实时荧光定量PCR技术在湖南油菜根肿病菌检测中的应用  

Application of real-time fluorescence quantitative PCR technology in detection of pathogen of rapeseed clubroot in Hunan

作　　者：周游 谢芳玲 肖蓉 张政兵 周鑫钰[1] 朱宏建[1] ZHOU You;XIE Fangling;XIAO Rong;ZHANG Zhengbing;ZHOU Xinyu;ZHU Hongjian(College of Plant Protection,Hunan Agricultural University,Changsha 410128,China;Hunan Institute of Microbiology,Changsha 410009,China;Plant Protection and Plant Inspection Station,Department of Agriculture and Rural Affairs of Hunan Province,Changsha 410005,China)

机构地区：[1]湖南农业大学植物保护学院,长沙410128 [2]湖南省微生物研究院,长沙410009 [3]湖南省农业农村厅植保植检站,长沙410005

出　　处：《植物保护》2023年第4期233-238,275,共7页Plant Protection

基　　金：湖南省自然科学基金(2021JJ30356);湖南省高校科技创新平台建设经费(20K071)。

摘　　要：为完善湖南油菜根肿病的监测预警体系,分析油菜根肿病的发生与芸薹根肿菌休眠孢子量的关系,防止湖南油菜根肿病进一步蔓延。本研究依据芸薹根肿菌ITS序列设计了一对特异性引物,通过制备标准质粒构建了快速、精准的实时荧光定量PCR检测方法。其检测灵敏度为3×10^(-10)μg/μL,比常规PCR高100倍;利用建立的方法检测了来自湖南各地土壤样品中芸薹根肿菌的休眠孢子数量,64份土样中的休眠孢子含量基本大于104个/g,所检测的土样中孢子含量最高为2.22×10^(7)个/g。据实地调查,当土壤中休眠孢子数量大于104个/g时,油菜发生根肿病的风险较大,休眠孢子含量较高的田块其发病程度也较为严重。结果表明,建立的实时荧光定量PCR检测方法能对油菜根肿病进行早期诊断,为湖南油菜根肿病发生的监测预警提供有效技术参考。In order to improve the monitoring and early warning system of rape clubroot in Hunan,the relationship between the occurrence of rape clubroot and the amount of dormant spores of P.brassicae was analyzed,so as to prevent the further spread of rape clubroot in Hunan.In this study,a pair of specific primers were designed based on the ITS sequence of P.brassicae,and a rapid and accurate real-time fluorescent quantitative PCR detection method was constructed by preparing standard plasmids.The detection sensitivity was 3×10^(-10)μg/μL,which was 100 times higher than that of conventional PCR.The established method was used to detect the number of dormant spores of P.brassicae in soil samples from all over Hunan.The number of dormant spores in 64 soil samples was almost greater than 104 spores/g,and the highest number reached to 2.22×10^(7)spores/g.According to field investigations,when the number of dormant spores in the soil was greater than 104 spores/g,the risk of clubroot disease in rapeseed was higher,and the incidence of field plots with higher content of dormant spores was also more serious.The results show that the established real-time quantitative PCR detection method can make early diagnosis of rape clubroot,and provide an effective technical reference for the monitoring and early warning of rapeseed clubroot in Hunan.

关 键 词：油菜根肿病 实时荧光定量PCR 休眠孢子 检测 

分 类 号：S432.44[农业科学—植物病理学]