Fungal species within Aspergillus section Flavi contaminate food and feed with aflatoxins. These toxic fungal metabolites compromise human and animal health and disrupt trade. Genotypically and phenotypically diverse species co-infect crops, but temporal and spatial variation in frequencies of different lineages suggests that environmental factors such as temperature may influence structure of aflatoxin-producing fungal communities. Furthermore, though most species within Aspergillus section Flavi produce sclerotia, divergent sclerotial morphologies (small or S-type sclerotia vs. large or L-type sclerotia) and differences in types and quantities of aflatoxins produced suggest lineages are adapted to different life strategies. Temperature is a key parameter influencing pre- and post-harvest aflatoxin contamination of crops. We tested the hypothesis that species of aflatoxin-producing fungi that differ in sclerotial morphology will vary in competitive ability and that outcomes of competition and aflatoxin production will be modulated by temperature. Paired competition experiments between highly aflatoxigenic S-type species (A. aflatoxiformans and Lethal Aflatoxicosis Fungus) and L-type species (A. flavus L morphotype and A. parasiticus) were conducted on maize kernels at 25 and 30°C. Proportions of each isolate growing within and sporulating on kernels were measured using quantitative pyrosequencing. At 30°C, S-type fungi were more effective at host colonization compared to L-type isolates. Total aflatoxins and the proportion of B vs. G aflatoxins were greater at 30°C compared to 25°C. Sporulation by L-type isolates was reduced during competition with S-type fungi at 30°C, while relative quantities of conidia produced by S-type species either increased or did not change during competition. Results indicate that both species interactions and temperature can shape population structure of Aspergillus section Flavi, with warmer temperatures favoring growth and dispersal of highly toxigenic species with S-type sclerotia.

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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.

Aspergillus section Flavi species co-occur and contaminate crops, including maize and groundnuts, with aflatoxins. Competition among A. flavus genotypes is influenced by crop host, but competition between Aspergillus species has not been examined. Objectives of the current study were to 1) assess competition among four aflatoxin-producing species on maize and groundnuts, and 2) evaluate within-species variation in competitive ability during co-infection with another species on the two crops. For Objective 1, maize and groundnut kernels were co-inoculated with all possible pairs of A. flavus, A. parasiticus, A. aflatoxiformans, and an unnamed taxon known as the Lethal Aflatoxicosis Fungus (LAF). For Objective 2, three isolates from each of the four species were co-inoculated with a representative isolate of a competing species on the two hosts. In all experiments, isolates were co-cultured for 7 days at 30°C and then aflatoxins and total conidia were measured, and percentages of each species within a treatment were assessed with quantitative pyrosequencing. Maize kernels supported greater aflatoxin production than groundnuts while groundnuts supported greater sporulation than maize. Hosts differentially influenced competition between species with A. flavus generally more competitive on maize and LAF more competitive on groundnuts. Overall, A. flavus and LAF were the most competitive species while A. parasiticus was the least competitive. However, isolates within a species varied in competitive ability and in their response to host and competing species. Results suggest that though crop hosts influence Aspergillus community composition, within-species variability makes it difficult to predict outcomes of competition on a particular crop.

Aspergillus flavus infects a wide range of crops, including pistachios, and subsequent aflatoxin contamination results in significant economic losses. Application of biocontrol products based on non-aflatoxigenic (atoxigenic) strains of A. flavus is one of the most effective tactics for controlling aflatoxins in crops. Both risk of aflatoxin contamination and effectiveness of biocontrol are influenced by the extent to which A. flavus spores move into pistachio tree canopies during periods of nut development. Thus, the purpose of this study was to evaluate spatial and temporal population dynamics of A. flavus, including the applied biocontrol strain AF36, in canopies of pistachio orchards in Arizona. Propagule densities of A. flavus were quantified on leaf samples collected from lower, middle, and upper canopies from spring through harvest in 2018 and 2019. Aspergillus flavus propagule densities peaked during periods of high temperature and rainfall in 2018 (up to 600 CFU/g) and 2019 (up to 23 CFU/g), which coincided with nut development and maturation. The applied biocontrol strain AF36 was detected at all canopy heights, but overall propagule densities were greater in the upper and middle canopy (mean = 70 CFU/g) compared to the lower canopy (mean = 47 CFU/g). Results suggest June to August is the period during which A. flavus inoculum increases in Arizona pistachio orchards, and to most effectively displace aflatoxin-producing fungi in tree canopies, biocontrol applications should precede this period. In addition, this study demonstrates that soil-applied biocontrol strains can successfully disperse throughout the canopies of commercial tree nut orchards.