泰安樱桃根癌病致病菌分离及生物型鉴定

Identification and characterization of pathogenic bacteria isolated from crown galls on cherry trees in Tai’an

由致病性土壤杆菌侵染引起的根癌病严重影响了樱桃树的生长。利用选择性培养基,以阿糖醇和赤藓糖醇作为不同碳源,从山东泰安食用樱桃根部瘤状组织和感病根际土壤中分离到土壤杆菌100余株,通过胡萝卜切片法和番茄茎部针刺接种法从中筛选到31株有致瘤活性的菌株。经16S rDNA和recA基因序列分析,结合生物型检测鉴定出96%以上的致病菌属原生物Ⅱ型(Agrobacterium rhizogenes),只有1株致病菌为原生物Ⅰ型(A.tumefaciens)。根据Ti质粒上致病相关基因保守序列设计3对引物,12株有致瘤活性菌株中11株PCR扩增到目的条带。经分子生物学测定和高压纸电泳检测,所有致病菌株的质粒均为胭脂碱型。建立了致病型土壤杆菌的分子生物学快速鉴定方法,为定向选择生防菌株提供基础。

The crown gall disease caused by infection of the pathogenic Agrobacterium seriously affected the growth of cherry trees.More than 100 bacterial strains were isolated from the tumor tissues and rhizosphere soil of edible cherry trees in Tai’an,Shandong province,using selective media with arabinitol or erythritol as the carbon sources.Pathogenicity tests on carrot disks and tomato stems showed that 31 of the 40 strains were virulent.Based on the 16S rDNA and recA gene sequence analysis combined with identification of biovars,more than 96%of pathogenic bacteria were biovarⅡ(A.rhizogenes)and only one pathogenic bacteria was biovar I(A.tumefaciens).Three pairs of primers were designed based on the conserved sequences of disease-causing genes on the Ti plasmid,and the target bands were amplified from 11 of 12 virulent strains by PCR.All strains investigated harbored nopaline-type of Ti plasmid based on molecular biology and high voltage paper electrophoresis.A rapid molecular identification method for the pathogenic Agrobacterium was established,which can provide a basis for the selection of biocontrol strains.