拮抗辣椒疫霉菌海洋细菌菌株SH-27的筛选鉴定及其防病促生作用
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摘要
【背景】由辣椒疫霉引起的辣椒疫病是全球辣椒生产中一种毁灭性的病害。近年来生物防治因其具有对环境友好、对人畜安全的特性而倍受关注。【目的】筛选对辣椒具有防病促生作用的海洋细菌菌株SH-27并鉴定其分类地位。【方法】采用稀释分离法和平板对峙法筛选拮抗辣椒疫霉菌的海洋细菌菌株,以发酵液灌根法测定海洋细菌SH-27菌株对辣椒盆栽的防病促生作用;通过形态特征、生理生化测试及多基因序列分析对海洋细菌SH-27菌株进行鉴定。【结果】从分离的142株海洋细菌中筛选获得11株对辣椒疫霉菌具有较强抑制作用的细菌菌株,其中以来自珊瑚的SH-27菌株的抑菌作用最强、抑菌谱广;室内盆栽试验结果表明,SH-27菌株发酵液处理后的辣椒植株根长、株高、茎粗、鲜重、干重均显著高于对照处理。SH-27菌株发酵液灌根处理后,对辣椒疫病4、6和9 d的防效分别为70.81%、66.55%和48.20%。经形态特征、生理生化测试及16S rRNA、gyrA基因序列分析,鉴定SH-27菌株为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。【结论】海洋细菌SH-27菌株对辣椒具有较好的防病促生效果,具有开发为微生物农药及菌肥的潜力。
[Background] Pepper Phytophthora blight, caused by Phytophthora capsici, is one of the most devastating diseases of pepper growing areas worldwide. Biological control has drawn more and more attention in recent years for its safety to environment, human and animal. [Objective] To screen and identify a marine bacterium strain SH-27 against Phytophthora capsici and study its effects on disease control and growth promotion on pepper. [Methods] Antagonistic marine bacteria were isolated by serial dilution and dual culture. The effects of controlling disease and growth promotion on pepper seedlings were studied by using the fermentation broth of strain SH-27. Strain SH-27 was identified by morphological characteristics, physiological and biochemical tests and 16 S r RNA and gyr A gene sequence analysis. [Results] A total of 142 bacterial isolates were obtained. Among them, 11 strains displayed the antagonistic activity to Phytophthora capsici. Especially strain SH-27 isolated from corals displayed high antagonistic activity and broad antimicrobial spectrum. Pot tests showed that the root length, plant height, stem diameter, fresh weight and dry weight of pepper plant treated with SH-27 fermentation broth were promoted significantly when compared to the control treatment. The control efficacy of strain SH-27 to pepper phytophthora blight were 70.81%, 66.55% and 48.20% when the pathogens were inoculated after 4, 6 and 9 days respectively. Based on characteristics in morphology, physiology, biochemistry, 16 S r RNA and gyr A gene sequence analysis, strain SH-27 was identified as Bacillus amyloliquefaciens. [Conclusion] Due to its effects of disease control and growth promotion on pepper, strain SH-27 is possible to be further developed as an excellent strain for microbial fertilizer and fungicide.
[Background] Pepper Phytophthora blight, caused by Phytophthora capsici, is one of the most devastating diseases of pepper growing areas worldwide. Biological control has drawn more and more attention in recent years for its safety to environment, human and animal. [Objective] To screen and identify a marine bacterium strain SH-27 against Phytophthora capsici and study its effects on disease control and growth promotion on pepper. [Methods] Antagonistic marine bacteria were isolated by serial dilution and dual culture. The effects of controlling disease and growth promotion on pepper seedlings were studied by using the fermentation broth of strain SH-27. Strain SH-27 was identified by morphological characteristics, physiological and biochemical tests and 16 S r RNA and gyr A gene sequence analysis. [Results] A total of 142 bacterial isolates were obtained. Among them, 11 strains displayed the antagonistic activity to Phytophthora capsici. Especially strain SH-27 isolated from corals displayed high antagonistic activity and broad antimicrobial spectrum. Pot tests showed that the root length, plant height, stem diameter, fresh weight and dry weight of pepper plant treated with SH-27 fermentation broth were promoted significantly when compared to the control treatment. The control efficacy of strain SH-27 to pepper phytophthora blight were 70.81%, 66.55% and 48.20% when the pathogens were inoculated after 4, 6 and 9 days respectively. Based on characteristics in morphology, physiology, biochemistry, 16 S r RNA and gyr A gene sequence analysis, strain SH-27 was identified as Bacillus amyloliquefaciens. [Conclusion] Due to its effects of disease control and growth promotion on pepper, strain SH-27 is possible to be further developed as an excellent strain for microbial fertilizer and fungicide.
引文
[1]Wang ZY,Langston DB,Csinos AS,et al.Development of an improved isolation approach and simple sequence repeat markers to characterize Phytophthora capsici populations in irrigation ponds in southern Georgia[J].Applied and Environmental Microbiology,2009,75(17):5467-5473
[2]Zhou Q,Li BT,Tang LM.A study on fungicidal activity of allicin against Colletotrichum capsici and Phytophthora capsici in the laboratory and its efficacy in the field[J].Acta Prataculturae Sinica,2014,23(3):262-268(in Chinese)周清,李保同,汤丼梅.大蒜素对辣椒炭疽病和辣椒疫病病菌的室内抑制活性测定及田间防效研究[J].草业学报,2014,23(3):262-268
[3]Liu YL,Yin CL,Tian YH,et al.Identification of the antagonistic fungus strain HTC and its potential for biocontrol of pepper phytophthora blight[J].Acta Phytophylacica Sinica,2013,40(5):437-444(in Chinese)刘永亮,尹成林,田叶韩,等.拮抗真菌HTC的鉴定及其对辣椒疫病的生物防治潜力[J].植物保护学报,2013,40(5):437-444
[4]Yuan SZ,Zhou MG.Screening and root colonization of biocontrol agents against Phytophthora capsici[J].Journal of Yangzhou University(Agricultural and Life Sciences Edition),2006,27(4):93-97(in Chinese)袁树忠,周明国.辣椒疫病生物防治菌株的筛选与定殖[J].扬州大学学报:农业与生命科学版,2006,27(4):93-97
[5]Fu SY,Chen SL,Yan SZ.Colonization dynamics of endophytic bacteria in Capsicum annuum and their control effects on pepper Phytophthora blight[J].Chinese Journal of Biological Control,2013,29(4):561-568(in Chinese)付思娅,陈双林,闫淑珍.植物内生细菌在辣椒体内的定殖动态及对辣椒疫病的防治效果[J].中国生物防治学报,2013,29(4):561-568
[6]Yang RX,Tao YF,Song MX,et al.Inhibition of endophytic bacterial strains isolated from Ginkgo biloba against phytophthora blight of pepper[J].Chinese Journal of Biological Control,2012,28(4):552-559(in Chinese)杨瑞先,陶玉凤,宋美仙,等.银杏内生细菌防治辣椒疫病研究[J].中国生物防治学报,2012,28(4):552-559
[7]Woo SM,Kim SD.Structural identification of siderophore(AH18)from Bacillus subtilis AH18,a biocontrol agent of Phytophthora blight disease in red-pepper[J].Korean Society for Microbiology and Biotechnology,2008,36(4):326-335
[8]Segarra G,Avilés M,Casanova E,et al.Effectiveness of biological control of Phytophthora capsici in pepper by Trichoderma asperellum strain T34[J].Phytopathologia Mediterranea,2013,52(1):77-83
[9]Tian L,Gu ZF,Chen J,et al.Inhibitor substance of marine bacterium and effects on pathogenic fungi[J].Acta P hytopathologica Sinica,2003,33(1):77-80(in Chinese)田黎,顾振芳,陈杰,等.海洋细菌B-9987菌株产生的抑菌物质及对几种植物病原真菌的作用[J].植物病理学报[J].2003,33(1):77-80
[10]Hu JC,Xue DL,Wang SJ,et al.Obstacles of soybean continuous cropping III.mechanism of soybean yield increment by marine actinomyces MB-97[J].Chinese Journal of Applied Ecology,2002,13(9):1095-1098(in Chinese)胡江春,薛德林,王书锦,等.大豆连作障碍研究III.海洋放线菌MB-97促迚连作大豆增产机理[J].应用生态学报,2002,13(9):1095-1098
[11]He H,Ou XC,Wang LC,et al.Bio-control efficacy of a marine bacterium CIII-1 from mangrove on capsicum bacterial wilt[J].Acta Phytophylacica Sinica,2008,35(6):497-500(in Chinese)何红,欧雄常,王立才,等.红树内生海洋细菌CIII-1菌株对辣椒青枯病的防病效果[J].植物保护学报,2008,35(6):497-500
[12]Ou XC,Liu F,Zhan RL,et al.Screen of mangrove endophytic bacteria antagonists against Phytophthora capsici and identification of strain RS261[J].Microbiology China,2009,36(2):175-180(in Chinese)欧雄常,柳凤,詹儒林,等.拮抗辣椒疫病菌的红树内生细菌筛选及RS261菌株鉴定[J].微生物学通报,2009,36(2):175-180
[13]Mao AJ,Fu Q,Geng SX.Research on inoculation and identification technology of Phytophthora capsici[J].Beijing Agricultural Sciences,1998,16(2):21-24(in Chinese)毛爱军,胡洽,耿三省.辣椒疫病菌接种鉴定技术研究[J].北京农业科学,1998,16(2):21-24
[14]Qin JJ,Yan SZ,Liu J.The growth-promotion on pepper and control of Phytophthora capsici by endophytic bacterium agents[J].Acta Phytophylacica Sinica,2010,37(4):325-330(in Chinese)秦娟娟,闫淑珍,刘佳.植物内生细菌固体菌剂对辣椒的促生和防病作用[J].植物保护学报,2010,37(4):325-330
[15]Dong XZ,Cai MY.Common Bacteria System Classification and Appraisal Method[M].Beijing:Science Press,2001(in Chinese)东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001
[16]Liu LX,Coenye T,Burns JL,et al.Ribosomal DNA directed PCR for identification of Achromobacter(Alcaligenes)xylosoxidans recovered from sputum samples from cystic fibrosis patients[J].Journal of Clinical Microbiology,2002,40(4):1210-1213
[17]Chun J,Bae KS.Phylogenetic analysis of Bacillus subtilis and related taxa based on partial gyr A gene sequences[J].Antonie van Leeuwenhoek,2000,78(2):123-127
[18]Bao ZH,Jiang Q.Isolation and selection of marine bacterium against plant pathogenic fungi[J].Journal of Henan Agricultural Sciences,2007,36(3):49-52(in Chinese)暴增海,蒋茜.3株抗植物病原真菌的海洋细菌的分离筛选[J].河南农业科学,2007,36(3):49-52
[19]Liu F,Pan CB,He H,et al.Suppression of pepper phytophthora blight by endophytic bacteria from mangroves[J].Chinese Journal of Biological Control,2008,24(4):379-381(in Chinese)柳凤,潘朝勃,何红,等.海洋细菌对辣椒疫霉和辣椒疫病的抑制作用[J].中国生物防治,2008,24(4):379-381
[20]Liu F,Ou XC,He H,et al.Control of capsicum phytophthora blight by endophytic bacteria RS261 from mangrove[J].Acta Phytopathologica Sinica,2009,39(3):333-336(in Chinese)柳凤,欧雄常,何红,等.红树内生细菌RS261菌株防治辣椒疫病的初步研究[J].植物病理学报,2009,39(3):333-336
[21]Liu F,Ou XC,He H,et al.Mechanism of biological control of phytophthora blight in pepper by mangrove endophytic bacterium strain RS261[J].Acta Phytopathologica Sinica,2010,40(1):74-80(in Chinese)柳凤,欧雄常,何红,等.红树内生细菌RS261防治辣椒疫病机理的初步研究[J].植物病理学报,2010,40(1):74-80
[22]Zhang XF,Liu F,He H,et al.Identification of endophytic bacterium CIII-1 strain from mangrove and isolation of its antimicrobial proteins[J].Microbiology China,2010,37(2):222-227(in Chinese)张关锋,柳凤,何红,等.红树内生细菌CⅢ-1菌株鉴定及其胞外抗菌蛋白性质[J].微生物学通报,2010,37(2):222-227
[23]He PQ,Wang HM,Shen JH,et al.Antarctic Bacillus sp.107growth characteristics and inhibition against plant pathogenic fungi[J].Advances in Marine Science,2011,29(2):179-185(in Chinese)何培青,王红梅,沈继红,等.南极芽孢杆菌Bacillus sp.107生长特性及抑制植物病原真菌的研究[J].海洋科学迚展,2011,29(2):179-185
[24]Tareq FS,Hasan MC,Lee HS,et al.Gageopeptins A and B,new inhibitors of zoospore motility of the phytopathogen Phytophthora capsici from a marine-derived bacterium Bacillus sp.109GGC020[J].Bioorganic&Medicinal Chemistry Letters,2015,25(16):3325-3329
[25]Saha M,Ghosh D Jr,Ghosh D,et al.Studies on the production and purification of an antimicrobial compound and taxonomy of the producer isolated from the marine environment of the Sundarbans[J].Applied Microbiology and Biotechnology,2005,66(5):497-505
[26]Mouchka ME,Hewson I,Harvell CD.Coral-associated bacterial assemblages:current knowledge and the potential for climate-driven impacts[J].Integrative and Comparative Biology,2010,50(4):662-674
[2]Zhou Q,Li BT,Tang LM.A study on fungicidal activity of allicin against Colletotrichum capsici and Phytophthora capsici in the laboratory and its efficacy in the field[J].Acta Prataculturae Sinica,2014,23(3):262-268(in Chinese)周清,李保同,汤丼梅.大蒜素对辣椒炭疽病和辣椒疫病病菌的室内抑制活性测定及田间防效研究[J].草业学报,2014,23(3):262-268
[3]Liu YL,Yin CL,Tian YH,et al.Identification of the antagonistic fungus strain HTC and its potential for biocontrol of pepper phytophthora blight[J].Acta Phytophylacica Sinica,2013,40(5):437-444(in Chinese)刘永亮,尹成林,田叶韩,等.拮抗真菌HTC的鉴定及其对辣椒疫病的生物防治潜力[J].植物保护学报,2013,40(5):437-444
[4]Yuan SZ,Zhou MG.Screening and root colonization of biocontrol agents against Phytophthora capsici[J].Journal of Yangzhou University(Agricultural and Life Sciences Edition),2006,27(4):93-97(in Chinese)袁树忠,周明国.辣椒疫病生物防治菌株的筛选与定殖[J].扬州大学学报:农业与生命科学版,2006,27(4):93-97
[5]Fu SY,Chen SL,Yan SZ.Colonization dynamics of endophytic bacteria in Capsicum annuum and their control effects on pepper Phytophthora blight[J].Chinese Journal of Biological Control,2013,29(4):561-568(in Chinese)付思娅,陈双林,闫淑珍.植物内生细菌在辣椒体内的定殖动态及对辣椒疫病的防治效果[J].中国生物防治学报,2013,29(4):561-568
[6]Yang RX,Tao YF,Song MX,et al.Inhibition of endophytic bacterial strains isolated from Ginkgo biloba against phytophthora blight of pepper[J].Chinese Journal of Biological Control,2012,28(4):552-559(in Chinese)杨瑞先,陶玉凤,宋美仙,等.银杏内生细菌防治辣椒疫病研究[J].中国生物防治学报,2012,28(4):552-559
[7]Woo SM,Kim SD.Structural identification of siderophore(AH18)from Bacillus subtilis AH18,a biocontrol agent of Phytophthora blight disease in red-pepper[J].Korean Society for Microbiology and Biotechnology,2008,36(4):326-335
[8]Segarra G,Avilés M,Casanova E,et al.Effectiveness of biological control of Phytophthora capsici in pepper by Trichoderma asperellum strain T34[J].Phytopathologia Mediterranea,2013,52(1):77-83
[9]Tian L,Gu ZF,Chen J,et al.Inhibitor substance of marine bacterium and effects on pathogenic fungi[J].Acta P hytopathologica Sinica,2003,33(1):77-80(in Chinese)田黎,顾振芳,陈杰,等.海洋细菌B-9987菌株产生的抑菌物质及对几种植物病原真菌的作用[J].植物病理学报[J].2003,33(1):77-80
[10]Hu JC,Xue DL,Wang SJ,et al.Obstacles of soybean continuous cropping III.mechanism of soybean yield increment by marine actinomyces MB-97[J].Chinese Journal of Applied Ecology,2002,13(9):1095-1098(in Chinese)胡江春,薛德林,王书锦,等.大豆连作障碍研究III.海洋放线菌MB-97促迚连作大豆增产机理[J].应用生态学报,2002,13(9):1095-1098
[11]He H,Ou XC,Wang LC,et al.Bio-control efficacy of a marine bacterium CIII-1 from mangrove on capsicum bacterial wilt[J].Acta Phytophylacica Sinica,2008,35(6):497-500(in Chinese)何红,欧雄常,王立才,等.红树内生海洋细菌CIII-1菌株对辣椒青枯病的防病效果[J].植物保护学报,2008,35(6):497-500
[12]Ou XC,Liu F,Zhan RL,et al.Screen of mangrove endophytic bacteria antagonists against Phytophthora capsici and identification of strain RS261[J].Microbiology China,2009,36(2):175-180(in Chinese)欧雄常,柳凤,詹儒林,等.拮抗辣椒疫病菌的红树内生细菌筛选及RS261菌株鉴定[J].微生物学通报,2009,36(2):175-180
[13]Mao AJ,Fu Q,Geng SX.Research on inoculation and identification technology of Phytophthora capsici[J].Beijing Agricultural Sciences,1998,16(2):21-24(in Chinese)毛爱军,胡洽,耿三省.辣椒疫病菌接种鉴定技术研究[J].北京农业科学,1998,16(2):21-24
[14]Qin JJ,Yan SZ,Liu J.The growth-promotion on pepper and control of Phytophthora capsici by endophytic bacterium agents[J].Acta Phytophylacica Sinica,2010,37(4):325-330(in Chinese)秦娟娟,闫淑珍,刘佳.植物内生细菌固体菌剂对辣椒的促生和防病作用[J].植物保护学报,2010,37(4):325-330
[15]Dong XZ,Cai MY.Common Bacteria System Classification and Appraisal Method[M].Beijing:Science Press,2001(in Chinese)东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001
[16]Liu LX,Coenye T,Burns JL,et al.Ribosomal DNA directed PCR for identification of Achromobacter(Alcaligenes)xylosoxidans recovered from sputum samples from cystic fibrosis patients[J].Journal of Clinical Microbiology,2002,40(4):1210-1213
[17]Chun J,Bae KS.Phylogenetic analysis of Bacillus subtilis and related taxa based on partial gyr A gene sequences[J].Antonie van Leeuwenhoek,2000,78(2):123-127
[18]Bao ZH,Jiang Q.Isolation and selection of marine bacterium against plant pathogenic fungi[J].Journal of Henan Agricultural Sciences,2007,36(3):49-52(in Chinese)暴增海,蒋茜.3株抗植物病原真菌的海洋细菌的分离筛选[J].河南农业科学,2007,36(3):49-52
[19]Liu F,Pan CB,He H,et al.Suppression of pepper phytophthora blight by endophytic bacteria from mangroves[J].Chinese Journal of Biological Control,2008,24(4):379-381(in Chinese)柳凤,潘朝勃,何红,等.海洋细菌对辣椒疫霉和辣椒疫病的抑制作用[J].中国生物防治,2008,24(4):379-381
[20]Liu F,Ou XC,He H,et al.Control of capsicum phytophthora blight by endophytic bacteria RS261 from mangrove[J].Acta Phytopathologica Sinica,2009,39(3):333-336(in Chinese)柳凤,欧雄常,何红,等.红树内生细菌RS261菌株防治辣椒疫病的初步研究[J].植物病理学报,2009,39(3):333-336
[21]Liu F,Ou XC,He H,et al.Mechanism of biological control of phytophthora blight in pepper by mangrove endophytic bacterium strain RS261[J].Acta Phytopathologica Sinica,2010,40(1):74-80(in Chinese)柳凤,欧雄常,何红,等.红树内生细菌RS261防治辣椒疫病机理的初步研究[J].植物病理学报,2010,40(1):74-80
[22]Zhang XF,Liu F,He H,et al.Identification of endophytic bacterium CIII-1 strain from mangrove and isolation of its antimicrobial proteins[J].Microbiology China,2010,37(2):222-227(in Chinese)张关锋,柳凤,何红,等.红树内生细菌CⅢ-1菌株鉴定及其胞外抗菌蛋白性质[J].微生物学通报,2010,37(2):222-227
[23]He PQ,Wang HM,Shen JH,et al.Antarctic Bacillus sp.107growth characteristics and inhibition against plant pathogenic fungi[J].Advances in Marine Science,2011,29(2):179-185(in Chinese)何培青,王红梅,沈继红,等.南极芽孢杆菌Bacillus sp.107生长特性及抑制植物病原真菌的研究[J].海洋科学迚展,2011,29(2):179-185
[24]Tareq FS,Hasan MC,Lee HS,et al.Gageopeptins A and B,new inhibitors of zoospore motility of the phytopathogen Phytophthora capsici from a marine-derived bacterium Bacillus sp.109GGC020[J].Bioorganic&Medicinal Chemistry Letters,2015,25(16):3325-3329
[25]Saha M,Ghosh D Jr,Ghosh D,et al.Studies on the production and purification of an antimicrobial compound and taxonomy of the producer isolated from the marine environment of the Sundarbans[J].Applied Microbiology and Biotechnology,2005,66(5):497-505
[26]Mouchka ME,Hewson I,Harvell CD.Coral-associated bacterial assemblages:current knowledge and the potential for climate-driven impacts[J].Integrative and Comparative Biology,2010,50(4):662-674