Bioresources And Biodiversity Of Some Epichloid Endophytes Native To China

Epichloid endophytes including Epichloe species (Ascomycota, Clavicipitaceae) and their asexual anamorphs Neotyphodium colonize the intercellular spaces within the leaves, culms, leaf sheaths and seeds of host plants. They are symbiotic with grasses (Poaceae). Until now, at least280grass species belong to80genera have been reported to be infected by fungal endophytes. These plants mostly distributed in Europe and North America, while few in China.From2011to2012,2001plants of at least15species,9genera were collected from Jiangsu, Anhui, Zhejiang, Liaoning, Inner Mongolia and Beijing. Thirty-five point four percent of these plants were detected to harbor fungal endophytes. The infection level was different among genera and species, indicating host specific of epichloid endophytes. No epichloid endophyte infection was confirmed within Bromus spp. except in samples of B. magnus from Huangshan, Anhui. While B. magnus collected from Nanjing are not infected. The infected B. magnus plants were rarely detected in eastern China.Forty-four grass plants with stromata were collected from Tongliao, Inner Mongolia. Combination of trnT-trnL intergenic space analysis, leaf epidermis and morphological characteristics, these stromata-bearing plants were identified as Leymus chinensis Trin.. The host specific fungal endophyte which developed stromata on L. chinensis had no record.In this study, we analyzed fungal endophytes within plants of B. magnus and L. chinensis. Thirty-one epichloid endophyte strains were isolated on PDA plates, these strains were divided into FG (9strains) and SG groups (22strains) depended on growth rate on PDA plates. Colonies of FG strains could grow to48.1-56.3mm/21d, while those of SG strains were12.9～15.9mm/21d. FG strains had bigger conidia (4.5-5.1×2.9-3.6μm) and longer phialides (15.8～24.6μm) than those of SG strains. Strains of FG and SG exhibited diversity although they were isolated from the same site and same kind of host plant. Phylogenetic tree based on tefA and tubB gene fragments revealed that strains of FG and SG clustered together in a distinct subclude within the E. bromicola E. yangzii clade, with posterior probability of1for tefA and0.84for tubB. Combination of host specificity, morphological and phylogenetic characteristics of fungal endophytes, we propose Neotyphodium magnicola H. Zhang, Z. Wang et Y. Ji, sp. nov. as a new fungal endophyte in Bromus magnicola Keng.On the sampling date of Sep.2011, Stromata surrounded the flag leaf sheathes of L. chinensis, white, at the final stage of development. Perithecia without asci were observed under microscope. Nine epichloid endophyte strains were obtained from samples grown in urban district, and16from Horqin district of Tongliao city. Morphological and phylogentic properties of25strains isolated from L. chinensis were investigated. They were similar with each other in morphology. Colony grow28.5～41.1mm/21d; phialides24.1～26.4μm long,2.1～2.5μm at base, tapering to less than1.0μm at tip; conidia reniform to elliptical,4.7～5.5×2.8～3.5μm. These morphological properties were typical of Epichloe endophytes. Based on the analysis of tefA and tubB gene fragments, all the6selected strains formed a distinct subclade in E. bromolical E. yangzii clade, both with posterior probability of1. They exhibited phylogenetic difference with epichloid endophytes have been reported. Considering morphological and phylogentic properties, we tend to determine strains isolated from L. chinensis as a new taxonomic group. Their taxon should be confirmed by mating tests in future.Sequence-related amplified polymorphism (SRAP) technique was conducted to evalute genetic diversity of22epichloid strains. Genetic distance among twenty-six strains ranged from0.000to0.876, reveraled diversity among strains from different host. Strains from Roegneria spp. and Elymus clindricus showed comparatively complicated. Strains from L. chinensis, B. magnus, Festuca parvigluma, Melica scabrosa and Achnatherum sibiricum clustered respectively. These results indicated significant diverse in genetic diversity among endophytic strains obatined from grasses grown in China. In this study we surveyed epichloid endophytes within grasses (Poaceae) in China, this study contribute to understand the whole fungal endophytes resources, and for application of this new microbial resources.