Isolation and identification of endophytes infecting Stipa purpurea, a dominant grass in meadows of the Qinghai-Tibet Plateau

Abstract

Stipa purpurea is a dominant grass species in alpine grassland of the Qinghai-Tibet Plateau. Research on this species to date has focused on defining genetic diversity, community classification, ecological function, physiological traits and grassland community characteristics; however, isolation and identification of endophyte fungi from S. purpurea has seldom been attempted. Endophyte was isolated from S. purpurea by leaf surface sterilization and axenic culture, and hyphae, colony physical characteristics, and conidial morphology were observed. Endophyte nucleotide sequences were cloned by tubB, tefA, and actG specific primers, and representative endophyte sequences were downloaded from Genbank to determine homology. A maximum likelihood method was applied to construct a phylogenetic tree. It was found that endophyte occurrence in S. purpurea was relatively high in Qinghai province, compared to other sites. Colony morphology characteristics, growth speed and morphology of conidia were identical to those of Epichloё spp. The results of tubB and tefA phylogeny indicated that four endophyte strains isolated from S. purpurea were most closely related to Epichloё chisosa from North American Achnatherum eminens and formed two distinct branches. Four other endophyte strains isolated from S. purpurea were most closely related to Epichloё indbrians and Epichloё gansuensis, which infect Achnatherum inebrians and these strains formed another distinct branch. In addition, analysis of actG phylogeny indicated that four clarify further endophyte strains isolated from S. purpurea were also most closely related to Epichloё chisosa from North American A. eminens and formed another distinct branch. Other endophyte strains isolated were most closely related to Epichloё gansuensis from China (Xinjiang) and formed another distinct branch. Our results suggest that the host-specificity might not occur in endophytes infecting S. purpurea and S. purpurea appears to be infected by various Epichloё species.