rDNA ITS区间PCR-RFLP在腐霉属11菌株快速鉴别中的应用

The Application of PCR-RFLP Method of rDNA ITS in the Quick Distinguishing of 11 Strains of Pythium

目的:将灭蚊腐霉Pythiumsp.与从猝倒、茎腐植株分离到的9个病原真菌相鉴别。方法:以瓜果腐霉作阳性对照。以ITS1和ITS4为引物,采用PCR方法对待测菌株的rDNA ITS区进行了扩增,再用4种限制性内切酶(TaqI,H infⅠ,H in6Ⅰ,MboⅠ)进行酶切,经琼脂糖凝胶电泳后得到RFLP图谱,利用Ne i et al的片段比较方法计算菌株间相似程度,NTSYS-PC软件包进行聚类分析。结果:对11个菌株进行了快速鉴别,证明从受感染的植物菌株上分离到的真菌主要是瓜果腐霉,与灭蚊腐霉Pythiumsp.有明显区别。结论:rDNA ITS区的PCR-RFLP可用于真菌的快速鉴别。

Objective： To quickly distinguish a strain of mosquito-killing fungus, Pythium sp.（MKP）, from other 9 strains of Pythium isolated from sick plants. Methods： Internal transcription spacer （ITS） of ribosomal DNA from MKP and the 9 strains of fungi was amplified using polymerase chain reaction （PCR） with universal primers ITS1 and ITS4. PCR products were digested with four restriction enzymes （Hinf Ⅰ, Cfo Ⅰ, Mbo Ⅰ, and Taq Ⅰ）. Their electrophoresis patterns of restriction digestion were achieved. Similarity between strains was analyzed with fragment comparing method of Nei et al and NTSYS-PC software. Results： The 9 strains of fungi were different from MKP. Conclusions： 1） The 9 strains of fungi isolated from sick plants are not MKP; 2） PCR-RFLP method based on rDNA ITS regions is useful in quick distinguishing of fungi.