BACKGROUND: Colletotrichum fungi infect a wide diversity of monocot and dicot hosts, causing diseases on almost all economically important plants worldwide. Colletotrichum is also a suitable model for studying gene family evolution on a fine scale to uncover events in the genome associated with biological changes.
RESULTS: Here we present the genome sequences of 30 Colletotrichum species covering the diversity within the genus. Evolutionary analyses revealed that the Colletotrichum ancestor diverged in the late Cretaceous in parallel with the diversification of flowering plants. We provide evidence of independent host jumps from dicots to monocots during the evolution of Colletotrichum, coinciding with a progressive shrinking of the plant cell wall degradative arsenal and expansions in lineage-specific gene families. Comparative transcriptomics of 4 species adapted to different hosts revealed similarity in gene content but high diversity in the modulation of their transcription profiles on different plant substrates. Combining genomics and transcriptomics, we identified a set of core genes such as specific transcription factors, putatively involved in plant cell wall degradation.
CONCLUSIONS: These results indicate that the ancestral Colletotrichum were associated with dicot plants and certain branches progressively adapted to different monocot hosts, reshaping the gene content and its regulation.

We sequenced and annotated the genomes of the ascomycete fungi Trichoderma harzianum, Trichoderma aggressivum f. europaeum, and Purpureocillium lilacinum. Moreover, we developed a website to allow users to interactively analyze the assemblies, gene predictions, and functional annotations of these species and 70+ previously sequenced fungi.

A.fumigatus is a deadly fungal pathogen, responsible for >400,000 infections/year and high mortality rates. A. fumigatus strains exhibit variation in infection-relevant traits, including in their virulence. However, most A. fumigatus protein-coding genes, including those that modulate its virulence, are shared between A. fumigatus strains and closely related non-pathogenic relatives. We hypothesized that A. fumigatus genes exhibit substantial genetic variation in the non-coding regions immediately upstream to the start codons of genes, which could reflect differences in gene regulation between strains. To begin testing this hypothesis, we identified 5,812 single-copy orthologs across the genomes of 263 A. fumigatus strains. A. fumigatus non-coding regions showed higher levels of sequence variation compared to their corresponding protein-coding regions. Specifically, we found that 1,274 non-coding regions exhibited <75% nucleotide sequence similarity (compared to 928 protein-coding regions) and 3,721 non-coding regions exhibited between 75% and 99% similarity (compared to 2,482 protein-coding regions) across strains. Only 817 non-coding regions exhibited ≥99% sequence similarity compared to 2,402 protein-coding regions. By examining 2,482 genes whose protein-coding sequence identity scores ranged between 75% and 99%, we identified 478 total genes with signatures of positive selection only in their non-coding regions and 65 total genes with signatures only in their protein-coding regions. 28 of the 478 non-coding regions and 5 of the 65 protein-coding regions under selection are associated with genes known to modulate A. fumigatus virulence. Non-coding region variation between A. fumigatus strains included single nucleotide polymorphisms and insertions or deletions of at least a few nucleotides. These results show that non-coding regions of A. fumigatus genes harbor greater sequence variation than protein-coding regions, raising the hypothesis that this variation may contribute to A. fumigatus phenotypic heterogeneity.

BACKGROUND: Filamentous fungi are prolific producers of bioactive molecules and enzymes with important applications in industry. Yet, the vast majority of fungal species remain undiscovered or uncharacterized. Here we focus our attention to a wild fungal isolate that we identified as Anthostomella pinea. The fungus belongs to a complex polyphyletic genus in the family of Xylariaceae, which is known to comprise endophytic and pathogenic fungi that produce a plethora of interesting secondary metabolites. Despite that, Anthostomella is largely understudied and only two species have been fully sequenced and characterized at a genomic level.
RESULTS: In this work, we used long-read sequencing to obtain the complete 53.7 Mb genome sequence including the full mitochondrial DNA. We performed extensive structural and functional annotation of coding sequences, including genes encoding enzymes with potential applications in biotechnology. Among others, we found that the genome of A. pinea encodes 91 biosynthetic gene clusters, more than 600 CAZymes, and 164 P450s. Furthermore, untargeted metabolomics and molecular networking analysis of the cultivation extracts revealed a rich secondary metabolism, and in particular an abundance of sesquiterpenoids and sesquiterpene lactones. We also identified the polyketide antibiotic xanthoepocin, to which we attribute the anti-Gram-positive effect of the extracts that we observed in antibacterial plate assays.
CONCLUSIONS: Taken together, our results provide a first glimpse into the potential of Anthstomella pinea to provide new bioactive molecules and biocatalysts and will facilitate future research into these valuable metabolites.

Candida albicans is an opportunistic fungal pathogen of humans, yet the within-host dynamics of C. albicans infection are not clear. While C. albicans is commonly diploid, it exhibits a range of ploidies, including tetraploidy. Previous work found that tetraploid C. albicans populations exhibited rapid adaptation and significant genome instability when evolved in vitro. Host immune function alters the rate and magnitude of C. albicans virulence evolution, but the effects of the host immunity on tetraploid C. albicans populations are unclear. Here, we tested the effects of the host immunity on genome stability and virulence evolution of tetraploid C. albicans using experimental evolution. We selected for C. albicans increased virulence within either immunocompetent or immunocompromised Caenorhabditis elegans hosts. After nine passages we observed a response to selection for increased virulence. Both populations exposed to either immunocompetent or immunocompromised hosts increased virulence after passage through C. elegans hosts. However, the C. albicans populations passaged through immunocompetent hosts under selection exhibited unique temporal dynamics, a rapid increase in virulence and then subsequent loss of virulence. Most C. albicans populations exhibited genome size reduction within six passages, however populations exposed to immunocompetent hosts exhibited the most rapid transition to ~diploid. Therefore, we found that tetraploids rapidly increase in virulence and decrease genome size within host environments. Further, the combination of selection for greater virulence in the presence of immunocompetent hosts results in major virulence fluctuations and genome size changes. Thus, host immunity significantly impacts the evolutionary trajectories of tetraploid C. albicans.

Understanding the origins of variation in agricultural pathogens is of fundamental interest and practical importance, especially for diseases that threaten food security. Fusarium oxysporum is among the most important of soil-borne pathogens, with a global distribution and an extensive host range. The pathogen is considered to be asexual, with horizontal transfer of chromosomes providing an analog of assortment by meiotic recombination. Here, we challenge those assumptions based on the results of population genomic analyses, describing the pathogen\'s diversity and inferring its origins and functional consequences in the context of a single, long-standing agricultural system. We identify simultaneously low nucleotide distance among strains, and unexpectedly high levels of genetic and genomic variability. We determine that these features arise from a combination of genome-scale recombination, best explained by widespread sexual reproduction, and presence-absence variation consistent with chromosomal rearrangement. Pangenome analyses document an accessory genome more than twice the size of the core genome, with contrasting evolutionary dynamics. The core genome is stable, with low diversity and high genetic differentiation across geographic space, while the accessory genome is paradoxically more diverse and unstable but with lower genetic differentiation and hallmarks of contemporary gene flow at local scales. We suggest a model in which episodic sexual reproduction generates haplotypes that are selected and then maintained through clone-like dynamics, followed by contemporary genomic rearrangements that reassort the accessory genome among sympatric strains. Taken together, these processes contribute unique genome content, including reassortment of virulence determinants that may explain observed variation in pathogenic potential.

UNASSIGNED: Nosema is a diverse genus of unicellular microsporidian parasites of insects and other arthropods. Nosema muscidifuracis infects parasitoid wasp species of Muscidifurax zaraptor and M. raptor (Hymenoptera: Pteromalidae), causing ~50% reduction in longevity and ~90% reduction in fecundity.
UNASSIGNED: Here, we report the first assembly of the N. muscidifuracis genome (14,397,169 bp in 28 contigs) of high continuity (contig N50 544.3 Kb) and completeness (BUSCO score 97.0%). A total of 2,782 protein-coding genes were annotated, with 66.2% of the genes having two copies and 24.0% of genes having three copies. These duplicated genes are highly similar, with a sequence identity of 99.3%. The complex pattern suggests extensive gene duplications and rearrangements across the genome. We annotated 57 rDNA loci, which are highly GC-rich (37%) in a GC-poor genome (25% genome average). Nosema-specific qPCR primer sets were designed based on 18S rDNA annotation as a diagnostic tool to determine its titer in host samples. We discovered high Nosema titers in Nosema-cured M. raptor and M. zaraptor using heat treatment in 2017 and 2019, suggesting that the remedy did not completely eliminate the Nosema infection. Cytogenetic analyses revealed heavy infections of N. muscidifuracis within the ovaries of M. raptor and M. zaraptor, consistent with the titer determined by qPCR and suggesting a heritable component of infection and per ovum vertical transmission.
UNASSIGNED: The parasitoids-Nosema system is laboratory tractable and, therefore, can serve as a model to inform future genome manipulations of Nosema-host system for investigations of Nosemosis.

Genomic analyses are widely applied to epidemiological, population genetic and experimental studies of pathogenic fungi. A wide range of methods are employed to carry out these analyses, typically without including controls that gauge the accuracy of variant prediction. The importance of tracking outbreaks at a global scale has raised the urgency of establishing high-accuracy pipelines that generate consistent results between research groups. To evaluate currently employed methods for whole-genome variant detection and elaborate best practices for fungal pathogens, we compared how 14 independent variant calling pipelines performed across 35 Candida auris isolates from 4 distinct clades and evaluated the performance of variant calling, single-nucleotide polymorphism (SNP) counts and phylogenetic inference results. Although these pipelines used different variant callers and filtering criteria, we found high overall agreement of SNPs from each pipeline. This concordance correlated with site quality, as SNPs discovered by a few pipelines tended to show lower mapping quality scores and depth of coverage than those recovered by all pipelines. We observed that the major differences between pipelines were due to variation in read trimming strategies, SNP calling methods and parameters, and downstream filtration criteria. We calculated specificity and sensitivity for each pipeline by aligning three isolates with chromosomal level assemblies and found that the GATK-based pipelines were well balanced between these metrics. Selection of trimming methods had a greater impact on SAMtools-based pipelines than those using GATK. Phylogenetic trees inferred by each pipeline showed high consistency at the clade level, but there was more variability between isolates from a single outbreak, with pipelines that used more stringent cutoffs having lower resolution. This project generated two truth datasets useful for routine benchmarking of C. auris variant calling, a consensus VCF of genotypes discovered by 10 or more pipelines across these 35 diverse isolates and variants for 2 samples identified from whole-genome alignments. This study provides a foundation for evaluating SNP calling pipelines and developing best practices for future fungal genomic studies.

Invasive aspergillosis is a deadly fungal disease; more than 400,000 patients are infected worldwide each year and the mortality rate can be as high as 50-95%. Of the ~450 species in the genus Aspergillus only a few are known to be clinically relevant, with the major pathogen Aspergillus fumigatus being responsible for ~50% of all invasive mold infections. Genomic comparisons between A. fumigatus and other Aspergillus species have historically focused on protein-coding regions. However, most A. fumigatus genes, including those that modulate its virulence, are also present in other pathogenic and non-pathogenic closely related species. Our hypothesis is that differential gene regulation - mediated through the non-coding regions upstream of genes\' first codon - contributes to A. fumigatus pathogenicity. To begin testing this, we compared non-coding regions upstream of the first codon of single-copy orthologous genes from the two A. fumigatus reference strains Af293 and A1163 and eight closely related Aspergillus section Fumigati species. We found that these non-coding regions showed extensive sequence variation and lack of homology across species. By examining the evolutionary rates of both protein-coding and non-coding regions in a subset of orthologous genes with highly conserved non-coding regions across the phylogeny, we identified 418 genes, including 25 genes known to modulate A. fumigatus virulence, whose non-coding regions exhibit a different rate of evolution in A. fumigatus. Examination of sequence alignments of these non-coding regions revealed numerous instances of insertions, deletions, and other types of mutations of at least a few nucleotides in A. fumigatus compared to its close relatives. These results show that closely related Aspergillus species that vary greatly in their pathogenicity exhibit extensive non-coding sequence variation and identify numerous changes in non-coding regions of A. fumigatus genes known to contribute to virulence.

Using a legacy of genetic mutants of Neurospora crassa, paired with resequencing efforts through JGI, we have identified the gene responsible for the \'scumbo\' mutant. This early morphological mutant was described as \"Irregular flat, spreading growth with knobby protrusions and abnormal conidiation, but no free conidia. Mycelium usually appears yellowish rather than orange. Female fertile.\" (Perkins, Radford et al. 2000). Our further investigation has found new insights into the identity and associated functions of scumbo as a ceramide C9 methyltransferase, previously annotated as \"similar to cyclopropane-fatty-acyl-phospholipidsynthase\", encoded by the gene NCU07859. This enzyme performs a fungal-specific methyl modification of glycosyl-ceramides and has implications for membrane homeostasis and hyphal polarity in filamentous fungi.