Study On The Isolation, Identification, Secondary Metabolites Of Endophytic Fungi Of Vaccinium Corymbosum L.

Endophytic fungi exist widely in the body of plants.The people have been paying more attention to them.As a big group of microbe inhabiting in the living tissues of plants,endophytic fungi have no significantly negative effects on the host plants during their whole life history or some phases.There are different kinds and quantity of endophytic fungi in different plants.It was reported that endophytic fungi can produce the same or similar compound with their host.In this scientific dissertation,have studied on resources of endophytic fungi toward Vaccinium corymbosum L..And have been identified the strains through the methods of morphology and molecular biology.The secondary metabolites of strain H2 were also isolated and purified.The results were as follows:1.By tissue surface-sterilized,isolation media were PDA and TWA,and cultured at 25℃in the incubator. And 54 strains endophytic fungi were isolated from health leaves;24 strains were belonged to Alternaria sp. and 21 strains were belonged to Pestalotiopsis sp.,the isolated rates were 46%and 41%respectively,higher than others.2.Through determined the growth curve of strain H1 and strain H2,the results showed that the growth curve were nearly "s" pattern.On the normal culture,15 days could overgrow the culture capsule.The growth curve provided the best extraction period for secondary metabolites of endophytic fungi.3.The phylogentic taxonomy of endophytic fungi was studied by modern molecular biology technique.This study extracted DNA by CTAB method.Used ITS1 and ITS4 to be primers,PCR amplification the DNA that extract from H1,sequencing the amplification products later.The result showed that:this endophytic fungi has highly homology(99%) with Alternaria azukiaein genebank.4.Separated and purified the secondary metabolites of H2.There were five chemical compositions in the eluant of CHCl₃:MeOH(10:1 to 10:10) by SG column chromatography.Through the detection of TLC,C-2 and C-6 had the similar Rf value to plants' portion in acetidin.Through the TLC,MS,IR,NMR and compared the documents,the compound C-6 was a sort of sesquiterpene lactone,molecular formula is C₁₅O₂H₂₂.This study have been established the foundation for secondary metabolites of endophytic fungi Vaccinium corymbosum L..To sum up,the achievements of this research are as follows:It's the first time to isolate the endophytic fungi from health leaves of Vaccinium corymbosum L.to use tissue separation method,and determined the dominant species were Alternaria sp.and Pestalotiopsis sp..It firstly to study on the secondary metabolite of strain H2,and through the chromatographic,it demonstrated that the secondary metabolites were similar to plants of Vaccinium corymbosum L..