侵染广西辣椒的两种单组分菜豆金色花叶病毒属病毒的分子特征

Molecular characterization of two monopartite begomovirus infecting Capsicum annuum L.in Guangxi

【目的】明确引起广西百色市辣椒呈现叶片上卷、皱缩等症状的病毒病原,并对各病毒分离物的遗传进化关系进行分析,旨在为辣椒病毒病的防控提供科学依据。【方法】从广西百色市采集4份疑似被菜豆金色花叶病毒属病毒感染的辣椒样品,利用PCR、分段克隆、核苷酸序列比对分析、进化树构建等方法对疑似样品进行鉴定及遗传进化分析。【结果】PCR结果显示,4个样品中均能扩增出约570 bp目标PCR条带,将PCR产物直接送样测序,所得序列进行blastn分析发现,4条序列分别与已登录GenBank的TYLCV和PaLCuCNV各分离物的序列具有较高的核苷酸相似性,证实所采集的辣椒植株受到菜豆金色花叶病毒属病毒侵染。参考已登录GenBank的TYLCV(GenBank登录号:MG904859和KY783940)、PaLCuCNV(GenBank登录号:KU892661和MW779523)的序列设计2对背靠背引物,随机选择2份阳性样品进行全基因组扩增和序列测定,将所得PCR产物纯化后克隆至pMD18T载体上,挑选阳性克隆进行测序,从2份阳性样品中共获得3条病毒全长基因组序列,将所得序列在GenBank中进行blastn分析,发现其中2条序列与已登录GenBank的部分TYLCV分离物的核苷酸相似性达到91%以上,1条序列与已登录GenBank的部分PaLCuCNV分离物核苷酸相似性在91%以上。根据双生病毒的分类标准,确定侵染广西辣椒的双生病毒为TYLCV和PaLCuCNV的不同分离物。进化树分析发现,TYLCV广西辣椒分离物BS66-1与TYLCVSG1分离物(GenBank登录号:MT969010,寄主为番茄)具有较近的亲缘关系,而TYLCV广西辣椒分离物BS67-1则与TYLCV GX和TYLCV BS3分离物(GenBank登录号:MW389934、MT375610,寄主均为番茄)具有较近的亲缘关系;PaLCuCNV广西辣椒分离物BS66-2则与PaLCuCNV GX01分离物(GenBank登录号:MW779523,寄主为番茄)具有较近的亲缘关系。【结论】侵染广西辣椒呈现叶片上卷、皱缩等症状的病原为TYLCV和PaLCuCNV,本研究为菜豆金色花叶病毒属病毒侵染广西辣椒的首次报道。

【Objective】The study aimed to identify the viral pathogen of Capsicum annuum L.in Baise,Guangxi province and the results would provide theoretical basis for the effective prevention and control of the pepper viral disease.【Method】Four samples of C.annuum L.with leaf rolling and crinkling symptoms were collected from Baise,Guangxi province in 2022.The samples were identified and analyzed by polymerase chain reaction(PCR),gene cloning,nucleotide comparison,phylogentic tree construction.【Result】The PCR amplicons with ex⁃pected size of 570 bp were obtained from 4 samples,the PCR products were sequenced and the sequences were analyzed in GenBank,the four sequences showed high nucleotide identities with tomato yellow leaf curl virus(TYLCV)and papaya leaf curl China virus(PaLCuCNV)isolates avilable in GenBank,respectively,and the results above confirmed that all of the four samples were infected by begomoviruses.To obtained the whole genome of the virus,two pairs of primers were designed based on the TYLCV(GenBank No.:MG904859 and KY783940)and PaLCuCNV(GenBank No.:KU892661 and MW779523)isolates available in GenBank.The PCR products were purified and cloned into the pMD18T vector,positive clones were selected for gene sequencing.Three whole genome sequences were obtained from two positive samples.Two of the three sequences had 91%nucleotide identities with some of isolates of TYLCV available in GenBank,and one sequence had 91%nucleotide identities with the some of isolates of PaLCuCNV available in GenBank.Following the guidelines of the classification of begomoviruses,the viruses infecting pepper in Guangxi were identified as TYLCV and PaLCuCNV.Phylogenetic tree analy⁃sis showed that the TYLCV Guangxi pepper isolate BS66⁃1 branched with TYLCV SG1(Accession No.:MT969010,host:tomato)isolate,indicating their close evolutionary relationship;and BS67⁃1 branched with GX isolate and BS3 isolate(Accession No.:MW389934 and MT375610,host:tomato),indicating their close evolutionary relationship;The PaLCuCNV Guangxi pepper isolate BS66⁃2 clustered with PaLCuCNV GX01(Accession No.:MW779523,host:tomato)isolate in a small branch,indicating their close evolutionary relationship.【Conclusion】TYLCV and PaLCuCNV are the pathogens that infect C.annuum(L.)in Guangxi.And this is the first report of the begomo⁃viruses infecting C.annuum L.in Guangxi.