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Quickly understanding the genomic changes that lead to pathogen emergence is necessary to launch mitigation efforts and reduce harm. Often the evolutionary events that result in an epidemic typically remain elusive long after an outbreak, which is particularly true for plant pathogens. To rapidly define the consequential evolutionary events result in pathogen emergence, we tracked in real-time a 2022 bacterial plant disease outbreak in US geranium (Pelargoniumxhortorum) caused by Xhp2022, a novel lineage ofXanthomonas hortorum. Genomes from 31 Xhp2022 isolates from seven states showed limited chromosomal variation, and all contained a single plasmid (p93). Time tree and SNP whole genome analysis estimated that Xhp2022 emerged in the early 2020s. Phylogenomic analysis determined that p93 resulted from cointegration of three plasmids (p31, p45, and p66) present in a 2012 outbreak. p31, p45 and p66 were individually found in varying abundance acrossX. hortorumisolates from historical outbreaks dating to 1974 suggesting these plasmids were maintained in the broader metapopulation. p93 specifically arose from two co-integration events from homologous and Tn3and XerC-mediated site-specific recombination. Although p93 suffered a 49kb nucleotide reduction, it maintained critical fitness gene functions encoding, for example, metal resistance and virulence factors, which were likely selected by the ornamental production system. Overall we demonstrate how rapid sequencing of current and historical isolates track the evolutionary history of an emerging, ongoing threat. We show a recent, tractable event of genome reduction for niche adaptation typically observed over millenia in obligate and fastidious pathogens.SignificanceGenome-resolved epidemiology is rapidly changing how we track pathogens in real-time to support stakeholders and health. This research highlights how we responded to a current disease outbreak of geranium. Our work revealed that a new group of the bacterial plant pathogenXanthomonas horotrumemerged in 2022 as a result of a recent genome reduction. We determined that three distinct plasmids were present in the broaderX. hortorummetapopulation since 1974. In 2012, the three plasmids were altogether present in individual isolates; then in 2022, all three plasmids co-integrated while maintaining critical fitness genes but losing extraneous genomic material. This parallels genome efficiency and reduction that we see across millenia or even millions of years with obligate parasites with increased niche-specificity.

Trichodermais 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 likeTrichoderma, 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-eightTrichodermagenomes representing the full breadth of environmentalTrichoderma’s diverse lifestyles and nutritional modes. We generated four newTrichoderma endophyticumgenomes to improve the sampling of endophytic isolates from this genus. As predicted, endophyticTrichodermagenomes 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 theTrichodermaendophytic lifestyle. Several mycoparasitism genes, but no chitinase genes, were associated with endophyticTrichodermagenomes. Most genomic differences betweenTrichodermalifestyles 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 toTrichoderma.

10 p.-6 fig., Lentinula is a broadly distributed group of fungi that contains the cultivated shiitake mushroom, L. edodes. We sequenced 24 genomes representing eight described species and several unnamed lineages of Lentinula from 15 countries on four continents. Lentinula comprises four major clades that arose in the Oligocene, three in the Americas and one in Asia–Australasia. To expand sampling of shiitake mushrooms, we assembled 60 genomes of L. edodes from China that were previously published as raw Illumina reads and added them to our dataset. Lentinula edodes sensu lato (s. lat.) contains three lineages that may warrant recognition as species, one including a single isolate from Nepal that is the sister group to the rest of L. edodes s. lat., a second with 20 cultivars and 12 wild isolates from China, Japan, Korea, and the Russian Far East, and a third with 28 wild isolates from China, Thailand, and Vietnam. Two additional lineages in China have arisen by hybridization among the second and third groups. Genes encoding cysteine sulfoxide lyase (lecsl) and γ-glutamyl transpeptidase (leggt), which are implicated in biosynthesis of the organosulfur flavor compound lenthionine, have diversified in Lentinula. Paralogs of both genes that are unique to Lentinula (lecsl 3 and leggt 5b) are coordinately up-regulated in fruiting bodies of L. edodes. The pangenome of L. edodes s. lat. contains 20,308 groups of orthologous genes, but only 6,438 orthogroups (32%) are shared among all strains, whereas 3,444 orthogroups (17%) are found only in wild populations, which should be targeted for conservation., This research was supported by the NSF awards DEB-1456588 to D.H. and DEB-1442113 to A.R., the Department of Energy award 0000238431 to D.H., the GENOBIOREF (BIO2017-86559-R) project of the Spanish Ministry of Science and Innovation (cofinanced by FEDER funds) to F.J.R.-D., S.C., and A.T.M., grant PID2019-103901GB-I00 of the Spanish Ministry of Science and Innovation to P.F., and the NIH award R56AI146096 to A.R. J.L.S. and A.R. were supported by the HHMI and the Burroughs Wellcome Fund. The work (proposal: 10.46936/10.25585/60001048) conducted by the US Department of Energy JGI (https://ror.org/04xm1d337), a DOE Office of Science User Facility, was supported by the Office of Science of the US Department of Energy operated under contract no. DE-AC02-05CH11231.

Accessory genes are variably present among members of a species and are a reservoir of adaptive functions. In bacteria, differences in gene distributions among individuals largely result from mobile elements that acquire and disperse accessory genes as cargo. In contrast, the impact of cargo-carrying elements on eukaryotic evolution remains largely unknown. Here, we show that variation in genome content within multiple fungal species is facilitated by Starships, a novel group of massive mobile elements that are 110 kb long on average, share conserved components, and carry diverse arrays of accessory genes. We identified hundreds of Starship-like regions across every major class of filamentous Ascomycetes, including 28 distinct Starships that range from 27-393 kb and last shared a common ancestor ca. 400 mya. Using new long-read assemblies of the plant pathogen Macrophomina phaseolina, we characterize 4 additional Starships whose past and ongoing activities contribute to standing variation in genome structure and content. One of these elements, Voyager, inserts into 5S rDNA and contains a candidate virulence factor whose increasing copy number has contrasting associations with pathogenic and saprophytic growth, suggesting Voyager’s activity underlies an ecological trade-off. We propose that Starships are eukaryotic analogs of bacterial integrative and conjugative elements based on parallels between their conserved components and may therefore represent the first known agents of active gene transfer in eukaryotes. Our results suggest that Starships have shaped the content and structure of fungal genomes for millions of years and reveal a new concerted route for evolution throughout an entire eukaryotic phylum.

Global, large-scale surveys of phylogenetically diverse plant and lichen hosts have revealed an extremely high richness of endophytes in the Xylariales, one of the largest clades of filamentous fungi and a significant source of novel secondary metabolites (SMs). Endophytes may produce host protective antimicrobial or insecticidal SMs, as well as compounds that facilitate symbiotic establishment through suppression or degradation of host immune response, but the ecological roles of most SMs are unknown. Here we characterized metabolic gene clusters in 96 genomes of endophytes and closely related saprotrophs and pathogens in two clades of Xylariales (Xylariaceae s.l. and Hypoxylaceae). Hundreds of genes appear horizontally transferred to xylarialean fungi from distantly related fungi and bacteria, including numerous genes in secondary metabolite gene clusters (SMGCs). Although all xylarialean genomes contain hyperabundant SMGCs, we show that increased gene duplications, horizontal gene transfers (HGTs), and SMGC content in Xylariaceae s.l. taxa are linked to greater phylogenetic host breadth, larger biogeographic distributions, and increased capacity for lignocellulose decomposition compared to Hypoxylaceae taxa. Overall, our results suggest that xylarialean endophytes capable of dual ecological modes (symbiotic and saprotrophic) experience greater selection to diversify SMGCs to both increase competitiveness within microbial communities and facilitate diverse symbiotic interactions.

Atheliales is a diverse order of crust-forming Basidiomycota fungi. Here, we report the draft genome of the “cuckoo fungus,” Athelia (Fibularhizoctonia) sp. strain TMB (Atheliales), which forms termite-egg-mimicking sclerotia for which termites care. We further compare its repertoire of psilocybin gene homologs to homologs previously reported for Fibularhizoctonia psychrophila., Atheliales is a diverse order of crust-forming Basidiomycota fungi. Here, we report the draft genome of the “cuckoo fungus,” Athelia (Fibularhizoctonia) sp. TMB strain TB5 (Atheliales), which forms termite-egg-mimicking sclerotia that termites tend. We further compare its repertoire of psilocybin gene homologs to homologs previously reported for Fibularhizoctonia psychrophila.

Atheliales is a diverse order of crust-forming Basidiomycota fungi. Here, we report the draft genome of the “cuckoo fungus,” Athelia ( Fibularhizoctonia ) sp. strain TMB (Atheliales), which forms termite-egg-mimicking sclerotia for which termites care. We further compare its repertoire of psilocybin gene homologs to homologs previously reported for Fibularhizoctonia psychrophila .