The Iturin Lipopeptides as Key Compounds in Antagonism of Bacillus subtilis UTB96 toward Aspergillus flavus

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Abstract

Members of the genus Bacillus are among the most promising candidates for biological control of plant pathogenic bacteria. In this study, cell free supernatants of three strains B. subtilis UTB96, B. subtilis UTB1 and B. subtilis UTB70 against Aspergillus flavus R5 were investigated using dual culture assay. Strain B. subtilis UTB96 showed the highest inhibition effect. Thin lyer chromatography in extracted lipopeptides of the bacterial cell free supernatants proved the presence of the three families of lipopeptides iturin, fengycin and surfactin. Bioautography analysis of the chromatogarams against A. flavus R5 revealed the antifungal potential of iturins in strain B. subtilis UTB96. Gene bymB (the third gene in iturin biosynthesis) was disrupted usisng targeted mutagenesis to confirm role of iturins in antifungal activity. Molecular study (PCR analysis and sequencing the amplified fragment) of the mutants revealed that the targeted gene (bmyB) was replaced by spectinomycin resistance gene, hence the gene was disrupted. The phenotypical comparison of the bmyB mutants with the parental strain demonstrated that the antifungal activity of cell free supernatants and extracted lipopeptides was decreased interestingly in a dual culture assay with A. flavus, bioautography analysis and also on pistachio nuts. To sum up, these findings allowed us to conclude that iturin-like lipopeptides are indispensable for the biocontrol ability of the strain UTB96 against A. flavus R5.

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Ahimou F, Jacques P, Deleu M (2000) Surfactin and iturin A effects on Bacillus subtilis surface hydrophobicity. Enzyme and Microbial Technology 27: 749-754.
Amaike S, Keller NP (2011) Aspergillus flavus. Annual Review of Phytopathology 49: 107-133.
Arrebola E, Jacobs R, Korsten L (2010) Iturin A is the principal inhibitor in the biocontrol activity of Bacillus amyloliquefaciens PPCB004 against postharvest fungal pathogens. Journal of Applied Microbiology 108: 386-395.
Arguelles-Arias A, Ongena M, Halimi B, Lara Y, Brans A, Joris B, Fickers P (2009) Bacillus amyloliquefaciens GA1 as a source of potent antibiotics and other secondary metabolites for biocontrol of plant pathogens. Microbial Cell Factories 8: 63.
Asaka O, Shoda M (1996) Biocontrol of Rhizoctonia solani damping-off of tomato with Bacillus subtilis RB14. Applied and Environmental Microbiology 62: 4081-4085.
Avis TJ, Bélanger RR (2002) Mechanisms and means of detection of biocontrol activity of Pseudozima yeast against plant-pathogenic fungi. FEMS Yeast Research 2: 5-8.
Cho KM, Math RK, Hong SU, Asraful Islam SM, Mandanna DK et al. (2009) Iturin produced by Bacilluspumilus HY1 from Korean soybean sauce (kanjang) inhibits growth of aflatoxin producing fungi. Food Control 20: 402-406.
Cotty PJ, Mellon JE (2006) Ecology of aflatoxin producing fungi and biocontrol of aflatoxin contamination. Mycotoxin Research 22: 110-117.
DeLucca AJ, Bland JM, Jacks TJ, Grimm C, Cleveland TE, Walsh TJ (1997) Fungicidal activity of cecropin A. Antimicrobial Agents and Chemotherapy 41: 481-483.
Dorner JW (2004) Biological control of aflatoxin contamination of crops. Journal of Toxicology 23: 425-450.
Droby S (2006) Improving quality and safety of fresh fruits and vegetables after harvest by the use of biocontrol agents and natural materials. ISHS Acta Horticulturae 709: 45-51.
Dvorockova I (1999) Aflatoxins and human health. CRC Press. Boca Raton.
 EC (European Commission) (2010) Commission regulation (EC) no. 178/2010 of 2 March 2010 amending regulation (EC) No 401/2006 as regards groundnuts (peanuts), other oilseeds, tree nuts, apricot kernels, liquoric and vegetable oil. Official Journal of the European Union, L52, 32-43, 3.3.2010
Fravel DR (2005) Commercialization and implementation of biocontrol. Annual Review of Phytopathology  43: 337-359.
Hesseltine CW (1965) A millennium of fungi, food, and fermentation. Mycologia 57: 149-197.
Janisiewicz W, Korsten L (2002) Biological control of postharvest diseases of fruit. Annual Review of Phytopathology 40: 411-441.
Kildea S, Ransbotyn V, Khan MR, Fagan B, Leonard G, Mullins E, Doohan FM (2008) Bacillus megaterium shows potential for the biocontrol of Septoria tritici blotch of wheat. Biological Control 47: 37-45.
Klich MA, Lax AR, Bland JM, Scharfenstein LL (1993) Influence of iturin A on mycelial weight and aflatoxin production by Aspergillus flavus and Aspergillus parasiticus in shake culture. Mycopathologia 123: 35-38.
Koumoutsi A, Chen, XH, Henne A, Liesegang H, Hitzeroth G, Franke P, Vater J, Borriss R (2004). Structural and functional characterization of gene clusters directing nonribosomal synthesis of bioactive cyclic lipopeptides in Bacillus amyloliquefaciens strain FZB42. Journal of Bacteriology 186: 1084-1096.
Krebs B, Höding B, Kübart S, Workie MA, Junge H, Schmiedeknecht G, Grosch R, Bochow H, Hevesi M (1998) Use of Bacillus subtilis as biological control agent. I. Activities and characterisation of Bacillus subtilis strains. Journal of Plant Diseases and Protection 105: 181-197.
Latoud C, Peypoux F, Michel G (1990) Interaction of iturin A, a lipopeptide antibiotic, with Saccharomyces cerevisiae cells: Influence of the sterol membrane composition. Canadian Journal of Microbiology 36: 384-389.
Maget-Dana R, Thimon L, Peypoux F, Ptak M (1992) Surfactin/iturin A interactions may explain the synergistic effect of surfactin on the biological properties of iturin A. Biochimie 74: 1047-1051.
Malina A, Shai Y (2005) Conjugation of fatty acids with different lengths modulates the antibacterial and antifungal activity of a cationic biologically inactive peptide Biochemical Journal 390: 695-702.
Mojtahedi H, Rabie CJ, Lubben A, Steyn M, Danesh D (1979) Toxic Aspergilli from pistachio nuts. Mycopathologia67: 123-127.
Moyne AL, Shelby R, Cleveland TE, Tuzun S (2001) Bacillomycin D: an iturin with antifungal activity against Aspergillus flavus. Journal of Applied Microbiology 90: 622-629.
Moyne AL, Cleveland TE, Tuzun S (2004) Molecular characterization and analysis of the operon encoding the antifungal lipopeptide bacillomycin D. FEMS Microbiology Letters 234: 43-49.
Ongena M, Henry G, Thonart P (2009) The roles of cyclic lipopeptides in the biocontrol activity of Bacillus subtilis, in Gisi, U., Chet, L., Gullino, M. L.  (Eds.), Recent developments in management of plant diseases, plant pathology in the 21st century. (Paper presented at the 9th International Congress of Plant Pathology, Berlin).
Ongena M, Jacques P (2008) Bacillus lipopeptides: Versatile weapons for plant disease control. Trends in Microbiology  16: 115-125.
Ono M, Kimura N (1991) Antifungal peptides produced by Bacillus subtilis for the biological control of aflatoxin contamination. Proceedings of Japan Association Mycotoxicology 34: 23-28.
Pérez-García A, Romero D, de Vicente A (2011) Plant protection and growth stimulation by microorganisms: Biotechnological applications of Bacilli in agriculture. Current Opinion in Biotechnology  22: 187-193.
Pittet A (1998) Natural occurrence of mycotoxins in foods and feeds- an updated review. Revue de Medecine Veterinaire 149: 479-492.
Quentin MJ, Besson F, Peypoux F, Michel G (1982) Action of peptidolipidic antibiotics of the iturin group on erythrocytes. Effect of some lipids on hemolysis. Biochimica et Biophysica Acta  684: 207-211.
Razafindralambo H, Paquot M, Hbid C, Jacques P, Destain J, Thonart P (1993) Purification of antifungal lipopeptides by reversed-phase high performance liquid chromatography. Journal of Chromatography A  639: 81-85.
Romero D, de Vicente A, Olmos JL, Dávila JC, Pérez-García A (2007a) Effect of lipopeptides of antagonistic strains of Bacillus subtilis on the morphology and ultrastructure of the cucurbit fungal pathogen Podosphaera fusca. Journal of Applied Microbiology 103: 969-976.
Romero D, de Vicente A, Rakotoalay RH, Dufour SE, Veening JW, Arrebola A, Cazorla FM, Kuipers OP, Paquot M, Pérez-García A (2007b) The iturin and fengycin families of lipopeptides are key factors in antagonism of Bacillus subtilis toward Podosphaera fusca. Molecular Plant-Microbe Interactions Journal  20: 430-440.
Romero D, Pe´rez-Garcı´a A, Rivera ME, Cazorla FM, de Vicente A (2004) Isolation and evaluation of antagonistic bacteria towards the cucurbit powdery mildew fungus Podosphaera fusca. Applied Microbiology and Biotechnology 64: 263-269.
Sinha KK, Bhatnagar D (1998) Mycotoxin in agriculture and food safety. New York: Marcel Dekker Inc.
Shoda M (2000) Bacterial control of plant diseases. Journal of Bioscience and Bioengineering  89: 515-521.
Spurr H, Jr W (1981) Introduction of microbial antagonists for the control of foliar plant pathogens.  Pages 323-332. In Biological Control in Crop Production (BARC Symposium No.5).  G.C.  Papavizas, [ed.], Allanheld, Osum Co., Totowa, N. J.
Stein T (2005) Bacillus subtilis antibiotics: structures, syntheses and specific functions. Molecular Microbiology  56: 845-857.
Vanittanakom N, Loeffler W, Koch U, Jung G (1986) Fengycin -a novel antifungal lipopeptide antibiotic produced by Bacillus subtilis F-29-3. Journal of Antibiotics 39: 888-901.
Wulff EG, Mguni CM, Mansfeld-Giese K, Fels J, Lübeck M, Hockenhull J (2002) Biochemical and molecular characterization of Bacillus amyloliquefaciens, B. subtilis and B. pumilus isolates with distinct antagonistic potential against Xanthomonas campestris pv. campestris. Plant Patholology51: 574-584.
Yánez-Mendizábal V,  Zeriouh H, Viñas I, Torres R, Usall J, de Vicente A, Pérez-García A, Teixidó N (2011) Biological control of peach brown rot (Monilinia spp.) by Bacillus subtilis CPA-8 is based on production of fengycin-like lipopeptides. Eur. Journal of Plant Pathology 132: 609-619.
Yazgan A, Ozcengiz G, Marahiel A (2001) Tn10 insertional mutations of Bacillus subtilis that block the biosynthesis of bacilysin. Biochimica et Biophysica Acta  1518: 87-94.
Yu GY, Sinclair JB, Hartman GL, Bertagnolli BL (2002) Production of iturin A by Bacillus amyloliquefaciens suppressing Rhizoctonia solani. Soil Biology and Biochemistry 7: 955-963.
Zeriouh H, Romero D, García-Gutiérrez L, Cazorla FM, de Vicente A, Pérez-García A (2011) The iturin-like lipopeptides are essential components in the biological control arsenal of Bacillus subtilis against bacterial diseases of cucurbits. Molecular Plant-Microbe Interactions Journal 24: 1540-1552.
Zhang T, Shi ZQ, Hu LB, Cheng LG, Wang F (2008). Antifungal compound from Bacillus subtilis B-FS06 inhibiting the growth of Aspergillus flavus. World Journal of Microbiology and Biotechnology 24: 783-788.

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