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对从北京地区芹菜软腐组织中分离到的37株细菌性软腐病菌株,进行了微生物碳源利用(BiologTM)、生化特性、特异性PCR、ITS-RFLP带型以及基于16S rDNA完整序列遗传关系分析。结果如下:菌株革兰氏染色阴性、具周生鞭毛;可在5%和7%NaCl及28℃和37℃条件下生长;可分解柠檬酸盐及液化明胶;Biolog分析将这些菌株均鉴定为Pectobacterium carotovorum subsp.carotovorum(Pcc)。生化特性分析发现,其中36株菌株能利用D-山梨醇、D-阿拉伯糖醇、异麦芽酮糖和α-甲基葡萄糖苷,与对照菌株P.carotovorum subsp.odoriferum(Pco)S7一致;另外1株菌株Q34不能利用上述糖醇,与对照菌株P.carotovorum subsp.brasilience(Pcb)BC1和P.carotovorum subsp.carotovorum(Pcc)ECC71表现一致。利用pel(pectate lyase)基因特异性引物Y1/Y2在所有的37株菌株中均扩增出预期片段(434 bp),表明其基因组中均含有果胶酶基因。Pcb特异性PCR引物BR1f/L1r仅在Q34菌株与Pcb BC1中扩增出预期片段(322 bp),而Pcc特异性PCR引物EXPCCF/EXPCCR则在除Pcb BC1外所有菌株中均扩增出预期片段(550 bp)。Rsa I酶切16S-23S rDNA ITS片段结果显示,Q34酶切带型与Pcb BC1相同,其余36株带型与Pcc ECC71和Pco S7相同。基于16S rDNA基因完整序列,以Dickeya dadantii菌株582为外群,该37株菌株与已发表的Pectobacterium菌株系统发育树聚类分析结果表明,Q34与已发表的其他Pcb菌株形成了明显的Pcb类群,其余36株菌株则与已发表的其他Pco菌株形成了明显的Pco类群。综合多种鉴定结果,36株被鉴定为Pco,Q34被鉴定为Pcb。菌株致病力测试结果显示,36株Pco菌株中仅Q47表现为低等的致病力,其余24株和11株分别表现为中等和高等致病力;Pcb菌株Q34则表现出中等致病力。
Thirty-seven bacterial strains of soft rot isolated from soft rot of celery in Beijing area were biologically biochemically characterized, biochemically characterized, characterized by PCR, ITS-RFLP bands, and sequenced based on the complete 16S rDNA sequence Relationship analysis. The results are as follows: Strain Gram-negative with periplasmic flagella; grows in 5% and 7% NaCl at 28 ° C and 37 ° C; decomposes citrate and liquefied gelatin; Biolog analysis identifies these strains as Pectobacterium carotovorum subsp. Carotovorum (Pcc). The results of biochemical analysis showed that 36 strains could utilize D-sorbitol, D-arabitol, isomaltulose and α-methylglucoside, which were consistent with the control strain P. carotovorum subsp. Odoriferum (Pco) S7. One strain Q34 was unable to utilize the above sugar alcohol and was consistent with the control strains P.carotovorum subsp. Brasilience (Pcb) BC1 and P.carotovorum subsp. Carotovorum (Pcc) ECC71. The predicted fragment (434 bp) was amplified from all 37 strains using the pel (pectate lyase) gene-specific primer Y1 / Y2, indicating that the genome contains pectinase genes. The Pcb-specific PCR primer BR1f / L1r only amplified the expected fragment (322 bp) in Q34 strain and Pcb BC1, whereas the Pcc-specific PCR primer EXPCCF / EXPCCR amplified the expected fragment in all strains except Pcb BC1 (550 bp). The results of Rsa I digestion of 16S-23S rDNA ITS fragment showed that the Q34 digestion pattern was the same as that of Pcb BC1 and the remaining 36 bands were the same as Pcc ECC71 and Pco S7. Based on the complete sequence of 16S rDNA gene and Dickeya dadantii strain 582 as the outer group, the phylogenetic tree cluster analysis of the 37 strains with the published Pectobacterium strains showed that Q34 formed obvious Pcb groups with other published Pcb strains, The remaining 36 strains formed obvious Pco groups with other published Pco strains. Based on a variety of identification results, 36 strains were identified as Pco, and Q34 was identified as Pcb. The pathogenicity test results of the strains showed that only Q47 showed low pathogenicity among the 36 Pco strains, and the remaining 24 and 11 strains showed medium and high pathogenicity respectively. The Pcb strain Q34 showed moderate pathogenicity .