基于小RNA深度测序技术鉴定侵染我国部分省市草莓种苗的病毒种类

来源:用户上传      作者:王佳 白莹雪 祝宁 齐长红 任俊达 韩成贵 尚巧霞

　　摘要 随着草莓保护地栽培面积的增加和无性繁殖种苗的繁殖与调运，草莓病毒病的发生与流行日益严重。为明确侵染我国部分省市草莓种苗的病毒种类，应用小RNA深度测序技术进行检测，并利用RT-PCR技术对结果进行验证及序列分析。Y果表明，从来自我国7省市的41株具有典型病毒病症状的草莓种苗样品中检测到草莓斑驳病毒strawberry mottle virus （SMoV）、草莓镶脉病毒strawberry vein banding virus（SVBV）和草莓轻型黄边病毒strawberry mild yellow edge virus （SMYEV）3种。SMoV、SVBV和SMYEV的检出率分别为34.1%、24.4%和2.4%。选取不同产地草莓种苗上检出的不同病毒进行部分序列测定和分析，获得了3个SMoV分离物（四川分离物schhy13、辽宁分离物lnhy23和河北分离物hbhy28）的部分RNA1 3′端非编码区606 bp核苷酸序列，其一致性为98.12%～99.34%。测定并获得了5个SVBV分离物（辽宁分离物lnhy15、lnhy17、lnhy24、河北分离物hbhy28和陕西分离物shaxhy35）的ORF6的部分基因及其5′端非编码区的核苷酸序列，其中部分ORF6基因序列的一致性为97.98%～99.8%。仅1个辽宁样品中检测到SMYEV，获得了辽宁分离物lnhy26，其CP基因序列与中国甘肃分离物sy02的一致性为99.02%。病毒侵染草莓植株产生的典型症状有叶片畸形、变色和植株矮化。存在2种病毒和3种病毒复合侵染的情况，占所有阳性样品的41.2%。辽宁和河北的‘红颜’种苗中检出SMoV和SVBV或SMoV、SVBV和SMYEV的复合侵染情况;四川和陕西的‘红颜’种苗仅检出SMoV或SVBV，而其他省市和品种均未检出病毒。研究结果可以为草莓病毒的检测以及草莓病毒病的科学防治提供依据。
　　关键词 草莓病毒病;小RNA深度测序技术;RT-PCR;病毒检测
　　中图分类号： S436.639
　　文献标识码： A
　　DOI： 10.16688/j.zwbh.2021079
　　Abstract With the increase of protected cultivation area， propagation and transportation of strawberry asexual reproductive seedlings between different regions are increasingly frequent， and the occurrence and prevalence of strawberry viral disease are also becoming more and more serious. To identify the viruses infecting strawberry plants from different provinces of China， small RNA deep sequencing assay was used to detect the viruses in strawberry seedling samples， and then the detected viruses were further verified by RT-PCR and sequence analysis. The results showed that strawberry mottle virus （SMoV）， strawberry vein banding virus （SVBV） and strawberry mild yellow edge virus （SMYEV） were detected in 41 strawberry seedlings with typical symptoms from seven provinces of China. The positive rates of SMoV， SVBV and SMYEV detection were 34.1%， 24.4% and 2.4%， respectively. Strawberry seedling samples infected by different viruses from different regions were selected， from which the partial viral nucleotide sequences of the detected viruses were sequenced and analyzed. The partial 3′ UTR nucleotide sequences of RNA1 of three samples infected by SMoV from Sichuan， Liaoning and Hebei were sequenced， which was 606 bp in length. The partial SMoV nucleotide sequences among Sichuan isolate schhy13， Liaoning isolate lnhy23 and Hebei isolate hbhy28 shared identity from 98.12% to 99.34%. The sequences of partial ORF6 gene and its 5′ UTR of SVBV Liaoning isolates lnhy15， lnhy17， lnhy24， Hebei isolate hbhy28 and Shaanxi isolate shaxhy35 were obtained. The partial ORF6 gene nucleotide sequences of five SVBV samples shared identity from 97.98% to 99.8%. Only one sample with SMYEV was detected from Liaoning， and the CP gene sequence of Liaoning isolate lnhy26 shared identity of 99.02% with that of Gansu isolate sy02. Symptoms of strawberry plants infected by virus included leaf deformity， leaf discoloration and dwarf. The detection results showed that combined infection by two or three viruses existed， and the detection rate accounted for 41.2% of virus-positive samples. ‘Benihoppe’ seedlings from Liaoning and Hebei were simultaneously infected by SMoV and SVBV or SMoV， SVBV and SMYEV. ‘Benihoppe’ seedlings from Sichuan and Shaanxi were only infected by SMoV or SVBV， but no virus was detected in strawberry seedlings sampled from other provinces or in other varieties. This study provided important information and techniques for virus detection and strawberry viral disease management.

nlc202206091429

转载注明来源:https://www.xzbu.com/1/view-15433114.htm