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.香柱菌属（Epichloë）物种（隶属于子囊菌门（Ascomycota）麦角菌科（Clavicipitaceae））是众多冷季型草本植物的内生共生体。香柱菌与宿主草本植物间的多数互作可促进植物生长、抵御多种病原菌与昆虫害虫，并提升植物对干旱胁迫的耐受性。此前已有研究表明，与大麦属（Hordeum）物种相关的内生菌对昆虫取食的抗性，会因内生菌-草本-昆虫组合的不同而存在差异。本研究针对野生大麦属物种携带的内生菌的遗传与化学型多样性展开探究。我们分析了取自美国农业部（USDA）国家植物种质系统（NPGS）的布格大麦（Hordeum bogdanii）、短芒大麦（H. brevisubulatum）与花大麦（H. comosum）的种子，其中部分种子此前已被报道存在内生菌侵染。通过使用针对香柱菌属的特异性引物开展聚合酶链式反应（PCR），我们在56份植物引种（PI）系的种子中鉴定出17份携带内生菌，其中仅9份植物引种系可获得具有生活力的种子。对持家基因、生物碱生物合成基因以及交配型基因的系统发育分析显示，受侵染的PI系所携带的内生菌可划分为5个分类群：布罗米科拉香柱菌（Epichloë bromicola）、坦布拉达香柱菌（Epichloë tembladerae），以及3个尚未定名的种间杂交物种。其中1份PI系所携带的内生菌被归为一个新的分类群：香柱菌未定种（Epichloë sp.）HboTG-3（布格大麦分类群3，H. bogdanii Taxonomic Group 3）。对取自布格大麦与短芒大麦的种间杂交内生菌的系统发育分析表明，这些分类群均携带E. bromicola等位基因，但其第二等位基因存在变异。我们对可获得生活力种子的5个基因型开展了植株体内生物碱合成量的验证。同时分析了从可产生生活力种子的大麦属物种中分离得到的菌株的形态特征，包括其在培养基与植株体内的生长表现。在植株体内的观察中，我们发现了叶表生长现象，部分受侵染植株的叶片还出现了产孢现象。