广藿香青枯病菌rep-PCR基因指纹图谱分析
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摘要
目的对广藿香青枯病病原菌进行鉴定,并利用rep-PCR基因指纹图谱技术,对广藿香青枯病菌进行遗传多样性分析,以了解青枯病菌的遗传结构及菌株间的遗传分化情况。方法利用青枯菌特异性引物OLI1/Y2对广藿香青枯菌疑似菌株进行PCR鉴定;利用rep-PCR技术,分别用3组引物(BOX A1R;ERIC 1R,ERIC2;REP 1R,REP 2-1)对广藿香青枯病菌进行PCR扩增及基因指纹图谱分析。结果共有12株广藿香青枯菌疑似菌株扩增出288 bp大小的目标条带;BOX-PCR、ERIC-PCR及REP-PCR指纹图谱分析结果表明,供试菌株在相似系数为0.5处,分别分成6,7,8个簇群;在相似系数为0.8处,均分为8个簇群,其中簇群Ⅰ均包括5个亲缘关系相近的菌株。结论 rep-PCR基因指纹图谱技术能有效地区分供试菌株间的遗传差异;广藿香青枯病菌具有较丰富的遗传多样性,部分菌株间遗传相似度较高,组成优势菌群。
Objective To identify Ralstonia solanacearum strains isolated from Pogostemon cablin Benth.,and then to study the genetic diversity of the Ralstonia solanacearum strains by repetitive sequence-based polymerase chain reaction(rep-PCR).Methods The identification of the Ralstonia solanacearum strains was conducted via PCR amplification,using specific primer OLI1/Y2 designed based on 16 S r RNA gene sequences of R.solanacearum strain.The genetic diversity among R.solanacearum strains was analyzed by Rep-PCR fingerprinting with three groups of primers which were BOX and A1 R,ERIC 1R and ERIC 2,and REP 1R and REP 2-1.Results The objective band with 288 bp was obtained from 12 strains which were identified as R.solanacearum.The results of BOX-PCR,ERIC-PCR and REP-PCR genetic fingerprinting showed that R.solanacearum strains were divided into 6,7,8 clusters at 0.5similarity level,and were similarly divided into 8 clusters at 0.8 similarity level,respectively.At 0.8 similarity level,clusterⅠwas predominant,including 5 strains which possessed high genetic similarity.Conclusion Rep-PCR is an effective technique in discriminating genetically related R.solanacearum strains isolated from Pogostemon cablin Benth..Significantly genetic diversity exists among the tested strains.Some dominant strains were also found in this study.
Objective To identify Ralstonia solanacearum strains isolated from Pogostemon cablin Benth.,and then to study the genetic diversity of the Ralstonia solanacearum strains by repetitive sequence-based polymerase chain reaction(rep-PCR).Methods The identification of the Ralstonia solanacearum strains was conducted via PCR amplification,using specific primer OLI1/Y2 designed based on 16 S r RNA gene sequences of R.solanacearum strain.The genetic diversity among R.solanacearum strains was analyzed by Rep-PCR fingerprinting with three groups of primers which were BOX and A1 R,ERIC 1R and ERIC 2,and REP 1R and REP 2-1.Results The objective band with 288 bp was obtained from 12 strains which were identified as R.solanacearum.The results of BOX-PCR,ERIC-PCR and REP-PCR genetic fingerprinting showed that R.solanacearum strains were divided into 6,7,8 clusters at 0.5similarity level,and were similarly divided into 8 clusters at 0.8 similarity level,respectively.At 0.8 similarity level,clusterⅠwas predominant,including 5 strains which possessed high genetic similarity.Conclusion Rep-PCR is an effective technique in discriminating genetically related R.solanacearum strains isolated from Pogostemon cablin Benth..Significantly genetic diversity exists among the tested strains.Some dominant strains were also found in this study.
引文
[1]文衍堂,郑小波.海南岛广藿香、沙姜青枯病病原菌的鉴定[J].热带作物学报,1984,5(2):113-119.
[2]Versalovic J,Koeuth T,Lupski JR.Distribution of repetitive DNA sequence in eubacteria and application to fingerprinting of bacterial genomes[J].Nucleic Acid Research,1991,19(24):6823-6831.
[3]Lee CM,Sieo CC,Cheah YK,et al.Discrimination of probiotic Lactobacillus strains for poultry by repetitive sequenced-based PCR fingerprinting[J].Journal of the Science of Food and Agriculture,2012,92(3):660-666.
[4]Ishii S,Michael JS.Applications of the rep-PCR DNA fingerprinting technique to study microbial diversity,ecology and evolution[J].Environmental Microbiology,2009,11(4):733-740.
[5]Di Giovanni GD,Watrud LS,Seidler RJ,et al.Fingerprinting of mixed bacterial strains and BIOLOG Gram-negative(GN)substrate communities by enterobacterial repetitive intergenic consensus sequence-PCR(ERIC-PCR)[J].Current Microbiology,1999,38(4):217-223.
[6]Pangallo D,Drahovska H,Harichova J,et al.Evaluation of different PCR-based approaches for the identification and typing of environmental enterococci[J].Antonie Van Leeuwenhoek,2008,93(1-2):193-203.
[7]Seal SE,Jackson LA,Young JPW,et al.Differentiation of Pseudomonas solanacearum,Pseudomonas syzygii,Pseudomonas pickettii and the blood disease bacterium by partial 16S r RNA sequencing:construction of oligonucleotide primers for sensitive detection by polymerase chain reaction[J].Journal of General Microbiology,1993,139(7):1587-1594.
[8]Louws FJ,Fulbright DW,Stephens CT,et al.Specific genomic fingerprints of Phytopathogenic Xanthomonas and Pseudomonas pathovars and strains generated with repetitive sequences and PCR[J].Applied and Environmental Microbiology,1994,60(7):2286-2295.
[9]刘丹,贺红,黄海波,等.广藿香青枯病菌的分离培养及致病性测定[J].中草药,2011,42(8):1596-1599.
[10]Allen C,Prior P,Hayward AC.Book Review:Bacterial wilt disease and the Ralstonia solanacearum species complex[J].European Journal of Plant Pathology,2006,114(2):227-228.
[11]杨玉秀,贺红,徐燃,等.广藿香青枯菌的PCR鉴定与寄主专化性研究[J].广州中医药大学学报,2013,30(4):566-603.
[2]Versalovic J,Koeuth T,Lupski JR.Distribution of repetitive DNA sequence in eubacteria and application to fingerprinting of bacterial genomes[J].Nucleic Acid Research,1991,19(24):6823-6831.
[3]Lee CM,Sieo CC,Cheah YK,et al.Discrimination of probiotic Lactobacillus strains for poultry by repetitive sequenced-based PCR fingerprinting[J].Journal of the Science of Food and Agriculture,2012,92(3):660-666.
[4]Ishii S,Michael JS.Applications of the rep-PCR DNA fingerprinting technique to study microbial diversity,ecology and evolution[J].Environmental Microbiology,2009,11(4):733-740.
[5]Di Giovanni GD,Watrud LS,Seidler RJ,et al.Fingerprinting of mixed bacterial strains and BIOLOG Gram-negative(GN)substrate communities by enterobacterial repetitive intergenic consensus sequence-PCR(ERIC-PCR)[J].Current Microbiology,1999,38(4):217-223.
[6]Pangallo D,Drahovska H,Harichova J,et al.Evaluation of different PCR-based approaches for the identification and typing of environmental enterococci[J].Antonie Van Leeuwenhoek,2008,93(1-2):193-203.
[7]Seal SE,Jackson LA,Young JPW,et al.Differentiation of Pseudomonas solanacearum,Pseudomonas syzygii,Pseudomonas pickettii and the blood disease bacterium by partial 16S r RNA sequencing:construction of oligonucleotide primers for sensitive detection by polymerase chain reaction[J].Journal of General Microbiology,1993,139(7):1587-1594.
[8]Louws FJ,Fulbright DW,Stephens CT,et al.Specific genomic fingerprints of Phytopathogenic Xanthomonas and Pseudomonas pathovars and strains generated with repetitive sequences and PCR[J].Applied and Environmental Microbiology,1994,60(7):2286-2295.
[9]刘丹,贺红,黄海波,等.广藿香青枯病菌的分离培养及致病性测定[J].中草药,2011,42(8):1596-1599.
[10]Allen C,Prior P,Hayward AC.Book Review:Bacterial wilt disease and the Ralstonia solanacearum species complex[J].European Journal of Plant Pathology,2006,114(2):227-228.
[11]杨玉秀,贺红,徐燃,等.广藿香青枯菌的PCR鉴定与寄主专化性研究[J].广州中医药大学学报,2013,30(4):566-603.