Frontiers in Fungal Biology

Trichoderma is a cosmopolitan genus with diverse lifestyles and nutritional modes, including mycotrophy, saprophytism, and endophytism. Previous research has reported greater metabolic gene repertoires in endophytic fungal species compared to closely-related non-endophytes. However, the extent of this ecological trend and its underlying mechanisms are unclear. Some endophytic fungi may also be mycotrophs and have one or more mycoparasitism mechanisms. Mycotrophic endophytes are prominent in certain genera like Trichoderma, therefore, the mechanisms that enable these fungi to colonize both living plants and fungi may be the result of expanded metabolic gene repertoires. Our objective was to determine what, if any, genomic features are overrepresented in endophytic fungi genomes in order to undercover the genomic underpinning of the fungal endophytic lifestyle. Here we compared metabolic gene cluster and mycoparasitism gene diversity across a dataset of thirty-eight Trichoderma genomes representing the full breadth of environmental Trichodermas diverse lifestyles and nutritional modes. We generated four new Trichoderma endophyticum genomes to improve the sampling of endophytic isolates from this genus. As predicted, endophytic Trichoderma genomes contained, on average, more total biosynthetic and degradative gene clusters than non-endophytic isolates, suggesting that the ability to create/modify a diversity of metabolites potential is beneficial or necessary to the endophytic fungi. Still, once the phylogenetic signal was taken in consideration, no particular class of metabolic gene cluster was independently associated with the Trichoderma endophytic lifestyle. Several mycoparasitism genes, but no chitinase genes, were associated with endophytic Trichoderma genomes. Most genomic differences between Trichoderma lifestyles and nutritional modes are difficult to disentangle from phylogenetic divergences among species, suggesting that Trichoderma genomes maybe particularly well-equipped for lifestyle plasticity. We also consider the role of endophytism in diversifying secondary metabolism after identifying the horizontal transfer of the ergot alkaloid gene cluster to Trichoderma.

Several asexual filamentous fungal pathogens contain supernumerary chromosomes carrying secondary metabolite (SM) or pathogenicity genes as well as transposons. Supernumerary chromosomes have been shown in in vitro experiments to transfer from pathogenic isolates to non-pathogenic ones and between isolates whose fusion can result in vegetative or heterokaryon incompatibility (HET). However, much is still unknown about the extent of horizontal transfer of supernumerary chromosomes within and between asexual pathogenic populations in adaptation to their hosts. We investigated several asexual fungal pathogens for genomic elements involved in maintaining telomeres for supernumerary and core chromosomes during vegetative reproduction. We found that in vegetative populations or lineages with a nearly complete telomere-to-telomere genome assembly (e.g. Fusarium equiseti and five formae speciales of the F. oxysporum species complex), core and supernumerary chromosomes were flanked by highly similar subtelomeric sequences on the 3 side and by their reverse complements on the 5 side. This subtelomere sequence structure was specific to the host. We detected instances of recent horizontal transfer of regions of a supernumerary chromosome between distant populations in the F. oxysporum species complex, and we also found field isolates with two structurally different copies of a supernumerary chromosome in a young asexual population, raising the possibility that those copies originated from different lineages by intrastrain anastomosis. A large number of HET domain genes were located in SM/pathogenicity gene clusters, with a potential role in marking these gene clusters during vegetative reproduction. The emergence of novel asexual pathogenic populations by horizontal transfer of transposon-rich supernumerary chromosomes within and between populations poses challenges to the control and management of these pathogens.

Several asexual species of filamentous fungal pathogens contain supernumerary chromosomes carrying secondary metabolite (SM) or pathogenicity genes. Supernumerary chromosomes have been shown in in vitro experiments to transfer from pathogenic isolates to non-pathogenic ones and between isolates whose fusion can result in vegetative or heterokaryon incompatibility (HET). However, much is still unknown about the acquisition and maintenance of SM/pathogenicity gene clusters in the adaptation of these asexual pathogens to their hosts. We investigated several asexual fungal pathogens for genomic elements involved in maintaining telomeres for supernumerary and core chromosomes during vegetative reproduction. We found that in vegetative species or lineages with a nearly complete telomere-to-telomere genome assembly (e.g. Fusarium equiseti and five formae speciales of the F. oxysporum species complex), core and super-numerary chromosomes were flanked by highly similar subtelomeric sequences on the 3 side and by their reverse complements on the 5 side. This subtelomere sequence structure was preserved in isolates from the same species or from polyphyletic lineages in the same forma specialis (f.sp.) of the F. oxysporum species complex. Moreover, between some isolates within F. oxysporum f.sp. lycopersici, the mean rate of single nucleotide polymorphisms (SNPs) in a supernumerary chromosome was at least 300 times lower than those in core chromosomes. And a large number of HET domain genes were located in SM/pathogenicity gene clusters, with a potential role in maintaining these gene clusters during vegetative reproduction.

Nakaseomyces glabratus is a globally distributed opportunistic fungal pathogen. An ongoing discussion in studies of N. glabratus population structure has been whether genetic clusters are best defined using multilocus sequence typing (MLST) or short-read whole-genome sequencing (WGS). To assess the concordance between MLST- and WGS-based phylogenies, we analyzed a dataset of 548 N. glabratus WGS sequences from 12 countries. Clusters identified from WGS largely recapitulated the MLST-defined sequence type (ST) groups: fourteen WGS clusters were composed of a single MLST ST, and the remaining contained STs with very closely related MLST profiles. We thus propose a pragmatic naming convention, consistent with the system used in other microbial species, which specifies WGS cluster labels based on the primary ST. From the large WGS isolate dataset, we determined the prevalence of admixture and genomic variants. Interestingly, seven of the nine singleton isolates were admixed, in addition to 58 isolates from six different clusters. Aneuploidy was detected in 4% of isolates, most commonly in chrE, which contains ERG11, the gene encoding the enzyme targeted by azole antifungals. Aneuploid chromosomes did not exhibit elevated heterozygosity relative to the sequencing error rate, consistent with instability of extra chromosome copies. Copy number variants were found in 3% of the isolates; some of the CNVs co-occurred with aneuploidies, and were primarily identified on chrD, chrE, chrI, and chrM. Our findings demonstrate that deep splits between clusters preserve the utility of MLST ST designations for clade-level designation, yet underscore the utility of WGS for high-resolution genomic analyses. Article SummaryThere is an ongoing debate in studies on Nakaseomyces glabratus about whether traditional MLST analysis is sufficient to determine population structure, or whether the precision of whole genome sequencing (WGS) is necessary. We analyzed WGS data from 548 isolates from around the world. We found a very strong agreement between the two methods. We propose a hybrid naming system, where cluster names are based on the dominant MLST group. We used the WGS data to show that admixed isolates, and those with extra chromosomes or CNVs are rare (<7% of isolates in each class) and are distributed throughout the phylogeny.

Peanut smut, caused by the fungus Thecaphora frezzii, is a significant disease of peanuts in Argentina. Infected plants have seeds replaced by a mass of dark teliospores, reducing yield and seed quality. To prevent the spread of the pathogen, several countries have limited import of raw peanuts from Argentina, a major grower and exporter. Following successful in vitro culture of the fungus in its haploid stage, we produced a chromosome-level genome assembly of the species for the first time. We compare this genome with those of 49 other species of true smut fungi, or Ustilaginomycetes, including species of medical, agricultural, and industrial importance, some of which are known as pathogens and others only as saprotrophic yeasts. At almost 39 Mb, T. frezzii has the largest genome of the smut fungi sequenced to date and the highest repetitive content. While it shares some core effectors with species of the distantly related and better studied Ustilago and related fungi, predicted effectors only found in T. frezzii or in Thecaphora suggest unique infection strategies. Comparisons among the 50 smut genomes also show that the 14 smut fungi observed only as yeasts share genomic traits such as low repeat content and generally smaller genomes. This supports the hypothesis that some smut fungi are adapted to saprotrophic growth as yeasts. The high-quality, annotated genome for T. frezzii will be a valuable resource for investigating the population dynamics and evolution of an economically important pathogen, as well as illuminating an understudied clade of smut fungi. Article summaryPeanut smut is a destructive and costly disease of peanuts in Argentina. For the first time, a high quality, annotated genome is presented for the causal agent, Thecaphora frezzii. This fungus has the largest and most repetitive genome of the true smut fungi, thus prompting comparison with 49 other species of smut fungi with available genomes, including non-pathogenic ones. While it shares some likely pathogenicity factors with well-studied smuts, it has many unique genes, a trait reflective of its evolutionary distance and likely novel mechanism for infection. This important genomic resource will benefit research regarding the evolution and adaption of T. frezzii, the development of diagnostic tools that enable rapid detection of it, and the study of smut fungi broadly.

Balancing selection, an evolutionary force that retains genetic diversity, has been detected in multiple genes and organisms, such as the sexual mating loci in fungi. However, to quantify the strength of balancing selection and define the mating-related genes require a large number of specimens. In tetrapolar basidiomycete fungi, sexual type is determined by two unlinked loci, MATA and MATB. Genes in both loci defines mating type identity, control successful mating and completion of the life cycle. These loci are usually highly diverse. Previous studies have speculated, based on culture crosses, that species of the non-model genus Trichaptum (Hymenochaetales, Basidiomycota) possess a tetrapolar mating system, with multiple alleles. Here, we sequenced a hundred and eighty specimens of three Trichaptum species. We characterized the chromosomal location of MATA and MATB, the molecular structure of MAT regions and their allelic richness. Our sequencing effort was sufficient to molecularly characterize multiple MAT alleles segregating before the speciation event of Trichaptum species. Our analyses suggested that long-term balancing selection has generated trans-species polymorphisms. Mating sequences were classified in different allelic classes based on an amino acid identity (AAI) threshold supported by phylogenetics. The inferred allelic information mirrored the outcome of in vitro crosses, thus allowing us to support the degree of allelic divergence needed for successful mating. Even with the high amount of divergence, key amino acids in functional domains are conserved. The observed allelic classes could potentially generate 14,560 different mating types. We conclude that the genetic diversity of mating in Trichaptum loci is due to long-term balancing selection, with limited recombination and duplication activity. Our large number of sequenced specimens highlighted the importance of sequencing multiple individuals from different species to detect the mating-related genes, the mechanisms generating diversity and the evolutionary forces maintaining them. Author summaryFungi have complex mating systems, and basidiomycete fungi can encode thousands of mating types. Individuals with the same mating type cannot mate. This sexual system has evolved to facilitate sexual mating, increasing the chances to recombine into advantageous allelic combination and prune deleterious alleles. We explored the genomes of hundred and eighty specimens, combined with experimental mating studies of selected specimens, from a non-model organism (Trichaptum). We characterized the genomic regions controlling sex. The mating ability of the specimens confirmed the role of the mating alleles observed in the genomic data. The detailed analyses of many specimens allowed us to observe gene duplication and rearrangements within the mating loci, increasing the diversity within these loci. We supported previous suggestions of balancing selection in this region, an evolutionary force that maintains genomic diversity. These results supports that our fungal specimens are prone to outcross, which might facilitate the adaptation to new conditions.

How genomic differences contribute to phenotypic differences across species is a major question in biology. The recently characterized genomes, isolation environments, and qualitative patterns of growth on 122 sources and conditions of 1,154 strains from 1,049 fungal species (nearly all known) in the subphylum Saccharomycotina provide a powerful, yet complex, dataset for addressing this question. In recent years, machine learning has been successfully used in diverse analyses of biological big data. Using a random forest classification algorithm trained on these genomic, metabolic, and/or environmental data, we predicted growth on several carbon sources and conditions with high accuracy from presence/absence patterns of genes and of growth in other conditions. Known structural genes involved in assimilation of these sources were important features contributing to prediction accuracy, whereas isolation environmental data were poor predictors. By further examining growth on galactose, we found that it can be predicted with high accuracy from either genomic (92.6%) or growth data in 120 other conditions (83.3%) but not from isolation environment data (65.7%). When we combined genomic and growth data, we noted that prediction accuracy was even higher (93.4%) and that, after the GALactose utilization genes, the most important feature for predicting growth on galactose was growth on galactitol. These data raised the hypothesis that several species in two orders, Serinales and Pichiales (containing Candida auris and the genus Ogataea, respectively), have an alternative galactose utilization pathway because they lack the GAL genes. Growth and biochemical assays of several of these species confirmed that they utilize galactose through an oxidoreductive D-galactose pathway, rather than the canonical GAL pathway. We conclude that machine learning is a powerful tool for investigating the evolution of the yeast genotype-phenotype map and that it can help uncover novel biology, even in well-studied traits.

Fungal plant pathogens constantly evolve and deploy novel peptide and metabolite effectors to break down plant resistance and adapt to new host plants. The blast fungal pathogen Pyricularia oryzae is a single species subdivided into multiple host-specific lineages that have evolved through gain and/or loss of virulence and/or effector related genes through chromosomal rearrangement. Here, we mined 68 genomes of P. oryzae, belonging to six host-specific lineages, to identify secondary metabolite (SM) biosynthetic gene clusters (BGCs) likely associated with potential metabolite effectors involved in host specialization. A similarity network analysis grouped a total of 4501 BGCs into 283 gene cluster families (GCFs), based on the content and architecture of the BGCs. While most of the GCFs were present in all the P. oryzae lineages, two (BGC-O1 and BGC-O2) were found specifically in the Oryza lineage and one (BGC-TLE) was found in the lineage specific to Triticum, Lolium and Eleusine hosts. Further analysis of the phylogenetic relationships between core biosynthetic genes confirmed that BGC-O1, which comprises a reducing polyketide synthase gene (MGG_08236) and four putative tailoring genes, was present only in the Oryza lineage. Importantly, most genes, including MGG_08236, from the BGC-O1 were expressed specifically during pathogenesis. We propose that the Oryza lineage-specific BGC-O1 produces a metabolite effector likely involved in specialization of P. oryzae to the rice host. In addition, we identified five SM genes under positive or balancing selection only in the Oryza lineage, suggesting a role in the interaction with rice specifically. Our findings highlight the importance of further mining novel metabolite effectors in specialization and virulence of the blast fungus to different cereal hosts.

Mycena citricolor is a fungus that causes the American Leaf Spot (ALS) disease in multiple hosts, including coffee and avocado. This hemibiotroph penetrates the plant through damage induced by oxalic acid. This can cause 20-90% crop losses in coffee depending on the environmental and production conditions. M. citricolor is the only known pathogenic species in the Mycena genus, a large group of saprophytic mushrooms. Comparing the saprophytic and pathogenic genomes can allow us to identify genetic machinery associated with the pathogens genome-wide functional acquisitions to cause disease. To identify pathogenicity-related genes in M. citricolor, we analysed protein family copy-number variation, secretome prediction, and homology to known virulence factors in two M. citricolor assemblies, including a newly assembled and annotated long-read genome. We found that the pathogenic M. citricolor had a higher proportion of secreted genes expanded in copy-number, and expanded gene copies homologous to known virulence factors than the saprophytic Mycena. We shortlisted over 300 candidate genes in each M. citricolor assembly. Focusing on genes strongly regulated during plant interaction, we found over 100 candidates, primarily from multiple copies (up to 4-3 times) of 42 well-known virulence factors (e.g. MFS1, CUTA, NoxA/B, OLE1, NorA), plus a few clade-specific uncharacterised genes. M. citricolor transition to a pathogenic lifestyle reflected genome-wide functional changes. M. citricolor seems to primarily depend on well-known virulence factors in large copy numbers, suggesting the molecular plant-interaction processes involved are like those of better-studied fungi. Hypothetically, the development of ALS resistance could mirror studied responses to these virulence factors.

Pathogens responses to sudden temperature fluctuations, spanning various temporal scales, are critical determinants of their survival, growth, reproduction, and homeostasis. Here, we combined phenotyping, transcriptomics, and genome-wide association approaches to investigate how the wheat pathogen Zymoseptoria tritici responds to and recovers from temperature shocks. Survival emerged as the most significantly affected trait immediately following temperature shocks across 122 geographically diverse strains. In contrast, post-recovery phenotypic traits, including growth rate and melanization, showed no significant deviations from control conditions. Transcriptomic analyses of a reference strain revealed temperature stress-specific gene expression patterns, with genes involved in protein folding, redox homeostasis, membrane stabilization, and cell-wall remodeling playing central roles in the response. A multi-reference k-mer genome-wide association study (GWAS) identified six loci significantly associated with cold shock responses. Among these, two loci emerged as strong candidates for near-freezing temperature adaptation, including a 60S ribosomal protein gene involved in protein synthesis and stress recovery, and an NADH oxidoreductase gene implicated in redox homeostasis and oxidative stress tolerance. These findings shed light on the distinct molecular strategies Z. tritici employs to adapt to temperature stress and provide novel insights into fungal resilience under dynamic environmental conditions. Author summaryTemperature fluctuations, an inherent aspect of natural environments, are increasingly exacerbated by climate change, intensifying challenges for organisms to maintain homeostasis amid more frequent and severe extreme weather events. This study reveals distinct phenotypic, transcriptomic, and genetic mechanisms underlying Z. triticis responses to short-term temperature shocks. Survival-related phenotypic traits were significantly reduced by heat and cold shocks, while other traits measured after a recovery period demonstrated the resilience of Z. tritici strains to temperature stress, reflecting efficient recovery mechanisms. Transcriptomic analyses uncovered temperature-specific gene expression patterns, emphasizing unique regulatory strategies, which mostly return to baseline levels after a recovery period. The discovery of novel loci associated with cold shock responses provides valuable insights into the genetic basis of resilience to short-term temperature stress, offering a foundation for future research on pathogen adaptation to fluctuating environments.

Members of the Colletotrichum gloeosporioides species complex are causal agents of anthracnose in a wide range of commercially important plants. To provide an in-depth overview of its diversity, we sequenced the genomes of fungi belonging to this group, including multiple strains of C. fructicola (Cf) and C. siamense (Cs), as well as representatives of three previously unsequenced species, C. aenigma (Ca), C. tropicale and C. viniferum. Comparisons between multiple Cf and Cs strains led to the identification of accessory regions that show variable conservation in both lineages. These accessory regions encode effector candidate genes, including homologs of previously characterized effectors, organized in clusters of conserved synteny with copy number variations in different strains of Cf, Cs and Ca. Analysis of highly contiguous assemblies of Cf, Cs and Ca strains revealed the association of such accessory effector gene clusters with subtelomeric regions and repeat-rich minichromosomes and provided evidence of gene transfer between these two genomic compartments. In addition, expression analysis indicated that paralogs associated with clusters of conserved synteny showed a tendency for correlated gene expression. These data highlight the importance of subtelomeric regions and repeat-rich chromosomes to the genome plasticity of Colletotrichum fungi.

The genomic diversity of many fungal pathogens is driven by rapidly evolving regions harbouring significant amounts of transposable elements. Such genomic regions often contain genes important for pathogenicity and may be sequestered from the core genome on accessory chromosomes, which are variably present in isolates of a species. In this study, we present a pangenomic analysis of a fungal insect pathogen, Metarhizium acridum, using seven chromosome-scale genomes from four different continents. We show that approximately 25% of the M. acridum pangenome is comprised of genes only present in one isolate (singletons) or more than one but not all isolates (accessory). These genes are enriched for functions in secondary metabolite production, nutrient transport, and chromosome organization. We also find evidence of functional compartmentalization of the genome, as the core genome of M. acridum is enriched in carbohydrate-active enzymes, while the accessory components are enriched in effectors that are located in gene-sparse regions of the genome. Furthermore, we identified the first naturally recovered accessory chromosome in M. acridum, which does not harbor effector or secondary metabolite proteins related to host-insect interactions but is enriched in functions related to sexual reproduction. Within the genus Metarhizium, M. acridum is one of few species considered to regularly reproduce sexually. We show that the accessory chromosome contains genes from both the MAT1-1 and MAT1-2 idiomorphs, in addition to the MAT locus found on the core chromosomes. Our findings suggest that the presence of this accessory chromosome may facilitate sexual reproduction, possibly even primary homothallism, in the otherwise heterothallic M. acridum. Overall, our study presents a putative novel mechanism whereby this fungal pathogen may acquire gene recombination and ensure maintenance of the accessory chromosome.

Arbuscular mycorrhizal (AM) fungi form obligate symbiosis with the roots of the majority of land plants and are found in all terrestrial ecosystems. The source and structure of genetic variation in AM fungi has remained an enigma due to difficulties in the axenic cultivation and generation of high-quality genome assemblies from most species. Furthermore, how AM fungi survives long-term without a single nuclear stage per cell life history is puzzling, prompting hypotheses on selection at the nuclear level which functions to purge deleterious mutations. In this study, we aimed to characterize both intra- and inter-organismal genetic variation in AM fungi by analyzing genomic information from individual nuclei of three strains from two species of the genus Claroideoglomus. We observed overall low levels of genetic variation within the strains, most of which represent rare variants likely kept at low frequency by purifying selection. We also observed variants that have been maintained as polymorphic across both strains and species. The results in this study affirm our conceptual understanding that nuclei in AM fungal strains function as populations of asexually reproducing units. Further, we propose that selection acts on different levels within the organism, with strong signals of purifying selection on nuclei within strain.

Fungal and bacterial symbiosis is an important adaptation that has occurred within many insect species, which usually results in the relaxation of selection across the symbiont genome. However, the evolutionary pressures and genomic consequences associated with this transition are not well understood. Pathogenic fungi of the genus Ophiocordyceps have undergone multiple, independent transitions from pathogen to associate, infecting soft-scale insects trans-generationally without killing them. To gain an understanding of the genomic adaptations underlying this transition, long-read sequencing was utilized to assemble the genomes of both Parthenolecanium corni and its Ophiocordyceps associate from a single insect. A highly contiguous haploid assembly was obtained for Part. corni, representing the first assembly from a single Coccoidea insect, in which 97% of its 227.8 Mb genome was contained within 24 contigs. Metagenomic-based binning produced a chromosome-level genome for Part. cornis Ophiocordyceps associate. The associate genome contained 524 gene loss events compared to free-living pathogenic Ophiocordyceps relatives, with predicted roles in hyphal growth, cell wall integrity, metabolism, gene regulation and toxin production. Contrasting patterns of selection were observed between the nuclear and mitochondrial genomes specific to the associate lineage. Intensified selection was most frequently observed across nuclear orthologs, while selection on mitochondrial genes was found to be relaxed. Furthermore, scans for diversifying selection identified associate specific selection within three adjacent enzymes catalyzing acetoacetates metabolism to acetyl-COA. This work provides insight into the adaptive landscape during the transition to an associate life history, along with a base for future research into the genomic mechanisms underpinning the evolution of Ophiocordyceps.

Mushroom-forming fungi (Agaricomycetes) are emerging as pivotal players in several fields, as drivers of nutrient cycling, sources of novel applications, and the group includes some of the most morphologically complex multicellular fungi. Genomic data for Agaricomycetes are accumulating at a steady pace, however, this is not paralleled by improvements in the quality of genome sequence and associated functional gene annotations, which leaves gene function notoriously poorly understood in comparison with other fungi and model eukaryotes. We set out to improve our functional understanding of the model mushroom Coprinopsis cinerea by integrating a new, chromosome-level assembly with high-quality gene predictions and functional information derived from gene-expression profiling data across 67 developmental, stress, and light conditions. The new annotation has considerably improved quality metrics and includes 5- and 3-untranslated regions (UTRs), polyadenylation sites (PAS), upstream ORFs (uORFs), splicing isoforms, conserved sequence motifs (e.g., TATA and Kozak boxes) and microexons. We found that alternative polyadenylation is widespread in C. cinerea, but that it is not specifically regulated across the various conditions used here. Transcriptome profiling allowed us to delineate core gene sets corresponding to carbon starvation, light-response, and hyphal differentiation, and uncover new aspects of the light-regulated phases of life cycle. As a result, the genome of C. cinerea has now become the most comprehensively annotated genome among mushroom-forming fungi, which will contribute to multiple rapidly expanding fields, including research on their life history, light and stress responses, as well as multicellular development.

Maltose is one of the most abundant sugars in brewers wort, and its efficient utilization is critical for successful fermentation. However, maltose consumption varies naturally among Saccharomyces eubayanus strains isolated from different host trees, such as Quercus and Nothofagus. To identify the genetic determinants underlying these phenotypic differences, we performed bulk segregant analysis (BSA) and quantitative trait loci (QTL) mapping using an F2 offspring derived from QC18 (Quercus-associated) and CL467.1 (Nothofagus-associated) strains. QTL mapping identified two significant genomic regions on subtelomeric loci of chromosomes V-R and XVI-L, each containing complete MAL loci composed of MAL32 (encoding maltase), MAL31 (transporter), and MAL33 (transcriptional activator) genes. Comparative polymorphism analyses identified mutations in MAL32 and MAL33 of QC18, including frameshift mutations resulting in premature stop codons. Functional validation demonstrated that the heterologous expression of MAL33ChrV from CL467.1 fully restored maltose utilization in QC18, indicating the functional presence of MAL33 cis-regulatory sequences and MAL32 and MAL31 genes in QC18. While structural protein predictions identified truncation and impaired functionality in the maltose-responsive activation domain of Mal33p from QC18, overexpression of QC18s own MAL33ChrV allele also improved maltose metabolism, suggesting dosage-dependent transcriptional limitations rather than complete functional loss. These results indicate that allelic variations in the maltose-responsive activation domain of Mal33p lead to differences in maltose consumption between strains. We hypothesized that reduced maltose metabolism in QC18 is an adaptive response to the distinct sugar composition in Quercus robur bark, contrasting with the starch-rich environment of Nothofagus pumilio. These findings highlight subtelomeric MAL gene diversity as a reservoir of evolutionary plasticity, representing a key evolutionary mechanism that influences maltose adaptation among natural Saccharomyces isolates.

Fungal pathogens have dramatically altered forests worldwide, yet the mechanisms underlying their virulence remain poorly understood. From the 1970s to the early 2000s, dogwood anthracnose, caused by Discula destructiva Redlin, devastated flowering and Pacific dogwoods (Cornus florida L. and C. nuttallii Aud., respectively). Despite the impacts of D. destructiva and other phytopathogens on forest ecosystems, genomic resources remain limited, hindering efforts to understand pathogenicity. The goal of this study was to evaluate the historical D. destructiva epidemic through a modern genomics lens by uncovering virulence- associated genes that likely contributed to its rapid spread across native dogwoods. We therefore utilized PacBio HiFi and Proximo Hi-C sequencing to assemble the first telomere-to-telomere, chromosome-scale genome for D. destructiva isolate AS111. The resulting 46.655 Mb assembly comprised eight chromosomes with an overall BUSCO completeness of 97.64%. We also identified 10,373 predicted gene models with an overall BUSCO completeness of 96.45%. To investigate gene expression across distinct life cycle phases, reproductive (sporulating) and vegetative (nonsporulating), we conducted RNA sequencing and identified 240 differentially expressed genes (padj < 0.05). GO enrichment revealed 162 upregulated genes during sporulation linked to plant cell wall degradation and sugar metabolism, whereas 78 downregulated genes were linked to electron carrier activity and redox balance. Among these 240 genes, 117 genes had predicted protein sequences that were also identified as a candidate virulence factor, including signal peptides, carbohydrate-active enzymes (CAZymes), and effectors, highlighting the role of sporulation-associated gene expression in D. destructiva virulence. Together, these findings suggest that the reproductive phase primes D. destructiva for host invasion and ecological persistence, which may influence its success as a forest pathogen. Author SummaryForest pathogens threaten ecosystems worldwide and cause extensive ecological and economic damage. Since the 1970s, native dogwood populations in North America have been devastated by dogwood anthracnose, caused by the exotic fungal pathogen Discula destructiva. Discula destructiva is just one of many destructive fungal pathogens in the order Diaporthales, which includes other noteworthy pathogens that cause chestnut blight (Cryphonectria parasitica) and butternut canker (Ophiognomonia clavigignenti-juglandacearum). Despite the widespread impact of fungal diseases, little is known about the genetic factors that drive their spread and severity. To help address this gap, we generated the first telomere-to-telomere, chromosome- scale genome assembly of D. destructiva and analyzed gene expression across distinct life cycle phases: reproductive (sporulating) and vegetative (nonsporulating) growth. Our findings revealed key sporulation-associated genes and virulence factors that may facilitate host infection and underscore the importance of sporulation in the pathogenicity of D. destructiva. These insights improve our understanding of the mechanisms that drive disease development, influence how fungal pathogens such as D. destructiva establish and persist in forest ecosystems, and provide a foundation for future comparative genomics among other devastating pathogens in Diaporthales.

To complete its parasitic lifecycle, Salmacisia buchloeana, a biotrophic fungus, manipulates reproductive organ development, meristem determinacy, and resource allocation in its dioecious plant host, buffalograss (Bouteloua dactyloides; Poaceae). To gain insight into S. buchloeanas ability to manipulate its host, we sequenced and assembled the 20.1 Mb genome of S. buchloeana into 22 chromosome-level pseudomolecules. Phylogenetic analysis suggests that S. buchloeana is nested within the genus Tilletia and diverged from T. caries and T. walkeri [~]40 million years ago. We find that S. buchloeana has a novel chromosome arm with no syntenic relationship to other publicly available Tilletia genomes and that genes on the novel arm are upregulated upon infection, suggesting that this unique chromosomal segment may have played a critical role in S. buchloeanas evolution and host specificity. Salmacisia buchloeana has one of the largest fractions of serine peptidases (1.53% of the proteome) and one of the highest GC contents (62.3%) in all classified fungi. Analysis of codon base composition indicated that GC content is controlled more by selective constraints than directional mutation and that S. buchloeana has a unique bias for the serine codon UCG. Finally, we identify three inteins within the S. buchloeana genome, two of which are located in a gene often used in fungal taxonomy. The genomic and transcriptomic resources generated here will aid plant pathologists and breeders by providing insight into the extracellular components contributing to sex determination in dioecious grasses.

BackgroundFungi use the accessory segments of their pan-genomes to adapt to their environments. While gene presence-absence variation (PAV) contributes to shaping these accessory gene reservoirs, whether these events happen in specific genomic contexts remains unclear. Additionally, since pan-genome studies often group together all members of the same species, it is uncertain whether genomic or epigenomic features shaping pan-genome evolution are consistent across populations within the same species. Fungal plant pathogens are useful models for answering these questions because members of the same species often infect distinct hosts, and they frequently rely on gene PAV to adapt to these hosts. ResultsWe analyzed gene PAV in the rice and wheat blast fungus, Magnaporthe oryzae, and found that PAV of disease-causing effectors, antibiotic production, and non-self-recognition genes may drive the adaptation of the fungus to its environment. We then analyzed genomic and epigenomic features and data from available datasets for patterns that might help explain these PAV events. We observed that proximity to transposable elements (TEs), gene GC content, gene length, expression level in the host, and histone H3K27me3 marks were different between PAV genes and conserved genes, among other features. We used these features to construct a random forest classifier that was able to predict whether a gene is likely to experience PAV with high precision (86.06%) and recall (92.88%) in rice-infecting M. oryzae. Finally, we found that PAV in wheat- and rice-infecting pathotypes of M. oryzae differed in their number and their genomic context. ConclusionsOur results suggest that genomic and epigenomic features of gene PAV can be used to better understand and even predict fungal pan-genome evolution. We also show that substantial intra-species variation can exist in these features.

Cryptic fungal pathogens pose significant identification and disease management challenges due to their morphological resemblance to known pathogenic species while harboring genetic and (often) infection-relevant trait differences. The cryptic fungal pathogen Aspergillus latus, an allodiploid hybrid originating from Aspergillus spinulosporus and an unknown close relative of Aspergillus quadrilineatus within section Nidulantes, remains poorly understood. The absence of accurate diagnostics for A. latus has led to misidentifications, hindering epidemiological studies and the design of effective treatment plans. We conducted an in-depth investigation of the genomes and phenotypes of 44 globally distributed isolates (41 clinical isolates and three type strains) from Aspergillus section Nidulantes. We found that 21 clinical isolates were A. latus; notably, standard methods of pathogen identification misidentified all A. latus isolates. The remaining isolates were identified as A. spinulosporus (8), A. quadrilineatus (1), or A. nidulans (11). Phylogenomic analyses shed light on the origin of A. latus, indicating one or two hybridization events gave rise to the species during the Miocene, approximately 15.4 to 8.8 million years ago. Characterizing the A. latus pangenome uncovered substantial genetic diversity within gene families and biosynthetic gene clusters. Transcriptomic analysis revealed that both parental genomes are actively expressed in nearly equal proportions and respond to environmental stimuli. Further investigation into infection-relevant chemical and physiological traits, including drug resistance profiles, growth under oxidative stress conditions, and secondary metabolite biosynthesis, highlight distinct phenotypic profiles of the hybrid A. latus compared to its parental and closely related species. Leveraging our comprehensive genomic and phenotypic analyses, we propose five genomic and phenotypic markers as diagnostics for A. latus species identification. These findings provide valuable insights into the evolutionary origin, genomic outcome, and phenotypic implications of hybridization in a cryptic fungal pathogen, thus enhancing our understanding of the underlying processes contributing to fungal pathogenesis. Furthermore, our study underscores the effectiveness of extensive genomic and phenotypic analyses as a promising approach for developing diagnostics applicable to future investigations of cryptic and emerging pathogens.