Endophytes Alleviate Drought-Derived Oxidative Damage in Achnatherum inebrians Plants Through Increasing Antioxidants and Regulating Host Stress Responses.

Endophytes generally increase antioxidant contents of plants subjected to environmental stresses. However, the mechanisms by which endophytes alter the accumulation of antioxidants in plant tissues are not entirely clear. We hypothesized that, in stress situations, endophytes would simultaneously reduce oxidative damage and increase antioxidant contents of plants and that the accumulation of antioxidants would be a consequence of the endophyte ability to regulate the expression of plant antioxidant genes. We investigated the effects of the fungal endophyte Epichloë gansuensis (C.J. Li & Nan) on oxidative damage, antioxidant contents, and expression of representative genes associated with antioxidant pathways in Achnatherum inebrians (Hance) Keng plants subjected to low (15%) and high (60%) soil moisture conditions. Gene expression levels were measured using RNA-seq. As expected, the endophyte reduced the oxidative damage by 17.55% and increased the antioxidant contents by 53.14% (on average) in plants subjected to low soil moisture. In line with the accumulation of antioxidants in plant tissues, the endophyte increased the expression of most plant genes associated with the biosynthesis of antioxidants (e.g., MIOX, crtB, gpx) while it reduced the expression of plant genes related to the metabolization of antioxidants (e.g., GST, PRODH, ALDH). Our findings suggest that endophyte ability of increasing antioxidant contents in plants may reduce the oxidative damage caused by stresses and that the fungal regulation of plant antioxidants would partly explain the accumulation of these compounds in plant tissues.

Two-speed genomes of Epichloe fungal pathogens show contrasting signatures of selection between species and across populations.

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.

Epichloë fungal endophyte interactions in perennial ryegrass (Lolium perenne L.) modified to accumulate foliar lipids for increased energy density.

BACKGROUND: Commercial cultivars of perennial ryegrass infected with selected Epichloë fungal endophytes are highly desirable in certain pastures as the resulting mutualistic association has the capacity to confer agronomic benefits (such as invertebrate pest deterrence) largely due to fungal produced secondary metabolites (e.g., alkaloids). In this study, we investigated T2 segregating populations derived from two independent transformation events expressing diacylglycerol acyltransferase (DGAT) and cysteine oleosin (CO) genes designed to increase foliar lipid and biomass accumulation. These populations were either infected with Epichloë festucae var. lolii strain AR1 or Epichloë sp. LpTG-3 strain AR37 to examine relationships between the introduced trait and the endophytic association. Here we report on experiments designed to investigate if expression of the DGAT + CO trait in foliar tissues of perennial ryegrass could negatively impact the grass-endophyte association and vice versa. Both endophyte and plant characters were measured under controlled environment and field conditions. RESULTS: Expected relative increases in total fatty acids of 17-58% accrued as a result of DGAT + CO expression with no significant difference between the endophyte-infected and non-infected progeny. Hyphal growth in association with DGAT + CO expression appeared normal when compared to control plants in a growth chamber. There was no significant difference in mycelial biomass for both strains AR1 and AR37, however, Epichloë-derived alkaloid concentrations were significantly lower on some occasions in the DGAT + CO plants compared to the corresponding null-segregant progenies, although these remained within the reported range for bioactivity. CONCLUSIONS: These results suggest that the mutualistic association formed between perennial ryegrass and selected Epichloë strains does not influence expression of the host DGAT + CO technology, but that endophyte performance may be reduced under some circumstances. Further investigation will now be required to determine the preferred genetic backgrounds for introgression of the DGAT + CO trait in combination with selected endophyte strains, as grass host genetics is a major determinant to the success of the grass-endophyte association in this species.

Characterization of the Biosynthetic Gene Cluster for the Ribosomally Synthesized Cyclic Peptide Epichloëcyclins in Epichloë festucae.

The various grass-induced epichloëcyclins of the Epichloë spp. are ribosomally synthesized and post-translationally modified peptides (RiPPs), produced as small, secreted cyclopeptides from a single gene, gigA. Here, four clustered and coregulated genes (gigA, gigB, gigC, and kexB) with predicted roles in epichloëcyclin production in Epichloë festucae were evaluated through gene disruption. Subsequent chemical analysis indicates that GigB is a DUF3328 domain-containing protein associated with cyclization of epichloëcyclins; GigC is a methyltransferase enzyme responsible for N-methylation of desmethylepichloëcyclins; and KexB is a subtilisin-like enzyme, partly responsible for the propeptide cleavage of epichloëcyclin intermediates. Symbiotic effects on the host phenotype were not observed for gigA, gigC, or kexB mutants, although ΔgigB infection correlated with increased host tiller height and biomass, while only ΔkexB exhibited an effect on endophyte morphology. Disrupting epichloëcyclin biosynthesis showed negligible influence on the biosynthesis of E. festucae-associated alkaloids. Epichloëcyclins may perform other secondary metabolism functions in Epichloë and other fungi.

Host Genetic Background Effect on Vertical Seed Transmission of Epichloë Endophyte Strains in Tall Fescue.

Tall fescue (Lolium arundinaceum (Schreb.) Darbysh.) is a cool-season perennial grass widely grown for forage and turf. Tall fescue lives in association with a fungal endophyte that helps the grass overcome abiotic and biotic stressors. The endophyte is asexual and transmits vertically from the tall fescue plant to the next generation through the seed. Producers of endophyte-infected tall fescue must have endophyte infection in at least 70% of their seed. Therefore, endophyte seed transmission is vital in breeding and seed production. Transfer of endophytes from their native host to different backgrounds of elite tall fescue cultivars can lead to a low seed transmission of the endophyte to the seed. This study screened 23 previously uncharacterized endophyte strains for transmissibility when artificially inoculated into continental and Mediterranean-type host tall fescue. We found no correlation between the rate of successful inoculation and the seed transmission rate of the endophyte in the new host. Nor did the seed transmission rate of the endophyte strains in their native host correlate with the seed transmission rate of the endophyte in the new host. Five strains exhibited seed transmission above 70% in both Mediterranean and Continental host backgrounds and will be characterized further for potential use in cultivar development.

Mechanisms of Epichloë bromicola to Promote Plant Growth and Its Potential Application for Coix lacryma-jobi L. Cultivation.

Endophytic fungi play important roles in regulating plant growth and development and usually used as a promising strategy to enhance the biosynthesis of host valuable secondary metabolite, but the underlying growth-promoting mechanisms are only partly understood. In this study, the wild-type Arabidopsis thaliana seedlings co-cultured with fungal endophyte Epichloë bromicola showed auxin (IAA)-stimulated phenotypes, and the growth-promoting effects caused by E. bromicola were further verified by the experiments of spatially separated co-culture and fungal extract treatment. IAA was detected and identified in the extract of E. bromicola culture by LC-HRMS/MS, whereas 2,3-butanediol was confirmed to be the predominant volatile active compound in the diethyl ether and ethyl acetate extracts by GC-MS. Further study observed that IAA-related genes including synthesis key enzyme genes (CYP79B2, CYP79B3, NIT1, TAA1 and YUCCA1) and controlling polar transport genes (AUX1, BIG, EIR1, AXR3 and ARF1), were highly expressed at different periods after E. bromicola inoculation. More importantly, the introduction of fungal endophyte E. bromicola could effectively promote the growth and accumulation of coixol in Coix under soil conditions. Our study showed that endophytic fungus E. bromicola might be considered as a potential inoculant for improving medicinal plant growth.

Genetic Covariation Between the Vertically Transmitted Endophyte Epichloë canadensis and Its Host Canada Wildrye.

Symbiotic mutualisms are thought to be stabilized by correlations between the interacting genotypes which may be strengthened via vertical transmission and/or reduced genetic variability within each species. Vertical transmission, however, may weaken interactions over time as the endosymbionts would acquire mutations that could not be purged. Additionally, temporal variation in a conditional mutualism could create genetic variation and increased variation in the interaction outcome. In this study, we assessed genetic variation in both members of a symbiosis, the endosymbiotic fungal endophyte Epichloë canadensis and its grass host Canada wildrye (Elymus canadensis). Both species exhibited comparable levels of diversity, mostly within populations rather than between. There were significant differences between populations, although not in the same pattern for the two species, and the differences were not correlated with geographic distance for either species. Interindividual genetic distance matrices for the two species were significantly correlated, although all combinations of discriminant analysis of principle components (DAPC) defined multilocus genotype groups were found suggesting that strict genotype matching is not necessary. Variation in interaction outcome is common in grass/endophyte interactions, and our results suggest that the accumulation of mutations overtime combined with temporal variation in selection pressures increasing genetic variation in the symbiosis may be the cause.

Infection by Endophytic Epichloë sibirica Was Associated with Activation of Defense Hormone Signal Transduction Pathways and Enhanced Pathogen Resistance in the Grass Achnatherum sibiricum.

Epichloë endophytes can improve the resistance of host grasses to pathogenic fungi, but the underlying mechanisms remain largely unknown. Here, we used phytohormone quantifications, gene expression analysis, and pathogenicity experiments to investigate the effect of Epichloë sibirica on the resistance of Achnatherum sibiricum to Curvularia lunata pathogens. Comparison of gene expression patterns between endophyte-infected and endophyte-free leaves revealed that endophyte infection was associated with significant induction of 1,758 and 765 differentially expressed genes in the host before and after pathogen inoculation, respectively. Functional analysis of the differentially expressed genes suggested that endophyte infection could activate the constitutive resistance of the host by increasing photosynthesis, enhancing the ability to scavenge reactive oxygen species, and actively regulating the expression of genes with function related to disease resistance. We found that endophyte infection was associated with induction of the expression of genes involved in the biosynthesis pathways of jasmonic acid, ethylene, and pipecolic acid and amplified the defense response of the jasmonic acid/ethylene co-regulated EIN/ERF1 transduction pathway and Pip-mediated TGA transduction pathway. Phytohormone quantifications showed that endophyte infection was associated with significant accumulation of jasmonic acid, ethylene, and pipecolic acid after pathogen inoculation. Exogenous phytohormone treatments confirmed that the disease index of plants was negatively related to both jasmonic acid and ethylene concentrations. Our results demonstrate that endophyte infection can not only improve the constitutive resistance of the host to phytopathogens before pathogen inoculation but also be associated with enhanced systemic resistance of the host to necrotrophs after C. lunata inoculation.

Cross-species transcriptomics identifies core regulatory changes differentiating the asymptomatic asexual and virulent sexual life cycles of grass-symbiotic Epichloë fungi.

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.

Chromosome-level genomes provide insights into genome evolution, organization and size in Epichloe fungi.

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.

Response of sheep rumen fermentation and microbial communities to feed infected with the endophyte Epichloë gansuensis as evaluated with rumen-simulating technology.

Achnatherum inebrians, a perennial grass, is widely distributed in China. When infected by the endophyte Epichloë gansuensis, A. inebrians produces an abundance of alkaloids that enhance plant survival but are toxic to animals. Here we used in vitro fermentation to study the impact of endophyte- infected A. inebrians (E+) addition on rumen fermentation characteristics and on microbial community and diversity as assessed with amplicon sequencing technology. We examined E+ addition at five levels, E0, E25, E50, E75, and E100, corresponding to 0%, 25%, 50%, 75%, and 100% of the fermentation substrate, respectively. Both the fermentation characteristics and rumen microbial community structure differed significantly among treatments. E100 resulted in the highest values for pH, the Shannon index, Kiritimatiellaeota, and Lentisphaerae levels relative to the other treatments. In contrast, E25 was associated with higher levels of ammonia nitrogen, total volatile fatty acid, propionate, butyrate, isobutyrate, valerate, of the phyla Bacteroidetes and Firmicutes, and of the genus Prevotella_1, Succiniclasticum, Family_XIII_AD3011_group, Rikenellaceae_RC9_gut_group, Prevotellaceae_UCG-001, and Pyramidobacter as compared with other treatments. E50 resulted in the greatest values for the abundance-based coverage estimator (ACE) and the Chao1 index as compared with other treatments. E0 resulted in the greatest values for digestibility of dry matter, gas production, acetate, and Ruminobacter as compared with other treatments. This approach avoided animal toxicity experiments and confirmed that rumen fermentation characteristics and rumen microbiota were affected by E+ toxin. Therefore, E25 showed higher abundance in Prevotella_1, Prevotellaceae_ UCG-001, and Lachnospiraceae_XPB1014_group that implied they should play significant roles in E+ alkaloids degradation. And then, we can infer that rumen microorganisms should function as an antidote with respect to this poisoning reaction at moderate dietary percentages of E+.

Chitin Deacetylases Are Required for Epichloë festucae Endophytic Cell Wall Remodeling During Establishment of a Mutualistic Symbiotic Interaction with Lolium perenne.

Epichloë festucae forms a mutualistic symbiotic association with Lolium perenne. This biotrophic fungus systemically colonizes the intercellular spaces of aerial tissues to form an endophytic hyphal network and also grows as an epiphyte. However, little is known about the cell wall-remodeling mechanisms required to avoid host defense and maintain intercalary growth within the host. Here, we use a suite of molecular probes to show that the E. festucae cell wall is remodeled by conversion of chitin to chitosan during infection of L. perenne seedlings, as the hyphae switch from free-living to endophytic growth. When hyphae transition from endophytic to epiphytic growth, the cell wall is remodeled from predominantly chitosan to chitin. This conversion from chitin to chitosan is catalyzed by chitin deacetylase. The genome of E. festucae encodes three putative chitin deacetylases, two of which (cdaA and cdaB) are expressed in planta. Deletion of either of these genes results in disruption of fungal intercalary growth in the intercellular spaces of plants infected with these mutants. These results establish that these two genes are required for maintenance of the mutualistic symbiotic interaction between E. festucae and L. perenne.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Non-Transgenic CRISPR-Mediated Knockout of Entire Ergot Alkaloid Gene Clusters in Slow-Growing Asexual Polyploid Fungi.

The Epichloë species of fungi include seed-borne symbionts (endophytes) of cool-season grasses that enhance plant fitness, although some also produce alkaloids that are toxic to livestock. Selected or mutated toxin-free endophytes can be introduced into forage cultivars for improved livestock performance. Long-read genome sequencing revealed clusters of ergot alkaloid biosynthesis (EAS) genes in Epichloë coenophiala strain e19 from tall fescue (Lolium arundinaceum) and Epichloë hybrida Lp1 from perennial ryegrass (Lolium perenne). The two homeologous clusters in E. coenophiala-a triploid hybrid species-were 196 kb (EAS1) and 75 kb (EAS2), and the E. hybrida EAS cluster was 83 kb. As a CRISPR-based approach to target these clusters, the fungi were transformed with ribonucleoprotein (RNP) complexes of modified Cas9 nuclease (Cas9-2NLS) and pairs of single guide RNAs (sgRNAs), plus a transiently selected plasmid. In E. coenophiala, the procedure generated deletions of EAS1 and EAS2 separately, as well as both clusters simultaneously. The technique also gave deletions of the EAS cluster in E. hybrida and of individual alkaloid biosynthesis genes (dmaW and lolC) that had previously proved difficult to delete in E. coenophiala. Thus, this facile CRISPR RNP approach readily generates non-transgenic endophytes without toxin genes for use in research and forage cultivar improvement.

The Impact of Alkaloid-Producing Epichloë Endophyte on Forage Ryegrass Breeding: A New Zealand Perspective.

For 30 years, forage ryegrass breeding has known that the germplasm may contain a maternally inherited symbiotic Epichloë endophyte. These endophytes produce a suite of secondary alkaloid compounds, dependent upon strain. Many produce ergot and other alkaloids, which are associated with both insect deterrence and livestock health issues. The levels of alkaloids and other endophyte characteristics are influenced by strain, host germplasm, and environmental conditions. Some strains in the right host germplasm can confer an advantage over biotic and abiotic stressors, thus acting as a maternally inherited desirable 'trait'. Through seed production, these mutualistic endophytes do not transmit into 100% of the crop seed and are less vigorous than the grass seed itself. This causes stability and longevity issues for seed production and storage should the 'trait' be desired in the germplasm. This makes understanding the precise nature of the relationship vitally important to the plant breeder. These Epichloë endophytes cannot be 'bred' in the conventional sense, as they are asexual. Instead, the breeder may modulate endophyte characteristics through selection of host germplasm, a sort of breeding by proxy. This article explores, from a forage seed company perspective, the issues that endophyte characteristics and breeding them by proxy have on ryegrass breeding, and outlines the methods used to assess the 'trait', and the application of these through the breeding, production, and deployment processes. Finally, this article investigates opportunities for enhancing the utilisation of alkaloid-producing endophytes within pastures, with a focus on balancing alkaloid levels to further enhance pest deterrence and improving livestock outcomes.

Genetic Manipulation of the Ergot Alkaloid Pathway in Epichloë festucae var. lolii and Its Effect on Black Beetle Feeding Deterrence.

Epichloë endophytes are filamentous fungi (family Clavicipitaceae) that live in symbiotic associations with grasses in the sub family Poöideae. In New Zealand, E. festucae var. lolii confers significant resistance to perennial ryegrass (Lolium perenne) against insect and animal herbivory and is an essential component of pastoral agriculture, where ryegrass is a major forage species. The fungus produces in planta a range of bioactive secondary metabolites, including ergovaline, which has demonstrated bioactivity against the important pasture pest black beetle, but can also cause mammalian toxicosis. We genetically modified E. festucae var. lolii strain AR5 to eliminate key enzymatic steps in the ergovaline pathway to determine if intermediate ergot alkaloid compounds can still provide insecticidal benefits in the absence of the toxic end product ergovaline. Four genes (dmaW, easG, cloA, and lpsB) spanning the pathway were deleted and each deletion mutant was inoculated into five different plant genotypes of perennial ryegrass, which were later harvested for a full chemical analysis of the ergot alkaloid compounds produced. These associations were also used in a black beetle feeding deterrence study. Deterrence was seen with just chanoclavine present, but was cumulative as more intermediate compounds in the pathway were made available. Ergovaline was not detected in any of the deletion associations, indicating that bioactivity towards black beetle can be obtained in the absence of this mammalian toxin.

Regulation of host-infection ability in the grass-symbiotic fungus Epichloë festucae by histone H3K9 and H3K36 methyltransferases.

Recent studies have identified key genes that control the symbiotic interaction between Epichloë festucae and Lolium perenne. Here we report on the identification of specific E. festucae genes that control host infection. Deletion of setB, which encodes a homologue of the H3K36 histone methyltransferase Set2/KMT3, reduced histone H3K36 trimethylation and led to severe defects in colony growth and hyphal development. The E. festucae ΔclrD mutant, which lacks the gene encoding the homologue of the H3K9 methyltransferase KMT1, displays similar developmental defects. Both mutants are completely defective in their ability to infect L. perenne. Alleles that complement the culture and plant phenotypes of both mutants also complement the histone methylation defects. Co-inoculation of either ΔsetB or ΔclrD with the wild-type strain enables these mutants to colonize the host. However, successful colonization by the mutants resulted in death or stunting of the host plant. Transcriptome analysis at the early infection stage identified four fungal candidate genes, three of which encode small-secreted proteins, that are differentially regulated in these mutants compared to wild type. Deletion of crbA, which encodes a putative carbohydrate binding protein, resulted in significantly reduced host infection rates by E. festucae.

Fungus-originated genes in the genomes of cereal and pasture grasses acquired through ancient lateral transfer.

Evidence for ancestral gene transfer between Epichloë fungal endophyte ancestors and their host grass species is described. From genomes of cool-season grasses (the Poeae tribe), two Epichloë-originated genes were identified through DNA sequence similarity analysis. The two genes showed 96% and 85% DNA sequence identities between the corresponding Epichloë genes. One of the genes was specific to the Loliinae sub-tribe. The other gene was more widely conserved in the Poeae and Triticeae tribes, including wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.). The genes were independently transferred during the last 39 million years. The transferred genes were expressed in plant tissues, presumably retaining molecular functions. Multiple gene transfer events between the specific plant and fungal lineages are unique. A range of cereal crops is included in the Poeae and Triticeae tribes, and the Loliinae sub-tribe is consisted of economically important pasture and forage crops. Identification and characterisation of the 'natural' adaptation transgenes in the genomes of cereals, and pasture and forage grasses, that worldwide underpin the production of major foods, such as bread, meat, and milk, may change the 'unnatural' perception status of transgenic and gene-edited plants.

A nuclear protein NsiA from Epichloë festucae interacts with a MAP kinase MpkB and regulates the expression of genes required for symbiotic infection and hyphal cell fusion.

The endophytic fungus Epichloë festucae systemically colonizes the intercellular spaces of cool-season grasses to establish a mutualistic symbiosis. Hyphal growth of the endophyte within the host plant is tightly regulated and synchronized with the growth of the host plant. A genetic screen to identify symbiotic genes identified mutant FR405 that had an antagonistic interaction with the host plant. Perennial ryegrass infected with the FR405 mutant were stunted and underwent premature senescence and death. The disrupted gene in FR405 encodes a nuclear-localized protein, designated as NsiA for nuclear protein for symbiotic infection. Like previously isolated symbiotic mutants the nsiA mutant is defective in hyphal cell fusion. NsiA interacts with Ste12, a C2H2 zinc-finger transcription factor, and a MAP kinase MpkB. Both are known as essential components for cell fusion in other fungal species. In E. festucae, MpkB, but not Ste12, is essential for cell fusion. Expression of several genes required for cell fusion and symbiosis, including proA/adv-1, pro41/ham-6, ham7, ham8, and ham9 were downregulated in the nsiA mutant. However, the NsiA ortholog in Neurospora crassa was not essential for hyphal cell fusion. These results demonstrate that the roles of NsiA and Ste12 orthologs in hyphal cell fusion are distinctive between fungal species.

Role of Epichloë Endophytes in Improving Host Grass Resistance Ability and Soil Properties.

The past decade has witnessed significant advances in understanding the interaction between grasses and systemic fungal endophytes of the genus Epichloë, with evidence that plants have evolved multiple strategies to cope with abiotic stresses by reprogramming physiological responses. Soil nutrients directly affect plant growth, while soil microbes are also closely connected to plant growth and health. Epichloë endophytes could affect soil fertility by modifying soil nutrient contents and soil microbial diversity. Therefore, we analyze recent advances in our understanding of the role of Epichloë endophytes under the various abiotic stresses and the role of grass-Epichloë symbiosis on soil fertility. Various cool-season grasses are infected by Epichloë species, which contribute to health, growth, persistence, and seed survival of host grasses by regulating key systems, including photosynthesis, osmotic regulation, and antioxidants and activity of key enzymes of host physiology processes under abiotic stresses. The Epichloë endophyte offers significant prospects to magnify the crop yield, plant resistance, and food safety in ecological systems by modulating soil physiochemical properties and soil microbes. The enhancing resistance of host grasses to abiotic stresses by an Epichloë endophyte is a complex manifestation of different physiological and biochemical events through regulating soil properties and soil microbes by the fungal endophyte. The Epichloë-mediated mechanisms underlying regulation of abiotic stress responses are involved in osmotic adjustment, antioxidant machinery, photosynthetic system, and activity of key enzymes critical in developing plant adaptation strategies to abiotic stress. Therefore, the Epichloë endophytes are an attractive choice in increasing resistance of plants to abiotic stresses and are also a good candidate for improving soil fertility and regulating microbial diversity to improve plant growth.

Mycelial biomass and concentration of loline alkaloids driven by complex population structure in Epichloë uncinata and meadow fescue (Schedonorus pratensis).

Many efforts have been made to select and isolate naturally occurring animal-friendly Epichloë strains for later reinfection into elite cultivars. Often this process involves large-scale screening of Epichloë-infected wild grass populations where strains are characterized and alkaloids measured. Here, we describe for the first time the use of genotyping-by-sequencing (GBS) on a collection of 217 Epichloë-infected grasses (7 S. arundinaceum, 4 L. perenne, and 206 S. pratensis). This genotyping strategy is cheaper than complete genome sequencing, is suitable for a large number of individuals, and, when applied to endophyte-infected grasses, conveniently genotypes both organisms. In total, 6273 single nucleotide polymorphisms (SNPs) in the endophyte data set and 38 323 SNPs in the host data set were obtained. Our findings reveal a composite structure with three distinct endophyte clusters unrelated to the three main S. pratensis gene pools that have most likely spread from different glacial refugia in Eurasia. All three gene pools can establish symbiosis with E. uncinata. A comparison of the endophyte clusters with microsatellite-based fingerprinting of the same samples allows a quick test to discriminate between these clusters using two simple sequence repeats (SSRs). Concentrations of loline alkaloids and mycelial biomass are correlated and differ significantly among the plant and endophyte subpopulations; one endophyte strain has higher levels of lolines than others, and one specific host genotype is particularly suitable to host E. uncinata. These findings pave the way for targeted artificial inoculations of specific host-endophyte combinations to boost loline production in the symbiota and for genome association studies with the aim of isolating genes involved in the compatibility between meadow fescue and E. uncinata.