Scientia Agricultura Sinica

Evaluation of Heat Tolerance in Wheat Germplasm Resources

WANG XiaoBo,GUAN PanFeng,XIN MingMing,WANG YongFa,CHEN XiYong,ZHAO AiJu,LIU ManShuang,LI HongXia,ZHANG MingYi,LU LaHu,WEI YiQin,LIU WangQing,ZHANG JinBo,NI ZhongFu,YAO YingYin,HU ZhaoRong,PENG HuiRu,SUN QiXin

Scientia Agricultura Sinica. 2019, 52(23): 4191-4200. doi:10.3864/j.issn.0578-1752.2019.23.001

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【Objective】 High-throughput evaluation of winter and spring wheat accessions for heat tolerance via heat susceptibility index (HSI) could provide the potentially superior accessions for heat-tolerant breeding programs. 【Method】 In order to expose plants to high temperatures during grain filling period, winter wheat accessions were sown in normal and late seasons, and spring wheat accessions were sown in different geographical environments with contrasting temperatures. The thousand grain weight (TGW) of winter and spring wheat accessions were measured under normal and heat stress environments, respectively. HSI was calculated from the TGW data of two different conditions. Using heat susceptibility index, 1 325 wheat germplasms from different wheat ecological zones of China, and international areas and organizations, including 688 winter wheat accessions and 637 spring wheat accessions, were evaluated for heat tolerance. Genotypes were classified into four tolerant grades, i.e. highly heat-tolerant (HSI<0.50), medium heat-tolerant (0.5≤HSI<1), medium heat-susceptible (1≤HSI<1.5) and highly heat-susceptible (HSI>1.5). 【Result】 The average maximum temperature at grain filling stage under heat stress condition was higher than that of the controls by 1.91℃ for winter wheat and 7.09℃ for spring wheat, respectively. TGW under heat stress condition was significantly lower than that of the corresponding control. According to the grading evaluation results of HSI, thirty-one and 48 highly heat-tolerant winter and spring wheat accessions accounted for 4.51% and 7.54% of the test materials, 19 and 58 highly heat-susceptible winter and spring wheat accessions accounted for 2.76% and 9.11% of the tested materials, and the rest were medium germplasms (medium heat-tolerant and medium heat-susceptible). According to the geographical distribution of wheat ecological regions, winter wheat from the southern wheat region (Southwestern Winter Wheat Zone, Qinghai Tibetan Plateau Spring and Winter Wheat Zone, and Middle and Lower Yangtze Valley Winter Wheat Zone) were more tolerant than that from northern wheat region (Northern Winter Wheat Zone, and Yellow and Huai River Winter Wheat Zone). For spring wheat, the average HSI of accessions from Xinjiang Spring and Winter Wheat Zone was 0.70, which was the most heat-tolerant, and 88.00% of the accessions belong to heat-tolerant (highly heat-tolerant or medium heat-tolerant) germplasms. In addition, the average HSI of spring wheat from the International Center for Agricultural Research in the Dry Areas (ICARDA) with 0.88 showed heat-tolerant. The synthetic hexaploid wheats from CIMMYT had the weakest heat tolerance, with an average HSI of 1.18, of which 69.58% were heat-susceptible germplasms (medium heat-susceptible and highly heat-susceptible). 【Conclusion】 Delayed sowing or planting in environment with high temperatures can make wheat encounter high temperature stress at grain filling stage. High-throughput method based on the HSI of TGW was performed to evaluate heat tolerance of 1 325 winter and spring wheat germplasms. Overall, one hundred and three heat-tolerant germplasms with high yield potential were identified, which could be used as parents developing heat-tolerant wheat varieties.

Genetic Diversity Analysis and Comprehensive Evaluation of Phenotypic Traits in Hulless Barley Germplasm Resources

BAI YiXiong, ZHENG XueQing, YAO YouHua, YAO XiaoHua, WU KunLun

Scientia Agricultura Sinica. 2019, 52(23): 4201-4214. doi:10.3864/j.issn.0578-1752.2019.23.002

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【Objective】The genetic diversity in the hulless barley (Hordeum vulgare L. var. nudum Hook. f.) germplasm resources can be screened to improve phenotypic appearance traits. 【Method】Shannon-Wiener diversity index was used to analyze the genetic diversity of 15 phenotypic traits in 205 hulless barley accessions. The distribution frequency of all phenotypic traits in test germplasm resources were analyzed, which contained the data of Xining and Haibei experiment point. Correlation analysis was used to identify the relationship among all traits; cluster analysis was carried out to clarify its classification of the tested germplasm. Principal component analysis was used to construct a comprehensive evaluation system of hulless barley germplasm resources, and the system was verified by linear regression analysis. Screening excellent hulless barley germplasm resources based on the results of comprehensive evaluation and high yield and stability analysis. Correlation analysis, principal component analysis, cluster analysis and high yield and stability analysis were used to evaluate the germplasms. 【Result】The genetic variation level of lodging rate was the richest, the genetic variation level of the center of gravity is the most deficient, the genetic variation of the Haibei test plot showed higher than that of Xining point. The genetic diversity of panicle weight was the most abundant, the genetic uniformity of lodging rate was the highest. Except for the lodging rate, the additional traits showed normal or skewed distribution, and the distribution frequency showed the trend of higher middle and lower sides. The spike length and spike weight showed normally distributed in each genotype. Spike length and panicle weight have normal distributions among the hulless barley accessions. The phenotypic traits showed highly significant differences related to environmental, genotypes, and years factors. Genotype and environment (G×E) factors, genotype and year (G×Y) factors and genotype × environment× year (G×E×Y) interactions produced highly significant differences in the phenotypic traits. There was a significant correlation between the indicators among the roots, stems and panicles of the hulless barley, and there was also a significant correlation between the agronomic traits among the tissues. Well-developed root system, greater stem bending resistance, and stronger mechanical retention, reduced the lodging. Susceptibility to lodging of hulless barley limits the growth and development of spikes and reduce spike length, grain number per ear, grain size, and ear weight. This greatly reduces hulless barley yield. The clustering results demonstrated that the germplasm could be divided into three categories. Category one had a high center of gravity and was susceptible to lodging, but the remaining traits were intermediate. Category two contained germplasm with good agronomic qualities such as dwarf, stalk with a low center of gravity and other useful traits. Category three contained germplasm with high plant height, underdeveloped roots, easily to folded stems, and poor panicle performance. The F value results and yield stability analysis identified five types of barley hull germplasms with excellent overall traits, high yield and stability. 【Conclusion】 High genetic diversity exists in the barley germplasm resources. The spike length and spike weight were normally distributed within each genotype. With the exception of the lodging rate, 12 other traits had skewed distributed within the genotypes. Eight traits including root dry weight, center of gravity, stem wall thickness, main stem diameter, stem strength, spike length, kernels per spike and yield are useful indicators for evaluating barley germplasms.

Response and the Expression of Pi-Responsive Genes in Leymus chinensis Under Inorganic Phosphate Treatment

WAN DongLi,HOU XiangYang,DING Yong,REN WeiBo,WANG Kai,LI XiLiang,WAN YongQing

Scientia Agricultura Sinica. 2019, 52(23): 4215-4227. doi:10.3864/j.issn.0578-1752.2019.23.003

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【Objective】Phosphorus is the nutrient elements that is essential for plant growth. It would provide the basic data for molecular mechanism investigation of Leymus chinensis responding to inorganic phosphate (Pi) treatment, via analyzing the response under Pi stress in L. chinensis, and selection of the reference genes for qRT-PCR analysis, along with relative expression analysis of Pi-responsive genes. 【Method】Using L. chinensis seedlings as materials, the length of shoots and roots were measured, and the contents of Pi were examined via vanadium molybdenum yellow colorimetric method following different concentration of Pi treatments. Based on transcriptome data of L. chinensis, as well as NCBI nucleic acid sequences database, 7 and 1 candidate reference genes were selected, respectively. Total RNA was isolated from each sample after different concentration of Pi treatment. Expression level of candidate reference genes was examined through qRT-PCR, and the stability was calculated by geNorm, NormFinder and Bestkeeper. According to the most stable reference genes selected, the relative expression of Pi-responsive genes was analyzed according to the qRT-PCR results. 【Result】The phenotype observation showed that both lower and higher concentration of Pi stresses reduced the growth of L. chinensis, and the shoot growth was more sensitive to low Pi (or Pi deficiency) stress, whereas root length was more sensitive to high Pi stress. In addition, the accumulation of Pi was enhanced along with the increased concentration of Pi treatments. The melting curve analysis showed that single peak of every candidate reference gene was observed, and gene expression profiles indicated that their CT values ranged from 17.16 to 26.61, among which LcGAPDH exhibited the highest expression abundance with CT values ranged form 17.16 to 20.22, and Lc18SrRNA showed the lowest expression level with CT values ranged form 23.28 to 26.61. LcARPT (2.09%) showed the smallest expression variation, and LcTUA (6.8%) had the largest expression variation. Finally, according to the stability ranking of geNorm, NormFinder and Bestkeeper, the comprehensive stability ranking of 8 candidate reference genes were obtained via calculation geometric mean, among which the top three stable genes were Lc18SrRNA, LcCAP and LcEF1α, and the most unstable two genes were LcTUA and LcTUB. Using Lc18SrRNA, LcCAP and LcEF1α as reference genes, qRT-PCR results showed that, compared with the control, the expression of LcPHO1-2 was induced by low Pi or complete deprive of Pi, LcPAP2 was induced by high Pi, while the expressions of LcPAP27 was induced by both low and high Pi treatments.【Conclusion】Both lower and higher concentration of Pi stresses blocked the L. chinensis growth, and different responsive patterns were observed between the shoots and roots. Three relatively stable expression genes Lc18SrRNA, LcCAP and LcEF1α were selected and could be used as reference genes for qRT-PCR analysis under Pi stress. LcPHO1-2 and LcPAP2 were involved in the response of L. chinensis to low Pi and high Pi treatment, respectively, while LcPAP27 was participated in the responsive process of both low Pi and high Pi treatments.

Effects of Side Deep Fertilization on Yield Formation and Nitrogen Utilization of Mechanized Transplanting Rice

ZHU CongHua, ZHANG YuPing, XIANG Jing, ZHANG YiKai, WU Hui, WANG YaLiang, ZHU DeFeng, CHEN HuiZhe

Scientia Agricultura Sinica. 2019, 52(23): 4228-4239. doi:10.3864/j.issn.0578-1752.2019.23.004

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【Objective】Mechanized transplanting of rice with synchronous side deep application of fertilizer is a new and advanced technology that is still developing rapidly. In-depth studies on the effects of mechanized side deep placement of different types of nitrogen (N) fertilizer on the grain yield and N utilization efficiency of mechanized transplanted rice will be helpful for devising strategies to improve the mechanization of planting and fertilization, and to provide a theoretical basis for reducing costs and increasing fertilization efficiency in rice production. 【Method】Field experiments were conducted in 2017 and 2018 with a randomized complete block design, with five N fertilizer application treatments: N0-plots without N fertilizer; CUB-manual surface broadcast of urea (CU); CUM-mechanized side deep placement of CU; CRUB-manual surface broadcast of controlled release urea (CRU); and CRUM-mechanized side deep placement of CRU. The characteristics of matter production, as well as N uptake and distribution, N use efficiency, yield, and yield components of rice were determined. 【Result】Each N fertilizer application treatment had similar effects on yield formation and N use efficiency in the two years. Compared with the CU treatment, the CRU treatment significantly improved dry matter accumulation, N uptake, N utilization efficiency, and grain yield. The dry matter accumulation and N uptake at maturity, N recovery efficiency (NRE), N agronomy efficiency (NAE), and grain yield were higher in the CRU treatment than in the CU treatment by 3.22%, 17.50%, 46.00%, 17.79%, and 3.72%, respectively, in 2017; and by 8.77%, 13.27%, 32.07%, 12.74%, and 3.32%, respectively, in 2018. Compared with surface broadcasting, mechanized deep placement of N fertilizer, regardless of the type of N fertilizer, significantly enhanced N use efficiency, and increased NRE and NAE by 17.91%-43.14% and 19.61%-37.39% respectively, in 2017; and by 53.80%-54.10% and 21.11%-35.11%, respectively, in 2018. Compared with surface broadcasting, mechanized deep placement of N fertilizer (CU or CRU) increased the grain yields in 2017 and 2018 by 4.46%-6.95% and 5.55%-8.11%, respectively, because of increased numbers of effective panicles and spikelets. The N uptake in stems-sheaths and leaves and the apparent amount of N translocated in stems-sheaths and leaves (TNT) were significantly higher in the CRUM treatment than in any other N application treatments from the heading stage to the maturity stage. Compared with the other N fertilizer treatments, the CRUM treatment also increased N uptake, SPAD values, and total aboveground biomass at the panicle initiation stage and full heading stage. 【Conclusion】Mechanized side deep placement of controlled release urea is an efficient fertilization method to increase the grain yield and N use efficiency of mechanized transplanted rice.

Effect of Pot-Mat Seedling on the Quality of Machined Transplanting and Yield Formation of Super Early Rice

CHEN HuiZhe, XU YiCheng, ZHANG YuPing, XIANG Jing, ZHANG YiKai, ZHU DeFeng

Scientia Agricultura Sinica. 2019, 52(23): 4240-4250. doi:10.3864/j.issn.0578-1752.2019.23.005

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【Objective】Seedling raising in pot-mat tray was the key point of pot-mat seedling mechanized transplanting technology system. This technology was conducted in early rice season to study the effects of pot-mat seedling on mechanized transplanting quality and yield of super early rice.【Method】In this study, the super early rice varieties Zhongzao39 and Zhongjiazao17 were sown in pot-mat seedling tray (BT) and traditional flat tray (CK), and then the seedling emergence rate, seedling quality, root morphology and distribution, transplanting quality and yield were investigated.【Result】There were no significant differences of seedling emergence rate between pot-mat seedling and traditional flat-mat seedling. The root system of pot-mat seedling form bowl shape, and root surface area, root diameter and root volume increased compared with traditional mat seedling. 56.03% root of BT seedling was in the bottom bowl, and the upper root was 43.97%, while that of the control seedling was 37.86% at the bottom and the upper root was 62.14%. The root-cutting rate of Zhongzao 39 and Zhongjiazao 17 pot-mat seedlings were 25.06% and 14.24%, respectively, and lower than that of flat seedling. Under the same sowing, the seedling missing-transplanting rate of pot-mat seedling treatment decreased significantly, and Zhongzao39 decreased 1.67%-3.89%, Zhongjiazao17 decreased 1.66%-2.22%. Besides, the percentage of turnover seedling, floated and injured seedlings when seedling mechanized transplant decreased compared with that of the control. The plant height, and weight of leaves, stem, root, shoot and content of chlorophyll increased at 14 days after transplanting, indicating that it was helpful to promote the early emergence and rapid growth of seedlings. The grain yield of BT treatment was significantly higher than that of control, and the yield of Zhongzao 39 and Zhongjia Zao 17 increased by 6.35%-7.66% and 8.99%-10.87%, respectively. The increase in yield was mainly achieved by the increase in the number of effective panicles. The number of effective panicles treated by pot-mat seedlings machine transplanting of Zhongzao 39 and Zhongjiazao 17 increased by 2.14%-6.01% and 4.76%-6.98%, respectively.【Conclusion】Pot-mat seedling transplanting technology improved the quality of mechanical transplanting, reduced the missing-transplanting seedling rate and injury root rate of seedling. The technique could promote early emergence of tillers, increase the number of effective panicles, and achieve high yield.

Characteristics of Grain Filling and Dehydration in Wheat

ZHU DongMei, WANG Hui, LIU DaTong, GAO DeRong, Lü GuoFeng, WANG JunChan, GAO ZhiFu, LU ChengBin

Scientia Agricultura Sinica. 2019, 52(23): 4251-4261. doi:10.3864/j.issn.0578-1752.2019.23.006

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【Objective】The characteristic of grain filling and dehydration in wheat was studied, which provided a selection method and a theoretical basis for breeding wheat variety with fast filling and dehydration, and its grain with safety moisture content without drying process at harvesting date. 【Method】 In 2015 and 2016, 7 main wheat varieties in the middle and lower of the Yangtze River Valley were used as tested materials. The grain filling traits, dehydration rate and grain moisture content at physiological maturity and harvest date were measured to explore their profiles by Logistic Growth Equation (LGE) fitting analysis and multiple comparison and correlation coefficient method. 【Result】 The results indicated that grain-filling of 7 varieties fitted LGE best, with an S-like breakthrough curve of slow-fast-slow trend, but there were significant differences in the maximum grain-filling rate, average grain-filling rate and grain-filling duration in different varieties. The maximum grain-filling rates and average grain-filling rates of Yangmai 11, Yangmai 158 and Yangmai 16 were higher, whose dry grain weights at 30 d after anthesis were more than 35 g and the grain-filling durations were shorter; The grain filling rate of Yangmai 15 was the fourth fastest, but the grain-filling duration was the longest among the 7 wheat genotypes; Ningmai 13, Yangmai 20 and Yangmai 22 had the smaller filling rates, respectively. The maximum grain-filling rate, average grain-filling rate, R1, R2 and R3 were significantly positively correlated with 1000-grain weight. The rate of grain filling was R2>R1>R3 in the three filling stages. The filling grain of wheat was basically finished at 30 d after anthesis. After the grain filling stage, it started the dehydration and drying stage. There were significant differences among the 7 genotypes in the grain moisture content at physiological maturity and harvest date, as well as the grain dehydration rate. The grain dehydration rates of Yangmai 11, Yangmai 158 and Yangmai 16 were higher than the others, whereas Yangmai15 was the lowest. The grain moisture content at harvest date was significantly (P<0.01, 0.05) correlated with the grain moisture content at physiological maturity date, the average dehydration rate after physiological maturity, and the grain dehydration rate at 2 d after physiological maturity.【Conclusion】In Yangmai 11, Yangmai 158 and Yangmai 16, the grain-filling was faster and completed earlier, and the grain dehydrated more quickly. The grain weight >35 g at 30 d after anthesis could be used as the selection parameter of the grain-filling rate. The average grain dehydration rate after physiological maturity could be a selection parameter that evaluates the dehydration property of wheat.

Detection and Analysis of Magnaporthe oryzae Avirulence Genes AVR-Pib, AVR-Pik and AvrPiz-t in Heilongjiang Province

MENG Feng,ZHANG YaLing,JIN XueHui,ZHANG XiaoYu,JIANG Jun

Scientia Agricultura Sinica. 2019, 52(23): 4262-4273. doi:10.3864/j.issn.0578-1752.2019.23.007

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【Objective】The objective of this study is to investigate the distribution and variation mechanism of avirulence genes AVR-Pib, AVR-Pik and AvrPiz-t in Magnaporthe oryzae strains from different regions and years in Heilongjiang Province, to understand the pathogenic phenotypes of different avirulence gene alleles, and to provide a reference for utilization and distribution of resistance cultivars in Heilongjiang Province.【Method】Based on the avirulence gene sequences published in NCBI, specific primers were designed to amplify full length and the coding sequence (CDS) regions of three genes, respectively. From 2016 to 2017, 335 M. oryzae strains in different regions of Heilongjiang Province were collected and isolated, and their DNA was PCR-amplified using avirulence genes primers and analyzed by agarose gel electrophoresis. The PCR products with different band patterns and from representative strains of different regions were selected for sequencing. The sequencing results were compared with the corresponding avirulence gene sequences for base and amino acid. The pathogenic phenotype of M. oryzae strains with different variant types was determined based on the rice resistance to single-gene lines.【Result】The specific bands of AVR-Pib, AVR-Pik and AvrPiz-t were detected in PCR detection and appeared in different distribution frequencies and mutation types, indicating that these 3 avirulence genes were all distributed in Heilongjiang Province. The average amplification frequency of the 3 avirulence genes was 75.52%, 87.16% and 85.67%, respectively. Among them, 4 types of band (bandless, high band, mid to high band and low band) of AVR-Pib were detected by electrophoresis analysis and 5 variant types AVR-Pib (1-1, 1-2, 2, 3, 3-1) were detected by PCR product sequencing. The genotypes AVR-Pib-1-1, AVR-Pib-1-2, AVR-Pib-2 and AVR-Pib-3-1 are newly discovered variant types, of which genotypes AVR-Pib-1-1 and AVR-Pib-1-2 are insertions of transposon Pot2 but with different insertion sites. The genotype AVR-Pib-2 has a small fragment insertion in the upstream of CDS region. The genotype AVR-Pib-3-1 base sequence has 4 differences from the original sequence, namely 32 (C/G) 35 (T/A) 36 (T/A) 38 (T/A), and the amino acid translation was terminated prematurely. Pathogenic analysis showed that except for the normal genotype AVR-Pib-3, the other alleles lost their avirulence functions. Seven AVR-Pik alleles (D, A, B, C, E, F, F2) were detected after PCR product sequencing, and the alterations in the nucleotide sequences of these alleles all resulted in amino acid missense mutations. The 7 AVR-Pik alleles have been reported previously. The avirulence gene AvrPiz-t was analyzed by electrophoresis and sequencing of PCR products, and 2 types of band (high band and normal band type) and 4 genotypes of AvrPiz-t (A, B, C, D) were revealed. Among them, AvrPiz-t-A is the original genotype, while AvrPiz-t-B has a base A insertion at position 191, causing premature termination of amino acid translation. Genotype AvrPiz-t-C is a newly discovered allelic type, characterized by the presence of a nucleotide variation at position 17 (T/C) and the insertion of base C at position 19 compared with type A, leading to the frameshift mutation and premature translation termination. The high band type avirulent genotype AvrPiz-t-D was sequenced and verified as having an insertion of the Pot3 transposon. Rice single-gene lines infection showed that the strains with AvrPiz-t-A were avirulent to Piz-t line due to Piz-t recognition, whereas the strains with AvrPiz-t (B, C, D) were virulent to Piz-t line due to lost the ability recognized by Piz-t.【Conclusion】The avirulence genes AVR-Pib, AVR-Pik and AvrPiz-t of M. oryzae in Heilongjiang Province are widely distributed and the types of variation are abundant. The results of this study can provide a reference for breeding and popularizing rice cultivars with corresponding disease-resistance genes.

Identification of Genes Encoding Secretory Proteins Related to the Pathogenicity of Sclerotinia sclerotiorum Using TRV-HIGS

YUAN JunHu,DING YiJuan,YANG WenJing,YAN BaoQin,CHAI YaRu,MEI JiaQin,QIAN Wei

Scientia Agricultura Sinica. 2019, 52(23): 4274-4284. doi:10.3864/j.issn.0578-1752.2019.23.008

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【Objective】 Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum is the main problem in rapeseed planting in China, which causes serious yield and quality loss. Secretory proteins play an important role in the pathogenesis of pathogens. The genome of S. sclerotiorum contains a large number of genes encoding secretory proteins. The objective of this study is to identify and screen the secretory protein genes related to pathogenicity, reveal the pathogenic mechanism of S. sclerotiorum, and to provide an important target for the prevention and control of SSR. 【Method】 SMART software was used to analyze the protein domains of 8 candidate genes with signal peptides that were differentially expressed in the process of S. sclerotiorum infecting the susceptible and resistant Brassica oleracea lines, then the domains obtained by SMART analysis were annotated in SCOP, Pfam and PDB databases. The fragment with the length of around 300 bp in the encoding region of these genes was cloned into pTRV2 vector together with the GFP fragment. The suspension of pTRV1 was mixed equally with pTRV2-Gene and pTRV2-GFP, respectively. After 3 hours at room temperature, pTRV2-Gene vector and control (pTRV2-GFP) were transformed into 5-6 week-old leaves of Nicotiana benthamiana using syringe infiltration method. Subsequently, the infiltrated plants were cultured in dark for 48 hours and then grown in the normal light for 7 days. PDA mycelium blocks of S. sclerotiorum with a diameter of 6 mm were used to inoculate the infiltrated leaves of tobacco at the 9th day after transformation in vivo, in which the carrying surface was close to the leaves. After 48 hours of inoculation, the lesion size was measured and RNA from necrotic and infected tissues (around 1 cm from the edge of necrotic tissue) was extracted. qRT-PCR analysis was carried out to estimate the relative expression of target gene in N. benthamiana lines carrying TRV-HIGS vector. 【Result】 The putative functions of these 8 genes predicated with SMART and domain annotation were involved in the hydrolysis of proteins, nucleic acids or polysaccharides, the immunity response of host plants, and the tolerance to drugs and biotin synthesis of S. sclerotiorum. The average lesion area of control carrying TRV-GFP was 3.44 cm ² at 48 hours post inoculation of S. sclerotiorum. Except for one line (SS1G_07655), the lesion area of other 7 lines carrying TRV-HIGS vector was significantly lower than that of the control plants (P≤0.05), ranging from 1.63 to 2.61 cm ². qRT-PCR analysis showed that the gene expression level of these 7 genes in the TRV-HIGS lines was significantly lower than that of the control (P≤0.05). 【Conclusion】 Eight secretory protein genes with unknown function in S. sclerotiorum were successfully identified by TRV-HIGS technique. Seven genes related to the pathogenicity of S. sclerotiorum were screened out. Among them, SS1G_03146 with the strongest effect on the pathogenicity of S. sclerotiorum may be involved in the synthesis of biotin, and SS1G_04343 and SS1G_11912 may be involved in the immune response of host.

Effects of Lime Content on Soil Acidity, Soil Nutrients and Crop Growth in Rice-Rape Rotation System

YAN ZhiHao,HU ZhiHua,WANG ShiChao,HUAI ShengChang,WU HongLiang,WANG JinYu,XING TingTing,YU XiChu,LI DaMing,LU ChangAi

Scientia Agricultura Sinica. 2019, 52(23): 4285-4295. doi:10.3864/j.issn.0578-1752.2019.23.009

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【Objective】Soil acidification is becoming more and more serious, which reducing crop yield in rice-rape rotation system of southern of China. In this study, the effects of lime application on soil nutrients and crop growth in acidic soil were studied, and the response relationship between soil available nutrients, yield and crop nutrient uptake to soil pH was clarified, so as to provide theoretical basis for the improvement of acidified soil in paddy fields. 【Method】 From 2015 to 2018, the paddy field with soil pH 4.5 was selected in Jinxian county, Jiangxi province, and hydrated lime was used as acid soil modifier. Through laboratory simulation, the amount of hydrated lime under different soil pH values was calculated. Then field experiments were carried out with six soil pH gradients of pH 4.5, pH 5.0, pH 5.6, pH 6.3, pH 6.8, and pH 7.3. In 2015, in order to ensure that the pH value of the treated soil was basically consistent with the measured pH value, one year after the soil was uniformly planted, the hydrated lime was used for quantitative adjustment with a period of one year. 【Result】 (1) With the amounts of lime and soil pH increase, the contents of soil available nitrogen increased first and then decreased, the content of soil exchangeable Ca ²⁺ and exchangeable Mg ²⁺ increased significantly, and the content of soil available potassium and available phosphorus decreased significantly. (2) With the increase of lime contents and soil pH, crop yield first increased and then decreased. At pH 6.4 (equivalent to the amount of 6 145 kg·hm ^-2hydrated lime), the yield of rape reached the highest; compared with pH 4.5, the yield increased by 202.2%. At pH 6.8 (equivalent to the amount of 7 474 kg·hm ^-2hydrated lime), the rice yield reached the highest; compared with pH 4.5, the yield increased by 61.2%. When the yield was reduced by 50%, the soil pH thresholds of rape and rice were 4.7 and 4.2, respectively. (3) Soil pH significantly affected crop nutrient uptake content. With the increase of the amount of hydrated lime, the nitrogen, phosphorus and potassium uptake contents in rape increased first and then decreased. The average increase of nitrogen, phosphorus and potassium uptake contents in rape from 2016 to 2018 was 59.5%-181.4%, 36.2%-188.8% and 65.7%-198.9%, respectively. The nitrogen, phosphorus and potassium uptake contents in rice first increased and then decreased. The uptake content of rice was the highest at pH 6.8. Compared with that without lime application the average increase of nitrogen, phosphorus and potassium uptake of rice from 2016 to 2018 was 11.1%-88.6%, 13.5%-68.5% and 9.7%-66.1%, respectively.【Conclusion】Under the application of lime conditioner, the contents of soil available nitrogen and exchangeable Ca ²⁺ and exchangeable Mg ²⁺ were increased with the increase of soil pH, which promoted the uptake of nitrogen, phosphorus and potassium nutrients of crops and increased the crop yield. Under the experiments conditions, the optimal dosage of lime in acid soil (pH 4.5) of rice -rape rotation system was about 6 500 kg·hm ^-2, which could obtain stable and high yield of crops in rice-rape rotation system of southern China.

Analysis of Drought Characteristics and Its Trend Change in Shaanxi Province Based on SPEI and MI

DING YiBo,XU JiaTun,LI Liang,CAI HuanJie,SUN YaNan

Scientia Agricultura Sinica. 2019, 52(23): 4296-4308. doi:10.3864/j.issn.0578-1752.2019.23.010

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【Objective】 At present, most drought studies were based on historical drought events to analyze the causes and trends. This paper sought to simulate the drought index method when outputting future meteorological data based on CMIP5 model, and explored the characteristics of past and future drought changes in Shaanxi Province, which could provide a basis for the future management of agricultural water resources in Shaanxi Province. 【Method】Based on the historical data of 18 meteorological stations in Shaanxi Province and CMIP5 model, the future meteorological data were output. The reference crop evapotranspiration (ET₀) was simulated by comparing three kinds of models. The standard precipitation evaporation index (SPEI) and relative moisture index (MI) were calculated based on the reference crop ET₀ and precipitation data to reflect the drought degree. The spatial and temporal characteristics of drought in the past (1958-2017) and in the future (2018-2100) were compared.【Result】Multiple linear regression (MLR) simulation could accurately predict the reference crop ET₀ (RMSE=0.457 mm·d ^-1). In the RCP2.6 and RCP8.5 scenarios, the future drought index showed an upward trend. Under the RCP8.5 scenario, there was a sudden change in the drought index in the 1940s. The degree of drought would decrease in the future of Shaanxi Province, and the distribution of drought would be more uneven during the year. In the future, the degree of drought would decrease during summer maize growth season, and the degree of drought would increase during winter wheat growth season.【Conclusion】The characteristics and extent of drought change were different under different RCP scenarios. The changes in drought characteristics reflected by SPEI and MI were basically the same, but there were differences in the changes in some time periods. In order to effectively cope with the negative impact of climate change on dry crop yields, it was necessary to enhance soil water storage and conservation capacity, especially to strengthen drought resistance during the winter wheat growing season.

Spatial-Temporal Characteristics and Economic Relevance of Agricultural Carbon Emissions in Hubei Province

LI Bo,DU JianGuo,LIU XueQi

Scientia Agricultura Sinica. 2019, 52(23): 4309-4319. doi:10.3864/j.issn.0578-1752.2019.23.011

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【Objective】Hubei Province is a large agricultural province, and the carbon emissions from agricultural production account for a large proportion of the total carbon emissions. Environmental problems such as greenhouse effect caused by carbon emission and non-point source pollution caused by agricultural production cannot be ignored. In this study, the co-integration relationship between agricultural economic growth and agricultural carbon emissions was analyzed, and the error correction was carried out, which provided an important theoretical basis and reference for the development of carbon emission reduction in Hubei Province. 【Method】Based on six kinds of main carbon sources from the agricultural inputting and production, the agricultural carbon emission load from 1993 to 2017 was calculated, and then the temporal and spatial characteristics of agricultural carbon emission in Hubei Province were analyzed. Furtherly, Kernel density estimation demonstrated that the regional gap of agricultural carbon emissions in Hubei province. Finally, the integrated use of co-order error correction model was discussed as an evidence of Hubei Province's agricultural economic growth and agricultural carbon emissions. 【Result】The total amount and intensity of agricultural carbon emissions in Hubei Province showed a trend of rising first and then later. The average annual growth rate of agriculture carbon emissions was 2.32%, while the average annual growth rate of intensity was 2.21%. The chain growth of which was general in the stage of decline. Fertilizers, pesticides, agricultural film, agricultural diesel, real tillage and agricultural irrigation as a result of carbon emissions, average annual increase rate was 2.23%, 2.44%, 2.40%, 3.32%, 0.44%, and 2.32%, respectively. Kernel density estimation demonstrated that the regional gap of agricultural carbon emissions in Hubei Province was widening. The integrated use of co-order error correction model was discussed as an evidence of Hubei Province's agricultural economic growth and agricultural carbon emissions. The results showed that: for every 1% increase in per capita agricultural output value, the total carbon intensity of pesticides, agricultural film, agricultural diesel, agricultural irrigation and other carbon sources of carbon emission intensity increased by 0.58%, 0.59%, 0.25% and 0.15%, respectively, and the total agricultural carbon intensity increased by 0.19%.【Conclusion】Different agricultural economic development, production conditions and regional development strategies in Hubei Province led to more and more obvious agricultural carbon emission gap between regions. There was a long-term stable relationship between agricultural economic growth and agricultural carbon emission in Hubei Province, which indicated that Hubei Province was also in a critical period of transition from traditional farming mode to green and low-carbon farming mode, and this development mode had existed for a long time.

Strengthen the Research of Molecular Biology, Promote the Sustainable Development of Apple Industry

CONG PeiHua,ZHANG CaiXia,HAN XiaoLei,ZHANG LiYi

Scientia Agricultura Sinica. 2019, 52(23): 4320-4321. doi:10.3864/j.issn.0578-1752.2019.23.012

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Bioinformatics and Expression Analysis of the LIM Gene Family in Apple

YUAN GaoPeng, HAN XiaoLei, BIAN ShuXun, ZHANG LiYi, TIAN Yi, ZHANG CaiXia, CONG PeiHua

Scientia Agricultura Sinica. 2019, 52(23): 4322-4332. doi:10.3864/j.issn.0578-1752.2019.23.013

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【Objective】 In order to lay a basis for the further functional research and application of MdLIM genes, this study were carried out to analyze the bioinformatics (e.g promoter action element, conserved domain, gene clustering, gene structure, chromosome localization) and expression of the LIM gene family in apple. 【Method】Based on the apple genome database GDR and PLAZA, the members of LIM gene were identified. The MdLIM amino acid sequence prediction, subcellular localization prediction, LIM domain analysis, and phylogenetic tree the gene structure were completed by ExPASy Proteomics Server, Cell-PLoc, CD-Search Tool, MEGA7, and GSDS, respectively. In addition, the expression pattern of MdLIM genes in different tissues and in peels with different degree of fruit russeting in samples was analyzed by real-time qRT-PCR.【Result】A total of eleven MdLIM genes were identified from apple genome. These MdLIM proteins contained 96-222 amino acid residues with isoelectric points ranging from 6.14 to 9.01. The results of subcellular localization showed that the apple LIM proteins were distributed in the nucleus. Analysis of promoter showed these 11 MdLIM genes contained cis-acting elements related to hormone responses, environmental adaptability and adversity induction. Conserved domains showed that ten MdLIM proteins had double LIM domains except MdLIM8. According to the phylogeny relationship, MdLIM genes were divided into four categories. The expression patterns of the 11 MdLIM genes in flowers, leaves, fruit peels and stems were determined by real-time RT-PCR, and the results showed that their diverse and specific expression could be detected in all of the four tissues, suggesting that they might play different roles in different tissues. 【Conclusion】Eleven MdLIM genes were identified from the whole genome of apple, and they could be divided into four groups, and distributed on 7 chromosomes with diverse and specific tissues expression patterns.

Genome-Wide Identification and Expression Pattern Analysis of NLP (Nin-Like Protein) Transcription Factor Gene Family in Apple

WANG Xun, CHEN XiXia, LI HongLiang, ZHANG FuJun, ZHAO XianYan, HAN YuePeng, WANG XiaoFei, HAO YuJin

Scientia Agricultura Sinica. 2019, 52(23): 4333-4349. doi:10.3864/j.issn.0578-1752.2019.23.014

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【Objective】The study was carried out to explore the whole genome characteristics and expression patterns of NLP transcription factors in apple, and further understand its structural characteristics and mechanism.【Method】Based on the local BLAST database and Pfam database, the members of NLP transcription factor family in the whole genome of apple were identified by using two query strategies with blastp and hmmsearch. Through strict filtration and confirmation, the results were used for further analysis, and analysis mainly divides into three parts, including NLP proteins analysis, analysis of NLP genes and NLP expression analysis in apple. Programs or softwares, such as ProtParam, Clustal Omega, MEGA7, MEME 5.0.2, SOPMA, Phyre ², WoLF PSORT and STRING, were used for protein analysis, online tools included MG2C, GSDS2.0, PlantCARE, psRNATarget, were used for gene analysis, and the expression of MdNLP gene was quantitatively detected by qRT-PCR.【Result】6 NLP members were identified from apple protein databases, which were classified into three categories by phylogenetic analysis: I, II and III. The protein secondary structure was dominated by random coil, followed by alpha-helix, and the smallest proportion was beta-turn. The prediction of subcellular localization was located in the nucleus, which was consistent with the characteristics of transcription factors. Chromosome localization showed that five genes (except MDP0000584547) were located on four chromosomes. Promoter analysis revealed a large number of cis-acting elements related to hormone and stress response, suggesting that MdNLP genes might be involved in the regulation of hormone and stress signals. In addition, a nitrogen-responsive GCN4 element was also identified, further indicating that such transcription factors were closely related to nitrogen. By quantitative detection, the pattern which NLP family had high expression in stem and leaf of apple was revealed. And the expression analysis results also confirmed that MdNLP genes responded to nitrogen starvation and drought stress, and so on.【Conclusion】Through the apple genome analysis, 6 NLP transcription factors were found; the analysis of structure and conserved domain of protein for 6 gene suggested that there had a very high similarity and conservation between them, at the same time, they were different in a way; the associated protein network of NLP family in Arabidopsis thaliana were used for MdNLPs, MDP0000132856, which had the highest homology with AtNLP7, might also have complicated features and functions.

Screening and Expression Analysis of Co Candidate Genes in Columnar Apple

BAI TuanHui,LI Li,ZHENG XianBo,WANG MiaoMiao,SONG ShangWei,JIAO Jian,SONG ChunHui

Scientia Agricultura Sinica. 2019, 52(23): 4350-4363. doi:10.3864/j.issn.0578-1752.2019.23.015

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【Objective】Columnar growth in apple (Malus × domestica Borkh.) is a special type of dwarf mutation. Due to short internodes and lateral branches, a limited canopy and minimal pruning, columnar apple trees are well adapted to high density plantings. Based on the fine mapping of Co, the genes in the localization interval were screened, which laid a foundation for elucidating the molecular mechanism of columnar apple formation and breeding new varieties of columnar apples.【Method】Based on the latest apple genome data and transcriptome information, the buds, stem tips and leaves of columnar apple Wujia, Runtai No.1 and standard apple Fuji and Huashuo were used as test materials, and the genes between the fine mapping Co gene in interval from 27.66 Mb to 29.05 Mb were annotated and predicted. The coding sequence of the target gene was selected to detect primer specificity by RT-PCR, the real-time quantitative PCR was used to analyze the expression characteristics of target genes in different tissues and organs, and differential genes were screened out as candidate genes.【Result】The results showed that there were 67 genes between 27.66 Mb and 29.05 Mb in chromosome 10, 12 of which were non-coding RNAs (ncRNA), and the rest were genes with encoding proteins. According to the columnar and standard apple RNA-seq, there were 25 genes with more than 1 fold difference, 13 of which were up-regulated, and 12 genes were down-regulated in columnar apples. Among the 14 predicted genes, there were significant differences in the relative expression of the four genes MD10G1184100, MD10G1185400, MD10G1185600 and MD10G1190500 between shoot tips and lateral shoot tips in columnar and standard apples. The relative expression levels of MD10G1184100 and MD10G1185600 in the tip of terminal bud of two columnar apples were significantly higher than those of both standard apples. The relative expression of MD10G1185400 and MD10G1190500 genes in the tip of lateral bud of two columnar apples was significantly higher than that of two standard apples, while the expression of MD10G1184100 gene in two columnar apples was significantly lower than that of standard apples. The gene expression patterns of different tissues or organs of four candidate genes were analyzed. The result showed that the expression of MD10G1184100 gene in the roots of columnar apples was significantly higher than that in other tissues. The MD10G1185400 and D10G1185600 genes were significantly expressed in lateral tips in columnar apples, while MD10G1190500 gene was prominently expressed in the terminal bud of columnar apples.【Conclusion】4 genes with significant differences in columnar and standard apple could be regarded as Co candidate genes, which laid a foundation for gene cloning and functional verification and apple tree-oriented genetic improvement.

Cloning and Functional Analysis of U6 Promoter in Apple

BIAN ShuXun,HAN XiaoLei,YUAN GaoPeng,ZHANG LiYi,TIAN Yi,ZHANG CaiXia,CONG PeiHua

Scientia Agricultura Sinica. 2019, 52(23): 4364-4373. doi:10.3864/j.issn.0578-1752.2019.23.016

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【Objective】U6 promoter is an important element for the transcription of sgRNA in the CRISPR/Cas9 genome editing system. There may be species-specific factors in U6 promoter, and the activity of U6 promoter would be altered when its length changed. So far, in apple (Malus×domestica), the transcriptional characterization of U6 promoters has not been reported. Therefore, selecting an apple U6 promoter with high transcriptional activity and suitable fragment size would provide a basis for optimizing apple CRISPR/Cas9 gene editing technology system. 【Method】 DNAMAN, promoter cis element online predicting website PLACE and plant CARE were used to do the comparative analysis of apple U6 promoters; U6 promoters were cloned and constructed into firefly luciferase vector; the apple callus and tobacco (Nicotiana benthamiana) leaves were transformed via Agrobacterium-mediated transient transformation; the transcriptional activity of U6 promoter was determined according to the luciferase activity. 【Result】 There were six alternative U6 promoters in apple genome (E-value<3e ^-40), which were located on chr 6, chr 7, chr 9, chr 10, chr 15 and chr 17, respectively. 27 bp snRNA at 5′ end and its upstream 1 500 bp were selected as candidate U6 promoter. Sequence analysis results showed that the upstream sequence element (USE) and TATA-like elements were contained in six U6 promoters of apple, the same as Arabidopsis. After transient transformation, the luciferase activity assay showed that, the U6 promoter on chromosome 10 had the highest transcriptional activity. Among all U6 promoters which were shortened at 5′ end (1 500 bp, 959 bp, 275 bp, and 116 bp) on chromosome 10, the 275 bp one had the highest transcriptional activity. In addition, compared with the Arabidopsis U6 promoter, in apple callus, the transcriptional activity of the apple U6 promoter was higher. 【Conclusion】Six U6 promoters were cloned from the apple genome, and a U6 promoter with high transcriptional activity and suitable sequence fragment was obtained.

Analysis of Apple Ethylene Response Factor MdERF72 to Abiotic Stresses

WANG JiaHui,GU KaiDi,WANG ChuKun,YOU ChunXiang,HU DaGang,HAO YuJin

Scientia Agricultura Sinica. 2019, 52(23): 4374-4385. doi:10.3864/j.issn.0578-1752.2019.23.017

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【Objective】Ethylene response factor (ERF) , a plant-specific transcription factor, is involved in the growth and development of root formation, hypocotyl elongation, fruit ripening, and organ senescence. It also plays a vital role in regulating responses of plant biological and abiotic stress, as well as fruit qualities. In this study, we cloned the apple ethylene response factor MdERF72. Subsequently, a series of expression analysis and functional identification of transgenic apple calli were performed to study its role in abiotic stress responses. These results provided a theoretical basis for exploring the functions of MdERF72 in plant growth and development.【Method】Using Orin apple calli (Malus calli stica Borkh.) as the test material, the MdERF72 was cloned from apple fruits by RT-PCR assay. Bioinformatics methods were used to analyze its amino acid sequence, physicochemical properties genetic relationship, and spatial structure. MEGA5.0 was used to construct the phylogenetic tree for analyzing the homology of its protein sequence with ERF-B2 subfamily in Arabidopsis. The real-time fluorescence PCR (qRT-PCR) assays were performed to analyse the expression of MdERF72 in different organs and tissues of apple, as well as in apple fruits during different developmental stages. Meanwhile, the expression of MdERF72 in Gala apple tissue culture seedlings treated with ACC, NaCl and low-temperature was detected by qRT-PCR assay. We also constructed its overexpression vector and obtained stable overexpression apple calli through Agrobacterium-mediated genetic transformation. The fresh weight, malondialdehyde content, electrical conductivity, hydrogen peroxide content and superoxide anion content of the wild type and transgenic apple calli were detected after NaCl and low temperature treatment. 【Result】 MdERF72 was located on chromosome 13 in apple genome, which had an AP2/ERF domain, unique to ERF family. Phylogenetic tree analyses indicated that the apple MdERF72 exhibited the highest sequence similarity to Arabidopsis AtERF72, and belonged to the B2 subfamily of ERF family. Analysis of amino acid physicochemical properties indicated that MdERF72 encodes 253 amino acids, and its protein molecular weight was predicted as 27.61 kD, the isoelectric point (pI) was 5.10. In addition, the pro-hydrophobic prediction showed that the hydrophobic portion of MdERF72 was larger than the hydrophilic portion, indicating that it belonged to a hydrophobic protein. Phosphorylation site analysis revealed that MdERF72 had only one threonine phosphorylation site, suggesting that the protein might be regulated by phosphorylation. The results revealed that the MdERF72 promoter sequence contains cis-acting elements associated with jasmonic acid (JA), auxin and drought signals. qRT-PCR analysis showed that MdERF72 was a positive regulatory transcription factor of ethylene, which was expressed in all tissues of apple. Its expression in fruits and stems was relatively high, and gradually increased with the fruit ripening. The expression of MdERF72 in apple tissue culture seedlings was significantly induced by high salt and low temperature. Under the treatment of high salt and low temperature stresses, the MdERF72- overexpressing apple calli had stronger growth potential than the wild type control, and the conductivity, malondialdehyde, hydrogen peroxide and superoxide anion content were lower than the wild type control, indicating that MdERF72 increased the resistance to salt and low temperature stresses.【Conclusion】MdERF72 played an important role in the regulation of high salt and low temperature stresses. Overexpression of MdERF72 could increase the resistance of apple calli to high salt and low temperature stresses.

Promoting the Research on Prevention and Control of Waterfowl Infectious Disease in China

LIU YueHuan

Scientia Agricultura Sinica. 2019, 52(23): 4386-4389. doi:10.3864/j.issn.0578-1752.2019.23.018

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The Effects of Downstream 3513bp of UL56 on Characterization of Duck Enteritis Virus

MAO YaQing,ZHANG Bing,WANG TuanJie,HOU LiDan,HUANG XiaoJie,LIU Dan,ZHAO JunJie,LI QiHong,WANG LeYuan,LI JunPing,YANG ChengHuai

Scientia Agricultura Sinica. 2019, 52(23): 4390-4397. doi:10.3864/j.issn.0578-1752.2019.23.019

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【Objective】Duck enteritis virus (DEV) taxonomically belongs to family Herpesviridae and infects ducks, geese, and swans, which results in high mortality and decreased egg production in domestic and wild waterfowl. Several DEV whole genomic sequences were published, which contained 158-162 kb. Compared with DEV vaccine strain, the virulent DEV strain for vaccine evaluation had a 3513-bp insertion, resulting to UL56 frameshift mutation. At present, there are few papers about DEV gene function and pathogenic mechanism. To study the effect of the 3513-bp insertion on DEV characterization, a recombinant DEV with the 3513-bp deletion was constructed.【Method】The extracted DEV genomic DNA was used as template to amplify the UL56-u and UL56-d of the upstream and downstream ends of UL56 gene, respectively. The two homologous arm fragments were cloned into pMD18T-Simple vector, and the recombinant plasmid pT-UL56ud was obtained. The recombinant plasmid pT-UL56ud was cut by MluI enzyme, and after electrophoretic recovery and dephosphorylation, the recombinant plasmid pT-UL56-RFP was obtained by inserting RFP expression box into pT-UL56ud. After DEV was inoculated with duck embryo fibroblast (DEF) (MOI=0.1) for 1 to 2 h, the purified plasmid pT-UL56-RFP was transfected according to Lipofectamine 2000 instructions, and then purified by plaque. A 3513-bp deletion mutant, DEVΔ3513-RFP, was generated by targeted homologous recombination, in which red fluorescent protein (RFP) as a reporter replaced the 3 513 bp. The RFP was then removed to generate DEV△3513. A rescue mutant, DEVΔ3513(R), was constructed by reinserting the 3 513 bp into the genome of DEVΔ3513-RFP. The recombinant virus and its parent virus were inoculated in DEF (MOI =0.01), the virus titer was measured and the growth curve was plotted. The recombinant virus and its parent virus were diluted properly and inoculated into monolayers DEF, covered with M199 agarose and cultured 5-7 days. Twenty plaques were selected randomly and the area of plaques was assayed. Six-week old susceptible ducks were inoculated with 10 ^5.0TCID₅₀ of the 3513-bp deletion mutant, rescue mutant and its parental virus, respectively. After 10 days, all the surviving ducks were euthanized. The liver tissues were taken from all the animals and fixed to 4% poly Formaldehyde Solution; the pathological sections were prepared by routine procedures and stained with HE. The recombinant virus and the Duck Plague Live Vaccine (CVCC AV1222) were injected with 10 ^3.0TCID₅₀ in muscles for 6 weeks of age susceptible ducks to be tested for immunogenicity. After 14 days, all vaccinated ducks were challenged with 10 ^3.0MLD of lethal DEV (CVCC AV1221).【Result】The recombinant viruses, DEVΔ3513 and DEVΔ3513(R), and their parental virus possessed similar growth kinetics, and their titer peaked at 72 hours with the peak titer 10 ^6.2-6.5TCID₅₀/0.1mL. Their average plaques sizes were not significantly different; DEVΔ3513 was avirulent in 6-week ducks; the ducks vaccinated with 10 ³TCID₅₀ were protected against subsequent challenge with lethal DEV.【Conclusion】We successfully constructed a DEV mutant with 3 513 bp deletion, and firstly confirmed that the deletion of the 3 513 bp had no effect on virus replication in cells and immunogenicity in ducks. Moreover, the 3 513 bp was associated with DEV virulence.

Construction of a Recombinant Duck Enteritis Virus Expressing Hemagglutinin of H9N2 Avian Influenza Virus

SUN Ying,ZHANG Bing,LI Ling,HUANG XiaoJie,HOU LiDan,LIU Dan,LI QiHong,LI JunPing,WANG LeYuan,LI HuiJiao,YANG ChengHuai

Scientia Agricultura Sinica. 2019, 52(23): 4398-4405. doi:10.3864/j.issn.0578-1752.2019.23.020

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【Background】The H9N2 avian influenza virus (AIV) pathogenicity and transmissibility have recently showed an increasing trend. Moreover, it donates partial or even whole cassette of internal genes to generate novel reassortants, which is serious threat to poultry industry and public health. Waterfowls are considered as the natural host and reservoirs of AIVs and play an important role in the spread and mutation of AIV. Therefore, successful control of the spread of H9N2 in waterfowls contributes significantly to poultry industry and public health. Duck enteritis virus (DEV) taxonomically belongs to family Herpesviridae and infects ducks, geese, and swans, which results in high mortality and decreased egg production in domestic and wild waterfowl. DEV may be a promising candidate viral vector for aquatic poultry vaccination because it has a large genome and good immunogenicity. 【Objective】 In this study, we constructed a recombinant DEV expressing the hemagglutinin (HA) gene of a H9N2 virus that was inserted into the deleted viral gE gene, and then its characterization to explore the feasibility of the recombinant DEV as a live vectored vaccine was studied.【Method】 The HA gene of H9N2 was cloned to construct the transfer vector pT-gE-HA. Plasmid pT-gE-HA and rDEV-△gE-GFP were co-transfected into CEF cells. After plaque-purification, we obtained a pure recombinant virus which expressed H9N2 AIV HA protein, and named as rDEV-△gE-HA; PCR and sequencing assay were used to identify the recombinant virus. The recombinant virus was passaged in primary CEF 20 times to evaluate the genetic stability of the foreign gene in the recombinant virus. Ducks were inoculated with 10 ³EID₅₀ rDEV-△gE-HA, then challenged with lethal DEV. Ducks were vaccinated intramuscularly with 10 ³-10 ⁶ TCID₅₀ of rDEV-△gE-HA. At 14, 21, and 28 days post-vaccination (d.p.v.), sera were obtained from all ducks to monitor HI antibody against H9N2 AIV. At 28 d.p.v. all ducks were challenged with 10 ⁸ EID₅₀ H9N2 (A/duck/GD/08) by intravenous injection. Oropharyngeal swabs were collected from H9N2 virus challenged ducks to detect viral shedding.【Result】The recombinant expression vector pT-gE-HA was constructed and transfected with rDEV-△gE-GFP in chicken embryo fibroblasts (CEF). After 3 rounds of plaque-purification, the purified rDEV-△gE-HA was obtained. The results of the PCR and sequencing indicated that the HA expression cassette had already successfully been inserted into the DEV. The HA gene were stably maintained after the recombinant was passaged 20 times in CEF. Ducks inoculated with 10 ³ TCID₅₀ of rDEV-△gE-HA were protected against lethal DEV. HI antibody was detected in all vaccinated ducks at 14 d.p.v. and slightly increased at 21 d.p.v.. Challenge with H9N2 at 28 d.p.v., ducks inoculated with 10 ³, 10 ⁴ and 10 ⁶TCID₅₀were completely protected from challenge, as evidenced by the finding that no virus was recovered from collected oropharyngeal swabs, while 80% ducks (4/5) inoculated with 10 ⁵TCID₅₀ were protected.【Conclusion】In this research, we successfully constructed a stable recombinant DEV expressing the HA of H9N2 AIV. The recombinant DEV remained the protective efficacy of the parental virus against lethal DEV parental virus. Moreover, it could induce HI antibody in ducks and protect no less 80% ducks against H9N2 AIV challenge, although the titer of HI antibody was not too high. This study laid a foundation for developing bivalent vaccine controlling DEV and AIV infection.

Virulence, E Gene Sequence and Antigenic Difference of 4 Duck Tembusu Virus Isolations

YANG ZhiYuan,DUAN HuiJuan,WANG XiaoLei,LIU LiXin,ZHAO JiCheng,PAN Jie,LIU YueHuan,LIN Jian

Scientia Agricultura Sinica. 2019, 52(23): 4406-4414. doi:10.3864/j.issn.0578-1752.2019.23.021

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【Objective】The research aimed to compare the virulence of Duck Tembusu Virus (DTMUV), and to analysis its envelope protein gene sequence analysis and antigenic difference. 【Method】Susceptible 10-day-old duck embryos were inoculated with four different DTMUV strains, including DTMUV-HB isolated in 2011, DTMUV-AH isolated in 2014, DTMUV-GX1 isolated in 2012 and DTMUV-GX2 isolated in 2015. The median embryo lethal dose (ELD₅₀) of the four strains was measured with 6-day-old SPF chicken embryos. According to that, forty 180-day-old healthy Peking duck were challenged respectively with four strains which were diluted into 100 ELD₅₀/0.5 mL. The clinical, virological, pathological features of different DTMUV strains infection in ducks were characterized. The viral RNA of eight DTMUV strains were extracted from the allantoic fluid, and then the E gene were amplified by RT-PCR and sequenced. Then the similarity analysis of nucleotide and amino acid sequence and phylogenetic analysis were carried out. The HI titers of 4 antisera against 4 DTMUV strains were determined with duck Tembusu virus hemagglutination inhibition test. The neutralization titers of 4 antisera against 4 DTMUV strains were determined by neutralization assay using C6/36 cell lines. We analyzed the antigenic difference of 4 DTMUV strains by R value, which contained cross hemagglutination inhibition test and cell cross neutralization test. 【Result】(1) The median embryo lethal dose (ELD₅₀) were 10 ^-4.7-10 ^-5.3/0.1ml. The artificial infection test suggested that, feed intake and egg production of the challenged group decreased significantly on 3 days post inoculation (dpi), and the virus positive isolation rate were more than 85% on 2 dpi. The gross lesions of the reproductive system were mainly deformed and hemorrhaged follicular by necropsying on 8 dpi. (2) The results of sequence analysis showed that nucleotide sequence similarity was 95.7% - 100%, and the similarity of amino acid sequence was 98.2%. Genetic evolutionary analysis illustrated that all the DTMUV isolates in this study gathered into the same clade. (3) The R value showed antigenic difference of cross hemagglutination inhibition test were 0.79-1.12, and that of cell cross neutralization test were 0.79-1.20. 【Conclusion】There were no significant difference in virulence, E gene sequence and antigenicity of four DTMUV strains isolated in this study.

Hemagglutinating Activity of Duck Tembusu Virus

WANG XiaoLei, LIU YueHuan, DUAN HuiJuan, LIU LiXin, YANG ZhiYuan, ZHAO JiCheng, PAN Jie, LIU RuiHua, ZHAO WenQi, TIAN FangJie, Lü JinBao, LIN Jian

Scientia Agricultura Sinica. 2019, 52(23): 4415-4422. doi:10.3864/j.issn.0578-1752.2019.23.022

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【Objective】 The objective of this paper was to study the hemagglutinating activity of duck Tembusu virus (DTMUV). 【Method】The DTMUV was inoculated intracerebrally in the 1-3-day-old sucking mice of BALB/c in different dilution to observe the morbidity. The brains of sucking mice were collected and purified according to the referenced methods. The purified virus fluid was inactivated by suitable inactivator, followed by the inactivated inspection in 6-day-old SPF chicken embryo. The virus fluid was evaluated the hemagglutinating activity with the erythrocyte suspension of chicken, duck, goose, pigeon and swine. The concentration of the erythrocyte suspension used in the hemagglutination test was compared to select the proper concentration. The pH value of the reaction system between 6.0-7.0 and the reaction temperature including 4℃, room temperature and 37℃ were compared. 【Result】 In the 6 day after inoculation , the sucking mice inoculated with 1:10 dilution appeared severe clinical symptoms including twitch, paralysis, and most of them were in agonal stage, while the symptoms of the sucking mice inoculated with 1:50 and 1:100 dilution were slighter. The purified DTMUV fluid was pinkey and clarified. It could be inactivated by formaldehyde but not β-propiolactone that would produce lots of flocks and change the property of the fluid. The DTMUV could hemagglutinate with the erythrocyte suspension of chicken, duck, goose, pigeon and swine, and the agglutination graphics was clear and stable when the concentration of the erythrocyte suspension was 0.33%. The hemagglutinating activity of DTMUV could be showed when the reaction system pH value was 6.0-6.8, while the highest hemagglutination titer appeared during the pH 6.0-6.2. Furthermore, the hemagglutinating activity could be appeared at 4℃, room temperature and 37℃, respectively, although the time required for hemagglutination at 4℃ was much longer. 【Conclusion】The DTMUV proliferated in the sucking mice of BALB/c had a broad spectrum of hemagglutinating activity that could hemagglutinate with the erythrocyte suspension of chicken, duck, goose, pigeon and swine. The hemagglutinating activity was stable and could hemagglutinate with the goose erythrocyte at 4℃, room temperature and 37℃. The suitable reaction conditions were 0.33% concentration of the erythrocyte suspension and pH 6.0-6.2 of the reaction system.

Correlation Research of the Serological Test (HI) and Challenge Test of Duck Tembusu Virus Inactivated Vaccine

WANG XiaoLei,LIN Jian,YANG ZhiYuan,LIU LiXin,DUAN HuiJuan,CHENG HuiMin,ZHAO JiCheng,PAN Jie,LIU YueHuan

Scientia Agricultura Sinica. 2019, 52(23): 4423-4428. doi:10.3864/j.issn.0578-1752.2019.23.023

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【Objective】The objective of this paper was to evaluate the correlation of the serological test (HI) and challenge test of duck Tembusu virus inactivated vaccine.【Method】Three batches of vaccines ( laboratory trial products) were used to immunize the 51-day-old young ducks and 260-day-old laying ducks by intramuscular injection in 0.15, 0.3, 0.5 and 1.0 mL, respectively. Secondary immunization was proceeded in the same doses and route at 14 day after primary immunization. Ducks were regrouped according to the HI titer and challenged with 0.5 mL (100 DID₅₀) DTMUV at day 28 after secondary immunization. Serum samples were collected at 2 days later for virus isolation and laying ducks were dissected at 8 days later to observe the ovarian lesions. All the results were counted to evaluate the immune potency of vaccines. The correlation of HI titer and immune protection rate was analyzed.【Result】The protection rate of immunized young ducks with HI titer of 1:5 or lower, 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640 was 50% (20/40), 43.8% (7/16), 74.3% (26/35), 86.7% (26/30), 96.6% (57/59), 92.7% (51/55), 95% (38/40) and 100% (11/11), respectively. The protection rate was 48.2% with HI titer lower than 1:20, while the rate was 74.3% with HI titer of 1:20 and 93.8% with HI titer of 1:40 or greater. In laying ducks, the protection rate of flocks with HI titer of 1:5 or lower, 1:20, 1:40 and 1:80 was 66.7% (12/18), 83.3% (10/12), 95.2% (20/21) and 100% (15/15), respectively. The protected proportion of ducks with HI titer of 1:10, 1:160, 1:320 and 1:640 was 2/3, 7/7, 1/1 and 2/2, respectively. The protection rate was 66.7% with HI titer of 1:10 or below, while the rate was 83.3% with HI titer of 1:20 and 97.8% with HI titer of 1:40 or greater. There were significant positive correlations between the HI titer and the immune protection rate both in young ducks and laying ducks. The Spearman’s correlation coefficient was 0.905 (P<0.01) and 0.932 (P<0.01), respectively. 【Conclusion】There were positive correlations between the HI titer and the immune protection rate of duck Tembusu virus inactivated vaccine (strain HB). The HI detection could be used to evaluate the efficacy of duck Tembusu virus inactivated vaccine (strain HB) instead of challenge test. The HI titer of 1:20 could be defined as the criteria of protection for the duck Tembusu virus inactivated vaccine after immunization.

Prokaryotic Expression and Polyclonal Antibody Preparation of Duck Oligoadenylate Synthase-Like Protein

BI KeRan,LI Yin,HAN KaiKai,ZHAO DongMin,LIU QingTao,LIU YuZhuo,HUANG XinMei,YANG Jing

Scientia Agricultura Sinica. 2019, 52(23): 4429-4436. doi:10.3864/j.issn.0578-1752.2019.23.024

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【Objective】In previous study, we have obtained the full-length gene of duck oligoadenylate synthase-like protein (duOASL) and confirmed that duOASL could inhibit DTMUV replication. To further investigate the inhibitory effect of duOASL on DTMUV replication, the recombinant protein (pGEX-OASL) with GST tag and polyclonal antibody of duOASL protein was obtained by prokaryotic expression and polyclonal antibody preparation technology. These results would lay a foundation for performing further studies to determine mechanism(s) underlying the antiviral activity duOASL at the molecular level. 【Method】Primers were designed based on the ORF sequence of duOASL gene from healthy Cherry Valley ducks. Total RNA was extracted from the spleen tissues of duckling by using HP Total RNA Kits. The cDNA was synthesized using random primers and M-MLV reverse transcriptase. Full-length of duOASL gene was obtained by synthesising pGEX-OAS-F and pGEX-OAS-R primers by using the cDNA. The PCR results were detected by 1.5% agarose electrophoresis. The sequences of the same size as the target band were purified and cloned to pEASY-T1 vector. The cloned gene ORF was inserted into the expression vector pGEX-4t-1 with GST tag and by BamH I and Xho I digestion. The recombinant plasmid was transformed into E. coli BL21 and expressed under the induction with 0.5 mmol·L ^-1 IPTG for 4 h. The expression of the fusion protein was detected by SDS-PAGE and Western-blotting. Then the polyclonal antibody was obtained from rabbits which had immunized by the purified duOASL recombinant protein. The purity and titer of the polyclonal antibody were determined by SDS-PAGE and ELISA.【Result】The recombinant protein was a soluble protein and the molecular weight was 84 kD. The purity was 0.5 mg·mL ^-1. The antibody titer of polyclonal antibodies was 1:512 000.【Conclusion】Above results would lay a foundation for further studies of duOASL inhibiting DTMUV replication.

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