Study On The Diversity Of Endophyte From Roots Of Vaccinium Dunalianum And The Chemical Biology Of The Dominant Fungus

Vaccinium dunalianum Wight(Ericaceae)is an evergreen shrub mainly distributed in the Southwest of China.The local people of its growing area in Yunnan use the leaves of this plant as a folk medicine for the treatment of articular rheumatism,and the dried leaf buds as a local herbal tea.The 6'-O-caffeoylarbutin(CA)was the major component in this plant,expressed a stronger anti-melanin activity in a dose-dependent manner with about a two-fold more than that of arbutin,but with less toxicity about a two-fold lower than that of arbutin.Through the first time studies on the diversity of endophyte from roots of V.dunalianum and the drought resistance and chemical constituents of the dominant fungus,we expect to make a certain contribution to develop and use of V.dunalianum and its endophytic fungi.The results were as follows:1.This study explored the biodiversity and phylogeny of endophytic fungi and endophytic bacteria isolated from the roots of V.dunalianum.A total of 284 endophytic fungi obtained were grouped into 38 species,and 21 genera,which were authenticated by molecular identification based on r DNA internal transcribed spacer(ITS)sequence analysis.Of the 284 explicit isolates,141 isolates majority of endophytic fungi isolated from the roots of V.dunalianum are included in the Ascomycota and within four classes:Sordariomycetes,Eurotiomycetes,Leotiomycetes and Dothideomycetes.There are only2 isolates were clustered in the Zygomycota within the Mucoromycotina,which includes Umbelopsis ramanniana(1 species,16 isolate)and Umbelopsis dimorpha(1species,127 isolate).Only 1 isolate was clustered in the Basidiomycota within the Agaricomycetes.The results suggested that U.dimorpha is the dominant fungus in roots of the V.dunalianum,which had the highest isolation frequency(44.7%).Furthermore,a total of 57 endophytic culturable endophytic bacteria were obtained from roots of the V.dunalianum,all of which were classified in Actinobacteria,Firmicutes and Proteobacteria,5 classes,7 orders,12 families and 13 genera.Here,for the first time,this study systematically demonstrated the constitution of endophytic fungi and endophytic bacteria associated with the roots of V.dunalianum,initiated us into the endophytic microecosystem of this plant,and laid a foundation for the further study of the resistance to stress and the relationship between the plant and endophytic fingi.2.To explore the effect of symbiotic microorganisms on plant drought resistant properties,this study used dominant fungus isolated from the roots of V.dunalianum inoculated into the model plant Arabidopsis thaliana and the tissue culture plantlets of V.dunalianum respectively,researched the influence of the symbiosis for drought resistance of A.thaliana and V.dunalianum.The major results are as follows:Inoculated endophyte had significant effect on the germination of A.thaliana seeds at concentration 0%PEG;at15%PEG stress make the germination rate increased from 81.10%to 92.10%;at 20%PEG stress make the germination rate increased from 38.00%to 51.95%;at 25%PEG stress make the germination rate increased from 0.0%to 4.81%.The root length of A.thaliana seeds inoculated with dominant fungal was 2 times than that of the uninoculated one.The results showed that the dominant endophytic fungi in the roots of V.dunalianum could not only promote the germination and root growth of A.thaliana seeds under normal water content,but also improve the seed vigor and germination rate under drought stress,and significantly promote the seed root length.Furthermore,under drought stress,the dominant endophytic fungi inoculated could rapidly and significantly enhance the activity of POD in plants,which was conducive to the rapid regulation of CAT activity in plantlets,enhanced the activity of catalase and the content of proline in plantlets more rapidly and significantly.At the same time,it can also improve the response speed of the regulation of soluble sugar content in plantlets and contribute to the accumulation of soluble sugar in plantlets under drought stress.The experimental results showed that the dominant fungal in the roots of V.dunalianum could enhance the drought resistance of the host.3.Using the phytochemical method to study the secondary metabolites from the fermentation medium of U.dimorpha.The fermentation medium was extracted with methanol and ethyl acetate,successively.Thirty-four compounds were isolated from the methanol layer and ethyl acetate layer by silica gle and Sephadex LH-20 column chromatographies.On the basis of the MS,¹H and ¹³C NMR spectroscopic analyses,the chemical structures of fourteen compounds were elucidated as 2'-O-methyladenosine(7),4-hydroxyhippuric acid methyl ester(8),(S)-2-hydroxy-3-(4?-hydroxyphenyl)propanoic acid(10),latifolicinin C(11),adenosine(13),1-(?-D-ribofuranosyl)-1H-1,2,4-triazone(14),thymidine(20),adenosine(23),2'-O-methylguanosine(27),6'-O-caffeoylarbutin(32),methyl 2,4-dihydroxy-3,5,6-trimethylbenzoate(33)and three novel compounds dimorpharins A(5*),B(17*)and C(18*),respectively.All of the compounds were isolated from the U.dimorpha for the first time,and the structural elucidation of the other isolated compounds are in progress.