Advertisement

Mycopathologia

, Volume 171, Issue 2, pp 133–138 | Cite as

Fluorescent Viability Stains to Probe the Metabolic Status of Aflatoxigenic Fungus in Dual Culture of Aspergillus flavus and Pichia anomala

  • Sui Sheng T. Hua
  • Maria T. Brandl
  • Bradley Hernlem
  • Jeffrey G. Eng
  • Siov Bouy L. Sarreal
Article

Abstract

The metabolic activity of the aflatoxigenic fungus, Aspergillus flavus co-cultured with the biocontrol yeast, Pichia anomala was examined using several viability stains. Both the FUN-1 stain and the combined use of DiBAC4(5) with CDFA-AM stains were applied in this study. The results suggest that the ATP-generating system in A. flavus was inactivated as the ratio of yeasts to fungi increased in the dual culture. A decrease in hyphal membrane potential and esterase activity was substantiated by the combined stains of DiBAC4(5) and CDFA-AM. Reduced metabolic function in conjunction with cell wall damage of A. flavus hindered the growth and biomass production of this fungus. Viability stains such as FUN-1 and DiBAC4(5) with CDFA-AM may assist in elucidating the biocontrol mechanism by allowing for the visualization of the antagonistic effect of yeast species on target fungi in situ, as well as for screening potent biocontrol yeast agents against fungal pathogens.

Keywords

Aflatoxin Yeast Fungus Fluorescence Mechanism of biocontrol 

Notes

Acknowledgments

We thank Thomas McKeon. Glenn Barley and Bruce C. Campbell for their comments and reviews of the manuscript.

References

  1. 1.
    Hickey PC, Swift SR, Roca MG, Read ND. Live-cell imaging of filamentous fungi using vital fluorescent dyes and confocal microscopy. Method Microbiol. 2004;34:63–87.CrossRefGoogle Scholar
  2. 2.
    Lloyd D, Hayes AJ. Vigour, vitality and viability of microorganisms. FEMS Microbiol Lett. 1995;133:1–7.CrossRefGoogle Scholar
  3. 3.
    Millard PJ, Roth BL, Thai HP, Yu ST, Haugland RP. Development of the FUN-1 family of fluorescent probes for vacuole labeling and viability testing of yeasts. Appl Environ Microbiol. 1997;63:2897–905.PubMedGoogle Scholar
  4. 4.
    Lass-Flörl C, Nagl M, Speth C, Ulmer H, Dierich MP, Würzner R. Studies of in vitro activities of Voriconazole and Itraconazole against Aspergillus hyphae using viability staining. Antimicrob Agents Chemother. 2001;45:124–8.CrossRefPubMedGoogle Scholar
  5. 5.
    Marr KA, Koudadoust M, Black M, Balajee SA. Early events in macrophage killing of Aspergillus fumigatus conidia: new flow cytometric viability assay. Clin Diag Lab Immunol. 2001;8:1240–7.Google Scholar
  6. 6.
    Balajee SA, Marr KA. Conidial viability assay for rapid susceptibility testing of Aspergillus species. J Clin Microbiol. 2002;40:2741–5.CrossRefPubMedGoogle Scholar
  7. 7.
    Bräuner T, Hülser DF, Strasser RJ. Comparative measurement of membrane potentials with microelectrodes and voltage-sensitive dyes. Biochimi Biophys Acta. 1984;771:208–16.CrossRefGoogle Scholar
  8. 8.
    Janisiewicz W, Korsten L. Biological control of postharvest diseases of fruits. Ann Rev Phytopathol. 2002;40:411–41.CrossRefGoogle Scholar
  9. 9.
    Spadaro D, Gullino ML. State of the art and future prospects of the biological control of postharvest fruit diseases. Int J Food Microbiol. 2004;91:185–94.CrossRefPubMedGoogle Scholar
  10. 10.
    Jijakli MH, Lepoivre P. Characterization of an exo-β-1, 3 glucanase produced by Pichia anomala K, an antagonist of Botrytis cinerea on apples. Phytopathology. 1998;88:335–43.CrossRefPubMedGoogle Scholar
  11. 11.
    Petersson S, Schnurer J. Biocontrol of mold growth in high-moisture wheat stored under airtight conditions by Pichia anomala, Pichia guillermondii, and Saccharomyces cerevisiae. Appl Environ Microbiol. 1995;61:1027–31.PubMedGoogle Scholar
  12. 12.
    Petersson S, Scnurer J. Pichia anomala as a biocontrol agent of Penicillium roqueforti in high-moisture wheat, rye, barley, and oats stored under airtight conditions. Can J Microbiol. 1998;44:471–6.CrossRefGoogle Scholar
  13. 13.
    Hua SST, Baker JL, Flores-Espiritu M. Interactions of saprophytic yeasts with a nor mutant of Aspergillus flavus. Appl Environ Microbiol. 1999;65:2738–40.PubMedGoogle Scholar
  14. 14.
    Hua SST. Progress in prevention of aflatoxin contamination in food by preharvest application of Pichia anomala WRL-076. In: Mendez-Vilas A, editor. Recent advances in multidisciplinary applied microbiology. Weinheim: Wiley-VCH Verlag GmbH&Co. KGaA; 2006. p. 322–6.CrossRefGoogle Scholar
  15. 15.
    Scudamore KA. Aspergillus toxin in food and animal feeding stuffs. In: Powell KA, Renwick A, Peberdy JF, editors. The genus Aspergillus. New York: Plenum Press; 1994. p. 59–71.Google Scholar
  16. 16.
    Commission of the European Community. Commission Directive 98/53/EC of July 1998 laying down the sampling methods and the methods of analysis for the official control of the levels of certain contaminants in food stuffs. Off Eur Communities Legislation. 1998; L201:93–101.Google Scholar
  17. 17.
    U. S. Food and Drug Administration. Compliance policy guides manual. Washington, DC: U. S. FDA; 1996. Sec. 555.400, 268; Sec. 570.500, 299.Google Scholar

Copyright information

© U.S. Government 2010

Authors and Affiliations

  • Sui Sheng T. Hua
    • 1
  • Maria T. Brandl
    • 1
  • Bradley Hernlem
    • 1
  • Jeffrey G. Eng
    • 1
  • Siov Bouy L. Sarreal
    • 1
  1. 1.U. S. Department of Agriculture, Agricultural Research ServiceWestern Regional Research CenterAlbanyUSA

Personalised recommendations