玉米大斑病菌和小斑病菌交配型多重PCR检测方法的建立与应用  

Establishment and Application of a Multiple PCR Method to Detect Mating Types of Exserohilum turcicum and Bipolaris maydis

作　　者：代玉立[1] 甘林[1] 滕振勇 杨静民 祁月月 石妞妞[1] 陈福如[1] 杨秀娟[1] DAI YuLi;GAN Lin;TENG ZhenYong;YANG JingMin;QI YueYue;SHI NiuNiu;CHEN FuRu;YANG XiuJuan(Institute of Plant Protection,Fujian Academy of Agricultural Sciences/Fujian Key Laboratory for Monitoring and Integrated Management of Crop Pests,Fuzhou 350013;Fujian Seed Management Station,Fuzhou 350001;Jianou Municipal Bureau of Agriculture and Rural Affairs,Jianou 353100,Fujian;Zhejiang Tianfeng Biological Science Co.Ltd,Jinhua 321000,Zhejiang)

机构地区：[1]福建省农业科学院植物保护研究所/福建省作物有害生物监测与治理重点实验室,福州350013 [2]福建省种子管理总站,福州350001 [3]建瓯市农业农村局,福建建瓯353100 [4]浙江天丰生物科学有限公司,浙江金华321000

出　　处：《中国农业科学》2020年第3期527-538,共12页Scientia Agricultura Sinica

基　　金：福建省属公益类科研院所专项（2017R1025-3,2018R1025-1）;国家重点研发计划（2018YFD0200706）;福建省农业科学院青年自由探索项目（AA2018-14）;福建省农业科学院青年科技英才百人计划（YC2016-4）;福建省农业科学院植物保护创新团队（STIT2017-1-8）

摘　　要：【目的】由大斑突脐蠕孢(Exserohilum turcicum)和玉蜀黍平脐蠕孢(Bipolaris maydis)引起的玉米大斑病和小斑病是玉米生产上重要的叶部真菌病害,本研究旨在建立玉米大斑病菌和小斑病菌交配型多重PCR检测方法,为玉米大斑病菌和小斑病菌的交配型田间分布和有性生殖研究提供技术方法。【方法】根据GenBank中已登录的玉米大斑病菌(登录号MAT1-1:GU997138和MAT1-2:GU997137)和小斑病菌(登录号MAT1-1:X68399和MAT1-2:X68398)交配型基因序列,利用Primer Premier 5.0软件设计2种病原菌交配型多重PCR检测特异性引物,采用单因素法对引物的退火温度以及扩增程序中延伸时间和循环数等重要参数进行优化,建立玉米大斑病菌和小斑病菌交配型多重PCR检测方法,并对2种病原菌的交配型多重PCR检测灵敏度和特异性进行检验。同时,对田间采集的129株玉米大斑病菌和194株玉米小斑病菌单孢菌株的交配型进行多重PCR检测,以明确建立的交配型多重PCR检测方法的适用性。【结果】建立的多重PCR方法和设计的交配型特异引物StMAT01-2F/R、StMAT02-3F/R、ChMAT01-3F/R和ChMAT02-2F/R可分别扩增出MAT1-1、MAT1-2型菌株大小为816、132 bp(大病斑菌)与490、136 bp(小病斑菌)的特异性目的条带。25μL多重PCR扩增体系:2×Multiplex PCR Mix 12.5μL,引物各10 pmol,DNA模板100 ng,退火温度为57.2℃(大病斑菌)和55.0℃(小斑病菌),35个循环。该多重PCR对玉米大斑病菌MAT1-1、MAT1-2型单孢菌株的检测灵敏度分别为0.1、0.01 ng基因组DNA,而对玉米小斑病菌MAT1-1、MAT1-2交配型的检测灵敏度均为0.1 ng基因组DNA。该交配型多重PCR检测方法对玉米大斑病菌和小斑病菌特异性很强,能够很好地区分玉米大斑病菌和小斑病菌相应的近缘种和14株其他真菌菌株。不同地理来源的玉米大斑病菌和小斑病菌交配型检测结果表明,该多重PCR能够准确地检出129株玉米大斑病菌和19【Objective】 Northern corn leaf blight(NCLB) and southern corn leaf blight(SCLB), caused by Exserohilum turcicum and Bipolaris maydis, respectively, are the most important foliar fungal diseases affecting maize production.The objective of this study is to establish a multiple PCR method to detect mating types of E.turcicum and B.maydis, and to provide a technical method for the study of mating type distribution in the field and sexual reproduction of E.turcicum and B.maydis.【Method】Mating type-specific primers for the two pathogens were designed on the basis of mating type gene sequences of E.turcicum(accession numbers: GU997138 for MAT1-1;GU997137 for MAT1-2) and B.maydis(accession numbers: X68399 for MAT1-1;X68398 for MAT1-2) obtained from GenBank, and the important parameters of primer annealing temperatures, extension times and amplification cycles in the amplification program were optimized using the single factor method.A multiple PCR method was established to detect mating types of E.turcicum and B.maydis, and the sensitivity and specificity of the multiple PCR were also assessed.Meanwhile, the mating types of 129 strains of E.turcicum and 194 strains of B.maydis from field-collections were detected by the multiple PCR to determine the adaptability of the established method.【Result】The expected 816, 132 bp(E.turcicum), and 490, 136 bp(B.maydis) target fragments were amplified specifically using the multiple PCR with mating type-specific primers of StMAT01-2 F/R, StMAT02-3 F/R, and ChMAT01-3 F/R, ChMAT02-2 F/R from MAT1-1 and MAT1-2 strains, respectively.A 25 μL PCR reaction system consisted of 12.5 μL 2×Multiplex PCR Mix, 10 pmol each primer, and 100 ng DNA template.The annealing temperatures for E.turcicum and B.maydis were 57.2℃ and 55.0℃, respectively, and the number of amplification cycles was 35.The multiple PCR method could reliably detect mating types of E.turcicum at 0.1 ng genomic DNA for MAT1-1 or 0.01 ng DNA for MAT1-2 from single-spore strains, while the sensitivity of the mul

关 键 词：玉米大斑病菌 玉米小斑病菌 交配型 多重PCR 灵敏度 特异性 

分 类 号：S435.131[农业科学—农业昆虫与害虫防治]