Primary Study On The Endophytic Fungi Of Vitis Vinifera And Their Secendary Metabolites

Since 1990s,the range of plant endophytic fungi study has been expanded,not being limited gramineous plants only,but tending to isolate endophytic fungi from woody plants to explore the biological diversity,biology influence,endophytic mechanism,relation with host,and the exploration of new bioactive compounds,etc.This thesis focused on the endophytic fungi of Vitis vinifera in an introduction experimental vineyard,analyzed the community constituents and the phylogenetic relation,supplied a preliminary study on the inhibitory activity of the strains to phytopathogenic and human pathogen,investigated the constituents and the inhibitory activity to phytopathogenic fungi of the compounds which were traced and isolated from the metabolites of active strain A23,and inspected whether the wine brewed from the fruits in this vineyard was polluted or not by the endophytic fungi's metabolites harmful to human beings.455 endophytic fungi strains were isolated from the roots,branches, leaves,and fruits of Vitis viniferas.The statistics indicated that the types and quantities of the endophytic fungi were related with host's health situation.The separation rate of the endophytic fungi from plants with high survival ratio was relevantly high,and the vice versa.The analysis of community constituents and phylogenetic relation demonstrated that Alternaria,Penicillium,Fusarium were dominant species,but among which there were some potential phytopathogenic fungi,such as Diaporthe phaseolorum,Aspergillus oryzae, Fusarium equiseti,Pestalotiopsis uvicola,etc.Taked Colletotrichum lagenarium(Passerini),Verticillium dahliae Kleb,and Phytophthora parasitica Destur as indicator fungi,the primary screening employed antagonistic method to check the antiseptic activity of 103 endophytic fungi,while rescreening used filter paper method.Thirty active strains were obtained from the primary screening,accounting for 29.1%of the total strains.The rescreening displayed that there were eight strains whose inhibition to Bacillus pumilus and Bacillus subtilis surpassed ampicillin,and thirteen strains,among which seven strains belonged to dominant species,had resistance to the three above-mentioned indicator fungi,which indicated a certain broad spectrum resistance.The results demonstrated that Vitis vinifera consisted of abundant endophytic fungi. Through the interspecific competition,the dominant species created various active metabolites to improve the host's anti-disease and anti-adversity ability, which proved the view of mutual choice and mutual adaptation between host and endophytic fungi,and "The Host Relatedness Hypothesis".One endophytic fungus was found out to belong to Cordyceps fungi during the research of endophytic fungi phylogenetic relation,and the phylogenetic tree was created together with insect Cordyceps fungi reported in papers.The result indicated that the Cordyceps fungi in plants were different with those in insects;they had become independent species;and a "leap" from plant to insects' host had happened through the gene regulation during the developmental process.The Vitis vinifera belonged to one of the segments.The strain A23 belonged to Fusarium tricinctum(Corda) through the molecular identification.The separation methods,such as solid culture, extraction,Silica Column Chromatography,Dextran gel Column Chromatography(HPLC),etc.were employed in this thesis to get five pure compounds from the fermentation production.The spectral analysis,such as NMR,displayed that the structure of two compounds was the same,that was, Enniatin B.Its inhibitory activity of phytopathogen indicated that Enniatin B had broad spectrum resistance and good activity:the MIC for Colletotrichum lagenarium was 7μg/mL,for Verticillium dahliae,the MIC was 3μg/mL,and for Phytophthora parasitica MIC was 15μg/mL.Combined with pot experiment test,Enniatin B was proved to have the potentiality to be qualified agriculture antibiotics.To check whether the wine was polluted by the endophytic fungi's metabolites that was harmful to human beings,two kinds of wine(red wine and white wine) brewed from the fruits in the same vineyard were analyzed by HPLC.The result proved that red wine dides not comprise Enniatin B,while white wine comprises it,reaching to 11.04μg/mL.It illustrated that the plant endophytic fungi and their metabolites may entered into plant production through some certain ways,which may generated food safety problems.The present study proved that with abundant endophytic fungi,Vitis vinifera was a good way to investigate endophytic fungi and host phylogenetic relation.And with large quantity of active compounds,it was also an effective way to find out new agricultural chemicals and human antibiotics.However, harmful toxinum may be producted and taken into human food,generating food safety problems.On the one hand,plant endophytic fungi can improve host's anti-adversity ability,supply useful active compounds for human beings, and make a contribution to agriculture,forestry and livestock production,on the other hand,they may reduce the output value of agriculture,forestry and livestock,and threaten food safety.They have the double-response to human life.