广西烟草病毒病发生情况调查和病原病毒的鉴定

2010~2011年对广西百色市、河池市和贺州市的主要烤烟产区进行烟草病毒病发生情况和种类调查。结果显示,广西烟草病毒病以由烟草花叶病毒和黄瓜花叶病毒引起的花叶类型症状为主,一般发病率1.0%~10.0%,个别严重发病田块发病率达到82.0%~100.0%;马铃薯Y病毒病、番茄斑萎病毒病和曲叶病则为局部发生病害,其中马铃薯Y病毒病在靖西县、南丹县、富川县和钟山县零星发生,发病率0.1%~5.0%,个别田块达到20.0%;番茄斑萎病毒病主要在靖西县、隆林县、田林县和南丹县发生;曲叶病仅在靖西县、隆林县、田林县和乐业县等百色市烟区发现。采用间接ELISA、PCR和RT-PCR等方法对田间采集的185个各类疑似病毒病样进行病毒种类检测,其中145个样品检测出病毒,分别是烟草花叶病毒、黄瓜花叶病毒、马铃薯Y病毒、番茄环纹斑点病毒、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2种病毒与云南辣椒曲叶病毒重组而来的2种重组病毒,其中以TMV的检出率最高,占检出样品的86.7%,是广西烟草病毒病的优势病原病毒。     

恩施地区烟草病毒病的电镜诊断与RT-PCR鉴定

采集湖北省恩施地区疑似感染烟草病毒病的新鲜烟叶样品,提纯获取混合样品病毒粒子悬浮液,采用悬滴法进行负染,通过电镜观察对病原物进行初步鉴定。结果显示,病毒粒子悬浮液中存在大量线状、杆状与球状形态病毒粒子。进一步提取混合样品总RNA,使用马铃薯Y病毒属通用引物,TMV、CMV外壳蛋白序列特异性引物进行PCR扩增,将扩增产物回收克隆用于测序,经BLAST程序与GenBank进行比对,感病样品中检出TMV、CMV、PVY,与电镜所观察到的病毒粒子形态结构一致。     

利用小RNA测序技术鉴定吉林省甘薯病毒

在吉林省7个主要甘薯种植区共采集85份甘薯叶片样品,利用小RNA深度测序技术对混合样品进行检测,经RT-PCR和测序验证,鉴定出样品中存在10种病毒,包括6种RNA病毒和4种DNA病毒。分别是马铃薯Y病毒科马铃薯Y病毒属的甘薯羽状斑驳病毒Sweet potato feathery mottle virus (SPFMV)、甘薯潜隐病毒Sweet potato latent virus (SPLV)、甘薯G病毒Sweet potato virus G (SPVG)、甘薯C病毒Sweet potato virus C (SPVC)、甘薯2号病毒Sweet potato virus 2 (SPV2);长线形病毒科毛形病毒属的甘薯褪绿矮化病毒Sweet potato chlorotic stunt virus (SPCSV);双生病毒科菜豆金色花叶病毒属的甘薯曲叶病毒Sweet potato leaf curl virus(SPLCV);玉米线条病毒属的甘薯无症状1号病毒Sweet potato symptomless virus 1 (SPSMV1);花椰菜花叶病毒科杆状DNA病毒属的甘薯杆状DNA病毒B Sweet potato badnavirus B (SPBV-B)和甘薯隐症病毒Sweet potato pakakuy virus (SPPV)。     

一套适合烤烟3种RNA病毒的RT-PCR检测方法

烤烟是贵州省的重要经济作物,但是烟草花叶病毒(TMV)、黄瓜花叶病毒(CMV)和马铃薯Y病毒(PVY) 3种RNA病毒在贵州烤烟产区造成巨大损失。3种病毒引起的症状相似性造成了田间诊断的困难。针对贵州烟区的样品,开发了一套包含了靶向3种病毒外壳蛋白基因特异性片段的RT-PCR检测方法。3对引物涵盖的基因片段长度分别为:TMV-120 bp,CMV-240 bp,PVY-660 bp,退火温度均为55°C。通过1次PCR反应,就可以根据扩增条带的大小判断病毒的种类,还可以检测复合侵染的样品中2种病毒与3种病毒混合DNA的种类。该方法非常适合基层检测站点快速和高效地检测大批量烟叶样品,指导后续防控工作的开展。     

深度测序发现贵阳发生的辣椒病毒病由多种病毒复合侵染所致

2016年9月贵州省贵阳市发生严重的辣椒病毒病,症状复杂,主要表现为植株矮化,叶片黄化、花叶、皱缩、畸形以及枯死斑,果实有坏死斑等,根据症状难以判断病毒种类。本文采用小RNA深度测序技术对田间自然发病的2株辣椒标样进行了毒源鉴定,发现样品1由蚕豆萎蔫病毒2号(Broad bean wilt virus2,BBWV2)、辣椒脉斑驳病毒(Chilli veinal mottle virus,ChiVMV)、黄瓜花叶病毒(Cucumber mosaic virus,CMV)和辣椒内源RNA病毒(Bell pepper endornavirus,BPEV)4种病原复合侵染;样品2中除鉴定到上述4种病毒外,还检测到马铃薯Y病毒(Potato virusY,PVY)。进一步通过反转录PCR(RT-PCR)对深度测序结果进行了验证,证明其准确可靠。其中4个辣椒标样中均有的辣椒内源RNA病毒(BPEV)为贵州省首次报道。多种病毒复合侵染是辣椒产量和品质的重要限制因素之一,是辣椒生产的主要威胁。     

五种烟草病毒TMV、CMV、TEV、PVY及TVBMV的多重RT-PCR同步检测

 我国烟草病毒主要有烟草花叶病毒(TMV)、黄瓜花叶病毒(CMV)、烟草蚀纹病毒(TEV)、马铃薯Y病毒(PVY)和烟草脉带花叶病毒(TVBMV),通常发生复合侵染。本研究对我国5种烟草病毒的外壳蛋白基因部分序列设计引物,通过优化引物和模板浓度,摸索扩增参数,在一个体系中成功对5种病毒复合侵染的烟草材料进行多重RT-PCR扩增,得到237、273、347、456和547 bp共5条特异性条带,建立了能同时检测TMV、CMV、TEV、PVY和TVBMV的多重RT-PCR检测体系。对田间样品检测结果证明,多重RT-PCR体系能够同时检测5种病毒,并且灵敏度高。     

马铃薯Y病毒云南昭通烟草分离物的检测及年度间差异比较

利用DAS-ELISA检测试剂盒检测昭通烟草脉斑病样品,结果表明存在马铃薯Y病毒O株系和马铃薯Y病毒N株系两个不同株系病毒。利用Sprimer和M4引物扩增、克隆、测序,得到长度为1 771 bp目的片段,该片段包含病毒的外壳蛋白基因序列、3′ UTR序列以及部分Nib基因序列;序列分析表明与湖南HN 2分离物（GenBank No. GQ200836）和美国NE 11分离物（GenBank No. DQ157180）核苷酸序列相似性均为98%,系统进化分析表明其具有较近的亲缘关系。     

云南、福建、湖南烟区烟草花叶病主要病毒种类检测及黄瓜花叶病毒亚组鉴定

 采用抗原直接包被和双抗体夹心酶联免疫吸附测定法(ELISA)对采自云南、福建、湖南烟区烟草花叶病样品进行了病毒种类检测,利用三抗体夹心ELISA对黄瓜花叶病毒(Cucumber mosaic virus,CMV)的亚组类型进行了鉴定。在云南采集的520个花叶病样品中,烟草花叶病毒(Tobacco mosaic virus,TMV)、CMV和马铃薯Y病毒(Potato virus Y,PVY)总检出率分别为71.74%、55.01%和6.35%;在福建采集的150个花叶病样品中,TMV、CMV和PVY的总检出率分别为94%、24.66%和8.00%;在湖南采集的74个花叶病样品中,TMV、CMV和PVY的总检出率分别为58.11%、51.35%和2.70%。部分样品为2种以上病毒复合侵染。云南、福建和湖南采集的64个CMV阳性样品中,属亚组Ⅰ的样品为57个,占89.1%;属亚组Ⅱ的样品为10个,占15.6%;其中3个样品为亚组Ⅰ和亚组Ⅱ的复合侵染。     

利用siRNA高通量测序技术检测烟草病毒

<正>烟草是我国的主要经济作物之一,病毒病是烟草生产上的重要病害。常见的烟草病毒主要有马铃薯Y病毒(Potato virus Y,PVY)、黄瓜花叶病毒(Cucumber mosaic virus,CMV)、烟草花叶病毒(Tobacco mosaic virus,TMV)、烟草脉带花叶病毒(Tobacco vein banding mosaic virus,TVMBV)等[1]。此外,近年来在烟草上还发生一些新的病毒病害,如南美红辣椒脉斑驳病毒(Chilli veinal mottle virus,ChiVMV)[2]、番茄环纹斑点病毒(Tomato zonate spot virus,ZTSV)[3]和番茄斑萎病毒(Tomato spotted wilt virus,TSWV)[4]。这些病毒     

siRNA对烟草原生质体中TMV RNA积累的干扰作用研究(英文)

 RNA干扰被认为是转录后基因沉默的一种机制。RNA干扰通过小干扰RNA特异性降解目标mRNA来沉默基因表达。本文以烟草花叶病毒126kD蛋白为靶蛋白,在原生质体水平上研究了小干扰RNA对病毒侵染的干扰和抑制作用。ELISA和Northern杂交的实验结果表明,共转染小干扰RNA和TMV的原生质体内检测到较低的病毒含量。在枯斑寄主上,叶片接种小干扰RNA和TMV共转染原生质体后,与对照叶片相比,仅有很少量的病斑产生。这说明,小干扰RNA能够在原生质体水平对病毒起到干扰和抑制作用。因此认为,烟草原生质体系统有利于快速和定量分析小干扰RNA介导对植物病毒的抑制作用。     

河南省烟草病毒的小RNA测序检测

To identify viruses in Henan tobacco-planting areas, from 2015 to 2017 and 2019, 288 symptomatic tobacco samples were collected and then subjected to small RNA sequencing. Results showed that at least 7 viruses were detected from these samples which including four previously reported viruses, cucumber mosaic virus (CMV), tobacco mosaic virus (TMV), potato virus Y (PVY) and tobacco vein banding mosaic virus (TVBMV). Other three viruses, wild tomato mosaic virus (WTMV), brassica yellows virus (BrYV), and cycas necrotic stunt virus (CNSV) were firstly detected in Henan province. However, tobacco etch virus (TEV) and tobacco ringspot virus (TRSV) were not detected from these samples. In addition, CMV, TMV, PVY, TVBMV, and BrYV were the dominant viruses infecting tobacco in Henan Province.     

基于小RNA深度测序和RT-PCR检测侵染广东省冬种马铃薯的病毒

为明确侵染广东省冬种马铃薯的病毒种类及优势病毒,结合小RNA深度测序技术及RTPCR检测方法,对采集于广东省冬种马铃薯7个主产区的189份疑似病样进行检测分析。结果表明,经小RNA深度测序技术检测马铃薯病毒病混合样,发现存在马铃薯Y病毒(Potato virus Y,PVY)、马铃薯S病毒(Potato virus S,PVS)和马铃薯卷叶病毒(Potato leaf-roll virus,PLRV)3种病毒。进一步设计3种病毒的特异性引物并利用国内已报道的其它5种马铃薯病毒的特异性引物进行RT-PCR检测,发现189份马铃薯病毒病疑似病样中仅检测到PVY、PVS和PLRV这3种病毒,检出率依次为75.13%、10.05%和4.76%,且3种病毒在马铃薯上还存在复合侵染,复合侵染率为14.19%,其中PVY在各马铃薯产区均可检测到。表明侵染广东省冬种马铃薯的病毒为PVY、PVS和PLRV,其中PVY是优势病毒。     

2011年云南烟草病毒病发生动态及复合侵染分析

 烟草病毒病发生普遍,在我国部分烟区已上升为烟草的第一大病害。烟草是云南省主要经济支柱之一,然而,病毒病的发生严重影响烟叶的产量和质量,造成一定经济损失。20世纪80～90年代,我国系统地调查了全国烟草侵染性病害,并对侵染我国烟草的病毒种类和严重影响生产的主要种类进行鉴定检测。2005年Zhang等^［1］对云南烟草病毒病及病原进行了研究。但是随着种植业结构的调整,品种的更换,烟草病毒种类出现新的变化,在田间病毒侵染情况也会发生变化。因此,有必要重新调查和鉴定云南烟草病毒种类,弄清影响云南烟草的病毒种类和优势种类,明确其复合侵染情况,从而指导病毒病的防控。     

Incidence and identification of virus diseases of tobacco in three provinces of Zambia

Nine tobacco fields of small- and large-scale farmers in Central, Lusaka and Southern provinces of Zambia with an experimental area in the range of 4–52 ha were surveyed for the incidence, prevalence and identification of virus diseases during the growing season of 1997. Samples were collected from three tobacco fields in each of the three provinces, and a total of 72 samples was analysed. Virus identification was based on field disease syndrome, host range studies, DAS-ELISA and electron microscopy of virus particles in some cases. The study demonstrated the occurrence of Tobacco mosaic virus (TMV), Potato virus Y (PVY), Alfalfa mosaic virus (AMV) and Tobacco ringspot virus (TRSV). TMV and PVY occurred widely and were common in all three provinces, while AMV and TRSV were relatively less common. The prevalence of the four viruses was TMV 78%, PVY 67%, AMV 33% and TRSV 22%. Serological tests for Tomato spotted wilt virus (TSWV) and Cucumber mosaic virus (CMV) showed that these viruses were not present in the tobacco samples analysed.     

Identification of the principal viruses infecting tomato crops in Tunisia

Serological tests have been used to detect viruses associated with tomato in 257 samples collected in different regions in Tunisia, Cap-Bon, Sahel and South during successive seasons. The viruses detected were cucumber mosaic cucumovirus (CMV) and tomato aspermy cucumovirus (TAV), potato Y potyvirus (PVY), tobacco etch potyvirus (TEV) and pepper veinal mottle potyvirus (PVMV), tomato mosaic tobamovirus (ToMV) and tobacco mosaic tobamovirus (TMV), tobacco rattle tobravirus (TRV), alfalfa mosaic alfamovirus (AMV), tomato spotted wilt tospovirus (TSWV), tomato ringspot nepovirus (TomRSV) and potato X potexvirus (PVX). Some were detected in all three regions surveyed, at variable frequencies: TMV, CMV, TEV, PVY, ToMV, AMV, TAV, TSWV and TRV. Others were only detected in two regions (PVMV in Cap-Bon and Sahel and PVX in Sahel and in the south) or one region (TomRSV in Cap-Bon). Movement of individual viruses from one region to another may be due to movement of specific vectors, as in the case of the thrips-transmitted TSWV moving from the south to the north. Some of these viruses were found for the first time in Tunisia.     

马铃薯Y 病毒贵州黔西烟草分离物P1 基因序列分析

 Potato virus Y (PVY) is the type member of the genus Potyvirus and infects several important solanaceous crops, including tobacco. PVY is one of the most widespread and economically destructive viruses.The P1 region of PVY had been used to determine the diversity, evolution and the homology based on the
geographic location of the virus. The P1 region of PVY tobacco isolate from Qianxi in Guizhou Province was cloned and sequenced. Database searches and multiple sequence alignment showed subtle variation within the same strain, while apparent variation between O strain and the other three strains, N, NTN and N∶O. Moreover, variation in P1 region of Qianxi isolate derived from the gene mutation rather than the recombination among genomes. Phylogenic analysis revealed that the clustering only discriminated O strain from others. Consequently, sequence analysis in P1 region is unable to afford strain determination.     

Biological characterization of French Potato virus Y (PVY) isolates collected from PVY‐susceptible or ‐resistant tobacco plants possessing the recessive resistance gene va

Improved tobacco cultivars introgressed with alleles of the recessive resistance va gene have been widely deployed in France to limit agronomical consequences associated with Potato virus Y (PVY) infections. Unfortunately, necrotic symptoms associated with PVY have been reported on these cultivars suggesting that PVY is able to overcome the resistance. A field survey was performed in France in 2007 to (i) estimate the prevalence of PVY in tobacco plants showing symptoms and (ii) characterize PVY isolates present in susceptible and va‐derived tobacco cultivars. A serological typing procedure, applied to 556 leaves collected from different French tobacco growing areas, was performed using polyclonal antisera raised against different viral species including PVY. Viral species were detected in 80·8% of leaves and PVY was present in 83·5% of infected samples. However, statistical analysis confirmed that the probability of a tobacco plant being infected with PVY is reduced in va hosts. Eighty‐six PVY isolates were mechanically inoculated on one susceptible and three va‐derived tobacco cultivars used as indicator hosts to define virulence of these isolates against alleles 0, 1 and 2 of the va gene. Both qualitative and quantitative analyses showed that 55 PVY isolates were able to overcome the three va alleles. Moreover, the monitored biological diversity of PVY isolates was higher in the susceptible tobacco hosts than in the va‐derived ones. This study helps to understand consequences of the deployment of the va gene in tobacco on diversity and virulence of PVY isolates.     

利用RNA介导的抗病性获得高度抗马铃薯Y病毒的转基因烟草

 以马铃薯Y病毒坏死株系(PVY^N)的RNA为模板,应用反转录-聚合酶链式反应(RT-PCR)方法,扩增出长度为801 bp的非翻译的马铃薯Y病毒外壳蛋白基因。将扩增的片段克隆到pBSK的BamHI和KpnI之间并进行了序列测定。用BamHI和KpnI从重组克隆载体上切下该基因并插入到质粒pROKII内得到植物表达载体pPVYCP。通过根癌农杆菌(LBA4404)介导的方法转化烟草NC89,经卡那霉素抗性筛选、PCR和Southern blot检测,获得82株转基因植株。Northern blot和Western blot分析表明,转基因植株只在RNA水平上得到了表达。抗病性试验表明转基因植株之间抗性水平存在着差异,其中有7株是对PVY^N高度抗病性的植株。转基因植株抗病特异性试验初步表明,对PVY^N表现高度抗病的植株对PVY^O也具有高度抗病性。