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Asexual fungal endophytes of the genus Epichloë form mutually beneficial associations with cool-season pasture grasses such as ryegrasses (Lolium spp.). Alkaloid production by the fungus confers both beneficial (deterrence of invertebrate herbivory) and detrimental (toxicity to mammalian livestock) attributes. A few novel strains with desirable metabolite profiles have been advanced into commercial production by inoculation of perennial ryegrass cultivars. In the present study, an integrated process for discovery of novel endophytes based on exploitation of genotypic information has been designed and implemented. A survey of genetic diversity was performed on a large-scale, customised germplasm collection (containing 244 accessions) in order to identify previously uncharacterised endophyte genotypes. Preliminary qualitative metabolic profiling in the endogenous genetic background permitted elimination of undesirable combinations, and definition of a subset of priority candidates. A novel method was developed for inoculation of endophytes into meristem-culture-derived callus tissue of single genotypes from multiple perennial ryegrass cultivars, in order to allow isogenic comparisons with respect to both host and endophyte genotype. Beneficial toxin profiles were confirmed for associations formed with the grass genotypic panel, and semi-quantitative metabolite analysis provided evidence for genotype-specific effects of both host and genotype on levels of alkaloid production. Vegetative stability was also assessed over both shorter and longer terms. A final set of three prioritised candidates was obtained, two of which (belonging to the known taxa E. festucae var. lolii and LpTG-2) produce the alkaloids ergovaline and peramine. The third candidate endophyte, belonging to a putative novel taxon, solely produces representatives of an additional alkaloid class, the epoxy-janthitrems.

A number of pasture and turf grass species form mutually beneficial symbiotic associations with endophytic fungal species. Within the fescue grasses, diploid meadow fescue (Festuca pratensisHuds.) interacts with Neotyphodium uncinatumwhile allohexaploid tall fescue (Festuca arundinaceaSchreb.) has been reported to associate with Neotyphodium coenophialumand two other morphologically distinct taxa (Festuca arundinaceataxonomic groups 2 and 3 [FaTG‐2 and FaTG‐3]). The evolutionary history of hexaploid tall fescue is complex, as part of a species group with varying ploidy levels and exhibiting distinct ecogeographical morphotypes. To evaluate both naturally occurring variation and host grass taxon specificity, diversity was determined in collections representing multiple meadow fescue and tall fescue accessions. Initial screening with a minimal set of endophyte‐specific simple sequence repeat (SSR) genetic markers detected endophyte incidence in 33% of 701 tested accessions. Subsequent analysis identified N. coenophialumgenotypes within Continental and rhizomatous hexaploid and octoploid tall fescue [F. arundinaceasubsp. atlantigena(St.‐Yves) Auquier] accessions. Festuca arundinaceataxonomic group 2 and FaTG‐3 endophytes appeared to be restricted to Mediterranean hexaploid and decaploid tall fescue [F. arundinacea cirtensis(St.‐Yves) Gamisans] hosts. Endophytes of meadow fescue were confirmed as belonging to N. uncinatum. This study has elucidated host specificity of fescue endophyte taxa and supported models for host–symbiont coevolution. A substantial number of candidate novel endophytes have been identified that are suitable for metabolic characterization and deployment by inoculation in fescue breeding programs.