Influence of Neighboring Clonal-Colonies on Aflatoxin Production by Aspergillus flavus

Abstract

Aspergillus flavus is an ascomycete fungus that infects and contaminates corn, peanuts, cottonseed, and treenuts with acutely toxic and carcinogenic aflatoxins. The ecological function of aflatoxin production is not well understood; though not phytotoxic, aflatoxin may be involved in resisting oxidative stress responses from infection or drought stress in plants. Observation of aflatoxin stimulation in 48-well plates in response to increasing inoculated wells sparked an investigation to determine if A. flavus volatiles influence aflatoxin production in neighboring colonies. Experiments controlling several culture conditions demonstrated a stimulation of aflatoxin production with increased well occupancy independent of pH buffer, moisture, or isolate. However, even with all wells inoculated, aflatoxin production was less in interior wells. Only one isolate stimulated aflatoxin production in a large Petri-dish format containing eight small Petri dishes with shared headspace. Other isolates consistently inhibited aflatoxin production when all eight Petri dishes were inoculated with A. flavus. No contact between cultures and only shared headspace implied the fungus produced inhibitory and stimulatory gases. Adding activated charcoal between wells and dishes prevented inhibition but not stimulation indicating stimulatory and inhibitory gases are different and/or gas is inhibitory at high concentration and stimulatory at lower concentrations. Characterizing stimulatory and inhibitory effects of gases in A. flavus headspace as well as the apparently opposing results in the two systems deserves further investigation. Determining how gases contribute to quorum sensing and communication could facilitate managing or using the gases in modified atmospheres during grain storage to minimize aflatoxin contamination.

Keywords: Aspergillus flavus; aflatoxin; quorum sensing; volatile chemical; volatile sensing.

FIGURE 1

Number of inoculated wells and well-location changed aflatoxin production by Aspergillus flavus. 48-well plates were inoculated with a single isolate 53 of A. flavus in the four arrays depicted. Centrally located wells produced more conidia and growth appeared less dense when all wells were inoculated. Aflatoxin production was less in the interior wells and greatest when all wells were inoculated with A. flavus.

FIGURE 2

Changing the number of inoculated open Petri-dishes within a closed dish alters aflatoxin production. Either 1, 4 or 8 small Petri-dishes were center-point inoculated and enclosed in a larger dish. All produced similar colonies with dense hyphal growth and minimal conidia; occasionally aerial hyphae could be observed for those with eight inoculated dishes.

FIGURE 3

Model for aflatoxin inhibition and stimulation caused by changing density of identical Aspergillus flavus cultures with/in a closed system. Individual fungal mycelial cultures are represented by line drawings. Relative aflatoxin production is represented by red shade, darker red indicate greater aflatoxin production. Clouds represent gas production during fungal growth. Arrows represent aflatoxin production, blunt ends represent blocked aflatoxin production and pointed ends represent production.