西瓜枯萎病菌快速LAMP检测体系的建立  

Establishment of a Rapid LAMP Detection Assay for Fusarium oxysporum f.sp.niveum

作　　者：杨科 肖姬玲 张屹 魏林 梁志怀 YANG Ke;XIAO Ji-Ling;ZHANG Yi;WEI Lin;LIANG Zhi-Huai(Longping Branch,Graduate School of Hunan University,Changsha 410125;Hunan Agricultural Biotechnology Research Institute,Changsha 410125;Hunan Institute of Plant Protection,Changsha 410125)

机构地区：[1]湖南大学研究生院隆平分院,长沙410125 [2]湖南省农业生物技术研究所,长沙410125 [3]湖南省植物保护研究所,长沙410125

出　　处：《农业生物技术学报》2021年第9期1836-1844,共9页Journal of Agricultural Biotechnology

基　　金：国家重点研发计划(2017YFD0200606);湖南省自然科学基金(2019JJ40160);湖南省农业科技创新资金(2020CX59)。

摘　　要：由尖孢镰刀菌西瓜专化型(Fusarium oxysporum f.sp.niveum,FON)引起的西瓜枯萎病(Fusarium wilt of watermelon)是西瓜(Citrullus lanatus)上的主要病害,在我国西瓜种植区均有发生,建立西瓜枯萎病菌的快速检测方法对该病的诊断和防控十分重要。FON是造成西瓜枯萎病的主要病原真菌。本研究以FON的特异DNA片段为靶标,设计了一套灵敏、特异的快速环介导等温技术(loop-mediated isothermal amplification,LAMP)引物,并结合DNA快速提取方法,建立了西瓜枯萎病菌的LAMP检测体系。引物特异性实验结果显示,唯有FON-0、FON-1、FON-2的LAMP检测结果呈现黄绿色,且LAMP产物琼脂糖凝胶电泳结果呈现梯形条带,说明引物具有较高特异性。灵敏度检测结果表明,此LAMP体系对FON-1基因组DNA的检测下限为10 pg/μL,对植物带菌量的检测下限为5×10^(2)孢子/g。盆栽实验结果表明,以根部组织为样品,此体系能对西瓜幼苗是否被FON侵染作出正确判断。该体系从DNA提取到获得检测结果仅需90 min左右,且病原菌菌丝和植物组织均可作为检测材料。反应结果肉眼可辨,具有操作简单、特异性强、灵敏度高、稳定性强、应用性广的特点。本检测体系可为田间西瓜枯萎病菌的快速诊断提供技术支撑。Fusarium wilt of watermelon caused by Fusarium oxysporum f.sp.niveum(FON)is the main disease of watermelon across all major watermelon-growing regions in China.Therefore,the establishment of a rapid detection method of FON is quite important for the diagnosis,prevention and control of Fusarium wilt of watermelon.In this study,using the specific DNA sequences of FON as the target,a set of sensitive and specific loop-mediated isothermal amplification(LAMP)primers were designed,and combined with the rapid DNA extraction method,a rapid LAMP detection assay for FON was established.The results of primer specificity experiments showed that only FON-0,FON-1 and FON-2 showed yellow-green color for LAMP detection,and the agarose gel electrophoresis results of LAMP products showed trapezoidal bands,indicating that the primers had high specificity.The sensitivity detection results showed that the lower limit of detection of the LAMP assay for FON-1 genomic DNA was 10 pg/μL,and for FON-1 content in plants was 5×10^(2) spores/g.The results of potting experiments showed that the assay could make a correct judgment on whether watermelon seedlings were infested by FON,using root tissues as samples.In this assay,from the beginning of DNA extraction to the detection results,only about 90 min was needed,and the pathogenic mycelia,the tissues of the plants could be used as detection materials.The results of LAMP reaction were discernible to the naked eye,with simple operation,strong specificity,high sensitivity,stability and wide applicability.It provides technical support for rapid molecular diagnosis of FON in the field.

关 键 词：西瓜 尖孢镰刀菌西瓜专化型(FON) 环介导等温技术(LAMP) DNA简提法 

分 类 号：S436.5[农业科学—农业昆虫与害虫防治]