四川莴笋上番茄斑萎病毒的电镜观察与小RNA测序鉴定  

Identification and Sequence Analysis of Tomato Spotted Wilt Orthotospovirus Infecting Lettuce in Sichuan

作　　者：韩帅[1,2] 吴婕[1,2] 张河庆[1,2] 席亚东[1,2] HAN Shuai;WU Jie;ZHANG Heqing;and XI Yadong(Key Laboratory of Integrated Pest Management on Crops in Southwest,Ministry of Agriculture and Rural Affairs,Institute of Plant Protection,Sichuan Academy of Agriculture Sciences,Chengdu 610066,China;Vegetable Germplasm Innovation and Variety Improvement Key Laboratory of Sichuan Province,Horticulture of Research Institute,Sichuan Academy of Agriculture Sciences,Chengdu 610066,China)

机构地区：[1]四川省农业科学院植物保护研究所,农业农村部西南作物有害生物综合治理重点实验室,成都610066 [2]四川省农业科学院园艺研究所,蔬菜种质与品种创新四川省重点实验室,成都610066

出　　处：《园艺学报》2022年第9期2007-2016,共10页Acta Horticulturae Sinica

基　　金：四川省科技计划项目(2021YFYZ0021);四川省农业科学院现代农业学科建设推进工程项目(2021XKJS087);四川省农业科学院科技成果中试熟化与示范转化工程项目(2021ZSSF021)。

摘　　要：为明确引起四川彭州莴笋病毒病的病毒种类,通过生物接种和电子显微镜方法,结合小RNA测序技术和反转录PCR对采集病样进行鉴定和分析。摩擦接种试验表明回接莴笋的发病症状与田间相似,矮牵牛叶片出现局部枯斑;利用电镜可观察到叶肉细胞中有大量病毒粒体聚集于内质网池,其形态特征与正番茄斑萎病毒属(Orthotospovirus)病毒相似;通过小RNA测序技术获得了番茄斑萎病毒(tomato spotted wilt orthotospovirus,TSWV)接近全长的序列,表明侵染该批次莴笋样品的病原物可能是TSWV。利用RT-PCR方法确定田间和回接莴笋及枯斑寄主中均存在TSWV,进一步证明四川彭州莴笋病毒病是由TSWV侵染引起的,这是四川首次发现TSWV为害莴笋。对拼接序列和N基因序列分别构建系统发育树可知,彭州分离物TSWV-SCPZ分别与韩国分离物(YY和K)和美国分离物(MR-01)亲缘关系最近。Biological inoculation,electron microscopy and small RNA sequencing were employed to identify the virus infecting lettuce in Pengzhou,Sichuan Province,followed by reverse transcription(RT)-PCR for verification. Mechanical inoculation showed similar symptom on back inoculated lettuce to field samples,and necrosis was observed on Petunia hybrida leaves. Numerous virions which had similar morphology characteristics to members of the genus Orthotospovirus were observed,and they clustered in the cisternae of endoplasmic reticulum in the mesophyll cells. Nearly complete sequence of tomato spotted wilt orthotospovirus(TSWV)was obtained by small RNA sequencing,revealing that the pathogen in infected samples might be TSWV. RT-PCR results indicated the presence of TSWV in both field and inoculated lettuce plants and indicator plant,further conformed that TSWV was the pathogen causing lettuce virus disease in Pengzhou. This was the first report on TSWV infecting lettuce in Sichuan. The phylogenetic tree constructed based on assemble sequences showed that Pengzhou isolate(TSWV-SCPZ)had close relationship with Korea isolate(YY and K),whereas more close related to America isolate(MR-01)based on N gene.These isolates shared a similar evolutionary pattern with other isolates present in Korea and America.

关 键 词：莴笋 番茄斑萎病毒 小RNA测序 电子显微技术 

分 类 号：S636.2[农业科学—蔬菜学]