Morphological, phylogenetic, and alkaloid profile analyses of a new Epichloë species symbiotic with Elymus kamoji in China.

The aboveground endophytes of the genus Epichloë 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 Elymus kamoji in China, exhibiting morphological characteristics typical of Epichloë species. Phylogenetic analyses using maximum likelihood method on tubB and tefA gene sequences revealed that two strains from Elymus kamoji represent a novel Epichloë interspecific hybrid species. Allele 1 grouped within Epichloë bromicola, whereas allele 2 grouped within Epichloë calamagrostidis. We propose the name Epichloë tibetica, extending the diversity of Epichloë species known to colonize Elymus kamoji. Both isolates are mating type B (MTB), and no sexual structures or epiphyllous growth was observed on Epichloë-infected El. kamoji. 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 (ppzA) profiles, the hybrid E. tibetica was found to contain both the ppzA and ppzA-∆R alleles. The ppzA-∆R allele is characterized by large deletions spanning the ppzA-M and ppzA-T2 domains. Within an alternate ppzA allele, we have localized a region downstream of ppzA-A2 whose structural properties block amplification of the ppzA-A2 region using conserved domain-specific primers. These traits characteristics may position E. tibetica as a viable model for studying ppzA allele diversity. Our findings further highlight the necessity of employing diverse primer combinations to elucidate the profiles of alkaloid synthesis genes across hybrid Epichloë species.