M60家族肽酶参与调控蜡质芽胞杆菌AR156防治南方根结线虫病的作用机制
|
摘要
【背景】南方根结线虫(Meloidogyne incognita)寄主范围广泛,能侵染茄科、豆科、葫芦科等蔬菜,对世界农业生产造成了严重危害,每年造成的损失可高达数百亿美元。蜡质芽胞杆菌(Bacillus cereus) AR156是一种革兰氏阳性杆状细菌,对于南方根结线虫引起的土传病害有较好的防治效果,并且已经完成了全基因组测序。【目的】了解AR156对南方根结线虫的生防机理,寻找与生防功能相关的基因,进行遗传功能基因的分析。【方法】构建蜡质芽胞杆菌AR156 miniTn10随机插入突变体文库,通过对南方根结线虫致死率和温室防病试验进行筛选,找到与AR156生防能力相关的突变体,鉴定相关功能基因,并对产蛋白酶活性、定殖能力、生物膜形成和游动性等方面进行检测。【结果】与AR156野生型相比,转座子插入位点在M60家族肽酶的突变体BC41产蛋白酶活性下降,植物根围定殖能力减弱,生物膜形成能力和游动性受到影响,从而导致其防治南方根结线虫的能力降低。【结论】通过对生防相关功能基因的分析,初步明确M60家族肽酶在蜡质芽胞杆菌AR156防治南方根结线虫的过程中发挥重要作用。
[Background] Meloidogyne incognita has a wide range of hosts to infect including Solanaceae, Leguminosae, Cucurbitaceae and other vegetables. It causes serious damage and tens of billions of losses to the global agricultural production every year. Bacillus cereus AR156 is a gram-positive and rod-shaped bacterium that can be used as a biocontrol agent to control M. incognita, and has complete genome-wide sequencing. [Objective] In order to understand its biocontrol mechanism against M. incognita and to search for the related gene of biocontrol function, we analyzed the functional gene of B. cereus AR156. [Methods] We screened mortality of M. incognita and biocontrol efficiency under greenhouse in the AR156 miniTn10 random insertion transposon library to find mutants associated with biocontrol ability in AR156. Gene involved in biocontrol in AR156 was identified. And we compared producing-protease ability, colonization, biofilm formation and swarming motility. [Results] Compared with the AR156 wild-type, BC41 had reduced production of protease, colonization of plant rhizosphere, formation of biofilm and swarming motility. Its insertion site is the M60 family peptidase, which decreased the ability to biocontrol M. incognita. [Conclusion] M60 family peptidase plays an important role in the biocontrol activity of B. cereus AR156 by the analysis of biocontrol-related functional gene.
[Background] Meloidogyne incognita has a wide range of hosts to infect including Solanaceae, Leguminosae, Cucurbitaceae and other vegetables. It causes serious damage and tens of billions of losses to the global agricultural production every year. Bacillus cereus AR156 is a gram-positive and rod-shaped bacterium that can be used as a biocontrol agent to control M. incognita, and has complete genome-wide sequencing. [Objective] In order to understand its biocontrol mechanism against M. incognita and to search for the related gene of biocontrol function, we analyzed the functional gene of B. cereus AR156. [Methods] We screened mortality of M. incognita and biocontrol efficiency under greenhouse in the AR156 miniTn10 random insertion transposon library to find mutants associated with biocontrol ability in AR156. Gene involved in biocontrol in AR156 was identified. And we compared producing-protease ability, colonization, biofilm formation and swarming motility. [Results] Compared with the AR156 wild-type, BC41 had reduced production of protease, colonization of plant rhizosphere, formation of biofilm and swarming motility. Its insertion site is the M60 family peptidase, which decreased the ability to biocontrol M. incognita. [Conclusion] M60 family peptidase plays an important role in the biocontrol activity of B. cereus AR156 by the analysis of biocontrol-related functional gene.
引文
[1]Yang XJ,Chen FR,Xie SY.Advances in occurrence and control of Citrus Bacterial Canker Diseases[J].China Fruits,2002(5):46-50(in Chinese)杨秀娟,陈福如,谢世勇.柑桔溃疡病发生与防治研究迚展[J].中国果树,2002(5):46-50
[2]Ding GC,Fu P,Li HM,et al.Biocontrol evaluation of Bacillus subtilus strain AR11 against Meloidogyne incognita[J].Journal of Nanjing Agricultural University,2005,28(2):46-49(in Chinese)丁国春,付鹏,李红梅,等.枯草芽孢杆菌AR11菌株对南方根结线虫的生物防治[J].南京农业大学学报,2005,28(2):46-49
[3]Duan YX,Qu ZL,Wang YY,et al.Screening the strains of Fusarium against Meloidogyne incognita[J].Agrochemicals,2010,49(8):607-609,620(in Chinese)段玉玺,曲泽岚,王媛媛,等.南方根结线虫生防镰刀菌菌株筛选[J].农药,2010,49(8):607-609,620
[4]Cai JX.Study on control technology of Meloidogyne incognita in tomato[D].Hangzhou:Master’s Thesis of Zhejiang University,2016(in Chinese)蔡加星.番茄南方根结线虫防控技术的研究[D].杭州:浙江大学硕士学位论文,2016
[5]Liu GK,Xiao S,Hong CF,et al.Parasitism of isolates of Fusarium spp.to Meloidogyne incognita eggs[J].Journal of Fujian Agriculture and Forestry University(Natural Science Edition),2006,35(5):459-462(in Chinese)刘国坤,肖顺,洪彩凤,等.镰刀菌对南方根结线虫卵的寄生特性[J].福建农林大学学报:自然科学版,2006,35(5):459-462
[6]Zhu XF,Duan YX,Chen LJ,et al.Study on the toxicity test and control effect of Aspergillus niger Snf009 fermentation liquid on Meloidogyne incognita[J].Journal of Henan Agricultural Sciences,2009,38(4):84-85(in Chinese)朱晓峰,段玉玺,陈立杰,等.黑曲霉Snf009发酵液对根结线虫的毒性测定及温室防效研究[J].河南农业科学,2009,38(4):84-85
[7]Liu JS,Li Y,Li YM,et al.Study on control techniques of cucumber root-knot nematode pesticides in greenhouse[J].Shaanxi Journal of Agricultural Sciences,2010,56(1):74-77(in Chinese)刘俊生,李媛,李英梅,等.温室黄瓜根结线虫病药剂防治技术研究[J].陕西农业科学,2010,56(1):74-77
[8]Becker JO,Zavaleta-Mejia E,Colbert SF,et al.Effects of rhizobacteria on root-knot nematodes and gall formation[J].Phytopathology,1998,78:1466-1469
[9]Xu ZG.General Plant Pathology[M].4th Edition.Beijing:Higher Education Press,2009:230(in Chinese)许志刚.普通植物病理学[M].4版.北京:高等教育出版社,2009:230
[10]Yan F.Characterization of novel genes involved in biocontrol and multicellularity in Bacillus[D].Nanjing:Doctoral Dissertation of Nanjing Agricultural University,2015(in Chinese)严芳.蜡质芽胞杆菌AR156生防功能基因和调控生物膜形成功能基因研究[D].南京:南京农业大学博士学位论文,2015
[11]Yang JH,Liu HX,Zhu GM,et al.Diversity analysis of antagonists from rice-associated bacteria and their application in biocontrol of rice diseases[J].Journal of Applied Microbiology,2008,104(1):91-104
[12]Kearns DB,Chu F,Rudner R,et al.Genes governing swarming in Bacillus subtilis and evidence for a phase variation mechanism controlling surface motility[J].Molecular Microbiology,2004,52(2):357-369
[13]Chen LJ,Chen Y,Zhang GD,et al.Nematicidal activity of extraction and fermentation filtrate of basidiomycetes collected in Liaoning province,China[J].Chinese Journal of Biological Control,2010,26(4):467-473(in Chinese)陈立杰,陈越,张国栋,等.辽宁省大型真菌浸提液及发酵液对线虫的毒力作用[J].中国生物防治,2010,26(4):467-473
[14]Bridge J,Page SLJ.Estimation of root-knot nematode infestation levels on roots using a rating chart[J].Tropical Pest Management,1980,26(3):296-298
[15]Kearns DB,Losick R.Swarming motility in undomesticated Bacillus subtilis[J].Molecular Microbiology,2003,49(3):581-590
[16]Griffin AS,West SA,Buckling A.Cooperation and competition in pathogenic bacteria[J].Nature,2004,430(7003):1024-1027
[17]Siddiqui IA,Haas D,Heeb S.Extracellular protease of Pseudomonas fluorescens CHA0,a biocontrol factor with activity against the root-knot nematode Meloidogyne incognita[J].Applied and Environmental Microbiology,2005,71(9):5646-5649
[18]Moyne AL,Shelby R,Cleveland TE,et al.Bacillomycin D:an iturin with antifungal activity against Aspergillus flavus[J].Journal of Applied Microbiology,2001,90(4):622-629
[19]Huang XW,Zhao NH,Zhang KQ.Extracellular enzymes serving as virulence factors in nematophagous fungi involved in infection of the host[J].Research in Microbiology,2004,155(10):811-816
[20]Huang Y,Xu CK,Ma L,et al.Characterisation of volatiles produced from Bacillus megaterium YFM3.25 and their nematicidal activity against Meloidogyne incognita[J].European Journal of Plant Pathology,2010,126(3):417-422
[21]Niu QH,Huang XW,Zhang L,et al.A neutral protease from Bacillus nematocida,another potential virulence factor in the infection against nematodes[J].Archives of Microbiology,2006,185(6):439-448
[22]Lian LH,Tian BY,Xiong R,et al.Proteases from Bacillus:a new insight into the mechanism of action for rhizobacterial suppression of nematode populations[J].Letters in Applied Microbiology,2007,45(3):262-269
[23]Wei LH,Xue QY,Wei BQ,et al.Screening of antagonistic bacterial strains against Meloidogyne incognita using protease activity[J].Biocontrol Science and Technology,2010,20(7):739-750
[24]Wu JZ.Enzymatic hydrolysis of soybean protein and its antioxidant activity[D].Guangzhou:Doctoral Dissertation of South China University of Technology,2003(in Chinese)吴建中.大豆蛋白的酶法水解及产物抗氧化活性的研究[D].广州:华南理工大学博士学位论文,2003
[25]Wang P,Granados RR.An intestinal mucin is the target substrate for a baculovirus enhancin[J].Proceedings of the National Academy of Sciences of the United States of America,1997,94(13):6977-6982
[26]Nakjang S,Ndeh DA,Wipat A,et al.A novel extracellular metallopeptidase domain shared by animal host-associated mutualistic and pathogenic microbes[J].PLoS One,2012,7(1):e30287
[27]Barbara N,Maria V,Angela S,et al.SslE elicits functional antibodies that impair in vitro mucinase activity and in vivo colonization by both intestinal and extraintestinal Escherichia coli strains[J].PLoS Pathogens,2014,10(5):e1004124
[28]Costerton JW,Lewandowski Z,Caldwell DE,et al.Microbial biofilms[J].Annual Review of Microbiology,1995,49(1):711-745
[29]O’Toole GA,Gibbs KA,Hager PW,et al.The global carbon metabolism regulator Crc is a component of a signal transduction pathway required for biofilm development by Pseudomonas aeruginosa[J].Journal of Bacteriology,2000,182(2):425-431
[30]Hall-Stoodley L,Stoodley P.Evolving concepts in biofilm infections[J].Cellular Microbiology,2009,11(7):1034-1043
[31]Emmert EAB,Handelsman J.Biocontrol of plant disease:a(Gram-)positive perspective[J].FEMS Microbiology Letters,1999,171(1):1-9
[32]Chandramohan L,Ahn JS,Weaver KE,et al.An overlap between the control of programmed cell death in Bacillus anthracis and sporulation[J].Journal of Bacteriology,2009,191(13):4103-4110
[33]Shemesh M,Chai YR.A combination of glycerol and manganese promotes biofilm formation in Bacillus subtilis via histidine kinase KinD signaling[J].Journal of Bacteriology,2013,195(12):2747-2754
[2]Ding GC,Fu P,Li HM,et al.Biocontrol evaluation of Bacillus subtilus strain AR11 against Meloidogyne incognita[J].Journal of Nanjing Agricultural University,2005,28(2):46-49(in Chinese)丁国春,付鹏,李红梅,等.枯草芽孢杆菌AR11菌株对南方根结线虫的生物防治[J].南京农业大学学报,2005,28(2):46-49
[3]Duan YX,Qu ZL,Wang YY,et al.Screening the strains of Fusarium against Meloidogyne incognita[J].Agrochemicals,2010,49(8):607-609,620(in Chinese)段玉玺,曲泽岚,王媛媛,等.南方根结线虫生防镰刀菌菌株筛选[J].农药,2010,49(8):607-609,620
[4]Cai JX.Study on control technology of Meloidogyne incognita in tomato[D].Hangzhou:Master’s Thesis of Zhejiang University,2016(in Chinese)蔡加星.番茄南方根结线虫防控技术的研究[D].杭州:浙江大学硕士学位论文,2016
[5]Liu GK,Xiao S,Hong CF,et al.Parasitism of isolates of Fusarium spp.to Meloidogyne incognita eggs[J].Journal of Fujian Agriculture and Forestry University(Natural Science Edition),2006,35(5):459-462(in Chinese)刘国坤,肖顺,洪彩凤,等.镰刀菌对南方根结线虫卵的寄生特性[J].福建农林大学学报:自然科学版,2006,35(5):459-462
[6]Zhu XF,Duan YX,Chen LJ,et al.Study on the toxicity test and control effect of Aspergillus niger Snf009 fermentation liquid on Meloidogyne incognita[J].Journal of Henan Agricultural Sciences,2009,38(4):84-85(in Chinese)朱晓峰,段玉玺,陈立杰,等.黑曲霉Snf009发酵液对根结线虫的毒性测定及温室防效研究[J].河南农业科学,2009,38(4):84-85
[7]Liu JS,Li Y,Li YM,et al.Study on control techniques of cucumber root-knot nematode pesticides in greenhouse[J].Shaanxi Journal of Agricultural Sciences,2010,56(1):74-77(in Chinese)刘俊生,李媛,李英梅,等.温室黄瓜根结线虫病药剂防治技术研究[J].陕西农业科学,2010,56(1):74-77
[8]Becker JO,Zavaleta-Mejia E,Colbert SF,et al.Effects of rhizobacteria on root-knot nematodes and gall formation[J].Phytopathology,1998,78:1466-1469
[9]Xu ZG.General Plant Pathology[M].4th Edition.Beijing:Higher Education Press,2009:230(in Chinese)许志刚.普通植物病理学[M].4版.北京:高等教育出版社,2009:230
[10]Yan F.Characterization of novel genes involved in biocontrol and multicellularity in Bacillus[D].Nanjing:Doctoral Dissertation of Nanjing Agricultural University,2015(in Chinese)严芳.蜡质芽胞杆菌AR156生防功能基因和调控生物膜形成功能基因研究[D].南京:南京农业大学博士学位论文,2015
[11]Yang JH,Liu HX,Zhu GM,et al.Diversity analysis of antagonists from rice-associated bacteria and their application in biocontrol of rice diseases[J].Journal of Applied Microbiology,2008,104(1):91-104
[12]Kearns DB,Chu F,Rudner R,et al.Genes governing swarming in Bacillus subtilis and evidence for a phase variation mechanism controlling surface motility[J].Molecular Microbiology,2004,52(2):357-369
[13]Chen LJ,Chen Y,Zhang GD,et al.Nematicidal activity of extraction and fermentation filtrate of basidiomycetes collected in Liaoning province,China[J].Chinese Journal of Biological Control,2010,26(4):467-473(in Chinese)陈立杰,陈越,张国栋,等.辽宁省大型真菌浸提液及发酵液对线虫的毒力作用[J].中国生物防治,2010,26(4):467-473
[14]Bridge J,Page SLJ.Estimation of root-knot nematode infestation levels on roots using a rating chart[J].Tropical Pest Management,1980,26(3):296-298
[15]Kearns DB,Losick R.Swarming motility in undomesticated Bacillus subtilis[J].Molecular Microbiology,2003,49(3):581-590
[16]Griffin AS,West SA,Buckling A.Cooperation and competition in pathogenic bacteria[J].Nature,2004,430(7003):1024-1027
[17]Siddiqui IA,Haas D,Heeb S.Extracellular protease of Pseudomonas fluorescens CHA0,a biocontrol factor with activity against the root-knot nematode Meloidogyne incognita[J].Applied and Environmental Microbiology,2005,71(9):5646-5649
[18]Moyne AL,Shelby R,Cleveland TE,et al.Bacillomycin D:an iturin with antifungal activity against Aspergillus flavus[J].Journal of Applied Microbiology,2001,90(4):622-629
[19]Huang XW,Zhao NH,Zhang KQ.Extracellular enzymes serving as virulence factors in nematophagous fungi involved in infection of the host[J].Research in Microbiology,2004,155(10):811-816
[20]Huang Y,Xu CK,Ma L,et al.Characterisation of volatiles produced from Bacillus megaterium YFM3.25 and their nematicidal activity against Meloidogyne incognita[J].European Journal of Plant Pathology,2010,126(3):417-422
[21]Niu QH,Huang XW,Zhang L,et al.A neutral protease from Bacillus nematocida,another potential virulence factor in the infection against nematodes[J].Archives of Microbiology,2006,185(6):439-448
[22]Lian LH,Tian BY,Xiong R,et al.Proteases from Bacillus:a new insight into the mechanism of action for rhizobacterial suppression of nematode populations[J].Letters in Applied Microbiology,2007,45(3):262-269
[23]Wei LH,Xue QY,Wei BQ,et al.Screening of antagonistic bacterial strains against Meloidogyne incognita using protease activity[J].Biocontrol Science and Technology,2010,20(7):739-750
[24]Wu JZ.Enzymatic hydrolysis of soybean protein and its antioxidant activity[D].Guangzhou:Doctoral Dissertation of South China University of Technology,2003(in Chinese)吴建中.大豆蛋白的酶法水解及产物抗氧化活性的研究[D].广州:华南理工大学博士学位论文,2003
[25]Wang P,Granados RR.An intestinal mucin is the target substrate for a baculovirus enhancin[J].Proceedings of the National Academy of Sciences of the United States of America,1997,94(13):6977-6982
[26]Nakjang S,Ndeh DA,Wipat A,et al.A novel extracellular metallopeptidase domain shared by animal host-associated mutualistic and pathogenic microbes[J].PLoS One,2012,7(1):e30287
[27]Barbara N,Maria V,Angela S,et al.SslE elicits functional antibodies that impair in vitro mucinase activity and in vivo colonization by both intestinal and extraintestinal Escherichia coli strains[J].PLoS Pathogens,2014,10(5):e1004124
[28]Costerton JW,Lewandowski Z,Caldwell DE,et al.Microbial biofilms[J].Annual Review of Microbiology,1995,49(1):711-745
[29]O’Toole GA,Gibbs KA,Hager PW,et al.The global carbon metabolism regulator Crc is a component of a signal transduction pathway required for biofilm development by Pseudomonas aeruginosa[J].Journal of Bacteriology,2000,182(2):425-431
[30]Hall-Stoodley L,Stoodley P.Evolving concepts in biofilm infections[J].Cellular Microbiology,2009,11(7):1034-1043
[31]Emmert EAB,Handelsman J.Biocontrol of plant disease:a(Gram-)positive perspective[J].FEMS Microbiology Letters,1999,171(1):1-9
[32]Chandramohan L,Ahn JS,Weaver KE,et al.An overlap between the control of programmed cell death in Bacillus anthracis and sporulation[J].Journal of Bacteriology,2009,191(13):4103-4110
[33]Shemesh M,Chai YR.A combination of glycerol and manganese promotes biofilm formation in Bacillus subtilis via histidine kinase KinD signaling[J].Journal of Bacteriology,2013,195(12):2747-2754