黄花菜叶斑病菌鉴定、生物学特性分析、防治药剂筛选及LAMP检测体系的建立  

Identification,biological characteristics,screening of fungicides and establishment of loop-mediated isothermal amplification for the pathogen causing leaf spot of citron daylily Hemerocallis citrina

作　　者：朱俊子 邱泽澜 高必达[1] 钟杰[1] 李晓刚[1] Zhu Junzi;Qiu Zelan;Gao Bida;Zhong Jie;Li Xiaogang(College of Plant Protection,Hunan Agricultural University,Changsha 410128,Hunan Province,China)

机构地区：[1]湖南农业大学植物保护学院,长沙410128

出　　处：《植物保护学报》2022年第6期1631-1641,共11页Journal of Plant Protection

基　　金：湖南省自然科学基金(2018JJ3229);湖南省教育厅科研基金重点项目(20A257)。

摘　　要：为明确浙江省黄花菜叶斑病病原菌的种类,探究其对杀菌剂的敏感性并建立快捷检测方法,采用组织分离法获得黄花菜叶斑病菌,通过形态学特征观察和分子生物学技术对病原菌进行鉴定,测定其致病力及生物学特性,分析6种杀菌剂对病原菌的生长抑制效果,并以ITS和EF-1α序列为靶标设计特异性引物建立环介导等温扩增(loop-mediated isothermal amplification,LAMP)检测方法。结果表明,根据形态学特征以及ITS与EF-1α序列分析结果确定黄花菜叶斑病菌为杏黄镰孢Fusarium armeniacum。该病原菌菌丝生长适宜温度为20~30℃,pH 7时菌丝生长最快,以葡萄糖为碳源、硝酸钠为氮源时利用率最高。96%己唑醇对菌丝生长的抑制作用最强,EC50为0.119μg/mL。本研究所建立的LAMP体系在60℃条件下反应60 min,琼脂糖凝胶电泳和SYBR Green I染料染色均可判断检测结果。该方法可特异性地检测出杏黄镰孢,最低检测灵敏度为100 pg/μL,是常规PCR检测灵敏度的10倍。表明本研究所建立的LAMP检测体系可快速检测出人工接种和实际发病黄花菜叶片中的病原菌。In order to identify the causal agent of leaf spot on citron daylily Hemerocallis citrina in Zhejiang Province of China,to determine its sensitivity to six fungicides and to construct a fast detection method,the pathogen was obtained with tissue separation method and identified based on morphological characteristics,molecular biological characteristics and pathogenicity tests.The biological characteristics of the pathogen were studied.The inhibitory effects of six fungicides on the pathogen were determined in laboratory.A loop-mediated isothermal amplification(LAMP)detection method was established by designing the specific primers based on ITS and EF-1αsequences.The pathogen was identified as F.armeniacum according to morphological characteristics as well as the sequence analysis of ITS and EF-1α.The optimum temperature for the growth of the was 20-30℃,and the hyphal growth rate was the fastest at pH 7.Among all carbon and nitrogen sources tested,the mycelial growth of the pathogen was fastest using glucose and sodium nitrate as carbon and nitrogen sources,respectively.Among the six fungicides,96%hexakonazol had the best inhibition effect on hyphal growth,with a smallest EC50value of 0.119μg/mL.The LAMP detection system could detect F.armeniacum specifically with a minimum limit of 100 pg/μL,which was ten times higher than that of the conventional PCR.This LAMP method could be used to detect pathogen in artificial inoculated and naturally infected H.citrina leaves.

关 键 词：杏黄镰孢 黄花菜 叶斑病 分子鉴定 生物学特性 抑制效果 LAMP体系 

分 类 号：S436.44[农业科学—农业昆虫与害虫防治]