Study On The Taxonomy,diversity And Metabolites Of Epichlo(?) Endophyte In Festuca Sinensis

Festuca sinensis,a Chinese native species,is one of the high-quality forages in Qinghai-Tibet Plateau,which plays an increasingly important role in restoring degraded grassland and improving ecosystem and environment.It often forms mutually beneficial symbiosis with Epichlo(?) endophyte;however,there is no report on the toxicity of this symbiosis to livestock.In addition,the taxonomy,biological and ecological characteristics of this Epichlo(?) endophyte have not been identified.In the present study,F.sinensis seed lots were collected from 12 geographical locations in Qinghai,Gansu and Sichuan provinces in Qinghai-Tibet Plateau to isolate fungal endophytes.The taxonomy and biological characteristics of the endophyte and the biology activity and metabolites of its fermentation broth were identified using morphological,molecular and mass-spectrometric technology to provide basic information for further studies of F.sinensis-endophyte symbiosis.The main findings are as follows:(1)One new Epichlo(?) species was named with Epichlo(?) sinensis sp.nov.Through morphological observation,41 Epichlo(?) strains had typical morphological characteristics of the Epichlo(?) genus.Endophyte hyphae were long,thin,septate,a little curved,and orientated longitudinally and parallel to each other without branching in the intercellular spaces of leaf sheaths.On potato dextrose agar(PDA),colonies were smooth,cottony,and white.Conidiogenous cell was phialidic,sparse,arising solitarily and perpendicularly from aerial hyphae,unbranched,hyaline,18.5-31.5 ?m.Conidia were navicular or asymmetric,hyaline,4-7 ?m × 1.5-4.0 ?m.Phylogenetic relationships based on sequences of ?-tubulin(Tub B)and translation elongation factor1-?(Tef1-?)showed that this endophyte had two gene copies which have the same origin with E.poae and E.sibirica.(2)Epichlo(?) sinensis had the chemotype of Epichlo(?) endophyte.Based on PCR analysis of 30 genes in alkaloid synthesis pathway and SSR(Simple Sequence Repeats)polymorphisms,all 41 Epichlo(?) strains had 2 genes in peramine synthesis,8 genes in indolditerpene synthesis and 1 gene in pyrrolizidine synthesis.However,they lacked the genes in ergot synthesis.These strains did not show genetic polymorphism.(3)The biological characteristics of Epichlo(?) sinensis were identified.The optimal temperature for the Epichlo(?) sinensis strains on PDA growth was 22-25?.The optimal p H value for them was 4-7.The optimal medium for these strains was PDA and the average growth rate was 4.0 mm/week.The most efficient carbon and nitrogen source for these strains were sorbitol and peptone,with the average growth rate being5.0 and 5.1 mm/week,respectively.The growth rate of these 41 strains ranged from 1.0to 6.0 mm/week.Under the same medium,different endophyte strains had significantly(P < 0.05)different growth rates.Further analysis did not suggest significant correlations between the growth rate of endophyte strains under different temperatures and host plant growth environmental conditions such as altitude,annual precipitation and annual temperature.(4)Fermentation broth of Epichlo(?) sinensis had biological activities.The fermentation broth of 3 strains collected from PDB significantly promoted Elymus nutans seed germination and growth.After being treated with 100% fermentation broth,the germination rate and germination potential of E.nutans seeds increased by 30.3%and 47.5%(P <0.05).With treatment of 60% fermentation broth,the radicle and plumule length of E.nutans seeds increased by 43.9% and 16.2%(P < 0.05).Fermentation broth of 3 endophyte strains(1,2,84C)had inhibition effects on the pathogens including Fusarium sp.,Bipolaris sp.and Alternaria sp.The inhibition effects increased with increasing concentration of fermentation broth and the average inhibation rate was 24.5%,22.3% and 44.3%.(5)The metabolite categories of Epichlo(?) sinensis were identified.Three E.sinensis endophyte strains(1,2 and 84C)produced abundant metabolites under PDA and PDB culture conditions,which were tested by gas chromatography-mass spectrometry(GC-MS)and liquid chromatography-mass spectrometry(LC-MS).There were a total of 1027 compounds that were distributed in a similar manner in these three endophytes strains.There were 36 compounds tested by GC-MS,which belonged to 7classes such as amino acids,saccharides,alcohols,carboxylic acids;and there were 991 compounds tested by LC-MS which belonged to 28 classes such as carboxylic acids,amino acids and derivatives,heterocyclic compounds,esters,saccharides.Culture conditions had significant impacts on the types of metabolites.Both the types and the number of metabolites in mycelia in PDA,mycelia in PDB and fermentation broth of the 3 tested strains varied.There were many different compounds among different strains.The different compounds had significant effects on the metabolic pathway and metabolite synthesis of the endophyte.