河南省小麦全蚀病菌的鉴定、进化分析及标志基因筛选

为了解河南省郑州市中牟市小麦全蚀病菌的变种类型及进化情况,筛选全蚀病菌侵染小麦的分子标记,采用形态学观察、科赫氏法则验证、ITS序列分析和系统进化树构建等方法对分离的小麦全蚀病菌进行鉴定,并对其侵染后小麦病原相关蛋白(pathogen-related protein,PR)基因的表达进行实时定量PCR分析。结果表明,显微形态观察初步确定分离的小麦全蚀病菌为禾顶囊壳Gaeumannomyces graminis(Sacc.)v. Arx.Olivier,经科赫氏法则验证、ITS序列比对及系统进化树分析进一步证明该病菌均为禾顶囊壳。本研究分离的小麦全蚀病菌与地域相距较远的英国、美国的小麦全蚀病菌间的同源性比与来自中国陕西省的小麦全蚀病菌的同源性更高。采用禾顶囊壳4个变种的特异性引物进行PCR扩增,所有分离病菌均扩增出了小麦变种的特异性条带,证实分离菌株为禾顶囊壳小麦变种G. graminis var. tritici。实时定量PCR分析发现,病原相关蛋白基因PR4a、PR4b、PR2、PR10受到小麦全蚀病菌侵染后表达量均显著上调,在侵染后第5～6天达到最高峰,表明这些基因可作为小麦全蚀病菌侵染小麦的标志性基因。

Phylogenetic analysis, identification and marker genes screening of wheat take-all fungus in Henan Province

In order to completely identify the variant types and evolutionary relationship of wheat takeall disease in Zhongmou of Zhengzhou City of Henan Province, and screening for the molecular marker genes of wheat, a series of experiments were carried out in this research. The isolated pathogens were identified with morphological characters, the ribosomal DNA internal transcribed spacer (ITS) sequence, the expression patterns of pathogenesis-related (PR) proteins were characterized after pathogen inoculation, for the screening of the marker genes of take-all fungus infection. Microscopic morphological observation results showed that the pathogen was Gaeumannomyces graminis (Sacc.) Arx & Olivier, and molecular identification and phylogenetic tree results further confirmed it. The phylogenetic tree results had also verified that the isolated fungus had closer homology with the isolates from UK and USA than that from Shaanxi Province of China. All the isolated pathogens in the four pairs of variety-specific primers used to identify the fungus turned out to be G. graminis var. tritici, according to the results of PCR amplification. The expression level of PR4a, PR4b, PR2 and PR10 post infected with G. graminis, analyzed by quantitative real-time PCR (q RT-PCR), had increased significantly and reached a peak on the fifth and sixth days after inoculated with the pathogen. In conclusion, all the above PR genes could be served as marker genes of G. graminis.