北京地区四种百合病毒的检测及病毒序列分析  

Detection and Viral Sequence Analysis of Four Lily-infecting Viruses in Beijing

作　　者：鲁婧童 孔祥凤 李介文 贾桂霞 LU Jing-Tong;KONG Xiang-Feng;LI Jie-Wen;JIA Gui-Xia(School of Landscape Architecture/Urban and Rural Ecological Environment Beijing Laboratory/Ministry of Education Forest and Flower Breeding Laboratory/Beijing Key Laboratory of Flower Germplasm Innovation and Molecular Breeding/National Flower Engineering Technology Research Center,Beijing Forestry University,Beijing 100083,China)

机构地区：[1]北京林业大学园林学院/城乡生态环境北京实验室/林木花卉遗传育种教育部重点实验室/花卉种质创新与分子育种北京市重点实验室/国家花卉工程技术研究中心,北京100083

出　　处：《农业生物技术学报》2024年第10期2424-2436,共13页Journal of Agricultural Biotechnology

基　　金：国家重点研发计划(2023YFD1200105);河北省重点研发计划(21326317D)。

摘　　要：百合属植物(Lilium spp.)易受多种病毒复合侵染,病毒检测与序列分析有助于明确感染病毒种类、揭示病毒变异情况。本研究基于百合无症病毒(Lily symptomless virus,LSV)、百合斑驳病毒(Lily mottle virus,LMoV)、黄瓜花叶病毒(Cucumber mosaic virus,CMV)和车前草花叶病毒(Plantago asiatica mosaic virus,PLAMV)中保守基因序列的4对引物,通过优化PCR延伸时间建立上述4种病毒的多重RT-PCR检测方法,并利用该方法抽检了北京地区田间种植百合的病毒感染情况,进一步分离4种病毒产物进行测序与比对,构建系统进化树。结果表明,在多重RT-PCR反应中,增加延伸反应时间至90 s时可扩增出全部4种病毒及18S rRNA(内参基因)条带;抽样检测结果显示,田间种植的百合多受2种及以上病毒复合侵染,其中病毒检出率由大到小依次为LSV(100%)>CMV(92.31%)>LMoV(30.77%)>PLAMV(15.38%);序列分析发现,4种病毒分离物与其各自同种病毒序列平均相似性在84.4%以上,证明检测所得4种病毒为目标病毒;4种病毒变异分析发现,CMV分离物以百合为寄主时单独聚为一个分支,具有寄主差异性;LMoV在系统发育关系上可分为2个分支,实验中的分离物属于分支Ⅱ;LSV和PLAMV未见明显分支模式。本研究为百合病毒病的常规分子诊断与研究提供技术支持,为病害防治提供基础参考。Lilies(Lilium spp.)are susceptible to complex viral infections.Virus detection and sequence analysis are helpful in identifying the types of infecting viruses and revealing viral variations.In this study,based on the conservative gene sequences of Lily symptomless virus(LSV),Lily mottle virus(LMoV),Cucumber mosaic virus(CMV),and Plantago asiatica mosaic virus(PLAMV),4 pairs of primers were used to establish a multiplex RT-PCR detection method for these 4 viruses by optimizing the PCR extension time.This method was used to sample virus infections in lilies grown in the fields in Beijing.Furthermore,the 4 viral products were isolated for sequencing and alignment to construct a systematic evolutionary tree.The results showed that in the quintuplex RT-PCR reaction,extending the extension reaction time to 90 s resulted in the amplification of all 4 viruses and the 18S rRNA(reference gene).Sampling results indicated that lilies grown in the fields were often co-infected with 2 or more viruses,with detection rates in the order of LSV(100%)>CMV(92.31%)>LMoV(30.77%)>PLAMV(15.38%).Sequence analysis revealed that the average similarity of the isolated viral products to their respective viral sequences was above 84.4%,confirming the identification of the 4 target viruses.Variability analysis of the 4 viruses showed that when CMV was hosted by lilies,it formed a distinct branch,indicating host specificity.LMoV could be divided into 2 branches in the evolutionary relationship,with the experimental isolates belonging to branchⅡ.No clear branching patterns were observed for LSV and PLAMV.This study provides technical support for the routine molecular diagnosis and research of lily viral diseases,offering a foundational reference for disease prevention and control.

关 键 词：百合 病毒检测 多重RT-PCR 序列分析 

分 类 号：S682[农业科学—观赏园艺]