重庆黄瓜病毒病病原分子鉴定及序列分析  

Molecular Identification and Sequence Analysis of the Pathogen Causing Cucumber (Cucumis sativus) Virus Disease in Chongqing

作　　者：熊艳[1] 孙淼[2] 王鹤冰[1] 向华丰[1] 陈新中[1] 张洪成[1] 青玲[2] 

机构地区：[1]重庆市农业科学院蔬菜花卉所,重庆401329 [2]西南大学植物保护学院/植物病害生物学重庆市高校级重点实验室,重庆400716

出　　处：《农业生物技术学报》2017年第4期650-658,共9页Journal of Agricultural Biotechnology

基　　金：公益性行业(农业)科研专项(No.201303028-04);重庆市基本科研业务费专项(No.2016csct-jbky-00510)

摘　　要：黄瓜(Cucumis sativus)是世界范围内广泛分布的蔬菜,也是重庆主要经济作物之一。然而2013年以来,在重庆主要黄瓜种植区保护地及露地栽培的黄瓜上陆续发现病毒病症状植株。为明确采自重庆的5份表现植株矮化、叶片皱缩并有花叶、叶片扭曲的黄瓜病毒病的病原,利用11种常见蔬菜病毒特异引物,对样品进行了RT-PCR检测。检测结果表明,从黄瓜样品中扩增到了黄瓜花叶病毒(Cucumber mosaic virus,CMV)、南瓜花叶病毒(Squash mosaic virus,Sq MV)、芜菁花叶病毒(Turnip mosaic virus,Tu MV)、西瓜花叶病毒(Watermelon mosaic virus,WMV)和小西葫芦黄花叶病毒(Zucchini yellow mosaic virus,ZYMV)5种病毒,且均为4种及以上病毒的复合侵染。通过PCR、克隆和测序等技术获得WMV和ZYMV外壳蛋白(coat protein,CP)核苷酸序列,对序列进行了系统进化分析。结果表明,WMV重庆分离物WMV-H3与已报道的WMV各分离物CP核苷酸相似性在92.3%~96.9%,并与新疆哈密瓜(Cucumis melo var.saccharinus)上WMV-T-2-9相似性最高,为96.9%;ZYMV重庆分离物ZYMV-H3与安徽合肥分离物ZYMV-Hefei核苷酸相似性最高,为97.7%,与国内外已报道的ZYMV各分离物CP核苷酸相似性在93.0%~97.7%。系统进化树分析表明,44个WMV分离物被划分为5个基因型,WMV-H3属于基因型Ⅲ,WMV遗传变异与地理来源及寄主均存在一定的相关性,且与地域的相关性更强;80个ZYMV分离物被划分为6个基因型,ZYMV-H3属于基因型Ⅲ,ZYMV分离物分布具有一定地域分化。本研究首次研究了Sq MV、WMV和ZYMV在重庆地区黄瓜上的侵染危害,明确了重庆地区黄瓜上的病毒种类及进化起源,为进一步研究黄瓜病毒病分布及有效防控提供了理论依据。Cucumber （Cucumis sativus）, a worldwide distributed vegetable, is an economically important？crop in Chongqing. However, viruslike symptoms were observed in greenhouse- and field-grown in the major cucurbit cultivation regions in Chongqing from 2013. In this study, 11 common vegetable viruses specific primers were used to detect the pathogenic agents associated with 5 diseased cucumber plants exhibiting typical stunting, wrinkled, mosaic and distortion symptoms collected from Chongqing. The detection results showed that 5 cucumber plants were mix-infected with more than 4 viruses including Cucumber mosaic virus（CMV）, Squash mosaic virus（SqMV）, Turnip mosaic virus（TuMV）, Watermelon mosaic virus（WMV） and Zucchini yellow mosaic virus（ZYMV）. Using PCR, cloning, and sequencing techniques coat protein （CP） gene of WMV （WMV-H3） and ZYMV （ZYMV-H3） were identified for phylogenetic analysis. Compared with reported sequences of different geographic origins, the CP genes of WMV-H3 and ZYMV-H3 shared similarity of 92.3%~96.9%, 93.0%~97.7% at nucleotide level, respectively. WMV-H3 had the highest sequence identity with WMV-T-2-9 and ZYMV-H3 shared the highest sequence identity with ZYMV-Hefei. The phylogenetic trees constructed with CP genes showed that 44 WMV isolates were clustered into 5 groups and WMV-H3 belonged to group Ⅲ, the genetic variation of which was related to geographical distribution and host origins; 80 ZYMV isolates were divided into 6 groups and ZYMV-H3 belonged to groupⅢ, the distribution of which exhibited a distinct geographical differentiation. This is the first report of SqMV, WMV and ZYMV infecting cucumber in Chongqing. The results provide theoretical basis for further study on the distribution of the disease in cucurbit-growing regions and the disease management.

关 键 词：黄瓜病毒病 RT-PCR 复合侵染 外壳蛋白(CP)基因 系统进化分析 

分 类 号：S436.5[农业科学—农业昆虫与害虫防治]