Epichloë spp. are naturally occurring fungal endophytic symbionts of many cool-season grasses. Infection by the fungal endophytes often confers biotic and abiotic stress tolerance to their hosts. Endophyte-mediated disease resistance is well-established in the fine fescue grass Festuca rubra subsp. rubra (strong creeping red fescue) infected with E. festucae. Resistance to fungal pathogens is not an established effect of endophyte infection of other grass species, and may therefore be unique to the fine fescues. The underlying mechanism of the disease resistance is unknown. E. festucae produces a secreted antifungal protein that is highly expressed in the infected plant tissues and may therefore be involved in the disease resistance. Most Epichloë spp. do not have a gene for a similar antifungal protein. Here we report the characterization of the E. festucae antifungal protein, designated Efe-AfpA. The antifungal protein partially purified from the apoplastic proteins of endophyte-infected plant tissue and the recombinant protein expressed in the yeast Pichia pastoris was found to have activity against the important plant pathogen Sclerotinia homoeocarpa. Efe-AfpA may therefore be a component of the disease resistance seen in endophyte-infected strong creeping red fescue.

Epichloë spp. are symbiotic fungal endophytes of many cool season grasses. The presence of the fungal endophytes often confers insect, drought, and disease tolerance to the host grasses. The presence of the fungal endophytes within the host plants does not elicit host defense responses. The molecular basis for this phenomenon is not known. Epichloë festucae, the endophyte of Festuca rubra, expresses a salicylate hydroxylase similar to NahG from the bacterium Pseudomonas putida. Few fungal salicylate hydroxylase enzymes have been reported. The in planta expression of an endophyte salicylate hydroxylase raised the possibility that degradation of plant-produced salicylic acid is a factor in the mechanism of how the endophyte avoids eliciting host plant defenses. Here we report the characterization of the E. festucae salicylate hydroxylase, designated Efe-shyA. Although the fungal enzyme has the expected activity, based on salicylic acid levels in endophyte-free and endophyte-infected plants it is unlikely that expression of the endophyte salicylate hydroxylase is a factor in the lack of a host defense response to the presence of the fungal endophyte.

Strong creeping red fescue (Festuca rubra ssp. rubra) is an important cool season turfgrass species. Cultivars are often infected with the fungal endophyte Epichloë festucae. Endophyte infection is known to confer insect and disease resistance to the plants. The effect of endophyte infection on drought or heat stress tolerance of strong creeping red fescue is not yet established. The objectives of this controlled-environment study were to determine if endophyte infection had any effect on physiological parameters associated with plant tolerance to drought or heat stress or the combination of the two stresses. In this study, endophyte status had no effect on turf quality (TQ), relative water content (RWC), photochemical efficiency, chlorophyll content, electrolyte leakage (EL), or malondialdehyde (MDA) content of the plants under any of the stress treatments. Our results suggested that E. festucae infection had no physiological effects on improving drought, heat or the combined stress tolerance in strong creeping red fescue.

Dollar spot disease, caused by the fungal pathogen Clarireedia jacksonii, is a major problem in many turfgrass species, particularly creeping bentgrass (Agrostis stolonifera). It is well-established that strong creeping red fescue (Festuca rubra subsp. rubra) exhibits good dollar spot resistance when infected by the fungal endophyte Epichloë festucae. This endophyte-mediated disease resistance is unique to the fine fescues and has not been observed in other grass species infected with other Epichloë spp. The mechanism underlying the unique endophyte-mediated disease resistance in strong creeping red fescue has not yet been established. We pursued the possibility that it may be due to the presence of an abundant secreted antifungal protein produced by E. festucae. Here, we compare the activity of the antifungal protein expressed in Escherichia coli, Pichia pastoris, and Penicillium chrysogenum. Active protein was recovered from all systems, with the best activity being from Pe. chrysogenum. In greenhouse assays, topical application of the purified antifungal protein to creeping bentgrass and endophyte-free strong creeping red fescue protected the plants from developing severe symptoms caused by C. jacksonii. These results support the hypothesis that Efe-AfpA is a major contributor to the dollar spot resistance observed with E. festucae-infected strong creeping red fescue in the field, and that this protein could be developed as an alternative or complement to fungicides for the management of this disease on turfgrasses.

Festuca rubra L. subsp. rubra (strong creeping red fescue) exhibits endophyte‐mediated fungal disease resistance. Epichloë festucae Leuchtm., Schardl & M.R. Siegel, the fungal endophyte of F. rubra subsp. rubra, produces an abundant transcript encoding a potential secreted antifungal protein. The objective of this work was to confirm the presence of the antifungal protein in the apoplastic proteins and to express it in the yeast Pichia pastoris for future tests for activity against turfgrass fungal pathogens.