The N-terminal 62 amino acid residues of the coat protein of <Emphasis Type="Italic">Tomato yellow leaf curl Thailand virus</Emphasis> are responsible for DNA binding

The DNA-binding activity and DNA-binding domain of Tomato yellow leaf curl Thailand virus coat protein were investigated. A full-length coat protein (CP) and two truncated derivatives lacking the amino (CPΔ1-62) and carboxyl (CPΔ126-257) termini were produced in Escherichia coli as fusion proteins to glutathione-S-transferase (GST). Southwestern analysis showed that GST-CP bound both single-stranded (ss) and double-stranded (ds) DNA, while GST-CPΔ126-257 interacted only with ssDNA. Neither ss nor dsDNA bound to GST-CPΔ1-62. The results suggested that a putative DNA-binding domain is located at the N-terminal 1-62 amino residues. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AF141922 (TYLCTHV-[2]), AF220561 (RTBVCN), X01633 (MSV) and AF295401 (ToLCBV-[Ban5])     

Evidence of a new Tomato yellow leaf curl virus in Japan and its detection using PCR

A new isolate of Tomato yellow leaf curl virus (TYLCV) has been identified from tomato plants in Kochi Prefecture in Japan and designated TYLCV-[Tosa]. The complete nucleotide sequence of the isolate was determined and found to consist of 2781 nt. In phylogenetic analyses of entire nucleotide sequences, TYLCV-[Tosa] was delineated as a single branch and was more closely related to TYLCV-[Almeria] than TYLCV isolates Ng, Sz, or Ai reported in Japan, which had spread since 1996. Isolate TYLCV-[Tosa] is suggested to be a newly introduced, novel isolate of TYLCV that dispersed into Kochi Prefecture. In addition, a rapid method using the polymerase chain reaction to separate TYLCV isolates into four genetic groups was established. This method would be useful for reliable diagnosis based on genetic differences among isolates of TYLCV.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB192965 and AB192966     

应用纳米磁珠实时荧光定量PCR方法检测番茄黄化曲叶病毒

In order to develop a rapid, sensitive and specific qPCR assay for detection and quantification of Tomato yellow leaf curl virus (TYLCV), a pair of primers and TaqMan probe were designed according to the conserved sequence of known TYLCV isolates. Combining with MNP technique, a novel MNP-qPCR detection method was established and verified based on specificity, sensitivity and reproducibility tests. The results indicated that the Ct value of plotted standard curve showed good linear relationship(R² =0.9994)with the log of copy number of template. The established method showed a high specificity for TYLCV detection without crossing reaction with Tomato severe leaf curl virus and Tomato yellow leaf curl Sadinia virus, and was 10-fold more sensitive than routine PCR. Both coefficients of variation were less than 2%, indicating a good reproducibility. We have provided a novel method for detection of TYLCV in plant samples rapidly and quantitatively.     

伴随卫星分子的两种单组分双生病毒TbCSV和TYLCCNV之间的拮抗互作

为明确烟草曲茎病毒(Tobacco curly shoot virus,TbCSV)和中国番茄黄化曲叶病毒(Tomato yellow leafcurl China virus,TYLCCNV)在不同寄主中的相互作用,将含有TbCSV分离物Y35(Y35)和TYLCCNV分离物Y10(Y10)及伴随卫星DNAβ(Y35β或Y10β)侵染性克隆的农杆菌按不同组合接种本氏烟和心叶烟,定期观察植株症状。结果表明,当Y10和Y35同时伴随Y10β或Y35β时,接种植株60天后,在本氏烟中引起的曲叶症状减轻,而在心叶烟中曲叶症状减轻的现象在侵染早期较明显,且植株表现的症状与Y35+Y10β或Y35+Y35β引起的症状相似。PCR及Southern印迹检测结果显示,在本氏烟植株中,两种病毒及卫星在整个侵染过程中均能复制,且Y10和Y35的复制水平在侵染后期均降低。在接种后15天的心叶烟植株中能检测到所有DNA组分,但接种100天后在这些植株中均不能检测到Y10的复制,而Y35的复制水平与单独伴随卫星相比差异不大。表明TbCSV与TYLCCNV在症状形成和复制水平上存在拮抗互作,且这种拮抗作用随寄主不同表现各异。     

不同番茄品种对番茄黄化曲叶病毒的抗病性鉴定

为评估生产上常用番茄栽培品种对番茄黄化曲叶病毒(Tomato yellow leaf curl virus,TYLCV)的抗性水平,采用田间大棚自然传毒的方式,通过对各品种的发病时间、发病率及病情指数等参数的比较,结合PCR及ELISA对TYLCV的检测结果,综合分析了20个番茄品种对番茄黄化曲叶病毒的抗病性。不同品种对番茄黄化曲叶病毒的抗性差异较大,试验筛选出仙客6号和金棚1号2份感病材料,发病率和病情指数在90%和60.9以上;佳红8号、10-秋展47和红罗曼2号3份高抗材料,发病率和病情指数均为0;其它表现不同程度抗、耐病水平的材料15份,发病率和病情指数分别在10.0%～85.0%和1.0～40.6之间。     

关中地区温室越冬茬番茄黄化曲叶病毒病分子鉴定及流行规律

为了明确关中地区越冬茬番茄黄化曲叶病毒病发生和流行规律,通过分析该病发生与番茄品种、定植期及传播介体烟粉虱之间的关系,并采用PCR技术对田间病原进行分子鉴定。结果表明,番茄黄化曲叶病毒病在8月中下旬至11月上中旬开始侵染,翌年3月中下旬发生再侵染,秋季病情减轻;烟粉虱种群数量与病害发生程度呈线性正相关;不同番茄品种对番茄黄化曲叶病毒(Tomato yellow leaf curl virus,TYLCV)的抗性差异显著,其中大番茄品种布鲁尼1288和DRW7728,小番茄品种千禧和美红对该病表现为免疫;分子检测结果表明,4个样品中均扩增出543 bp的特异片段,与NCBI数据库Gen Bank的TYLCV序列(登录号为GU084381、KC138544.1、KC138543.1和JX456642.1)的相似性达99%。研究表明,关中地区番茄病毒病为番茄黄化曲叶病毒病,番茄品种、定植期及烟粉虱发生动态是影响该病发生的主要因素。     

南疆温室番茄黄化曲叶病病毒种类的分子鉴定

为明确南疆温室番茄黄化曲叶病的病毒种类,利用双生病毒的兼并引物通过PCR扩增,对采集的20个番茄病株进行了分子检测.从20个病株中均扩增到约500 bp的目标片段,对其中4株进行克隆和测序,其相互间序列同源性为97.1％ ～99.3％,与番茄黄化曲叶病毒(Tomato yellow leaf curl virus,TYLCV)的同源性较高,为98.6％ ～ 99.5％.随机选取莎车分离物KS2-5进行全基因组的克隆和测序,KS2-5 DNA全长为2781 nt(序列号:JQ807735),具有典型的双生病毒基因组特征,与TYLCV其它分离物同源性达到98.9％～99.5％,而与其它粉虱传双生病毒的序列同源性较低,为68.3％ ～75.5％,表明危害南疆温室番茄的病毒种类为番茄黄化曲叶病毒TYLCV.     

普通烟对烟粉虱取食及其与TYLCCNV共侵染的生理生化反应

为明确普通烟遭受烟粉虱Bemisia tabaci取食及与中国番茄黄化曲叶病毒(Tomato yellow leaf curl China virus,TYLCCNV)共侵染时的响应机制,采用常规生理生化指标测定法分析比较MED隐种烟粉虱取食及其与TYLCCNV共侵染处理对普通烟植株中营养组分、防御物质积累及主要氧化酶活性的影响。结果显示,与对照植株相比,烟粉虱取食及其与TYLCCNV共侵染均可导致普通烟植株中可溶性糖和可溶性蛋白含量明显降低,多酚含量显著升高,过氧化氢酶(CAT)活性显著升高,超氧化物歧化酶(SOD)和过氧化物酶(POD)活性呈现先升后降的变化趋势。与烟粉虱取食植株相比,烟粉虱与TYLCCNV共侵染后1 d时普通烟植株中可溶糖含量显著降低了21.05%,3、7和9 d时可溶性蛋白含量分别显著降低了27.40%、26.84%和20.90%;共侵染后的1、3、5和9 d时多酚含量分别显著降低了34.60%、14.30%、3.07%和15.19%;共侵染后的1、3、5和7 d时CAT活性分别提高了3.04倍、1.42倍、1.68倍和1.96倍;共侵染后7 d时POD活性显著降低,共侵染后9 d时SOD活性显著升高。表明普通烟可通过改变其营养组分及积累防御物质或提高氧化酶活性来防御烟粉虱取食及其与TYLCCNV的共侵染,而烟粉虱与TYLCCNV共侵染可诱导植株内产生较单独取食时更少的多酚类防御物质,以提高其在宿主植物上的适应性。     

烟粉虱传播的番茄褪绿病毒和番茄黄化曲叶病毒对不同番茄品种的复合侵染

为明确烟粉虱传播的番茄褪绿病毒(Tomato chlorosis virus,ToCV)与番茄黄化曲叶病毒(Tomato yellow leaf curl virus,TYLCV)对不同番茄品种的复合侵染情况,于2015年11月在山东省寿光市温室内采集13个番茄品种共390份疑似发病植株叶片,对不同番茄品种的TYLCV抗性和2种病毒的复合侵染以及温室内发病番茄植株上烟粉虱成虫的带毒率进行检测。结果表明,采集的13个番茄品种经分子标记检测鉴定均为TYLCV杂合抗性;不同番茄品种ToCV与TYLCV的复合侵染率存在明显差异,大果番茄粉宴和贝瑞上复合侵染率最高可达73.3%,而樱桃番茄八喜上未检测到这2种病毒的复合侵染。此外,在发病番茄植株上采集的烟粉虱成虫体内可检测到2种病毒,其中烟粉虱ToCV带毒率为90.7%,TYLCV带毒率为80.0%,同时检测到ToCV与TYLCV的概率为71.3%。表明ToCV和TYLCV的复合侵染在山东省番茄生产中普遍发生,烟粉虱可同时携带这2种病毒并广泛传播。     

Three distinct groups of isolates of <Emphasis Type="Italic">Tomato yellow leaf curl virus</Emphasis> in Japan and construction of an infectious clone

Complete nucleotide sequences of eight Japanese isolates of Tomato yellow leaf curl virus (TYLCV) were determined and compared with four TYLCV isolates already reported. These isolates separated into three groups – Shizuoka (Sz), Aichi (Ai), Nagasaki (Ng) – and had 99% identities within the groups. Full-length molecules of DNA-A of group Sz consist of 2791nt and those of group Ai contain 2787nt. Both were closely related to TYLCV-Is.M, although those of group Ng had 2793nt and were more closely related to TYLCV-Is. Comparison of common sequences of isolates belonging to groups Sz and Ai had substitutions of 4nt in the intergenic region and nonsynonymous substitutions at open reading frames between the groups. None of the isolates tested had DNA molecules. Agroinfection of four plant species with a DNA-A dimeric infectious clone of TYLCV-SzY, a member of group Sz, resulted in systemic infection. Tomato plants then developed typical yellow leaf curl symptoms.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB116629, AB116630, AB116631, AB116632, AB116633, AB116634, AB116635, and AB116636     

北京地区番茄黄化曲叶病病毒分离物测定及株系的初步鉴定

 番茄黄化曲叶病毒病是番茄生产中的一种毁灭性病毒病害,2009年传入北京。利用烟粉虱传双生病毒简并引物PA/PB对2010年~2011年采集自北京市5个区县的53个番茄样品进行检测,30个表现典型黄化曲叶病症状的样品均扩增得到约500 bp的特异条带,测定了其中7个样品的部分序列,经序列比对分析表明其为番茄黄化曲叶病毒（Tomato yellow leaf curl virus, TYLCV）。利用TYLCV特异引物TJ-F/TJ-R、TY-F/TY-R对样品BJDXXY、BJFS02、BJFS03、BJMY2231进行TYLCV基因组克隆和序列测定,经分析4个样品携带的TYLCV基因组长度均为2 781碱基,编码6个蛋白。基因组序列比较发现,这4个分离物与TYLCV-Israel株系同源性达到98%以上;通过建立系统发育树,发现BJDXXY、BJFS02、BJFS03与河北分离物（HBLF4）、山东分离物（SDSG）亲缘关系较近,BJMY2231与上海分离物（TYLCV-Israel）、江苏分离物（JSNJ1）亲缘关系较近。     

番茄黄化曲叶病毒V 2基因原核表达及多克隆抗体制备

 通过PCR的方法,从番茄黄化曲叶病毒江苏分离物DNA中扩增到V2基因,并克隆到pMD18-T载体,序列测定结果显示其与报道的XH2分离物序列完全一致,核苷酸序列全长351 bp,编码115个氨基酸,推测分子量约13 kD。将该V2基因亚克隆到原核表达载体PET32a中获得重组表达载体PET32a-V2,转化大肠杆菌BL21（DE3）,IPTG可诱导1个分子量约为32 kDa的融合蛋白（含His标签）表达,经Ni⁺ NTA亲和柱纯化,获得纯化的重组蛋白,免疫家兔制备TYLCV-V2蛋白的多克隆抗体Anti-V2,间接ELISA测定Anti-V2的效价达1∶655 360,Western blot及DIBA分析结果表明Anti-V2可与V2蛋白发生特异血清反应,可为进一步研究V2蛋白的功能提供参考。     

复合侵染水茄的两种菜豆金色花叶病毒属病毒基因组结构特征分析

为明确水茄Solanum torvum植株叶片邹缩、褪绿是否由菜豆金色花叶病毒属病毒侵染引起,从云南省西双版纳傣族自治州田间采集具有疑似感染症状的水茄植株叶片样品,应用菜豆金色花叶病毒属病毒简并引物和特异性引物进行PCR扩增、克隆和测序,通过生物信息软件分析比较其核苷酸序列特征,并对其进行系统发育分析。结果显示,从采集的疑似病叶中共克隆获得了5条菜豆金色花叶病毒属病毒DNA-A全序列和3条DNA-B全序列,经全序列分析发现,侵染水茄的2种菜豆金色花叶病毒属病毒分离物分别属于中国南瓜曲叶病毒(squash leaf curl China virus,SLCCNV)和野茼蒿黄脉病毒(Crassocephalum yellow vein virus,CraYVV)。SLCCNV水茄分离物的基因组具有典型的菜豆金色花叶病毒属病毒双组分结构特征,与来自泰国的SLCCNV分离物(AB330078)亲缘关系最近,相似性最高达到99.0%;CraYVV水茄分离物的基因组具有典型的菜豆金色花叶病毒属病毒单组分结构特征,与来自云南省景洪市的CraYVV分离物(EF165536)亲缘关系最近,相似性最高达到97.6%。表明水茄是这2种菜豆金色花叶病毒属病毒的新寄主,并首次发现双组分和单组分菜豆金色花叶病毒属病毒可复合侵染水茄。     

2种单组份双生病毒与异源卫星DNA的互作

 烟草曲茎病毒(Tobacco curly shoot virus,TbCSV) Y35分离物(Y35)和中国番茄黄化曲叶病毒(Tomato yellow leaf curl China virus,TYLCCNV) Y10分离物(Y10)是从云南烟草上分离的2种单组份双生病毒,分别伴随卫星DNAβ(Y35β及Y10β)分子。将Y35和Y10分别伴随其同源或异源卫星(Y35β或Y10β)接种本氏烟和心叶烟,症状观察表明,无论Y10还是Y35伴随异源卫星DNAβ时引起的症状均与其伴随同源DNAβ引起的症状明显不同,且Y10β伴随Y35致病性最强。Southern印迹结果表明,Y10β及Y35β均可被异源辅助病毒稳定复制,但其复制水平明显低于伴随同源病毒时的水平,且植株中病毒和卫星的积累量并不完全与症状严重程度直接相关。     

河北省番茄黄化曲叶病毒病的分子鉴定初报

双生病毒是一类具有孪生颗粒形态的植物单链病毒,目前双生病毒病害已在多个国家和地区的作物上造成严重危害。近年来,我国多省报道作物上有这类病毒的发生,且有逐年加重和扩散的趋势。根据危害番茄的双生病毒DNA A序列保守区设计简并引物,对2009年4月采自河北省魏县表现叶片黄化、曲叶症状的4个番茄样品进行PCR检测,均为双生病毒阳性,对样品的扩增片断进行了克隆测序,经序列比对分析,与番茄黄化曲叶病毒（Tomato leaf curl virus,TYLCV）山东分离物（FJ646611.1）序列相似性为99.25%~99.55%,说明河北番茄黄化曲叶病由TYLCV引起。     

湖南省番茄和牵牛花上双生病毒的分子鉴定

双生病毒是一类在全世界范围内广泛发生的单链环状DNA病毒。本文对从湖南采集到的6例(洋姜、番茄、萝卜、赛葵、甘薯、牵牛花)疑似双生病毒侵染的植物叶片进行了分子鉴定。利用滚环扩增技术(RCA)对样品DNA进行扩增,分别对其RCA产物进行酶切,并将酶切得到的片段测序后进行BLAST比对,结果显示番茄样品中的病毒分离物与番茄黄化曲叶病毒相似性最高(99%),牵牛花样品中的病毒分离物与甘薯卷叶病毒相似性最高(99%),证明这两个分离物是单组分DNA-A双生病毒。这是在湖南省首次发现并报道双生病毒的全核酸序列。     

Panel of real‐time PCRs for the multiplexed detection of two tomato‐infecting begomoviruses and their cognate whitefly vector species

A new approach for the simultaneous identification of the viruses and vectors responsible for tomato yellow leaf curl disease (TYLCD) epidemics is presented. A panel of quantitative multiplexed real‐time PCR assays was developed for the sensitive and reliable detection of Tomato yellow leaf curl virus‐Israel (TYLCV‐IL), Tomato leaf curl virus (ToLCV), Bemisia tabaci Middle East Asia Minor 1 species (MEAM1, B biotype) and B. tabaci Mediterranean species (MED, Q biotype) from either plant or whitefly samples. For quality‐assurance purposes, two internal control assays were included in the assay panel for the co‐amplification of solanaceous plant DNA or B. tabaci DNA. All assays were shown to be specific and reproducible. The multiplexed assays were able to reliably detect as few as 10 plasmid copies of TYLCV‐IL, 100 plasmid copies of ToLCV, 500 fg B. tabaci MEAM1 and 300 fg B. tabaci MED DNA. Evaluated methods for routine testing of field‐collected whiteflies are presented, including protocols for processing B. tabaci captured on yellow sticky traps and for bulking of multiple B. tabaci individuals prior to DNA extraction. This work assembles all of the essential features of a validated and quality‐assured diagnostic method for the identification and discrimination of tomato‐infecting begomovirus and B. tabaci vector species in Australia. This flexible panel of assays will facilitate improved quarantine, biosecurity and disease‐management programmes both in Australia and worldwide.     

广西部分烟区烟草曲叶病病原的分子鉴定

[目的]明确广西西部地区靖西(JX)、凌云(LY)、德保(DB)和乐业(LeY)等4个县市烟草曲叶病的病原。[方法]2010年5-6月分别从广西靖西、凌云、德保和乐业等县市采集具有典型曲叶症状的烟草叶片,用基于双生病毒DNA保守序列设计简并引物Bego-1和Bego-6对病叶组织总DNA抽提物进行PCR扩增和对PCR产物进行序列测定,用BLAST、Vector NTI、MEGA 4.0和Simplot program 3.2软件等进行病毒序列分析、系统进化树构建和病毒重组分析。[结果]从选取的9个表现典型曲叶症状的样品叶组织总DNA抽提物中均可扩增出约1500bp与预期大小相符的DNA片段。测序和序列比对分析显示,9个样品扩增产物核苷酸序列相似性为73.7%～99.2%,与已报道的双生病毒具较高的相似性。其中,JX-2与中国番茄曲叶病毒广西番茄分离物(G32)的相似性最高,达99.2%;JX-3和JX-5与云南胡椒曲叶病毒云南辣椒分离物(YN323)相似性最高,分别为92.5%和93.4%;LeY-1、LY-1、DB-1、JX-1、JX-4和JX-6则与中国番茄黄化曲叶病毒中国番茄分离物(CHI)和广西烟草分离物(G102)的相似性最高,均高于95.0%。基于PCR扩增产物及已报道的双生病毒属代表种相应核苷酸序列构建的系统进化树分析表明,9个广西烟草分离物分属3个簇群:中国番茄曲叶病毒簇、云南辣椒曲叶病毒簇和中国番茄黄化曲叶病毒簇。重组分析结果表明:JX-3是云南辣椒曲叶病毒和中国番茄曲叶病毒的重组病毒,JX-5是云南辣椒曲叶病毒和中国番茄黄化曲叶病毒的重组病毒。[结论]9个广西烟草分离物分属于4种双生病毒:中国番茄曲叶病毒和中国番茄黄化曲叶病毒,以及分别由上述两种病毒与云南辣椒曲叶病毒重组而来的2种重组病毒。其中,中国番茄曲叶病毒自然侵染烟草、云南辣椒曲叶病毒和中国番茄曲叶病毒及中国番茄黄化曲叶病毒的重组病毒等结果此前均未见报道。     

广东省番茄黄化曲叶病毒的分子鉴定及序列分析

番茄黄化曲叶病毒病是世界番茄生产上一种毁灭性病害,番茄黄化曲叶病毒Tomato yellow leaf curl virus(TYLCV)是引起该病害的主要病原病毒之一。本文采用滚环扩增及基因克隆方法,获得了侵染广东佛山和肇庆番茄的TYLCV 4个分离物全基因组;它们均为2 781 nt,编码6个ORF,其中病毒链上编码AV1和AV2,互补链上编码AC1、AC2、AC3和AC4。同源性比较结果表明,4个广东分离物基因组序列两两间同源性为99%以上;与已报道的TYLCV各分离物同源性在90%以上,而与来自中国不同地区的TYLCV分离物的同源率均在98%以上。系统进化分析显示,广东分离物与来自中国不同地区的TYLCV分离物亲缘关系较近,并聚类在一个分支。因此,侵染引起广东佛山和肇庆番茄黄化曲叶病的病毒应来自国内其他地区。本研究是对TYLCV广东分离物分子特征的首次报道。     

侵染假酸浆的泰国番茄黄化曲叶病毒全基因组结构特征

为明确假酸浆Nicandra physalodes叶片黄化、皱缩症状是否由菜豆金色花叶病毒属病毒侵染引起,本研究利用分子检测方法和生物信息学技术鉴定了假酸浆样品中的病毒种类。从采集的病样中克隆并获得了2条菜豆金色花叶病毒属病毒DNA-A全序列和1条beta卫星全序列,经全序列分析发现,该双生病毒的两条DNA-A全序列与泰国番茄黄化曲叶病毒(tomato yellow leaf curl Thailand virus, TYLCTHV)云南分离物TYLCTHV-YN1732一致性最高,达99.3%,亲缘关系较近;beta卫星的全序列与云南番茄曲叶beta卫星(tomato leaf curl Yunnan betasatellite, TLCYnB)的分离物YN5230一致性最高,达99.3%,亲缘关系较近。重组分析显示,假酸浆上分离的TYLCTHV-YN5735-12是一个重组病毒,有两个重组事件,一个主要发生在AV1的编码区,由中国番茄黄化曲叶病毒(tomato yellow leaf curl China virus, TYLCCNV)和广西大戟曲叶病毒(euphorbia lea...