Isolation of Bacterial Antagonists of Aspergillus flavus from Almonds


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.

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  1. 1.

    Bayman, P, Baker, JL, Mahoney, NE (2002) Aspergillus on tree nuts: incidence and associations. Mycopathologia 155: 161–169

    PubMed  Article  Google 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: 623–626

    CAS  Google 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: 151–157

    Article  Google 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: 747–754

    PubMed  Article  CAS  Google 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–D296

    PubMed  Article  CAS  Google 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: 41–46

    PubMed  Article  CAS  Google 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: 4197–4201

    PubMed  Article  CAS  Google 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: 1270–1277

    Article  Google Scholar 

  9. 9.

    Cotty, PJ, Lee, LS (1989) Aflatoxin contamination of cottonseed: comparison of pink bollworm damaged and undamaged bolls. Trop Sci 29: 273–276

    Google 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: 249–270

    CAS  Google 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: 888–892

    CAS  Google 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: 171–176

    Article  Google Scholar 

  13. 13.

    Dorner, JW, Cole, RJ, Wicklow, DT (1999) Aflatoxin reduction in corn through field application of competitive fungi. J Food Prot 62: 650–656

    PubMed  CAS  Google Scholar 

  14. 14.

    Doster, MA, Michailides, TJ (1994) Aspergillus molds and aflatoxins in pistachio nut in California. Phytopathology 84: 583–590

    Article  CAS  Google Scholar 

  15. 15.

    Gradziel, TM, Wang, D (1994) Susceptibility of California almond cultivars to aflatoxigenic Aspergillus flavus. HortScience 29: 33–35

    Google Scholar 

  16. 16.

    Gregersen, T (1978) Rapid method for distinction of Gram-negative from Gram-positive bacteria. Eur J Appl Microbiol Biotechnol 5: 123–127

    Article  Google 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: 179–191

    PubMed  Article  CAS  Google 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: 2738–2740

    PubMed  CAS  Google 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: 423–441

    PubMed  Article  CAS  Google Scholar 

  20. 20.

    Klich, MA (2002) Identification of common Aspergillus species. Centraalbureau voor Schimmelcultures, 1st edn. Utrecht, Netherlands, pp 122

  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: 650–657

    CAS  Google 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: 1149–1156

    Article  Google 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: 455–459

    Article  CAS  Google 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 115–175

    Google Scholar 

  25. 25.

    Leben, C (1965) Epiphytic microorganisms in relation to plant disease. Annu Rev Phytopathol 3: 209–230

    Article  Google 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: 2023–2031

    PubMed  Article  CAS  Google 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: 97–108

    PubMed  CAS  Google Scholar 

  28. 28.

    Lingappa, Y, Lockwood, JL (1962) Chitin media for selective isolation and culture of actinomycetes. Phytopathology 52: 317–323

    Google 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: 622–629

    PubMed  Article  CAS  Google Scholar 

  30. 30.

    Munimbazi, C, Bullerman, LB (1997) Inhibition of aflatoxin production of Aspergillus parasiticus NRRL 2999 by Bacillus pumilus. Mycopathologia 140: 163–169

    Article  Google Scholar 

  31. 31.

    Munimbazi, C, Bullerman, LB (1998) Isolation and partial characterization of antifungal metabolites of Bacillus pumilus. J Appl Microbiol 84: 959–968

    PubMed  Article  CAS  Google 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: 772–780

    PubMed  Article  CAS  Google 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: 548–572

    PubMed  Article  CAS  Google 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: 111–118

    CAS  Google 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: 4362–4370

    PubMed  Article  CAS  Google Scholar 

  36. 36.

    Papa, KE (1984) Genetics of Aspergillus flavus: linkage of aflatoxin mutants. Can J Microbiol 30: 68–73

    PubMed  CAS  Article  Google 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: 1201–1206

    Article  Google Scholar 

  38. 38.

    Pitt, JI, Hocking, AD (1999) Fungi and Food Spoilage, 2nd edn. Aspen Publishers, Inc., Gaithersburg, MD, pp 593

    Google Scholar 

  39. 39.

    Taylor, WJ, Draughon, FA (2001) Nannocystis exedens: a potential biocompetitive agent against Aspergillus flavus and Aspergillus parasiticus. J Food Prot 64: 1030–1034

    PubMed  CAS  Google Scholar 

  40. 40.

    van Egmond, HP (1995) Mycotoxins: regulations, quality assurance and reference materials. Food Addit Contam 12: 321–330

    PubMed  Google Scholar 

  41. 41.

    Wilson, M, Lindow, SE (1994) Coexistence among epiphytic bacterial populations mediated through nutritional resource partitioning. Appl Environ Microbiol 60: 4468–4477

    PubMed  CAS  Google 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: 7466–7473

    PubMed  Article  CAS  Google Scholar 

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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.

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Correspondence to Jeffrey D. Palumbo.

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Palumbo, J.D., Baker, J.L. & Mahoney, N.E. Isolation of Bacterial Antagonists of Aspergillus flavus from Almonds. Microb Ecol 52, 45–52 (2006).

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  • Aflatoxin
  • Antifungal Activity
  • Aflatoxin Production
  • Aflatoxin Contamination
  • Yeast Cell Wall