Study On The Inhibitory Effect Of Chemical Components Of Vanderbylia Robiniophila Fermentation Broth On Ginseng Pathogenic Fungi

Soil borne diseases seriously affect the quality and yield of ginseng.Traditional chemical control will destroy the ecological environment,along with people’s increasing attention to ecological environment protection,biological control has become a research hotspot because of its advantages of environmental friendliness and no pesticide residues.At present,many natural metabolites with antifungal activity have been screened.[Vanderbylia robiniophila（Murrill）B.K.Cui&Y.C.Dai]is an important medicinal fungus.It is mainly distributed in Liaoning,Shandong,Hebei,Beijing,Shanxi and Henan.Wild V.robiniaphila resources are scarce,artificial cultivation is difficult,a large number of stable secondary metabolites can be obtained by liquid fermentation technology.At present,the research on V.robiniaphila is mostly focused on anti-cancer treatment,there is no report on the antibacterial activity against ginseng pathogenic fungi and fermentation broth chemical constituents of ginseng.Therefore,this paper studies the inhibitory effect of plant fermentation broth on ginseng pathogenic fungi was studied and the chemical separation was carried out systematically,and find out the active ingredients which can inhibit ginseng pathogenic fungi,In order to lay a foundation for the application of V.robiniaphila in the biological control of ginseng pathogenic fungi,it also provides a theoretical basis for the development and utilization of V.robiniaphila in natural fungicides,and the active components with inhibitory effect on Sclerotinia ginseng were obtained.In this study,V.robiniaphila as the research object,through single factor and orthogonal test to optimize its liquid culture conditions,on this basis,the effect of ethyl acetate extract of V.robiniaphila fermentation broth on the growth of ginseng pathogenic fungi mycelia was studied,and its inhibition mechanism was determined,and its chemical composition and antifungal activity of monomer compounds were determined.The main results are as follows:1.Optimization of liquid culture conditions of V.robiniaphilaThe suitable carbon source for the growth of V.robiniaphila mycelium was soluble starch,followed by fructose and sucrose by single factor test;The suitable nitrogen source was yeast extract,followed by yeast extract and beef extract;The composition of V.robiniophila medium was optimized by orthogonal experiment,subsequently,the optimal medium formula is obtained:sucrose 20 g,yeast extract 2 g,KH₂PO₄2 g,Mg SO₄1 g,distilled water 1 L;The optimal liquid culture conditions of V.robiniaphila was obtained through the selection of temperature,initial p H and inoculum amount:temperature 22℃,initial p H 6.0 and inoculum amount 12%.2.Inhibitory effect of ethyl acetate extract from V.robiniaphila fermentation broth on ginseng pathogenic fungiIn the preliminary screening experiment,the extract had the best inhibitory effect on the mycelial growth of S.ginseng.When the concentration of the extract was 20 mg/m L,the inhibition rate was 100%.Along with the increase of the concentration of the extract,the inhibition gradually increased,MIC was 2.2 mg/m L,MFC was 10.00 mg/m L.When the extract concentration was MFC,the spore germination rate of S.ginseng was 2.2%,and the inhibition rate of spore germination was 93.6%;When the concentration of the extract was MIC,the spore germination rate of S.ginseng germination was 10.0%,and the inhibition rate of spore germination was 88.4%.The results showed that the extract had an obvious inhibitory effect on mycelial growth and spore germination of S.ginseng.Under the light microscope,the extract can make the mycelia of S.ginseng grow abnormally,wilt and shrivel in shape,accompanied by the phenomenon of hyphal cell rupture,and a large number of internal solutes leak out.After 1 h of culture,the conductivity and nucleic acid content of different treatment groups（MIC and MFC）were 1.2 and 1.8 times higher than those of the control group,respectively;After 12 h treatment,MDA content of different treatment groups increased by 28.89%and 61.74%respectively,and H₂O₂ Content of different treatment groups increased by 47.27%and 62.39%respectively.After 48 hours of treatment,SOD activity of different treatment groups decreased by 10.04%and 44.17%,POD activity decreased by 16.88%and 37.66%,respectively.The results showed that the ethyl acetate extract of V.robiniaphila fermentation broth could make the mycelium growth of S.ginseng abnormal,damage the cell membrane,destroy the integrity of the membrane,and eventually lead to the leakage of the contents of the cell tissue and inhibit the mycelium growth.3.Chemical constituents and monomer activity of ethyl acetate extract from V.robiniaphila fermentation brothThe ethyl acetate part of V.robiniaphila fermentation broth was isolated using chromatographic techniques and identified by NMR.Sixteen compounds were isolated and identified as uracil（1）,methyl 3,4-dihydroxybenzoate（2）,3,4-dihydro-xybenzoicacid（3）,1-phenyl-1,2-propanediol（4）,cyclo（Leu-Pro）（5）,cyclo（Phe-Ala）（6）,cyclo（4-S-hydroxy-R-Pro-R-isoleucine）,（7）,cyclo（Phe-Gly）（8）,cyclo（D-Hpro-L-Phe）（9）,cyclo（L-Phe-L-Hpro）（10）,methyl 4-hydroxybenzoate（11）,p-hydroxybenzoicacid（12）,p-hydroxybenzaldehyde（13）,ethyl 3,4-dihydroxybenzoate（14）,cyclo（Ala-Ile）（15）,cyclo（D-Leu-trans-4-hydroxy-L-Pro）（16）.The above compounds were isolated from the fungus fermentation liquid for the first time.Among them,compounds 2,3,9～14 had good inhibitory effect on S.ginseng.