枯草芽胞杆菌M29产抑菌物质培养条件的优化及抑菌物质组成初探

doi:10.16409/j.cnki.2095-039x.2019.06.021

摘要/Abstract

摘要： 前期研究发现枯草芽胞杆菌Bacillus subtilis M29对黄瓜枯萎病具有较好的防治效果，然而其抑菌物质组成和抑菌机制尚不清楚。本文首先确定了菌株M29产抑菌物质的抑菌效果最强时的培养条件，进一步结合热处理、酶处理、基因检测和质谱检测初步分析此抑菌物质的组成。结果表明，以Landy培养基为基础、初始pH为7、培养温度在30℃、培养36～48 h时，菌株M29产生的抑菌物质的抑菌效果最好。菌株M29抑菌物质粗提液的抑菌活性具有热稳定性和酶稳定性。此外，在菌株M29的基因组DNA中扩增出合成fengycin的调控基因fenB，结合质谱检测到Fengycin B的分子量，推测菌株M29分泌物中可能存在脂肽抗生素Fengycin B。因此，枯草芽胞杆菌M29可能通过分泌脂肽抗生素来抑制病原菌生长，通过优化培养条件可以显著提高该菌的抑菌能力，本研究不仅有助于更深入地理解菌株M29的抑菌机制，同时也将为该菌在未来的实际应用提供理论指导。

关键词: 枯草芽胞杆菌Bacillussubtilis M29, 培养条件, 抑菌物质, 脂肽化合物

Abstract: Our previous work found that Bacillus subtilis M29 has a good control effect on cucumber Fusarium wilt. However, the composition and mechanism of its antifungal substances are still unclear. Here, we will screen the optimal culture conditions of this strain, and determine the composition of antifungal substances produced by the strain. We determined the culture conditions based on the antifungal effect of the substances, optimal conditions (e.g., temperature and pH) were selected when the stain displayed the strongest antifungal effect. Then, we analyzed the composition of antifungal substances using heat treatment, enzyme treatment, gene detection and mass spectrometry. The results showed that the antifungal effect of the antifungal substances produced by strain M29 was strongest when Landy-based medium was used with an initial pH of 7, a culture temperature of 30℃ and a culture time of 36-48 h. The crude extract of the antimicrobial substance still had antifungal activity after heat treatment and enzyme treatment. In addition, we amplified the synthetic gene fenB of fengycin from the genome DNA of strain M29 and detected the molecular weight of fengycin B by mass spectrometry. Therefore, we speculate that the lipopeptide antibiotic fengycin B may be present in the secretions of strain M29. We concluded that Bacillus subtilis M29 may inhibit the growth of pathogenic fungal by secreting lipopeptide antibiotics, and the antifungal ability can be significantly improved by optimizing the culture conditions. This study will not only contribute to a better understanding of the antimicrobial mechanism of the strain, but also provide theoretical guidance for the practical application of this strain in the future.

Key words: Bacillus subtilis M29, culture conditions, antifungal substances, lipopeptide compounds

中图分类号:

S476

季冠宁, 汪志鹏, 焦加国, 胡锋, 李辉信. 枯草芽胞杆菌M29产抑菌物质培养条件的优化及抑菌物质组成初探[J]. 中国生物防治学报, 2019, 35(6): 930-939.

JI Guanning, WANG Zhipeng, JIAO Jiaguo, HU Feng, LI Huixin. Optimization of Culture Conditions for Antifungal Substances Produced by Bacillus subtilis M29 and Preliminary Study on the Composition of Antifungal Substances[J]. Chinese Journal Of Biological Control, 2019, 35(6): 930-939.

参考文献

[1] 叶晶晶, 曹宁宁, 吴建梅, 等. 生防芽胞杆菌的应用研究进展[J]. 西北农林科技大学学报(自然科学版), 2014, 42(8):185-190.
[2] Calvo H, Marco P, Blanco D, et al. Potential of a new strain of Bacillus amyloliquefaciens BUZ-14 as a biocontrol agent of postharvest fruit diseases[J]. Food Microbiology, 2017, 63:101-110.
[3] 马佳, 李颖, 胡栋, 等. 芽胞杆菌生物防治作用机理与应用研究进展[J]. 中国生物防治学报, 2018, 34(4):639-648.
[4] 张洁, 汤蒙蒙, 夏明聪, 等. 枯草芽胞杆菌YB-05与申嗪霉素复配防治小麦茎基腐病[J]. 中国生物防治学报, 2018, 34(6):866-872.
[5] Shafi J, Tian H, Ji M. Bacillus species as versatile weapons for plant pathogens:a review[J]. Biotechnology and Biotechnological Equipment, 2017, 31(3):446-459.
[6] Hill D S, Stein J I, Torkewitz N R, et al. Cloning of genes involved in the synthesis of pyrrolnitrin from pseudomonas-fluorescens and role of pyrrolnitrin synthesis in biological-control of plant-disease[J]. Applied and Environmental Microbiology, 1994, 60(1):78-85.
[7] 凡肖, 束长龙, 关雄, 等. 细菌生物被膜在生物防治中的作用[J]. 中国生物防治学报, 2018, 34(5):791-801.
[8] 陈志谊, 刘永峰, 刘邮洲, 等. 植物病害生防芽胞杆菌研究进展[J]. 江苏农业学报, 2012, 28(5):999-1006.
[9] 乔俊卿, 张心宁, 梁雪杰, 等. 枯草芽胞杆菌PTS-394诱导番茄对灰霉病的系统抗性[J]. 中国生物防治学报, 2017, 33(2):219-225.
[10] Shan H, Zhao M, Chen D, et al. Biocontrol of rice blast by the phenaminomethylacetic acid producer of Bacillus methylotrophicus strain BC79[J]. Crop Protection, 2013, 44:29-37.
[11] Liu Y, Cheng Z, Ng T B, et al. Bacisubin, an antifungal protein with ribonuclease and hemagglutinating activities from Bacillus subtilis strain B-916[J]. Peptides, 2007, 28:553-559.
[12] 别小妹, 吕凤霞, 陆兆新, 等. Bacillus subtilis fmbJ脂肽类抗菌物质的分离和鉴定[J]. 生物工程学报, 2006(4):644-649.
[13] 张晓云, 郭庆港, 鹿秀云, 等. 枯草芽胞杆菌NCD-2菌株抑菌蛋白的分离及鉴定[J]. 植物病理学报, 2018, 48(3):395-401.
[14] Mu J, Li X, Jiao J, et al. Biocontrol potential of vermicompost through antifungal volatiles produced by indigenous bacteria[J]. Biological Control, 2017, 112:49-54.
[15] Cao Y, Xu Z, Ling N, et al. Isolation and identification of lipopeptides produced by B. subtilis SQR 9 for suppressing Fusarium wilt of cucumber[J]. Scientia Horticulturae, 2012, 135(1):32-39.
[16] 向亚萍, 陈志谊, 罗楚平, 等. 芽胞杆菌的抑菌活性与其产脂肽类抗生素的相关性[J]. 中国农业科学, 2015, 48(20):4064-4076.
[17] Ye Y F, Li Q Q, Fu G, et al. Identification of antifungal substance (Iturin A2) produced by Bacillus subtilis B47 and its effect on southern corn leaf blight[J]. Journal of Integrative Agriculture, 2012, 11(1):90-99.
[18] Wang J, Liu J, Wang X, et al. Application of electrospray ionization mass spectrometry in rapid typing of fengycin homologues produced by Bacillus subtilis[J]. Letters in Applied Microbiology, 2010, 39(1):98-102.
[19] 孙力军, 陆兆新, 孙德坤. Bacillus amyloliquefaciens ES-2液体发酵抗菌脂肽培养基及其主要影响因子筛选[J]. 食品工业科技, 2008(5):60-63.
[20] 孙沙沙, 夏振远, 吴德喜. 枯草芽胞杆菌CG24发酵条件优化[J]. 云南农业大学学报(自然科学版), 2013, 28(1):36-43.
[21] 张雯, 卞丹, 沈燕秋, 等. 枯草芽胞杆菌抑菌活性物质鉴定、抑菌特性及发酵条件优化[J]. 中国食品学报, 2017, 17(12):105-115.
[22] Moyne A L, Cleveland T E, Tuzun S. Molecular characterization and analysis of the operon encoding the antifungal lipopeptide bacillomycin D[J]. FEMS Microbiology Letters, 2004, 234(1):43-49.
[23] 刘颖, 徐庆, 陈章良. 抗真菌肽LP-1的分离纯化及特性分析[J]. 微生物学报, 1999, 23(5):441-447.
[24] 邹秋霞, 任佐华, 高诗涵, 等. 枯草芽胞杆菌YN145分离鉴定及抑菌活性[J]. 中国生物防治报, 2017, 33(3):421-426.
[25] 王祺, 张一名, 赵君, 等. 枯草芽胞杆菌S-16抑菌活性物质的理化性质及培养条件的研究[J]. 中国生物防治学报, 2015, 31(3):439-444.
[26] Ohno A, Ano T, Shoda M. Effect of temperature change and aeration on the production of antifungal peptide antibiotic iturin by Bacillus subtilis NB22 in liquid cultivation[J]. Journal of Fermentations and Bioengineering, 1993, 75(6):463-465.
[27] Akpa E, Jacques P, Fuchs R, et al. Influence of the culture conditions on lipopeptide production by Bacillus subtilis[C]//Symposium of Biotechnology for Fuels & Chemicals, 2000.
[28] Arima K, Kakinuma A, Tamura G. Surfactin, a crystalline peptidelipid surfactant produced by Bacillus stubilis:isolation, characterization and its inhibition of fibrin clot formation[J]. Biochemical and Biophysical Research Communication, 1968, 31(3):488-494.
[29] 邓建良, 刘红彦, 王鹏涛, 等. 生防芽胞杆菌脂肽抗生素研究进展[J]. 植物保护, 2010, 36(3):20-25.
[30] Cawoy H, Debois D, Franzil L, et al. Lipopeptides as main ingredients for inhibition of fungal phytopathogens by Bacillus subtilis/amyloliquefaciens[J]. Microbial Biotechnology, 2015, 8(2):281-295.
[31] Fan H, Ru J, Zhang Y, et al. Fengycin produced by Bacillus subtilis 9407 plays a major role in the biocontrol of apple ring rot disease[J]. Microbiological Research, 2017, 199:89-97.
[32] Zohora U S, Ano T, Rahman M S. Biocontrol of Rhizoctonia solani K1 by iturin A producer Bacillus subtilis RB14 seed treatment in tomato plants[J]. Advances in Microbiology, 2016, 6(6):424.
[33] Mnif I, Grau-Campistany A, Coronel-León J, et al. Purification and identification of Bacillus subtilis SPB1 lipopeptide biosurfactant exhibiting antifungal activity against Rhizoctonia bataticola and Rhizoctonia solani[J]. Environmental Science and Pollution Research, 2016, 23(7):6690-6699.