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Summary Drought stress conditions decrease maize growth and yield, and aggravate preharvest aflatoxin contamination. While several studies have been performed on mature kernels responding to drought stress, the metabolic profiles of developing kernels are not as well characterized, particularly in germplasm with contrasting resistance to both drought and mycotoxin contamination. Here, following screening for drought tolerance, a drought‐sensitive line, B73, and a drought‐tolerant line, Lo964, were selected and stressed beginning at 14 days after pollination. Developing kernels were sampled 7 and 14 days after drought induction (DAI) from both stressed and irrigated plants. Comparative biochemical and metabolomic analyses profiled 409 differentially accumulated metabolites. Multivariate statistics and pathway analyses showed that drought stress induced an accumulation of simple sugars and polyunsaturated fatty acids and a decrease in amines, polyamines and dipeptides in B73. Conversely, sphingolipid, sterol, phenylpropanoid and dipeptide metabolites accumulated in Lo964 under drought stress. Drought stress also resulted in the greater accumulation of reactive oxygen species (ROS) and aflatoxin in kernels of B73 in comparison with Lo964 implying a correlation in their production. Overall, field drought treatments disordered a cascade of normal metabolic programming during development of maize kernels and subsequently caused oxidative stress. The glutathione and urea cycles along with the metabolism of carbohydrates and lipids for osmoprotection, membrane maintenance and antioxidant protection were central among the drought stress responses observed in developing kernels. These results also provide novel targets to enhance host drought tolerance and disease resistance through the use of biotechnologies such as transgenics and genome editing.

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.Author 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.

Drought stress decreases crop growth, yield, and can further exacerbate pre-harvest aflatoxin contamination. Tolerance and adaptation to drought stress is an important trait of agricultural crops like maize. However, maize genotypes with contrasting drought tolerances have been shown to possess both common and genotype-specific adaptations to cope with drought stress. In this research, the physiological and metabolic response patterns in the leaves of maize seedlings subjected to drought stress were investigated using six maize genotypes including: A638, B73, Grace-E5, Lo964, Lo1016, and Va35. During drought treatments, drought-sensitive maize seedlings displayed more severe symptoms such as chlorosis and wilting, exhibited significant decreases in photosynthetic parameters, and accumulated significantly more reactive oxygen species (ROS) and reactive nitrogen species (RNS) than tolerant genotypes. Sensitive genotypes also showed rapid increases in enzyme activities involved in ROS and RNS metabolism. However, the measured antioxidant enzyme activities were higher in the tolerant genotypes than in the sensitive genotypes in which increased rapidly following drought stress. The results suggest that drought stress causes differential responses to oxidative and nitrosative stress in maize genotypes with tolerant genotypes with slower reaction and less ROS and RNS production than sensitive ones. These differential patterns may be utilized as potential biological markers for use in marker assisted breeding.

Aflatoxin contamination in the field is known to be influenced by numerous factors. Drought and high temperatures are conducive to Aspergillus flavus infection and aflatoxin contamination. This article will review the application of new molecular tools to study host resistance to