番茄早疫病菌dsRNA多样性分析

Diversity analysis of dsRNA in Alternaria solani

【目的】研究番茄早疫病菌携带dsRNA的多样性,为筛选具有生防作用的真菌病毒提供依据。【方法】从石河子周边加工番茄种植区采集典型症状的组织样品,通过组织分离和单孢分离法获得纯培养;提取dsRNA及琼脂糖凝胶电泳明确携带dsRNA的情况。选择6个菌株,通过随机引物RT-PCR、序列分析,研究Blast对携带病毒种类及分类。【结果】从分离到的191株番茄早疫病菌菌株中提取dsRNA,有43株中检测到1-4条dsRNA条带,带毒率为22.5%。不同菌株携带dsRNA的大小不尽相同。对6株菌株的dsRNA进行序列测定、组装得到Contigs,Blast比对、分析,鉴定出7种dsRNA病毒和2种+ssRNA病毒,分别与整体病毒科(Totiviridae)、双分病毒科(Partitiviridae)、产黄青霉病毒科(Chrysoviridae)、芜菁黄花叶病毒科(Tymoviridae)和裸露病毒科(Narnaviridae)成员存在序列同源性。【结论】番茄早疫病菌携带dsRNA存在多样性,不仅携带多种病毒,而且还存在复合侵染现象。已经检测到的9种病毒分属于Totiviridae、Partitiviridae、Tymoviridae、Tymoviridae和Narnaviridae,有些病毒是已经报道的病毒或其新株系,有些则是未被揭示的新病毒。

【Objective】The objective of this study is to investigate the diversity of dsRNA in A.solani.,and to provide new biological control resources for the disease and new understanding of virus diversity and evolutionary.【Methods】Tissue samples with typical symptom were collected from tomato growing areas around Shihezi and pure culture was obtained by tissue isolation and single spore separation.After that,the diversity of dsRNA carried by A.solani.was determined by dsRNA extraction and gel electrophoresis.In the end by RT-PCR using random primers,and molecular cloning,and sequencing techniques,the phylogenetics for the mycovirus carried by 6 strain were analyzed.【Results】The pure culture of 191 strains of A.solani.was obtained and 42 strains with obvious dsRNA bands(from 1 to 4)were found.The mycovirus strains were accounting for 22%of the pure culture strains.The band's sizes were different among the dsRNA carried strains.The Contigs were obtained by sequence and assemble from 6 strains.The blast results showed that the 7 dsRNA virues and 2+ssRNA virues were shared sequence homology with isolates among the Totiviridae,Partitiviridae,Tymoviridae,Tymoviridae,and(or)Narnaviridae respectively.【Conclusion】There are diverse dsRNAs in A.solani.The strains of A.solani not only carry many mycoviruses,but also mixed infections with two or more unrelated viruses.The 9 mycovirues identified can be divided into 5 known families(Totiviridae,Partitiviridae,Tymoviridae,Tymoviridae,and Narnaviridae).Some are reported viruses or new strains while others are new viruses that have not yet been discovered.