豇豆叶片生防细菌的鉴定及其拮抗作用测定
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摘要
为明确由豇豆叶片分离出的20株生防内生菌的生理生化性质及其潜在生防价值,本研究通过形态学、平板对峙、PCR技术、生物信息学等方法对20株生防细菌进行鉴定研究,并以4株病原真菌为靶标病菌,测定20株生防菌对病原真菌的抑制效果。结果显示,20株内生细菌中有3株属于假单胞菌属(Pseudomonas spp.);12株为枯草芽孢杆菌(Bacillus subtilis);剩余5株为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。经过革兰氏染色得知2号、16号、19号三株假单胞菌株为革兰氏阴性菌株,其余为阳性菌;通过解淀粉测定,2、16、19三株菌株没有形成不变色透明圈,无解淀粉功能,其余中间形成明显的透明圈,有解淀粉功能;经过V-P测定,发现1号、2号、11号、15号、16号、18号共6株菌株呈阴性,其余呈阳性;在MR甲基红测定中,1号、6号、7号、10号、11号共5株菌株测定呈阳性,其余呈阴性;由氧化酶测定可知2号、16号、17号、19号4株菌株氧化酶测定为阳性,其余为阴性;由接触酶测定得知20株细菌均为阳性;在抑菌试验中20株生防细菌对4株供试的植物病原真菌均产生了拮抗作用,其抑菌率为29.58%～80.28%,其中抑菌率最高的是18号菌株枯草芽孢杆菌对真菌橡胶树炭疽病菌(Colletotrichum gloeosporioides),抑菌率为80.28%,抑菌率最低的是2号菌株假单胞菌对芒果炭疽病菌(Colletotrichum gloeosporioiles),抑菌率为29.58%。不同菌株之间抑制效果差异较大,大部分菌株对供试的病原真菌具有良好的抑制效果。本研究结果为后续生防机理的研究、植物病害的防治和生物药剂的开发提供理论依据。
In order to clarify the potential bio-control value of 20 strains of endophytic bacteria isolated from cowpea leaves and the evolutionary relationship between them, twenty strains of biocontrol bacteria were studied by morphological method, plate confrontation method, PCR technique and bioinformatics method. The pathogenic fungi were tested for their inhibitory effects against various pathogenic fungi. The results showed that 20 strains of endophytic bacteria were identified as Bacillus subtilis, Pseudomonas ssp. and Bacillus amyloliquefaciens, 12 strains of 20 endophyti c bacteria were identified as Bacillus subtilis, 3 strains were Pseudomonas ssp., 5 strains were Bacillus amyloliquefaciens. All of the biocontrol bacteria were found to inhibit the growth of 4 tested plant pathogens antagonism produced by fungi. Among all of them, the highest inhibition rate was No.18 Bacillus subtilis against No.8 Colletotrichum gloeosporioides, the antibacterial rate was 80.28%, and the worst effect was Bacteria No.2 pseudomonas against the fungus No.4 Colletotrichum gloeosporioiles, had an antibacterial rate of29.58%. Bio-control effects vary greatly among different strains, and some strains have significant biocontrol effects. The results of this study provide a theoretical basis for the study of the subsequent biocontrol mechanism,prevention of plant diseases, and the development of biopharmaceuticals.
In order to clarify the potential bio-control value of 20 strains of endophytic bacteria isolated from cowpea leaves and the evolutionary relationship between them, twenty strains of biocontrol bacteria were studied by morphological method, plate confrontation method, PCR technique and bioinformatics method. The pathogenic fungi were tested for their inhibitory effects against various pathogenic fungi. The results showed that 20 strains of endophytic bacteria were identified as Bacillus subtilis, Pseudomonas ssp. and Bacillus amyloliquefaciens, 12 strains of 20 endophyti c bacteria were identified as Bacillus subtilis, 3 strains were Pseudomonas ssp., 5 strains were Bacillus amyloliquefaciens. All of the biocontrol bacteria were found to inhibit the growth of 4 tested plant pathogens antagonism produced by fungi. Among all of them, the highest inhibition rate was No.18 Bacillus subtilis against No.8 Colletotrichum gloeosporioides, the antibacterial rate was 80.28%, and the worst effect was Bacteria No.2 pseudomonas against the fungus No.4 Colletotrichum gloeosporioiles, had an antibacterial rate of29.58%. Bio-control effects vary greatly among different strains, and some strains have significant biocontrol effects. The results of this study provide a theoretical basis for the study of the subsequent biocontrol mechanism,prevention of plant diseases, and the development of biopharmaceuticals.
引文
Chen X.H.,Ko umoutsi A.,Scholz R.,Eisenreich A.,Schneider K.,Heinemeyer I.,Morgenstern B.,Voss B.,Hess W.R.,Reva O.,Junge H.,Voigt B.,Jungblut P.R.,Vater J.,Süssmuth R.,Liesegang H.,Strittmatter A.,Gottschalk G.,and Borriss R.,2007,Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42,Nat.Biotechnol.,25(9):1007-1014
Compant S.,Duffy B.,Nowak J.,Clément C.,and Barka E.A.,2005,Use of plant-growth promoting bacteria for biocontrol of plant diseases:principles,mechanisms of action,and future prospects,Appl.Environ.Microbiol.,71(9):4951-4959
Han C.Z.,2015,Research status and development trend of microbial antagonists against plant diseases,Zhongguo Senlin Bingchong(Forest Pest and Disease),34(1):33-37(韩长志2015,植物病害生防菌的研究现状及发展趋势,中国森林病虫,34(1):33-37)
Hu G.P.,Zheng X.F.,You M.S.,and Liu B.,2010,Recent advances in research on endophytes,Fujian Nongye Xuebao(Fujian Journal of Agricultural Sciences),25(2):226-234(胡桂萍,郑雪芳,尤民生,刘波,2010,植物内生菌的研究进展,福建农业学报,25(2):226-234)
Huang Y.,ed.,2010,Biological control of plant diseases,Science Press,Beijing,China,pp.250-288(黄云,编著,2010,植物病害生物防治学,科学出版社,中国,北京,pp.250-288)
Kloepper J.W.,Leong J.,Teintze M.,and Schroth M.N.,1980Enhancing plant growth by siderophores produced by plan growth-promoting rhizobacteria,Nature,286:885-886
Li M.,Shuang B.,Li H.T.,Wang Q.,and Gao J.G.,2009,Progress and application of Bacillus subtilis in different fields,Dongbei Nongye Daxue Xuebao(Journal of Northeast Agricultural University),40(9):111-114(李明,双宝,李海涛,王晴高继国,2009,枯草芽孢杆菌的研究与应用,东北农业大学学报,40(9):111-114)
Li R.F.,Zhao Y.F.,Wei J.,Xue W.W.,and Liu B.,2010,Isolation and identification of antagonistic Bacillus subtilis strain against Magnaporthe grisea,Henan Nongye Kexue(Journa of Henan Agricultural Sciences),39(3):63-65(李瑞芳,赵玉峰,魏建,薛雯雯,刘冰,2010,具有稻瘟病菌拮抗活性的枯草芽孢杆菌的分离与鉴定,河南农业科学,39(3):63-65)
Liang Z.N.,Zhu H.,Lai K.P.,and Chen L.,2014,Isolation of endophytic fungi from medicinal plant Brucea javanica and their microbial inhibition activity,Zhongyaocai(Journal of Chinese Medicinal Materials),37(4):564-568(梁子宁,朱华,赖开平,陈龙,2014,药用植物鸦胆子内生真菌分离及其抑菌活性初步研究,中药材,37(4):564-568)
Ouyang D.S.,2005,Plant disease management in sustainable agrieulture,Dissertation for Ph.D.,Fujian Agriculture and Forestry University,Supervisor:Xie L.H.,and Lin Q.,pp8-15(欧阳迪莎,2005,可持续农业中的植物病害管理,博士学位论文,福建农林大学,导师:谢联辉,林卿,pp.8-15)
Song Y.J.,2017,Isolation and identification of endophytes from medicinal plants(Polygonatum sibiricum and Astragalus)and study on the structure and biological activity of exopolysaccharide,Dissertation for Ph.D.,Shandong University,Supervisor:Zhang H.C.,pp.24-27(宋颖娟,2017,药用植物(黄精和黄芪)内生菌的的分离鉴定及胞外多糖结构和生物活性的研究,博士学位论文,山东大学,导师:张厚程,pp.24-27)
Xu M.L.,Duan Y.P.,Wu Z.J.,Xie L.Y.,Xie L.H.,and Lin Q.Y.,2009,Inhibitory effect and mechanism of two Bacillus sp.strains on Colletotrichum musae,Yunnan Nongye Daxue Xuebao(Journal of Yunnan Agricultural University),24(4):524-527(许曼琳,段永平,吴祖建,谢荔岩,谢联辉,林奇英,2009,芽孢杆菌两菌株对香蕉炭疽病菌的抑制作用及其机制,云南农业大学学报,24(4):524-527)
Yuan Z.,2015,The research on arthropod community in cottom field and bio-control of cotton red spider mite,Thesis for M.S.,Hunan Agricultural University,Supervisor:Zeng A.P.,pp.2-8(袁准,2015,棉田节肢动物群落及棉红蜘蛛生防研究,硕士学位论文,湖南农业大学,导师:曾爱平,pp.2-8)
Zhan R.L.,Tan W.Q.,Song Y.,Zhang S.Q.,HO H.H.,Xu T.W.,and Huang J.S.,2007,Isolation and identification of Metarhizium anisopliae from natural infections of coconut hispid beetle Brontispa longissima(Gestro)(Coleoptera:Chrysomelidae),and preliminary studies on against this pest in Hainan island,Shengtai Xuebao(China Acta Ecologica Sinica),27(4):1558-1564(詹儒林,覃伟权,宋妍,张世清,HO H.H.,许天委,黄俊生,2007,海南椰心叶甲病原菌金龟子绿僵菌的分离、鉴定及其生防潜力,生态学报,27(4):1558-1564)
Zhang Y.J.,Luo J.C.,Wu Y.P.,and Yang H.T.,2009,Research advancement of the biological control bacteria B.subtilies,Shnegming Kexue Yiqi(Life Science Instruments),7(4):19-23(张彦杰,罗俊彩,武燕萍,杨合同,2009,生防枯草芽孢杆菌研究进展,生命科学仪器,7(4):19-23)
Zhang Z.F.,2010,16S r DNA identification and diversity function of endophytic Bacillus subtilis from forage grasses,Thesis for M.S.,Gansu Agricultural University,Supervisor:Chen X.R.,pp.7-10(张振粉,2010,牧草内生枯草芽孢杆菌的功能多样性及其16S r DNA鉴定,硕士学位论文,甘肃农业大学,导师:陈秀蓉,pp.7-10)
Zou W.X.,and Tan R.X.,2001,Recent advances on endophyte research,Zhiwu Xuebao(Bulletin of Botany),43(9):881-892(邹文欣,谭仁祥,2001,植物内生菌研究新进展,植物学报,43(9):881-892)
Compant S.,Duffy B.,Nowak J.,Clément C.,and Barka E.A.,2005,Use of plant-growth promoting bacteria for biocontrol of plant diseases:principles,mechanisms of action,and future prospects,Appl.Environ.Microbiol.,71(9):4951-4959
Han C.Z.,2015,Research status and development trend of microbial antagonists against plant diseases,Zhongguo Senlin Bingchong(Forest Pest and Disease),34(1):33-37(韩长志2015,植物病害生防菌的研究现状及发展趋势,中国森林病虫,34(1):33-37)
Hu G.P.,Zheng X.F.,You M.S.,and Liu B.,2010,Recent advances in research on endophytes,Fujian Nongye Xuebao(Fujian Journal of Agricultural Sciences),25(2):226-234(胡桂萍,郑雪芳,尤民生,刘波,2010,植物内生菌的研究进展,福建农业学报,25(2):226-234)
Huang Y.,ed.,2010,Biological control of plant diseases,Science Press,Beijing,China,pp.250-288(黄云,编著,2010,植物病害生物防治学,科学出版社,中国,北京,pp.250-288)
Kloepper J.W.,Leong J.,Teintze M.,and Schroth M.N.,1980Enhancing plant growth by siderophores produced by plan growth-promoting rhizobacteria,Nature,286:885-886
Li M.,Shuang B.,Li H.T.,Wang Q.,and Gao J.G.,2009,Progress and application of Bacillus subtilis in different fields,Dongbei Nongye Daxue Xuebao(Journal of Northeast Agricultural University),40(9):111-114(李明,双宝,李海涛,王晴高继国,2009,枯草芽孢杆菌的研究与应用,东北农业大学学报,40(9):111-114)
Li R.F.,Zhao Y.F.,Wei J.,Xue W.W.,and Liu B.,2010,Isolation and identification of antagonistic Bacillus subtilis strain against Magnaporthe grisea,Henan Nongye Kexue(Journa of Henan Agricultural Sciences),39(3):63-65(李瑞芳,赵玉峰,魏建,薛雯雯,刘冰,2010,具有稻瘟病菌拮抗活性的枯草芽孢杆菌的分离与鉴定,河南农业科学,39(3):63-65)
Liang Z.N.,Zhu H.,Lai K.P.,and Chen L.,2014,Isolation of endophytic fungi from medicinal plant Brucea javanica and their microbial inhibition activity,Zhongyaocai(Journal of Chinese Medicinal Materials),37(4):564-568(梁子宁,朱华,赖开平,陈龙,2014,药用植物鸦胆子内生真菌分离及其抑菌活性初步研究,中药材,37(4):564-568)
Ouyang D.S.,2005,Plant disease management in sustainable agrieulture,Dissertation for Ph.D.,Fujian Agriculture and Forestry University,Supervisor:Xie L.H.,and Lin Q.,pp8-15(欧阳迪莎,2005,可持续农业中的植物病害管理,博士学位论文,福建农林大学,导师:谢联辉,林卿,pp.8-15)
Song Y.J.,2017,Isolation and identification of endophytes from medicinal plants(Polygonatum sibiricum and Astragalus)and study on the structure and biological activity of exopolysaccharide,Dissertation for Ph.D.,Shandong University,Supervisor:Zhang H.C.,pp.24-27(宋颖娟,2017,药用植物(黄精和黄芪)内生菌的的分离鉴定及胞外多糖结构和生物活性的研究,博士学位论文,山东大学,导师:张厚程,pp.24-27)
Xu M.L.,Duan Y.P.,Wu Z.J.,Xie L.Y.,Xie L.H.,and Lin Q.Y.,2009,Inhibitory effect and mechanism of two Bacillus sp.strains on Colletotrichum musae,Yunnan Nongye Daxue Xuebao(Journal of Yunnan Agricultural University),24(4):524-527(许曼琳,段永平,吴祖建,谢荔岩,谢联辉,林奇英,2009,芽孢杆菌两菌株对香蕉炭疽病菌的抑制作用及其机制,云南农业大学学报,24(4):524-527)
Yuan Z.,2015,The research on arthropod community in cottom field and bio-control of cotton red spider mite,Thesis for M.S.,Hunan Agricultural University,Supervisor:Zeng A.P.,pp.2-8(袁准,2015,棉田节肢动物群落及棉红蜘蛛生防研究,硕士学位论文,湖南农业大学,导师:曾爱平,pp.2-8)
Zhan R.L.,Tan W.Q.,Song Y.,Zhang S.Q.,HO H.H.,Xu T.W.,and Huang J.S.,2007,Isolation and identification of Metarhizium anisopliae from natural infections of coconut hispid beetle Brontispa longissima(Gestro)(Coleoptera:Chrysomelidae),and preliminary studies on against this pest in Hainan island,Shengtai Xuebao(China Acta Ecologica Sinica),27(4):1558-1564(詹儒林,覃伟权,宋妍,张世清,HO H.H.,许天委,黄俊生,2007,海南椰心叶甲病原菌金龟子绿僵菌的分离、鉴定及其生防潜力,生态学报,27(4):1558-1564)
Zhang Y.J.,Luo J.C.,Wu Y.P.,and Yang H.T.,2009,Research advancement of the biological control bacteria B.subtilies,Shnegming Kexue Yiqi(Life Science Instruments),7(4):19-23(张彦杰,罗俊彩,武燕萍,杨合同,2009,生防枯草芽孢杆菌研究进展,生命科学仪器,7(4):19-23)
Zhang Z.F.,2010,16S r DNA identification and diversity function of endophytic Bacillus subtilis from forage grasses,Thesis for M.S.,Gansu Agricultural University,Supervisor:Chen X.R.,pp.7-10(张振粉,2010,牧草内生枯草芽孢杆菌的功能多样性及其16S r DNA鉴定,硕士学位论文,甘肃农业大学,导师:陈秀蓉,pp.7-10)
Zou W.X.,and Tan R.X.,2001,Recent advances on endophyte research,Zhiwu Xuebao(Bulletin of Botany),43(9):881-892(邹文欣,谭仁祥,2001,植物内生菌研究新进展,植物学报,43(9):881-892)