黄芪根腐病尖孢镰刀菌LAMP可视化快速检测方法的建立  

Establishment of LAMP Visual Rapid Detection for Fusarium oxysporum of Root Rot of Astragalus membranaceus

作　　者：梁伊菲 王春伟 LIANG Yifei;WANG Chunwei(College of Plant Protection,Shanxi Agricultural University,Taigu 030801,China)

机构地区：[1]山西农业大学植物保护学院,山西太谷030801

出　　处：《山西农业科学》2023年第4期420-426,共7页Journal of Shanxi Agricultural Sciences

基　　金：山西省现代农业产业技术体系建设专项(2021);山西省高等学校大学生创新创业训练计划项目(20210130)。

摘　　要：由于黄芪的轮作周期缩短、连作面积连续增加,导致黄芪根腐病的发病率大大增加。目前,黄芪根腐病成为限制黄芪产量的主要因素之一,严重制约着黄芪产业的持续发展。虽然多种镰刀菌在整个种植季均可侵染黄芪,但尖孢镰刀菌(Fusarium oxysporum)是造成黄芪根部腐烂最主要的病原菌之一。镰刀菌在不同的培养基上形态不同,由多种镰刀菌引起的黄芪根腐症状通常很难根据其形态学的特征加以区分,需要通过一些基因测序手段。翻译延伸因子(TEF-1alpha)已被广泛应用于研究镰刀菌属的种内和种间变异和系统发育。因此,可以针对TEF-1alpha区域为靶点设计LAMP引物。为了给尖孢镰刀菌的野外和现场快速检测提供新思路,为黄芪根腐病病害的田间快速诊断提供一个简便快速的新技术,研究针对尖孢镰刀菌翻译延伸因子(TEF-1alpha)序列设计引物,优化环介导等温扩增(LAMP)反应时间和温度,测试LAMP反应的灵敏性和特异性,构建黄芪根腐病尖孢镰刀菌的LAMP快速检测方法。结果表明,反应最适温度为62℃,最适时间为30 min;在最佳反应条件下,黄芪根腐病尖孢镰刀菌的LAMP快速检测方法对尖孢镰刀菌具有良好的特异性,灵敏度可达到10 pg/μL;在反应开始前加入SYBR Green I显色剂,可在反应结束后观察检测结果。Due to the rotation cycle of Astragalus membranaceus was shortened,and the continuous cropping area was increased,the incidence rate of root rot of Astragalus membranaceus was greatly increased.Now,root rot has become one of the main factors limiting the yield of Astragalus membranaceus,and seriously restricts the sustainable development of the Astragalus membranaceus industry.Although a variety of Fusarium can infect Astragalus membranaceus throughout the planting season,Fusarium oxysporum is one of the main pathogens causing root rot of Astragalus membranaceus.The morphology of Fusarium on different media is different,and the symptoms of root rot caused by various Fusarium are often difficult to distinguish according to their morphological characteristics,so some genetic sequencing methods are needed.Translation extension factor(TEF-1alpha)has been widely used to study intraspecific and interspecific variation and phylogeny of Fusarium.Therefore,loop-mediated isothermal amplification(LAMP)primers can be designed for taking TEF-1alpha regions as the target.In order to provide a novel idea for the rapid detection of Fusarium oxysporum in the fields and scenes,and provide a simple and fast new technology for the rapid diagnosis of root rot of Astragalus membranaceus in the fields,in this study,primers were designed aming at the translation extension factor(TEF-1alpha)of Fusarium oxysporum.A LAMP rapid detection for Fusarium oxysporum of root rot of Astragalus membranaceus was established.The LAMP reaction time and temperature were optimized to test the sensitivity and specificity of LAMP assay.The results showed that the optimum temperature was 62℃and the optimum time was 30 min.Under the optimal conditions,the LAMP rapid detection for Fusarium oxysporum of root rot of Astragalus membranaceus had good specificity for Fusarium oxysporum,and the detection sensitivity could reach 10 pg/μL.The chromogenic agent of SYBR Green I was added before the reaction began and the detection results could be observed after the

关 键 词：环介导等温扩增(LAMP) 尖孢镰刀菌 黄芪 根腐病 可视化 快速检测 

分 类 号：S435.67[农业科学—农业昆虫与害虫防治]