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The fungus Aspergillus nidulans produces secondary metabolites during sexual development to protect itself from predators.The fungus Aspergillus nidulans produces secondary metabolites during sexual development to protect itself from predators.

Most asexual species of fungi have either lost sexuality recently, or they experience recombination by cryptic sexual reproduction. Verticillium dahliae is a plant-pathogenic, ascomycete fungus with no known sexual stage, even though related genera have well-described sexual reproduction. V. dahliae reproduces mitotically and its population structure is highly clonal. However, previously described discrepancies in phylogenetic relationships among clonal lineages may be explained more parsimoniously by recombination than mutation; therefore, we looked for evidence of recombination within and between clonal lineages. Genotyping by sequencing was performed on 141 V. dahliae isolates from diverse geographic and host origins, resulting in 26,748 single-nucleotide polymorphisms (SNPs). We found a strongly clonal population structure with the same lineages as described previously by vegetative compatibility groups (VCGs) and molecular markers. We detected 443 recombination events, evenly distributed throughout the genome. Most recombination events detected were between clonal lineages, with relatively few recombinant haplotypes detected within lineages. The only three isolates with mating type MAT1-1 had recombinant SNP haplotypes; all other isolates had mating type MAT1-2. We found homologs of eight meiosis-specific genes in the V. dahliae genome, all with conserved or partially conserved protein domains. The extent of recombination and molecular signs of sex in (mating-type and meiosis-specific genes) suggest that V. dahliae clonal lineages arose by recombination, even though the current population structure is markedly clonal. Moreover, the detection of new lineages may be evidence that sexual reproduction has occurred recently and may potentially occur under some circumstances. We speculate that the current clonal population structure, despite the sexual origin of lineages, has arisen, in part, as a consequence of agriculture and selection for adaptation to agricultural cropping systems.

Fusarium graminearum is a primary cause of Fusarium head blight (FHB) on wheat and barley. The fungus produces trichothecene mycotoxins that render grain unsuitable for food, feed, or malt. Isolates of F. graminearum can differ in trichothecene production phenotypes (chemotypes), with individuals producing predominantly one of four toxins: 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, nivalenol, or NX-2. Molecular tools to diagnose chemotypes remain inefficient. This study aimed to develop a single-tube, multiplex molecular assay that can predict the four F. graminearum chemotypes. Conserved functional regions of three trichothecene biosynthetic genes ( TRI1 , TRI8 , and TRI13 ) were targeted to develop a high-resolution melting (HRM) assay. Multiplex HRM analysis produced unique melting profiles for each chemotype, and was validated on a panel of 80 isolates. We applied machine learning-based linear discriminant analysis (LDA) to automate the classification of chemotypes from the HRM data, achieving a prediction accuracy of over 99%. The assay is sensitive, with a limit of detection below 0.02 ng of fungal DNA. The HRM analysis also differentiated chemotypes from a small sample of F. gerlachii , F. asiaticum , and F. vorosii isolates. Together, our results demonstrate that this simple, rapid, and accurate assay can be applied to F. graminearum molecular diagnostics and population surveillance programs.

Aspergillus flavus is a clinically and agriculturally important saprotrophic fungus responsible for severe human infections and extensive crop losses. Here, we analyze genomic data from 300 (117 clinical and 183 environmental) A. flavus isolates from 13 countries, including 82 clinical isolates sequenced in this study, to examine population and pan-genome structure and their relationship to pathogenicity. We use single nucleotide polymorphisms to build a phylogeny, analyze admixture, and perform discriminant analysis of principal components. We identify five A. flavus populations, including a new population, D, corresponding to distinct clades in the genome-wide phylogeny. Strikingly, > 75% of clinical isolates were in population D and <5% in population B. We also use orthogroup clustering to identify core and accessory genes within the pan-genome. Accessory genes, including genes within biosynthetic gene clusters, were significantly more common in some populations but rare in others. Our functional annotations show that population D is enriched for genes associated with carbohydrate metabolism, lipid metabolism and certain types of hydrolase activity, whereas a non-clinical population is depleted in genes related to zinc ion binding. In contrast to previous results from the major human pathogen Aspergillus fumigatus , isolation of A. flavus from human specimens is associated with population structure, providing a promising system for future investigations into the contributions of population-specific genetic differences to human infection. Genetic diversity between clinical and environmental fungal isolates of Aspergillus flavus is poorly studied. Here, the authors analysed genomic data from a global set of clinical and environmental A. flavus isolates and report that clinical prevalence is associated with population structure.

Differences in the relative frequencies of sexual and asexual reproduction have profound implications for the accumulation of deleterious mutations (Muller’s ratchet), but little is known about how these differences impact the evolution of ecologically important phenotypes. Aspergillus flavus is the main producer of aflatoxin, a notoriously potent carcinogen that often contaminates food. We investigated if differences in the levels of production of aflatoxin by A. flavus could be explained by the accumulation of deleterious mutations due to a lack of recombination. Despite differences in the extent of recombination, variation in aflatoxin production is better explained by the demography and history of specific populations and may suggest important differences in the ecological roles of aflatoxin among populations. Furthermore, the association of aflatoxin production and populations provides a means of predicting the risk of aflatoxin contamination by determining the frequencies of isolates from low- and high-production populations. The apparent rarity of sex in many fungal species has raised questions about how much sex is needed to purge deleterious mutations and how differences in frequency of sex impact fungal evolution. We sought to determine how differences in the extent of recombination between populations of Aspergillus flavus impact the evolution of genes associated with the synthesis of aflatoxin, a notoriously potent carcinogen. We sequenced the genomes of, and quantified aflatoxin production in, 94 isolates of A. flavus sampled from seven states in eastern and central latitudinal transects of the United States. The overall population is subdivided into three genetically differentiated populations (A, B, and C) that differ greatly in their extent of recombination, diversity, and aflatoxin-producing ability. Estimates of the number of recombination events and linkage disequilibrium decay suggest relatively frequent sex only in population A. Population B is sympatric with population A but produces significantly less aflatoxin and is the only population where the inability of nonaflatoxigenic isolates to produce aflatoxin was explained by multiple gene deletions. Population expansion evident in population B suggests a recent introduction or range expansion. Population C is largely nonaflatoxigenic and restricted mainly to northern sampling locations through restricted migration and/or selection. Despite differences in the number and type of mutations in the aflatoxin gene cluster, codon optimization and site frequency differences in synonymous and nonsynonymous mutations suggest that low levels of recombination in some A. flavus populations are sufficient to purge deleterious mutations. IMPORTANCE Differences in the relative frequencies of sexual and asexual reproduction have profound implications for the accumulation of deleterious mutations (Muller’s ratchet), but little is known about how these differences impact the evolution of ecologically important phenotypes. Aspergillus flavus is the main producer of aflatoxin, a notoriously potent carcinogen that often contaminates food. We investigated if differences in the levels of production of aflatoxin by A. flavus could be explained by the accumulation of deleterious mutations due to a lack of recombination. Despite differences in the extent of recombination, variation in aflatoxin production is better explained by the demography and history of specific populations and may suggest important differences in the ecological roles of aflatoxin among populations. Furthermore, the association of aflatoxin production and populations provides a means of predicting the risk of aflatoxin contamination by determining the frequencies of isolates from low- and high-production populations.

Background Tar spot of corn is a significant and spreading disease in the continental U.S. and Canada caused by the obligate biotrophic fungus Phyllachora maydis. As of 2023, tar spot had been reported in 18 U.S. states and one Canadian Province. The symptoms of tar spot include chlorotic flecking followed by the formation of black stromata where conidia and ascospores are produced. Advancements in research and management for tar spot have been limited by a need for a reliable method to inoculate plants to enable the study of the disease. The goal of this study was to develop a reliable method to induce tar spot in controlled conditions. Results We induced infection of corn by P. maydis in 100% of inoculated plants with a new inoculation method. This method includes the use of vacuum-collection tools to extract ascospores from field-infected corn leaves, application of spores to leaves, and induction of the disease in the dark at high humidity and moderate temperatures. Infection and disease development were consistently achieved in four independent experiments on different corn hybrids and under different environmental conditions in a greenhouse and growth chamber. Disease induction was impacted by the source and storage conditions of spores, as tar spot was not induced with ascospores from leaves stored dry at 25 ºC for 5 months but was induced using ascospores from infected leaves stored at -20 ºC for 5 months. The time from inoculation to stromata formation was 10 to 12 days and ascospores were present 19 days after inoculation throughout our experiments. In addition to providing techniques that enable in-vitro experimentation, our research also provides fundamental insights into the conditions that favor tar spot epidemics. Conclusions We developed a method to reliably inoculate corn with P. maydis . The method was validated by multiple independent experiments in which infection was induced in 100% of the plants, demonstrating its consistency in controlled conditions. This new method facilitates research on tar spot and provides opportunities to study the biology of P. maydis , the epidemiology of tar spot, and for identifying host resistance.

Aflatoxin, produced by the fungus Aspergillus flavus , is an extremely potent hepatotoxin that causes acute toxicosis and cancer, and it incurs hundreds of millions of dollars annually in agricultural losses. Despite the importance of this toxin to humans, it has remained unclear what the fungus gains by producing aflatoxin. In fact, not all strains of A. flavus produce aflatoxin. Previous work has shown an advantage to producing aflatoxin in the presence of some insects. Our current work demonstrates the first evidence of a disadvantage to A. flavus in producing aflatoxin when competing with soil microbes. Together, these opposing evolutionary forces could explain the persistence of both aflatoxigenic and nonaflatoxigenic strains through evolutionary time. Selective forces that maintain the polymorphism for aflatoxigenic and nonaflatoxigenic individuals of Aspergillus flavus are largely unknown. As soils are widely considered the natural habitat of A. flavus , we hypothesized that aflatoxin production would confer a fitness advantage in the soil environment. To test this hypothesis, we used A. flavus DNA quantified by quantitative PCR (qPCR) as a proxy for fitness of aflatoxigenic and nonaflatoxigenic field isolates grown in soil microcosms. Contrary to predictions, aflatoxigenic isolates had significantly lower fitness than did nonaflatoxigenic isolates in natural soils across three temperatures (25, 37, and 42°C). The addition of aflatoxin to soils (500 ng/g) had no effect on the growth of A. flavus . Amplicon sequencing showed that neither the aflatoxin-producing ability of the fungus nor the addition of aflatoxin had a significant effect on the composition of fungal or bacterial communities in soil. We argue that the fitness disadvantage of aflatoxigenic isolates is most likely explained by the metabolic cost of producing aflatoxin. Coupled with a previous report of a selective advantage of aflatoxin production in the presence of some insects, our findings give an ecological explanation for balancing selection resulting in persistent polymorphisms in aflatoxin production. IMPORTANCE Aflatoxin, produced by the fungus Aspergillus flavus , is an extremely potent hepatotoxin that causes acute toxicosis and cancer, and it incurs hundreds of millions of dollars annually in agricultural losses. Despite the importance of this toxin to humans, it has remained unclear what the fungus gains by producing aflatoxin. In fact, not all strains of A. flavus produce aflatoxin. Previous work has shown an advantage to producing aflatoxin in the presence of some insects. Our current work demonstrates the first evidence of a disadvantage to A. flavus in producing aflatoxin when competing with soil microbes. Together, these opposing evolutionary forces could explain the persistence of both aflatoxigenic and nonaflatoxigenic strains through evolutionary time.

Cheese rinds are economically important microbial communities where fungi can impact food quality and aesthetics. The specific mechanisms by which fungi can regulate bacterial community assembly in cheeses, other fermented foods, and microbiomes in general are largely unknown. Potent antimicrobial metabolites are produced by filamentous fungi in pure culture, but their ecological functions in nature are often unknown. Using an antibacterial Penicillium isolate and a cheese rind microbial community, we demonstrate that a fungal specialized metabolite can regulate the diversity of bacterial communities. Inactivation of the global regulator, LaeA, resulted in the loss of antibacterial activity in the Penicillium isolate. Cheese rind bacterial communities assembled with the laeA deletion strain had significantly higher bacterial abundances than the wild-type strain. RNA-sequencing and metabolite profiling demonstrated a striking reduction in the expression and production of the natural product pseurotin in the laeA deletion strain. Inactivation of a core gene in the pseurotin biosynthetic cluster restored bacterial community composition, confirming the role of pseurotins in mediating bacterial community assembly. Our discovery demonstrates how global regulators of fungal transcription can control the assembly of bacterial communities and highlights an ecological role for a widespread class of fungal specialized metabolites. IMPORTANCE Cheese rinds are economically important microbial communities where fungi can impact food quality and aesthetics. The specific mechanisms by which fungi can regulate bacterial community assembly in cheeses, other fermented foods, and microbiomes in general are largely unknown. Our study highlights how specialized metabolites secreted by a Penicillium species can mediate cheese rind development via differential inhibition of bacterial community members. Because LaeA regulates specialized metabolites and other ecologically relevant traits in a wide range of filamentous fungi, this global regulator may have similar impacts in other fungus-dominated microbiomes.

Developing disease-resistant crops is a critical strategy for reducing chemical treatments and mitigating plant disease outbreaks, particularly amid global environmental changes. Fusarium head blight (FHB), caused by a complex of Fusarium species, is one of the most devastating cereal diseases, leading to significant economic losses and contamination of grain with harmful mycotoxins that threaten global cereal production and human health. The high variability in virulence within the complex of Fusarium spp and the lack of efficient high-throughput screening methods have impeded the development of resistant cultivars and made large-scale virulence testing labor-intensive and time-consuming. This study evaluates the efficacy of detached leaf, coleoptile, and seedling assays as high-throughput alternatives to the standard head infection assay for assessing the virulence of Fusarium species and differentiating wheat genotypes by resistance or susceptibility. Two near-isogenic wheat lines, one carrying FHB resistance loci and the other without, were used to assess the virulence of four Fusarium species. The seedling and coleoptile assays showed strong concordance with the traditional head infection assay, accurately reflecting differences in disease severity across Fusarium species and between wheat lines. Conversely, the detached leaf assay provided some differentiation among species but was inconsistent in identifying differences between plant genotypes. Across all assays, F. graminearum consistently exhibited the highest virulence, causing severe disease in leaves, stems, seedlings, and heads, while F. poae was the least virulent. Interestingly, F. culmorum and F. avenaceum displayed tissue-specific variability. These findings establish the coleoptile and seedling assays as rapid, high-throughput alternatives for breeding programs, accelerating the identification of FHB-resistant genotypes and reducing the reliance on the labor-intensive head assay.

Fungi are prolific producers of natural products, compounds which have had a large societal impact as pharmaceuticals, mycotoxins, and agrochemicals. Despite the availability of over 1,000 fungal genomes and several decades of compound discovery efforts from fungi, the biosynthetic gene clusters (BGCs) encoded by these genomes and the associated chemical space have yet to be analyzed systematically. Here, we provide detailed annotation and analyses of fungal biosynthetic and chemical space to enable genome mining and discovery of fungal natural products. Using 1,037 genomes from species across the fungal kingdom (e.g., Ascomycota, Basidiomycota, and non-Dikarya taxa), 36,399 predicted BGCs were organized into a network of 12,067 gene cluster families (GCFs). Anchoring these GCFs with reference BGCs enabled automated annotation of 2,026 BGCs with predicted metabolite scaffolds. We performed parallel analyses of the chemical repertoire of fungi, organizing 15,213 fungal compounds into 2,945 molecular families (MFs). The taxonomic landscape of fungal GCFs is largely species specific, though select families such as the equisetin GCF are present across vast phylogenetic distances with parallel diversifications in the GCF and MF. We compare these fungal datasets with a set of 5,453 bacterial genomes and their BGCs and 9,382 bacterial compounds, revealing dramatic differences between bacterial and fungal biosynthetic logic and chemical space. These genomics and cheminformatics analyses reveal the large extent to which fungal and bacterial sources represent distinct compound reservoirs. With a >10-fold increase in the number of interpreted strains and annotated BGCs, this work better regularizes the biosynthetic potential of fungi for rational compound discovery.