Discovery and Characterization of Epichloë Fungal Endophytes from Elymus spp. in Northwest China.

Epichloë fungal endophytes hold promise in sustainable agriculture by fortifying cool-season grasses such as Elymus spp. against various stresses. Elymus spp. are widely distributed in Northwest China with a high incidence of endophyte infections. In this study, we identified 20 Epichloë endophytic fungal strains carried by five Elymus spp. from five areas of Northwest China and systematically characterized their morphology, molecular phylogeny, mating type, and alkaloid diversity for the first time. The morphological characterization underscores strain diversity, with variable colony textures and growth rates. A phylogenetic analysis confirms all strains are E. bromicola, emphasizing their taxonomic status. Alkaloid-encoding gene profiling delineates distinct alkaloid synthesis capabilities among the strains, which are crucial for host adaptability and resistance. A mating-type analysis reveals uniformity (mtAC) across the Epichloë strains, simplifying breeding strategies. Notably, the Epichloë strains exhibit diverse alkaloid synthesis gene profiles, impacting host interactions. This research emphasizes the ecological significance of Epichloë endophytes in Elymus spp. ecosystems, offering insights into their genetic diversity and potential applications in sustainable agriculture.

Discovery of Epichloë as novel endophytes of Psathyrostachys lanuginosa in China and their alkaloid profiling.

The Epichloë genus represents a significant group of above-ground endophytes extensively researched for their potential applications in agriculture and ecology. Additionally, Epichloë species synthesize bioactive alkaloids, which generally cause health problems in livestock and have detrimental effects on the performance of insect herbivores. Psathyrostachys lanuginosa serves as a valuable forage grass for livestock owing to its high nutritional value and resilience in adverse environmental conditions. Nevertheless, to date, no reports have documented Epichloë as endophytes of P. lanuginosa. In this study, four strains (PF5, PF9, QG2, and QG4) were isolated and identified through morphological, molecular, and phylogenetic analyses as endophytes of P. lanuginosa. Morphological analysis indicated colony characteristics and conidia features consistent with symbiotic Epichloë, with no significant differences observed in growth rates or conidia dimensions among the four strains. Phylogenetic analysis confirmed all strains as E. bromicola. Additionally, alkaloid biosynthetic genes were detected, revealing differences in the potential synthesis of peramine and indole diterpenoid alkaloids among strains from different geographic origins. However, all four E. bromicola strains exhibited similar potential for synthesizing ergot alkaloids, but not loline alkaloids. Overall, this study identified P. lanuginosa as a novel host for E. bromicola and provided insights into the alkaloid profiles of these strains, laying a solid foundation for the scientific and rational utilization of Epichloë resources.

Alien Chromatin from Hordeeae Grasses Enhances the Compatibility of Epichloë Endophyte Symbiosis with the Hexaploid Wheat Triticum aestivum.

The inoculation of Epichloë endophytes into modern cereals, resulting in systemic infection, depends on the genetics of both the host and the endophyte strain deployed. Until very recently, the only modern cereal to have been infected with Epichloë, in which normal phenotype seed-transmitted associations were achieved, is rye (Secale cereale). Whilst minor in-roads have been achieved in infecting hexaploid wheat (Triticum aestivum), the phenotypes of these associations have all been extremely poor, including host death and stunting. To identify host genetic factors that may impact the compatibility of Epichloë infection in wheat, wheat-alien chromosome addition/substitution lines were inoculated with Epichloë, and the phenotypes of infected plants were assessed. Symbioses were identified whereby infected wheat plants were phenotypically like uninfected controls. These plants completed their full lifecycle, including the vertical transmission of Epichloë into the next generation of grain, and represent the first ever compatible wheat-Epichloë associations to be created.

Grass-Endophyte Interactions and Their Associated Alkaloids as a Potential Management Strategy for Plant Parasitic Nematodes.

Claviceptaceous endophytic fungi in the genus Epichloë mostly form a symbiotic relationship with cool-season grasses. Epichloë spp. are capable of producing bioactive alkaloids such as peramines, lolines, ergot alkaloids, and indole-diterpenes, which protect the host plant from herbivory by animals, insects, and nematodes. The host also benefits from enhanced tolerance to abiotic stresses, such as salt, drought, waterlogging, cold, heavy metals, and low nitrogen stress. The bioactive alkaloids produced can have both direct and indirect effects towards plant parasitic nematodes. Direct interaction with nematodes' motile stages can cause paralysis (nematostatic effect) or death (nematicidal effect). Indirectly, the metabolites may induce host immunity which inhibits feeding and subsequent nematode development. This review highlights the different mechanisms through which this interaction and the metabolites produced have been explored in the suppression of plant parasitic nematodes and also how the specific interactions between different grass genotypes and endophyte strains result in variable suppression of different nematode species. An understanding of the different grass-endophyte interactions and their successes and failures in suppressing various nematode species is essential to enable the proper selection of grass-endophyte combinations to identify the alkaloids produced, concentrations required, and determine which nematodes are sensitive to which specific alkaloids.

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.

Microbial symbionts buffer hosts from the demographic costs of environmental stochasticity.

Species' persistence in increasingly variable climates will depend on resilience against the fitness costs of environmental stochasticity. Most organisms host microbiota that shield against stressors. Here, we test the hypothesis that, by limiting exposure to temporally variable stressors, microbial symbionts reduce hosts' demographic variance. We parameterized stochastic population models using data from a 14-year symbiont-removal experiment including seven grass species that host Epichloë fungal endophytes. Results provide novel evidence that symbiotic benefits arise not only through improved mean fitness, but also through dampened inter-annual variance. Hosts with "fast" life-history traits benefited most from symbiont-mediated demographic buffering. Under current climate conditions, contributions of demographic buffering were modest compared to benefits to mean fitness. However, simulations of increased stochasticity amplified benefits of demographic buffering and made it the more important pathway of host-symbiont mutualism. Microbial-mediated variance buffering is likely an important, yet cryptic, mechanism of resilience in an increasingly variable world.

The Impact of Aboveground Epichloë Endophytic Fungi on the Rhizosphere Microbial Functions of the Host Melica transsilvanica.

In nature, the symbiotic relationship between plants and microorganisms is crucial for ecosystem balance and plant growth. This study investigates the impact of Epichloë endophytic fungi, which are exclusively present aboveground, on the rhizosphere microbial functions of the host Melica transsilvanica. Using metagenomic methods, we analyzed the differences in microbial functional groups and functional genes in the rhizosphere soil between symbiotic (EI) and non-symbiotic (EF) plants. The results reveal that the presence of Epichloë altered the community structure of carbon and nitrogen cycling-related microbial populations in the host's rhizosphere, significantly increasing the abundance of the genes (porA, porG, IDH1) involved in the rTCA cycle of the carbon fixation pathway, as well as the abundance of nxrAB genes related to nitrification in the nitrogen-cycling pathway. Furthermore, the presence of Epichloë reduces the enrichment of virulence factors in the host rhizosphere microbiome, while significantly increasing the accumulation of resistance genes against heavy metals such as Zn, Sb, and Pb. This study provides new insights into the interactions among endophytic fungi, host plants, and rhizosphere microorganisms, and offers potential applications for utilizing endophytic fungi resources to improve plant growth and soil health.

The growth promotion in endophyte symbiotic plants does not penalise the resistance to herbivores and bacterial microbiota.

A trade-off between growth and defence against biotic stresses is common in plants. Fungal endophytes of the genus Epichloë may relieve this trade-off in their host grasses since they can simultaneously induce plant growth and produce antiherbivore alkaloids that circumvent the need for host defence. The Epichloë ability to decouple the growth-defence trade-off was evaluated by subjecting ryegrass with and without Epichloë endophytes to an exogenous treatment with gibberellin (GA) followed by a challenge with Rhopalosiphum padi aphids. In agreement with the endophyte-mediated trade-off decoupling hypothesis, the GA-derived promotion of plant growth increased the susceptibility to aphids in endophyte-free plants but did not affect the insect resistance in endophyte-symbiotic plants. In line with the unaltered insect resistance, the GA treatment did not reduce the concentration of Epichloë-derived alkaloids. The Epichloë mycelial biomass was transiently increased by the GA treatment but at the expense of hyphal integrity. The response of the phyllosphere bacterial microbiota to both GA treatment and Epichloë was also evaluated. Only Epichloë, and not the GA treatment, altered the composition of the phyllosphere microbiota and the abundance of certain bacterial taxa. Our findings clearly demonstrate that Epichloë does indeed relieve the plant growth-defence trade-off.

Interactions between Epichloë endophyte and the plant microbiome impact nitrogen responses in host Achnatherum inebrians plants.

The clavicipitaceous fungus Epichloë gansuensis forms symbiotic associations with drunken horse grass (Achnatherum inebrians), providing biotic and abiotic stress protection to its host. However, it is unclear how E. gansuensis affects the assembly of host plant-associated bacterial communities after ammonium nitrogen (NH4+-N) treatment. We examined the shoot- and root-associated bacterial microbiota and root metabolites of A. inebrians when infected (I) or uninfected (F) with E. gansuensis endophyte. The results showed more pronounced NH4+-N-induced microbial and metabolic changes in the endophyte-infected plants compared to the endophyte-free plants. E. gansuensis significantly altered bacterial community composition and ß-diversity in shoots and roots and increased bacterial α-diversity under NH4+-N treatment. The relative abundance of 117 and 157 root metabolites significantly changed with E. gansuensis infection under water and NH4+-N treatment compared to endophyte-free plants. Root bacterial community composition was significantly related to the abundance of the top 30 metabolites [variable importance in the projection (VIP) > 2 and VIP > 3] contributing to differences between I and F plants, especially alkaloids. The correlation network between root microbiome and metabolites was complex. Microorganisms in the Proteobacteria and Firmicutes phyla were significantly associated with the R00693 metabolic reaction of cysteine and methionine metabolism. Co-metabolism network analysis revealed common metabolites between host plants and microorganisms.IMPORTANCEOur results suggest that the effect of endophyte infection is sensitive to nitrogen availability. Endophyte symbiosis altered the composition of shoot and root bacterial communities, increasing bacterial diversity. There was also a change in the class and relative abundance of metabolites. We found a complex co-occurrence network between root microorganisms and metabolites, with some metabolites shared between the host plant and its microbiome. The precise ecological function of the metabolites produced in response to endophyte infection remains unknown. However, some of these compounds may facilitate plant-microbe symbiosis by increasing the uptake of beneficial soil bacteria into plant tissues. Overall, these findings advance our understanding of the interactions between the microbiome, metabolome, and endophyte symbiosis in grasses. The results provide critical insight into the mechanisms by which the plant microbiome responds to nutrient stress in the presence of fungal endophytes.

Ruminal ergovaline and volatile fatty acid dynamics: Association with poor performance and a key growth regulator in steers grazing toxic tall fescue.

Fescue toxicosis (FT) is produced by an ergot alkaloid (i.e., ergovaline [EV])-producing fungus residing in toxic fescue plants. Associations between EV, decreased weight gain and ruminal volatile fatty acids are unclear. Feces, rumen fluid, and blood were collected from 12 steers that grazed non-toxic (NT) or toxic (E +) fescue for 28 days. The E + group exhibited decreased propionate (P), increased acetate (A), and increased ruminal A:P ratio, with similar trends in feces. Plasma GASP-1 (G-Protein-Coupled-Receptor-Associated-Sorting-Protein), a myostatin inhibitor, decreased (day 14) only in E + steers. Ergovaline was present only in E + ruminal fluid and peaked on day 14. The lower ruminal propionate and higher A:P ratio might contribute to FT while reduced GASP-1 might be a new mechanism linked to E + -related weight gain reduction. Day 14 ergovaline zenith likely reflects ruminal adaptations favoring EV breakdown and its presence only in rumen points to local, rather than systemic effects.

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.

Epichloë Endophyte Enhanced Insect Resistance of Host Grass Leymus Chinensis by Affecting Volatile Organic Compound Emissions.

In plant-herbivore interactions, plant volatile organic compounds (VOCs) play an important role in anti-herbivore defense. Grasses and Epichloë endophytes often form defensive mutualistic symbioses. Most Epichloë species produce alkaloids to protect hosts from herbivores, but there is no strong evidence that endophytes can affect the insect resistance of their hosts by altering VOC emissions. In this study, a native dominant grass, sheepgrass (Leymus chinensis), and its herbivore, oriental migratory locust (Locusta migratoria), were used as experimental materials. We studied the effect of endophyte-associated VOC emissions on the insect resistance of L. chinensis. The results showed that endophyte infection enhanced insect resistance of the host, and locusts preferred the odor of endophyte-free (EF) leaves to that of endophyte-infected (EI) leaves. We determined the VOC profile of L. chinensis using gas chromatography-mass spectrometry (GC-MS), and found that endophyte infection decreased the pentadecane (an alkane) emission from uneaten plants, and increased the nonanal (an aldehyde) emission from eaten plants. The olfactory response experiment showed that locusts were attracted by high concentration of pentadecane, while repelled by high concentration of nonanal, indicating that Epichloë endophytes may increase locust resistance of L. chinensis by decreasing pentadecane while increasing nonanal emission. Our results suggest that endophytes can induce VOC-mediated defense in hosts in addition to producing alkaloids, contributing to a better understanding the endophyte-plant-herbivore interactions.

Effect of Epichloë Endophyte on the Growth and Carbon Allocation of Its Host Plant Stipa purpurea under Hemiparasitic Root Stress.

Epichloë endophytes not only affect the growth and resistance of their host plants but also confer nutrient benefits to parasitized hosts. In this study, we used Pedicularis kansuensis to parasitize Stipa purpurea, both with and without endophytic fungi, and to establish a parasitic system. In this study, endophytic fungal infection was found to increase the dry weight of the leaf, stem, and leaf sheath, as well as the plant height, root length, tiller number, aboveground biomass, and underground biomass of S. purpurea under root hemiparasitic stress. Meanwhile, the 13C allocation of the leaf sheaths and roots of S. purpurea increased as the density of P. kansuensis increased, while the 13C allocation of the leaf sheaths and roots of E+ S. purpurea was lower than that of E- S. purpurea. The 13C allocation of the stem, leaf sheath, and root of E+ S. purpurea was higher than that of its E- counterpart. Furthermore, the content of photosynthetic 13C and the 13C partition rate of the stems, leaves, roots, and entire plant of S. purpurea and P. kansuensis transferred from S. purpurea increased as the density of P. kansuensis increased. These results will generate new insights into the potential role of symbiotic microorganisms in regulating the interaction between root hemiparasites and their hosts.

Corrigendum: Methylome changes in Lolium perenne associated with long-term colonisation by the endophytic fungus Epichloë sp. LpTG-3 strain AR37.

[This corrects the article DOI: 10.3389/fpls.2023.1258100.].

Methylome changes in Lolium perenne associated with long-term colonisation by the endophytic fungus Epichloë sp. LpTG-3 strain AR37.

Epichloë spp. often form mutualistic interactions with cool-season grasses, such as Lolium perenne. However, the molecular mechanisms underlying this interaction remain poorly understood. In this study, we employed reduced representation bisulfite sequencing method (epiGBS) to investigate the impact of the Epichloë sp. LpTG-3 strain AR37 on the methylome of L. perenne across multiple grass generations and under drought stress conditions. Our results showed that the presence of the endophyte leads to a decrease in DNA methylation across genomic features, with differentially methylated regions primarily located in intergenic regions and CHH contexts. The presence of the endophyte was consistently associated with hypomethylation in plants across generations. This research sheds new light on the molecular mechanisms governing the mutualistic interaction between Epichloë sp. LpTG-3 strain AR37 and L. perenne. It underscores the role of methylation changes associated with endophyte infection and suggests that the observed global DNA hypomethylation in L. perenne may be influenced by factors such as the duration of the endophyte-plant association and the accumulation of genetic and epigenetic changes over time.

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.

Behavioral and Physiological Alterations in Angus Steers Grazing Endophyte-Infected Toxic Fescue during Late Fall.

Fescue toxicosis is caused by grazing ergot alkaloid-producing endophyte (Epichloë coenophiala)-infected tall fescue (E+). Summer grazing of E+ leads to decreased productivity, associated impaired thermoregulation, and altered behavior. The goal of this study was to determine the role of E+ grazing-climate interaction on animal behavior and thermoregulation during late fall. Eighteen Angus steers were placed on nontoxic (NT), toxic (E+) and endophyte-free (E-) fescue pastures for 28 days. Physiological parameters, such as rectal temperature (RT), respiration rate (RR), ear and ankle surface temperature (ET, AT), and body weights, were measured. Skin surface temperature (SST) and animal activity were recorded continuously with temperature and behavioral activity sensors, respectively. Environmental conditions were collected using paddocks-placed data loggers. Across the trial, steers on E+ gained about 60% less weight than the other two groups. E+ steers also had higher RT than E- and NT, and lower SST than NT post-pasture placement. Importantly, animals grazing E+ spent more time lying, less time standing, and took more steps. These data suggest that late fall E+ grazing impairs core and surface temperature regulation and increases non-productive lying time, which may be partly responsible for the observed decreased weight gains.

Nitrogen addition and fungal symbiosis alter early dune plant succession.

Anthropogenic nitrogen (N) enrichment can have complex effects on plant communities. In low-nutrient, primary successional systems such as sand dunes, N enrichment may alter the trajectory of plant community assembly or the dominance of foundational, ecosystem-engineering plants. Predicting the consequences of N enrichment may be complicated by plant interactions with microbial symbionts because increases in a limiting resource, such as N, could alter the costs and benefits of symbiosis. To evaluate the direct and interactive effects of microbial symbiosis and N addition on plant succession, we established a long-term field experiment in Michigan, USA, manipulating the presence of the symbiotic fungal endophyte Epichloë amarillans in Ammophila breviligulata, a dominant ecosystem-engineering dune grass species. From 2016 to 2020, we implemented N fertilization treatments (control, low, high) in a subset of the long-term experiment. N addition suppressed the accumulation of plant diversity over time mainly by reducing species richness of colonizing plants. However, this suppression occurred only when the endophyte was present in Ammophila. Although Epichloë enhanced Ammophila tiller density over time, N addition did not strongly interact with Epichloë symbiosis to influence vegetative growth of Ammophila. Instead, N addition directly altered plant community composition by increasing the abundance of efficient colonizers, especially C4 grasses. In conclusion, hidden microbial symbionts can alter the consequences of N enrichment on plant primary succession.

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.

Effect of Epichloë gansuensis Endophyte on Seed-Borne Microbes and Seed Metabolites in Achnatherum inebrians.

The seed-borne microbiota and seed metabolites of the grass Achnatherum inebrians, either host to Epichloë gansuensis (endophyte infected [EI]) or endophyte free (EF), were investigated. This study determined the microbial communities both within the seed (endophytic) and on the seed surface (epiphytic) and of the protective glumes by using Illumina sequencing technology. Epichloë gansuensis decreased the richness of the seed-borne microbiota except for the epiphytic fungi of glumes and also decreased the diversity of seed-borne microbiota. In addition, metabolites of seeds and glumes were detected using liquid chromatography-mass spectrometry (LC-MS). Unlike with the seeds of EF plants, the presence of E. gansuensis resulted in significant changes in the content of 108 seed and 31 glume metabolites. A total of 319 significant correlations occurred between seed-borne microbiota and seed metabolites; these correlations comprised 163 (147 bacterial and 16 fungal) positive correlations and 156 (136 bacterial and 20 fungal) negative correlations. Meanwhile, there were 42 significant correlations between glume microbiota and metabolites; these correlations comprised 28 positive (10 bacterial and 18 fungal) and 14 negative (9 bacterial and 5 fungal) correlations. The presence of E. gansuensis endophyte altered the communities and diversities of seed-borne microbes and altered the composition and content of seed metabolites, and there were many close and complex relationships between microbes and metabolites. IMPORTANCE The present study was to investigate seed-borne microbiota and seed metabolites in Achnatherum inebrians using high-throughput sequencing and LC-MS technology. Epichloë gansuensis decreased the richness of the seed-borne microbiota except for the epiphytic fungi of glumes and also decreased the diversity of seed-borne microbiota. Compared with endophyte-free plants, the content of 108 seed and 31 glume metabolites of endophyte-infected plants was significantly changed. There were 319 significant correlations between seed-borne microbiota and seed metabolites and 42 significant correlations between glume microbiota and metabolites.