Frontiers in Fungal Biology

Agarikon (Laricifomes officinalis syn. Fomitopsis officinalis) is an endangered fungus belonging to a unique lineage in the Polyporales (Basidiomycota) with a growing body of evidence supporting its medicinal value. In this study, we report the hybrid de novo assembly and annotation of the first L. officinalis nuclear and mitochondrial genome sequences, with a nuclear genome size of 28.76 Mb assembled across 66 scaffolds (51.96% GC content; BUSCO completeness of 99.4%), and a complete core mitochondrial genome size of 197.67 kb. Structural and functional annotation of the nuclear genome yielded 8,717 predicted genes including 8,604 protein-coding genes, with 310 genes in 27 biosynthetic gene clusters. We characterized the mating type loci matA and matB, consistent with a tetrapolar mating system, and identified genes encoding key enzymes involved in triterpenoid and polyketide biosynthetic pathways that lead to the production of a diverse array of secondary metabolites. Additionally, we conducted maximum likelihood phylogenomic analysis to confirm the taxonomic position of L. officinalis among 21 species in Polyporales using protein sequences for 860 shared BUSCO genes. This high-quality annotated genome of L. officinalis will serve as a foundation for further investigations into the evolutionary history of this distinct fungal lineage, provide a reference for future population genomic analyses, and elucidate mechanisms underlying the synthesis of the bioactive compounds responsible for agarikons wide-ranging medicinal benefits.

Fungal genomics has expanded rapidly over the past 30 years, and recently the pace and breath has further quickened for many taxa, although many taxonomic gaps persist. With three decades of rapid growth, fungal genomics now merits a re-examination of its history, progress, and unresolved taxonomic gaps. Here, we review the development of fungal genomics from early efforts such as the Fungal Genome Initiative to current progress driven by third-generation long-read sequencing. We have compiled and summarised publicly available fungal genomes to highlight trends in assembly quality, adoption of long-read technologies, and taxonomic representation. Notably, substantial phylogenetic gaps remain, particularly outside Dikarya, and significant challenges persist for unculturable taxa. This review identifies priorities for the fungal community, including: (1) coordinated efforts to close major taxonomic gaps across the fungal tree of life; (2) improved repository metrics to facilitate identification of high-quality assemblies; and (3) improved and standardised genome annotation which is lacking for most assemblies. Together, these steps will support the development of reliable genomic resources that capture the full breadth of diversity across the fungal kingdom, generating foundational data for comparative genomics, evolutionary biology, functional studies, genetic studies and applied research.

Accessory genes are thought to contribute to fungal adaptation and pathogenicity by modulating host immunity, while core genes play crucial roles in maintaining fundamental biological processes. Rust fungi (order Pucciniales) are obligate biotrophic plant-pathogens and infect economically relevant crops. Here, we characterize core and accessory gene repertoires across rust fungi, with a particular focus on Phakopsora pachyrhizi, the causal agent of Asian soybean rust. Across Pucciniales genomes, accessory genes represented the largest fraction of gene content (~44.6% on average), whereas core genes accounted for a smaller proportion (~18-35%). Notably, variations in accessory gene content among rust fungi are perhaps attributed to lineage-specific gene expansions and losses. Core gene content was positively correlated with total gene number across Pucciniales genomes, suggesting retention after gene duplication events, consistent with their essential biological functions. Among P. pachyrhizi genes expressed during soybean infection, core effectors were associated with cysteine-rich proteins, pectin-degrading enzymes, and SPFH/Band 7 family, while accessory effectors included phosphatidylethanolamine-binding proteins, trehalose phosphatases, and CFEM domain-containing proteins. The in-plant induced core and accessory genes in P. pachyrhizi also comprised multiple families of CAZymes (GH5/GH7 cellulases, CE5 cutinases, CE8 pectinesterases, CE4/GH18 chitin-modifying enzymes); proteases (aspartyl proteases, serine carboxypeptidases, alpha/beta hydrolases); transporters (amino acid permeases, ferric reductase-like transmembrane proteins, and OPT oligopeptide transporter), and transcription factors (bZIP, GATA zinc finger, STE-like, and homeobox KN). Our study highlights that core and accessory gene families have shaped P. pachyrhizi-soybean interactions, identifying promising targets for functional studies aimed at elucidating host-adaptation mechanisms in rust fungi.

Trichoderma species are widely used as biological control agents due to their ability to parasitize plant pathogens. However, substantial variability in mycoparasitic performance exists among strains, even within the same species, and the underlying molecular mechanisms remain poorly understood. Here, we performed comparative genomic and transcriptomic analyses of six Trichoderma atroviride strains exhibiting contrasting mycoparasitic performance (weakly or highly parasitic; WP or HP) against Alternaria brassicicola, Rhizoctonia solani, and Globisporangium ultimum. Comparative genomics revealed limited strain-specific differences, mainly restricted to NLR (NOD-like receptor) repertoires, with certain NLR-coding genes absent from WP strain genomes compared to HP strains, while overall genomic variation remained low. In contrast, transcriptomic analyses revealed strong differences in gene expression dynamics between HP and WP strains. Co-expression network analysis identified two modules associated with mycoparasitic performance. The first was specifically induced in response to pathogen contact and was enriched in genes encoding cell wall-degrading enzymes, with stronger expression in HP strains. The second module was more broadly overexpressed in HP strains across all conditions and included genes involved in detoxification and defense-related pathways. In addition, this module encompassed genes involved in specialized metabolite biosynthesis and effector-like protein secretion, with WP and HP strains differentially expressing distinct gene subsets within these categories. Together, these results provide a comprehensive framework for identifying the molecular drivers of mycoparasitic performance in T. atroviride. This study deepens our understanding of the functional diversity within the species and establishes a robust foundation for the future development of molecular markers to predict strain efficiency.

High-quality fungal reference genomes are essential for comparative, functional, and evolutionary studies, yet fungal genome features such as repeats, structural rearrangements, accessory chromosomes, and intron-rich genes can complicate genome assembly and the selection of cost-effective sequencing strategies. Here, we benchmark fungal genome assembly performance using simulated and empirical short- and long-read datasets to evaluate how sequencing depth, assembler choice, and genome characteristics influence contiguity, completeness, accuracy, and computational requirements. Using simulated reads from complete fungal genomes spanning diverse sizes and compositions, we evaluated short-read, long-read, hybrid, and polished long-read assemblies across sequencing depths from 10X to 100X. Key trends were validated using empirical sequencing data from 10 fungal isolates assembled with multiple strategies, including different Flye assembler parameter sensitivity and short-read polishing. Across datasets, long reads produced the largest improvements in contiguity, with most gains achieved at [~]20-40X coverage and diminishing returns beyond moderate depth. Short-read polishing substantially improved base-level accuracy at relatively low cost, with [~]10-20X coverage often sufficient to approach maximal error reduction. Hybrid assemblers showed strong algorithmic variability, with trade-offs between contiguity, error rates, and computational demand. Genome architecture also influenced outcomes, as larger and more feature-dense genomes benefited more from long-read data while GC content had limited impact. Overall, our results suggest that moderate long-read coverage ([~]30-40X) combined with modest short-read polishing ([~]10-20X), particularly using Flye plus Polypolish, provides a strong balance of contiguity, completeness, accuracy, and resource efficiency for generating high-quality fungal genome assemblies. Impact statementFungal genome sequencing is expanding rapidly across ecology, plant pathology, biotechnology, and clinical and veterinary microbiology, yet experimental design decisions regarding sequencing depth, assembler selection, and hybrid workflows are still largely guided by bacterial benchmarking studies or limited single-species comparisons. Because fungal genomes vary widely in size, repeat content, and gene architecture, these assumptions can lead to inefficient sequencing strategies, increased computational costs, and suboptimal assemblies. Here, we develop a reproducible assembly benchmarking framework that combines large-scale simulations from 66 complete fungal genomes spanning plant, animal, and human-associated taxa with newly generated short- and long-read sequencing data from 10 field-collected isolates. This approach enables evaluation of assembler performance across diverse genome architectures and tests whether patterns identified in simulations translate to real biological datasets. Across both simulated and empirical datasets, we show that reliable fungal genome reconstruction can be achieved without excessive sequencing depth by identifying consistent performance thresholds. Assembly contiguity and completeness stabilize at moderate long-read coverage, after which improvements depend more strongly on assembler choice and genome structure than on additional data volume. Hybrid workflows show trade-offs in accuracy, contiguity, and computational demand, whereas targeted short-read polishing provides an efficient strategy for improving base-level accuracy. These findings offer practical guidance for fungal genome assembly and support more robust downstream genomic analyses across non-model microbial systems, including ecologically, agriculturally, and clinically important fungi. Data summaryThe reference fungal genomes used for simulation are available in the NCBI Assembly database under the accession numbers listed in Supplementary Table S1. Empirical raw sequencing data generated for this study are deposited in the NCBI Sequence Read Archive (SRA) under accession PRJNA1474061. All scripts used for read simulation, assembly, polishing, benchmarking, and statistical analyses are available in the project GitHub repository (github.com/bielasilva/fungi_assembly_benchmarking). Software versions, parameters, and workflow configurations are provided within the repository and detailed in the Methods.

The contribution of accessory or conditionally dispensable chromosomes to host-specific virulence was first demonstrated in members of the Fusarium solani species complex (FSSC) that are pathogens of garden pea, Pisum sativum L. The phenomenon has since been shown to exist in many fungal plant pathogens, including the closely related F. oxysporum species complex (FOSC). Genome analysis of members of the FSSC and FOSC pathogenic on pea revealed a diverse size range of the accessory genome of these fungi. Despite the ~65 million years of diverging time, regions on a chromosome known to carry host-specific virulence factors for pea, including the cytochrome P450 pisatin demethylase (PDA) and other pea pathogenicity (PEP) genes, were present in all genomes of these pea pathogens. Genes directly involved in virulence on pea - PEP2, PDA, and PEP5- were the most frequently clustered together. Transcriptome analysis of fungal mycelia treated with the pea phytoalexin pisatin, identified 1,155 differentially expressed genes where many were involved in cellular stress responses. As wilt pathogens that invade host xylem, members of the FOSC encode more putative effectors, when compared to those in the FSSC, and several FOSC effectors were identified to confer race specificity. The conservation of part of the accessory genomes across two evolutionarily diverged species complexes suggests a common origin. Horizontal transfer of accessory chromosomes containing genetic loci involved in pathogenesis for garden pea offers a parsimonious explanation of the polyphyletic origin of host specificity.

The ability to opportunistically infect humans has evolved multiple times across fungi and is a major burden to public health. Opportunistic pathogenicity requires the confluence of pre-existing traits in the fungus that facilitate host colonization (e.g., the ability to grow at 37C) and the existence of host immune filters that permit the survival of some colonizers (e.g., inborn errors of immunity). Numerous studies have previously shown that fungal pathogens can exhibit extensive strain-to-strain variation in the ability to cause disease. Moreover, it is also well established that non-pathogenic fungi can occasionally cause severe infections. Together, these observations provoke the question: what differentiates opportunistic fungal pathogens from non-pathogens? To empirically address this, we directly compared phenotypic, metabolomic, and genomic variation between Aspergillus fumigatus, an organism responsible for more than 300,000 infections per year, and Aspergillus fischeri, a close relative of A. fumigatus that is not considered clinically relevant. By examining 26 phenotypic traits across 16 representative strains of A. fumigatus and 16 of A. fischeri, we find that infection-relevant traits measured under in vitro monoculture conditions show species-specific distributions, whereas traits measured under in vitro coculture with murine macrophages overlap in their distributions. Strikingly, strains of the two species also overlap in their virulence profiles in an immunocompromised murine model of pulmonary aspergillosis; three strains of A. fischeri exhibit lethality rates of >50% while two A. fumigatus strains were among the least virulent of all 32 strains tested. Consistent with the observed overlap, we could not statistically associate variation in virulence to variation in the presence of specific genomic elements, phenotypic traits, or secondary metabolites. Our results raise the hypothesis that opportunistic pathogenicity can extend beyond the boundaries of individual species. We propose a conceptual model where the opportunistic pathogenic potential of any fungal strain is the product of complex interactions among numerous genomic, ecological, and host immunity factors.

C. glabrata (syn. Nakaseomyces glabratus) is a major fungal pathogen, typically isolated as a haploid but occasionally found in diploid form. We isolated a spontaneous diploid variant of the type strain CBS138 and performed experimental evolution under fluconazole. At physiologically relevant concentrations of fluconazole, we found that haploids and diploids both acquired PDR1 mutations, as is commonly observed in C. glabrata. Diploids additionally acquired heterozygous ERG11 and ERG25 mutations, and were more likely to acquire aneuploidies. Despite an ancestral fitness advantage for haploids, after [~]200 generations the highest-fitness clone, as measured in competitive assays with and without fluconazole, was a diploid PDR1 V329F/+ ERG11 K152E/+ double heterozygote. Diploid clones also had higher MICs. In a follow-up experiment in which we rapidly increased the fluconazole concentration to [~]1 mg/mL, haploids and diploids adapted via entirely different paths: haploids via co-mutation in ERG3 and CgOSH3, a previously unreported path to fluconazole resistance, and diploids via heterozygous mutation in ERG25 coupled with trisomies of chrF, chrG, and chrI (in all clones) and chrC (in most).

The fungal pathogen Pyricularia oryzae is notorious for causing blast disease in various important cereal crops, including wheat, rice, millet, and oat. Whole-genome-informed data on this pathogen are necessary to better understand the host adaptability of the fungus, including identifying key determinants of infection to enable more precise disease control. Here, we report highly contiguous genome sequences (using long-read PacBio technology) of two isolates from rice paddies in Portugal, M22.7 and T22.2, which exhibit distinctly aggressive symptoms in rice. Both mitochondrial and nuclear sequences were characterised in this study. The resulting nuclear genomes have assembly lengths of 46.4 Mb for M22.7 (198x coverage) and 46.3 Mb for T22.2 (163x coverage), with near-complete BUSCO completeness (98.8%) and a 0% contamination score (EukCC). Phenotypic analysis showed M22.7 to be more virulent than T22.2, which may be explained by the lower number of predicted effector genes and higher transposable element content in M22.7 relative to T22.2. This announcement represents the first genome resource for natural isolates of P. oryzae from Portugal in over 20 years, filling an important data gap from a major European rice-producing country that produces locally adapted rice varieties under specific agro-environmental conditions (near the Atlantic coast).

Dikaryotic rust fungi maintain two distinct haploid nuclei for most of their life cycle, making their large, repeat-rich genomes difficult to assemble and phase. Here we present haplotype-phased, near chromosome-scale genome assemblies for the poplar rust pathogens Melampsora larici-populina 98AG31 and Melampsora allii-populina 12AY07, generated using PacBio HiFi sequencing and Hi-C-guided scaffolding. For each species, we resolved 18 chromosomes per haplotype, providing the first near chromosome-level representations of poplar rust fungal species. M. larici-populina diploid assembly spans ~203 Mb, while M. allii-populina reaches ~416 Mb, with high completeness and strong collinearity between haplotypes. Compared with previous fragmented or collapsed references, these assemblies greatly improve contiguity, recover centromeric and telomeric features, and support the transposable element-driven genome size expansion in M. allii-populina. The haplotype-aware annotations of genes and predicted effectors derived from these resources will enable detailed analyses of genome architecture, repeat dynamics, and key loci such as avirulence genes. Together, these assemblies provide a robust genomic resource for investigating host adaptation, virulence evolution, and population diversity in poplar rust fungi.

While transposable elements (TEs) are recognized as major drivers of fungal genome structure, evidence of their direct involvement in the interaction with host plants and the environment is only beginning to emerge. Retrotransposons can generate small RNA (sRNA) that act through cross-kingdom RNA interference, while giant DNA TEs called Starships carry dozens of cargo genes that enrich the accessory gene compartment of fungal genomes. In the polyphagous pathogen Botrytis cinerea, the Vv3 strain and other strains specialized on grapevine display a specific repertoire of TEs, including the retrotransposons BcCopia4, BcGypsy6, and BcGypsy7. This study first explored the putative role of sRNA generated from these retrotransposons in the interaction between the Vv3 strain and its host of origin, grapevine. Putative targets were identified among the host mRNAs, but predicted cleavage sites could not be experimentally validated. Moreover, Dicer mutants unable to produce retrotransposons-derived sRNA remained fully pathogenic on grapevine, indicating that these sRNAs do not act as virulence factors on this host. In parallel, this study provides an updated RNA-seq-based annotation of the accessory genes of the Vv3 strain, which revealed a new 93 kb-Starship harboring 43 cargo genes, some of which are related to arsenic resistance. A formal genetic approach confirmed that this locus confers resistance to this metalloid. This giant TE, named Ariane, was also detected in additional grapevine-specialized strains resistant to arsenic but not in strains isolated from other hosts such as tomato. In conclusion, this study highlights how a Starship giant transposon shaped the accessory genes compartment of the polyphagous fungus B. cinerea and may have contributed to its adaptation to vine cultivation by conferring resistance to arsenic, a compound widely used in vineyards during the last century. IMPACT STATEMENTFungal genomes contain many families of transposons whose functional role in adaptation to the environment and in biotic interactions remained hidden for a long time. In the grey mold fungus Botrytis cinerea, strains specialized on grapevine, such as Vv3, carry a specific repertoire of transposons which provides a valuable opportunity to investigate their role in niche adaptation. In this study, we first investigated retrotransposon-derived small RNA, previously described as effectors capable of manipulating the immunity of the model plant Arabidopsis thaliana. Although in silico analysis of the specific repertoire of small RNAs of the Vv3 strain suggested that some could target the expression of grapevine genes, a genetic approach demonstrated that they do not play a significant role in virulence on this host. In contrast, this study identified a new transposon, named Ariane, that carries 43 cargo genes and confers a selective advantage to the Vv3 strain. Ariane belongs to a family of giant transposons called Starships, recently discovered in fungi and considered to be responsible for horizontal genes transfers between unrelated species. Ariane was detected only in some B. cinerea strains isolated from grapevine, and a genetic cross showed that it provides these strains with the ability to grow in presence of arsenic. Arsenic was used in vineyards until the beginning of the 21st century to control fungal trunk diseases and insect pests. Therefore, Ariane appears to have played an important role in the adaptation of B. cinerea strains to cultivated grapevine. Overall, these results underline the importance of considering Starships when predicting emergence of resistance to antifungal compounds. DATA SUMMARYThe novel data described in this study, i.e., RNA-Seq data and the Starship element are accessible under NCBI GEO accession GSE327899 and at https://doi.org/10.57745/HYWRNM, respectively. All information related to Botrytis cinerea genomes used in this study are centralized and kept up to date at the Bioinfo Bioger genomic web portal: https://bioinfo.bioger.inrae.fr/portal/genome-portal/. Direct links to individual portals are respectively https://bioinfo.bioger.inrae.fr/portal/genome-portal/3/ for B. cinerea Vv3 genome, https://bioinfo.bioger.inrae.fr/portal/genome-portal/2/ for B. cinerea Sl3 genome, and https://bioinfo.bioger.inrae.fr/portal/genome-portal/4/ for B. cinerea populations isolated on tomato or grapevine. Each portal provides: (i) a centralized access to public genomic resources, including the genome, transposon, and RNA repositories; (ii) a data browser to download the genomic files; (iii) a genome browser that enables visualization of features within their genomic context, along with associated expression data. As a summary, the prior main public B. cinerea genomic accessions and resources used in this study are: GCA_039644125 for VV3 genome, GCA_022560135 for Sl3 genome, GCA_000143535 for B05.10 genome, PRJNA624742 for populations, https://doi.org/10.57745/HYWRNM for transposons, and GSE181592 for small RNAs. Furthermore, table S1 summarizes the list and characteristics of the 64 B. cinerea genomes publicly available to date. The genomic data for Vitis vinifera genome PN40024.v4 used in this study are available at: https://integrape.eu/resources/genes-genomes/genome-accessions/.

The Agaricomycotina accounts for roughly a third of all described fungi. They are important due to their wide range of lifestyles and economic and environmental relevance. Certain agaricomycetes act as lignocellulose degraders, playing a significant role in forest ecosystems and bioremediation processes. These wood-decaying fungi have historically been classified as mostly white- or brown-rot based on their ability to degrade lignin, with white-rot fungi possessing a collection of lignocellulose-degrading enzymes, which are reduced or absent in brown-rot fungi. Here, we sequenced and annotated the genome of the agaricomycete Crepidotus cesatii CBS 511.95 and explored its genome and predicted enzymatic content in a comparative context. The 36.04 Mbp genome is in 235 scaffolds, with 3.34% repeat content and 12,891 predicted genes. We found that the PFAM distributions of identified orthogroups suggested that C. cesatii shows patterns more similar to white-rot fungi compared to brown-rot fungi. Additionally, C. cesatii contained multiple copies of CAZymes CBM1 and AA9 involved in hydrolysis of lignocellulose, similar to white-rot fungi. On the other hand, according to the Conserved Unique Peptide Patterns (CUPP) data for AA2 peroxidases, the key enzymes in lignin degradation, C. cesatii is more similar to brown-rot fungi. Based on our analyses we predict that C. cesatii is another representation of the continuum of wood decaying modes between white and brown rot fungi combining genetic features of both types of fungi.

Pseudomonas aeruginosa is an opportunistic pathogen that can cause severe infections in immunocompromised individuals, such as patients with cystic fibrosis where it commonly forms biofilms. Ciprofloxacin is used extensively to treat P. aeruginosa infections, but its effectiveness can be significantly reduced due to biofilm formation. Although many individual genes associated with biofilm formation or ciprofloxacin resistance have been characterised, the genetic basis of P. aeruginosa biofilm fitness related to antibiotic challenge remains incompletely understood. In this study we employed a whole genome screen to assay the impact of gene disruptions or altered gene expression on survival of P. aeruginosa biofilms exposed to different concentrations of ciprofloxacin. Genes impacting fitness in the biofilm context were identified by comparing the biofilm samples to planktonic samples harvested at 12h, 24h and 48h with and without ciprofloxacin. Genes associated with c-di-GMP regulation and Gac/Rsm signalling were identified as primary regulators for biofilm formation in the presence and absence of ciprofloxacin. In addition, a group of genes involved in respiration, metabolism (especially polyamine metabolism), and various transporter and efflux systems were identified as important for biofilm fitness. Ciprofloxacin specifically imposed a selective pressure on flagellar function and Psl production which were essential for survival in early biofilms. Moreover, transposon insertions within the CPA gene clusters (PA5448-PA5451 and PA5455-PA5456) and the salvage peptidoglycan recycling pathway showed reduced fitness in late biofilms at high concentration of ciprofloxacin, indicating that cell envelope integrity is beneficial for mature biofilms. This study identifies important determinants of survival for biofilms at different stages of maturity in the presence and absence of ciprofloxacin and implicates potential therapeutic targets for antibiofilm drug development.

Cytochrome P450 monooxygenases (CYP450s) are key oxidative enzymes that diversify plant specialized metabolites and play a central role in the biosynthesis of bioactive withanolides in Withania somnifera (L.) Dunal. Despite their importance, genome-wide information on CYP450s in W. somnifera has remained elusive. Herein, the first high-quality genome assembly (2.2 Gb, scaffold N50: 47.4 kb) of an Indian W. somnifera cultivar was generated using a hybrid Oxford Nanopore-Illumina sequencing strategy. Comparative analysis with the NCBI reference genome revealed moderate SNP and indel variations, reflecting intraspecific genetic diversity. A comprehensive CYP450 catalog was established and analyzed phylogenomically across nine plant genomes, encompassing both withanolide-producing and non-producing Solanaceae and non-Solanaceae species. Unique CYP families (CYP450A, CYP1194, and CYP705A) were detected exclusively in W. somnifera, suggesting lineage-specific metabolic innovations, while Solanaceae-restricted (CYP82E/M) and absent (CYP81B, CYP6) lineages highlight taxonomic divergence. Across all analyzed genomes, 36 conserved CYP450 subfamilies, including triterpenoid-associated members, were identified, suggesting a shared oxidative framework adaptable to specialized metabolism. Moreover, potential candidate genes in the triterpenoid pathway, including CYP72A692_1, CYP72A560_4, CYP716A48, CYP724B2, and CYP51G1, were identified through phylogenetic integration with functionally validated triterpenoid-modifying enzymes from other plant species. Gene family evolution analysis further revealed contraction of monoterpenoid-related subfamilies (CYP76A), implying a metabolic shift toward triterpenoid specialization. The comprehensive genome assembly and CYPome of W. somnifera offer a valuable resource for functional characterization, evolutionary analysis, and the identification of genes underlying its specialized metabolism. Furthermore, the study advances our understanding of CYP450 diversity and evolution, revealing lineage-specific innovations, conserved subfamilies, and key candidate genes involved in triterpenoid biosynthesis. Together, these findings lay a foundation for future functional studies and pathway engineering aimed at optimizing the metabolic potential of this important medicinal plant.

Hybridization is a major driver of fungal evolution, yet knowledge of the molecular mechanisms underpinning hybridization and its genomic impact remain limited. Here, we analyse 197 Cryptococcus neoformans genomic sequences, including 13 newly sequenced strains, identifying three genetically clustered and distinct hybrid groups (H1, H2 and H3) each with unique parental origins and ecological associations. Using phylogenomics, population structure analyses, and long-read genome assemblies, we identified hybrid genomes with chromosome-wide loss of heterozygosity (LOH), inheritance of large intact parental haplotype blocks and widespread aneuploidy within and across genomes. These patterns are also observed when progeny were generated with spo11{Delta} parents, indicating these features are a result of a meiotic-independent process such as parasex. This is further underscored by discovery of haploid and near-haploid recombinants in both spo11{Delta} mutant progeny and reanalysed the wild-type hybrid progeny. We hypothesise these haploid and near-haploid recombinants are generated through ploidy reduction via independent chromosome assortment because of concerted chromosomal loss, which is a key feature of the parasexual cycle. Phenotypic assays demonstrated that several hybrid isolates have diverse growth and virulence patterns, underscoring functional consequences of genome plasticity. Together, our work suggests a non-meiotic reproductive process contributes to shape the genotypic and phenotypic diversity of Cryptococcus neoformans. Significance statementHybridisation in fungal pathogens has been linked to novel genotypes, some of which have enhanced virulence. Cryptococcus is a human pathogen responsible for approximately 180,000 deaths annually worldwide. However, the mechanisms by which Cryptococcus generates genotypic diversity remains understudied. By analysing 197 C. neoformans genome sequences, we identified three distinct groups of hybrids, defined by their ancestral inheritance. In each of these 3 groups, we discovered novel features of hybrid isolates including uneven chromosome numbers in most but not all hybrids, chromosome-wide loss of heterozygosity consistent with whole chromosome inheritance from a single parent, and whole-chromosome-wide haplotype inheritance. These genomic features are consistent with a parasexual form of reproduction, which is a non-meiotic process involving cell and nuclear fusion, followed by gradual chromosome loss and sometimes mitotic recombination. Surprisingly, similar genomic patterns were observed in spo11{Delta} mutant crosses, supporting the hypothesis that a non-canonical process such as parasex is facilitating cryptococcal hybridisation. Our results expand our understanding of fungal reproduction and highlight new routes for the emergence of virulence and antifungal resistance.

A genome of Pyricularia oryzae (synonym Magnaporthe oryzae), the fungus that causes blast disease on diverse grass species, has seven core chromosomes and may contain supernumerary mini-chromosomes. The P. oryzae Triticum (PoT) pathotype is the phylogenetic lineage responsible for devastating epidemics of wheat blast disease. Genomic analysis of wheat blast field isolates from the initial outbreak in 1985 in Brazil through recent field isolates in South America revealed dynamic presence and structure of mini-chromosomes. Two "earliest" field isolates representing founder lineages for the Triticum pathotype contain similar mini-chromosomes. Another PoT founder isolate from 1986 and 37 out of 39 Triticum field isolates collected between 1986 and 1992 lack mini-chromosomes. Mini-chromosomes present in the founder strains each contain two copies of the PWT7 wheat blast avirulence gene, and PWT7 was lost from subsequent early strains through mini-chromosome loss. Almost all PoT field isolates from 2005 to 2020 have regained mini-chromosomes in which PWT7 sequences have been replaced by other sequences. Telomere-to-telomere assemblies of 11 mini-chromosomes identified two major mini-chromosome types in the South American PoT population, and demonstrated significant within-mini-chromosome sequence alterations as well as recombination with other mini-chromosomes or core chromosome ends. Additionally, our data indicate horizontal mini-chromosome transfer between Pyricularia species, resulting in nearly identical genomic fragments shared between P. oryzae and Pyricularia pennisetigena isolates in the PWT4 avirulence gene region. Our genomic analysis depicts the dynamic mini-chromosome compartment in the diverse South American Triticum field population through time, indicating important roles for mini-chromosomes in pathogen adaptation and pathogenicity.

The fungal crop pathogen Blumeria hordei, causal agent of powdery mildew on barley, presents life-history and epidemiological characteristics, as well as and selective pressures due to modern agriculture leading to expected sweepstakes reproduction, that is highly skewed offspring distributions. Using genome-wide polymorphism data and population genomics inferences, we aim to 1) infer the past demographic history and the strength of sweepstakes reproduction in B. hordei, and 2) quantify the contributions of these selective and neutral processes in the genome. An new inference method based on Neural Posterior Estimation and diversity and linkage disequilibrium statistics was developed and tested on simulated and B. hordei genomic data. We confirm that B. hordei exhibits a moderate sweepstakes reproduction (-parameter of 1.6). We highlight that the Site Frequency Spectrum (SFS) appears sensitive to the joint occurrence of sweepstakes and recent demographic changes, which may caution on the reliability of the SFS to infer sweepstakes reproduction. We then scan the genome for selective sweeps, adjusting the significance thresholds of the methods for demographic history and sweepstakes reproduction, thereby yielding a counterintuitive result. When conditioning the significance threshold for sweep detection on simulations under sweepstakes and demography, a very large number of putatively selected regions is found (11.6% of the genome). We suggest that sweepstakes reproduction in B. hordei is due to 1) neutrality (clonal/sexual phases and Boom-and-Bust cycles) generating a genome-wide level of background noise in the coalescent genealogies, and 2) selective sweepstakes due to pervasive positive selection. Our findings have important implications for both population genomic methodology and our understanding of pathogen evolution.

Bacterial spot is a consistent threat to global tomato and pepper productions; however, Ohios fresh market production currently lacks the updated surveillance data necessary to provide accurate management solutions. While traditional diagnostics focus on identification of a single causal agent, shotgun metagenomic sequencing (MGS) offers a comprehensive view of the infection court. An assignment-first MGS workflow was developed and validated in this study, utilizing Kraken2 databases to extract Xanthomonas species associated with bacterial spot and to characterize the microbial communities of bacterial spot in Ohio production systems. Through in silico spiking experiments, thresholds were established for bacterial spot identification. Species and pathovar identification via average nucleotide identity (ANI) remained accurate at abundance as low as 0.1%. A minimum of 2% Xanthomonas reads were required for high genome completeness (BUSCO >90%) and 3% for reliable type III secretion system (T3SS) effector profiling. Analysis of 63 samples from fresh-market production fields identified Xanthomonas hortorum pv. gardneri, Xanthomonas euvesicatoria pv. euvesicatoria, and Xanthomonas arboricola residing in symptomatic samples, alongside other taxa including Pseudomonas and Stenotrophomonas. Phylogenetic comparisons of metagenome-assembled genomes (MAGs) were comparable to whole genome sequences (WGS) from the same samples, supporting the reliability of culture-independent diagnostics. These results provide a robust framework for utilizing metagenomics as a diagnostic tool, expanding our knowledge of bacterial spot population structure in Ohio, and uncovering the bacterial communities associated with bacterial spot.

Magnaporthe oryzae, the rice blast fungus, plays a role as a model organism for molecular plant-microbe interaction research. Studies on the pathogenic mechanism of this fungus revealed many genes involved in signaling pathways. As multi-omics data are being available, genomic-level researches have been conducted to uncover the underlying biological processes during the pathogenesis of M. oryzae. Identifying the genome-wide protein-protein interaction (PPI) network is one of the omics-level approaches, which helps to understand signaling and regulatory pathways. However, existing biological network resources of M. oryzae are not sufficient to decipher pathogenesis mechanisms due to the abundance of false positives/negatives. In this study, a reliable PPI network database of M. oryzae, MagNet, was constructed with three methods, including homology-based Interolog search, co-expression network construction, and domain-domain interaction (DDI)-based prediction. With three approaches altogether, the pan-network with 5,600,976 interactions was generated, including 217,531 highly confident interactions supported by all three methods. Experimental data on M. oryzae PPIs supported that our PPI network can predict PPIs with higher accuracy compared to the previously constructed databases. MagNet would provide integrated biological network data, which can help to understand the molecular mechanisms of the rice blast fungus. The PPI data can be accessed via https:/magnet.scnu.ac.kr.

ObjectivesModel species are essential for fundamental research in biology. While a complete genomic sequence is a prerequisite for genetic studies, it is not enough on its own. Understanding the three-dimensional organization of the genome is also important, allowing researchers to gain a more realistic understanding of the mechanisms governing genome function. In the fungal model Podospora anserina, although the genomic sequence has been established for a long time, the three-dimensional organization remained unknown. Here we obtained the first Hi-C datasets and present associated 3D models, providing the research community with a valuable resource for better multi-omics data integration. Data descriptionHi-C experiments were performed in duplicate, using nuclei purified from wild-type fungal mycelium. Four FASTQ files were obtained (two per replicate) and used as inputs for the 3DGB workflow with four different output resolutions, to observe the genome organization of P. anserina at different levels of detail (50 kb, 20 kb, 10 kb, and 5 kb). In a context where researchers already have, for this species, a large amount of traditional omics data (ChIP-seq, RNA-seq, etc.), these 3D models are helpful for complementing the linear representation of the genome, which is traditionally used in bioinformatic analyses.