Molecular identification and characterization of endophytes from uncultivated barley

<p><i>Epichloë</i> species (Clavicipitaceae, Ascomycota) are endophytic symbionts of many cool-season grasses. Many interactions between <i>Epichloë</i> 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 <i>Hordeum</i> 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 <i>Hordeum</i> species. We analyzed seeds of <i>Hordeum bogdanii, H. brevisubulatum</i>, and <i>H. comosum</i> 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 <i>Epichloë</i> 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: <i>Epichloë bromicola, Epichloë tembladerae</i>, and three unnamed interspecific hybrid species. One PI line contained an endophyte that is considered a new taxonomic group, <i>Epichloë</i> sp. HboTG-3 (<i>H. bogdanii</i> Taxonomic Group 3). Phylogenetic analyses of the interspecific hybrid endophytes from <i>H. bogdanii</i> and <i>H. brevisubulatum</i> indicate that these taxa all have an <i>E. bromicola</i> 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 <i>Hordeum</i> 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.