Taxonomic And Physiological Studies On Endophytic Fungi Of Calamagrosits Spp. In China

Neotyphodium species(Clavicipitaceae,Hypocreales,Ascomycota) as plant endophytes are biotrophic symbionts of many cool-season grasses(subfamily Po(o|¨)ideae). Neotyphodium spp.,known as asexual type,never cause any symptom on host plants.Most of them were interspecific hybrids among Epichlo(e|¨) spp..Epichlo(e|¨) spp.usually develop stromata on the culms of host plants.A fly-mediated mating between stromata of opposite mating type results in production of ascospores that mediate horizontal transmission to florets of neighboring uninfected plants.Because more researches have shown that Epichlo(e|¨) species and their asexual relatives(Neotyphodium species)--collectively called epichlo(e|¨) endophytes.Recently,17 species and 5 variaties in genus Neotyphodium had been respectively described all over the world.In this study,we had investigated distribution of Neotyphodium spp.in Calamagrostis spp.Adans.grow in China.From these plants 22 fungal isolates were obtained.Morphological characters,host specificity,interfertility tests and molecular phylogenetic evidences were studied for taxonomy of these fungal isolates. Then,biological and physiological characteristics of some fungal isolates were investigated.Firstly,437 Calamagrostis spp.were collected from Xinjiang,Qinghai,Gansu,Jiangsu, Anhui,Zhejiang and Shanghai.All the 305 samples from west China were detected to be free of endophyte,while non-inflorences Calamagrostis sp.grew in Nanjing and C. epigeios Roth.grew in Huangshan were 100%infected by endophyte.In contrast,C. epigeios in Nanjing harbored no endophyte.The level of endophyte concentration varied with geographical distribution and host species.Secondly,host plants found in Nanjing were upstanding and 90-120cm in height with aggressive stolons,but did not emerge inflorescences during the two heading periods. Mainly basing on morphology,leaf epidermis micromorphology and rDNA-ITS phylogenetic analysis,the host plant was identified as Calamagrostis sp..Thirdly,according to morphological properties of asexual structures,the isolates were identified as Neotyphodium spp.Botanical properties of host plant and development of non-fertilize stromata of the endophyte indicate this symbiosis found in Nanjing and Huangshan should be two brand new host-endophyte associations.Morphological properties also indicated that Neotyphodium spp.in native Calamagrostis spp.exhibited abundant diversity.Fourthly,DNA fragments of tubB genes from 4 and tefA genes from 5 fungal isolates obtained from Calamagrostis sp.in Nanjing were amplified and directly sequenced.DNA sequences of all 4 tubB fragments or 5 tefA fragments were completely identical. Phylogenetic analysis strongly supported that these isolates should be significantly different from other epichlo(e|¨) endophytes species,they are a new phylogenetic species.The species and distribution of host,morphological and molecular phylogenetic evidences,especially the production of infertile and extremely long stromata and absences of sexual structures,supported erectation ofNeotyphodium stromatolongus Y.Ji,L.Zhan et Z.Wang as a new species.Fifthly,biological and physiological characteristics of Neotyphodium sp.from Nanjing and Huangshan were examined by mycelium growth in vitro at a range ofpH value,carbon and nitrogen amended media.The optimal pH for isolates from Nanjing and Huangshan were 5.0.The Neotyphodium endophytes varied in their ability to grow on media containing different carbon and nitrogen nutrients.The preference of the isolates from Nangjing for carbon source was glucose,sucrose,and the preference of the isolates from Huangshan for carbon source was glucose,sucrose.Growth of all three isolates tested was improved by peptone,casein,potassium nitrate and ammonium chloride.Peptone and casein significantly improved growth of the isolates from Nanjing,and peptone,casein and potassium nitrate improved growth of the isolates from Huangshan.Isolates from Nanjing grew more rapidly than isolates from Huangshan in each test.They are differenent in physiological characteristics.