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

【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194株玉米小斑病菌的交配型,且检测结果与随机抽取的菌株杂交验证结果完全吻合。【结论】构建的玉米大斑病菌和小斑病菌交配型多重PCR检测方法灵敏度高、特异性好、操作简便,能够准确、快速地检测出玉米大斑病菌和小斑病菌的交配型,为玉米大斑病菌和小斑病菌的交配型田间分布和监测及有性生殖研究提供了可靠的技术方法。

【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 multiple PCR to detect mating types of B.maydis was 0.1 ng genomic DNA for both MAT1-1 and MAT1-2 from pure culture strains.The method exhibited specificity in differentiating mating types of E.turcicum and B.maydis from their closely-related species, as well as 14 other fungal genera.The results of mating types detection of E.turcicum and B.maydis strains from different geographical origins indicated that the multiple PCR could reliably detect mating types of 129 and 194 strains of E.turcicum and B.maydis, respectively.These results were consistent with the verification results of laboratorial cross assays with random selected strains from different locations.【Conclusion】The established multiple PCR method for mating types detection of E.turcicum and B.maydis in this study was characterized as high sensitivity, specificity and user-friendly control, it could accurately and rapidly detect mating types of E.turcicum and B.maydis.This study provides a reliable technique and approach for the study of distribution and monitoring of mating types in the field and sexual reproduction of E.turcicum and B.maydis.