大豆疫霉菌ITS分子检测程序的建立及其应用

Molecular Detection of Phytophthora sojae

依据GenBank中登录的大豆疫霉菌(Phytophthora sojae)、近缘种及相似种rDNA的ITS区序列差异,进行多重比较后设计合成一对大豆疫霉菌特异引物,并在PCR反应体系和扩增条件优化的基础上,对包括大豆疫霉菌在内的共140个菌株进行PCR检测。结果表明,电泳后只有大豆疫霉菌扩增出一条288bp的特异性条带。运用设计的大豆疫霉菌专用引物(专利申请号200610089105.4)及建立的检测程序对大豆疫霉菌纯培养游动孢子、接种于土壤中的游动孢子和卵孢子以及接种发病的大豆染病组织进行了检测应用,结果显示该检测程序对接种于土壤中的大豆疫霉菌游动孢子和卵孢子的检测理论精度分别达0.3和0.06个孢子,对染病组织检测也表现出了较高的灵敏度。

An oligonueleotide primer pair was designed and synthesized after comparison and homologieal analysis of rDNA ITS sequences among Phytophthora sojae, its related Phytophthora species, and allied fungal and bacterial species from GenBank. PCR amplifications were carried out for 140 isolates including Phytophthora sojae .It showed that only isolates of Phytophthora sojae can be amplified and a special fragment of 288bp were produced by the primers. These primers were used to detect Phytophthora sojae in pure culture,inoculated diseased soybean plants, and inoculated soil samples. The detection protocol has good sensitivity to diseased tissues.