Fungi from the genus Epichloë form systemic and seed-transmissible endophytic infections of grasses in a symbiosis that is considered predominately mutualistic. During asymptomatic growth these Epichloë fungi produce a variety of secondary metabolites that benefit their host by providing protection against herbivory and disease. However, the Epichloë sexual cycle is antagonistic to its host, characterised by the proliferation of hyphae on reproductive tillers to form fungal stromata that encase and sterilise developing host inflorescences. Production of known host-protective Epichloë-derived secondary metabolites is suppressed in these stromata; however, we describe here the pyrrolodiazipic acids, a novel class of fungal alkaloids that are specifically produced in Epichloë stromata. Transcriptomic analysis identified the five-gene PZA cluster that, unlike other Epichloë biosynthetic gene clusters, is specifically expressed in stromata. Reconstruction of this PZA cluster in a heterologous host enabled the function of the encoded enzymes to be defined, key biosynthetic intermediates to be identified, and the final product characterised – stromatine, a pyrrolodiazepine with carboxylic acid functionality. Analysis of material from many different Epichloë-host associations revealed that stromatine was universally and exclusively found in stroma tissues. Stromatine was also produced in exceptionally large amounts, contributing almost 1% of the total stroma dry mass in some grass-endophyte associations. PZA gene deletion analyses across three different Epichloë species yielded interesting results; stromatine appears to be required for host infection by some Epichloë species, while the accumulation of PZA pathway intermediates appears to be detrimental to symbiosis. Genomic analyses reveal the PZA cluster is conserved among sexual Epichloë species but is dispensable for asexual strains. Combined with the tissue specificity and high levels of production, this suggests that stromatine plays a critical role in the Epichloë sexual cycle, with potential bioactivities and functions to be dissected further.

Epichlo & euml; species are systemic, often seed-transmissible symbionts (endophytes) of cool-season grasses (Poaceae subfam. Po & ouml;ideae) that produce up to four classes of bioprotective alkaloids. Whereas haploid Epichlo & euml; species may reproduce sexually and transmit between host plants (horizontally), many Epichlo & euml; species are polyploid hybrids that are exclusively transmitted via seeds (vertically). Therefore, the generation of, and selection on, chemotypic (alkaloid) profiles and diversity should differ between haploids and hybrids. We undertook a genome-level analysis of haploids and polyploid hybrids, emphasizing hybrids that produce lolines, which are potent broad-spectrum anti-invertebrate alkaloids that can accumulate to levels up to 2% of plant dry mass. Prior phylogenetic analysis had indicated that loline alkaloid gene clusters (LOL) in many hybrids are from the haploid species Epichlo & euml; bromicola, but no LOL-containing E. bromicola strains were previously identified. We discovered LOL-containing E. bromicola from host grasses Bromus tomentellus and Melica persica in a Mediterranean-climate region (MCR) in Isfahan Province, Iran, and from Thinopyrum intermedium in Poland. The isolates from B. tomentellus and M. persica were closely related and had nearly identical alkaloid gene profiles, and their LOL clusters were most closely related to those of several Epichlo & euml; hybrids. In contrast, several LOL genes in the isolate from T. intermedium were phylogenetically more basal in genus Epichlo & euml;, indicating trans-species polymorphism. While identifying likely hybrid ancestors, this study also revealed novel host ranges in central Iran, with the first observation of E. bromicola in host tribe Meliceae and of Epichlo & euml; festucae in host tribe Bromeae. We discuss the possibility that MCRs may be hotspots for diversification of grass-Epichlo & euml; symbioses via extended host ranges and interspecific hybridization of the symbionts.

Antagonistic selection between pathogens and their hosts can drive rapid evolutionary change and leave distinct molecular footprints of past and ongoing selection in the genomes of the interacting species. Despite an increasing availability of tools able to identify signatures of selection, the genetic mechanisms underlying coevolutionary interactions and the specific genes involved are still poorly understood, especially in heterogeneous natural environments. We searched the genomes of two species of Epichloe plant pathogen for evidence of recent selection. The Epichloe genus includes highly host-specific species that can sterilize their grass hosts. We performed selection scans using genome-wide SNP data from seven natural populations of two co-occurring Epichloe sibling species specialized on different hosts. We found evidence of recent (and ongoing) selective sweeps across the genome in both species. However, selective sweeps were more abundant in the species with a larger effective population size. Sweep regions often overlapped with highly polymorphic AT-rich regions supporting the role of these genome compartments in adaptive evolution. Although most loci under selection were specific to individual populations, we could also identify several candidate genes targeted by selection in sweep regions shared among populations. The genes encoded small secreted proteins typical of fungal effectors and cell wall-degrading enzymes. By investigating the genomic signatures of selection across multiple populations and species, this study contributes to our understanding of complex adaptive processes in natural plant pathogen systems.

Understanding the population genetic processes driving the evolution of plant pathogens is of central interest to plant pathologists and evolutionary biologists alike. However, most studies focus on host-pathogen associations in agricultural systems of high genetic and environmental homogeneity and less is known about the genetic structure of pathogen populations infecting wild plants in natural ecosystems. We performed parallel population sampling of two pathogenic Epichloe species occurring sympatrically on different host grasses in natural and seminatural grasslands in Europe: E. typhina infecting Dactylis glomerata and E. clarkii infecting Holcus lanatus . We sequenced 422 haploid isolates and generated genome-wide SNP datasets to investigate genetic diversity and population structure. In both species geographically separated populations formed genetically distinct groups, however population separation was less distinct in E. typhina compared to E. clarkii . The patterns of among population admixture also differed between species across the same geographic range: we found higher levels of population genetic differentiation and a stronger effect of isolation by distance in E. clarkii compared to E. typhina , consistent with lower levels of gene flow in the former. This pattern may be explained by the different dispersal abilities of the two pathogens and is expected to be influenced by the genetic structure of host populations. In addition, genetic diversity was higher in E. typhina populations compared to E. clarkii , indicative of higher effective population size in E. typhina . These results suggest that the effect of genetic drift and the efficacy of selection may differ in the two species. Our study provides evidence of how ecologically similar species occupying the same geographical space can experience different evolutionary contexts, which could influence local adaptation and coevolutionary dynamics of these fungal pathogens.

Fungi of genus Epichloë (Ascomycota, Clavicipitaceae) are common endophytic symbionts of Poaceae, including wild and agronomically important cool-season grass species (subfam. Poöideae). Here, we examined the genetic diversity of Epichloë from three European species of Brachypodium (B. sylvaticum, B. pinnatum and B. phoenicoides) and three species of Calamagrostis (C. arundinacea, C. purpurea and C. villosa), using DNA sequences of tubB and tefA genes. In addition, microsatellite markers were obtained from a larger set of isolates from B. sylvaticum sampled across Europe. Based on phylogenetic analyses the isolates from Brachypodium hosts were placed in three different subclades within the Epichloë typhina complex (ETC) but did not strictly group according to host grass species, suggesting that the host does not always select for particular endophyte genotypes. Analysis of microsatellite markers confirmed the presence of genetically distinct lineages of Epichloësylvatica on B. sylvaticum, which appeared to be tied to different modes of reproduction (sexual or asexual). Among isolates from Calamagrostis hosts, two subclades were detected which were placed outside ETC. These endophyte lineages are recognized as distinct species for which we propose the names E. calamagrostidis Leuchtm. & Schardl, sp. nov. and E. ftanensis Leuchtm. & A.D. Treindl, sp. nov. This study extends knowledge of the phylogeny and evolutionary diversification of Epichloë endophytes that are symbionts of wild Brachypodium and Calamagrostis host grasses.

Genome rearrangements in filamentous fungi are prevalent but little is known about the modalities of their evolution, in part because few complete genomes are available within a single genus. To address this, we have generated and compared 15 complete telomere-to-telomere genomes across the phylogeny of a single genus of filamentous fungi, Epichloë. We find that the striking distinction between gene-rich and repeat-rich regions previously reported for isolated species is ubiquitous across the Epichloë genus. We built a species phylogeny from single-copy gene orthologs to provide a comparative framing to study chromosome composition and structural change through evolutionary time. All Epichloë genomes have exactly seven nuclear chromosomes, but despite this conserved ploidy, analyses reveal low synteny and substantial rearrangement of gene content across the genus. These rearrangements are highly lineage-dependent, with most occurring over short evolutionary distances, with long periods of structural stasis. Quantification of chromosomal rearrangements shows they are uncorrelated with numbers of substitutions and evolutionary distances, suggesting that different modes of evolution are acting to create nucleotide and chromosome-scale changes.

Epichloë (Ascomycota: Clavicipitaceae) fungi can form an intriguing interaction with Botanophila flies. The fungi live within above-ground shoots of grasses. Some species (type I) only reproduce sexually by forming stromata on all host culms (choke disease). Stromata produce haploid spores (spermatia) that fertilize stromata of opposite mating type to form dikaryotic cells. A second category of Epichloë species (type II) produces stromata on only some of the host culms; culms without choke produce flowers and seeds. These Epichloë can reproduce asexually by invading host seed, as well as sexually. Female Botanophila flies visit stromata for feeding and oviposition. Spermatia pass through the gut of Botanophila intact and viable. Flies can cross-fertilize the fungus during defecation after egg laying. Hence, we described the interaction as a mutualism similar to pollination. Yet, subsequent work by others and ourselves showed that visitation by Botanophila flies was not necessary for cross fertilization of Epichloë. We believe these contradictory results can be reconciled from an evolutionary perspective, if one takes into account the reproductive mode of the fungus. We explore a novel hypothesis to reconcile this contradiction, its predictions and discuss ways in which to test them.

Abstract Fungi from the genus Epichloë form systemic endobiotic infections of cool season grasses, producing a range of host-protective natural products in return for access to nutrients. These infections are asymptomatic during vegetative host growth, with associations between asexual Epichloë spp. and their hosts considered mutualistic. However, the sexual cycle of Epichloë spp. involves virulent growth, characterized by the envelopment and sterilization of a developing host inflorescence by a dense sheath of mycelia known as a stroma. Microscopic analysis of stromata revealed a dramatic increase in hyphal propagation and host degradation compared with asymptomatic tissues. RNAseq was used to identify differentially expressed genes in asymptomatic vs stromatized tissues from 3 diverse Epichloë–host associations. Comparative analysis identified a core set of 135 differentially expressed genes that exhibited conserved transcriptional changes across all 3 associations. The core differentially expressed genes more strongly expressed during virulent growth encode proteins associated with host suppression, digestion, adaptation to the external environment, a biosynthetic gene cluster, and 5 transcription factors that may regulate Epichloë stroma formation. An additional 5 transcription factor encoding differentially expressed genes were suppressed during virulent growth, suggesting they regulate mutualistic processes. Expression of biosynthetic gene clusters for natural products that suppress herbivory was universally suppressed during virulent growth, and additional biosynthetic gene clusters that may encode production of novel host-protective natural products were identified. A comparative analysis of 26 Epichloë genomes found a general decrease in core differentially expressed gene conservation among asexual species, and a specific decrease in conservation for the biosynthetic gene cluster expressed during virulent growth and an unusual uncharacterized gene.

Epichloe fungi are endophytes of cool season grasses, both wild species and commercial cultivars, where they may exhibit mutualistic or pathogenic lifestyles. The Epichloe-grass symbiosis is of great interest to agricultural research for the fungal bioprotective properties conferred to host grasses but also serves as an ideal system to study the evolution of fungal plant-pathogens in natural environments. Here, we assembled and annotated gapless chromosome-level genomes of two pathogenic Epichloe sibling species. Both genomes have a bipartite genome organization, with blocks of highly syntenic gene-rich regions separated by blocks of AT-rich DNA. The AT-rich regions show an extensive signature of RIP (repeat-induced point mutation) and the expansion of this compartment accounts for the large difference in genome size between the two species. This study reveals how the rapid evolution of repeat structure can drive divergence between closely related taxa and highlights the evolutionary role of dynamic compartments in fungal genomes.

Nonribosomal peptide synthetases (NRPSs) generate the core peptide scaffolds of many natural products. These include small cyclic dipeptides such as the insect feeding deterrent peramine, which is a pyrrolopyrazine (PPZ) produced by grass-endophytic Epichloë fungi. Biosynthesis of peramine is catalyzed by the 2-module NRPS, PpzA-1, which has a C-terminal reductase (R) domain that is required for reductive release and cyclization of the NRPS-tethered dipeptidyl-thioester intermediate. However, some PpzA variants lack this R domain due to insertion of a transposable element into the 3' end of ppzA We demonstrate here that these truncated PpzA variants utilize nonenzymatic cyclization of the dipeptidyl thioester to a 2,5-diketopiperazine (DKP) to synthesize a range of novel PPZ products. Truncation of the R domain is sufficient to subfunctionalize PpzA-1 into a dedicated DKP synthetase, exemplified by the truncated variant, PpzA-2, which has also evolved altered substrate specificity and reduced N-methyltransferase activity relative to PpzA-1. Further allelic diversity has been generated by recombination-mediated domain shuffling between ppzA-1 and ppzA-2, resulting in the ppzA-3 and ppzA-4 alleles, each of which encodes synthesis of a unique PPZ metabolite. This research establishes that efficient NRPS-catalyzed DKP biosynthesis can occur in vivo through nonenzymatic dipeptidyl cyclization and presents a remarkably clean example of NRPS evolution through recombinant exchange of functionally divergent domains. This work highlights that allelic variants of a single NRPS can result in a surprising level of secondary metabolite diversity comparable to that observed for some gene clusters.

Species belonging to the Festuca-Lolium complex are often naturally infected with endophytic fungi of genus Epichloë. Recent studies on endophytes have shown the beneficial roles of host-endophyte associations as protection against insect herbivores in agriculturally important grasses. However, large-scale screenings are crucial to identify animal friendly strains suitable for agricultural use. In this study we analyzed collected populations of meadow fescue (Schedonorus pratensis) from 135 different locations across Europe, 255 accessions from the United States Department of Agriculture and 96 accessions from The Nordic Genetic Resource Centre. The analysis also included representatives of S. arundinaceus, S. giganteus, and Lolium perenne. All plants were screened for the presence of Epichloë endophytes, resulting in a nursery of about 2500 infected plants from 176 different locations. Genetic diversity was investigated on 250 isolates using a microsatellite-based PCR fingerprinting assay at 7 loci, 5 of which were uncharacterized for these species. Phylogenetic and principal components analysis showed a strong interspecific genetic differentiation among isolates, and, with E. uncinata isolates, a small but significant correlation between genetic diversity and geographical effect (r = 0.227) was detected. Concentrations of loline alkaloids were measured in 218 infected meadow fescue plants. Average amount of total loline and the proportions of the single loline alkaloids differed significantly among endophyte haplotypes (P < 0.005). This study provides insight into endophyte genetic diversity and geographic variation in Europe and a reference database of allele sizes for fast discrimination of isolates. We also discuss the possibility of multiple hybridization events as a source of genetic and alkaloid variation observed in E. uncinata.

The ectomycorrhizal fungus Boletus edulis, commonly known as king bolete, Steinpilz, porcini or cep, is one of the most popular edible mushrooms in Europe, North America and Asia. To produce fruiting bodies, it usually relies on the symbiotic association with deciduous or coniferous trees. Here, we report on an exceptional finding of B. edulis at an altitude of 2440 m a.s.l. in the Swiss Alps and document for the first time its ectomycorrhizal association with Salix herbacea (dwarf willow) based on molecular markers and microscopic observations.

Epichloë fungi are endophytes within grasses that can form stromata on culms of their hosts. Botanophila flies visit the stromata for egg laying and in the process can vector spermatial spores, thereby cross fertilising the fungus. Following egg hatch, larval flies consume fungal tissue and spores. Thus, Epichloë individuals with traits that limit larval consumption could be at a selective advantage. We assessed Botanophila fly larvae from sites within the United States and Europe for infection by the bacterial sexual parasite Wolbachia through amplification of the Wolbachia surface protein gene (wsp). Nearly 70% of fly larvae in our samples were infected by Wolbachia. This is the first record of infection by Wolbachia within Botanophila and could have far reaching effects on not only the fly host, but also the Epichloë fungi upon which Botanophila feeds as well as the grass host within which the fungi live. For example, infection by Wolbachia could limit consumption of Epichloë spores by Botanophila larvae if the bacteria promoted premature larval death.

Epichloe endophytes (Clavicipitaceae) infect pooid grass genera worldwide but predominantly in the Northern Hemisphere, but appear to be rare in native grasses of the Southern Hemisphere. Because of benefits that hosts may receive from the symbiosis, Epichloe endophytes have been extensively studied and are considered important components of sustainable agriculture. There are only a few studies available on the incidence of endophyte infection in grasses of the Southern Hemisphere and most grass species have never been examined. Here we report on a survey of native grasses of New Zealand including 25 endemic or indigenous species. We sampled up to 10 plants per species at different sites from both the North and South Island of New Zealand and examined tissues microscopically for endophyte infection. Overall, only two species were found to be infected, Poa matthewsii (Matthew's bluegrass) and Dichelachne micrantha (short-hair plume grass). Based on analyses of tefA and tubB genes, both endophytes were found to be interspecific hybrids. The endophyte of the new host D. micrantha was previously described as Epichloe australiensis, while the endophyte of P. matthewsii is a new species named here E. novae-zelandiae. The new species is a hybrid derived from E. amarillans, E. bromicola and E. typhina subsp. poae. Alkaloid analyses in planta suggested that E. novae-zelandiae can produce small amounts of peramine, early pathway indole-diterpenes and ergot alkaloids, but no lolines or lolitrems. Target specific primers suggested the presence of genes for ergot alkaloids and peramine, but genes of only early pathway steps for the other alkaloids. Furthermore, genes for both mating type idiomorphs (MTA and MTB) were present, a single copy of MTA and two copies of MTB. Endophytes of native grasses may provide a genetic resource that could be exploited for developing pasture grass cultivars with improved performance.

Entomopathogenic fungi are used for biological control of insect pests. Metarhizium brunneum Petch (Hypocreales) has potential to control Diabrotica virgifera virgifera LeConte (Chrysomelidae), which is a major pest of maize in North America and has recently invaded Europe. The inundative application of an entomopathogenic fungal strain in biological control results in high densities of fungal propagules in the soil which can potentially affect soil microbial communities and their multiple functions in soil. The objective of the present study was to assess potential effects of M. brunneum on soil fungal and prokaryotic communities in a pot experiment over a time course of 4 months using high-throughput sequencing (HTS) of ribosomal markers. The application of M. brunneum formulated as fungus colonised barley kernels (FCBK) led to a significant increase of the applied strain in soil, as assessed by cultivation-dependent (plating on selective medium followed by genotyping of Metarhizium isolates) and cultivation-independent (HTS of ribosomal markers) approaches. Data revealed that soil fungal and prokaryotic community structures did not change after the application of M. brunneum. Temporal changes of the fungal and prokaryotic communities were observed and the prokaryotic communities showed minor changes to barley kernels (BK), the matrix of the formulation. Results of this study are in accordance with other investigations lacking any evidence for adverse effects on microbial communities caused by applied entomopathogenic fungi.

Mate recognition mechanisms resulting in assortative mating constitute an effective reproductive barrier that may promote sexual isolation and speciation. While such mechanisms are widely documented for animals and plants, they remain poorly studied in fungi. We used two interfertile species of Epichloë (Clavicipitaceae, Ascomycota), E. typhina and E. clarkii, which are host-specific endophytes of two sympatrically occurring grasses. The life cycle of these obligatory outcrossing fungi entails dispersal of gametes by a fly vector among external fungal structures (stromata). To test for assortative mating, we mimicked the natural fertilization process by applying mixtures of spermatia from both species and examined their reproductive success. Our trials revealed that fertilization is non-random and preferentially takes place between conspecific mating partners, which is indicative of assortative mating. Additionally, the viability of hybrid and non-hybrid ascospore offspring was assessed. Germination rates were lower in E. clarkii than in E. typhina and were reduced in ascospore progeny from treatments with high proportions of heterospecific spermatia. The preferential mating between conspecific genotypes and reduced hybrid viability represent important reproductive barriers that have not been documented before in Epichloë. Insights from fungal systems will deepen our understanding of the evolutionary mechanisms leading to reproductive isolation and speciation.

Host specialization is a key process in ecological divergence and speciation of plant‐associated fungi. The underlying determinants of host specialization are generally poorly understood, especially in endophytes, which constitute one of the most abundant components of the plant microbiome. We addressed the genetic basis of host specialization in two sympatric subspecies of grass‐endophytic fungi from the Epichloë typhina complex: subsp. typhina and clarkii. The life cycle of these fungi entails unrestricted dispersal of gametes and sexual reproduction before infection of a new host, implying that the host imposes a selective barrier on viability of the progeny. We aimed to detect genes under divergent selection between subspecies, experiencing restricted gene flow due to adaptation to different hosts. Using pooled whole‐genome sequencing data, we combined FST and DXY population statistics in genome scans and detected 57 outlier genes showing strong differentiation between the two subspecies. Genomewide analyses of nucleotide diversity (π), Tajima's D and dN/dS ratios indicated that these genes have evolved under positive selection. Genes encoding secreted proteins were enriched among the genes showing evidence of positive selection, suggesting that molecular plant–fungus interactions are strong drivers of endophyte divergence. We focused on five genes encoding secreted proteins, which were further sequenced in 28 additional isolates collected across Europe to assess genetic variation in a larger sample size. Signature of positive selection in these isolates and putative identification of pathogenic function supports our findings that these genes represent strong candidates for host specialization determinants in Epichloë endophytes. Our results highlight the role of secreted proteins as key determinants of host specialization.

The release of large quantities of microorganisms to soil for purposes such as pest control or plant growth promotion may affect the indigenous soil microbial communities. In our study, we investigated potential effects of Metarhizium brunneum ART2825 on soil fungi and prokaryota in bulk soil using high-throughput sequencing of ribosomal markers. Different formulations of this strain, and combinations of the fungus with garlic as efficacy-enhancing agent, were tested over 4 months in a pot and a field experiment carried out for biological control of Agriotes spp. in potatoes. A biocontrol effect was observed only in the pot experiment, i.e. the application of FCBK resulted in 77% efficacy. Colony counts combined with genotyping and marker sequence abundance confirmed the successful establishment of the applied strain. Only the formulated applied strain caused small shifts in fungal communities in the pot experiment. Treatment effects were in the same range as the effects caused by barley kernels, the carrier of the FCBK formulation and temporal effects. Garlic treatments and time affected prokaryotic communities. In the field experiment, only spatial differences affected fungal and prokaryotic communities. Our findings suggest that M. brunneum may not adversely affect soil microbial communities.