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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. [ABSTRACT FROM AUTHOR]

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 FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Symbiosis is the typical state for large organisms, either plant or animal. Advances in endophyte molecular biology promise to shed light on the mechanisms of the symbioses and host benefits.

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. • Mating preferentially takes place between conspecific individuals of Epichloë. • Hybrid ascospores have reduced viability. • Assortative mating and inferior hybrid vigor both act as reproductive barriers. • Pre- and postzygotic mechanisms underlie speciation processes in Epichloë species. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [ABSTRACT FROM AUTHOR]

As the depth of soil petroleum contamination can vary substantially under field conditions, a rhizotron experiment was performed to investigate the influence of endophyte,P. indica, on maize growth and degradation of petroleum components in a shallow and a deep-reaching subsurface layer of a soil. For control, a treatment without soil contamination was also included. The degree in contamination and the depth to which it extended had a strong effect on the growth of the plant roots. Contaminated soil layers severely inhibited root growth thus many roots preferred to bypass the shallow contaminated layer and grow in the uncontaminated soil. While the length and branching pattern of these roots were similar to those of uncontaminated treatment. Inoculation of maize withP. indicacould improve root distribution and root and shoot growth in all three contamination treatments. This inoculation also enhanced petroleum degradation in soil, especially in the treatment with deep-reaching contamination, consequently the accumulation of petroleum hydrocarbons (PAHs) in the plant tissues were increased. [ABSTRACT FROM PUBLISHER]

The anthomyiid genus Botanophila consists of over 200, mostly phytophagous, species. One species group maintains a mutualistic relationship with Epichloë fungi, thereby acting as vector of fungal spermatia required for fertilization, similar to pollinating parasites. The phylogenetic relationship of these flies with other Botanophila and the related Chiastocheta species (obligatory associated with globeflowers) remains largely unresolved. In this study, we obtained new sequences of the mitochondrial genes COI and COII from a representative sample of 17 European Botanophila and Chiastocheta species including all six Epichloë-associated species, as well as from four outgroup taxa. Phylogenetic analyses indicated that Epichloë-associated Botanophila form a distinct clade suggesting that ancestral Botanophila may have expanded its niche to European fungal hosts once and then successfully radiated while exploiting fungal tissue as additional food source. Furthermore, the three included Chiastocheta species formed a distinct clade nested within Botanophila, leaving the genus paraphyletic as currently circumscribed. [ABSTRACT FROM AUTHOR]