Molecular characterization of a Cucumber mosaic virus isolate associated with mosaic disease of banana in India

Severe mosaic accompanied by leaf and fruit deformation symptoms was observed on banana plants growing in three banana farms of Uttar Pradesh, India. The disease incidence was approximately 18–25% at these locations during the three successive years from 2005 to 2007. The occurrence of Cucumber mosaic virus (CMV) was initially detected by bioassay, electron microscopic observations, Western blot immunoassay and RT-PCR. For molecular identification of virus, the RNA 1a, RNA 2b and RNA 3 genomic fragments were amplified by RT-PCR and sequenced. The sequence analysis of these genomic fragments revealed its highest identities and close relationships with Indian strains of CMV of subgroup IB; therefore, virus associated with the mosaic disease of banana was identified as an isolate of CMV of subgroup IB. In the limited reports existing from India, which provided preliminary serological or only coat protein-based identification of CMV infecting banana but the comprehensive studies were lacking. In the present communication, we present a detailed biological, serological and molecular characterization of CMV-Banana for the first time from India.     

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.     

Molecular identification of a new isolate of <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> subgroup II from basil (<Emphasis Type="Italic">Ocimum sanctum</Emphasis>) in India

Natural occurrence of mosaic disease was observed on basil (Ocimum sanctum L.) in Aligarh, U. P., India, during 2008. The disease could be transmitted by sap inoculations from naturally infected O. sanctum to O. sanctum and some test plant species. Cucumber mosaic virus (CMV) was detected by RT-PCR using coat protein gene specific primers of CMV (Acc. AM180922 & AM180923), which resulted in the expected size ~650 bp amplicon in infected samples. The amplicon was cloned, sequenced and data were deposited in GenBank Acc. EU600216. The sequence data analysis revealed 97–99% identities at both nucleotide and amino acid levels with the CMV strains of subgroup II reported worldwide. Based on the high sequence identities and close phylogenetic relationships with CMV subgroup II strains, the virus under study has been identified as a new isolate of CMV subgroup II and designated as CMV-Basil.     

黄瓜花叶病毒NASBA检测技术的建立

 以香蕉花叶病病样为材料,初步建立了黄瓜花叶病毒核酸序列依赖性扩增(Nucleic acid sequence based amplifica-tion,NASBA)的检测技术。通过以香蕉叶片总RNA为模板,在黄瓜花叶病毒(Cucumber mosaic virus,CMV)亚组ⅠRNA 2高保守区设计特异引物,进行NASBA反应,经5%琼脂糖凝胶电泳检测,阳性样品中出现了预期大小为310 bp的条带,而阴性和空白对照中均未出现。并对11份香蕉样品分别进行NASBA反应,并经过斑点杂交验证与RT-PCR检测比较,两者的检测结果一致,灵敏度相当,检出限量可达100 pg。     

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。     

A Cucumber mosaic virus isolated from Momordica charantia L.

Momordica charantia L. plants systemically infected with Cucumber mosaic virus (CMV) were found in Oita Prefecture. The virus isolated from the host plant was characterized by biological, serological, and molecular biological methods. The purified virus was used to mechanically inoculate the host and produced green mottle, green mosaic, and/or chlorotic spots in the noninoculated upper leaves of the host. The virus was identified as an isolate of CMV containing genomic RNA3 derived from subgroup IA by several lines of evidence based on electron microscopy, serological detection, host range, symptoms, and the entire nucleotide sequence of RNA3.     

Coat protein-mediated transgenic resistance in tomato against a IB subgroup Cucumber mosaic virus strain

Transgenic tomato plants containing the coat protein (CP) gene of Cucumber mosaic virus (CMV) of subgroup IB were developed through Agrobacterium-mediated transformations. The progenies of transgenic plants showed the presence of transgene, its expression and translation of 26 KDa CP. The T₁ and T₂ generation plants were evaluated for resistance against challenge inoculations by a homologous strain of CMV. Visual observations of challenged transgenic plants categorized them into resistant, tolerant and susceptible as compared with untransformed control plants. Out of 33 plants of the T₁ generation, 36.3% showed resistance and remained symptomless throughout their life, 48.4% showed tolerance which developed delayed symptoms of mild mosaic, and 15.1% showed susceptibility to CMV which developed severe systemic mosaic and leaf distortion symptoms after 30?days of virus challenge. Out of 120 plants of the T₂ generation, 60% showed resistance, 26.6% were tolerant and only 13.3% were found susceptible to challenge inoculations of CMV. Resistant transgenic plants also showed less CP accumulation in systemic upper leaves as compared with challenged untransformed plants. In this study, CP of a CMV subgroup IB strain has demonstrated a significant level of resistance in transgenic tomato plants against the CMV strain. The strategy may be applied for better quality and productivity of tomato crops.     

白术矮化病毒病病原的分子鉴定和部分序列分析

 本试验在生物学接种的基础上,利用非序列依赖性PCR扩增(sequence-independent amplification,SIA)对白术矮化病毒病病原进行了分子鉴定,序列测定及分析。结果发现,具有矮化症状的白术为黄瓜花叶病毒(Cucumber mosaic virus,CMV)所侵染。为明确CMV白术分离物(CMV-Am)的分类地位,本研究进一步克隆了CMV-Am的外壳蛋白基因(CP)和移动蛋白基因(MP)全序列。序列比对分析表明,CMV-Am与CMV亚组ⅠB中株系XJ2序列同源性最高,核苷酸、氨基酸序列同源性分别为98.9%、99.1%。氨基酸序列同源性聚类分析表明,CMV-Am与中国大多数CMV分离物一样,属于CMV亚组ⅠB。     

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。     

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。     

烟草花叶病毒丁香分离物的分离与鉴定

 从表现花叶症状的丁香病株上获得一病毒分离物,其在电镜下为约300 nm×18nm的杆状粒子;电泳分析表明感病组织中ds RNA大约为6.4kbp,而其外壳蛋白分子量约为17.6k Da。以上实验结果初步将该病毒分离物鉴定为烟草花叶病毒属(Tobamovirus)。根据该属病毒复制酶基因序列设计通用引物,进行RT-PCR检测,扩增出约1000 bp的预期特异片段(Gen Bank AY566703)。将PCR产物克隆后测序,序列分析表明,与从蚕豆中分离的TMV-B株系序列(Gen Bank AJ011933.1)同源性为99.90%。根据烟草花叶病毒(Tobacco mosaic virus,TMV)的RNA CP基因序列设计引物,进行RT-PCR,扩增出约800 bp的预期特异片段(Gen Bank AY56672),序列分析表明,与TMV-B株系序列(Gen Bank AJ011933.1)同源性达99%,上述实验结果表明,该病毒分离物为TMV。由于该分离物与TMV-B在指示植物上的症状存在明显差异,所以,作者把该分离物暂命名为TMV-S。     

Biological, serological and molecular characterization of a cucumber mosaic virus isolate from India

A. virus causing mosaic and leaf deformation of Physalis minima has been identified as an isolate of cucumber mosaic virus (CMV) on the basis of its transmission by aphids in a non-persistent manner, polyhedral particles of 29 nm diameter, molecular weight of coat protein subunits us 24-5 kDa. serological relationship with a CMV isolate and a tripartite single-stranded RNA genome with a subgenomic RNA4- Furthermore. cDNA representing coat protein gene was synthesized and cloned. Complete nucleotide sequences (890 nt) were obtained which showed a coat protein gene open reading frame of 657 residues. THE nucleotide sequences provided the 218 amino ACID sequences of the coat protein. Nucleotide as well as amino acid sequences revealed more than 90% identity with the CMV subgroup I strains.     

Molecular identification of an Ageratum enation virus isolate associated with mosaic disease of pointed gourd (Trichosanthes dioica) in India

A severe mosaic disease of pointed gourd (Trichosanthes dioica Roxb.) was observed with significant disease incidence in Gopalganj, India, during 2008. Begomovirus was detected from symptomatic leaf samples by polymerase chain reaction (PCR) using coat protein gene-specific primers of a well characterized begomovirus which revealed positive amplification of expected size ~800 bp DNA band. To confirm begomovirus association, the complete DNA-A was amplified using three sets of begomovirus DNA-A primers. The amplicons were cloned, sequenced, and sequence of the complete DNA-A (2757 nt) was determined by combining the sequence data of all amplicons (Accession no. GQ268327). The sequence data showed 99–93% sequence identities and close phylogenetic relationships with isolates of Ageratum enation virus (AgEV). The begomovirus associated with mosaic disease of T. dioica was identified as an isolate of Ageratum enation virus, which is a new record from India.     

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。     

黄瓜花叶病毒浙江白术分离物全基因组序列测定与分析

正白术(Atractylodes macrocephala Koidz.)为菊科(Compositae)苍术属(Atractylodes)多年生草本植物,其根茎入药具补脾健胃、燥湿利水、止汗安胎等功效,在我国广泛栽培,并以于术(浙江临安于潜)品质最佳[1]。近年来白术生产由于品种单一,长期连作等因素导致病毒病害日趋严重。据报道,黄瓜花叶病毒(Cucumber mosaic virus,CMV)~([2])、蚕豆萎蔫病毒2号(Broad bean wilt virus 2,