Table of Content

16 July 2016, Volume 49 Issue 14

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

The Function Expression of Salt-Tolerant Yeast Gene Halotolerance ( HAL1 ) in Cotton

MU Min, SHU Na, WANG Shuai, GUO Li-xue, FAN Wei-li, YIN Zu-jun, WANG Jun-juan, WANG De-long, YE Wu-wei

Scientia Agricultura Sinica. 2016, 49(14):  2651-2661.  doi:10.3864/j.issn.0578-1752.2016.14.001

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【Objective】 The objective of this study is to clone Saccharomyces cerevisiae halotolerance (ScHAL1) gene and transformed into cotton , explore the function of the gene in cotton, to further explore function of the salt-tolerant yeast genes in higher plants. 【Method】According to total length of mRNA sequence information of ScHAL1 in NCBI, the gene was cloned using RT-PCR technology from Saccharomyces cerevisiae As2.375, double enzyme digestion was made with XbaⅠand SmaⅠfor pBI121::GFP, and pBI121-ScHAL1::GFP fusion expression vector was constructed by In-Fusion technique. With weak auto-fluorescence upland cotton varieties, Y-2067, ZA-23 and GZ-2 pollen as materials, using the gene bombarding technique to study transient expression of cotton pollen. The expression vector pBI121-ScHAL1::GFP was transformed into cotton salt-sensitive material Zhong s9612 with gene gun in vivo conversion technology, T0 generation cotton genetically seeds were modified. Solution with 100 mmol·L-1 NaCl was used to test the salt resistance of seeds in the transgenic T0 generation seed germination experiment, molecular detection was carried out, and semal salt resistance of transgenic plants was analysis. 【Result】 ScHAL1 cloned from Saccharomyces cerevisiae As2.375 and ScHAL1 is 885 bp in length, which encoding 294 amino acids. After its sequence analysis, it was found that the largest proportion of the whole HAL1 protein is serine, and it is alkaline and positively charged and it is a hydrophilic protein. According to the results of protein secondary structure prediction, it is speculated that the structure of the protein function domain may mainly made up of random curl and beta sheet. Cotton pollen instantaneous expression results showed that after conversion of HAL1 gene, three land cotton powder green fluorescence were obviously enhanced, suggesting that the gene expressed in these three upland cotton pollen. With 100 mmol·L^-1 NaCl, transgenic T0 generation seed germination ability obviously stronger than receptor s9612 selfing seed material, which preliminary showed that HAL1 could also improve the seed salt resistance. According to the gene nucleotide sequences, two pairs of primers were designed for molecular detection of T0 generation. Direct sequencing of PCR product was conducted and the sequencing results preliminarily evidence that the transgene is successful. With the semal salt resistance analysis, it was found that the chlorophyll contents of transgenic plants in 600 mmol·L^-1 NaCl and 400 mmol·L^-1 NaCl were higher than the control, and the chlorophyll contents of transgenic plants in 600 mmol·L-1 NaCl were higher than that in 400 mmol·L^-1 NaCl. 【Conclusion】HAL1 gene was Successfully cloned from Saccharomyces cerevisiae, yeast HAL1 gene plays an important role in improving cotton salt resistance.

Cloning and Expression Analysis of Strigolactones Biosynthesis- Related Gene ScCCD8 in Sugarcane

WU Zhuan-di, LIU Xin-long, LIU Jia-yong, ZAN Feng-gang, ZHAO Pei-fang, LIN Xiu-qin, CHEN Xue-kuan, SU Huo-sheng, LIU Hong-bo, WU Cai-wen

Scientia Agricultura Sinica. 2016, 49(14):  2662-2674.  doi:10.3864/j.issn.0578-1752.2016.14.002

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【Objective】The gene of Carotenoid Cleavage Dioxygenase8 (CCD8) from sugarcane (Saccharum officinarum L.) was cloned, then the sequence signature and functions were investigated, furthermore the gene expression in different tissues, different abiotic stresses and different growth times were analyzed. The objectives of the present study were to provide theoretical supports for the application of the ScCCD8 gene in sugarcane genetic engineering breeding.【Method】Using homologous cloning method to obtain the sequence of ScCCD8 gene from ROC22, reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends PCR (RACE-PCR) were used to clone the full-length sequence of ScCCD8. The bioinformatic characteristics of the ScCCD8 was analyzed using online service. The expression profiles of ScCCD8 in various tissues, in response to different stress treatments and different growth times were investigated using quantitative real-time PCR (qRT-PCR). 【Result】CCD8 was isolated from sugarcane, named ScCCD8, which was submitted to GenBank with accession number KP742973.1. It has 2 016 bp in length containing a 1 623 bp open reading frame (ORF) and encoding 540 amino acid residues. The molecular weights of ScCCD8 encoding protein were 59.534 kD, it is not a transmembrane protein that did not contain the signal peptide sites, indicating that it is not the secretory protein. Subcellular localization prediction showed that ScCCD8 might localize in chloroplast, and it contains several active sites such as phosphorylation sites and glycosylation sites. Comparison of protein sequences similarity analysis showed that ScCCD8 had more similarity with CCD8 from different plants. Phylogenetic tree analysis showed that ScCCD8 had the closest genetic relationship with Sorghum bicolor. Expression quantity of ScCCD8 was the highest in root, which is 18 times more than in old leaves. Analysis of the expression patterns in response to abiotic stress revealed that ScCCD8 is up-regulated by PEG（20% PEG）, NaCl（200 mmol·L-1 NaCl） and phosphorus deficiency （1/8 mmol·L^-1）and nutritional deficiency（cultured in pure water） in stem tip, and the obviously increase expression was found after treated 24 hours. The expression of ScCCD8 in stem tip were various in different growth times, moreover the expression was higher in germination stage then in tillering stage.【Conclusion】Sugarcane strigolactones biosynthesis-related gene ScCCD8 was cloned from ROC22, which is the member of CCD8 gene family. It is speculated that ScCCD8 might participated in plant resistance to abiotic stresses.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY

Effects of Supplemental Irrigation with Micro-Sprinkling Hoses on Flag Leaves Senescence and Photosynthetic Characteristics, Grain Yield and Water Use Efficiency in Winter Wheat

XU Xue-xin, WANG Dong

Scientia Agricultura Sinica. 2016, 49(14):  2675-2686.  doi:10.3864/j.issn.0578-1752.2016.14.003

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【Objective】The objective of the experiment is to study the effects of supplemental irrigation with micro-sprinkling hoses on flag leaves senescence, photosynthetic rate, grain filling rate, grain yield and water use efficiency in winter wheat. 【Method】 Field experiments were carried out in 2011-2013 growth seasons, using high-yield wheat cultivar Jimai 22. Three irrigation treatments were arranged with no irrigation during the whole growth stage (W0), supplemental irrigation with micro-sprinkling hoses (W1), traditional border irrigation (W2), to explore the changes of winter wheat under different treatments in leaf water potential, activities of superoxide dismutase (SOD) and catalase (CAT), chlorophyll fluorescence parameters, canopy apparent photosynthetic rate, grain filling rate, and so on. The irrigation stage in W1 was the same as that in W2. they were all irrigated once at jointing stage and anthesis stage respectively. W1 was irrigated with the micro-sprinkling hoses special for wheat (ZL201220356553.7). The soil water content was measured before irrigation. The target relative soil moisture content in the 0–140 cm soil layer after supplemental irrigation at jointing was set as 70% of field water capacity in 2011-2012 and 2012-2013. The target relative soil moisture content in the 0–140 cm soil layer after supplemental irrigation at anthesis was set as 70% and 65% of field water capacity in 2011-2012 and 2012-2013, respectively. The amount of the supplemental irrigation was calculated according to the irrigation quota formula. W2 was irrigated by the traditional border irrigation method. The inflow cutoff was set as 90% of border length, namely, stopping irrigation when the water reached 90% of the border length. The amount of irrigation was measured by water meter. W1 was the same as W2 in the specifications of experiment plot. In each experimental plot, the border width (the vertical distance between the center of two adjacent border ridges) was 2 m; the border ridge width was 0.4 m; the border length was 60 m and the plot area was 120 m². A 1.0 m wide unirrigated zone was maintained between adjacent plots to minimize the effects of adjacent treatments. Eight rows of winter wheat were planted in each experimental plot with row spacing of 22.9 cm. The micro-sprinkling hose was laid between the fourth and the fifth rows of wheat. A pressure-regulated valve and a flow-meter were installed at the head of each micro-sprinkling irrigation hose. The working pressure of each micro-sprinkling irrigation hose was 0.02 MPa. The irrigation water was pumped from well and then was transported to the inlet of micro-sprinkling irrigation hose or border through the PVC belt. The discharge per unit width of border irrigation was 4.6-5.2 L·m^-1·s^-1.【Result】During the two growth seasons, the supplemental irrigation amounts of W1 were 21.3-96.0 mm at jointing and 29.0-38.5 mm at anthesis. The irrigation water distribution uniformity of W1 reached 82.7%-97.0% after irrigation, not lower than that of the border irrigation with inflow cutoff designed as 90% (W2). The total irrigation amount of W1 reduced by 33.2-70.8 mm, saving 21.0%-54.2% of irrigation water, compared to that of W2. In contrast, there was no significant difference between W1 and W2 in the flag leaf water potential, the activities of SOD and CAT, the content of methane dicarboxylic aldehyde, the flag leaf maximum photochemical efficiency, actual photochemical efficiency, canopy apparent photosynthetic rate, grain filling rate, and grain yield. The water use efficiency of W1 increased by 2.1-2.9 kg·hm^-2·mm^-1 and reached 21.6-23.2 kg·hm^-2·mm^-1. 【Conclusion】 The irrigation amount applied at jointing and anthesis can be adjusted according to the precipitation and soil water content before irrigation by supplemental irrigation with micro-sprinkling hoses, to moderately supply the physiological water requirement of winter wheat for high-yield, and the irrigation water also can be uniformly and accurately sprayed into the field. This technology can excavate greater potential of winter wheat for water-saving.

Relationship Between Rhizosphere Soil Properties and Yield of Maize at Different Nitrogen Levels

ZHANG Xue-lin, XU Jun, AN Ting-ting, HOU Xiao-pan, LI Chao-hai

Scientia Agricultura Sinica. 2016, 49(14):  2687-2699.  doi:10.3864/j.issn.0578-1752.2016.14.004

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【Objective】Making the relationship between rhizosphere soil properties and maize grain yield clear could help managing nitrogen (N) fertilizer application, improving N use efficiency and reducing environmental pollution. 【Method】A field experiment with three N fertilizer treatments (Control: CK; 180 kg N·hm^-2:N180; 240 kg N·hm^-2: N240; 300 kg N·hm^-2: N300 and 360 kg N·hm^-2: N360) was established in 2012, and was carried out during maize growth periods in 2012, 2013, and 2014, respectively. Both rhizosphere soil and bulk soil NH₄⁺-N, NO₃^--N, urease, catalase, pH value, root and aboveground biomass and their N accumulation were measured at critical stages during maize growth periods, and their relationships with maize grain yield were analyzed. 【Result】In comparison with CK, the maize grain yield of four N fertilizer treatments increased by 23.85%, 36.40%, 39.87% and 34.78% for the three annual average, respectively, and their aboveground N accumulation was significantly higher than that of CK. rhizosphere soil NO₃^--N content of four N fertilizer treatments for their three years average increased by 23.38%, 57.13%, 57.87% and 69.74% in comparison with the CK, and 59.49%, 92.01%, 132.08% and 179.35% for their bulk soil NO₃^--N content, respectively. With the N fertilizer rate increasing, the bulk soil NH₄⁺-N content increased by 4.27%, 3.51%, 5.04% and 26.26%, respectively. Both rhizosphere soil and bulk soil pH decreased with the increasing of N application rate, their ranges were 4.5-6.7 and 5.5-7.2, and the bulk soil pH value was 5% higher than that of rhizosphere soil. Rhizosphere soil urease activity increased with the N fertilizer rate increasing, and bulk soil urease activities of four N treatments for their three years average increased by 4.02%, 14.73%, 24.55% and 19.64%, respectively, in comparison with the CK. Bulk soil catalase activities decreased with the N fertilizer rate increasing, and reduced by 3.03%, 5.09%, 8.24% and 12.67% in four N fertilizer treatments in comparison with CK. The rhizosphere soil and bulk soil characteristics of CK, N240 and N360 treatments were used to analyze their relationship with maize yield. Pearson correlation analysis showed that rhizosphere soil NO₃^- -N content at jointing stage; rhizosphere and bulk soil NO₃^- -N, rhizosphere soil NH₄⁺-N and bulk soil pH at silking stage were all significantly and positively correlated with grain yield. Rhizosphere soil urease enzyme activity and pH at silking stage in 2013 and 2014 were significantly correlated with grain yield, and rhizosphere soil and bulk soil NO₃^- -N content at maturity stage in 2013 and 2014 were also significantly correlated with the yield. Principal component analysis indicated that rhizosphere soil NO₃^- -N content, catalase activity, aboveground biomass and their N accumulation at jointing stage; rhizosphere soil and bulk soil NO₃^- -N content, rhizosphere soil NH₄⁺-N, bulk soil pH, aboveground biomass and their N accumulation at silking stage, and aboveground biomass and their N accumulation at maturity stage were all significantly correlated with grain yield.【Conclusion】All of these results indicated that according to the relationship between rhizosphere soil properties at different stages and maize grain yield, a suitable N fertilizer methods should be taken to ensure the soil available N supply, create a favorable rhizosphere soil environment, improve N utilization efficiency, and increase grain yield.

PLANT PROTECTION

Preparation of 8% Fenpropathrin·Cyflumetofen Nano-Emulsion and Its Performance

ZHAO Heng-ke, LAN Yue, NAN Can, HU Yue, RAO Ping, TIAN Ya, YAN Wei, QIAN Kun, HE Lin

Scientia Agricultura Sinica. 2016, 49(14):  2700-2710.  doi:10.3864/j.issn.0578-1752.2016.14.005

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【Objective】Pesticide mixtures can expand controlling spectrum, decrease pesticide dosage and the cost of production, and prolong the service life of insecticides. To a certain extent, nano-emulsion with low energy consumption and good stability can be developed. The effective components of the pesticide can produce biological effects better by pesticide formulation.【Method】Toxicities of fenpropathrin and cyflumetofen (two types of insecticide and acaricide) to Tetranychus cinnabarinus were determined by residual contact vial method. co-toxicity factor was used to evaluate the formulation synergism and co-toxicity coefficient method was adopted to screen the optimum ratio of pesticides, thus a mathematical model equation was fitted by using the method of optimum ratio and co-toxicity coefficient. Based on the optimum ratio, the 8% fenpropathrin·cyflumetofen nano-emulsion was processed by low-energy emulsification method. According to the characteristics of the nano-emulsion and the basic standards formulated by FAO, the quality control indexes of the product was developed. Finally, the performance of the nano-emulsion was primarily determined by the measurements of contact angle and adhesion work.【Result】After 24 h treatment, the LC₅₀ of fenpropathrin and cyflumetofen against T. cinnabarinus female adults was 711.62 and 4.32 mg·L^-1, respectively. Co-toxicity factors method showed that fenpropathrin and cyflumetofen complex had a synergistic effect. Results of the experiment showed that synergism was observed when the ratio of fenpropathrin to cyflumetofen was in range of 18﹕1 to 165﹕1. The synergetic ratio range of fenpropathrin to cyflumetofen is larger and it seems that this pesticide mixtures is feasible. Cotoxicity coefficient method showed the best synergistic effect of fenpropathrin﹕cyflumetofen was 50﹕1, co-toxicity coefficients was up to 209.96. The ratio mathematical model of fenpropathrin﹕cyflumetofen is y=-216.86x²+19201x-424807, R²=0.864, the optimum ratio in theory is 39﹕1, CTC=211.91. Through further cotoxicity coefficient and fitting equation analysis, it showed that toxicity regression equation is y=0.66x+3.8, r=0.9757, LC₅₀=60.96 mg·L^-1, the CTC value is 215.36 as the ratio of fenpropathrin to cyflumetofen was 39﹕1. Finally, this ratio of fenpropathrin to cyflumetofen (39﹕1) was selected as the optimum ratio for preparing nano-emulsion. Through optimization of solvent, emulsifier and water quality, the optimum formula is as follows: fenpropathrin 7.8%, cyflumetofen 0.2%, solvent 10% (ratio of 150# solvent naphtha﹕xylene=4﹕1), emulsifier 9%-11% (ratio of calciumalkylaromaticsulfonate﹕ polyoxyethylene aliphatate=2﹕3), propanetriol 2%, and water up to 100%. The 8% fenpropathrin·cyflumetofen nano-emulsion showed excellent performance, and the 100-fold dilution was pale blue homogeneous translucent liquid system with good dispersity. The experiment of contact angle showed that the 8% fenpropathrin·cyflumetofen nano-emulsion had the smaller contact angle and the 1arger adhesion work which was conducive to the absorption of plant leaves to liquid medicine and thus improve efficacy. 【Conclusion】 Optimal proportions was screened by means of co-toxicity factor, cotoxicity coefficient and fitting equation, which can be more comprehensively and objectively reflect the efficiency of the two compound agents, as well as to provide certain references for pesticide mixtures. It will provide some instructive significance to combined pesticides.

Preparation and Application of Monoclonal Antibodies Against Watermelon mosaic virus (WMV)

CHEN Zhe, SONG Ge, ZHOU Xue-ping, WU Jian-xiang

Scientia Agricultura Sinica. 2016, 49(14):  2711-2724.  doi:10.3864/j.issn.0578-1752.2016.14.006

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【Objective】The aim of this study is to prepare monoclonal antibodies (MAbs) against Watermelon mosaic virus (WMV) and develop effective serological assays for rapid and reliable virus detection, and to provide technology and materiel for diagnosis and detection, forecast and early warning and establishment of a scientific prevention and control system of the WMV disease.【Method】Using the purified WMV particles as an immunogen, hybridoma lines secreting MAbs specific for WMV were obtained via cell fusion, cell culture, antibody detection and cell cloning. The hybridomas were injected intraperitoneally into BALB/c mice to produce MAb-containing ascitic fluids. Based on the prepared MAbs, five detection assays, ACP-ELISA, DAS-ELISA, dot-ELISA, Tissue blot-ELISA and IC-RT-PCR were developed for accurately, sensitively and specifically detecting WMV in field plant samples. Besides, a dot-ELISA for specifically detecting WMV in individual viruliferous aphid was established.【Result】Three hybridoma lines (2C8, 15A8 and 16C12) steadily secreting MAbs specific for WMV and their MAb-containing ascitic fluids were produced. The titers of ascitic fluids of MAbs were up to 10-6 by indirect-ELISA. All these MAbs belong to IgG1 isotype, κ light chain. Western blot analyses indicated that all these three MAbs could specifically react with the coat protein of WMV. The ACP-ELISA, DAS-ELISA, dot-ELISA and IC-RT-PCR could detect WMV in infected plant crude extracts diluted up to 1﹕163 840, 1﹕327 680, 1﹕5 120 and 1﹕1 310 720 (w/v, g/mL), respectively. And the specificity analyses demonstrated that the developed ACP-ELISA, DAS-ELISA, dot-ELISA, Tissue blot-ELISA and IC-RT-PCR had strongly positive immune reactions with WMV-infected plant tissues, but had negative reactions with healthy, ZYMV-, CMV-, CGMMV-infected cucurbitaceous plant tissues or PVY-infected tobacco plant tissues. Besides, the developed dot-ELISA for detecting vector sample had a strongly positive immune reaction with individual viruliferous aphid, and negative reactions with non-viruliferous aphids. A total of 275 cucurbitaceous plant samples showing virus-like symptoms from Zhejiang, Jiangsu, Shandong and Hainan provinces in China were screened for the presence of WMV using the developed assays, and the detection results showed that 187 of the 275 plant samples were infected by WMV and the incidence rate was up to 68%, demonstrating that WMV is prevalent in field cucurbitaceous plants in China. And the detection results of serological assays were in agreement with those of RT-PCR. The sequences of PCR-amplified products were sequenced and compared with the WMV CP sequences. The results indicated that the nucleotide sequences of the PCR-amplified products were WMV CP gene segment, demonstrating that the positive samples tested by serological assays were really infected with WMV.【Conclusion】Three specific and sensitive MAbs against WMV and the five developed assays based on prepared MAbs in this study could accurately, sensitively and reliably detect WMV in field plant or vector samples, which would provide technology and materiel for rapid detection and diagnoses of field large-scale samples, forecast and early warning, scientific prevention and control of WMV disease in China.

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

Bioavailability and Fate of Nitrogen from ¹⁵N-labeled Corn Straw as Affected by Nitrogen Management and Straw Microbial Inoculants

DING Wen-cheng, LI Shu-tian, HUANG Shao-min

Scientia Agricultura Sinica. 2016, 49(14):  2725-2736.  doi:10.3864/j.issn.0578-1752.2016.14.007

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【Objective】The purpose of this study was to demonstrate the effect of rate of nitrogen fertilization, fertilizer N combined with manure N and straw microbial inoculants on the transformation and bioavailability of nitrogen (N) from crop straw for providing scientific information and guidelines on N management under straw returning conditions. 【Method】Pot experiments were carried out using ¹⁵N isotope techniques by continuously planting one season of winter wheat and two seasons of corn to study N availability from ¹⁵N labeled corn straw (¹⁵N-straw) and its contribution to plant and soil. The recommended N rate was 210 kg N·hm^-2, equivalent to 0.1 g N·kg^-1 soil. The crushed straw was incorporated into each pot at the rate of 3.0 g·kg^-1 soil. There were four N levels: control without N (CK), 100% fertilizer N, 80% fertilizer N and 80% fertilizer N plus 20% manure N. Each above treatment had two levels of microbial inoculants: 0 and 0.1 g·kg^-1 soil. A total of eight treatments with four replications for each were designed. 【Result】The percentage of N derived from ¹⁵N-straw (%Ndfs) in winter wheat plant was 6.30% to 14.25%, reduced by N application compared with CK. Combination of fertilizer N and manure N resulted in higher winter wheat %Ndfs than fertilizer N alone. Addition of microbial inoculants did not significantly influence winter wheat %Ndfs compared with treatments without microbial inoculants. The %Ndfs in the following 1st and 2nd corn plant was, respectively, 1.13% to 3.73% and 1.67% to 5.97%, reduced by N application but no difference existed between N treatments. Addition of microbial inoculants reduced corn plant %Ndfs from residual ¹⁵N-straw. Recovery of ¹⁵N (RE_N) from ¹⁵N-straw by winter wheat was 7.14% to 10.32%, while the residual RE_N from ¹⁵N-straw by the 1st and 2nd following corn was 3.75% to 5.51% and 2.28% to 3.18%, respectively. Total of 13.13% to 18.60% of ¹⁵N-straw N was recovered, 55.63% to 69.16% remained in soil and 17.26% to 26.09% lost after three times of cropping. N application increased RE_N compared with CK. N management did not influence RE_N from ¹⁵N-straw by winter wheat and the 2^nd corn, but 80% recommended fertilizer N decreased RE_N by the 1st corn and three crops, while increased when applied with manure. Addition of microbial inoculants significantly improved RE_N from ¹⁵N-straw by winter wheat and the 1st season corn and total of three cropping, reduced residual and loss of N from ¹⁵N-straw. The content of mineral N (N_min) and microbial biomass N (MBN) after crop harvests varied greatly, but the %Ndfs in N_min and MBN was all less than 3% which was not greatly influenced by N fertilizer management. While, addition of microbial inoculants increased %Ndfs in soil N_min as well as decreased %Ndfs in soil MBN after winter wheat and 1st corn，but no effect after 2nd corn. The percentage of remained ¹⁵N-straw in N_min and MBN after three crops was less than 3%, suggesting that the remained ¹⁵N-straw N in soil after three cropping was in organic form. 【Conclusion】Combination of chemical fertilizer N with manure N and addition of microbial inoculants is the recommended practice to increase N availability of straw under the conditions of straw returning to field.

Effects of Partial Water and Nitrogen Resupplies on Maize Root Nitrogen Absorbing Capacity and Distribution

NIU Xiao-li, HU Tian-tian, ZHANG Fu-cang, WANG Li, LIU Jie, FENG Pu-yu, YANG Shuo-huan, SONG Xue

Scientia Agricultura Sinica. 2016, 49(14):  2737-2750.  doi:10.3864/j.issn.0578-1752.2016.14.008

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【Objective】Water and nitrogen (N) resupplies can significantly enhance root absorbing capacity. Partial water and N supplies can stimulate the compensation effect of root absorbing capacity at the non-stressed sub-root zone. The objective of this study is to identify the dynamics and influencing factors of the compensation effect of maize roots (Zea mays L. hybrid cv. Aoyu No. 3007) N absorbing capacity under partial resupply after previous water and N stresses.【Method】With the split-root technology, a hydroponic experiment was conducted to analyze the root zone water and N stresses, where the water stress was stimulated by the osmotic potential of a nutrient solution (PEG 6000) and N stress was stimulated by different N levels. There were three water and N stress levels, i.e., mild, moderate, severe water and N stresses and a control treatment (CK, both sides of the root zone supplied with sufficient water and N). The root N inflow rate, N content and accumulation of each root zone were measured at 0, 1, 3, 5, 7 and 9 d of resupplying water and N in half of root-zone 6 d after water and N stresses.【Result】Compared with non-stressed sub-root, the root N inflow rate, N content and accumulation in stressed sub-root were significantly decreased under partial resupply after water and N stresses. During 1-3 days after treatment (DAT), the root N inflow rate in stressed sub-root reduced by 38.2% and 48.7%, respectively, and was 84.9% and 86.4% lower than that in non-stressed sub-root. For non-stressed sub-root, partial water and N resupplies significantly enhanced the root N inflow rate compared with previous water and N stresses during 0-1 DAT. When water and N stresses did not exceed moderate stress level, partial water and N resupplies significantly increased root N inflow rate compared with control treatment during 0-1 and 7-9 DAT. However, during 3-7 DAT, the root N inflow rate was similar to or lowers than control treatment. The root N content and accumulation in mild and moderate stress treatments returned to control level at 1 and 5 DAT, respectively, which resulted in similar plant N use efficiency to control treatment. Moreover, partial water and N resupplies significantly increased the percentage of ¹⁵N-fertilizer-N allocation in shoot compared with control treatment, and the increment reduced with the severity of water and N stresses. For non-stressed sub-root, the percentage of ¹⁵N-fertilizer-N allocation showed a reverse trend. For mild stress treatment, the percentage of ¹⁵N-fertilizer-N allocation of stressed sub-root had no significant difference compared with that of control treatment. The percentage of ¹⁵N-fertilizer-N allocation of stressed sub-root at moderate and severe stress treatments was significantly higher than that of control treatment, at 3-9 DAT, although there was no significant difference between moderate and severe stress treatments.【Conclusion】When previous water and N stresses did not exceed moderate stress level, the compensation effect of root N absorbing capacity in the non-stressed sub-root can be effectively stimulated by partial water and N resupplies. The compensation effect was affected by the severity and duration of the water and N stresses. Percentage of ¹⁵N-fertilizer-N allocation in different organs is closely related to the severity and duration of the water an N stresses. Thus, the above conclusion provides theoretical supports for regulating the interaction between plants and soil environment and making use of the potential plant response to soil water and nutrient stresses.

Sensitivity and Contribution Rate Analysis of the Influencing Factors of Spring Wheat Water Footprint in Hetao Irrigation District

SUN Shi-kun, LIU Wen-yan, LIU jing, WANG Yu-bao, CHEN Di-yi, WU Pu-te

Scientia Agricultura Sinica. 2016, 49(14):  2751-2762.  doi:10.3864/j.issn.0578-1752.2016.14.009

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【Objective】The efficient utilization of agricultural water resources is a key measure to guarantee national food and water security. Crop water use efficiency evaluation is one of the main research fields of agricultural water management. Water footprint provides a new index for agricultural water use evaluation, and the quantitative evaluation on the influencing factors of crop water footprint will be helpful to the implementation of water footprint control and improvement of the agricultural water use efficiency. 【Method】Based on the concept of water footprint, the water footprint of spring wheat in Hetao irrigation district was quantified by using an improved calculation method and the temporal variation was analyzed. Sensitivity and contribution rate analysis were used to quantify the relationship between crop water footprint and its influencing factors. 【Result】The results show that water footprint of wheat declined significantly during the study period. It decreased from 4.71 m³·kg^-1 in 1981 to 1.52 m³·kg^-1 in 2010. The variation of water footprint of wheat displayed an obvious stage characteristic. It can be divided into three stages: fluctuate declining stage (1981-1987), rapid declining stage (1988-1995) and slow declining stage (1996-2010). And this variation characteristic was consistent with the variation of agricultural production and irrigation level in Hetao irrigation district. The blue water footprint accounted for the larger proportion (more than 90%), while for the share of green water footprint it was relatively small. Therefore, the production of wheat in the Hetao irrigation district mainly depended on blue water (irrigation water). Sensitivity analysis shows that the difference of sensitivity between the influencing factors was significant. The variations of water footprint of spring wheat was ±30%, ±24%, ±2%, ±63% and ±4% when sunshine hours, relative humidity, precipitation, irrigation water use coefficient and fertilizer rate per unit area varied at ±20%. Irrigation water use coefficient is the most sensitivity factor of wheat water footprint, following by sunshine hours, relative humidity, fertilizer usage and precipitation. Contribution analysis results show that the decline of relative humidity and the increase of precipitation led to the increase of wheat water footprint. On the contrary, the decrease of sunshine hours combined with the increase of fertilizer usage and irrigation water use efficiency led to the decrease of wheat water footprint. The contribution rates of fertilizer and irrigation water use coefficient were -36.89% and -39.42%, respectively, while the contribution rate of the total climatic factors was 2.80%. The increase of utilization coefficient of irrigation water had the largest contribution rate to the decrease of wheat water footprint during the study period, followed by fertilizer usage. The relative humidity, sunshine hours and precipitation had similar contribution rate to the variation of wheat water footprint. 【Conclusion】 The main kinds of influencing factors of crop water footprint are climate, agricultural production inputs and water use efficiency. As for Hetao irrigation district, the improvement of agricultural production and water use efficiency are the major driving forces that cause the variation of wheat water footprint in Hetao irrigation district, while the climate factors have little effect on wheat water footprint. The results of this study could provide reference for the water footprint control.

HORTICULTURE

The Role of CBF Cold Response Pathway Gene in Heat Treatment-Induced Chilling Tolerance in Banana Fruits

WANG Hai-bo, LI Lu, SU Xin-guo, ZHANG Zhao-qi, PANG Xue-qun

Scientia Agricultura Sinica. 2016, 49(14):  2763-2771.  doi:10.3864/j.issn.0578-1752.2016.14.010

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【Objective】The objective of this study is to investigate the role of C-repeat binding transcription factor (CBF) cold-resistance pathway in heat treatment -induced chilling tolerance in banana fruits and to provide reference for the study of the signal transduction of CBF cold response pathway in banana fruits.【Method】The sequences of 7 genes related to CBF cold response pathway were selected from the banana genome database (http://banana-genome-hub.southgreen.fr/), and then specific primers were designed respectively. Analysis of these 7 genes’ expression patterns of the heat-induced chilling tolerance in banana fruits was conducted using quantitative real-time PCR.【Result】The expression of MaICE gene of banana fruit increased rapidly and reached the maximum level at 7℃ for 1 hour. The expression of DREB (MaDREB1D, MaDREB1E, MaDREB1G and MaDREB3) genes displayed a peak at 7℃ for 1 hour. The expression of MaCOR413 gene had a peak at 7℃ for 4 hours. These results indicated that the CBF cold response pathway (ICE-CBF-COR pathway) exist in banana fruit during cold storage at 7℃. The expression of 7 gene related to CBF cold response pathway reached the maximum levels at 0.5 hour after heat treatment (52℃ 3 min). Furthermore, when the heat treated banana fruits storage at 7℃ for 5 days, the expressions of MaDREB1D, MaDREB1E, MaDREB2C, MaDREB3 and MaCOR413 genes were higher than the non-heated control.【Conclusion】 MaICE, DREB, and MaCOR413 increased in turn when banana fruits stored at 7℃. The CBF cold response pathway exist in banana fruits during cold storage at 7℃. Enhancement of gene expression related to CBF cold response pathway may be involved in chilling tolerance induced by heat treatment in banana fruits.

Genetic Diversity and Structure of 255 Cultivars of Ziziphus jujuba Mill.

LIU Xiu-yun, LI Hui, LIU Zhi-guo, ZHAO Jin, LIU Meng-jun

Scientia Agricultura Sinica. 2016, 49(14):  2772-2791.  doi:10.3864/j.issn.0578-1752.2016.14.011

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【Objective】 There are abundant jujube germplasm resources in China. A total of 255 cultivars of Ziziphus jujuba Mill. from 22 provenances were used as materials to reveal their genetic diversity and phylogenetic relationship by SSR analysis, and the results of analysis would help us to manage jujube germplasm resources and offer references for molecular maker-assisted breeding.【Method】 Good genomic DNA was extracted from young leaves of jujube germplasm resources following the improved CTAB method, and then were amplified by simple sequence repeat molecular markers to analyze genetic diversity and genetic structure with the selected high-efficiency primer pairs which were excavated based on the genome sequencing. Separation of the amplified fragments was performed on 8% denaturing polyacrylamide gels and the gels were stained with AgNO3 for visualizing the SSR fragments. The data were counted by presence or absence of the band and the percentage of polymorphic loci (PIC) was calculated. UPGMA cluster analysis was carried by software NTSYS, the optimal number of groups and population genetic structure was analyzed by software Structure.【Result】Totally, 117 polymorphic alleles were revealed with 23 primer pairs which was selected from 64 primer pairs, each primer amplified polymorphic loci ranged from 2 to 10, with an average of 5.09 for each primer pairs. Polymorphism information content (PIC) values for the primer pairs ranged from 0.359 to 0.727, with an average of 0.548, these polymorphisms primers could be further applied to other study. The fingerprint for some jujube cultivars was established with 1-2 markers, providing a reference for the management of jujube germplasm. Meanwhile, based on the UPGMA cluster analysis, 255 cultivars were divided into fifteen subgroups, which included four big groups and eleven small groups. Similarity coefficients among the cultivars were between 0.71 to 1.00, ‘Beijinghuashengzao’ was clustered into one separate group, which has a distant relationship with other cultivars. The similarity coefficients of ‘Fengjiejidanzao’ and ‘Xupujidanzao’, ‘Shannxinaizao’ and ‘Tianjindamayazao’ were both 1.00. In some subgroups the genetic relationship between cultivars and their provenances has a significant positive correlation, but the cultivars and their uses has no significant correlation. Based on K and ΔK values, 255 jujube cultivars were also divided into fifteen populations by the population genetic structure analysis. The kinship among cultivars in the same population was relatively simple, and a few cultivars contained genetic component of other groups. The cultivars from Shanxi or Shannxi were distributed in most populations, indicating jujube cultivars of the two provinces played important roles in the gene exchange among populations. The jujube cultivars from Hunan of the South region formed a relatively alone population, indicating that the cultivars might be from the same source, or in the long-term cultivation few times of gene exchange were happened in Hunan cultivars with other populations. Different geographical environment played a key role in the evolution of jujube germplasm populations, some cultivars were selected from the same geographical environment and the others were selected by genetic recombination among those cultivars from the various geographical environment. Meanwhile, the consistency of the two different methods was further verifed the accuracy of the results, which provide useful clues and reference for the genetic diversity and structure of jujube germplasm.【Conclusion】Geographical environment play significant roles in the population evolution of jujube cultivars, affecting the genetic structure composition between different habitats.

Promoting the Research on Prevention & Control Technology of Waterfowl Infectious Disease and the Technical Reserves

LIU Yue-huan

Scientia Agricultura Sinica. 2016, 49(14):  2792-2795.  doi:10.3864/j.issn.0578-1752.2016.14.012

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VETERINARY SCIENCE

Preparation of Monoclonal Antibodies Against DPV and Development of Colloidal Gold Strip for DPV Detection

ZHAO Dan-dan, YANG Guo-ping, DIAO You-xiang, CHEN Hao, TI Jin-feng, ZHANG Lu, ZHANG Ying, LI Chuan-chuan

Scientia Agricultura Sinica. 2016, 49(14):  2796-2804.  doi:10.3864/j.issn.0578-1752.2016.14.013

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【Objective】 Duck plague（DP） is an acute, septic contagion, caused by duck plague virus（DPV）, with the characteristics of head and neck swelling, the mucosa of esophageal and cloacal bleeding and yellowish-white ulcer, head and neck skin has a yellowish-white gelatin sample. Once an outbreak of this disease manifested by with high morbidity and high mortality, it would cause serious harm to the duck industry. The aim of this assay is to establish a method of colloidal gold strip for the detection of duck plague virus (DPV) rapidly. 【Method】 The main antigenic domain of DPV was chosen and analyzed by using of the Protean Biology software to design a pair of primer to amplify the aim gene by PCR. Then the fragment was inserted into prokaryotic expression vector pET-28a to construct recombinant plasmid. Then it was transformed into Rosetta for expression. During the experiment, the authors have groped the concentration of the IPTG and the induction time. After purification, the concentration of the aim protein was tested and was also analyzed and identified by Western blotting. Hybridoma cell lines stably secreting monoclonal antibody against gB protein of DPV were generated by fusing SP2/0 myeloma cells with splenocytes from the immunized mice, which used the gB protein of DPV, expressed and purified with prokaryotic, as the antigen. The monoclonal antibody-based colloidal gold immunochromatography strip was developed for the detection of DPV. The purified DPV-gB monoclonal antibody, named H6F6, was labeled with colloidal gold, with the appropriate pH between 8.0 and 8.5 and the concentration was 15 times dilution. The purified A8D7 monoclonal antibody, with the concentration of twice dilution, and the goat anti-mouse immunoglobulin G (IgG) antibody, with the concentration of ten times dilution, were blotted on nitrocellulose membrane as test line and control line, respectively. 【Result】 Hybridoma cell lines designated as A8D9, E6C3, H11F8, H6A10, stably secreting monoclonal antibody against gB protein of DPV. The titres of ascitic fluid was1:10³, 1:10³, 1:10⁵, 1:10³, respectively by indirect ELISA and the immunoglobulin subtype of the monoclonal antibodies was IgG2b, IgG2a, IgG2b, IgG1,with the light chain of kappa. The result of western blot showed that the four monoclonal antibodies were able to specifically recognize gB protein of DPV. The result of IFA showed that the four monoclonal antibodies were specific to DPV. The detection results indicated that the strip was specific to DPV and had no cross reaction with DRV, EDS-76V, AIV-H9N2, and TMUV. The detection limit of DPV were 50 times dilution. 38 clinical suspected samples were simultaneously detected by immunochromatography strip and PCR while the results showed 91.6 % accuracy between them. The monoclonal antibodies-based colloidal gold strip was highly specific and sensitive and more convenient for the clinical diagnosis of DPV.【Conclusion】The colloidal gold strip was highly specific and sensitive and more convenient for the clinical diagnosis of DPV.

Construction and Characterization of Recombinant Duck Enteritis Virus Expressing the Green Fluorescent Protein

SUN Ying, LI Jun-ping, HUANG Xiao-jie, LI Ling, CAO Ming-hui, LI Qi-hong, LI Hui-jiao, YANG Cheng-huai

Scientia Agricultura Sinica. 2016, 49(14):  2805-2812.  doi:10.3864/j.issn.0578-1752.2016.14.014

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【Objective】Compared with duck enteritis virus(DEV) virulent strain, the vaccine strain has a 528 bp deletions at the UL2, resulting to a 176 aa deletion after amino acids 65. To study the effect of UL2 gene on virus biological properties and explore the feasibility of the DEV as a carrier to express foreign gene, a recombinant DEV expressing the green fluorescent protein (GFP) were constructed;【Method】In this study, the UL2 gene of DEV was chosen as a target site and homologous arm for recombination. Two fragments of UL2 gene were amplified by polymerase chain reaction (PCR) with DNA of DEV cell-adapted strain as template, and were cloned into the pMD-18T vector. The expression cassette including GFP gene and gpt gene controlled by CMV promoter was cloned into UL2 gene as a transfer vector pT-UL2-GFP-gpt. Confluent CEF monolayers were transfected with DEV and Lipofectamine 2000 was used as the transfer vector. When the cytopathic effect (CPE) was observed, the total supernatant and cells were harvested. The infected virus was diluted and then plated on the fresh CEF, and overlaid with M199-FBS containing 1% agarose. When green fluorescent plaques were observed, plaque-purification was carried out to obtain a green fluorescent plaque population termed rDEV-△UL2-GFP-gpt, PCR and sequencing assay were used to identify the recombinant virus. CEF cells cultured in 25cm2 flasks were inoculated with recombinant virus at an MOI of 0.01. The cells and supernatants were harvested respectively every 12 hours, the titer of virus were measured and the one-step growth analyses was performed; To evaluate the genetic stability of GFP gene in the recombinant virus, the virus was passaged in primary CEF 20 times. Four-week-old specific- pathogen-free (SPF) ducks were inoculated intramuscularly with the recombinant virus, and the ducks were challenged with lethal DEV (CVCC AV1221) by intramuscular injection at 14 days post vaccination, then the ducks were observed for symptom of disease and death.【Result】The recombinant expression vector pT-UL2-GFP-gpt was correctly constructed, identified by double-enzyme digestion. After 8 hours of transfection, spindle cells with green fluorescent were appeared. After 8 rounds of plaque-purification, the purified rDEV-△UL2-GFP-gpt were obtained. The results of the PCR and sequencing indicated that the GFP expression cassette has already successfully insert into the DEV genome, which replaced 196-723 nucleotide of UL2. The recombinant virus possessed growth kinetics were similar to that of the parental virus, the cell titer peaked at 36 hours with the peak titer 106.2TCID50/0.1mL, and the supernatant titer peaked at 72 hours with the peak titer 105.5TCID50/0.1mL. The virus were passaged in CEF cells 20 times, the GFP gene was stably maintained in 1st to 5th passages, however, from the 6th passage, there was little CPE without green fluorescent, and in 15th to 20th passages, most CPE had no green fluorescent, GFP mutated during subculture. All immunized animals were protected against subsequent challenge with lethal DEV, the insertion of the GFP gene did not alter the protective efficacy of parental virus. 【Conclusion】In this research, the recombinant DEV expressing the green fluorescent protein were successfully constructed, and firstly has confirmed that the deletion of UL2 gene has no effect on virus replication in cells and the immunogenicity in ducks. This study laid a foundation for the research of the function of the DEV UL2 gene and the DEV vector vaccine.

Construction and Characterization of a Recombinant Duck Enteritis Virus Expressing VP2 Gene of Goose Parvovirus

CHEN Liu, YU Bin, NI Zheng, HUA Jiong-gang, YE Wei-cheng, YUN Tao, ZHANG Cun

Scientia Agricultura Sinica. 2016, 49(14):  2813-2821.  doi:10.3864/j.issn.0578-1752.2016.14.015

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【Objective】Duck enteritis virus (DEV) and goose parvovirus (GPV) are considered to be two of the most important and widespread viruses infecting ducklings, Muscovy ducklings and goslings. According to the most recent virus taxonomy reported in 2012 by the International Committee on Taxonomy of Viruses (ICTV), DEV (also referred to Anatid herpesvirus 1) is classified into the genus Mardivirus, the subfamily Alphaherpesvirinae of Herpesviridae. Many herpesviruses, such as Pseudorabies virus (PRV), Marek's disease virus (MDV), Herpesvirus of turkey（HVT）have been widely made as live viral vector for the expression of foreign antigens, and there were some reports about DEV as live viral vector in recent years. To control DEV and GPV infection, a recombinant vectored DEV expressing GPV VP2 was constructed in this study based on the bacterial artificial chromosome (BAC) clone pDEV-EF1 which carries DEV full-length genome (Chen L, et al. , 2015), and then the biological characteristics of the obtained recombinant virus rDEV-VP2 were analyzed to explore the possibility of rDEV-VP2 as duplex live carrier vaccine. 【Method】 The recombinant BAC clone pDEV-VP2 carrying GPV VP2 gene was generated by two-step Red/ET recombination in E. coli. pDEV-VP2 was constructed by inserting codon optimized-GPV VP2 expression cassette between DEV US7 and US8 genes on pDEV-EF1. The recombinant viruses rDEV-VP2 and rDEV-VP2-Cre without BAC sequence were rescued from chicken embryo fibroblasts (CEFs) by calcium phosphate precipitation. And the growth curve in vitro, plaque size and expression of GPV VP2 in CEFs were analyzed. The antibody level of GPV VP2 in sera of rDEV-VP2-incoculated ducklings was detected by an indirect-ELISA method based on the GPV VP2 protein. 【Result】 The recombinant viruses rDEV-VP2 and rDEV-VP2-Cre were rescued from chicken embryo fibroblasts (CEFs) by calcium phosphate precipitation. Growth curves show that the growth kinetics of rDEV-VP2 was basically consistent with those of parental virus in vitro. And the plaque size of rDEV-VP2 was slightly increased compared to the parental virus rDEV-BAC. Immunofluorescence assay and Western blot analysis showed that GPV VP2 protein is expressed in recombinant virus-infected CEFs. And the rDEV-VP2 infection could induce 7-day-old Muscovy ducklings to produce antibody specific for GPV VP2. 【Conclusion】 In this study, the antigen gene VP2 of GPV was inserted into the genome of DEV US7 and US8, and an recombinant infectious BAC clone of DEV was successfully constructed. Then the corresponding recombinant virus rDEV-VP2 was rescued, and its cellular growth characteristics were basically consistent with those of parental virus, and rDEV-VP2 could induce Muscovy ducklings to produce VP2-specific antibody. These studies have laid a foundation for developing bivalent vaccine controlling DEV and GPV infection.

Selection of a Live Chicken Embryo Attenuated Duck Tembusu Virus Vaccine

YU Ke-xiang, MA Xiu-li, YUAN Xiao-yuan, LIU Cun-xia, HU Feng, LING Hong-li, LI Yu-feng, HUANG Bing

Scientia Agricultura Sinica. 2016, 49(14):  2822-2829.  doi:10.3864/j.issn.0578-1752.2016.14.016

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【Objective】 Tembusu virus BZ-2010 strain was continuously passaged in specific-pathogen-free embryonic (SPF) eggs in order to select a live attenuated vaccine candidate of good safety and immunogenicity properties. 【Method】 Tembusu virus BZ-2010 strain was cultured for 120 passages in SPF eggs. The safety of the 120th passage viral strain was evaluated with 1-day-old SPF ducklings and 30-week-old egg-laying ducks. The property of virulent return of VC2 viral strain was evaluated with 1-day-old SPF ducklings. The neutralizing antibodies were detected after the 18-week-old breeding ducks were immunized with VC2 strain. The protective effects were evaluated after the 25-week-old breeding ducks were immunized with VC2 strain. E gene and NS4A gene of BZ_2010 and VC2 strains were amplified by RT-PCR and sequenced. 【Result】 The average death time of SPF eggs was shortened by passage virus and viral titer was increased with the escalation of passage times in SPF chicken embryonic eggs. ELD₅₀ of the 20th virus was 10-5.3/0.1mL and ELD₅₀ of the 120th virus was 10-5.8/0.1mL.The viral titer reached the plateau at passage 80 and remained unchanged further passages. The experimental ducks showed no clinical symptoms after 1-day-old ducklings and 30-week-old breeding ducks were immunized with VC2 strain by subcutaneous injection and by intramuscular injection, respectively. The results showed that VC2 strain had a good safety. No symptoms appeared in 1-day-old ducklings in which VC2 strain were cultured for 5 passages. 1-day-old ducklings were infected with the 5th tissue suspension and no symptoms were observed in liver pathological section by microscope. The results showed that VC2 strain had a good stability. Sequence analysis revealed that the E protein of Tembusu VC2 evolved amino acid changes in positions 86, 157, 189, 301, and 302, respectively. The NS4A protein of Tembusu VC2 only had one amino acid change in position 54 in that phenylalanine was replaced by Leucine. The level of antibodies rose very quickly, reached the plateau at the 4th week and remained a long time. Ducks were challenged by TMUV virulent strain at 2 and 50 weeks after immunization with VC2 strain in the experimental group. There was no symptom, normal stool, and regular egg production in the vaccinated group after challenge of virulent strain. The results showed that VC2 strain could provide complete protection for the challenge of TMUV virulent strain.【Conclusions】 An attenuated strain of TMUV with good immunogenicity and high safety was acquired through serial passages of SPF chicken embryos. The level of antibodies rose very quickly and remained a long time after immunization of the VC2 attenuated strain. The toxicity attack experiments showed that VC2 could provide complete protection for the challenge of TMUV virulent strain.

Criterion of Ovarian Lesions of Duck Tembusu Virus Disease

LIN Jian, YANG Zhi-yuan, HE Ping-you, DUAN Hui-juan, ZOU Li-hong, YANG Bao-shou, ZHAO Ji-cheng, PAN Jie, WANG Xiao-lei, LIU Li-xin, LIU Yue-huan

Scientia Agricultura Sinica. 2016, 49(14):  2830-2836.  doi:10.3864/j.issn.0578-1752.2016.14.017

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【Objective】The objective of this study is to understand the process and regularities of ovarian lesions on ducks infected with duck Tembusu virus, and to provide data for the efficiency evaluation of vaccine using laying ducks ovary pathological inspection.【Method】Seventy 260-day-old laying cherry ducks were inoculated intramuscularly with duck Tembusu virus (HB strain) at dosage of 0.5ml(100 DID₅₀)/duck. Clinical symptoms were observed, and the egg production and feed intake were recorded daily. Serum samples were collected from all ducks for virus isolation via wing vein on 2 days post inoculation (dpi). Each serum sample was inoculated into five 6-day-old SPF chicken embryos via yolk-sac route at the inoculum of 0.1 ml per embryo. Then they were hatched at 37℃ further. The chicken embryos died in 24h were abandoned. The viral nucleic acid was detected by RT-PCR in the death chicken embryos during 24-72 h. If there were more than one (including one) death chicken embryos, and the nucleic acid testing was positive, then it was concluded that virus isolation was positive. On 4-10 dpi, 10 ducks were necropsied and the gross lesions of the reproductive system were observed every day. The pathologic rate was calculated, and the gross lesions of the reproductive system were conducted including whether there were eggs in the fallopian tube, whether the follicle was deformed, hemorrhaged or ruptured or not. According to the statistical pathologic rate, the time, the content of the examination and the criterions for determining lesion ovary were confirmed. 【Result】 (1) Feed intake and egg production decreased significantly on 3-6 dpi. The mental state of the duck on 7 dpi improved, and feed intake began to rise on 8 dpi. (2) Virus isolation of 70 ducks were all positive except one duck. The virus positive isolation rate was 98.6% (69/70) on 2 dpi. (3) On 4-10 dpi, a total of 64 laying duck reproductive organs can be determined. (4) On 4-10 dpi, the ovarian lesions rate were 66.7% (6/9), 100% (10/10), 100% (10/10), 100% (9/9), 100% (9/9), 100% (9/9) and 100% (8/8) respectively. (5) Eggs were found in fallopian tube of 2 ducks among 10 ducks that were necropsied on 4 dpi, and there was no egg found in fallopian tube of the remaining 62 ducks. The egg negative rate was 96.9(62/64). (6) On 4-10 dpi, the proportion of deformed follicular duck and hemorrhaged follicular duck were both 96.9% (62/64), and the proportion of deformed and hemorrhaged follicular duck was 95.3%（61/64）, while the proportion of ruptured follicular duck was 34.4% (22/64).【Conclusion】 (1) The time for the examination of the pathological changes of ovary was determined as 7 to 8 dpi. (2) The criterion of abnormal follicle is that one of the lesions of deformation and hemorrhage or both were found. The criterion of lesion ovary is that three abnormal follicles or more appeared and no egg in the fallopian tube.

Dynamic Study on Maternal Antibody of Duck Tembusu Virus Disease Inactivated Vaccine (HB Strain)

HAN Chun-hua, ZHAO Ji-cheng, DUAN Hui-juan, LIN Jian,YANG Zhi-yuan, XIE Jia, PAN Jie, WANG Xiao-lei, LIU Li-xin, LIU Yue-huan

Scientia Agricultura Sinica. 2016, 49(14):  2837-2843.  doi:10.3864/j.issn.0578-1752.2016.14.018

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【Objective】The objective of this study is to evaluate the efficacy of maternal antibodies induced by Duck Tembusu Virus Disease Inactivated Vaccine and to determine the age of optimal initial immunity.【Method】Fertilized eggs were collected at random from the Cherry Valley Duck farm which was 135 days post-vaccination with Duck Tembusu Virus Disease Inactivated Vaccine (HB strain), ten progeny ducklings from the immunized breed ducks and 5 progeny ducklings from non-immunized breed ducks were randomly selected when they were 5 ,7 ,10, and 15 days old. Serum samples were collected from all ducks for the detection of maternal antibody, then the ducks were challenged with Duck Tembusu virus (HB strain) at 0.1ml（100DID₅₀）/duck intramuscularly. Clinical symptoms of the challenged ducks were observed within 10 days, such as food intake, feces, abnormal clinical sighs and death. Serum samples were collected from all ducks for virus isolation via jugular vein on 2 days post inoculation (DPI). Each serum sample was inoculated into five 6-day-old SPF chicken embryos at the inoculum of 0.1 ml per embryo. Then they were hatched at 37℃ for 168h. The chicken embryos died within 24h were discarded. If more than one (including one) death chicken embryos were obsearved, then it was concluded that virus isolation was positive. The rate of protection of ducklings with maternal antibody and the morbidity of ducklings without maternal antibody were calculated. On 5 dpi, all ducklings were weighed respectively, and the average daily gain was calculated. The effect of maternal antibody on the weight gain of ducklings were analyzed by T test for paired samples. The efficacy of maternal antibodies was evaluated by neutralizing antibody titer, body weight changes and virus isolation.【Result】 (1) The number of positive maternal antibody titers peaked in 1 day old ducklings was 56.1%（37/66）, then fell to 40% (4/10) in ducklings on day 5, 50% (5/10) on day 7, 30% (3/10) on day 10, and 0% (0/10) on day 15. (2) On viral challenge, the control group showed signs of depression (20/20), neurologic disturbances (6/20) and death (2/20). Ducklings with positive maternal antibody titers showed mild depression. (3) On 5 dpi, the average daily gain of 5-, 7-, 10- and 15-day old ducklings with maternal antibody were 115.5, 142.8, 177.8 and 162.2g, respectively, and that of the ducklings without maternal antibody were 54.5, 91, 165 and 118.8g, respectively. (4) The rate of protection against challenge with DTMUV of 5-, 7-, 10- and 15-day old ducklings with maternal antibody were 50%(5/10), 60%(6/10), 20%(2/10) and 0%(0/10), respectively. The morbidity of 5-, 7-, 10- and 15-day old ducklings without maternal antibody were all 100%. (5) The average weight gain and efficacy reached a peak in 5-day old and 7-day old ducklings, which were 50% (5/10) and 60% (6/10), respectively. Although the maternal antibodies decreased between 10 days old and 15 days old ducklings (20% and 0%), it still has protective effect compared with the control group. 【Conclusion】(1) Duck Tembusu Virus Disease killed vaccine maternal antibodies, so it play an important role in the protection of 10-day-old ducklings against virus infection; (2) Vaccination age is optimized between 7 to 10 days of age.

The Pathogenicity of Duck Reovirus on SPF Chicken Embryo

LIU Xiao-li, LIU Ting, LIU Bo, CHENG Guo-fu, GU Chang-qin, ZHANG Wan-po, HU Xue-ying

Scientia Agricultura Sinica. 2016, 49(14):  2844-2849.  doi:10.3864/j.issn.0578-1752.2016.14.019

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【Objective】Previous studies showed that avian reovirus could infect vertically through egg, and avian reovirus can cause avian viral arthritis, respiratory and intestinal disease, myocarditis, hepatitis, immune suppression and so on. HP080421 strain of duck reovirus(DRV) isolated in the authors’ laboratory can cause soft duck feet as the main clinical features, and sick duck showed that a lot of white necrotic stove was on the surface of liver and spleen, and also kidney swelling and bleeding as the main pathological features. In this study, the pathogenicity of DRV to chicken embryo was investigated and whether the isolated HP080421 strain could infect chickens through the pathological changes of chicken was discussed, in order to provide a theoretical basis for the prevention and control of DRV infection.【Method】The infection model of SPF chicken to DRV was established through allantoic cavity inoculation SPF chicken embryos by using the isolated and identified HP080421 strains of DRV isolated in the lab. After chicks hatched from embryos, pathological examination methods such as clinical observation, pathological section examination, HE staining and immunohistochemical staining were used to study the pathobiology and pathogenicity of the SPF chicken embryo infected by DRV.【Result】Clinical observation found that chicken embryos were able to peck the shell after 22 - 23 days, but could not go out from the eggshell by themselves compared to the control group. At necropsy, liver and spleen were breakable and slightly swelling, many different size and yellow-white necrotic foci were consistently observed in the spleen and liver of the experimental group; the brain tissue was slightly swelling with few bleeding spots covered on it. Histopathological examination of H.E staining revealed necrotic foci in spleen and liver, which consisted of a necrotic center with lymphocytes infiltration at the periphery; in the Bursa of Fabricus, as well as in the thymus, lymphocyte depletion was apparent and cavities had developed in medulla. Besides, other organs such as lung, brain and kidney, showed different degrees of congestion and edema. Immunohistochemical detection showed that liver, lung, spleen and bursa of fabriciusa had positive signals, and were located in the cytoplasm and nucleus of epithelial cells and macrophages.【Conclusion】The results showed that the virus strain HP08421 could infect SPF chicken embryo and cause some specific pathologic changes. The pathological changes mainly focus on the liver and lymphoid organs, so DRV can be infected by vertical transmission of chickens, and can also lead to immune suppression. This study has expounded the possibility of infection of chickens by DRV, through the infection model of SPF chicken to DRV, as a matter of fact, in the actual process of production, farmers should prevent chicken embryo pollution by DRV to cut off the route of transmission, to achieve the purpose of preventing DRV infection.

RESEARCH NOTES

Analysis of Resistance of Different Tomato Varieties to Tomato yellow leaf curl virus in Tomato

TIAN Zhao-feng, LIU Wei-cheng, HOU Ling-yu, XIE Hua, LUO Chen, CHAI Min

Scientia Agricultura Sinica. 2016, 49(14):  2850-2856.  doi:10.3864/j.issn.0578-1752.2016.14.020

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【Objective】Tomato yellow leaf curl virus (TYLCV) disease is a serious threat to tomato production worldwide, breeding resistant varieties is the most economical, effective and friendly way to control this disease. Most of the tomato varieties resistant to TYLCV used in production of China were introduced from abroad. Many new tomato varieties resistant to TYLCV have been obtained by breeding, but the resistance is unknown. In this paper, the resistance levels of the new tomato varieties resistant to TYLCV bred by Vegetable Research Center, Beijing Academy of Agriculture and Forestry Sciences were assessed and the expression of resistance genes Ty-1/Ty-3a and the pathogenesis-related proteins glucanase/chitinase were analyzed. The purpose is to provide a reference for rational utilization of germplasm resources and reduce the harm of TYLCV.【Method】Four tomato varieties Qiuguang 285, Peng 137×246, Qiuguang 120 and Qiuguang 81 were used as the experimental materials. TYLCV disease incidence was observed in the field. The disease resistance of these tomato varieties was examined by investigating the disease incidence and index. The resistance levels of the new tomato varieties resistant to TYLCV were assessed and the expression of resistance genes and the pathogenesis-related proteins were analyzed by RT-PCR. 【Result】 The resistance of the hybrid was no obvious differences with that of the pure and the resistance of the two genes was a little higher than that of single resistance genes, but yet no significant differences. Semi-quantitative RT-PCR results showed that the expression of genes Ty-3a and Ty-1 increased with the increase of the incidence of TYLCCV in different tomato varieties. The expression level of Ty-3a in four tomato varieties from weak to strong is Qiuguang 285, Peng 137×246, Qiuguang 120 and Qiuguang 81. The expressionof Ty-1 in Qiuguang 81 was obviously higher than that of in Qiuguang 120. The gene expression of glucanase and chitinase in vivo was significantly higher than that in the control. The expression of the two genes increased with the increase of the time. In the 20 and 30 days after appearing of disease, the expression of glucanase gene in Qiuguang 81 with the Ty-1+Ty-3a was significantly higher than that in other varieties.【Conclusion】The resistance of four tomato varieties to TYLCV from weak to strong is Qiuguang 285, Peng 137×246, Qiuguang 120 and Qiuguang 81. After the infection of TYLCV, the expression levels of resistance genes increased with the time of disease in different tomato varieties. The expression levels of glucanase and chitinase genes in vivo were significantly higher than those in the control. The results suggested that the high resistance of variety to TYLCV is related to the high expression of resistance gene and pathogenesis-related proteins.