2.10.4
BIOCONTROL OF ASPERGILLUS FLAVUS BY SAPROPHYTIC YEASTS

SS HUA

Western Regional Research Center, Albany, CA 94710, USA

Background and objectives
The phyllosphere is colonized by various saprophytic and pathogenic microorganisms. Yeasts are common on the surface of plant leaves and fruits. It has been demonstrated that some yeasts compete well with many post-harvest fungal pathogens such as Penicillium expansum and Botrytis cinerea by depleting nutrients necessary for germination and hyphal growth [1]. Aspergillus flavus appears to be the most common aflatoxin-producing fungus on tree-nuts such as almonds, pistachios and walnuts. The fungus infects tree-nuts damaged by insects, animals, early splits and mechanical harvest. Any established infection will result in rapid accumulation of aflatoxin in the harvested nuts in warm conditions. Aflatoxin B1 is the most potent carcinogen known. The objectives of this research were to isolate the yeasts occurring on California tree-nuts and to investigate interactions of saprophytic yeasts and A. ;flavus that may lead to successful reduction of aflatoxin contamination in the edible nuts. The NOR mutant of A. ;flavus has a block in the reductase and is unable to complete aflatoxin biosynthesis. Instead, it accumulates norsolorinic acid, a bright red-orange compound [2]. A. ;flavus 827, a NOR mutant, has been used in this study as an indicator strain for a visual assay to monitor the interactions of yeasts and A. flavus.

Materials and methods
Yeasts were isolated from the surface of tree-nut fruits and identified by morphological, biochemical and physiological criteria. Selected yeast isolates were used to prepare suspensions at concentrations of 109cells/ml and spore suspensions of the A. ;flavus 827 were prepared to 104 spores/ml. Twenty microlitres of yeast suspension were applied to each side of PDA plates, 1 ;cm from the centre, to generate two streaks of yeast inocula. Four hours later, 20 ;l of spore suspension were inoculated along the centre line of the PDA. The Petri dishes were incubated at 28C for 10 ;days. The antagonistic effects of yeast isolates on A. ;flavus were evaluated by visualizing the amount of red-orange colour formed and counting the number of conidia.

Results and conclusion

Selected yeasts inhibited fungal growth, as shown in the biocontrol tests. A.. ;flavus 827 was restricted to within the boundary of the two streaks of yeast zones. Agar discs of fungi were cored from PDA. Conidia were eluted from the discs and counted. The number of conidia was reduced by 50-90% compared with the control, depending on the yeast strain used in the biocontrol test. The selected yeasts also inhibited production of the red-orange colour. The rationale for using the NOR mutant as an indicator strain is as follows: norsolorinic acid is an intermediate in the early stage of aflatoxin biosynthesis so, (1) if the mutant produces a red-orange colour in the growth medium, it implies that the aflatoxin biosythetic pathway is turned on; (2) if the NOR mutant stops producing the red-orange colour, it implies that the pathway is turned off. The interactions between the yeast isolates and A.. ;flavus are complex. It is likely that the yeasts produce diffusible compounds which inhibit aflatoxin biosynthesis and reduce the growth as well as sporulation of the fungus.

References

1. Wilson CL, Wisniewski ME, 1989. Annual Review of Phytopathology 27, 425-51.
2. Keller NP, Butchko RAE, Sarr B, Phillips TD, 1994. Phytopathology 84, 483-88.