Morphological, phylogenetic, and alkaloid profile analyses of a new <i>Epichloë</i> species symbiotic with <i>Elymus kamoji</i> in China

The aboveground endophytes of the genus <i>Epichloë</i> are notable for their mutualistic association with the Poaceae family, conferring benefits such as increased stress tolerance, competitiveness, and ecological dominance to host plants. Here, two endophytic fungal strains were isolated from <i>Elymus kamoji</i> in China, exhibiting morphological characteristics typical of <i>Epichloë</i> species. Phylogenetic analyses using maximum likelihood method on <i>tubB</i> and <i>tefA</i> gene sequences revealed that two strains from <i>Elymus kamoji</i> represent a novel <i>Epichloë</i> interspecific hybrid species. Allele 1 grouped within <i>Epichloë bromicola</i>, whereas allele 2 grouped within <i>Epichloë calamagrostidis</i>. We propose the name <i>Epichloë tibetica</i>, extending the diversity of <i>Epichloë</i> species known to colonize <i>Elymus kamoji</i>. Both isolates are mating type B (MTB), and no sexual structures or epiphyllous growth was observed on <i>Epichloë</i>-infected <i>El. kamoji</i>. DNA analysis revealed the absence of genes responsible for the biosynthesis of ergot alkaloids, indole-diterpenes, and 1-aminopyrrolizidines in both isolates. Regarding the pyrrolopyrazine synthetase A gene (<i>ppzA</i>) profiles, the hybrid <i>E. tibetica</i> was found to contain both the <i>ppzA</i> and <i>ppzA</i>-∆R alleles. The <i>ppzA</i>-∆R allele is characterized by large deletions spanning the <i>ppzA</i>-M and <i>ppzA</i>-T2 domains. Within an alternate <i>ppzA</i> allele, we have localized a region downstream of <i>ppzA</i>-A2 whose structural properties block amplification of the <i>ppzA</i>-A2 region using conserved domain-specific primers. These traits characteristics may position <i>E. tibetica</i> as a viable model for studying <i>ppzA</i> allele diversity. Our findings further highlight the necessity of employing diverse primer combinations to elucidate the profiles of alkaloid synthesis genes across hybrid <i>Epichloë</i> species.