山东省辣椒主要病毒种类的分子检测与鉴定

doi:10.3864/j.issn.0578-1752.2017.14.009

摘要/Abstract

摘要： 【目的】鉴定山东省辣椒上的主要病毒种类，明确该地区辣椒上的主要病毒病原。【方法】2014—2015年，在山东省临沂、日照、青岛、烟台、潍坊、淄博、济宁、菏泽、聊城、德州共10个市（区）采集253份疑似感病的辣椒植株叶片，提取叶片总RNA和总DNA，利用双生病毒的通用引物（PA/PB）、马铃薯卷叶病毒属通用引物（POL-F/POL-R）及已报道侵染辣椒的主要病毒的检测引物对样品进行PCR、RT-PCR分子检测与鉴定，将扩增得到的目的条带经凝胶回收试剂盒回收纯化后连接到pMD18-T载体上，再送至公司进行克隆测序。分别将所得的PMMoV、PeVYV和BWYV序列在NCBI上利用BLAST进行检索，利用DNAStar软件中的Megalign将所得序列与GenBank中已登录的世界各地有代表性的序列进行同源性比对，利用软件MEGA 5.05的Clustal W法进行多序列比对分析以及邻接法（neighbor-joining，NJ）构建系统进化树，系统进化树中各分支置信度（Bootstrap）进行1 000次重复分析。【结果】 PMMoV、CMV总检出率分别为61.66%、60.08%；TMGMV、BBWV-2、BWYV、TMV发生也比较普遍，检出率为41.90%、34.78%、33.20%和24.90%；PCV-2、ToMV、TYLCV、PVY、MABYV、PeVYV、PCV-1、ChiVMV、AMV发生侵染现象较少，检出率分别为11.86%、9.88%、9.09%、6.72%、5.53%、3.56%、3.16%、0.79%和0.40%；未检测到CaCV、PSV、ChiRSV、TSWV和ToMMV。通过对病毒复合侵染现象进行分析发现，病毒复合侵染率高达89.92%，其中3种病毒复合侵染现象最多，所占比例达28.63%，4种病毒复合侵染率为25.00%，2种病毒复合侵染率为21.77%，5种病毒复合侵染率为13.31%，一个辣椒样品最多可同时感染6种病毒，侵染率为1.21%；对山东省10个地区的辣椒病毒检测，结果表明在辣椒上检出病毒种类最多的为临沂、济宁，检测到12种病毒，其次是烟台、聊城，检测到11种病毒，潍坊、菏泽检测到9种病毒，青岛、德州检测到8种病毒，日照检测到7种病毒，淄博检测到6种病毒，日照地区辣椒病毒复合侵染率最高，为100%，菏泽地区复合侵染率最低，为69.23%。分别根据PMMoV的部分CP基因序列、PeVYV部分RdRp、CP、MP基因序列以及BWYV部分CP、MP基因序列构建系统发育树，结果表明本研究PMMoV山东分离物SD60与中国贵州分离物的亲缘关系最近，PeVYV山东分离物SDRZ31-1与意大利IT83分离物的亲缘关系最近，BWYV山东分离物SD与韩国分离物LS的亲缘关系最近。【结论】在山东省采集的253份辣椒样品可被15种病毒侵染，PMMoV、CMV为主要病毒种类；在山东新检测到的病毒有MABYV、PeVYV、BWYV、PCV-1和PCV-2；明确了山东省主要辣椒产区病毒病发生情况以及病毒种类的主次关系。

关键词: 辣椒病毒病, 辣椒轻斑驳病毒, 甜菜西方黄化病毒, 甜瓜蚜传黄化病毒, 复合侵染

Abstract: 【Objective】 The objective of this study is to identify the main viruses causing pepper virus diseases in Shandong Province, China.【Method】In 2014-2015, a total of 253 susceptible pepper plant samples were collected from 10 urban areas (Linyi, Rizhao, Qingdao, Yantai, Weifang, Zibo, Jining, Heze, Liaocheng, Dezhou) in Shandong Province. Total RNA and DNA were extracted from the leaves of pepper samples, and were subjected to detect viruses with Geminivirus universal primers (PA/PB), Polerovirus universal primers (POL-F/POL-R) and specific primers of main viruses infecting pepper have been reported by PCR and reverse transcription-PCR (RT-PCR). Each amplified fragment was purified by DNA gel extraction kit, cloned into pMD18-T vector and sequenced. The obtained sequences of PMMoV, PeVYV and BWYV were subjected to BLAST search, and compared with the representative sequences of GenBank which had been registered around the world using the Megalign in DNAStar software. Clustal W method of MEGA 5.05 was used for multiple sequence alignment analysis and the phylogenetic tree were constructed by neighbor-joining (NJ) of MEGA 5.05, with the bootstrap of each branch of phylogenetic tree analyzed 1 000 times. 【Result】The PMMoV and CMV had the highest detection rate, were 61.66% and 60.08%, respectively. TMGMV, BBWV-2, BWYV and TMV were popular with the detection rate of 41.90%, 34.78%, 33.20% and 24.90%, respectively. The detection rates of PCV-2, ToMV, TYLCV, PVY, MABYV, PeVYV, PCV-1, ChiVMV and AMV were 11.86%, 9.88%, 9.09%, 6.72%, 5.53%, 3.56%, 3.16%, 0.79% and 0.40%, respectively. No CaCV, PSV, ChiRSV, TSWV and ToMMV were detected. The complex infection phenomenon showed that co-infections were up to 89.92%, three kinds of viruses mixed infection occurs the highest rate, up to 28.63%, followed by four kinds of viruses mixed infection, up to 25.00%, two kinds of viruses mixed infection, up to 21.77%, five kinds of viruses mixed infection up to 13.31%, and 6 viruses detected in one sample is the extreme case, with the mixed infection rate up to 1.21%. Phylogenetic trees were constructed with partial CP of PMMoV, partial RdRp, CP and MP genes of PeVYV and partial CP and MP genes of BWYV, respectively. PMMoV Shandong isolate SD60 and the China Guizhou isolate had the closest relationship, PeVYV-SDRZ31-1 and Italy isolate IT83 shared the closest relationship, BWYV Shandong isolate SD clustered with Korean isolate LS. 【Conclusion】 Fifteen viruses were detected, among which PMMoV and CMV were the main pathogens. The newly detected viruses are MABYV, PeVYV, BWYV, MABYV, PCV-1 and PCV-2 in pepper in Shandong Province. The occurrence of viral diseases and the primary and secondary relationship of the main types of pepper viruses in Shandong Province were clarified.

Key words: pepper virus diseases, Pepper mild mottle virus (PMMoV), Beet western yellows virus (BWYV), Melon aphid-borne yellows virus (MABYV), complex infection

王少立，谭玮萍，杨园园，代惠洁，孙晓辉，乔宁，竺晓平. 山东省辣椒主要病毒种类的分子检测与鉴定[J]. 中国农业科学, 2017, 50(14): 2728-2738.

WANG ShaoLi, TAN WeiPing, YANG YuanYuan, DAI HuiJie, SUN XiaoHui, QIAO Ning, ZHU XiaoPing. Molecular Detection and Identification of Main Viruses on Pepper in Shandong Province[J]. Scientia Agricultura Sinica, 2017, 50(14): 2728-2738.

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