Advertisement

Microbial Ecology

, Volume 52, Issue 1, pp 45–52 | Cite as

Isolation of Bacterial Antagonists of Aspergillus flavus from Almonds

  • Jeffrey D. PalumboEmail author
  • James L. Baker
  • Noreen E. Mahoney
Article

Abstract

Bacteria were isolated from California almond orchard samples to evaluate their potential antifungal activity against aflatoxin-producing Aspergillus flavus. Fungal populations from the same samples were examined to determine the incidence of aflatoxigenic Aspergillus species. Antagonistic activities of the isolated bacterial strains were screened against a nonaflatoxigenic nor mutant of A. flavus, which accumulates the pigmented aflatoxin precursor norsolorinic acid (NOR) under conditions conducive to aflatoxin production. Using solid and liquid media in coculture assays, 171 bacteria isolated from almond flowers, immature nut fruits, and mature nut fruits showed inhibition of A. flavus growth and/or inhibition of NOR accumulation. Bacterial isolates were further characterized for production of extracellular enzymes capable of hydrolyzing chitin or yeast cell walls. Molecular and physiological identification of the bacterial strains indicated that the predominant genera isolated were Bacillus, Pseudomonas, Ralstonia, and Burkholderia, as well as several plant-associated enteric and nonenteric bacteria. A set of 20 isolates was selected for further study based on their species identification, antifungal phenotypes, and extracellular enzyme production. Quantitative assays using these isolates in liquid coculture with a wild-type, aflatoxin-producing A. flavus strain showed that a number of strains completely inhibited fungal growth in three different media. These results indicate the potential for development of bacterial antagonists as biological control agents against aflatoxigenic aspergilli on almonds.

Keywords

Aflatoxin Antifungal Activity Aflatoxin Production Aflatoxin Contamination Yeast Cell Wall 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgments

We thank K. Chan and L. Lazo for technical assistance. This work was supported by U.S. Department of Agriculture Agricultural Research Service CRIS project 5325-42000-031-00D.

References

  1. 1.
    Bayman, P, Baker, JL, Mahoney, NE (2002) Aspergillus on tree nuts: incidence and associations. Mycopathologia 155: 161–169PubMedCrossRefGoogle Scholar
  2. 2.
    Brown, RL, Cotty, PJ, Cleveland, TE (1991) Reduction in aflatoxin content of maize by atoxigenic strains of Aspergillus flavus. J Food Prot 54: 623–626Google Scholar
  3. 3.
    Burr, TJ, Matteson, MC, Smith, CA, Corral-Garcia, MR, Huang, T-C (1996) Effectiveness of bacteria and yeasts from apple orchards as biological control agents of apple scab. Biol Control 6: 151–157CrossRefGoogle Scholar
  4. 4.
    Cavaglieri, L, Passone, A, Etcheverry, M (2004) Screening procedures for selecting rhizobacteria with biocontrol effects upon Fusarium verticillioides growth and fumonisin B1 production. Res Microbiol 155: 747–754PubMedCrossRefGoogle Scholar
  5. 5.
    Cole, JR, Chai, B, Farris, RJ, Wang, Q, Kulam, SA, McGarrell, DM, Garrity, GM, Tiedje, JM (2005) The Ribosomal Database Project (RDP-II): sequences and tools for high-throughput rRNA analysis. Nucleic Acids Res 33: D294–D296PubMedCrossRefGoogle Scholar
  6. 6.
    Cole, RJ, Sanders, TH, Hill, RA, Blankenship, PD (1985) Mean geocarposphere temperatures that induce preharvest aflatoxin contamination of peanuts under drought stress. Mycopathologia 91: 41–46PubMedCrossRefGoogle Scholar
  7. 7.
    Cook, RJ, Thomashow, LS, Weller, DM, Fujimoto, D, Mazzola, M, Bangera, G, Kim, DS (1995) Molecular mechanisms of defense by rhizobacteria against root disease. Proc Natl Acad Sci USA 92: 4197–4201PubMedCrossRefGoogle Scholar
  8. 8.
    Cotty, PJ (1994) Influence of field application of an atoxigenic strain of Aspergillus flavus on the populations of A. flavus infecting cotton bolls and on aflatoxin content of cottonseed. Phytopathology 84: 1270–1277CrossRefGoogle Scholar
  9. 9.
    Cotty, PJ, Lee, LS (1989) Aflatoxin contamination of cottonseed: comparison of pink bollworm damaged and undamaged bolls. Trop Sci 29: 273–276Google Scholar
  10. 10.
    Diener, UL, Cole, RJ, Sanders, TH, Payne, GA, Lee, LS, Klich, MA (1987) Epidemiology of aflatoxin formation by Aspergillus flavus. Annu Rev Phytopathol 25: 249–270Google Scholar
  11. 11.
    Dorner, JW, Cole, RJ, Blankenship, PD (1992) Use of a biocompetitive agent to control preharvest aflatoxin in drought stressed peanuts. J Food Prot 55: 888–892Google Scholar
  12. 12.
    Dorner, JW, Cole, RJ, Blankenship, PD (1998) Effect of inoculum rate of biocontrol agents on preharvest aflatoxin contamination of peanuts. Biol Control 12: 171–176CrossRefGoogle Scholar
  13. 13.
    Dorner, JW, Cole, RJ, Wicklow, DT (1999) Aflatoxin reduction in corn through field application of competitive fungi. J Food Prot 62: 650–656PubMedGoogle Scholar
  14. 14.
    Doster, MA, Michailides, TJ (1994) Aspergillus molds and aflatoxins in pistachio nut in California. Phytopathology 84: 583–590CrossRefGoogle Scholar
  15. 15.
    Gradziel, TM, Wang, D (1994) Susceptibility of California almond cultivars to aflatoxigenic Aspergillus flavus. HortScience 29: 33–35Google Scholar
  16. 16.
    Gregersen, T (1978) Rapid method for distinction of Gram-negative from Gram-positive bacteria. Eur J Appl Microbiol Biotechnol 5: 123–127CrossRefGoogle Scholar
  17. 17.
    Horn, BW, Dorner, JW, Greene, RL, Blankenship, PD, Cole, RJ (1994) Effect of Aspergillus parasiticus soil inoculum on invasion of peanut seeds. Mycopathologia 125: 179–191PubMedCrossRefGoogle Scholar
  18. 18.
    Hua, SS, Baker, JL, Flores-Espiritu, M (1999) Interactions of saprophytic yeasts with a nor mutant of Aspergillus flavus. Appl Environ Microbiol 65: 2738–2740PubMedGoogle Scholar
  19. 19.
    Kerry, BR (2000) Rhizosphere interactions and the exploitation of microbial agents for the biological control of plant-parasitic nematodes. Annu Rev Phytopathol 38: 423–441PubMedCrossRefGoogle Scholar
  20. 20.
    Klich, MA (2002) Identification of common Aspergillus species. Centraalbureau voor Schimmelcultures, 1st edn. Utrecht, Netherlands, pp 122Google Scholar
  21. 21.
    Kondo, T, Sakurada, M, Okamoto, S, Ono, M, Tsukigi, H, Suzuki, A, Nagasawa, H, Sakuda, S (2001) Effects of aflastatin A, an inhibitor of aflatoxin production, on aflatoxin biosynthetic pathway and glucose metabolism in Aspergillus parasiticus. J Antibiot (Tokyo) 54: 650–657Google Scholar
  22. 22.
    Korsten, L, De Jager, ES, De Villiers, EE, Lourens, A, Kotzé, JM, Wehner, FC (1995) Evaluation of bacterial epiphytes isolated from avocado leaf and fruit surfaces for biocontrol of avocado postharvest diseases. Plant Dis 79: 1149–1156CrossRefGoogle Scholar
  23. 23.
    Korsten, L, De Villiers, EE, Wehner, FC, Kotzé, JM (1997) Field sprays of Bacillus subtilis and fungicides for control of preharvest fruit diseases of avocado in South Africa. Plant Dis 81: 455–459CrossRefGoogle Scholar
  24. 24.
    Lane, DJ (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow, M (eds.) Nucleic Acid Techniques in Bacterial Systematics, John Wiley & Sons, Ltd., New York, pp 115–175Google Scholar
  25. 25.
    Leben, C (1965) Epiphytic microorganisms in relation to plant disease. Annu Rev Phytopathol 3: 209–230CrossRefGoogle Scholar
  26. 26.
    Lee, JY, Moon, SS, Hwang, BK (2003) Isolation and antifungal and antioomycete activities of aerugine produced by Pseudomonas fluorescens strain MM-B16. Appl Environ Microbiol 69: 2023–2031PubMedCrossRefGoogle Scholar
  27. 27.
    Leifert, C, Li, H, Chidburee, S, Hampson, S, Workman, S, Sigee, D, Epton, HAS, Harbour, A (1995) Antibiotic production and biocontrol activity by Bacillus subtilis CL27 and Bacillus pumilus CL45. J Appl Bacteriol 78: 97–108PubMedGoogle Scholar
  28. 28.
    Lingappa, Y, Lockwood, JL (1962) Chitin media for selective isolation and culture of actinomycetes. Phytopathology 52: 317–323Google Scholar
  29. 29.
    Moyne, A-L, Shelby, R, Cleveland, TE, Tuzun, S (2001) Bacillomycin D: an iturin with antifungal activity against Aspergillus flavus. J Appl Microbiol 90: 622–629PubMedCrossRefGoogle Scholar
  30. 30.
    Munimbazi, C, Bullerman, LB (1997) Inhibition of aflatoxin production of Aspergillus parasiticus NRRL 2999 by Bacillus pumilus. Mycopathologia 140: 163–169CrossRefGoogle Scholar
  31. 31.
    Munimbazi, C, Bullerman, LB (1998) Isolation and partial characterization of antifungal metabolites of Bacillus pumilus. J Appl Microbiol 84: 959–968PubMedCrossRefGoogle Scholar
  32. 32.
    Nair, JR, Singh, G, Sekar, V (2002) Isolation and characterization of a novel Bacillus strain from coffee phyllosphere showing antifungal activity. J Appl Microbiol 93: 772–780PubMedCrossRefGoogle Scholar
  33. 33.
    Nicholson, WL, Munakata, N, Horneck, G, Melosh, HJ, Setlow, P (2000) Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments. Microbiol Mol Biol Rev 64: 548–572PubMedCrossRefGoogle Scholar
  34. 34.
    Ono, M, Sakuda, S, Suzuki, A, Isogai, A (1997) Aflastatin A, a novel inhibitor of aflatoxin production by aflatoxigenic fungi. J Antibiot (Tokyo) 50: 111–118Google Scholar
  35. 35.
    Palumbo, JD, Sullivan, RF, Kobayashi, DY (2003) Molecular characterization and expression in Escherichia coli of three β-1,3-glucanase genes from Lysobacter enzymogenes strain N4-7. J Bacteriol 185: 4362–4370PubMedCrossRefGoogle Scholar
  36. 36.
    Papa, KE (1984) Genetics of Aspergillus flavus: linkage of aflatoxin mutants. Can J Microbiol 30: 68–73PubMedCrossRefGoogle Scholar
  37. 37.
    Paster, N, Drody, S, Chalutz, E, Menasherov, M, Nitzan, R, Wilson, CL (1993) Evaluation of the potential of the yeast Pichia guilliermondii as a biocontrol agent against Aspergillus flavus and fungi of stored soya beans. Mycol Res 97: 1201–1206CrossRefGoogle Scholar
  38. 38.
    Pitt, JI, Hocking, AD (1999) Fungi and Food Spoilage, 2nd edn. Aspen Publishers, Inc., Gaithersburg, MD, pp 593Google Scholar
  39. 39.
    Taylor, WJ, Draughon, FA (2001) Nannocystis exedens: a potential biocompetitive agent against Aspergillus flavus and Aspergillus parasiticus. J Food Prot 64: 1030–1034PubMedGoogle Scholar
  40. 40.
    van Egmond, HP (1995) Mycotoxins: regulations, quality assurance and reference materials. Food Addit Contam 12: 321–330PubMedGoogle Scholar
  41. 41.
    Wilson, M, Lindow, SE (1994) Coexistence among epiphytic bacterial populations mediated through nutritional resource partitioning. Appl Environ Microbiol 60: 4468–4477PubMedGoogle Scholar
  42. 42.
    Yan, PS, Song, Y, Sakuno, E, Nakajima, H, Nakagawa, H, Yabe, K (2004) Cyclo(l-leucyl-l-prolyl) produced by Achromobacter xylosoxidans inhibits aflatoxin production by Aspergillus parasiticus. Appl Environ Microbiol 70: 7466–7473PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2006

Authors and Affiliations

  • Jeffrey D. Palumbo
    • 1
    Email author
  • James L. Baker
    • 1
  • Noreen E. Mahoney
    • 1
  1. 1.Plant Mycotoxin Research Unit, Western Regional Research Center, Agricultural Research ServiceU.S. Department of AgricultureAlbanyUSA

Personalised recommendations