Seeds of aflatoxin-resistant and aflatoxin-susceptible maize lines were inoculated with conidia of aflatoxin-producing Aspergillus flavus or A. parasiticus isolates or isogenic non-producing mutants. Conidia were recovered from resistant maize seed after seven days in significantly lower amounts for aflatoxin-producing A. flavus and A. parasiticus strains than for isogenic mutants incapable of aflatoxin production. This result helps to explain why, in currently used biocontrol strategies for aflatoxin elimination, non-aflatoxigenic isolates are able to out-compete aflatoxin-producing isolates for invasion of the seed.
Because fungi rarely grow in pure culture in natural environments [1-6], their ability to elaborate secondary metabolites is affected by competition with other microorganisms [7-9]. Contamination of maize, cotton, peanuts and tree nuts by the toxic and carcinogenic secondary metabolites of A. flavus, the aflatoxins, is a persistent problem in the United States and other countries [
Seed contamination by A. flavus depends on many factors including the seed’s innate susceptibility, environmental factors that contribute to that susceptibility, the fungal community structure in the soil, and the ability of the fungus to reach and penetrate the seed [
Two niaD– mutants, an A. parasiticus isolate BN009E (BN9) [
Fungal isolates were grown on 5% V8 juice, 2% agar plates in the dark at 30˚C. After seven days, the conidia were harvested in 0.1% Triton × 100 and vortexed for one min with glass beads (3 mm mean diameter) to obtain an even suspension. The conidial concentration was adjusted to 1 × 107 ml–1 in 0.1% Triton × 100. Spore density was estimated both by hemocytometer counting and plating for colony-forming units (CFU/ml). The resulting conidial suspension was used as the inoculum for both susceptible and resistant varieties of maize (see below).
Kernels of an aflatoxin-susceptible variety of maize (Pioneer hybrid 3142, Brown, unpublished data) were obtained from Pioneer Hi-Bred International, Inc. (Johnston, IA). Kernels of another variety of aflatoxin‑susceptible maize (SC212M) were obtained from the, Maize Host Plant Resistance Research Unit, USDA-ARS, Mississippi State University, University, MS. Kernels of an aflatoxin-resistant variety of inbred maize (MI82) [
The kernel screening assay (KSA) previously used to determine the ability of A. flavus to invade maize kernels [
Standard ANOVA with single factor and T-tests were conducted using Microsoft Excel to compare responses between the wild-type and isogenic mutants. Differences in responses were considered significant if P < 0.05.
A kernel screening assay was used previously to screen maize varieties for resistance to aflatoxin accumulation by A. flavus [26,27] . For aflatoxin to accumulate in the seed, the conidia must contact the seed, germinate, penetrate the seed, and reproduce within the seed. The resulting conidial population density recovered from a seed, therefore, provides a measure of the fungus’s ability to invade and colonize the seed.
ered after incubation with the two varieties of maize. For both A. parasiticus and A. flavus, significantly (P < 0.01) lower spore recovery was found for the aflatoxin-producing parental strain compared to the isogenic nonaflatoxin-producing mutant on the resistant maize variety (MI82). No significant difference in spore recovery was found for either of the susceptible varieties of maize (P3142 or SC212) inoculated with the same isolates. Therefore, production of aflatoxin is not only not necessary for contamination of the maize seed but can provide a slight, but, at least for the resistant maize variety, significant burden on the isolate regarding its ability to contaminate the seed.
A previous study using isogenic mutants of A. nidulans as the test organism found that spore recovery from maize was proportional to production of sterigmatocystin (a precursor of aflatoxin) and other precursor metabolites [
Both fungal development, as measured by spore formation, and secondary metabolism, as measu-red by sterigmatocystin production, are later stages in fungal growth and require some of the same regulatory machinery [29-31]. Production of secondary metabolites is an energy intensive process [32-34]. This expenditure of energy for aflatoxin production could come at the expense of the energy needed for conidial development and invasion of the plant. The increased ability of the nonaflatoxin producing strains to invade and colonize the resistant maize seeds could be due to an increased availability of energy that would normally be expended on aflatoxin production. The lack of a significant difference in the ability of the fungus to invade the aflatoxin-susceptible maize varieties suggests that less metabolic energy may be required for the Aspergillus isolates to mount an invasion of these varieties of seed.
Alternatively, the resistant variety, but not the susceptible variety, of maize may produce fungal inhibitory factors that ward off invasion by the fungus depending on its ability or inability to make aflatoxins. Since aflatoxins are transported from the fungal cell, the plant response to the presence of the fungus may be only experienced if it is able to respond to this secondary metabolite. The opposite results observed in A. nidulans could be a result of inherent differences in the two species with regard their use of metabolic energy in sterigmatocystin production and conidial development or to the fact that sterigmatocystin is predominantly intracellular and does not initiate a plant defensive response. In either case, our results suggest that A. flavus non-aflatoxin-producing strains have an equal ability or slight advantage over aflatoxin-producing strains in their ability to invade the seed and displace an aflatoxin-producing strain. Therefore, the strategy of introducing non-aflatoxin-producing A. flavus to limit aflatoxin contamination of maize and cotton by A. flavus is supported by our results.