我国部分省市草莓种苗中黄瓜花叶病毒的检测分析

 为明确黄瓜花叶病毒(cucumber mosaic virus,CMV)在草莓上的发生和危害情况,利用RT-PCR方法对不同产地的不同品种草莓种苗进行检测,并对CMV草莓分离物的部分核苷酸序列进行了分析。结果表明,CMV侵染草莓后产生的症状主要为植株不同部位的畸形、变色,但有些无明显症状的植株也可以检测出CMV。共检测了我国7个不同省市的220个草莓种苗样品,其中北京、云南、辽宁、河北、四川和陕西的草莓种苗样品中均可检出CMV,内蒙古的种苗中未检出CMV。检测的6个不同草莓品种均含CMV,但北京和内蒙古的‘红颜'种苗未检出CMV,云南的‘圣诞红'种苗CMV检出率仅为2.6%。利用RT-PCR技术扩增草莓种苗中CMV的特异性核苷酸片段并对PCR产物测序,得到包含部分外壳蛋白基因及3'端非编码区共430 bp的2个草莓分离物序列。获得的2个分离物序列的核苷酸序列同源性为90.97%,序列比对分析结果表明2个分离物分属于CMV不同亚组,其中北京草莓分离物Bjcmz归属于亚组IA,河北分离物Hbcmc归属于亚组IB。     

基于小RNA深度测序技术鉴定侵染我国部分省市草莓种苗的病毒种类

随着草莓保护地栽培面积的增加和无性繁殖种苗的繁殖与调运,草莓病毒病的发生与流行日益严重。为明确侵染我国部分省市草莓种苗的病毒种类,应用小RNA深度测序技术进行检测,并利用RT-PCR技术对结果进行验证及序列分析。结果表明,从来自我国7省市的41株具有典型病毒病症状的草莓种苗样品中检测到草莓斑驳病毒strawberry mottle virus (SMoV)、草莓镶脉病毒strawberry vein banding virus(SVBV)和草莓轻型黄边病毒strawberry mild yellow edge virus (SMYEV)3种。SMoV、SVBV和SMYEV的检出率分别为34.1%、24.4%和2.4%。选取不同产地草莓种苗上检出的不同病毒进行部分序列测定和分析,获得了3个SMoV分离物(四川分离物schhy13、辽宁分离物lnhy23和河北分离物hbhy28)的部分RNA1 3′端非编码区606 bp核苷酸序列,其一致性为98.12%～99.34%。测定并获得了5个SVBV分离物(辽宁分离物lnhy15、lnhy17、lnhy24、河北分离物hbhy28和陕西分离物sh...     

黄瓜花叶病毒分离物核酸酶保护试验(RPA)方法的株系分化研究

 采用黄瓜花叶病毒((CMV)亚组Ⅰ株系Fny-CMV及亚组Ⅱ株系Ls-CMV的RNA₂的特定序列片段的cDNA克隆,体外转录,同时掺入³²P标记制备负链RNA探针,再与纯化的甜椒上的CMV中国分离物的RNA进行杂交,检测其与探针之间的同源性。共检测样品分离物3份。试验结果表明:河南新乡和北京密云的CMV甜椒分离物与Fny-CMV的核苷酸有高度同源性,隶属于Fny-CMV为代表的亚组Ⅰ株系。来自福建的样品与亚组Ⅱ的Ls-CMV株系有高度同源性,隶属于CMV亚组Ⅱ株系。本试验同时利用源于我国CMV亚组Ⅰ的K株系的RNA₂两个EcoR Ⅰ位点间1657-2125 nt的核苷酸序列为探针,同样与以上3份CMV中国分离物进行RNA杂交,进一步比较分析了这几个分离物与我国亚组Ⅰ的K-CMV株系的关系,证明了我国CMV存在亚组与株系分化。     

侵染百合的黄瓜花叶病毒亚组Ⅱ分离物cp基因克隆和序列分析

 从云南大理的东方型百合上得到黄瓜花叶病毒分离物(CMV-DL), ELISA检测初步确定为CMV亚组Ⅱ分离物, 设计并合成CMV亚组Ⅱ的特异引物, RT-PCR扩增得到1条约800 nt的特异片段, 经克隆及序列测定, 该片段长828 nt, 包含的外壳蛋白(CP)基因由657 nt组成。将该分离物的cp基因与其它14个CMV分离物进行同源性比较, 在核苷酸水平上与CMV亚组I和亚组Ⅱ的同源性分别为76.8%~78.1%和98.6%~99.2%;在氨基酸水平上与CMV亚组I和亚组Ⅱ的同源性分别为82.0%~84.3%和95.9%~100.0%。结果表明CMV-DL为CMV亚组Ⅱ成员。     

新疆石河子、伊宁地区黄瓜花叶病毒株系分化

为了研究新疆黄瓜花叶病毒(Cucumber mosaic virus,CMV)分子变异及株系分化,对从石河子和伊宁地区采集的205个加工番茄、23个辣椒、4个番茄、2个南瓜样品进行酶联免疫检测和RT-PCR检测.在4种寄主上均检出了亚组Ⅰ型CMV,其中加工番茄的感染率高达74.15％.进一步对来自辣椒的YN-6、LJ-4、L-10,加工番茄的S1-1、S1-14,番茄的YN-2,以及南瓜的YN-9等7个样品CP、RNA3、MP核苷酸序列进行相似性和进化树分析.7个样品与CMV亚组ⅠB株系分离物的相似性较高、亲缘关系最近,均可归为CMV亚组ⅠB.而来自辣椒的LJ-4、L-10与其余5个样品的序列相似性较低,亲缘关系较远,在进化树上形成独立分支.说明在新疆加工番茄及其它蔬菜上广泛流行的CMV存在分子变异.     

我国部分地区常见农作物上黄瓜花叶病毒分离物核酸多样性分析

为明确我国黄瓜花叶病毒株系分化及系统进化基本情况,从湖南、新疆、青海和海南4省区采集1 367个样品对其进行酶联免疫和RT-PCR检测,并对分离获得的15个黄瓜花叶病毒(Cucumber mosaic virus,CMV)纯化分离物CP、MP、2b核苷酸序列进行相似性和进化树分析及生物学性状比较。结果表明,辣椒、龙葵和黄瓜的CMV阳性检出率较高,分别为54.13%、29.19%和18.46%。进化树分析显示CMV-Q5与CMV亚组II的亲缘性较高;CMV-N7为新发现的重组株系,其CP、2b基因属于CMV亚组IB,MP基因却属于CMV亚组II;其余13个分离物均属于CMV亚组IB。CMV-N7和CMV-Q5在系统寄主心叶烟和枯斑寄主苋色藜上引发的症状相似,但比对照株系CMV-P3613(IB)的发病时间要晚1~2 d,系统花叶较温和,枯斑较小。表明在以上4省区常见农作物上广泛流行的CMV存在分子变异。     

桃树上啤酒花矮化类病毒(Hop stunt viroid)的检测及序列分析

 2005年8月和2006年2月从中国北京、陕西、河北、山东、广西等地共采集76个无明显症状的桃树样品,经斑点杂交、RT-PCR以及生物学鉴定检测,来自北京和陕西的11个样品中检测到啤酒花矮化类病毒(Hop stunt viroid,HSVd),总感染率达14.5%。上述3种方法检测桃树上的HSVd具有一致性。将5个样品中的HSVd进行克隆测序,得到12条不同HSVd核酸序列,与GenBank中D13764序列(日本桃果实HSVd分离物)同源性为93.29%~100%。可以看出,国内桃树HSVd分离物核酸序列变异比较小,地域和品种间核酸序列无明显差异。这是首次比较系统地检测中国桃树上HSVd发生情况的报道。     

我国部分省市甜椒黄瓜花叶病毒的亚组鉴定

 CMV是我国甜椒上的主要毒原种类,并有着许多不伺的株系,近年来,一些CMV株系被分为2个亚组^[1,2]。同一亚组株系的核苷酸序列同源性在95%以上,在寄主范围、致病性等方面有一定的相似性;而不同亚组株系在寄主范围、致病性、复制及传播等方面均有差异^[3]。所以,以快速、准确的分子生物学方法鉴定我国甜椒CMV分离物的亚组地位,可以为抗病毒基因工程育种、品种合理布局以及防治措施的实施提供依据。     

黄瓜花叶病毒2个分离物的亚组鉴定及株系分化研究

 基于黄瓜花叶病毒甜菜分离物CMV-XJ1和CMV-XJ2独特的生物学特性,利用RT-PCR对接种寄主植物进行了检测。RT-PCR产物的RFLP结果显示,2个病毒分离物的MspⅠ酶切图谱与CMV亚组Ⅰ病毒的酶切图谱很相似,但经EcoRⅠ酶切后出现显著差异;进一步对2个分离物的外壳蛋白基因进行了克隆,序列分析表明,CMV-XJ1和CMV-XJ2归属CMVIB亚组,二者存在着株系分化的趋势。     

Genetic diversity,anastomosis groups and virulence of <Emphasis Type="Italic">Rhizoctonia</Emphasis> spp. from strawberry

Virulent Rhizoctonia spp. isolated from strawberry in Israel belonged to anastomosis groups (AG) of: binucleate Rhizoctonia (BNR) AG-A, AG-G, AG-K and AG-F, and to multinucleate Rhizoctonia (MNR) AG 4 subgroup HG-I. In addition, a soil isolate of AG 4 subgroup HG-III was also found to be virulent on strawberry. None of the Israeli isolates obtained in the present study belonged to BNR AG-I, or other MNR AGs. In the cluster analysis of rDNA-ITS sequences, all of the isolate sequences consistently clustered according to their known AGs and subgroups. One AG-F cluster included sequences of 10 strawberry isolates, while another AG-F cluster included sequences of two isolates submitted to GenBank. Additional work is needed to determine whether the isolates of these two clusters may belong to different AG-F subgroups. The current virulence bioassay used for Rhizoctonia spp. isolates on strawberry is based on inoculation of stolon-derived daughter plants with the isolates and estimation of the reduction in plant biomass, rather than on specific distinct disease severity symptoms. The duration of this test is relatively long (ca. 5 weeks or more) and the availability of daughter plants from runners is naturally limited to a certain season. Among the possible alternative methods evaluated in the present study (inoculation of fruits or seedlings developed from germinated strawberry seeds), the method based on seedlings was best. This method has a potential to replace the currently used stolon-daughter plant inoculation bioassay for testing virulence of strawberry root pathogens. This is the first report indicating that Rhizoctonia spp. isolates that belong to AG-F, AG-K, AG 4 HG-I and AG 4 HG-III are virulent to strawberry.     

我国不同CMV分离物2b基因片段的RT-PCR扩增及其序列比较

 本研究根据黄瓜花叶病毒2b基因的保守序列设计引物,利用一步RT-PCR技术对多个不同寄主和不同地理来源的黄瓜花叶病毒分离物的2b基因片段进行扩增,获得了含该基因全长约90%的cDNA片段(300bp)。序列测定与比较分析结果表明,国内不同CMV分离物2b基因片段核酸序列同源性达93%以上,推测的氨基酸序列同源性超过90%,且均属于亚组Ⅰ。     

Survey of viruses infecting open‐field pepper crops in Côte d'Ivoire and diversity of Pepper veinal mottle virus and Cucumber mosaic virus

The prevalence of viruses in pepper crops grown in open fields in the different agro‐ecological zones (AEZs) of Côte d'Ivoire was surveyed. Pepper veinal mottle virus (PVMV; genus Potyvirus) and Cucumber mosaic virus (CMV; genus Cucumovirus) were the most frequent viruses among those surveyed, while tobamoviruses (genus Tobamovirus) were detected at low frequency. PVMV showed a high heterogeneity across AEZs, which may be related to climatic, ecological or agronomical conditions, whereas CMV was more homogeneously distributed. The molecular diversity of CMV and PVMV were analysed from partial genome sequences. Despite the low number of CMV isolates characterized, two molecular groups were revealed, one corresponding to subgroup IA and the other to reassortants between subgroups IA and IB. RNAs 1 and 3 of the reassortants clustered with the IB subgroup of CMV isolates, whereas their RNA 2 clustered with the IA subgroup. Importantly, RNA 1 of CMV isolates of the IB subgroup has been shown to be responsible for adaptation to pepper resistance. The diversity of PVMV in the VPg‐ and coat protein‐coding regions revealed multiple clades. The central part of the VPg showed a high level of amino acid diversity and evidence of positive selection, which may be a signature of adaptation to plant recessive resistance. As a consequence, for efficient deployment of resistant pepper cultivars, it would be desirable to examine the occurrence of virulent isolates in the CMV or PVMV populations in Côte d'Ivoire and to follow their evolution as the resistance becomes more widely deployed.     

Phylogeny of Egyptian isolates of <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> (CMV) and <Emphasis Type="Italic">Tomato mosaic virus</Emphasis> (ToMV) infecting <Emphasis Type="Italic">Solanum lycopersicum</Emphasis>

Four Cucumber mosaic virus (CMV) (CMV-HM 1–4) and nine Tomato mosaic virus (ToMV) (ToMV AH 1–9) isolates detected in tomato samples collected from different governorates in Egypt during 2014, were here characterized. According to the coat protein gene sequence and to the complete nucleotide sequence of total genomic RNA1, RNA2 and RNA3 of CMV-HM3 the new Egyptian isolates are related to members of the CMV subgroup IB. The nine ToMV Egyptian isolates were characterized by sequence analysis of the coat protein and the movement protein genes. All isolates were grouped within the same branch and showed high relatedness to all considered isolates (98–99%). Complete nucleotide sequence of total genomic RNA of ToMV AH4 isolate was obtained and its comparison showed a closer degree of relatedness to isolate 99–1 from the USA (99%). To our knowledge, this is the first report of CMV isolates from subgroup IB in Egypt and the first full length sequencing of an ToMV Egyptian isolate.     

青蒿中番茄斑萎病毒和烟草花叶病毒的分子鉴定及相关序列分析

番茄斑萎病毒(tomato spotted wilt virus, TSWV)和烟草花叶病毒(tobacco mosaic virus, TMV)是2种重要的植物病原病毒, 对多种经济作物的产量和品质均造成严重影响。2021年-2022年, 在云南省丽江市烟草种植区不同烟区采集叶片黄化、皱缩以及无症状的青蒿Artemisia caruifolia样品共计14份, 利用免疫金标速测卡和RT-PCR对其病原病毒进行检测。利用免疫金标速测卡检测结果显示, 在所检样品中有9份样品检测出TSWV, 检出率为64.28%, 有3份样品检测出TMV, 检出率为21.43%, 2种病毒复合侵染的检出率同样为21.43%;利用RT-PCR对复合侵染的3份样品进行分子检测, 结果显示, 在3份复合侵染青蒿样品中获得3条TSWV N基因序列、3条TMV cp基因序列和2条TMV RdRp部分序列。TSWV青蒿分离物与分离自云南的TSWV-2分离物相似性最高, 为99.6%;TMV青蒿分离物与分离自辽宁的TMV-Shenyang分离物和分离自云南的TMV-Yongren-1相似性最高, 均大于99.4%。这是首次发现TSWV和TMV 2种不同属病毒复合侵染青蒿。     

侵染牡丹的烟草脆裂病毒的检测鉴定及序列分析

为明确江苏省牡丹上烟草脆裂病毒(Tobacco rattle virus,TRV)的发生情况,利用ELISA和RT-PCR方法对采集自扬州市的40份牡丹叶片样品进行了检测鉴定,测定了其中一个分离物Peony-11中TRV RNA1分子的部分蛋白编码区序列,并结合Gen Bank中已报道的相关序列对其进行了多样性和系统发生分析。结果显示,江苏省扬州市牡丹上TRV的检出率高达62.5%;序列分析表明本研究获得的TRV牡丹分离物Peony-11与Gen Bank中其它63个分离物的核苷酸序列一致率为90.5%~99.7%;系统发生和遗传距离分析表明TRV可以分成2个组10个亚组,组间、亚组间具有较为清晰的地理和寄主特异性,其中Peony-11分离物位于亚组I-1。