四川大白菜细菌性软腐病新病原菌Pectobacterium versatile的鉴定

Identification of Pectobacterium versatile,a new pathogen causing bacterial soft rot of Chinese cabbage(Brassica rapa L.ssp.pekinensis)in Sichuan province

为明确引起四川理县大白菜软腐病的病原菌种类,从理县梭罗沟村田间采集具有典型软腐症状的大白菜病样,采用组织分离、致病力测定、生理生化分析、形态学和分子生物学方法进行鉴定。结果表明,分离获得的5株细菌在NA培养基上形成的菌落呈圆形、半透明、乳白色、中心突起、边缘平滑,在半选择性培养基CVP上培养48 h后产生凹陷。5株菌均能使大白菜、胡萝卜、马铃薯和芹菜的离体组织腐烂,接种于白菜苗后叶片呈湿腐萎蔫症状。通过大白菜叶柄接种部位形成的病斑长径来比较菌株致病力强弱,各菌株的致病力有显著差异。基于看家基因pgi、rpoS、mdh、proA、mtlD和icdA(NCBI登录号为OL963562~OL963571)的多基因联合系统发育树,分离菌株与Pectobacterium versatile聚为一簇,dnaX-leuS-recA(NCBI登录号为OL963572~OL963576)的多位点序列分析(MLSA)进一步支持以上结果,且准确性更高。分离菌株的生理生化特征与P.versatile模式菌株一致,综上将其鉴定为P.versatile。这是我国首次报道P.versatile引起大白菜软腐病。

To determine the pathogens causing soft rot disease of Chinese cabbage,diseased cabbage samples with typical symptoms were collected from Suoluogou village of Lixian county,Sichuan province,and the pathogens were identified by using tissue isolation,pathogenicity tests,physiological and biochemical analysis,morphological and molecular methods.The results showed that five bacterial strains isolated from Chinese cabbage formed round,translucent,creamy white colonies with a raised center and smooth surfaces on nutrient agar(NA)and produced pits within 48 h on crystal violet pectate medium(CVP).All strains induced maceration on Chinese cabbage petioles,carrot roots,potato tubers and celery stems,and produced soft rot symptoms on inoculated Chinese cabbage plants.The pathogenicity test by measuring diameters of disease spot formed at the inoculation site reveal that the significant different pathogenicity of these strains.The polygenic phylogenetic tree based on six housekeeping genes(pgi,rpoS,mdh,proA,mtlD and icdA)(acc.no.OL963562-OL963571)show that tested strains with four known Pectobacterium versatile strains clustered together.And multilocus sequence analysis(MLSA)with concatenated gene sequences of dnaX-leuS-recA(OL963572-OL963576)further supported this result and can more accurately identify the strains belonging to the genus Pectobacterium.Meanwhile,the physiological characteristics of these strains were consistent with the description of P.versatile type strain.It is the first report that P.versatile causes soft rot disease in Chinese cabbage in China.