板栗实腐病菌rDNA-ITS 的RFLP和测序分析

摘要

为了明确孝感本地板栗实腐病菌的种类及其多样性，对分离自孝感的11株板栗实腐病菌的ITS进行了测序和RFLP分析。用通用引物ITS4和ITS5扩增病原菌的ITS1-5.8S-ITS2区域，PCR扩增产物经EcoRⅠ、HaeⅢ和TaqⅠ分别酶切，酶切结果用软件NTSYSpc2.1作聚类图。经ITS-RFLP分析得到了11条多态性DNA条带，在遗传相似系数0.72水平分为4个类群。并对PCR产物进行测序和BLAST分析，相似序列用MEGA5.1构建系统发育树。经形态学和ITS双向测序和比对分析，病原菌主要来源于3个属：Botryosphaeria（无性态Diplodia）、Neofusicoccum和Fusarium（有性态Gibberella），其中Neofusicoccum为引起板栗实腐病菌的新报道属。%To define species and diversity of Castanea mollissima seed rot pathogens in Xiaogan, ITS in 11 strains isolated from the local area were analyzed by sequencing and RFLP. The ITS1-5.8S-ITS2 region was amplified by universal primers ITS4 and ITS5. The products were digested by using EcoRⅠ, HaeⅢand TaqⅠrespectively, and the dendrogram was draw by NTSYSpc2.1 software. Total 11 polymorphic DNA brands are obtained through ITS-RFLP analysis, and the strains can be divided into four groups at 0.72 genetic similarity coefifcient. Furthermore, PCR productions were analyzed by sequencing and BLAST, and the phylogenetic tree of similar sequences was constructed by MEGA5.1. Based on morphology and ITS bidirectional sequencing, the pathogens mainly belong to three genus:Botryosphaeria (anamorph Diplodia), Neofusicoccum and Fusarium (teleomorph Gibberella), and Neofusicoccum is ifrstly reported in the disease.