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Table of Content

    16 October 2017, Volume 50 Issue 20
    CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS
    Phenotypical Analysis and Gene Mapping of Abnormal Floral Organ Number Mutant afon1 in Rice (Oryza sativa L.)
    YANG ChengCong, LIANG Rong, QIN Ran, ZENG DongDong, JIN XiaoLi, SHI ChunHai
    Scientia Agricultura Sinica. 2017, 50(20):  3837-3847.  doi:10.3864/j.issn.0578-1752.2017.20.001
    Abstract ( 224 )   HTML ( 3 )   PDF (4541KB) ( 347 )   Save
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    【Objective】The abnormal floral organ number mutant of rice was used to study the molecular mechanisms of floral organ development, and to identify the related genes of floral organ in rice. 【Method】In present study, a rice mutant, abnormal floral organ number1 (afon1) was isolated from an indica cultivar Zhenong 34 M2 population by mutagenesis with ethyl methane sulfonate (EMS). At flowering stage, five panicles from afon1 and Zhenong 34 were randomly selected to observe the morphological phenotype, cytological features and pollen fertility by histology analysis and scanning electron microscopy, respectively. At mature stage, ten plants from afon1 and Zhenong 34 were randomly chosen for measuring the main agronomic traits, such as plant height, number of tillering, panicle length, number of floret per panicle, number of filled grain per panicle and 1 000-grain weight. One hundred plump seeds were selected for calculating the germination potential and germination rate of the mutant afon1 and wild type. The F2 population from crossing of afon1 with WT Zhenong 34 and Zhenongda 104 were used for genetic analysis and gene mapping, respectively. Then, the DNA sequencing was conducted, the model of AFON1 protein was built and the space structure for AFON1 protein was analyzed. Moreover, the expression of candidate gene and floral organ number associated genes were detected by real-time PCR. 【Result】Compared with the wild type, the floral organ number of 59.64% spikelet was abnormal in mutant afon1, which most of them had a glume-like organ on the side of the leman and there were 2-4 rounds of floral organ number increased at the same time in others. Furthermore, the plant height and 1 000-grain weight of afon1 were visibly higher, while the seed setting rate was significantly reduced. The results of the genetic analysis showed that the phenotype of F1 population from the crossing of afon1 with Zhenong 34 was normal and the segregation ratio of wild-type and mutant phenotype plants from F2 population fitted a ratio of 3﹕1, which revealed that the mutant trait of afon1 was controlled by a single recessive nuclear gene. The afon1 was further mapped on the arm of chromosome 1 between InDel markers 1M5 and 1M18 with a physical distance of 73 kb, where there were 6 annotated genes. The sequencing results between the mutant afon1 and the wild type illustrated that there was a single base-pair substitution of T (565th) to A on the exon of LOC_Os01g67430 resulting in the mutation of Trp (189th) to Arg. Protein sequencing and structure analysis revealed that there was a Lipase_3 domain and the mutation in the region changed the space structure of AFON1 obviously. The real-time PCR result showed that the expression of LOC_Os01g67430 in young panicle increased significantly, while the expression had no obvious difference in root, stem and leaf. Additionally, the expression of floral organ number related genes FON1 and FON2/4 was obviously increased in floral organ at young panicle developmental stage. 【Conclusion】The LOC_Os01g67430 was speculated as the gene afon1 regulating the number of floral organ by influencing the expression of corresponding genes which determined the number of floral organ.
    Evaluation of Genetic Diversity of Common Millet (Panicum miliaceum) Germplasm Available in China Using High Motif Nucleotide Repeat SSR Markers
    WANG RuiYun, LIU XiaoYu, WANG HaiGang, LU Ping, LIU MinXuan, CHEN Ling, QIAO ZhiJun
    Scientia Agricultura Sinica. 2017, 50(20):  3848-3859.  doi:10.3864/j.issn.0578-1752.2017.20.002
    Abstract ( 244 )   HTML ( 4 )   PDF (2396KB) ( 287 )   Save
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    【Objective】The objective of this study to develop SSRs with high motif (tetra-, penta- and hexa-) nucleotide repeat and use them to evaluate the genetic diversity of common millet germplasms, and to provide significant implications for future linkage maps construction and evolution research of common millet.【Method】Comprehensive evaluation of phenotypic diversity was carried out based on membership function, principal component analysis (PCA) and cluster analysis. SSR primers developed in author’s laboratory by high-throughput sequencing were used to identify polymorphisms in different common millet accessions from various geographic origins. PowerMarker 3.25 and PopGen 1.32 were used to calculate the measures of genetic diversity and Nei’s genetic distance, respectively. MEGA 5.0 and Structure 2.2 were used to carry out cluster analysis and identify genetic groups.【Result】A set of 96 common millet accessions were used and seven phenotypic traits were investigated. Results showed that the genetic variation of tiller numbers and plant height were the most abundant. A total of 85 pairs of amplified primers (56.29%) showed high polymorphism among the 96 genotypes. A total of 71 tetra-nucleotide repeat SSRs were detected, accounted for 83.53%. Meanwhile, 10 (11.76%) penta- and 4 (4.7%) hexa- nucleotide repeat SSRs were identified, respectively. The size of 85 SSRs ranged from 100 to 450 bp with an average value of polymorphism information content (PIC) of 0.51. Resolving power (Rp) value varied from 1.00 to 5.75 (mean = 3.15). The average Rp value of tetra-, penta- and hexa- nucleotide repeat SSRs was 3.15, 2.8 and 4.0, respectively. Evaluating the distribution frequency of 85 SSRs based on Rp, it was observed that their ranges were 0-1, 1-2, 2-3, 3-4, 4-5 and 5-6, with 1 (1.18%), 15 (17.65%), 31 (36.47%), 20 (23.53%), 12 (14.12%) and 6 (7.06%) markers at each interval, respectively. Fifty-one markers accounting for 60% distributed at intervals of 2-3 and 3-4. A total of 232 alleles were amplified among 96 accessions by the 85 SSR markers. 2-3 alleles were generated by each locus, with an average of 2.7294 alleles. Sixty-two markers produced 3 alleles, and 23 markers produced 2 alleles. The range of gene diversity was 0.2842-1.0633, with an average of 0.7708. The range of PIC was 0.0400-0.7281, with an average of 0.4723. Genetic distance and genetic identity of common millet resources with different ecotopes were 0.0093-0.5052 (average=0.1798) and 0.6034-0.9907 (average=0.8485). Cluster analysis based on unweighted pair group method of mathematical averages (UPGMA) separated the 96 accessions into four groups (Northeast spring-sowing, Northern spring-sowing, Northern summer-sowing and Loess Plateau spring & summer-sowing ecotopes). A genetic structure assay indicated a close correlation between geographical region and genetic diversity. 【Conclusion】The present work developed a set of 85 tetra-, penta- and hexa- nucleotide repeat SSRs in common millet. These high motif microsatellite markers with high Rp value can distinguish different genotypes, generate high polymorphisms in polymerase chain reaction amplification. Based on the above molecular markers, the genetic diversity of Chinese common millet germplasms was assessed and those accessions from Northern spring-sowing and Loess Plateau spring & summer-sowing ecotopes are highly and genetically diverse.
    Salt and Drought Tolerance in Heterologous-Expression of irrE Transgenic Tobacco
    ZHAO LeiLin, FAN Xin, NIE Xing, LIANG ChengZhen, ZHANG Rui, SUN GuoQing, MENG ZhiGang, LIN Min, WANG Yuan, GUO SanDui
    Scientia Agricultura Sinica. 2017, 50(20):  3860-3870.  doi:10.3864/j.issn.0578-1752.2017.20.003
    Abstract ( 225 )   HTML ( 4 )   PDF (3910KB) ( 252 )   Save
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    【Objective】 IrrE is a protein found in Deinococcus radiodurans, and it process the transcription regulating function mainly through repairing DNA damage in conditions of strong radiations and other adverse environments. It is of great significance to study the tolerance of optimized irrE against drought and salt in transgenic plants and to lay a theoretical basis of crops resistance breeding.【Method】The bacteria irrE codons were optimized with the plant preferences and the gene was synthesized. Efficient plant expression vector was constructed by using the method of enzyme digestion and connection. Leaf disc method was used for transforming tobacco, and the positive regenerated seedlings were screened with PCR. The expression level of irrE in transgenic strain was analyzed. The IrrE protein was tested using Western blot. Pure transgenic generations were recruited and treated with the mannitol and NaCl treatments, which simulated drought and salt conditions. Physiological tests were conducted to determine drought and salt tolerance.【Result】The irrE was modified according to plant codes, and 241 codes were optimized. The efficient plant expression vector GBI-IE was constructed. The regenerated seedlings were screened by herbicide glyphosate, and 15 independent transgenic strains were obtained using PCR, and two highest expression lines, GO1 and GO2, were used for subsequent analysis of tolerance under adverse environmental conditions. Western blot proved that protein IrrE encoded in GO1 and GO2 could be translated correctly. Salt and drought tolerance of transgenic tobacco was analyzed. Germinating experiments displayed that there was no obvious difference between transgenic lines (GO1 and GO2) and non-GMO wild type (WT) in terms of germination rate in 1/2 MS culture medium, however, in medium containing 250 mmol·L-1 NaCl, germination rate of GO1 and GO2 was 78.8% and 90.0%, respectively, while the one of the WT was 10.3%, increased by 68.5% and 79.7%, respectively. Similarly, germination rate of WT was 39.7% in medium containing 300 mmol·L-1 mannitol, the rate of GO1 and GO2 increased by 42.9% and 50.8%, respectively. Normally germinated seedlings were transplanted into 250 mmol·L-1 NaCl and 300 mmol·L-1 mannitol environment for 12 days, and root length, lateral roots number and fresh weight of those two transgenic lines were markedly higher than the ones of WT. Tobacco seedlings of 30 days normal growth in the greenhouse were treated with 250 mmol·L-1 NaCl of which transgenic tobacco SOD and CAT activity was more 48.80% and 88.55% than WT, respectively, and the MDA content was less 61.61% than WT, In addition, the expression level of drought responsive gene in GO1 and GO2, like NtABF2, NtLEA5, Ntzfp, NtCDPK2, was significantly higher than WT. Similarly, the changes of physiological and biochemical indexes in transgenetic lines in 300 mmol·L-1 mannitol treatment were higher than the ones in WT.【Conclusion】Expression of irrE from D. radiodurans remarkably improved the tolerance of tobacco to drought and salt stresses in which the pleiotropy could be beneficial gene resource of gene engineering about resistance to stress in plant.
    TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY
    Effects of Exogenous 6-BA on Photosynthetic Characteristics and Endogenous Hormone Content in Wheat Leaves Under Two Nitrogen Application Levels at Seedling Stage
    YANG DongQing, DONG WenHua, LUO YongLi, SONG WenTing, CAI Te, LI Yong, YIN YanPing, WANG ZhenLin
    Scientia Agricultura Sinica. 2017, 50(20):  3871-3884.  doi:10.3864/j.issn.0578-1752.2017.20.004
    Abstract ( 316 )   HTML ( 10 )   PDF (650KB) ( 480 )   Save
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    【Objective】The purpose of this study was to test the effects of nitrogen concentrations and spraying exogenous cytokinin (6-BA) on photosynthetic characteristics, chlorophyll a fluorescence, nitrate reductase (NR) and glutamine synthetase (GS) activities, changes of endogenous hormones in leaves of wheat at seedling stage and grain yield, and then provide a theoretical basis for improving the development of winter wheat seedlings. 【Method】Wheat (Jimai 22) was sown in the field and pot culture experiments with two nitrogen levels ( N1, 120 kg·hm-2; N2, 240 kg·hm-2) and modified Hoagland nutrient solutions containing two nitrogen levels (HN, 3.75 mmol·L-1; LN, 0.63 mmol·L-1), respectively. 6-BA (30 mg·L-1) and lovastatin (300 mg·L-1) were sprayed to the whole plants at a rate of 100 mL·m-2 at the three-leaf stage. Ten wheat plants were sampled at 3 d intervals to test chlorophyll contents, NR and GS activities, photosynthetic parameters (Gs, Tr, Ci and Pn), chlorophyll fluorescence parameters (Wk, Vj, Ψo and PIabs), and endogenous hormones. In addition, grain yield, kernels per spike, 1000-grain weight, and ear numbers were determined at maturity stage.【Result】The above ground biomass (AGBM) was significantly increased by exogenous 6-BA treatment in the pot experiment. Conversely, application of lovastatin significantly decreased AGBM. Compared with HN treatment, AGBM under HN+6-BA treatment increased by 21.39% at 12 days after treatment (DAT), and that of LN+6-BA treatment increased by 43.92%, compared with LN treatment. Application of 6-BA significantly increased values of Gs, Tr, Ci and Pn under high nitrogen condition. Especially, Gs, Tr, Pn and Ci increased by 68.32%、58.66%、30.72%、51.61% at 12 DAT, respectively. Chl a increased by application of 6-BA at 9 to 12 DAT, while content of Chl b has significantly increased by exogenous 6-BA from 3 to 12 DAT. Conversely, both Chl a and Chl b have significantly decreased by application of lovastatin. Compared with HN treatment, HN+6-BA treatment significantly increased the activity of nitrate reductase (NR) and glutamine synthetase (GS). While application of lovastatin significantly decreased NR and GS activity. Exogenous 6-BA treatments changed the fast chlorophyll fluorescence induction kinetics curves. Application of 6-BA significantly increased Ψo and PIabs, and decreased Wk and Vj. Wk and Vj decreased by 22.09% and 36.05%, respectively, under LN+6-BA treatment, compared with LN treatment. Application of 6-BA significantly increased Zt content from 3 to 12 DAT, increased IAA content from 6 to 12 DAT, and decreased ABA content. However, spraying exogenous lovastatin obviously increased ABA content. Application of 6-BA significantly increased grain yield in the field experiment. Compared with N1 and N2, grain yield of N1+6-BA and N2+6-BA increased by 10.48% and 16.61%, respectively. 【Conclusion】Exogenous 6-BA increased aboveground biomass due to increasing the leaf photosynthesis and nitrogen assimilation through regulating endogenous hormones contents to enhance chlorophyll content and NR and GS activity, and to improve the electron transfer capability of both the donor and the acceptor sides at PSII reaction center resulting in improved PSII performance. Grain yield has significantly increased under 6-BA combined with nitrogen application treatments.
    Effects of Nitrogen-Reducing and Suitable Soil Moisture on Nitrate Nitrogen Distribution in Soil, Nitrogen Absorption and Utilization of Winter Wheat
    ZHANG Man, ZHOU SuMei, YANG XiWen, ZHOU Yan, YANG Rui, ZHANG KeKe, HE DeXian, YIN Jun
    Scientia Agricultura Sinica. 2017, 50(20):  3885-3897.  doi:10.3864/j.issn.0578-1752.2017.20.005
    Abstract ( 268 )   HTML ( 7 )   PDF (506KB) ( 312 )   Save
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    【Objective】The objective of this experiment was to study the effect of reduced nitrogen(N) application under different soil moisture on the nitrate nitrogen (NO3-N) distribution in soil, N absorption, use efficiency in plant and grain yield of winter wheat. This study will provide a scientific basis for rational N application in Huang-Huai plain.【Method】To determine NO3-N content in different soil layers of 0-100 cm soil depth, N accumulation, N translocation amount and grain yield of winter wheat, we established a split-plot experiment for two consecutive years (2014-2015 and 2015-2016) in field, i.e. three levels of soil moisture, water deficits to no irrigation (W1), water-savingirrigation to 70%±5% of soil relative moisture after jointing stage (W2) and adequate irrigation to 80%±5% of soil relative moisture after jointing stage (W3); three levels of nitrogen: 0 (N1), 195 kg·hm-2 (N2) and 270 kg·hm-2 (N3).【Result】The content of NO3-N in 0-60 cm soil layer decreased with the layer's deepening, and soil water content and N application input amount increased; whereas the NO3-N content in 60-100 cm soil layers increased, especially under W3 treatment. Besides, soil NO3-N content of 80-100 cm soil layer at N2 and N3 under high supplemental irrigation condition (W3) increased by 3.8 mg·kg-1 and 4.2 mg·kg-1, respectively, compared with that under no irrigation (W1). N2 treatment had higher NO3-N content of 0-20 cm soil layer at anthesis, increased by 2.2 mg·kg-1 than that at maturity; whereas there was more NO3-N residue under high N application (N3) at post-harvest. The results also showed that N translocation amount in vegetative organs ordered by leaf > stem> sheath. In addition, W2 with N2 treatment had the largest N accumulation amount in plant at anthesis, pre-antheis N translocation amount (N accumulation in vegetative organs at anthesis minus the N accumulation in vegetative organs at maturity, NTA) and the contribution of N remobilized to grain (NTA/ N accumulation in vegetative organs at maturity stage, NRC) from vegetative organs after anthesis. Similarly, the grain yield and N accumulation amount in grain under W2 with N2 treatment were 15.4% and 27.3% higher than that at other treatments, respectively. Those increases would improve N uptake efficiency and N fertilizer productive efficiency under W2 with N2 treatment (P<0.05).【Conclusion】Considering the yield, N uptake efficiency, N fertilizer productive efficiency and soil nitrogen balance, reducing N application from 270 to 195 kg·hm-2 under suitable soil moisture (water-saving) is optimal in Huang-Huai Rivers Valley wheat region.
    Analysis of Morphological and Physiological Responses to Low Pi Stress in Different Alfalfas
    LI ZhenYi, ZHANG QiXin, TONG ZongYong, LI Yue, XU HongYu, WAN XiuFu, BI ShuYi, CAO Jing, HE Feng, WAN LiQiang, LI XiangLin
    Scientia Agricultura Sinica. 2017, 50(20):  3898-3907.  doi:10.3864/j.issn.0578-1752.2017.20.006
    Abstract ( 256 )   HTML ( 10 )   PDF (641KB) ( 641 )   Save
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    【Objective】 The objective of this study is to screen low Pi (inorganic phosphate) tolerant alfalfa and related characters, and to provide the theoretical basis for deficient Pi tolerance mechanism and then for application in production. 【Method】 In the condition of nutrient solution, twenty 24-day-old alfalfa seedlings were cultured in nutrient solution with sufficient phosphate (500 μmol·L1 KH2PO4) and deficient phosphate (5 μmol·L1 KH2PO4). After 30 days of treatment, a variety of physiological indexes, including dry weight of stems and leaves (SLW), plant height (PH), dry weight of root (RW), ratio of root to shoot (RS), total root length (TRL), root square area (RSA), total Pi content (TP), phosphate utilization efficiency (PUE), acid phosphatase activity (ACPA), were measured, and coefficients of resistance to deficient-Pi of each index were calculated. Furthermore, the correlation was carried out on the deficient-Pi coefficient of each index. Based on principal component analysis, subordinate function analysis, regression analysis and cluster analysis, deficient-Pi tolerance of different varieties were evaluated, and the indexes closely related to deficient-Pi tolerance were screened. At the same time, the evaluation model of deficient-Pi tolerance was established. 【Result】 Under deficient-Pi stress, the aboveground growth of all varieties were inhibited. However, the root system and the content of total Pi were decreased (P<0.01). Through principal component analysis, nine primary indexes could be transformed into four new mutual independent comprehensive indexes, which covered 92.04% of information. Then four new indexes were used to comprehensively assess the characters of deficient-Pi tolerance by subordinate function analysis. Combined with the clusters result, 20 alfalfa varieties were divided into three clusters. The varieties resistant to deficient-Pi include Aohan, Xinmu NO.1, Magnum Salt, Phabulous; varieties moderate resistant to deficient-Pi include nine varieties, such as Knight T, Caribou, Amerigraze 37CR, Longmu 801 and so on; varieties sensitive to deficient-Pi include seven varieties, such as Magnum II, Zhongmu NO. 2, Concept and so on. Furthermore, the mathematic evaluation model of resistance to deficient-Pi, D=﹣0.7997+0.3856 SLW+0.2025 PH+0.3789 RW+0.1051 TRL+0.4188 TP+0.1347 ACPA (R2 = 0.9982).【Conclusion】The analysis of the ability of deficient-Pi tolerance in different alfalfa varieties showed Aohan, Xinmu NO.1, Magnum Salt and Phabulous have strong resistance to deficient-Pi stress. And the dry weight of stems and leaves, plant height, root dry weight, total root length, total Pi content and acid phosphatase activity were the most suitable indicators for the tolerance to deficient-Pi.
    PLANT PROTECTION
    Molecular identification of FHB resistance gene in varieties derived from common wheat-Thinopyrum ponticum partial amphiploid
    LIU XinLun, WANG Chao, NIU LiHua, LIU ZhiLi, ZHANG LuDe, CHEN ChunHuan, ZHANG RongQi, ZHANG Hong, WANG ChangYou, WANG YaJuan, TIAN ZengRong, JI WanQuan
    Scientia Agricultura Sinica. 2017, 50(20):  3908-3917.  doi:10.3864/j.issn.0578-1752.2017.20.007
    Abstract ( 313 )   HTML ( 5 )   PDF (1669KB) ( 675 )   Save
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    【Objective】 Fusarium head blight (FHB) is one of the most devastating diseases of wheat. Thinopyrum ponticum is one of the important source resistance cultivars of wheat disease resistance breeding, with high tolerance to FHB. Three derived varieties of common wheat-Th. ponticum Xinong 509, Xinong 511 and Xinong 529 showed moderate resistance to FHB in field. The objective of this study is to clarify the genetic constitution of resistance gene in three varieties.【Method】 Single drop injection with Fusarium graminearum was performed to detect the reaction of the three derived varieties of common wheat-Th. ponticum to FHB. Specific molecular markers located on homologous group 1 to 7 of Thinopyrum E chromosome were employed to detect the sources of resistance to FHB, screening the three derived varieties of common wheat-Th. ponticum, and their major donor parents Xiaoyan 693, Xiaoyan 597 and Th. ponticum. Specific molecular markers closely linked with ?anking Fhb7 were used to analyze the relationship between the gene conferring resistance to FHB and Fhb7.【Result】 Three derived varieties were moderately resistant to FHB in the field experiment, and had no much difference compared with the medium-resistant control variety. Seven of the 105 chromosome E specific molecular markers of Thinopyrum could be amplified polymorphism bands in the three derived varieties and Th. ponticum, which located on the chromosome 7E. A total of 97 specific markers located on the chromosome 7E,20 showed specific bands of Th. ponticum in the three derived varieties of /span>common wheat-Th. ponticum, and their major donor parents Xiaoyan 693, Xiaoyan 597. The results indicate that the 89 cM genetic fragment derived from Th. ponticum 7E chromosome. Of them, six molecular markers (XsdauK8, XsdauK144, XsdauK27, XsdauK99, Xcfa2040 and XsdauK116), which were located on 7EL 149.00-7EL 153.77, tightly linked to Fhb7. However, XsdauK60 (7EL 153.77), Xmag1932 (7EL 154.70), Xcfa2240 (7EL 156.27), XsdauK66 (7EL 158.02), XsdauK71 (7EL 158.97) and Xsews19 (7EL 160.00),which are next to Fhb7, showed no specific bands ofTh. ponticum among the three derived varieties of common wheat-Th. ponticum and Xiaoyan 597. 【Conclusion】 Xinong 509, Xinong 511 and Xinong 529, derived from Th. ponticum with good FHB resistance, possess a novel FHB resistance gene, which derived from Th. ponticum and was different from Fhb7.
    Screening and identification of peach endophytic bacteria with antagonism against Agrobacterium tumefaciens
    LI YuJia, LI Qian, ZHANG ZhiXiang, LI ShiFang
    Scientia Agricultura Sinica. 2017, 50(20):  3918-3929.  doi:10.3864/j.issn.0578-1752.2017.20.008
    Abstract ( 349 )   HTML ( 8 )   PDF (2529KB) ( 812 )   Save
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    【Objective】The objectives of this study are to identify the population composition of cultivable endophytes in peach twigs of ‘Xibei 13-1’ cultivar, and to screen the new antagonists for biological control of crown gall disease. 【Method】 Peach twigs of ‘Xibei 13-1’ cultivar were inoculated with Agrobacterium tumefaciens, and mock inoculation of the control was performed in a similar manner, but sterilized distilled water was used instead of the bacterial suspension. Twigs with different treatments were collected at different time points (before inoculation, 10 and 60 days after inoculation, respectively). Surface sterilization, endophytes isolation and counts, and 16S rDNA sequencing, were conducted to analyze the quantity and diversity of endophytic bacteria among samples. Their antagonisms against the causal agent of crown gall disease, A. tumefaciens, were tested by pair co-culturing method, and the efficacy of the antagonists in suppressing crown gall disease was further evaluated in greenhouse using sunflowers as a susceptible plant. The taxonomic status was clarified by physiological and biochemical and molecular methods. In order to reveal the antagonistic mechanism, the plasmid of pBBR1MCS-2 that contains green fluorescent protein gene (GFP) was transformed into the antagonistic strains by electroporation. Antibacterial activities, dynamic analysis and stability of the GFP-labeled strain were tested. The labeled strain suspension was inoculated using root irrigation method, then the A. tumefaciens suspension was inoculated at the 2nd day. Their re-colonization on the tomato roots was examined by fluorescence microscope and plate dilution method. 【Result】A total of 108 endophytic bacterial isolates were obtained from peach twigs of ‘Xibei 13-1’ cultivar. All isolates were identified as 17 genera of the 5 bacterial groups-phylogenetically based on 16S rDNA. The 5 groups are Gammaproteobacteria, Alpharoteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes. This result showed the biodiversity of endophytic bacteria from peach twigs of ‘Xibei 13-1’ cultivar. Among them, genera Enterobacter, Pantoea, and Rhizobium were the most abundant and significantly increased following inoculation of A. tumefaciens. In addition, most of the antagonists belong to these three genera as well (10/14). In vivo, strains 10DM4-1 and 10DI2-2 showed good performances in disease control with the efficacy of 86.08% and 89.87%, respectively. Strains 10DM4-1 and 10DI2-2 were then identified as Pantoea deleyi and Enterobacter cowanii by their 16S rDNA sequences in combination with their biochemical and physiological characteristics, respectively. The population of transformants 10DM4-1-gfp and 10DI2-2-gfp on tomato roots decreased sharply in the first 10 days, then declined slowly after 10th day and remained constant at lower level (104 CFU/g), indicating that they could stably colonize and survive in the intercellular of tomato roots. 【Conclusion】The resistance of peach ‘Xibei 13-1’ cultivar against crown gall disease may be related to its endogenous Enterobacter, Pantoea and Rhizobium. P. deleyi 10DM4-1 and E. cowanii 10DI2-2 can effectively suppress crown gall disease caused by A. tumefaciens, produce antagonistic substances, colonize the favorable ecological niche and has high potential application value for biological control of crown gall disease.
    SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT
    Effects of Slow-Release Nitrogen Fertilizer and Urea Blending on Maize Growth and Nitrogen Uptake Under Different Nitrogen Application Rates
    GUO JinJin, ZHANG FuCang, WANG HaiDong, YAN ShiCheng, ZHENG Jing, CHEN DongFeng, LI ZhiJun
    Scientia Agricultura Sinica. 2017, 50(20):  3930-3943.  doi:10.3864/j.issn.0578-1752.2017.20.009
    Abstract ( 275 )   HTML ( 7 )   PDF (549KB) ( 571 )   Save
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    【Objective】Understanding of the effects of mixed slow-release nitrogen fertilizer on crop growth can serve as a guide for optimal N management by improving N use efficiency and grain yield and reducing soil NO3-- N residue.【Method】An experiment was conducted in Guanzhong Plain to investigate the effects of a mixture of conventional urea and slow-release fertilizer (N ratios 3∶7, respectively) and N application rates on maize plant height, stem diameter, leaf area index, dry matter accumulation , yield, yield components and nitrogen use with a high yielding ‘Zhengdan 958’in a randomized complete block design. Three nitrogen fertilizer types (U: conventional urea, S: slow- release urea and SU: a mixture of slow-release and conventional urea with N ratio 7∶3) and four N application rates N1 (90 kg·hm-2), N2 (120 kg·hm-2), N3 (180 kg·hm-2) and N4 (240 kg·hm-2) were applied in the study. No nitrogen plot was designed as the control (CK). There were 13 treatments were established.【Result】The interactions of nitrogen fertilizer types and rates on maize growth index, dry matter accumulation, yield and yield components were significant. Compared with other treatments, the maximum dry matter and nitrogen accumulation (17 927.9 kg·hm-2and 156.1 kg·hm-2) with SU (N3) were increased by 16.0%-61.7% and 8.1%-45.2%, respectively .The highest grain yield (6 200 kg·hm-2) was achieved in the treatment of SU (N3), increased by 19.8% and 20.7% in comparison with the treatments of U (N3) and S (N2), respectively. There was no significant difference in yield among S (N2), SU (N2) and U (N3). The results also indicated that the yield did not increase with increasing N application rate. In the N3 treatment, the yields with U and SU were, respectively, increased by 19.7% and 19.0%, compared with N4. Moreover, the yield with S (N2) application was increased by 10.9% and 26.5% compared with S under N3 and N4, respectively. Compared with the U (N3) and S (N2) treatments, the nitrogen translocation amount with SU (N3) was highest, increased by 14.7% and 8.2%, respectively. It was conducive to promoting the grain yield. The accumulation of nitrate nitrogen in soil increased with higher N application rate, but applying mixture (SU) reduced the NO3--N accumulation by 21.2% and 9.5% compared with monotypic conventional or slow- release urea. Regardless of application rate, the mixed urea treatments reduced the leaching N in the soil profile by augmenting NO3--N contents at 0-40 cm soil layers.【Conclusion】The results demonstrate that moderate amount (180 kg·hm-2) of application of the mixture of slow-release and conventional urea is recommended for gaining greater yields of maize and N use efficiency in Guanzhong Plain.
    Effects of Reduced Phosphorus Fertilization on Soil Phosphorus Accumulation, Leaching and Utilization in Greenhouse Vegetable Production
    LI RuoNan, WU XuePing, ZHANG YanCai, WANG LiYing, ZHAI FengZhi, CHEN LiLi, SHI JianShuo, XU QiangSheng, HUANG ShaoWen
    Scientia Agricultura Sinica. 2017, 50(20):  3944-3952.  doi:10.3864/j.issn.0578-1752.2017.20.010
    Abstract ( 290 )   HTML ( 10 )   PDF (600KB) ( 237 )   Save
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    【Objective】Excessive application of phosphorus fertilizer is commonly observed in vegetable production in China. The purpose of this study was to investigate the potential of phosphorus fertilization reduction in vegetable production and recommend appropriate phosphorus rates for cucumber and tomato.【Method】Cucumber and tomato were widely cultivated in North China, and thus were taken as the research objects. In this study, cropping model was the winter-spring cucumber and autumn-winter tomato double cropping system. The basic Olsen-P level was 40.2 mg·kg-1. Three treatments were designed in this experiment, including a non-phosphorus control (P0), a reduced phosphorus rate (P1) and a farmers’ conventional phosphorus rate (P2). In cucumber season, the amounts of P2O5 applied in P0, P1 and P2 treatments were 0, 300, 675 kg·hm-2, respectively. The corresponding phosphorus amounts in the tomato season were P2O5 0, 225, 675 kg·hm-2, respectively. The apparent phosphorus balances, soil phosphorus availability and movement and yield changes among 3 years were collected and analyzed to find out the appropriate phosphorus rates.【Result】(1) Phosphorus surplus was P 480.0 kg·hm-2·a-1 and the average Olsen-P content in 0-20 cm soil was 70.2 mg·kg-1 in P2 treatment during 3 years. Due to the surplus application, the Olsen-P in 0-20 cm soil increased by 2.7 mg·kg-1 when phosphorus surplus increased by P 100 kg·hm-2. The DPSM3 was 80% at 0-20 cm depth in P2 treatment and phosphorus leached obviously to deeper soil. (2) Compared to P2 treatment, the phosphorus application rate decreased by 61.1% and the phosphorus surplus among 3 years reduced by 71.0%-77.3% in P1 treatment; the Olsen-P contents decreased by 18.6%-43.5% with the average of 49.3 mg·kg-1 among 3 years, which was close to the critical value of the fruit vegetable production; the average of Olsen-P content decreased by 9.3%-30.1% at 20-60 cm soil depth, and the DPSM3 decreased by 21 percentage point, which indicated lower phosphorus leaching in P1 treatment, and no yield reduction was observed. (3) The Olsen-P in 0-20 cm soil decreased by 3.4 mg·kg-1 when phosphorus uptake increased by P 100 kg·hm-2 in P0 treatment. The average Olsen-P content in 0-20 cm soil was 30.5 mg·kg-1 in P0 treatment. Although no yield decrease was observed, the phosphorus uptake in 2008 tomato season decreased by 19.8%-30.0% in P0 treatment. (4) Based on this results, when the soil Olsen-P was above 40 mg·kg-1, less than 300 kg·hm-2 P2O5 was recommended for cucumber with yield level of 170 t·hm-2, and not more than 225 kg·hm-2 P2O5 was recommended for tomato with yield level of 100 t·hm-2.【Conclusion】The reduction of phosphorus fertilizer exhibited a great potential in the greenhouse vegetable production in North China Plain. For greenhouse planted for more than 3 years, 60% phosphorus could be saved and significantly decrease the phosphorus surplus, lessen available phosphorus accumulation, reduce phosphorus leaching and keep vegetable yield at high level.
    The Spatial Flow and Pattern Optimization of Carbon Sequestration Ecosystem Service in Guanzhong-Tianshui Economical Region
    LI Ting, LI Jing, WANG YanZe, ZENG Li
    Scientia Agricultura Sinica. 2017, 50(20):  3953-3969.  doi:10.3864/j.issn.0578-1752.2017.20.011
    Abstract ( 265 )   HTML ( 14 )   PDF (8135KB) ( 441 )   Save
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    【Objective】The aims of this paper were to quantify the balance of supply and demand situation of carbon sequestration service in regional ecosystem(hereinafter referred to as "carbon sequestration service"), simulate the spatial flow of carbon sequestration service, reveal the function change spatial rule of carbon sequestration service in regional ecosystem, and give the regional spatial layout optimization strategy which will provide an intuitive scientific reference for guiding the low carbon development of regions.【Method】Population spatial density was simulated with a multi-source data fusion model, and then the demand quantity of carbon sequestration service was estimated in Guanzhong-Tianshui Economic Region. The supply quantity of biologic carbon sequestration service in study area was calculated using CASA model, the soil carbon sequestration service supply was estimated using remote sensing model of carbon cycle. Based on these, the regional carbon balance was quantified with current ratio and the flow process of carbon sequestration service from generation to the use of land was elaborated with spatial visualization method. The conditional probability of environmental variables to the supply of carbon sequestration was calculated rely on Bayes principle, and the key variables were screened by entropy reduction model, the uncertainty of carbon sequestration pattern was discussed by using the distribution of key factor subsets with optimal state, and at last, the carbon sequestration space layout optimization strategy was given.【Result】(1) Carbon sequestration service in the study area overall oversupply, the spatial difference of balance between supply and demand is obvious, the high value areas of demand are mainly distributed in the Guanzhong plain high population areas, the high value areas of supply are mainly distributed in the areas along the Qinling Mountains and the Beishan mountains. (2) The spatial flow of carbon sequestration service in ecosystems is obviously different. According to the current ratio distribution, the study area can be divided into three carbon source concentration centers: Tianshui carbon source concentration center (Ri>0.04), Binxian carbon source concentration center (Ri>0.04), and Xi’an multi-level carbon source concentration center, with the highest value (Ri>0.20). The carbon sequestration service space flow in the study area can be divided into four groups: the flow from the Middle and East of Qinling Mountains, Yongshou County in Beishan Muontain to Guanzhong City Group, the flow from the west of Qinling Mountains to Tianshui city, the flow from Linyou county and Xunyi County to Binxian County, the flow from Tongchuan City, Chengcheng County, Hua County to Pucheng County. (3) According to the conditional probability and entropy calculation, taking set{DEM=3, PET=1} as a key variable optimal state subset of biology carbon pool, which is mainly distributed in Baoji City in southern Qinling Mountains and the southwest corner of Tianshui, where the optimal biological carbon fixation probability can reach 54.36%; taking set {NPP=3, DEM=3} as a key variable in the optimal state subset of soil carbon pool, which is mainly distributed in the Qinling Mountains, along the southwest corner of Tianshui and the northeast corner of the city of Xianyang, where the optimal probability is as high as 92.84%. The suitable areas of biology carbon pool mainly distributed in Tianshui and Wushan County, Qinzhou District and the suitable areas of soil carbon pool are mainly distributed in the middle part of Qinling Mountains district.【Conclusion】In general, carbon sequestration service demand is less than the supply in study area with obvious spatial flow. Taking Tianshui City and the middle areas of Qinling Mountains as the main region to optimize the carbon sequestration function can get a higher probability of good carbon sequestration pattern.
    HORTICULTURE
    Identification of MdWRKY40 Promoter Specific Response to Salicylic Acid by Transcriptome Sequencing
    QIU HuaRong, ZHOU QianQian, HE XiaoWen, ZHANG ZongYing, ZHANG ShiZhong, CHEN XueSen, WU ShuJing
    Scientia Agricultura Sinica. 2017, 50(20):  3970-3990.  doi:10.3864/j.issn.0578-1752.2017.20.012
    Abstract ( 299 )   HTML ( 9 )   PDF (5676KB) ( 506 )   Save
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    【Objective】 In order to explain the theoretical basis for disease resistance molecular mechanism mediated by SA, the influence of transcriptional regulation of apple leaves response to salicylic acid was studied, and the SA signaling pathway and its regulated genes were identified.【Method】 The leaves of tissue culture apple ‘gala’ seedling growing under 24℃ for 30 d were treated with 2 mmol?L-1 SA (0.2% ethanol treatment as control) for 12 h. Then the transcriptome libraries was constructed by using Illumina HiSeqTM 2000 sequencing technique, and the regulated genes of SA signaling pathway were screened by integrated bioinformatics analysis which included differential genes screening, condition specificity analysis, GO classification and KEGG enrichment analysis. The promotor of the gene which was specific response to SA was cloned and promotor activity was identified by using protoplast transformation technique. The specific response of different nucleotide fragment of promotor was verified.【Result】The original data of 750 439 459 and 751 596 153 bp sequences was obtained from CTRL and SA treatment samples, Which were 44.77% and 43.88% perfect match with the ‘golden delicious’ apple genome sequence, respectively. The transcriptome data of SA treated samples suggested that 3 329 genes were significantly expressed, including the genes related to biosynthesis of secondary metabolites pathway (the key enzyme of lignin synthetic pathway CAD, cytochrome P450, β-1,3-glucanase related to fungal resistance), the important functional genes involved in plant-pathogen pathway (calmodulin CaM, disease resistance protein RPM1, heat shock protein HSP90, WRKY transcription factors) and 33 condition specificity genes (NAC transcription factors, NIM1, WRKY40, Ethylene responsive factors and so on). Among them, 1 085 genes were up-regulated and 2 244 genes were down-regulated. Differentially expressed genes were participated in cellular process, metabolic process, binding, catalytic activity and so on. According to the transcriptome data, the promotor of MdWRKY40 was cloned into the expression vector and placed in the upstream of the luciferase gene. After transforming the vector to apple protoplasts, the luciferase activity of SA treated samples was 20.6 times of the control samples, and the SA treated samples did not affected by ABA, JA or ACC, only showed the specific response to SA. The results suggested that the promotor sequence was specially response to SA in apple. Different segments of the promoter with different response ability to SA. The region 500-1 000 bp of the promoter, which located in the upstream of the WRKY40 transcription start site ATG, only was response to high concentrations of SA and no response to low concentrations of SA. For 1 500 bp sequence, the response ability to high SA concentrations was enhanced significantly, but just slightly increased with low SA concentration. The length of the 2 000 bp nucleotide fragment had a significant response to both the high and low concentrations of SA and achieved the strongest ability level. Compared to 2 000 bp fragment, the 2 500 bp fragment didn’t show further enhanced response to SA. Overexpression of the MdWRKY40 protein inhibited its own transcription.【Conclusion】 The DEGs obtained under 2 mmol?L-1 SA treatment in apple leaves were involved in biosynthesis of phenylpropanoid and flavonoid, plant-pathogen interaction and plant hormone signal transduction pathways. The 2 500 bp nucleotide promotor sequence upstream of WRKY40 open reading frame was specific response to SA. There were unknown nucleotide sequences between 1 000-1 500 bp and 1 500-2 000 bp that significantly enhanced the sensitivity of the promoter response to SA, and the transcription regulation of MdWRKY40 existed a feedback suppression mechanism.
    Analysis of MYB Transcription Factor Family Based on Transcriptome Sequencing in Lycium ruthenicum Murr.
    YAN Li, WANG CuiPing, CHEN JianWei, QIAO GaiXia, LI Jian
    Scientia Agricultura Sinica. 2017, 50(20):  3991-4002.  doi:10.3864/j.issn.0578-1752.2017.20.013
    Abstract ( 354 )   HTML ( 12 )   PDF (3800KB) ( 687 )   Save
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    【Objective】 MYB is one of the most common transcription factor families in plants. It is widely involved in plant growth, and metabolic regulations. So far, there is no systematic analysis of the MYB transcription factor family of tree crops. Analysis of MYB family based on the transcriptome data in Lycium ruthenicum Murr. was conducted in this study, which laid a foundation for the research on biological function, and mechanism of metabolic regulations of MYB genes. 【Method】 Based on the transcriptome sequencing (RNA-Seq) data, the NR, NT, Swiss-Prot, PFAM and NCBI sites were used at the same time to screen and classify the MYB genes of L. ruthenicum Murr. The Web Logo3, Prot Comp 9.0, and MEGA5.0 were also applied to conservative structure prediction, subcellular localization, and phylogenetic analysis. The expression pattern of MYB genes related to fruit development was obtained and Real-time fluorescence quantitative PCR was used to detect the specific expression of those genes. 【Result】 Based on the transcriptome sequencing (RNA-Seq) data, 83 transcription factors of MYB family were annotated, selected, and divided into four categories (R2R3-MYB, 1R-MYB, 3R-MYB and 4R-MYB) according to their structural characteristics. The R2 MYB motif of the R2R3-MYB transcription factor contains three highly conserved tryptophan residues, and the first tryptophan residue in the R3 MYB motif is replaced by some hydrophobic amino acids. The phylogenetic trees of MYB family of L. ruthenicum Murr. and Arabidopsis thaliana were constructed, which showed that the MYB family of L. ruthenicum Murr. contained three major branches, and six evolutionary branches. The result of the subcellular localization demonstrated that 44 MYB transcription factors were located in the cytoplasm, and 37 MYB transcription factors were located in the nucleus. The analysis of differential expression of MYB genes of L. ruthenicum Murr. based on transcriptome sequencing (RNA-Seq) showed that MYB genes might be involved in the regulation of anthocyanin in three fruit development periods. Additionally, differential expression data based on fluorescence quantitative PCR confirmed that some MYB transcription factors might play a role in the regulation of anthocyanin synthesis in different fruit development periods of L. ruthenicum Murr.. 【Conclusion】 83 transcription factors of MYB family were annotated of L. ruthenicum Murr. The findings have laid a foundation for further studies of the structures and biological functions of MYB family.
    FOOD SCIENCE AND ENGINEERING
    Postharvest ABA Treatment Promote Wound Healing of Potato ‘Longshu No.3’ Tubers
    LI Xue, WU JueTian, WANG Yi, JIANG Hong, BI Yang, SI Min, ZHANG JingRong, XU Jie
    Scientia Agricultura Sinica. 2017, 50(20):  4003-4011.  doi:10.3864/j.issn.0578-1752.2017.20.014
    Abstract ( 290 )   HTML ( 4 )   PDF (509KB) ( 241 )   Save
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    【Objective】The experiment was conducted to study the effects of exogenous abscisic acid (ABA) treatment on wound healing of the potato tubers ‘Longshu No.3’, to analyze the role of phenylpropane metabolism and antioxidant enzymes in wound healing. 【Method】The potato cultivar ‘Longshu No.3’ was used as materials. The tubers were artificially damaged, then treated with different concentrations ABA, and healed under dark condition ((20±3)℃, RH (80±5)%). The healing ability of treated tubers was evaluated. The changes of activity of phenylpropane metabolism key enzymes, peroxidase and polyphenol oxidase were determined. The content of metabolites was determined at wounded site of tubers treated with 100mg·L-1ABA during healing. The formation of healing tissue was observed by scanning electron microscopy. 【Result】ABA treatment at different concentrations effectively promoted wound healing of tubers, the screened optimum concentration was 100 mg·L-1. ABA treatment at 100 mg·L-1 significantly increased the activity of phenylalnine ammonialyase, cinnamic acid hydroxylase, 4-coumaroyl-coenzyme A-ligase and cinnamyl alcohol dehydrogenase, sped the accumulation of total phenols, flavonoids and lignin at wounded site of tubers. In which, the activity of phenylalanine ammonialyase showed bimodal change, the treatment was 253.07% and 125.18% higher than the control after 3 days and 9 days of healing. The content of total phenols and flavonoids of the treatment showed 48.60% and 50.42% higher than that of the control. Moreover, the activities of peroxidase and polyphenol oxidase were significantly enhanced at wounded site of tubers. Observation of scanning electron microscopy showed that the formation of closing layer and wound periderm of tubers were accelerated after ABA treatment. 【Conclusion】Exogenous ABA treatment accelerates the formation of wound periderm of potato tubers by promoting the phenylpropane metabolism, and increasing the activities of peroxidase and polyphenol oxidase.
    ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT
    Effects of Different Carriers on Near Infrared Quantitative Model of Vitamin E in Premix
    WANG YanNi, CHEN YueYan, ZHA ShanShan, WANG Shi, XIAO ZhiMing, LI ShouXue, FAN Xia
    Scientia Agricultura Sinica. 2017, 50(20):  4012-4020.  doi:10.3864/j.issn.0578-1752.2017.20.015
    Abstract ( 229 )   HTML ( 8 )   PDF (1401KB) ( 250 )   Save
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    【Objective】Vitamin E is an important feed additive, the accurate determination of its content is of great significance for the preparation of dietary. In this paper, the rapid quantitative method of vitamin E in different carrier premixed feeds was studied. The aim of this paper was to describe the applicability of near infrared spectroscopy to different carrier premixed feeds, and to establish a near-infrared rapid measuring method for vitamin content in premixed feed.【Method】Commercial vitamin E powder was collected and mixed with the silica, defatted rice bran and stone powder. The premix of vitamin E in the concentration range of 5-250 IU·g-1was prepared by blending and collected its near-infrared spectra. This study compared the effects of different scanning times and resolution on the quality of the near infrared spectroscopy, compared the characteristic peak of vitamin E in premix of different carrier and different spectral preprocessing methods quantitative accuracy of PLS model and the difference of the model was analyzed from the point of view of VIP Score in PLS model. 【Result】 The results showed that 16 cm-1 resolution and 32 scans were suitable for spectral acquisition. There were significant differences in NIR spectra of premix with different carriers. The vitamin E absorption peaks of silica, defatted rice bran and stone powder as the carriers of the premix were 8 700-8 200, 7 100- 7 450, 6 020-5 550, 4 440-4 200, 5 210-4 760, 8 550-8 050, 7 410, 5 950-5 200, 4 400-4 050, 4 750-4 600 cm-1 and 8 550-8 050,   7 250, 5 950-5 200, 4 600-4 000, and 4 800-4 700 cm-1, respectively. The optimal spectral pretreatment methods for the near-infrared quantitative models were SNV+1stDeriv(2,15), MSC+1stDeriv(2,21)and SNV+2stDeriv(2,15), respectively, the calibration (Rc2)were 0.988, 0.949 and 0.941, respectively, the standard error of calibration (SEP)were 8.86, 10.36 and 23.37, respectively, the validation (Rp2) are 0.989, 0.980 and 0.921 respectively, the standard error of prediction(SEP)were 8.86, 10.36 and 23.37 respectively, and ratio of performance to standard deviation(RPD)were 8.10, 6.90 and 3.07 respectively. The VIP Scores value of PLS model with three different carrier diluents were extracted, and the contribution of the VIP Score values of silica were significant and that associated with vitamin E characteristic peaks was the highest, then was the skim rice bran and followed by stone powder.【Conclusion】Near-infrared spectroscopy for the analysis of vitamin E premixed feed, with the silica and skim rice bran as the carriers of the pre-mixed feed is better, with stone powder as the carrier of premixed feed, applicability of the measuring method is poor.
    Pharmacokinetics of Florfenicol in Ducks Infected with Riemerella anatipestifer After Intramuscular Administration
    MEI Xian, WAN Peng, HAN DongDong, PEI PengFei, ZENG QingLin, LI XiaoHong, ZENG ZhenLing
    Scientia Agricultura Sinica. 2017, 50(20):  4021-4027.  doi:10.3864/j.issn.0578-1752.2017.20.016
    Abstract ( 259 )   HTML ( 2 )   PDF (406KB) ( 238 )   Save
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    【Objective】The pharmacokinetic properties of florfenicol were studied and compared in both healthy ducks and ducks infected with Riemerella anatipestifer after intramuscular administration.【Method】A total of 240 ducks were randomly divided into 2 equal groups of 120 ducks each, one groups infected with Riemerella anatipestifer in the thigh. The two groups were injected with florfenicol injection following intramuscular (i.m.) administration at a single dosage of 30 mg·kg-1 body weight, respectively. Plasma samples were collected at 0, 5, 10, 15, 20, 30, 45 minutes and 1, 1.5, 2, 4, 6, 8, 10, and 14 hours after administration. The florfenicol concentration in plasma samples was analyzed using a high performance liquid chromatography (HPLC) method, with UV detector at 223 nm. The mobile phase (acetonitrile:water v/v 26/74) was delivered at a constant flow rate of 1mL·min-1. The column used was an Agilent C18 (4.6 mm×150 mm, 5 μm) column. A 20 μL aliquot of the reconstituted sample was injected onto the HPLC column. The drug concentration-time data were analyzed by non-compartmental analysis of pharmacokinetic software WinNonlin 5.2.1. Statistical analysis of pharmacokinetic parameters was carried out by statistical software SPSS17.0.【Result】The main pharmacokinetic parameters in healthy and infected ducks were as follows : the elimination half-life (t1/2β) was (2.57±0.51) h and (2.74±0.54) h. the peak time (Tmax) was (0.54±0.14) h and (0.55±0.18) h. the peak drug concentration (Cmax) was (22.88±3.11) μg·mL-1 and (19.77±1.82) μg·mL-1; the apparent distribution volume (Vd/F) was (2.39±0.81) L·kg-1 and (2.44±0.46) L·kg-1; the total body clearance (ClB/F) was (0.64±0.11) L·h-1·kg-1 and (0.63±0.08) L·h-1·kg-1; the area under the drug concentration-time curve (AUClast) was (47.28±7.87) μg·mL-1·h and (48.11±6.62) μg·mL-1·h, respectively. The pharmacokinetic parameters of florfenicol in healthy ducks and infected ducks were analyzed statistically. The results showed that there was no significant difference between the other parameters except Cmax (P<0.05).【Conclusion】The peak drug concentration (Cmax) in infected ducks was significantly lower (P<0.05) than that in healthy ducks. There was no significant difference between the other pharmacokinetic parameters. The characteristics of rapid absorption, high plasma concentration, wide distribution and faster elimination were showed following im florfenicol injection with a single dosage of 30 mg·kg-1 body weight in healthy and infected ducks.
    Identification the key areas of Bombyx mori Nucleopolyhedrovirus LEF-11 self-interaction
    JIANG YaMing, DONG ZhanQi, CHEN TingTing, HU Nan, DONG FeiFan, HUANG Liang, TANG LiangTong, PAN MinHui
    Scientia Agricultura Sinica. 2017, 50(20):  4028-4035.  doi:10.3864/j.issn.0578-1752.2017.20.017
    Abstract ( 191 )   HTML ( 2 )   PDF (1800KB) ( 313 )   Save
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    【Objective】Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the most common and most serious pathogenic microorganisms in sericulture, and LEF-11, one of the late expression factors of baculovirus, has been verified essential for virus proliferation. Related research proves that LEF-11 has the ability to form oligomers by self-interaction. the objective of this study is to explore the key areas of LEF-11 self-interaction via gradually truncated. 【Method】Bimolecular fluorescence complementation (BiFC) and fluorescent co-localization were utilized in this study. Firstly, the primers were designed based on the sequence of BmNPV lef-11 each truncated fragment as well as fluorescent protein sequence, all of the fragments were treated with restriction endonuclease after PCR. Then, pIZ/V5-His, the insect cell expression vector, was used to construct fluorescence fusion protein vector. Next, each truncated fragment fluorescence fusion protein vector was transfected together with pIZ-LEF11-GFP in BmN-SWU1, respectively. Cells were fixed after 48 h of the replacement of ordinary medium. Finally, the expression and localization of fluorescent fusion protein were observed under fluorescence confocal microscopy. As for the BiFC essay, the construction of vectors and the way of post processing were the same with fluorescence fusion protein.【Result】Both the 2-61 and 62-112 of the baculovirus LEF-11 protein could interact with the full length of the LEF-11 protein, and co-located in cytoplasm and nucleus, respectively. In the process of gradual truncation, LEF-11 protein N-terminal 2-61, 12-61, 22-61, 32-61, 42-61 and 2-51 could co-located with the full length of LEF-11 protein in cytoplasm, but N-terminal 52-61 and 2-41 did not co-locate. LEF-11 protein C-terminal 62-112, 72-112, 82-112, 72-101 and 72-91 could co-located with the full length of LEF-11 protein, but C-terminal 92-112 and 72-81 did not co-locate. The result of BiFC essay indicated that LEF-11 protein N-terminal 2-61, 12-61, 22-61, 32-61, 42-61 and 2-51 could interact with the full length of LEF-11 protein, but the 2-41 and 52-61 truncated fragments did not interact with the full length of LEF-11 protein. It was compatible with the result of N-terminal fragments fluorescence localization.【Conclusion】The 42-51 and 82-91 truncated fragments were identified as the key areas for the baculovirus LEF-11 protein self-interaction and could be used in LEF-11 protein related functional studies.
    RESEARCH NOTES
    Inhibitory Activity of Fludioxonil to Four Pathogenic Fungi of Peony Leaves
    XU JianQiang, PING ZhongLiang, LIU Ying, MA ShiChuang, XU DaoChao, YANG Lan, ZHENG Wei, LIU ShengMing, XIA YanFei, LIN XiaoMin
    Scientia Agricultura Sinica. 2017, 50(20):  4036-4045.  doi:10.3864/j.issn.0578-1752.2017.20.018
    Abstract ( 280 )   HTML ( 3 )   PDF (1319KB) ( 205 )   Save
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    【Objective】The objective of this study is to make clear the activity of fludioxonil on mycelial growth, conidia germination, germ tube elongation and sporulation of four peony foliar pathogenic fungi, including Alternaria suffruticosae, Phyllosticta commonsii, Hainesia lythri and Cladosporium paeoniae, and to analyze whether fludioxonil could be used in the chemical control of peony disease. 【Method】The inhibitory activity of fludioxonil against mycelial growth was measured through a mycelial linear growth test and the efficacy of fludioxonil on conidial germination, germ tube elongation and sporulation was determined on the PSA or KDA amended with fludioxonil at various concentrations. 【Result】Fludioxonil had a strongest inhibition on mycelial growth of H. lythri with the EC50 of 0.01 μg·mL-1, followed by 0.07 μg·mL-1 of A. suffruticosae and 0.35 μg·mL-1 of P. commonsii. Fludioxonil could strongly inhibit the conidia germination of four peony pathogenic fungi and the inhibitory activity on H. lythri was the strongest, with the EC50 of1.26 μg·mL-1. The EC50 to other three fungi ranged from 3.27 to 3.45 μg·mL-1. Fludioxonil could also strongly inhibit the germ tube elongation of four fungi, and the suppression ratio of 0.1 μg·mL-1 fludioxonil reached 40% to 70%. The germ tube of H. lythri was the most sensitive to fludioxonil, and the EC50 was 0.04 μg·mL-1. The EC50 of fludioxonil to other three fungi’s germ tube elongation varied from 0.08 to 0.22 μg·mL-1. Fludioxonil could cause the abnormality of conidia and the germ tube of A. suffruticosae and P. commonsii, including expanding of spore and germ tube and excessively branching of germ tube. However, the effect of fludioxonil on that of H. lythri and C. paeoniae was unconspicuous. Fludioxonil could delay sporulation of A. suffruticosae, P. commonsii and C. paeoniae. The EC50 of A. suffruticosae sporulation was 0.05 μg·mL-1, followed by the 0.38 μg·mL-1 of C. paeoniae, whereas fludioxonil could promote the sporulation of H. lythri.【Conclusion】Fludioxonil had a strong inhibitory activity on the mycelial growth, conidia germination and germ tube elongation of A. suffruticosae and H. lythri, but could promote the sporulation of H. lythri. Fludioxonil could also inhibit powerfully conidia germination and germ tube elongation of P. commonsii and C. paeoniae, mycelial growth of P. commonsii and sporulation of C. paeoniae.Fludioxonil should be used preferentially as a curative fungicide to avoid the pathogenic fungi infection, as it could not be absorbed by the plant tissue and inhibit the fungi that had penetrated and parasitized the plant.
    Molecular identification and sequence analysis of Ornithogalum mosaic virus in saffron (Crocus sativus) corms
    LIAO FuRong, LIN WuZhen, CHEN XiHong, CHEN Qing, CHEN HongYun, HUANG PengYing, FANG ZhiPeng, WU Yuan, SHEN JianGuo, LIN ShiMing
    Scientia Agricultura Sinica. 2017, 50(20):  4046-4054.  doi:10.3864/j.issn.0578-1752.2017.20.019
    Abstract ( 327 )   HTML ( 3 )   PDF (557KB) ( 426 )   Save
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    【Objective】The objective of this study is to identify viruses in saffron (Crocus sativus) corms imported from the Netherlands and to prevent the entry of dangerous virus into China. 【Method】Using the Potyviridae universal primers (Sprimer and M4T), the saffron corms samples were detected by reverse transcription polymerase chain reaction (RT-PCR) assay. After cloning and sequencing of the RT-PCR products, the Basic Local Alignment Search Tool (BLAST) and Multiple Sequence Comparison by Log-Expectation (MUSCLE) methods were used for multiple sequence alignment. The maximum likelihood method was used to construct the phylogenetic tree according to the coat protein (CP) nucleotide sequences. 【Result】The about 1.7 kb RT-PCR products were amplified from the samples, indicating a member of Potyviridae virus was present in the samples of saffron corms. A sequence of 1 666 bp segments was obtained from a positive sample (designated 2677-NL) and it contained a portion of nuclear inclusion b gene (NIb), the complete CP and 3′ untranslated region (3′-UTR). This isolate shared the highest nucleotide sequence identity (99.6%) with the SW3.3 isolate of Ornithogalum mosaic virus (OrMV) and shared 99.1% nucleotide sequence identity with the Reference genomic sequences (RefSeq) of OrMV (GenBank accession number: NC_019409). Of those, the CP of the 2677-NL isolate shared 71.5%-77.3% and 65.7%-79.7% nucleotide and amino acid identities, respectively, with those of six OrMV isolates from India (GenBank accession number: JQ686722, JQ686720, JN692498, JN692497, JN692496 and JF682235) whereas 77.9%-99.5% and 82.9%-99.2%, respectively, with the other 32 OrMV isolates. Phylogenetic analysis based on the CP genes showed that the OrMV could be divided into two groups, in which 2677-NL was clustered together with five OrMV isolates from Australia (GenBank accession number: JQ807995, JQ807996, NC_019409, AF185964 and AF185965), suggesting these OrMV were highly homologous in the phylogenetic relationship. 【Conclusion】According to the species demarcation criteria of the family Potyviridae on the International Committee on Taxonomy (ICTV), the 2677-NL was an isolate of OrMV, and it showed that the saffron corms sample was infected by OrMV. this is the first report on OrMV infecting C. sativus in the world.