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大豆霜霉病菌是引起大豆病害的重要病原之一,采用真菌18～28 S间的内转录间隔区(Internal transcribed spacer,ITS)通用引物ITS1和ITS4扩增大豆霜霉病菌和其他外群真菌的基因组DNA,扩增出约500 bp的片段;通过克隆测序大豆霜霉病菌的ITS全序列并与GenBank中霜霉菌属其他种的ITS序列比对,设计出大豆霜霉病菌的特异性引物PM1和PM2。用此特异引物可以从大豆霜霉菌株中扩增出380 bp的特异性片段,而其余9个参试菌株和大豆组织的PCR反应结果为阴性,灵敏度试验证明,可以检测到目标DNA的浓度为0.1 pg。该方法可用于快速、准确和灵敏地检测大豆霜霉病菌,为快速监测组织中霜霉病菌潜伏侵染并及早采取防治措施提供积极的指导作用。
Soybean downy mildew is one of the most important pathogens causing soybean disease. ITS1 and ITS4 were amplified by internal transcribed spacer (ITS) between 18 and 28 S of fungi. Genomic DNA, a fragment of about 500 bp was amplified. Through sequencing of ITS sequence of Plasmopara viticola and ITS sequence alignment with other species of Plasmopara in GenBank, PM1 and PM1 PM2. With this specific primer, a 380 bp specific fragment was amplified from the soybean downy mildew strain, while PCR results of the remaining nine tested strains and soybean tissues were negative. The sensitivity test proved that the target DNA concentration can be detected as 0.1 pg. The method can be used for rapid, accurate and sensitive detection of downy mildew of soybean and provides a positive guide for rapidly monitoring latent infection of downy mildew in tissues and taking prevention and treatment measures as early as possible.