Many cool-season grasses have symbiotic relationships with Epichloë (Ascomycota, Clavicipitaceae) fungal endophytes that inhabit the intercellular spaces of the above-ground parts of the host plants. The presence of the Epichloë endophytes is generally beneficial to the hosts due to enhanced tolerance to biotic and abiotic stresses conferred by the endophytes. Many Epichloë spp. are asexual, and those infections always remain asymptomatic. However, some Epichloë spp. have a sexual stage and produce a macroscopic fruiting body, a stroma, that envelops the developing inflorescence causing a syndrome termed “choke disease”. Here, we report a fungal and plant gene expression analysis of choke stroma tissue and asymptomatic inflorescence tissue of Epichloë festucae-infected strong creeping red fescue (Festuca rubra subsp. rubra). Hundreds of fungal genes and over 10% of the plant genes were differentially expressed when comparing the two tissue types. The differentially expressed fungal genes in the choke stroma tissue indicated a change in carbohydrate and lipid metabolism, as well as a change in expression of numerous genes for candidate effector proteins. Plant stress-related genes were up-regulated in the stroma tissue, suggesting the plant host was responding to the epiphytic stage of E. festucae as a pathogen.

Strong creeping red fescue (Festuca rubra subsp. rubra) is a commercially important low-maintenance turfgrass and is often naturally infected with the fungal endophyte Epichloëfestucae. Epichloë spp. are endophytes of several cool-season grass species, often conferring insect resistance to the grass hosts due to the production of toxic alkaloids. In addition to insect resistance, a unique feature of the strong creeping red fescue/E. festucae symbiosis is the endophyte-mediated disease resistance to the fungal pathogen Clarireedia jacksonii, the causal agent of dollar spot disease. Such disease resistance is not a general feature of other grass/ Epichloë interactions. E. festucae isolates infecting red fescue have an antifungal protein gene Efe-afpA, whereas most other Epichloë spp. do not have a similar gene. The uniqueness of this gene suggests it may, therefore, be a component of the unique disease resistance seen in endophyte-infected red fescue. Here, we report the generation of CRISPR-Cas9 Efe-afpA gene knockouts with the goal of determining if absence of the protein in endophyte-infected Festuca rubra leads to disease susceptibility. However, it was not possible to infect plants with the knockout isolates, although infection was possible with the wild type E. festucae and with complemented isolates. This raises the interesting possibility that, in addition to having antifungal activity, the protein is required for the symbiotic interaction. The antifungal protein is a small secreted protein with high expression in planta relative to its expression in culture, all characteristics consistent with effector proteins. If Efe-AfpA is an effector protein it must be specific to certain interactions, since most Epichloë spp. do not have such a gene in their genomes.

Fungal plant pathogens can present major problems for most crop species. Currently, control of fungal diseases relies heavily on the use of fungicides. However, there are problems associated with fungicide use, including potential toxicity to non-target organisms and the development of resistance in the target fungus. New strategies are being sought to reduce fungicide use. One area of active research is the potential use of antifungal proteins from various fungal species as alternatives or complements to traditional fungicides. An antifungal protein, Efe-AfpA, from the fungal endophyte Epichloë festucae was previously found to protect plants from the pathogen Clarireedia jacksonii, the causal agent of dollar spot disease. Here we report that Efe-AfpA also has inhibitory activity against other important plant pathogens. These results suggest that it may be possible to develop Efe-AfpA as a biofungicide to target a broad range of destructive plant pathogens.

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.