Journal of Chemical Ecology

, Volume 44, Issue 4, pp 374–383 | Cite as

Fungal Competitors Affect Production of Antimicrobial Lipopeptides in Bacillus subtilis Strain B9–5

  • Stefanie DeFilippi
  • Emma Groulx
  • Merna Megalla
  • Rowida Mohamed
  • Tyler J. Avis


Bacillus subtilis has shown success in antagonizing plant pathogens where strains of the bacterium produce antimicrobial cyclic lipopeptides (CLPs) in response to microbial competitors in their ecological niche. To gain insight into the inhibitory role of these CLPs, B. subtilis strain B9–5 was co-cultured with three pathogenic fungi. Inhibition of mycelial growth and spore germination was assessed and CLPs produced by B. subtilis B9–5 were quantified over the entire period of microbial interaction. B. subtilis B9–5 significantly inhibited mycelial growth and spore germination of Fusarium sambucinum and Verticillium dahliae, but not Rhizopus stolonifer. LC-MS analysis revealed that B. subtilis differentially produced fengycin and surfactin homologs depending on the competitor. CLP quantification suggested that the presence of Verticillium dahliae, a fungus highly sensitive to the compounds, caused an increase followed by a decrease in CLP production by the bacterium. In co-cultures with Fusarium sambucinum, a moderately sensitive fungus, CLP production increased more gradually, possibly because of its slower rate of spore germination. With co-cultures of the tolerant fungus Rhizopus stolonifer, B. subtilis produced high amounts of CLPs (per bacterial cell) for the duration of the interaction. Variations in CLP production could be explained, in part, by the pathogens’ overall sensitivities to the bacterial lipopeptides and/or the relative growth rates between the plant pathogen and B. subtilis. CLP production varied substantially temporally depending on the targeted fungus, which provides valuable insight concerning the effectiveness of B. subtilis B9–5 protecting its ecological niche against the ingress of these pathogens.


Antimicrobials Bacillus subtilis Fengycin Fungal pathogens Phytopathogen Plant disease Lipopeptides Surfactin 



The authors thank Dr. J. David Miller for use of LC-MS and Blake Green for technical assistance. This work was supported by research grant RGPIN-2015-05679 from the Natural Sciences and Engineering Research Council (NSERC) of Canada to T.J.A.


  1. Akpa E, Jacques P, Wathelet B, Paquot M, Fuchs R, Budzikiewicz H, Thonart P (2001) Influence of culture conditions on lipopeptide production by Bacillus subtilis. Appl Biochem Biotechnol 91-3:551–561. CrossRefGoogle Scholar
  2. Ali GS, El-Sayed ASA, Patel JS, Green KB, Ali M, Brennan M, Norman D (2016) Ex vivo application of secreted metabolites produced by soil-inhabiting Bacillus spp. efficiently controls foliar diseases caused by Alternaria spp. Appl Environ Microbiol 82:478–490. CrossRefPubMedPubMedCentralGoogle Scholar
  3. Bautista-Baños S, Bosquez-Molina E, Barrera-Necha LL (2014) Rhizopus stolonifer (soft rot). In: Bautista-Baños S (ed) Postharvest Decay. Elsevier, Inc., London, pp 1–37Google Scholar
  4. Bonmatin JM, Laprevote O, Peypoux F (2003) Diversity among microbial cyclic lipopeptides: iturins and surfactins. Activity-structure relationships to design new bioactive agents. Comb Chem High Throughput Screen 6:541–556. CrossRefPubMedGoogle Scholar
  5. Cao Y, Xu Z, Ling N, Yuan Y, Yang X, Chen L, Shen B, Shen Q (2012) Isolation and identification of lipopeptides produced by B. subtilis SQR 9 for suppressing Fusarium wilt of cucumber. Sci Hortic 135:32–39. CrossRefGoogle Scholar
  6. Chan Y-K, Savard ME, Reid LM, Cyr T, McCormick WA, Seguin C (2009) Identification of lipopeptide antibiotics of a Bacillus subtilis isolate and their control of Fusarium graminearum diseases in maize and wheat. BioControl 54:567–574. CrossRefGoogle Scholar
  7. Deleu M, Paquot M, Nylander T (2005) Fengycin interaction with lipid monolayers at the air-aqueous interface - implications for the effect of fengycin on biological membranes. J Colloid Interface Sci 283:358–365. CrossRefPubMedGoogle Scholar
  8. Dunlap CA, Schisler DA, Price NP, Vaughn SF (2011) Cyclic lipopeptide profile of three Bacillus subtilis strains; antagonists of Fusarium head blight. J Microbiol 49:603–609. CrossRefPubMedGoogle Scholar
  9. Eeman M, Berquand A, Dufrene YF, Paquot M, Dufour S, Deleu M (2006) Penetration of surfactin into phospholipid monolayers: nanoscale interfacial organization. Langmuir 22:11337–11345. CrossRefPubMedGoogle Scholar
  10. Falardeau J, Wise C, Novitsky L, Avis TJ (2013) Ecological and mechanistic insights into the direct and indirect antimicrobial properties of Bacillus subtilis lipopeptides on plant pathogens. J Chem Ecol 39:869–878. CrossRefPubMedGoogle Scholar
  11. Farace G, Fernandez O, Jacquens L, Coutte F, Krier F, Jacques P, Clément C, Barka EA, Jacquard C, Dorey S (2015) Cyclic lipopeptides from Bacillus subtilis activate distinct patterns of defence responses in grapevine. Mol Plant Pathol 16:177–187. CrossRefPubMedGoogle Scholar
  12. Fiedler S, Heerklotz H (2015) Vesicle leakage reflects the target selectivity of antimicrobial lipopeptides from Bacillus subtilis. Biophys J 109:2079–2089. CrossRefPubMedPubMedCentralGoogle Scholar
  13. Grover M, Nain L, Singh SB, Saxena AK (2010) Molecular and biochemical approaches for characterization of antifungal trait of a potent biocontrol agent Bacillus subtilis RP24. Curr Microbiol 60:99–106. CrossRefPubMedGoogle Scholar
  14. Hamdache A, Lamarti A, Aleu J, Collado IG (2011) Non-peptide metabolites from the genus Bacillus. J Nat Prod 74:893–899. CrossRefPubMedGoogle Scholar
  15. Kharwar RN, Upadhyay RS, Dubey NK, Raghuwanshi R (2014) Microbial diversity and biotechnology in food security. Springer, New DelhiCrossRefGoogle Scholar
  16. Liu JJ, Hagberg I, Novitsky L, Hadj-Moussa H, Avis TJ (2014) Interaction of antimicrobial cyclic lipopeptides from Bacillus subtilis influences their effect on spore germination and membrane permeability in fungal plant pathogens. Fungal Biol 118:855–861. CrossRefPubMedGoogle Scholar
  17. Malfanova N, Franzil L, Lugtenberg B, Chebotar V, Ongena M (2012) Cyclic lipopeptide profile of the plant-beneficial endophytic bacterium Bacillus subtilis HC8. Arch Microbiol 194:893–899. CrossRefPubMedPubMedCentralGoogle Scholar
  18. Mohamed R, Groulx E, Defilippi S, Erak T, Tambong JT, Tweddell RJ, Tsopmo A, Avis TJ (2017) Physiological and molecular characterization of compost bacteria antagonistic to soil-borne plant pathogens. Can J Microbiol 63:411–426. CrossRefPubMedGoogle Scholar
  19. Ongena M, Jacques P (2008) Bacillus lipopeptides: versatile weapons for plant disease biocontrol. Trends Microbiol 16:115–125. CrossRefPubMedGoogle Scholar
  20. Ongena M, Jacques P, Toure Y, Destain J, Jabrane A, Thonart P (2005) Involvement of fengycin-type lipopeptides in the multifaceted biocontrol potential of Bacillus subtilis. Appl Microbiol Biotechnol 69:29–38. CrossRefPubMedGoogle Scholar
  21. Perez-Garcia A, Romero D, de Vicente A (2011) Plant protection and growth stimulation by microorganisms: biotechnological applications of Bacilli in agriculture. Curr Opin Biotechnol 22:187–193. CrossRefPubMedGoogle Scholar
  22. Posada-Uribe LF, Romero-Tabarez M, Villegas-Escobar V (2015) Effect of medium components and culture conditions in Bacillus subtilis EA-CB0575 spore production. Bioprocess Biosyst Eng 38:1879–1888. CrossRefPubMedGoogle Scholar
  23. Rebib H, Hedi A, Rousset M, Boudabous A, Limam F, Sadfi-Zouaoui N (2012) Biological control of Fusarium foot rot of wheat using fengycin-producing Bacillus subtilis isolated from salty soil. Afr J Biotechnol 11:8464–8475Google Scholar
  24. Romero D, de Vicente A, Rakotoaly RH, Dufour SE, Veening JW, Arrebola E, Cazorla FM, Kuipers OP, Paquot M, Pérez-García A (2007) The iturin and fengycin families of lipopeptides are key factors in antagonism of Bacillus subtilis toward Podosphaera fusca. Mol Plant-Microbe Interact 20:430–440. CrossRefPubMedGoogle Scholar
  25. Stein T (2005) Bacillus subtilis antibiotics: structures, syntheses and specific functions. Mol Microbiol 56:845–857. CrossRefPubMedGoogle Scholar
  26. Tao Y, Bie XM, Lv FX, Zhao HZ, Lu ZX (2011) Antifungal activity and mechanism of fengycin in the presence and absence of commercial surfactin against Rhizopus stolonifer. J Microbiol 49:146–150. CrossRefPubMedGoogle Scholar
  27. Walker R, Powell AA, Seddon B (1998) Bacillus isolates from the spermosphere of peas and dwarf French beans with antifungal activity against Botrytis cinerea and Pythium species. J Appl Microbiol 84:791–801. CrossRefPubMedGoogle Scholar
  28. Wise C, Novitsky L, Tsopmo A, Avis TJ (2012) Production and antimicrobial activity of 3-hydroxypropionaldehyde from Bacillus subtilis strain CU12. J Chem Ecol 38:1521–1527. CrossRefPubMedGoogle Scholar
  29. Wise C, Falardeau J, Hagberg I, Avis TJ (2014) Cellular lipid composition affects sensitivity of plant pathogens to fengycin, an antifungal compound produced by Bacillus subtilis strain CU12. Phytopathology 104:1036–1041. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Stefanie DeFilippi
    • 1
  • Emma Groulx
    • 1
  • Merna Megalla
    • 1
  • Rowida Mohamed
    • 1
  • Tyler J. Avis
    • 1
  1. 1.Department of ChemistryCarleton UniversityOttawaCanada

Personalised recommendations