A High Throughput Screen for Epichloë Endophyte Detection in Plants.

Epichloë endophytes are agriculturally important fungal symbionts that associate with cool season grasses. They consist of both sexual (Epichloë) and asexual (Neotyphodium) species. Neotyphodium coenophialum and N. lolii are beneficial to the host, helping to alleviate biotic and abiotic stresses.  However the sexual Epichloë spp. can choke the inflorescence resulting in loss of seed production. We have developed a high throughput screen to detect epichloë endophytes in tillers and seeds to aid our grass breeding program. A PCR-based screen was designed to detect epichloë endophytes in cool season grasses such as tall fescue (Festuca arundinacea), Canada wildrye (Elymus canadensis), perennial ryegrass (Lolium perenne), western wheatgrass (Pascopyrum smithii), orchard grass (Dactylis glomerata), barley (Hordeum sp.), and bentgrass (Agrostis sp.). DNA was extracted from plant material using a high throughput system and the presence of the endophyte was detected by PCR using primers specific to all epichloë endophytes.  The PCR results compared well with other endophyte detection methods such as microscopy and immunoblotting.  The PCR-based screen was more suitable for endophyte-infected grasses from tribes such as Aveneae and Triticeae than microscopy and immunoblotting. Microsatellite fingerprinting of DNA from seeds and tillers was used to distinguish novel endophyte associations from the common toxic endophyte based on marker differences between the strains.  This assay provides a rapid and robust analysis for epichloë endophyte-infected grasses.