Carbohydrate, glutathione, and polyamine metabolism are central to Aspergillus flavus oxidative stress responses over time

Abstract

The primary and secondary metabolites of fungi are critical for adaptation to environmental stresses, host pathogenicity, competition with other microbes, and reproductive fitness. Drought-derived reactive oxygen species (ROS) have been shown to stimulate aflatoxin production and regulate development in Aspergillus flavus, and may function in signaling with host plants. Here, we have performed global, untargeted metabolomics to better understand the role of aflatoxin production in oxidative stress responses, and also explore isolate-specific oxidative stress responses over time. Two field isolates of A. flavus, AF13 and NRRL3357, possessing high and moderate aflatoxin production, respectively, were cultured in medium with and without supplementation with 15mM H2O2, and mycelia were collected following 4 and 7 days in culture for global metabolomics. Overall, 389 compounds were described in the analysis which were examined for differential accumulation. Significant differences were observed in both isolates in response to oxidative stress and when comparing sampling time points. The moderate aflatoxin-producing isolate, NRRL3357, showed extensive stimulation of antioxidant mechanisms and pathways including polyamines metabolism, glutathione metabolism, TCA cycle, and lipid metabolism while the highly aflatoxigenic isolate, AF13, showed a less vigorous response to stress. Carbohydrate pathway levels also imply that carbohydrate repression and starvation may influence metabolite accumulation at the later timepoint. Higher conidial oxidative stress tolerance and antioxidant capacity in AF13 compared to NRRL3357, inferred from their metabolomic profiles and growth curves over time, may be connected to aflatoxin production capability and aflatoxin-related antioxidant accumulation. The coincidence of several of the detected metabolites in H2O2-stressed A. flavus and drought-stressed hosts suggests their potential role in the interaction between these organisms and their use as markers/targets to enhance host resistance through biomarker selection or genetic engineering.\n\nAuthor SummaryAspergillus flavus is a fungal pathogen of several important crops including maize and peanut. This pathogen produces carcinogenic mycotoxins known as aflatoxins during infection of plant materials, and is particularly severe under drought stress conditions. This results in significant losses in crop value and poses a threat to food safety and security globally. To combat this, understanding how this fungus responds to environmental stresses related to drought can allow us to identify novel methods of mitigating aflatoxin contamination. Here, we analyzed the accumulation of a broad series of metabolites over time in two isolates of A. flavus with differing stress tolerance and aflatoxin production capabilities in response to drought-related oxidative stress. We identified several metabolites and mechanisms in A. flavus which allow it to cope with environmental oxidative stress and may influence aflatoxin production and fungal growth. These may serve as potential targets for selection in breeding programs for the development of new cultivars, or for alteration using genetic engineering approaches to mitigate excessive aflatoxin contamination under drought stress.