麦冬主要病害病原菌巢式多重PCR检测方法的建立  

Nested multiplex PCR to detect two major fungal pathogens of mondo grass Ophiopogon japonicus

作　　者：杨怡华 王明郧 曹瑱艳 申屠旭萍 俞晓平 Yang Yihua;Wang Mingyun;Cao Zhenyan;Shentu Xuping;Yu Xiaoping(Zhejiang Provincial Key Laboratory of Biometrology and Inspection&Quarantine,College of Life Science,China Jiliang University,Hangzhou 310018,Zhejiang Province,China)

机构地区：[1]中国计量大学生命科学学院,浙江省生物计量及检验检疫技术重点实验室,杭州310018

出　　处：《植物保护学报》2021年第4期742-747,共6页Journal of Plant Protection

基　　金：浙江省公益技术研究项目(2017C32006,2018C02030);中国计量大学学生科技创新专项(2021YW95)。

摘　　要：为准确快速地诊断浙江省慈溪市麦冬常见真菌病害——炭疽病和黑斑病,基于巢式多重PCR技术,针对核糖体内转录间隔区(ITS)序列分别设计特异性引物,并优化巢式多重PCR反应条件,建立可同时快速准确检测炭疽病病原菌山麦冬炭疽菌Colletotrichum liriopes和黑斑病病原菌互隔交链孢菌Alternaria alternata的方法。结果表明,建立的巢式多重PCR检测体系特异性好,同时检测2种病原菌的灵敏度高达100 pg DNA/μL,对10个田间病样进行检测,有5个病样检测到2种病原菌,3个病样检测到其中1种病原菌,2个样品未检测到目标病原菌,验证了该体系的可行性与准确性。表明所建立的巢氏多重PCR技术可用于快速准确检测麦冬2种主要病害的病原菌。In order to accurately and quickly diagnose two major diseases of Ophiopogon japonicus in Cixi City of Zhejiang Province, anthracnose and black spot, specific primer sets were designed based on the internal transcribed spacer(ITS) region sequences of the two pathogens, Colletotrichum liriopes and Alternaria alternata and conditions of a nested multiplex PCR assay were optimized for reliable, rapid and simultaneous detection of C. liriopes and A. alternata. The results showed that the nested multiplex PCR system had a high specificity and could detect C. liriopes and A. alternata in infected plant tissues rapidly with a sensitivity at 100 pg DNA/μL. To verify the feasibility and accuracy of the detection system, ten disease samples were randomly collected from the field. The two pathogenic fungi were simultaneously detected in five samples, either of two pathogenic fungi was detected in three samples, and no target pathogenic fungi was found in the other two samples. These findings indicated that the nested multiplex PCR method established in this study could provide reliable and informative identification of two major pathogens of O. japonicus.

关 键 词：麦冬 山麦冬炭疽菌 互隔交链孢菌 巢式多重PCR 

分 类 号：S435.672[农业科学—农业昆虫与害虫防治]