摘要
【目的】明确引起广西南宁市和钦州市的牛大力呈现花叶斑驳的病原病毒种类,为牛大力病毒病的防控提供理论依据。【方法】以2022—2024年从南宁市和钦州市采集的疑似被病毒感染的牛大力植株叶片样品为材料,通过小RNA深度测序、反转录聚合酶链式反应(RT-PCR)检测、分段克隆、序列分析、系统发育树构建等方法对疑似感病样品进行病毒种类鉴定和系统进化分析。【结果】小RNA深度测序获得49个contigs被注释为病毒基因组,其中25个contigs被注释为豇豆轻斑驳病毒(Cowpea mild mottle virus,CpMMV)、2个contigs被注释为瓜类褪绿黄化病毒(Cucurbit chlorotic yellows virus,CCYV)、22个contigs被注释为莴苣褪绿病毒(Lettuce chlorosis virus,LCV),表明采集的牛大力样品可能受到CpMMV、CCYV和LCV 3种病毒侵染。RT-PCR检测结果显示,样品中仅能扩增出CpMMV的目的条带,未能扩增出LCV和CCYV的目的条带,证实所采集的牛大力样品受到CpMMV的侵染。随机选择1份CpMMV检测阳性的南宁市牛大力样品,利用分段克隆的方法成功获得1条全长为8194 bp的CpMMV全基因组序列,命名为CpMMVGXNiuDaLi;将该序列在GenBank上进行BLASTn比对分析,发现该序列与已登录GenBank的CpMMV各分离物具有较高的核苷酸相似性,其中与CpMMVNJJS(GenBank登录号:OK625819.1)和CpMMVJSNJ(GenBank登录号:MW354944.1)的核苷酸相似性最高,均为97.46%;基于CpMMV全基因组序列和CP基因序列构建的系统发育树分析发现,用于分析的各分离物分为2个大的分支,其中国内的分离物处于一个大分支,国外各分离物除印度新德里的分离物New Delhi(GenBank登录号:OQ791180.1)外均处于另一大分支,说明这些分离物具有地域分布特点;而CpMMVGXNiuDaLi与国内各分离物处于一个大分支,又与CpMMVNJJS和CpMMVJSNJ同处于一个小的分支,表明3个分离物之间具有较近的亲缘关系。【结论】广西南宁市和钦州市呈现花叶斑驳症状的牛大力样品受到CpMMV的侵染。
【Objective】To clarify the viral pathogens which caused Millettia speciosa Champ.exhibiting mottled mo‐saic symptom in Nanning and Qinzhou of Guangxi,which could provide scientific basis for prevention and control of Mil‐lettia speciosa Champ.mosaic disease.【Method】During 2022-2024,leaf samples of Millettia speciosa Champ.suspected infected by viruses were collected from Nanning and Qinzhou.The methods of small RNA deep sequencing(RNA-Seq),reverse transcription polymerase chain reaction(RT-PCR),segmental cloning,sequence analysis and alignment,phylo‐genetic tree construction were applied for virus identification and phylogenetic analysis on suspected infectious samples.【Result】The results showed that 49 contigs obtained by RNA-seq were annotated as viral genome,of which 25 contigs were annotated as cowpea mild mottle virus(CpMMV),2 contigs were annotated as cucurbit chlorotic yellows virus(CCYV)and 22 contigs were annotated as lettuce chlorosis virus(LCV).These results above indicated that the Millettia speciosa Champ.samples might infected by the 3 viruses:CpMMV,CCYV and LCV.The RT-PCR test results showed that only the target band of CpMMV could be amplified in the samples,while LCV and CCYV failed to amplify the target band,confirming that the collected Millettia speciosa Champ.samples were infected by CpMMV.One sample of Millettia speciosa Champ.from Nanning with positive CpMMV detection was randomly selected.A CpMMV complete genome se‐quence with a total length of 8194 bp was successfully obtained by the method of segmental cloning and named CpMMV_GX_NiuDaLi.BLASTn analysis of the sequence in GenBank showed that the sequence had a high nucleotide similarity with each isolate of CpMMV registered in GenBank,nucleotide similarity of CpMMV_NJ_JS(GenBank acces‐sion number:OK625819.1)and CpMMV_JS_NJ(GenBank accession number:MW354944.1)was the highest(97.46%).Analysis of the phylogenetic evolutionary tree constructed based on the complete genome sequence of CpMMV and the sequence of the CP gene showed that the isolates used for the analysis were divided into 2 large branches,the isolates from China in a big branch and all foreign isolates,except for the New Delhi isolate from New Delhi,India(GenBank accession number:OQ791180.1),were in another major branch.It indicated that these isolates have regional distribution characteristics.CpMMV_GX_NiuDaLi was in a large branch with various isolates from China,and was also in a small branch with CpMMV_NJ_JS and CpMMV_JS_NJ,indicating that there was close genetic rela‐tionship among the 3 isolates.【Conclusion】The samples of Millettia speciosa Champ.with mottled leaves in Nanning and Qinzhou of Guangxi are infected by CpMMV.
作者
李祐聪
莫翠萍
谢慧婷
陈锦清
秦碧霞
崔丽贤
蔡健和
李界秋
李战彪
LI You-cong;MO Cui-ping;XIE Hui-ting;CHEN Jin-qing;QIN Bi-xia;CUI Li-xian;CAI Jian-he;LI Jie-qiu;LI Zhan-biao(College of Agriculture,Guangxi University,Nanning,Guangxi 530004,China;Plant Protection Research Institute,Guangxi Academy of Agricultural Sciences/Key Laboratory of Green Prevention and Control on Fruits and Vegetables in South China,Ministry of Agriculture and Rural Affairs/Guangxi Key Laboratory of Biology for Crop Diseases and Insect Pests,Nanning,Guangxi 530007,China)
出处
《南方农业学报》
北大核心
2025年第4期1158-1170,共13页
Journal of Southern Agriculture
基金
广西自然科学基金项目(2020GXNSFAA259003)
广西重点研发计划项目(桂科AB23026068)
广西农业科学院基本科研业务专项(桂农科2021YT071,桂农科2024ZX08)。
关键词
牛大力
花叶病
小RNA深度测序
豇豆轻斑驳病毒
广西
Millettia speciosa Champ.
mosaic disease
small RNA deep sequencing
cowpea mild mottle virus
Guangxi
