一种土壤中烟草疫霉菌的快速分离鉴定及计数方法

A Fast Method of Isolation, Identification and Counting of Phytophthora nicotianae from Soil

为了建立一种土壤烟草疫霉菌快速计数方法,将稀释平板法与选择性培养基相结合,进行了微量真菌DNA提取及定性PCR,结果表明:10-2稀释浓度和选择性培养基能很好抑制绝大多非疫霉菌和所有细菌生长,有利于对土壤中烟草疫霉菌进行分离计数;微量真菌DNA提取方法提取的菌落DNA能够满足定性PCR的要求,采用的特异引物只能从疫霉菌中扩增到737 bp的特异性片段;利用真菌ITS验证了该方法确定的6个土壤烟草疫霉菌与已报道烟草疫霉菌相似度达99%以上;表明该方法完全适宜于土壤中烟草疫霉菌数量的鉴定及计数,具有相对准确、低成本、简单易推广的优点。

The aim was to establish a fast counting method for Phytophthora nicotianae in soil.The pour plate method and the application of selective culture medium were combined to count P.nicotianae in soil.The mini-preparation method of fungal DNA and the qualitative PCR were carried out.The results showed that,when cultured in selective medium with the concentration of 10-2,most of the non-P.nicotianae could be restricted form growing,which was in favor of separating the target fungi.The method of mini-preparation of fungal DNA met the requirements of qualitative PCR,and specific gene fragments of P.nicotianae could be amplified into a fragment with the length of 737 bp.6 strains of P.nicotianae were found by ITS method to be similar to the reported P.nicotianae with a degree of 99%.Thus,this method was suitable for the accounting of P.nicotianae in soil with the advantages of being accurate,low-cost and simple to implement.