进境加拿大豌豆中豌豆细菌性疫病菌的检测

 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。

Detection of Pseudomonas syringae pv. pisi from imported Canadian pea seeds

An isolate 1314 was obtained from imported Canadian pea seeds with semi-selective MT(milk tween agar) medium, and identified by PCR, 16S and 23S rRNA sequence analysis, multilocus sequence analysis (MLSA), Biolog test, hypersensitivity reaction on tobacco and pathogenicity. With the specific primer AN7F/AN7R of Pseudomonas syringae pv. pisi (Ppi), 272 bp products (100% sequence identity) were yielded with this isolate and the Ppi strain ATCC 11043, and the sequence was 99.57% identical to the Ppi isolate (X97405) in GenBank. Sequence analysis revealed that partial sequences of 16S and 23S rRNA and 16S-23S sequence of the isolate 1314 shared 100% identities with the Ppi strain ATCC 11043 in correspondence. MLSA and phylogenetic analysis were carried out with the sequences from four housekeeping genes including gap1, gltA, gyrB and ropD. The isolate 1314 was clustered with Ppi strains in the phylogenetic tree. The result of Biolog test showed that this isolate was Ppi with the similarity (SIM) of 0.72 and probability (PROB) of 0.898.The isolate 1314 can trigger hypersensitivity reaction on tobacco by artificial injection inoculation and cause typical water-soaked symptom on the stem of inoculated young pea plant. Based on these results above, this isolate was identified as Pseudomonas syringae pv. pisi.