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利用MT选择性培养基从进境加拿大豌豆样品上分离到一株细菌分离物(编号1314),对该分离物进行PCR检测、16S和23S rRNA序列扩增、多位点序列分析、Biolog测定、烟草过敏性反应和致病性测试。豌豆细菌性疫病菌(Pseudomonas syringae pv.pisi,Ppi)特异引物AN7F/AN7R扩增分离物1314和Ppi菌株ATCC 11043得到预期272 bp的条带,二者的PCR产物序列一致,与GenBank中Ppi(X97405)的序列相似性为99.57%。分离物1314的部分16S rRNA、23S rRNA序列以及16S-23S序列均与Ppi菌株ATCC 11043一致。选择gap1、gltA、gyrB和ropD 4个看家基因进行多位点序列分析,系统发育树显示分离物1314与Ppi菌株聚在同一组内。Biolog鉴定结果表明:分离物1314为Ppi,PROB值为0.898,SIM为0.72。该分离物人工接种豌豆茎秆能引起水渍状症斑,接种烟草叶片产生过敏性反应。基于上述试验结果,将分离物1314鉴定为豌豆细菌性疫病菌。
A strain of bacterial isolates (Accession No. 1314) was isolated from Canadian Pea samples using MT selective medium. The PCR products were subjected to PCR detection, 16S and 23S rRNA sequence amplification, multilocus sequence analysis, Biolog assay, Tobacco allergic reaction and pathogenicity test. The predicted 272 bp band was obtained from the Pseudomonas syringae pv. Pisi specific primer AN7F / AN7R amplified isolate 1314 and the Ppi strain ATCC 11043. The PCR products of both sequences were consistent with those of Ppi ( X97405) has a sequence similarity of 99.57%. The partial 16S rRNA, 23S rRNA sequence, and 16S-23S sequences of isolate 1314 were identical to the Ppi strain ATCC 11043, respectively. Four housekeeping genes, gap1, gltA, gyrB and ropD, were selected for multiple site sequence analysis. Phylogenetic tree showed that isolate 1314 and Ppi clustered in the same group. Biolog identification results showed that isolate 1314 was Ppi with a PROB value of 0.898 and a SIM of 0.72. Artificially inoculated pea stalks of the isolate can cause waterlogging plaque, inoculation of tobacco leaves produce allergic reactions. Based on the above test results, isolate 1314 was identified as a pea bacterial blight.