A Metabolomic Study of Epichloë Endophytes for Screening Antifungal Metabolites

Epichloë endophytes, fungal endosymbionts of Pooidae grasses, are commonly utilized in forage and turf industries because they produce beneficial metabolites that enhance resistance against environmental stressors such as insect feeding and disease caused by phytopathogen infection. In pastoral agriculture, phytopathogenic diseases impact both pasture quality and animal production. Recently, bioactive endophyte strains have been reported to secrete compounds that significantly inhibit the growth of phytopathogenic fungi in vitro. A screen of previously described Epichloë-produced antifeedant and toxic alkaloids determined that the antifungal bioactivity observed is not due to the production of these known metabolites, and so there is a need for methods to identify new bioactive metabolites. The process described here is applicable more generally for the identification of antifungals in new endophytes. This study aims to characterize the fungicidal potential of novel, ‘animal friendly’ Epichloë endophyte strains NEA12 and NEA23 that exhibit strong antifungal activity using an in vitro assay. Bioassay-guided fractionation, followed by metabolite analysis, identified 61 metabolites that, either singly or in combination, are responsible for the observed bioactivity. Analysis of the perennial ryegrass-endophyte symbiota confirmed that NEA12 and NEA23 produce the prospective antifungal metabolites in symbiotic association and thus are candidates for compounds that promote disease resistance in planta. The “known unknown” suite of antifungal metabolites identified in this study are potential biomarkers for the selection of strains that enhance pasture and turf production through better disease control.

Metabolic Potential of Epichloë Endophytes for Host Grass Fungal Disease Resistance

Asexual species of the genus Epichloë (Clavicipitaceae, Ascomycota) form endosymbiotic associations with Pooidae grasses. This association is important both ecologically and to the pasture and turf industries, as the endophytic fungi confer a multitude of benefits to their host plant that improve competitive ability and performance such as growth promotion, abiotic stress tolerance, pest deterrence and increased host disease resistance. Biotic stress tolerance conferred by the production of bioprotective metabolites has a critical role in an industry context. While the known antimammalian and insecticidal toxins are well characterized due to their impact on livestock welfare, antimicrobial metabolites are less studied. Both pasture and turf grasses are challenged by many phytopathogenic diseases that result in significant economic losses and impact livestock health. Further investigations of Epichloë endophytes as natural biocontrol agents can be conducted on strains that are safe for animals. With the additional benefits of possessing host disease resistance, these strains would increase their commercial importance. Field reports have indicated that pasture grasses associated with Epichloë endophytes are superior in resisting fungal pathogens. However, only a few antifungal compounds have been identified and chemically characterized, and these from sexual (pathogenic) Epichloë species, rather than those utilized to enhance performance in turf and pasture industries. This review provides insight into the various strategies reported in identifying antifungal activity from Epichloë endophytes and, where described, the associated antifungal metabolites responsible for the activity.

Endophytic fungi facilitate the coexistence of host grasses and neighboring legumes by changing rhizobial abundance

Purpose: Grass-endophyte and legume-rhizobium symbionts coexist in grasslands. However, the effects of endophyte infection on legume-rhizobium symbionts remain poorly understood, especially in natural grasslands. Methods: In this study, Achnatherum sibiricum - Epichloë endophytes and Medicago ruthenica -rhizobia were selected as materials to investigate whether and how endophyte infection affected the growth of legume-rhizobia symbionts. It was hypothesized that endophytes can facilitate the coexistence of grass-legume systems. Results: The results demonstrated that endophyte infection affected the growth of both rhizobia and M. ruthenica -rhizobia symbionts, and the results depended on rhizobial identity. Endophyte infection inhibited the growth of Mesorhizobium ciceri , which significantly promoted the growth of M. ruthenica , and promoted Sinorhizobium meliloti , which had no significant effect on the growth of M. ruthenica . Endophyte infection also changed the interaction between A. sibiricum and M. ruthenica . When inoculated with M. ciceri , endophyte infection weakened the promoting effect of A. sibiricum on M. ruthenica , while when inoculated with S. meliloti , endophyte infection enhanced the promoting effect. Endophyte infection affected the growth of M. ruthenica -rhizobia symbionts by affecting rhizobia abundance in roots and nitrogen content in plant leaves. Conclusion: In conclusion, endophyte infection was beneficial to biomass accumulation and species coexistence in grass-legume mixed planting systems. In this study, it was proposed that endophyte infection may change the growth of legume-rhizobia symbionts by affecting the growth and nitrogen fixation of rhizobia.

The Known Antimammalian and Insecticidal Alkaloids Are Not Responsible for the Antifungal Activity of Epichloë Endophytes

Asexual Epichloë sp. endophytes in association with pasture grasses produce agronomically important alkaloids (e.g., lolitrem B, epoxy-janthitrems, ergovaline, peramine, and lolines) that exhibit toxicity to grazing mammals and/or insect pests. Novel strains are primarily characterised for the presence of these compounds to ensure they are beneficial in an agronomical setting. Previous work identified endophyte strains that exhibit enhanced antifungal activity, which have the potential to improve pasture and turf quality as well as animal welfare through phytopathogen disease control. The contribution of endophyte-derived alkaloids to improving pasture and turf grass disease resistance has not been closely examined. To assess antifungal bioactivity, nine Epichloë related compounds, namely peramine hemisulfate, n-formylloline-d3, n-acetylloline hydrochloride, lolitrem B, janthitrem A, paxilline, terpendole E, terpendole C, and ergovaline, and four Claviceps purpurea ergot alkaloids, namely ergotamine, ergocornine, ergocryptine, and ergotaminine, were tested at concentrations higher than observed in planta in glasshouse and field settings using in vitro agar well diffusion assays against three common pasture and turf phytopathogens, namely Ceratobasidium sp., Drechslera sp., and Fusarium sp. Visual characterisation of bioactivity using pathogen growth area, mycelial density, and direction of growth indicated no inhibition of pathogen growth. This was confirmed by statistical analysis. The compounds responsible for antifungal bioactivity of Epichloë endophytes hence remain unknown and require further investigation.

Antagonism to Plant Pathogens by Epichloë Fungal Endophytes—A Review

Epichloë is a genus of filamentous fungal endophytes that has co-evolved with cool-season grasses with which they form long-term, symbiotic associations. The most agriculturally important associations for pasture persistence for grazing livestock are those between asexual vertically transmitted Epichloë strains and the pasture species, perennial ryegrass, and tall fescue. The fungus confers additional traits to their host grasses including invertebrate pest deterrence and drought tolerance. Selected strains of these mutualistic endophytes have been developed into highly efficacious biocontrol products and are widely utilized within the Americas, Australia, and New Zealand for pasture persistence. Less publicized is the antagonism Epichloë endophytes display towards multiple species of saprophytic and pathogenic microbes. This opinion piece will review the current literature on antimicrobial properties exhibited by this genus of endophyte and discuss the reasons why this trait has historically remained a research curiosity rather than a trait of commercial significance.

Interaction Between Grasses and Epichloe Endophytes and Its Significance to Biotic and Abiotic Stress Tolerance and the Rhi-Zosphere

Cool-season grasses are the most common forage types in livestock operations and amenities. Several of the cool-season grasses establish mutualistic associations with an endophytic fungus of the Epichloe genus. The grasses and endophytic fungi have evolved over a long period of time to form host-fungus specific relationships that confer protection for the grass against various stressors in exchange for housing and nutrients to the fungus. This review provides an overview of the mechanisms by which Epichloe endophytes and grasses interact, including molecular pathways for secondary metabolite production. It also outlines specific mechanisms by which the endophyte helps protect the plant from various abiotic and biotic stressors. Finally, the review provides information on how Epichloe infection of grass and stress affect the rhizosphere environment of the plant.

Influence of Interactions between Nitrogen, Phosphorus Supply and Epichloёbromicola on Growth of Wild Barley (Hordeum brevisubulatum)

Epichloë endophytes are biotrophic fungi that establish mutualistic symbiotic relationship with grasses and affect performance of the host under different environments. Wild barley (Hordeum brevisubulatum) is an important forage grass and often infected by Epichloë bromicola, thus showing tolerances to stresses. Since the plant growth correlates with both microbial infection and nutrient stoichiometry, this study was performed to investigate whether the function of Epichloë bromicola endophyte to improve host growth depend upon the nitrogen (N), phosphorus (P) fertilization. Epichloë-infected (E+) and Epichloë-free (E−) wild barley plants were subjected to nine types of mixed N (0.2 mM, 3 mM, 15 mM) and P (0.01 mM, 0.1 mM, 1.5 mM) levels treatments for 90 d to collect plant samples and determine multiple related indexes. We found that E. bromicola and N, P additions positively affected seed germination. Further, E. bromicola significantly enhanced chlorophyll content and root metabolic activity under N-deficiency, and meanwhile, might alter allocation of photosynthate under different conditions. The contents of N, P and stoichiometry of C:N:P of E+ plants were significantly higher than that of E− under nutrient deficiency, but contrary results were observed under adequate nutrients. Therefore, we propose that the growth-promoting ability of E. bromicola is closely correlated with N and P additional levels. Under low N, P additions, positive roles of endophyte are significant as opposed to negative roles under high N, P additions.

Epichloë endophytes – new perspectives on a key ingredient for resilient perennial grass pastures

The confirmation that Epichloë endophytes are important for pest resistance in New Zealand pastures led to the development of a range of novel endophyte strain × host plant combinations that enhance the persistence of the grass, while mitigating adverse effects on grazing animals. Successfully delivering these endophytes to the pastoral industry has required the development of a range of scientific and commercial capabilities. In March 2012, the New Zealand proprietary seed industry established the Endophyte Technical Committee. This is a cooperative forum to ensure that endophyte strains in commerce or under development are tested uniformly, and to publish industry-agreed descriptions of the animal safety and insect control of commercial endophyte strains each year.

The Response of the Associations of Grass and Epichloë Endophytes to the Increased Content of Heavy Metals in the Soil

The rapid development of civilization increases the area of land exposed to the accumulation of toxic compounds, including heavy metals, both in water and soil. Endophytic fungi associated with many species of grasses are related to the resistance of plants to biotic and abiotic stresses, which include heavy metals. This paper reviews different aspects of symbiotic interactions between grass species and fungal endophytes from the genera Epichloë with special attention paid to the elevated concentration of heavy metals in growing substrates. The evidence shows the high resistance variation of plant endophyte symbiosis on the heavy metals in soil outcome. The fungal endophytes confer high heavy metal tolerance, which is the key feature in its practical application with their host plants, i.e., grasses in phytoremediation.