Molecular identification and characterization of endophytes from uncultivated barley

Epichloë species (Clavicipitaceae, Ascomycota) are endophytic symbionts of many cool-season grasses. Many interactions between Epichloë and their host grasses contribute to plant growth promotion, protection from many pathogens and insect pests, and tolerance to drought stress. Resistance to insect herbivores by endophytes associated with Hordeum species has been previously shown to vary depending on the endophyte-grass-insect combination. We explored the genetic and chemotypic diversity of endophytes present in wild Hordeum species. We analyzed seeds of Hordeum bogdanii, H. brevisubulatum, and H. comosum obtained from the US Department of Agriculture’s (USDA) National Plant Germplasm System (NPGS), of which some have been reported as endophyte-infected. Using polymerase chain reaction (PCR) with primers specific to Epichloë species, we were able to identify endophytes in seeds from 17 of the 56 Plant Introduction (PI) lines, of which only 9 lines yielded viable seed. Phylogenetic analyses of housekeeping, alkaloid biosynthesis, and mating type genes suggest that the endophytes of the infected PI lines separate into five taxa: Epichloë bromicola, Epichloë tembladerae, and three unnamed interspecific hybrid species. One PI line contained an endophyte that is considered a new taxonomic group, Epichloë sp. HboTG-3 (H. bogdanii Taxonomic Group 3). Phylogenetic analyses of the interspecific hybrid endophytes from H. bogdanii and H. brevisubulatum indicate that these taxa all have an E. bromicola allele but the second allele varies. We verified in planta alkaloid production from the five genotypes yielding viable seed. Morphological characteristics of the isolates from the viable Hordeum species were analyzed for their features in culture and in planta. In the latter, we observed epiphyllous growth and in some cases sporulation on leaves of infected plants.