进境澳大利亚绿豆中菜豆细菌性萎蔫病菌的检测

Detection of Curtobacterium flaccumfaciens pv.flaccumfaciens in Vigna radiata imported from Australia

利用NA培养基从澳大利亚进境绿豆样品中分离到一株疑似菜豆细菌性萎蔫病菌(Curtobacterium flaccumfaciens pv.flaccumfaciens,Cff)的细菌分离物2000-1,对该分离物进行革兰染色试验、PCR检测、多位点序列分析和致病性测试。分离物在NA平板上菌落浅黄色,圆形,隆起,黏性,有光泽且边缘整齐,革兰染色阳性。特异引物CffFOR2/CffREV4可扩增出306 bp的预期产物。分离物2000-1的16S rRNA序列与Cff菌株ATCC 51876完全一致,与GenBank中Cff菌株P990的序列相似性为99.58%,序列差异为3 nt。基于6个看家基因(atpD、dnaK、gyrB、ppK、recA和rpoB)的系统发育树显示,分离物2000-1与Cff相关种处于同一分支,与其中巴西大麦Cff分离物3185 UNESP亲缘关系最近,而与菌株ATCC 51876处于不同分支。分离物人工接种可引起大豆叶片水渍状病斑,大豆叶片出现萎蔫和焦枯,病斑周围伴有金黄色晕圈。根据以上测试结果,将分离物2000-1鉴定为菜豆细菌性萎蔫病菌(Curtobacterium flaccumfaciens pv.flaccumfaciens)。这是全国首次从进境绿豆中截获Cff。

An isolate 2000-1 similar to Curtobacterium flaccumfaciens pv.flaccumfaciens(Cff)was obtained from Australia Vigna radiata with NA(Nutrient Agar)medium,and tested by gram staining,PCR detection,multilocus sequence analysis and pathogenicity.The colony of isolate 2000-1 on NA plate was light yellow,round,swell,viscous,shiny and with regular edge and positive for gram staining.An expected product of 306 bp was amplified by specific primers CffFOR2/CffREV4.The 16 S rRNA sequence of isolate 2000-1 was identical with Cff strain ATCC51876,and the sequence had 99.58%of similarity to the Cff strain P990 in GenBank with 3 nt difference.Phylogenetic tree analysis based on six housekeeping genes(atpD,dnaK,gyrB,ppK,recA and rpoB)showed that isolate 2000-1 was in the same sub-cluster with Cff related species,and was most closely related to the Cff isolate 3185 UNESP from Brazilian barley,while strain ATCC51876 was in a different sub-cluster.Wound inoculation could cause wilting,scorch with golden halo and water-soaked symptoms on soybean leaf.Based on these results,the isolate 2000-1 was identified as the Curtobacterium flaccumfaciens pv.flaccumfaciens,and this is the first report of Cff intercepted from imported Vigna radiata in China.