Since the early 1960s, the fungal pathogen Aspergillus flavus (Link ex Fr.) has been the focus of intensive research due to the production of carcinogenic and highly toxic secondary metabolites collectively known as aflatoxins following pre-harvest colonization of crops. Given this recurrent problem and the occurrence of a severe aflatoxin outbreak in maize (Zea mays L.), particularly in the Southeast U.S. in the 1977 growing season, a significant research effort has been put forth to determine the nature of the interaction occurring between aflatoxin production, A. flavus, environment and its various hosts before harvest. Many studies have investigated this interaction at the genetic, transcript, and protein levels, and in terms of fungal biology at either pre- or post-harvest time points. Later experiments have indicated that the interaction and overall resistance phenotype of the host is a quantitative trait with a relatively low heritability. In addition, a high degree of environmental interaction has been noted, particularly with sources of abiotic stress for either the host or the fungus such as drought or heat stresses. Here, we review the history of research into this complex interaction and propose future directions for elucidating the relationship between resistance and susceptibility to A. flavus colonization, abiotic stress, and its relationship to oxidative stress in which aflatoxin production may function as a form of antioxidant protection to the producing fungus.

The infection of maize and peanut with Aspergillus flavus and subsequent contamination with aflatoxin pose a threat to global food safety and human health, and is exacerbated by drought stress. Drought stress-responding compounds such as reactive oxygen species (ROS) are associated with fungal stress responsive signaling and secondary metabolite production, and can stimulate the production of aflatoxin by A. flavus in vitro. These secondary metabolites have been shown to possess diverse functions in soil-borne fungi including antibiosis, competitive inhibition of other microbes, and abiotic stress alleviation. Previously, we observed that isolates of A. flavus showed differences in oxidative stress tolerance which correlated with their aflatoxin production capabilities. In order to better understand these isolate-specific oxidative stress responses, we examined the transcriptional responses of field isolates of A. flavus with varying levels of aflatoxin production (NRRL3357, AF13, and Tox4) to H2O2-induced oxidative stress using an RNA sequencing approach. These isolates were cultured in an aflatoxin-production conducive medium amended with various levels of H2O2. Whole transcriptomes were sequenced using an Illumina HiSeq platform with an average of 40.43 million filtered paired-end reads generated for each sample. The obtained transcriptomes were then used for differential expression, gene ontology, pathway, and co-expression analyses. Isolates which produced higher levels of aflatoxin tended to exhibit fewer differentially expressed genes than isolates with lower levels of production. Genes found to be differentially expressed in response to increasing oxidative stress included antioxidant enzymes, primary metabolism components, antibiosis-related genes, and secondary metabolite biosynthetic components specifically for aflatoxin, aflatrem, and kojic acid. The expression of fungal development-related genes including aminobenzoate degradation genes and conidiation regulators were found to be regulated in response to increasing stress. Aflatoxin biosynthetic genes and antioxidant enzyme genes were also found to be co-expressed and highly correlated with fungal biomass under stress. This suggests that these secondary metabolites may be produced as part of coordinated oxidative stress responses in A. flavus along with antioxidant enzyme gene expression and developmental regulation.

The colonization of maize (Zea mays L.) and peanut (Arachis hypogaea L.) by the fungal pathogen Aspergillus flavus results in the contamination of kernels with carcinogenic mycotoxins known as aflatoxins leading to economic losses and potential health threats to humans. The regulation of aflatoxin biosynthesis in various Aspergillus spp. has been extensively studied, and has been shown to be related to oxidative stress responses. Given that environmental stresses such as drought and heat stress result in the accumulation of reactive oxygen species (ROS) within host plant tissues, host-derived ROS may play an important role in cross-kingdom communication between host plants and A. flavus. Recent technological advances in plant breeding have provided the tools necessary to study and apply knowledge derived from metabolomic, proteomic, and transcriptomic studies in the context of productive breeding populations. Here, we review the current understanding of the potential roles of environmental stress, ROS, and aflatoxin in the interaction between A.flavus and its host plants, and the current status in molecular breeding and marker discovery for resistance to A. flavus colonization and aflatoxin contamination in maize and peanut. We will also propose future directions and a working model for continuing research efforts linking environmental stress tolerance and aflatoxin contamination resistance in maize and peanut.

SummaryDrought 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.

Drought stress is a major factor that contributes to disease susceptibility and yield loss in agricultural crops. To identify drought responsive proteins and explore metabolic pathways involved in maize tolerance to drought stress, two maize lines (B73 and Lo964) with contrasting drought sensitivity were examined. The treatments of drought and well water were applied at 14 days after pollination (DAP), and protein profiles were investigated in developing kernels (35 DAP) using iTRAQ (isobaric tags for relative and absolute quantitation). Proteomic analysis showed that 70 and 36 proteins were significantly altered in their expression under drought treatments in B73 and Lo964, respectively. The numbers and levels of differentially expressed proteins were generally higher in the sensitive genotype, B73, implying an increased sensitivity to drought given the function of the observed differentially expressed proteins, such as redox homeostasis, cell rescue/defense, hormone regulation and protein biosynthesis and degradation. Lo964 possessed a more stable status with fewer differentially expressed proteins. However, B73 seems to rapidly initiate signaling pathways in response to drought through adjusting diverse defense pathways. These changes in protein expression allow for the production of a drought stress-responsive network in maize kernels.