蚕豆萎蔫病毒2号安徽分离物全基因组序列测定与分析

利用酶联免疫和RT-PCR技术对采自安徽地区的蚕豆病株进行检测,确定其病原为蚕豆萎蔫病毒2号Broad bean wilt virus 2(BBWV2)。为明确BBWV2安徽分离物(BBWV2-AH)的分类地位,克隆了该分离物的全基因组序列,分析了其基因组特征。结果表明,BBWV2-AH RNA1全长为5 944 bp(GenBank登录号:KY606992),含有1个ORF;BBWV2-AH RNA2全长3 587bp(GenBank登录号:KY606993),含有1个ORF。全序列核苷酸和氨基酸相似性分析显示,BBWV2-AH RNA1与BBWV2其他分离物的核苷酸、氨基酸相似性分别为78.4%~96%和87.1%~99%;BBWV2-AH RNA2与BBWV2其他分离物的核苷酸、氨基酸相似性分别为76.8%~95.5%和88.2%~98.3%。全基因组核苷酸序列系统发育分析显示,BBWV2-AH RNA1与中国的BBWV2-Hunan RNA1的亲缘关系最近,而BBWV2-AH RNA2与韩国的多个分离物聚集在一起,再与中国的分离物BBWV2-B935形成一个分支。     

引起辣椒花叶、枯顶的一个病毒分离物的鉴定

 从河北望都辣椒地分离到一株辣椒病毒分离物.测定表明能侵染茄科、苋科、豆科、菊科、藜科的25种植物,不能侵染葫芦科和十字花科等13种植物.桃蚜（Myzus persicae）非持久性传毒;钝化温度65—70℃,稀释限点10^-4—10^-5体外保毒期6天（20—22℃）;病毒颗粒呈球形,直径约25nm;ISEM测定它与蚕豆萎蔫病毒（BBWV）关系密切;电镜下观察到BBWV所特有的长管状内含体和布纹状结晶体,认为该分离物是BBWV.但寄主范围和寄主反应与文献报道的BBWV各分离物有所不同,可能是另一毒株。     

Molecular and biological characterization of Potato mop‐top virus (PMTV,Pomovirus) isolates from the potato‐growing regions of Colombia

Potato mop‐top virus (PMTV) causes necrotic flecks inside and on tubers in temperate countries. In South America, these symptoms have not been observed, although the presence of the virus has been confirmed in the Andes and in Central America. To characterize PMTV isolates from the Andes, soil samples were taken from the main potato‐producing regions in Colombia and virus was recovered by planting Nicotiana benthamiana as bait plants. The complete genomes of five isolates were sequenced and three of the isolates were inoculated to four different indicator plants. Based on sequence comparisons, three types of RNA‐CP (RNA2) and RNA‐TGB (RNA3) were found. The isolates from the centre of the country (CO3 and CO4) were similar to isolates from Europe. The genomes of CO1, CO2 and CO5 differ from other PMTV isolates, placing them in a separate clade in phylogenetic trees. The three Colombian isolates (CO1, CO2 and CO5) only induced slightly different symptoms in the indicator plants. However, the isolate from the northwest of the country (CO1) induced stronger symptoms in N. benthamiana including severe stunting. A correlation between the genotype of the isolates and the symptoms they induced on indicator plants was not found.     

Tomato necrotic ring virus (TNRV), a recently described tospovirus species infecting tomato and pepper in Thailand

Two tospovirus isolates collected from tomato and bell pepper in Thailand were studied. The isolates induced severe necrotic mottling and/or necrotic spots and rings on the leaves and fruits of the respective plants as confirmed by back-inoculation. A polyclonal antiserum raised against its nucleocapsid (N) protein reacted only with an extract from plants infected with the homologous virus. Analysis of the nucleocapsid (N) gene sequence and its deduced amino acid sequence (Mw ∼31 kDa) showed 99% amino acid sequence homology with that of Tomato necrotic ring virus (TNRV). The nucleotide sequence of the 5΄ untranslated region and intergenic region flanking the N gene revealed typical features of the S RNA segment of tospoviruses. Mechanical inoculation of the virus on some plant species showed that most of the tested solanaceous species were susceptible to this virus. The biological, serological and molecular data presented here indicate that both isolates are identical to TNRV, a recently described tospovirus species in Thailand.     

Molecular characterization of the CP gene and 3'UTR of Chilli veinal mottle virus from South and Southeast Asia

Twenty-four isolates of Chilli veinal mottle virus (ChiVMV) from China, India, Indonesia, Taiwan and Thailand were analysed to determine their genetic relatedness. Pathogenicity of virus isolates was confirmed by induction of systemic mosaic and/or necrotic ringspot symptoms on Capsicum annuum after mechanical inoculation. The 3' terminal sequences of the viral genomic RNA were determined. The coat protein (CP) coding regions ranged from 858 to 864 nucleotides and the 3' untranslated regions (3'UTR) from 275 to 289 nucleotides in length. All isolates had the inverted repeat sequence GUGGNNNCCAC in the 3'UTR. The DAG motif, conserved in aphid-transmitted potyviruses, was observed in all isolates. All 24 isolates were considered as belonging to ChiVMV because of their high CP amino acid and nucleotide identity (more than 94·8 and 89·5%, respectively) with the reported ChiVMV isolates including the pepper vein banding virus (PVBV), the chilli vein-banding mottle virus (CVbMV) and the CVbMV Chiengmai isolate (CVbMV-CM1). Based on phylogenetic analysis, ChiVMV isolates including all 24 isolates tested, PVBV, CVbMV and CVbMV-CM1 can be classified into three groups. In addition, a conserved region of 204 amino acids with more than 90·2% identity was identified in the C terminal of the CP gene of ChiVMV and Pepper veinal mottle virus (PVMV), and may explain the serological cross reaction between these two viruses. The conserved region may also provide useful information for developing transgenic resistance to both ChiVMV and PVMV.     

侵染广西甜椒的西瓜银斑驳病毒分子鉴定

During a survey in 2019, sweet pepper plants (GXTJ) showing symptoms of ring spots and chlorotic on leaves and fruits were collected in Wuming district of Guangxi province. Serological tests by DAS-ELISA demonstrated that the GXTJ was reacted positively against to antisera of watermelon silver mottle virus (WSMoV), but negatively against to antisera of the tomato spotted wilt virus (TSWV), cucumber mosaic virus (CMV) and tobacco mosaic virus (TMV). Total RNA was extracted and 2 pairs specific primers were designed to amplify the WSMoV N gene sequence by RT-PCR. Two expected fragments (769 bp and 654 bp) were obtained from GXTJ, The results of sequence analysis showed that the sequence of N gene was 828 nt and sharing 100% identity with WSMoV isolates from Taiwan (X78556, U78734). Base on the phylogenetic analysis, the N gene sequence of WSMoV-GXTJ was grouped in the same clades as WSMoV isolates from Taiwan. These results indicate that the virus isolate from sweet pepper in Guangxi is an isolate of WSMoV.     

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。     

豌豆病毒病病原研究

 1986年至1990年,从豌豆田中采集了150余份病毒病样本,鉴定出蚕豆萎蔫病毒(BB-WV)、芜菁花叶病毒(TuMV)、马铃薯Y病毒组分离物、黄瓜花叶病毒(CMV)、莴苣花叶病毒(LMV)、大豆花叶病毒(SMV)、豌豆花叶病毒(PMV)、菜豆黄花叶病毒(BYMV)和苜蓿花叶病毒(AMV)等9种病毒。样本中,BBWV所占的比例最高,达59.2%,其次为CMV,占15.5%。BBWV常与CMV复合侵染豌豆,LMV发生也较普遍。田间调查表明,豌豆病毒病发病率因种植地区及品种不同而有差异,平均发病率为12.4%。     

水蜡A病毒在呼和浩特市和哈尔滨市紫丁香上的发生及其基因组分子特征分析

为明确侵染紫丁香Syringa oblata并引起褪绿花叶症状的病毒种类及其基因组分子特征,利用透射电子显微镜对分离自呼和浩特市和哈尔滨市的紫丁香病样中的病毒粒子进行观察,并通过小RNA高通量测序和RT-PCR技术对其进行检测分析。结果表明,在紫丁香显症叶片的病毒粗提液中观察到长约600 nm、宽约13 nm的线状病毒粒子。利用小RNA高通量测序和RT-PCR技术从病样中检测到水蜡A病毒（Ligustrum virus A,LVA）,发病率为3.7%。呼和浩特市紫丁香分离物LVA-Sob的基因组序列全长8 525 nt,包含6个开放阅读框,分别编码Rep（1 968 aa）、TGB1（229 aa）、TGB2（107 aa）、TGB3（60 aa）、CP（294 aa）和NABP（119 aa）共6个蛋白。序列一致性分析表明,分离物LVA-Sob与韩国水蜡树分离物LVA-SK的基因组序列一致率高达97.9%,而与我国辽宁省暴马丁香分离物LVA-DX的基因组序列一致率仅为73.6%。在这3个LVA分离物基因组中没有检测到重组事件;基于基因组和cp基因序列的系统发育树显示这3个LVA分离物形成一个分支,并与瑞香S病毒（daphne virus S,DVS）有较近的亲缘关系。     

侵染扶桑的烟草花叶病毒分离物鉴定

从表现叶斑驳症状的扶桑病株上获得一病毒分离物,电镜下可见约300 nm×18 nm的杆状粒子,其与烟草花叶病毒抗血清呈明显的阳性反应,dsRNA约为6.4 kbp。根据烟草花叶病毒(tobacco.mosaic virus,TMV)的RNA序列设计引物,进行RT-PCR检测,扩增出约800 bp的预期特异片段。将PCR产物连接pMD18-T载体,转化大肠杆菌DH5α,得到了含有目的片段的重组子。序列分析表明,与周雪平等报道的序列(GenBank AJ011933.1)同源性达99％。通过生物学、病毒粒子观察、血清学以及分子生物学实验结果,确定该病毒分离物为TMV。     

苹果褪绿叶斑病毒生物学及生化特性研究

 对从苹果和扁桃上获得2个苹果褪绿叶斑病毒的分离物ACLSV-C和ACLSV-B的主要生物学和生化特性进行了比较。人工接种5科19种草本植物,发现两者均能侵染苋色藜(Chenopodium amaranticolor)、昆诺藜(Ch.quinoa)和西方烟(Nicotiana occidentalis),产生局部侵染斑和系统褪绿斑。但症状反应存在差异,后者在这3种植物上引起叶片反卷等较强症状反应,还可潜伏侵染笋瓜(Cucurbita maxima cv.Buttercup Burgess)。经SDS-聚丙烯酰胺凝胶电泳结果显示,ACLSV-B衣壳蛋白的迁移率较ACLSV-C快。两者的RNA分子量及双链RNA数量无明显差异。根据已报道的核苷酸系列设计合成引物,采用PCR法检测ACLSV分离物,均获得特异性扩增产物。     

辣椒上烟草轻型绿花叶病毒的鉴定

 辣椒源自南美洲，属于茄科辣椒属（Capsicum L.），为重要经济作物。病毒病是影响辣椒生产的主要病害，侵染辣椒的病毒有40余种［1］，烟草轻型绿花叶病毒（Tobacco mild green mosaic virus, TMGMV）是近年来在辣椒上发生和危害报道较多的病毒之一。TMGMV是McKinney［2］于1935年在烟草属植物（Nicotiana gluanca）上首次发现的，为烟草花叶病毒属（Tobamovirus）成员。除辣椒外，番茄［3］、凤仙花、蓝眼菊和矮牵牛也是TMGMV的自然寄主。     

Development and use of detection methods specific for <Emphasis Type="Italic">Cucumber vein yellowing virus</Emphasis> (CVYV)

Two methods for the detection of Cucumber vein yellowing virus (CVYV) on infected plants were developed, based on the information provided by cDNA clones covering the 3-end of the genome of a Spanish isolate (CVYV-AILM). The sequenced portion of the CVYV-AILM genome showed a 96.6% aminoacid identity with that of a reported sequence of another CVYV isolate from Israel (Lecoq et al., 2000). The first detection method used a RNA specific probe for hybridization with nucleic acids extracted from infected plants. The probe was complementary to a portion of the CVYV genome including the C-terminal part of the NIb and most of the coat protein (CP) coding regions. The second detection method employed polyclonal antisera raised against recombinant viral CP expressed in bacteria. The specific antibodies were used to detect the presence of virus particles in plant extracts. Both procedures resulted in a highly specific detection of CVYV in plants infected with different isolates of the virus. No interference was observed with other cucurbit-infecting viruses. Sensitivities achieved were sufficient for routine diagnosis of the presence of the virus in plants.     

番茄斑驳花叶病毒在广东省的首次报道

番茄斑驳花叶病毒(tomato mottle mosaic virus, ToMMV)是2013年发现的烟草花叶病毒属一个新种,目前在多国(地区)有发生。本文采用小RNA深度测序及RT-PCR检测方法在广东省广州市南沙区辣椒疑似病样中检测到ToMMV,命名为番茄斑驳花叶病毒广东分离物(ToMMV-GD-2020)。采用RT-PCR分段扩增获得了ToMMV-GD-2020的基因组全序列,该分离物基因组全长6 399 nt,包含4个开放阅读框,分别编码4个蛋白。序列相似性分析表明,ToMMV-GD-2020与已登录GenBank的14个ToMMV分离物基因组序列相似性分别为99.0%～99.7%,其中与中国辽宁分离物ToMMV-LN(GenBank登录号：MN853592)的相似性最高(99.7%),与危害我国番茄、同属烟草花叶病毒属的番茄花叶病毒(tomato mosaic virus, ToMV)、番茄褐色皱果病毒(tomato brown rugose fruit virus, ToBRFV)代表分离物的相似性分别为84.6%和81.0%。系统进化分析表明,ToMMV-GD-2020...     

北京地区侵染茄子的番茄斑萎病毒分子鉴定与分析

调查发现北京地区一温室栽培茄子Solanum melongena L.出现严重病毒病。利用基于小RNA的高通量测序技术和RT-PCR方法,明确了引起茄子病害的病毒种类为番茄斑萎病毒,将其命名为TSWV-eggplant分离物。进一步克隆了该病毒的基因组全长(S RNA、M RNA、L RNA),并构建其系统发育树。结果表明,该分离物的S RNA与美国分离物亲缘关系较近,M RNA与中国分离物亲缘关系较近,而L RNA与韩国分离物亲缘关系较近。因此,本研究发现的TSWV分离物与国内已发生报道的分离物不同,该分离物是否存在不同分离物之间基因组的重组需要进一步研究。     

菜豆普通花叶病毒RT-PCR检测及3′末端序列分析

根据哈尔滨地区豇豆感病植株的症状,初步鉴定感染豇豆的病毒为菜豆普通花叶病毒(Bean common mosaic virus, BCMV)。利用马铃薯Y病毒属通用引物扩增出病毒基因组3′末端序列,经BLAST检索表明该病毒为BCMV,将该序列与GenBank上的21个BCMV分离物的3′末端序列进行比较,显示其核苷酸序列与其他分离物的序列同源性为91.7%～97.3%。系统进化分析显示不同分离物可聚为5个类群,并显示出一定的寄主相关性。哈尔滨分离物BCMV X与2个浙江分离物、1个澳大利亚分离物聚为一支,且该4株分离物的寄主均为豇豆。RNA二级结构分析显示BCMV基因组3′末端非编码区形成4个茎环结构,不同分离物的序列变化并未引起茎环结构的明显变化。     

侵染勿忘我花卉的蚕豆萎蔫病毒的鉴定

从勿忘我（Ｍｙｏｓｏｔｉｓｓｉｌｖａｔｉｃａ）上采集到１病株，接种昆诺阿藜，接种叶褪绿斑，系统顶枯，最后整株枯死。蚕豆接种叶呈黑色坏死斑，主茎变褐、全株萎蔫。矮牵牛接种叶褪绿斑，系统花叶。用诱捕修饰法制片，观察到病毒粒体球形，并在病毒粒体外面包被着一层抗体。琼脂双扩散试验呈阳性反应     

新疆辣椒中BPEV和PCV2的鉴定与分析

 为明确新疆石河子辣椒感染病毒的种类,对表现明显皱缩、褪绿和卷叶等症状的辣椒样本进行长链非编码RNA(Long noncoding RNA, LncRNA)测序。根据LncRNA测序和RT-PCR检测,发现甜椒内源RNA病毒(Bell pepper endornavirus, BPEV)、辣椒隐症病毒2(Pepper cryptic virus 2, PCV2)和辣椒轻斑驳病毒(Pepper mild mottle virus, PMMoV)3种病毒。RT-PCR检测数据显示,新疆石河子的辣椒普遍感染BPEV和PMMoV。将BPEV的RdRp基因序列和PCV2-RNA1包含RdRp基因的序列分别进行核苷酸序列相似性和系统进化分析。结果表明本研究中BPEV-XJ和PCV2-XJ与其它分离物的相似性分别为89.1%~97.3%和96.9%~99.7%。BPEV-XJ与BPEV-TW分离物亲缘关系最近;PCV2-XJ与PCV2-DR分离物亲缘关系最近。辣椒中检测到BPEV和PCV2为新疆首次报道。