Scientia Agricultura Sinica ›› 2015, Vol. 48 ›› Issue (20): 4064-4076.doi: 10.3864/j.issn.0578-1752.2015.20.008

• PLANT PROTECTION • Previous Articles     Next Articles

The Antifungal Activities of Bacillus spp. and Its Relationship with Lipopeptide Antibiotics Produced by Bacillus spp.

 XIANG Ya-ping1,2, CHEN Zhi-yi1,2, LUO Chu-ping1,2, ZHOU Hua-fei1,2, LIU Yong-feng2
  

  1. 1College of Plant Protection, Nanjing Agricultural University, Nanjing 210095
    2Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014
  • Received:2015-04-13 Online:2015-10-20 Published:2015-10-20

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Abstract:

【Objective】 The objective of this study is to clear the inhibition effect of 55 Bacillus spp. strains to pathogenic fungi, Rhizoctonia cerealis, Fusarium graminearum and Fusarium oxysporum f. sp. niveum, and the qualitative and quantitative relationship withlipopeptide antibiotics produced by the Bacillus strains.【Method】Inhibition rates of the 55 Bacillus spp. strains and their crude lipopeptides extracts against three pathogenic fungi were tested, respectively, by dual culture on plate. Three pairs of primers designed based on the known lipopeptide genes were used to amplify the corresponding genes, srfA, ituA and fenA, from the genome of the 55 strains. The lipopeptides antibiotics were identified by high performance liquid chromatography (HPLC) and mass spectrum (MS) techniques.【Result】 The 55 strains of Bacillus spp. and their crude lipopeptides extracts had certain biological activities to the tested pathogenic fungi. The crude lipopeptides extracts of 16.5% strains had higher antifungal activities than the Bacillus strains in vivo, the antifungal activities against the pathogenic fungi of crude lipopeptides extracts of 55.8% strains were weaker than living cells of the strains in vivo, while the antifungal activities of 27.7% strains did not change basically. The results of PCR amplification showed that among the 55 Bacillu strains, the genomes of 89.1% Bacillus strains contained srfA, 87.3% strains contained ituA, only 56.4% strains contained fenA. Among the 55 Bacillus strains, 53 Bacillus strains were identified with surfactins in the crude lipopeptides extracts by the MS analysis, 48 strains were identified with iturins, only Bs916 (B. subtilis 916) was identified having fenfycins in the crude lipopeptides extracts. Those strains that were not identified having the three kinds of lipopeptides had weaker or no antifungal activity. There were 48 strains which were found to have iturins all identified with bacillomycin D, only 2 strains were identified with bacillomycin L, 12 strains were identified with bacillomycin F, iturin A or mycosubtilin. Among the 55 Bacillus strains, 53 strains were detected with suefactins by HPLC, 51 strains were detected with iturins, only Bs916 strain was detected with fengycins and 2 strains were not detected with the 3 kinds of lipopeptide antibiotics. According to the time and type of chromatographic peak, iturins were divided into 4 types, named as types 1, 2, 3, and 4. The antifungal activities intensity of 4 types of iturins were type 1＞type 2＞type 3＞type 4. There were two unknown lipopeptides (named as Unknowns: Unknown1, 2 ) which were speculated as new lipopeptides were detected with MS analysis in 3 strains JCC-1, and JCC-9 and JCC-18. 【Conclusion】 Lipopeptides antibiotics produced by Bacillus spp. were the main composition against plant pathogenic fungi and had diversity characteristics. The structure changes and secretion of iturins had certain relevance to antifungal activities. There were some unknown new resisted resources in lipopeptides compounds from Bacillus spp..

Key words: Bacillus spp., antifungal activity, lipopeptides, correlation

[1]    Tsuge K, Akiyama T, Shoda M. Cloning, sequencing and characterization of the iturin A operon. Journal of Bacteriology, 2001, 183(21): 6265-6273.
[2]    Moyne A L, Cleveland T E, Tuzun S. Molecular characterization and analysis of the operon encoding the antifugal lipopeptide bacillomycin D. FEMS Microbiology Letters, 2004, 234(1): 43-49.
[3]    Koumoutsi A, Chen X H, Henne A, Liesegang H, Hitzeroth G, Franke P, Vater J, Borriss R. Structural and functional characterization of gene clusters directing nonribosomal synthesis of bioactive cyclic lipopeptides in Bacillus amyloliquefaciens strain FZB42. Journal of Bacteriology, 2004, 186(4): 1084-1096.
[4]    Chen X H, Vater J, Piel J, Franke P, Scholz R, Scheider K, Koumoutsi A, Hitzeroth G, Grammel N, Strittmatter A W, Gottschalk G, Süssmuth R D, Borriss R. Structural and functional characterization of three polyketide synthase gene clusters in Bacillus amyloliquefaciens strains FZB42. Journal of Bacteriology, 2006, 188(11): 4024-4036.
[5]    王帅. 芽孢杆菌及其脂肽类化合物防治植物病害和促进植物生长的研究[D]. 南京: 南京农业大学, 2009.
Wang S. Plant growth promotion and control of plant disease with Bacillus spp. and lipopeptide[D]. Nanjing: Nanjing Agricultural University, 2009. (in Chinese)
[6]    王帅, 高圣风, 高学文, 王娜, 王光强. 枯草芽孢杆菌脂肽类抗生素发酵和提取条件. 中国生物防治, 2007, 23(4): 342-347.
Wang S, Gao S F, Gao X W, Wang N, Wang G Q. Fermentation optimization in lipopetide productivity of Bacillus subtilis G1. Chinese Journal of Biological Control, 2007, 23(4): 342-347. (in Chinese)
[7]    陈华, 袁成凌, 蔡克周, 郑之明, 余增亮. 枯草芽孢杆菌JA产生脂肽类抗生素-iturinA的纯化及电喷雾质谱鉴定. 微生物学报, 2008, 48(1): 116-120.
Chen H, Yuan C L, Cai K Z, Zheng Z M, Yu Z L. Purification and identification of iturin A from Bacillus subtilis JA by electrospray ionization mass spectrometry. Acta Microbiologica Sinica, 2008, 48(1): 116-120. (in Chinese)
[8]    高学文, 姚仕义, Pham H, Vater J, 王金生. 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. 微生物学报, 2003, 43(5): 647-652.
Gao X W, Yao S Y, Pham H, Vater J, Wang J S. Purification and identification of surfactin isoforms produced by Bacillus subtilis B2 strain. Acta Microbiologica Sinica, 2003, 43(5): 647-652. (in Chinese)
[9]    Romero D, Pérez-García A, Rivera M E, Cazorla F M, De Vicente A. Isolation and evaluation of antagonistic bacteria towards the cucurbit powdery mildew fungus Podosphaera fusca. Applied Microbiology and Biotechnology, 2004, 64: 263-269.
[10]   Romero D, De Vicente A, Rakotoaly R H, Dufour S E, Veening J W, Arrebola E, Cazorla F M, Kuiper O P, Paquot M,Pérez-García A. The iturin and fengycin families of lipopeptides are key factors in antagonism of Bacillus subtilis toward Podosphaera fusca. Molecular Plant- Microbe Interactions, 2007, 20(4): 430-440.
[11]   Zeriouh H, Romero D, Garcia-Gutierrez L, Cazorla F M,de Vicente A,Pérez-García A.The iturin-like lipopeptides are essential components in the biological control arsenal of Bacillus subtilis against bacterial diseases of cucurbits. Molecular Plant-Microbe Interactions, 2011,24(12):1540-1552.
[12]   罗楚平, 刘邮洲, 吴荷芳, 王晓宇, 刘永锋, 聂亚锋, 张荣胜, 陈志谊. 脂肽类化合物bacillomycin L抗真菌活性及其对水稻病害的防治. 中国生物防治学报, 2011, 27(1): 76-81.
Luo C P, Liu Y Z, Wu H F, Wang X Y, Liu Y F, Nie Y F, Zhang R S, Chen Z Y. Antifungal activity and rice disease biocontrol performance of lipopeptide antibiotic bacillomycin L. Chinese Journal of Biological Control, 2011, 27(1): 76-81. (in Chinese)
[13]   Meena K R, Kanwar S S. Lipopeptides as the antifungal and antibacterial agents: applications in food safety and therapeutics. BioMed Research International, 2015, 2015: Article ID 473050.
[14]   Gordillo M A, Maldonado M C. Purification of peptides fromBacillus strainswith biological activity. Chromatography and Its Applications, 2012, 11: 201-225.
[15]   Luo C, Liu X, Zhou H, Wang X, Chen Z. Nonribosomal peptide synthase gene clusters for lipopeptide biosynthesis in Bacillus subtilis 916 and their phenotypic functions. Applied and Environmental Microbiology, 2015, 81(1): 422-431.
[16]   Lee H, Churey J J, Worobo R W. Purification and structural characterization of bacillomycin F produced by a bacterial honey isolate active against Byssochlamys fulva H25. Journal of Applied Microbiology, 2008, 105: 663-673.
[17]   Duitman E H, Hamoen L W, Rembold M, Venema G, Seitz H. The mycosubtilin synthetase of Bacillus subtilis ATCC6633: A multifunctional hybrid between a peptide synthetase, an amino transferase, and a fatty acid synthase. Proceedings of the National Academy of Sciences of the United States of America, 1999, 96(23): 13294-13299.
[18]   罗楚平, 王晓宇, 周华飞, 刘邮洲, 陈志谊. 生防菌Bs916合成脂肽抗生素泛革素的操纵子结构功能及生物活性. 中国农业科学, 2013, 46(24): 5142-5149.
Luo C P, Wang X Y, Zhou H F, Liu Y Z, Chen Z Y. The operon, structure and biological activities of the lipopeptide fengycin produced by Bacillus subtilis 916. Scientia Agricultura Sinica, 2013, 46(24): 5142-5149. (in Chinese)
[19]   Vater J, Gao X W, Hitzeroth G, Wilde C, Franke P. “Whole cell”-matrix-assisted laser desorption ionization-time of flight-mass spectrometry, an emerging technique for efficient screening of biocombinatorial libraries of natural compounds-present state of research. Combinatorial Chemistry & High Throughput Screening, 2003, 6(6): 557-567.
[20]   何美玉. 现代有机与生物质谱. 北京: 北京大学出版社, 2002.
He M Y. Modern Organic and Biological Mass Spectrometry. Beijing: Beijing University Press, 2002. (in Chinese)
[21]   Kopp F, Marahiel M A. Macrocyclization strategies in polyketide and nonribosomal peptide biosynthesis. Natural Product Reports, 2007, 24: 735-749.
[22]   Raaijmakers J M, Vlami M, De Souza J T. Antibiotic production by bacterial biocontrol agents. Antonie van Leeuwenhoek, 2002, 81(1/4): 537-547.
[23]   Ongena M, Jacques P, Touré Y, Destain J, Jabrane A, Thonart P. Involvement of fengycin-type lipopeptides in the multifaceted biocontrol potential of Bacillus subtilis. Applied Microbiology and Biotechnology, 2005, 69(1): 29-38.
[24]   Zhao X, Zhou Z J, Han Y, Wang Z Z, Fan J, Xiao H Z. Isolation and identification of antifungal peptides fromBacillus BH072, a novel bacterium isolated from honey.Microbiological Research, 2013, 168(9): 598-606.
[25]   李荣. 枯草芽孢杆菌R31对香蕉枯萎病生防机制研究[D]. 武汉: 华中农业大学, 2013.
Li R. Investigation on the biocontrol mechenism of Bacillus subtilis strain R31 against banana fusarium wilt[D]. Wuhan: Huazhong Agricultural University, 2013. (in Chinese)
[26]   Zhao P, Quan C, Jin L, Wang L, Guo X, Fan S. Sequence characterization and computational analysis of the non-ribosomal peptide synthetases controlling biosynthesis of lipopeptides, fengycins and bacillomycin D, from Bacillus amyloliquefaciens Q-426. Biotechnology Letters, 2013, 35: 2155-2163.
[27]   Stein T. Bacillus subtilis antibiotics: structures, syntheses and specific functions. Molecular Microbiology, 2005, 56(4): 845-857.
[28]   Hamoen L W, Venema G, Kuipers O P. Controlling competence in Bacillus subtilis: shared use of regulators. Microbiology, 2003, 149: 9-17.
[29]   Lazazzera B A, Solomon J M, Grossman A D. An exported peptide functions intracellularly to contribute to cell density signaling in B. subtilis. Cell, 1997, 89: 917-925.
[30]   Ongena M, Jacques P. Bacillus lipopeptides: versatile weapons for plant disease biocontrol. Trends in Microbiology, 2007, 16(3): 115-125.
[31]   Kunst F, Ogasawara N, Moszer I, Albertini A M, Alloni G, Azevedo V. The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature, 1997, 390: 249-256.
[32]   Nakano M M, Corbell N, Besson J, Zuber P. Isolation and characterization of sfp: a gene that functions in the production of the lipopeptide biosurfactant, surfactin, in Bacillus subtilis. Molecular Genetics and Genomics, 1992, 232: 313-321.
[33]   Tsuge K, Ano T, Hirai M, Nakamura Y, Shoda M. The genes degQ, pps, and lpa-8 (sfp) are responsible for conversion of Bacillus subtilis 168 to plipastatin production. Antimicrob Agents Chemother, 1999, 43: 2183-2192.
[34]   Joshi R, McSpadden G B. Identification and characterization of novel genetic markers associated with biological control activities in Bacillus subtilis. Phytopathology, 2006, 96(2): 145-154.
[35]   罗楚平, 陈志谊, 刘永锋, 张杰, 刘邮洲, 王晓宇, 聂亚锋. 生防菌Bs-916合成脂肽类化合物Bac操纵子突变株构建及功能. 微生物学报, 2009, 49(4): 445-452.
Luo C P, Chen Z Y, Liu Y F, Zhang J, Liu Y Z, Wang X Y, Nie Y F. Construction and function analysis of Bac operon mutants of bio-control strain Bacillus subtilis Bs-916. Acta Microbiologica Sinica, 2009, 49(4): 445-452. (in Chinese)
[36]   Ajesh K, Sudarslal S, Arunan C, Sreejith K. Kannurin, a novel lipopeptide fromBacillus cereus strain AK1: isolation, structural evaluation and antifungal activities.Journal of Applied Microbiology, 2013, 115(6): 1287-1296.
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