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.

Practical considerations will ensure the continued success of pre-harvest biocontrol using non-aflatoxigenic Aspergillus flavus strains.

There is an important reason for the accelerated use of non-aflatoxigenic Aspergillus flavus to mitigate pre-harvest aflatoxin contamination… it effectively addresses the imperative need for safer food and feed. Now that we have decades of proof of the effectiveness of A. flavus as biocontrol, it is time to improve several aspects of this strategy. If we are to continue relying heavily on this form of aflatoxin mitigation, there are considerations we must acknowledge, and actions we must take, to ensure that we are best wielding this strategy to our advantage. These include its: (1) potential to produce other mycotoxins, (2) persistence in the field in light of several ecological factors, (3) its reproductive and genetic stability, (4) the mechanism(s) employed that allow it to elicit control over aflatoxigenic strains and species of agricultural importance and (5) supplemental alternatives that increase its effectiveness. There is a need to be consistent, practical and thoughtful when it comes to implementing this method of mycotoxin mitigation since these fungi are living organisms that have been adapting, evolving and surviving on this planet for tens-of-millions of years. This document will serve as a critical review of the literature regarding pre-harvest A. flavus biocontrol and will discuss opportunities for improvements.

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.

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.

Evolution of polyketide synthesis in a Dothideomycete forest pathogen.

Fungal secondary metabolites have many important biological roles and some, like the toxic polyketide aflatoxin, have been intensively studied at the genetic level. Complete sets of polyketide synthase (PKS) genes can now be identified in fungal pathogens by whole genome sequencing and studied in order to predict the biosynthetic potential of those fungi. The pine needle pathogen Dothistroma septosporum is predicted to have only three functional PKS genes, a small number for a hemibiotrophic fungus. One of these genes is required for production of dothistromin, a polyketide virulence factor related to aflatoxin, whose biosynthetic genes are dispersed across one chromosome rather than being clustered. Here we evaluated the evolution of the other two genes, and their predicted gene clusters, using phylogenetic and population analyses. DsPks1 and its gene cluster are quite conserved amongst related fungi, whilst DsPks2 appears to be novel. The DsPks1 protein was predicted to be required for dihydroxynaphthalene (DHN) melanin biosynthesis but functional analysis of DsPks1 mutants showed that D. septosporum produced mainly dihydroxyphenylalanine (DOPA) melanin, which is produced by a PKS-independent pathway. Although the secondary metabolites made by these two PKS genes are not known, comparisons between strains of D. septosporum from different regions of the world revealed that both PKS core genes are under negative selection and we suggest they may have important cryptic roles in planta.

Genomic sequence of the aflatoxigenic filamentous fungus Aspergillus nomius.

BACKGROUND: Aspergillus nomius is an opportunistic pathogen and one of the three most important producers of aflatoxins in section Flavi. This fungus has been reported to contaminate agricultural commodities, but it has also been sampled in non-agricultural areas so the host range is not well known. Having a similar mycotoxin profile as A. parasiticus, isolates of A. nomius are capable of secreting B- and G- aflatoxins. RESULTS: In this study we discovered that the A. nomius type strain (NRRL 13137) has a genome size of approximately 36 Mb which is comparable to other Aspergilli whose genomes have been sequenced. Its genome encompasses 11,918 predicted genes, 72% of which were assigned GO terms using BLAST2GO. More than 1,200 of those predicted genes were identified as unique to A. nomius, and the most significantly enriched GO category among the unique genes was oxidoreducatase activity. Phylogenomic inference shows NRRL 13137 as ancestral to the other aflatoxigenic species examined from section Flavi. This strain contains a single mating-type idiomorph designated as MAT1-1. CONCLUSIONS: This study provides a preliminary analysis of the A. nomius genome. Given the recently discovered potential for A. nomius to undergo sexual recombination, and based on our findings, this genome sequence provides an additional evolutionary reference point for studying the genetics and biology of aflatoxin production.

Sexuality generates diversity in the aflatoxin gene cluster: evidence on a global scale.

Aflatoxins are produced by Aspergillus flavus and A. parasiticus in oil-rich seed and grain crops and are a serious problem in agriculture, with aflatoxin B1 being the most carcinogenic natural compound known. Sexual reproduction in these species occurs between individuals belonging to different vegetative compatibility groups (VCGs). We examined natural genetic variation in 758 isolates of A. flavus, A. parasiticus and A. minisclerotigenes sampled from single peanut fields in the United States (Georgia), Africa (Benin), Argentina (Córdoba), Australia (Queensland) and India (Karnataka). Analysis of DNA sequence variation across multiple intergenic regions in the aflatoxin gene clusters of A. flavus, A. parasiticus and A. minisclerotigenes revealed significant linkage disequilibrium (LD) organized into distinct blocks that are conserved across different localities, suggesting that genetic recombination is nonrandom and a global occurrence. To assess the contributions of asexual and sexual reproduction to fixation and maintenance of toxin chemotype diversity in populations from each locality/species, we tested the null hypothesis of an equal number of MAT1-1 and MAT1-2 mating-type individuals, which is indicative of a sexually recombining population. All samples were clone-corrected using multi-locus sequence typing which associates closely with VCG. For both A. flavus and A. parasiticus, when the proportions of MAT1-1 and MAT1-2 were significantly different, there was more extensive LD in the aflatoxin cluster and populations were fixed for specific toxin chemotype classes, either the non-aflatoxigenic class in A. flavus or the B1-dominant and G1-dominant classes in A. parasiticus. A mating type ratio close to 1∶1 in A. flavus, A. parasiticus and A. minisclerotigenes was associated with higher recombination rates in the aflatoxin cluster and less pronounced chemotype differences in populations. This work shows that the reproductive nature of the population (more sexual versus more asexual) is predictive of aflatoxin chemotype diversity in these agriculturally important fungi.

Enhanced diversity and aflatoxigenicity in interspecific hybrids of Aspergillus flavus and Aspergillus parasiticus.

Aspergillus flavus and A. parasiticus are the two most important aflatoxin-producing fungi responsible for the contamination of agricultural commodities worldwide. Both species are heterothallic and undergo sexual reproduction in laboratory crosses. Here we examine the possibility of interspecific matings between A. flavus and A. parasiticus. These species can be distinguished morphologically and genetically, as well as by their mycotoxin profiles. Aspergillus flavus produces both B aflatoxins and cyclopiazonic acid (CPA), B aflatoxins or CPA alone, or neither mycotoxin; Aspergillus parasiticus produces B and G aflatoxins or the aflatoxin precursor O-methylsterigmatocystin, but not CPA. Only four of forty-five attempted interspecific crosses between opposite mating types of A. flavus and A. parasiticus were fertile and produced viable ascospores. Single ascospore strains from each cross were shown to be recombinant hybrids using multilocus genotyping and array comparative genome hybridization. Conidia of parents and their hybrid progeny were haploid and predominantly monokaryons and dikaryons based on flow cytometry. Multilocus phylogenetic inference showed that experimental hybrid progeny were grouped with naturally occurring A. flavus L strain and A. parasiticus. Higher total aflatoxin concentrations in some F1 progeny strains compared to midpoint parent aflatoxin levels indicate synergism in aflatoxin production; moreover, three progeny strains synthesized G aflatoxins that were not produced by the parents, and there was evidence of allopolyploidization in one strain. These results suggest that hybridization is an important diversifying force resulting in the genesis of novel toxin profiles in these agriculturally important fungi.

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.

Fragmentation of an aflatoxin-like gene cluster in a forest pathogen.

Plant pathogens use a complex arsenal of weapons, such as toxic secondary metabolites, to invade and destroy their hosts. Knowledge of how secondary metabolite pathways evolved is central to understanding the evolution of host specificity. The secondary metabolite dothistromin is structurally similar to aflatoxins and is produced by the fungal pine pathogen Dothistroma septosporum. Our study focused on dothistromin genes, which are widely dispersed across one chromosome, to determine whether this unusual distributed arrangement evolved from an ancestral cluster. We combined comparative genomics and population genetics approaches to elucidate the origins of the dispersed arrangement of dothistromin genes over a broad evolutionary time-scale at the phylum, class and species levels. Orthologs of dothistromin genes were found in two major classes of fungi. Their organization is consistent with clustering of core pathway genes in a common ancestor, but with intermediate cluster fragmentation states in the Dothideomycetes fungi. Recombination hotspots in a D. septosporum population matched sites of gene acquisition and cluster fragmentation at higher evolutionary levels. The results suggest that fragmentation of a larger ancestral cluster gave rise to the arrangement seen in D. septosporum. We propose that cluster fragmentation may facilitate metabolic retooling and subsequent host adaptation of plant pathogens.

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.

Functional and phylogenetic analysis of the Aspergillus ochraceoroseus aflQ (ordA) gene ortholog.

Within the Aspergillus parasiticus and A. flavus aflatoxin (AF) biosynthetic gene cluster the aflQ (ordA) and aflP (omtA) genes encode respectively an oxidoreductase and methyltransferase. These genes are required for the final steps in the conversion of sterigmatocystin (ST) to aflatoxin B(1) (AFB(1)). Aspergillus nidulans harbors a gene cluster that produces ST, as the aflQ and aflP orthologs are either non-functional or absent in the genome. Aspergillus ochraceoroseus produces both AF and ST, and it harbors an AF/ST biosynthetic gene cluster that is organized much like the A. nidulans ST cluster. The A. ochraceoroseus cluster also does not contain aflQ or aflP orthologs. However the ability of A. ochraceoroseus to produce AF would indicate that functional aflQ and aflP orthologs are present within the genome. Utilizing degenerate primers based on conserved regions of the A. flavus aflQ gene and an A. nidulans gene demonstrating the highest level of homology to aflQ, a putative aflQ ortholog was PCR amplified from A. ochraceoroseus genomic DNA. The A. ochraceoroseus aflQ ortholog demonstrated 57% amino acid identity to A. flavus AflQ. Transformation of an O-methylsterigmatocystin (OMST)-accumulating A. parasiticus aflQ mutant with the putative A. ochraceoroseus aflQ gene restored AF production. Although the aflQ gene does not reside in the AF/ST cluster it appears to be regulated in a manner similar to other A. ochraceoroseus AF/ST cluster genes. Phylogenetic analysis of AflQ and AflQ-like proteins from a number of ST- and AF-producing Aspergilli indicates that A. ochraceoroseus might be ancestral to A. nidulans and A. flavus.

Cumulative Effects of Non-Aflatoxigenic Aspergillus flavus Volatile Organic Compounds to Abate Toxin Production by Mycotoxigenic Aspergilli.

Previously, authors reported that individual volatile organic compounds (VOCs) emitted by non-aflatoxigenic Aspergillus flavus could act as a mechanism of biocontrol to significantly reduce aflatoxins and cyclopiazonic acid (CPA) produced by toxigenic strains. In this study, various combinations and volumes of three mycotoxin-reductive VOCs (2,3-dihydrofuran, 3-octanone and decane) were assessed for their cumulative impacts on four Aspergillus strains (LA1-LA4), which were then analyzed for changes in growth, as well as the production of mycotoxins, including aflatoxins, CPA and multiple indole diterpenes. Fungal growth remained minimally inhibited when exposed to various combinations of VOCs. No single combination was able to consistently, or completely, inhibit aflatoxin or CPA across all toxigenic strains tested. However, the combination of 2,3-dihydrofuran and 3-octanone offered the greatest overall reductions in aflatoxin and CPA production. Despite no elimination of their production, findings showed that combining VOCs produced solely by non-aflatoxigenic A. flavus still inhibited several agriculturally important mycotoxins, including B and G aflatoxins and CPA. Therefore, other VOC combinations are worth testing as post-harvest biocontrol treatments to ensure the prolonged effectiveness of pre-harvest biocontrol efforts.

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.

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.

Multi-kingdom microbiota analyses identify bacterial-fungal interactions and biomarkers of colorectal cancer across cohorts.

Despite recent progress in our understanding of the association between the gut microbiome and colorectal cancer (CRC), multi-kingdom gut microbiome dysbiosis in CRC across cohorts is unexplored. We investigated four-kingdom microbiota alterations using CRC metagenomic datasets of 1,368 samples from 8 distinct geographical cohorts. Integrated analysis identified 20 archaeal, 27 bacterial, 20 fungal and 21 viral species for each single-kingdom diagnostic model. However, our data revealed superior diagnostic accuracy for models constructed with multi-kingdom markers, in particular the addition of fungal species. Specifically, 16 multi-kingdom markers including 11 bacterial, 4 fungal and 1 archaeal feature, achieved good performance in diagnosing patients with CRC (area under the receiver operating characteristic curve (AUROC) = 0.83) and maintained accuracy across 3 independent cohorts. Coabundance analysis of the ecological network revealed associations between bacterial and fungal species, such as Talaromyces islandicus and Clostridium saccharobutylicum. Using metagenome shotgun sequencing data, the predictive power of the microbial functional potential was explored and elevated D-amino acid metabolism and butanoate metabolism were observed in CRC. Interestingly, the diagnostic model based on functional EggNOG genes achieved high accuracy (AUROC = 0.86). Collectively, our findings uncovered CRC-associated microbiota common across cohorts and demonstrate the applicability of multi-kingdom and functional markers as CRC diagnostic tools and, potentially, as therapeutic targets for the treatment of CRC.

Sexual reproduction in aflatoxin-producing Aspergillus nomius.

Sexual reproduction was examined in the aflatoxin-producing fungus Aspergillus nomius. Crosses between sexually compatible strains resulted in the formation of multiple nonostiolate ascocarps within stromata, which places the teleomorph in genus Petromyces. Ascocarp and ascospore morphology in Petromyces nomius were similar to that in P. flavus and P. parasiticus, and differences between teleomorphs were insufficient for species separation. Formation of mature ascocarps was infrequent, with only 24% of the 83 crosses producing viable ascospores. The majority of P. nomius strains contained a single mating-type gene (MAT1-1 or MAT1-2), but several strains contained both genes. MAT1-1/MAT1-2 strains were self-sterile and capable of mating with both MAT1-1 and MAT1-2 strains; hence P. nomius appears to be functionally heterothallic.

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.

Draft Genome Sequences of 20 Aspergillus flavus Isolates from Corn Kernels and Cornfield Soils in Louisiana.

Aspergillus flavus is a common saprophyte and opportunistic fungal pathogen that infects plants, animals, and humans. It also produces numerous toxic and nontoxic secondary metabolites. Here, we report the draft genome sequences of 20 A. flavus isolates, belonging to 16 vegetative compatibility groups, from Louisiana corn kernels and cornfield soils.

Characterization of morphological changes within stromata during sexual reproduction in Aspergillus flavus.

Aspergillus flavus contaminates agricultural products worldwide with carcinogenic aflatoxins that pose a serious health risk to humans and animals. The fungus survives adverse environmental conditions through production of sclerotia. When fertilized by a compatible conidium of an opposite mating type, a sclerotium transforms into a stroma within which ascocarps, asci, and ascospores are formed. However, the transition from a sclerotium to a stroma during sexual reproduction in A. flavus is not well understood. Early events during the interaction between sexually compatible strains of A. flavus were visualized using conidia of a green fluorescent protein (GFP)-labeled MAT1-1 strain and sclerotia of an mCherry-labeled MAT1-2 strain. Both conidia and sclerotia of transformed strains germinated to produce hyphae within 24 h of incubation. Hyphal growth of these two strains produced what appeared to be a network of interlocking hyphal strands that were observed at the base of the mCherry-labeled sclerotia (i.e., region in contact with agar surface) after 72 h of incubation. At 5 wk following incubation, intracellular green-fluorescent hyphal strands were observed within the stromatal matrix of the mCherry-labeled strain. Scanning electron microscopy of stromata from a high- and low-fertility cross and unmated sclerotia was used to visualize the formation and development of sexual structures within the stromatal and sclerotial matrices, starting at the time of crossing and thereafter every 2 wk until 8 wk of incubation. Morphological differences between sclerotia and stromata became apparent at 4 wk of incubation. Internal hyphae and croziers were detected inside multiple ascocarps that developed within the stromatal matrix of the high-fertility cross but were not detected in the matrix of the low-fertility cross or the unmated sclerotia. At 6 to 8 wk of incubation, hyphal tips produced numerous asci, each containing one to eight ascospores that emerged out of an ascus following the breakdown of the ascus wall. These observations broaden our knowledge of early events during sexual reproduction and suggest that hyphae from the conidium-producing strain may be involved in the early stages of sexual reproduction in A. flavus. When combined with omics data, these findings could be useful in further exploration of the molecular and biochemical mechanisms underlying sexual reproduction in A. flavus.