我国梨腐烂病病原菌的初步鉴定及序列分析

Sequence analysis and preliminary identification for the pathogen of pear Valsa canker in China

【目的】为了对我国梨腐烂病病原菌进行初步鉴定和序列分析,【方法】从我国15个省（市）梨产区采集腐烂病样品并观察其田间危害症状,通过组织分离法分离获得168份梨腐烂病菌分离株,从中选取72份进行单孢纯化,共获得79份梨腐烂病菌纯化分离株;观察在PDA、25℃黑暗条件下病原菌菌落形态以及产孢体形态,并对其在梨枝条上产生的分生孢子器徒手切片置显微镜下观察其结构特征和分生孢子形态;采用菌丝块接种法测定梨腐烂病菌在‘翠冠’梨离体枝条上的致病力。对部分菌株rDNA-ITS进行PCR扩增、测序,利用BLAST软件与GenBank数据库进行序列相似性分析,并用MEGA 4.1和邻接法构建系统发育树。【结果】根据梨腐烂病菌各分离株在PDA上的菌落形态特征可分为Ⅰ型和Ⅱ型两种菌落类型,不同梨腐烂病菌分离株在PDA上产生多种类型的产孢体,不同梨腐烂病菌菌株在离体梨树枝条上的致病力存在差异,我国梨腐烂病菌的rDNA-ITS核苷酸序列一致率为99.98%~100%,与苹果腐烂病菌分别聚在同一亚组的两个分支。【结论】我国梨腐烂病病原菌存在不同的菌落类型,其rDNA-ITS核苷酸序列分析显示均为V.mali var.pyri。

【Objective】The preliminary identification and sequence analysis for the pathogens of pear Valsa canker in China were carried out in this study.【Methods】The symptoms of pear Valsa canker disease in field were observed and recorded in 15 provinces（cities）.Total 168 pathogen isolates of pear Valsa canker were isolated with organizational separation methods.Total 79 isolates were obtained by isolating single spore and their sporulation bodies were observed at 25 ℃ in darkness on PDA medium.The conidia and pycnidia of the pathogens on pear twigs were observed by hand sectioning under the microscope.The pathogenicity for the isolates from pear Valsa canker were determined with one-year in vitro branches inoculated by the pathogen,and the data were analyzed by Statistic Package for Social Science（SPSS）.The rDNA-ITS genes for pathogen isolates of pear Valsa canker from different regions were amplified with apple Valsa canker as references.Multiple alignments of nucleotide sequences of rDNA-ITS genes were obtained by using the program Clustal X1.8 and DNAMAN.Phylogenic analysis were done by using the minimum evolution method of phylogenetic interference with 1000 bootstrap replicates and neighbor-joining（NJ） method incorporated in the 4.1 version of the Molecular Evolution Genetics Analysis（MEGA） software.【Results】The results showed that there were two colony types,designated as type I and II,in the pathogen isolates of pear Valsa canker.The pathogen isolates of pear Valsa canker formed different sporulation bodies on PDA medium.The pathogenicity of the pathogen isolates was different on in vitro branches of pear.The nucleotide sequences of rDNA-ITS from different isolates showed high identities,ranging from 99.98% to 100%.Phylogenetic analysis on nucleotide（nt） sequences of rDNA-ITS genes indicated that the pathogen isolates of pear Valsa canker and apple Valsa canker obtained in the study were clustered into two branches of the same subgroup.【Conclusion】The pathogen isolates of pear Valsa canker in China have different colony types,which belonged to V.mali var.Pyri based on analysis of nucleotide sequences of rDNA-ITS genes.