青藏高原高寒草地优势禾草—紫花针茅内生真菌分离和鉴定

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

紫花针茅作为青藏高原高寒草原群落中主要优势禾草之一,关于该植物所感染禾草内生真菌的形态和分类研究尚未见报道。本研究采用分离培养获取不同样点的紫花针茅内生真菌菌落,利用特异性引物克隆紫花针茅内生真菌序列,并与Genbank中下载的序列共同构建系统进化树。结果表明,青海省紫花针茅样品带菌率高达100%,而其他区域样品均不带菌。分离的内生真菌菌落从菌落形态、生长速度及分生孢子形态等特征均与Epichloё属内生真菌的形态特征相似。系统进化关系表明,它们分别与北美洲竖针茅体内无性态内生真菌Epichloёchisosa、醉马草内生真菌Epichloёinebrians及甘肃内生真菌Epichloёgansuensis具有较近的亲缘关系,进一步说明紫花针茅所感染内生真菌与宿主间未体现出严格的宿主特异性。

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 morpholo-gy 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 like-lihood method was applied to construct a phylogenetic tree.It was found that endophyte occurrence in S .pur-purea was relatively high in Qinghai province,compared to other sites.Colony morphology characteristics, growth speed and morphology of conidia were identical to those of Epichloёspp.The results of tubB and tefA phylogeny indicated that four endophyte strains isolated from S .purpurea were most closely related to Epichloёchisosa from North American Achnatherum eminens and formed two distinct branches.Four other en-dophyte strains isolated from S .purpurea were most closely related to Epichloёindbrians and Epichloёgan-suensis ,which infect Achnatherum inebrians and these strains formed another distinct branch.In addition,a-nalysis of actG phylogeny indicated that four clarify further endophyte strains isolated from S .purpurea were also most closely related to Epichloёchisosa from North American A.eminens and formed another distinct branch.Other endophyte strains isolated were most closely related to Epichloёgansuensis from China (Xin-jiang)and formed another distinct branch.Our results suggest that the host-specificity might not occur in en-dophytes infecting S .purpurea and S .purpurea appears to be infected by various Epichloёspecies.