油茶炭疽病拮抗细菌P-14的拮抗物质分析
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摘要
[目的]探索解淀粉芽孢杆菌P-14对油茶炭疽病病原菌的拮抗物质,以期为油茶炭疽病的生物农药开发利用提供理论依据。[方法]利用酸沉淀法和高效液相色谱法分离解淀粉芽孢杆菌P-14发酵液中的拮抗物质,通过顶空固相微萃取法和气相色谱-质谱法对解淀粉芽孢杆菌P-14的挥发性物质进行了分离鉴定。[结果]分离后的组分a对油茶炭疽病的病原菌具有良好的抑制作用,其主要成分为C15 bacillomycin D(杆菌霉素D)。此外,共分离得到35种挥发性化合物,其相对抑菌率为(26.19±3.82)%,其中包含苯类物质(benzenes)10种、烷基类物质(alkyls)8种、醇类物质(alcohols)2种、酮类物质(ketones)11种、醛类物质(aldehydes)3种和1种酯类物质(esters)。[结论]解淀粉芽孢杆菌P-14能产生对油茶炭疽病的病原菌具有明显的拮抗作用的物质,并确定了其拮抗物质种类,为油茶炭疽病的生物农药开发提供了可靠的研究内容。
[Objective] The purpose of this study is to explore the metabolic antagonists against Colletotrichum camellia produced by antagonistic bacteria P-14, in order to provide a theoretical basis for the development and utilization of biopesticides of Camellia oleifera. [Method] The acid precipitation and high-performance liquid chromatography was used to identify the lipopeptide antibiotics produced by strain P-14, and the volatile compounds were analyzed by gas chromatography-mass spectrometry. [Result] The component a containing C15 bacillomycin D showed the strongest inhibitory effect against Colletotrichum camellia. In addition, a total of 35 volatile compounds were separated, the relative bacteriostatic rate was(26.19±3.82)%, including 10 benzenes, 8 alkyls, 2 alcohols, 11 ketones, 3 aldehydes, and 1 esters. [Conclusion] Bacillus amyloliquefaciens P-14 could produce metabolic antagonists against Colletotrichum camellia, and the metabolic antagonists has been identified, which can provide with reliable research data for the development of biopesticides against C. oleifera.
[Objective] The purpose of this study is to explore the metabolic antagonists against Colletotrichum camellia produced by antagonistic bacteria P-14, in order to provide a theoretical basis for the development and utilization of biopesticides of Camellia oleifera. [Method] The acid precipitation and high-performance liquid chromatography was used to identify the lipopeptide antibiotics produced by strain P-14, and the volatile compounds were analyzed by gas chromatography-mass spectrometry. [Result] The component a containing C15 bacillomycin D showed the strongest inhibitory effect against Colletotrichum camellia. In addition, a total of 35 volatile compounds were separated, the relative bacteriostatic rate was(26.19±3.82)%, including 10 benzenes, 8 alkyls, 2 alcohols, 11 ketones, 3 aldehydes, and 1 esters. [Conclusion] Bacillus amyloliquefaciens P-14 could produce metabolic antagonists against Colletotrichum camellia, and the metabolic antagonists has been identified, which can provide with reliable research data for the development of biopesticides against C. oleifera.
引文
[1] 庄瑞林. 中国油茶[M]. 北京, 中国林业出版社, 2012.
[2] 周建宏. 油茶主要病害的植物源药剂研究[D].长沙:中南林业科技大学, 2011.
[3] 喻锦秀, 聂云安, 周刚, 等.湖南省油茶主要病害发生规律研究[J]. 湖南林业科技, 2014, 41(1): 94-97.
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[5] 邓鑫州, 黄连桂, 邓荫伟. 5种药剂对油茶炭疽病的防治效果研究[J].安徽农业科学, 2011, 39(16): 9653-9654.
[6] 刘军根, 吴军平, 陈刚, 等. 生物制剂果力士防治油茶炭疽病药效试验[J]. 林业实用技术, 2011(6): 46-47.
[7] 罗洪, 梁勇, 肖琴琳, 等. 果力士防治油茶嫁接苗炭疽病试验[J]. 中国森林病虫, 2011, 30(3): 46.
[8] Damalas C A. Understanding benefits and risks of pesticide use[J]. Scientific Research & Essays, 2009, 4(10):945-949.
[9] Hvistendahl M. In rural asia, locking up poisons to prevent suicides[J]. Science, 2013, 341(6147):738.
[10] Borriss R. Use of plant-associated bacillus strains as biofertilizers and biocontrol agents in agriculture[M]//Mahshwari. Bacteria in Agrobiology: Plant Growth Responses.Springer, 2011:41-76.
[11] Erlacher A, Cardinale M, Grosch R, et al. The impact of the pathogen Rhizoctonia solani and its beneficial counterpart Bacillus amyloliquefaciens on the indigenous lettuce microbiome[J]. Front Microbiol, 2014, 5(175):175.
[12] Chen X H, Koumoutsi A, Scholz R, et al. Genome analysis of Bacillus amyloliquefaciens FZB42 reveals its potential for biocontrol of plant pathogens[J]. Journal of Biotechnology, 2009, 140(1):27-37.
[13] Chowdhury S P, Hartmann A, Gao X W, et al. Biocontrol mechanism by root-associated Bacillus amyloliquefaciens FZB42-a review[J]. Frontiers in Microbiology, 2015, 6(780):780.
[14] 高学文, 姚仕义, Huong, 等.枯草芽孢杆菌B2菌株产生的表面活性素变异体的纯化和鉴定[J]. 微生物学报, 2003, 43(5): 647-652.
[15] Chen X H, Koumoutsi A, Scholz R, et al. Comparative analysis of the complete genome sequence of the plant growth promoting bacterium Bacillus amyloliquefaciens FZB42[J]. Nature Biotechnology, 2007, 25(9):1007-1014.
[16] Yuan J, Raza W, Shen Q, et al. Antifungal activity of Bacillus amyloliquefaciens NJN-6 volatile compounds against Fusarium oxysporum f. sp. cubense[J]. Applied & Environmental Microbiology, 2012, 78(16):5942-5944.
[17] Grosch R, Junge H, Krebs B, et al. Use of Bacillus subtilis as a biocontrol agent. III. Influence of Bacillus subtilis on fungal root diseases and on yield in soilless culture[J]. Journal of Plant Diseases & Protection, 1999, 106(6):568-580.
[18] Yao A V, Bochow Dr H, Karimov S, et al. Effect of FZB 24~? Bacillus subtilis as a biofertilizer on cotton yields in field tests[J]. Archives of Phytopathology & Plant Protection, 2006, 39(4):323-328.
[19] Gül A, Kidoglu F, Tüzel Y, et al. Effects of nutrition and Bacillus amyloliquefaciens on tomato (Solanum lycopersicum L.) growing in perlite[J]. 2008, 6(3):422-429.
[20] Scholz R, Molohon K J, Nachtigall J, et al. Plantazolicin, a novel microcin B17/streptolysin S-like natural product from Bacillus amyloliquefaciens FZB42. [J]. Journal of Bacteriology, 2011, 193(1):215-224.
[21] Scholz R, Vater J, Budiharjo A, et al. Amylocyclicin, a novel circular bacteriocin produced by Bacillus amyloliquefaciens FZB42[J]. Journal of Bacteriology, 2014, 196(10):1842-1852.
[22] Chen X H, Scholz R, Borriss M, et al. Difficidin and bacilysin produced by plant-associated Bacillus amyloliquefaciens, are efficient in controlling fire blight disease[J]. Journal of Biotechnology, 2009, 140(1–2):38-44.
[23] Koumoutsi A, Chen X H, Henne A, et al. Structural and functional characterization of gene clusters directing nonribosomal synthesis of bioactive cyclic lipopeptides in Bacillus amyloliquefaciens strain FZB42[J]. Journal of Bacteriology, 2004, 186(4):1084.
[24] Chen X H, Vater J, Piel J, et al. Structural and functional characterization of three polyketide synthase gene clusters in Bacillus amyloliquefaciens FZB42[J]. Journal of Bacteriology, 2006, 188(11):4024.
[25] F?ldes T, Bánhegyi I, Herpai Z, et al. Isolation of Bacillus strains from the rhizosphere of cereals and in vitro screening for antagonism against phytopathogenic, food-borne pathogenic and spoilage micro-organisms[J]. Journal of Applied Microbiology, 2000, 89(5):840-846.
[26] Romero D, De V A, Olmos J L, et al. Effect of lipopeptides of antagonistic strains of Bacillus subtilis, on the morphology and ultrastructure of the cucurbit fungal pathogen Podosphaera fusca[J]. Journal of Applied Microbiology, 2007, 103(4):969–976.
[27] 李宝庆, 鹿秀云, 郭庆港, 等. 枯草芽孢杆菌BAB-1产脂肽类及挥发性物质的分离和鉴定[J].中国农业科学, 2010, 43(17): 3547-3554.
[28] Ongena M, Jacques P. Bacillus lipopeptides: versatile weapons for plant disease biocontrol[J]. Trends in Microbiology, 2008, 16(3):115-125.
[29] Xu Z, Shao J, Li B, et al. Contribution of bacillomycin D in Bacillus amyloliquefaciens SQR9 to antifungal activity and biofilm formation[J]. Applied & Environmental Microbiology, 2013, 79(3):808-815.
[30] Romero D, De V A, Rakotoaly R H, et al. The iturin and fengycin families of lipopeptides are key factors in antagonism of Bacillus subtilis toward Podosphaera fusca[J]. Molecular Plant-microbe Interactions: MPMI, 2007, 20(4):430.
[31] Moyne A L, Shelby R, Cleveland T E, et al. Bacillomycin D: an iturin with antifungal activity against Aspergillus flavus[J]. Journal of Applied Microbiology, 2001, 90(4):622.
[32] 信珊珊.解淀粉芽胞杆菌WH1的发酵优化及其产物对油茶炭疽病和肿瘤的抑制作用研究[D]. 武汉:华中农业大学, 2011.
[33] 信珊珊, 祁高富, 朱发银, 等.1株解淀粉芽孢杆菌发酵条件的优化及其对油茶炭疽病的防效[J]. 华中农业大学学报, 2011, 30(4): 411-415.
[34] 孟庆敏, 周国英, 刘君昂, 等.油茶炭疽病拮抗细菌Y13主要抑菌物质分离纯化及作用方式[J]. 植物保护, 2014, 40(2): 36-42
[35] 龚庆伟.芽孢杆菌抗菌脂肽分离纯化及Bacillomycin D抑制黄曲霉作用的研究[D]. 南京:南京农业大学, 2012.
[36] W G Dilantha F, Ramarathnam R, Krishnamoorthy A S, et al. Identification and use of potential bacterial organic antifungal volatiles in biocontrol[J]. Soil Biology & Biochemistry, 2005, 37(5):955-964.
[37] Kai M, Effmert U, Berg G, et al. Volatiles of bacterial antagonists inhibit mycelial growth of the plant pathogen Rhizoctonia solani[J]. Archives of Microbiology, 2007, 187(5):351-360.
[38] Almenar E, Del V V, Catala R, et al. Active package for wild strawberry fruit (Fragaria vesca L.)[J]. Journal of Agricultural & Food Chemistry, 2007, 55(6):2240-2245.
[39] Raza W, Wang J, Wu Y, et al. Effects of volatile organic compounds produced by Bacillus amyloliquefaciens, on the growth and virulence traits of tomato bacterial wilt pathogen Ralstonia solanacearum[J]. Applied Microbiology & Biotechnology, 2016, 100(17):1-12.
[2] 周建宏. 油茶主要病害的植物源药剂研究[D].长沙:中南林业科技大学, 2011.
[3] 喻锦秀, 聂云安, 周刚, 等.湖南省油茶主要病害发生规律研究[J]. 湖南林业科技, 2014, 41(1): 94-97.
[4] 黄新华. 百菌清等3种药剂防治油茶炭疽病药效试验[J]. 江西林业科技, 2000(2): 18-19.
[5] 邓鑫州, 黄连桂, 邓荫伟. 5种药剂对油茶炭疽病的防治效果研究[J].安徽农业科学, 2011, 39(16): 9653-9654.
[6] 刘军根, 吴军平, 陈刚, 等. 生物制剂果力士防治油茶炭疽病药效试验[J]. 林业实用技术, 2011(6): 46-47.
[7] 罗洪, 梁勇, 肖琴琳, 等. 果力士防治油茶嫁接苗炭疽病试验[J]. 中国森林病虫, 2011, 30(3): 46.
[8] Damalas C A. Understanding benefits and risks of pesticide use[J]. Scientific Research & Essays, 2009, 4(10):945-949.
[9] Hvistendahl M. In rural asia, locking up poisons to prevent suicides[J]. Science, 2013, 341(6147):738.
[10] Borriss R. Use of plant-associated bacillus strains as biofertilizers and biocontrol agents in agriculture[M]//Mahshwari. Bacteria in Agrobiology: Plant Growth Responses.Springer, 2011:41-76.
[11] Erlacher A, Cardinale M, Grosch R, et al. The impact of the pathogen Rhizoctonia solani and its beneficial counterpart Bacillus amyloliquefaciens on the indigenous lettuce microbiome[J]. Front Microbiol, 2014, 5(175):175.
[12] Chen X H, Koumoutsi A, Scholz R, et al. Genome analysis of Bacillus amyloliquefaciens FZB42 reveals its potential for biocontrol of plant pathogens[J]. Journal of Biotechnology, 2009, 140(1):27-37.
[13] Chowdhury S P, Hartmann A, Gao X W, et al. Biocontrol mechanism by root-associated Bacillus amyloliquefaciens FZB42-a review[J]. Frontiers in Microbiology, 2015, 6(780):780.
[14] 高学文, 姚仕义, Huong, 等.枯草芽孢杆菌B2菌株产生的表面活性素变异体的纯化和鉴定[J]. 微生物学报, 2003, 43(5): 647-652.
[15] Chen X H, Koumoutsi A, Scholz R, et al. Comparative analysis of the complete genome sequence of the plant growth promoting bacterium Bacillus amyloliquefaciens FZB42[J]. Nature Biotechnology, 2007, 25(9):1007-1014.
[16] Yuan J, Raza W, Shen Q, et al. Antifungal activity of Bacillus amyloliquefaciens NJN-6 volatile compounds against Fusarium oxysporum f. sp. cubense[J]. Applied & Environmental Microbiology, 2012, 78(16):5942-5944.
[17] Grosch R, Junge H, Krebs B, et al. Use of Bacillus subtilis as a biocontrol agent. III. Influence of Bacillus subtilis on fungal root diseases and on yield in soilless culture[J]. Journal of Plant Diseases & Protection, 1999, 106(6):568-580.
[18] Yao A V, Bochow Dr H, Karimov S, et al. Effect of FZB 24~? Bacillus subtilis as a biofertilizer on cotton yields in field tests[J]. Archives of Phytopathology & Plant Protection, 2006, 39(4):323-328.
[19] Gül A, Kidoglu F, Tüzel Y, et al. Effects of nutrition and Bacillus amyloliquefaciens on tomato (Solanum lycopersicum L.) growing in perlite[J]. 2008, 6(3):422-429.
[20] Scholz R, Molohon K J, Nachtigall J, et al. Plantazolicin, a novel microcin B17/streptolysin S-like natural product from Bacillus amyloliquefaciens FZB42. [J]. Journal of Bacteriology, 2011, 193(1):215-224.
[21] Scholz R, Vater J, Budiharjo A, et al. Amylocyclicin, a novel circular bacteriocin produced by Bacillus amyloliquefaciens FZB42[J]. Journal of Bacteriology, 2014, 196(10):1842-1852.
[22] Chen X H, Scholz R, Borriss M, et al. Difficidin and bacilysin produced by plant-associated Bacillus amyloliquefaciens, are efficient in controlling fire blight disease[J]. Journal of Biotechnology, 2009, 140(1–2):38-44.
[23] Koumoutsi A, Chen X H, Henne A, et al. Structural and functional characterization of gene clusters directing nonribosomal synthesis of bioactive cyclic lipopeptides in Bacillus amyloliquefaciens strain FZB42[J]. Journal of Bacteriology, 2004, 186(4):1084.
[24] Chen X H, Vater J, Piel J, et al. Structural and functional characterization of three polyketide synthase gene clusters in Bacillus amyloliquefaciens FZB42[J]. Journal of Bacteriology, 2006, 188(11):4024.
[25] F?ldes T, Bánhegyi I, Herpai Z, et al. Isolation of Bacillus strains from the rhizosphere of cereals and in vitro screening for antagonism against phytopathogenic, food-borne pathogenic and spoilage micro-organisms[J]. Journal of Applied Microbiology, 2000, 89(5):840-846.
[26] Romero D, De V A, Olmos J L, et al. Effect of lipopeptides of antagonistic strains of Bacillus subtilis, on the morphology and ultrastructure of the cucurbit fungal pathogen Podosphaera fusca[J]. Journal of Applied Microbiology, 2007, 103(4):969–976.
[27] 李宝庆, 鹿秀云, 郭庆港, 等. 枯草芽孢杆菌BAB-1产脂肽类及挥发性物质的分离和鉴定[J].中国农业科学, 2010, 43(17): 3547-3554.
[28] Ongena M, Jacques P. Bacillus lipopeptides: versatile weapons for plant disease biocontrol[J]. Trends in Microbiology, 2008, 16(3):115-125.
[29] Xu Z, Shao J, Li B, et al. Contribution of bacillomycin D in Bacillus amyloliquefaciens SQR9 to antifungal activity and biofilm formation[J]. Applied & Environmental Microbiology, 2013, 79(3):808-815.
[30] Romero D, De V A, Rakotoaly R H, et al. The iturin and fengycin families of lipopeptides are key factors in antagonism of Bacillus subtilis toward Podosphaera fusca[J]. Molecular Plant-microbe Interactions: MPMI, 2007, 20(4):430.
[31] Moyne A L, Shelby R, Cleveland T E, et al. Bacillomycin D: an iturin with antifungal activity against Aspergillus flavus[J]. Journal of Applied Microbiology, 2001, 90(4):622.
[32] 信珊珊.解淀粉芽胞杆菌WH1的发酵优化及其产物对油茶炭疽病和肿瘤的抑制作用研究[D]. 武汉:华中农业大学, 2011.
[33] 信珊珊, 祁高富, 朱发银, 等.1株解淀粉芽孢杆菌发酵条件的优化及其对油茶炭疽病的防效[J]. 华中农业大学学报, 2011, 30(4): 411-415.
[34] 孟庆敏, 周国英, 刘君昂, 等.油茶炭疽病拮抗细菌Y13主要抑菌物质分离纯化及作用方式[J]. 植物保护, 2014, 40(2): 36-42
[35] 龚庆伟.芽孢杆菌抗菌脂肽分离纯化及Bacillomycin D抑制黄曲霉作用的研究[D]. 南京:南京农业大学, 2012.
[36] W G Dilantha F, Ramarathnam R, Krishnamoorthy A S, et al. Identification and use of potential bacterial organic antifungal volatiles in biocontrol[J]. Soil Biology & Biochemistry, 2005, 37(5):955-964.
[37] Kai M, Effmert U, Berg G, et al. Volatiles of bacterial antagonists inhibit mycelial growth of the plant pathogen Rhizoctonia solani[J]. Archives of Microbiology, 2007, 187(5):351-360.
[38] Almenar E, Del V V, Catala R, et al. Active package for wild strawberry fruit (Fragaria vesca L.)[J]. Journal of Agricultural & Food Chemistry, 2007, 55(6):2240-2245.
[39] Raza W, Wang J, Wu Y, et al. Effects of volatile organic compounds produced by Bacillus amyloliquefaciens, on the growth and virulence traits of tomato bacterial wilt pathogen Ralstonia solanacearum[J]. Applied Microbiology & Biotechnology, 2016, 100(17):1-12.