Divergent Aspergillus flavus corn population is composed of prolific conidium producers: Implications for saprophytic disease cycle.

The ascomycete fungus Aspergillus flavus infects and contaminates corn, peanuts, cottonseed, and tree nuts with toxic and carcinogenic aflatoxins. Subdivision between soil and host plant populations suggests that certain A. flavus strains are specialized to infect peanut, cotton, and corn despite having a broad host range. In this study, the ability of strains isolated from corn and/or soil in 11 Louisiana fields to produce conidia (field inoculum and male gamete) and sclerotia (resting bodies and female gamete) was assessed and compared with genotypic single-nucleotide polymorphism (SNP) differences between whole genomes. Corn strains produced upward of 47× more conidia than strains restricted to soil. Conversely, corn strains produced as much as 3000× fewer sclerotia than soil strains. Aspergillus flavus strains, typified by sclerotium diameter (small S-strains, <400 µm; large L-strains, >400 µm), belonged to separate clades. Several strains produced a mixture (M) of S and L sclerotia, and an intermediate number of conidia and sclerotia, compared with typical S-strains (minimal conidia, copious sclerotia) and L-strains (copious conidia, minimal sclerotia). They also belonged to a unique phylogenetic mixed (M) clade. Migration from soil to corn positively correlated with conidium production and negatively correlated with sclerotium production. Genetic differences correlated with differences in conidium and sclerotium production. Opposite skews in female (sclerotia) or male (conidia) gametic production by soil or corn strains, respectively, resulted in reduced effective breeding population sizes when comparing male:female gamete ratio with mating type distribution. Combining both soil and corn populations increased the effective breeding population, presumably due to contribution of male gametes from corn, which fertilize sclerotia on the soil surface. Incongruencies between aflatoxin clusters, strain morphotype designation, and whole genome phylogenies suggest a history of sexual reproduction within this Louisiana population, demonstrating the importance of conidium production, as infectious propagules and as fertilizers of the A. flavus soil population.

Genomic and metabolomic diversity within a familial population of Aspergillus flavus.

Aspergillus flavus is an agriculturally significant micro-fungus having potential to contaminate food and feed crops with toxic secondary metabolites such as aflatoxin (AF) and cyclopiazonic acid (CPA). Research has shown A. flavus strains can overcome heterokaryon incompatibility and undergo meiotic recombination as teleomorphs. Although evidence of recombination in the AF gene cluster has been reported, the impacts of recombination on genotype and metabolomic phenotype in a single generation are lacking. In previous studies, we paired an aflatoxigenic MAT1-1 A. flavus strain with a non-aflatoxigenic MAT1-2 A. flavus strain that had been tagged with green fluorescent protein and then 10 F1 progenies (a mix of fluorescent and non-fluorescent) were randomly selected from single-ascospore colonies and broadly examined for evidence of recombination. In this study, we determined four of those 10 F1 progenies were recombinants because they were not vegetatively compatible with either parent or their siblings, and they exhibited other distinctive traits that could only result from meiotic recombination. The other six progenies examined shared genomic identity with the non-aflatoxigenic, fluorescent, and MAT1-2 parent, but were metabolically distinct. This study highlights phenotypic and genomic changes that may occur in a single generation from the outcrossing of sexually compatible strains of A. flavus.

Microbiota of maize kernels as influenced by Aspergillus flavus infection in susceptible and resistant inbreds.

Background: Nearly everything on Earth harbors a microbiome. A microbiome is a community of microbes (bacteria, fungi, and viruses) with potential to form complex networks that involve mutualistic and antagonistic interactions. Resident microbiota on/in an organism are determined by the external environment, both biotic and abiotic, and the intrinsic adaptability of each organism. Although the maize microbiome has been characterized, community changes that result from the application of fungal biocontrol strains, such as non-aflatoxigenic Aspergillus flavus, have not. Methods: We silk channel inoculated field-grown maize separately with a non-aflatoxigenic biocontrol strain (K49), a highly toxigenic strain (Tox4), and a combination of both A. flavus strains. Two maize inbreds were treated, A. flavus-susceptible B73 and A. flavus-resistant CML322. We then assessed the impacts of A. flavus introduction on the epibiota and endobiota of their maize kernels. Results: We found that the native microbial communities were significantly affected, irrespective of genotype or sampled tissue. Overall, bacteriomes exhibited greater diversity of genera than mycobiomes. The abundance of certain genera was unchanged by treatment, including genera of bacteria (e.g., Enterobacter, Pantoea) and fungi (e.g., Sarocladium, Meyerozyma) that are known to be beneficial, antagonistic, or both on plant growth and health. Conclusion: Beneficial microbes like Sarocladium that responded well to A. flavus biocontrol strains are expected to enhance biocontrol efficacy, while also displacing/antagonizing harmful microbes.

Complete genome of the toxic mold Aspergillus pseudotamarii isolate NRRL 25517 reveals genomic instability of the aflatoxin biosynthesis cluster.

Fungi can synthesize a broad array of secondary metabolite chemicals. The genes underpinning their biosynthesis are typically arranged in tightly linked clusters in the genome. For example, ∼25 genes responsible for the biosynthesis of carcinogenic aflatoxins by Aspergillus section Flavi species are grouped in a ∼70 Kb cluster. Assembly fragmentation prevents assessment of the role of structural genomic variation in secondary metabolite evolution in this clade. More comprehensive analyses of secondary metabolite evolution will be possible by working with more complete and accurate genomes of taxonomically diverse Aspergillus species. Here, we combined short- and long-read DNA sequencing to generate a highly contiguous genome of the aflatoxigenic fungus, Aspergillus pseudotamarii (isolate NRRL 25517 = CBS 766.97; scaffold N50 = 5.5 Mb). The nuclear genome is 39.4 Mb, encompassing 12,639 putative protein-encoding genes and 74-97 candidate secondary metabolite biosynthesis gene clusters. The circular mitogenome is 29.7 Kb and contains 14 protein-encoding genes that are highly conserved across the genus. This highly contiguous A. pseudotamarii genome assembly enables comparisons of genomic rearrangements between Aspergillus section Flavi series Kitamyces and series Flavi. Although the aflatoxin biosynthesis gene cluster of A. pseudotamarii is conserved with Aspergillus flavus, the cluster has an inverted orientation relative to the telomere and occurs on a different chromosome.

Putative Core Transcription Factors Affecting Virulence in Aspergillus flavus during Infection of Maize.

Aspergillus flavus is an opportunistic pathogen responsible for millions of dollars in crop losses annually and negative health impacts on crop consumers globally. A. flavus strains have the potential to produce aflatoxin and other toxic secondary metabolites, which often increase during plant colonization. To mitigate the impacts of this international issue, we employ a range of strategies to directly impact fungal physiology, growth and development, thus requiring knowledge on the underlying molecular mechanisms driving these processes. Here we utilize RNA-sequencing data that are obtained from in situ assays, whereby Zea mays kernels are inoculated with A. flavus strains, to select transcription factors putatively driving virulence-related gene networks. We demonstrate, through growth, sporulation, oxidative stress-response and aflatoxin/CPA analysis, that three A. flavus strains with knockout mutations for the putative transcription factors AFLA_089270, AFLA_112760, and AFLA_031450 demonstrate characteristics such as reduced growth capacity and decreased aflatoxin/CPA accumulation in kernels consistent with decreased fungal pathogenicity. Furthermore, AFLA_089270, also known as HacA, eliminates CPA production and impacts the fungus's capacity to respond to highly oxidative conditions, indicating an impact on plant colonization. Taken together, these data provide a sound foundation for elucidating the downstream molecular pathways potentially contributing to fungal virulence.

Dataset for transcriptomic profiles associated with development of sexual structures in Aspergillus flavus.

Information on the transcriptomic changes that occur within sclerotia of Aspergillus flavus during its sexual cycle is very limited and warrants further research. The findings will broaden our knowledge of the biology of A. flavus and can provide valuable insights in the development or deployment of non-toxigenic strains as biocontrol agents against aflatoxigenic strains. This article presents transcriptomic datasets included in our research article entitled, "Development of sexual structures influences metabolomic and transcriptomic profiles in Aspergillus flavus" [1], which utilized transcriptomics to identify possible genes and gene clusters associated with sexual reproduction and fertilization in A. flavus. RNA was extracted from sclerotia of a high fertility cross (Hi-Fert-Mated), a low fertility cross (Lo-Fert-Mated), and unmated strains (Hi-Fert-Unmated and Lo-Fert-Unmated) of A. flavus collected immediately after crossing and at every two weeks until eight weeks of incubation on mixed cereal agar at 30 °C in continuous darkness (n = 4 replicates from each treatment for each time point; 80 total). Raw sequencing reads obtained on an Illumina NovaSeq 6000 were deposited in NCBI's Sequence Read Archive (SRA) repository under BioProject accession number PRJNA789260. Reads were mapped to the A. flavus NRRL 3357 genome (assembly JCVI-afl1-v2.0; GCA_000006275.2) using STAR software. Differential gene expression analyses, functional analyses, and weighted gene co-expression network analysis were performed using DESeq2 R packages. The raw and analyzed data presented in this article could be reused for comparisons with other datasets to obtain transcriptional differences among strains of A. flavus or closely related species. The data can also be used for further investigation of the molecular basis of different processes involved in sexual reproduction and sclerotia fertility in A. flavus.

Development of sexual structures influences metabolomic and transcriptomic profiles in Aspergillus flavus.

Sclerotium (female) fertility, the ability of a strain to produce ascocarps, influences internal morphological changes during sexual reproduction in Aspergillus flavus. Although sclerotial morphogenesis has been linked to secondary metabolite (SM) biosynthesis, metabolic and transcriptomic changes within A. flavus sclerotia during sexual development are not known. Successful mating between compatible strains may result in relatively high or low numbers of ascocarps being produced. Sclerotia from a high fertility cross (Hi-Fert-Mated), a low fertility cross (Lo-Fert-Mated), unmated strains (Hi-Fert-Unmated and Lo-Fert-Unmated) were harvested immediately after crosses were made and every two weeks until 8 weeks of incubation, then subjected to targeted metabolomics (n = 106) and transcriptomics analyses (n = 80). Aflatoxin B1 production varied between Hi-Fert-Mated and Hi-Fert-Unmated sclerotia, while it remained low or was undetected in Lo-Fert-Mated and Lo-Fert-Unmated sclerotia. Profiling of 14 SMs showed elevated production of an aflavazole analog, an aflavinine isomer, and hydroxyaflavinine in Hi-Fert-Mated sclerotia at 4 to 8 weeks. Similarly, genes ayg1, hxtA, MAT1, asd-3, preA and preB, and genes in uncharacterized SM gene clusters 30 and 44 showed increased expression in Hi-Fert-Mated sclerotia at these time points. These results broaden our knowledge of the biochemical and transcriptional processes during sexual development in A. flavus.

Small NRPS-like enzymes in Aspergillus sections Flavi and Circumdati selectively form substituted pyrazinone metabolites.

Aspergillus fungi produce mycotoxins that are detrimental to human and animal health. Two sections of aspergilli are of particular importance to cereal food crops such as corn and barley. Aspergillus section Flavi species like A. flavus and A. parasiticus produce aflatoxins, while section Circumdati species like A. ochraceus and A. sclerotiorum produce ochratoxin A. Mitigating these toxins in food and feed is a critical and ongoing worldwide effort. We have previously investigated biosynthetic gene clusters in Aspergillus flavus that are linked to fungal virulence in corn. We found that one such cluster, asa, is responsible for the production of aspergillic acid, an iron-binding, hydroxamic acid-containing pyrazinone metabolite. Furthermore, we found that the asa gene cluster is present in many other aflatoxin- and ochratoxin-producing aspergilli. The core gene in the asa cluster encodes the small nonribosomal peptide synthetase-like (NRPS-like) protein AsaC. We have swapped the asaC ortholog from A. sclerotiorum into A. flavus, replacing its native copy, and have also cloned both asaC orthologs into Saccharomyces cerevisiae. We show that AsaC orthologs in section Flavi and section Circumdati, while only containing adenylation-thiolation-reductase (ATR) domains, can selectively biosynthesize distinct pyrazinone natural products: deoxyaspergillic acid and flavacol, respectively. Because pyrazinone natural products and the gene clusters responsible for their production are implicated in a variety of important microbe-host interactions, uncovering the function and selectivity of the enzymes involved could lead to strategies that ultimately benefit human health.

Flavonoids Modulate the Accumulation of Toxins From Aspergillus flavus in Maize Kernels.

Aspergillus flavus is an opportunistic fungal pathogen capable of producing aflatoxins, potent carcinogenic toxins that accumulate in maize kernels after infection. To better understand the molecular mechanisms of maize resistance to A. flavus growth and aflatoxin accumulation, we performed a high-throughput transcriptomic study in situ using maize kernels infected with A. flavus strain 3357. Three maize lines were evaluated: aflatoxin-contamination resistant line TZAR102, semi-resistant MI82, and susceptible line Va35. A modified genotype-environment association method (GEA) used to detect loci under selection via redundancy analysis (RDA) was used with the transcriptomic data to detect genes significantly influenced by maize line, fungal treatment, and duration of infection. Gene ontology enrichment analysis of genes highly expressed in infected kernels identified molecular pathways associated with defense responses to fungi and other microbes such as production of pathogenesis-related (PR) proteins and lipid bilayer formation. To further identify novel genes of interest, we incorporated genomic and phenotypic field data from a genome wide association analysis with gene expression data, allowing us to detect significantly expressed quantitative trait loci (eQTL). These results identified significant association between flavonoid biosynthetic pathway genes and infection by A. flavus. In planta fungal infections showed that the resistant line, TZAR102, has a higher fold increase of the metabolites naringenin and luteolin than the susceptible line, Va35, when comparing untreated and fungal infected plants. These results suggest flavonoids contribute to plant resistance mechanisms against aflatoxin contamination through modulation of toxin accumulation in maize kernels.

Genetic Responses and Aflatoxin Inhibition during Co-Culture of Aflatoxigenic and Non-Aflatoxigenic Aspergillus flavus.

Aflatoxin is a carcinogenic mycotoxin produced by Aspergillus flavus. Non-aflatoxigenic (Non-tox) A. flavus isolates are deployed in corn fields as biocontrol because they substantially reduce aflatoxin contamination via direct replacement and additionally via direct contact or touch with toxigenic (Tox) isolates and secretion of inhibitory/degradative chemicals. To understand touch inhibition, HPLC analysis and RNA sequencing examined aflatoxin production and gene expression of Non-tox isolate 17 and Tox isolate 53 mono-cultures and during their interaction in co-culture. Aflatoxin production was reduced by 99.7% in 72 h co-cultures. Fewer than expected unique reads were assigned to Tox 53 during co-culture, indicating its growth and/or gene expression was inhibited in response to Non-tox 17. Predicted secreted proteins and genes involved in oxidation/reduction were enriched in Non-tox 17 and co-cultures compared to Tox 53. Five secondary metabolite (SM) gene clusters and kojic acid synthesis genes were upregulated in Non-tox 17 compared to Tox 53 and a few were further upregulated in co-cultures in response to touch. These results suggest Non-tox strains can inhibit growth and aflatoxin gene cluster expression in Tox strains through touch. Additionally, upregulation of other SM genes and redox genes during the biocontrol interaction demonstrates a potential role of inhibitory SMs and antioxidants as additional biocontrol mechanisms and deserves further exploration to improve biocontrol formulations.

Chromosome-Level Aspergillus flavus Strain CA14 Genome Assembly.

We report here a chromosome-level genome assembly of the aflatoxigenic fungus Aspergillus flavus strain CA14. This strain is the basis for numerous studies in fungal physiology and secondary metabolism. This full-length assembly will aid in subsequent genomics research.

Genome Sequences of 20 Georeferenced Aspergillus flavus Isolates.

Several agricultural commodities can be infected by Aspergillus flavus, a fungus that can produce the carcinogen aflatoxin. Here, we report the whole-genome sequences for 20 georeferenced isolates collected from soil and corn under field conditions. This information contributes to an understanding of A. flavus population structure and dynamics in a field environment.

Development of Genomic Resources for the Powdery Mildew, Erysiphe pulchra.

Powdery mildews (PMs) are important plant pathogens causing widespread damage. Here, we report the first draft genome of Erysiphe pulchra, the causative agent of PM of flowering dogwood, Cornus florida. The assembled genome was 63.5 Mbp and resulted in formation of 19,442 contigs (N50 = 11,686 bp) that contained an estimated 6,860 genes with a genome coverage of 62×. We found 102 candidate secreted effector proteins (CSEPs) in E. pulchra similar to E. necator genes that are potentially involved in disease development. This draft genome is an initial step for understanding the evolutionary history of the PMs and will also provide insight into evolutionary strategies that led to the wide host expansion and environmental adaptations so effectively employed by the PM lineages.

The Transcriptional Regulator Hbx1 Affects the Expression of Thousands of Genes in the Aflatoxin-Producing Fungus Aspergillus flavus.

In filamentous fungi, homeobox proteins are conserved transcriptional regulators described to control conidiogenesis and fruiting body formation. Eight homeobox (hbx) genes are found in the genome of the aflatoxin-producing ascomycete, Aspergillus flavus While loss-of-function of seven of the eight genes had little to no effect on fungal growth and development, disruption of hbx1, resulted in aconidial colonies and lack of sclerotial production. Furthermore, the hbx1 mutant was unable to produce aflatoxins B1 and B2, cyclopiazonic acid and aflatrem. In the present study, hbx1 transcriptome analysis revealed that hbx1 has a broad effect on A. flavus gene expression, and the effect of hbx1 increases overtime, impacting more than five thousand protein-coding genes. Among the affected genes, those in the category of secondary metabolism (SM), followed by that of cellular transport, were the most affected. Specifically, regarding the effect of hbx1 on SM, we found that genes in 44 SM gene clusters where upregulated while 49 were downregulated in the absence of hbx1, including genes in the SM clusters responsible for the synthesis of asparasone, piperazine and aflavarin, all known to be associated with sclerotia. In addition, our study revealed that hbx1 affects the expression of other transcription factor genes involved in development, including the conidiation central regulatory pathway and flb genes.

Whole genome comparison of Aspergillus flavus L-morphotype strain NRRL 3357 (type) and S-morphotype strain AF70.

Aspergillus flavus is a saprophytic fungus that infects corn, peanuts, tree nuts and other agriculturally important crops. Once the crop is infected the fungus has the potential to secrete one or more mycotoxins, the most carcinogenic of which is aflatoxin. Aflatoxin contaminated crops are deemed unfit for human or animal consumption, which results in both food and economic losses. Within A. flavus, two morphotypes exist: the S strains (small sclerotia) and L strains (large sclerotia). Significant morphological and physiological differences exist between the two morphotypes. For example, the S-morphotypes produces sclerotia that are smaller (< 400 µm), greater in quantity, and contain higher concentrations of aflatoxin than the L-morphotypes (>400 µm). The morphotypes also differ in pigmentation, pH homeostasis in culture and the number of spores produced. Here we report the first full genome sequence of an A. flavus S morphotype, strain AF70. We provide a comprehensive comparison of the A. flavus S-morphotype genome sequence with a previously sequenced genome of an L-morphotype strain (NRRL 3357), including an in-depth analysis of secondary metabolic clusters and the identification SNPs within their aflatoxin gene clusters.

Identification and functional analysis of the aspergillic acid gene cluster in Aspergillus flavus.

Aspergillus flavus can colonize important food staples and produce aflatoxins, a group of toxic and carcinogenic secondary metabolites. Previous in silico analysis of the A. flavus genome revealed 56 gene clusters predicted to be involved in the biosynthesis of secondary metabolites. A. flavus secondary metabolites produced during infection of maize seed are of particular interest, especially with respect to their roles in the biology of the fungus. A predicted nonribosomal peptide synthetase-like (NRPS-like) gene, designated asaC (AFLA_023020), present in the uncharacterized A. flavus secondary metabolite gene cluster 11 was previously shown to be expressed during the earliest stages of maize kernel infection. Cluster 11 is composed of six additional genes encoding a number of putative decorating enzymes as well as a transporter and transcription factor. We generated knock-out mutants of the seven predicted cluster 11 genes. LC-MS analysis of extracts from knockout mutants of these genes showed that they were responsible for the synthesis of the previously characterized antimicrobial mycotoxin aspergillic acid. Extracts of the asaC mutant showed no production of aspergillic acid or its precursors. Knockout of the cluster 11 P450 oxidoreductase afforded a pyrazinone metabolite, the aspergillic acid precursor deoxyaspergillic acid. The formation of hydroxyaspergillic acid was abolished in a desaturase/hydroxylase mutant. The hydroxamic acid functional group in aspergillic acid allows the molecule to bind to iron resulting in the production of a red pigment in A. flavus identified previously as ferriaspergillin. A reduction of aflatoxin B1 and cyclopiazonic acid that correlated with reduced fungal growth was observed in maize kernel infection assays when aspergillic acid biosynthesis in A. flavus is halted.

Genome sequence of an aflatoxigenic pathogen of Argentinian peanut, Aspergillus arachidicola.

BACKGROUND: Aspergillus arachidicola is an aflatoxigenic fungal species, first isolated from the leaves of a wild peanut species native to Argentina. It has since been reported in maize, Brazil nut and human sputum samples. This aflatoxigenic species is capable of secreting both B and G aflatoxins, similar to A. parasiticus and A. nomius. It has other characteristics that may result in its misidentification as one of several other section Flavi species. This study offers a preliminary analysis of the A. arachidicola genome. RESULTS: In this study we sequenced the genome of the A. arachidicola type strain (CBS 117610) and found its genome size to be 38.9 Mb, and its number of predicted genes to be 12,091, which are values comparable to those in other sequenced Aspergilli. A comparison of 57 known Aspergillus secondary metabolite gene clusters, among closely-related aflatoxigenic species, revealed nearly half were predicted to exist in the type strain of A. arachidicola. Of its predicted genes, 691 were identified as unique to the species and 60% were assigned Gene Ontology terms using BLAST2GO. Phylogenomic inference shows CBS 117610 sharing a most recent common ancestor with A. parasiticus. Finally, BLAST query of A. flavus mating-type idiomorph sequences to this strain revealed the presence of a single mating-type (MAT1-1) idiomorph. CONCLUSIONS: Based on A. arachidicola morphological, genetic and chemotype similarities with A. flavus and A. parasiticus, sequencing the genome of A. arachidicola will contribute to our understanding of the evolutionary relatedness among aflatoxigenic fungi.

Carbon Dioxide Mediates the Response to Temperature and Water Activity Levels in Aspergillus flavus during Infection of Maize Kernels.

Aspergillus flavus is a saprophytic fungus that may colonize several important crops, including cotton, maize, peanuts and tree nuts. Concomitant with A. flavus colonization is its potential to secrete mycotoxins, of which the most prominent is aflatoxin. Temperature, water activity (aw) and carbon dioxide (CO2) are three environmental factors shown to influence the fungus-plant interaction, which are predicted to undergo significant changes in the next century. In this study, we used RNA sequencing to better understand the transcriptomic response of the fungus to aw, temperature, and elevated CO2 levels. We demonstrate that aflatoxin (AFB1) production on maize grain was altered by water availability, temperature and CO2. RNA-Sequencing data indicated that several genes, and in particular those involved in the biosynthesis of secondary metabolites, exhibit different responses to water availability or temperature stress depending on the atmospheric CO2 content. Other gene categories affected by CO2 levels alone (350 ppm vs. 1000 ppm at 30 °C/0.99 aw), included amino acid metabolism and folate biosynthesis. Finally, we identified two gene networks significantly influenced by changes in CO2 levels that contain several genes related to cellular replication and transcription. These results demonstrate that changes in atmospheric CO2 under climate change scenarios greatly influences the response of A. flavus to water and temperature when colonizing maize grain.

The Aspergillus flavus Homeobox Gene, hbx1, is Required for Development and Aflatoxin Production.

Homeobox proteins, a class of well conserved transcription factors, regulate the expression of targeted genes, especially those involved in development. In filamentous fungi, homeobox genes are required for normal conidiogenesis and fruiting body formation. In the present study, we identified eight homeobox (hbx) genes in the aflatoxin-producing ascomycete, Aspergillus flavus, and determined their respective role in growth, conidiation and sclerotial production. Disruption of seven of the eight genes had little to no effect on fungal growth and development. However, disruption of the homeobox gene AFLA_069100, designated as hbx1, in two morphologically different A. flavus strains, CA14 and AF70, resulted in complete loss of production of conidia and sclerotia as well as aflatoxins B1 and B2, cyclopiazonic acid and aflatrem. Microscopic examination showed that the Δhbx1 mutants did not produce conidiophores. The inability of Δhbx1 mutants to produce conidia was related to downregulation of brlA (bristle) and abaA (abacus), regulatory genes for conidiophore development. These mutants also had significant downregulation of the aflatoxin pathway biosynthetic genes aflC, aflD, aflM and the cluster-specific regulatory gene, aflR. Our results demonstrate that hbx1 not only plays a significant role in controlling A. flavus development but is also critical for the production of secondary metabolites, such as aflatoxins.

Interactions between water activity and temperature on the Aspergillus flavus transcriptome and aflatoxin B1 production.

Effects of Aspergillus flavus colonization of maize kernels under different water activities (aw; 0.99 and 0.91) and temperatures (30, 37°C) on (a) aflatoxin B1 (AFB1) production and (b) the transcriptome using RNAseq were examined. There was no significant difference (p=0.05) in AFB1 production at 30 and 37°C and 0.99 aw. However, there was a significant (p=0.05) increase in AFB1 at 0.91 aw at 37°C when compared with 30°C/0.99 aw. Environmental stress effects using gene ontology enrichment analysis of the RNA-seq results for increasing temperature at 0.99 and 0.91 aw showed differential expression of 2224 and 481 genes, respectively. With decreasing water availability, 4307 were affected at 30°C and 702 genes at 37°C. Increasing temperature from 30 to 37°C at both aw levels resulted in 12 biological processes being upregulated and 9 significantly downregulated. Decreasing aw at both temperatures resulted in 22 biological processes significantly upregulated and 25 downregulated. The interacting environmental factors influenced functioning of the secondary metabolite gene clusters for aflatoxins and cyclopiazonic acid (CPA). An elevated number of genes were co-regulated by both aw and temperature. An interaction effect for 4 of the 25 AFB1 genes, including regulatory and transcription activators occurred. For CPA, all 5 biosynthetic genes were affected by aw stress, regardless of temperature. The molecular regulation of A. flavus in maize is discussed.