广东省番茄白尖曲叶病病原鉴定及其致病性分析  

Identification of Pathogen Causing Tomato White Tip and Curl Leaf Disease and Its Pathogenicity in Guangdong Province

作　　者：王愿 都梦丹 李正刚 佘小漫[2] 于琳 蓝国兵[2] 丁善文 何自福[2] 汤亚飞[2] WANG Yuan;DU MengDan;LI ZhengGang;SHE XiaoMan;YU Lin;LAN GuoBing;DING ShanWen;HE ZiFu;TANG YaFei(College of Plant Protection,South China Agricultural University,Guangzhou 510642;Plant Protection Research Institute,Guangdong Academy of Agricultural Sciences/South China Key Laboratory for Green Prevention and Control of Fruits and Vegetables,Ministry of Agriculture and Rural Affairs/Guangdong Provincial Key Laboratory of High Technology for Plant Protection,Guangzhou 510640)

机构地区：[1]华南农业大学植物保护学院,广州510642 [2]广东省农业科学院植物保护研究所/农业农村部华南果蔬绿色防控重点实验室/广东省植物保护新技术重点实验室,广州510640

出　　处：《中国农业科学》2024年第12期2350-2363,共14页Scientia Agricultura Sinica

基　　金：国家自然科学基金(32072392);广东省现代农业产业技术体系创新团队项目(2020KJ134);广东省农业科学院“十四五”学科团队建设项目(202105TD);广东省农业科学院协同创新中心项目(XT202210)。

摘　　要：【目的】洋桔梗耳突曲叶病毒(lisianthus enation leaf curl virus,LELCV)是2015年发现的菜豆金色黄花叶病毒属(Begomovirus)病毒新种。论文旨在探究入侵中国内地的首个LELCV分离物Tomato-2022的分子特征、亲缘关系、致病性及有效传播介体,为防控该病毒病提供理论依据。【方法】2022年,在广东省广州市增城区发生一种番茄新病害,病株表现叶小、卷曲、叶尖白化症状,采集两份病样提取总DNA,利用Begomovirus通用引物AV494/CoPR进行PCR检测。选取PCR检测为阳性的样品进行RCA扩增、酶切、克隆及测序,获得分离物Tomato-2022的基因组全长序列。采用BLASTn程序对病毒序列进行相似性检索并下载相似性较高的代表分离物序列,进一步用SDTv1.2的MUSCLE alignment方法进行序列相似性比对;利用软件MEGA7.0对获得的Tomato-2022基因组序列及已报道的相似性较高的代表分离物进行系统发育分析。应用酶切连接法构建Tomato-2022的侵染性克隆pGreenII0229-1.6A,通过农杆菌介导注射接种本氏烟叶片和番茄茎秆,测定其致病性。采取室内人工传毒方法,测定烟粉虱MEAM1隐种对分离物Tomato-2022的传播效率。利用LELCV的PCR特异引物对2021—2023年采集于广东省番茄主产区的135份番茄病样进行检测,明确该病毒在广东省的分布。【结果】PCR检测结果表明,两份表现叶小、卷曲、叶尖白化症状的番茄病样感染了菜豆金色黄花叶病毒属病毒。基因克隆与序列分析显示,该病毒分离物Tomato-2022基因组仅含DNA-A,大小为2757 nt,含有6个ORF。序列相似性比较表明,分离物Tomato-2022基因组序列与已登录GenBank的LELCV各分离物相似性较高,其中与LELCV中国台湾洋桔梗分离物(LC091539、LC091538)相似性最高,为98.33%。系统发育分析显示,Tomato-2022与LELCV的19个分离物聚在一个独立的分支,亲缘关系较近。利用构建的Tomato-2022侵染性克隆pGreenII0229-1.6A,农杆菌介【Objective】Lisianthus enation leaf curl virus(LELCV)is a new species of the genus Begomovirus discovered in 2015.The objective of this study is to explore the molecular characteristics,genetic relationship,pathogenicity and effective transmission vector of the first isolate Tomato-2022 of LELCV invading China's Mainland,and to provide a theoretical basis for the prevention and control of the virus disease.【Method】In 2022,a new disease of tomato occurred in Zengcheng District,Guangzhou City,Guangdong Province.The diseased plants exhibited small leaf,curling and leaf tips whitening symptoms.Total DNA was extracted from two diseased samples,respectively,and used as template for PCR detection with degenerate Begomovirus primers AV494/CoPR.The full genome sequence of isolate Tomato-2022 was obtained by RCA amplification,gene cloning and sequencing from positive samples by PCR detection.The virus sequence was analyzed with BLASTn program and the representative isolate sequences with high similarity were downloaded.Further the sequence similarity was compared using MUSCLE alignment of SDTv1.2 software.Phylogenetic analysis between isolate Tomato-2022 and the reported representative isolates with high similarity was performed using MEGA7.0.Infectious clone pGreenII0229-1.6A of isolate Tomato-2022 was constructed using restriction enzyme digestion and ligation.The infectious clone was inoculated to Nicotiana benthamiana leaves and tomato stems by Agrobacterium-mediated injection to determine its pathogenicity.The efficiency of Bemisia tabaci MEAM1 cryptic species transmitting isolate Tomato-2022 was determined by indoor artificial virus transmission method.PCR specific primers of LELCV were used to detect 135 diseased tomato samples collected from the main tomato producing areas of Guangdong Province from 2021 to 2023 to clarify the distribution of LELCV in Guangdong Province.【Result】The PCR detection result showed that two diseased tomato samples with small leaf,curling and leaf tips whitening symptoms were i

关 键 词：洋桔梗耳突曲叶病毒 番茄 侵染性克隆 致病性 介体传毒效率 烟粉虱 

分 类 号：S436.412.1[农业科学—农业昆虫与害虫防治]