番茄溃疡病furA和katA基因突变体的构建
|
摘要
番茄溃疡病(Clavibacter michiganensis subsp. Michiganensis,简称Cmm)是番茄(Lycopersicon culentum Mill.)生产中最为严重、具有毁灭性的病害之一。该病自从1909年首次在美国密执安州的温室番茄上发现以来,现已广泛分布于美国各番茄产区,并逐渐成为世界性病害。目前番茄溃疡病在我国北京、黑龙江、吉林、内蒙古、新疆、河南、河北、山西、山东、上海、海南等省、市、自治区都有发生,使许多地区番茄生产受到了不同程度的影响。有研究表明,furA基因与katA基因可能与番茄溃疡病原菌(Cmm)致病性相关。为验证Cmm中furA基因与katA基因之间的相互关系,推测其致病机理,为抗病育种提供理论依据,我们构建了番茄溃疡病furA和katA基因的不同缺失突变体,通过电击转化的方法将其转入Cmm野生型菌株中,并分别进行了分子生物学检测和生理指标的检测。
以Cmm野生菌株的基因组DNA为模板,根据furA和katA基因序列的设计特异引物,进行PCR扩增反应。PCR扩增产物电泳分析后,回收目的条带,与pMD18-T载体连接,分别命名为Cmm 1号,2号,3号,4号片段和123-T。利用BamHⅠ位点,将经过序列测定正确的目的片段从pMD18-T载体上酶切回收。用T_4 DNA连接酶分别连接1号和2号片段,3号和4号片段。之后通过PCR扩增对不同缺失片段加KpnⅠ接头,回收目的条带,连pMD18-T或pEASY-T1载体;利用KpnⅠ位点,将测序正确的目的片段从测序载体上酶切回收,用T4 DNA连接酶连接分别获得1234,12’3,12’34;上述片段PCR扩增加EcoRⅠ接头,连pMD18-T或pEASY-T1载体经EcoRⅠ酶切;同时将pHN216表达载体线性化,回收的条带。将回收的EcoRⅠ酶切1234,12’3,12’34,123加EcoRⅠ接头片段和pHN216载体片段用T4 DNA连接酶连接,连接产物转化到大肠杆菌感受态细胞(DH5α)中,挑取阳性克隆进行鉴定。随后通过电击转化的方法将上述鉴定正确的转化子转入Cmm野生型菌株中。
结果发现,在培养基中Fe~(2+)浓度分别为0和0.5mM的情况下,Cmm野生菌株和各突变菌株,以及空载体对照的过氧化氢消耗量,显示出不同的差异。当培养基中Fe~(2+)浓度为0 mM时,各处理间差异不显著;当培养基中加入0.5mM Fe~(2+)时,各处理间差异显著:12'3-2(furA-katA-)和12'34-2(furA-katA+)是野生型(furA+katA+)的1.5倍;123-2(furA+katA-)是野生型(furA+katA+)的0.5倍;空载体对照与野生型相比,差异不显著。
Bacterial canker of tomato is one of the most serious and devastating diseases in tomato production.. It is now widely distributed in the tomato-producing areas of the United States and gradually become a global disease from it was first found in Michigan in 1909. Now, the bacterial canker of tomato has occurred in Beijing, Heilongjiang, Jilin, Inner Mongolia, Xinjiang, Henan, Hebei, Shanxi, Shandong, Shanghai, Hainan and other provinces in China,, and it affects the tomato production in varying degrees in most regions. Previous study showed that gene furA katA may be associated with the pathogenicity of Cmm. In order to study the relationship between gene furA and katA, speculate its pathogenesis and provide a theoretical basis for the resistance breeding,we constructed the different gene furA and katA deletion mutants of Cmm, and transferred them into Cmm wild-type strains by electroporation method, and carried out molecular biological and physiological testing.
Use Cmm genome of wild strains as the template DNA, according to furA and katA gene sequence specific primers, the PCR were performed. After electrophoresis, target PCR product was recovered, then linked with pMD18-T vector, named Cmm 1, 2, 3, 4 clips and 123-T. Digestion by BamHⅠ, the correct sequencing of the target fragments were gotten from the pMD18-T vector. And then 1 and 2 fragment,3 and 4 fragments were linked by T_4 DNA lingase. Followed by PCR to add KpnⅠto different fragments, target bands were recycled, then were linked with pMD18-T or pEASY-T1 vector. After digested by KpnⅠ, the target sequencing fragments were recovered from the sequencing vector,then linked with each other by T4 DNA ligase, acquired 1234,12 '3, 12'34;add the EcoRⅠsites to each fragment through PCR,linked with pMD18-T or pEASY-T1 vector,then digested by EcoRⅠ; while pHN216 vector was linearized by EcoRⅠ,Then the recovery of 1234,12 '3, 12'34, 123 fragment and pHN216 vector fragment were linked by T4 DNA lingase. Next,the linked product was transformed into Escherichia coli competent cell(DH5α), the positive clones were picked out and identified. Finally, with the method of electroporation,the correct recombinant were transformed into the wild-type strains of Cmm.
The results showed that,when the concentration of Fe~(2+) in the medium was 0 mM and 0.5mM, the consumption of hydrogen peroxide for the wild strain and the mutant strains of Cmm, and the control of vector, showed various differences. When the concentration of Fe~(2+) was 0mM, the various treatments had no significant differences; While the concentration of Fe~(2+) was 0.5mM, the treatments showed significant differences: 12'3-2 (furA-katA-) and 12'34-2 (furA-katA+) is 1.5 times of the wild type (furA+katA+); 123-2 (furA+katA-) is 0.5 times of the wild-type (furA+katA+); the vector control compared with the wild type, the difference was not significant.
以Cmm野生菌株的基因组DNA为模板,根据furA和katA基因序列的设计特异引物,进行PCR扩增反应。PCR扩增产物电泳分析后,回收目的条带,与pMD18-T载体连接,分别命名为Cmm 1号,2号,3号,4号片段和123-T。利用BamHⅠ位点,将经过序列测定正确的目的片段从pMD18-T载体上酶切回收。用T_4 DNA连接酶分别连接1号和2号片段,3号和4号片段。之后通过PCR扩增对不同缺失片段加KpnⅠ接头,回收目的条带,连pMD18-T或pEASY-T1载体;利用KpnⅠ位点,将测序正确的目的片段从测序载体上酶切回收,用T4 DNA连接酶连接分别获得1234,12’3,12’34;上述片段PCR扩增加EcoRⅠ接头,连pMD18-T或pEASY-T1载体经EcoRⅠ酶切;同时将pHN216表达载体线性化,回收的条带。将回收的EcoRⅠ酶切1234,12’3,12’34,123加EcoRⅠ接头片段和pHN216载体片段用T4 DNA连接酶连接,连接产物转化到大肠杆菌感受态细胞(DH5α)中,挑取阳性克隆进行鉴定。随后通过电击转化的方法将上述鉴定正确的转化子转入Cmm野生型菌株中。
结果发现,在培养基中Fe~(2+)浓度分别为0和0.5mM的情况下,Cmm野生菌株和各突变菌株,以及空载体对照的过氧化氢消耗量,显示出不同的差异。当培养基中Fe~(2+)浓度为0 mM时,各处理间差异不显著;当培养基中加入0.5mM Fe~(2+)时,各处理间差异显著:12'3-2(furA-katA-)和12'34-2(furA-katA+)是野生型(furA+katA+)的1.5倍;123-2(furA+katA-)是野生型(furA+katA+)的0.5倍;空载体对照与野生型相比,差异不显著。
Bacterial canker of tomato is one of the most serious and devastating diseases in tomato production.. It is now widely distributed in the tomato-producing areas of the United States and gradually become a global disease from it was first found in Michigan in 1909. Now, the bacterial canker of tomato has occurred in Beijing, Heilongjiang, Jilin, Inner Mongolia, Xinjiang, Henan, Hebei, Shanxi, Shandong, Shanghai, Hainan and other provinces in China,, and it affects the tomato production in varying degrees in most regions. Previous study showed that gene furA katA may be associated with the pathogenicity of Cmm. In order to study the relationship between gene furA and katA, speculate its pathogenesis and provide a theoretical basis for the resistance breeding,we constructed the different gene furA and katA deletion mutants of Cmm, and transferred them into Cmm wild-type strains by electroporation method, and carried out molecular biological and physiological testing.
Use Cmm genome of wild strains as the template DNA, according to furA and katA gene sequence specific primers, the PCR were performed. After electrophoresis, target PCR product was recovered, then linked with pMD18-T vector, named Cmm 1, 2, 3, 4 clips and 123-T. Digestion by BamHⅠ, the correct sequencing of the target fragments were gotten from the pMD18-T vector. And then 1 and 2 fragment,3 and 4 fragments were linked by T_4 DNA lingase. Followed by PCR to add KpnⅠto different fragments, target bands were recycled, then were linked with pMD18-T or pEASY-T1 vector. After digested by KpnⅠ, the target sequencing fragments were recovered from the sequencing vector,then linked with each other by T4 DNA ligase, acquired 1234,12 '3, 12'34;add the EcoRⅠsites to each fragment through PCR,linked with pMD18-T or pEASY-T1 vector,then digested by EcoRⅠ; while pHN216 vector was linearized by EcoRⅠ,Then the recovery of 1234,12 '3, 12'34, 123 fragment and pHN216 vector fragment were linked by T4 DNA lingase. Next,the linked product was transformed into Escherichia coli competent cell(DH5α), the positive clones were picked out and identified. Finally, with the method of electroporation,the correct recombinant were transformed into the wild-type strains of Cmm.
The results showed that,when the concentration of Fe~(2+) in the medium was 0 mM and 0.5mM, the consumption of hydrogen peroxide for the wild strain and the mutant strains of Cmm, and the control of vector, showed various differences. When the concentration of Fe~(2+) was 0mM, the various treatments had no significant differences; While the concentration of Fe~(2+) was 0.5mM, the treatments showed significant differences: 12'3-2 (furA-katA-) and 12'34-2 (furA-katA+) is 1.5 times of the wild type (furA+katA+); 123-2 (furA+katA-) is 0.5 times of the wild-type (furA+katA+); the vector control compared with the wild type, the difference was not significant.
引文
[1] Gleason M L.Gitatis Ron D.Ricker Mark D.Recent progress in understanding and controlling bacterial canker of tomato in eastern north America[J].Plant Disease,1993,77:1069-1076.
[2] Francis D M,Kabelka E.Resistance to bacterial canker in tomato and its progeny derived from crossed to L.esculentum[J].Plant Disease,2001,85:1171-1176.
[3]胡俊,塔娜,胡宁宝,康志强,张国贤.内蒙古西部地区番茄溃疡病发生特点及防治对策[J].内蒙古农业科技,1998, (增刊):147-148.
[4]赵廷昌,王克,白金恺,金兆兰,谢森全.东北地区番茄细菌性溃疡病的发生和病原菌鉴定研究[J].植物病理学报,1993,23(1):29-34.
[5] Mekina- Mora C M,HausBeck M K,Fulbright D W.Birds eye lesions of tomato fruit produced by aerosol and direct application of Clavibacter michiganensis subsp. michiganensis[J].Plant Disease 2001,85:88-91.
[6]罗来鑫,赵廷昌.番茄细菌性溃疡病研究进展[J].中国农业科学,2004,37(8):1144-1150.
[7]韦成礼.警惕番茄溃疡病传入广西[J].广西植保,1998,(3):33-35.
[8] Fatmi M,Schaad N W.Survival of Clavibacter michiganensis subsp.michigaanensis in infected tomato stems under natural field conditions in California,ohio and Morocco[J].Plant Pathology,2002,51:149-154.
[9] Dhanvantari B N.Comparison of selective media for isolation of Clavibacter michiganensis subsp.michigaanensis. Phytopathology,1987,77:1694.
[10] Chang R J,Ries S M,Pataky J K.Dissemination of Clavibacter michiganensis subsp. michiganensis by practices used to produce tomato transplants[J].Phytopathology,1991,81:1276- 1281.
[11] Werner N A,Fulbright D W,Podolsky R.Limiting populations and spread of Clavubacter michiganensis subsp.michiganensis on seedling tomatoes in the greenhouse[J].Plant Disease,2002,86:535-542.
[12] Jens Dreier,Andreas Bermpohl,Rudolf Eichenlaub.Southern Hybridization and PCR for specific detection of phytopathogenic Clavibacter michiganensis subsp. michiganensis[J]. Phytopatohology,1995,85:462-468.
[13] Mirza M S,Rademaker J L W,Janse J D.Specific 16s ribosomal targeted oligonucleotide probeagainst Clavibacter michiganensis subsp. michiganensis[J].Canada Journal of Microbiology,1993, 39:1029-1034.
[14] Saettler A W,Schaad N W,Roth D A.Detaction of Bacteria in Seed and Other Planting Material[J].American Phytopathological Society,1989:45-49.
[15] Gitatis R D,Beaver R W.Characteriaztion of fatty acid methyiester content of Clavibacter michiganens subsp.michiganensis[J].Phytopathology,1990,80:318-321.
[16] Sasser J M,Fieldhouse D J,Carter C N.Computer assisted identification of bacteria based on fatty acid analysis[J].Phytopathology,1984,74:882.
[17] Louws F J,Bell J,Medina-Mora C M,Smart C D.Rep-PCR-Mediated genomic fingerprienting:A rapid and effective method to identify Clavibacter michiganens subsp.michiganensis[J].Phytopathology,1998,88:862-868.
[18] Bell J,Medina C,Louwes F.Varulence among field isolation of Clavibacter michiganensis subsp.michiganensis[J].Proceeding of the 11th annaul,Tomato Disease Workshop,1995:9-39..
[19] M K Hausbeck.J Bell,C M Medina-Mora,R Podolsky,D W Fulbright.Effect of bacteicides on population sizes spread of Clavibacter michiganensis subsp.Michiganensis on tomatoes in the greenhouse and on disease development and crop yield in the field [J] .Phytopathology,2000,90: 38-44.
[20]李春,金潜.新疆发生番茄溃疡病[J].植物检疫,1994,8(6):345.
[21] Kabelka E,Franchino B,Francis D M.Two loci from lycopersicon hirsutum LA407 confer resistance to strains of Clavibacter michiganensis subsp.michiganensis[J].phytopatohology,2002, 92:504-510.
[22] Francis D M,Kabelka E.Resistance to bacterial canker in tomato (lycoper siconhirsutum LA407)and its progeny derived from crossed to L.esculeentum[J].Plant Disease,2001,85:1171-1175.
[23] Gleason M L,Braun E J,Cariton W M,Peterson R H.Survival and dissemination of Clavibacter michiganensis subsp.michigaanensis in tomatoes[J].Phytopathology,1991,81(1): 1519-1523.
[24]魏亚东.番茄溃疡病[J].天津农林科技,1996,(4):42-43.
[25] Daly,J.M.,Ladlen,P.,Seevers,P.Physiology Plant Pathology[M].1971,1:397-401.[26]耿福利.番茄溃疡病的发生与综合防治[J].蔬菜,1999,(12):25.
[27] Chang R J,Ries S M,Pataky J K.Dissemination of Clavibacter michiganensis subsp.Michiganensis by practices used to produce tomato transplants[J]. Phytopathology, 1991,81:1276-1281.
[28] Marko J.Laine,Hassan Nakhei,Jens Dreier,Katja Lehtila,Dietmar Meletzus,Rudolf Eichenlaub,Maryc Metzler.Stable Transformation of the Gram-Positive Phytopathogenic Bacterium Clavibacter michiganensis subsp. Sepedonicus with Several Cloning Vectors[J].Applied and environmental microbiology,1996,5(62):1500-1506.
[29] Antoniou P P,Tjamos E C,Panagopoulos C G.Use of soil solarization for controlling bacterial canker of tomato in plastic houses in Greece [J].Plant Pathology,1995,44:438-447.
[30]刘泮华等.番茄溃疡病的研究[J].植物检疫,1989,3,(1):31-33.
[31]李春等.新疆番茄溃疡病的发生及其病原菌鉴定[J].新疆农业科学,1995,5:221-223.
[32]李春,金潜.密执安棒形杆菌密执安亚种菌株分化的研究[J].石河子农学院学报,1996,4(36): 21-24.
[33]罗来鑫,李健强,Hasan Bolkan.番茄细菌性溃疡病苗期接种新方法的研究[J].植物病理学报,2005,35(2):123-128.
[34]付鹏等.番茄溃疡病菌分子检测技术[J].江苏农业学报,2005,21(2):118-122.
[35]吴兴海等.应用分子生物学方法快速检测番茄溃疡病菌的研究[J].江西农业学报,2006, 18(2):5-7.
[36]王欢,刘箐.番茄溃疡病一步法快速检测技术研究[J].甘肃农业科技,2006,5:12-15.
[37]夏明星等.番茄细菌性溃疡病菌的实时荧光PCR检测[J].植物病理学报,2006,36(2):152- 157.
[38]许志刚,沈秀萍,胡白石.番茄上两种细菌性病害的诊断与防治[J].植物检疫,2007,1(21): 53-56.
[39] Gitatis R D,Beaver R W.Characteriaztion of fatty acid methyiester content of Clavibacter michiganens subsp.michiganensis[J]. Phytopathology,1990,80:318-321.
[40] Jense Dreier,Andreas Bermpohl,Rudolf Eichenlaub.Southem hybridization and PCR forspecifc detection of Phytopathologenic Clavibacter michiganensis subsp.michiganens[J]. Phytopathology,1995,85:462-468.
[41] Mirza M S,Rademaker J L W,Janse J D.Specific 16s ribosomal targetsed oligonleotide probe against Clavibacter michiganensis subsp.sepedonicus[J].Canada Journal of Microbiology,1993, 39:1029-1034.
[42] C.Alaron,J.Castro,F.munoz.Proteins from Gram-positive bacterium Clavibacter michiganensis subsp.michiganensis induces hypersensitive reponse in plants[J].Phytopathology,1998,88:306-310.
[43] Louws F J,Bell J,Medina-Mora C M,Smart C D.Rep-PCR-Mediated genomic fingerprienting:A rapid and effective method to identify Clavibacter michiganens subsp.michiganensis[J].Phytopathology,1998,88:862-868.
[44] Bell J,Medina C,Louwes F.Varulence among field isolation of Clavibacter michiganensis subsp.michiganensis[J].Proceeding of the 11th annaul,Tomato Disease Workshop,1995:9-39.
[45] Holger Jahr,Jens Dreier,Dietmar Meletzus.The endo-β-1,4-glucanase celA of Clavibacter michiganensis subsp.michiganensis is a pathogenicity determinant required for induction of bacterial wilt of tomato[J].The American Phytopathological Society,2000,13:703-714.
[46] Holtsmark,D.Mantzilas,V G H.Eilsink and M.B.Brurberg.The tomato pathogen Clavibacter michiganensis subsp.michiganensis:produrcer of several antimicrobial substances[J].Journal of Applied Microbiology,2006:1-8.
[47] D.Burokiene.Early detection of Clavibacter michiganensis subsp.michiganensis in tomato seeding[J].Agronomy Research,2006,4:l5l-l54.
[48] R.J.Chang,S.M.Ries,Effect of temperature,plant age,inoculum concentration,and cultivar on the incubation period and severity of bacterial canker of tomato[J].Plant Disease,1992,76:1150- 1155.
[49] Yang W C and Francis D M. Genetics and Breeeding for resistance to bacterial diseases in tomato: prospects for marker-assisted selection[J].Genetic Improvement of Solanaceous Crops,2007,2:379-420.
[50] Karl-Heinz Gartemann,Birte Abt,Thomas Bekel.The genome sequence of the tomato-pathogenic actinomycete Clavibacter michiganensis subsp. Michiganensis NCPPB382 reveals a large island involved in pathogenicity[J].Journal of Bacteriology,2008,190(6):2138–2149.
[51] Francesca Mariani,Giulia Cappelli,Giovanna Riccardi,Vittorio Colizzi.Mycobacterium tuberculosis H37Rv comparative gene-expression analysis in synthetic medium and human macrophage[J]. Gene,2000, 253:281–291.
[52] Junko Amemura-Maekawa,Sakurako Mishima-Abe,Fumiaki Kura,Tomoko Takahashi,Haruo Watanabe.Identication of a novel periplasmic catalase-peroxidase KatA of Legionella pneumophila[J].FEMS Microbiology Letters 1999,176:339-344.
[53] Claudia Sala,Francesca Forti,Elisabetta Di Florio,Fabio Canneva,Anna Milano,Giovanna Riccardi,and Daniela Ghisotti1.Mycobacterium Tuberculosis FurA Autoregulates Its Own Expression[J].Journal of bacteriology,2003,9:5357-5362.
[54] V. Deretic, W. Philipp, S. Dhandayuthapani.Mycobacterium tuberculosis is a natural mutant with an inactivated oxidative-stress regulatory gene:implications for sensitivity to isoniazid[J]. Molecular Microbiology,1995,17(5):889-900.
[55] Thomas C. Zahrt, Jian Song,Jessica Siple and Vojo Deretic.Mycobacterial FurA is a negative regulator of catalase-peroxidase gene katG[J]. Molecular Microbiology,200,39(5):1174-1185.
[56] Ludmil Benov, Fatima Sequeira.Escherichia coli ?fur mutant displays low HPII catalase activity in stationary phase[J]. Redox Report,2003,8(6):379-383.
[57] Tarrik Zaid, Trivandrum Sukumaran Nair Srikumar and Ludmil Benov.Growth of Escherichia coli in Iron-enriched Medium Increases HPI Catalase Activity[J].Journal of Biochemistry and Molecular Biology, 2003, 36(6):608-610.
[58] Touati, D. Iron and oxidative stress in bacteria[J]. Arch.Biochem. Biophys.2000,373: 1-6.
[59] Keyer, K. and Imlay, J. A. Superoxide accelerates DNAdamage by elevating free-iron levels[J]. Proc. Natl. Acad. Sci.USA1996,93:13635-13640.
[2] Francis D M,Kabelka E.Resistance to bacterial canker in tomato and its progeny derived from crossed to L.esculentum[J].Plant Disease,2001,85:1171-1176.
[3]胡俊,塔娜,胡宁宝,康志强,张国贤.内蒙古西部地区番茄溃疡病发生特点及防治对策[J].内蒙古农业科技,1998, (增刊):147-148.
[4]赵廷昌,王克,白金恺,金兆兰,谢森全.东北地区番茄细菌性溃疡病的发生和病原菌鉴定研究[J].植物病理学报,1993,23(1):29-34.
[5] Mekina- Mora C M,HausBeck M K,Fulbright D W.Birds eye lesions of tomato fruit produced by aerosol and direct application of Clavibacter michiganensis subsp. michiganensis[J].Plant Disease 2001,85:88-91.
[6]罗来鑫,赵廷昌.番茄细菌性溃疡病研究进展[J].中国农业科学,2004,37(8):1144-1150.
[7]韦成礼.警惕番茄溃疡病传入广西[J].广西植保,1998,(3):33-35.
[8] Fatmi M,Schaad N W.Survival of Clavibacter michiganensis subsp.michigaanensis in infected tomato stems under natural field conditions in California,ohio and Morocco[J].Plant Pathology,2002,51:149-154.
[9] Dhanvantari B N.Comparison of selective media for isolation of Clavibacter michiganensis subsp.michigaanensis. Phytopathology,1987,77:1694.
[10] Chang R J,Ries S M,Pataky J K.Dissemination of Clavibacter michiganensis subsp. michiganensis by practices used to produce tomato transplants[J].Phytopathology,1991,81:1276- 1281.
[11] Werner N A,Fulbright D W,Podolsky R.Limiting populations and spread of Clavubacter michiganensis subsp.michiganensis on seedling tomatoes in the greenhouse[J].Plant Disease,2002,86:535-542.
[12] Jens Dreier,Andreas Bermpohl,Rudolf Eichenlaub.Southern Hybridization and PCR for specific detection of phytopathogenic Clavibacter michiganensis subsp. michiganensis[J]. Phytopatohology,1995,85:462-468.
[13] Mirza M S,Rademaker J L W,Janse J D.Specific 16s ribosomal targeted oligonucleotide probeagainst Clavibacter michiganensis subsp. michiganensis[J].Canada Journal of Microbiology,1993, 39:1029-1034.
[14] Saettler A W,Schaad N W,Roth D A.Detaction of Bacteria in Seed and Other Planting Material[J].American Phytopathological Society,1989:45-49.
[15] Gitatis R D,Beaver R W.Characteriaztion of fatty acid methyiester content of Clavibacter michiganens subsp.michiganensis[J].Phytopathology,1990,80:318-321.
[16] Sasser J M,Fieldhouse D J,Carter C N.Computer assisted identification of bacteria based on fatty acid analysis[J].Phytopathology,1984,74:882.
[17] Louws F J,Bell J,Medina-Mora C M,Smart C D.Rep-PCR-Mediated genomic fingerprienting:A rapid and effective method to identify Clavibacter michiganens subsp.michiganensis[J].Phytopathology,1998,88:862-868.
[18] Bell J,Medina C,Louwes F.Varulence among field isolation of Clavibacter michiganensis subsp.michiganensis[J].Proceeding of the 11th annaul,Tomato Disease Workshop,1995:9-39..
[19] M K Hausbeck.J Bell,C M Medina-Mora,R Podolsky,D W Fulbright.Effect of bacteicides on population sizes spread of Clavibacter michiganensis subsp.Michiganensis on tomatoes in the greenhouse and on disease development and crop yield in the field [J] .Phytopathology,2000,90: 38-44.
[20]李春,金潜.新疆发生番茄溃疡病[J].植物检疫,1994,8(6):345.
[21] Kabelka E,Franchino B,Francis D M.Two loci from lycopersicon hirsutum LA407 confer resistance to strains of Clavibacter michiganensis subsp.michiganensis[J].phytopatohology,2002, 92:504-510.
[22] Francis D M,Kabelka E.Resistance to bacterial canker in tomato (lycoper siconhirsutum LA407)and its progeny derived from crossed to L.esculeentum[J].Plant Disease,2001,85:1171-1175.
[23] Gleason M L,Braun E J,Cariton W M,Peterson R H.Survival and dissemination of Clavibacter michiganensis subsp.michigaanensis in tomatoes[J].Phytopathology,1991,81(1): 1519-1523.
[24]魏亚东.番茄溃疡病[J].天津农林科技,1996,(4):42-43.
[25] Daly,J.M.,Ladlen,P.,Seevers,P.Physiology Plant Pathology[M].1971,1:397-401.[26]耿福利.番茄溃疡病的发生与综合防治[J].蔬菜,1999,(12):25.
[27] Chang R J,Ries S M,Pataky J K.Dissemination of Clavibacter michiganensis subsp.Michiganensis by practices used to produce tomato transplants[J]. Phytopathology, 1991,81:1276-1281.
[28] Marko J.Laine,Hassan Nakhei,Jens Dreier,Katja Lehtila,Dietmar Meletzus,Rudolf Eichenlaub,Maryc Metzler.Stable Transformation of the Gram-Positive Phytopathogenic Bacterium Clavibacter michiganensis subsp. Sepedonicus with Several Cloning Vectors[J].Applied and environmental microbiology,1996,5(62):1500-1506.
[29] Antoniou P P,Tjamos E C,Panagopoulos C G.Use of soil solarization for controlling bacterial canker of tomato in plastic houses in Greece [J].Plant Pathology,1995,44:438-447.
[30]刘泮华等.番茄溃疡病的研究[J].植物检疫,1989,3,(1):31-33.
[31]李春等.新疆番茄溃疡病的发生及其病原菌鉴定[J].新疆农业科学,1995,5:221-223.
[32]李春,金潜.密执安棒形杆菌密执安亚种菌株分化的研究[J].石河子农学院学报,1996,4(36): 21-24.
[33]罗来鑫,李健强,Hasan Bolkan.番茄细菌性溃疡病苗期接种新方法的研究[J].植物病理学报,2005,35(2):123-128.
[34]付鹏等.番茄溃疡病菌分子检测技术[J].江苏农业学报,2005,21(2):118-122.
[35]吴兴海等.应用分子生物学方法快速检测番茄溃疡病菌的研究[J].江西农业学报,2006, 18(2):5-7.
[36]王欢,刘箐.番茄溃疡病一步法快速检测技术研究[J].甘肃农业科技,2006,5:12-15.
[37]夏明星等.番茄细菌性溃疡病菌的实时荧光PCR检测[J].植物病理学报,2006,36(2):152- 157.
[38]许志刚,沈秀萍,胡白石.番茄上两种细菌性病害的诊断与防治[J].植物检疫,2007,1(21): 53-56.
[39] Gitatis R D,Beaver R W.Characteriaztion of fatty acid methyiester content of Clavibacter michiganens subsp.michiganensis[J]. Phytopathology,1990,80:318-321.
[40] Jense Dreier,Andreas Bermpohl,Rudolf Eichenlaub.Southem hybridization and PCR forspecifc detection of Phytopathologenic Clavibacter michiganensis subsp.michiganens[J]. Phytopathology,1995,85:462-468.
[41] Mirza M S,Rademaker J L W,Janse J D.Specific 16s ribosomal targetsed oligonleotide probe against Clavibacter michiganensis subsp.sepedonicus[J].Canada Journal of Microbiology,1993, 39:1029-1034.
[42] C.Alaron,J.Castro,F.munoz.Proteins from Gram-positive bacterium Clavibacter michiganensis subsp.michiganensis induces hypersensitive reponse in plants[J].Phytopathology,1998,88:306-310.
[43] Louws F J,Bell J,Medina-Mora C M,Smart C D.Rep-PCR-Mediated genomic fingerprienting:A rapid and effective method to identify Clavibacter michiganens subsp.michiganensis[J].Phytopathology,1998,88:862-868.
[44] Bell J,Medina C,Louwes F.Varulence among field isolation of Clavibacter michiganensis subsp.michiganensis[J].Proceeding of the 11th annaul,Tomato Disease Workshop,1995:9-39.
[45] Holger Jahr,Jens Dreier,Dietmar Meletzus.The endo-β-1,4-glucanase celA of Clavibacter michiganensis subsp.michiganensis is a pathogenicity determinant required for induction of bacterial wilt of tomato[J].The American Phytopathological Society,2000,13:703-714.
[46] Holtsmark,D.Mantzilas,V G H.Eilsink and M.B.Brurberg.The tomato pathogen Clavibacter michiganensis subsp.michiganensis:produrcer of several antimicrobial substances[J].Journal of Applied Microbiology,2006:1-8.
[47] D.Burokiene.Early detection of Clavibacter michiganensis subsp.michiganensis in tomato seeding[J].Agronomy Research,2006,4:l5l-l54.
[48] R.J.Chang,S.M.Ries,Effect of temperature,plant age,inoculum concentration,and cultivar on the incubation period and severity of bacterial canker of tomato[J].Plant Disease,1992,76:1150- 1155.
[49] Yang W C and Francis D M. Genetics and Breeeding for resistance to bacterial diseases in tomato: prospects for marker-assisted selection[J].Genetic Improvement of Solanaceous Crops,2007,2:379-420.
[50] Karl-Heinz Gartemann,Birte Abt,Thomas Bekel.The genome sequence of the tomato-pathogenic actinomycete Clavibacter michiganensis subsp. Michiganensis NCPPB382 reveals a large island involved in pathogenicity[J].Journal of Bacteriology,2008,190(6):2138–2149.
[51] Francesca Mariani,Giulia Cappelli,Giovanna Riccardi,Vittorio Colizzi.Mycobacterium tuberculosis H37Rv comparative gene-expression analysis in synthetic medium and human macrophage[J]. Gene,2000, 253:281–291.
[52] Junko Amemura-Maekawa,Sakurako Mishima-Abe,Fumiaki Kura,Tomoko Takahashi,Haruo Watanabe.Identication of a novel periplasmic catalase-peroxidase KatA of Legionella pneumophila[J].FEMS Microbiology Letters 1999,176:339-344.
[53] Claudia Sala,Francesca Forti,Elisabetta Di Florio,Fabio Canneva,Anna Milano,Giovanna Riccardi,and Daniela Ghisotti1.Mycobacterium Tuberculosis FurA Autoregulates Its Own Expression[J].Journal of bacteriology,2003,9:5357-5362.
[54] V. Deretic, W. Philipp, S. Dhandayuthapani.Mycobacterium tuberculosis is a natural mutant with an inactivated oxidative-stress regulatory gene:implications for sensitivity to isoniazid[J]. Molecular Microbiology,1995,17(5):889-900.
[55] Thomas C. Zahrt, Jian Song,Jessica Siple and Vojo Deretic.Mycobacterial FurA is a negative regulator of catalase-peroxidase gene katG[J]. Molecular Microbiology,200,39(5):1174-1185.
[56] Ludmil Benov, Fatima Sequeira.Escherichia coli ?fur mutant displays low HPII catalase activity in stationary phase[J]. Redox Report,2003,8(6):379-383.
[57] Tarrik Zaid, Trivandrum Sukumaran Nair Srikumar and Ludmil Benov.Growth of Escherichia coli in Iron-enriched Medium Increases HPI Catalase Activity[J].Journal of Biochemistry and Molecular Biology, 2003, 36(6):608-610.
[58] Touati, D. Iron and oxidative stress in bacteria[J]. Arch.Biochem. Biophys.2000,373: 1-6.
[59] Keyer, K. and Imlay, J. A. Superoxide accelerates DNAdamage by elevating free-iron levels[J]. Proc. Natl. Acad. Sci.USA1996,93:13635-13640.