH level (ROBINSON et al., 2000;RICHTER;EVANS, 2002). Thus, we observed a decrease in percentage of cows that ovulated, probably due to lack of a GnRH/LH surge in some cows with P4 above 0.1 ng/mL. In contrast, induction of ovulation by using GnRH is likely to occur, even in the presence of low concentrations of P4, however, the threshold that inhibits gamete transport, a second mechanism that reduces fertility (DAY; POLGE, 1968;HUNTER, 1968), may be reached when circulating P4 exceeds 0.3 to 0.5 ng/mL. In our study if we evaluated only those cows that ovulated at the end of the protocol, there was still a tendency for reduced fertility (>20% reduction) in cows with a small elevation in circulating P4. Thus, other mechanisms, in addition to inhibition of ovulation, are likely to underlie part of the reduction in fertility in cows with slightly elevated P4 near AI. A major problem that we and others (SOUZA et al., 2009;PEREIRA et al., 2013aPEREIRA et al., , 2013b have observed in the E2/P4-based FTAI protocols is that some cows do not ovulate at the end of the protocol. In our study more than 20% of cows did not ovulate at the end of the protocol and none of these cows became pregnant to the FTAI. As discussed above, over half of the cows that did not ovulate at the end of the protocol (13/23 in experiment 2), had elevated P4 that probably inhibited the ECP-induced GnRH/LH surge. In addition, some cows (9/28, both experiments) appeared to have late emergence or no emergence of a follicular wave during the protocol and therefore the dominant follicle was still small at the time of ECP treatment. A previous study in lactating Holstein dairy cows reported that the dominant follicle did not acquire ovulatory capacity until it reached a diameter of more than 10.0 mm (SARTORI et al., 2001). One focus in future studies to improve E2/P4-based FTAI protocols should be to increase ovulation at the end of the protocol. One surprising observation was that the percentage of cows with synchronized wave emergence and ovulation of a single follicle was below 50% in both of our experiments. One reason was that about 20% of cows did not have a new follicular wave and another 20% of cows did not have ovulation at the end of the protocol, as discussed above. However, the multiple ovulation rate was also surprisingly high in these studies, particularly in experiment 1. (KINSEL et al., 1998;DEL RIO et al., 2007). In conclusion, although FTAI protocols using EB and P4 produce satisfactory results for lactating dairy cows, they still need improvements to provide a more consistent wave emergence synchronization, higher circulating P4 during the protocol, successful induction of complete luteolysis, as well as higher ovulation rates. Presynchronization with GnRH or increasing the dose of EB to 3 mg were not effective in providing those improvements. Theriogenology, Stoneham, v. 76, p. 1568Stoneham, v. 76, p. -1582Stoneham, v. 76, p. , 2011. SUBJECTED TO AI OR ET Abstract In order to evaluate the effect of progesterone (P4) supplementation starting during metestrus on the formation of the corpus luteum (CL), and on the fertility of lactating dairy cows subjected to fixed time artificial insemination (FTAI) or embryo transfer (ET) were carried out three experiments. In Exp 1, 42 Holstein cows were allocated randomly to untreated (Control), or a controlled internal drug release implant containing 1.9 g of P4 from d 3 to 20 after FTAI (CIDR). The FTAI protocol consisted of placing a CIDR and administering 2 mg estradiol benzoate on d -10. On d -3, 25 mg dinoprost (PGF) and on d-2, 1 mg estradiol cypionate with CIDR removal. Blood samples were collected on d 3, 4, 7, 11, 14, 17, 20 and 21 for plasma concentration of P4 by RIA. Ultrasound scans were performed at d 4, 7, 11, 14 and 20 to calculate the CL volume. In Exp 2, 668 Holstein and crossbred cows were subjected to FTAI and allocated randomly to untreated (AI-Control) or to receive a CIDR from d 3 to 17 (AI-CIDR) after FTAI. In Exp 3, 360 Holstein cows were treated with PGF and after heat detection (d 0), they were allocated to untreated (ET-Control), or to receive a CIDR from d 4 ± 1 to 8 ± 1 (ET-CIDR-4) or a CIDR from 4 ± 1 to 18 ± 1 (ET-CIDR-14). In vitro-produced embryos were transferred on d 8 ± 1. Pregnancy diagnoses were performed by ultrasound. In Exp 1 there was interaction between treatment and day in relation to plasma P4 on d 4 and d 7 due to supplementation. Independent of treatment, pregnant cows had higher plasma P4 from d 14 to d 21. Supplementation did not seem to compromise CL development. In Exp 2, there was no effect of supplementation of P4 on pregnancy per AI (P/AI) on d 32 (32.0% vs. 31.8%, for AI-Control and AI-CIDR, respectively) or pregnancy loss (15.6% vs. 17.6%, for AI-Control and AI-CIDR, respectively). In Exp 3, there was no interaction between treatment and day. However, P4 supplementation compromised pregnancy/ET (P/ET) on d 32 in both supplemented groups (39.7% vs. 21.3% vs. 15.2% for ET-Control, ET-CIDR-4, and ET-CIDR-14, respectively), with no effect on pregnancy loss. Therefore, although CIDR insertion on d 3 after FTAI did not affect CL function and increased circulating P4, it did not increase P/AI in lactating dairy cows submitted to FTAI. Moreover, P4 supplementation decreased P/ET in lactating recipient cows. Introduction A decline on fertility of dairy cows has been observed on the last 40 years, in terms of increased number of artificial insemination (AI) per pregnant cow (LUCY, 2001;WASHBURN et al., 2002). This was caused by several factors, such as changes in the physiology related to higher milk production. For example, dairy cows have lesser duration and events of estrus (LOPEZ; SATTER; WILTBANK, 2004). One feature on the high-producing lactating dairy cow is high dry matter intake (HARRISON et al., 1990). Dry matter intake is negatively correlated with plasma concentration of progesterone (P4) and estradiol (E2) (SANGSRITAVONG et al., 2002), due to increased liver blood flow SANGSRITAVONG et al., 2002). Nevertheless, to improve reproductive efficiency in dairy cattle, management programs have been adopted, such as fixed time AI (FTAI), that has been used wide world (BISINOTTO; RIBEIRO; SANTOS, 2014). Moreover, and in spite of the negative effects of the high milk production and heat stress on the oocyte quality (SARTORI; BASTOS; WILTBANK, 2010), some studies have shown increased fertility when embryo transfer (ET) in dairy cattle was used in substitution to AI (DEMETRIO et al., 2007;STEWART et al., 2011). In lactating dairy cows, high circulating P4 during follicle development and after AI (PARR et al., 2012) has been shown to be associated with increased fertility. Supplemental P4 has been used before and after AI (WILTBANK et al., 1956;ROSS;FOURT, 1958;LARSON;BUTLER;CURRIE, 2007;MONTEIRO et al., 2014) either by the use of intravaginal P4 implants (WILTBANK et al., 1956;JOHNSON et al., 1958;LARSON et al., 2007;MONTEIRO et al., 2014) or by induction of accessory corpus luteum (CL) (SANTOS et al., 2001;NASCIMENTO et al., 2013). This strategy has been associated with increased pregnancy/AI (P/AI). Additionally, cows subjected to P4 The objectives of the present study were to investigate the effects of P4 supplementation, post ovulation, on CL function, on P/AI and on pregnancy/ET (P/ET). Therefore, three experiments were conducted to test the hypotheses that P4 supplementation 3 d after FTAI do not interfere with CL development and function, and increases fertility in dairy cows. In addition, we hypothesized that P4 supplementation 4 d before ET improves P/ET. Materials and Methods All procedures with cows were approved by the Ethics Committee for Animal Use of the University of São Paulo/ESALQ. Cows and Housing This study was conducted on a dairy farm in southeastern region of Brazil using 64 lactating Holstein cows. Primiparous (n = 27) and multiparous (n = 37) cows (35.3 ± 10.60 kg/d of milk, 170.6 ± 180.97 DIM, and body condition score [BCS] of 2.71 ± 0.30 [mean ± standard deviation]) were enrolled in this experiment. Cows were housed in free-stall barns, milked three times daily, and fed TMR typical of high producing herds. This experiment was conducted during the end of winter and beginning of spring (August and September). Experimental Design, Ultrasonography Evaluation and Treatments All cows were synchronized and received FTAI consisted of 2.0 mg, im, of estradiol benzoate (EB, Gonadiol, MSD Saúde Animal, São Paulo, Brazil) associated with a controlled internal drug release (CIDR, Zoetis, São Paulo, Brazil) insert containing 1.9 g of P4 (d -10). Samples were centrifuged (1,700 x g) for 15 min, and plasma were harvested and stored at -20 °C until assayed for P4 using a solid-phase, no-extraction radioimmunoassay (Coat-a-Count Progesterone, Diagnostic Products Corp., Los Angeles, CA). Only a single assay was performed and the intra-assay CV was 4.2%. Pregnancy Diagnosis Thirty-one d after FTAI, pregnancy diagnoses were performed by ultrasound (DP-2200, Experimental Design and Treatments On all farms, cows were synchronized and received FTAI consisted of 2.0 mg of EB, im, associated to a CIDR (d -10). Seven d later (d -3) cows received PGF, im, and 1.5 d after (d -1.5), cows received 1.0 mg of EB, im, associated to a second dose of PGF, im, and CIDR was removed. All cows were inseminated on d 0 (Figure 3.2). Pregnancy Diagnosis In all farms, 32 d after FTAI, pregnancy diagnoses were performed by transrectal ultrasound (DP-2200, Mindray). Cows with embryonic vesicle were eligible to be pregnant. All pregnant cows were reconfirmed at 60 d after FTAI on all three farms by transrectal palpation of the uterus. Cows and Housing Three-hundred and sixty lactating Holstein cows (28.9 ± 10.11 kg/d of milk and 206.8 ± 141.19 DIM [mean ± SD]), primiparous (n = 184) and multiparous (n = 176), milked three times a day, from a dairy farm in the southeastern region of Brazil were assigned in this study from June to December. Cows were housed in free-stall barns and fed a TMR containing corn silage, finely ground corn, soybean meal, whole cottonseed, minerals and vitamins. Donors and Ovum Pick-Up One hundred eighty five lactating dairy cows were subjected to ovum pick-up (OPU) sessions in the morning of d 0 of the recipients' protocol and had their perineal area cleaned with water and 70% ethanol. Epidural anesthesia was performed with 5 mL of 2% lidocaine hydrochloride (Xylestesin, Cristália, Itapira, Brazil) to facilitate the handling of the ovaries through the rectum. All follicle greater than 2.0 mm were aspirated by ultrasound (SSD 500, Female sex-sorted semen were thawed for 30 s at 35 °C and deposited on a 90% to 45% Percoll gradient prepared with sperm wash medium (modified Tyrode medium) and centrifuged at 320 x g for 30 min to separate the motile sperm. Sperm pellet was evaluated for motility and concentration. The final concentration used was 1.0 x 10 6 live sperm/mL, and each fertilization droplet received 5 µL (5,000 sperm cells). Cumulus oocyte complexes and sperm were incubated at 38.5 °C in an atmosphere of 5% CO2 for 18 to 20 h. After 18 h of the insemination, presumptive zygotes had their cumulus cells removed by mechanical pipetting and were cultured to 100 µL drops of embryo culture medium (Synthetic Oviduct Fluid containing 8 mg/mL bovine albumin serum and 1 mM glutamine) under 38.5 °C in an atmosphere of 5% CO2 for 18 to 20 h. Until transfer, the embryos were kept under these conditions. Experimental Design, Recipient's Protocol and Treatments Recipient cows were synchronized with a single dose of PGF, im, 11 d before ET, and only cows that showed estrus signs between 3 and 5 d after PGF (d 0 to d -2) received ET. Therefore, these cows were on d 7, d 8 or d 9 of the estrous cycle. Four d before ET, cows were randomly assigned to one of three treatments: ET-Control (n = 132), cows were not supplemented with P4; ET-CIDR-4 (n = 119), cows received a CIDR 4 days before ET and CIDR was removed at the time of ET, 4 d of P4 supplementation; or ET-CIDR-14 (n = 109) cows received a single CIDR 4 d before ET until 10 d after ET, 14 d of P4 supplementation (Figure 3.3). Prior to ET, recipient cows received an epidural anesthesia with 5 mL of 2% lidocaine hydrochloride and fresh embryos were transferred nonsurgically into the uterine horn ipsilateral to the CL. Cows that showed estrus on d 0 to d 2 were randomly assigned on the study into three treatments, ET-Control (n = 132), cows did not receive a controlled internal drugrelease (CIDR) implant containing 1.9 g of progesterone (P4); ET-CIDR-4 (n = 119), cows received a CIDR from d 4 ± 1 until the day of the embryo transfer (ET); or ET-CIDR-14 (n = 109), cows received a CIDR from d 4 ± 1 until 18 ± 1. All recipient cows received embryo on d 8 ± 1 Pregnancy Diagnosis After 25 Categorical data of experiments 2 and 3 were analyzed using the GLIMMIX procedure of SAS fitting a binary distribution. For experiment 2, the model included the fixed effects of treatment, parity, breed categorized (Holstein or crossbred Holstein x Gyr), farm, sire, DIM categorized as above or below the mean value, milk yield categorized during the week of d 0 as above or below the mean value, and interaction between parity and treatment, as well as the random effects of cows. For experiment 3, the model included the fixed effects of treatment, parity, DIM categorized as above or below the mean value, milk yield categorized during the week of d 0 as above or below the mean value, day of estrous cycle of recipients, d 7, d 8, or d 9, and interaction between treatment and day of estrous cycles, as well as the random effects of cows. Only variables with P < 0.20 were kept in the final model, unless the variable was essential, for example treatment, day, day of estrous cycle (experiment 3), interaction between treatment and day (experiment 1). The Kenward-Roger method was used to calculate the denominator degrees of freedom to approximate the F test in the mixed models. Differences with P ≤ 0.05 were considered significant and those with 0.05 < P ≤ 0.10 were considered tendencies. Results are shown as least squares means and standard error. Mean concentrations of P4 was similar between the two treatment groups throughout the experimental period (P = 0.14), but there were effects of day (P < 0.001) and interaction among treatment x day (P < 0.001; Figure 4A). Supplemented cows had concentration of P4 on d 4 (P < 0.001) and d 7 (P < 0.01) after FTAI higher than cows that did not receive supplementation. For the remainder days, the concentrations of P4 did not differ between treatments. As anticipated, concentrations of P4 did not differ between treatments on d 3. (Figure 3.4A). Inclusion of a CIDR on d 3 after FTAI increased concentrations of There was not effect of treatment (P = 0.66) and interaction between treatment and day (P = 0.31), but independent of treatment, there was effect of day on the CL volume (P < 0.001; Figure 4B). The CL on d 4 was smaller than on the other days. No effect (P = 0.19) was observed of pregnancy status on CL volume (Figure 3.4B). Considering only ovulated cows, the P/AI on d 31 ( with supplemented primiparous or multiparous cows. There was no effect of treatment or interaction between treatment and parity on P/AI at d 60 (Table 1). There was also no effect of treatment ( for AI-Control and AI-CIDR, respectively) on pregnancy loss between d 32 and d 60, but there was a tendency (P = 0.08) for interaction between treatment and parity. Supplemented primiparous cows tended to present higher pregnancy loss than supplemented multiparous cows (Table 1). (○) cows did not receive a CIDR and were not pregnant on d 31 after FTAI (n = 11); (▲) cows received a CIDR from d 3 to 20 after FTAI and were pregnant on d 31 after FTAI (n = 14); (∆) cows received a CIDR from d 3 to 20 after FTAI and were not pregnant on d 31 after FTAI (n = 7). Only ovulated cows were enrolled on the experiment. There was no effect of treatment (P = 0.14), but there was effect of day (P < 0.001) and interaction between treatment and day (P < 0.01) on the concentration of P4. There was no effect of treatment (P = 0.66) and no interaction between treatment and day (P = 0.31), but there was effect of day (P < 0.001). *Within a day, effect of treatment on the concentration of P4. δ Within a day, effect of pregnancy status on the concentration of P4 Experiment 3 Both supplemented groups, ET-CIDR-4 and ET-CIDR-14, had lower proportion of pregnant cows on d 32 (P < 0.001) and d 88 (P = 0.001) than ET-Control. However, pregnancy loss between d 32 and d 88 did not differ among treatments (Table 3). There was no interaction between treatment and parity on P/ET on d 32 and d 88, neither on pregnancy loss. Although other studies (WILTBANK et al., 1956;JOHNSON et al., 1958;SANTOS et al., 2001;LARSON et al., 2007) have shown benefits of supplemental P4 after AI, in the present study, no effect was observed on the fertility of dairy cows. In fact, an interaction and a tendency for interaction between supplementation of P4 and parity was observed on the P/AI at d 32 and pregnancy loss, respectively. Cows supplemented with P4 after ovulation had an improvement on embryo development O'HARA et al., 2014), and although nonpregnant cows presented a shortened estrous cycle than not supplemented cows, there was a positive effect of P4 supplementation at d 4 after FTAI on P/AI (MONTEIRO et al., 2014). Additionally, when pregnant primiparous cows were compared to pregnant multiparous cows, they had greater expression of stimulated IFN-τ genes on d 19 than multiparous . This is an indicative that the IFN-τ secreted by the elongating conceptus may inhibit the luteolytic mechanism (SPENCER et al., 1995) in primiparous cows because it happens before -probably some hours or a day before -than in multiparous cows. Nevertheless, due to the advancement of luteolytic mechanism in supplemented cows (LAWSON;CAHILL, 1983;MONTEIRO et al., 2014), probably, in multiparous cows, luteolysis had started before the time of maternal recognition of pregnancy. On the other hand, in primiparous cows, the embryo had time to inhibit luteolysis. However, a tendency observed on the unexpected increased pregnancy loss in P4-supplemented primiparous cows resulted in similar P/AI on d 60 among groups. Although IVP embryo is a model widely used to study the effect of P4 supplementation on embryo development (MANN et al., 2006;LONERGAN et al., 2007;CARTER et al., 2008;, in our study supplementation of P4 for lactating recipient dairy cows had negative effect on fertility. Recipients supplemented with P4 had 50% or less P/ET than the control group. The exact mechanism related to P4 supplementation and decreased fertility in recipients is still unknown, but we believe that this may be associated with a delayed expression of IFN-τ of in vitro as compared with in vivo-produced embryos (BERTOLINI et al., 2002). Similarly to what happened with FTAI multiparouscows, embryo recipient dairy cows may have had luteolysis before enough IFN-τ secretion by the embryo. An alternative hypothesis for the decreased fertility in P4-supplemented embryo recipients is vaginitis caused due to the presence of the intravaginal P4 device at the time of embryo transfer, potentially resulting in uterine contamination. In conclusion, although supplementation of P4 to lactating dairy cows with intravaginal inserts post ovulation increased the concentration of P4 on d 4 and d 7 without compromising CL volume and circulating P4, it did not improve P/AI in cows subjected to FTAI. Moreover, P4 supplementation to embryo recipient lactating dairy cows decreased fertility. Champaign, v. 39, p. 456-461, 1956. Abstract Objectives were to evaluate the effects of supplemental progesterone after artificial insemination (AI) on expression of interferon-stimulated genes (ISG) in blood leukocytes and fertility in lactating dairy cows. Weekly cohorts of Holstein cows were blocked by parity (575 primiparous and 923 multiparous) and method of insemination as timed AI or AI on estrus and allocated randomly within each block to untreated controls, a controlled-internal drug release (CIDR) containing 1.38 g of progesterone from d 4 to 18 after AI (CIDR4), or a CIDR on d 4 and another on d 7 after AI and both removed on d 18 (CIDR4+7). Blood was sampled to quantify progesterone concentrations in plasma and mRNA expression in of leukocytes for the ubiquitin-like IFN-stimulated gene 15-kDa protein (ISG15) and receptor transporter protein-4 (RTP4) genes. Pregnancy was diagnosed on d 34 ± 3 and 62 ± 3 after AI. Treatment increased progesterone concentrations between d 5 and 18 after AI in a dose-dependent manner (control = 3.42, CIDR4 = 4.97, and CIDR4+7 = 5.46 ng/mL). Cows supplemented with progesterone tended to have increased luteolysis by d 19 after AI (control = 17.2 vs. CIDR4 = 29.1 vs. CIDR4+7 = 30.2%), which resulted in a shorter AI interval for those reinseminated after study d 18. Pregnancy upregulated expression of ISG in leukocytes on d 19 of gestation, but supplementing progesterone did not increase mRNA abundance for ISG15 and RTP4 on d 16 after insemination and tended to reduce mRNA expression on d 19 after AI. For RTP4 on d 19, the negative effect of supplemental progesterone was observed only in the nonpregnant cows. No overall effect of treatment was observed on P/AI on d 62 after insemination and averaged 28.6, 32.7, and 29.5% for control, CIDR4 and CIDR4+7, respectively. Interestingly, an interaction between level of supplemental progesterone and method of AI was observed for P/AI. For cows receiving exogenous progesterone, the lower supplementation with CIDR4 increased P/AI on d 62 in cows inseminated following timed AI (CIDR4 = 39.2 vs. CIDR4+7 = 27.5%) but, in those inseminated following detection of estrus, the use of a second insert on d 7 resulted in greater P/AI (CIDR4 = 26.9 vs. CIDR4+7 = 31.5%). Pregnancy loss did not differ among treatments. Supplemental progesterone post-AI using a single intravaginal insert on d 4 was beneficial to pregnancy in cows inseminated following timed AI, but incremental progesterone with a second insert on d 7 did not improve fertility of dairy cows. Introduction Progesterone is pivotal for successful pregnancy in ruminants (SPENCER et al., 2007), and lactating dairy cows are known to have reduced systemic concentrations of progesterone during diestrus compared with dairy heifers . It is thought that inadequate progesterone concentrations during early development of the conceptus might be one of the reasons for reduced fertility observed in high-producing dairy cows (WILTBANK et al., 2011), in part because catabolism of steroids increases with increased feed intake associated with high production WILTBANK et al., 2011). In fact, supplemental progesterone after AI from an exogenous source (STEVENSON et al., 2007;WILTBANK et al., 2011) resulted in small increases in pregnancy per AI (P/AI). Compared with untreated controls, beef heifers treated with exogenous progesterone starting on d 3 of the estrous cycle had a larger conceptus on d 17 (CARTER et al., 2008. Timed AI protocols result in variable sizes of ovulatory follicles in dairy cows SANTOS et al., 2010). Induction of ovulation of small follicles resulted in reduced P/AI in beef (PERRY et al., 2005) and dairy cows , and increased risk of pregnancy loss (PERRY et al., 2005). Embryo quality in cows synchronized for timed AI is of equal or better quality than that of cows inseminated after detected estrus (CERRI et al., 2009b); however, cows subjected to timed AI protocols that are induced to ovulate small follicles have a small resulting corpus luteum (CL) with reduced ability to increase peripheral concentrations of progesterone (VASCONCELOS et al., 2001), thereby potentially reducing P/AI (PARR et al., 2012). Therefore, benefits from progesterone supplementation might be greater in cows inseminated following timed AI programs. Interferon-τ secreted by the trophectoderm of the conceptus is the main signal for pregnancy recognition, initiating the process to block the luteolytic cascade and preventing the demise of the CL (MEYER et al., 1995). In the bovine conceptus, mRNA for IFN-τ is detected in trophoblast on d 12 of gestation with peaks occurring between d 15 and 16 (FARIN et al., 1990). Interferon gene mRNA expression in conceptuses is activated with developmental stage of the blastocyst, and progesterone plays a pivotal role in stimulating conceptus development in utero . In general, it is thought that stimulation of embryo development by an early rise in progesterone should benefit fertility (STRONGE et al., 2005;PARR et al., 2012), possibly because of advancing conceptus development. Nevertheless, fertility responses to exogenous progesterone seem to be greater when supplementation occurs before than after (STEVENSON et al., 2007) AI. In most studies with post-AI progesterone supplementation to lactating dairy cows no apparent attempt was made to mimic the normal rise in progesterone observed in heifers, which is greater and faster than that of cows ). An exception is the recent work by Nascimento et al. (2013) in which lactating dairy cows receiving and injection of 3,300 IU of human chorionic gonadotropin (hCG) on d 5 concurrent with insertion of a controlled internal drug release containing progesterone had progesterone profiles during diestrus similar to those of dairy heifers. The authors speculated that such manipulation that mimics the progesterone concentrations in heifers might benefit fertility of lactating dairy cows. Therefore, it is possible that the limited benefit to post-AI progesterone supplementation on pregnancy might be the result of insufficient supplementation or inability to mimic the continuous rise and incremental difference during diestrus between groups known to have low (lactating cows) and those of high fertility (heifers). Interferon-τ binds type I IFN receptor (ROBERTS et al., 1999), which leads to downregulation of oxytocin receptor expression on superficial glandular and luminal epithelia in sheep (ROBERTS et al., 1999;SPENCER et al., 2007), and this mechanism is thought to be similar among all domestic ruminants. The down-regulation of oxytocin receptors ultimately inhibits pulsatile release of PGF2α responsible for the demise of the CL (MEYER et al., 1995;SPENCER et al., 2007). Production of IFN-τ by the conceptus induces IFN-stimulated genes (ISG) in the endometrium such as myxovirus (influenza virus) resistance 1 (Mx1) and receptor transporter protein 4 (RTP4; HICKS et al., 2003;GIFFORD et al., 2008). Blood leukocytes harvested on d 16 or 19 after AI had increased expression of the ISG ubiquitin-like IFNstimulated gene 15-kDa protein (IGS15), Mx1, Mx2, and RTP4 (GIFFORD et al., 2007;, and leukocyte mRNA for ISG was correlated with the amount of IFNτ in the uterus (MATSUYAMA et al., 2012). Interestingly, stimulation of conceptus development during pre-and peri-implantation resulted in increased expression of ISG in blood leukocytes and increased P/AI in lactating dairy cows . The main hypothesis of the present study was that supplemental progesterone starting during metestrus would improve P/AI in dairy cows, particularly in those synchronized for timed AI. It was thought that supplemental progesterone would increase concentrations of progesterone in plasma in a dose-dependent manner, which would stimulate mRNA abundance for ISG in peripheral blood leukocytes, as a measure of improved embryonic-maternal crosstalk, thereby supporting improved pregnancy. Therefore, the main objective of the present study was to investigate the effects of supplemental progesterone starting during metestrus on P/AI when cows are inseminated following detected estrus or timed AI. Additional objectives were to characterize concentrations of progesterone in plasma, luteal lifespan, and abundance of mRNA for ISG in leukocytes in lactating dairy cows supplemented with progesterone. Materials and methods All procedures involving animals in this study were approved by the University of Florida Non-Regulatory Animal Research Committee. Cows, Housing and Diets The study was conducted on a dairy farm in Central Florida milking 5,400 cows with a yearly rolling herd average milk yield of 10,700 kg during the study period. Weekly cohorts of cows were enrolled during 7 consecutive weeks and all inseminations were performed from March 25 to May 9, 2013. Primiparous (n = 575) and multiparous (n = 923) cows were housed separately in free-stall barns equipped with sprinklers and fans. Cows received the same TMR to meet or exceed the nutrient requirements for a lactating Holstein cow producing 45 kg of milk per d with 3.5% fat and 3.1% true protein when DM intake is 24 kg/d (NRC, 2001). Diet consisted of rye grass silage, corn silage, corn earlage, ground corn, citrus pulp, solvent extracted soybean meal, expeller soybean meal, corn gluten feed, molasses, minerals and vitamins. Cows were fed twice and milked thrice daily. Sample Size and Experimental Design The sample size was calculated using the MINITAB statistical software ver. 16 using a one-sided test to provide sufficient experimental units to detect statistical significance (α = 0.05; β = 0.20) when P/AI increased 6 percentage units (e.g. 30 vs. 36%) with supplemental progesterone. Approximately 488 cows per treatment were deemed necessary, or a total of 1,464. Because of potential attrition during the study, a total of 1,498 were randomly assigned to treatments in a randomized complete block design. Two blocking criteria were used before randomization to treatments, method of AI, as detected-estrus or timed AI, and parity, as primiparous or multiparous. Four randomization forms were pre-prepared, one for each combination of blocks: primiparous inseminated at detected estrus, primiparous inseminated at fixed time, multiparous inseminated at detected estrus, and multiparous inseminated at fixed time. Within each form, treatments were randomized within a block containing three cows such that each block had each of the three treatments represented. Cows were enrolled in sequence of availability as they were found in the farm on d 4 after AI in one of the four forms according to the blocking criteria, and day of insemination was considered study d 0. gonadorelin diacetate tetrahydrate equivalent to 43 µg of gonadorelin/mL, Merial Ltd., Duluth, GA], d 74 and 75 PGF2α, d 77 GnRH and timed AI). All cows not returning to estrus spontaneously received in advance an injection of GnRH injection for pre-enrollment for resynchronization on d 29  3 after AI. Cows diagnosed non-pregnant on d 34  3 after AI resumed the 5-d timed AI protocol, and timed AI was performed on d 37  3 d after the previous insemination. Throughout the study, after 57 DIM, cows had their tailheads painted using paintsticks (All-Weather Paintstik; LA-CO Industries Inc., Chicago, IL), and detection of estrus was evaluated daily, in the morning, based on removal of the tail paint. Cows identified in estrus were inseminated on the same morning. Blood Sampling and Analysis of Progesterone in Plasma Blood was sampled from a subset of 20 randomly selected blocks of cows (60 cows), 20 controls, 20 CIDR4, and 20 CIDR4+7 on study d 4, immediately before progesterone administration, and then again in the mornings of d 5, 7, immediately before placement of the second progesterone insert in CIDR4+7,8,11,14,16,18 and 19. A second subset of 60 randomly selected blocks of cows were also sampled, 60 controls, 60 CIDR4, and 60 CIDR4+7 on study d 8, 16, and 19. Blood was sampled by puncture of the coccygeal vein or artery into evacuated tubes containing K2 EDTA (Vacutainer; Becton Dickinson, Franklin Lakes, NJ). Immediately upon collection, tubes with blood were placed in ice and kept refrigerated until transported to the laboratory within 4 to 5 h for processing. Blood tubes were centrifuged at 1,500 x g for 15 min at 4ºC for plasma separation. Aliquots of plasma were frozen at -20ºC until assayed. Concentrations of progesterone were analyzed in plasma by RIA using a commercial kit (Coata-Count, Siemens Healthcare Diagnostics, Los Angeles, CA). Three assays were performed and the sensitivity of assays 1, 2 and 3 was at least 0.05 ng/mL when calculated as 2 SD below the mean counts per minute at maximum binding. Samples with low and moderate concentrations of progesterone, 1.3 and 4.0 ng/mL, respectively, were incorporated into each assay multiple times for quality control and for calculation of intra and inter-assay CV. The intra-assay CV for the low and moderate progesterone samples were, respectively, 8.3 and 3.5% in assay 1, 4.7 and 5.1% in assay 2, and 3.7 and 4.0% in assay 3. The inter-assay CV for the low and moderate samples were 1.1 and 1.4%, respectively. Leukocyte Isolation and mRNA Extraction Blood sampled on d 16 and 19 from 58 blocks of cows from which progesterone concentrations were measured were also used for leukocyte isolation as described by . The pellets of isolated leukocytes were suspended with 0.8 mL of Trizol, transferred to microcentrifuge tubes and stored at -80ºC until RNA extraction. the ABI 7300 Real Time PCR System (Applied Biosystems). After an initial activation at 60 o C for 2 min followed by denaturation at 95 o C for 10 min, the amplification protocol followed 40 cycles of 95°C for 15 sec and 60°C for 1 min. Each sample was evaluated in triplicate, and the specificity for amplification was verified by melting curve analysis. Four genes were investigated (Table 4.1), including the two reference genes, beta-actin (ACTB) and ribosomal protein L19 (RPL19), and two target genes, ISG15 and RTP4. Pregnancy loss was calculated as the number of cows that lost a pregnancy between d 34  3 and 62  3 after AI divided by the number of cows diagnosed pregnant on d 34  3 after AI. Cows that were detected in estrus before pregnancy diagnosis were re-inseminated and considered as non-pregnant. Body Condition Score and Milk Yield The body condition of all cows was scored on study d 4 according to Ferguson et al. (1994) using the Elanco BCS chart (Elanco, 2009). For statistical analysis, BCS was categorized as low, when BCS ≤ 2.75, or moderate, when BCS ≥ 3.00. Yields of milk were recorded for individual cows once monthly using on-farm milk meters (Tru-Test Ltd., Manukau, New Zealand). The production on the month of insemination was categorized as above or below the mean milk yield within primiparous and within multiparous cows in the study and were included in the statistical models for data analyses. Statistical Analysis Categorical data were analyzed by logistic regression using the GLIMMIX procedure of SAS version 9.3 (SAS/STAT, SAS Institute Inc., Cary, NC) fitting a binary distribution. The models included the fixed effects of treatment, parity, type of AI, BCS category, number of AI (first AI vs. resynchronized AI), categorized milk yield within parity in the month of AI as above or below the mean value, and the interactions between treatment and parity, treatment and type of AI, and treatment and number of AI, and the random effect of block. For P/AI and pregnancy loss, the fixed effects of sire and technician were also included in the models. The Kenward-Roger method was used to calculate the denominator degrees of freedom to approximate the F tests in the mixed models. Model fitting was evaluated using the fit statistics. The estimates were back-transformed using the ILINK function of SAS to generate the adjusted proportions. An additional analysis of pregnancy on d 34 was performed in the 240 cows with progesterone concentration on d 8 after AI to model the effect of progesterone concentration on P/AI. The model included the fixed effects of treatment, progesterone concentration on d 8 as a linear or quadratic term, and the random effect of block. The logistic function was used to model the probability of pregnancy as a function of progesterone concentration. Continuous data with repeated measures over time were analyzed using the GLIMMIX procedure of SAS with models fitting a Gaussian distribution. Data were tested for normality of residuals, and data with residuals not normally distributed were transformed before analysis. The models included the fixed effects of treatment, day of measurement, parity, type of AI, interactions between treatment and day, treatment and parity, treatment and type of AI, and treatment and number of AI, and the random effects of cows nested within treatment and block. The effect of pregnancy on d 34 and the interaction between treatment and pregnancy on d 34 were also included for the analysis of progesterone concentrations in plasma. When the F-test for an interaction was significant, means were then partitioned using the SLICE command in SAS. The covariance structure that resulted in the smallest Akaike's information criterion was selected for the model. When time intervals between measurements were unequal, then the spatial power covariance structure was used. Model fitting was evaluated using the fit statistics. Quantitative PCR data are presented using the comparative method developed by Livak and Schmittgen (2001) using nonpregnant cows from the control group as the reference for relative expression of mRNA abundance, which was set to the relative value of 1. The delta cycle threshold (∆CT) values for each target gene were obtained after normalization of CT value of the gene with the geometric mean of CT values from the two reference genes according to Vandesompele et al. (2002). Data were analyzed using the ΔCT for d 16 or d 19 with the Mixed procedure of SAS fitting a model with the fixed effects of treatment, pregnancy on d 34, and the interaction between treatment and pregnancy on d 34, and the random effect of block. The ∆∆CT were obtained from ∆CT LSM differences of pairwise comparisons among treatments and the reference group control nonpregnant cows (YUAN et al., 2006). The relative expression values were obtained by raising the PCR amplification efficiency (E = 2) to the power ∆∆CT (YUAN et al., 2006). Confidence limits for graphical representation of relative expression were generated from the lower and upper CI obtained for ∆CT LSM differences as described by Yuan et al. (2006). Orthogonal comparisons were used to determine the effects of supplementing progesterone with CIDR (control vs. CIDR4 + CIDR4+7) and the effects of amount of progesterone supplemented (CIDR4 vs. CIDR4+7). Contrasts for the interactions between type of AI and supplemental progesterone or the amount of supplemental progesterone were also tested. Differences with P ≤ 0.05 were considered significant and those with 0.05 < P ≤ 0.10 were considered tendencies. Results Of the 504 CIDR4 cows, 19 lost the insert before d 18 of the study (3.8%). Of the 495 CIDR4+7 cows, 28 lost at least 1 insert (5.6%), of which 20 lost a single insert (4.0%) and 8 lost both inserts (1.6%) before d 18. Twelve of the 1,498 initially enrolled cows were excluded from the data analyses (4 control, 4 CIDR4, 4 CIDR4+7) because of errors during treatment administration or because they received another AI on study d 4, concurrent with treatment administration. Another 18 cows were excluded from the analysis of P/AI because they either died or were sold before the day of pregnancy diagnosis. Therefore, of the initial 1,498 cows, 1,468 were used for statistical analyses of the data. Milk yield on the month of enrollment did not differ (P = 0.47) among treatments and averaged 38.3 ± 0.5, 39.0 ± 0.5, and 38.2 ± 0.5 kg/d for control, CIDR4, and CIDR4+7, respectively. Multiparous cows had greater (P < 0.01) milk production than primiparous cows (41.9 ± 0.4 vs. 35.1 ± 0.5 kg/d). No difference in milk yield was observed for cows inseminated in estrus or timed AI, and averaged 38.5 kg/d. The DIM at AI for all cows in the study did not differ (P = 0.66) among treatments and averaged 114.7 ± 2.1, 114.9 ± 2.1, and 117.0 ± 2.1 for control, CIDR4, and CIDR4+7. The proportions of control, CIDR4, and CIDR4+7 cows receiving first and resynchronized AI, respectively, were 32. 5 and 67.5, 35.5 and 64.5, and 34.7 and 65.4%. The mean and median numbers of AI for cows enrolled in the study were 2.7 ± 0.1 and 2.0, and both did not differ among treatments. The median BCS of cows in the study was less (P = 0.04) for control than CIDR4 and CIDR4+7 (2.75 vs. 3.00 vs. 3.00). Because of this difference in median BCS, a tendency (P = 0.08) was observed for more control cows to have BCS < 3.00 compared with cows receiving CIDR4 and CIDR4+7 (50.7 vs. 43.9 vs. 46.8%). Concentrations of Progesterone and Luteolysis by d 19 Mean concentrations of progesterone in the 20 blocks of cows sampled throughout the treatment period increased (P < 0.02) with supplemental progesterone, but only a numerical increase was observed between means for the CIDR4 and CIDR4+7 (Figure 4. Supplementing progesterone tended (P = 0.07) to increase the incidence of luteolysis on d 19 after AI (Table 4.2). In fact, from all nonpregnant cows on d 34, a greater (P = 0.01) proportion of progesterone-supplemented cows had undergone luteolysis by d 19. Level of progesterone supplementation did not affect the risk of luteolysis by d 19 after AI. Method of AI or interaction between treatment and method of AI did not influence the risk of luteal regression by d 19 (Table 4.3). Re-insemination in Estrus, Pregnancy per AI, and Pregnancy Loss The proportion of cows reinseminated on estrus before pregnancy diagnosis on d 34 was not affected by supplemental progesterone, but it was greater (P = 0.04) for CIDR4+7 than CIDR4 (Table 4.2). When all nonpregnant cows were considered, including those reinseminated on or before d 18, then the interval between pre-enrollment and post-enrollment AI tended (P = 0.06) to be longer for cows supplemented with progesterone, but there was no effect of level of progesterone supplementation. With the exception of 3 cows in CIDR4 that returned to estrus on or before d 18, the CIDR, as expected, prevented cows from returning to estrus until the removal of the inserts (Figure 4.5). One cow in CIDR4 lost the CIDR and returned to estrus on d 15, whereas the other two had the CIDR when detected in estrus, one on d 13 and another on d 18. Interestingly, when only cows inseminated after d 18 were considered, which coincided with the removal of the intravaginal inserts in CIDR4 and CIDR4+7, then interval between AI was longer (P = 0.02) for control cows than for cows supplemented with progesterone. Pregnancy per AI on d 34 and 62 after insemination did not differ with supplemental progesterone or level of supplementation (Table 4.2). However, an interaction (P < 0.02) between level of supplemental progesterone and method of AI was observed for P/AI on d 34 and 62 (Table 4.3). For cows inseminated following detection of estrus, P/AI increased with administration of two intravaginal inserts in CIDR4+7 compared with CIDR4; however, for those inseminated following timed AI, administering a single insert with CIDR4 increased P/AI. For cows inseminated following timed AI, treatment with a single progesterone insert improved P/AI compared with control or CIDR4+7 (Table 4.3). When data were analyzed with the 240 cows in which plasma was quantified for concentrations of progesterone on d 8 after AI, a quadratic relationship (P = 0.08) was observed between progesterone concentration and the probability of pregnancy on d 34. The same analysis was performed separately for cows inseminated after detected estrus or following timed AI (Figure 4.6). For cows inseminated at detected estrus, the relationship was quadratic (P = 0.09); however, for cows receiving timed AI the same relationship was linear (P < 0.001). Milk yield or BCS were not associated with P/AI on d 34 or 62 after insemination. (Table 4.2). Similarly, no interaction was observed between method of AI and supplemental progesterone or amount of progesterone supplemented for pregnancy loss (Table 4.3). Cows inseminated following detection of estrus tended (P = 0.10) to have increased pregnancy loss compared with those Discussion The main hypothesis of the present study was that progesterone rise after insemination was an important limiting factor for pregnancy in lactating dairy cows. Supplementing progesterone to mimic concentrations that typically are observed in dairy heifers, which were anticipated to length exposure of the conceptus and endometrium to concentrations compatible An additional hypothesis was that the beneficial effects of supplementing progesterone on fertility would be exacerbated in cows inseminated following timed AI. Although properly implemented timed AI protocols result in good to excellent embryo quality (CERRI et al., 2009b), synchronizing ovulation also results in variable size ovulatory follicles SANTOS et al., 2010), and inducing ovulation of small follicles reduces concentrations of progesterone during the subsequent diestrus (VASCONCELOS et al., 2001) and P/AI (PERRY et al., 2005;SOUZA et al., 2007). No interaction between supplemental progesterone and method of AI was observed, indicating that exogenous progesterone as CIDR4 and CIDR4+7 did not have a differential effect on pregnancy in cows inseminated either on estrus or following timed AI; however, the single insert on d 4 benefited pregnancy in cows inseminated following timed AI. In fact, when only supplemented cows were considered, an interaction between level of progesterone supplementation and method of AI was observed. Within estrus-detected cows, addition of a second progesterone insert with CIDR4+7 numerically increased P/AI, but for timed AI cows, the single insert in CIDR4 resulted in greater P/AI than using two sequential inserts in CIDR4+7. Perhaps, cows inseminated on detected estrus had less incidence of multiple ovulation and had some benefit from increased progesterone supplementation. On the other hand, 25 to 30% of the cows inseminated following timed AI develop the ovulatory follicle under low concentrations of progesterone , which results in more multiple ovulations and, perhaps, less need for larger amounts of supplemental progesterone. Cows inseminated following timed AI or after detected estrus have many distinct physiological differences, including expression of estrus and size of the ovulatory follicle, and it is possible that they are differentially responsive to two methods of progesterone supplementation used in the current study. In general, increments in progesterone concentrations in early diestrus are linked with improved P/AI (PARR et al., 2012;STRONGE et al., 2005). In the current study, when all cows were analyzed together, and a quadratic association between progesterone on d 8 and pregnancy was observed in the current study, as suggested by others in dairy heifers (PARR et al., 2012). Nonetheless, the relationship between progesterone and pregnancy differed slightly when data from cows inseminated on estrus were analyzed separately from those of cows subjected to fixed time AI. For cows inseminated following estrus, a positive quadratic relationship was observed indicating that as progesterone increased, so did P/AI, but the benefit declined as concentrations became very high. On the other hand, for timed AI cows the relationship was linear indicating that the incremental benefits of increasing progesterone during diestrus to P/AI were similar at all ranges of progesterone concentrations observed. The rationale for supplementing progesterone starting on d 4 after AI was based on the results from Mann and Lamming (1999) that observed improved P/AI when supplementation initiated during late metestrus or early diestrus, but not after d 6 post-AI. It is thought that lactation, with associated increases in DM intake, enhances the catabolism of progesterone by the splanchnic tissues, primarily the liver WILTBANK et al., 2011). A reduction in progesterone could limit proper uterine priming and conceptus development (CARTER et al., 2008;, thereby reducing P/AI in dairy cows (PARR et al., 2012;STRONGE et al., 2005). Dairy heifers, which are known to have high fertility, have a steeper rise in post-ovulation progesterone concentrations starting on d 4 after AI compared with lactating dairy cows , and the differences in concentrations approximate 2 ng/mL starting on d 7 after AI. Nascimento et al. (2013) demonstrated that concurrent use of a CIDR and treatment with 3,300 IU of hCG on d 5 of the estrous cycle resulted in progesterone profiles in lactating dairy cows that were similar to those of dairy heifers; however, the same authors demonstrated that use of a single CIDR or only hCG on d 5 did not result in progesterone profiles in lactating dairy cows that mimic those of heifers. In the current study, addition of a single intravaginal insert in CIDR4 increased progesterone concentrations by approximately 1.5 ng/mL between d 5 and 18 after AI, and the inclusion of a second insert on d 7 in CIDR4+7 resulted in an additional 0.5 ng/mL increment in progesterone concentration during the same period. When a larger number of cows was evaluated, but only on d 8 and 16 of the study, then progesterone concentrations increased from 4.7 ng/mL in controls to 5.3 in CIDR4 and to 6.5 ng/mL in CIDR4+7. Therefore, the increments in progesterone concentrations with addition of the CIDR were compatible with the findings of others in which each insert results in approximately 0.8 to 1.0 ng/mL additional progesterone in plasma (CERRI et al., 2009a;. Furthermore, the changes in the present study mimic increases in progesterone associated with higher fertility in heifers . Overall, P/AI did not differ between controls and cows supplemented with progesterone, LAMMING, 1999). Interestingly, Stevenson et al. (2007) also demonstrated a 5 percentage units, from 28.3 to 32.7%, increment in P/AI in dairy cows when receiving supplemental progesterone though CIDR inserts, but contrary to the findings of Mann and Lamming (1999), all the benefit occurred when the device was inserted after d 6. In the current study, stimulation of P/AI with exogenous progesterone was only observed when a single insert was administered on d 4 to cows inseminated following timed AI. In general, increasing progesterone concentrations early in the estrous cycle stimulates GIFFORD, 2010). Interferon-τ exits the uterus and reaches the maternal circulation (Oliveira et al., 2008), which induces expression of genes in blood leukocytes (GIFFORD et al., 2008;OLIVEIRA et al., 2008). In fact, mRNA abundance for ISG in leukocytes parallels the concentrations of IFN-τ in utero (MATSUYAMA et al., 2012). On d 16, expression of ISG in leukocytes was small and indistinguishable between cows diagnosed nonpregnant and pregnant at day 34. This probably reflected the reduced concentrations of IFN-τ in utero at that stage of gestation in cows; however, by d 19, mRNA expression increased substantially in pregnant cows. Nevertheless, exogenous progesterone did not increase mRNA expression for ISG in leukocytes on d 16 and 19 after AI. The lack of positive effect of treatment on ISG in leukocytes might be related to the increase in luteolysis by d 19 in cows that were later found to be nonpregnant. Also, at approximately d 16 of gestation, uterine IFN-τ in bovine peaks and then declines (FARIN et al., 1990). It is possible that the advancements in patterns of endometrial gene expression observed with prolonged exposure to progesterone with supplementation (FORDE et al., 2009) might have also advanced the reduction in mRNA expression of IFN-τ by conceptus trophoblast (FARIN et al., 1999) and, therefore, induced an earlier decline in concentrations in utero. Two concerns with supplementing progesterone during metestrus are the potential interference with CL formation and function and/or advancement of the luteolytic signals that might induce premature luteolysis. Based on concentrations of progesterone depicted in Figure 4.3A in pregnant cows on d 19, after treatments had ceased, it is plausible to suggest that CIDR4 and CIDR4+7 did not impair the ability of the CL to produce progesterone. Conversely, 1983), which may explain the reductions in pregnancy in heifers supplemented with progesterone within 2 d after AI ( VAN CLEEFF et al., 1996). Prolonged exposure to progesterone with supplementation in early diestrus may have advanced the peaks of IFN-τ expression in cows (FARIN et al., 1990;ROBERTS et al., 1999). Increased luteolysis by d 19 in cows later found nonpregnant and potential advancements in peaks of IFN-τ might have precluded the detection of increments in mRNA expression on d 19 for ISG15 or RTP4 with progesterone supplementation. Furthermore, it is suggested that reduced pregnancy around the time of conceptus-endometrium cross-talk probably explains the reductions in gene expression of RTP4 in leukocytes in cows found nonpregnant on d 34. It is unclear why supplemental progesterone did not result in further stimulation of mRNA expression of ISG in leukocytes of pregnant cows compared with untreated controls. It was thought that pregnant cows would have advanced conceptus development with supplemental progesterone (CARTER et al., 2008), and the latter were expected to have an earlier and more robust increment in expression of ISG because of increased IFN-τ in utero (MATSUYAMA et al., 2012). The mRNA abundance in blood leukocytes of the two genes investigated in the present study were previously described to be associated with improved conceptus development and increased fertility in lactating dairy cows . Nonetheless, exogenous progesterone might have attenuated some of the effects of IFN-τ on leukocytes. Addition of progesterone to leukocytes cultured in vitro blunted the effect of IFNα in stimulating mRNA expression for myxovirus resistance protein A (MxA; TAYEL et al., 2013), an ISG of the same family as Mx1 typically quantified in leukocytes of cattle (GIFFORD et al., 2007;. Interferon-τ and IFN-α are both type I IFN that interact with the same IFN receptor (ROBERTS et al., 1999). Therefore, it is plausible to suggest that the positive effects of increasing systemic progesterone on conceptus development in CIDR4 and CIDR4+7 were not evident in blood leukocytes because of direct immunomodulation of supplemental progesterone on ISG (TAYEL et al., 2013). In fact, responses of blood leukocytes to IFN-τ during early pregnancy are believed to counter-balance the immunosuppressive effects of progesterone on the maternal immune system (OTT; GIFFORD, 2010). A study supplementing somatotropin that stimulated conceptus development, without changes in progesterone concentrations, resulted in increased expression of ISG in leukocytes and P/AI in dairy cows . In the present study, if advanced conceptus development was successfully obtained with exogenous progesterone as observed by others (CARTER et al., 2008;, then the lack of responses in ISG in blood leukocytes and P/AI bring new insights to the interactions among progesterone concentrations, conceptus development, IFN-τ, and maternal immune system during establishment of pregnancy in dairy cattle. Conclusion Supplementing progesterone to lactating dairy cows with intravaginal inserts starting on d 4 after AI increased concentrations of progesterone in plasma in a dose-dependent manner, but did not increase mRNA expression of IFN-induced genes in leukocytes. Although P/AI was associated with concentrations of progesterone on d 8 after insemination, the use of intravaginal inserts to increase progesterone, up to 2 ng/mL, post-AI did not have an overall beneficial effect on maintenance of pregnancy in lactating dairy cows. When cows were inseminated following timed AI, then a single insert improved P/AI. Furthermore, results indicate that supplemental progesterone during early diestrus increased luteolysis by d 19 in cows found nonpregnant on d 34. Exogenous progesterone supplementation through CIDR was unable to stimulate expression of genes stimulated by interferon, but some benefits were observed in P/AI when a single insert was used in cows inseminated following timed AI. General conclusions The objectives of these studies were to evaluate the hormone supplementation to increase fertility in dairy cows. To improve the response of E2/P4-based FTAI protocol, studies with presynchronization protocols and increasing the EB dose at the beginning of FTAI protocol were carried out. Moreover, it was evaluated the effect of P4 supplementation on AI after estrus detection and in cows subjected to FTAI (E2/P4 or GnRH-based protocols). Finally, the effect of P4 supplementation after in vitro embryo transfer on lactating recipient cows was studied. Several hypotheses were tested, and the outcomes are presented below: 1. Higher EB dose would increase the synchronization of follicular wave emergence and thereby increase synchronization during the protocol. The EB dose of 2.0 mg would be insufficient, thus could explain the P/AI around 30% on the FTAI E2/P4-based protocols (Souza, Viechnieski, Lima, Silva, Araujo, Bo, Wiltbank, and Baruselli, 2009). By increasing the EB dose to 3.0 mg increased cows with premature CL regression and did not improve synchronization of the follicular wave. 2. Due to the success of presynchronization programs on fertility in GnRH-based protocols (Moreira, Orlandi, Risco, Mattos, Lopes, and Thatcher, 2001;, we hypothesized that presynchronization with a single GnRH treatment would synchronize the stage of the follicular wave at the beginning of the E2/P4 protocol and induce a greater synchronization rate. It was observed that, regardless of the follicular wave stage a proportion of cows still failed to have a synchronized emerge a new follicular wave. 3. The most interesting findings of the experiments above were not hypothesized, but when daily ultrasound exams were analyzed, we were able to understand some of the reason that led to lack of synchronization during the protocols. Based on data of emergence of a new follicle wave and ovulation at the end of the protocol, only 60% of the cows were considered to be synchronized. In these cows, the P/AI was about 60%. 4. P4 supplementation post ovulation would not interfere in the CL volume and function. In fact, plasma concentration of P4 was increased between d 4 and 7, and there was no effect on CL volume. Nevertheless, the lifespan of the CL was affected when cows were supplemented 4 days after AI, therefore a higher proportion of these cows had concentration of P4 on d 19 below 1.0 ng/mL. 5. Cows subjected to P4 supplementation should present increased ISG expression. This hypothesis was rejected due to the fact that ISG expression, in general, was similar between control and treated cows. 6. Supplemental P4 would increase fertility in dairy cows after AI by estrus detection or FTAI protocols. Neither cows inseminated after estrus detection nor by E2/P4-based FTAI protocols had increased P/AI. Nevertheless, cows subjected to GnRH-based FTAI protocol had increased P/AI when supplemented with a single P4 intravaginal device. 7. Progesterone supplementation 4 days before ET would improve P/ET. This hypothesis was rejected, considering that, independent of number of supplementation days (4 or 14 days) lactating recipient cows supplemented with P4 had decreased P/ET. Other study Because the incidence of cows that did not synchronize the emergence of a new follicular wave was about 26.2% and 22.2% failed to ovulate at the end E2/P4-based protocolwe hypothesized that GnRH would be better than EB for synchronization of follicular wave emergence. The first reason was because EB-treated cows had premature luteolysis. High circulating P4 during follicle development is important for greater P/AI . In our study, pregnant cows had higher P4 concentration during the protocol than nonpregnant cows. The second reason was due to the number of cows that ovulated persistent follicle. Thus, GnRH must be used to improve the follicular wave emergence rate. In relation to ovulation inductor, ECP has been widely used in Brazil, nevertheless, it has longer half-life and lower peak of E2 than EB (SOUZA et al., 2005). Additionally, EB has serum E2 profile more similar to physiology than ECP (SOUZA et al., 2005). Based on these findings, we performed an applied manipulative study that is not part of this thesis document. In order to increase the P/AI in cows subjected to FTAI protocols which consisted of EB or GnRH at the beginning of protocol associated with P4 intravaginal device (d -10) to synchronize wave emergence. Cows were treated with PGF on d -3 and d -2. The P4 device was removed with the second dose of PGF. Ovulation was induced with ECP on d -2 or EB on d -1. All cows were inseminated on d 0. A total of 418 cows was used in a 2 x 2 factorial design. In this study, when compared EB and GnRH, it was observed that more cows treated with EB on d -10 had greater proportion of luteolysis between d -10 and d -3 (49.5% ± 5.04 treatments. There was no interaction (P > 0.10) between the treatments on d -10 and ovulation induction. Although GnRH on d 0 provided better results than EB, as increased circulating P4 and increased ovulatory follicle size, there was no increase on fertility. Maybe if GnRH had higher ovulation rate, considering that only 29% of cows ovulated, could the outcomes might have been different. Future studies Although the results of the experiments did not contribute directly to increase fertility in dairy cows, they signal a pathway for that. Thus, based on the deficiencies identified on the E2/P4-based FTAI protocols, studies can be done in order to understand the possible reasons of some cows not emerging a new follicular wave. Moreover, further studies must be performed to evaluate the effect of P4 supplementation associated with antiluteolytic strategies delaying luteolysis in order to improve maternal recognition of pregnancy. In cows, when combining E2 with P4, normally it is observed the emergence of a new follicular wave (BO et al., 1993). According to our studies, chapter 1, some cows did not emerge a new wave. Increase of EB dose to 3.0 mg, also showed the same problem. Thus, it is concluded that this finding was not caused by low E2. Probably, the problem is associated with the P4 concentration. FTAI protocols that ensure high concentration of P4 on the beginning of the protocol associated with EB may resolve this problem. Another study can be done to understand the synchronization failures and lack of ovulation in FTAI protocols based on E2/P4 and these failures may be associated with diseases in dairy cows. Clinical diseases decrease the fertility in dairy cows , and in Brazil there were no studies associating disease with fertility, neither by estrus detection nor in cows subjected to FTAI protocols. Supplementation with P4 is associated with greater embryo size ). Nevertheless, it was observed that cows supplemented with P4 did not have increased fertility when subjected to AI. Moreover, when P4 supplementation was used in lactating recipient dairy cows a decreased fertility was observed. As discussed on chapter 2 and 3 of this present thesis, probably the benefit caused to increase conceptus is lost due to induced early luteolysis. This signals the need to study P4 supplementation associated with an antiluteolytic strategy. Thereby, it is likely that the beneficial effects of P4 supplementation on fertility of dairy cows can be obtained.

v3-fos

2016-05-04T20:20:58.661Z

2015-09-08T00:00:00.000Z

8631721

s2

Efficacy Study of Broken Rice Maltodextrin in In Vitro Wound Healing Assay Maltodextrins that contain both simple sugars and polymers of saccharides have been widely used as ingredients in food products and pharmaceutical delivery systems. To date, no much work has been reported on the applications of maltodextrin from broken rice (RB) sources. Therefore, the objective of this work was to investigate the in vitro wound healing efficacy of RB maltodextrin at different conditions. Wounds treated with lower dextrose equivalent (DE) range (DE 10–14) of maltodextrins at a concentration of 10% obtained from RB were found to be able to heal the wounds significantly faster (p < 0.01) than maltodextrin with higher DE ranges (DE 15–19 and DE 20–24) and concentrations of 5% and 20%. The findings from both BrdU and MTT assay further confirmed its wound healing properties as the NIH 3T3 fibroblast wounded cells were able to proliferate without causing cytotoxic effect when wounded cell was treated with maltodextrin. All these findings indicated that the RB maltodextrin could perform better than the commercial maltodextrin at the same DE range. This study showed that RB maltodextrins had better functionality properties than other maltodextrin sources and played a beneficial role in wound healing application. Introduction Rice constitutes the world's principal source of food. For example, it is the major source of dietary energy and protein for 80% of the population in Southeast Asia [1]. About 14% of broken rice (RB) is generated during rice milling processing leading to a direct economic loss to millers [2]. In the past, RB was used in beer making [3], and now, RB is used for commercial broilers to reduce the cost of poultry production and sparing maize for other uses [4]. This low valued byproduct from rice milling industry should be used for applications with better economic returns [5]. Rice is rich in starch, containing about 88% on average [2]. According to a study conducted by Guenoun et al. [6], broken rice constitutes 82.31% of starch yield. This rich in starch source is an ideal source to produce a high quality grade of maltodextrin for the application in food and pharmaceutical industries. to the underlying granulation tissue. Low DE maltodextrin is semipermeable to gas and fluids and thus provides an ideal protective cover to reduce the loss of fluid and plasma and the invasion of pathogenic microorganisms [12]. Moreover, a gradual release of small amount of glucose content in low DE maltodextrin is particularly effective to provide topical nutrition to the wound site, creating a natural wound healing environment [12]. Wound healing process consists of a series of recovery steps: (a) injured tissue is repaired; (b) specialized tissue is regenerated, and (c) new tissue is reorganized [13]. When cells are injured or killed from a wound, a wound healing step is required to resuscitate the injured cells and produce new cells to replace the dead cells. The healing process requires the reversal of cytotoxicity, the suppression of inflammation, and the stimulation of cellular viability and proliferation [14]. Diseases such as diabetes, immunocompromised, ischemia, and other conditions like malnourishments, ageing, local infections, and local tissue damaged wounds could cause a delay in the healing process [15]. Such conditions certainly require the use of healing agents to facilitate the wound healing process. One of the major problems with many known film forming agents is that they are rarely capable of enhancing the wound healing process. Therefore, in the wound of any substantial size, skin grafting will always be required [16]. Most published wound healing studies focused on microfluidic wound healing treatment [17], wound healing comparative studies [18], radiation therapy treatment [19], or topical ointment treatment [20]. Up to date, there is no information on the use of RB maltodextrin as a wound healing agent, reported. Thus, RB maltodextrin with different DE groups was produced and subjected to an in vitro wound healing and proliferation assay on NIH 3T3 cell line. The main objective of this study is to examine the wound healing efficiency of RB maltodextrin using an in vitro model on NIH 3T3 fibroblast cells and at the same time, comparison of its wound recovery rate with a commercial maltodextrin will be carried out to confirm the quality of maltodextrin produced from RB sources. Materials. Mature but unripened RB (blends of local varieties, MR 219 and MRR 220) were purchased from the local market (Serdang, Selangor). All starches were prepared at the laboratory scale [21,22]. RB maltodextrin of different DE group (DE 10-14, was supplied by MARDI (Serdang). A 10% of commercial (COM) maltodextrin (DE [10][11][12][13][14] was used as a reference as it was known to produce high quality maltodextrin [22]. Multidex, a known commercial wound dressing agent containing maltodextrin, was used as a comparison purpose . Culture of Cell Lines. NIH 3T3 cell line was obtained from ATCC, USA, and cultured in RPMI 1640 medium supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum (FBS) in a humidified incubator with 5% CO 2 . Cell line was detached from the culture flasks using a trypsin-EDTA solution (0.25-0.025%) and resuspended as a single cell suspension in RPMI 1640 culture medium. In Vitro Wound Scratch Assay and Microscopy Evaluation. NIH 3T3 cells were seeded in a tissue culture 6-well plate at an initial density of 2.4 × 10 5 cells/cm 2 overnight. A micropipette tip was used to create a wound in the monolayer by scraping. A total of 10 (%, w/v) RB maltodextrin were added in each treatment well with or without an addition of 100 ppm of various additives including aloe vera, curcumin, hydroxyproline, ascorbic acid, L-arginine, lactic acid, and kojic acid, which were added separately to each well. Another two wells were treated with Multidex and media only (control), respectively. Wound closure was observed by phase-contrast microscopy (NIKON, Japan) and digital images were taken at the interval time of 3 h up to 24 h. Determination of NIH 3T3 Cell Viability via Trypan Blue Cell Count Assay. Trypan blue cell count was carried out to identify the amount of viable cells present in each sample. After 24 h incubation period, harvested cell suspension (10 L) was added with equal volume of 0.4% trypan blue stain. Hemocytometer was used for cell counting under inverted light microscope (NIKON, Japan). Viable cells are those excluded from the stain. 5-Bromo-2 -deoxyuridine (BrdU) ELISA Cell Proliferation Assay. Maltodextrin treated and untreated NIH 3T3 cell proliferation was measured using the Bromodeoxyuridine (BrdU) Cell Proliferation Kit (Merck, USA). The cells were seeded in a 96-well plate at a concentration of 0.8 × 10 5 cells/mL overnight. A total of 10% RB maltodextrin were added separately with or without the addition of 100 ppm of various additives, including aloe vera, curcumin, and hydroxyproline and incubated for 24, 48, and 72 h, respectively, at 37 ∘ C and 5% CO 2 . Another two wells were treated with Multidex and media only to serve as untreated control. After the corresponding period, BrdU label was added into all wells and incubated for an additional 24 h. At the respected incubation hours, the cells were fixed and incubated at 4 ∘ C for approximately 30 min. After that, the plates were washed twice, added with 100 L detector antibodies into each well, and incubated for 1 h. Then, 100 L of goat anti-mouse Ig G-HRP conjugated was added and incubated for 30 min. Then, the plates were incubated with 100 L of 3, 3 , 5, 5 -Tetramethylbenzidine (TMB) substrate for another 30 min. Finally, 100 L of stop solution (sulfuric acid) was added and the absorbance was measured at 450 nm, using an ELISA microplate reader (Biotech Instruments, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl Tetrazolium Bromide (MTT) Cell Viability Assay on NIH 3T3 Cells. NIH 3T3 cells were seeded on 96-well microtiter plates overnight. A respective amount of 5% and 10% of RB maltodextrin was added in each treatment well separately. An addition of 100 ppm of various additives including aloe vera, curcumin, and hydroxyproline was added separately to each well. Both Multidex and media acted as control. The fibroblast cells treated with various samples were exposed to the culture BioMed Research International 3 medium up to 72 h. At each interval time of 24 h, a total of 20 L/well of MTT solution (Calbhiochem, USA) were added, followed by incubation at 37 ∘ C for a period of 4 h in an atmosphere of air with 5% CO 2 . After that, supernatants were removed from the wells and 100 L/well of dimethyl sulfoxide (DMSO) (Fisher, USA) was added to solubilize formazan. The absorbance was quantified at 570 nm using ELISA microplate reader (Biotech Instruments, USA). In Vitro Red Blood Cell (RBC) Irritation Assay. Approximately 2 mL of RBC obtained from a volunteer (Pusat Kesihatan, UPM) and was washed in PBS in a ratio of 1 : 10, followed by centrifuged for 10 min at 1500 rpm with a controlled temperature of 10 ∘ C, and this step was repeated triplicate. The RBC was then diluted with PBS to a 1% concentration. Each 100 L of RB, COM maltodextrin, and Multidex were loaded into the first well in a 96-well plate separately, followed by 50 L of PBS loaded from the 2nd to the 12th well. A serial dilution was carried out from the 1st well to the 11th well and the 12th well was treated as the positive control. Then, a total of 50 L of 1% RBC were added to each well before being incubated at room temperature for 30 min. At the end of incubation, the suspension was centrifuged at 500 rpm at 10 ∘ C for 10 min. Statistical Analysis. Data was statistically analyzed by one-way analysis of variance (SPSS statistics version 16). Significant differences ( < 0.01) between means were determined by Duncan's multiple range test. A Preliminary Wound Healing Comparison Study on RB Maltodextrin with the Addition of 100 ppm of Various Additives. In vitro wound healing process starts with the spreading of individual cells at the wound edge and the synthesis of matrix fibrils (e.g., fibronectin), followed by cell migration (translocation) along the fibronectin and cell proliferation process [23]. Table 1 shows the results of a preliminary study of in vitro wound healing on RB maltodextrin. In this study, the NIH 3T3 cells were seeded in sixwell culture plates to test the wound healing effect of RB maltodextrin with and without 100 ppm of various additives. Overall, this study consisted of three parameters with the aims to examine the wound healing effect of (a) different concentration of additives choice; (b) different concentration of RB maltodextrin; and (c) different DE grades of RB maltodextrin. These factors were studied to identify the optimum condition of RB maltodextrin to assist the wound healing of NIH 3T3 cells. Various additives including curcumin, hydroxyproline, ascorbic acid, L-arginine, lactic acid, and kojic acid were selected based on previous publications on their significance to promote wound healing properties [24][25][26][27][28]. Aloe vera is a tropical cactus which has been reported to have therapeutic potential in a variety of soft tissue injuries, medical and cosmetic purposes, and general health [29]. A study carried out by Vera [30] reported that 50.8% improvement in wound closure was observed in mice when treated with topical aloe vera. Heggers [31] study confirmed the therapeutic effects of aloe vera, whereby it showed the progressive prevention of tissue loss in dermal ischemia caused by burns, frostbite, electrical injury, distal dying flap, and intra-arterial drug abuse in both man and animal models. Therefore, 100 ppm of aloe vera was added to examine its proliferation effect on NIH 3T3 wounded cells. According to Schreier et al. [23], the addition of additives (platelet-derived growth factor) was noted to increase the cell migration to the "wounded" area compared to cells in the absence of additives. Furthermore, active additives may promote the process of wound healing by increasing the viability of collagen fibrils and the strength of collagen fibers, either by increasing the circulation or by preventing the cell damage or by promoting the DNA synthesis [23]. Multidex, one of the popular commercial wound dressings, was used as a comparative control while the cells containing only fresh media without any treatment acted as the control. Additives act as growth factors to promote the tissue repair, migrating cell into the wound site and stimulating cell proliferation [32]. Initially, a study of using different additives concentration effect on the wound closure was conducted to determine its healing capability of NIH 3T3 cells. Based on the results (Table 1(a)), the response obtained from each NIH 3T3 cells treatment exposed to different concentrations of additives was distinguishable ( < 0.01) than responses received in the control group. In brief, the use of 50 ppm additives was noted to be insufficient to heal the wounds within 24 h relative to 100 ppm additives. It is important to identify the concentration at which maltodextrin can perform the best healing capability [33]. Therefore, different concentrations of RB maltodextrin (5%, 10%, and 20%) with the same DE value of 10-14 were conducted (Table 1(b)). Addition of 100 ppm additives to each maltodextrin was studied also in the wound healing process of NIH 3T3 cells. The response obtained from each NIH 3T3 cells after exposure to different concentrations of maltodextrin was distinguishable ( < 0.01) than responses received in the control group. In Table 1(b), treatments with either 5% or 20% RB maltodextrin were not able to heal the wounds completely within 24 h. These two concentrations did not show significant improvement ( < 0.01) in the percentage of wound closure relative to the control group. The addition of 5% RB maltodextrin into NIH 3T3 wounded cell was found to be too liquefied and could not provide sufficient nutrients to the cells leading to poor cell migration. On the other hand, high concentration of RB maltodextrin (20%) was not suitable to be used in wound healing as it was too concentrated and yield a syrup-like characteristic due to high solid content [9]. These results indicated that high viscosity of RB maltodextrin prevents the absorbance of nutrients to the cells to enhance the proliferation and migration of the cells. Only 10% concentration of RB maltodextrin was capable of achieving 100% recovery within 24 h as shown in Tables 1(b)(ii) and 2. It was also noted that 10% RB maltodextrin alone showed the best wound healing of NIH 3T3 at 12 h (61.13%), followed by 10% RB maltodextrin with 100 ppm curcumin (52.97%), 10% RB maltodextrin with 100 ppm hydroxyproline (51.52%), 10% RB maltodextrin with 100 ppm ascorbic acid (47.78%), 10% RB maltodextrin with 100 ppm Larginine (47.66%), 10% RB maltodextrin with 100 ppm lactic acid (44.94%), and 10% RB maltodextrin with 100 ppm kojic acid (32.98%). Multidex was found to cause a cytotoxicity rather than wound healing effect on the NIH 3T3, whereby the cells were observed to be unhealthy and unable to migrate and died within 12 h as shown in Table 2 Maltodextrin with a lower DE value is preferable to facilitate the exposure of dermatological agents added to improve the healing and facilitates the contact to all areas of the wound [16]. The DE value of maltodextrin affects the viscosity, sugar composition, and the characteristics of the maltodextrin [34]. According to Sun et al. [35], the molecular composition of maltodextrin differs at different DE values. Maltodextrin with a lower DE range possessed higher molecular weight and longer chain of glucose polymers [35]. The presence of short chain sugar molecules in the maltodextrin DE 10-14 provided sufficient nutrient and showed better stimulation and migration of the cells [10]. On the other hand, a higher DE value of maltodextrin (DE 15-19 and 20-24) tends to increase the viscosity and provides a more sugary characteristic, subsequently affects the cell migration, and thus decreases the ability of maltodextrin to diffuse into the cell monolayer [10]. Overall, 10% RB maltodextrin DE 10-14 had shown the best performance in healing NIH 3T3 wounded cells relative to 5%, 20% RB maltodextrin concentrations, and also RB maltodextrin with higher DE value ( . In comparison to various additives effect on the percentage wound closure of NIH 3T3 fibroblast cell, both lactic acid and kojic acid performed significantly the poorest ( < 0.01) compared to the other additives. Lactic acid and kojic acid are mostly used in the cosmetic area and may not be very suitable in the wound healing of NIH 3T3 cells as these additives may have the potential to cause irritations if not used at a suitable amount [36]. However, the wounded cells treated with curcumin and hydroxyproline showed significantly higher ( < 0.01) healing power compared to other additives. This phenomenon may be caused by their attribution to increase the stimulation of the fibroblast cells proliferation [29]. Curcumin, the active ingredient in the spice, turmeric, has been found to be effective in the skin injury treatment, whereas hydroxyproline, an amino acid, is unique for collagen that helps in the tissue recovery of wound area [27,37]. The addition of 100 ppm additives did improve the percentage wound closure compared to media alone. However, 10% RB maltodextrin DE 10-14 alone had shown significantly the best recovery rate ( < 0.01) in the wound healing compared to the treatment with addition of various additives. The purpose of adding various types of additives was to compare the healing effects of these additives with RB maltodextrin. This finding indicated that 10% RB maltodextrin DE 10-14 was the best wound healing agent and proved its effectiveness to facilitate wound healing process of NIH 3T3 cells. To further investigate the wound healing capability of other maltodextrin source on the NIH 3T3 wounded cell, a commercial (COM) maltodextrin from cassava source was studied under the same condition. Cassava maltodextrin was selected as it was known of its high quality maltodextrin and widely used in the food and pharmaceutical applications. A previous comparison study between RB and cassava starch by Koh and Long [22] had confirmed their differences in the physicochemical properties, and eventually it will affect their functional property when these starches were hydrolyzed to produce maltodextrin. Figure 1 shows the wound healing performance of 10% RB maltodextrin, 10% COM and 10% Multidex. Based on the graph, it showed that the percentage wound closure of NIH 3T3 wounded cell when treated with RB maltodextrin was higher than COM maltodextrins. Although RB and COM maltodextrins were under the same DE range, the time taken for each cell migration in the wound closure study was found to vary, significantly depending on its maltodextrin source. This is proportional to the earlier described statement; maltodextrin with the same DE ranges from different starchy sources has different functional and physiochemical properties, which is highly dependent on the starch molecular structure itself [34,35]. As reported herein, RB maltodextrin could significantly stimulate the proliferation of NIH 3T3 cells better than COM maltodextrin. One of the possible pros reason to explain the less efficient performance of COM maltodextrin in the wound healing relative to RB maltodextrins was due to its molecule size. According to Koh and Long [22], cassava starch possessed the largest molecule size compared to RB starches as shown by a micrograph study. The larger molecule size of COM maltodextrin may have led to poor nutrient supplement to the NIH 3T3 cells and thus slowed down the rate of cell migration. Overall, it was clearly shown that RB maltodextrins had positive influence on its characteristics in the wound healing functional property, which substantiates it as a wound healing agent. In this study, we have proven that RB maltodextrins have performed significantly better compared to the COM maltodextrin as wound healing agent. An occlusive dressing may facilitate a moist wound environment and retains the wound fluid and its various components; however, it also keep oxygen away from the tissues at the same time. Oxygen plays an important role in the collagen synthesis [38]. One of the important functions of maltodextrin in the wound healing application is the formation of a film, which is intimately adhered to the underlying granulation tissue. This film is semipermeable to gas and fluids, providing an ideal protective cover to reduce the loss of fluid and plasma and invasion by pathogenic bacteria [12]. This finding indicated that our newly produced RB maltodextrins not only enhance the speed recovery rate of wound healing but also are capable of forming a thin protective layer over the wound that subsequently allowed the exchange of oxygen and retaining the required sugars (from maltodextrin) as a nutritious source to the wounded cells for the proliferation of new cells. Even though our study was conducted using in vitro model on NIH 3T3 fibroblast cell, Heng's [38] study had supported our findings and they claimed that sugar dressings (maltodextrin) tested on wounded dogs and cats in a veterinary clinical study capably drew macrophages into the wound and accelerated sloughing of necrotic tissue which enhanced the recovery rate of wound healing process. In addition, their study also reported that the supply of simple sugar to the wounded site acted as a local nutrient source which decreased the incidence of inflammatory edema and subsequently sped up the cell granulation and epithelialization process. Determination of NIH 3T3 Cell Viability via Trypan Blue Cell Count Assay. A well-recognized essential requirement of most biological investigations using cellular preparations is the assertion of cell viability [39]. The most widely applied criterion for investigation of cell permeability is the exclusion by cell of dyes with higher molecular weights (vital stains) such as trypan blue [39]. Trypan blue is a vital dye in which its chromophore is negatively charged and does not interact with the cells unless the membrane is damaged [40]. Therefore, cells which exclude the dye are considered to be viable. Figure 2 represents the trypan blue cell count assay of NIH 3T3 cell treated with RB maltodextrin, to evaluate the percentage of viable cells during in vitro wound healing study. In general, all trypan blue cell count results were noted to tally with the percentage of wound healing findings. It was found that those treatments had shown an improvement in the wound closure possessed higher percentage of cell viability. Based on the cell counts findings, the cell viability of all maltodextrin treatment groups achieved 100% viability rate, except for RBLA treatment, which only showed the cell viability at the rate of 93%. This phenomenon indicated that the presence of lactic acid had an effect on the cell viability of NIH 3T3 fibroblast cell. Although RB maltodextrin alone was shown to be able to heal the wounds better than the same treatment with addition of other additives, most of the cells treated with additives were able to maintain 100% viability. In all of the results presented, it was found that Multidex, a commercial wound dressing, showed zero percentage of cell viability. This finding indicated that the presence of Multidex in the NIH 3T3 cells most probably had caused substantial cell damage over time after treatment, leading to the increase in the cell death [6]. Generally, the trypan blue cell count assay had supported the findings of wound healing capability of RB maltodextrins with DE value of 10-14 as confirmed in the percentage of wound closure observed in the NIH 3T3 wounded cells. [41] has shown that proliferation of fibroblast can be observed by BrdU incorporation. Thus, BrdU ELISA cell proliferation assay was used to evaluate the proliferative effect of maltodextrin on NIH 3T3 fibroblast cell in vitro. Cultured cells that entered the log growth phase are pulsed to be labelled with the nonradioactive BrdU [42]. The proliferation of NIH 3T3 cells treated either with 10% RB maltodextrin with or without additives compared to the controlled NIH 3T3 cells that are cultured with media only is shown in Figure 3. Generally, the proliferation rate of NIH 3T3 cells was noted to increase gradually across time comparing to the controlled cells, except for COM and Multidex treatment, whereby the percentage of cell proliferation decreased significantly ( < 0.01) from 24 to 72 h. BrdU ELISA Cell Proliferation Assay. Freshney Overall, all treated NIH 3T3 cells (except COM and Multidex) achieved the highest percentage of cell proliferation after 72 h of incubation. Increase in the percentage of cell proliferation may be attributed to the stimulation by maltodextrin to promote the propagation of fibroblast cells. Cells treated with 10% RB maltodextrin with or without additives showed a higher percentage of cell proliferation than the control group. In general, the highest percentage of cell proliferation at 72 h was RB maltodextrin, which was 47.33% higher in proliferation compared to the control group, followed by RB maltodextrin with 100 ppm curcumin (23.53%), RB maltodextrin with 100 ppm hydroxyproline (13.91%), and RB maltodextrin with 100 ppm aloe vera extract (5.60%). However, cells treated with COM and Multidex maltodextrin possessed a lower proliferation rate compared to the control group across time. Overall, our newly developed RB based maltodextrin alone had proven to have higher cell proliferation rate, which was also confirmed through the findings that indicated its better functional property in the wound healing application. This effect may be contributed by the presence of glucose in maltodextrin, which supplied energy to the metabolism and proliferation of the cell [10]. Thus, this finding confirmed that NIH 3T3 wounded cells that were treated with nutrient rich maltodextrin improved the proliferation of cells; therefore it showed better performance in the proliferation of cells as opposed to the untreated cells. MTT Cell Viability Assay on NIH 3T3 Cells. MTT assay was performed to determine the cell viability of NIH 3T3 cell after being treated with maltodextrin. This test involves the conversion of tetrazolium salt, 3-(4,5-dimethylthiazol-2yl)-2, 5 diphenyl tetrazolium bromide (MTT) to an insoluble formazan product, which is quantitated by spectrophotometric method [27]. Figure 4 represents the cell viability of NIH 3T3 cells treated with RB maltodextrin at (a) 5% and (b) 10% concentrations, with or without the addition of 100 ppm additives, respectively. Generally, the cell viability of RB maltodextrins at 5% and 10% concentration was observed increased significantly ( < 0.01) with the incubation time. The increase in the cell viability percentage had indicated that the cells were still viable even after culture up to 72 h. In comparison to 5% maltodextrin, the cell viability of NIH 3T3 cells treated with 10% RB maltodextrins was shown significantly ( < 0.01) to be higher. Similar to BrdU proliferation assay's findings, MTT cell viability assay also showed a significant decrease ( < 0.01) in the percentage of cell viability in Multidex treated group, as observed at 24, 48, and 72 h. Toxicity appeared in the cells treated with either 5 or 10% Multidex which had caused a decrease in the percentage of cell viability from 61.46% to 54.22% for 5% Multidex and 53.85% to 44.83% for 10% Multidex after 72 h of incubation, respectively. Briefly, the NIH 3T3 cell treated with 10% RB maltodextrin emerged to have the highest percentage of cell viability after 72 h (121.70%), followed by COM maltodextrin (120.96%), RB maltodextrin with 100 ppm curcumin (116.60%), RB maltodextrin with 100 ppm aloe vera extract (111.21%), and RB maltodextrin with 100 ppm hydroxyproline (108.94%). Similar to the findings as reported in the BrdU cell proliferation assay, RB maltodextrin alone possessed higher cell viability relative to other treatments. In Vitro RBC Irritation Assay. The criticism of the classical in vivo methods to predict skin or ocular irritation brings up the development of a number of replacement in vitro methods, which have been recently reviewed to reduce the use of animal for this purpose [37]. In vitro studies using RBC are an alternative technique to the in vivo eye irritation test since it is an inexpensive and rapid and provides reliable results with good reproducibility method that reduces and even avoids the use of experimental animals for this kind of test [31]. When the RBC is in contact and in circular shape, it indicates that the tested sample does not cause irritation to the cell and thus the sample is safe to be used on human skin. If the RBC lyse, this indicates that the sample at the tested concentration had irritation effects. Figure 5 shows the RBC hemolysis assay results when tested with various concentrations of RB maltodextrin, COM maltodextrin, and Multidex. Wells consisting either PBS or RBC were served as positive control. When the maltodextrin treatments were added to the erythrocyte suspension in aqueous medium, they will be distributed between the erythrocyte membrane and the solution by absorption first until the equilibrium is reached [43]. Hemolysis probably begins when the erythrocyte membranes are saturated with the treatment molecules [43]. Based on the results, all treatments had no lysis effect on the hemolytic activity. This finding proven that maltodextrin is safe to be used topically and would not cause any irritation to the human skin. Multidex also did not cause the RBC to lyse although it caused cell death in the in vitro wound healing. The presence of preservative in Multidex reduced the cell viability on NIH 3T3 cells, which were sensitive to the cells. This finding showed that the preservatives in Multidex are safe to use on the human skin but not safe to be consumed orally or injected into the skin. In conclusion, RB maltodextrin was confirmed to be safe and proven to have no hemolytic effects even at a high concentration treatment. Conclusion RB maltodextrins with low DE group (DE [10][11][12][13][14] showed better improvement of the wound closure compared to high DE group ( as proven in the in vitro wound healing model. Interestingly, RB maltodextrin with low DE value alone treatment had exhibited the best recovery rate in wound healing treatment by an evidence shown in the wound scratch assay, trypan blue, MTT cytotoxic, and BrdU cell proliferation assay compared with those treatment containing the addition of additives. When compared to the COM maltodextrin, RB maltodextrins exhibited higher healing efficiency as shown in the wound healing, MTT cell viability, and BrdU cell proliferation assays. More importantly, RB maltodextrins also did not induce irritation effect in the RBC assay. In conclusion, RB maltodextrin has successfully shown a positive influence to speed up in vitro wound closure and play a beneficial role in wound healing.

v3-fos

2018-04-03T04:38:09.791Z

2015-08-26T00:00:00.000Z

12336741

s2

Sensory quality of soymilk and tofu from soybeans lacking lipoxygenases Abstract The oxidation of unsaturated lipids by lipoxygenases in soybeans causes undesirable flavors in soy foods. Using a traditional and a nontraditional soy food user group, we examined the cultural difference in perceiving the sensory characteristics of soymilk and tofu produced from soybeans with or without lipoxygenases (Lx123). The two groups described the samples using similar terms. The traditional users preferred the control soy milk and lipoxygenase‐free tofu while the nontraditional users preferred the lipoxygenase‐free soymilk with no preference for tofu. In a separate study, a trained descriptive taste panel compared the odor of soymilk and tofu from control soybeans or those lacking lipoxygenase‐1 and lipoxygenase‐2 (Lx12) or all three isomers (Lx123). The rancid/grassy odor was rated the lowest in Lx123 products, followed by Lx12 products with the control products given the highest rating. The Lx12 and Lx123 products were also sweeter and less bitter than the controls. Taken together, our results demonstrated that soybeans lacking lipoxygenases can produce soy foods with less undesirable aromas and are therefore likely more acceptable to the consumers. Introduction Soy foods produced from whole soybean seeds such as soymilk and tofu have been part of the traditional foods in East Asia for a long time. Soybean is also an important ingredient in many processed foods. Its production and use has increased rapidly due to its high nutritional value and potential health benefits. Utilization of soybean seeds as food materials, however, has sometimes been limited, particularly in Western societies, because of their "grassy/ beany" flavor (Krishna et al. 2003) and certain consumers prefer a bland or neutral flavor in soy products. The consumers from traditional soy food consumption countries, on the other hand, generally favorably associate beany flavor with soy products such as soymilk and tofu. Cross-cultural variations in food preferences are well known and cultural differences in consumption of soy products might account for some differences in perception of sensory attributes. These undesirable flavors, characterized as beany, green, grassy, painty, astringent, and bitter, have been associated with off-flavors from the oxidation of polyunsaturated lipids by lipoxygenases (LOX) present in soybeans. Soybeans are known to be a rich source of LOX and mature soybean seeds contain three major lipoxygenases: LOX-1 (lipoxygenase-1), LOX-2 (lipoxygenase-2), and LOX-3 (lipoxygenase-3). Soybean also contains high amount ORIGINAL RESEARCH Sensory quality of soymilk and tofu from soybeans lacking lipoxygenases of polyunsaturated fatty acids (PUFA), predominantly linoleic acid (C18:2) at around 50% and linolenic acid (C18:3) up to 11% (King et al. 1998;Gerde and White 2008). Both C18:2 and C18:3 contain a cis,cis-1,4-pentadiene structure potentially leading to the production of hydroperoxides when oxidized, which in turn are converted into volatile compounds associated with undesirable flavors (Kitamura 1984). Full-fat soy flour is especially prone to such deterioration and has a disagreeable taste that is difficult to mask. Before beans are crushed or ground, LOX and PUFA are separated within the cell, but following soaking and homogenization in the process of making soymilk, they are mixed and begin to react to form the oxidation products and volatile compounds. Among the volatile compounds detectable in soy products, hexanal is primarily responsible for the objectionable flavor or aroma of soy foods and has a very low detection threshold (Wilkens and Lin 1970). The removal of all or some of the LOX present in soybeans through breeding has produced soybean lines lacking LOX and subsequent lower levels of volatiles responsible for the rancid flavor, which could be useful in improving the acceptance of Western consumers. While LOX-free soybean has been shown to produce lower hydroperoxide and hexanal levels than that with LOX or lacking LOX-1, -2, or -3 isozyme (Hildebrand et al. 1991;Kobayashi et al. 1995;Furuta et al. 1996), there have also been mixed results in the literature on the impact of these LOX-free soybean lines on controlling the beany flavor of soy foods produced. For example, using GC-MS, Kobayashi et al. (1995) showed that almost all volatiles in soymilk produced from mutants lacking LOX were markedly lower than those from a normal variety. Wilson (1996) reported that tofu made from LOX-2 null lines of soybeans were less beany than their respective controls as evaluated by a sensory panel. However, while LOX-free soybeans reduced the beany flavor in certain types of soy products such as soymilk and tofu (Wilson 1996;Torres-Penaranda et al. 1998), they showed no effect in oils, breads, and meat patties (King et al. 1998(King et al. , 2001Liu et al. 2008). Besides improved flavor and aroma, soybeans null in LOX could be more resistant to adverse storage conditions than the normal soybeans with regard to changes in pH and solid content of the soymilk (Lambrecht et al. 1996), although Torres-Penaranda and Reitmeier (2001) reported significant sensory differences between soymilks from soybeans lacking LOX stored for 3 months and 15 months and no difference for the soymilks from the normal soybeans stored for these times. The LOX-null lines, including those developed in Australia, have been shown to have similar agronomic traits including yield Reinprecht et al. 2006) (A. James, 2013. However, the LOXfree lines from Australia have not been evaluated for their effect on storage stability, beany flavor, and other quality attributes in soy foods. There are two types of commercial soymilk in Australia, one made from imported soy protein isolates and one made from domestically sourced whole soybean. The grassy/beany flavor of the whole bean soymilk is reported to be a limitation on the uptake of this type by the market. Therefore, soybean without lipoxygenase may offer an opportunity to expand the market for Australian-grown soybeans. Studies using panels from different cultural backgrounds to evaluate the sample product may give some indication as to whether such differences should be considered. The Australian market for soy products has consumers of many cultural and ethnic backgrounds. The objectives of this study were therefore to compare soymilk and silken tofu from normal and lipoxygenase-free soybeans using a predominantly native Chinese panel ("traditional soy users") and an Anglo-Australian panel ("nontraditional soy users") in Australia and to investigate the impact of LOX-free soybeans developed in Australia on sensory attributes of soy products in order to improve our understanding of the potential utility of these traits for the Australian market. Soybeans, soymilk and tofu Normal soybeans and soybean lines selected for LOX-null in Australia were used in these studies. All the lines used were grown, harvested, and stored together before use. In the preliminary study, a commercial line and a genotype lacking all three major LOX isozymes (Lx123) were used. In the following sensory study, normal soybeans and lines lacking LOX-1 and LOX-2 (Lx12) or all three isomers (Lx123) were selected. The LOX-1 and LOX-2 genes are tightly linked and inherited together, while the LOX-3 gene is independently inherited (Davies and Nielsen 1986). All the materials were kept at around 22°C until use. All soybean lines were checked for LOX using a screening method (Suda et al. 1995;Narvel et al. 2000) and SDS-PAGE (sodium dodecyl-polyacrylamide gel electrophoresis) (Yang and James 2013) to confirm the absence of LOX-1 and LOX-2 or all three LOX isomers. A typical SDS-PAGE profile of soybean seed proteins with or without LOX isozymes is presented in Figure 1. Soymilk and silken tofu were produced according to the procedure optimized by Yang and James (2013) and stored at 4°C overnight before use in sensory and focus group assessments. Briefly, the beans were soaked overnight before they were ground in a domestic blender. The slurry was cooked at 98°C and then filtered using a juice extractor to obtain soymilk. The required amount of the soymilk for assessment was transferred to 4°C. The remaining soymilk was made into silken tofu by adding 0.3% coagulant (nigari, mostly magnesium chloride) and allowing the curd to form at 85°C. The soymilk and tofu were kept at 4°C until use. Preliminary study of soymilk and tofu from control and LOX-free soybeans Sensory evaluation using panels of "traditional" and "nontraditional" soy users Two semiformal focus groups were held (each of 1 h duration) involving two target groups namely "nontraditional soy users" (the Australian panel) and "traditional soy users" (the Chinese panel). The profiles of the participants for each group are given in Table 1. The participants in each group were presented samples of soy milk and silken tofu made from commercial beans (control) and lipoxygenasefree soybeans (LOX-free) together with paper questionnaires. Participants were asked to describe the appearance, aroma, texture and mouth feel, flavor, and aftertaste of samples and to rate the overall acceptability of each sample on an unstructured 15-cm hedonic line scale anchored from "dislike extremely" to "like extremely". The individual assessments were made under controlled conditions (light, temperature) and samples were labeled with 3-digit codes. Presentation order of samples was identical across the participants as follows: control soy milk, LOX-free soy milk, control silken tofu, and LOX-free tofu. Samples (15-20 g each) were presented in disposable plastic pots (tofu) and cups (milks). Participants were also asked to complete a short demographic questionnaire and indicate their soy product knowledge and consumption behavior. Following the taste session, a focus group discussion was held in which participants were asked to share and discuss their thoughts on the sensory properties and acceptability of the samples and their past soy product experiences and consumption behavior. Instrumental analysis of volatile compounds by GC-MS Replicated samples of soy milk and silken tofu (control and LOX-free) after 1 day and 8 days stored at 4°C were sampled and frozen (−19°C) for volatile analysis. Aliquots of 5 mL of soy milk or 5 g of silken tofu were placed in 25 mL glass vials for SPME (solid phase microextraction) and immediately closed with screw caps with PTFE/ silicone septa (Supelco, Bellefonte, PA). The method used for volatile analysis was a modified version of the method reported by Achouri et al. (2006). Samples were analyzed with a 6890N GC (gas chromatograph) equipped with a 5975 MSD (mass spectrometric detector) (Agilent Technologies, Palo Alto, CA). The GC was fitted with a DB-WAX column (J&W Science, i.d. = 0.25 μm, length = 30.0 m, film thickness = 0.25 μm), and helium (BOC gases, ultrahigh purity) was used as a carrier gas at a linear velocity of 56 cm/min and at a flow rate of 2.4 mL/min. The polydivinylbenzene and carbowax and polydimethylsiloxane (DVB-CAR-PDMS, Gray, 50/30 μm, Supelco) fiber was introduced into the headspace of the sample vial and was allowed to incubate at 45°C for 20 min. After the incubation, the fiber was retracted into a needle and desorbed into the injection port of the GC for 6 min. The fiber remained in the injection port for 6 min to eliminate the possible residues on the fiber. Extraction was supported by magnetic stirring at 250 rpm/s and a fiber blank experiment was performed. The mass spectrometer was operated in the electron impact ion mode with a source temperature of 250°C. The electron energy was 70 eV and the mass range was 35-300 m/z. The GC oven temperature started at 35°C for 3 min, was increased at 6.0°C/min to 220°C, and was held for 10 min. Data analysis was carried out with MSD ChemStation Data Analysis software (Agilent Technologies) to obtain peak responses of volatiles. Peak identification was achieved by comparison of spectra and retention times with authentic reference standards. Sensory study on comparing control, Lx12, and Lx123 soy products Soybean seed characterization The moisture, oil, and protein content of the soybeans were determined as described by Yang and James (2013). The seeds were analyzed for their fatty acid composition using GC. Briefly, soybean seeds were ground to a fine powder and oil was extracted twice with n-hexane. The combined hexane extractions were evaporated under nitrogen. The oil, 0.1 g, was methylated with 0.5 mol/L sodium methoxide solution at 80°C for 10 min. After cooling to room temperature, 0.05 mL of glacial acetic acid, 2.5 mL of distilled water, and 2.5 mL of petroleum spirit (or hexane) were added. The mixture was vortexed and 0.5 μL of the clear upper phase was used for analysis. The separation and distribution of fatty acid methyl esters were obtained using GC (Perkin Elmer Autosystem Gas Chromatograph, MA, USA) fitted with a FID (flame ionization detector) and a capillary column (SGE BPX70, 25 m × 0.22 mm). The carrier gas was helium at 15 psi. The injector temperature was 240°C and detector temperature was 280°C. The oven temperature started at 150°C before reaching 200°C. The ground full-fat soybean meal was also stored at 37°C for 1 week and the total volatiles were measured using a SPME-GC-FID procedure. Sensory evaluation Three replicates of each silken tofu and soy milk sample were prepared from the composite samples of soybeans the day prior to the commencement of sensory testing and training and formal assessments were completed within a 3 day period to ensure freshness of samples. Sensory descriptive analysis techniques were applied to assess and quantify the major sensory properties of six samples including tofu and soy milk made from commercial soybeans (control), from triple-null soybeans (Lx123), and from double-null soybeans (Lx12). A total of 11 panelists (four male, seven female), who were experienced in descriptive sensory studies and who were staff and students at the Health and Food Sciences Precinct, Coopers Plains, Queensland, participated in the study. Their profiles are listed in Table 1. Two training sessions were conducted in a board-room style round table suitable for discussions. The first training session involved presentation of all samples to panelists, individual aroma and taste assessments, and vocabulary development through discussion ( Table 2). The second training session involved practicing rating attributes for each of the samples and developing a concise list of attributes, scales, and definitions. Attributes selected during training, together with their definitions, are shown in Table 3. Formal assessments were conducted in the sensory laboratory of the Health and Food Sciences Precinct, Coopers Plains, Queensland. The laboratory consists of individual tasting booths equipped with computers, daylight equivalent lighting, and temperature control. Three replicate sessions were held whereby all six samples were presented and assessed according to a balanced presentation design. Data were collected using sensory software Compusense five (version 5.0, Compusense Inc., Guelph, ON, Canada) and data were exported and analyzed using XLSTAT (version 2014.6.05, Addinsoft 1995-2014. During training and formal sessions, samples of tofu and milk (~10-15 g) were presented to panelists in small plastic cups covered with plastic lids. Milks were freshly stirred and tofu was freshly sliced prior to serving. Table 2. Sensory descriptors and vocabulary from the training session in the preliminary evaluation of control and LOX-free soymilk and tofu. Statistical analysis All measurements in this study were replicated three times. The data were subject to analysis of variance to examine the effect of genotypes on each of the sensory attributes examined in soymilk and tofu. The means were separated using least square difference at the 5% significance level. Results Preliminary study using panels of "traditional" and "nontraditional" soy users Typically, the participants from each target group described the samples using similar terms. The control soy milk was paler in color than the LOX-free soy milk. Both groups described the LOX-free milk as being less intense and more subtle with more floury, cooked pasta-type aromas which also came through on the palate together with some sweet green snow pea-like flavors. The LOXfree milk was also described as sweeter than the control and less intense in flavor overall. The control soy milk was stronger in odor with raw green pea/bean, stale-earthy, and rancid-type aromas. The flavor was also stronger, raw, and grassy with some rancid oil, painty, and bittertype flavors. Similarly for the silken tofu samples, the control was described overall as being more intense in odor and flavor. The LOX-free tofu was described as having fresher/cleaner, more subtle attributes, while the control was described as being rancid (not fresh) and more intense aroma and flavor. Interestingly, the Chinese panel ("traditional soy users") described the control soy milk as "typical" Chinese-style whereas they described the LOXfree milk as unusual. Overall, the Chinese panel preferred the control soymilk and the LOX-free tofu while the Australian panel ("nontraditional soy users") preferred the LOX-free soymilk and did not seem to like either of the tofu products (Fig. 2). The presence or absence of the three major lipoxygenases was confirmed using SDS-PAGE (Fig. 1) and a screening method. The soymilk from the control and LOX-free soybean had similar solid content in soymilk (~11%). The major volatiles in these products as determined by GC-MS were hexanal with some 1-hexanol and butanol. A GC-MS profile showing the difference in the major volatiles in soymilk stored at 4°C for 1 day is presented in Figure 3. The peak area data (×100,000) revealed that all three volatiles in the control products were much higher than that in the LOX-free products, 1.85, 2.75, and 1.22 for control soymilk and 0.60, 2.05, and 0.42 for LOX-free soymilk for hexanal, 1-hexanol, and butanol, respectively; 4.5, 2.15, and 1.03 for control tofu and 1.25, 1.75, and 0.40 for LOX-free tofu for hexanal, 1-hexanol, and butanol, respectively. The difference in the major volatile, hexanal, between control and LOXfree products seemed to have increased after 8 days of refrigerated storage (Fig. 4). Sensory study on comparing control and Lx12 and Lx123 soy products The seed characteristics of the soybeans containing the three major lipoxygenases, lacking LOX-1 and LOX-2 (Lx12) or lacking all three isomers (Lx123) are presented in Table 4. The similar genetic background of the three soybean types, except for lipoxygenases, was expected to yield products with similar characteristics other than the beany aroma/flavor. While the seeds of Lx12 were smaller and, compared with the controls, contained more protein, Table 3. Sensory attributes and definitions developed and used in the sensory descriptive study of control and LOX-free soymilk and tofu. Aroma intensity The overall intensity of the samples aroma from subtle to intense Fresh pea aroma A fresh pea, cucumber, melon-like or lettuce aroma Grassy rancid aroma A grassy rancid aroma, sappy, sour, raw and painty, like rancid oil Floury aroma A floury, starchy, potato-like aroma, reminiscent of clay and earth Flavor intensity The overall flavor intensity of the sample when in the mouth Sweetness The perceived sweetness of samples experienced in the mouth Bitterness The perceived bitterness of samples experienced in the mouth the three groups had similar oil content and fatty acids profiles, in particular total saturated fatty acids, total PUFA, and the ratio of linoleic to linolenic fatty acids. As rancidity results from the oxidation of PUFA, the similar fatty acid composition provided an important basis for comparing differences in the sensory attributes or storage stability. After 1 week's storage at 37°C, the total peak area for the major volatiles detected using SPME-GC-FID increased over fourfold for the control full-fat soybean meal while the increase for double-null and triple-null samples was only 1.3-fold (P < 0.05). The sensory analysis of the soy products made from control soybeans or those lacking LOX-1 and LOX-2 (Lx12) or all three isomers (Lx123) showed clear and significant differences in the major aroma notes between these three groups for soymilk (Fig. 5A) or silken tofu (Fig. 5B). The objectionable rancid/grassy odors were rated the lowest by the taste panel in both soymilk and tofu from Lx123 soybeans, followed by Lx12 products with the control products given the highest rancid odor rating. The difference between these three soybeans was statistically significant for both soymilk and tofu (P < 0.05). Bitterness, another negative sensory note, was rated significantly lower in the soymilk and tofu made from the soybeans lacking two or three lipoxygenases compared with those made from the soybean containing all three isozymes (P < 0.05). Aroma intensity was also scored significantly lower for tofu produced from Lx12 and Lx123 soybeans, although this difference was not statistically significant for soymilk. On the other hand, sweetness, a positive sensory note, was rated significantly higher in the Lx12 and Lx123 products than the controls for both soymilk and tofu (P < 0.05). Fresh pea aroma was also rated higher in the Lx12 and Lx123 products than the controls, although this difference was only significant for tofu. Flavor intensity was rated differently by the taste panels for soymilk and tofu. It was scored lower for the control soymilk (P < 0.05) and higher for the control tofu, although this difference was not statistically different. Discussion Cultural difference and preference, as a result of longterm or traditional consumption of soy foods, has been reported in the perception of sensory attributes of soy foods, although the results are not clear-cut (Torres-Penaranda et al. 1998). The preference for control soymilk from an almost-native Chinese panel in this study was therefore expected as "traditional" soy users would associate the "beany" flavor with the typical flavor of soy milk. It is possible that consumers of traditional soy foods such as soymilk and tofu may find the products without lipoxygenases too bland due to the absence of the familiar beany flavor and taste. As a result, while soybean varieties lacking lipoxygenases have been developed in countries of traditional soy food consumption such as Korea and China, these varieties have not been widely taken up (K. Lee 2014 and M. Zhang 2013, pers. comm.). For "nontraditional" soy users, the traditional tofu is usually too bland in flavor and mushy in texture to their liking. They may still prefer the seasoned and harder products irrespective of lipoxygenases. Among the volatile compounds contributing to the beany/ rancid/off-flavor in soy products, hexanal is the major volatile responsible and has been studied most. The lower hexanal level observed in this study in the soymilk and tofu made from the LOX-free soybeans was consistent with the results from many studies which reported that LOX-free soybeans produce lower hydroxyperoxide and hexanal levels than those with LOX or lacking the LOX-1, -2, or -3 isozyme alone (Hildebrand et al. 1991;Kobayashi et al. 1995;Furuta et al. 1996;King et al. 1998;Ma et al. 2002Ma et al. , 2015. There have also been attempts to link soybean oil profile, lipoxygenase status, and off-flavor development in soy foods and a direct relationship between the degree of oil polyunsaturation, volatile compounds, hexanal level in particular, and off-flavor determining parameters has been reported (Furuta et al. 1996;Yuan and Chang 2007;Mandal et al. 2014). The significantly larger increase in the total volatiles in the full-fat soy meal after 1 week's storage at 37°C was similar to those reported by Kobayashi et al. (1995), although King et al. (1998) observed no difference in hexanal level between control and LOX-free oils and an increased hexanal level and peroxide values in LOX-free oil after 2 weeks' storage at 35°C and 50% humidity. The authors attributed these results to the differences in initial fatty acid composition. The effects of removing lipoxygenases from soybeans on the sensory attributes, in particular on the objectionable beany/grass flavor, of soy products are mixed and seem to be product-dependent. For example, Torres-Penaranda et al. (1998) reported significant less cooked beany aroma and flavor in soy milk made from the LOXfree soybean than that from the normal line, although no difference was observed in tofu in the same study. They also found significant interactions between soybean types and panelist ethnic backgrounds for raw beany aroma and flavor, which was attributed to the low intensity of the raw aroma and flavor making differentiation between soymilk from normal and LOX-free soybeans difficult. Wilson (1996) also reported that tofu made from LOX-2 null lines of soybeans were less beany than their respective controls as evaluated by a sensory panel. LOX-free soybeans, however, showed no effect in oils, breads, and meat patties (King et al. 1998(King et al. , 2001Liu et al. 2008). It is possible that the beany flavor was being masked by other stronger sensory notes in these products. In evaluating 70 genotypes including two series of near isogenic lines with or without lipoxygenase isozymes, no effect of lipoxygenase absence was observed on soymilk flavor parameters (Ma et al. 2015). Our results clearly showed that soybeans null in two or three of the major lipoxygenases positively reduced the negative sensory notes including rancidity, bitterness, and aroma intensity and improved the positive sensory notes such as sweetness and fresh pea aroma in soymilk and tofu. Soybeans lacking all three major isomers seemed to have an additive effect on reducing the negative notes in these soy products. Taken together, our results demonstrated that soybean seeds lacking lipoxygenases can produce whole-bean-based soy foods such as soymilk and tofu with improved flavor and aroma which are likely more acceptable to the consumers, particularly "nontraditional" soy users, and perhaps allow their utilization into other products. The soybean lines lacking lipoxygenases will also provide the Australian food industry with additional ingredients that would broaden and increase the utilization of soybeans and soy proteins in a wide range of food applications.

v3-fos

2018-12-27T08:37:47.042Z

2015-10-28T00:00:00.000Z

59403271

s2

Surveillance of Antibiotic Resistant Staphylococcus aureus in Agricultural Production Chain of Mongolia Monitoring of food borne pathogens in food is the primary tool for the implementation of food safety systems. It is necessary to monitor the prevalence of food borne pathogens for effective food safety planning and targeted interventions. Staphylococcus aureus is considered as the third largest cause of food related illness in worldwide. The present study aimed at surveillance of S. aureus contamination of meat on meat supply chain stages, which is a common benchmark of meat market in Mongolia, and characterization of isolated and collected strains from other agricultural sources. The cultural and polymerase chain reaction (PCR) methods were used for isolation, identification and characterization of S. aureus. In 216 cultures of S. aureus among 634 Staphylococci isolates obtained from different sources throughout the agricultural production chain in this study, common gene for S. aureus (98.74%), and nuc (97.47%), mecA (44.12%), msrA (9.66%), gyrA (32.77%) and ermC (29.41%) genes were identified. As seen in the surveillance result, the prevalence of methicillin-resistance S. aureus (MRSA) is 44% among S. aureus isolates from agricultural production chain. Confirmed cases of food-borne infections and intoxications caused by S. aureus should be considered as one of mean criteria of food safety issues in Mongolia, and special attentions should be paid on antibiotic resistant bacteria, such as S. aureus. S. aureus is considered as the third largest cause of food related illness in worldwide [4]. Monitoring the presence of food borne pathogens in food is the primary tool for the implementation of food safety systems. It is necessary to monitor the prevalence of food borne pathogens for effective food safety planning and targeted interventions [5]. Methicillin-resistant S. aureus (MRSA) that is resistant to virtually all β-lactam antibiotics is mediated by the chromosomally located mecA gene [6]. Livestock constitutes a potential reservoir of MRSA isolates belonging to a recently derived lineage within clonal complex 398 (MRSA CC398-IIa). Since its discovery in the early 2000s, this lineage has become a major cause of human disease in Europe, posing a serious public health challenge in countries with intensive livestock production. Various studies suggest that environmental contamination of air and D DAVID PUBLISHING Surveillance of Antibiotic Resistant Staphylococcus aureus in Agricultural Production Chain of Mongolia 702 contacted surfaces may also contribute to MRSA CC398 transmission [7][8][9][10]. Moreover, MRSA CC398 is a relatively common contaminant of retail meat in Europe, and food-borne transmission has been hypothesized as a possible source of infections in people with no livestock contact. However, epidemiological data suggest that food-borne transmission is rare [11]. Results of the studies in last years demonstrate that the use of antibiotics is now out of control and antibiotics resistance of bacteria is broadening in Mongolia [12]. Among cases of food-borne infections and intoxications in Mongolia, it caused by S. aureus is not rare. For instance, intoxications occurrence increased two times during last two years according to the Bacteriological Laboratory of National Center for Communicable Disease (NCCD), and a total of 216 coagulase-positive Staphylococci cultures were determined, as all cultures were sensitive to cefazolin and 79% to ciprofloxacin, but 60% were resistant to penicillin and ampicillin in 2012. Also an outbreak among soldiers in the Army Unit 167 in Umnugovi aimag was caused by S. aureus and S. aureus were detected in textbooks of school children in Orkhon aimag [13]. With development of molecular techniques, polymerase chain reaction (PCR) has become recently an important tool for detecting pathogenic microorganisms in food products by replacing the time-consuming culture-based classical techniques [14]. It is rapid, easy to handle, sensitive and specific, and constitutes very valuable tools for microbiological applications. Therefore, it has been essentially important to detect bacteria resistance to antibiotics, conduct surveillance of them, make risk assessments, improve diagnostic capacity and take control on veterinary drug use. Thus, the present study aimed to conduct surveillance of S. aureus, detect its virulence and antibiotic resistance and improve their diagnostic technology and proficiency testing. Sampling and Strains Collection Isolates, cultures and strains from six sources were used for studying antibiotic resistance of S. aureus as shown in Table 1 Isolation and Identification by Cultural Methods Specimen from animal products and fomites were planted on both nutrient and nutrient agar, a total of 225 mL of tryptic soya broth (TSB; Oxoid, Basingstoke, Hampshire, UK) containing 10% NaCl was added to 25 g of aseptically ground sample in a stomacher bag. Bags were stomached using a Stomacher 400 circulator (Seward, Inc., London, UK) at 230 rpm for 2 min, then incubated at 35 °C for 24 h [15]. Then, smear was prepared and stained by Gram's method and Gram positive clustered cocci were selected. Colonies were selected based on whether the cocci cause beta hemolysis on blood agar and form black colonies on Baird Parker selective agar. In order to differentiate staphylococci from other cocci, catalase test was used, while coagulase test was used to identify S. aureus from other Staphylococci [15]. Biochemical Test For identification of Staphylococci by biochemical characteristics, API Staph test kit (BioMerieux) was used as described in the manufacturers instruction [16]. Briefly, the following steps and procedures were used. The first step in this procedure is to make a saline suspension of the organism from an isolated colony. A Cultures taken from laboratories, such as NCCD Bacteriological Laboratory, SCVL, VLUC, NRLFS, FSHL-IVM and LIDI-IVM, and samples collected for last 3-4 years in these laboratories from the above mentioned sources. -: unknown numbers of samples. staph strip is then placed in a tray that has a small amount of water added to it to provide humidity during incubation. Next, a sterile pipette is used to dispense 2-3 drops of the bacterial suspension to each micro cupule. The inoculated tray is covered and incubated aerobically for 18-24 h at 35-37 °C. Finally, a seven-digit profile number is obtained and used to identify the bacteria. Antibiotic Resistance Test Antibiotic resistance and susceptibility of 216 S. min. Supernatant was removed and 200 µL distilled water was added into the precipitate, followed by mixing in vortex. Then, the mixture was placed in boiling water for 15 min and template was prepared by centrifuging at 1,0000 rpm for 10 min. DNA was extracted from blood and nasal swabs using the QIAamp DNA mini kit from Qiagen. The QIAamp DNA mini kit was used for the protocol of commercial guideline. To isolate DNA from meat, phenol-chloroform extraction method was used. Yield and purity of isolated DNA were measured by spectrophotometer at 260 nm and 280 nm wavelength and the purity ranged between 1.72 to 1.94. PCR Method For the surveillance of genes of S. aureus, which is resistant to both β-lactam and non β-lactam antibiotics, including oxacillin, methiciliin and erythromycin, the following primers shown in Table 2 and both of PCR and multiplex PCR methods for surveillance of antibiotics resistant genes were used [25]. Amplification In total of 35 cycles, there were such steps as initialization at 95 °C for 7 min, denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, elongation at 72 °C for 1 min and final elongation at 72 °C for 7 min, which was 10 min for multiplex PCR [25]. Gel Electrophoresis Mixture of 8 µL PCR product and 2 µL loading buffer was loaded in wells on the gel, and run in 1.5%-2% agarose gel depending on its DNA length. The gel was stained by ethidium bromide for 15 min and DNA fragments were visualized on transilluminator at 320 nm wavelength. Prevalence and Identification of S. aureus Of 861 samples taken from meat production and distribution chain, coagulase-positive Staphylococcus samples represent 10% by microbiological method, whilst 12% were coagulase positive Staphylococcus by PCR method (Table 3). In these positive samples, S. aureus was detected in 3.8% of animal samples, 5.8% of meat samples, 9.6% of environmental swabs and 3.8% of patients (Table 3, Fig. 1). For samples taken from food markets, S. aureus was detected in 14.42% of meat, 3.4% in carriers and 30.4% in fomite surface swabs (Fig. 2). Of 156 strains and cultures identified by laboratory examinations in the last three years, 83.7% were positive for mecA S. aureus. Furthermore, 3.6% of S. aureus identified from 360 horse samples (nasal swab and blood) of Selenge, Darkhan, Orkhon-Uul and Bulgan provinces were positive for mecA S. aureus (Table 4). Result of API Staph Test Isolates from fomites and animal products accounted for 61.3% and 28.7%, respectively, in total isolates and serotyping of staphylococci by API test. When serotype of 634 cultures of Staphylococcus by their biochemistry and enzyme activity identified, there were S. aureus (35.3%), S. xylosus (29.4%), S. hominis (17.6%) and S. saprophyticus (8.8%) (Fig. 3). Results of the study demonstrated that portion of S. aureus, which is the cause of infection and intoxication, was greater than other types and S. aureus is seen to be indicator of fomite borne infection (Fig. 4). Result of PCR Analysis on Virulence and Antibiotic Resistance Genes of S. aureus Although there were some differences in primers used for detection of antibiotics resistance gene and antibiotics discs for some antibiotics in the study. Types of both β-lactam and non β-lactam antibiotics were consistent, and result of antibiotics disc test for oxacillin and erythromycin was also consistent with that for PCR. Results were summarized in Table 5. β-lactams are preferred antibiotics used to treat serious S. aureus infections [26]. However, since 1961, when methicillin was introduced for clinical use, the occurrence of MRSA strains has steadily increased and MRSA infection have become a serious problem internationally [27,28]. Identification of MRSA strains in food animals led to concerns regarding food-borne contamination, and MRSA has been identified in retail meat in Europe, Asia, and North America [29][30][31]. The prevalence of methicillin-resistance is known to more than 70% among S. aureus isolates from hospitals in Korea [32]. According to the present study, MRSA accounts for 44.7% of all S. aureus cultures in Mongolia. Many MRSA isolates exhibit multiple resistance to the commonly used antimicrobial agents amikacin, oxacillin, penicillin, erythromycin and tetracycline [33,34]. PCR Sensitivity Test Result The comparative study using McNemar's test showed that PCR has sensitivity of 96.2%, whereas culturing method was 78.4% sensitive. There was discrepancy of 16.4% between both methods (Fig. 12) and that means microbiological analysis for processed and stored samples will be more reliable and less time-consuming if it is done at DNA level. In the present study, direct PCR was shown to be very effective in detection of the pathogens from meat sample homogenates, indicating that it is a robust method for rapid detection in comparison with culture technique which provides a significant contribution to both regulatory agencies and meat. Especially, differences of testing results for carriers can depend on the presence of a number of issues, such as human nutrition, immunity and use of medicines, which affect bacterial viability. As well, it is observed that difference between both methods is probable to depend on the genera of bacteria. Conclusions Results of this surveillances of mecA positive S. aureus in the present study and confirmed cases of food-borne infections and intoxications caused by S. aureus should be considered as one of mean criteria of food safety issues in Mongolia, and special attentions should be paid on antibiotic resistant bacteria, such as S. aureus. Due to the diversity of these resistance mechanisms and the constant appearance of new patterns, antibiotic utilization in developing countries should be under strict control and should be monitored to avoid the exhaustion of the antibiotic arsenal that is under intense use.

v3-fos

2019-04-04T13:02:14.745Z

2015-01-01T00:00:00.000Z

44226131

s2

Effect of Pre-treatments and Drying Methods on Dehydration and Rehydration Characteristics of Carrot The study was conducted to evaluate the effect of pre-treatments (0.1%KMS, 0.2%KMS, 0.3%KMS and blanching) and drying methods (mechanical drying and solar drying) on the dehydration and rehydration characteristics of carrot. The two drying methods yielded dehydrated products with different dehydration ratio, rehydration ratio, co-efficient of reconstitution and moisture content in both the dehydrated and rehydrated materials. It was seen that the drying time has been influenced by pre-treatments and drying methods. Pre-treatments increased drying time (for 0.3%KMS) and higher drying time (for 0.3%KMS) was required in solar drying. Pre-treatment, drying methods and boiling time affected the various rehydration properties. Highest rehydration ratio (3.70) and co-efficient of reconstitution (0.48) values were found for 0.1% KMS pre-treated mechanically dried and 0.2% KMS pre-treated solar dried carrots, respectively. Mechanical drying method was found better both for dehydration and rehydration properties for all the pre-treatments for example, 0.1%KMS pre-treated carrot gave co-efficient of reconstitution 0.48, while this value was 0.45 for same pre-treatment in solar drying. Introduction The carrot (Daucus carota) is a root vegetable, usually orange, purple, red, white or yellow in color, with a crisp texture when fresh. It is a rich source of β-carotene and contains other vitamins, like thiamine, riboflavin, vitamin B-complex and minerals. The consumption of carrot mainly as raw, juice, salads, cooked vegetable, sweet dishes etc [1]. In recent years, the consumption of carrot and its products have increased steadily due to their recognition as an important source of natural antioxidants besides, anticancer activity of β-carotene being a precursor of vitamin A [2,3]. Carrot being perishable and seasonal, it is not possible to make it readily available throughout the year. Dehydration of carrot during the main growing season is one of the important alternatives of preservation to develop further value added products throughout the year. Processing of carrots into products like canned slices, juice, concentrate, pickle, preserve, cake, and halwa are some of the methods to make this important vegetable available throughout the year. Drying of vegetable is an important means of preservation. Out of various methods available to extend the shelf life of perishable crops, dehydration is one of the easy and less expensive processes. Drying is a complex process involving transient heat and mass transfer and various factors should be taken into account [4]. For an optimized dryer and process design and improved drying process parameters and hence quality, heat and mass transfers in the product during drying should be analyzed. A number of internal and external parameters influence drying behavior. External parameters include temperature, velocity and relative humidity of the drying medium (air), while internal parameters include density, permeability, porosity, sorption-desorption characteristics and thermo-physical properties of the material being dried [4]. Pre-treatment improves nutritional, sensorial and functional properties of the dehydrated food without changing its integrity. It also improves the texture as well as stability of the pigment during dehydration and the storage of dehydrated product [5,6]. Carrot can be dried in either a mechanical dryer or a solar dryer. In mechanical dryer temperature usually remains higher than solar dryer. This leads to high production rates and improved quality products due to shorter drying time and reduction of the risk of insect infestation and microbial spoilage. Since mechanical drying is not dependent on solar light, it can be done as and when necessary. Many reports have been available regarding the procedures for rehydration and cooking of vegetables [7,8]. However, information required for standardization of the methods is scarce. Since quality of products may vary greatly with the methods of reconstitution, the importance or optimum conditions for reconstituting dehydrated product is evident. There are many factors, which affect the quality of dried fruits and vegetables for reconstitution. Soaking, period of soaking, temperature of soaking water, ratio of 24 Effect of Pre-treatments and Drying Methods on Dehydration and Rehydration Characteristics of Carrot water to dried products, rate of heating and length of cooking are some of the important factors for reconstitution [9]. But the effect of pre-treatments has not been studied yet. That is why, the present study has been conducted for analyzing the various effects of pre-treatments on the dehydration and rehydration characteristics of carrots and to observe the storage behavior of the dehydrated carrot. Materials and Methodology The experiment was conducted in the laboratory of the Department of Food Technology and Rural Industries (DFTRI), Bangladesh Agricultural University, Mymensingh, Bangladesh. The fresh and tender carrots (Daucus carota) were procured from local market. Chemicals used were of reagent grade and collected from laboratory stock of DFTRI. Medium density polyethylene bags were used for storage of dried samples throughout the experiment. Proximate Analysis The fresh carrots were analysed for moisture, protein, fat, carbohydrate, vitamin C, ash, titrable acidity, pH, total soluble solids and total carbohydrate content as per the methods summarized by [10]. Pre-treatments For this study fresh and tender carrots were selected and washed thoroughly with potable water. The carrots were then cut into 5 mm thick slices. For experimental studies the prepared carrot slices were blanched for 5 min. in boiling water. Three solutions were prepared in three different pans. These solution were contained 0.1% KMS (Potassium Meta bi-Sulphite), 0.2% KMS, and 0.3% KMS, respectively. Then prepared carrot slices were soaked in those solutions for 5 minutes. After 5 minutes, the carrot slices were separated from the solution and the surface water of carrot slices were removed by blotting with filter paper. Drying Methods Two types of drying were applied: Solar drying and Mechanical drying. Solar-drying Algate Solar Dryer (A.S.D) was used in this investigation. Algate dryer is a dryer in which black polythene is spread over a plane concrete or hard dried soil surface and the materials to be dried are placed on it. The black surface as well as the samples absorbs the solar radiation quickly, as a result, there is an increase in the heat inside the dryer which causes the faster removal of moisture from the product which are placed for drying. There is no temperature control system. So, the effect of temperature on the rate of drying cannot be determined. The blanched and sulphited (pre-treated) carrot slices were placed on a stainless steel tray to dry in the solar dryer. The tray load was 0.75 lb of prepared carrot slices per square ft. The time required for removal of moisture to safe level (5 to 7% moisture) was 16-20 hours and temperature was at a range of 30-60°C. The dried carrot slices were cooled, packed in polyethylene bags. After sealing the dried carrots were stored at room temperature in the laboratory for further investigations. Mechanical drying Cabinet drier, model OV-165 (Gallen Kamp Company) was used for the dehydration of carrot slices. The dryer consists of chamber in which trays of products were placed. Air was blown by a fan past a heater and then across the trays of products being dried. The carrots were dried with tray load of 0.75 lb/ft 2 at drier temperature of 60°C for periods upto 6-7hours. After drying to a safe level of moisture content (5-7%), the dried samples were cooled, packed in polyethylene bags and kept at room temperature in the laboratory. Procedure for Rehydration (Reconstitution) Rehydration is a process of refreshing the dried material in water. Both the solar-dried and mechanically dried carrots were reconstituted as follows: each sample was pre-soaked in water for 45 min. and then six beakers of each 500 ml capacity were taken and 150 ml water and 2g of dried sample were poured into each beaker. The contents were then boiled for 5, 10, 15, 20, 25, 30 minutes, respectively. The dried samples were added to the water when boiling started and counting of time began after that. It was necessary to add 20-30 ml of extra water for the last two tests to maintain the liquid level than for shorter boiling duration. After boiling, the liquid portion was drained off and excess water was removed by filter paper. The rehydrated materials were removed from the filter paper and weights were recorded separately and the following parameters were calculated. Percent Water in Rehydrated Material The percent water in rehydrated material was determined as per the methods of [10]. Studies on Storage Behavior of Dried Carrots Both solar dried and mechanically dried carrots were packed in sealed polyethylene bags (medium density film) and stored for 2 months at room temperature. Atmospheric temperatures and relative humidity over the storage period ranged from 27-34°C and 60-92%, respectively. The observations were made at 1-week intervals for moisture contents and organoleptic properties such as colour and flavour of dried products. Proximate Composition of Fresh Carrot The fresh carrot was analyzed for moisture, ash, fat, protein, vitamin C and total carbohydrate. The results are presented in Table 1. The results are almost similar to those reported by [11] except for moisture and carbohydrate content. They showed the nutritive value of carrot per 100 g edible portion as: moisture 86.00%, ash 0.70%, protein 1.70%, fat 0.30% and total carbohydrate 11.30%. The dry basis calculation showed that carrot is rich in protein content. It is seen that carrot contains higher amount protein than rice (9%db) and slightly lower than wheat (13.4%db). The variation in water content and carbohydrate content of carrot might be due to varietal difference, stage of maturity, the growing condition and the post-harvest storage condition of carrot. Drying Behavior of Carrot Experiments were conducted to determine the effects of pre-treatments and drying methods on dehydration and rehydration properties of carrot using a mechanical dryer and a solar dryer. The temperature of the mechanical dryer was 60°C, while the temperature in the solar dryer fluctuated from 30ºC to 60°C. This fluctuation was due to black body inside the drier which was covered by polyethylene and the intensity of sun light. Carrot slices were dried in the mechanical dryer (at 60ºC, at constant loading density, 0.75 lb/ft 2 ) and in the solar dryer (at 30-60ºC, at loading density 0.75 lb/ft 2 ) using single layer. The experimental data were analyzed by as per the Fick's 2nd law of diffusion and moisture ratio (MR) versus drying time (min) were plotted on a semi-log co-ordinate and regression lines and equations were obtained (Figure 1 Figure 1(a) was constructed to show first falling rate period taking only data for two hour period of drying although for complete drying 6-7 hours were required. From Figure 1(a) and the above developed equations it is seen that at constant loading density and constant temperature lower time is required for drying carrot slices with 0.1 %KMS pre-treatment than that required for 0.2% KMS and 0.3% KMS pre-treatment and higher time is required for 0.3% KMS pre-treatment to dry to a specific moisture ratio for mechanical drying. In case of 0.2% KMS pre-treatment time is required for higher than 0.1% KMS pre-treatment and lowers than 0.3% KMS pre-treatment. In other words, it can be said that KMS has profound influence on drying time and it offers higher resistance to both heat and mass transfer with resultant higher drying time for carrot with increasing KMS percentage and blanching also offers resistance on drying, as a result higher drying time is required in mechanical drying. From equations 1 to 4 it is seen that rate constant decreases gradually for drying of carrot with increasing percentage of KMS and also decreases during blanching but less than comparing with 0.3% KMS pre-treatment. This implies that at specific moisture ratio, more amount of water is evaporated per unit area for a given time from the samples of carrots with lower percentage of KMS than that of carrots with higher KMS when they are exposed to same drying atmosphere. Here, 3 rd highest rate constant was found for blanching pre-treatment. This behavior is attributed due to higher mass transfer resistance given by KMS and blanching treatment. From Figure 1(a), it is seen that the drying curves follow first order reaction kinetics and curves were drawn only to show first falling rate period. Similar characteristics were found by [12]. Here, rate constants decreases gradually with increasing percentage of KMS. It is also seen from Figure 1(a), KMS gives resistance to dry the pre-treated carrot. More percentage gives more resistance and takes more time to dry the sample to a specific moisture ratio. Similar properties were found by [13] and [12] where they showed that drying curves follow the first order kinetics and KMS increased the drying time. MR = 0.95e −0.0038t (For Blanching pre-treatment) (8) Figure 1(b) was constructed to show first falling rate period taking only data for two hour period of drying although for complete drying 16-20 hours were required. From Figure 1(b) and the above developed equations it is seen that at constant loading density and constant temperature lower time is required for drying carrot slices with 0.1% KMS pre-treatment than that required for 0.2% KMS and 0.3% KMS pre-treatments and higher time is required for 0.3% KMS pre-treatment to dry to a specific moisture ratio during solar drying. In case of 0.2% KMS pre-treatment time is required for higher than 0.1% KMS pre-treatment but lower than 0.3% KMS pre-treatment. In case of blanching pre-treatment, lowest time is required in comparison with 0.1% KMS, 0.2% KMS and 0.3% KMS pre-treatment to dry in solar drying. In other words, it can be said that KMS has profound influence on drying time and it offers higher resistance to both heat and mass transfer with resultant higher drying time for carrot with increasing KMS percentage and blanching offers least resistance on drying, resulting lower drying time is required in solar drying. From equations 5 to 8 it is seen that rate constant decreases gradually for drying of carrot with increasing percentage of KMS. This implies that at specific moisture ratio, more amount of water is evaporated per unit area for a given time from the samples of carrots with lower percentage of KMS than that of carrots with higher KMS when they are exposed to same drying atmosphere. Here, 2 nd highest rate constant was found for blanching pre-treatment. In mechanical drying method, for blanching, 2 nd highest rate constant was also found. This behavior is attributed due to higher mass transfer resistance given by KMS and blanching treatment. Similar behavior was found by [14]. In mechanical drying, the dryer took less time to dry the samples to a specific moisture ratio (MR) in compare to solar drying. Mechanical dryer also dried the samples more uniformly than solar dryer because of constant drying temperature. This was caused because in solar dryer temperature fluctuated from 30 to 60 o C. That is why, less uniformity was found in the solar dried samples. This characteristic influenced the rehydration properties of dried samples. Subsequently, diffusion co-efficient of mechanically dried and solar dried carrots were calculated using the equation (m = π 2 D e L 2 ). The D e values are listed in Table 2. It is seen from the Table 2 that, the Diffusion Co-efficient of mechanically dried and solar dried carrots decreases with increasing percentage of KMS. That means KMS has profound effect on Diffusion Co-efficient values. It is also seen from Table 2 that, mechanical drying method gave higher Diffusion Co-efficient value than solar drying method. Rehydration Characteristics of Carrots To investigate the rehydration characteristics, dried products were boiled for final reconstitution (the stage at which the absorption of water is maximum). It was found that there is difference between the rehydration characteristics of dried products with different pre-treatments even when the products were dried by the same drying method. The experiment (Table 3) showed that a maximum rehydration ratio for mechanically dried carrot (with 0.1% KMS pre-treatment) was 3.70 and for solar dried carrot (with 0.2% KMS pre-treatment) were 3.65. These were obtained for 25 min boiling in water bath which gives favorable condition for carrot. For solar dried carrot similar process was followed. It was seen that boiling up to 25 minutes gave the highest rehydration ratio (until disruption of structure) after which it was reduced a little bit. The various reconstitution data for mechanically dried and solar dried carrot with 0.1% KMS, 0.2% KMS, 0.3% KMS and blanching pre-treatments are shown in Table 3. For mechanically dried carrot, rehydration ratio with 0.1% KMS pre-treatment was higher than 0.2% KMS, 0.3% KMS pre-treated and blanching pre-treated carrot and they are 3.70, 3.50, 3.35 and 3.60, respectively. From this result, it is obvious that KMS and blanching have profound effect on rehydration of carrots. Higher percentage of KMS decreased the rehydration ratio and increased shrinkage. Cellular and structural disruption during blanching might have contribution to increase the rehydration rate of carrots and decreased shrinkage. It is also observed that after 25 minute boiling of the sample, the regained weight remained near about the same as the samples approached saturation condition. In case of solar dried carrots, rehydration ratio for 0.1% KMS, 0.2% KMS, 0.3% KMS and blanching pre-treatments was 3.50, 3.65, 3.30 and 2.70, respectively. There also have profound the effect of KMS and blanching on rehydration. These variations though very small, might have occurred due to the reasons that are mentioned for mechanically dried samples. The rehydration ratio values for solar dried samples are almost similar to those of mechanically dried samples, which are shown in Table 3. The rehydration ratio values were lower for solar dried samples than those of mechanically dried samples. It was also found that prolonged boiling reduces rehydration ratio which was due to the increase in leaching losses. On the other hand, shorter boiling time adversely affects rehydration ratio due to the inadequate absorption of solvents. For mechanically dried samples, the co-efficients of reconstitution were 0.47, 0.45, 0.44 and 0.46 for carrots with 0.1% KMS, 0.2% KMS, 0.3% KMS and blanching pre-treatments, respectively and are higher than those of solar dried samples 0.45, 0.48, 0.44 and 0.35 for 0.1% KMS, 0.2% KMS, 0.3% KMS and blanching pre-treatments, respectively. It indicates that mechanically dried carrots possess better reconstitution properties than solar dried counterparts. This behavior may be attributed to the change in rate of drying during two methods [15]. Mechanical drying gives higher rate of drying resulting in higher co-efficient of reconstitution than solar dried counterparts due to slower drying rate. As compared to the moisture content of the fresh carrots 87.77% (wb), rehydrated samples contained significantly lower moisture content (71.69-74.37%, wb) for mechanically dried and (65.44-74.43%,wb) for solar dried carrots, respectively. The low moisture content attained following rehydration due to the loss of water during drying process, with resultant increase in the concentration of dissolved substances in the tissue of vegetables. This may lead to the irreversible damage to the texture and these textural changes cause the tissues to shrink. As a result, upon reconstitution (depending on the conditions of drying), they were not able to regain their initial moisture content, volume (or weight) and tenderness. The dehydration ratio was found to be in the range from 7.66 to 7.83 for mechanically dried and 7.48 to 7.74 for solar dried carrots, respectively. Mechanical drying gives higher dehydration ratio than solar drying due to faster drying rate. Storage behavior of dried carrot Both solar dried and cabinet-dried carrots were packed in sealed polyethylene bags (medium density film) and stored for 2 months at room temperature. Atmospheric temperatures and relative humidity over the storage period were 27-34°C and 60-92% respectively. The observations were made at 1-week intervals for moisture contents and organoleptic properties such as colour and flavour of dried products. After one month stored products were remained almost unchanged without moisture content. After one month moisture content was found 6.25 and 7.92% (wb) for mechanical and solar dried carrots, respectively. At the time of packaging the moisture content of mechanically dried and solar dried samples was 5.16 and 6.68% (wb), respectively. The moisture content increased slightly after two month storage because the temperature and relative humidity varied Conclusions Carrot is a highly nutritious vegetable. During peak season due to lack of adequate processing facilities, farmers are bound to sell their produce at a very low price. But if farmers can process their produce by effective and economic ways, they will be able to get proper price and get encouraged to maximize production. Mechanical and solar drying systems along with different pretreatments may be used for both large scale and small industries.

v3-fos

2019-03-18T14:07:54.275Z

2015-01-27T00:00:00.000Z

55313991

s2

Field Evaluation of Sugarcane Orange Rust for First Clonal Stage of the CP Cultivar Development Program Corresponding Author: Duli Zhao USDA-ARS, Sugarcane Field Station, Canal Point, Florida, USA Tel: 011-1-561-924-5227 Fax: 011-1-561-924-6109 Email: duli.zhao@ars.usda.gov Abstract: Consistent development of high-yielding sugarcane (a complex hybrid of Saccharum spp.) cultivars with resistance or tolerance to biotic and abiotic stresses is critical to commercial sugarcane production. Currently, orange rust (caused by Puccinia kuehnii E.J. Butler) is a big challenge for the sugarcane production in Florida, USA. A better understanding of sugarcane genotypic variability in response to orange rust disease will help optimize breeding and selection strategies for disease resistance. Orange rust ratings, scaled from non-infection (0) to severe infection (4) with intervals of 0.5, were recorded from genotypes at the first clonal selection stage (Stage I) of the Canal Point sugarcane breeding and cultivar development program in Florida. Data were collected from all 14,272 and 12,661 genotypes and four replicated reference cultivars, CP 78-1628, CP 80-1743, CP 88-1762 and CP 89-2143, in July-August 2012 and 2013, respectively. Mean rust rating, % of rust infection and rust severity in each family (i.e., progeny of the cross from a female and male) and female parent and their Coefficients of Variation (CV) within and among families (females) were estimated. Results indicated that considerable variation exists in rust tolerance among families or females. The families or females for their progenies with the high susceptibility or resistance to orange rust were identified and ranked. The findings of this study are useful for evaluating sugarcane crosses and parents for rust disease and can help breeders use desirable parents for crossing and improve genotypic resistance to orange rust in the sugarcane breeding programs. Introduction Sugarcane (a complex hybrid of Saccharum spp.) is an important industrial crop in Florida, USA with an annual economic impact of more than $677 million (USDA-NASS, 2014). Consistent and continuous development of high-yielding sugarcane cultivars with resistance or tolerance to biotic and abiotic stresses is critical for commercial sugarcane production in South Florida (Zhao et al., 2010). The USDA-ARS Sugarcane Field Station at Canal Point (26.52° N; 80.36° W), Florida was initially established at its present site in 1920 to conduct sugarcane breeding and selection for Louisiana to make crosses and produce true sugarcane seed for the sugarcane industry. Since the 1960s, the Canal Point station has been developing sugarcane cultivars with CP prefixes for Florida under a three-party cooperative agreement among the USDA-ARS, the University of Florida and the Florida Sugar Cane League, Inc. Also, the Canal Point station still makes crosses for the USDA-ARS in Houma, Louisiana. The CP cultivars now account for more than 90% of the hectarage in Florida up from 14% in 1970. In 2012, the top six major sugarcane cultivars grown in Florida were 'CP 89-2143' (Glaz et al., 2000), 'CP 88-1762' (Tai et al., 1997), 'CP 00-1101' (Gilbert et al., 2008), 'CP 96-1252' , 'CL 88-4730' (a cultivar of the United States Sugar Corporation) and 'CP 78-1628' (Tai et al., 1991) and their percent hectares were 20.7, 19.2, 10.2, 8.6, 7.4 and 7.3%, respectively (Rice et al., 2013). The Canal Point sugarcane breeding and cultivar development program (CP program) consists of six stages, namely Crossing, Seedlings and Stages I, II, III and IV (Zhao et al., 2012). It takes at least eight years to release a cultivar from the time a cross is made (Tai and Miller, 1989). Cane yield (TCH, Mg ha −1 ), commercial recoverable sucrose (CRS, kg sucrose Mg −1 cane) and sucrose yield (TSH, Mg ha -1 ) with disease resistance are the major agronomic traits considered in advancing sugarcane clones during the selection stages. reported that CRS, TCH and TSH of Florida commercial sugarcane cultivars linearly increased by 26.0, 15.5 and 47.0%, respectively, from 1968 to 2000. Underscoring the critical need for cultivar development for the Florida sugarcane industry, about 69% of the sugar yield gain in Florida came from genetic improvement attributable to the CP program , indicating importance of the CP program in sugarcane production in Florida. Sugarcane orange rust, caused by P. kuehnii (W. Krüger) E.J. Butler, resulted in considerable economic loss in Australia (Braithwaite et al., 2009). This disease was recorded in Florida for the first time in 2007 . Sugarcane orange rust has caused significant yield losses in Florida because most widely planted commercial cultivars in the region are susceptible to this disease (Raid et al., 2013). Orange rust has also been found in sugarcane growing in the Central American countries of Guatemala (Ovalle et al., 2008), Costa Rica (Chavarría et al., 2009), Nicaragua (Chavarría et al., 2009), Mexico (Flores et al., 2009), El Salvador (Flores et al., 2009), Panama (Flores et al., 2009) and more recently in Brazil (Barbasso et al., 2010) and in Louisiana, USA (Grisham et al., 2013). Zhao et al. (2011) determined physiological mechanisms of the orange rustinduced reductions in sugarcane growth and yield by quantifying effects of this disease on leaf relative chlorophyll level, photosynthetic rate, dark respiration rate, photosynthetic radiation use efficiency, carbon use efficiency and the relationships between these leaf photosynthetic components and rust disease ratings. Currently, leaf orange rust and brown rust (caused by Puccinia melanocephala H. & P. Sydow) diseases are great challenges for sugarcane production in Florida. Most dominant commercial cultivars in Florida are susceptible to one or both rusts. Growers use fungicides to control the negative effects of rusts on yields, but the cost of three split applications of fungicides (at a hectarage level) during a growing season is equivalent to 3 tonns of cane yield lost per hectare. The frequent applications of fungicides and low net profits preclude their use for controlling sugarcane brown rust (Jiang, 1985) and orange rust (Staier et al., 2003), making cultivar resistance and cultural practices are the most viable alternatives. Therefore, development of rust resistant varieties is the first priority for sustaining sugarcane production in Florida. Scientists in the CP program and in industry are using multiple approaches to develop new cultivars with rust resistance and high yields. Cultivars developed in the CP program not only are used in the United States, but also many Central American sugarcane industries use them for either breeding or commercial production (Machado, 2013). Therefore, evaluation and screening of genotypes for resistance to rusts in the CP program are critical for sustainable sugarcane production in USA and other countries. Although Sood et al. (2009) developed a whorl inoculation method to more accurately and efficiently test sugarcane genotype resistance in brown rust (Sood et al., 2009) and orange rust (Zhao et al., 2011), it is still difficult to use the artificial inoculation test in Stage I of the CP program because of a large number (12,000-15,000) of genotypes in this stage due to limited resources (Zhao et al., 2012). Therefore, natural infection has been the primary means of assessing rust resistance in the Stage-I sugarcanes in the CP program. The selection of which sugarcane clones to be used as crossing parents is a crucial decision for breeders. Knowledge and better understanding of variability in rust infection and severity among genotypes may provide useful information for genotype advancement and for efficient use of parents in future crossing efforts for developing orange rust resistance. Thus, a study was conducted in the Stage I fields of the CP program at the USDA-ARS Sugarcane Field Station, Canal Point, Florida. Objectives of this study were to determine variability in orange rust ratings among crosses based on data collected from the Stage I clones of the CP program in 2012 and 2013 and to use the information for consideration of parental selection and cross appraisal. Overall, orange rust in the 2012 and 2013 sugarcane growing seasons in south Florida were the most severe in the last five years due to favorable environment conditions for rust development. The data of orange ratings in Stage I may be useful for achieving the specific breeding objectives for rust resistance. Plant Culture Individual stalks of 14,272 genotypes in 2012 and 12,661 genotypes in 2013 were visually selected from fields of the 2011 and 2012 Seedling stages and planted in single-row plots in the Stage I fields of the CP program in January of 2012 and 2013, respectively. All the visually selected genotypes in the Seedling stages did not show clear orange rust disease symptoms due to late transplanting (Mid May) in the CP program. To facilitate stalk transport and planting, two to five stalks (each stalk came from a true seed plant) in the Seedling fields were bundled and labeled by family (i.e., cross) prior to advancing them to Stage I (Zhao et al., 2012). These bundles were randomly distributed in the Stage I fields. One stalk was placed in each plot and cut into two sections (each approximately 0.9 m long). The two sections were placed in the center of the plot as double pieces of seedcane. The plot length was 2.4 m, with 1.5m between-row spacing. There was a 1.5-m gap between adjacent clones within a row to allow scientists to recognize individual clones during disease rating, growth vigor evaluation and selection and to easily distinguish individuals at selection time. Four commercial cultivars, CP 78-1628, CP 80-1743(Deren et al., 1991, CP 88-1762 and CP 89-2143, were used as checks each year and one check plot was randomly planted in approximately every 100 plots. There were approximately 40 replicated plots for each check cultivar each year. There was a 4.5 m alley every eight rows to facilitate field maintenance and genotype selection. Evaluation of Orange Rust During the grand growth (July-August) in 2012 and 2013, orange rust ratings were recorded in all plots of Stage I under natural infection using a scale from 0 (no rust infection) to 4 (most severe rust infection) with intervals of 0.5. The 0 to 4 scale levels were defined as: 0 = no rust, 1 = one to a few pustules, 2 = patching presented, 3 = patching widespread up into the upper canopy with some lower leaf death and 4 = massive amounts of rust pustules with heavy lower leaf death. In general, the plants with rust ratings of 0 to 1 were considered resistant or tolerant, with rating of 2 were considered moderately susceptible and with ratings of 3 and 4 were considered susceptible and highly susceptible. Additionally, all clones were visually evaluated for other diseases [brown rust, leaf scald (Xanthomonas albilineans), smut (Sporisorium scitamineum) and mosaic] and plant vigor and agronomic traits (Zhao et al., 2012) at the same time and in early September. A subjective plant vigor rating was determined for individual clones using a scale from 1 (worst) to 9 (best). All clones with a vigor rating ≥6 or better than that of check cultivars, acceptable rust resistance (rust rating ≤1) and no other disease symptoms were further assessed for Brix (an indicator of sucrose content) in early November. Approximately 1,500 best clones with the largest vigor × Brix products and proper disease resistance were advanced to Stage II. Recently, Zhao et al. (2012) reported the details of vigor rating, Brix and the Stage-I selection strategies in the CP program. Therefore, we mainly focused on orange rust on the basis of families and female parents in this study. Three variables of mean orange rust rating, percent of rust infection and rust severity were used to determine variation of rust diseases among families or females based on their progenies. These variables were defined and estimated using the following formulas: Mean orange rust rating = Σ (rust rating) ÷ total number of clones (including clones with 0 rating) Percent of infection = (the number of clones infected ÷ total number of clones) × 100 (2) Severity = Σ (rust rating) ÷ the number of clones infected (3) Data Analysis For the four check cultivars, replicated plots were completely randomized in the Stage-I field both years. The MIXED procedure of SAS (SAS Institute Inc., Cary, NC) was used to test differences of cultivar, year and their interaction for the mean rating of orange rust and for rust severity. If the hypothesis of equal means between the check cultivars was rejected by the F test, the trait means were separated with the LSD at P = 0.05. The LSD values were calculated with the SE values generated by the Diff option in the SAS MIXED procedure. For the Stage I clones, their parental combinations in the Crossing stage varied annually. Thus, the Stage I data were analyzed separately for each year. Orange rust distributions were determined by pooling data across all clones within a year. Data of orange rust were analyzed for each family and female parent. Data of male parents were not analyzed because many of the progeny in the 2012 and 2013 Stage I were developed from Poly crosses (i.e., where a female tassel received pollen from several different male tassels and their male parents could be any one of these males). Thus, the specific males were unknown for all the Poly crosses. For families and female parents that had ≥15 progeny clones planted in Stage I, the mean ratings and Coefficients of Variation (CVs) were determined for orange rust to assess variability. The means and CVs were calculated using PROC MEANS of SAS. Then, the CVs of amongfamilies (females) were obtained based on their means and the CVs of within-families (-females) were estimated by averaged CVs of individual families (females). Coefficients of variation for the rust in each family were calculated from the individual clonal values of rust rating from all clones within a family or female according to Zhao et al. (2012). The within-family or within-female CV for each parameter was estimated by calculating the overall mean CV of all individual family (female) CVs for that trait. The among-family CVs were estimated using the mean (rather than individual clone) values of each family (female). For example, to calculate the among-family CV of 20 families for orange rust, we would have calculated the CV based on the standard deviation and overall mean from the 20 mean rust rating values of each of the 20 families. The variability amongand within-families was described using respective CVs. The top 20 families that were most susceptible or most tolerant to orange rust in each year were further determined based on ranking their mean rust ratings. The top 20 females in which their progenies were most susceptible or most tolerant to the rust in each year were also determined based on mean ratings of orange rust. Orange Rusts of Check Cultivars There were significant differences among check cultivars and between years in mean ratings of orange rust and the cultivar × year interactions were also highly significant (Table 1). The mean rating of orange rust in 2012 was lower than that in 2013. Averaged across the four cultivars, mean ratings of orange rust in 2012 and 2013 were 1.0 and 2.0, respectively. CP 78-1628 had significantly lower mean rating of orange rust than other three cultivars in both years. The differences in % infection of orange rust among cultivars or between years were similar to those in the mean rust ratings. On the other hand, the differences in severity were relatively less among cultivars or between years compared with % infection (Table 1). Orange rust severity differed significantly among cultivars, but did not differ between years. There was no interaction of cultivar × year on orange rust severity (Table 1) Distributions of Orange Rust Ratings Numbers of clones used for orange rust ratings in Stage I of the CP program were 14,272 in 2012 and 12,661 in 2013 (Fig. 1). These did not include 158 and 166 replicated check plots in 2012 and 2013, respectively. The % infections of orange rust in 2012 and 2013 were 18.3 and 50.2%, respectively. The distributions of orange rust ratings based on the number of clones at each rating level are given in Fig. 1. Although the peak frequency of the rating distribution for orange rust was at 2 and the overall severity value was also approximately 2 in both years, the peak value of orange rust in 2012 was smaller than that in 2013 (Fig. 1). The CVs of orange rust ratings across the infected clones in 2012 (2,607) and 2013 (6,356) were 19.8 and 28.8%, respectively. Additionally, the % infection and mean rating of orange rust in 2013 were much greater than in 2012. The differences in severity between years were small (Fig. 1). Variability in Clonal Numbers and Rust Ratings among Families Total crosses (families) advanced to Stage I from the Seedling stage by individual selection were 576 and 455, respectively, in 2012 and 2013. Clone numbers among families ranged from 1 to 214 with a mean of 25 in 2012 and from 1 to 209 with a mean of 28 in 2013 and their CVs in two years were 113 and 100%, respectively (Table 2). Clone ( Large variability in the number of clones and in rust ratings (Table 2) among families in Stage I of the CP program suggested that Stage I data can be used to identify useful parental combinations and individual parents for their progenies to be resistant/tolerant to orange rust in the test years. The greater CV values for rust rating within families than among families suggested that placing more emphasis on both individual clonal evaluation and family-based evaluation in Stage I of the CP program may help improve our knowledge and ability to select genotypes with potential for eliminating rust effects. Correlations of Mean Rust Ratings with % Infection and Severity In 576 (2012) and 455 (2013) families planted in Stage I, 295 and 263 families, respectively, had ≥15 clones. From these families with ≥15 clones, the mean ratings, % infections and severity values of orange rusts for each family were calculated (Fig. 2). Overall, the mean ratings of orange rust ranged from 0.0 to 2.1; the values of % infection ranged from 2.3 to 100.0% and the values of severity (i.e., mean ratings calculated based on infected clones) ranged from 1.0 to 3.0. Averaged across years, the CV values of the mean rating, % infection and severity were 69.8, 41.8 and 11.8%, respectively among these families. The mean ratings of orange rust highly correlated with % infection (r 2 = 0.92-0.97), but the relationships between the mean ratings and severity values were poor (r 2 = 0.14-0.39) (Fig. 2). Consideration of Variability (CV) and correlation between the three rust variables, the rust severity might not be a good parameter to distinguish family differences in response to orange rust in the early selection stage of sugarcane breeding program. (16) Evaluation of Families Based on Mean Rust Ratings The families with ≥15 clones were also used to evaluate family tolerance to orange rust. The 295 and 263 families that had ≥15 clones planted in Stage I in 2012 and 2013, respectively, were ranked based on their mean ratings of orange rust (Tables 3 and 4). The mean orange rust ratings across families were 0.4 (ranged from 0.0 to 2.2) in 2012 and 1.0 (ranged from 0.1 to 2.1) in 2013 with the CV values of 94 and 47%, respectively. Top 20 families with the highest (Table 3) and 20 families with the lowest (Table 4) mean rust ratings in each year were identified. The % infection and severity of orange rust for the families and their parents are also listed in Tables 3 and 4. Orange rust had greater variability (CV) in the mean rating and in severity within the family than among families and the CV values of the mean orange rust rating were greater than those of orange rust severity. Parental information of these families might help breeders select proper parents for crossing to develop genotypes with resistance to orange rust. The results of orange rust suggest that using CP 94-2203, CP 04-1250 and CP 99-1896 as female parents will increase probability to produce progenies with high susceptibility to orange rust (Table 3). In contrast, using CP 06-2664, CP 92-1167 and CL 88-4730 as female parents may improve orange rust resistance of progeny (Table 4). Evaluation of Females Based on Mean Rust Ratings of Their Progenies When data were analyzed by female parents regardless of families and males, there were 204 and 157 females used for generating the Stage I clones, respectively, in 2012 and 2013. Of these females, 135 in 2012 and 113 in 2013 females had at least 15 progenies planted in the Stage I fields. The progenies of these females were sorted by the rust mean ratings. The 20 females with their progenies having the highest (Table 5) and 20 females with progenies having the lowest ( Table 6) mean rating of orange rust were further identified and ranked. Orange rust is one of the most devastated sugarcane diseases for sugarcane production in Florida. Using proper parents with resistance to orange rust to make crosses is the priority for the CP program. There was great variation in orange rust ratings among families (Tables 3 and 4) in Stage I of the CP program. The great variation in mean ratings of orange rust among female parents for their progenies was also detected (Tables 5 and 6). Clearly, using clones listed in Table 5 as female parents increased the risk to get progenies with high orange rust ratings. In both years, progenies from females CP 94-2203, CP 04-1250, CL 87-1630, CP 88-1762, CPCL 99-2103 and CP 00-2188 had high mean rating of orange rust and ranked in the top 20 (Table 5). These female parents should be limited in the CP program to develop genotypes with resistance to orange rust. Overall, the progenies developed from female parents listed in Table 6 had the lowest mean ratings of orange rust. Increase in proportion of these female clones for crossing might improve resistance to orange rust of progenies in Stage I of the CP program. Discussion Sugarcane orange rust is a devastating disease for sugarcane production in the United States because most commercial cultivars are susceptible to this disease and it caused substantial yield loss of susceptible varieties (Raid et al., 2013). Multiple approaches have been and are being used for eliminating rust negative effect on yield of sugarcane. These include disease monitoring, adjustment of management practices, fungicide applications (Raid et al., 2011) and development of new cultivars with resistance to orange rust by intensive screening in the breeding program (Zhao et al., 2013). The increase in frequent applications of fungicides results in high input cost and low net profits of sugarcane production (Jiang, 1985;Staier et al., 2003). Therefore, development of rust resistant varieties is important for sustaining sugarcane production and for improving production and profitability. In the present study, we investigated orange rust disease in the first colonal stage of the CP program based on three parameters of mean rust rating, percentage of infection and severity and found that great variability (CV) existed in both mean rust ratings and % infection across large numbers of clones and families. Therefore, these two parameters can be used to evaluate genotypes for orange rust resistance. The rust severity might not a good parameter to distinguish family differences in response to orange rust in the early selection stage of sugarcane breeding program because of relatively smaller CVs than the mean rust rating or % infection. It is important for sugarcane breeders to develop a data base of parental clones using agronomic and physiological traits and molecular markers for disease resistance and for yield and profit improvement. Virtudazo et al. (2001) conducted phylogenetic analysis of sugarcane rusts, including orange rust, based on rDNA sequences. Sugarcane brown rust resistance gene (Bru1) has been used as a marker to identify if sugarcane genotypes are potentially resistant to brown rust (Asnaghi et al., 2004). This technology was also utilized in the CP program to direct breeding strategies for brown rust resistance (Glynn et al., 2013). Scientists are working on developing a reference genomic map and identifying the markers linked to sugarcane orange rust resistance genes (Yang et al., 2014). However, the molecular markers for orange rust resistance are not commercially available yet. Traditional breeding approaches for rust resistance have involved a better understanding of inheritance of resistance in seedling populations (Ramdoyal et al., 2000) and selection and advancement of genotypes that are free from visual disease symptoms. To evaluate performance of sugarcane families in some specific traits, Wang et al. (2008) suggested that at least 10 to 20 clones per family were required. Therefore, the families that had ≥15 clones and females that had ≥15 progeny clones were used in the present study to assess orange rust resistance at the family and female parental levels based on the disease data collected from individual clones. Our results indicated that orange rust ratings collected in the first clonal stage can be used not only for advancing elite clones to next stage of the CP program as one of selecting criteria, but also for evaluating families and their parents for orange rust resistance. These findings could be useful for breeders to design cross combinations and to improve selection efficiency of new cultivars for rust resistance. Shanthi et al. (2008) suggested that parents producing progeny with a high frequency of transgressive recombination for agronomic traits should provide the best opportunity for sugarcane breeders to select clones superior to their parents. In addition to directly evaluating parental lines for disease resistant traits, therefore, parental evaluation based on their progeny performance for rust resistance and other agronomic traits in the early clonal stage of a sugarcane breeding program with large numbers of clones might help optimize parental selection and crossing combination. Quantitative analyses of sugarcane clonal data of rusts in Stage I of the CP program could help us evaluate not only crosses (families), but also their parents as described above. Studies have suggested that family selection is effective in improving sugarcane populations in early selection stages (Chang and Milligan, 1992;Cox and Hogarth, 1993;McRae et al., 1993;Shanthi et al., 2008) because it can identify those families that harbor the highest proportion of desirable clones and makes it possible to focus selection for superior clones (Shanthi et al., 2008). Availability of family data in rust diseases helps sugarcane breeders improve crossing combinations for developing genotypes with resistance to diseases and high yields. Conclusion Early stage selection methods and specific selection strategies of sugarcane cultivar development programs are dependent on environmental conditions and the unique goals of each selection program. Analyses of orange rust data collected from large numbers of individual clones in Stage I of the CP program in 2012 and 2013 revealed that there was great variation in orange rust ratings among genotypes and among families. Our results indicated that using the rust rating data along with individual selection data on plant vigor and stalk juice Brix (Zhao et al., 2012) for making comparisons of family performance and among-and within-family variability would improve our parental selection and optimize crosses among selected parents, which should then improve the progenies for rust resistance and yield potential in the sugarcane breeding programs.

v3-fos

2018-04-03T00:05:44.027Z

2015-03-31T00:00:00.000Z

5042661

s2

Effects of Functionalized and Raw Multi-Walled Carbon Nanotubes on Soil Bacterial Community Composition Carbon nanotubes (CNTs) are widely used in industry, but their environmental impacts on soil microbial communities are poorly known. In this paper, we compare the effect of both raw and acid treated or functionalized (fCNTs) multi-walled carbon nanotubes (MWCNTs) on soil bacterial communities, applying different concentrations of MWCNTs (0 µg/g, 50 µg/g, 500 µg/g and 5000 µg/g) to a soil microcosm system. Soil DNA was extracted at 0, 2 and 8 weeks and the V3 region of the 16S rRNA gene was PCR-amplified and sequenced using paired-end Illumina bar-coded sequencing. The results show that bacterial diversity was not affected by either type of MWCNT. However, overall soil bacterial community composition, as illustrated by NMDS, was affected only by fMWCNT at high concentrations. This effect, detectable at 2 weeks, remained equally strong by 8 weeks. In the case of fMWCNTs, overall changes in relative abundance of the dominant phyla were also found. The stronger effect of fMWCNTs could be explained by their intrinsically acidic nature, as the soil pH was lower at higher concentrations of fMWCNTs. Overall, this study suggests that fMWCNTs may at least temporarily alter microbial community composition on the timescale of at least weeks to months. It appears, by contrast, that raw MWCNTs do not affect soil microbial community composition. Introduction Carbon nanotubes (CNTs) are widely used in novel industrial materials because of their particular chemical and physical characteristics [1][2][3]. They are currently used for-or under development in-electron emission devices, energy storage devices, drug delivery mechanisms, and a range of other engineering applications [4][5][6][7][8][9]. Carbon nanotubes (CNTs) are single atom layers of hexagonal carbon rolled up into hollow cylinders. They are classified as single-walled Previous studies on CNTs in the soil system have been carried out by Chung et al. [2] who demonstrate that short-term exposure (20 days) to multi-walled CNTs can lower most enzymatic activities and overall microbial biomass in soils, at exposures of around 5000 μg of MWCNTs per gram of soil. The same group [32] has observed similar effect of CNTs on soil enzyme activities and microbial biomass at the concentration 300-1000 μg/g but using singlewalled CNTs (SWCNTs). Another short-term study indicates that SWCNTs may alter the structure of activated sludge microbial communities [37]. Furthermore, many of the previous studies used untreated CNTs, which are known to be hydrophobic, mixing poorly with soils. Only a subset of studies has used the far more easily miscible acid-treated CNTs (functionalized or fCNTs), which are also commonly used in industry [32], and which seem more likely to interact with the soil ecosystem. Here we set out to understand the effect of both treated (fMWCNTs) and untreated MWCNTs on soil bacterial communities at a range of taxonomic levels. We consider shifts in both relative abundance and diversity. The relative abundance of certain groups might be important because particular taxa are consistently associated with ecological functions in the soil, especially at the finer taxonomic level. Diversity may be important because it is widely thought that ecosystem resilience is affected by taxonomic diversity [38][39][40]. Furthermore, we continued our experiment for 8 weeks; most other studies ran for less than 4 weeks [2,32]. Given that soil bacteria are generally thought to be slow growing with a high proportion of dormant cells much of the time, a longer duration of study seems desirable, being more likely to show relevant shifts in ecology. Shrestha et al. [17] evaluated the impact of MWCNTs on soil microbial community structure and functioning in soil over 90 days of exposure and they found no effect on soil respiration, enzymatic activities and microbial community composition. However, at the highest (MWCNTs) concentration (10,000 mg/kg), shifts in microbial community composition and abundance of some bacterial genera were observed. Essentially our hypotheses were as follows: 1. That fMWCNTs will have a greater effect on soil bacteria than untreated MWCNTs, due to their greater ability to mix with soil water and interact directly with bacterial cells. This higher concentration of MWCNTs will also have stronger effects. 2. That given the relatively slow and long-term nature of soil processes, including bacterial community shifts, the 8-week timeframe will show a different result from the much shorter 2-3 week timeframe used in most previous studies. The effect may intensify, as more cells die or are unable to reproduce under the effect of the MWCNTs. In the present study, we use paired-end Illumina bar-coded sequencing of hypervariable V3 region of 16S rRNA gene to investigate the impact of multi-walled carbon nanotubes on soil bacteria. Soil sampling Soil samples were collected in June 2013 from an overgrown flowerbed on Seoul National University campus, which is located in the Gwanak Mountain area, south of Seoul. This sampling site was selected because it represents a typical and widespread type of soil (slightly acidic sandy loam) in South Korea. The upper 10 cm of soil was sieved and thoroughly mixed in a sterile container. The soil was divided into pots containing 200 g soil each. Soil pH was measured using a soil pH meter (Hanna Instruments HI 99121N Direct Soil pH Meter). Soil pH was around 6.1 and it contained 10.4% clay, 18.4% silt and 71.2% sand. Soil texture and organic matter content were measured at National Instrumentation Center for Environmental Management (NICEM, South Korea) following the standard protocol of SSSA (Soil Science Society of America). Preparation of MWCNTs Pure commercial MWCNTs were purchased from Hanwha Nanotech, Republic of Korea. Two forms of CNTs were used in the present experiment: raw and functionalized forms of MWCNTs. The powder MWCNTs were not treated, but used in the form received from the purchaser because this is a form that microorganisms might encounter if there is an accidental release from a manufacturing facility. The functionalized MWCNTs are more commonly used during fabrication processes of commercial products, so would more likely be released at those sites [32]. The MWCNTs were functionalized (fMWCNTs), following the protocol described by Saito et al. [41], by attaching carboxyl groups (-COOH) to their surfaces using acidic solutions [42]. A mixture of H 2 SO 4 :HNO 3 = 3:1 (v:v) were added to the raw MWCNTs at room temperature [43]. The mixture was bath sonicated for 24h, followed by vacuum filtration through 0.22 μm Millipore Teflon membrane (JGWP04700). Then, the membrane was thoroughly washed using deionized (DI) water, and was immersed in DI water according to established protocols [44]. The MWCNTs were then dried overnight in the oven at 60°C. Characterization of MWCNTs MWCNTs were characterized using Energy-Filtering transmission electron microscopy (EF-TEM: LIBRA 120, Carl Zeiss, Germany) and field-emission scanning electron microscopy (FE-SEM: S-4800, Hitachi, Japan). These techniques were effective in characterizing the internal structure (diameter and wall number) of MWCNTs. Raman spectra were taken to determine the diameter distribution using LabRam Aramis (Horiba Jobin-Yvon, France). The metal components in the MWCNTs are less than 5% in weight. They have been analyzed by the manufacturer (Hanwha Nanotech, Republic of Korea) and are aluminum, iron, and molybdenum. Soil incubation The soil was divided into plastic self-draining pots containing 200 g soil each, to give 3 replicates to be exposed to each concentration of raw MWCNTs or fMWCNTs. The concentrations of MWCNTs applied to soil were 0 (DI water only), 50, 500, and 5000 μg/g soil. The soils were then well mixed to ensure homogeneity before incubation in a BOD incubator at 25°C for 8 weeks. The pots were not covered to allow free gas exchange to the soil microbial community. The positions of replicate pots of different treatments were randomized and randomly interchanged each week. Soil moisture was adjusted to 60% water holding capacity. Soil moisture content was maintained by weighing the pots twice a week and adjusting to initial weight by regular addition of DI water. Samples of 3 g of soil were collected from each pot, at different time points (0, 2, and 8 weeks), to be used for DNA extraction. At T = 0 weeks, we took samples almost immediately (1 hour later) after adding MWCNTs to soil. DNA extraction and sequencing The soil DNA was extracted from 0.3 g of the mixed 3 g sample of soil, using the Power Soil DNA extraction kit (MO BIO Laboratories, Carlsbad, CA, USA) following the protocol described by the manufacturer. DNA isolated from each sample was amplified using primers 338F (5 = -XXXXXXXXGTACTCCTACGGGAGGCAGCAG-3 =) and 533R (5 = TTACCGCGGCTGCT GGCAC-3 =), targeting the V3 hypervariable regions of the bacterial 16S rRNA gene (the X sequence denotes a barcode sequence) [45]. The Polymerase chain reactions (PCR) were carried out under the following thermal profile: denaturation at 94°C for 2 min, followed by 25 cycles of amplification at 94°C for 30 s, 57°C for 30 s and 72°C for 30 s, followed by a final extension of 72°C for 5 min. PCR products were analyzed by electrophoresis in 1% agarose gels and were purified using Wizard SV Gel and PCR Clean-up System (Promega, USA). The paired-end sequencing was performed at Kim lab incorporation (Yonsei University, Seoul), using a paired 150-bp HiSeq 2000 sequencing system (Illumina) according to the manufacturer's instructions. Library preparation, sequencing and initial quality filtering were performed as described previously [46]. Quantitative PCR analysis Relative abundance of bacterial subunit rRNA gene copies was quantified using quantitative PCR (qPCR). Standard curves were created using a 6-fold serial dilution (10 -2 to 10 -7 ) of a plasmid containing a full-length copy of the Escherichia coli 16S rRNA gene, to estimate bacterial relative abundance. qPCR assays were conducted in 48-well plates. Each 10 μl reaction contained 5 μl of reaction mixture (2X Real-Time PCR Smart mix), 0.5 μl of forward and reverse primers (Eub 338 and Eub 518), and DNA-free water. PCR conditions were 2 min at 50°C, and 15 min at 95°C, followed by 40 cycles of 95°C for 60 s, 53°C for 30 s and 72°C for 45 s. Melting curve analyses was performed to confirm that the amplified products were of the appropriate size. Each plate included triplicate reactions per DNA sample. Data analysis The sequenced data were processed using the mothur platform [47]. Illumina sequencing data was pair-assembled using pandaseq [48] with an assembly quality score of 0.9, which is the most stringent option to reduce errors. Next, the sequences were aligned against the EzTaxonaligned reference [49]. Sequences were denoised using the 'pre.cluster' command in mothur, which applies a pseudo-single linkage algorithm with the goal of removing sequences that are likely due to pyrosequencing errors [50]. Putative chimeric sequences were detected and removed via the Chimera Uchime algorithm contained within mothur [51]. The taxonomic classification was performed using mothur's version of the RDP Bayesian classifier, using EzTaxon-e database for each sequence at 80% Naïve Bayesian bootstrap cutoff with 1000 iterations. The sequences used in this study have been deposited in the NCBI Sequence Read Archive under accession number SRP043977. Statistical analysis To perform the statistical analysis, all samples were standardized by random subsampling to 4,073 sequences per sample, using the sub.sample command (http://www.mothur.org/wiki/ Sub.sample) in mothur. To assess the relationship between soil bacteria richness/diversity and MWCNTs concentration, as well as with time incubation, the richness of OTUs and other diversity indices were calculated using the mothur platform [47]. We calculated weighted UniFrac which measures sequence difference between samples based on phylogenetic information to analyze the bacterial community similarity. We used a non-metric multidimensional scaling plot (NMDS) using the weighted UniFrac distance in Primer 6 to visualize the clustering of bacterial community composition over time. We then performed an analysis of similarity (ANOSIM) with pairwise weighted UniFrac distance as the response variable and MWCNTs concentration, time incubation and form as factors. We performed multiple regression analysis in R software package 2.15.2 using linear model (LM) for normal data, and a generalized linear model (GLM) for non-normal data to evaluate the effects of raw and functionalized MWCNTs concentrations, incubation time, and their interactions on bacterial richness and diversity, as well as on the relative abundance of dominant bacterial phyla. There were four different concentration treatments of raw and functionalized MWCNTs i.e. 0, 50, 500, and 5000 μg/g soil, and each treatment had three replicates. Aliquots of soil from each treatment were collected at different time points (0, 2, and 8 weeks). To test whether bacterial abundance (qPCR) was correlated with fMWCNTs and raw MWCNTs across different sampling time, we performed regression analysis using linear functions in Sig-maPlot. We used analysis of variance (ANOVA) to test the effect of fMWCNTs and raw MWCNTs on organic matter content of the soils. Results A total of 2,568,331 quality bacterial sequences were obtained from the 63 samples, with an average of 40,767 sequences per soil sample and with coverage ranging from 4,760 to 219,775 reads per sample (S1 Table). MWCNTs characterization The characterization of MWCNTs based on FE-SEM (Fig. 1A) and EF-TEM (Fig. 1B, 1C) images showed that the average diameter was around 13.4 nm and the number of walls was 11 in average. The size of CNTs is an important factor in toxicological studies [11,52]. In fact, the interactions between carbon nanotubes and living cells decreased with the size increase [11]. Raman spectrum showed that the D-band/G-band ratio was approximately 1.305 and this result showed that defects have been generated from pristine MWCNT which D-band/G-band ratio is 1.087 (Fig. 1D). Effect of MWCNTs on bacterial community diversity The effects of raw and functionalized MWCNTs concentrations, incubation time, and their interactions on bacterial richness and diversity were evaluated using multiple regression analyses. The results showed that the concentration of both fMWCNTs and raw MWCNTs did not show any correlation with OTUs richness and Chao index (All P > 0.05). In contrast, time was significantly correlated with OTUs richness and diversity for the two MWCNTs forms (All P < 0.05). Considering time and MWCNTs concentration together, there was an important correlation for OTUs richness and diversity indices (All P < 0.05), but only for functionalized carbon nanotubes (fMWCNTs) ( Table 1). Effect of MWCNTs on bacterial community composition OTU community composition did however differ between different concentrations of fMWCNTs, (fMWCNTs, R = 0.24, P = 0.001) but not with different concentrations of raw MWCNTs for Bray-Curtis dissimilarities. NMDS plots showed a clustering of soil samples according to MWCNT concentrations; nevertheless samples from soil with highest concentration of fMWCNTs showed a greater dissimilarity in their bacterial community composition compared to soil treated with raw MWCNTs (Fig. 2). Considering the variation in the bacterial community over time, the NMDS plot showed a clustering of the soils sampled at different sampling times according to different MWCNT concentrations used in this experiment for fMWCNTs, but not for raw MWCNTs (Fig. 3). An ANOSIM test confirmed this result as indicated by the test results ( Table 2). Effect of MWCNTs on bacterial community abundance Overall, the most abundant bacterial phyla were Proteobacteria with 29% of the sequences, followed by Acidobacteria (20%), Actinobacteria (15%), Chloroflexi (9%), and Bacteroidetes (7%); around 4% of the sequences were unclassified. Of the most abundant phyla, we found significant differences in relative abundance between different concentrations of fMWCNTs except for Proteobacteria, Actinobacteria, Chloroflexi, and Bacteriodetes (Fig. 4). The multiple regression analyses showed that time and fMWCNTs together have an effect on soil bacteria (Table 3). However, the samples treated with raw MWCNTs showed less correlation with both time and concentration for some phyla (Table 3 Table 1 . Multiple regression between richness (OTUs) and diversity indices with CNTs concentrations and incubation time for both acid treated (fMWCNTs) and raw MWCNTs. fMWCNTs OTUs (R 2 = 0.54***) Shannon (R 2 = 0.53***) Simpson (R 2 = 0.58***) Chao (R 2 = 0.52***) Intercept 1.745e +03 *** 6.5520*** 7.585e -02 *** 6086.9*** and Fig. 4). Consequently, the fMWCNTs showed a more highly significant change in the relative abundance of the dominant detected phyla. Changes in the relative abundance of the predominant bacterial genera in response to exposure to fMWCNTs and raw MWCNTs are illustrated in Fig. 5 using a heat map. The most abundant OTU across the various soil replicates/treatments was classified under the genus Blastocatella (Acidobacteria) represented by 5.6% of the total reads. This genus significantly decreased in abundance in the highest treatments with fMWCNTs at 2 weeks, but later in the experiment its abundance increased by the final time of sampling at 8 weeks. However, raw MWCNTs did not have any detectable effect on OTU relative abundances. Overall, even fMWCNTs did not have profound effects on the soil bacterial community, even though effects were detectable. Shifts in bacterial abundance were observed only for fMWCNTs with the greatest changes observed at highest concentrations (5000 μg/g). For all the qPCR assays, there was a linear relationship between the log of plasmid DNA copy number and the calculated threshold cycle value across the different concentration range (R 2 > 94 in all cases). The bacterial abundance, as determined using qPCR, did not show any Effects of Carbon Nanotubes on Soil Bacteria correlation with fMWCNTs or raw MWCNTs across different concentrations, except with fMWCNTs at zero time where P < 0.005 and R 2 = 0.54 (Fig. 6). Effect of MWCNTs on soil organic matter The ANOVA test results showed that the content of organic matter measured in the soil among different treatments with MWCNTs were highly significant (F 3,8 = 58.08, P < 0.001). High concentrations of both forms of MWCNTs increased the organic matter content of the soil. Effect of MWCNTs on soil bacterial community In our study, we had predicted that MWCNTs would significantly alter both the soil bacterial community and its diversity. Yet during the two month-long experiment, we found no differences in diversity as a result of exposure to either raw or fMWCNTs. Nevertheless, fMWCNTs showed an effect on bacterial community composition. Raw MWCNTs did not show any effect on community composition. Changes in the structure and abundance of the soil bacterial community in response to MWCNT exposure have been observed in previous studies [17,37]. Our results showed that soils treated with fMWCNTs exhibited shifts in bacterial community composition for different MWCNT concentrations at both of the sampling times (2 weeks and 8 weeks). The relative abundance of the most common bacterial phyla was affected by the presence of fMWCNTs and showed differing responses to exposure to fMWCNTs. The abundance of Proteobacteria, and TM7 was comparatively higher in the highest fMWCNTs treatment, while there was a decrease in Chloroflexi at the highest concentration of fMWCNTs. Acidobacteria, Bacteriodetes and Gemmatimonadetes showed an initial decrease (2 weeks) at the highest concentration of fMWCNTs then increased over time (8 weeks), whereas Actinobacteria increased at highest concentration of fMWCNTs, then later decreased. These findings suggest that the exposure of soil to fMWCNTs could in fact have some impact on carbon cycling by altering the microbial community [12]. For example, Actinobacteria (which undergo significant shifts in abundance) are important in the biogeochemical cycle of carbon in soils [53,54]. In particular, they play a major role in the degradation of cellulolytic and hemicellulolytic compounds in Effects of Carbon Nanotubes on Soil Bacteria soils [55,56]. Acidobacteria are ubiquitous and among the most abundant bacterial phyla in soil [57]: their relative abundance is generally negatively correlated with soil carbon availability [58]. The Chloroflexi are commonly found in soils, also playing an important role in the biogeochemical cycle of carbon and the CO 2 dynamics in soils [55,56,59]. Such changes in bacterial abundance may be explained in terms of a shift of microbial community towards bacterial species that are more tolerant of the effects of fMWCNTs and the decline of less tolerant species [60][61][62]. Despite the evident effects of exposure to fMWCNTs, overall it appears from our experiment that the soil bacterial community is quite resilient to the environmental perturbation caused by high concentrations of fMWCNTs. The community largely recovers from exposure to fMWCNTs by 8 weeks. It also appears that the soil bacterial community is resistant to perturbation from raw MWCNTs, with almost no observed effects on bacterial community. The observed lack of response to raw MWCNTs generally matches previous findings for untreated nanotubes and other carbon-based new materials [63,64]. For instance, Khodakovskaya et al. [65] observed no impact on soil bacterial diversity of MWCNTs added by watering into soil at concentrations up to 200 μg/ml. Other studies on the impact of a carbon-based nanomaterial, C 60 fullerene by Chung et al. [2] and Tong et al. [66] demonstrated no effect of toxicity on soil bacterial diversity even at 1000 mg/kg concentration. However, some studies have shown toxin-like effects of raw MWCNTs on bacteria. Rodrigues et al. [12] found that raw MWCNTs can negatively affect soil bacterial diversity. In fact, they observed a major effect of single-walled carbon nanotubes on the soil bacterial community after only 3 days of exposure, and then bacterial diversity recovered after 14 days' exposure. If this is the case, our observed lack of an effect from raw MWCNTs after 2 weeks may be due to the system having already recovered from an initial perturbation. MWCNTs are chemically Effects of Carbon Nanotubes on Soil Bacteria extremely inert, especially in relation to biological processes [42], and this could be one of the possible reasons that we did not find any effect of MWCNTs on soil bacterial communities. Why do fMWCNTs cause a shift in the soil bacterial community? fMWCNTs are acidic in nature: pure fMWCNTs, after thorough washing, have a pH around 3 due to carboxyl groups that cover their surface. Our measurements of soil pH showed that pH was around 4 just after adding fMWCNTs to soil at 0 weeks for the highest fMWCNTs concentration, two units lower than the control without nanotubes, around 4.8 at 2 weeks, and around 5.5 at 8 weeks. There is abundant evidence that pH is crucial to bacterial community structure [67][68][69]; in fact, pH seems the strongest factor of all in structuring soil bacterial communities on a global scale [70,71]. Thus, it is no surprise that fMWCNTs-with associated lowering of soil pH-caused significant changes in soil bacterial communities. One might hypothesize that fMWCNTs effects would decrease or even disappear on a time scale of months as their acidity becomes neutralized. Longer-term studies are warranted to confirm whether this is indeed the case. Aside from following the overall bacterial community, it will also be important to examine effects on biogeochemical processes, such as carbon and nitrogen cycling, in fMWCNTcontaminated soils. Conclusion The overall picture is of rather weak-but still detectable-effects from fMWCNTs on soil bacterial community structure, combined with the lack of any observable effects from raw MWCNTs. This gives a generally reassuring picture in terms of the effects of MWCNTs on the soil environment. Even at the high concentrations used here, fMWCNTs apparently do not have profound effects on soil bacterial communities. The observed effects of fMWCNTs would, however, warrant further experimental investigations for any changes in soil nutrient cycling processes, using functional metagenomics or observations of fluxes. Supporting Information S1 Table. Relative abundances of bacterial phyla classified against the Ribosomal Database Project (RDP) training dataset number 9 across all 63 soil replicates of different treatments with raw and acid treated MWCNTs. (XLSX)

v3-fos

2015-09-18T23:22:04.000Z

2015-09-01T00:00:00.000Z

3258571

s2

Solvent effects on phytochemical constituent profiles and antioxidant activities, using four different extraction formulations for analysis of Bucida buceras L. and Phoradendron californicum Background The present investigation evaluated 4 different solvent compositions for their relative capacity to extract total phenolic and total flavonoid (TF) components of the leaves, trunks, and stems of Bucida buceras L. (Combretaceae), and the stems of Phoradendron californicum (Viscaceae), plus mesquite and oak species endemic to the Southwestern United States, northern Mexico, and tropical regions of Central and South America, as well as to profile the composition of these plant materials and to measure their antioxidant capacity. Methods The total phenolic content of plant material used in the present investigation was measured using the Folin–Ciocalteau assay. Total flavonoids were assayed by AlCl3 and 2,4-dinitrophenylhydrazin colorimetry. Nitroblue tetrazolium was utilized for scavenging of superoxide anion, and in vitro antioxidant activity was evaluated using the 2, 2-diphenyl-1-picrylhydrazyl and Ferric Reducing/Antioxidant Power assays. Results Phytochemical screening of each plant extract evaluated revealed the following major results: (1) No evidence of alkaloids for each of the extraction phases tested was detected in the hexanic, ethanolic, or aqueous phases of Bucida buceras and Phoradendron californicum (oak and mesquite); (2) Analysis of the hexane phase of B. buceras and P. californicum (mesquite) extracts revealed the presence of carotenes, triterpenes/steroids, and lactonic groups; (3) Analysis of the ethanol and aqueous extraction phases for both plants revealed the presence of a diverse range of compounds, including tripterpenes/steroids, lactonics groups, saponins, phenols/tannins, amines and/or amino acids, and flavonoids/anthocyanins; and (4) The highest total phenolic and flavonoid content were observed in P. californicum (oak): 523.886 ± 51.457 µg GAE/mg extract and 409.651 ± 23.091 µg/mg of extract for methanol and aqueous fractions, respectively. The highest flavonoid content was 237.273 ± 21.250 µg PNE/mg extract in the acetone extract of Bucida buceras stems; while the flavonol content (260.685 ± 23.031 µg CE/mg extract) was higher in the ethanol extract of P. californicum (oak). The acetone extract of B. buceras trunk extract showed the highest levels of DPPH radical-scavenging activity (IC50 = 4.136 ± 0.446 µg/mL) and reducing power (4928.392 ± 281.427 µM AAE/mg extract). The highest superoxide radical scavenging activity (IC50) was 55.249 ± 9.829 µg/mL, observed in acetone extracts of B. buceras leaves. Conclusions The results of the present investigation demonstrated the effects of extraction solvent on phenolic and flavonoid content yield—and antioxidant activities by Bucida buceras and Phoradendron californicum. The present investigation further revealed that Bucida buceras exhibited optimal antioxidant capacity when acetone was used as extraction solvent; and the highest yield of phenols and flavonoids were obtained from the P. californicum oak, using methanol and aqueous solvents, respectively. Background Free radicals contribute to more than one hundred disorders in humans, including atherosclerosis, arthritis, ischemia and repercussion injury of many tissues, central nervous system injury, gastritis, and cancer [1]. Environmental pollutants, including radiation, chemicals, and dietary toxicants, along with physical trauma, cause dysregulation of immune activity, and may alter gene expression to induce expression of abnormal proteins. Oxidative processes, which trigger the production of free radicals, resulting in tissue damage, are a major contributor to diminished health, and manifested in a wide spectrum of disease [2]. Plant-derived antioxidants, especially polyphenolic compounds, have gained considerable importance due to their potential health benefits. Antioxidants are important compounds, which protect organisms from damage caused by free radical-induced oxidative stress [3]. The antioxidant activity of phenolic compounds is mainly due to their redox properties, which allow them to act as reducing agents, hydrogen donors, singlet oxygen quenchers and metal chelators [3][4][5]. Many plants respond to environmental stressors by producing antioxidants such as polyphenols. These absorb and neutralize free radicals, quenching singlet and triplet oxygen, or inducing expression of peroxides and other toxic metabolites [6,7]. The medicinal value of plants is related to their phytochemical component content and secondary metabolites, including: phenolic compounds, flavonoids, alkaloids, tannins, and other stress gene response products [8,9]. Several major groups of plant products with antioxidant and anti-inflammatory capacity have been identified. A particularly valuable class of health-enhancing plant compounds is flavonoids. These are polyphenolic molecules with properties that include free radical scavenging, inhibition of hydrolytic and oxidative enzymes, anti-inflammatory action, reduction of blood-lipids and glucose, and the enhancement of human immunity [10]. Anti-inflammatory plant metabolites are currently the focus of intensive research in the development of novel preventive and therapeutic strategies. Accordingly, methods of extracting these compounds from source material are a major focus of investigation. Current isolation and chemical purification methods used include solvent extraction processes that utilize solvent polarity as a major separation technique. These methods frequently include the use of ethyl acetate, phenol/chloroform, aqueous, and several other approaches [1,9,[11][12][13][14][15]. This study examines the effects of type solvents on extraction of bioactive compounds from Bucida buceras L., a timber and shade evergreen tree that belongs to the family Combretaceae. It is endemic to the tropical regions of Northern South America, and is commonly referred to as "the nonedible black olive tree" [16]. The plants in this family are used for the treatment of various diseases in humans, including abdominal pains, chest coughs, colds, conjunctivitis, diarrhea, earache, fever, infertility in women, leprosy, pneumonia, heart diseases, sore throat, and nose bleeds [17,18]. Diverse authors have found that Bucida buceras has high antifungal activity [19][20][21]. This investigation will also evaluate effects of the extraction of the solvent on Phoradendron californicum (known in United States as "dessert mistletoe" and "toji" in Mexico), which is an autotrophic hemiparasite, surviving at the expense of its higher vascular plant host [22][23][24]. The leaves and bark of this plant are used to treat stomachache and digestive disorders. It is also used with other plants in mixed herbal teas against venereal diseases. In a recent study, toji was found to have multiple biological activities, including anti-inflammatory and anti-proliferative effects [25]. Bucida buceras and Phoradendron californicum (mesquite and oak) are medicinal plants and both have been attributed with beneficial health effects, but there are only a few studies related to this topic. The aim of this work has been to investigate the effect of this solvent type on the yield and profile of component phenolics and flavonoids and antioxidant activity. Sample sources and collection The leaves, trunks and stems of Bucida buceras L. were collected in Hermosillo, Sonora, Mexico, during the summer of 2012. This genus grows in tropical areas and the tree blooms and fructifies irregularly throughout the year. The stems of Phoradendron californicum of Prosopis species (mesquite) and Quercus (oak) were collected from Carbo and Moctezuma towns, Sonora, Mexico, at the same year. Members of this genus grow in warm temperate and desert regions. Fruit ripening occurs in late winter to early spring. The plants were taxonomically identified by Dr. Jose Cosme Guerrero Ruiz at the Agronomy Department of Sonora University and assigned voucher specimen designations as follows: For Bucida buceras: RP2014-10, for Phoradendron californicum (mesquite): RP2014-11; and for Phoradendron californicum (oak): RP2014-12 deposited at the herbarium of Sonora University. All the plants were washed, dried and stored in polyethylene bags protected from light at 25 °C until analysis. Preparation of plant extracts The different parts of Bucida buceras and Phoradendron californicum were dried at room temperature for 7 days, finely ground and used for extraction. The powder obtained (500 g) was mixed with 1.5 L of organic solvents, including acetone, methanol and ethanol for leaves, trunks and stems of B. buceras; and for the stems of Phoradendron californicum (mesquite and oak). All the extracts were shaken mechanically for 3 days at room temperature in an orbital shaker. The supernatant was next filtered through Whatman No. 41 filter paper by vacuum and fresh solvent was added to the samples, which were extracted for another 3 days. For aqueous extracts of B. buceras and Phoradendron californicum (mesquite and oak), 100 g of dried plants were extracted with 1L of water at 100 °C for 30 min. The residues were subsequently extracted twice with fresh aqueous solvent (water) and the extracts were combined. Finally, the two supernatants of the different extracts of B. buceras and P. californicum were concentrated separately in a rotary evaporator (Buchi R-210, Switzerland) at 37 °C, then frozen at −77 °C and finally lyophilized at −46 °C. At last, the extracts were powdered in a coffee grinder and dissolved in dimethyl sulfoxide to a final concentration of 200 mg of dry matter/mL. All extracts were stored at −4 °C until use. Phytochemical screening A preliminary screenings of each extract was performed following the standard phytochemical analysis protocol described by Chhabra et al. [26], with some modifications. Briefly, 10 g of the collected materials were extracted successively with hexane, ethanol and water in a Soxhlet extractor for 18-20 h. The extracts were concentrated using a rotary evaporator (Buchi R-210, Switzerland) at 37 °C and preserved at 4 °C. All extracts were subjected to qualitative chemical tests for the identification of selected phytoconstituents such as alkaloids, using Mayer´s and Draggendorff´s test, carotenes, using the Carr-Price test, triterpenes/steroids using the Liebermann-Burchard test, quinines, using the Borntrager test, lactonic groups, using the Baljet test, lipids and essential oils, using the Sudan III test, reducing compounds, using the Fehling test, saponins, using the Frothing test, phenols/tanines, using the ferric chloride test, flavonoids, using the Shinona test, amines/amino acids, using the Ninhidrin test and cardiotonic glycosides, using the Kedde test. Determination of total phenols and flavonoids Total phenols The total phenolic content of plant extracts was determined using the Folin-Ciocalteu reagent described by Singleton and Rossi [27] with some modification, as reported by Iloki et al. [28]. Briefly, 500 µL of plant extract samples in a range of concentrations (25-500 µg/mL) were mixed with 500 µL of Folin-Ciocalteu reagent (1:4) and 500 µL of a 10 % sodium carbonate solution (w/v) were added. The sample was left standing at room temperature (~25 °C) for 2 h and the absorbance was measured at 760 nm in a microplate spectrophotometer reader (Thermo Scientific). Based in the calibration curve established as preparation for the experiments, (0.00625-0.1 mg/mL), the results were expressed as micrograms of gallic acid equivalent per milligram of extract (µg GAE/mg). Flavonoids The total flavonoid content of each batch of plant material was measured by two complementary colorimetric methods, one by the method aluminum chloride (AlCl 3 ) for the quantification of flavones and flavonols that react better with AlCl 3 and other by the method of 2,4-dinitrophenylhydrazin (DNP) for flavanones and flavanonols [29,30]. Aluminum chloride (AlCl 3 ) method The colorimetric aluminum chloride method was used to determine flavonoid content according to the method proposed by Zou et al. [31]. The method is based on quantification of the yellow-orange color produced by the interaction of flavonoid with AlCl 3 . A 50 μL aliquot of plant extract appropriately diluted, was mixed with 1250 μL of deionized water and 75 μL of 5 % sodium nitrite, after 6 min, 150 μL of 10 % AlCl 3 solution was added and the mixture was allowed to stand for 5 min; followed by addition of 500 μL of 1 M sodium hydroxide. After 30 min of reaction, absorbance was read at 510 nm. The flavonoid content was assessed by reference to a calibration curve of catechin (0.2-1.0 mg/mL) and expressed as μg of catechin equivalent per mg of extract (μg CE/ mg). 2,4-Dinitrophenylhidrazin (DNP) method Quantification of flavanones and flavanonols was accomplished using a spectrophotometric method proposed by Nagy and Grancai [32], which was based on the interaction of these compounds with DNP in acid medium, to form phenylhydrazones. 40 µL of sample were dissolved in 80 µL of DNP solution (For 5 mL: 50 mg of DNP in 100 µL of 96 % sulfuric acid and 4850 µL of methanol), mixed and incubated at 50 °C for 50 min in a water bath, cooled at room temperature and 280 µL of 10 % potassium hydroxide (KOH) in water were added. Finally, the absorbance was measured at 486 nm. The total content of flavanones was determined using a calibration curve based on pinocembrin (0.5-5.0 mg/mL) and expressed as μg of pinocembrin equivalent per milligram of extract (μg PNE/mg). Antioxidant activity Free-radical scavenging activity (DPPH assay) The free-radical scavenging activity of each plant material was measured as described previously with some modifications [33]. Briefly, 500 µL of (0.2 mM) 1,1-diphenyl-2-picrylhydrazyl (DPPH) in ethanol was added to 500 µL of selected concentrations (12-100 µg/mL) of the extracts and the mixture was incubated for 30 min in the dark, at room temperature. Absorbance of the mixture was measured using a microplate spectrophotometer reader (Thermo Scientific) at 517 nm. The assay vehicle without extract, served as an absolute control (A blank). The free radical scavenging of DPPH was calculated according to the following formula: where A blank is the absorbance of the control reaction (containing DPPH solution adequately diluted with ethanol) and A sample is the absorbance of the test compound. Extract concentrations mediating 50 % inhibition (IC 50 ) was calculated from a graphic plot of inhibition percentage versus extract concentration. The lower the IC 50 , the higher the antioxidant activity of the examined compound. Ferric reducing antioxidant power assay (FRAP assay) The FRAP reagent was prepared in acetate buffer (pH 3.6), 10 mmol 2,4,6-tripyridyl-s-triazine (TPTZ) solution in 40 mmol hydrochlorin acid and 20 mmol iron (III) chloride solution in proportions of 10:1:1 (v/v), respectively. The FRAP reagent was prepared daily. 5 µL of samples at 0.5 to 2 mg/mL diluted with 20 μL of distilled water were added to 150 μL of FRAP reagent [34]. The absorbance of the mixture was measured using microplate spectrophotometer reader Thermo Scientific at 593 nm after 8 min. The standard curve was prepared with ascorbic acid (AA) and the results were expressed as μmol AA Equivalent/mg polyphenol-rich extract. Superoxide radical scavenging Superoxide anion radical scavenging activity was determined by the method proposed by Beauchamp and Fridovich [35] with little modifications. The assay is based on the capacity of the sample to inhibit formazan blue formation by scavenging the superoxide radicals generated in the riboflavin-light-nitroblue tetrazolium (NBT) system. The reaction medium contains 625 µL phosphate buffer (pH 7.6), 25 µL riboflavin (0.2 mg/mL), 50 µL EDTA (12 mM), 25 µL NBT (1 mg/mL) and 25 µL of different concentration of sample. Illuminating the reaction mixture for 10 min started the reaction. Superoxide radical content of each sample was measured spectrophotometrically by the increase in the amount of the absorbance at 590 nm. Blank standardization was performed in the same way with 25 µL of ethanol in the place of samples being of tested. The concentration of test sample required to inhibit NBT reduction by 50 % (IC 50 ) was calculated from dose-inhibition curves. Statistical analysis All data were expressed as mean ± standard deviation. Statistical analysis was performed by analysis of variance (ANOVA). A post hoc test (Turkey) was carried out when the differences shown by data were significant (p < 0.05). NCSS (version 2007) statistical program was used for all analysis included the Pearson's correlation coefficient. Phytochemical screening Preliminary phytochemical screening of Bucida buceras L. and Phoradendron californicum extract show the presence of various bioactive components which are shown in Table 1. The results provide evidence of the presence of carotenes, triterpenes/steroids, lactonic groups, phenols/ tannins, amines or amino acids, flavonoids/anthocyanins, saponins, reducing compounds and lipids/essential oils in selected extracts of B. buceras and P. californicum (mesquite and oak), except the last group for B. buceras. The results also revealed the absence of alkaloids and quinines with the exception of P. californicum (mesquite) on the ethanolic phase in which the presence of quinines was detected. Some authors have found the presence of diterpenes and flavanones (flavonoids) in Bucida buceras [36,37], this result agree with some of the results obtained in this investigation. Meanwhile, Iloki et al. [33] found that P. californicum is a plant rich in flavonoids, saponins, phenols and/or tannins mainly. Major outcomes of the present investigation revealed that the samples tested contained high concentrations of health-enhancing phytochemical constituents, including flavonoids, phenols and/or tannins, saponins, amines and/or amino acids [25]. Plants endemic to Northwest Mexico such as Phoradendron californicum contained compounds with significant antioxidant and antiproliferative activities. Hayashi et al. [36,37] identified presence of potent cytotoxic agents including Bucidarasins and Buceracidins in Bucida buceras. Moreover, many flavonoids and terpenoids are potent antioxidants, with anti-inflammatory, antibacterial, antiviral and anticancer properties [38,39]. Other compounds previously identified in these plant materials include phenolics, which possess antioxidative, antidiabetic, anticarcinogenic, antimicrobial, antiallergic, anti-inflammatory and antimutagenic activities [40,41]. These plants also contain steroids, which are known to mediate cardiotonic activities and possess insecticidal and antimicrobial properties, while tannins, which are also found in these plants, are known to possess general antimicrobial and antioxidant activities [42]. The saponins fraction of extracts described here, are typically used in treatment of hypercholesterolemia, hyperglycemia, as antioxidants, anticancer, antifungal, antibacterial, anti-inflammatory and in weight loss [40,43]. Total phenolic and flavonoid content determination Total phenolic content (TPC) Sample content of phenolic compounds (µg GAE/mg of extract) in different solvent extracts of the leaves, stems and trunks of B. buceras L. and stems of P. californicum of mesquite and oak are summarized in Fig. 1. Results of the assays for phenolics described in the present report, indicated a wide variation in the total phenolic content in the different extracts, ranging from 82.818 to 523.886 µg GAE/mg of extract, showing the higher phenolic content the methanol, ethanol Oak toji extracts and acetone trunk and leaves of B. buceras extract. Results of these assays, demonstrated significant variability in total yield of phenolic compounds (p < 0.05). In the present study, methanol proved to be the most effective solvent for isolation of phenolic compounds (327.91-523.89 µg GAE/mg of extract) from samples of B. buceras and P. californicum, whereas much lower yields were obtained from samples extracted with acetone (82.82-480.11 µg GAE/mg). The order of effectiveness in extraction of phenolics was methanol (388.04 µg GAE/mg) >aqueous (321.05 µg GAE/mg) ≥ethanol (304.34 µg GAE/mg) ≥acetone (283.49 µg GAE/mg). In general, extractability of a particular component appeared to depend on extraction medium polarity and the ratio of solute to solvent. Moreover, recovery of phenolic compounds appeared dependent on the type of solvent used, its polarity index (PI) and the solubility of phenolic compounds in the extraction solvents. The solubility of polyphenols was observed to depend mainly on the presence and position of hydroxyl groups and the molecular size and the length of constituent hydrocarbon chains. Phenolic compounds are often extracted in higher amounts in more polar solvents. In such cases aqueous extract (PI = 9) was not more efficient in extracting polyphenolic compounds than methanol (PI = 6.6), a phenomenon that may be due at least in part, to the thermal decomposition of some phenolic antioxidants at the higher temperatures which were used for this extraction. This effect was evident, despite the observation that increased heating temperature may increase the yield of phenols in an extraction. Results shown here demonstrate that solvents with similar polarity ethanol (PI = 5.2) and acetone (PI = 5.4) have significantly different effects on polyphenol content, mainly for trunk and stem of B. buceras and Toji Oak. These Values as mean ± standard deviation (n = 3) × triplicate. Different lowercase letter indicate significant differences (p < 0.05) between different solvent type in the same plant part and different capital letter indicate significant differences (p < 0.05) between the same solvent type in different plant part for B. buceras and same solvent type in same part (stems) for P. californicum (oak and mesquite) significant variations indicated that change in phenolic solubility may be altered dependent on particular extraction conditions. Whereas certain compounds are common to most parts of a particular plant, none are ubiquitously abundant and most are restricted in distribution to a defined location of plant anatomy (e.g. seeds, stems, leaves, bark, etc.). The present study revealed that the distribution of polyphenols differed significantly between different of B. buceras parts for all solvents. For example, leaf content of these compounds was 417.41 µg GAE/mg of extract; and stems contained 355.53 µg GAE/mg of extract which were approximately 15 and 30 % more than the phenolic content of the trunks (314.24 µg GAE/mg). This difference was particularly marked in ethanol and aqueous solvents. Significant differences were found between extraction yields of phenolics, dependent on types of solvent used; and origin of a sample on plant anatomy. It was observed that the highest extraction yield of phenolic compounds was achieved in acetone for B. buceras leaves and trunk. Significant solvent-dependent differences in content of phenolic compounds were observed when samples from P. californicum (oak and mesquite) were evaluated. Contributing factors may be related to environments in which the plants lived. Oaks from highland forests are adapted to cold and acid soils. Environmental stressors to which the plants are subjected favor expression of stressresponse compounds, including polyphenols. In the present study, methanolic extraction protocols were the most efficient for recovery of phenols. Different parts of the same plant may synthesize and accumulate different compounds or different amounts of a particular compound due to their differential gene expression, which in turn, affects antioxidant activities and other biological properties of the plant extracts [44,45]. Many studies have confirmed that the amount and composition of phenolic and flavonoid compounds is diversified at the sub-cellular level and within plant tissues as well [46,47]. In general, the results of the study revealed that methanol extracted higher levels of phenolics from Toji Oak than the other solvent systems tested. These results were similar to those reported for Toji Oak by Iloki et al. [33]. In these investigations, methanolic extracts of Toji Oak and Mesquite contained the highest total amounts of phenolics, flavonoids and condensed tannin compounds, when compared to extraction yields of the other solvent systems: (acetone, methanol and water). Further, results shown here are similar to those reported by Sun and Ho [48] where methanol solvent was most effective in extracting phenolic components from oat bran. Methanol and ethanol have been proven as effective solvents for extraction of phenolic compounds [14]. Conversely, Jayaprakasha et al. [49] demonstrated that acetone, methanol and water were relatively ineffective for the extraction of total phenols from grapes seeds. The recovery of phenolic contents in different samples is influenced by the polarity of extracting solvents and the solubility of each compound in the solvent used for the extraction process [50,51]. Therefore, it is difficult to select an optimally appropriate solvent for extraction of phenolics from multiple plant material samples [52]. The profile and yield of polyphenol content and antioxidant activity appears higher in more polar solvents [16], hence these seem good choices for screening of multi-sample substances. In the context of these observations, it should be noted that the phenolic compounds are often associated with other biomolecules (polysaccharides, proteins, terpenes, chlorophyll, inorganic compounds) and a solvent suitable for the extraction of particular classes of compound must be used based on the structural features and related level of aqueous solubility of a particular target molecule [9]. Using this general strategy, the phenolic extraction efficiencies of the different parts of Bucida and Toji may be optimized, based on the relative polarities of target compounds. Thus different classes of solvent may therefore be required depending on the known distribution of target compounds in various plant anatomical locations. Total flavonoid content (TFC) Total flavonoid content was evaluated in the present study using two colorimetric methods. The flavonol/flavones content in the different extracts of plant materials was evaluated using catechin equivalent as a reference; and the flavanones/flavanonols content was evaluated in pinocembrin equivalent as a reference. Some insight into the molecular mechanisms contributing to solvent extraction efficiency may be gained by considering major features of target compounds. For example, the aluminum chloride method involves formation of stable acid complexes between the AlCl 3 reagent and the C-4 keto group; and either the C-3 or C-5 hydroxyl group of flavones and flavonols. In addition, aluminum chloride forms labile acid complexes with the ortho-dihydroxyl groups in the A-or B-ring of flavonoids. Of the plant materials studied here, the highest yields of flavanones and flavanonols were found in Bucida buceras. In trunk and stem samples, flavanones yields were 2-3 times greater than those of flavones/flavonols ( Table 2). The flavanone content in the trunk was greater than the leaves and stem, except in acetone extracts of the stem. Non-significant differences between solvents were observed in analysis of phenolic content of trunks, whereas for leaves and stems, ethanol and acetone contained the highest flavanone content. These findings suggest that acetone extraction appears superior to other solvents for recovery of flavonols/flavones and flavanones (total flavonoids) from Bucida buceras. Assays conducted on Phoradendron californicum revealed that the content of flavonols/flavones was approximately 3-fold higher than that of flavanones. Analysis of plant materials showed that Toji (Oak) contained the highest flavonol/flavone content compared to toji (Mesquite). In contrast to Bucida buceras, aqueous and methanol extracts exhibited the highest content of phenolic compounds; while the acetone solvent was less effective in extracting flavonols/flavones components from Toji (oak and mesquite). These outcomes indicate that polyphenols from Phoradendron californicum were extracted most efficiently in more polar solvents. The amount of total flavonoids extracted from Bucida buceras and Phoradendron californicum using different solvents type ranged from 68.788 ± 6.022 to 409.651 ± 23.091 µg/mg of extract, with the highest amount of total flavonoids observed in the aqueous extract of oak, whereas the lowest yield was obtained in the acetone extract of mesquite. Results of the present study demonstrated higher total flavonoid content in Phoradendron californicum (mesquite) than was previously reported by Jiménez et al. [25]. It has been established that phenolics are the major plant compounds with antioxidant activity, a property derived from their redox abilities. Phenolic compounds are a class of antioxidant agents, which can quench and neutralize the free radicals [53]. Flavonoids and flavonols are two other major classes of health-enhancing plant compounds and are among the most widely distributed groups of compound found in the plants with known benefit to vertebrate viability. Both flavonoids and flavonols possess antioxidant activity as a result of a native scavenging or chelating attribute [54]. Evaluation of the antioxidant activity DPPH radical scavenging assay DPPH radical scavenging activity of the various Bucida buceras and Phoradendron californicum (mesquite and oak) extracts evaluated in the present investigation are shown in Fig. 2. The extracts of each plant examined in Table 2 Flavonoids content by the two colorimetric methods on B. buceras and P. californicum in different solvent type The results are expressed as mean ± standard deviation (n = 3) of triplicate samples. Values in the same column followed by a different lowercase letter are significantly different (p < 0.05) in the same plant and part with different solvent. Different capital letter indicate significant differences (p < 0.05) between different part of the plant in the same solvent for B. buceras and same part and solvent for P. californicum (oak and mesquite) For the purpose of the present investigation, acetone extracts of leaves and trunk of Bucida buceras exhibited potent scavenging capacity against the free radical DPPH. Moreover, with this same solvent (acetone), the highest content of polyphenols was observed. Significant differences (p < 0.05) in free radical scavenging capacity between extracts using the different solvent were observed. In decreasing order of scavenging effectiveness, this included: acetone > methanol ≈ aqueous > ethanol (Fig. 2). These outcomes demonstrated that high-polarity solvents (aqueous and methanol) were less effective for extraction of antioxidants with efficient free radical scavenging properties versus an intermediate-polarity solvent (acetone) but not ethanol. Acetone extracts of Bucida buceras trunk showed the highest DPPH radicalscavenging activity (4.136 ± 0.446 µg/mL), which can be attributed to their high phenolic contents. These results were much better than those obtained in an acetone extract of trunks of Delonix regia (44.64 ± 1.8 µg/mL) conducted by Shabir et al. [55]. Meanwhile, Masoko and Eloff [56] found a strong antioxidant activity in different plants of the family Combretaceae in acetone solvent. Also, this value was higher than the values of standards like ascorbic acid and rutin (10.0 and 29.0 µg/mL respectively) [57] showing high scavenging of DPPH radical. Significant influence of the polarity solvent on antioxidant activity was observed for different parts of Bucida buceras plant anatomy. While the DPPH scavenging activity was not significantly different when comparisons were made between leaves, trunk and stem in high polarity solvents (water and methanol), significant differences emerged when moderately polar solvents (acetone and ethanol) were used to extract Bucida buceras stem samples. Sultana et al. [15] found that the ethanolic extract of Terminalia arjuna showed the highest scavenging activity. Contrary, Jegadeesware et al. [58] found the lowest IC 50 value in ethyl acetate, a solvent much less polar (PI = 4.4). Conversely, methanol, ethanol and aqueous extracts from Phoradendron californicum-oak exhibited higher total phenolic and flavonol/flavone content than Bucida Values as mean ± standard deviation (n = 3) × triplicate. Different lowercase letter indicate significant differences (p < 0.05) between different solvent type in the same plant part and different capital letter indicate significant differences (p < 0.05) between the same solvent type in different plant part for B. buceras and same solvent type in same part (stems) for P. californicum (oak and mesquite) buceras extracts, but not antioxidant activity. It is possible in this context phenolic compounds (flavanones) produced by Bucida buceras possess an ideal structure for the scavenging of free radicals since structure of these molecules includes a number of hydroxyls acting as hydrogen donators which makes them very powerful antioxidant agents. The higher efficiency of free radical inhibition of the Bucida buceras extracts can be related to its higher flavanone content, rather than to flavonols/ flavones, which are predominant in the extract. In addition, a moderate and significant correlation (r = −0.52, p < 0.001) between total phenolic and free radical scavenging was observed for this plant. Also the high abundance of carotene detected by phytochemical screening of Bucida buceras may account for this activity. Among the variants of Phoradendron californicum, toji-Oak exhibited 2-4 times greater antioxidant activity than toji-Mesquite. The observed ratio of oak-mesquite activity was more variable: approximately 2:1 in the aqueous and methanol extracts, 3:1 in the acetone and 4.5:1 in the ethanol extract. Results of the present study reveal high and significant correlations between the content of total phenolic (r = −0.78, p < 0.001) and flavonols/flavones (r = −0.73, p < 0.001) with the antiradical activity of Phoradendron californicum-oak. Similar results were reported on Zingiber officinale Roscoe (ginger). DPPH activities vary with high levels of total phenols and total flavonoids and exhibit high free radical scavenging activity [59,60]. Previous investigations have demonstrated that alteration in solvent polarity may be used to differentially precipitate selected antioxidant compounds [1,61]. In the present study, significant differences in free radical scavenging ability for extracts of Phoradendron californicum-mesquite were observed dependent on the kind of extraction solvent used, while in Phoradendron californicum-oak the effect of solvent was minimized. Aqueous and methanol extracts showed the strongest scavenging ability, followed by acetone and ethanol extract. Ferric reducing antioxidant power assay (FRAP assay) The reducing power of the plant extracts ranged from 283.899 ± 3.912 to 4928 ± 281.427 µM AAE/mg of extract. Figure 3 shows the ferric reducing antioxidant power of the different extracts of B. buceras and P. californicum (oak and mesquite). The highest reducing power was exhibited by the acetone trunk Bucida buceras wish is also high in phenolic content (480.113 ± 26.044 µg GAE/mg of extract) and the acetone P. californicum (mesquite) showed lowest activity. Diverse studies have shown that acetone or its combinations with water show Fig. 3 Reducing power of different extracts from B. buceras and P. californicum of mesquite and oak. Values as mean ± standard deviation (n = 3) × triplicate. Different lowercase letter indicate significant differences (p < 0.05) between different solvent type in the same plant part and different capital letter indicate significant differences (p < 0.05) between the same solvent type in different plant part for B. buceras and same solvent type in same part (stems) for P. californicum (oak and mesquite) better reducing antioxidant power and partially accord with the results obtained in this investigation [52,62,63]. In this assay, the presence of antioxidants in the extracts reduced Fe +3 /TPTZ complex to the ferrous form. The intensity of the blue color formation is proportional to the concentration of the ferrous form and the antioxidant capacity of the extract. Antioxidant compounds that exhibit antioxidant capacity in FRAP assay are usually electron donors. FRAP assay had a similar trend to DPPH radical assay. A significant correlation was found between IC 50 values of DPPH and FRAP (r = −0.69, p < 0.001). The reducing capacity of the extracts may serve as an indicator of potential antioxidant activities through the action of breaking the free radical chain by donating hydrogen atom [64]. Results shown here indicate that methanol solvent exhibits comparatively high reducing power for different parts of B. buceras and type of P. californicum due to the high polarity of the solvent system (Fig. 3). These results agree with the results obtained by Barchan et al. [65] were the methanolic extract of Mentha tree showed high reducing power. The reducing potential of antioxidant components is closely associated with their total phenolic content. The extracts with higher levels of total phenolics, also exhibit greater ferric reducing power. In the aforementioned examples (B. buceras and type of P. californicum), the correlation between FRAP with phenolic and flavonoid contents was 0.806 (p < 0.001) and 0.590 (p < 0.001) respectively. In B. buceras among different parts, trunk and leaves exhibited the highest ferric reducing antioxidant power with respect to stem samples-an effect that might be due to the greater polyphenol content in these parts. P. californicum-oak exhibited higher reducing power than P. californicum-mesquite mainly in aqueous and ethanolic extracts. Superoxide radical scavenging activity (O 2-) Superoxide radical is a potent reactive oxygen species and is highly toxic at fairly low tissue concentrations [66]. Moreover, although this anion is a weak oxidant, it gives reacts with other molecules in tissues to form highly toxic hydroxyl radicals as well as single oxygen, both of which are significant contributors to oxidative stress [67]. Results of O 2-activity assays conducted in this study, yielded, values ranging between 55.249 ± 9.829 and 1062.17 ± 116.172 µg/mL. Figure 4 shows the results of assays for superoxide radical scavenging activity of the different extracts of Bucida buceras and Phoradendron californicum (mesquite and oak). The acetone leaves and trunk Bucida buceras showed the best superoxide radical scavenging activity and the aqueous P. californicum gave the lowest. Values as mean ± standard deviation (n = 3) × triplicate. Different lowercase letter indicate significant differences (p < 0.05) between different solvent type in the same plant part and different capital letter indicate significant differences (p < 0.05) between the same solvent type in different plant part for B. buceras and same solvent type in same part (stems) for P. californicum (oak and mesquite) The low IC 50 values in Bucida buceras followed by Phoradendron californicum oak show a high capacity for superoxide scavenging of these extracts while Phoradendron californicum-mesquite exhibited a fivefold less activity. Similar results were observed in assays for the ability to scavenge the DPPH radical and significant association was observed between these antioxidant activities (r = 0.7444, p < 0.001). Methanolic extracts exhibited the most robust superoxide scavenging ability for phenolic compounds (r = −0.760, p < 0.001). Extracts of trunk and leaves from Bucida buceras were found to possess a strong and potent superoxide scavenging capacity with respect to analyses for these parameters in stems. The scavenging activities of leaves and trunk of Bucida buceras were similar to that of standard reference compounds such as scopoletin (54.5 µg/mL), quercetin (70.3 µg/mL) and catechin (73.3 µg/mL). Conclusions The present study revealed the presence of different phytochemicals such as phenolic compounds, carotenes, saponines, aminoacids among others; and the absence of alkaloids and quinines in the plant materials analyzed. The in vitro antioxidant activity (DPPH, FRAP, O 2 -) and content of phenolic compounds for four solvent extracts of Bucida buceras and Phoradendron californicum (mesquite and oak) were evaluated. The solvent effects identified in this study, revealed that the most efficient extraction medium for phenolic compounds was methanol for P. californicum oak; while acetone extraction of P. californicum mesquite showed the lowest content of phenolic compounds. Comparison of solvent effects in characterization of total flavonoid content revealed that aqueous P. californicum oak was the most efficient extraction medium. The acetone extract of trunk samples revealed that the highest radical scavenging activity and ferric-reducing antioxidant power activity and the highest superoxide scavenging activity was obtained in acetone extracts of Bucida buceras leaves. The various extracts of Bucida buceras exhibited potent scavenging activity and indeed, were superior to commercial standards such as ascorbic acid or the flavonoid rutin. Many plants contain phytochemicals that are beneficial for general health. Investigations such as the present study are progressively characterizing bioactivities of plant products and expanding their use in healthcare.

v3-fos

2018-04-03T00:25:44.637Z

2015-09-01T00:00:00.000Z

11547421

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A Comprehensive Review of Tropical Milky White Mushroom (Calocybe indica P&C) A compressive description of tropical milky white mushroom (Calocybe indica P&C var. APK2) is provided in this review. This mushroom variety was first identified in the eastern Indian state of West Bengal and can be cultivated on a wide variety of substrates, at a high temperature range (30~38℃). However, no commercial cultivation was made until 1998. Krishnamoorthy 1997 rediscovered the fungus from Tamil Nadu, India and standardized the commercial production techniques for the first time in the world. This edible mushroom has a long shelf life (5~7 days) compared to other commercially available counterparts. A comprehensive and critical review on physiological and nutritional requirements viz., pH, temperature, carbon to nitrogen ratio, best carbon source, best nitrogen source, growth period, growth promoters for mycelia biomass production; substrate preparation; spawn inoculation; different supplementation and casing requirements to increase the yield of mushrooms has been outlined. Innovative and inexpensive methods developed to commercially cultivate milky white mushrooms on different lignocellulosic biomass is also described in this review. The composition profiles of milky white mushroom, its mineral contents and non-enzymatic antioxidants are provided in comparison with button mushroom (Agaricus bisporus) and oyster mushroom (Pleurotus ostreatus). Antioxidant assay results using methanol extract of milky white mushroom has been provided along with the information about the compounds that are responsible for flavor profile both in fresh and dry mushrooms. Milky white mushroom extracts are known to have anti-hyperglycemic effect and anti-lipid peroxidation effect. The advantage of growing at elevated temperature creates newer avenues to explore milky white mushroom cultivation economically around the world, especially, in humid tropical and sub-tropical zones. Because of its incomparable productivity and shelf life to any other cultivated mushrooms in the world, milky white mushroom could play an important role in satisfying the growing market demands for edible mushrooms in the near future. INTRODUCTION TO MILKY WHITE MUSHROOMS The world population is currently 7 billion and it is increasing at a faster rate. By the year 2050 the global population is expected to reach 9 billion and during 2100 it could be 20 billion [1]. Shortage of food and diminishing quality of human health will be growing concerns because of the population increase and urbanization, with a concomitant reduction in arable land. Converting lignocellulosic agricultural and forest residues into protein-rich mushrooms is one of the most economically viable and sustainable biotechnology processes to address world food demand, especially protein demand [2]. Consumption of edible fungi to fulfill human nutritional needs has been a common denominator in the history of mankind [3,4]. Since most of these edible mushrooms have favorable growth conditions at lower temperatures (< 25 o C), creation of infrastructure for commercial cultivation, especially in warm humid tropical regions, is always expensive [5]. Identification and cultivation of warm-weather (30~38 o C) varieties of edible mushrooms has been scientifically challenging. Milky white (Calocybe indica var. APK2) is one of such mushroom varieties ( Fig. 1), where complete commercial production techniques have been standardized [6]. The first ever milky white mushroom variety (Calocybe indica P&C) var. APK2 was released from Tamil Nadu Agricultural University, Coimbatore, India during 1998. Over a decade, commercial production of this mushroom variety has assumed greater impetus in India, uplifting rural livelihood [6]. The first report on wild occurrence of Calocybe indica P&C, commonly called "Dhuth chatta" (means "Milky white mushroom" originated from India). For several decades, people from West Bengal (Eastern Indian State) have collected these mushrooms and sold in local markets. Its milky white color and robust nature are appealing to consumers ( Fig. 1) [7]. In nature, milky white mushrooms are seen grown on humus rich soil in agricultural fields or along the roadside in tropical and subtropical parts of India, especially in the plains of Tamil Nadu (South Indian State) and in Rajasthan (located in the western edge of India) [8]. These mushrooms grow every year between the months of May and August, which normally coincides with sufficient showers after a prolonged dry spell. C. indica is mainly a grassland species, saprophytic (organisms which obtain nutrients from dead organic matter) in nature and sometimes ectomycorrhizal (symbiotic relationship with root of some plants) with Cocos nucifera, Borassus flabellifer, Tamarindus indicus, and Peltophorum ferruginum. Detailed studies on preferential physiological and cultural requirements viz., pH, temperature, carbon and nitrogen source ratio (C : N), light and substrate requirement for growing C. Indica were reported [7][8][9]. Since this mushrooms is morphologically similar to Agaricus bisporus (button mushroom), it has been quite popular in southern Indian states and slowly getting popular in other countries (China, Malaysia, and Singapore). Small scale mushroom growers prefer to grow this tropical mushroom due to the following reasons: (1) ideally suited to warm humid climate (30~38 o C; 80% to 85% humidity), (2) its longer shelf life without any refrigeration (can be stored up to 7 days at room temperature), (3) retains fresh look and does not turn brown or dark black like that of button mushrooms, (4) lesser contamination due to competitor molds and insects during crop production under controlled conditions, (5) infrastructure needed to grow this mushroom is very much affordable and cost of production is comparatively low, which means industrial production could be attractive, and (6) has a short crop cycle (7~8 wk) and good biological efficiency of 140% (140 kg fresh mushroom/100 kg dry substrate). Methods have been developed to cultivate this mushroom on commercial scale since late nineties [6]. This review mainly focuses on the various aspects of milky white mushroom cultivation (viz., growth conditions, casing requirements, yield on different substrates), proximate composition of milky white mushrooms, flavor producing ingredients, medicinal value of the mushroom and its future prospects with indications for certain researchable issues. PHYSIOLOGY AND CULTURAL REQUIREMENTS In general, mycelia growth profile and sporophore production of any cultivated mushroom is a function of time, temperature, pH, available C : N ratio, light, CO 2 and O 2 requirements 186 Subbiah and Balan during morphogenesis. Considerable attention has been given by various authors to understanding the optimal physiological and culture requirements of C. indica for mycelia growth, tissue culture, spawn production and cultivation [6,[9][10][11]. The majority of the results indicate that the time required for maximum mycelia growth in culture media like potato dextrose agar or malt extract agar is 8 to 10 days. The pH requirement has been reported to have a wide optimal range, between 5.5 and 8.5. The optimum temperature for mycelia growth and mushroom production has been reported to be around 30~35 o C ( Fig. 2). At temperatures below 25 o C or above 38 o C did not support the growth of C. indica. Cumulative results on growing mycelia are summarized in Table 1. As far as the mycelia production in liquid broth is concerned, potato dextrose broth yielded the maximum of 0.22 g in 100 mL of broth, while malt extract broth produced 0.19 g per 100 mL in 8 days at pH 6 when incubated at 30 o C [11]. Among the various water soluble carbon sources studied, including glucose, starch, sucrose, lactose, xylose, fructose and maltose, xylose gave the best results (0.27 g of biomass/ 100 mL). Similarly, among the different nitrogen sources tested (NaNO 3 , Urea, (NH 4 ) 2 SO 4 , yeast extract, peptone, NH 4 Cl), yeast extract was the most beneficial to mycelial growth when combined with xylose (0.25 g of mycelial mat/100 mL). The optimum nitrogen concentration with yeast extract as nitrogen source was found to be 2.5% (0.32 g/100 mL); and the optimum C : N ratio that favored the maximum growth was found to be 12 : 1 (0.35 g/100 mL) ( Table 1). Light intensity, duration and wavelength are the important components of sporophore initiation. This effect is known as tropic effect. It has long been known that low light intensity, or absence of light, may result in sporophores of curious shape, often with elongated stripes and poor pileus development. Purkayastha and Chandra [12] reported that cultures of C. inidica kept continuously in dark, showed no initiation of fruit bodies. But, when exposed to diffused light, elongation of stipe to a considerable extent occurred. Low light intensity of 800 lux and below favored spawn run. But, increased mushroom yield was obtained only at higher light intensity of about 1,600 lux (455 g per bed containing 500 g of paddy straw). According to the availability of light, the size of the mushroom also varied. At higher light intensity, the stipe length was significantly reduced, whereas pileus width increased substantially. Spawn and spawning. Sorghum or wheat grains were found to be the best substrates for C. indica spawn production [12,13]. During preparation of the spawn culture, these substrates are half cooked in water for about 30 min and the excess water is usually drained before the grains are slightly air-dried and mixed thoroughly with 2 wt% calcium carbonate [14]. This wet substrate is then transferred to autoclavable polypropylene bags (usually 30 × 12 cm), which should filled up to 75% volume and sterilized at 1.42 kg/ cm 2 pressure for 2 hr. After cooling to ambient temperature, the bags should be aseptically inoculated with the mushroom mycelia, closed and incubated at 30 o C. After 15 to 20 days of incubation, complete colonization of the substrate by the mushroom mycelia should be observed, meaning that they can be used for culture bed inoculation [15,16]. The age of spawn is an important factor that influences the flushing pattern and yield of milky white mushroom. An interesting study developed by Pani [17], who prepared spawns with wheat grains and stored for different periods (14~60 days), revealed that the best milky white mushroom yields were Nitrogen concentration: Six concentrations of yeast extract (YE) and sodium nitrate (best N sources), i.e., 0.05%, 0.1%, 0.15%, 0.20%, 0.25%, and 0.30% were tried using Maltova broth with 1% xylose. d C : N ratio: the medium was prepared with different C : N ratios, 6 : 1 to 14 : 1, using yeast extract at 2.5 g (best concentration from previous experiment) and different amounts of xylose. obtained using 21-day-old spawn. Prolonged storage of spawn reduced the productivity and total yield. Studies conducted on the amount of grain spawn to wet substrate by various authors revealed that 2% spawning is good for the best spawn run and crop production. Any further increase in the inoculum showed only marginal improvements in mushroom yield. Layer spawning reduced the colonization time in the substrate (15 days) as compared to through spawning (more than 20 days). SUBSTRATES AND SUPPLEMENTS A variety of substrates were tested for the cultivation of C. indica [12,18], but, with limited success. These authors tried to induce fruit bodies in a number of growth media, including soil-sand, soil-sand-maize meal and soil-sandpulse powder. In soil-sand-meal, primordial fungus appeared in 5~6 wk, but another 2~3 wk were required to have a matured well differentiated fruiting body (stipe 4.2 cm long; pileus 4.0 cm dia. and fresh weight 6.11 g). Later attempts were made to develop suitable, low cost synthetic compost for the production of more fruit bodies. By 1981, it had become possible to grow C. indica on unsterilized, paddy straw-maize or wheat bran substrate [19]. Purkayastha [8] used chopped rice straw, pre-soaked for 18 to 24 hr in water and put in hot water for 2~3 hr. This substrate was filled in trays and seeded with spawn. Doshi and his group evaluated wheat straw, maize stalks, sorghum stalks, maize meal, rice meal, sorghum meal, and wheat bran as basal substrates for the production of C. indica [20,21]. The results indicated that wheat straw was the best substrate for fruit body production. Addition of different supplements to the substrate also influences the spawn run, days for pinning, number of pinhead initiation, flushing pattern and overall mushroom yield. Krishnamoorthy [22] concluded that substrates like paddy straw and sorghum stalks were colonized more quickly by the milky white mushroom fungus compared to black gram hay, soybean hay, maize stalks and finger millet straw [6]. The study also indicated that substrates like coconut coir pith compost, paddy straw compost and saw dust did not favor the growth of C. indica. In addition, supplementation of paddy straw with neem (Azadirachta indica) cake, black gram husk and red gram husk, followed by cotton seed cake (5 wt% of the wet weight of the substrate), significantly improved the yield of milky white mushrooms (300~380 g increase over control). Interestingly, the average weight of individual sporophores (spore-producing hyphae, which may be loosely arranged) was always found to be high when the beds were supplemented with black gram husk, red gram husk and neem cake. While, minimum weight of individual mushrooms was recorded in the sporophores harvested from beds that were supplemented with wheat bran. Several other lignocellulosic agricultural residues like sunflower stalks and hulled head, jackfruit rind, cotton stalks, waste cotton, sugar cane bagasse, jute waste, corn cobs, groundnut hulls, coffee/tea waste have also been tried by many workers with limited success [23][24][25][26]. In a separate study, Kumar et al. [27] evaluated 11 different supplements viz., wheat bran, soybean flour, pigeon pea powder, green gram powder, cotton cake, mustard cake, neem cake and lentil powder. Alam et al. [28] have used 30% maize powder to supplement paddy straw substrate in order to increase mushroom yields. More promisingly, supplements like soybean and cotton seed cake gave the highest absolute mushroom yields (64.8% and 59.2% increased biological efficiency over control). Milky white mushroom production. Currently, the milky white mushroom farming is done manually. The cultivation process is labor intensive and fairly energy demanding. The crop production process involves six different steps viz., (1) spawn production, (2) substrate pretreatment, (3) mushroom bed preparation, (4) cropping room maintenance during spawn run and mushroom production, (5) harvesting and packaging, and (6) management of spent mushroom substrate (Fig. 3). Farmers normally use milled paddy straw as substrate (2~4 cm) The milled straw is soaked in water for 4~5 hr, prior to hot water (80 o C) or steaming treatment for 45~60 min. After pre-treatment, the materials are shade dried to get appropriate moisture condition (60~70%) before bed preparation. Polypropylene bags (60 × 30 cm) are normally used as containers for bed production and layer spawning with grain spawn is typically the adopted technique. For spawn run, the bags are kept in clean rooms maintained at 25~30 o C and 80~85% humidity for 15~20 days. At this stage, steam-treated casing soil is applied on half-cut beds to a depth of 1.5~2.0 cm. The beds are then transferred to cropping rooms (polythene sheet covered rooms) maintained at 30~35 o C, with humidity of higher than 80%. Sufficient natural light should be made available inside the cropping room. Pin heads will appear on the casing surface within a week and the mushrooms attain harvesting maturity in a couple of days. The first flush of mushrooms will normally appear within 24~30 days of bed preparation. Over a period of 40~45 days, mushrooms could be harvested in three to four flushes [6]. Several cases of less organized cultivation in thatched houses or under tarpaulin roofs surrounded by brick walls have also been reported with limited success [29]. These sheds are not properly insulated and the growth requirements, including temperature and relative humidity, could fluctuate depending on the external environment. A one-year study on the yield of milky white mushrooms in such sheds, using rice straw as substrate, showed the best performance during the months of May and June (peak summer season in South India) [27]. Temperature ranges of less than 25 o C, degenerated the mushroom growth. Other hindrances for milky mushroom production include over-matured spawn, insect infestations, contaminant fungi and bacteria due to unhygienic conditions and poor farm maintenance. Casing requirements. Casing is an important agronomic practice in the cultivation of any humicolous mushroom (that grow on soil) and milky white mushroom is not an exception. Casing triggers off the change from vegetative to reproductive phase. Compact casing interfaces impede the diffusion of harmful metabolic gases on mushroom bed surface. Thus accumulation of high concentrations of carbon dioxide in the soil during fructification usually results in yield depression [30]. Smerdon defined clearly the qualities of casing soil and said that it should have a high water holding capacity, besides retaining a good air space ratio to facilitate gaseous exchange [31]. They also concluded that the pH of such soil must be neutral to alkaline. Singh et al. [32] concluded that steam sterilized casing soil produced better yield than the chemically treated with formalin or using heat sterilization. Casing was found to be an absolute requirement for proper fructification in C. indica by several workers. Purkayastha [8] used loamy soil or garden soil and sand (1 : 1) mixed thoroughly with calcium carbonate at 12% level (pH 7) on milky white mushroom beds. Krishnamoorthy et al. [6,10] have concluded that partially steamed clay loam soil (pH 8.4) generates maximum yields and a higher number of buttons than other media, including peat soil, sand, biogas slurry, farm yard manure and coir pith compost. In sandy Table 2. List of growth regulators and their structure that increase milky white mycelia and mushroom yield [15,33] Mycelial dry weight (mg) Mushroom production soil and farm yard manure, the fungus took more than 10 days for the production of pinheads and attained harvesting maturity after 10.6 and 9.2 days, respectively. In clay loam soil and peat, the buttons appeared almost 2 days earlier when compared to all other casing media tried. Interestingly, the clay loam soil had the quality to absorb moisture quickly and release it slowly. In this soil, less water was needed to maintain the required moisture level and a delay in spraying did not lead to the total drying of bed surface. Using vermin-compost as casing substrate was also reported with limited success. In addition to its composition, pH, EC, water holding capacity, porosity and bulk density of casing mixture are some of the important factors to be considered while selecting substrates for casing [34]. Role of growth regulators in crop production. Several growth regulators like indole acetic acid, indole-3-butryic acid, gibberllic acid (GA), and kinetin were tested for their effect on the sporophore size and yield of milky white mushroom [35]. The results clearly indicated that GA at 40 ppm increased the yield of sporophores ( Table 2). The average weight of individual mushrooms was found to be high when GA and kinetin were sprayed. Pileus (cap of mushroom) diameter and its weight were found to be slightly higher in the above treatments, but none of the growth regulators showed a significant influence on the stipe length and its weight. Pani has also reported that spraying GA at 50 ppm at the time of pinhead formation had greatly influenced the size of sporophores and yield of milky white mushrooms (82% as compared to 68% in control) [35]. CHEMICAL COMPOSITION AND NUTRITIONAL VALUE The six major constituents of mushrooms are water, proteins, carbohydrates, dietary fiber, fat, and ash [33]. The moisture content of mushrooms is usually determined by drying at 105 o C in a hot air oven overnight to a constant weight. The difference in weight before and after drying is expressed in terms of percentage. The protein content is determined by Kjeldahl method and the lipids are estimated by Twisselman method using extractive solvent like diethyl ether [36]. The lipids in mushrooms include free fatty acids, mono-, di-, and triglycerides, sterols, sterol esters and phospholipids. The sporophore samples are incubated in a muffle furnace at 500 o C to estimate the ash content which normally contains potassium and phosphorous. Total carbohydrate content in a given mushroom sample is calculated using the formula, 100 − moisture (%) − protein (%) − crude fat (%) − ash (%) and expressed as g/100 g of fresh or dry sample [37]. According to Crisan and Sands, the energy content in mushrooms is influenced by the composition of crude protein, fat and carbohydrates whose conversion factors are 2.62, 8.37, and 3.50 kcal/g of the individual components, respectively [38]. These conversion factors are slightly lower than the actual conversion factors used for other food ingredients because they are estimated as crude components. Sivaprakasam and Doshi et al. [38,39], recorded 20.2% protein from the caps of milky white mushroom (on dry weight basis). Krishnamoorthy et al. [6] reported 32.2% protein (dry weight basis) in a medium sized milky mushroom. Interestingly, among the three mushrooms viz., button, oyster, and milky white mushrooms, the latter has been reported by several authors to contain more protein [6]. Milky white mushroom also has lower moisture content (85.6%) and increased fiber content (61.1% on dry weight basis). Total carbohydrates and crude fat were also found to be more abundant in milky mushrooms (59.9% and 0.67%, respectively) when compared to oyster mushroom varieties (P. eous and P. sajor caju). These differences could be due to variations in mushroom culture conditions, time of sampling and substrates used. Saranya et al. [40] have reported that the type of substrates and supplements used for mushroom cultivation had greatly influenced the proximate composition (carbohydrate, protein, fat, fiber, ash content, and moisture content) including antioxidants. Based on the availability of soluble sugars. These authors also reported increased levels of calcium, phosphorus and iron in the milky white mushroom. Similarly, total lipids, carbohydrates and ash contents in milky white mushroom were also found to be comparatively higher, ultimately increasing its total calorific value (50.03 kcal/100 g). The dietary fiber (fungal cell wall components mostly, chitin-N-acetyl-glycosamine units) and protein contents of milky white mushrooms are higher than button mushroom, but lower than oyster mushroom (Table 3). It has been reported that chitosan from C. indica sporophores ranged from 2.5% to 2.9% on dry weight basis [41]. In case of P. florida, an oyster mushroom species, it ranged from 2.0% to 2.3%. The FT-IR spectrum of the fungal chitosan obtained from C. indica, P. florida and shrimp chitosan exhibited eight major peaks at the ranges of 3,000~3,500/cm, 3,426/cm, 2,885/ The beta-glycans present in dietary fibers of mushrooms are reported to have stimulatory effect on immune system with anti-mutagenic, anticancer and antitumor activities [37]. Mushrooms are good sources of minerals (Ca, K, Mg, Na, and P), trace elements (Cu, Fe, Mn, and Zn) and sometimes, toxic heavy metals (Cd and Pb) as compared to vegetables [43]. The mineral components of milky white mushrooms [44,45], as reported in the literature are given in Table 4. NON-ENZYMATIC ANTIOXIDANT Several chronic diseases like rheumatoid arthritis, cirrhosis and life threatening diseases like cancer are caused due to reactive oxygen species and free radicals. Enzymes like superoxide dismutase, catalase and chemicals compounds like vitamin E, C, polyphenols, carotenoids, and glutathione play important role in neutralizing free radicals. Mushrooms are a good source of some of these biologically active compounds that protect the human body against several chronic and degenerative diseases. Most of the mushrooms are rich in vitamins and minerals, particularly, B complex vitamins (thiamine, riboflavin, pridoxine, pantotene acid, nicotinic acid, nicotinamide, folic acid, and cobalamin); as well as ergosterol and biotin, vitamin A in fresh and dry milky white mushrooms have been reported to be 0.35 mg and 0.275 mg per g respectively (Table 4) [46]. Water soluble vitamin C (a free radical scavenger and a well-known antioxidant and inhibitor of lipid peroxidation [LPO]) has been reported in fresh and dry milky white mushrooms (1.03 and 0.4 mg/100 g, respectively) [42]. Similarly, vitamin E (tocopherol), an antioxidant that protects membranes, lipids and lipoproteins [43] has also been reported in fresh and dry milky white mushroom samples (2.8 mg/g and 0.80 mg/g, respectively). The most abundant non-protein thiol (organic chemical compounds similar to the alcohols and phenols but containing a sulfur atom in place of the oxygen atom) in animal cells is glutathione. Glutathione exist is both reduced (GSH) and oxidized (GSSG) state. GSH will be able to donate a reducing equivalent (H + + e − ) to other unstable molecules, such as reactive oxygen species and at high concentration they self-react and form GSSG. GSH can be regenerated from GSSG by the enzyme glutathione reductase [47]. The GSH is essential for protein and DNA synthesis, regulation of enzyme activities and protection against free radicals [42]. Presence of GSH in fresh and dry milky white mushroom samples were found to be 0.15 and 0.025 nmole/g respectively. The vitamin C content in milky white mushroom was higher than oyster mushroom, which contains higher levels of vitamin E [42,48]. In general, fresh mushrooms contain more antioxidants compared to dried samples. Mahfuz and co-workers used methanolic extract of commercially cultivated mushrooms to quantify the amount of antioxidants [49]. Some other interesting in vitro antioxidant studies include (1) 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity; (2) ferrous ion chelating activity; (3) reducing power using potassium fericyanide and ferric chloride mixture and measuring absorbance at 700 nm; (4) reduction of ferric tripyridyltriazene (Fe 3+ -TPTZ) complex to ferrous (Fe 2+ ) ion determined by measuring absorbance at 595 nm; (5) hydrogen peroxide scavenging activity using luminescence spectrophotometer; (6) estimation of phenolic contents; and (7) estimation of flavonoids. Table 4. Comparison of mineral and non-enzymatic reducing compounds composition milky white mushroom with two other widely popular mushrooms (white button and oyster) both in fresh and dry form (/100 g) Button mushrooms [37] (Agaricus bisporus) Oyster mushrooms [45] (Pleurotus ostreatus) Milky white mushrooms [42,47,48] Similarly, methanol extracts of cap and stem of C. indica have been reported [50] for the presence of antioxidants. Comparative estimates of antioxidant properties of methanolic extracts of A. bisporus and C. indica are presented in Table 5. Mirunalini et al. [48] and Babu and Rao [50], have reported in vitro antioxidant activities of C. indica extracts. The results showed that the DPPH scavenging activity, reducing power, chelation, and hydrogen peroxide scavenging activity were higher in C. indica when compared to Agaricus bisporus. Interestingly, the stipe of C. indica exhibited more chelation, hydrogen peroxide scavenging activity, flavonoid and total phenolic contents as compared to its cap. VOLATILE AND FLAVOUR COMPOUNDS Mushrooms are known to produce a wide range of volatile and flavor compounds with a distinctive profiles that vary according to species, variety and sometimes cultural conditions [52,53]. The flavor profile also changes when mushrooms are dried, primarily due to the high level of oxidation [54]. Usually, these compounds are extracted using a combination of polar and non-polar solvents including water and diethyl ether. Apart from this, vacuum distillation, nitrogen flow conveyance, capillary gas chromatography and use of carbon tetrachloride are also followed routinely and the concentration of the flavor compounds significantly change depending upon the extraction method [53]. Most of these flavor compounds include alcohols, aldehydes, ketones and oxides [55][56][57]. In C. indica, a total of 20 compounds have been identified as listed [58] in Table 6. Two of the most abundant compounds present in fresh C. indica sporophores include eight carbon containing volatiles like 1-octen-3-ol (58.3%) and n-octanol (17.9%) of the total volatile fractions. The concentration of these compounds decreased to 10.6% and 2.4% respectively after drying [58]. In addition, eight carbon volatiles including 1-octen-3-one, 3-octanone and 3-octanol have been also reported in several mushrooms, which are normally produced due to the enzymatic interaction of hydro-peroxide lyase utilizing fatty acids, more specifically linoleic acid as precursor. Noticeably, benzyl alcohol and n-Hexanal present in trace amounts in fresh mushrooms, increased to about 10.2% and 15.3%, respectively, after drying. Also, during the process of drying, total alcohol content decreased from 81.9% to 41%, while the concentration of aldehydes increased from 3.1% to 16.9%. In addition, drying decreased the total ketone levels from 4.3% to 1.5% and increased the oxide concentration from 1.2% to 2.1%. The number of unknown compounds is substantially higher in dried mushrooms as compared to fresh ones. OTHER MEDICINAL PROPERTIES Nutritional quality of mushroom is influenced by the substrate used, organic supplementation and other additive effect [51,59]. Medicinal mushrooms are known to be abundant source of nutraceuticals which could decrease/ reverse the progression of several diseases. One such disease is diabetes mellitus, which is otherwise characterized as hyperglycemia associated with insulin deficiency. Complication of this disease includes hypertension, atherosclerosis, microcirculatory disorder and changes in large and small blood vessels. Along with medicinal herbs, mushrooms are believed to play an important role in treating diabetic patients without any harmful side effect. Both cold and hot water extracts of milky white mushroom powder was tested for anti-hyperglycemic effect on diabetes induced rats (using streptozotocin) [60]. The rats were orally given with mushroom extract for 45 days and tested for insulin and glycosylated hemoglobin levels. The results indicated normal values at the end of treatment. In addition, positive effect on hematological parameters including increase in lymphocytes, platelets and red blood cell (RBC) counts were observed. The life span of treated rats was also found to be increased. LPO inhibition is a process that deteriorates polyunsaturated lipids to release several toxic compounds through oxygen free radicals, which are otherwise known to induce cellular damage [61]. In an interesting study, Selvi et al. [61] used two different membrane model systems viz., goat liver homogenate and RBC ghost, which have different lipid compositions; and C. indica mushroom extract to evaluate LPO inhibition. A higher level of LPO inhibition (71.3%) was observed for RBC ghost when compared to goat liver homogenate (59%). CONCLUSIONS Worldwide mushroom production technology has been emerging as a multibillion dollar industry [3,4,62]. Current world mushroom production is about 30.2 million tons and (worth $ 2,800 billion). Mushroom production in India for the year 2013 is roughly 40 thousand tons contributing approximately 1% of total world production [5]. Milky white mushrooms are highly suitable for commercial production in humid tropical and subtropical regions of the world where, the average temperature falls between 25 o C and 35 o C throughout the year [63]. Apart from India, several regions in Africa, North America, South America, Middle East, South East Asia, and Australia where the cost of labor is also comparatively cheaper are suitable for growing milky white mushrooms. In the tropical region, infrastructure required (cooling and heating) to grow milky white mushroom is comparatively less expensive when compared to button mushroom production. Since milky white mushroom resembles button mushroom in several aspects, with higher shelf life, increased productivity, appealing milky white color, it will have greater stake hold in the world market. The mushrooms are robust and flexible for production in varied sizes from a small button (average weight, 35~40 g) to large caps depending upon consumer demand, which is normally not possible in any other cultivated mushrooms around the world. The demand statistics, indicate a 3~5% increase in world mushroom consumption every year; and milky white mushroom cultivator could explore this opportunity to establish newer markets in developed countries like US and Europe where, the demand for mushrooms is quite high [7]. Industrialization of the new concept by mechanization and controlled environment production will be required to further reduce the cost of production and increase profit margins to mushroom producers. Mass production of mushrooms will pay way to produce processed mushrooms (dry, canned, pickled, and fried) will increase the shelf life that could be marketed in different regions of the world.

v3-fos

2018-04-03T04:50:33.958Z

2015-03-26T00:00:00.000Z

17577421

s2

Genetics, structure, and prevalence of FP967 (CDC Triffid) T-DNA in flax The detection of T-DNA from a genetically modified flaxseed line (FP967, formally CDC Triffid) in a shipment of Canadian flaxseed exported to Europe resulted in a large decrease in the amount of flax planted in Canada. The Canadian flaxseed industry undertook major changes to ensure the removal of FP967 from the supply chain. This study aimed to resolve the genetics and structure of the FP967 transfer DNA (T-DNA). The FP967 T-DNA is thought to be inserted in at single genomic locus. The junction between the T-DNA and genomic DNA consisted of two inverted Right Borders with no Left Border (LB) flanking genomic DNA sequences recovered. This information was used to develop an event-specific quantitative PCR (qPCR) assay. This assay and an existing assay specific to the T-DNA construct were used to determine the genetics and prevalence of the FP967 T-DNA. These data supported the hypothesis that the T-DNA is present at a single location in the genome. The FP967 T-DNA is present at a low level (between 0.01 and 0.1%) in breeder seed lots from 2009 and 2010. None of the 11,000 and 16,000 lines selected for advancement through the Flax Breeding Program in 2010 and 2011, respectively, tested positive for the FP967 T-DNA, however. Most of the FP967 T-DNA sequence was resolved via PCR cloning and next generation sequencing. A 3,720 bp duplication of an internal portion of the T-DNA (including a Right Border) was discovered between the flanking genomic DNA and the LB. An event-specific assay, SAT2-LB, was developed for the junction between this repeat and the LB. Introduction In April 2009, transgenic flaxseed was detected in two 5,000 tonne shipments of flax during preprocessing in Europe (Flax Council of Canada 2009a). Shipments of Canadian flax have also tested positive for transgenes in Japan and Brazil (Flax Council of Canada 2009b). The EU and these countries have regulations regarding the detection of genetically modified (GM) flax in shipments and have zero tolerance for its presence. As a consequence, Canadian flax shipments to Europe fell dramatically from 2010-2012; Europe imported 80% of Canadian flaxseed prior to 2009 but only 20% in 2011 (personal communication, William Hill, President, Flax Council of Canada). The Canadian industry responded to this reduction in flax exports to Europe by introducing measures to eliminate the presence of GM flax. These included the reconstitution of popular flax varieties (CDC Bethune, CDC Sorrel) from reserved GM-free seed stocks; encouragement by commodity groups to sell stored grain, clean out grain bins, and purchase fresh seed; and increased screening for the GM construct at all levels of production (Booker and Lamb 2012). As a consequence, the estimated incidence of the GM construct has fallen from a high of 0.004% in the 2009 and 2010 crop years to 0.0001% in the 2012 and 2013 crop years (Booker et al. 2014). Positive results for GM flax presence in some Crop Development Centre (CDC; University of Saskatchewan, Saskatoon, Canada) breeder seed lots occurred when testing was done at or below the 0.01% level (CDC 2010). The Flax Council of Canada has proposed testing all certified seed for the presence of GM before it is used for planting (Flax Council of Canada 2011). Further development of valid methods to detect transgenes in flax and vigorous screening of flax breeding material for transgenes is urgently needed and will contribute towards understanding the inheritance of transgenes in the flax germplasm and, importantly, the restoration of export markets. The presence of transgenes in flax shipments from Canada relates to the mid-1990s introduction of GM line FP967 (registered as CDC Triffid in 1997) (McHughen and Holm, 1994;McHughen et al. 1997). This variety was deregistered in 2001 due to concerns about the effect of production of GM flax on export markets (Ryan and Smyth 2012). Here, we describe the development of an eventspecific assay to detect the presence of FP967 transfer DNA (T-DNA). The assay developed was used to determine the number of loci containing the T-DNA in the FP967 genome. In addition, a retrospective look at breeder seed produced by the CDC to isolate potential sources of the GM construct was performed, as well as a detailed examination of lines currently passing through the CDC's Flax Breeding Program. Cloning of genomic fragments from the T-DNA and next generation sequencing (NGS) of the FP967 line were done to obtain better knowledge of the GM construct. Sampling of breeder seed A total of 27 samples of CDC flax breeder seed were obtained for registered CDC flax varieties. Subsamples were taken from each of these samples. Each subsample weighed approximately 5.7 g and contained about 950 seeds. A positive control sample, consisting of approximately 5.7 g of CDC Bethune seed spiked with a single heat-killed FP967 seed, was incorporated in each set of extracts. Between three and nine subsamples were examined from each sample of seed, with the number varying due to availability. DNA extraction breeder seed DNA was extracted from these subsamples using a modified guanidinium hydrochloride-silica matrix extraction protocol. Whole seeds were ground at 1200 rpm for 4 min using a vertical ball mill (Genogrinder from Spex SamplePrep, Metuchen, NJ, USA) in 20 mL of 5 M NaCl with three 12 mm diameter zirconium-yttrium ceramic cylinders (Inframat Advanced Materials, Manchester, CT, USA) in a 50 mL tube. The resulting homogenate was centrifuged at 1250 × G for 10 min and 2 × 1.2 mL of supernatant drawn off and transferred to fresh tubes containing 20 μL of SilMag slurry (silica-coated iron powder, Chemicell GmbH, Berlin, Germany). The mixture was left to stand at room temperature, with occasional agitation, for 5 min. Subsequent washing and elution steps were performed using a magnetic rack (for tubes). The iron particles were collected and washed once with 1.2 mL of 4 M guanidinium hydrochloride in 50% ethanol and twice with 10 mM Tris HCl, pH 8.0 in 80% ethanol. Excess wash solution was drawn off using a pipette and the pellet allowed to air dry for 10-15 min. DNA was eluted in 100 uL sterile distilled water. Construct-and event-specific assays Two Taqman assays were used to determine the presence of the FP967 T-DNA (Tables 1 and 2, Figure 1). The first assay is construct specific and is used in commercial testing laboratories to detect the presence of FP967 (Anon 2009;Grohmann et al. 2011). It consists of a pair of primers (Table 2) designed to amplify a 105 bp NOS-terminator to DHFR fragment of the FP967 T-DNA. A second pair of primers and a probe are used to detect a 68 bp fragment of stearoyl-acyl carrier protein desaturase (SAD) as a reference gene. The second assay was designed as an event-specific assay (Tables 1 and 2). Primers (P13, P14, and P15) were designed using inverse-PCR generated sequence of the right border region of the T-DNA (Figures 1 and 2). The presence of the T-DNA produces two fragments of 215 and 186 bp from FFS1 and FFS2, respectively. Both transgenic fragments are detected with the same probe. If the T-DNA is absent, a 202 bp fragment of scaffold261 is amplified and detected with a probe complementary to the region of DNA eliminated by insertion of the T-DNA. In this assay, homozygous plants are detected by one or the other probe while hemizygous plants produce both fragments (Table 1). Assay conditions are provided in the supplementary data archive (http://dx.doi.org/ 10.6070/H498851J). The construct specific assay is able to distinguish between WT and transgenic plants, while the event specific assay can also distinguish between hemizygous and homozygous individuals. Sequences of the primers and probes are shown in Table 2, while their approximate location is shown on Figure 2. The sequences and orientations of the LB and flanking region, the pBR322 fragments and the LIH had not been confirmed. The construct specific assay, which detects the DHFR fragment form E. coli and the Nos terminator, is indicated (P3, P4 and prb2). The event specific assay, developed in this project, is also shown. It uses a primer in Norlin gDNA (P13 or P14), a primer in the RB (P15) and a probe in the RB (prb5) to detect the TDNA. C) Deduced T-DNA structure after NGS and PCR fragment cloning. The inverted portion of the TDNA inserted between FFS1 and the LB is indicated, as is the new event-specific assay, which spans the junction between the SAT2 gene of the SpecR cassette and the LB (P85, P86 and prb28). The orientation and sequence of the LIH, AtALS, NPTII, SpecR cassette and internal Nos gene were deduced. Inverted sections were found to be oriented in the reverse direction. Sampling of breeding lines Two seeds from each line selected for advancement through the CDC Flax Breeding Program in 2010 were grown on cotton in 96-well trays in the lab for 7-9 d. Each hypocotyl section was excised and hydrolyzed in 40 μL of 0.25 M NaOH at 95°C for 45 s before being neutralized with 60 μL of 0.5 M Tris pH 8.0. A second incubation at 95°C for 3 min was performed and the extracts allowed to cool to room temperature. A one microliter aliquot of each extract was used in the construct-specific assay (16 μL total volume). A single FP967 seedling was used as a positive control in each tray. A different protocol was used on seed from the 2011 nursery. Ten seeds from each line selected for advancement through the program were homogenized in 4 M guanidine hydrochloride in 50% ethanol by a 5 mm zirconium-yttrium ceramic bead in a deep well microtitre plate using a vertical ball mill. Debris was pelleted by centrifugation at 3700 rpm for 10 min and 700 μL drawn off to a fresh deep well microtitre plate containing 15 μL of MagSil slurry. The subsequent wash steps were performed using a Kingfisher apparatus (ThermoFisher) and followed the same procedures as described for the bulk seed DNA extraction. A single microliter of the DNA extract was used in both the construct-specific and event-specific assays. A single heat-killed FP967 seed, along with nine CDC Bethune seeds, was used as a positive control in each plate. The original papers describing the development of FP967 did not definitively state the number of copies of the T-DNA present in the line (McHughen 1989;McSheffrey et al. 1992). Accordingly, we used both qPCR assays to determine the number of copies of the T-DNA present within FP967. Both F2 and BC2 lines from Norlin × FP967 crosses were examined. Genomic DNA was extracted from FP967 root tips and sequenced using Illumina HiSeq chemistry. Both mate-paired and paired-end reactions were performed, using 36 and 100 cycles, respectively. The resultant reads were assembled against the flax reference genome (version 1.0 at Phytozome.net, accessed 8 Feb 2015; Wang et al. 2012) and unaligned reads identified. Bowtie2 and Ray were used to assemble scaffolds and construct putative sequences for the FP967 T-DNA. In some instances, a reduced dataset consisting of reads not aligning with the L. usitatissimum reference sequence, collected using Bowtie2 (Boisvert et al. 2010), was used in de novo assembly using Ray (Langmead and Salzberg 2012) or SOAPdenovo. A wide range of kmers and parameters were used to remove the majority of non-relevant reads from the data. In addition to this approach, reads were aligned against known fragments of the T-DNA and the PCR-generated clone sequences using Bowtie2 (Fraley et al. 1983). Assembly and alignment of the reads and contigs were performed on a local machine using Geneious or using Hermes, a WestGrid server that is a part of Compute Canada Calcul Canada. Results and discussion Development of an event-specific assay for FP967 T-DNA Inverse PCR from the Right Border (RB) of the T-DNA showed two copies of this sequence present in scaf-fold261 from the Linum usitatissimum genome assembly v1.0 (Figure 2). These two RB fragments were in an inverted orientation relative to one another and replaced 12 bp of scaffold261. Attempts to identify genomic regions adjacent to the Left Border (LB) were unsuccessful. These results suggest at least one insertion of the T-DNA or a duplication of a fragment of the T-DNA occurred. The propensity of T-DNA to undergo rearrangement and/or duplication, including inverted repeats, has been noted previously, e.g., (De Buck et al. 1999). The inverted and repeated structure of the T-DNA RBs means that an event-specific assay could be developed. qPCR primers and probes were developed (Table 2, Figures 1 and 2) to detect both the intact Norlin scaf-fold261 sequence (named FFS1 and FFS2 for FP967 Flanking Sequence 1 and 2 on either side of the insertion site, respectively) as well as simultaneously detect both RB sequences. The event-specific assay is designed to produce one of two mutually exclusive products in homozygous individuals; that is, either the scaffold261 or the FFS/RB PCR product is synthesized and detected. In heterozygous individuals, or DNA admixtures containing both scaffold261 and the T-DNA, both products are amplified and detected. The detection of these two DNA fragments occurs in a single tube. The eventspecific qPCR assay is complementary to the constructspecific assay used in commercial test laboratories (Anon 2009;Grohmann et al. 2011) and the assay designed to detect the mutated Arabidopsis acetolactate synthase (AtALS) gene that provides resistance to sulphonylurea herbicides (Nakamura et al. 2010). The event-specific assay was tested on genomic DNA extracted from individual FP967 plants as well as from mixtures of FP967 and CDC Bethune seeds. The developed assay was specific to FP967 and could detect the T-DNA in DNA extracted from seed admixtures (results not shown). In our hands, the event-specific assay was sensitive to one FP967 seed in admixtures containing~5,000 seeds. The described event-specific assay is not as sensitive as the construct-specific assay. The construct specific promoter may be more sensitive than the event specific assay as it was discovered that there is a duplication of the Nos gene-Streptomycin resistance cassette region of the TDNA containing the target of the construct specific assay (amplified by P3, P4 and prb2). This means that double the number of copies of the construct specific assay target are present. The event-specific assay has the advantage of having one less primer than the commercial test. The assay also produces both a positive and a negative polymerase chain reaction (PCR) product, eliminating the need for a PCR control reaction (stearoyl-acyl carrier protein desaturase (SAD) in the commercial tests). As a result, this assay halves the number of PCR assays required compared to assays where the T-DNA and SAD are detected in separate reactions. However, because the eventspecific assay is not as sensitive as the commercial assay, it is not as useful for detecting the presence of FP967 T-DNA in bulk samples. Genetics and prevalence of the FP967 T-DNA The event-specific assay may be used to detect heterozygous individuals as it produces a PCR product from both the T-DNA and the non-transformed genomic DNA sequence. The inheritance of FP967 T-DNA was investigated using both the event-and construct-specific assays. Using the construct-specific assay, we detected the presence of FP967 T-DNA in 55 of 80 F 2 plants, suggesting a single locus for the T-DNA (χ 2 = 1.67, 1df, p =0.13). Using the event-specific assay to test the same F 2 individuals, we observed 24 FP967 homozygotes, 31 heterozygotes, and 25 Norlin homozygotes in the same population, once again suggesting a single T-DNA locus (χ 2 = 4.08, 2df, p = 0.07). This conclusion is further supported by the 15:17 ratio (T-DNA with scaffold261: scaf-fold261) of individuals in the BC1 generation (χ 2 = 0.13, 1df, p =0.47). The results from these two independent populations indicate that a single insertion of the T-DNA is present in the Norlin genome, supporting previous work based on progeny tolerance to chlorosulfuron in vitro (McHughen 1989). Our results also clarify previous work using Southern blots that suggests one to three copies of the T-DNA are present in the FP967 genome (personal communication, Alan McHughen, University of California Center Sacramento). These results, along with the cloning and sequencing of the T-DNA strongly support the hypothesis that the T-DNA is present at a single locus in FP967. The event-and construct-specific assays were used to detect the presence of the FP967 T-DNA in CDC breeder seed. A total of two assays tested positive for FP967 T-DNA in breeder seed sampled in 2009 (out of 45 performed over 15 samples) and three in breeder seed sampled in 2010 (out of 72 performed over 12 samples). The positive samples corresponded to CDC Arras and CDC Normandy. Possible trace levels of FP967 T-DNA were detected in a sample of CDC Mons breeder seed; however, this result was not replicated in repeated assays of the extracted DNA (Table 3). Breeder seed from other varieties (CDC Bethune, CDC Gold, CDC Sanctuary, CDC Sorrel, CDC Valour, Flanders, Somme, and Vimy) were negative for the T-DNA in our tests (Table 3); however, commercial testing of these sources returned positive results (Lamb and Booker 2011). The discrepancy between in-house and commercial testing results may be due to differences in sensitivity and the size of the replicates assayed. Commercial testing currently examines four 60 g replicates and records a positive result if the T-DNA is detected in a single replicate; the subsample size used in this study was much smaller. As the level of FP967 present in these seed batches is low, it is likely that the smaller sample size used in our in-house assays simply did not contain any FP967 seeds, thus making providing a negative result. Overall, the level of FP967 present in the breeder seed samples tested was low, between the 0.1% threshold detectable in the event-specific assay and the 0.01% standard of the commercial assay. As the level of FP967 is so low, modifications to our in house sampling procedure are required. Breeding lines currently advancing through the CDC Flax Breeding Program were tested for the presence of the construct found in CDC Triffid to prevent further introduction of the FP967 T-DNA into the Canadian flaxseed supply. Lines selected for advancement through the CDC Flax Breeding Program have been examined since 2010. Approximately 11,000 and 16,000 lines were examined in 2010 and 2011, respectively, for the presence of the FP967 T-DNA, none of which tested positive. In light of the zero detected incidence of FP967 T-DNA in the CDC Flax Breeding Program breeding lines, the current GM testing protocol consists of testing 1) all individual plants used in crosses, 2) all individual plants grown in growth chambers for increasing seed volumes for the field, 3) a random selection of 10% of the single plant selections for advancement in any year, and 4) all breeder seed leaving the program for seed growers and advanced breeding lines in regional cooperative trials or populations for winter nursery increase. Determining FP967 T-DNA sequence There were several reasons to determine the sequence of the FP967 T-DNA. A complete sequence of the original construct was not required for registration when FP967 was developed, and providing this information would help fill a knowledge gap with respect to this deregistered variety. A complete sequence of the construct could also lead to the development of other event-specific assays as well as more specific or robust construct-specific assays; the current commercial assay contains a primer located in the dihydrofolate reductase (DHFR) region of E. coli integron 2, and shares similarity to the flax orthologue. In addition, we wanted to try to identify the flanking regions adjacent to the LB of the T-DNA to better understand the structure of this rearrangement ( Figure 1B). A number of seed samples of CDC varieties were sampled in 2009 and 2010. These samples were tested at a small scale (5.7 g) in-house using the construct-and event-specific assays. Commercial laboratories using the construct-specific assay also tested these same samples. The commercial assay consisted of either a single or four independent 60 g subsamples. Results from the commercial tests were reported as either positive/negative for the subsamples as a whole, or positive/ negative/trace for each subsample. As some four-subsample tests were reported as positive or negative, it is not possible to report the number of positive assays. The single positive in-house assay for CDC Mons was very weak and is reported as trace. A large number of PCR clones were developed from the FP967 T-DNA. These were aligned against a draft version of the putative FP967 T-DNA generated from literature published about the AtALS mutant (Haughn et al. 1988;Sathasivan et al. 1990;McSheffrey et al. 1992) and cointegrate Ti plasmid construction (Zambryski et al. 1983;Fraley et al. 1985;Sanders et al. 1987). Seventy-nine sequence fragments or contigs derived from the PCR clone sequences aligned against the FP967 draft, comprising~120 kb of sequence and covering 89% of the putative T-DNA sequence. An additional 13 fragments contained the FFS1 or FFS2 flanking regions from scaffold261 and the RB fragment of the T-DNA. The sequences of these clones has been placed in the Labarchives online repository at http://dx.doi.org/10.6070/ H498851J. Several sequence gaps were present due to challenges associated with cloning long DNA fragments. In addition, plasmids containing the pBR322 ori site could not be cloned into pBluescript as these two plasmids have identical origins of replication. Several attempts at plasmid rescue from FP967 genomic DNA using PCR-and ligation-based approaches were attempted, but only flax genomic fragments unrelated to the insertion site were recovered. NGS was used to further determine the FP967 T-DNA sequence. Both mate-pair (MP) and paired-end (PE) sequencing were used. The four lanes of MP reads (2.3 and 2.8 kbp fractions) and seven lanes of PE data were assembled against a putative T-DNA sequence using Bowtie2 (Boisvert et al. 2010). Another approach was to extract T-DNA specific sequences from the NGS reads. Reads not aligning to the L. usitatissimum reference sequence (Wang et al. 2012) were collected using Bowtie2 and de novo assembled using Ray (Langmead and Salzberg 2012) or SOAPdenovo. Contigs containing known FP967 T-DNA sequences were identified from this assembly and aligned against the draft sequence. Using these two approaches, approximately 32,000 reads aligned against the FP967 T-DNA as well as the cloned PCR fragment sequences (available at DOI: http://dx.doi.org/10.6070/ H498851J). A putative structure for the T-DNA was developed ( Figure 1C and supplementary data online) using these data. Further cloning work using primers designed on either side of gaps in the putative sequence was performed. T-DNA components apparent in the putative sequence ( Figure 1C) include the entire LB region with an adjacent ampicillin resistance gene from pBR322, a large fragment incorporating the NOS gene, the spectinomycin resistance/DHFR from E. coli, a chimeric NPTII gene (with NOS promoter and terminator), and the AtALS gene with an adjacent LIH (left inside homology) region from the T-DNA. Both of the repeated pBR322 fragments and the three NOS promoter and terminator regions were challenging to resolve, even with the NGS data. Attempts to identify the junctions of the NOS terminators and promoters and the pBR322 sections required manual analysis and alignment of putative contigs. Using two assembly processes (one using unpaired MP reads aligning to known DNA fragments in the T-DNA and the other using paired MP reads that did not align to the flax reference genome) resulted in two opposite orientations for the T-DNA components between the LB and RB regions. The orientation of this central part of the T-DNA (from one of the NOS genes to the LIH) was resolved by examining the sequence of the PCR clones spanning the junctions of this segment. The order of the components along the T-DNA suggests the homologous recombination that introduced the intermediate cloning vector, pGH6 (Haughn et al. 1988), into the disarmed Ti-plasmid, pGV3850 (Zambryski et al. 1983), occurred between the pBR322 fragments, rather than at the LIH. This is expected as the LIH in pGH6 is derived from an octopine Ti plasmid, pTiA6, and does not share homology with the LB region present in pGV3850 (Fraley et al. 1985). Evidence suggests a repeat of an internal portion of the T-DNA is located between FFS1 and the LB of the T-DNA ( Figure 1C). The repeated fragment starts at the NOS promoter of the Nos gene and extends to just past the streptothricin acetyltransferase 2 (SAT2) gene. (This gene is incorporated in the fragment of DNA used to provide streptomycin resistance (aminoglycoside adenyltransferase A (AADA)) in the intermediate cloning vectors (Fraley et al. 1983).) The evidence supporting this possibility includes six PCR clones obtained using primers located in FFS1 and near AADA and an assembly contig that contains 184 bp of LB adjacent to the SAT2 gene. In addition, PCR reactions using one primer located in the duplicated region (P30 or P86) and another in the LB (P20 or P85) DNA yielded fragments of the expected size. Sequencing of these fragments revealed the expected sequence (Figure 3). The sequence of~1 kb of this junction region is archived in the supplementary data (http://dx.doi.org/10.6070/H498851J). Together, these data show that a duplication of a portion of the T-DNA resulted in the generation of the two RB regions with flanking FFS regions and an additional copy of the chimeric Nos to SAT2 fragment. This event prevented the LB from having flanking sequences in the flax genome. We estimate that the duplicated region between FFS1 and the LB (from the chimeric NPTII to the SAT2/AADA fragment) is 3,720 bp in length. The presence of this repeat was used to develop a simple assay, SAT2-LB, that we assume is event specific as it is highly unlikely that a similar rearrangement and duplication would have occurred in other transformations. The assay developed contains a primer in the SAT2 gene (P86), one in the LB region of the T-DNA (P85), and a probe that spans the junction between these two components (prb28; Figure 1C). This qPCR assay only detects FP967 and not Norlin gDNA, even at a low stringency (54°annealing/extension in a two-step reaction). The assay was also tested on crude gDNA extractions from seedling hypocotyls and mixtures of DNA extracted from a single FP967 seed along with nine CDC Bethune seeds. The event-specific assay was able to detect the FP967 T-DNA in both of these admixtures (data not shown). Conclusions An event-specific assay was developed to detect FP967 T-DNA based on the inverted repeat structure of the two right border sequences adjacent to FFS1 and FFS2, two flax genomic DNA regions. Using the assay, we determined the existence of a single locus for the FP967 T-DNA in both F2 and BC populations. In addition, we identified FP967 T-DNA fragments in seed obtained from CDC flax breeder seed at levels of detection between 0.01 and 0.1%. Breeding lines currently advancing through the CDC Flax Breeding Program are free of the FP967 event. To determine the structure of the FP967 T-DNA, PCR cloning and NGS were utilized to identify the order of genes along the T-DNA and the sequence of most of the construct. Evidence points to a repeat of an internal T-DNA fragment, which led to the unusual inverted-repeat structure of the right border/FFS regions. The unique sequence at the T-DNA LB junction was used to design a second event-specific assay, SAT2-LB, that is specific to this site and is simpler than the one using primers situated in the flanking genomic DNA sequences. Sequence adjacent to the LB region in FP967 and SAT2-LB event specific assay. Our analysis indicated a region of the T-DNA between the NOS promoter of NPTII and SAT2 was duplicated between the FFS1 region of the flax genome and the LB of the T-DNA. A qPCR assay was developed to detect this event specific fragment (P85, P86 and prb28). The sequence of these primers and probe is indicated with green and red blocks, respectively. The location of the Spectinomycin resistance gene AADA and SAT2 are shown in yellow and the LB region in grey. The sequence of this fragment was confirmed by PCR sequencing of P30-P85 and P30-P20 fragments.

v3-fos

2016-05-12T22:15:10.714Z

2015-06-19T00:00:00.000Z

27782461

s2

Correction: One-Seeded Fruits in the Core Caryophyllales: Their Origin and Structural Diversity This article was republished on May 27, 2015 to correct words that were inadvertently merged during the typesetting process. The publisher apologizes for the errors. Also, grammatical errors, hyperlinks and the author contributions were corrected. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference. Introduction The Caryophyllales belong to the core eudicots [1] and comprises approximately 12 000 species distributed worldwide [2]. Molecular phylogeny divides the order into two large clades. The first clade, which is often referred to as the core Caryophyllales or caryophyllids, includes taxa in the traditional order circumscription (Caryophyllaceae, Amaranthaceae s.str., Chenopodiaceae, widely accepted embryological data, several authors [39][40][41] suggested that fruit/seed characters became simplified over time (Table 1). Some authors [26] [42] instead hypothesized that the fruit/seed structure became more complex. The indehiscent or irregularly dehiscent fruits in the Caryophyllaceae are now considered to be an ancestral trait retained in Сorrigioleae (or Corrigiolaceae) and Paronychieae, with subsequent radiations to multi-seeded fruits inferred in various lineages, including the majority of Caryophyllaceae (Plurcaryophyllaceae clade: see [43]). The aim of the present study is to determine the origin and evolution of one-seeded fruits and their structural diversity within the core Caryophyllales. Specifically we attempt to (1) reconstruct the origin and diversification of one-seeded fruits with molecular phylogenetic analysis using a combined rbcl and matK matrix; (2) investigate or ascertain the carpological characters of one-seeded fruits with implications for the recent taxonomy of the core Caryophyllales and to discover the possible peculiarities in the fruit/seed structure within the families in their current circumscription; (3) reconstruct evolutionary history in fine carpological characters to understand their origin and possible changes based on a molecular phylogeny derived from published data for rbcL and matK. Origin of material and its preparation The carpology of *460 representatives of the core Caryophyllales were investigated. About 260 species from Chenopodiaceae were included in previous studies [31], [44][45][46][47][48][49][50]. A list of additional plants studied here for the first time is given in S1 Appendix. Specimens were collected by the authors in many parts of Eurasia and Africa and are stored at the following herbaria: B, E, G, H, LE, MW, W, PUC, with simultaneous preservation of the fruits in 70% ethyl alcohol. No specific permissions were required for the locations of the material, and field studies did not involve endangered or protected species (weed species investigated were collected from the families Amaranthaceae, Caryophyllaceae, Chenopodiaceae and Nyctaginaceae in European Russia, Nepal, South Africa and Israel). Other material (mostly fallen fruits) was obtained from herbarium specimens (with permission) and soaked in a mixture of ethyl alcohol, water and glycerine in equal proportions. Anatomical cross-sections were made by hand or with a microtome. For the majority of the families, the pericarp or seed structure did not depend on the topology of the cross-sections. However, we argued that the fully developed pericarp structure in some Amaranthaceae s.str. (Gomphrenoideae, Achyranthoids) and Chenopodiaceae (Salsoloideae) is represented in the upper part of the fruit, while the lower parts are characterized by reduced zones and layers. This topographically dependent inequality of pericarp layers also occurs in one-seeded Caryophyllaceae, as noted by Rohweder [39] and Schiman-Czeika [51]. The most valuable data sources in these three families were therefore obtained from cross-sections made from the upper part of the fruit. For tissue staining, the following solutions were used: 0.2% aqueous toluidine blue or 1% aqueous solutions of Safranin + 1% Light Green in concentrated picric acid were used for general tissue staining, Sudan IV for revealing fatty substances and Lugol's iodine for revealing starch. To detect crystals, sections were viewed under polarized light. Prior to scanning electron microscopy (SEM), the material was dehydrated in aqueous ethyl alcohol solutions of increasing concentration, followed by alcohol-acetone solutions and pure acetone. SEM observations were made with a JSM-6380 (JEOL Ltd., Japan) at 15 kV after critical point drying and sputtercoating with gold-palladium. The carpological terms used are those of Werker [52]. DNA sequence data for two plastid loci, matK and rbcL, were taken from the GenBank/ EMBL databases (S1 Table), concatenated and analyzed as a single dataset using Maximum Likelihood (ML) (RAxML 7.0.4; [53]) and Bayesian analyses (BI) (MrBayes 3.1.2; [54]), as described in Crowl et al. [55]. Two runs with four chains each (three heated and one cold) were run for 15 million generations; the chains were sampled every 1000 generations with default parameters. The first 1000 Bayesian trees were discarded as burn-in, and posterior probabilities were calculated from the majority-rule consensus (50%) of the remaining trees sampled in both runs. At the end of the runs, the standard deviation of split frequencies between the two runs had fallen to 0.0060 [55] for the alignment strategy. Maximum parsimony reconstructions of the histories of the characters were performed using Mesquite v. 2.75 [56]. The topography of the taxa in the combined tree ( Fig. 1) is almost the same as fin recent investigations [5], [8]. The most significant change concerned Microtea and Macarthuria. In contrast to the previous data that indicated Macarthuria as the sister clade of Microtea and other clades (incl. the 'Globular Inclusion' and AAC+CC clades), our analysis recovered Macarthuria as the sister group of the 'Globular Inclusion' clade. The genera Limonium, Polygonum and Drosera (polygonids) were chosen as outgroups. Results Origin of one-seeded fruits in the core Caryophyllales (Fig. 2) For the analysis, the number of seeds in the fruits were classified into three types: 0one-seeded fruits, 1multi-seeded fruits, 2conjoined (both one-and many-seeded) fruits within an individual. The one-seeded fruit type is the ancestral character state for all the Caryophyllales (core Caryophyllales + Polygonids). Earlier diverging lineages. One-seeded fruit type is the initial character state in all the clades of the 'Earlier diverging' lineages (Rhabdodendron, Physena, Asteropeia, Microtea). Simmondsia, however, as the first lineage, is characterized by a labile seed (1 to 3) number. AAC+CC clade. The one-seeded fruit type is the ancestral character state for the AAC+CC alliance. Many-seeded fruits mostly evolved in the Caryophyllaceae+Corrigiolaceae families. This tendency is seen in the Corrigiolaceae (gen. Telephium) and in the tribe Polycarpaeae. The multi-seeded fruit type is the ancestral character state in the large Plurcaryophyllaceae clade (Sperguleae, Sagineae, Sclerantheae, Arenarieae, Caryophylleae and Sileneae), with rare cases of reversion to a one-seeded character state in Scleranthus (Sclerantheae), some Stellaria (Alsineae) and (not shown in the tree) in Sileneae (Silene ampullata) and Caryophylleae (Scleranthopsis, Saponaria, and Acanthophyllum). In the ААС clade, the conjoined fruit types are the ancestral state only in tribe Celosieae (Amaranthaceae), with a further change to form stable multi-seeded capsules (Pleuropetalum). The data presented here provide the most valuable information about the fruit and seed structure of the families investigated. Based on these features, we compared fruit and seed data for many groups of the core Caryophyllales, with further evolutionary reconstructions of several carpological characters using a plastid-based phylogeny. Discussion Fruit and seed anatomy of the families investigated and its taxonomic importance Earlier Diverging lineages. Almost all the families of the 'Earlier diverging' lineages-the monotypic Rhabdodendraceae, Simmondsiaceae, Physenaceae and Asteropeiaceae-clearly demonstrate a distinctive carpological structure in contrast to the other members of the order. This is expressed in the possession of a thick pericarp divided into several topographic zones, and in their unusual seed-coat structure. The seed coat typically consists of many layers, but they are all are equal in size. Differentiation into a thick one-layered exotesta and thin one-to several-layered endotegmen was reported to be typical for core Caryophyllales [20], but was not observed in any of our representatives. The perisperm was not found in the ripe seeds. Rhabdodendraceae. This is a monotypic family with several species in tropical America. Its systematic position within Caryophyllales was based on important anatomical and palynological data [57]. Investigations concerning the characters of Rhabdodendron [57][58][59][60][61], however, showed that the genus is distinct in many details from other members of the order. This is also true for its gynoecium structure. The style appears to be in a lateral position (anacrostyly) and thus the fruit axis is displaced horizontally. Three topographic zones can be distinguished in the fruit: an exocarp with thickened walls, a tangentially elongated thin-walled parenchyma, and a very hard pyrena consisting of many-layered stone cells filled with tannins. The seed coat is brownish and parchment-like, of three to four thin layers, with equal cells havingbar-thickened walls. The seed embryo appears horizontal but is actually vertical due to displacement of the fruit axis. Only traces of nutritive tissue are found in the seed. Simmondsiaceae. This family consists of a single species, Simmondsia chinensis, with a narrow distribution in the Sonoran desert [62]. Its systematic position was uncertain (e.g., [63][64][65][66]), but many of its anatomical characters do not correspond with families earler considered to be relatives such as Buxaceae or Euphorbiaceae [20], [67][68][69][70]. Recently Simmondsiaceae was placed as diverging near the root of the core Caryophyllales [3], [4]. Only wood anatomy in this family appears to be similar to the other core Caryophyllales and was evaluated as primitive in all dicots [71]. Its fruit and seed anatomy ( Fig. 3A-B) are indeed very unusual within core Caryophyllales. Despite reports that only one seed is present in the capsule [23], [72], sometimes with several rudimentary ovules [73], we also found two or three completely developed seeds in the fruit. Anatomically, the pericarp possesses a one-layered exocarp comprising radially elongated sclereids. Spherical cells of underlying parenchyma (0.7-1.3 mm thick), partially filled with tannin-like substances, form at least 15 chaotically arrangedlayers. In its lower half, this thinwalled parenchyma is intermixed with brachysclereids. The next fewthinlayers of parenchyma consist of crumpled, tangentially elongated cells without tanniniferous content. The inner epidermis comprises spherical cells impregnated with tannins. Both seed integuments consist of *20 layers [20] with no crushing in the ripe seed and cells are filled with tannins. Our investigation confirms previous observations on seed anatomy [20], [23], [67] with some amendments: (1) the number of layers in the dorsal part of the seed is increased in contrast to the ventral surface, and (2) the outer seed-coat layer may consist of both radially elongated and tiny (8-12 μm) thick-walled cells. The peculiarities of the seed coat are connected with their significant thickness (from 180 μm) and its subdivision into several topographical zones (sclerified layers; loose isodiametric, mesotestal layers; and inner epidermis). The seed cavity is completely filled with a straight embryo that is oriented vertically. The highly diversified and thick fruit and seed covers, the presence of the tannins in many (vegetative and reproductive) organs [68] and liquid wax in the seeds [74] all serve to distinguish Simmondsia from almost all other members of core Caryophyllales. Physenaceae and Asteropeiaceae. These two monotypic Madagascan families were only two decades ago placed in an extended Caryophyllales [75]. The combined rbcL/matK analysis supports their position within the root of the core order as sister groups [3]. As in Simmondsia, the wood anatomy is the more primitive type seen in angiosperms [76]. It also appliesto the pericarp of Physena and Asteropeia, which is robust (0.4-0.6 mm thick in Physena madagascariensis, 0.6-0.8 mm in P. sessiliflora, and greater than 1 mm in Asteropeia), and consists of many layers that are divided into two topographic zones ( Fig. 3C-D): (1) mechanical tissue and (2) thin-walled parenchyma. In both species of Physena, the outermost layer consists of radially elongated sclereids with cristate cell cavities that are substituted below by a many-layered sclerenchymatous parenchyma (more than 20 cells). The cells of the mechanical sheath of Asteropeia densiflora have even thickness, and they often have a tannin-like content. Asteropeia multiflora has an outermost layer of radially elongated cells, and several additional layers with rounded cells. The parenchymatous layers form a robust many-layered zone with discontinuous vascular bundles. The fruit bears only a single ripe seed [77]. The seed coat is especially thick (more than 350 μm) in Physena and consists of many layers whose cells are impregnated with tannins ( Fig. 3E). This robust seed coat looks similar to that in Simmondsia, but there are air cavities in the mesotestal layers (P. madagascariensis) or hair-like outgrowths of the exotesta cells (P. sessiliflora). In contrast, the seed coat of Asteropeia is thinner and consists of two to five unequal cells that are also filled with tannins. The seed embryo is straight with curved imbricate cotyledons. Only traces of nutritive tissue are found in the representatives of both families. Macarthuriaceae. It is a recently described monotypic family [78] restricted to Australia that consists of about 10 herbaceous to shrubby species. The systematic position of Macarthuria within the core Caryophyllales has been labile [79] (with references herein). Most authors placed the genus in Molluginaceae (e.g. [80]), despite some differences in the general characteristics, including the presence of the funicular seed aril [81]. Recently the genus was included in one of the deepest lineages of core Caryophyllales based on both rbcL and matK markers [4], [5], [82]. Anatomically, the pericarp of Macarthuria is distinct in that both the thick-walled exocarp and endocarp are impregnated with tannins (M. australis), and in having a two-to four-layered thin-walled mesocarp (Fig. 3F). Microteaceae. This family was recently established [2] and comprises a single genus with several annual species that are distributed in the tropical parts of America. It is distinguished by its possession of small (1-3 mm), spherical, and indehiscent fruits containing a single seed. The pericarp varies among species: its surface can be smooth or alveolate, or contains prickles or acute outgrowths ( Fig. 4A-B). The presence of projections was one of the reasons used to transfer M. maypurensis into a separate genus, Ancistrocarpus [83]. However, all investigated Microtea species share the same carpological traits: (1) the presence of a parenchymatous pericarp that is tightly adjoined to the seed coat, (2) a thick testa, and (3) a thin tegmen with barthickened walls. The embryo is vertical, annular or (in M. maypurensis) slightly bent. Two lineages, Microtea and Macarthuria, are the first clades sharing carpological characters that are typical for the majority of the members of the order. They have: (1) a relatively small (1.5-5 mm) fruit and seed, (2) a non-multiplicative black seed coat with a crustaceous (thick) single-layered testa (30-50 μm), and one to several barely noticeable endotegmen layer(s), and (3) the central position of the perisperm in the seed and annular embryo shape. AAC+ CC clade. The Caryophyllaceae alliance (Caryophyllaceae s.str. + Corrigiolaceae)-This alliance consists of *2200 species distributed worldwide (Caryophyllaceae s.l. in [84]) and is one of the deepest, but highly diversified clades in the core Caryophyllales [3] with some archaic traits in stem anatomy [85]. The former classification of Caryophyllaceae into three subfamilies based on reproductive character sets appears to be unreliable [86], [87]. Further reconstructions of the character states within the new taxonomic rearrangement show that the representatives with the indehiscent or irregularly dehiscing one-seeded fruits first evolved in the deepest clades (Corrigioleae, or Corrigiolaceae; Paronychieae, and partially Polycarpaeae), and the evolution of capsules from a one-seeded fruit type is a common trend in many clades [43]. A tendency of reduction in seed number is assumed in all tribes in which one of several to many ovules becomes the surviving seed (e.g., [39], [51]), but this trend is exceptionally rare in the remainder of Caryophyllaceae (Caryophylleae: Acanthophyllum, Scleranthopsis, some Saponaria, and Sileneae: Silene ampullata). Investigations have shown that Saponaria species, especially S. ocymoides, usually considered as having one seed in the capsule, can in fact develop one to four seeds [88]. Although many members of Caryophyllaceae s.str.are thought to be well-studiedcarpologically, especially with regard to their seeds, we uncovered additional carpological variation that has not attracted attention so far. The first clade in this allianceis Corrigiolaceae (or Corrigioleae), consisting of two genera-Corrigiola and Telephium [43], [89]. We recognizeCorrigioleae atthe familial level, as proposed by Dumortier [90],based on its position as the sister clade of other Caryophyllaceae s.str. An unusual trigonous pericarp shape is found in both Corrigiola and Telephium (see also [91]), but Corrigiola andina and C. africana possess almost orbicular fruits, hardly sharp-edged and then only at the apex. Another peculiarity of both genera is an almost completely sclerified pericarp consisting of many (7-10) layers. The sole exception is Telephium imperati, which has two (-three) outermost sclerenchymatous layers. The unlignified cells substitute the sclerenchyma only beneath as 1-3(-5) innermost, often crushed layer(s). However, these genera differ from each other by the fruit type, which can be a many-seeded (dehiscent) capsule (Telephium) or a one-seeded utricle (Corrigola), contrasting with previous reports of a one-seeded utricle as the only fruit type [43]. Although the fruit of Corrigiola is always described as indehiscent, Rohweder [39] stated that its loculicidal ('ribbed') dehiscence occurs only at seed germination. In contrast to Corrigiolaceae, the pericarp of Caryophyllaceae s.str.is usually not very thick, and cell-wall sclerification is found in the epidermis and (facultatively) in one or several subepidermal cells only (as in Telephium imperati). A similarthinner pericarp with the same topography is found in multi-seeded fruits in the representatives of Sileneae [92], and in the present study in Sperguleae, Polycarpaeae(Ortegia), Sclerantheae (gen. Schiedea) as well as in one-seeded fruits of Minuartia hamata (Sagineae), Acanthophyllum (Caryophylleae), and Cometes (not included in the molecular analysis). The second type of pericarp zonal division, in Scleranthus (Sclerantheae), involves sclerification of the cell walls of the inner layers at the apexof the fruit that is anchored. However, the sclerification of the pericarp is totally absent in most members with one-seeded fruits, and the thin pericarp consists of parenchymatous layer (s) (Herniaria, Anychia, Gymnocarpos, Pteranthus, some Paronychia: [93]). Rarely, the supporting tissue is absent in many-seeded fruits in which the outermost layer consists of living cells with more or less thickened walls (Sclerantheae: Wilhelmsia physodes, Honckenya peploides; Sileneae: Silene baccifera). In such fruits, which have a parenchymatous pericarp, the innermost layer consists of cells with thick anticlinal and inner periclinal walls, but with no fine crystalliferous content. The parenchymatous pericarp of Illecebrum verticillatum consists of parenchymatous cells with bar-thickened walls. Of all the Paronychieae, Paronychia appears to be the most complicated genus. Molecular investigations suggest that Paronychia is not monophyletic and is divided into two different clades within the Paronychieae [43]. From a carpological point of view, some species possess a parenchymatous one-to two-layered pericarp (P. argentea, P. amani), but P. kurdica, P. capitata, P. chionaea and P. chlorothyrsa have a thin-walled exocarp and endocarp and sclerified cell walls with a crystal-filled mesocarp. The seeds are quite diverse. Keeled seeds are rare but are found in Telephium imperati(Corrigiolaceae) and Herniaria(Caryophyllaceae) species, for example. The seeds can take two forms (especially in Corrigiolaceae): smooth, with equally thick testa and tegmen (Corrigiola), or clearly mamillate (Fig. 4C), with a thick testa and a thin tegmen (Telephium). The smooth pattern is most common in Paronychieae and Sclerantheae, whereas the sculptured (mamillate, alveolate or finger-like) testa is quite well developed in the cladesSperguleae, Arenarieae, Alsineae, Caryophylleae, and Sileneae. However, the representatives of Acanthophyllum have only one-seeded fruits that can have various (smooth, alveolate or finger-like) types of seed coat, and Stellaria monosperma is characterized by having one-seeded fruits with a smooth seed surface, in contrast to other Stellaria taxawhose fruits contain many seeds and have mamillate sculpturing. The majority of the alliance's representatives have curved (annular) embryos, but we agree with Pal [94]that bent or straight embryos are not rare in many taxa in Caryophyllaceae s.str. (e.g. Achyronychia, Polycarpon, Illecebrum, Pteranthus, Pollichia). One additional embryo type is known in several members: in Spergula and Drypis [84] it is coiled in 1.5 turns. In spite of different degrees of curvature of the embryo, it is oriented vertically in all the members. Achatocarpaceae. This family comprises two American shrubby genera: Achatocarpus (about 15 species) and the monotypic Phaulothamnus. The small female flowers produce translucent berries which turn black when dry. In the past, Achatocarpaceae was considered to be part of Phytolaccaceae which has an uncertain position in the family [95], [96]. The distinct wood anatomy [97] and the absence of anomalous secondary thickening inthe stem [9] have supported its familial status, as proposed by Heimerl [98]. The twogenera share similar pollen structure [99], [100] and carpological characters described here. The pericarp is divided into four topographic zones: (one-) two-to five-layered outer epidermis, a rare trait in core Caryophyllales, (2) underlying multi-layered soaked parenchyma, (3) one to two layers of U-shaped cells with fine crystalliferous content, and (4) one-layered inner epidermis. The seed coat consists of a robust testa and thinner tegmen that have bar-thickened walls; theembryo is vertical. Abundant starch-granule conglomerates are found in the perisperm, and this carbohydrate type hasapparently not beenfound in other core Caryophyllales so far. In addition to this peculiarity, we note that Achatocarpaceae is the first lineage in AAC clade which has evolved a pericarp with a multi-layered outer epidermis and a layer with U-shaped cells with fine crystalliferous content. A similar multi-layered outer epidermis is also found in the pericarp of restricted Chenopodiaceae members of different taxonomic position: in three Anabasis species [31] and Holmbergia tweedii [101]. Amaranthaceae s.str. This is one of largest families (*700 sp.) distributed in the Tropics. It is the sister clade of Chenopodiaceae [102]. Most species have one-seeded fruits, with a single exception: tribe Celosieae has several-or many-seeded fruits. The fruit and seed anatomy of Amaranthaceae are poorly investigated. The fruit anatomy is known in the closely related genera Amaranthus and Chamissoa only. Costea et al. [103]discovered a four-to eight-layered pericarp in some Amaranthus consisting of unlignified cells. In Chamissoa, the pericarp is composed of both epidermal parenchyma and sclerenchymatous mesocarp [104]. The seed coat is reported to be thick [105]. However, none ofthese descriptionsrepresents the main carpological structure in Amaranthaceae (see below). The groups in the family are named according to Müller and Borsch [106] whose circumscription is generally confirmed by recent studies [107], [108]. The first grades of Amaranthaceae include subf. Polycnemoideae [109] and the genera Bosea and Charpentiera. Subfam. Polycnemoideae. This group was transferred from Chenopodiaceae to Amaranthaceae [102], [107] and consists of four small genera [109] with a disjunct range pattern: Nitrophila (America), Polycnemum (temperate Eurasia + North Africa), Hemichroa and Surreya (both from Australia). Polycnemum and Hemichroa are investigated here, and many traits (including pericarp outlines, pericarp adherence to the seed coat) are found to be diverse (S2 Table). Both genera, however, have a homocellular parenchymatous pericarp and black seeds that have a more or less hard testa, with stalactites in the outer cell wall (Fig. 5A). One peculiarity in H. pentandra is that one seed margin is keeled and the other is rounded. Bosea. This is a genus of three shrubby species with a small anddisjunctrange pattern: B. amherstiana occurs in NW Himalaya, while B. cypria and B. yervamora are found in the Mediterranean area (Cyprus and Canary Islands, respectively). There are no differences in their carpological characters. The fruits are red-coloured berries with a multilayered pericarp that consists of (1) a one-layered outer epidermis; (2) a robust parenchyma that accounts forthe fruit'sfleshiness; (3) U-shaped cells with fine crystalliferous content, and; (4) an inner epidermis. There are no data on the dispersal of Bosea fruits [110], [111], but they should be welladapted to bird dispersal, as they have an attractive pericarp color and a hard testa 30-100 μm thick that should protect the embryo after digestion (Fig. 5B). The embryo is mostly curved, but can be almost straight in B. yervamora. The main pericarp type in Amaranthaceae (Bosea, Charpentiera, Achyranthoids, Gomphrenoideae, Celosieae, and some Amaranthoids) is observed to have zonality. Only a few details distinguish the pericarp of Bosea from most other representatives ofthe family. It has a thick subepidermal parenchyma that is colored, and a robust seed coat. Together with morphological data [112], [113], these carpological results confirm the similarity of Bosea to Amaranthaceae and not to Anacardiaceae that is proposed by Kunkel [114], the members of whichhave other traits in fruit/seed structure, as studied by Plisko [115]. Charpentiera. Eight tree-like species are locally distributed in Polynesia [116]. Two species investigated here have a dry, thin pericarp of four to five layers (C. obovata) or many layers (6-10 layers in C. australis), but with the same topography as Bosea. The seeds have a crustaceous testa (50-60 μm) with stalactites in the outer cell walls and compressed protoplast. The embryo is sometimes underdeveloped ( [117], and present investigation). Amaranthoids (Amaranthus, Chamissoa, Pleuropterantha). In general, all three genera have similar pericarp structures that differ only marginally. The core genus Amaranthus often possesses a pericarp with an alveolate or rough surface, and its mesocarp has radially elongated, parenchymatous cells located in three to eight layers, often with largeintercellular spaces [103]. The pericarp of Chamissoaaltissima consists of several (four to six) layers that are tightly packed but without any intercellular cavities. In contrast to a previous investigation [104], we found that this sclerenchymatous layer (the innermost layer of mesocarp) located above the inner (often crushed) pericarp epidermis consists of large U-shaped cells with a fine crystalliferous content. The innermost mesocarp layer of Pleuropterantha revoilii is distinguished in having equally thickened (O-shaped) cells with monocrystals (Fig. 5C). The monocrystals are not found in Amaranthus or Chamissoa, but scattered cells with a fine crystalliferous content in the upper part of the fruitare present in some members of Amaranthus (e.g., A. muricatus, A. viridis). The seeds mostly have a thick, crustaceous testa with vertical stalactites in the outer cell walls (Amaranthus, Chamissoa). In some Amaranthus, the evident structural heterospermy is evolved with predominating one of two (dark or yellow) seed types, the latter lacking stalactites. Pleuropterantha is distinguished by having thin, almost equal seed coat layers that lack stalactites in the testa cells. Achyranthoids (of which Achyranthes, Centemopsis, Cyphocarpa, Mechowia, Cyathula, Pandiaka, Sericocomopsis, Sericostachys, Pupalia were studied). The pericarp structure is the main pericarp type of Amaranthaceae (i.e. a parenchymatous exocarp, subepidermal parenchyma facultatively present when the pericarp layers are more than two, crystalliferous layer with U-shaped walls and finely divided content or several small prismatic crystals, and easily visible inner epidermis). Rarely, the U-shaped cells are substituted by cells with completely sclerified walls and several rhombic crystalls (Mechowia), or the pericarp is homocellular (Fig. 5D). The seed coat is mostly thin, and the thickness of the testa is either twice that of the tegmen, or testa and tegmen are equally thick. The testa of Pupalia lappacea is unusualin being thick, black, and crustaceous. Aervoids (Aerva, Ptilotus). Both genera investigated here correspond to achyranthoids in their pericarp structure, but in Aerva the seed coat is thicker (12-25 μm) with a stripe-like protoplast and stalactites in the outer cell walls. Subfam. Gomphrenoideae (Gomphrena, Blutaparon, Froelichia, Alternanthera, Tidestromia and Pseudoplantago are studied here). The fruit and seed coat structure of nearly all thesegenera is similar to that ofthe achyranthoids and consists of two to three, orrarely many (Froelichia gracilis) layers. The crystalliferous layer in the pericarp is mostly represented by cells that have thickened walls with rhombic monocrystals in the cell content, or that rarely are fine crystals (Blutaparon). U-shaped cells with finely divided crystalliferous content are found in Tidestromiaoblongifolia. The seed coat does not differ from that of most achyranthoids (except in Pupalia, see above). Tribe Celosieae (Celosia, Hermbstaedtia, Deeringia, Pleuropetalum). This group is distinguished by dehiscent fruit containing several or many seeds inserted basally (Fig. 5E). The one-seeded fruits have been noted onlyin some Celosia [118], but they are not an exception to the rule and have been occasionally found by us in Hermbstaedtia glauca and Deeringia amaranthoides. The pericarp of Celosia, Hermbstaedtia and Deeringia is divided into four topographic zones, as in Bosea. Both species of Deeringia investigated here (Asian D. amaranthoides and Madagascan D. mirabilis) have the same fruit and seed anatomy as other Deeringia members that can be used as an additional character to include the latter species within Deeringia as part of Celosieae-Amaranthaceae, asproposed by Applequist and Pratt [119]. The seed resembles Amaranthus in that it has a thick testa with stalactites in its outer cell walls. Chenopodiaceae. Chenopodiaceae is one of the largestfamilies within core Caryophyllales and comprises *1600 species distributed worldwide butmostlyoutside tropical regions. Until now, only the leaf structure of Chenopodiaceae could be assigned to the recent molecular classification with subdivisions into several clades [102] that generally corresponds with the subfamilial system as proposed by Ulbrich [120]. The fruit and seed anatomy of the family (and their taxonomic implications) have beenintensively studied [31], [44], [46], [47], [49], [50], [121]. We proposehere that a different set of carpological characters is found for everysubfamily, and suggest that our data are of particular importance for delimiting of each of them (S2 Table, Table 2; Fig. 6A-E.). Chenopodiaceae-Amaranthaceae alliance: carpological similarities and differences. A close relationship between the twofamilies wassuggestedafter the discovery of a special form of their sieve-element plastids [122], and some similarities in their pollen morphology [123], [124]. The twofamilies also share many carpological features. The Chenopodioideae (mostly Chenopodieae tribe) and Amaranthus species share the simple parenchymatous pericarp and seeds with hard testa containing stalactites, and Salsoloideae (Chenopodiaceae) resemble the majority of Amaranthaceae in pericarp anatomy [31] (inner epidermis, parenchyma, cells with crystalliferous content, and inner epidermis). In both families the complicated pericarp histology has evolved in the upper part of the fruit, whileits structure in the lower half comprises homocellular thin-walled cells. The function of the crystalliferous layer is not clear, but it may impede the transmission of sunlight to the underlying tissue [125]. In contrast to Amaranthaceae s.str., the solitary prismatic crystals in the U-shaped cells of the pericarp were found within Chenopodiaceae only in Fadenia zygophylloides, before this wastransferred to the genus Salsola [126]. No cells with equally thickened walls (O-shaped cells) and containing prismatic crystals have beenfound in the pericarp of Chenopodiaceae. Such monocrystals are especially common in the pericarp of Amaranthaceae-Gomphrenoideae, but seem to be rare in the vegetative organs of Amaranthaceae [127], or else the crystals are deposited in the form of sand [112]. This feature is also characteristic ofthe pericarp of Pleuropterantha, which was originally included in Chenopodiaceae ( [128] sub Salsolaceae), and in the enigmatic Chinese genus Baolia, known from only one location near the border of Sichuan and Gansu provinces [129]. The presence of leaf stipulae and thedetails of its fruit anatomy do not confirm the previous placement of Baolia within the tribe Chenopodieae, as proposed by Zhu [130]. The classification of Baolia within subf. Polycnemoideae as a part of Chenopodiaceae [131], that is now considered to be a member of Amaranthaceae s.str. [102], [109], is also weakly supported by two unique carpological characters: pericarp alveolation One-Seeded Fruits in the Core Caryophyllales resulting from the rupturing of the thin outer cell wall of the outer pericarp layer, and the presence of monocrystals in the O-shaped cells in the innermost mesocarp layer. A second difference between the two families concerns the diverse spatial embryo position in the seeds. The emergence of the horizontallyoriented embryo in many Chenopodiaceae groups is not found in theAmaranthaceae or in almost all the other one-seeded members of core Caryophyllales. Some groups of Chenopodiaceae can also be distinguished by spatial heterospermy in the partial inflorescences, which likely evolved mixed horizontal and vertical embryo positions within one individual plant [49], [132]. 'Globular Inclusion' clade. Sarcobataceae. This monotypic family (1-2 shrubby species in North America), which was established by Behnke [133]on the basis of thedistinctive sieve-element plastids, is well supported by molecular results [3]. However, some recent authors [37], [134], [135] still accept its placement within the Chenopodiaceae [120], [136]. We postulate here that the development of a radial wing from the middle portion of the pericarp of Sarcobatus (Fig. 6F) is a unique case in the core Caryophyllales, in contrast withthe "winged diaspores" in Chenopodiaceae, which (when present) are formed mostly from theperianth, or rarely themarginal parts of the fruit. The pericarp anatomy of Sarcobatus sets it apart from all Chenopodiaceae studied to date [31], [44], [46], [49], [137], [138]. Except for the wing area, the pericarp is ribbed, flattened in the lower half (up to thewing), and unequally thick, from 100-125 μm in the ribs, to 50-60 μm between them, and it is differentiated into several topographic zones (Fig. 7A): (1) a unicellular epidermis possessing scattered T-shaped, stellate and simple trichomes; (2) parenchyma cells that contain druses; (3) mechanical tissue of 1-2 layers arrangedparallel to the fruit axis; (4) an inner epidermis. Vascular bundles are present in the ribs. The cells of the inner epidermis in the rib zone sometimes bearshort cylindrical papillae. The wing consists of parenchyma and fibers with the cells from 1 mm long. The two-layered seed coat does not adhere but tightly adjoins the pericarp. Its cells are thin-walled, equal in size, or (between the ribs) the outer layer consists of spongy cells. The seed embryo is vertical, spirally coiled in two turns; the perisperm is scarious, one-to two-layered, and is located peripherally near the seed coat. Although the family belongs to the so-called 'Raphide clade' [4], only druses are found in the pericarp parenchyma. Nyctaginaceae. The Nyctaginaceae is mostly an American family (*400 sp.) with a few members found in Africa, Asia and Australia [139], [140]. A diagnostic character of Nyctaginaceae is the persistent lower part of the perianth that usually appears to be accrescent, fully enveloping the fruit and as a rule containing mucilage [141][142][143]. In contrast to the taxonomic importance in the family of the anthocarp structure [98], [144][145][146], the pericarp and seed coat seem to be less diversified. The pericarp is dry, smooth, rarely ribbed(Guapira graciliflora) and not papillate, but can havestellate trichomes onits surface (andother organs), a feature that is peculiar to the Leucastereae [147]. The pericarp is one-to two-(three)-layered, rarely four-to five-layered and not divided into different topographic zones but tightly adjoining the seed coat. According to the embryological studies, the one-layered, thin pericarp in Nyctaginaceae results from the obliteration of all of the innermost layers [148], [149]. However, some genera are characterized by having a pericarp that consists of several or many layers that can be rather transparent, although sometimes they maybe brownish due to the presence of tannin-like substances. The robust 5-12-layered parenchymatous pericarp in the ripe fruits is found in Andradea as part of tribe Leucastereae and in Bougainvillea (Bougainvilleeae). Although the pericarp of Andradea possesses vascular bundles with bar-thickened walls that are not found in other genera, it is exceptionally brittle. The alveolate fruit wall of Cryptocarpus and Salpianthus bears three to six layers of dead, tannin-filled cells with inclusions of raphides located subepidermally (Cryptocarpus) or as theinnermost pericarp layer (Salpianthus). A similar pericarp structure is observed in Boldoa purpurascens, but the fruit wall is thinner, and the raphides are visible with the naked eye as white striae. A multi-layered pericarp was found in one of the samplesof Pisonia aculeata. The seed coat consists of two to four membranous layers formed by both integuments (testa and tegmen), but they often appear to be equally thick, or the testa is insignificantly thicker than the inner layer(s). In the tribes Leucastereae, Colignonieae and Boldoeae the seed coat is described as crustaceous [98]. These groups are distinguished by having a thick seed coat, but their structure is not equivalent. Colignonieae and Leucastereae have largetesta cells with welldeveloped intermediate layers, but there isno decrease of the protoplast or impregnation of the cell walls with the stalactites (Fig. 7B). The seed coat is brittle in Leucastereae. Boldoa purpurascens (Boldoeae), Cryptocarpus pyriformis and Salpianthus aequalis have black seeds with a robust testa possessing stalactites in its outer cell walls which influence the compression of the cell content (Fig. 7C). The stalactites in the testa are not mentioned in other Nyctaginaceae tribes. The majority of representatives in the other tribes clearly have a thinner seed coat with tangentially elongated testa and tegmen cells (Fig. 7D). The embryo is vertical and usually curved with perisperm often present. Only in the tribe Pisonieae in its recent circumscription [150] do the seedshave a straight embryo that fills the seed, and in most cases the perisperm is lost. Aslightly bent and small embryo is found in Colignonia scandens. We observed raphides in the embryonic cotyledons of Commicarpus species. The family is distinguished by three peculiarities in their fruit/seed anatomy within all oneseeded core Caryophyllales: (1) the presence of scattered raphides in the pericarp, seed coat, and even the embryo (e.g.Commicarpus), mentioned by Wilson [151]for the testa cells of some Abronia, where they were called 'aleurone grains'. These crystalliferous inclusions are also present in vegetative organs [127], [152][153][154][155][156]; (2) in many of the Nyctaginaceaethe pericarp is onelayered and very thin (a few mm thick) tightly adjoining the seed coat; (3) the perisperm (if present) is two-lobed or consists of two separatedparts; it is entire only in Boldoa, Salpianthus and Cryptocarpus. Basellaceae. This is a small family of four genera (Basella, Ullucus, Anredera, Tournonia) with diversity in the Tropics, predominantly of the New World. Until now, the pericarp of Basella has sometimes beenconsidered to befleshy [23], [157]. We agree with investigations by Franz [158] and Lacroix [159], and with the exact morphological descriptions of the Madagascan endemics of Basella [160], who noted that the consistency and colour of the diaspore is mostly provided by water-filled floral structures (subtending bracts and tepals) that tightly envelop the fruit, while the fruit itself is hard in all members of the family. The exocarp of Basella, represented by a sclerified layer, can also be slightly pink [18] due to pigmentation of thecell contents. In Basella alba and B. rubra, the exocarp cells are radially elongated, but in B. paniculata some parts of the fruit may contain two sclerified layers (exocarp and outermost mesocarp layer) with no cell elongation. The underlying 2-4 mesocarp layers can consist of unlignified parenchyma, but in some fruits these layers are represented only by fibers that are rounded in cross-section. The seed coat tightly adjoins the pericarp and comprises several layers with a prominent development of the testa [23]. In other genera these elongated exocarp cells are not found. The outer sclerenchymatous layer is present in both Anredera brachystachya and A. scandens with a parenchymatous layer beneath. However, in the basal part of the fruit of both mentioned Anredera species investigated and especially in its upper portion (near stylodia), the pericarp layers increase to five to seven layers due to the emergence of parenchyma covering the sclerenchymatous layer from above, whilesuch parenchyma is lacking in the middle or lower parts of the fruit. Within the core Caryophyllales, this structure seems to be a unique case in which the epidermal layer transitionsfrom parenchyma to sclerenchyma depending on the fruit topography. Anrederacordifolia, formerly placedwithin the genus Boussingaultia (B. gracilis) and now recognizedas a member of Anredera [161], possesses parenchymatous pericarp layers. The same topography is found in Ullucustuberosus, although its pericarp appears to be alveolate. Tournonia hookeriana has a many-layered pericarp made up entirely of sclerenchyma (Fig. 7E). The most recognizable carpological characters of Basellaceae are: (1) globose fruits, (2) significant tanniniferous impregnation of the outer cell wall of the testa in contrast to other seed-coat cell walls (the inner cell wall of the tegmen is also impregnated with tannins in Basella), and (3) embryos with a tendency to be spirally twisted [162]. Its new wider circumscription includes the Madagascan members Didierea, Alluaudia, and Decarya, and the South and East African Ceraria, Calyptrotheca and Portulacaria [163], [164]. Almost all taxa have one-seeded fruits, including Calyptrotheca that have unusual splitting of the capsule from the base upwards [165], although we have observed one to three normally developed seeds in Calyptrotheca somalensis. Some of the genera (Didierea, Decarya) are distinguished by trigonous fruit outlines that resemble those of some Polygonaceae, and Ceraria possesses flattened fruits that sometimes have large simple trichomes (C. namaquensis). The general pericarp topography (Fig. 7F) is found to be the same in almost all genera investigated (except Ceraria): sclerenchyma as outer layer(s), and unlignified, sometimes hardly noticeable or crushed cells beneath with prominently expressed mesocarp layer(s). A one-layered sclerenchyma (exocarp) is found in Didierea, Alluaudia and Decarya (subfam. Didiereiodeae), whereas Calyptrotheca (Calyptrothecoideae) possesses several lignified layers substituted below by tangentially elongated and sometimes crushed parenchyma. Two investigated Ceraria species are distinct in their pericarp anatomy: C. namaquensis has a multi-layered pericarp consisting of 7-10 layers of parenchyma and 1-2 sclerenchyma layers beneath, with largeair cavities in the marginal parts supported by a sclerenchymatous sheath, and C. longipedunculata possesses a 3-4-layered, thin fruit wall with anindistinct exocarp with scattered glandular trichomes as well as an endocarp, and a prominent intermediate cell layer filled with tannins. The seed is supplied with an aril (except Ceraria), and there is noperisperm; the embryo is in a vertical position. The seed-coat testa can be robust, especially in Calyptrotheca, sometimes with a prominent intermediate layer between the testa and tegmen. The tegmen is always easilyvisible with the cell having remarkable bar-thickened walls. Carpology of the Phytolaccaceae cohort with predominantly one-seeded groups. Currently this large family is considered to be polyphyletic. It is actively beingre-evaluated and has been placed in several different positions in the trees of the core Caryophyllales [2], [3], [6], [166][167][168]. The placements of some members are unstable, e.g. Monococcus [168], but some cohort members (e.g., Rivinaceae and Petiveriaceae) seem to be closely related based on molecular results [5] and share a similar wood anatomy [169]. Lophiocarpaceae. Both genera Lophiocarpus (*12 spp., South Africa) and Corbichonia (2 spp. in South and Eastern Africa, one of them, C. decumbens, has radiated into the Arabian floristic province) together form a separate lineage that should be excluded from Phytolaccaceae s.str. and Molluginaceae [3]. They were later united in their own family Lophiocarpaceae [170]. They are, however, not closely related to each other embryologically [171][172][173]orin flower morphology [42], [172], fruit type (many-seeded capsule in Corbichonia vs. one-seeded indehiscent fruit in Lophiocarpus), seed-coat testa that can be alveolate in Lophiocarpus or with short papilla-like outgrowths in Corbichonia [174], or the presence of a seed aril (only Corbichonia). We provide additional differences in the fine carpology of both genera (Table 3; Fig. 8A-C). Due to important differencesin the reproductive characters, the placement of Corbichonia into Lophiocarpaceae needs further investigations. Rivinoideae clade (Rivina, Trichostigma, Hilleria, Petiveria, Gallesia and Seguieria are investigated here). Carpologically, the representatives are clearly split into two groups. The first comprises Rivina, Trichostigma, and Hilleria with more or less fleshy fruits, a homocellular pericarp and a hard seed-coat testa (Rivinaceae s.str.), sometimes with acicular outgrowths of the testa cells (some Rivina : Fig. 8D). The second group (Fig. 8E-F) unites the representatives with dry fruits anda pericarp differentiatedinto several topographic zones (outer epiderm, underlying parenchymatous layers, thick sclerenchymatous sheath, inner parenchyma of one to three cell layers), and a thin seed coat with no importantdifferences in the thickness of the layers (Petiveria, Seguieria, Gallesia). However, Petiveria (Petiveriaceae s.str.) and both Seguieria and Gallesia (Seguieriaceae s.str.) disagree in several main characters. The fruits of Petiveria terminate in (mostly) four reflexed aristae that probably facilitate epizoochoric dispersal. Both members of Seguieriaceae are characterized by the flattened wing-like stylodium of the fruit that enables anemochorous dissemination. The perisperm is easilyvisible only in Petiveria, and is almost absent in both Seguieria and Gallesia. The embryo is straight with plicate cotyledons (Petiveria), or annular in the Seguieriaceae. Stegnospermataceae. A monotypic family of several shrubby species distributed in the Tropics of the New World. In contrast to Phytolaccaceae s.str., some characters are distinctive, for example, a diffuse axial parenchyma in the stem [175], sieve-element plastids [122], flower morphology [176], embryology [177], a two-loculed capsule as fruit type with 1-2 seeds in each locule, and the presence of a seed aril [21]. The valves of the capsule in Stegnosperma cubense are leathery, from 300 to 600 μm, with subdivisions into three topographic zones: (1) one-layered, radially elongated, sclerenchymatous parenchyma, (2) several-layered, thin-walled parenchymatous cells, and (3) four to eight layers of tangentially elongated fibers. The seeds are typical for the core Caryophyllales in having a large testa (100 μm) with an easily visible cell content and a thin striate tegmen layers with bar-thickening of the radial walls. Both molecular and carpological data suggest that Stegnospermataceae cannot be considered as a family with primitive individual characters within core Caryophyllales as proposed by Hofmann [178]. Bar-thickening of the endotegmen cells-one additional parameter in the description of the core Caryophyllales From the nine characters that unite many of the core Caryophyllales, three traits belong to the seed structure (curved ovules, seed coat composed of exotesta and endotegmen, and well developed perisperm [23]). Another character that can be added to the existing description of this group is the presence of bar-thickening in the tegmen cell walls. The first comprehensive data about this peculiarity were provided by Kowal [179], [180] for Chenopodium, Atriplex (Chenopodiaceae), and Amaranthus (Amaranthaceae). Indeed, many members of the Chenopodiaceae-Amaranthaceae alliance are found to have bar-thickened tegmen (S2 Table): Chenopodiaceae-Chenopodioideae (Chenopodiums.str., Atriplex, Lipandra, Oxybasis, Blitum), Chenopodiaceae-Suaedoideae (Suaeda) and many genera of Amaranthaceae s.str. of different taxonomic position. Besides Chenopodiaceae and Amaranthaceae, this feature was also discovered in the present study in the tegmen cell walls of Achatocarpaceae, Rivinaceae s.str. (Fig. 9), Stegnospermataceae, Seguieriaceae, Petiveriaceae, Microteaceae, Lophiocarpaceae, Macarthuriaceae, gen. Adenogramma, only a part of Nyctaginaceae and Caryophyllaceae. The Rhabdodendraceae is set apart from all the members of the alliance by having all (three to four) seed coat layers with bar-thickened walls. Amongmany-seeded fruits, such endotegmen cell walls are reported in the seeds of Aizoaceae, including Tetragoniaceae, Molluginaceae, Portulacaceae, Didiereaceae, Basellaceae, Cactaceae [23], and Anacampserotaceae [181] and were found in the present study in Macarthuria sp. (Macarthuriaceae), Gisekia pharnaceoides (Gisekiaceae) and Limeum obovatum (Limeaceae). Does the origin of one-seeded fruits based on molecular phylogeny coincide with the embryological data? The discussion about the origin of the one-seeded nuts from many-seeded capsules is wellknown but only in the Phytolaccaceae s.l. and Caryophyllaceae. In previous investigations, Phytolacaceae was considered in a broader sense to include the one-seeded Rivinaceae, Seguieriaceae and Petiveriaceae, and it was suggested that the origin of the one-seeded fruits in Phytolaccaceae s.l. was connected with a reduction of the carpels [182][183][184][185][186]. According to the One-Seeded Fruits in the Core Caryophyllales molecular phylogeny, the origin of the one-seeded fruits in Rivinaceae and its relatives cannot be directly assigned on the basis ofPhytolaccaceae s.str., but we conclude that the multi-seeded fruit type is an ancestral state in the Phytolaccaceae s.str. clade and relatives, and that it has been converted into the one-seeded state in the clades Agdestiaceae + Sarcobataceae / Nyctaginaceae + Seguieriaceae + Rivinaceae + Petiveriaceae. This conclusion supportsthe results of other investigations [14], [187]. Recent statement concerning the ancestral characters of one-seeded fruits in Caryophyllaceae + Corrigiolaceae [43] are still contradicted by embryological data that mostly suggest the derivation of the one-seeded fruits from syncarpous capsules [40], [188], [189], with reduction of the central fruit column bearing the seeds with their placentas and septa. In the light of the molecular phylogeny, the increase of seed number in the fruit might be connected with the following basic transitions: (1) one basal ovule in the fruit as an ancestral character state (as in Corrigiola); (2) an increase in ovule numbers with maintenance of free funiculi. This is a common trend traced in many groups including Telephium (Corrigiolaceae) to several higher clades of Caryophyllaceae s.str. (Sperguleae, Sagineae, Arenarieae, Alsineae, a part of Polycarpaeae and Sclerantheae); (3) emergence of the central fruit column formed from the concrescent lower parts of the funiculi that can be extended to the fruit apex in the clades Caryophylleae and Sileneae. In these possible changes in fruit structure, only the evolutionary emergence of the septa between the central column and fruit margins whose fusion is postgenital [190] remains unclear and should be clarified by further detailed investigations. Thus the syncarpy-to-lysicarpy paradigm in the family needs to be reinterpreted with respect to most of the family. Reconstruction of the carpological characters For the reconstructions of the seed and fruit ancestral states we have chosen the most important characters concerned with the changes in the fruit and seed structure. All (one-, manyseeded, or associated) fruit types have been taken into account. Pericarp succulence (Fig. 10). All the major clades, including the Polygonids, possess a dry pericarp. Fleshy and (often) coloured pericarp fruits originatedindependently in the AAC clade (Achatocarpaceae), some representatives of Amaranthaceae (Bosea, Pleuropetalum and Deeringia) and Chenopodiaceae (Anabasis). The mixed pericarp resulting from heterocarpy within one individual originated at least twice in Chenopodiaceae-Holmbergia tweedi and Chenopodiumnutans and relatives (Chenopodium sect. Rhagodia and Einadia), and is connected with changes in the consistency of dry fruit types. In the 'Globular Inclusion' clade, fleshy fruits have originated several times at least in Phytollacaceae (Ercilla, Phytollaca), Rivinaceae (Hilleria, Rivina, Trichostigma), and also in the large family Cactaceae. There are no reversions to the dry fruit types. Pericarp layers (Fig. 11). Pericarps consisting of more than three layers are the ancestral character state in almost all the clades, and all deepest groups in the core Caryophyllales (Simmondsiaceae, Physenaceae, Asteropeaceae, Microteaceae), as well as the majority of the Caryophyllaceae, and the Amaranthaceae s.str. are characterized by a multi-layered pericarp. The simplification of the pericarpto one to three layers evolved independently in both theAAC +CC and the 'Globular Inclusion' clades. In the latter clade, this trend is anchored in the large family Nyctaginaceae (the clades after Bougainvillea: Neea, Guapira, Allionia, Mirabilis). Reduction of the number oflayers is also traced in Amaranthaceae (Polycnemoideae) and Herniaria (Caryophyllaceae). In Chenopodiaceae, the multi-layered pericarp is the ancestral state in Salsoloideae, including Camphorosmeae (Anabasis, Bassia), Betoideae and Corispermoideae. However, a reduction in numberof pericarp layers evolved independently in Suaedoideae + Salicornioideae and almost all Chenopodioideae, with further reversion to an increase in the pericarp layers in Chenopodium sect. Rhagodia, genera Holmbergia and Manochlamys. Pericarp topography (Fig. 12). The reconstruction of the pericarp topology suggests that the ancestral state for the core Caryophyllales + polygonids, as well as for 'Earlier Diverging' lineages, both theAAC+CC clade and the 'Globular Inclusion' clade, is a pericarp divided into two zones: sclerenchyma as outer layers with thin-walled parenchyma below. Such a fruit-wall arrangement exists in the Physenaceae and Asteropeiaceae (as well as in some polygonids) and it is typical in Corrigiolaceae and many members of Caryophyllaceae. However, Rhabdodendraceae and Simmondsiaceae evolved the distinct pericarp topography that is typical for drupes (Rhabdodendron) or similar to other deepest Caryophyllales but with complicated structure (Simmondsia). In the AAC+CC clade, the pericarp topography has radically changedfrom an initial sclerenchymatous-parenchymatous pericarp into two different types. The least frequent change is to parenchyma as the outermost layer(s) with sclerenchyma beneath, and is traced in Scleranthus (Caryophyllaceae-Sclerantheae). Simplification of the fruit-wall structure to one or several homocellular layers occurred independently in several clades; it is especially common in some Paronychieae and Polycarpaeae, and is found as rare exceptions in Plurcaryophyllaceae (e.g. Stellaria monosperma). The conversion of the pericarp topography into several different zones evolved in the AAC clade, with further changes in pericarp structure expressed in its simplification in Suaedoideae + Salicornioideae, and conversion to parenchyma as the outermost layers with sclerenchyma below in Betoideae + Corispermoideae. The ancestral character state in subfamily Chenopodioideae might be characterized by both homocellular and parenchymatous-sclerenchymatous pericarp, but many lineages of Chenopodioideae (Chenopodieae and a part of Axyrideae: Krascheninnikovia + Ceratocarpus) have independently evolved the homocellular pericarp. In the 'Globular Inclusion' clade, the ancestral pericarp zonation remains in many groups in the"Portulaceous alliance" (Didieriaceae, Talinaceae, Portulacaceae), while the members of the 'Raphide clade' evolved a complicated (Petiveria, Sarcobatus, Gallesia and Seguieria) or simplified (Nyctaginaceae, Phytolaccaceae s.str., Rivinaceae) pericarp structure. In some families with many-seeded fruits (Cactaceae, Aizoaceae), the pericarp topography has not yet been studied. Embryo orientation (Fig. 13). The vertical embryo position in the one-seeded fruits is an ancestral character state in all the large clades of the core Caryophyllales. Such a position was also retained when the multi-seeded fruit types (first originated from one-seeded fruit types) were decreased to single-seeded nuts in 'Globular Inclusion' clade and Caryophyllaceae. The changeto the horizontal position occurred independently in some Chenopodiaceae (Salsoloideae, Betoideae, Chenopodioideae), with laterreversion to the vertical positionin Grayia, Manochlamis, Holmbergia, Archiatriplex, Atriplex, Microgynoecium, and Suckleya (all Chenopodioideae). The mixed-embryo statewithin a single individualplantmay have originated in two ways: from a horizontal position in some Dysphania, and from a vertical position in Atriplex and Blitum. Conclusions The one-seeded fruits in the core Caryophyllales are inferred to be an ancestral character state in the 'Earlier Diverging' lineages (Rhabdodendraceae, Simmondsiaceae, Asteropeiaceae, Physenaceae) and AAC+CC clade. The origin of the one-seeded fruits is a reversion from multiseeded fruits in the 'Globular Inclusion' clade. The dry and multi-layered pericarp, topographically divided into sclerenchyma as outermost layer(s) and parenchyma below, is an initial state in the core Caryophyllales, with a tendency to reduction in the number of layers in many clades, especially in large families such as Nyctaginaceae and Chenopodiaceae. The wing-like outgrowths of the pericarp that enable anemochory are rare and known only in Sarcobataceae, Seguieriaceae and some representatives of the Chenopodiaceae (subfam. Corispermoideae). MP reconstruction of the evolutionary history of pericarp topography of combined plastid dataset. Character states: 0-no drastic differences in the consistency of all layers (cells parenchymatous, non-lignified or rarely sclerenchymatous), or only one layer is present; 1-differentiated into parenchyma as outermost layer(s) and sclerenchyma beneath (at least in some fruits, if heterocarpic); 2-differentiated into sclerenchyma (or sclerenchymatous parenchyma) as outermost layer(s) and thin-walled parenchyma; 3-differentiated into: (a) outer parenchymatous epidermis; (b)-thinwalled parenchyma (but sometimes reduced); (c)-sclerenchyma present as O-shaped cells (with equally thickened walls) or U-shaped cells (unequally thickened walls) that often contain crystals in the protoplast; (d)-inner epidermis (sometimes obliterated); 4-differentiated into: (a) outer sclereid layers; (b) thin-walled parenchyma intermixed with brachysclereids; (c) crumpled parenchyma; (d) inner epidermis; 5-differentiated into: (a) 1 or several layers with thick walls; (b) thin-walled parenchyma; (c) brachysclereids with walls filled with tannins (fruit is a typical drupe); 6-divided into (a) thick outer epidermis, (b) thin-walled parenchyma, and (c) thick inner epidermis; 7-divided into (a) parenchyma as outermost layer(s), b-sclerenchymatous layer(s), c-parenchyma layer(s); 8-divided into (a) sclerenchymatous parenchyma, (b) thin-walled parenchyma, (c) multilayered fibers. The position 9 (see text) is not shown on the tree due to lacking of the samplings of both Anredera brachystachya and A. scandens. Morphological characters treated as unordered. Ambiguities recoded as polymorphic states. Fleshy and coloured fruits evolved several times from dry fruits. The families Rhabdodendraceae, Simmondsiaceae, Physenaceae and Asteropeiaceae are set apart from the other core Caryophyllales in having a thick multilayered fruit wall that is divided into several topographic zones, as well as a distinct seed coat structure. These families also share carpological characters such as massive fruits and a lack of nutritive tissue (perisperm and endosperm) in the ripe seed. The first lineage in the core Caryophyllales with typical "centrospermous" fruit and seedcoat structure is (depending on the topology of the molecular trees) either the American Microteaceae or the Australian Macarthuriaceae. The U-shaped cells with fine-grain crystalliferous content in the pericarp above the inner epidermis are mostly known in the Achatocarpaceae, Amaranthaceae, and Chenopodiaceae (AAC clade). The hard seed-coat testa appears to be typical of the majority of the core Caryophyllales. The bar-thickening of the endotegmen layer can be added as a basic character for many core Caryophyllales. All the members with one-seeded fruits arranged their embryos vertically, and many Chenopodiaceae representatives of different taxonomic position evolved a horizontally oriented embryo as a synapomorphy. Unique, taxonomically important carpological characters justifying family rank have been proposed for Achatocarpaceae (polystarch grains in the seeds) and Sarcobataceae (general pericarp structure), and also for Seguieriaceae and Petiveriaceae (pericarp structure), the latter previously being considered part of Phytolaccaceae. The presence of raphides in the pericarp and seed coats, which is hardly noticeable in the one-layered pericarp, and the seed perisperm that is divided into two parts, are the carpological peculiarities of the Nyctaginaceae. The majority of the small families (Lophiocarpaceae, Microteaceae, Rivinaceae) do not show relevant differences in their pericarp and seed coat structure, although some Rivina species are distinguished by acicular outgrowths of the seed-coat testa. Supporting Information S1 Appendix. Origin of the material used in the carpological investigation in the present article. (DOC) S1 Table. List of taxa with GenBank-EMBL accession numbers used in the analyses. (DOC) S2 Table. Diversity of the carpological characters in one-seeded fruits. Abbreviation: n/acharacter not applicable (characters 4, 6 and 24 in many-seeded fruits). For more, see text. (XLSX)

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2018-04-03T04:47:14.161Z

2015-11-01T00:00:00.000Z

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Coevolution between Cancer Activities and Food Structure of Human Being from Southwest China Yunnan and Tibet are the lowest cancer mortality and the largest producer for anticancer crops (brown rice, barley, buckwheat, tea, walnut, mushrooms, and so forth). Shanghai and Jiangsu province in China have the highest mortality of cancers, which are associated with the sharp decline of barley. Introduction Natural products are very popular to combat various physiological threats [1]. Vegetables, fruits, spices, herbs, and beverages have opened up new avenue for the role of phytochemicals in the prevention of human chronic diseases like cancer [2]. Functional foods not only are natural bioactive products with food value and promising cancer prevention and therapy [3], but also prevent diseases, suppress aging, enhance biodefense, bioregulation, and so forth [4]. The biopolymers of edible mushrooms make them very good candidates for formulation of novel functional foods for anticancer and so forth [5]. Greater consumption of fruits and vegetables, as well as whole grain products, appears to lower the risk of multimorbidity [6]. Cancer is becoming the most important public health burden in the world. Its incidence is varying among geographical regions, for example, esophageal cancer high in China, lung cancer in USA, and gallbladder cancer in Chile [7]. Each year 11,844 to 121,442 additional cases of lung cancer, 9,129 to 119,176 cases of bladder cancer, and 10,729 to 110,015 cases of skin cancer worldwide are attributable to inorganic arsenic in food [8]. A diet with higher total diversity may reduce the risk of bladder cancer [9]. The dietary factors are the primary cause of nasopharyngeal cancer [10]. The phytochemicals for anticancer drug design from the green husk of Juglans regia L. have gained attention worldwide [11]. -glucans from the cell walls of barley, oat, mushrooms, yeast, seaweeds, algae, and bacteria are essential for new therapeutic strategies against cancer [12]. Southwest China is the only geographical area where functional crop production has significant anticancer effects on humans [13]. Yunnan has not only the largest biodiversity center (higher plants >18,000 species; over 500 cultivated plants and 650 species of wild crops), but also the largest reserves of Al, Pb, Zn, Ti, Sn, Cu, and Ni in China [14] and the lowest incidence and mortality of cancers in China. Modern humans originated from Africa between ∼100 and 200 Ka, Asia as early as ∼130 Ka, and Northern Eurasia by ∼50 Ka [15]. Hunger due to food shortage with climate change is the cause of early humans' evolution from Africa to Asia and later into Eurasia. Hunger was the cause of migration for early humans' evolution; however, disease prevention of early human migrations was associated with food structure from centers of crop origin, but coevolution between anticancer activities and food structure of human based on crop origin center from Southwest China is unclear. The Lowest Cancer Mortality Associated with Anticancer Crops in Yunnan Yunnan province in China spans approximately 394,000 Km 2 . It borders Vietnam, Laos, and Myanmar. Kunming is the provincial capital of Yunnan, and its elevation is 1894 m. Yunnan is not only the cradle of human childhood, a transitional region among East Asia continent, South Asian subcontinent, and Indo-China Peninsula, but also a core integration area of Chinese culture, Indian culture, and Mid-south Peninsula culture which all merge with the local culture [30]. Yunnan province is renowned for three kingdoms of plants, animal, and nonferrous metals, parallel evolution of crop adaptation to nonferrous metals, and anticancer foods for human being are mostly cultivated in this region. Yunnan is not only the region with lowest incidence and mortality of cancers (0.541‰) in China, especially esophageal cancer, gastric cancer, liver cancer, leukemia, female breast cancer, and cervical cancer [17] (see Table 1), but also the largest center of origin and diversity of cultivated crops [13]. The foods/plant extracts of turmeric and Chinese goldthread are more likely to be beneficial against cancer [31]. The cysteine-conjugated metabolites of shogaols are novel dietary colon cancer preventive agents [32]. Consumption of herbal tea is associated with reduced risk of colon cancer, but iced coffee increases rectal cancer risk [33]. Yunnan is not only a core integration area of Vavilov's three centers of crop origin, including Chinese Center, Indian Center, and Central Asiatic Center, but also the largest center of origin and diversity of anticancer crop. Major anticancer food structures for crop are as follows. Association of Brown Rice with Anticancer Activities. Rice originates from a single domestication 8.2-13.5 Kya in the Southwest China [34]. In 2013, global rice yield was 700.7 million tons, but Chinese rice yield was 203.3 million tons. Yunnan is a region not only presenting great genetic diversity, but also the center of genetic differentiation of indica and japonica subspecies of Asian cultivated rice; however, pigmented rice with similar wild rice with 2384 accessions accounts for 45.1% of rice landrace in Yunnan [35], but white rice for present day human consumption accounts for 95% rice cultivars in China. MGN-3 from rice bran may represent a novel adjuvant for the treatment of metastatic breast cancer [36]. The momilactone B in rice bran caused G1 cell cycle arrest and apoptosis in U937 cells, which may be related to anticancer activity [37]. Atractylenolide I might contribute to the anticancer effect of germinated brown rice [38]. The purple rice extract could be developed for functional foods for colon cancer prevention [39]. Human HepG2 cell apoptosis induced by Methanolic-Payao-Purple rice extracts and vinblastine was mediated through a mitochondrial pathway [40]. -Oryzanol, proanthocyanidin, and -tocotrienol in red rice extract may have a potential to serve as food-derived chemotherapeutic agents for cancer patients [41]. Thai purple rice cooked under sterilization could be a potential source of protocatechuic acid exerting high antiproliferative activity [42]. Treatments with peonidin-3-glucoside and cyaniding-3-glucoside from black rice extracts significantly reduced the tumor size and volume in vivo [43]. China and India are the world's largest producers of rice, which account for 26% and 20% of all world rice production, respectively. Therefore, Southwest China and North India have the lowest incidence and mortality of cancers associated with origin center of rice, especially pigmented rice. Association of Barley with Anticancer Activities. Barley is the most important crop among functional foods [13]. Yunnan is the center of second origin for two naked barleys and the largest diversity center in China [14], as well as the largest Chinese producer. All -glucans differ by their length and branching structures, which are considered biological response modifiers with health beneficial effects including anticancer activities [44]. The genotypes of vitamin E 31.5 g/g dry weight while being of ascorbic acid equivalent antioxidant capacity 158.1 mg AEAC/100 g fresh weight are potential candidates for breeding of barley cultivars with high vitamin E content or antioxidant capacity at harvest, even after storage [45]. The content (mg/kg) of tocotrienols for anticancer activities in barley is higher; that is, barley (910) > rice bran (465) > oat (210) > maize (200) > wheat germ oil (189) > rye (92) [46]. Green barley extract induced preferential antiproliferative and proapoptotic signals within B-lineage leukemia/lymphoma cells [47]. The bioactive compounds in germinated barley and other cereals may reduce the risk of diabetic agents and colon cancer [48]. Protocatechualdehyde present in barley suppressed cell growth and induced apoptosis, which may be a result of deacetylase 2-mediated cyclin D1 suppression [49]. Lunasin present in barley has been observed to prevent skin cancer, which could play an important role in the prevention of cancer in humans [50]. Remarkably high reduction of tumorigenesis and induction of apoptosis in the liver section were achieved in the mouse models with barley-Shochu distillation remnants [51]. Germinated barley foodstuff significantly increased the production of a tumor suppressor gene, which showed promising antineoplastic effects [52]. Association of Buckwheat with Anticancer Activities. Yunnan is not only the center of origins and evolution for buckwheat [13,14], but also the largest Chinese producer of Tartary buckwheat, which accounts for 60.1% in China. Rutin (2215.5 mg/100 g at 7 days) and quercitrin (2301.0 mg/100 g at 8 days) contents after sowing of buckwheat sprouts were approximately 35 and 65 times higher than those of buckwheat seeds [53]. Yunnan golden buckwheat has unique anticancer effects, and its product, "Weimaining" capsules, is the national second-class anticancer drug [54]. TBWSP31 from Tartary buckwheat water-soluble extracts is a novel antitumor protein and apoptosis inducer [55], and also quercetin from its seeds and bran exhibited the strongest cytotoxic effects against the human hepatoma cell line [56]. Tatariside G may be an effective candidate for chemotherapy against cervical cancer [57]. Association of Tea with Anticancer Activities. After water, tea is the most widely consumed beverage [58]; tea is cultivated in Asia which is producing more than 91% of the world. Green tea and quercetin enhanced the therapeutic effect of docetaxel in castration-resistant prostate cancer cells [59]. Green tea polyphenols have strong antioxidants and the inhibition of 16 cancers [13], such as (−)-epigallocatechin-3-O-gallate, induces apoptosis in acute myeloid leukemia cells [60]. The articles on the association of tea with cancer are 3214 according to PubMed literature database. Yunnan province in China is center of origin for Camellia sinensis, which has 35 species and 3 varieties, accounting for 76.6% of Camellia sinensis in the world [13,14,61]. There are more than 500 compounds identified. Tea in Yunnan has 15 accessions for 35.0%-46.8% polyphenols and 14 accessions for 5% caffeine [61]. Epigallocatechin-gallate in green tea has been shown to have antiproliferative activity in colon cancer cells [62]. Catechins of green tea are flavanols, which have many health related characteristics; they especially lower the cytotoxicity and cost of anticancer treatment, inhibit proliferation of breast cancer cells, and block carcinogenesis [1,63]. Humans would be able to achieve consistent cancer prevention effects provided there is timely intervention of green tea catechins at appropriate high-dose levels [58]. Green tea and coffee consumption has protective effects on esophageal cancer [64]. Therefore, green tea is the most economic and effective method for anticancer treatment. Association of Walnut with Anticancer Activities. Yunnan is not only the center of origins and evolution for fruits, which has 66 families, 134 genus, and 499 species [65], but also the Chinese largest producer of walnut (2679,000 ha), which accounts for 50.2% of China in 2013. Walnuts are rich in -3 fatty acids, tocopherols, -sitosterol, and pedunculagin, which slow down the growth of prostate, colon breast, and renal cancers [66]. The tumor size in mice having walnut in their diet was one-fourth than that of the control diet [67]. The -linolenic acid and -sitosterol from walnut oil decreased proliferation of MCF-7 cells [68]. Changes in the miRNA expression profiles likely affect target gene transcripts involved in pathways of anti-inflammation, antivascularization, antiproliferation, and apoptosis [69]. Walnuts decrease the risk of these chronic diseases (cancer, type 2 diabetes, cardiovascular disease, and visceral obesity via inflammation) [70]. Dietary walnut can reduce cancer growth and development by its anticancer mechanism of suppressing the activation of NF B [71]. The dihydroxy-3,4 -dimethoxyflavone and regiolone from walnut leaves can induce apoptosis in human breast adenocarcinoma cells [72]. The bioactive compounds in walnut green husks are capable of killing prostate carcinoma cells by inducing apoptosis [11]. Association of Mushrooms with Anticancer Activities. Yunnan is richest in having species of wild mushrooms in China, which accounts for 90.2% in China, and 44.1% in the world. It has 670 species of edible mushrooms, which accounts for 72.4% in China, including boletes (224 species) and edible boletes (144 species) accounting for 57.4% and 72.4% in China, respectively [73]. The export of boletes and Tricholoma matsutake, having anticancer activities, from Yunnan province is up to 91,780,000 and 57,380,000 USD in 2011 [13]. Medicinal mushrooms have been used to treat cancer, fungal infections, hypertension, diabetes, inflammation, and renal disorders [74]. The most potent extract identified from Ganoderma lucidum inhibited the growth of a gastric cancer cell line by interfering with cellular autophagy and cell cycle [75]. Intake of mushrooms seems to be inversely associated with the epithelial ovarian cancer [76]. Polysaccharides from mushrooms have been widely used in far Asia as antitumor, immunostimulating, antimicrobial, hypocholesterolemic, hypoglycemic, and health-promoting agents [5]. The water extract of Umbilicaria esculenta has a great potential to be developed into an anticancer agent that targets telomerase [77]. Association of Panax notoginseng with Anticancer Activities. Yunnan is the Chinese largest producer of Panax notoginseng, which accounts for 97% of the production of China, and more than 400 products were made from it by 1302 companies in China. P. notoginseng is a promising candidate in preventing and treating inflammation-associated colon carcinogenesis [78]. Macroporous resin from the leaves of P. notoginseng is enriched with low polarity PPD group saponins of 85% ethanol fraction, which is a new alternative source of anticancer saponins [79]. A new protopanaxadioltype ginsenoside, 6-O--d-glucopyranosyl-20-O--d-glucopyranosyl-20(S)-protopanaxadiol-3-one (1), was isolated from the roots of P. notoginseng, which exhibited cytotoxic activity against five human cancer cell lines [80]. An arabinogalactan RN1 from flowers of Panax notoginseng had an antiangiogenic effect via BMP2 signaling and could be a potential novel inhibitor of angiogenesis [81]. The major saponins in P. notoginseng saponin extract were ginsenosides Rg1 (31.1%) and Rb1 (34.4%), which may provide significant natural defense against human colon cancer [82]. In addition, Yunnan is the richest in species of Amorphophalms knojac (66.7%) and its largest producer in China. The konjac glucomannans are associated with a range of health applications which include decreasing the risk of gut cancer and colon carcinogenesis through reduced toxicity of faecal water and precancerous risk factors of human colon cancer [83]. Lower Cancer Mortality Associated with Anticancerous Food in Tibet Tibet in China spans approximately over 1,200,000 Km 2 . It borders Myanmar, Bhutan, and Nepal. Tibetans have been adapted to an altitude exceeding 3,500 m in the early Upper Paleolithic [84]. Tibet is one of the regions with lowest cancer incidence and mortality of cancers (0.643‰) in China, especially colon cancer, lung cancer, nasopharyngeal cancer, and leukemia [17] (see Table 1). Major anticancer food structures are as follows. Association of Naked Barley with Anticancer Activities. Tibet and its vicinity are not only the centers of domestication of cultivated barley [85,86], but also the world's largest producer of naked barley for major food. Barley and its products are good sources of antioxidants [87], especially anticancer. The most common anthocyanin in the purple barley is cyanidin 3-glucoside, whereas delphinidin 3-glucoside is the most abundant anthocyanin in the blue and black groups [87]. Himalaya 292 for barley mutant has high contents ofglucan (9.7%) and protein (16.4%), as well as amylose starch (81.6%) [88]. The intrinsic differences of -glucans in barley and other cereals will elicit variable immune and anticancer responses. The molecular mechanisms of -glucan-induced signaling in immune cells are essential for the design of new therapeutic strategies against cancer [12,89]. -D-glucan from barley regulates breast cancer-relevant gene expression and may be useful for inhibiting endocrine-resistant breast cancer cell proliferation [90]. Whole hulless barley is a functional food that can reduce the prevalence of metabolic syndrome [91]. Association of Milk with Anticancer Activities. Milk consumption is prevalent in daily diets, and its lactase persistence is likely to have an independent origin in Tibetans [92]. The milk protein -casein would provide a more natural and nontoxic approach to the development of novel anticancer therapies [93]. In addition, Guizhou is not only one of the regions of lower cancer incidence and mortality of cancers (0.756‰) in China, but also one of the cradles of human childhood. The Highest Cancer Mortality Associated with Food in Some Province of China Human chronic disease outbreak owes its origin to consumption of brown rice and barley, which were the staple diet of the ancient people, whereas white rice and wheat white flour are now consumed as staple foods of modern people [13]. Yunnan Province is the lowest mortality of cancers (0.541‰) in China [17] or all over the world [94], which associated with the largest production base for barley and tea as well as walnut, and so forth in China. East China includes Shanghai, Jiangsu, Zhejiang, Shandong, Anhui, Fujian, and Jiangxi 7 provinces/city, and it is the highest mortality of cancers (1.6688‰) and the most cancer villages (76) in China, which mainly covers some lower reaches of rivers including the Yellow River, Huaihe River, and Yangtze River and also appears near the Dongting Lake and Poyang Lake; the number of cancer villages in China is in turn East China ( [96]. The lipidtransfer protein from barley grain has an affinity to bind Co 2+ and Pb 2+ but has no affinity towards Cd 2+ , Cu 2+ , Zn 2+ , and Cr 3+ [97]. Barley -glucan is a radioprotective agent, and it can enhance radioprotection in the human hepatoma cell line HepG2 [98]. Barley with polyphenols possesses many other anticarcinogenic activities, and high epicatechin may be related to a reduced risk of breast cancer [99] and colon cancer. Consumption of lunasin from barley could play an important role in cancer prevention [100], but the barley cultivated areas in China in 1935 (6,380,000 ha) were 5.1 times than that in 2012. Shanghai city in 1986 was 6.0 times barley cultivated more than in 2012; Jiangsu province in 1957 (1,401,400 ha) was 7.5 times than in 2012; however Zhejiang province in 1935 (283800 ha) was 9.5 times cultivated than in 2012. Shanghai city has not only the highest cancer mortality (2.171‰) in China, especially colon cancer, lung cancer, and female breast cancer [17] (see Table 1) but also has the lowest diversity of cultivated crops, and its elevation is 4.0 m. Colorectal cancer increases by 4.2% annually in Shanghai, which is faster than the average increasing rate of the world [101]. Lunasin from soybean cotyledon and barley, a peptide with 43 amino acid residues, demonstrated chemopreventive and anticancer properties against colon and breast cancers [100,102]. The consumption of barley rice has certain prevention and adjunctive dietary therapy functions for diabetes mellitus, cardiovascular disease, and cancers [103]. Breast cancer is the most common cancer among women in urban China, such as Shanghai, and so forth; however, soy food consumption is significantly associated with decreased risk of breast cancer and lung cancer [104], but cultivated area of soybean in Shanghai is the lowest in China. Fruit intake is inversely associated with the risk of colorectal cancer [105], but cultivated area of fruits in Shanghai is the lowest in China. The high intake of fruits, vegetables, milk, and eggs may reduce the risk of breast cancer, whereas high animal food intake may increase the risk [106]. Age seems to contribute to increased morbidity and mortality of colorectal carcinoma in Yangpu district of Shanghai, but the mortality of colorectal carcinoma appeared higher than the incidence [107]. Jiangsu province has not only the highest cancer mortality (1.936‰) in China, especially gastric cancer and liver cancer [17] (see Table 1), but also a province of lowest altitude (<50 m) in China. The elevation of Nanjing in Jiangsu province is 15.6 m. The barley with anticancer cultivated areas of Jiangsu province in China in 1957 (1,401,400 ha) was 7.5 times than in 2012. Cancer outbreak owes its origin to consumption of barley of the ancient people, which is replaced by wheat white flour of modern people. Cancer villages of main production regions of Chinese wheat are in turn Anhui (26) > Shangdong (16) > Henan (15) > Jiangsu (14) >Hebei (12) [95]. There are significant correlations between topsoil Pb concentration and gastric cancer, as well as grain Hg concentration with liver cancer in humans [108]. Natural lycopene shows a potential anticancer activity and reduces gastric cancer incidence [109]. The tricin from young green barley leaves on melanin production in B16 melanoma cells inhibits melanin biosynthesis with higher efficacy than arbutin, and it could be used as a whitening agent [110]. The annual average crude incidence and age-standardized incidence by world population were 2.52‰ and 1.79‰, respectively, but Jiangsu being an area with relatively low risk of female breast cancer presented cancer registry areas from 2006 to 2010 as 0.703‰ and 0.481‰, respectively [111]. Zhejiang province has very high cancer mortality (1.727‰) in China, especially for liver cancer [17] (see Table 1). The crude incidence of cancer registered in Zhejiang province in 2009 was 3.202‰; however, age-standardized incidence by Chinese and world standard population was separately 1.6199‰ and 2.0792‰; meanwhile, the crude mortality rate was 1.7697‰ and the age-standardized mortality by Chinese and world standard population were 0.7917‰ and 1.0702‰, respectively [112]. The highest soil concentrations in Zhejiang province were 70.36 mg/kg for Pb, 47.49 mg/kg for Cr, 13.51 mg/kg for As, 0.73 mg/kg for Cd, and 0.67 mg/kg for Hg, while Cd caused the greatest cancer risk [113]. The bioaccumulation of heavy metals in food tubers carries a considerable risk for human cancers [114]. The inhibition of cancer cell viability and apoptosis by protocatechualdehyde in barley leaves may be result of activating transcription factor 3 expression through ERK1/2 and p38-mediated transcriptional activation [115]. Conclusion and Future Prospects Chronic disease prevention of early human migrations was associated with food structures from crop origin centers, especially from Asia with four centers of crop origin, which account for 58% in the world population. The early modern human of Southwest China was related to many ancestors of Asians. Southwest China, richest in anticancer crop, not only is the most important evolution base of Asian and anticancer crops, but also has the lowest mortality and incidence of cancers in China. Yunnan, richest in anticancer crops, is the cradle of human childhood and the lowest cancer incidence as well as mortality of cancers (0.541‰) in China, especially esophageal cancer, gastric cancer, liver cancer, leukemia, female breast cancer, and cervical cancer, and also the largest center of origin and diversity of functional crops with anticancer activities (brown rice, barley, buckwheat, tea, walnut, mushrooms, Panax notoginseng, Knojac, etc.). Tibet is not only one of the regions of lowest incidence and mortality of cancers (0.643‰) in China, especially colon cancer, lung cancer, nasopharyngeal cancer, and leukemia, but also the largest center of origin and diversity of naked barley for major functional foods. Shanghai and Jiangsu, in China, have the highest mortality of cancers (1.936∼2.171‰), which are associated with barley cultivated areas dropped about 6.5 times and 7.5 times, respectively. These results further support that Southwest China (especially Yunnan and Tibet) is the center of lowest mortality of cancers (0.643‰) in China based on coevolution between human's anticancer activities and functional foods from crop origin center.

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2019-04-13T13:12:17.296Z

2014-11-23T00:00:00.000Z

55917711

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Goats: Imperatives for Developing the Champions of the Poor and the Landless Animal-agriculture production is pivotal for food security, economic growth and rural prosperity. In Asian agriculture, the goat is revered as the first animal to be domesticated, and has an important economic and ecological niche. It is very widely distributed, but the preferred environment is the semi-arid to arid agro-ecological zones (AEZs) [1] such as West Asia and North Africa (WANA) region [2], within-country in Rajasthan in India, Baluchistan in Pakistan, Harare in Zimbabwe, Chihuahua and San Louis Portosi in Mexico The value of goats increases in relation to its contributions, capacity to adapt to different rainfed less-favored areas (LFAs), cope with the effects of climate change, and respond to market opportunities and human dietary changes [3]. Introduction Animal-agriculture production is pivotal for food security, economic growth and rural prosperity. In Asian agriculture, the goat is revered as the first animal to be domesticated, and has an important economic and ecological niche. It is very widely distributed, but the preferred environment is the semi-arid to arid agro-ecological zones (AEZs) [1] such as West Asia and North Africa (WANA) region [2], within-country in Rajasthan in India, Baluchistan in Pakistan, Harare in Zimbabwe, Chihuahua and San Louis Portosi in Mexico The value of goats increases in relation to its contributions, capacity to adapt to different rainfed less-favored areas (LFAs), cope with the effects of climate change, and respond to market opportunities and human dietary changes [3]. Inherent Attributes of Goats Goats have many unique inherent attributes and features as follows:-• The small size of goats is significant, for many imputale reasons of socio-economic, managerial, biological traits food security, socioeconomic relevance, survival, and development for productivity enhancement ( Figure 1). Figure 1: Note in this photo from the Xian province in China, that three generations are involved: young boy, his parents, and behind them the grandfather (C Devendra). • Irrespective of location and quantum of milk used, more people drink goat milk than from any other species in the world These are attractive to the inquisitive feeding habits of goats and their inherent ability to use fibrous feeds more efficiently [4]. The situation favors the development underestimated silvopastoral systems [5] • Carbon is sequestered in agroforestry or silvopastoral systems. • Additionally, goat manure and urine provide precious nutrients for microorganisms the soils, especially in the LFAs. The development of agriculture and livestock assets is a most significant contribution by goats to the livelihoods of millions of small farmers and the landless. In India, the late Mahatma Gandhi championed the ownership of goats for socio-economic benefits, nutritional security, and the value of goat milk for good health, especially for the under-privileged and the malnourished rural poor. Current Trends in R and D An objective critique of research undertaken and their implications hitherto reflect some deep concerns inter alia as follows :-• Most developing countries do not accord adequate policy and priority funding to recognise and maximize potential productivity • Research thrusts continue to emphasise disciplines devoid of sustainability issues • Fundamental and applied efforts are the norm but more adaptive, interdisciplinary on-farm R and D, including the effects of climate change are scarce • The application of methodologies and yield-inducing R and D in whole -farm systems in response to needs and priorities is unclear and weak, and • Projects are inadequate that specifically focus on pro-poor initiatives that enhance livelihoods of small farmers and the landless are inadequate. Transforming Agriculture Technologically driven transformation is important to increase the productivity and contribution from goats. These include the following:-Changing the mind set (a) Ensure detailed identification of the major constraints and priorities for resolution (b) Determine yield-inducing technologies using systems perspectives and farm needs (c) Facilitate community-based participation to demonstrate impacts, and (d) Promote efficient management of natural resources [6], recognizing community participation and traditional knowledge, in the context of envionmentally sustainable production systems. Importance of interdisciplinary research A policy framework is very important and includes the following inter alia:-policy through advocacy, on gender, R and D investment, and through direct government action. Improved value chains Improved and efficient value chains are essential to support the marketing of clean goat meat. Marketing and transaction costs are major constraints for small farmers and need to be reduced. A significant development pathway for small farmers and the landless are intensification of production systems, specialization, commercialization and more aggressive participation in market competitiveness (Table 1). Region Potential export areas Table 1: Potential export target areas around the globe With improved production systems and yield inducing technologies, challenging opportunities exist for tapping into large scale goat meat markets. The first priority of course is to maximize domestic production to meet national needs in intensive and specialized systems. This will also promote trade within-and between regions, reflected very broadly below, notwithstanding addressing issues of logistics and trans-boundary diseases. These challenges and opportunities can pave the way for a vibrant export trade R and D Priorities and a Vision for the Future Improved agriculture and potential increased productivity will need assertive a number of key R and D investigations inter alia:-Wider use of indigenous breeds, especially the "improver breeds" and indigenous knowledge. Examples of improver breeds: Barbari and Malabar (India) and Bach Thou (Vietnam) Aggressive priority development of the rainfed areas or LFAs needs urgent attention and donor funding Generate more improved understanding of the available information is necessary on various aspects of the value chains e.g. methods of transportation, types of markets (assembling, distribution markets and weekly markets), characteristics of marketing systems; marketing channels and outlets etc. (iv) intensify gender equality and women's empowerment through education and training, participation in project formulation and contribution, improved understanding of the dynamics of the ownership and decision making, strengthen local women's organisations, and gender equality in livestock services and activities [7], and (v) Strategic partnerships with Asia can greatly enrich and complement R and D, and promote more rapid progress in Africa and Latin America [8]. Conclusions and the Continuing Dilemma A predominant feature about the dominance of goats in the developing world is that there is natural population increase, and the species has ensured conclusion`s survival in the most poverty-stricken areas. The justification for priority development in the future is associated with the following:-• Improved agricultural technologies in the past came from the developed countries, notably the USA, Australia, New Zealand, England, and South Africa. Developing countries will therefore have to be more self-reliant to pursue R and D using locally available inexpensive inputs that are appropriate to the local environment. • Community-based R and D that builds on indigenous resources and the meagre assets of the poor are more likely to be adopted than from elsewhere. • Donor communities have provided negligible funds for R and D on goats. Many of the projects have tended to be top down, without regard to community-based partnerships. • However, dairy production in goats has greatly benefitted from the developed countries, notably the USA, France and Germany. R and D in dairy science largely refers to dairy goat breeds, but developing countries also have to deal with dual-and triple purpose breed • Interdisciplinary approaches that address demographics, socioeconomics, resource allocation, value chains, trade and marketing, and self-reliance will be very useful. These awesome details emphasize how important it is to seek the highest efficiency in the use of the natural resources to the extent possible. Maximizing the contribution from goats is just one example of how they can be used as an entry point for the development of the LFAs. The real question is whether systems orientation and effective development policy can directly improve sustainable food production and environmental protection for small farmers and the landless who own goats, and whose enduring hope is of secure and sustained food supplies from -animal agriculture .The goal of technologically driven transformation is our collective responsibility and in the long term, hopefully the vision for the future is sustained food supplies fromanimal agriculture, decreased poverty, significantly improved livelihoods and self-reliance in the immediate future. Agriculture is under pressure to change from traditional structures to globalization, and in these circumstances direct assertive development is essential.. For the poor and the marginalized, the enduring dream for the future is a world where there is a peaceful agricultural countryside, where natural resources are environmentally sustainable, where there is no poverty and starvation, innovation and self-reliance are vital, and the agricultural landscape is in harmony with nature. These goals are achievable, but resourceful innovation and vision must lead the way in the future. Let me end by echoing an adaptation from an excellent Ethiopian proverb as follows:-"Rainfed land rich in goats is never poor and land poor in goats is never rich"

v3-fos

2016-05-15T18:15:38.425Z

2015-03-06T00:00:00.000Z

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Measurement of pH micro-heterogeneity in natural cheese matrices by fluorescence lifetime imaging Cheese, a product of microbial fermentation may be defined as a protein matrix entrapping fat, moisture, minerals and solutes as well as dispersed bacterial colonies. The growth and physiology of bacterial cells in these colonies may be influenced by the microenvironment around the colony, or alternatively the cells within the colony may modify the microenvironment (e.g., pH, redox potential) due to their metabolic activity. While cheese pH may be measured at macro level there remains a significant knowledge gap relating to the degree of micro-heterogeneity of pH within the cheese matrix and its relationship with microbial, enzymatic and physiochemical parameters and ultimately with cheese quality, consistency and ripening patterns. The pH of cheese samples was monitored both at macroscopic scale and at microscopic scale, using a non-destructive microscopic technique employing C-SNARF-4 and Oregon Green 488 fluorescent probes. The objectives of this work were to evaluate the suitability of these dyes for microscale pH measurements in natural cheese matrices and to enhance the sensitivity and extend the useful pH range of these probes using fluorescence lifetime imaging (FLIM). In particular, fluorescence lifetime of Oregon Green 488 proved to be sensitive probe to map pH micro heterogeneity within cheese matrices. Good agreement was observed between macroscopic scale pH measurement by FLIM and by traditional pH methods, but in addition considerable localized microheterogeneity in pH was evident within the curd matrix with pH range between 4.0 and 5.5. This technique provides significant potential to further investigate the relationship between cheese matrix physico-chemistry and bacterial metabolism during cheese manufacture and ripening. Introduction Cheese is a complex physiochemical and microbial system containing many interacting components. It may be defined as a protein matrix entrapping fat, moisture, minerals and other solutes as well as dispersed bacterial colonies. Irrespective of cheese type, both starter and non-starter bacteria are immobilized and isolated within the curd matrix during the coagulation step of cheese manufacture (Fox et al., 2000;McSweeney, 2004b;Jeanson et al., 2011). Each inoculated bacterial cell is assumed to grow, generating colonies which are dispersed within the cheese curd. Bacterial colonies within the cheese matrix have been shown to be located on or within milk fat globular membrane material (MFGM) and at fat-protein interfaces with the curd (Laloy et al., 1996;Lopez et al., 2006) and are considered to interact with the cheese matrix during ripening (Feeney et al., 2002;McSweeney, 2004a). The growth and physiology of bacterial cells in these colonies may be influenced by the microenvironment around the colony, or alternatively the cells within the colony may modify the microenvironment (e.g., pH, redox potential) due to their metabolic activity (McSweeney, 2004a). The pH of cheese curd is determined by the extent of acidification during manufacture, by the availability of substrate for fermentation, principally lactose, by the buffering capacity of the cheese curd and, in some cases, by the degree of deacidification during ripening. Cheese pH affects the texture of curd directly by influencing the degree of casein hydration, which in turn influences the visco-elastic behavior of the protein matrix (Euston et al., 2002;Kilcast and Angus, 2007). pH also affects texture and flavor indirectly by influencing the activity of enzymes important to ripening, e.g., plasmin (Grufferty and Fox, 1988) and, in the case of the coagulant, both the retention of and the activity of the enzyme in the curd during manufacture and subsequent ripening (Holmes et al., 1977;Stadhouders et al., 1977;Visser, 1977;Creamer et al., 1985;Garnot et al., 1987). The pH also influences the metabolic activity of lactic acid bacteria (Meldrum et al., 2003;Kajfasz and Quivey, 2011;Jeanson et al., 2013). The pH is a key factor influencing amino acid decarboxylase activity (Gardini et al., 2001) and bacteriocin production (Foulquié Moreno et al., 2003). Development of a method capable of precisely measuring pH at localized level within the cheese matrix will facilitate a new understanding of the relationship between cheese matrix physico-chemistry and bacterial metabolism during cheese manufacture and ripening. Until now different techniques have been used to monitor pH in and around colonies. Microelectrodes were first used to measure pH in and around submerged colonies of S. typhimurium (Wimpenny et al., 1995;Jeanson et al., 2013), however, these cannot map the spatial variation of pH to a resolution of a micrometer. Standard methodologies to measure the pH in cell biology include the use of pH sensitive fluorescent dyes-fluorescein based pH indicator, benzoxanthene dyes, cyanine-based pH indicators, etc. (Han and Burgess, 2010). In the vast majority of these experiments Confocal Laser Scanning Fluorescence Microscopy (CLSM) is employed, mainly due to its ability to perform optical sectioning and provide accurate three-dimensional representation of the fluorophore distribution (Inoue, 1995). Seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) is a long wavelength fluorescent pH indicator (Haugland, 2002). The dye exhibits two emission peaks whose intensities display different pH dependencies, thus it is suitable as a ratiometric fluorescent pH probe. The results of the fluorescence intensity ratio of these two peaks have been confirmed as a reliable indicator of pH at values 5.0 and above (Hunter and Beveridge, 2005). However, in more acidic environments the pH sensitivity of the fluorescence ratio decreases rapidly. Oregon Green 488 is a fluorinated analog of fluorescein. It exhibits higher photostability and lower pK a (pK a = 4.7 vs. 6.4 for fluorescein), making it a useful pH indicator in the weakly acidic range (pH 4-6). However, to use Oregon Green as a ratiometric pH probe, two excitation wavelengths are necessary . Oregon Green carboxylic acid has been widely employed to measure lysosomal and endosomal pH . Bioconjugate prepared from Oregon Green 488, dextran, has the advantage of high stability and low affinity to cheese matrix components (proteins). Besides fluorescence intensity, fluorescence lifetime, a probabilistic timescale of fluorescence emission (Sun et al., 2011), may provide additional information about the chemical environment (e.g., pH) of the probe. The most common example is fluorescein, whose fluorescence lifetime changes from 4 ns at pH 10 to 2 ns at pH 7 (Lakowicz, 2006), and many other fluorophores were probed for lifetime-pH dependence, including some of the SNARF family (Szmacinski and Lakowicz, 1993). Oregon Green dyes were also probed for lifetime-Ca 2+ sensitivity (Agronskaia et al., 2004). Fluorescence Lifetime Imaging (FLIM) Fluorescence of a fluorophore is characterized by its absorption spectrum (a probability that a photon of given wavelength is absorbed by the molecule), quantum yield (a probability that the excited molecule emits a photon during transition to the ground state), its emission spectrum (a distribution of wavelengths of the emitted photon) and the fluorescence lifetime (a characteristic time the molecule spends in the excited state before emitting a photon). Both excitation and emission spectrum are used extensively to distinguish different fluorophores and to sense the local environment of a fluorophore (local ion concentration, electric potential, pH, etc., Lakowicz, 2006). However, only a limited number of fluorophores, often specifically designed, display useful changes in excitation and emission spectra. Here we note that changes in fluorescence intensity alone cannot be exploited in most cases, because the fluorescence intensity depends predominantly on the concentration of the fluorescent molecules. When such fluorophores are not available or do not perform optimally in specific experimental conditions, one may explore the dependence of fluorescence lifetime on the local environment. The fluorescence lifetime has only recently been widely adopted, mainly because of the technical challenges associated with the measurement of such fast phenomena (typically in the nanosecond range). One of the most efficient precise and reliable methods used in conjunction with confocal microscopy is Time Correlated Single Photon Counting (TCSPC). This method relies on sub-nanosecond pulsed illumination and fast photoncounting detectors to measure the delay between the excitation pulse and the photon detection event (Becker, 2005). The intensity of fluorescence following a short excitation pulse (usually much shorter than 1 ns) decays exponentially on a nanosecond timescale ( Figure 1A). This decay, however, cannot be digitized directly with current instrumentation; moreover, in a typical confocal microscopy experiment there is less than one photon detected in a hundred of laser pulses. Instead, the time delay between the laser pulse and photon detection event is measured ( Figure 1B) for each photon and a histogram ( Figure 1C) is built from these delays in each pixel of a confocal image. By fitting an exponential model (Equation 1) to these decays, the information about the lifetime of the constituents is recovered. In simple cases (single molecular species with single ground and excited state configuration, or with configurations changing in sub-nanosecond timescale) single exponential decay completely describes the experimental data. However, often more exponential components are needed to achieve acceptable fit between the model (Equation 1) and the recorded decay. Although the detailed mechanism is often poorly understood, multi-exponential decay suggest the existence of more distinct fluorescent species or several stable configurations of the fluorophore on long timescales (relatively to the fluorescence lifetime). Despite the inherent difficulties and numerical instability of multi-exponential fitting, when performed cautiously, the individual lifetime components can provide valuable information regarding local environment of the fluorophore (Becker, 2005). The objectives of this study were (i) to assess the potential of C-SNARF-4 and Oregon Green 488 fluorescent dyes to perform as ratiometric pH indicators in cheese matrices at microscale, (ii) to extend the useful pH range of the probes using lifetime measurements, and (iii) to determine the level of pH microheterogeneity within a natural cheese matrix. To our knowledge it is the first time that advanced microscopic technique fluorescent lifetime imaging microscopy (FLIM) of C-SNARF-4 and Oregon Green 488 has been applied to determine localized pH of different types of natural cheese. Cheese Manufacture Two cheese making trials consisting of four vats of 454 kg cheese milk were undertaken over a 6 month period. Raw milk was obtained from a local dairy company, standardized to a protein to fat ratio of 1.01:1, held overnight at 4 • C, pasteurized at 72 • C for 15 s, and pumped at 32 • C into cylindrical, jacketed, stainless steel vats (500·l) with automated variable speed cutting and stirring equipment (APV) Schweiz AG, Worb, Switzerland). Cheesemilk was heated to 34 • C and inoculated with 0.1 g/l ST (10 6 cfu/ml) and 0.05 g/l LH (5·10 5 cfu/ml). After a 60 min ripening period, chymosin (Chymax plus, Chr. Hansens Ltd.), diluted 1:6 with deionized water, was added at a level of 18 ml per 100 kg milk. A coagulation time of 35 min was allowed prior to the cutting of the coagulum. After a 10 min healing period, the curd/whey mixture was cooked by steam injection into the jacket of the vat with constant stirring. Maximum scalds employed were 50 or 40 • C FIGURE 1 | The Fluorescence decay and its measurement. (A) The fluorescence decay of an ensemble of identical molecules after a laser pulse. The Instrumental Response Function (IRF) combines the excitation pulse duration and the time resolution of the detector and an associated hardware. The intensity of a single fluorescent species that can only assume single ground and excited state follows an exponential decay with a characteristic lifetime τ . (B) In a practical TCSPC experiment there is typically much less than one photon detected for every laser pulse. The time of photon arrival t within the laser pulse period T is measured for every photon. In our case of 80 MHz laser pulse repetition rate the period T = 12.5 ns. (C) A histogram of photon arrival times that is built from the single photon events approaches the theoretical ensemble decay curve of (A). This histogram can be built in each pixel of a confocal image and the lifetime information at each pixel is then extracted by fitting an exponential model (Equation 1). Brine salted (BS) cheeses were pre-pressed under whey at 5.4 kPa for 10 min after which the curd was placed in 10 kg round molds and pressed under increasing pressure up to 35.2 kPa. At pH 5.3, the cheeses were de-molded and placed for 21 h in a saturated brine solution [23% (w/w) NaCl, 0.2% (w/w) Ca, pH 5.2, 10 • C]. Dry salted (DS) cheeses were drained and the curd cheddared, milled at pH 5.3, salted at a rate of 1.45% (w/w) and pressed overnight on a horizontal press at 264.6 kPa in 20 kg blocks. All cheeses were vacuum packed post brine/pressing and ripened at 8 • C for up to 230 days. Macroscopic pH Measurement A standard procedure to measure the pH of cheese (BS 770-5:1976) was used. Briefly, a slurry was created from a cheese sample using distilled water at 20 • C and the pH of the cheese suspension was recorded with a standard laboratory electrode-based pH meter. All samples were measured in duplicate and the mean values are provided. Sample Preparation C-SNARF-4 calibration samples were prepared by mixing 10 µl of 1 mM C-SNARF-4 in DMSO stock solution with 90 µl of 100 mM sodium citrate buffer adjusted to pH 3.0-7.0. A 20 µl drop of the solution was placed on a glass coverslip and imaged with an inverted confocal microscope. C-SNARF-4 stained cheese samples were prepared by cutting a small piece (about 5 × 5× 0.5 mm) from the bulk of the cheese, the piece was then placed on a glass coverslip with a 20 µl drop of fluorophore solution (10 µl of 1 mM C-SNARF-4 in DMSO stock solution mixed with 90 µl of deionized water). Additionally, cheese samples without the fluorescent probe were prepared to assess the amount of cheese autofluorescence. Oregon Green 488 (Molecular Probes, D-7172) stained calibration samples were prepared by immersing a small piece of cheese in a 40 µl drop of Oregon Green 488 buffered solution (20 µl of 50 µM dye in deionized water, mixed with 20 µl of 100 mM sodium citrate buffer adjusted to pH 3.0-6.0) on a glass coverslip and imaged with an inverted microscope. For cheese pH measurements the same protocol was followed, but the citrate buffer was replaced by an equal amount of deionized water to maintain the same dye concentration as in the calibration samples. To assess the cheese autofluorescence, cheese samples without the fluorescent probe were also prepared. Fluorescence Image Acquisition Spectrally resolved fluorescence measurements and excitationemission spectra measurements of C-SNARF-4 were performed with Leica TCS SP8 X confocal laser scanning microscope based on Leica DMI6000 inverted fluorescent microscope (Leica Microsystems CMS GmbH) equipped with White Light Laser (freely tunable excitation in the spectral range 470-670 nm), acousto-optical beam splitter and hybrid detectors with a LightGate option. The Acousto-Optical Beam Modulator (AOBM) was set in the mode of constant output power over the whole working interval that allows the constant intensity of the excitation laser beam for all excitation wavelengths impinging on the sample. The emission spectra were collected sequentially with internal spectral detector, with spectral bandwidth of 20 nm and step size 20 nm in the range 500-720 nm with the starting offset 10 nm from the excitation wavelength. We did not take into account cross-excitation and possible fluorescence emitted in the range less than 10 nm from the excitation wavelength. The image acquisition and analysis were performed in software LAS AF 3.1.2 (Leica Microsystems GmbH). Data were averaged from 200 × 200 µm area, approximately 30 µm deep in the calibration solution with a 20×/0.75 immersion objective lens. Fluorescence Lifetime Measurements Lifetime measurements of C-SNARF-4 calibration samples and C-SNARF-4 stained cheese samples were performed with Olympus FV1200 confocal laser scanning microscope based on Olympus IX83 inverted fluorescence microscope (Olympus Europe GmbH) equipped with two single photon counting hybrid detectors, two channel time correlator and 485 nm pulsed picosecond laser (PicoQuant GmbH). The laser repetition rate was set to 40 MHz and the laser power was adjusted to keep the mean detected photon rate below 2% of the laser repetition rate. Two spectral ranges were measured simultaneously (channel 1 corresponding to 570-620 nm, channel 2 corresponding to 650-700 nm). Typically, calibration data were averaged from 100 × 100 µm area, approximately 30 µm deep in the calibration solution with a 60×/1.2 water immersion objective lens. Typically, 120 s acquisition was necessary to accumulate 10 5 detection events. When measuring cheese samples the focus was set to the cheese surface, i.e., the plane of the strongest fluorescence. The measured fluorescence decay curves were fitted to a two-or three-component exponential model (a sum of two or three exponential decays) according to I(t)= N n = 1 a n e −t/τ n + b (1) using the PicoQuant SymPhoTime64 TCSPC fitting software. Here I(t) is the model fluorescence intensity, N is the number of components of the model, I n and τ n are the intensity and the lifetime of the n-th component of the model respectively (n = 1, 2, 3; lifetimes τ n arranged in ascending order) and b is the mean background. The fluorescence images were acquired at 512 × 512 pixels that were binned to 256 × 256 pixels for lifetime calculations. Lifetime measurements of Oregon Green 488 stained cheese samples were performed with Zeiss LSM780 NLO confocal microscope based on Axio Observer. Z1 inverted microscope (Carl Zeiss MicroImaging GmbH) equipped with Chameleon Vision femtosecond pulsed IR laser (Coherent Inc.) tuned to 860 nm and Becker and Hickl hybrid detector (HPM-100-40) and TCSPC hardware and software (SPC-150, Becker & Hickl GmbH). The repetition rate of the pulsed laser was fixed to 80 MHz. Fluorescence was collected through an IR blocking filter and 580-640 nm emission filter with a Plan Apo 63×/1.4 oil IR objective lens. Fluorescence lifetimes were calculated from 512 × 512 pixel images binned to 256 × 256 pixels. The measured fluorescence decay curves were analyzed in terms of two-exponential model according to Equation (1) and the mean lifetime was then calculated according to Macroscopic pH Of Natural Cheese Samples The results obtained through the standard measurement with a pH electrode of homogenized slurry samples are summarized in Table 2. It is evident that the cheese pHs vary depending on the cheese manufacture treatments but all are within the range 4.99-5.31 and are typical of the pH of many natural cheese types However, what this method does not measure is the degree and pattern of variability in pH within each cheese matrix. It is possible to get approximate measurement by direct measurement with a pH electrode of points within the cheese matrix but this is not sufficiently accurate to create a full pH map of the matrix and to relate the pH map to localized compositional, biochemical and microbial differences within the cheese matrix. C-SNARF-4 Emission Spectra The pH dependence of the probe's emission spectrum is summarized in Figure 2 for two excitation wavelengths (480 nm-the wavelength used for subsequent lifetime measurements; and 530 nm-the wavelength of most efficient excitation) in the pH range 3.0-7.0. The pH dependence of the ratio of the intensities of the two emission peaks (centered at 590 and 660 nm, respectively) is shown in Figure 3. C-SNARF-4 Excitation-Emission Spectra The excitation-emission scans (Figure 4) indicate almost no pH sensitivity of the dye in the pH range of 3.7-5.0 (the Figures 4A-F show the same spectral footprint regardless of pH). At pH 5 and above (Figures 4F-J) there is an apparent trend in the spectra that manifests the pH sensitivity of the probe. However, introducing time gating (discarding photons that arrive within 1 ns of the excitation pulse) modifies the spectral footprint of the (Figure 5) suggesting that lifetime imaging (FLIM) may enhance pH sensitivity at lower pH. C-SNARF-4 Fluorescence Lifetimes In Aqueous Solution We performed calibration measurements of C-SNARF-4 dye in aqueous pH buffers in the range of pH 3.0-7.0 and measured the fluorescence lifetimes in two spectral bands: channel 1 corresponds to 570-620 nm, and channel 2 corresponds to 650-700 nm. These two spectral bands were chosen according to the two emission peaks of the dye used for ratiometric pH imaging. While a two-exponential model fits sufficiently well the decay curves of channel 2 (far red fluorescence), three-exponential model is necessary for analyzing channel 1 (orange fluorescence). The results of the multi-exponential fitting procedure, i.e., pH dependence of the individual lifetime components are shown in Figure 6A (channel 1) and Figure 6B (channel 2). While the microscopic origin of the individual components is unknown, they can be used empirically to measure local pH, as long as they display sufficient sensitivity (the lifetime changes monotonically with pH and this change is bigger than the statistical error of the measurement). None of the channel 1 lifetime components displayed pronounced sensitivity toward pH. On the other hand, the long lifetime component of the channel 2 decay curves shows .1, and (J) 6.5. Increase of pH value results in moving maximum emission from the blue part of spectra toward the red part of spectra with forming two peaks horizontally for pH 5.5 and quasi-symmetrical emission for pH 6.1 and 6.5. pronounced pH dependence in the whole measured pH range (the upper curve in Figure 6B). Mean fluorescence lifetime τ m calculated according to Equation (2) does not show pronounced pH dependence, thus two-exponential fitting is necessary to extract the pH information. Microscopic pH Of Cheese Samples Measured With C-SNARF-4 Based on calibration data we tried to utilize the pH dependence of C-SNARF-4 fluorescence in the red spectral channel for cheese pH measurements on the microscopic scale. However, both the fluorescence intensity and the fluorescence lifetime distribution were essentially homogeneous across the sample. We have also observed a strong affinity of the dye toward the cheese surface. We measured a total of 4 cheeses (designated vat 1 to vat 4), each multiple times. Surprisingly, a new decay component with relatively long lifetime (τ 3 ≥ 3.5 ns) appeared (Table 3). Moreover, the τ 2 component, which is assumed to correspond to the pH dependent component of the calibration data, has significantly shorter lifetime than the value deduced from calibration (expected value is about 2.2 ns for pH 4.5). Also the fastest component (τ 1 ≈ 0.7 ns) is significantly shorter, than the corresponding lifetime from calibration (about 0.9 ns). Thus, in our scenario the C-SNARF-4 probe did not prove itself as a useful probe of the local pH. Control experiments performed on cheese samples without fluorescent probe revealed, that the cheese autofluorescence is almost two orders of magnitude weaker than C-SNARF-4 fluorescence, and its lifetime is significantly longer (over 6 ns) than that of the fluorescent probe, so that the autofluorescence does not interfere with our measurements. Oregon Green 488 Fluorescence Lifetimes And Microscopic pH Measurements Oregon Green 488 displays single emission peak whose position is independent of local environment. Thus fluorescence lifetime (FLIM) must be employed in order to measure the local pH variation. The mean fluorescence lifetime of the Oregon Green 488 excited by 860 nm pulsed laser at 80 MHz and detected in the red emission band was determined to be 3.9 ± 0.1 ns in aqueous buffer at pH 7. When the dye is applied to the cheese matrix, the lifetime shortens significantly, but the pH sensitivity of the dye is preserved. Figure 7A shows representative FLIM images of cheese matrix buffered to pH 3.0-6.0 and stained with Oregon Green 488. The pH sensitivity of the dye is immediately apparent from the change of the pseudocolor encoding local fluorescence lifetime. The mean lifetime τ m calculated as a mean value from several fields of view is plotted against the buffer pH in Figure 7B (solid symbols and a linear least-squares fit). The mean lifetime of the dye changes by more than 1 ns in the studied pH range. To measure the local pH of cheese the dye is applied to the cheese matrix without a pH buffer and the same procedure is followed. From the acquired FLIM image and the linear fit of the calibration series the average pH of vat 2 cheese sample after 223 days of ripening was determined to 5.2 ± 0.2 ( Figure 7B, open symbol). The local variations of the fluorescence lifetime (and thus the pH) in a natural cheese sample are shown in greater detail in Figure 8. We note that application of the dye to the non-buffered cheese produced no measurable macroscopic pH change, as measured by a macroscopic pH electrode method. Discussion We have confirmed that the C-SNARF-4 fluorescent pH probe may be used as a reliable ratiometric pH indicator at pH 5.5 and above (Figure 3). Essentially, the emission spectra do not depend on the excitation wavelength in the range 480-530 nm, as illustrated in Figure 2. However, the range of interest in cheese research is usually at lower pH values, generally around 4.8-6.0 (with some exceptions). In this range the traditional ratiometric approach does not provide sufficient sensitivity. Excitation-emissions scans (Figure 4) show that the results of ratiometric pH measurements are largely insensitive to the excitation wavelength, but utilizing the lifetime information may extend the useful range of the dye toward lower pH's ( Figure 5). We have observed and measured the pH dependence of fluorescence lifetime of C-SNARF-4 dye, which extends through the pH range that can be sensed toward more acidic values (down to pH ≈ 3), part of this range which may be encountered in cheese research. From the calibration curve τ 2 in Figure 6B) one can clearly see the relationship between pH and fluorescence lifetime. But the calibrated lifetime-pH dependence performed in aqueous solutions is not directly applicable to cheese matrix results, as a new long-lifetime component appears τ 3 in Table 3) and the lifetimes of the other two components are significantly shortened in such a protein-rich environment. More specifically, the τ 2 component measured on cheese samples (about 1.7 ns, Table 3) would correspond to pH below 4.0 (according to the calibration curve τ 2 in Figure 6B), which is much lower than the reference macroscopic value (about pH 5.0, Table 2). A similar interaction of SNARF-based dye with proteins was described previously (Srivastava and Krishnamoorthy, 1997). We also note that we observed strong affinity of the fluorescent probe toward the protein-rich cheese surface, which changes the dye's photophysical properties significantly and alter its pH response. Both the spectral and lifetime signatures of the stained protein matrix do not correspond to the calibration values at any pH. Moreover, we observed that both the fluorescence intensity and lifetimes are essentially homogeneous across the sample surface (as shown by the small standard deviations in Table 3). The expected pH variation between samples is less than 0.5 and cannot be reliably detected by the C-SNARF-4 dye. Similarly, we also applied the same methodology to other pH sensitive fluorescent dyes, namely BCECF-AM, FITC, and Acridine Orange, but again we observed little to no lifetime-pH sensitivity when applied to cheese matrix (data not shown). On the other hand, our results suggest that applying the FLIM microscopic technique to the Oregon Green 488 fluorescent dye resulted in an accurate method of determination of pH in the cheese samples (the main results are summarized in Figure 7). The average value of pH 5.2 ± 0.2 is in good agreement with the macroscopic pH 5.3 ( Table 2). In addition micro-heterogeneity in pH is apparent in the cheese matrix as show in Figure 8 with pH ranging between 4.0 and 5.5 depending on location. This is particularly interesting as it shows the pH of cheese matrix is not homogenous but contains localized variation of pH. This may be due to localized differences in the aqueous phase or concentrations of constituents of the aqueous phase including lactose, lactate, minerals or salt. It may also be influenced by variations in buffering capacity of the surrounding cheese matrix. It also poses questions regarding the influence of microbial colonies on pH micro heterogeneity. Other authors (Lopez et al., 2006(Lopez et al., , 2007Pereira et al., 2009) reported bacterial colonies to be located at the protein fat interface, with the bacterial cells always being in contact with the fat globule membrane. Although the study of (Jeanson et al., 2013) showed no differences in pH around microbial colonies, that study differed in that it was conducted on an unripened non-fat UF model cheese system rather than on a ripened natural cheese matrix, and the microbial colonies were lactococci rather than thermophillic species. The current study has focused on applying the developed method to determining the micro heterogeneity within one of the cheese types (vat 2). It is envisaged that future work would focus on using the developed method to determine whether manufacture processes influence pH at local level within different cheese matrices and whether different cheese types may have different patterns of micro heterogeneity. We also expect that this methodology can be employed to examine also wide range of different types of food products with sufficient moisture content.

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Solid-state fermentation in multi-well plates to assess pretreatment efficiency of rot fungi on lignocellulose biomass The potential of fungal pretreatment to improve fermentable sugar yields from wheat straw or Miscanthus was investigated. We assessed 63 fungal strains including 53 white-rot and 10 brown-rot fungi belonging to the Basidiomycota phylum in an original 12 day small-scale solid-state fermentation (SSF) experiment using 24-well plates. This method offers the convenience of one-pot processing of samples from SSF to enzymatic hydrolysis. The comparison of the lignocellulolytic activity profiles of white-rot fungi and brown-rot fungi showed different behaviours. The hierarchical clustering according to glucose and reducing sugars released from each biomass after 72 h enzymatic hydrolysis splits the set of fungal strains into three groups: efficient, no-effect and detrimental-effect species. The efficient group contained 17 species belonging to seven white-rot genera and one brown-rot genus. The yield of sugar released increased significantly (max. 62%) compared with non-inoculated controls for both substrates. Introduction Fungi play a dominant role in lignocellulose conversion. Wood-decay fungi, including white-rot and brown-rot fungi (WRFs and BRFs), have been extensively studied for their abilities to efficiently modify, degrade and depolymerize major plant cell wall components. Among them, some WRFs are able to mineralize the more recalcitrant lignin with little consumption of cellulose (Martinez et al., 2005). This unique ability lends great potential interest for them in the pretreatment of biomass in many biotechnological applications, such as wood pulping and bleaching, and biofuel production. Solid-state fermentation (SSF) is defined by Barrios-Gonzalez (2012) as when a microbial culture develops on the surface and inside of a solid matrix in the absence of free water. SSF has been shown to be of interest in at least two types of applications: the production of lignocellulolytic enzymes and the degradation of lignocellulose itself. SSF mimics the natural habitat of the fungus to facilitate enzyme secretion. Industrial enzymes for biofuel applications have been successfully produced at the commercial level by SSF (Singhania et al., 2010), and differences in enzyme production by fungus between SSF and submerged fermentation (SmF) have been reviewed by Barrios-Gonzalez (2012). Enzymes are generally produced in much higher concentrations in SSF, some of them display higher optimal temperature or pH stability and some are secreted only in SSF. Regarding the degradation of lignocellulose, the enzymes secreted along mycelia are close to their substrates, and the rapid diffusion of oxygen in SSF creates favourable conditions to achieve high performance in the oxidative degradation of lignocellulose. These findings have led to fungi to be considered as a suitable alternative for lignocellulose pretreatment in the second-generation ethanol production processes due to their low cost, environmental friendliness and adaptability to both continuous and batch processes. Moreover, cellulose and hemicellulose become more accessible for hydrolysis by the breakdown of the lignin-carbohydrate complexes, and so avoid substantial holocellulose (cellulose and hemicellulose) loss, thereby increasing conversion rates and productivity of fermentable sugar. Recent studies have demonstrated the efficiency of some fungal strains [e.g. Ceriporiopsis subvermispora (Cianchetta et al., 2014), Pycnoporus cinnabarinus (Gupta et al., 2011), Trametes hirsuta (Saritha et al., 2012), Irpex lacteus , Gloeophyllum trabeum and Postia placenta (Schilling et al., 2012)] in such pretreatment steps for improving the production of bioethanol or biogas from various substrates. Two distinguishable patterns of biomass breakdown, namely simultaneous lignin and polysaccharide degradation or selective delignification have been described among WRFs (Martinez et al., 2005). The simultaneous attack of cellulose and lignin used as a preferred strategy by some strains could result in the concurrent production of cellulases, hemicellulases and ligninolytic activities; whereas fungal strains acting as selective delignifiers secrete very low levels of holocellulolytic enzymes. However, selective delignification ranges among fungal species according to pretreatment time and the type of lignocellulosic biomass (Wan and Li, 2012). A biodiversity screening step is a prerequisite in choosing the best fungus for efficient pretreatment. Natural fungal diversity offers a huge array of new fungal strains with high biotechnological potential (Blackwell, 2011;Berrin et al., 2012). Fungal biodiversity has been investigated previously for biomass pretreatment, but these studies were often restricted to a limited number of strains (Li et al., 2008;Salvachua et al., 2011). Traditional screening is performed with multiple fungal SSF in flasks and it is difficult to apply these culture conditions to large-scale strain screening. Scaled-down screening methods hold great promise for accelerating progress in biomass conversion as they lead to the selection of a narrower number of most effective strains to be later tested in a scaled-up process. This strategy has been successfully used in screening the transformation of various biomasses (Santoro et al., 2010) and biomass-degrading enzymes (Navarro et al., 2010;Cianchetta et al., 2012). In this paper, we screened biodiversity for fungal pretreatment of lignocellulosic biomasses to preselect promising candidates, based on the yields of fermentable sugar, for scaled-up fermentation. For this purpose, 63 strains of basidiomycetes were grown on wheat straw or on miscanthus using a medium-throughput SSF screening method. The ligninolytic and carbohydrate hydrolytic activities involved in biomass degradation were also examined. 24-well plate-based screening of basidiomycete strains Biological pretreatment using wood-rotting fungi is a wellresearched process for bioethanol production from lignocellulosic materials. The main fungal groups of interest for lignocellulose degradation are the WRFs and the BRFs belonging to the Basidiomycota phylum (Martinez et al., 2005). The new 24-well plate-based screening method was used to evaluate the potential of several strains of basidiomycetes simultaneously (Fig. 1). SSF of lignocellulosic substrates and subsequent enzymatic hydrolysis of the pretreated biomasses were performed. This allows one-pot multistep treatments including fungal growth, mild alkaline wash and enzymatic hydrolysis. This procedure was refined from to an experimental design previously described in studies on the evaluation of biological pretreatment (Tian et al., 2012;Lopez-Abelairas et al., 2013). A culture time of 12 days combined with a high inoculum loading allowed extensive and fast fungal colonization. The mild alkaline wash performed before enzymatic hydrolysis is reported to improve the enzymatic digestibility of the biopretreated substrate (Yu et al., 2010;Salvachua et al., 2011). The main effect of sodium hydroxide pretreatment on lignocellulosic biomass was shown to be delignification through the cleavage of the ester bonds cross-linking lignin and xylan, thus increasing the porosity of the biomass (McIntosh and Vancov, 2011;Maurya et al., 2015). In addition, fungal hyphae grow on the surface of the lignocellulosic materials forming a dense hyphal coat that hinders cellulases' access to cellulose. As suggested for washing or heating steps before enzymatic hydrolysis, the very mild alkaline conditions applied were expected to remove this biological barrier or inhibitors of saccharification (Shi et al., 2009). The additional effects of these actions were expected to render the biomass more susceptible and accessible to saccharification, and increase the production of fermentable sugar. Compared with traditional cultures grown in flasks or in packed columns (bioreactors), the 24-well plate-based method requires less 'hands-on' time and allows the simultaneous processing of multiple strains. It is therefore well adapted to screen a large number of fungi. Additionally, this method avoids excess substrate handling and losses, thus favouring a more accurate analysis. In this study, a total of 63 fungal strains comprising 53 WRFs and 10 BRFs were screened on wheat straw and miscanthus. The most effective strains for biopretreatment should consume little sugar for their own growth while promoting a high lignocellulose deconstruction to enhance enzymatic hydrolysis. Among those strains examined, 58 belonged to Polyporales, mainly from Polyporaceae (32), Ganodermataceae (13) and Fomitopsidaceae (10), representing the distribution of fungal diversity in the International Centre of Microbial Resources (CIRM-CF) collection (Table S1). Unlike the WRFs, the BRFs were exclusively collected from temperate forests: these fungi, described as less deeply involved in the decay of hardwood compared with softwood, dominate the decomposition of conifer wood in boreal forests (Eastwood et al., 2011). Some strains of the same species were selected to compare their performance in pretreatment according to the geographical zone where they were collected. Enzymatic hydrolysis of fungal pretreated lignocellulosic biomass Visual observation of the cultures indicated mycelial growth of all the strains regardless of the substrate. To evaluate the effect of fungal pretreatment on saccharification, released reducing sugars and glucose from pretreated miscanthus and wheat straw were determined after 72 h of enzymatic hydrolysis using cellulases of Trichoderma reesei supplemented with a β-glucosidase from Aspergillus niger. A hierarchical cluster analysis (HCA) was performed to characterize the grouping of fungal species according to their efficiency in enhancing enzymatic hydrolysis. A principal component analysis (PCA) was carried out prior to HCA to better highlight the main feature of the data set. The first two principal components showed the highest eigenvalues and accounted for 91% of the total variation; they were kept for the clustering. HCA was performed by calculating the mean Euclidian distances between the samples, followed by a hierarchical tree construction using Ward's criterion. The fungal species were split into three major groups according to their ability to improve enzymatic hydrolysis (Fig. 2): efficient (G1), no-effect (G2) and detrimental-effect (G3) species compared with the control. We noted that the 11 strains of Trametes, the 12 strains of Ganoderma and the six strains of Leiotrametes fell into different groups, while five strains of the Fomitopsis genus did not. Intraspecific differences were also globally observed for several strains of the same species, regard- less of their geographic origin. These results are consistent with previous works (Elisashvili et al., 2008;Simonic et al., 2010;Berrin et al., 2012), indicating intrageneric and intraspecific differences of efficiency in enhancing lignocellulosic biomass bioconversion. The fungal group giving the highest saccharification results (G1) included the genera Trametes (eight strains), Polyporus, Phlebia, Ganoderma, Lenzites (two strains), Dichostereum, Antrodia and Artolenzites. A noteworthy feature of the G1 strains was their remarkable ability to improve saccharification of miscanthus. Fungal pretreatment of this recalcitrant crop appears to be especially useful and could, in combination with the existing conventional physicochemical treatment, permit less drastic operating conditions than those currently required (Keller et al., 2003;Wang et al., 2010;2012;Li and Chen, 2014 BRFM 1123 proved to be particularly efficient in increasing the yields of fermentable sugar from wheat straw and miscanthus with notably a maximum improvement of up to 15% and 62% of glucose, respectively, compared with the control. Most of the genera in the cluster G1 (Polyporus, Trametes, Ganoderma, Antrodia, Lenzites and Phlebia) have already been reported to show potential for application in the biological pretreatment of several feedstocks (Saritha et al., 2012;Vaidya and Singh, 2012;Wang et al., 2012;Ryu et al., 2013;Deswal et al., 2014). We note that the sole remaining fungal genus identified in this work as being able to increase enzymatic hydrolysis of miscanthus, i.e. Dichostereum, has to our knowledge not yet been explored. Also, strains belonging to the genera Pycnoporus, Pleurotus and Gloeophyllum had previously been identified as good candidates for the bioprocessing of switchgrass, wheat straw and aspen, respectively (Schilling et al., 2012;Liu et al., 2013;Lopez-Abelairas et al., 2013), but were not highlighted in our screening. The discrepancy between our results and those of the literature could arise from: (i) strain differences at both inter-and intraspecific levels, (ii) culture conditions and (iii) nature of the substrate. The concept behind biomass degradation by fungi is the secretion of a complex mixture of cell wall-degrading enzymes, including cellulases, hemicellulases and lignindegrading enzymes. Fungi with high selectivity for lignin degradation, thereby leading to less holocellulose loss during growth, are important for a successful fungal pretreatment. The data provided by genomic, transcriptomic and secretomic analyses of wood-decay fungi show that the fungal enzymatic system and non-enzymatic oxidative mechanisms involved in lignocellulose depolymerization form a complex process that depends on many factors such as substrates, fungal strain and culture conditions (Vanden Wymelenberg et al., 2010;Grigoriev et al., 2011). Owing to differences in the chemical composition and structure of the substrates to be treated, the strains suitable for one substrate may not be always suitable for others. It is therefore essential to select the best-adapted fungal strain for a given substrate. This underlines the crucial importance of a biodiversity exploration step to define the best fungal strain/biomass combination to develop an efficient bioprocess. This study also reminds us that fungal biodiversity is a huge underexploited asset for innovation. It offers opportunities for the use of unexplored fungi with biotechnological potential in biorefining, such as the Dichostereum genera, which was highlighted as being efficient in pretreating miscanthus. Lignocellulolytic enzyme profiles during fungal SSF To gain insight into the lignocellulose-degrading enzymes secreted during fungal pretreatment, pattern and levels of ligninolytic and carbohydrate hydrolytic activities were analysed in the water-soluble extracts from the 12-day-old cultures of fungal strains growing on both substrates. Laccase and peroxidases related to lignin degradation, and the activities of endo-1,4-β-glucanase and endo-1,4β-xylanase, key enzymes in the degradation of holocellulose, were determined. In general, a low or zero level of endo-1,4-β-glucanase and endo-1,4-β-xylanase was found in the WRF cultures, whereas most of the BRFs tested produced high levels of carbohydrate hydrolytic activities on both substrates (Fig. 3). The highest production of hydrolytic activities found in BRF cultures, as described elsewhere (Machuca and Ferraz, 2001;Mathieu et al., 2013), could be explained by a rapid and extensive holocellulose depolymerization in the early stages of the decay process (Arantes et al., 2012). In addition, laccase and peroxidase activities were found mainly in the water extracts of WRF cultures (Fig. 3). In contrast to WRFs, most BRFs were shown to lack extracellular phenoloxidases, although a few of them have been reported to produce peroxidases (Huang et al., 2009;Yadav et al., 2011). It is known that BRFs modify lignin slightly, mostly by demethoxylation occurring through mediated Fenton reactions (Arantes et al., 2012). The general features observed for the patterns of lignocellulosic enzyme activities among BRFs and WRFs emphasize differences in the mechanisms involved in the lignocellulose breakdown between these two fungal groups. The hierarchical clustering based on ligno-and holocellulolytic activities classified the strains into two major groups for both substrates (Fig. 4). The larger group contains mainly WRFs belonging to the three clusters G1, G2 and G3 (except for Antrodia malicola BRFM 1200, Postia stiptica BRFM 1148, Fomitopsis pinicola BRFM 1291 and Fomitopsis rosea BRFM 1062), while the second group contains only BRFs belonging to G2. Despite the general features observed in the patterns of extracellular enzyme activities among BRFs and WRFs, some strains produce distinct enzymatic profiles. The BRFs A. malicola BRFM 1200 and P. stiptica BRFM 1148 show an enzymatic pattern close to the WRFs presenting low carbohydrate hydrolysing and ligninolytic activities as described elsewhere (Huang et al., 2009;Wei et al., 2010). The two BRF strains F. pinicola BRFM 1291 and F. rosea BRFM 1062 produce carbohydrate hydrolysing enzymes with lower levels compared with most of the BRFs. Regardless of the cluster the strains belong to, the fungal enzymatic profiles were not correlated with saccharification efficiency. However, it must be kept in mind that lignocellulose breakdown occurs through complex mechanisms involving an arsenal of plant cell walldegrading enzymes. The lignocellulolytic activities studied here give only a snapshot of this arsenal (only one time point at day 12); more detailed investigations to take into account the greater complexity of the multi-enzymatic process are necessary to increase our knowledge and understanding of the relationship between the fungal lignocellulolytic enzyme system and lignocellulose breakdown. This study highlights the importance of a systematic assessment of fungal biodiversity in order to identify promising fungal strains for the conversion of lignocellulosic biomass. The new and efficient multi-well plate SSF method developed here copes with the necessity of screening hundreds of fungi on different biomass feedstocks. To our knowledge, this is the first report of SSF culturing of filamentous fungi in a multi-well format with in situ post-treatments at a scale suitable for mediumthroughput screening. The ability to assess rapidly, accurately and reproducibly a wider range of fungi represents a breakthrough to better explore and exploit fungal biodiversity in a biorefinery concept to produce biofuels and value-added bioproducts. Conclusions A large number of WRFs and BRFs were simultaneously evaluated for lignocellulosic biomass conversion by a multi-well plate SSF method. Several fungi, mainly WRFs, were found to show high potential for biological pretreatments, as they enhanced enzymatic hydrolysis. A few strains were found to be efficient on both wheat straw and miscanthus, which makes them promising candidates deserving further investigation. The medium-throughput screening method described in this study is a powerful tool to preselect a restricted number of strains suited for a natural raw material/relevant industrial application combination. The preselected strains can then undergo scaled-up SSF to further select the best-adapted ones and to optimize the process. For this purpose, critical factors, not provided at a small scale, such as weight loss, carbohydrate preservation, quantification of fungal biomass, etc., need to be taken into account. The scaled down SSF method developed in this study could be useful to preselect relevant fungi for biorefining in White and Green Chemistry, and could be applied to a wide variety of microorganisms and substrates. The method could also be helpful for performing preliminary assays for culture condition optimization. Fungal strains The basidiomycetes fungi used in the present study (Table S1) were obtained from the CIRM-CF dedicated to filamentous fungi of biotechnological interest (http://www.inra.fr/crb-cirm/) at the French National Institute for Agricultural Research (Marseille, France). All the strains were authenticated by ITS sequencing. The screened fungi were isolated from tropical and temperate forests. As proposed by Welti and colleagues (2012) Growth conditions Miniaturized fungal pretreatment cultures were performed in polypropylene 24-well plates (Whatman INC, Piscataway, USA) under SSF conditions. Wheat straw (0.1 g, dry weight) or miscanthus and 4 ml deionized water were dispensed into each well of the plates. The plates were sealed with aluminum foil by an automatic plate sealer (PlateLoc, Agilent, USA) and autoclaved at 110°C for 30 min. After sterilization, the plates were centrifuged at 3500 r.p.m. for 5 min, and 2 ml of the supernatant were discarded. The substrate was then washed by adding 2 ml of sterilized water to each well, stirring at 450 r.p.m. for 10 min (Microtron, Infors AG, Switzerland) and removing 2 ml of the wash liquid after subsequent centrifuging. After repeating the washing steps three times, 2 ml of nutrient solution (40 g l −1 glucose and 3.68 g l −1 diammonium tartrate) were added per well and mixed. The excess liquid medium (i.e. 3 ml) was removed, leading to a final lignocellulosic substrate consistency of 10% dm (w/v). Three non-inoculated controls per plate were set up by replacing the nutrient solution with 2 ml of water. Finally, wells were inoculated in triplicate for each fungal strain, with one 5 mm diameter agar disc of 7-day-old mycelia crushed and mixed with substrates using a sterilized toothpick. The plates were covered with an adhesive membrane (Breathseal, Greiner Bio-One), which allows gas exchange, and were incubated at 25°C for 12 days under saturated humidity conditions. To allow an extensive fungal colonization on the substrates, the substrates embedded in fungal mycelium were turned upside down at day 6, leaving an air space at the bottom of the well. Fungal post-treatments All fungal post-treatments described below were performed in situ in the same 24-well plate culture (Fig. 1). Extraction of water-soluble components. Biopretreated and non-inoculated substrates after 12 days of incubation were washed with deionized water (1.5 ml well −1 ) for 2 h, at 50°C and 450 r.p.m. (Microtron) and then centrifuged at 2500 r.p.m. for 5 min. Three hundred microlitre aliquots of supernatants were harvested and dispensed into a 96-well filter-plate (1 μm) to be centrifuge-filtered (2500 r.p.m.; 5 min) for further analyses. Mild alkaline wash. The remaining solid fractions underwent mild alkali treatments as described by Salvachua and colleagues (2011), adapted to a 24-well plate with 0.1% (w/v) sodium hydroxide with a substrate consistency of 5% dm (w/v) at 50°C and 450 r.p.m. for 1 h. The substrate was then washed by adding 3 ml of deionized water to each well, incubating at 50°C and 450 r.p.m. for 10 min (Microtron) and removing 3 ml of the wash liquid after centrifugation at 3500 r.p.m. for 5 min. The washing steps were repeated four times to reach neutral pH in the last water wash. Enzymatic hydrolysis assays. Experiments were carried out at 2.5% dm (w/v) consistency by adding 3 ml of citrate phosphate buffer pH 4.8, 50 mM. The suspension was further supplemented with 12 FPU g −1 substrate (dry weight) of commercial cellulases GC220 from T. reesei (Genencor Danisco, NY, USA) and 60 U g −1 substrate (dry weight) of β-glucosidase from A. niger (Novozyme SP188). Tetracycline (150 mg l −1 ) and cycloheximide (40 mg l −1 ) were added to prevent any microbial contamination. The 24-well plates were then heat sealed with aluminum using a plate sealer (PlateLoc), and incubated at 50°C and 450 r.p.m. in a Microtron incubator for 72 h. The 24-well plates were centrifuged at 3500 r.p.m. for 10 min, and the resulting hydrolysis supernatants (250 μl) were taken from the reaction mixture and dispensed into 96-well plates. The reducing sugars and the glucose released were quantified respectively using the dinitrosalicylic acid method (Navarro et al., 2010) and the Glucose RTU kit (Biomérieux, Marcy-l'Etoile, France). All assays were carried out in triplicate. For all experiments, the variation coefficient was lower than 10%. Glucose and reducing sugar yields are expressed as quantity of glucose or reducing sugars released per gram of initial dry substrate. To calculate the percentage improvement, glucose and reducing sugar yields of non-inoculated controls were considered as 100%. Enzyme activity assays Endo-1,4-β-glucanase and endo-1,4-β-xylanase activities were assessed in a 96-well plate by measuring, respectively, the hydrolysis of 1% azo-CM-cellulose and 1% azo-xylan (Megazyme, Ireland) in citrate phosphate buffer pH 4.8, 50 mM. Ten microlitres of the water extract were added to 50 μl of azo-substrate solutions. The 96-well plates were then heat sealed with aluminum (PlateLoc) and incubated at 50°C for 3 h. One hundred fifty microlitres of precipitant solution at pH 5 [80% (v/v) ethanol with 0.29 M sodium acetate and 18 mM zinc acetate] were then added to precipitate undigested cellulose or xylan. The plates were stirred, incubated for 10 min at room temperature and centrifuged at 3500 r.p.m. for 5 min. Supernatants were collected and the optical density was measured at 590 nm. Activities were expressed as percentage of azo-substrate hydrolysed with the 100% value taken as the mean absorbance at 590 nm of three wells to which no ethanol and no water extract had been added. The laccase and peroxidase activities were determined using 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) and 2,6-dimethoxyphenol (DMP) as substrates respectively. All tests were conducted in 96-well plates. For laccase activity, the reaction mixture (total volume 210 μl) contained 20 μl of water extract and 0.476 mM of ABTS in 47.6 mM sodium tartrate buffer at pH 4.0. The reaction was monitored by measuring ABTS oxidation at 420 nm and 30°C for 0.5 min. For peroxidase activity, the reaction mixture (total volume 200 μl) contained 10 μl of water extract, 5 mM DMP in 100 mM sodium tartrate buffer, pH 5.0, 0.10 mM MnSO4 and 4 mM of sodium fluoride (laccase inhibitor) with or without 0.1 mM H2O2. The increase in absorption at 469 nm was monitored at 30°C for 5 min. The activities of peroxidases were obtained by subtracting values obtained in the presence and absence of H2O2. Ligninolytic enzyme activities were expressed in nkatal ml −1 , where one nkatal is defined as the amount of enzyme that oxidizes 1 nmole of substrate per second. Statistical analysis PCA and HCA were performed using the FactoMineR package in the R commander environment with R (R Devel-opment Core Team 2007), available from the Comprehensive R Archive Network at http://CRAN.R-project.org/.

v3-fos

2016-03-14T22:51:50.573Z

2015-04-01T00:00:00.000Z

7791421

s2

Economically Viable Components from Jerusalem Artichoke (Helianthus tuberosus L.) in a Biorefinery Concept Biorefinery applications are receiving growing interest due to climatic and waste disposal issues and lack of petroleum resources. Jerusalem artichoke (Helianthus tuberosus L.) is suitable for biorefinery applications due to high biomass production and limited cultivation requirements. This paper focuses on the potential of Jerusalem artichoke as a biorefinery crop and the most viable products in such a case. The carbohydrates in the tubers were found to have potential for production of platform chemicals, e.g., succinic acid. However, economic analysis showed that production of platform chemicals as a single product was too expensive to be competitive with petrochemically produced sugars. Therefore, production of several products from the same crop is a must. Additional products are protein based ones from tubers and leaves and biogas from residues, although both are of low value and amount. High bioactive activity was found in the young leaves of the crop, and the sesquiterpene lactones are of specific interest, as other compounds from this group have shown inhibitory effects on several human diseases. Thus, future focus should be on understanding the usefulness of small molecules, to develop methods for their extraction and purification and to further develop sustainable and viable methods for the production of platform chemicals. should be on understanding the usefulness of small molecules, to develop methods for their extraction and purification and to further develop sustainable and viable methods for the production of platform chemicals. Introduction-Characteristics of the Jerusalem Artichoke for Potential Biorefinery or Multipurpose Use A concept of rising interest for society is the development of biorefineries. A biorefinery is an analogue to today's petroleum-based refineries with the difference that the biorefinery is built on renewable biomass resources instead of the petroleum that is the feedstock for today's refineries. The main reason for the upcoming biorefineries is a wish to transfer from today's system of fossil fuel use, which is non-sustainable with rising prices in the long term due to depletion of resources [1,2]. At present, a large share of the energy carriers worldwide, as well as materials and chemicals produced, have their origin in fossil resources [1]. Thus, a sustainable society with economic growth and development requires novel solutions based on sustainable use of biological raw material, mitigating climate change and taking development, production and economy into consideration [1,3]. In a biorefinery there is in principal an opportunity to convert almost any type of biomass into almost any type of biofuel, biochemical or biomaterial, if only suitable biotechnological and chemical techniques are combined [1,3]. However, while considering biorefineries, not only production must be discussed but also how sustainable that production is from economic, resource use and social perspectives [3,4]. From the resource sustainability perspective, selection of biomass to be used in the biorefinery is an important aspect. Most literature related to biorefinery research is based on forest biomass, algae biomass, agricultural and/or food waste and crops cultivated on marginal land resources. Literature on crops to be cultivated for biorefinery use is scarce, most likely due to the ongoing debate related to agricultural land to be used for food or fuel production in a world which still sees starvation and malnutrition for part of its population [5,6]. Jerusalem artichoke has some interesting features which make it interesting as a biorefinery crop; it is resistant to most pests and diseases, it is frost and drought tolerant, it can grow on most soils and has low fertilizer requirements [7][8][9][10]. Thus, Jerusalem artichoke can grow on soils were many other food crops cannot grow, and it can be grown further north than many other food crops while still having the potential to yield well (5 Mg/ha dry weight of tubers; 58°20-40'N, Southern Norway) [11]. A recent study also showed that Jerusalem artichoke has the potential for higher dry matter yield (4-35 Mg/ha) from crop residues compared to other crop residues such as corn stover, rice straw, sugarcane bagasse, wheat straw and hemp stem (2-11 Mg/ha; 55°39'N, Southern Sweden) [12]. The high variation in dry matter yield reported (from 4 to 35 Mg/ha) was due to three harvest occasions over the season and 11 clones of Jerusalem artichoke evaluated, meaning that the selection of Jerusalem artichoke clone and harvest time is of utmost importance for high yield. In a study using one clone of Jerusalem artichoke harvested in early autumn, the crop was not found to have similar high potential when compared to a number of other vegetable biomass feed-stocks [13]. As in most research based field trials, the results comparing Jerusalem artichokes clones and harvest dates originated from small hand-harvested plots, and thus the results are not fully comparable to commercial field production of Jerusalem artichoke. Beside the crop residue dry matter yield, Jerusalem artichoke produces tubers with a dry matter yield of 0. 45-15.8 Mg/ha (calculations from raw data of Gunnarsson et al. 2014 [12]). Data from China reports dry weight tuber yield of 9.1-10.6 Mg/ha and aerial biomass dry matter yield of 18.1-31.3 Mg/ha [14]. Comparatively, grain production of cereals reaches 0.5-12 Mg/ha [15]. The combination of the hardiness of the crop and the high dry matter yield makes Jerusalem artichoke of specific interest as a biorefinery crop. The most envisioned approach at the moment when it comes to biorefineries is that they should focus on producing chemicals by depolymerization and/or fermentation of biopolymers [16]. One important aspect if the biorefinery is to become a competitive process is that it should produce at least one product of high value (such as a high value chemical or material). Beside chemicals, one energy product should also be produced [1]. Of specific interest are small bioactive molecules that are of use as dietary components in food, as flavors, fragrances, sweeteners, as natural pesticides and as pharmaceuticals [17]. Jerusalem artichoke is known to contain an interesting polysaccharide in its tubers, inulin, amounting to 10%-20% of fresh tuber weight [18][19][20][21], being a dietary fiber and also known to have prebiotic effects [22,23]. However, at present root chicory (Cichorium intybes L.) is the main crop for inulin production [24]. Jerusalem artichoke is also known to contain other high value chemicals and small bioactive components of bioeconomic interest if the crop is utilized in a biorefinery concept [9]. The fact that the crop, besides being hardy and high yielding, also contains inulin makes it relevant for further evaluation as a potential biorefinery crop. However, economic and environmental evaluations of the potentials of various components of Jerusalem artichoke have been limited. The present paper reviews data on Jerusalem artichoke. Additionally, results on variation in protein content in leaves and tubers, and antioxidant capacity in leaves of Jerusalem artichoke between clones and harvest times not previously published are presented. From the review data as well as the new additional data, potential products are discussed. Furthermore, preliminary economic evaluations in the present paper are shown as a concept to reveal the options for Jerusalem artichoke as a potential biorefinery crop. Carbohydrates-Types, Content and Potential Uses Jerusalem artichoke tubers primarily contain two types of carbohydrates, inulin and sugars (fructose and glucose) [12,25]. The main carbohydrates in the aerial biomass are cellulose and hemicellulose [12]. Inulin is an interesting compound from a biorefinery point of view, being a functional food ingredient [23,26]. It contributes to the organoleptic characteristics of food, improves stability of foams and emulsions, and when used as a gel in water it has fat like characteristics [26]. Inulin is degraded to oligofructose through hydrolysis by inulinase [27]. Inulin and oligofructose have been shown to stimulate the immune systems in the body, increase absorption of calcium, and decrease triglycerides and fatty acids content in blood serum; they modulate hormonal levels of insulin and glucagon and reduce the incidence of colon cancer [23]. Oligofructose has technological properties closely related to sugar and glucose syrup [26]. Yield of inulin in tubers of Jerusalem artichoke has been reported to vary between 0.36-12.6 Mg/ha (75.8-84.3 g/100 g dry weight) over the season and in different clones [12]. Inulin and oligofructose are commonly found in nature, being present in around 15% of all flowering plants [23,26]. However, at present there are mainly two species, Jerusalem artichoke and chicory, which are used by industry for the production of inulin [23]. Chicory dry matter yield has been reported of 5.6-7.8 Mg/ha [28] with an inulin content of 70%-80% [23]. Thus, the inulin yield per ha is often higher in Jerusalem artichoke than in chicory. One important aspect for the quality of the inulin is its degree of polymerization (DP). In general the DP was found higher (around 14) earlier in the season than later in Jerusalem artichoke [12]. DP of around 10-12 has been reported for standard inulin from chicory and Jerusalem artichoke, although high performance inulin with a DP of 25 has also been produced from chicory [12,26]. The sugar content of Jerusalem artichoke tubers has been reported to be around 4%-5% of the dry weight [12]. Tubers of Jerusalem artichoke have been evaluated both as substrate for ethanol and succinic acid production with yields of 48% [29,30]. Additionally, L-lactic acid, acetone-butanol, 2,3-butandiol, butyric acid, sorbitol and biodiesel are other products that have been obtained through fermentation processes of the tubers of Jerusalem artichoke [31]. In the aerial parts of Jerusalem artichoke a dry matter cellulose yield of up to 8.8 Mg/ha and a dry matter hemicellulose yield of up to 4.6 Mg/ha have been reported [12], making this part of the crop competitive with other cellulose rich crop residues (corn stover, rice or wheat straw, sugarcane bagasse or hemp stem having maximum dry matter yields of 6.8 Mg/ha for cellulose and 3 Mg/ha for hemicellulose) [12]. However, the relatively low content (measured as % dry weight) of cellulose (11.3-30.8 g/100 g dry weight) and hemicellulose (9.0-17.3 g/100 g dry weight) in the Jerusalem artichoke stalks, the chemical complexity of these compounds and also the high content of lignin in the stalks have limited their usefulness [32]. Recent studies indicate the possibility of using the whole plant of Jerusalem artichoke for ethanol [33] or 2,3-butanediol production [31]. An ethanol yield of 1800-3100 kg/ha from the whole plant has been reported [13,34]. Proteins-Types, Content and Potential Uses Protein content of 5.3%-10.4% (dry wt.) has been reported for Jerusalem artichoke tubers, while the aerial parts were reported to have a protein content of 1.1%-6.1% (dry wt.) [12]. While dividing the aerial part of the Jerusalem artichokes into different fractions (leaves, stalk, stump), and analyzing the protein content through the use of the Dumas method on a Flash 2000 NC Analyzer (N conversion factor 6.25 applied) [12,35], a low protein content was generally found in the stalk (1.6%-4.5% with the lowest values at late harvest) and stump (1.6%-2.6% with the lowest values at late harvest). Content of protein was found to be much higher in the leaves of the Jerusalem artichoke (7.1%-24.5%, also with lowest values at late harvest; Table 1). Over all of the seasons and in all different plant parts, the highest protein content was found in the leaves early during the season, with over 20% (dry wt.) protein in some of the clones (Table 1). Table 1. Mean protein content (% of dry matter) measured by the Dumas method on a Flash 2000 NC Analyzer and applying a nitrogen conversion factor of 6.25 [35] in leaves and tubers of 11 different clones of Jerusalem artichoke harvested at three different occasions during the season. For description of the plant material see ref. [12]. Limited information is available as to the protein composition in Jerusalem artichoke tubers. In general, the content of amino acids essential for humans is relatively high in Jerusalem artichoke, e.g., higher than in chicory and potatoes. Jerusalem artichoke tubers were also especially rich in sulfur containing amino acids, e.g., four times higher than chicory and potatoes [36]. The combination of Jerusalem artichoke tubers being rich both in essential amino acids and sulfur containing amino acids makes the proteins of this crop of some interest, to be evaluated both for food industry application and as an alternative for the plastics/materials industry. Nutritive value of proteins is always of relevance for the food industry. The content of sulfur containing amino acids may indicate that the proteins have good foaming ability (of importance for food industry but also for e.g., production of insulation materials) [37]. The proteins may also have the ability to form films of good properties as sulfur containing amino acids are related to the formation of disulphide bonds thus building polymeric proteins [38][39][40][41][42]. Besides the option to use the proteins directly in the food or materials industry, proteins are also an interesting source for production of platform chemicals through a first step of degrading the proteins to amino acids from which chemicals can be built [43]. The protein composition of the aerial parts of Jerusalem artichoke has also received limited attention. Two recent studies have focused on developing suitable protocols and methods for proteomic studies of the proteins of Jerusalem artichoke aerial parts [44,45]. These studies report high levels of rubisco in the leaves in Jerusalem artichoke. Rubisco is probably the most abundant protein on earth and makes up between 4% and 28% of the protein in green leaves [46]. Rubisco has a good nutritional profile, comparing favourably with eggs or meat [47,48]. In its purified form spinach rubisco has attractive functional properties with low thermal gelation temperature (approx. 75-85 °C) and relatively low gelation concentration (4% vs. 10% for whey proteins) with good foam formation, suggesting use as a replacement for dairy based foams [49]. The ability of alfalfa rubisco to form emulsions can be better than egg white, but its activity depends on the processing parameters [50]. Proteins from the aerial parts may also be further valorised utilizing their ability to form films. Plant proteins have previously been processed into biobased and biodegradable plastics using commercial plastic processing techniques [51,52], to our knowledge rubisco has not been investigated for these non-food applications. Bioactive Compounds-Type, Content and Potential Uses Mean antioxidant capacity of eleven different Jerusalem artichoke clones was found to be 10.2 mmol/100 g DW (mean values ranges 6.6-11.9 among clones) for the tubers and 41.1 mmol/100 g DW (mean value ranges 36.8-47.2 among clones) for the leaves at early harvest (Table 2) by the use of FRAP (ferric reducing ability of plasma) [53][54][55]. The antioxidant capacity in both leaves and tubers thereafter decreased steadily over the harvest season, resulting in values of around 1 mmol/100 g DW in the leaves at late harvest. The FRAP values found for the Jerusalem artichoke leaves at the first harvest ( Table 2) are well in accordance with what is reported in many fruits and berries, while those for tubers are lower. Actually, FRAP values found in Jerusalem artichoke leaves at the first harvest are higher than those reported for apple peel (25.2 mmol/100 g DW), and the berries of cranberry, lingonberry, raspberry, sea buckthorn and strawberry (13.9-36.0 mmol/100 g DW) [56]. On the other hand, higher FRAP values have been reported in some berries-bilberry, black currant, elderberry, purple chokeberry, rose hips and sloe (42.1-178.5 mmol/100 g DW) [56]-than in the Jerusalem artichoke green leaves. Berries are well known as sources of bioactive compounds, and the content of phenolic compounds, especially, have been related to human health [57]. Rose hips in particular have been reported in several studies as a rich source of bioactive compounds [58,59] and having high FRAP levels [59]. Vegetables and green leaves, also known to contain bioactive compounds, are normally reported to have lower FRAP activity levels than the berries, e.g., peppers were reported as having the highest FRAP levels among a number of vegetables with values of 15-19 mmol/100 g DW) [60]. For the leaves of Jerusalem artichokes, FRAP values were decreasing over the season and at a second harvest, mean levels had fallen to 23.0 mmol/100 g DW (Table 2). Thus, time of harvest of vegetables and green leaves might play a role for previously reported FRAP values from other sources. Since ancient times, Jerusalem artichoke has been known in folk medicine as being beneficial for the treatment of diseases including diabetes and rheumatism [61,62]. Bioactive compounds known to be present in Jerusalem artichoke are coumarins [63], polyacetylenes and their derivatives [62,64,65], and sesquiterpenes [66]. Aerial parts of Jerusalem artichoke have shown antimicrobial and antifungal activities [9,67]. Furthermore, recent studies have shown that germacrane sesquiterpene lactones from Jerusalem artichoke have anticancer properties and that these compounds are cytotoxic agents [67][68][69]. Sesquiterpene lactones are compounds known to exert a variety of biological activities, including anti-tumour, anti-inflammatory, cytotoxic, and anti-microbial effects [70]. Of specific recent interest has been the discovery of artemisinin, which is found in Artemisia annua, and has gained much popularity as an antimalarial drug, as resistance to other drugs has been growing. Several sesquiterpene lactones are also in clinical tests as anti-cancer drugs and for the prevention of cardiovascular diseases [71]. Economic Aspects of Jerusalem Artichoke Cultivation as a Biorefinery Crop As mentioned above, a biorefinery should focus on at least one high value chemical or material and one energy product [1]. Thus, for a crop to be a biorefinery/green chemical crop, a similar requirement persists. Besides that, the yield of each of the components is important for positive economics in cultivating the crop. As seen from above, Jerusalem artichoke fulfills many of these requirements making it relevant as a biorefinery/green chemicals crop. The highest value products do often come from small molecules, such as bioactive compounds that can be used as dietary components in food, as flavors, fragrances, sweeteners, natural pesticides and pharmaceuticals [17]. Jerusalem artichoke leaves show extremely high levels of antioxidant activity (Table 2), higher than has been reported in other vegetables or green leaves [60] and the levels are instead similar to many berries [56]. Thus, for economic purposes, harvests of bioactive compounds from the leaves should be taken into consideration while growing Jerusalem artichoke as a biorefinery/green chemical crop. Highest levels of antioxidant activities were found early during the season (September harvest, Sweden). Also, significant differences were found among the different clones investigated, with the highest levels occurring in clone 8 among the clones we investigated (Table 2). Therefore, if Jerusalem artichoke should be grown as a biorefinery/green chemicals crop, where the highest value product is one or several bioactive compounds, determination of harvest date and clone to be cultivated are important parameters. One type of bioactive compound known to be present in Jerusalem artichoke is sesquiterpene lactones. Sesquiterpene lactones from other sources are used as malaria medications and are in clinical tests as anti-cancer and anti-cardiovascular drugs. It might therefore be possible that the sesquiterpene lactones in Jerusalem artichoke are also the highest value products that can be produced from the crop, although neither commercial processes, nor extraction or purification have been developed, making economics difficult to define. Proteins, both the rubisco proteins from the leaves and the proteins of less known type from the tubers, are most likely high value products that can be sequentially extracted with the bioactive compounds from the crop. Recent research on lucerne has shown the opportunity to extract rubisco proteins from leaves at the same price as extraction of soy proteins [72,73]. Similar extraction procedures at a similar price are most likely possible for Jerusalem artichoke, which was shown to have a protein concentration in the leaves (20% DW, Table 1) similar to that found in lucerne [72]. Also, the rubisco protein has a better nutritional profile [49] than soy protein and probably better foaming properties [47]. Therefore, rubisco protein should most likely receive a higher price than is obtained for soy protein. The tubers of Jerusalem artichoke are rich in carbohydrates, while the aerial part also has a high yield per ha, but the % content is somewhat lower with a relatively high lignin content, making aerial parts more difficult to utilize. For the tubers, there are a number of possible uses after the extraction of proteins. The tubers can be utilized for production of inulin, biogas, ethanol or platform chemicals, e.g., succinic acid. Current prices of ethanol are 0.5 USD/kg (1.53 USD/GAL, 1 GAL = 3.79 L, 0.789 g/cm 3 ) [74], succinic acid 6-9 USD/kg [75], while the price for natural gas is 3.27 USD/GAL [76] and the price for biogas somewhat lower [77]. Prices for inulin products are around 3-4 USD/kg [78]. From the above numbers it is clearly shown that it is more beneficial to produce platform chemicals, such as succinic acid, with a higher price than the very cheapest ones, e.g., ethanol, if the production costs are relatively similar. Moreover, production of succinic acid is connected with use of CO2. Thus an additional environmental advantage in terms of abatement of CO2 emissions is achieved. Production of succinic acid by fermentation consumes 1 mol of CO2 per 1 mol of succinic acid produced. It has been estimated that CO2 emission savings in the range of 4.5-5 Mg per Mg succinic acid produced can be achieved [79]. Issues Related to the Multipurpose Use of Crops The multipurpose use of crops with an integrated approach to obtain several products at the same time creates certain demands on the extraction and production procedures [80]. Components need to be properly extracted without interacting negatively on other components that should be extracted or fermented later in the process or with the environment. The sesquiterpene lactone component that has been mostly investigated for extraction and purification purposes is artimisinin, utilized as an anti-malarial drug. Extractions of artimisinin have been carried out using hexane, supercritical carbon dioxide, hydrofluorocarbon HFC-134a, ionic liquids and ethanol [81]. Similar methods can most likely be used for extraction of sesquiterpene lactones from Jerusalem artichoke. However, among these methods hexane is the one that has been most widely used and this method might be the most cost-effective [81]. However, it is also seen as the worst with regard to safety and environmental impact, so if Jerusalem artichoke is to be used as a multipurpose sustainable biorefinery crop, the hexane method might not be the method to be used. Newer and greener methods for extraction of artimisinin are available [81] and might be considered for the extraction of sesquiterpene lactones from Jerusalem artichoke. As for proteins, extraction methods need to be selected in relation to what proteins are to be extracted. Albumin types of proteins are known to be soluble in water, globulins in salt, prolamins in alcohol and glutelins in acid or base [82]. The proteins in both leaves and tubers of Jerusalem artichoke are most likely primarily of the albumin and globulin types. However, recent studies have shown that it is possible to extract various portions of albumins and globulins from, e.g., Crambe by adjusting extraction and precipitation through various pHs [83]. Recent studies on the production of bio-succinic acid have shown the benefits of removing CO2 from biogas and converting it into bio-succinic acid through the use of the bacterial strain Actinobacillus succinogenes 130Z [84]. Thus with this system it is beneficial if the same crop can be used as a substrate for biogas and succinic acid as we are suggesting for Jerusalem artichoke. The fact that biogas can be simultaneously upgraded to vehicle fuel by this method as bio-succinic acid is produced increases the economic potential of the use of Jerusalem artichoke for these purposes. Furthermore, recent work has shown that Jerusalem artichoke tubers can be fermented into succinic acid without the use of enzymes, thus the tubers with their high carbohydrate content and relatively simple bioconversion is an attractive biomass feedstock, which also influences the production costs [30]. Preliminary Economic Analyses of the Use of Jerusalem Artichoke as a Biorefinery Crop To better understand if and how Jerusalem artichoke can act as a biorefinery crop, we have carried out a preliminary economic analysis on production of various products from Jerusalem artichoke harvested on various occasions. Due to lack of data as to what potential products can be produced from the bioactive compounds of Jerusalem artichoke and production costs/prices of these products, possible small molecule based products are omitted from the analysis. Thus, the economic analysis was carried out on production of rubisco from the aerial biomass, protein and succinic acid from tubers and energy from residues of both aerial biomass and tubers, and based on raw yield data on 11 clones of Jerusalem artichoke harvested at three occasions [12]. Processing efficiencies applied in the calculations have been adopted from the literature (Table 3). For calculation of energy yields, an estimation of maximum methane potential has been carried out based on the amount of process residues. Methane production potentials were calculated following previously described methods [88] and literature data for the different compounds (Table 4). Complete degradation of the compounds was assumed. Methane volumes were converted to energy units using the higher heating value for methane of 39.2 MJ/Nm 3 . Table 4. Assumed degradation and methane production potentials in anaerobic digestion. As to the production cost of Jerusalem artichoke for biorefinery purposes, production can be assumed to be similar to that of potatoes for industrial purposes. However, extra costs for more expensive seeds, extra mechanical row cleaning and cost for harvest and transport of the tops needs to be added on the production costs per hectare for Jerusalem artichoke as compared to potatoes. Production costs for potatoes for industrial utilization are around 4000-4400 €/ha [92], and thus Jerusalem artichoke production can be estimated to be 20% more expensive, i.e., around 4800 €/ha. As a sensitivity analysis, the cost range between 3800 and 6000 €/ha was tested. Based on literature data on processing costs ( Table 5) an estimation of gross margin for production of succinic acid and biogas from Jerusalem artichoke was calculated according to: Processing costs for fermentative succinic acid production were calculated based on previous investigations [95] assuming a sugar conversion efficiency of 91% [96] for profit margins between 10% and 30%. Based on the mentioned estimations, high yields of succinic acids was obtained from the tubers of all the 11 clones of Jerusalem artichoke with an increasing yield at late harvest dates (Figure 1a). Similarly, protein and energy yields from the tubers as well as energy yields from the aerial parts increased with later harvest dates (Figure 1a,b), although variation among clones was found for all four products. As to rubisco protein yields from the leaves, a decrease was noted with later harvest dates (Figure 1b,c), also here with large variation among clones. (a) (b) (c) Figure 1. Average yields of (a) protein and succinic acid from tubers, (b) energy from biogas production of fermentation and extraction residues from tubers and tops, and (c) rubisco protein from tops, for different clones of Jerusalem artichoke harvested at early (September), medium (October) and late (December) harvest date. From the present economic analysis, extracted protein from the tubers was the product of Jerusalem artichoke with highest impact on the income, contributing on average 29%, 39% and 45% of the total income at early, medium and late harvest, respectively. Succinic acid from the tubers contributed 28%, 41% and 42% and rubisco from the leaves contributed 21%, 11% and 9%, respectively. Biogas from tubers and aerial biomass process residues contributed 21%, 12% and 10%, respectively. Thus, succinic acid showed the second highest impact on the income. Despite that fact, sole production of succinic acid from Jerusalem artichoke, meaning that this product should bear the full production cost of the crop and of the processing, is hardly competitive compared to the use of other sugar feed-stocks for succinic acid production (Figure 2a). Production costs for production of succinic acid via catalytic hydrogenation of petro-chemically derived maleic acid or maleic anhydride are currently still lower than the succinic acid derived from carbohydrate fermentation [97,98]. However, bio-based succinic acid is becoming more competitive as prices for maleic acid are increasing (Figure 2a) [99,100]. Also, recent studies using Jerusalem artichoke as a feedstock have shown high yields from direct fermentation of the hydrolysis broth [84], and purification costs can likewise be lowered by carrying out subsequent conversions directly in the fermentation broth. [99,100]; In (b) grey markers represent average gross margin and black bars represent range according to variation in original chemical analyses and low/high variation for processing efficiencies and costs. Generally, average gross margins for biorefinery utilization of Jerusalem artichoke were highest at late harvest, with exception of clones 5 and 6, with highest gross margins at medium harvest (Figure 2b). Clone 1 showed an exceptionally high gross margin at late harvest, while results at early and medium harvest were comparable to the other clones. At medium cultivation costs and a 20% profit margin, refining of the clones 1, 4, 7 and 8 became economically viable starting with medium harvest, while the other clones were economically viable at all harvest dates. Conclusions-Can Jerusalem Artichoke Be Seen as a Potential Biorefinery Crop? Jerusalem artichoke can definitely be seen as a potential biorefinery crop. However, a multi-purpose use of the crop for sequential production of several products seems beneficial. For economic profit, the products of highest economical value from the crop have to be defined for a biorefinery utilization of the crop. Potential such high value products are those for medical uses, and bioactive compounds (in this crop probably sesquiterpene lactone mediated ones) from the leaves are specifically interesting. Furthermore, the rubisco protein from the leaves might be of relevance to be used in the food and materials industry. Suitable extraction methods to obtain the bioactive compounds and rubisco proteins in a pure, secure and suitable conformation and to a reasonable price need therefore to be worked out. The wastes after extraction of bioactive compounds and rubisco proteins from the leaves should preferably be used for biogas production, as should the rest of the aerial parts of the Jerusalem artichoke. The protein of the tubers might be of relevance to be utilized by the food industry and economic analyses indicated the tuber protein as a large part of the economic benefit of the crop. Suitable extraction procedures need to be developed here as well. However, the carbohydrates in the tubers must be seen as the main product of the tubers and an economically viable use of these is currently a necessity to succeed with a biorefinery use of the crop. The carbohydrates of the tubers should preferably be used in a biorefinery concept for succinic acid or other relatively high value platform chemicals production. Eventual residues can be added to the residues of the aerial parts of the Jerusalem artichoke and be utilized for biogas production. the same style and the style of the journal. Sven-Erik Svensson was responsible for the field experiments, Ingólfur Bragi Gunnarsson for the succinic acid experiments, Johansson for the protein experiments, Helena Persson Hovmalm for the bioactive activity experiments and Thomas Prade for the economic calculations. Conflicts of Interest The authors declare no conflict of interest.

v3-fos

2019-04-07T13:04:57.742Z

2015-09-10T00:00:00.000Z

19930821

s2

Identification of Erosion Hotspot Area using GIS and MCE Technique for Koga Watershed in the Upper Blue Nile Basin, Ethiopia Corresponding Author: Tewodros T. Assefa Energy and Environmental System, North Carolina A and T State University, Greensboro, USA Email: tassefa@aggies.ncat.edu Abstract: Soil erosion is a serious threat in Ethiopian highlands. Continuous land degradation resulted in loss of fertile top soil leading to low agricultural productivity. In addition, excessive soil erosion from Koga Watershed in upper catchment to an artificial reservoir (Koga Dam reservoir) is substantially reducing its service life. Community participatory based effective watershed management strategies may have tremendous potential to reduce soil erosion. However, it is not practical to implement management interventions in the entire basin. This study aims to identify and map erosion hotspot areas in Koga Watershed to assist local government decision towards implementing watershed management strategies. Multi Criteria Evaluation (MCE) technique was integrated with Geographic Information System (GIS). For these analysis four major factors: Topography, soil, land use and potential location of gullies were considered. Each of these was processed and analyzed for its potential contribution to erosion on a pixel by pixel basis. The factors were weighted using pair-wise comparison matrix and weights were combined using Weighted Overlay Tool of ArcGIS Spatial Analyst Toolbox to obtain the final erosion hotspot map. The results found that 2% (440 ha) to be highly sensitive, 43% (9,460 ha) to be moderately sensitive, 16% (3,520 ha) to be marginally sensitive and 32% (7,040 ha) currently not sensitive. The remaining 7% of the watershed area (22,000 ha) was constraint to erosion. The lowland area near the dam was found to be found most sensitive for erosion and sedimentation. Introduction Excessive soil erosion in the highlands of Ethiopia brought reduced agricultural productivity. Furthermore, natural and artificial reservoirs are suffering from sedimentation and constructed dams are ceased functioning before their service life. Lack of effective watershed management system and poor land use practices played significant role in land degradation in the region (Setegn et al., 2009). According to the Ethiopian highland reclamation study (Yilma and Awulachew, 2009), in the mid 1980's, 27 million hectare or almost 50% of the high land area was significantly eroded, 14 million hectare seriously eroded and over 2 million hectare were beyond reclamation. Rate of soil erosion is increasing alarmingly necessitating design of proper watershed management strategies. Recently constructed Koga dam reservoir is one of the affected reservoirs in the basin. Its storage volume is substantially reducing because of siltation. The siltation rate is high (100-200 Mt/ha/yr.) and 50% is supplied from agricultural lands in the form of sheet and rill erosion (Reynolds, 2012). This adversely results in constrained irrigation supply to farmlands. Field visit witnessed inability of Koga dam reservoir to serve some farmlands which are originally included in the design. Cultivation became infeasible in several parts of Koga Watershed; however and farmers continue to cultivate marginal lands (Mengstie, 2009). Current soil and water conservation practices in Koga Watershed are focused at the highland areas which are highly degraded and have limited production potential. However, this effort could not show significant erosion reduction to date (Mengstie, 2009). Strategic watershed management interventions should focus on erosion sensitive portion of the catchment to prevent further land degradation and siltation to the dam. Lack of finer resolution spatial data and closely installed hydrometeorological data hindered spatial analysis of erosion generation. Setegn et al. (2009) delineated erosion sensitive area for Lake Tana Basin considering steep slope as the main source of erosion (infiltration excess runoff mechanism). However, the saturated area could be the main source of erosion and the dominant runoff mechanism is uncertain for the wider basin. Understanding the dominant runoff mechanisms (infiltration excess or saturation) is essential to identify watershed management priority areas. Both mechanisms could happen in the same watershed while one dominate the other depending on hydrological properties, moisture content, slope and other factors. In a recent study conducted to nearby experimental watershed, Debremawi (Tilahun et al., 2014) showed that the infiltration capacity were exceeded by rainfall intensity during only 1.5 and 4% of the time in 2010 and 2011, respectively, indicating the dominance of saturation excess runoff mechanism. These findings indicate that the study area (Koga Watershed) is also dominated by saturation excess runoff mechanism. The present study utilized GIS capability to analyze topographical characteristics of the watershed in terms of wetness property of the land surface. Multi Criteria Evaluation (MCE) technique was integrated with GIS to rank/rate alternatives based on multiple criteria/factors that would affect erosion. MCE analysis of spatial information is an emerging approach and is efficient to analyze complex problems within a watershed (Setegn et al., 2009). MCE approach has been successfully used in many applications that involve decision making (Pereira and Duckstein, 1993;Malczewski, 2006;Hajkowicz and Higgins, 2008;Setegn et al., 2009;Greene et al., 2011). MCE technique is well assimilated in GIS environment. GIS is capable of efficiently storing, retrieving, transforming, displaying and analyzing spatial data. In this study, we used MCE technique within GIS environment to identify the actual source of erosion and map sensitive areas based on spatial dataset analysis. Weight of decision factors are assigned based on their relative effect to erosion process. Description of Study Area Koga River is a tributary to Gilgel Abay River which drains to Lake Tana in the Upper Blue Nile Basin. Koga dam (drainage area of around 22,000 ha) constructed downstream of the river, supply water for irrigating downstream farms. Koga dam is geographically located at 12 0 10'N latitude and 37 0 38'E longitude, within the Lake Tana Basin (Fig. 1). The annual rainfall is around 1480 mm based data available in an adjacent meteorological station, in Merawi. The dam was designed to store around 83 million metric cube of water to irrigate about 6,000 ha of command area (AfDB, 2001). The elevation of Koga watershed varies from 2,005 to 3,147 meter above sea level within the watershed based on Digital Elevation Model (DEM). Koga watershed falls under subtropical climate zone (Yeshaneh et al., 2013). The upstream of the watershed is narrow and mountainous while the downstream is wide and gentle slope. Data Requirement and Sources Data required for the MCE technique are spatial in nature. Four major types of data (Land use, DEM, Soil type and Rainfall) were collected. Data sources for each category presented in Table 1. There was no meteorological station found within the catchment. Nearby stations (Merawi, Adet, Dangila and Bahir Dar) were used to find the average rainfall using thiessen polygon method. The average rainfall was highly dominated (~97%) by a single adjacent station, Merawi. The variation of rainfall within the catchment was analyzed and found insignificant (with 95% confidence). This was because of absence of closely installed meteorological stations within the catchment. As a result, rainfall was not considered as one of MCE criteria in this research. After collecting the required data, spatial analysis were made to obtain MCE criteria map. Landsat 8 image together with intensive field point data collection were used to perform supervised land use classification in ArcGIS environment. The output map was validated and used to produce land use criteria map based on land use suitability classes. Soil map from Ministry of Water and Energy (MoWE) were directly used to produce soil criteria map based on soil type suitability classes. DEM with 30 m resolution was used to produce two criteria maps: Topographic wetness index and potential location of gullies. The wetness of the catchment (topographic wetness index) was predicted based on flow accumulation and slope of the particular pixel. Potential location of gullies was predicted based on threshold concept of two criteria: Wetness index and stream power index. Koga dam reservoir was considered as constraint. Each factor map (topography, potential locations of gullies, land use and soil) were reclassified based on sensitivity classes. The sensitivity classes for each factor were determined through discussion with experts (personal communication, T. Steenhuis, professor at Cornell University). Those experts have been doing intensive researches and experiments long on erosion and sediment transport process close to the study area. Relative weights were assigned to each factor depending on the relevance of each factor and experts opinions. The value (1 to 9 and its reciprocal) were assigned to each factors based on pairwise comparison criteria. Pairwise comparison method was used to get the final weight of each factor. Based on factors final weight, the reclassified map was overlaid to get the combined effect of all actors. The output map was multiplied with the constraint map to exclude the reservoir area and produce the final erosion hotspot map. Figure 2 below presented the procedure to obtain the final erosion hotspot map. The detail of sensitivity classes for each factor and criteria layer map discussed in result section. Factors Classification Approach Erosion sensitivity of the catchment is classified into two classes (i.e., sensitive and not-sensitive) based on FAO (1981) land classification framework for irrigation purpose. Sensitive classes are further classified depending on the degree of sensitivity while not sensitive classes are classified into two classes (currently not sensitive and permanently not sensitive) ( Table 2). In this study, permanently not sensitive classes are described as constraint to erosion. Important Factors for Soil Erosion Four significant factors that can potentially affect soil erosion were considered. The first factor was land cover which controls the detachability and transport of soil particles and infiltration of water in to soil. The second was topography in terms of the wetness of the land. Soil types are important factor to erosion and sediment transport process depending on their physical and chemical properties. Locations of gullies were also considered as one major factor promoting erosion. Preparation of Criteria Maps Field work was carried out to collect control points for Landsat 8 satellite image supervised land use classification. Accuracy assessments were performed on classified images to determine how well the classification process accomplished the task using error matrix. Ministry of Water and Energy (MoWE) soil classification comparatively has finer resolution for the region and hence chosen for this study. It has 5 soil classes for Koga Watershed. Digital Elevation Model (DEM) of 30 m resolution was used to extract topographical parameters and potential location of gullies as well. The potential locations of gullies were predicted when both of the following two conditions of the thresholds were satisfied (Lulseged and Vlek, 2005) where, A S is local upslope contributing area (m 2 ) from flow accumulation raster and is local slope angle (degree). Area covered by Koga dam reservoir were considered constraint, area that could not promote erosion because of physical limitations. The constraint map has a value of 0 and 1 (Fig. 7), where 0 indicates constraint area. Factors that cannot support erosion The weight of factors are assigned based on their relative effect on erosion and ranked according to their relative importance order (Wale et al., 2012). Pairwise comparison based on (Saaty, 1977) was adopted to calculate the total weight of each factor. It provides a powerful and simplified selection criterion by reducing bias in decision making (Wale et al., 2012). It refers to the process of comparing factors in pairs to evaluate which one is preferred. Equal interval range method was applied to distribute the overall weight of factor maps. Weighted Overlay Tool in ArcGIS Spatial Analyst Toolbox environment was used to identify erosion sensitive areas (Nyerges and Jankowski, 2009). Impact of Land Use on Erosion Land use map is one of the most important factors that affect surface runoff and erosion in a watershed. It enables to assess the resistance of terrain unit to erosion as a result of surface protection. High erosion and quick response to rainfall are resulted from poor surface cover. From field visit, eight land use/land cover categories were distinguished followed by supervised land use classification method and maximum likelihood algorithm. Post-classification processing was applied to the classified image such as filtering and smoothing class boundaries. Filtering is the process used to remove fixed isolated pixels from classified image. Smoothing class boundaries is the process used to clump the classes and smooth's the ragged edge. Accuracy assessments were performed on classified images to determine how well the classification process accomplished the task. It compares the classified image to an image which is considered to be correct (Google earth for this case) with the help of error matrix (Table 3). The error matrix compares the classified and correct image on a point by point basis. The points were generated randomly using ArcGIS random point generator tool (35 random points were generated for this case) within the boundary of Koga Watershed. The overall accuracy assessment resulted in acceptable range (~89%). In Table 3, numbers in the first row and first column represent land use classes which are: 1 (Bare land), 2 (Bush land), 3 (Forest), 4 (Grass land), 5 (Intensively cultivated), 6 (Moderately cultivated), 7 (Urban/Village), 8 (Water). Similar row and column numbers combination indicates that the land use class in the classified image (Fig. 3a) is the same as the land use in the correct map (Google earth). For instance, (a) represents how many random points that are bare land in Google earth and bare land in the classified image. But (b) represents how many random points are bare lands in Google earth but bush land in the classified image. Finally, the overall accuracy is the total number of random points that are the same in both images per total number of sample random points: (31*100/35) ~89%. According to Anderson et al. (1976), 85% accuracy is considered acceptable for image classification. Kappa coefficient provides an insight into land use classification whether or not better results are achieved than we would have achieved strictly by chance. Kappa coefficient is given below (Equation 3 (Table 4). Information from Table 3 was used to compute the value in Table 4. Column represented by "Total" from Table 3 multiplied with row represented by "Total" to produce the Kappa coefficient in Table 4. The product matrix was determined from the sum of the products of the diagonal matrix. Therefore, the Kappa coefficient is calculated as: Product matrix (8+6+2+4+32+140+1+81 = 274), cumulative sum product (1365), expected (274/1365 = 0.201) and finally Kappa coefficient (~86.5%). Kappa coefficient value interpreted as the classification was 86.5% better than it would have occurred strictly by chance. Percent distribution of land use/cover and sensitivity classes in Koga Watershed presented in Table 5. The re-classified land use map (Fig. 3b) indicated that 36.6% of the land use is highly sensitive; 34.6% moderately sensitive; 19.8% marginally sensitive; 4.4% currently not sensitive and about 7% constraint to erosion. Soil Type Impact on Erosion Soil type is one of the important factors that affect erosion process depending on the physical and chemical characteristics. It controls detachability of soil, soil particle transport and infiltration of water into the soil (Setegn et al., 2009). Soil texture is an important property which contributes to soil's erodibility. The watershed is dominated by Haplic Luvisols (38.0%) followed by Haplic Alisols (24.3%) which has moderately well drainage classes (Table 6). Figure 4a presented soil types in Koga Watershed. The reclassified soil map (Fig. 4b) indicated that 17.5% of the soil is highly sensitive; 16.7% is moderately sensitive; 59 marginally sensitive and the remaining (~7%) is constraint to erosion. Impact of Topography on Erosion Topography is the major surface parameter for soil erosion assessment. Although slope has a great impact on soil erosion, the presence of soil erosion and heavy runoff on gentle slopes indicate that this phenomenon can occur without any need for a steep slope; the action of rain is enough (Fauck, 1956). Topographic Wetness Index (TWI) was used to describe the effect of topography based on saturated excess runoff generation mechanism. It represents spatial distribution of surface saturation and surface runoff which is important factor for soil erosion simulation. The concept was originally developed in the TOPMODEL framework (Beven et al., 1984) and described in Equation 1. As the contributing area increases and slope gradient decreases, topographic wetness index and soil moisture increases, it has higher correlation with saturation (Easton et al., 2010). Figure 5a presented topographic index map of Koga watershed. Erosion sensitivity class for topographic wetness index is presented in Table 7. The reclassified TWI map (Fig. 5b) indicated that 12.6% is highly sensitive; 44.3% moderately sensitive; 36.1% marginally sensitive and about 7% constraint to erosion. Impact of Gully on Erosion Gullies are large and deep erosion depression or channels that normally occur in drainage ways and not much deep (Imasuen et al., 2011). It occurs where surface water flow has become trapped in a small concentrated stream and begins to erode channels in the ground surface. To predict the sensitivity of a particular field to gully formation, threshold concept has been adopted. Stream power is the rate of energy of flowing water which is exerted on the bed and bank of a channel. Stream Power Index (SPI) is the product of watershed area and slope that indicates possible source of erosion by concentrated flow detachment risk (Equation 2). The potential locations of gullies are predicted based on SPI and TWI combination (Fig. 6a). Table 7. Topographic wetness index sensitivity class TWI Erosion sensittivity group Up to 11.5 S3 11.5 to 16.5 S2 1.5 to High S1 Gully formation follows stream route and the map clearly shows that small gullies (plot level) were not capture by the threshold. Gully locations were given high sensitive class (S1) while no gully location less sensitive class (S3). The sensitivity classes were used to reclassify gully map (Fig. 6b). Constrain map (Fig. 7) was prepared considering Koga reservoir. Value zero was given to the reservoir area to indicate permanently not suitable class. However, the sensitivity class for value 1 varies depending on the weight of other factors. Combined Effect of all Factors Pairwise comparison matrix was prepared by comparing factors one to one based on Pairwise comparison scale which is broken down from 1 to 9. The highest value indicates absolute important and the reciprocal kept in the transpose position indicating absolute insignificant (Table 8). For example in Table 8 topographic index (on the left) is much more important from soil (on the top), then a value of 7 were assigned at their intersection topographic index row and soil column. Conversely, soil (on the left) of Table 8 is much less important than topographic index (on the top) therefore the reciprocal was assigned (i.e., 1/7). Among the major factors, soil type was considered as the least important factor. The eigenvector was calculated for each factor (rows). According to Podvezko (2009), eigenvector is defined as the n th root of product of rows. The weights of factors were computed after normalizing the Eigen vector by its cumulative and multiplied by 100%. The reliabilities of weights were checked by computing the consistency of comparison matrix which was found consistent. The percent value of each factor was divided into four using equal interval method to assign value for each sensitivity classes (S1, S2, S3, S4). Each map was then summed up using raster calculator so that each pixel in that map had the sum of the four map values based on sensitivity classes (overlay analysis) (Fig. 8a). Finally, the total raster value was re-classified equally into four regions: Highly sensitive, moderately sensitive, marginally sensitive and currently insensitive (Fig. 8b). Zero value indicated constraint map. The erosion sensitivity map indicated that 2% of the total watershed is highly sensitive (S1); 43% is moderately sensitive (S2); 16% marginally sensitive; 32% currently not sensitive and 7% constraint to erosion (reservoir) in Koga watershed. The result also indicated that major source of erosion is the low land area of the catchment. This is due to the gentle slope of catchment and especially areas near to the dam are flat and the ground water table is near to the surface (saturated areas). Saturated areas are found to be high source of erosion since the rainfall after saturation could not infiltrate more leading to high surface runoff. When there is high concentrated surface runoff the power of the stream will be higher to detach sediment particles and transport downstream. Furthermore, when the catchment is saturated the water inside has hydrostatic pressure head and needs only small reach like flow path to widen and slide the earth surface. The upland part of the watershed is found to be the least source of erosion. This is due to negligible saturation at the upland of the watershed and there is also source limit in some cases due to degraded and rocky areas. Furthermore, most of the gully formation which significantly affects erosion initiation is related to saturation while gully formation is negligible at the highland part of the watershed. Hence, it is essential to consider the saturated areas (low land) during design of watershed management strategies and implementation to effectively prevent soil loss from the catchment. Conclusion and Recommendation Multi-Criteria Evaluation (MCE) technique integrated with GIS has the potential to identify erosion hotspot area. The results in combination with proper field validation provide more accurate erosion sensitivity prediction. The threshold values for assessing the potential location of gullies (stream power index and wetness index threshold) were found to be high and hence did not capture catchment level small gully formations and hence further research on threshold value to capture catchment level gully formation would improve the result. The result could also be improved using higher resolution DEM. The overall research indicated that most erosion hotspots areas were found in the lowland (more than 75% of erosion hotspot area) of the catchment, indicating that it is extremely important to consider the saturated areas during design of watershed management strategies.

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2019-04-02T13:06:40.593Z

2015-12-18T00:00:00.000Z

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Influence of Airborne and Seed Inoculum in the Initiation of Leaf, Stem, and Root Infection by Systemic Botrytis cinerea in Lettuce (Lactuca sativa) Lettuce Lactuca sativa (Asteraceae; Compositae) is one of the World’s most important salad crops and horticulturists regard it as one of the most profitable to grow [1]. Leaf, stem, root and seed infections caused by systemic B. cinerea are common in lettuce grown in the whole of the UK [2], Infection of lettuce plants by B. cinerea is favoured by cool moist weather conditions [3,4]. However, the levels of disease in crops can alter from year to year, with the cost of losses in winter lettuce estimated at £257 million due to B. cinerea infection alone in fungicide-treated crops [5]. Initial infection can result from infected lettuce stubble and/or crop debris on the soil [6,7] and most commonly occurs when successive lettuce crops have been grown in the same field. Symptom development in the fields with no previous record of lettuce cropping can arise from the sowing of infected seed or by infection from air-dispersed spores. Once infection is established in a crop, the infected lettuce plants show abundant white, cottony mycelial growth and hard, black sclerotic bodies on the underside of the lower leaves and the basal part of the stem [4]. The head and stem of infected plants often disintegrates into a soft watery mass covered with sclerotia [8]. Introduction Lettuce Lactuca sativa (Asteraceae; Compositae) is one of the World's most important salad crops and horticulturists regard it as one of the most profitable to grow [1]. Leaf, stem, root and seed infections caused by systemic B. cinerea are common in lettuce grown in the whole of the UK [2], Infection of lettuce plants by B. cinerea is favoured by cool moist weather conditions [3,4]. However, the levels of disease in crops can alter from year to year, with the cost of losses in winter lettuce estimated at £257 million due to B. cinerea infection alone in fungicide-treated crops [5]. Initial infection can result from infected lettuce stubble and/or crop debris on the soil [6,7] and most commonly occurs when successive lettuce crops have been grown in the same field. Symptom development in the fields with no previous record of lettuce cropping can arise from the sowing of infected seed or by infection from air-dispersed spores. Once infection is established in a crop, the infected lettuce plants show abundant white, cottony mycelial growth and hard, black sclerotic bodies on the underside of the lower leaves and the basal part of the stem [4]. The head and stem of infected plants often disintegrates into a soft watery mass covered with sclerotia [8]. It has been reported by [1,2,9] that B. cinerea infection was seedborne, and it is thought that seed infection could be extremely important for the spread of B. cinerea into new areas with no record of lettuce cultivation [10,11]. In another study Shafia [12] found that the efficacy of disease transmission on seeds was dependent on the rate of seed germination and temperature, [1,10,13] showed that root, stem, leaf infection could develop from symptomless seeds. The symptoms of B. cinerea infection generally occur and can be identified by the presence of dense gray or light brown airborne spores (ascospores or conidia), which may require high humidity and free water for infection to occur [8,14,15]. Recent work using DNA-based markers has confirmed that B. cinerea populations are highly genetically variable, with the majority of variation distributed within individual fields [16,17]. These findings agree with the results of Ma and Michailides, Martin [18] who reported moderate genetic variation within B. cinerea isolates collected from fig plants in the same location in California however, differences between different hosts. Similarly, this was similarly Newton [19] reported that population of B. cinerea from different host's species (Strawberry, dandelion, black current, primula) sampled from different locations of the UK were genetically variable and most importantly highly variable within host plants. The objective of this study therefore, was to determine the role of seeds and airborne inoculum in the initiation of B. cinerea infection in leaf, stem, root and seed infection of lettuce plant in a field experiment where crop debris was excluded as a potential inoculum source. Covering the crop until disease symptoms appeared in the remaining plots was to test the hypothesis that conidia and ascospore release from plant material in autumn and winter could initiate leaf and stem infection in lettuce plant. Field trial The area designated for the research has been cropped with lettuce for the last five years, having been fallow for the last two years. The Nearest lettuce stubble field was at least 0.8 km away and the plots were about 500 m from grassland. The whole field measured 33 × 14 m and contained 12 small plots (3 × 3 m). The lettuce plots were either sown with 'clean' seed from Fothergills Seeds, New market, UK or from 'infected' seed harvested in the previous experiment from untreated plots at School of Biological Science, Reading. The certified lettuce seeds supplied by Fothergills Seeds, New market had been assessed experimentally and were found to be free of disease symptoms, whereas most of the infected seeds showed symptoms of B. cinerea both on the surface and interior of the seeds. All seeds were sown without the use of seed treatments on 2 January 2010. Specially designed tents made from aluminium tubing and acetate clear plastics were placed in the centre of selected plots to exclude airborne inoculum [1]. By covering the crop, the effect of excluding conidia and airborne spores as an inoculum source, which is an issue in other closely related species such as light leaf spot of oilseed rape (Pyrenopeziza brassicae), could be tested in the period of the growing season when ascospores release is anticipated. The covers were later removed to determine whether the clean lettuce plants which had been excluded from airborne inoculum could subsequently become infected. The use of Arabidopsis plants between the plots was to help minimise the spread of inoculum from plot to plot via rain splash. The mini tents were secured in the soil to a depth of 250 mm, covering all the plants in the centre square metre of each plot in a 'miniature greenhouse'. All the tents were thoroughly and regularly checked and any hole noticed was repaired using parcel tape until the development of disease symptoms, there after all the tents were removed from the trial plots. None of the plots in the field experiment received any fungicide treatments throughout the growing season, however, all other inputs were made according to standard farm practice with a spring application of 167 kg ha one of ammoniacal nitrogen 18.4%, nitric N 11.6% and watersoluble sulphur (SO3) 19.0% (Sulphur Gold, TERRA Nitrogen UK Ltd). Field trial sampling and visual assessments Assessments in the field were carried out every week. The study used conventional visual plant pathological techniques, in combination with spore trapping and quantitative real-time PCR which was developed previously [20] to check for the development of disease symptoms, until the time that they first appeared in multiple plots, symptoms were visible in all uncovered plots sown with infected seeds. All tents were then removed and plants were sampled from each of the nine, 1 m 2 subplots, contained in all 12 of the main plots. The visual assessments for B. cinerea in leaves and stems were carried out by counting the number of lesions on whole plants, and the plant growth stages were also recorded for each subset of 10 plants sampled. After assessment of the disease, all the plants were discarded except for the 10 plant samples from the central 1 m 2 subplot. Plants from this subplot were then further dissected, were each of the plant was separated into roots, white stems (colourless and located below the surface of the soil), green stems (found above the surface of the soil) and leaves (10 of each for each plant section, from three replicates). These were then frozen for future DNA extraction and PCR testing. The same procedure was used for the samples taken on 24 April 2010. Also visual assessments was performed on 10 plants per plot each month throughout the growing season. Trapping of airborne inoculum A seven-day recording volumetric spore trap Burkard (Burkard Manufacturing Co. Ltd.) was set up in the central strip of the field immediately after the crop was sown Arabidopsis. A 3 m radius directly surrounding the spore trap was kept clear to decrease rain splashdispersed spores being sucked into the trap. The spores were collected on Melinex polyester film tape (Burkard Manufacturing) coated with a mixture of paraffin wax and petroleum jelly. The tape was replaced every seven days. For analysis, the tape was cut into 48 mm sections, each representing a 24 h period. DNA extraction and quantification of B. cinerea DNA using real-time PCR DNA was extracted from all sampled plant and fungal material using the method of [1,21] except that the DNA extraction buffer was amended with 5 mM 1,10-phenanthroline monohydrate and 2% (wt/ vol) polyvinylpyrrolidone K30 (Sigma-Aldrich Chemie GmbH) to clean the DNA [22]. The DNA was quantified using the fluorescent dye thiazole orange (Sigma-Aldrich) as described previously [21]. Spore tape DNA extractions were done using Ballotini beads (Jencons Ltd.) in a Fast Prep machine (Savant Instruments) as described by Calderon et al. [23]. DNA was extracted from half of each 24-h tape sample and 1 lL from 200 lL final DNA solution was used in each PCR assay. Quantitative real-time PCR measurements were carried out in a Strata gene Mx3000P real-time PCR machine (Stratagene, La Jolla, California, USA) with the cytochrome b Locked Nucleic Acid (LNA) probe assay recently developed [20]. Determination of B. cinerea seed infection and its transmission in controlled environment Seeds from the two seed batches used in the field experiment were visually assessed and tested by real-time PCR to establish differences in seed infection levels and to determine whether symptomless seeds were infected with B. cinerea. Five seeds with and without symptoms were randomly selected on the grain surface of each seed batch and analysed. For the controlled environment study, five hundred seeds from each seed batch were sampled. Each seed was thoroughly examined with a hand lens and were separated into seed lots with and without symptoms. Symptomless seeds from the 'clean' seed batch and seeds with symptoms from the infected' seed batch were sown into autoclaved compost (John Innes, No 2) in propagator trays (50 × 20 cm) and incubated at separate controlled environment cabinets (Fisons 600H). The light intensity used was 200 l Einsteinm)2 s)1 PAR. After germination, the seedlings in the trays were watered daily, so that the soil remained damp regardless of the incubation temperature. Seedlings were sampled at growth stage 8-9 (coleoptiles breaking through soil surface) and at 14-15 (4 or 5 leaves unfolded). The samples were collected on different sampling dates, as the growth of the seedlings differ. The basic aluminium frame of the tent manufactured from 2.5 cm diameter tubing. Ten seedlings were sampled at each growth stage using ethanol-cleaned forceps. Seedlings were dissected into lower root, seed, upper root and above soil stem/leaf sections and B. cinerea infection levels determined using real-time PCR. Microscopy was carried out according to the methods developed [1,19] to examine the plant material for fungal structures of B. cinerea. Data analysis The visual observations and quantitative real-time PCR data sets were transformed using natural logarithms, ln (x+0.5) for visual data and ln (x+0.1) for PCR data, to normalize the residuals from fitted models. Results was examined with a 2-way analysis of variance (ANOVA) using Genstat, 14th edition, (VSN International, Ltd), with plant, plot and block as random factors. The weather data for the 2009-2010 growing season were obtained from the Meteorological Database. Effect of seed source and covering of plants on leaf infection The first symptom of leaf blotch, a single lesion in one of the plots drilled with infected seed, was seen on 3 February 2010. The tents were removed two weeks later when all uncovered plots sown with farm- (Table 1). However, covering the crop significantly reduced the formation of symptoms; at the time the tents were removed, there was no symptoms in any of the covered areas, whether drilled with symptomless infected or uninfected seed. Disease was recorded in seedlings surrounding the tents in the same plots (data not shown). However, after removal of the tents, disease levels in these previously covered areas remained lower than in plots that had not been covered. While in the covered plots, disease levels in plots drilled with uninfected clean seed remained much lower than in those drilled with farm-saved infected seed, and the same effect was also seen for the plots that were not covered. Results obtained from the ANOVA on all plots found that tenting greatly reduced symptoms of B. cinerea (P<0.001) and plots sown with uninfected clean seed had less disease than plots sown with infected seed (P < 0.001). Statistically, the interaction was significant (P < 0.001) because the covered plants were free of symptoms regardless of seed source. The difference between the seed types and the effect of covering the crop with tents both remained highly significant (P<0.001) for up to two months after the removal of the tents. However, four months after removal of the tents disease severity levels were relatively closer. The main effect differences were highly significant (P=0.006 for seed source, P<0.001 for tenting) and the interaction was not significant (P=0.4). Detection of B. cinerea in plant samples using real-time PCR All plant materials roots, stem and leaves sampled from plots which were not covered, had detectable amounts of B. cinerea. DNA in realtime PCR throughout the trial. However, plant samples from uncovered plots sown with an infected seeds had higher levels of B. cinerea than those collected from uncovered plots sown with uninfected clean seeds. The roots sampled from uncovered plots sown with an infected seeds contained around 6 pg B. cinerea DNA, compared to around 0.6 pg for the uninfected seeds. The PCR results from the February samples showed that no B. cinerea DNA could be detected in samples from plots sown with either the infected seed (plots 4, 7 and 11) or symptomless uninfected seeds when grown undercover. Nearly all plant parts sampled from all plots contained B. cinerea DNA April 2010. The highest levels of B. cinerea DNA were mostly found in the leaves and decreased towards the roots. B. cinerea DNA levels were lower in plots sown with uninfected seed (P<0.001) and in covered plots (P<0.001). The interaction between seed and cover was significant, showing that covering prevented disease development even when the seed was heavily infected with B. cinerea (P<0.001). The differences in B. cinerea DNA levels between the different plant parts (roots, seeds, stems and leaves) were also significant (P<0.001) as was the interaction between plant parts and cover (P<0.001). Patterns and levels of significance were similar on both sampling dates ( Table 1). Detection of airborne inoculum of B. cinerea Botrytis cinerea DNA was not detected in spore tape samples collected between October and December. However, small daily amounts of B. cinerea DNA, <2 pg, were detected between January and July during the period of the growing season when leaf symptoms was visible. Detection of airborne inoculum was not linked with rainfall events (data not shown). Assuming a detection threshold of 0.1 pg of pathogen DNA in a 24 h air sample is equivalent to approximately 3 spores m)3 [21] B. cinerea spore concentrations between 30 and 60 spores m)3 was only detected on three occasions. Anything detected below the threshold of 0.1 pg of pathogen is regarded as zero and not within the scope of this research. Seed infection and transmission of disease in controlled environment Seeds with symptoms had approximately twice as much B. cinerea DNA as randomly-selected seeds from the same batch (Table 2). Most seeds without symptoms tested positive for presence of B. cinerea in real-time PCR. However, the average infection level of seeds sampled from the symptomless clean seed batch was approximately 10-fold lower than seeds sampled from the farm-saved infected seed batch, irrespective of the sampling method. Most parts of plants produced from symptomless uninfected seed tested negative for B. cinerea DNA at all stages of growth and temperatures tested (data not shown). As expected, more samples were positive for B. cinerea infection when infected seeds were sown 6). The highest levels of pathogen DNA was detected after growth at 16_C. At this temperature, DNA of B. cinerea was detected in all plant parts sampled at growth stage 8-9 and 14-15. None of the plants grown in the controlled environment showed any symptoms of leaf infection at growth stage 14-15. No spores or hyphal ⁄ mycelium structures resembling B. cinerea was seen in any of the samples examined by light microscopy. Discussion The emergence of the lettuce seedling under the covers was quicker by about one week because of the warmer climate and thus the plants grow at a faster rate throughout the growing season. On the day that the tents were removed (growth stage [23][24], there were clear effects of both covering and seed batches on the epidemic development of B. cinerea (Table 1). Plants in symptomless open plots sown with uninfected seeds had significantly less disease than open plots sown with infected seed. These results were supported by the real-time PCR data. All plant parts (roots, stems and leaves) sampled from plots sown with infected seeds showed higher levels of B. cinerea DNA than equivalent samples taken from plots sown with uninfected seeds. Detection of B. cinerea in all of the plant parts sampled indicates a systemic infection spreading from the seed. However, the greater infection levels were always found in infected uncovered seed and these caused greater disease levels throughout the growing season when compared to all the other seed types and conditions. Botrytis cinerea was initially not detected in plants sampled from covered subplots, irrespective of seed source for reasons which are unclear. Suppression of disease development in the covered crop may have been due to the differences in soil water potential which could have influenced rhizosphere bacterial communities involved in disease suppression. Another possible explanation may be the environmental conditions for the covered crop may not have been conducive for the fungal growth as it may have been too warm and dry, but ideal for greater plant growth enabling disease escape. Also Shafia [12] demonstrated that the rate of germination of a seedling could prevent the infection occurring at increased temperature higher than the optimal 16_C) by causing the coleoptiles to grow away more rapidly from the infected area of the seed. Winter lettuce is now drilled as early as possible in the UK, when the soil temperature will be higher and therefore this should reduce the effective transmission of B. cinerea from seed to seedlings. However, during this experiment no data for the environmental impact of the covers were taken so it is not possible to determine what factors caused the lack of both visual disease and positive PCR results for samples taken from under covers in February from both infected and uninfected seed. Four weeks after the removal of the tents, B. cinerea DNA was detected in plant samples from all plots and this was eventually followed by leaf gray mold symptoms (Table 1). This may be due to a delayed spread of inoculum from the seed following a change in the environment, because highest levels of pathogen DNA were still recorded for plant parts (including roots) sampled from plots sown with the infected seed. However, it is also possible that infection may have developed from the outside plots following the removal of the covers and these plants could have been more susceptible to symptom development due to their earlier 'softer' growth environment containing elevated temperatures and humidity and reduced amounts of other outside factors such as wind and frost. The development of a similar disease pattern following the removal of the covers is probably the results of conidia spreading across all the plots with the exception of the infected uncovered seed plots which has initially higher pathogen levels because of the role of seed infection detection threshold were detected in spore tape samples from the end of January until the end of July 2010 with the occasional higher level of up to 1.9 pg of pathogen DNA. This pattern is not typical of other ascospore-producing fungi present in other arable crops such as Mycosphaerella graminicola, Pyrenopeziza brassicae and Oculimacula yallundae, which all tend to produce ascospores more abundantly from senesced plants and ⁄ or stubble during late summer to early winter. Moreover, these pathogens also produce some spores during late spring to summer [1,23,24]. The period in which positive tape samples were detected coincided largely with the period that symptoms were visible on the leaves and not during late summer to early winter. Because of the low level of detection in comparison with other ascospore producing pathogens, it is likely that the B. cinerea DNA detected originated from asexual spores, rather than from ascospores. Therefore, it was suggested that sexual recombination events may be common in populations of B. cinerea, and this may therefore, be the major reason for its epidemiology and adaptation to the environment [17,25,26]. Asexual spores of B. cinerea are relatively small in comparison with asexual spores of other fungi and therefore during humid conditions, water droplets containing spores could have been sucked directly into the spore trap. It was not possible to identify ascospores of B. cinerea by examining the spore tapes by microscopy since the morphology of the teleomorph is unknown. In order to identify ascospores in positive samples, work now needs to be carried out to develop an immunofluorescence microscopy assay using specific antibodies or in situ PCR to positively identify the source of fungal material that is causing a positive result for spore tape DNA samples. In the controlled environment study however, no root, stem or leaf symptoms were seen throughout the experiment. But, B. cinerea DNA was detected in seedlings from both infected and uninfected seeds at all temperatures used. Out of the temperatures tested, 16_C was optimal for B. cinerea, confirming earlier work [12]. However, [11] using competitive PCR demonstrated that visual assessments poorly corroborate with levels of B. cinerea DNA from the same seed. Plant roots were colonized by B. cinerea in both the controlled environment and the field experiments. There was as previous report of B. cinerea infection on the root system, and affected plants had reduced root length [27]. In 2003 [28] isolated B. cinerea from the roots of Primula plant, and recent work [29] has suggested that soil borne inoculum and root infection could be important in the development of rice blast disease, caused by Magnaporthe grisea. This fungus had previously been regarded as a foliar pathogen however, [29] were able to show that root colonization can lead to systemic invasion and development of disease on aerial parts of the plant. Unfortunately, no infection structure of B. cinerea was seen by light microscopy. Understanding which stages/structures of B. cinerea are important for the infection process and its life cycle could be investigated by green fluorescent protein technology to visualize by confocal microscopy, fungal development in plant [30]. The role of seed infection as primary inoculum for leaf and stem gray mold disease of B. cinerea in winter lettuce crops in the UK has probably been over looked because of the long latency of the disease. It is clear that B. cinerea can be transmitted from seeds, but may produce no symptoms in the plant for several months (latent infection), and currently almost all of the commercial seed treatments used by growers are not active against B. cinerea infection contained in the seed. But changes in the physiological condition of the plant and⁄or environmental conditions may trigger disease development. Seed borne infection may also contribute to the genetic variation of B. cinerea populations as batches of seed are often combined and travel for long distances before being sown. In addition to using resistant lettuce varieties and foliar fungicide applications, the testing of seed batches for B. cinerea by PCR, with rejection of severely contaminated batches, or additional seed treatments with fungicides might improve disease control. In addition to this more work is needed to establish what levels of seed infection can be tolerated before rejection should occur as most of the clean symptomless seeds used in this study were later found to be infected. Seed batches can also be tested for the presence of alleles conferring resistance to fungicides. Mutations in cytochrome b and b-tubulin encoding genes, targets for the quinone outside inhibitors (QoI) and methyl benzimidazole carbamate (MBC) fungicides, respectively, have been identified in populations of B. cinerea [31]. Seed infection may also be a reason behind the introduction of populations of B. cinerea, resistant to compounds such as QoI fungicides. The quinone outside inhibitors fungicide resistance that was developed in France during the summer of 2008 [32] may pose a greater risk to the UK growers if seed is imported from an infected area. Therefore, on the final note routine monitoring of seed and other source of B. cinerea inoculum using PCR should become vital for the long term viability of lettuce cropping in the UK.

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