我国苹果斑点落叶病菌携带dsRNA分析及一种dsRNA病毒的鉴定  

Analysis of dsRNA Carried by Alternaria alternata f.sp.mali in China and Identification of a dsRNA Virus

作　　者：曹钰晗 李紫腾 张静怡 张静娜 胡同乐[1] 王树桐[1] 王亚南[1] 曹克强[1] CAO YuHan;LI ZiTeng;ZHANG JingYi;ZHANG JingNa;HU TongLe;WANG ShuTong;WANG YaNan;CAO KeQiang(College of Plant Protection,Hebei Agricultural University,Baoding 071001,Hebei)

机构地区：[1]河北农业大学植物保护学院,河北保定071001

出　　处：《中国农业科学》2021年第22期4787-4799,共13页Scientia Agricultura Sinica

基　　金：国家现代农业产业技术体系建设专项(CARS-27);河北省自然科学基金(C2019204327);河北省重点研发计划(21326506D,19226508D,20327405D);河北省引进留学人员资助项目(C201839)。

摘　　要：【目的】苹果斑点落叶病在我国各苹果主产区均有发生,给苹果产业造成了严重的经济损失。苹果斑点落叶病是由链格孢苹果专化型(Alternaria alternata f. sp. mali)侵染引起的一种气传病害。本研究旨在探明我国苹果斑点落叶病菌携带dsRNA的情况,为田间防治斑点落叶病提供新的生防资源,并为病毒的多样性以及进化研究提供新的认识。【方法】从全国8个省份采集有典型症状的组织样本,通过组织分离和单孢分离法获得纯培养;通过dsRNA的提取及凝胶电泳明确我国苹果斑点落叶病菌携带dsRNA的情况;通过菌株培养特性、菌丝生长速率、在果实及叶片上的致病力测定揭示携带dsRNA病原菌的生物学性状;通过高通量测序技术、分子克隆技术对QY-2菌株携带的dsRNA病毒进行全基因组序列、基因组结构和系统进化分析。【结果】自我国苹果产区获得102株苹果斑点落叶病菌菌株的纯培养,通过dsRNA的提取及凝胶电泳明确了5个菌株带有明显的dsRNA条带,携带的dsRNA分为4种类型,类型Ⅰ(菌株YT-3-7)dsRNA在8、2.5和1.5 kb左右;类型Ⅱ(菌株SJZ-4)dsRNA在8 kb左右;类型Ⅲ(菌株QY-2)dsRNA在3和0.8 kb左右;类型Ⅳ(菌株CL-2-6和SQ-1-1)dsRNA在2.5和1.5 kb左右。含有dsRNA的菌株培养性状多样,菌落特征、生长速率与dsRNA携带与否及类型没有明显的关系,含有dsRNA的菌株大多属于弱致病力菌株。QY-2在叶片和果实上的致病力均最弱,确定其携带的dsRNA病毒基因组包括5个dsRNA片段,分别为dsRNA1-dsRNA5,片段大小依次为3 665、3 054、2 824、2 819、831 nt,提交至GenBank,登录号分别为MK672910、MK672913、MK672912、MK672911、MK836314。编码蛋白的分子量依次为124、83、84、83、13 kD。经系统进化关系分析,与Alternaria alternata chrysovirus 1遗传关系最近,确定其为产黄青霉病毒科(Chrysoviridae)、β产黄青霉病毒属(Betachrysovirus)的Alternaria alternata chrysovirus,【Objective】Apple Alternaria blotch occurs in all major apple producing areas in China, causing serious economic loss.Apple Alternaria blotch is a worldwide airborne disease caused by Alternaria alternata f. sp. mali. The objective of this study is to investigate the diversity of dsRNA of A. alternata f. sp. mali in China, and to provide new biological control resources for the disease and new understanding of virus diversity and evolutionary.【Method】Tissue samples with typical symptom were collected from eight provinces in China. Pure culture was obtained by tissue isolation and single spore separation. The diversity of dsRNA carried by A.alternata f. sp. mali in China was determined by ds RNA extraction and gel electrophoresis. The biological characteristics of dsRNA-carrying pathogens were determined by evaluation of culture characteristics, mycelial growth rate, and pathogenicity on fruits and leaves. By high throughput sequencing and molecular cloning techniques, the whole genome sequence, genome structure and phylogenetic for the mycovirus carried by QY-2 strain were analyzed.【Result】The pure culture of 102 strains of A. alternata f.sp. mali was obtained and five strains with obvious dsRNA bands were found. The dsRNA virus of A. alternata f. sp. mali in China could be divided into four types, there were about 8, 2.5 and 1.5 kb dsRNA in type Ⅰ(strain YT-3-7), about 8 kb dsRNA in type Ⅱ(strain SJZ-4), about 3 and 0.8 kb dsRNA in type Ⅲ(strain QY-2), and about 2.5 and 1.5 kb dsRNA in type Ⅳ(strains CL-2-6 and SQ-1-1). The strains carrying dsRNA had diverse culture characteristics, and there was no significant relationship between the colony characteristics, growth rate and whether dsRNA carrying or not and its type. The strains carrying ds RNA were mostly of low pathogenicity type. It was found that QY-2 strain showed weak pathogenicity on leaves and fruits, so QY-2 was selected for virus identification. The genome of the dsRNA virus carried by the strain QY-2 consists of five dsRNA fragment

关 键 词：苹果斑点落叶病 链格孢苹果专化型 DSRNA 生物学性状 致病力 生物防治 

分 类 号：S436.611.1[农业科学—农业昆虫与害虫防治]