Taxonomic Studies Of Neotyphodium Endophytes In Chinese Roegneria Grasses

The epichlo(e|¨) endophytes(Clavicipitaceae) are important filamentous fungi that habitat within the aerial tissues of cool-season grasses(subfamily Po(o|¨)ideae).This fungal group is comprised of sexual species(Epichlo(e|¨) Tul.spp.) and their asexual derivatives (Neotyphodium Glenn,Bacon,and Hanlin spp.).Within genus Neotyphodium,17 species have been formally described now.Distribution of the epichlo(e|¨) endophytes and their host plants were surveyed in Shanghai,Jiangsu,Anhui and Shanxi from March to July in 2006.In total 1,487 grass samples,54.3%were detected to be infected by epichlo(e|¨) endophytes.In Jiangsu and Shanxi, the infection rates were as high as 55.5%and 69.0%respectively.These indicated the wide distribution and abundant diversity of epichlo(e|¨) endophytes in native Chinese grasses. Filamentous fungal hyphae were observed within leaf sheath,pith and seed of grass but root.Vegetative hyphae ca.2μm wide,rarely branched,run almost parallel with each other in the intercellular spaces,did not cross each other in most cases within pith and sheath. However,hyphae were winding or convoluted in the aleurone layer of seed.From 2006 to 2007,1,126 Roegneria plants without stromata were collected in China, and 55.5%of them were detected to be infected by epichlo(e|¨) endophytes.From Roegneria plants were collected in 16 Provinces(city),about 80 fungal isolates were obtained, indicating a wide distribution of epichlo(e|¨) endophyte-Roegneria symbiotic combinations in China.Morphological characteristics of 6 isolates obtained from Roegneria plants in 4 provinces,out of the 80 isolates,were investigated in detail.On PDA plate,colonies white, cottony,reverse brown to pale brown,grow rapidly at a rate of 18.3-48.7mm/21 d.Conidia hyaline,reniform to elliptical,4.8-5.9μm×2.1-3.4μm.Phialides discrete,arising solitary from the aerial mycelia,septated at the base or middle,hyaline,smooth,16.1-24.2μm×1.8-2.5μm.These indicated that epichlo(e|¨) endophytes from Roegneria plants are quite diverse.To confirm Epichlo(e|¨) yangzii exists in the endophyte isolates obtained from Roegneria plants without stroma,mating tests between isolates of E.yangzii were firstly conducted and different mating types of isolates were confirmed.Spermatia obtained from 23 epichlo(e|¨) endophyte isolates of non-stromata producing Roegneria spp.were crossed with stromata of different mating types of E.yangzii.In the results,2 isolates were interfertile with E. yangzii(mat-1),while the remaining 21 isolates were not interfertile with both types of E. yangzii.These indicated that E.yangzii may be also involved in endopyte from Roegneria plants without stromata development.These also support that the mating test should be effective to distinguish Neotyphodium sp.from.E.yangzii obtained from the host plants without stroma development.DNA fragments ofβ-tubulin gene(tubB) and translation elongation factor 1-αgene (tefA) from fungal isolates obtained from Roegneria plants without stromata were cloned and sequenced.Sequences from representative strains of Epichlo(e|¨) spp.and Neotyphodium spp.obtained from GenBank were aligned and used for constructing phylogenetic trees with tubB and tefA sequenced in this study.Phylogenetic analysis based on tubB sequences of 8 isolates were used for confirming the isolates are Neotyphodium endopyte and verifying the result of mating test.Phylogenetic tree of 6 Neotyphodium isolate indicated that the sequences between these 6 isolates have two alleles.The first alleles of tefA and tubB clustered with bootstraps of 87 and then re-clustered with E.yangzii.Second tubB alleles of of Rxy6106,Rxy6107 and second tefA allele of Rdy4302 clustered with E. sylvatica in clade ETC with high bootstrap of 91 and 83 respectively.Therefore,these 6 isolates might be a new taxonomical group and originated from the hybridization of E. yangzii and E.sylvatica.Referring on host specificity,geographic distribution morphology,mating tests, molecular phylogenetic evidence,a new species,"Neotyphodium sinica Z.W.Wang,Y.L. Ji et Y.Kang,sp.nov.",was proposed.This new Neotyphodium species is the second member native in Asian continent,particularly in China.