从海南省杂草胜红蓟和假马鞭上检测到粉虱传双生病毒

 利用三抗体夹心ELISA(TAS-ELISA)及PCR检测的方法对采自海南的胜红蓟(Ageratum conyzoides)和假马鞭(Stachytarphetajamaicensis)的5个病样进行了检测,表明均为粉虱传双生病毒。PCR扩增产物克隆后进行序列测定,结果表明存在2类双生病毒,其中样品Hn2存在2类病毒的复合侵染。这是在海南首次报道存在有粉虱传双生病毒。     

粉虱传双生病毒的TAS-ELISA及PCR快速检测

 利用粉虱传双生病毒(WTGs)多克隆抗体及单克隆抗体,建立了三抗体夹心ELISA (TAS-ELISA)检测WTGs的方法,并发现了单克隆抗体SCR18可广泛用于我国WTGs的检测。利用根据WTGs基因组上基因间隔区及外壳蛋白基因保守序列设计的引物,建立了PCR特异检测WTGs的方法。对田间病样的TAS-ELISA和PCR检测表明,粉虱传双生病毒在云南省烟草、番茄和南瓜上均存在,2种方法的测定结果是一致的。     

烟粉虱中甘薯双生病毒(sweepoviruses)半巢式PCR快速检测技术的建立与应用

甘薯双生病毒(sweepoviruses)是侵染甘薯的一类重要病毒,通过烟粉虱以持久方式传播,我国甘薯上至少存在8种甘薯双生病毒.本研究根据我国已报道的8种甘薯双生病毒基因组保守区设计了一组引物,建立了单头烟粉虱中甘薯双生病毒的半巢式PCR快速检测方法.特异性和灵敏性分析结果表明,半巢式PCR具有较高的特异性和灵敏性,...     

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.     

利用内标为基础的RT-PCR技术检测草莓斑驳病毒

 利用改进的CTAB法提取出优质的草莓总RNA,可以稳定地进行RT PCR。针对草莓斑驳病毒(SMoV)基因组序列设计筛选引物,对病毒基因组的不同区域进行扩增,扩增产物经克隆、测序证明为SMoV的特异片段,与国外序列的同源性为91%~97%。为了监测RNA的质量和RT PCR反应的正常进行,在检测体系中引入线粒体NADH脱氢酶基因ND2亚基作为内标,内标引物跨越内含子区域,只对剪接后的mRNA进行特异扩增,可以较好地监测整个检测过程。在国内首次建立了SMoV的RT PCR检测体系,以优质的草莓总RNA为模板,结合扩增内标的检测体系,对草莓病毒指示植物和草莓栽培品种都可以进行快速稳定的检测。     

棉花皱叶病毒分子检测方法

棉花皱叶病毒(Cotton leaf crumple virus,CLCrV)是典型的双生病毒,含A和B两个组分。根据B组分的保守序列设计的一对普通PCR引物,得到约为636 bp的片段,实验结果显示能特异性地扩增CLCrV B组分,棉花曲叶病毒(Cotton leaf curl virus,CLCuV)、非洲木薯花叶病毒(African cassava mosaec vi-rus,ACMV)、花椰菜花叶病毒(Cauliflower mosaic virus,CaMV)及番茄黄化曲叶病毒(Tomato yellow leaf curl virus,TYLCV)不干扰普通RCR方法对CLCrV的检测。设计的实时荧光PCR引物和探针也能特异性地检测棉花皱叶病毒,而CLCuV、ACMV和TYLCV不影响实时荧光PCR对CLCrV的检测。     

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。     

四川米易赛葵上粉虱传双生病毒的分子鉴定及复合侵染检测

为了明确四川米易赛葵上的双生病毒种类及复合侵染情况,基于滚环扩增PCR（RCA-PCR）技术,利用双生病毒DNA-A、DNA-B及卫星DNA的通用引物对四川米易表现黄脉症状的12个赛葵样品进行了检测,并对其序列进行分析。双生病毒的检出率高达92%;共检出云南赛葵黄脉病毒（Malvastrum yellow vein Yunnan virus,MYVYNV）、赛葵黄脉病毒（Malvastrum yellow vein virus,MYVV）及中国番茄黄化曲叶病毒（Tomato yellow leaf curl China virus,TYLCCNV）等3种双生病毒,复合侵染率高达67%;11个阳性样品中共检测到两类卫星DNAβ分子,分别与MYVYNV伴随的DNAβ（MYVYNB-[Y160],98.4%～98.7%）和MYVV伴随的DNAβ（MYVB-[Y197],98.5%～98.7%）的序列相似性最高;未扩增到DNA-B及DNAα分子。表明病毒DNA-A与卫星DNAβ以病害复合体形式存在,且DNAβ可伴随异源辅助病毒。     

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。     

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。     

双生病毒——一类值得重视的植物病毒

 双生病毒是世界范围内广泛发生的一类植物单链DNA病毒,已在多种作物上引起严重危害。重组是导致双生病毒变异和病害流行的重要原因。近年来,在单组份双生病毒中发现有新型卫星DNA分子——DNAβ,DNAβ是病毒致病所必需的。本文就双生病毒的危害性、病毒的重组、DNAβ的发现及与致病性的关系、双生病毒与伴随的小分子DNA形成病害复合体、双生病毒DNA-A和小分子DNA的进化关系等问题进行了简要评述。     

Tomato dwarf leaf curl virus, a new bipartite geminivirus associated with tomatoes and peppers in Jamaica and mixed infection with tomato yellow leaf curl virus

Genomic characterization using nonradioactive probes, polymerase chain reaction with degenerate primers for whitefly transmitted geminiviruses and nucleotide sequencing were used to describe a new bipartite geminivirus, associated with dwarfing and leaf curling of tomatoes and peppers in Jamaica. Partial DNA-A and DNA-B clones were obtained. DNA sequence analysis showed that tomato and pepper samples have a similar geminivirus associated with them. Nucleotide sequence identity > 92% between the common regions of DNA-A and DNA-B confirmed the bipartite nature of the Jamaican geminivirus isolates. Nucleotide sequence comparisons of DNA-A and DNA-B with those of geminiviruses representing the major phylogenetic groups of Western Hemisphere geminiviruses showed the greatest similarity to potato yellow mosaic virus and members of the Abutilon mosaic virus cluster of geminiviruses. This new virus is given the name tomato dwarf leaf curl virus (TDLCV) because of the dwarfing and leaf curling symptoms associated with infected tomato plants. Polymerase chain reaction and Southern hybridization showed mixed infections of TDLCV with tomato yellow leaf curl virus from Israel in 16% of the field samples of tomatoes and peppers.     

侵染甜椒的番茄黄化曲叶病毒的分子鉴定和全序列分析

<正>番茄黄化曲叶病毒(Tomato yellow leaf curl virus,TYLCV)在世界范围内可危害多种作物,造成植株矮化、叶片皱缩变形、局部黄化等症状。该病毒自1964年首次报道以来已蔓延至世界多地。在我国2006年上海首次报道该病毒[1],随后江苏、山东、安徽、北京、河北、天津等地相继报道,危害严重。TYLCV为双生病毒科(Geminiviridae)菜豆金色花叶病毒属(Begomovirus)成员,基因组为单组     

trans-Dominant Inhibition of Geminiviral DNA Replication by Bean Golden Mosaic Geminivirus rep Gene Mutants

ABSTRACT Geminiviruses are a group of single-stranded DNA viruses that cause major losses on a number of important crops throughout the world. Bean golden mosaic virus (BGMV) is a typical bipartite, whitefly-transmitted geminivirus that causes a severe disease on beans (Phaseolus vulgaris) in the Western Hemisphere. The lack of natural resistance to geminiviruses has led to attempts to engineer resistance, particularly through the use of pathogen-derived resistance strategies. The rep gene contains several conserved domains including nucleoside triphosphate (NTP)-binding and DNA-nicking domains and is the only geminiviral gene necessary for replication. Previous analysis by our group and others has demonstrated that the NTP-binding and DNA-nicking domains are necessary for geminiviral DNA replication. The ability of the rep gene and rep gene mutants to interfere with geminiviral DNA replication, when expressed in trans, was examined using a transient assay in a tobacco suspension cell culture system. Wild-type (wt) and mutant rep genes were cloned into plasmids under the control of the cauliflower mosaic virus 35S promoter for in planta expression and coinoculated into tobacco cells with infectious clones of various geminiviruses. The wt rep gene from BGMV-GA was able to support replication of BGMV-GA DNA-B. Several different rep gene mutants, with function-abolishing mutations in the NTP-binding or DNA-nicking domains, were potent trans-dominant inhibitors of geminiviral DNA replication.     

高通量测序技术在鉴定木本植物双生病毒中的应用

高通量测序技术(next-generation sequencing,NGS)平台提供了一种高效、快速、低成本、深度测序DNA的解决方案,2009年该技术开始被应用于植物病毒学领域,包括新病毒的发现,病害病原的鉴定,病毒基因组多样性及进化的研究,显著加快了植物病毒学的发展进程。迄今,应用高通量测序技术已经成功鉴定了上百种新的植物病毒和类病毒。双生病毒是一类对多种作物造成毁灭性危害的DNA病毒,多发生于草本作物。然而利用高通量测序技术,从柑橘、葡萄、苹果和桑树等多种多年生木本植物中检测到了新的双生病毒,显示出了高通量测序技术所独有的、传统检测技术所不具备的优势。本文围绕高通量测度技术在植物病毒学领域的应用进行概述,重点阐述NGS用于检测木本植物双生病毒的几个实例。     

Tomato leaf curl geminivirus in Australia: occurrence, detection, sequence diversity and host range

The occurrence of whitefly transmitted geminiviruses in Australia was studied using a mixed DNA probe capable of detecting a range of distinct geminiviruses. The only geminivirus species detected was Tomato leaf curl virus (TLCV), which is spread across a vast geographical region of far-northern coastal Australia, an area inhabited by the Australasian-Oceania biotype of Bemisia tabaci . The newly introduced silverleaf whitefly, B. tabaci biotype B, forms high population densities in the eastern coastal region of Queensland and is currently located approximately 150 km from the nearest known TLCV-infected area. The viral host range appeared to be narrow and of 58 species of crop plants and weeds inoculated using the B biotype, only 11 became infected with the virus, including five that did not show foliar symptoms. A DNA fragment of 694 nt, including the complete C4 open reading frame (ORF), the overlapping N-terminal part of the C1 ORF and the viral iterons involved in replication, was amplified from 11 TLCV field isolates and sequenced. Sequence analysis revealed an overall sequence variation of up to 14% in this region, as well as the presence of distinct viral iterons.     

双生病毒的同义密码子用法及其进化分析

 对双生病毒编码基因的变异进行了密码子用法分析,推测双生病毒基因的表达水平普遍不高,而外壳蛋白基因的表达水平相对较高,它们在密码子第3位上偏好使用A或T。双生病毒的密码子用法主要受到突变偏好和翻译选择等因素的影响。双生病毒的密码子使用具有基因特异性和一定程度的寄主特异性。基于相对同义密码子使用频率的聚类分析可以很好反映侵染单子叶植物和双子叶植物双生病毒之间的差异,也在一定程度上反映出双生病毒之间的亲缘关系。     

A geminivirus causing vein yellowing of Ageratum conyzoides in Singapore

A vein-yellowing disease of Ageratum conyzoides in Singapore was shown to be caused by a geminivirus, here named ageratum yellow vein virus (AYVV), which was transmitted by the whitefly Bemisia tabaci but not by inoculation with sap or through seed. AYVV particles (30 × 20 nm) are serologically related to those of other whitefly-transmitted geminiviruses, and reacted with some monoclonal antibodies elicited by particles of African cassava mosaic or Indian cassava mosaic geminiviruses. However, the epitope profile of AYVV differed from the profiles of these viruses, and from those of geminiviruses from vein yellowing-affected A. conyzoides from India and from yellow leaf curl-affected tomato from either Singapore or India. The results provide further evidence of antigenic differences among geminiviruses that cause similar diseases in the same plant species in different geographical regions.     

The transmission of geminiviruses by Bemisia tabaci

The cosmopolitan whitefly species, Bemisia tabaci (Gennadius) and Trialeurodes taporariorum (Westwood) have always been regarded as pests to a large range of worldwide crops. Both species are capable of transmitting plant viruses, with T. vaporariorum being the vector of only a few ‘clostero’-like viruses and B. tabaci the vector of viruses in several groups. The largest group of viruses transmitted by B. tabaci are the geminiviruses and B. tabaci is known to transmit around 60 members. Until recently, B. tabaci had been associated with only a limited range of host plants within any one region, although its total potential host range was large. Virus transmission was confined within the plant host range of each regional population of B. tabaci. The emergence of the polyphagous ‘B’ biotype of B. tabaci with its increased host range of more than 600 plant species, has resulted in geminiviruses infecting previously unaffected crops. As the ‘B’ biotype spreads further into Europe, European field and glasshouse crops have been shown to be susceptible to whitefly-transmitted viruses already endemic to other parts of the world. More than 20 colonies of B. tabaci, including both ‘B’ and non-‘B’ biotypes from disparate global locations have been compared for their ability to transmit more than 20 geminiviruses. All but two highly host-specific colonies were capable of transmitting most geminiviruses tested. However, some viruses were transmitted more efficiently than others. The virus coat protein or capsid is essential for vector recognition and transmission. By comparing transmissible viruses at the molecular level to viruses that are no longer whitefly-transmissible, the active epitope on the virus coat protein could be identified for designing future virus control strategies.