单 位：
北京市农林科学院农业生物技术研究中心;北京市农林科学院北京农业生物技术研究中心农业基因资源与生物技术北京市重点实验室;北京农业生物技术研究中心
关键词：
芹菜(Apium graveolens L.);细菌性软腐病;Pectobacterium carotovorum subsp.odoriferum;Pectobacteriumcarotovorum subsp.brasilience;鉴定;致病力
摘 要：
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。
译 名：
Identification and pathogenicity analysis of soft rot bacteria on celery in Beijing
作 者：
TIAN Yu;MA Ya-li;HE Fu-xin;MA Rong-cai;XIE Hua;Beijing Agro-Biotechnology Research Center;Beijing Key Laboratory of Agricultural Genetic Resources and Biotechnology,Beijing Academy of Agriculture and Forestry Sciences;
关键词：
celery(Apium graveolens L.);;bacterial soft rot;;Pectobacterium carotovorum subsp. odoriferum;;Pectobacterium carotovorum subsp. brasilience;;identification;;pathogenicity
摘 要：
Carbon utilization analysis,biochemical characterization,subspecies-specific PCR analysis,ITSRFLP analysis,and phylogenetic analysis of the thirty-seven bacterial isolates from soft rot celeries in Beijing were conducted.We found that all the isolates were Gram-negative and capable of degrading citrate and liquefying gelatin,and growing at 28℃ and 37℃ and in 5% and 7% NaCl solution.Biolog analysis showed that they were Pectobacterium carotovorum subsp.carotovorum(Pec).Biochemical characterization analysis showed that 36 isolates from soft rotten celeries were capable of utilizing D-sorbitol,D-arabitol,palatinose and α-methylglucoside,which is in accordance with the control strain P.carotovorum subsp.odoriferum(Pco) S7.However,isolate Q34 cannot utilize these sugars,which is in accordance with the control strains P.carotovorum subsp.brasilienee(Pcb) BC1 and Pcc ECC71.PCR amplification with the pel specific primers(Y1/Y2) produced a434 bp product,indicating that each isolate had the pectate lyase-encoding gene.The expected 322 bp fragment was amplified only from isolate Q34 with Pcb specific primers Brlf/Llr.An expected 550-bp fragment was amplified with the Pcc specific primer set EXPCCF/EXPCCR from all the isolates except isolate Pcb BC1.After digestion of 16S-23 S rDNA intergenic transcribed spacer(ITS) with Rsa I,the band patterns of isolate Q34 and Pcb BCl were the same,and those of others 36 isolates,Pco S7 and Pcc ECC71 were the same.Phylogenetic analysis of the 16 S rDNA sequences showed that isolate Q34 was clustered in the same group with the control Pcb strains,and the other 36 isolates were clustered in the other group with the control Pco strains.Together,the isolate Q34 was identified as Pcb and the other 36 isolates were identified as Pco.Pathogenicity assay showed that among the 36 Pco isolates,only isolate Q47 exhibited low pathogenicity,and 24 and 11 Pco isolates showed medium and high pathogenicity,respectively,while Pcb isolate Q34 exhibited middle pathogenicity.
