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Characterization of the anti-fungal activity of a Bacillus spp. associated with sclerotia from Sclerotinia sclerotiorum


Sclerotinia sclerotiorum fruiting bodies (sclerotia) were found to harbour bacteria that possess anti-fungal activity. Among 1,140 bacterial isolates collected, 32 were found to inhibit the growth of four common fungal pathogens of canola, S. sclerotiorum, Rhizoctonia solani, Alternaria brassicae and Leptosphaeria maculans. One of these broad-spectrum isolates, LEV-006, was found to be closely related to Bacillus subtilis based on 16S rRNA analysis. The anti-fungal activities were purified and found to be associated with a low molecular weight peptide complex consisting mostly of the cyclic lipopeptide fengycin A and B, as revealed by matrix-assisted laser desorption/ionization time-of-flight and post-source decay analysis, as well as two proteins of 20 and 55 kDa. Peptide mass fingerprinting revealed that the 55-kDa protein was similar to vegetative catalase 1; however, when the enzyme was expressed in Escherichia coli, it exhibited catalase but not anti-fungal activity. The sequences of several peptides from the 20-kDa protein were obtained and indicated that it was a unique anti-fungal protein.

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  1. Bechard J, Eastwell KC, Sholberg PL, Mazza G, Skura B (1998) Isolation and partial characterization of an antimicrobial peptide produced by a strain of Bacillus subtilis. J Agric Food Chem 46:5355–5361

  2. Boland GJ, Inglis GD (1989) Antagonism of white mold (Sclerotinia sclerotiorum) of bean by fungi from bean and rapeseed flowers. Can J Bot 67:1775–1781

  3. Boyetchko SM (1999) Biological control agents of canola and rapeseed diseases—status and practical approaches. In: Mukerji KG, Chamola BP, Upadhyay K (eds) Biotechnological approaches in biocontrol of plant pathogens. Kluwer/Plenum, New York, pp 51–71

  4. Chan YK, McCormick WA, Seifert KA (2003) Characterization of an antifungal soil bacterium and its antagonistic activities against Fusarium species. Can J Microbiol 49:253–262

  5. Dufour M, Simmonds RS, Bremer PJ (2003) Development of a method to quantify the synergistic activity of natural antimicrobials. Int J Food Microbiol 85:249–258

  6. During K, Porsch P, Mahn A, Brinkmann O, Gieffers W (1999) The non-enzymatic microbicidal activity of lysozymes. FEBS Lett 449:93–100

  7. Fiddaman PJ, Rossall S (1993) The production of antifungal volatiles by Bacillus subtilis. J Appl Bacteriol 74:119–126

  8. Inglis GD, Boland GJ (1992) Evaluation of filamentous fungi isolated from petals of bean and rapeseed for suppression of white mold. Can J Microbiol 38:124–129

  9. Jenny K, Kappeli O, Fiechter A (1991) Biosurfactants from Bacillus licheniformis: structural analysis and characterization. Appl Microbiol Biotechnol 36:5–13

  10. Kharbanda PD, Tewari JP (1996) Integrated management of canola diseases using cultural methods. Can J Plant Pathol 18:168–175

  11. Kim PI, Chung KC (2004) Production of an antifungal protein for control of Colletotrichum lagenarium by Bacillus amyloliquefaciens MET0908. FEMS Microbiol Lett 234:177–183

  12. Kloepper JW, Ryu CM, Zhang S (2004) Induced systemic resistance and promotion of plant growth by Bacillus spp. Phytopathology 94:1259–1265

  13. Lin SC, Carswell KF, Sharma MM, Georgiou G (1994) Continuous production of the lipopetide biosurfactant from Bacillus licheniformis JF-2. Appl Microbiol Biotechnol 44:281–285

  14. Lin D, Qu LJ, Gu H, Chen Z (2001) A 3.1-kb genomic fragment of Bacillus subtilis encodes the protein inhibiting growth of Xanthomonas oryzae pv. oryzae. J Appl Microbiol 91:1044–1050

  15. Marten P, Smalla K, Berg G (2000) Genotypic and phenotypic differentiation of an antifungal biocontrol strain belonging to Bacillus subtilis. J Appl Microbiol 89:463–471

  16. Moyne AL, Shelby R, Cleveland TE, Tuzun S (2001) Bacillomycin D: an iturin with antifungal activity against Aspergillus flavus. J Appl Microbiol 90:622–629

  17. Mulligan CN, Gibbs BF (1990) Recovery of biosurfactants by ultrafiltration. J Chem Technol Biotech 47:23–29

  18. Ongena M, Duby F, Jourdan E, Beaudry T, Jadin V, Dommes J, Thonart P (2005) Bacillus subtilis M4 decreases plant susceptibility towards fungal pathogens by increasing host resistance associated with differential gene expression. Appl Microbiol Biotechnol 67:692–698

  19. Pedras MS, Ismail N, Quail JW, Boyetchko SM (2003) Structure, chemistry, and biological activity of pseudophomins A and B, new cyclic lipodepsipeptides isolated from the biocontrol bacterium Pseudomonas fluorescens. Phytochemistry 62:1105–1114

  20. Pelligrini A, Hulsmeier AJ, Hunziker P, Thomas U (2004) Proteolytic fragments of ovalbumin display antimicrobial activity. Biochim Biophys Acta 1672:76–85

  21. Perna NT, Plunkett G III, Burland V, Mau B, Glasner JD, Rose DJ, Mayhew GF, Evans PS, Gregor J, Kirkpatrick HA, Posfai G, Hackett J, Klink S, Boutin A, Shao Y, Miller L, Grotbeck EJ, Davis NW, Lim A, Dimalanta ET, Potamousis KD, Apodaca J, Anantharaman TS, Lin J, Yen G, Schwartz DC, Welch RA, Blattner FR (2001) Genome sequence of enterohaemorrhagic Escherichia coli O157:H7. Nature 409:463–466

  22. Quail JW, Ismail N, Pedras MS, Boyetchko SM (2002) Pseudophomins A and B, a class of cyclic lipodepsipeptides isolated from a Pseudomonas species. Acta Crystallogr C58:o268–o271

  23. Sambrook J, Russell DW (2003) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, New York

  24. Shoda M, Ano T (1994) Basic analysis of Bacillus subtilis NB22 and its application to biological control. Bioprocess Technol 19:641–664

  25. Sonenchein AL, Hoch JA, Losick R (2002) Bacillus subtilis and its closest relatives: from genes to cells. In: Sonenshein AL, Hoch JA, Losick R (eds) Bacillus subtilis and other gram-positive bacteria: biochemistry, physiology, and molecular genetics. American Society for Microbiology, Washington, DC

  26. Souto GI, Correa OS, Montecchia MS, Kerber NL, Pucheu NL, Bachur M, García AF (2004) Genetic and functional characterization of a Bacillus sp. strain excreting surfactin and antifungal metabolites partially identified as iturin-like compounds. J Appl Microbiol 97:1247–1256

  27. Stein T (2005) Bacillus subtilis antibiotics: structures, synthesis and specific functions. Mol Microbiol 56:845–857

  28. Touré Y, Ongena M, Jacques P, Guiro A, Thonart P (2004) Role of lipopeptides produced by Bacillus subtilis GA1 in the reduction of grey mould disease caused by Botrytis cinerea on apple. J Appl Microbiol 96:1151–1160

  29. Tschen JSM, Kuo WL (1985) Antibiotic inhibition and control of Rhizoctonia solani by Bacillus subtilis. Plant Prot Bull 27:95–103

  30. Turner JT, Backman PA (1991) Factors relating to peanut yield increases after seed treatment with Bacillus subtilis. Plant Dis 75:347–353

  31. Vater J, Kablitz B, Wilde C, Franke P, Mehta N, Cameotra SS (2002) Matrix assisted laser adsorption ionization-time of flight mass spectrometry of lipopeptide biosurfactants in whole cells and culture filtrates of Bacillus subtilis C-1 isolated from petroleum sludge. Appl Environ Microbiol 68:6210–6219

  32. Wang SY, Wu SJ, Thottappilly G, Locy RD, Singh NK (2001) Molecular cloning and structural analysis of the gene encoding Bacillus cereus exochitinase Chi36. J Biosci Bioeng 92:59–66

  33. Wang J, Liu J, Wang X, Yao J, Yu Z (2004) Application of electrospray ionization mass spectrometry in rapid typing of fengycin homologues produced by Bacillus subtilis. Lett Appl Microbiol 39:98–102

  34. Wintzingerode VF, Selent B, Hegemann W, Gobel UB (1999) Phylogenetic analysis of an anaerobic, trichlorobenzene-transforming microbial consortium. Appl Environ Microbiol 65:283–286

  35. Zuber P, Nakano NM, Mahariel MA (1993) Peptide antibiotics. In: Sonenshein AL, Hoch JA, Losick R (eds) Bacillus subtilis and other gram-positive bacteria: biochemistry, physiology, and molecular genetics. American Society for Microbiology, Washington, DC, pp 897–916

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This work was supported by a grant from the Saskatchewan Agricultural Development Fund. We are also grateful to C. Matsalla, D. Baldwin, D. Sutherland and W. Taylor for their technical assistance and advice.

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Correspondence to Dwayne Hegedus.

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Hou, X., Boyetchko, S.M., Brkic, M. et al. Characterization of the anti-fungal activity of a Bacillus spp. associated with sclerotia from Sclerotinia sclerotiorum . Appl Microbiol Biotechnol 72, 644–653 (2006).

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  • Bacterial Isolate
  • Biological Control Agent
  • Surfactin
  • Lipopeptides
  • Rhizoctonia Solani