兜兰萎蔫病病原菌的分离与鉴定

Isolation and Identification of Pathogen of Paphiopedilum Wilt Disease

【目的】明确引起兜兰萎蔫病(Paphiopedilum wilt disease)的病原菌种类,为兜兰萎蔫病的防控及兜兰产业的健康发展提供参考依据。【方法】以具有兜兰萎蔫病典型症状的病叶为材料,采用组织分离法获得纯化菌株,通过离体接种与活体接种相结合的方法进行分离菌株的致病性测定,采用形态学结合rDNA-ITS序列分子鉴定的方法进行病原菌种类鉴定。【结果】从兜兰萎蔫病病斑中分离纯化获得3株菌株,分别命名为WY-1、WY-2和WY-3。离体接种和活体接种试验结果显示,WY-1菌株和WY-3菌株无发病症状产生,WY-2菌株发病症状明显且引起的病害症状与田间表现一致。经显微镜观察,WY-2菌株产生的孢子形态为镰刀形、两端渐尖,在NCBI上进行BLAST在线比对发现,其rDNA-ITS测序序列与镰刀孢属尖孢镰刀菌(Fusarium oxysporum)相似性达99.0%。【结论】结合形态学和分子生物学分析可确定尖孢镰刀菌为兜兰萎蔫病致病菌。

【Objective】The pathogen species causing Paphiopedilum wilt disease are determined to provide reference for the prevention and control of Paphiopedilum wilt disease and the healthy development of Paphiopedilum industry.【Method】The purified strains were isolated from the typical Paphiopedilum wilt disease spots by the tissue isolation method, then the pathogenicity of the isolated strain is determined by combining in vitro and in vivo inoculation method and finally the pathogen species was identified by the morphological feature and rDNA-ITS sequence molecular systematics method.【Result】Three strains were isolated and purified from Paphiopedilum wilt disease spots, named WY-1, WY-2 and WY-3, respectively. The three strains were subjected to in vitro and in vivo inoculation tests, among which WY-1 and WY-3 strains had no symptoms after inoculation, and WY-2 strains had obvious symptoms and the disease symptoms caused were consistent with the field manifestations.The spores produced by WY-2 strain were observed by microscope as sickle-shaped and acuminate at both ends. BLAST alignment of the rDNA-ITS sequence of strain WY-2 on NCBI, the results showed that the similarity between strain WY-2 and Fusarium oxysporum reached 99 %.【Conclusion】Combined with morphological and molecular biological analysis, Fusarium oxysporum was identified as the pathogen of Paphiopedilum wilt disease.