Table of Content

16 July 2018, Volume 51 Issue 14

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Scientia Agricultura Sinica. 2018, 51(14):  0. 

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Using CRISPR/Cas9-Mediated Targeted Mutagenesis of qSH1 Reduces the Seed Shattering in Rice

SHENG XiaBing, TAN YanNing, SUN ZhiZhong, YU Dong, WANG XueFeng, YUAN GuiLong, YUAN DingYang, DUAN MeiJuan

Scientia Agricultura Sinica. 2018, 51(14):  2631-2641.  doi:10.3864/j.issn.0578-1752.2018.14.001

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【Objective】Seed shattering is not good for seeds harvest，nor suitable for mechanized production of rice. In this study, using the latest molecular breeding method-CRISPR/Cas9 system to site-directed edit of the rice seed shattering gene qSH1 and evaluated the improvement effect, in order to lay materials foundation and explore new method for creating stable-yielding rice germplasm suitable for mechanized seed production. 【Method】The sgRNA was designed based on the principle of CRISPR/Cas9 technology. After excluding non-specific target sites by analysis of sequence alignment in the rice genome database, the target sites of qSH1-T1 and qSH1-T5 were selected. The oligonucleotides of sgRNA were chemically synthesized, and then inserted into plasmid pYLgRNA-U3, pYLgRNA-U6a for constructing the U3-qSH1T5-gRNA and U6a-qSH1T1-gRNA expression-boxes respectively. And the pYLCRISPR/cas9-qsh1-t51 expression vector was constructed by linking expression-boxes to plasmid pYLCRISPR/Cas9. The vector of pYLCRISPR/cas9-qsh1-t51 was introduced into the callus induced from mature seeds of an indica rice variety HR1128 by the Agrobacterium-mediated transformation. At T₀ generation, the positive transgenic plants were selected by hygromycin-resistant, and the target site for each plant were detected via a test of PCR combined Sanger-sequencing for confirming whether it was mutated. Besides blasting sgRNA sequence with rice genome in NCBI (https://blast.ncbi.nlm.nih.gov), Gramene (http://ensembl.gramene.org), highly identical sites with more 15 matching bases and NGG site at 3′region were selected to assess off-target efficiency and specificity of sgRNA. By using hpt gene and target site PCR amplification, mark-free qsh1 mutants were selected and qsh1 mutation lines were constructed by qsh1 mutants self-cross, and the level of rice seed shattering, the expression level of qSH1 and amino acid sequence of gene production of qsh1 lines were analyzed.【Result】The pYLCRISPR/cas9-qsh1-t51 expression vector successfully actualized gene-specific editing of qSH1. We obtained 7 mutants in T₀ transgenic generation and the mutation type ratios of qSH1-T1, qSH1-T5 were 54.55%, 63.64% respectively. Their genotypes included homozygous, heterozygous, biallelic and chimeric mutations. The mutation types were mainly insertions, deletions and substitution. After analysis of T₁ transgenic plants by hpt gene and target site PCR amplification, we obtained 2 mutant types which were homozygous and mark-free, and 2 T₂ generation qsh1 mutation lines (namely as 17SZ01, 17SZ02) were constructed further. We examined three putative off-target sites in the rice genome that showed the highly similarity with our target sites. And any off-target mutations at these sites in 46 plants of T₀ and T₁ generations were not observed, which indicated the sgRNA was highly specificity. Compared with the wild type, the 2 lines showed a significant decreased level of seed shattering (P<0.05). Furthermore, the amino acid sequence were changed of mutation lines, and the qSH1 expression level also decreased significantly (P<0.05) of the 17SZ01 mutation line. 【Conclusion】The strategy of site-directed mutagenesis qSH1 by CRISPR/Cas9 works effective for reducing the seed shattering in rice.

GmHMADP Involved in Seed Vigor Formation of Soybean Under High Temperature and Humidity Stress and its Study Responsive to Copper and Cadmium Stress

ZHU YaJing, ZHOU YaLi, LIU SuShuang, WEI JiaPing, LIU XiaoLin, SHEN YingZi, ZHAO HaiHong, MA Hao

Scientia Agricultura Sinica. 2018, 51(14):  2642-2654.  doi:10.3864/j.issn.0578-1752.2018.14.002

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【Objective】Abiotic stress such as high temperature and humidity (HTH), heavy metal stress are important factors that restrict soybean production and affect seed development and quality. In this study, the expression level and function of GmHMADP gene under heavy metal cadmium, copper and the HTH stress were analyzed, so as to provide a theoretical and practical basis for breeding new soybean resistant cultivars.【Method】Primer Premier 5.0 was used to design primers, and the full length cDNA sequence of GmHMADP was isolated by using the cDNA of leaves of cv. Ningzhen No.1 and Xiangdou No.3 as template. The homologous amino acid sequences of GmHMADP were searched by BLAST of NCBI website, and the amino acid sequence alignment were carried out by DNAMAN. Via constructing 35S::GmHMADP-GFP fusion expression vector with XhoⅠand SalⅠrestriction sites, subcellular localization of GmHMADP was studied by transient expression analysis of tobacco mesophyll cells. In addition, the tissue-specific expression of GmHMADP gene and the expression pattern of GmHMADP gene under HTH, and CuSO₄ and CdCl₂ treatment were analyzed, respectively, by qRT-PCR. Simultaneously, pBI121 with XbaⅠand BamHⅠ restriction enzymes were used to construct fusion expression vectors. The transgenic Arabidopsis plants with over-expressing GmHMADP gene were generated, three transgenic lines were obtained and three independent homozygous T₃ transgenic lines were used for analysis.【Result】The full-length cDNA sequence of GmHMADP gene was isolated, which contained a complete open reading frame of 996 bp. Besides, the GmHMADP gene contained 4 exons and 3 introns. Amino acid sequence analysis showed that GmHMADP contains two heavy metal binding core motif (CXXC). The subcellular localization result showed that GmHMADP protein was located on cell membrane and nucleus. The results of tissue-specific analysis showed that GmHMADP gene had the higher expression levels in developing and mature seeds of both the cultivars. Compared to the corresponding controls, the expression levels of GmHMADP gene were significantly (P＜0.01) increased in seeds of cv. Ningzhen No. 1 at the HTH stress time points 48, 96, and 168h,and the expression level of GmHMADP gene were significantly (P＜0.01) increased in seeds of cv. Xiangdou No. 3 at the HTH stress time points 24, 48, 96, and 168h. In addition, GmHMADP were significantly (P＜0.01) increased by Cu²⁺ and Cd²⁺ treatment with different concentrations in seedling roots of cv. Xiangdou No. 3, while in that of cv. Ningzhen No. 1, the expression level of GmHMADP gene wre significantly (P＜0.01) increased only under 50 μmol·L^-1 Cu²⁺ and 200 μmol·L^-1 Cd²⁺ treatment. The germination potential, germination rate, seed vigor of overexpressed Arabidopsis thaliana were significantly (P＜0.01) higher than that of the wild type plants under HTH stress. The root length and dry weight of transgenic Arabidopsis were significantly (P＜0.05) higher than that of the wild type undercopper and cadmium stress.【Conclusion】 The GmHMADP was involved in the formation of seed vigor under HTH and responded to copper and cadmium stress. Moreover, overexpression of GmHMADP in Arabidopsis could enhance the seed vigor and the tolerance to copper and cadmium stress.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY

Effects of Light Intensity and Light Quality on Morphological and Photosynthetic Characteristics of Soybean Seedlings

CHENG YaJiao, CHEN JunXu, WANG ZhongLin, FAN YuanFang, CHEN SiYu, LI ZeLin, LIU QinLin, LI ZhongChuan, YANG Feng, YANG WenYu

Scientia Agricultura Sinica. 2018, 51(14):  2655-2663.  doi:10.3864/j.issn.0578-1752.2018.14.003

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【Objective】In the maize-soybean intercropping system, the shading by the taller maize crop modifies the light environment experienced by the lower soybean crop in terms of both light intensity and light quality, while these changes were affected by the intercropping configuration and crop architecture. The objective of this study was to provide a theoretical basis of physiological response for light intensity and light quality. 【Method】 The variety of soybean Nandou 12 used as test material, the experiment was conducted in controlled growth chamber by pot method. In this experiment, four treatments were set up, including A1 (normal light + far red light), A2 (weak light + far red light), A3 (weak light), and CK (normal illumination). The morphological and physiological parameters of soybean seedlings were determined. 【Result】When soybean growth under the normal light and the low ratio of red to far red light, compared with CK, the soybean plant height, biomass and net photosynthetic rate of A1 treatment were increased. On the contrary, the root length, root surface area, root volume, carotenoid content and the actual photochemical quantum efficiency of A1 treatment were decreased significantly, but the photochemical quenching coefficient was not significantly different (P>0.05). Compared with A3 treatment, the A2 treatment soybean plant height decreased, stem diameter increased, biomass increased by 9.8%, and chlorophyll a and total chlorophyll content decreased. The chlorophyll b content decreased by 6.48%, the net photosynthetic rate, maximum photochemical quantum yield, non-photochemical quenching, effective photochemical quantum yield of A2 treatment increased significantly, but the root characteristics was not significantly different (P>0.05). Under the invariant light quality and reduced light intensity, compared with A2 treatment, the plant height of A1 treatment soybean decreased significantly, root length, root surface area and root volume increased significantly, biomass increased by 62.87%. The leaf biomass of A1 treatment accounted for 54.96% of the whole plant, while the stem biomass accounted for 52.08%, the chlorophyll a, b, carotenoids and total chlorophyll content increased, the net photosynthetic rate increased by 16.22% and the chlorophyll fluorescence parameters of A2 treatment were not difference (P>0.05). Compared with CK, the A3 treatment soybean plant height increased significantly, while stem diameter, root length, root surface area and root volume decreased significantly. Meanwhile, the biomass decreased significantly by 61.09%, but the biomass of stem was increased significantly under A3 treatment. The A3 treatment accumulation of chlorophyll a, chlorophyll b and carotenoid increased by 29.41%, 4.17% and 41.51%, respectively, the net photosynthetic rate and actual photochemical quantum efficiency decreased significantly (P<0.05). 【Conclusion】 The results showed that light intensity directly influences the soybean morphogenesis and biomass accumulation. The low ratio of red to far red light could promote the photosynthetic rate and the substance accumulation of soybean.

Effects of Silicon Application Rate on Common Buckwheat Lodging and Yield

SHE HengZhi, NIE Jiao, LI YingShuang, ZHANG YuKe, HUANG KeHui, ZHANG YuanLi, FANG XiaoMei, RUAN RenWu, YI ZeLin

Scientia Agricultura Sinica. 2018, 51(14):  2664-2674.  doi:10.3864/j.issn.0578-1752.2018.14.004

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【Objective】 This study was aimed to obtain the optimum silicon fertilizer application rate by studying lodging and yield related factors of common buckwheat (F. esculentum), so as to provide a theoretical basis for productive, stable yield and lodging resistance cultivation. 【Method】 The field experiments with five levels of silicon fertilizer treatments (SiO₂ application rates of CK, S1, S2, S3 and S4 were 0, 100, 200, 300 and 400 kg·hm^-2) were conducted in 2015 and 2016 at the Xiema Experimental Station, Southwest University, China. Ningqiao 1, a moderate lodging resistance cultivar of common buckwheat, was used in this study. The silicon content in root and culm, lodging and yield related factors were measured and analyzed. 【Result】 (1) The silicon content in root was more than that of culm; the silicon content in root increased and then decreased from full bloom to maturity stage, and the maximum value obtained at filling stage. The silicon content in root increased gradually with the increase of silicon application rate. The silicon content in culm increased from full bloom to maturity stage and it was increased and then decreased with the increase of silicon application rate. (2) The total root length, total root surface area, average root diameter and number of root tips increased gradually from full bloom stage to maturity stage. The total root surface area increased with the increase of silicon application rate. The average root diameter and number of root tips increased and then decreased with the increase of silicon application rate, and the maximum value obtained under the S2 treatment. (3) Length, diameter, fresh weight and breaking resistance of the base second internode of culm increased from full bloom to maturity stage. The length and diameter of the base second internode of culm increased with the increase of silicon application rate. The fresh weight and breaking resistance of the base second internode of culm increased and then decreased with the increase of silicon application rate, and the maximum value was obtained under the S3 treatment. (4) The lignin content increased from full bloom to maturity stage. 4CL, PAL, and CAD (4-coumarate: CoA ligase, phenylalanine ammonia-lyase and cinnamyl alcohol dehydrogenase) activity increased and then decreased from full bloom to maturity stage, and the maximum value obtained at filling stage. The lignin content and 4CL, PAL, CAD activity increased and then decreased with the increase of silicon fertilizer application rate, and the maximum value obtained under the S3 treatment. (5) Silicon fertilizer reduced the lodging degree and decreased the lodging percentage significantly. The lodging stage of common buckwheat occurred at maturity when silicon fertilizer application rate ranged from 300 to 400 kg·hm^-2. (6) The yield, grain weight per plant and thousand grain weight increased and then decreased with the increase of silicon fertilizer application rate and grain number per plant increased with the increase of silicon fertilizer application rate. 【Conclusion】 In this research, the best silicon application rate was 300 kg·hm^-2.

Effects of Drought in Post-Flowering on Leaf Water Potential, Photosynthetic Physiology, Seed Phenotype and Yield of Hulless Barley in Tibet Plateau

HOU WeiHai, WANG JianLin, HU Dan, FENG XiBo

Scientia Agricultura Sinica. 2018, 51(14):  2675-2688.  doi:10.3864/j.issn.0578-1752.2018.14.005

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【Objective】 Study on a drought stress response model of highland barley after anthesis in the plateau environment was established and a rapid, and effective detection method for the drought degree of highland barley was developed, so as to provide theoretical basis and technical reference for water-saving and high-yield cultivation of highland barley.【Method】The pot experiment of highland barley was carried out in drought shed at after anthesis, and light drought stress treatment (75% of controlled irrigation amount, LD), moderate drought stress treatment (50% of controlled irrigation amount, MD) and heavy drought stress treatment (25% of controlled irrigation amount, HD) were conducted for 13 days. WP4C PotentiaMeter, LI-6400XT and OS5P portable pulse modulated chlorophyll fluorometer were used to measure the leaf water potential (LWP), leaf evaporative cooling value (?T), photosynthetic gas exchange parameter and chlorophyll fluorescence variable, respectively. After maturity, plant laboratory test was conducted to obtain production data. Moreover, precise quantitative analysis of grain phenotypes of different treatments was conducted by digital image analysis method and grains were divided into three grain levels: large, medium and small according to the two-dimensional grain size.【Result】Drought level was positively correlated with leaf water potential and negatively correlated with leaf evaporative cooling value (?T) (P<0.05). Both of them were sensitive to reflect the drought degree of highland barley. Compared with CK, for LD, MD and HD treatments, the net photosynthetic rate of flag leaves (Pn), stomatal conductance (g_s), intercellular CO₂ concentration (Ci), transpiration rate (Tr), maximum fluorescence (Fm), maximum quantum efficiency of PS II (Fv/Fm), actual light quantum yield of PS II (ΦPSII), photochemical quenching (qP), and relative rate of photosynthetic electron transport (ETR) decreased due to drought stress, while stomatal limitation (Ls), original fluorescence (Fo) and non-photochemical quenching (NPQ) were on the rise. The effects of MD and HD on the above parameters were more significant compared with CK. The plant laboratory test data showed that with the increase of drought stress, the decreasing trend of 1000-grain weight, grain yield, grain weight per plant, dry matter accumulation, and economic coefficient of highland barley was more obvious. The correlation analysis showed that the decrease of g_s induced by drought stress directly led to the increase of ?T and indirectly led to the decrease of FWP, which resulted in the increase of Fo, NPQ and the decrease of Fm, Fv/Fm, ΦPSII, ETR, qP and Pn, thus causing the decrease of two-dimensional grain size, grain diameter, grain perimeter, grain length and grain width and the increase of grain roundness. The proportion of small grains increased obviously, while the proportion of large grains decreased obviously.【Conclusion】LWP and ?T were sensitive to the drought stress of highland barley after anthesis, which could be used as an effective method to evaluate the drought degree. With the aggravation of drought stress, the changes of photosynthetic and chlorophyll fluorescence parameters in flag leaves of highland barley increased, causing the gradual decrease in phenotypic characters and grain levels of five grains, thus resulting in the decrease of 1000-grain weight, grain weight per plant, grain yield, dry matter accumulation and economic coefficient.

PLANT PROTECTION

Targeted Mutagenesis of NbbZIP28 and Its Stress Response to Virus Infection in Nicotiana benthamiana

HE QingYun, LIU XiaoWei, JIAO YuBing, YU FangFei, SHEN LiLi, YANG JinGuang, WANG FengLong

Scientia Agricultura Sinica. 2018, 51(14):  2689-2699.  doi:10.3864/j.issn.0578-1752.2018.14.006

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【Objective】 bZIP transcriptional factors are involved in plant developing, hormonal signal, disease resistance, and abiotic stress responsiveness. Previous study showed that Tobacco mosaic virus (TMV) or Cucumber mosaic virus (CMV) infection up-regulated the endoplasmic reticulum stress (ERs) gene NbbZIP28, and knock-down of NbbZIP28 increased more susceptibility to virus than wide type (wt). The mechanism of its stress response to virus infection still needs to be verified. 【Method】NbbZIP28 mutant plants were obtained by CRISPR/Cas9 gene editing technique and tobacco genetic transfection. The susceptibility of mutations to virus infection and the ER stress related UPR genes expression in mutations were compared to that in wide type by qRT-PCR. Then the fusion protein of NbbZIP28-GFP was expressed transiently in leaves of Nicotiana benthamiana in advance of inoculation with TMV 24 h or CMV 48 h, and its hydrolyzation induced by viral infection was checked by Western blot. The Cis-acting elements involved in defense and stress response in promoter region of NbbZIP28 were analyzed by PlantCARE. 【Result】The DNA deletion of 10 bp had been found in the target sites of NbbZIP28, leading to translation error and protein function changed. Under normal conditions, mutant plants showed no different phenotype from wt. Upon inoculation TMV-GFP, mutant plants showed more virus infected spots and faster expansion than WT. Upon inoculation CMV, the UPR related genes (BiP, PDI, CAM and NF-YC2) showed lower expression in mutants than that in wt plants at 12-48 hours post inoculation. in addition, mutant plants showed more virus accumulation than wt at 5-7 days, the symptoms of mosaic and shrinkage were more significant. protein sequence alignment analysis showed that NbbZIP28 had the same hydrolytic site of S1P and S2P as AbZIP28. Western blot detection found that TMV or CMV infection significantly promoted the full length of NbbZIP28-GFP to be cleaved at sites of S1P and S2P than inoculation water. There are five cis-acting elements involved in heat stress responsiveness (HSE), three cis-acting elements involved in low-temperature responsiveness (LTR) and one cis-acting element involved in defense and stress responsiveness (TC-rich repeats).【Conclusion】 Virus infection activated NbbZIP28 and up-regulated its related UPR genes. NbbZIP28 knock-out increased the susceptibility to virus than wt, and virus induced UPR gene expression is suppressed in mutant plants. NbbZIP28 is the regulating factor of UPR under virus infection stress and plays a role in suppression viral infection at early stage through up-regulating UPR signal and increasing plants basic defense response.

Monitoring of Resistance of Phytophthora infestans on Potato to Metalaxyl and the Control Efficacy of Alternative Fungicides

LU Fen, ZHAO JianJiang, LIU XiaoYun, MENG RunJie, WU Jie, HAN XiuYing, WANG WenQiao

Scientia Agricultura Sinica. 2018, 51(14):  2700-2710.  doi:10.3864/j.issn.0578-1752.2018.14.007

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【Objective】 The objective of this study is to clarify the dynamics of resistance to phenylamide fungicide metalaxyl of Phytophthora infestans from potato of Hebei Province, Inner Mongolia Autonomous Region and Jilin Province, screen for highly effective fungicides that can replace metalaxyl, assess the control efficacy against potato late blight of the alternate application of these alternative fungicides, and to provide the clues for control of potato late blight in the regions where P. infestans developed severe resistance to metalaxyl. 【Method】 A total of 634 single cystospore strains of P. infestans were isolated from major production areas of potato of Hebei, Inner Mongolia and Jilin. The sensitivity of 634 P. infestans strains to metalaxyl was determined by the mycelial growth inhibition test. The highly effective fungicides that can replace metalaxyl were screened through the control efficacy trials of fungicides against potato late blight in the fields, the control efficacy of alternate application of alternative fungicides against potato late blight in the area where metalaxy-resistant strains prevailed in P. infestans population was evaluated in the field trials.【Result】P. infestans widely developed resistance to metalaxyl in Hebei, Inner Mongolia and Jilin, the frequency of metalaxyl-resistant strains was up to 100% among 634 P. infestans strains collected from 2011-2016. The average resistance factor and resistance index to metalaxyl was 34 934 and 0.97, respectively. Moderately resistant or highly resistant strains predominated in all strains collected from 2011-2012, whereas the highly resistant strains predominated in all strains collected from 2013-2016. The average resistance factor of the strains from Jilin and Inner Mongolia was higher than that of the strains from Hebei. Resistance index of the strains collected from Jilin and Inner Mongolia in 2011-2012 was higher than resistance index of the strains collected from Hebei in 2011-2012. Resistance index of the strains collected from Hebei, Jilin and Inner Mongolia in 2013-2016 reached the maximum 1.00. In the field trials on control efficacy of fungicides applied 4 times at the recommended dosages, 25% metalaxyl SC, 68% mefenoxam·mancozeb WG, and 64% oxadixyl·mancozeb WP provided the efficacies of 37.0%, 67.5% and 50.9-65.8%, respectively, significantly less effective than 80% mancozeb WP (72.8%) in control of potato late blight. Whereas spray of 10% oxathiapiprolin OD, 50% LH-2010A WG, 50% dimethomorph WP, 687.5 g·L^-1 fluopicolide·propamocab hydrochloride SC, 16% oxathiapiprolin·azoxystrobin SC, or 26% oxathiapiprolin·mandipropamid SC gave good control efficacies (83.9%-90.3%) against potato late blight, undertaking of two alternate application programs consisting of 5 or 6 sprays of the highly effective fungicides (such as 10% oxathiapiprolin OD, 50% LH-2010A WG, 50% dimethomorph WP, 687.5 g·L^-1 fluopicolide·propamocab hydrochloride SC) with mancozeb, azoxystrobin, and flumorph provided good control efficacy of 88.3%-90.5%, and the yield in the districts of rotation application of the highly effective fungicides increased by 64.4%-78.3%, compared with the yield in the districts of rotation application of conventional fungicides (such as mancozeb, phenylamide fungicides and cymoxanil·mancozeb). 【Conclusion】 P. infestans widely developed resistance to metalaxyl in Hebei, Inner Mongolia and Jilin, which led to significant decrease of effectiveness of phenylamide fungicides including metalaxyl·mancozeb, mefenoxam·mancozeb, and oxadixyl·mancozeb. Spray use of the mixed fungicides containing metalaxyl, oxadixyl, or menfenoxam should be suspended in the regions where metalaxyl-resistant strains prevailed in P. infestans population. The fungicides including 10% oxathiapiprolin OD, 50% LH-2010A WG, 50% dimethomorph WP, 687.5 g·L^-1 fluopicolide·propamocab hydrochloride SC, 16% oxathiapiprolin·azoxystrobin SC, or 26% oxathiapiprolin·mandipropamid SC could replace metalaxyl. The contact fungicides with broad spectrum including mancozeb could be used in alternation with the effective systemic fungicides including azoxystrobin, dimethomorph, flumorph, oxathiapiprolin, and fluopicolide·propamocab hydrochloride for highly effective control of potato late blight.

Sensitivity of Sclerotinia sclerotiorum to Fluopyram and Evaluation of Its Application Potential in Controlling Sclerotinia Stem Rot

HUANG XuePing, SONG YuFei, LUO Jian, ZHAO ShiFeng, MU Wei, LIU Feng

Scientia Agricultura Sinica. 2018, 51(14):  2711-2718.  doi:10.3864/j.issn.0578-1752.2018.14.008

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【Objective】 The objective of this study is to evaluate the sensitivity of Sclerotinia sclerotiorum to fluopyram, a new succinic dehydrogenase inhibitor (SDHI), determine the virulence of fluopyram against S. sclerotiorum at different developmental stages, and the action mode and efficacy of fluopyram in managing Sclerotinia stem rot, and to guide the scientific application of fluopyram. 【Method】 A total of 173 S. sclerotiorum isolates were collected from 6 vegetables (including cucumber, tomato, eggplant, pumpkin, kidney bean and pepper). The sampling sites located in Changle, Shouguang, Qingzhou, Linqu and Taian of Shandong Province. The sensitivity of 173 S. sclerotiorum isolates to fluopyram was measured by mycelial growth rate method. The inhibitory activity of fluopyram on sclerotial production (number and dry weight), shape and myceliogenic germination of sclerotia was determined in laboratory. The action mode of fluopyram against S. sclerotiorum was tested on detached eggplant leaves. Field trials in two years were performed to evaluate the practical efficacy of fluopyram in controlling Sclerotinia stem rot. 【Result】Fluopyram had strong inhibitory activity on the mycelium growth of S. sclerotiorum and had little difference in EC₅₀ values against 173 S. sclerotiorum isolates, with the ranging from 0.02 to 0.30 μg·mL^-1, which suggested that these isolates could be used to analyze the sensitivity level of S. sclerotiorum to fluopyram. The frequency distribution of EC₅₀ showed a single peak skew normal curve, and the variation coefficient was small, indicating that the S. sclerotiorum isolates on vegetable in Shandong Province were sensitive to fluopyram. Additionally, fluopyram had high bioactivity in suppressing sclerotial production, sclerotial morphology and myceliogenic germination of sclerotia. After treated with fluopyram at 1.6 μg·mL^-1, the number and dry weight of sclerotia from three isolates significantly decreased, and the sclerotia became smaller, indicating that the fungicide could reduce the number of primary source of infection and suppress the activity of infection of this pathogen. After 3 consecutive days of observation, the inhibition rate of myceliogenic germination of sclerotia treated with fluopyram at 5 μg·mL^-1 was all above 95%, indicating that fluopyram could inhibit the infection approach of the fungus so as to protect the basal part of stem from infection. The inoculation test on the detached leaves of eggplant showed that fluopyram had protective and curative activities. Spray applications of fluopyram at 40 μg·mL^-1 provided protective efficacy of 100.00% and curative efficacy of 88.81%, respectively, which was significantly higher than that of carbendazim and dimethachlon at 40 μg·mL^-1. In addition, the protective efficacy of fluopyram was better than the curative efficacy, which suggested that fluopyram should be sprayed preventively or at the very early stage of disease occurrence to get a higher efficacy. In the field trials conducted in 2016 and 2017 under natural infection conditions, the control efficacy of 90.30% and 87.60% against Sclerotinia stem rot were given by application of fluopyram under the rate of 200 g a.i./hm², which was significantly higher than that of other treatments. 【Conclusion】 The novel SDHI fungicide fluopyram has high bioactivity in suppressing mycelial growth, sclerotial production, sclerotial morphology and myceliogenic germination of sclerotia and can effectively control the occurrence of Sclerotinia stem rot in the field, which means that the fungicide is an effective agent for preventing and controlling Sclerotinia stem rot, and can be served as an alternative fungicide to control the disease.

SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT

Guidance of Special Focus: Nutrient Management of Grain Crops and Green Agricultural Development

WANG ZhaoHui

Scientia Agricultura Sinica. 2018, 51(14):  2719-2721.  doi:10.3864/j.issn.0578-1752.2018.14.009

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Relationships of N, P and K Requirement to Wheat Grain Yield of Farmers in Major Wheat Production Regions of China

HUANG QianNan, WANG ZhaoHui, HUANG TingMiao, HOU SaiBin, ZHANG Xiang, MA QingXia, ZHANG XinXin

Scientia Agricultura Sinica. 2018, 51(14):  2722-2734.  doi:10.3864/j.issn.0578-1752.2018.14.010

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【Objective】It is of great significance to clarify relationships of NPK requirement to farmers’ wheat grain yield for reasonable fertilization and decreasing fertilizer application rates.【Method】A 2 years long on-farm survey combined with sample collection and analysis related to wheat production were carried out in the Spring Wheat (SW), Dryland Wheat (DW), Wheat-Maize (WM), Rice-Wheat regions (RW) of China, to study the relationships of NPK requirement to wheat grain yield of farmers.【Result】The mean grain yield of wheat was observed to be 6.4 t·hm^-2, and the differences were significant among the yield averages of different regions, with the grain yield average of 6.0, 4.0, 7.7, 5.5 t·hm^-2 in SW, DW, WM and RW region, respectively. The high yield regions usually had higher above-ground biomass and spike numbers, and harvest indexes increased with grain yields. The average N requirement was 28.1 kg·Mg^-1, with the average of 28.6, 28.3, 29.3 and 25.0 kg·Mg^-1 in SW, DW, WM and RW region, respectively, and the N requirement average decreased significantly by16.9% and 16.4% in DW and WM, tending to decrease but being not significant in SW and RW regions, when the yield was increased from the very low to the very high levels. The average P requirement was 4.0 kg·Mg^-1, with the average of 4.5, 3.2, 4.1 and 4.1 kg·Mg^-1 in SW, DW, WM and RW region, respectively. P requirement average decreased significantly by 11.4% and 17.8% in WM and RW, decreased by 8.6% but not significantin DW region, when the yield was increased from the very low to the very high levels, and even though the lowest P requirement of 3.7 kg·Mg^-1 occurred at the very low wheat yield level, it was still significantly decreased by 21.4%, when the yield was increased from the lower to the very high level. The average K requirement was 21.5 kg·Mg^-1, with the average of 26.5, 17.1, 23.3 and 18.8 kg·Mg^-1 respectively and significantly different in the four regions, and the K requirement average significantly decreased by 4.0%, 4.4%, 12.7% and 19.9% in SW, DW, WM and RW region, respectively, when the yield was increased from the very low to the very high levels, although the difference was only significant in the RW region. 【Conclusion】Wheat grain yield of farmers were significantly different among the main wheat production regions in China, relationships of NPK requirements to grain yields were also different with regions, and generally they tended to decrease with the grain yield increase. Therefore, fertilizer application recommendation should determine the reasonable nutrient requirement based on the yield levels, the crop nutrient requirement characteristics and the land soil nutrient supply capacities of farmers, in order to avoid over or insufficient fertilizer application in different regions of China.

Analysis on Yield Increasing and Nitrogen Efficiency Enhancing of Winter Wheat Under Film Mulching Cultivation in the Loess Plateau

LI TingLiang, XIE YingHe, GAO ZhiQiang, HONG JianPing, MENG LiXia, MA HongMei, MENG HuiSheng, JIA JunXiang

Scientia Agricultura Sinica. 2018, 51(14):  2735-2746.  doi:10.3864/j.issn.0578-1752.2018.14.011

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【Objective】The objective of the study was to determine the effects of film mulching cultivation on yield formation and nitrogen uptake-translocation of winter wheat in dry highland of Loess Plateau, so as to provide important basis for high yield and high efficiency in rainfed winter wheat production.【Method】An experiment was conducted to study the effects of interactions between different fertilization and film mulching practices on yield formation of winter wheat, aboveground nitrogen accumulation and translocation, soil nitrate-N residue and N balance in soil-plant system by four cultivation patterns, including farmer pattern (PF), farmer fertilization rate plus ridge mulching-furrow planting (RFSF1), monitoring fertilization plus ridge mulching-furrow planting (RFSF2) and monitoring fertilization plus whole field filming with soil covering and hole-seeding (WFFHS), during 2012-2016 in winter wheat growing region of dry highland in Southern Shanxi. 【Result】 Obtained results showed that average yield of winter wheat under traditional farmer pattern was 3 367 kg·hm^-2 , which could be increased to 4 491 kg·hm^-2 by optimized fertilization combined with mulching cultivation. The contribution rate of optimized fertilization on yield was 14.8%, and the synergy contribution rate of mulching and optimized fertilization was up to 24.7%-42.1%. The yield formation mainly depended on spike number per hectare, followed by thousand kernel weight in dry high land of Loess Plateau. Due to the reasonable population construction and favorable water and nutrient conditions, the spike number per hectare, 1000-grain weight and yield of WFFHS treatment were the highest, with the mean value of 5.81million·hm^-2, 44.3 g and 4 785 kg·hm^-2, respectively. In terms of aboveground nitrogen translocation, a significant positive correlation was observed between nitrogen translocation amount from vegetative organs to grains after anthesis and biomass or grain yield, the correlation coefficients were 0.959** and 0.960**, respectively. About three quarters of N in grain were from translocation of vegetative organs before anthesis, and a quarter from root uptake after anthesis under the PF treatment, optimized fertilization combined with mulching cultivation remarkably increased the nitrogen translocation amount from vegetative organs to grains, the translocation contribution rate was 81.4%-88.8%. In terms of soil nitrate-N residue, the accumulation amounts of nitrate nitrogen of 1 m soil layer had been over 100 kg·hm^-2 on account of long-term excessive fertilization in Loess Plateau, and the peak of nitrate nitrogen accumulation was 20-60 cm layer. After successive planting of winter wheat for 4 years, the accumulation amounts of nitrate nitrogen in 2 m soil layer under traditional fertilization rate had been up to 277 kg·hm^-2, of which 75% concentrated on 0-120 cm soil layer, with a 87.7% increase since pre-sowing of 2012. However, the amounts of nitrate nitrogen accumulation in 2 m soil layer under optimized fertilization combined with mulching cultivation was only 15.7%-24.2% higher than that at pre-sowing in 2012. Meanwhile, it was found that nitrate nitrogen accumulation in 120-200 cm at harvest stage in 2016 was 10.2%-133.7% higher than that at pre-sowing in 2012, which indicated that the residual nitrate nitrogen had a strong leaching downward trend. With considering of aftereffect of residual N, the soil N balance for 4 years was overall analyzed, and it was observed that the N utilization efficiency was 28.8%-56.7%, the N apparent residual rate was 12.1%-28.9%, and the N apparent loss rate was 31.2%-49.6% in dry highland of Loess Plateau. Optimized fertilization combined with mulching cultivation could reduce the apparent nitrogen loss and the residual Nmin, increase the apparent mineralized nitrogen. The WFFHS treatment could utilize the residual Nmin and mineralized nitrogen in a more great extent, which accumulated in the previous years, so the WFFHS treatment had lowest nitrogen apparent loss rate (31.2%) and N apparent residual rate (12.1%), highest N utilization efficiency (56.7%) among all the treatments.【Conclusion】it was concluded that whole field filming with soil covering and hole-seeding combined with monitoring fertilization cultivation could further improve the soil water and fertilizer condition, utilize the residual Nmin, increase the aboveground nitrogen accumulation and translocation, construct reasonable population, and obtain the remarkable yield increasing effect and higher N utilization efficiency finally. So it was considered as a recommendable cultivation mode in dryland of Loess Plateau.

Nitrogen Requirement and Saving Potential for Wheat and Maize in Henan Province

ZHAO YaNan, XU Xia, HUANG YuFang, SUN XiaoMei, YE YouLiang

Scientia Agricultura Sinica. 2018, 51(14):  2747-2757.  doi:10.3864/j.issn.0578-1752.2018.14.012

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【Objective】 Nitrogen (N) demand and saving potential under recommendation fertilization for the wheat and maize in Henan province were estimated to provide the basis for the policy of zero growth of chemical fertilizer in China.【Method】Based on the statistical data and the field trials, this paper studied the production and N consumption, estimated the N demand and furthermore the saving potential for the wheat and maize production of the whole province under the rational N fertilizer rate according to methods of fertilizer response mode, aboveground N accumulation and N requirements per 1000 kg grain yield.【Result】The N consumption for wheat and maize in Henan province was increased continuously, with the single N fertilizer consumption declining while the N fertilizer in compound and mixed fertilizer increasing. In 2015, the total N consumption for wheat and maize in the total province were 133.0×10⁴ t and 60.9×10⁴ t, respectively. The aboveground N accumulation for wheat and maize were averagely 209.4 and 183.7 kg·hm^-2, respectively, and N requirement per 1 000 kg grain were 29.1 and 23.0 kg, respectively. The regional mean optimal N fertilizer rate for wheat and maize were 171.0 and 202.5 kg·hm^-2 under the highest yield while 155.1 and 172.8 kg·hm^-2 under the highest economic profit, respectively. The N demand for wheat was 57.8×10⁴-67.7×10⁴ t, with N saving potential of 21.8×10⁴-48.8×10⁴ t or 16.4%-36.7% in Henan province; while the N demand for maize was 42.7×10⁴-67.7×10⁴ t, with the highest N saving potential 18.2×10⁴ t or 30.0%. 【Conclusion】The N consumption and demand for wheat and maize in Henan province were increased continuously, however, the actual N consumption exceeded the demand, representing a great potential for fertilizer saving under rational N fertilization in further.

The Ways to Reduce Chemical Fertilizer Input and Increase Fertilizer Use Efficiency in Maize in Northeast China

MI GuoHua, WU DaLi, CHEN YanLing, XIA TingTing, FENG GuoZhong, LI Qian, SHI DongFeng1, SU XiaoPo, GAO Qiang

Scientia Agricultura Sinica. 2018, 51(14):  2758-2770.  doi:10.3864/j.issn.0578-1752.2018.14.013

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To enhance the competitiveness of maize production and to protect the environment, it is necessary to reduce fertilizer input and to increase fertilizer use efficiency in China. Based on maize production in Northeast China, the ways to reduce fertilizer input and to increase fertilizer use efficiency are discussed from the viewpoints of the characteristics of maize nutrient requirements, the fertilizer-saving potential of nutrient-efficient cultivars, the 4R techniques for efficient fertilizer input and the alternative approaches by organic matter applications. To get 100 kg grain yield, the requirement for N, P₂O₅, K₂O in the plant was 1.56-1.89, 0.60-0.88, and 1.27-2.3 kg, respectively. Postsilking N and P uptake is 20%-30% and 20%-40% respectively, which contributes to the grain N and the grain P by 20%-30% and 30%-38%, respectively. At the current soil productivity condition, the N fertilizer requirement for a maize grain yield level of 12 000 kg·hm^-2 is around 180 kg·hm^-2. The application of various new-type fertilizer may save N fertilizer by 9-25 kg·hm^-2. Maize seedling growth can be improved by the application of starter fertilizer Diammonium phosphate and Ammonium sulphate + Calcium superphosphate. A high-clearance fertilizer applicator is required to apply fertilizer at later growth stage so as to match nutrient supply with plant nutrient demand. Fertigation techniques can be used in the sandy soil and/or under drought climate to increase maize yield by 19%-128%, with the highest yield level of 12 000-13 000 kg·hm^-2. Subsurface fertigation has similar effect as the surface fertigation and had a great potential for application. Greenseeker technique can be used to accurately estimate leaf area index, above-ground biomass, and N uptake at maize V5-V8 stage, and therefore to realize precision and variable recommendation of in-season N fertilizer input. Based on the climate and soil conditions in northeast China, various methods can be used to return maize straw residuals to the field so that fertilizer input can be reduced and soil quality is improved. Strip-till technique is expected to be applied greatly in this area because it combines both the advantages of traditional cultivation and no-till. The research in the future should take into account the farmers’ requirement and combines technique development with extension. The researchers should develop applicable technique protocols which target the specific cultivation and/or planting patterns and can be used in a large area now or in the future, so as to reduce fertilizer input and increase fertilizer efficiency in a regional level.

HORTICULTURE

Development of SNP Markers in Cabbage and Construction of DNA Fingerprinting of Main Varieties

LI ZhiYuan, YU HaiLong, FANG ZhiYuan, YANG LiMei, LIU YuMei, ZHUANG Mu, Lü HongHao, ZHANG YangYong

Scientia Agricultura Sinica. 2018, 51(14):  2771-2787.  doi:10.3864/j.issn.0578-1752.2018.14.014

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【Objective】The main cabbage varieties in production were collected and DNA fingerprinting of cabbage varieties were constructed with SNP markers to provide reference for variety distinctness and authenticity identification. 【Method】SNP loci were obtained by aligning resequencing data of 50 cabbage inbred lines to the reference genome of cabbage (02-12). The SNP loci were screened with two criteria: (i) high polymorphism information content (PIC), (ii) evenly distribution on each chromosome. And then KASP primers were designed based on these loci. The KASP platform was used to genotype the fifty-nine cabbage varieties. According to the result of genotyping, core primers were selected with high PIC value, no loci missing and even distribution on each chromosome. These core primers were used to establish SNP fingerprinting of fifty-nine cabbage varieties. Fifteen varieties randomly selected from the fifty-nine main varieties and five new unreleased combinations were mixed together to construct an artificial population. The artificially mixed population was used to validate the core primers in the variety distinctness and authenticity identification. 【Result】Two million five hundred and forty thousand SNP loci were obtained by aligning the resequencing data of fifty cabbage inbred lines to the reference genome of cabbage. Five hundred SNP markers were selected with high PIC value, low missing rates and even distribution on each chromosome, with 55.6 loci per chromosome. 442 of them were successfully transformed into KASP markers, occupying 88.4% of all the 500 SNP markers. The genotyping results showed that twenty-five pairs of KASP primers were unsuccessfully genotyped in more than five materials and were removed from further analysis. The PIC values among the 417 primers ranged from 0.12 to 0.38. The average PIC value was 0.36, showing moderate polymorphism. The number of varieties with heterozygous marker ratio greater than 30% was 57, occupying 88.4% of all the main varieties. The heterozygous marker ratio of ‘Yusheng Zaoshu Niuxin’ was the highest, which reaches 67.8%. Finally, fifty core markers were selected to construct DNA fingerprinting of 59 main varieties. PIC values of 50 core makers ranged from 0.35 to 0.38, and the average PIC was 0.36. The result of cluster analysis of core SNP markers indicated that the genetic similarity coefficient of 59 varieties varied from 0.43 to 0.98. The core SNP markers were validated by artificially mixed population and the results showed that SNP fingerprinting constructed with core markers could be used to identify the variety distinctness and authenticity effectively. 【Conclusion】Two million five hundred and forty thousand SNP loci were obtained by aligning resequencing data of cabbage inbred lines. Fifty pairs of core makers were selected and used to establish DNA fingerprint database of 59 cabbage varieties, and the core SNP markers were validated by artificially mixed population.

Effects of All-Organic Nutrient Solution and Water Coupling on Quality, Yield and Water Use Efficiency of Tomato

ZHANG JunHeng, MA LeLe, LI JianMing

Scientia Agricultura Sinica. 2018, 51(14):  2788-2798.  doi:10.3864/j.issn.0578-1752.2018.14.015

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【Objective】Under the organic substrate cultivation, the research on organic nutrient solution coupling with water is the important way to actualize the organic cultivation. 【Method】The cherry tomato was used as the test material, and the organic substrate in bag was selected as the cultivation method. In contrast to Japanese garden general formula (F3), 2 organic nutrient solutions (F1: Organic nutrient solution formula 1; F2: Organic nutrient solution formula 2) were set, and 2 irrigation amount levels (W1: 100% ET at seedling period and rainy days, 120% ET at other times; W2: 120% ET at seedling period and rainy days, 150% ET at other times) were set. 【Result】The results indicated that two organic nutrient solutions significantly increased the photosynthetic rate, stomatal conductance and transpiration rate of tomato leaves, and reduced the nitrate content in fruits, whereas the contents of soluble protein, soluble sugar and lycopene were improved, comparing with the inorganic nutrient solution. Meanwhile, with the increase of irrigation amount, the photosynthetic rate, stomatal conductance and transpiration rate of tomato leaves increased significantly, and the yield of tomato increased significantly too, while the fruit quality indexes showed the dilution effect. The comprehensive quality evaluation showed that F2W1 treatment was the best. Among different fertilizer and water treatments, F2W2 treatment had the highest yield, which was significantly higher than those under other treatments, except for the insignificant difference compared with F1W2 treatment. F2W1 treatment had the highest water use efficiency. 【Conclusion】Generally, F2W1 (Organic nutrient solution formula 2 with 100% ET at seedling period and rainy days, 120% ET at other times) was considered as the optimal fertilizer and water coupling treatment, which could be used as the all-organic nutrient solution and water management indicator.

FOOD SCIENCE AND ENGINEERING

Effects of Different Concentrations of Apple Polyphenols on the Physicochemical Properties of Chitosan Membrane Fluids

LIANG Di, YANG Xi, GUO YuRong

Scientia Agricultura Sinica. 2018, 51(14):  2799-2813.  doi:10.3864/j.issn.0578-1752.2018.14.016

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【Objective】The composite membrane liquids based on chitosan (5%, w/v) and varying apple polyphenols concentrations were developed in this work, and the composite membrane liquids with different polyphenol concentrations were fully investigated including their antioxidant activity, rheological properties and thermal stability. This result could provide theoretical basis for food preservation. 【Method】 The composite membrane liquids were prepared by adding different amounts of apple polyphenols (0.5%, 1.0% and 1.5%, respectively). Besides, effects of the different addition amounts of apple polyphenols on the antioxidant activity of the composite membrane fluids were measured. A controlled-strain rheometer was used to investigate static rheological properties of the composite membrane liquids, including the relationships between G′ and G″. The potential interactions between chitosan and apple polyphenols and the crystal structure of the composite membrane were characterized by Fourier transform infrared spectrum (FTIR) and X-ray diffraction, respectively. In addition, thermogravimetric (TG) and derivative thermogravimetric (DTG) analyses were conducted by using a thermal analysis equipment to characterize the thermal stability of the lyophilized composite membrane samples.【Result】The addition of apple polyphenols could significantly improve the antioxidant activities of the chitosan membrane liquid (P<0.01). The increase of apple polyphenols concentrations could lead to the increased antioxidant ability of the composite membrane liquids. When the apple polyphenols addition was 0.5%, the DPPH scavenging rate, inhibition capacity of lipid oxidation and OH radical scavenging rate were determined to be 41.33%, 56.32%, 35.44%, respectively. As the addition amount of apple polyphenols was increased to 1.5%, the antioxidant ability of the composite membrane liquids increased to 94.3%, 95.9%，98.4%, respectively, significantly higher than that of 0.5% polyphenol-incorporated composite membrane liquid (P<0.01). Compared to the BHT (1.5%), antioxidation properties of the composite membrane liquids with 1.5% apple polyphenols were higher (P<0.01). Moreover, the rheological analyses suggested that the composite membrane liquid was non-Newton pseudoplastic liquid (n<1，R2>0.99). With the increase of shear rate, the viscosity of the composite membrane liquids displayed an initially decrease but then a relatively stable trend (approximately 5 Pa·s). Meanwhile, the incorporation of apple polyphenols enhanced the viscoelastic property of the composite membrane liquids. Dynamic rheology of the composite membrane liquid transformed from G′G″. The results of FTIR and X-ray showed that the intermolecular interactions of apple polyphenols (-OH) and chitosan (-OH, -NH2) were formed, resulting in the slight change of absorption peak of the composite membrane at 3 400 cm-1, 1 545 cm-1 and 1 245 cm-1 and resulting in the reduced crystallinity of chitosan. According to the result of thermal analysis, thermal stability of the composite membrane liquids increased, probably due to the hydrogen bonds formed via the linkages between apple polyphenols and chitosan.【Conclusion】The composite membrane liquids prepared by adding apple polyphenols into the chitosan had higher antioxidant capacity, viscosity and thermal stability, thus could be used as a potential edible coating material for food preservation.

ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT

Study on the Relationship Between the Expression of Candidate miRNAs and the Developmental Characteristics in Different Patterns in Hu Sheep Lambskin

JIN ChengYan, Lü XiaoYang, GAO Wen, WANG Yue, CHEN WeiHao, SHENG ShuiXing, CHEN Ling, LIN Jie, SUN Wei

Scientia Agricultura Sinica. 2018, 51(14):  2814-2824.  doi:10.3864/j.issn.0578-1752.2018.14.017

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【Objective】 In the present study, high-throughput sequencing and bioinformatics analysis were initially employed to identify differentially expressed microRNAs (miRNAs) in hair follicles of different flower patterns with small, medium, and large waves of Hu sheep. Then 14 candidate miRNAs were screened. The purpose of this study was to study the amount of 14 nominated miRNA expression in different pattern groups and to find the correlation of different hair follicle index, and to further explore the relevance between candidate miRNAs and developmental characteristics of hair follicles and screen miRNAs for later functional validation.【Method】Firstly, utilizing high-throughput sequencing filtrated differentially expressed miRNAs in hair follicles of different flower patterns with small, medium, and large waves of Hu sheep. To confirm the reliability of sequencing results, five miRNAs were selected for RT-PCR verification. Paraffin sections of lambskin tissue were prepared to assess the structure of different hair follicles. Expression levels of 14 candidate miRNAs in different groups were analyzed by relative quantitation using fluorescent quantitative PCR. Combined with histological observation and microscopic observation technique, the correlation between expression levels of 14 candidate miRNAs and histological properties of hair follicles was analyzed by using SPSS 17.0.【Result】The distribution of hair follicles was mainly in the form of hair follicle groups, which distributed as a group of 3. The diameter of the primary follicles was larger than that of secondary follicles regardless of large, medium, or small waves. In the same field of view, no significant differences in primary follicles among the large, medium and small wave lambskins were observed (P＞0.05); the number of secondary follicles in the small waves was significantly higher than that in large and medium waves (P＜0.01), whereas the number of secondary follicles in the medium wave was similar to that in the small waves (P＞0.05). Consequently, the total number of hair follicles in the small waves was higher than that in the large and medium waves in the same unit area. The diameter of the primary and secondary follicles in the large waves were significantly higher than that in medium and small waves (P＜0.01) and the diameter of primary and secondary hair follicles were not significantly different between the medium and small waves groups (P＞0.05). The level of expression of miRNA-143 in the large waves was significantly lower than that in the large waves (P＜0.01). The expression level of miRNA-10a in the small waves was significantly higher than that in the large and medium waves (P＜0.05) and that of let-7i was significantly higher in the medium waves compared with that in the large waves (P＜0.05). In the differentially expressed novel miRNAs, the expression level of NW_004080184.1_6326 in medium waves was significantly lower than that in the large and small waves, respectively (P＜0.05; P＜0.01). The expression of NW_004080165.1_8572 in the medium waves was significantly different compared with that in the large and small waves (P＜0.01). NW_004080181.1_3961 was expressed at a higher level in small waves than in medium waves (P＜0.01). Highly significant differences in NW_004080190.1_13733 expression was observed between medium and large waves (P＜0.01). No significant differences in the expression levels of the rest of the miRNAs were observed among the large, medium, and small waves. The relative expression levels of 14 miRNAs were correlated with some indicators of hair follicles, indicating that all 14 miRNAs might be involved in the growth and development of hair follicles.【Conclusion】Overall, the present study showed that miRNA-143, miRNA-10a, let-7i, NW_004080184.1_6326, NW_004080165.1_8572, NW_004080181.1_3961, and NW_004080190.1_13733 could be considered as important candidate genes, indicating these seven miRNAs might play significant roles in hair follicle growth and development in Hu sheep lambskin, which would be confirmed in subsequent experiments.

Isolation and Identification of Lactic Acid Bacteria from Halostachys Caspica Silage

YIN Xue, GUO XueFeng, LIU JunFeng, ZHANG XiuPing, XI LinQiao, ZHANG SuJiang

Scientia Agricultura Sinica. 2018, 51(14):  2825-2834.  doi:10.3864/j.issn.0578-1752.2018.14.018

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【Objective】In order to make rational use of forage resources to provide high quality silage for animal husbandry in southern Xinjiang, the nutritional value and fermentation quality of Halostachys caspica silage after flowering and fruiting stage were discussed in this study, and the Lactobacillus resources were provided for screening silage fermentation quality. 【Method】A small dose natural fermented silage was made in the laboratory of flower and fruit period in the Akesu area of Xinjiang province. After 150 d fermentation, the contents of dry matter, crude protein, soluble sugar, neutral detergent fiber and acid washing fiber were compared before and after silage, and the contents of pH, ammonia nitrogen, lactic acid, acetic acid, propionic acid and butyric acid in silage were measured. Combining with the physiological and biochemical analysis, the pure culture method from silage microbial analysis was adopted to investigate the species, morphological, and physiological-biochemical characteristics of lactic acid bacteria from Halostachys caspica silage in Xinjiang. The DNA Blast technique was used to compare the measured sequence with the GenBank database, thus the species of the known classified status of the highest homology of the tested sequence were selected and downloaded. Together with the sequence of the strains tested in this test, the strains were compared with the Clustal, and the phylogenetic tree was plotted with MEGA5.0 to determine the classification status of the strains and to separate the isolates. As a result, the strains were identified. 【Result】Compared with pre silage, dry matter and soluble sugar content in raw materials decreased after lactic acid bacteria fermentation. At the same time, in the process of silage fermentation, the water-soluble carbohydrates, which were difficult to degrade in the stem, were degraded by lactic acid bacteria to the fibers those were easily used by domestic animals, which led to the increase of the content of neutral detergent fiber and acid washing fiber. Silage pH was 4.254—4.286, and lactic acid content was 60.923—61.283 mg·mL^-1, which belonged to the range of good quality silage. 9 strains of lactic acid bacteria were isolated and were mainly Enterococcus faecium, Enterococcus lactis, Bacillus cereus, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus buchneri. 9 strains of lactic acid bacteria were found in the range of pH 4—7, and grew well under the condition of 3% and 6.5% concentration of NaCl, at the same time, the bacteria also grew well between 10 °C and 45 °C. It could also use D- glucose, D- mannose, cellobiose, D- mannitol, D- fructose, sucrose and many other carbon sources. Among them, Lactobacillus pentosus and Lactobacillus buchneri had higher acid production rate and faster growth rate, which showed strong acid and salt tolerance, thrived in the 5—45 °C range, and could be applied to the preparation of silage additives. 【Conclusion】The nutrient content and fermentation quality of the Halostachys caspica were in good level after green storage. The 3 strains of Lactobacillus pentosus, Lactobacillus buchneri, and Lactobacillus plantarum could be selected as additives in the process of making silage.