Antifungal Activity And Mechanism Of Endophytic Fungus SYS-5-2 From Walnut

Walnut is a traditional dual-purpose plant for medicine and food in China.It is necessary to further improve the development of walnut resources and industrial added value because of its large planting amount and wide distribution.At the same time,the endophytic fungi of walnut are rich in biodiversity.The fermentation products and secondary metabolites of endophytic fungi have a good inhibitory effect on many pathogens,and also have a more obvious inhibitory or promoting effect on plant growth.In order to explore the possibility of endophytic fungi as a safe and efficient bioactive substance,this study comprehensively evaluated the endophytic fungi generation of walnut through the pathogen antagonistic test The bacteriostatic activity of the metabolite was used to select the highly active strains,and the bacteriostatic mechanism of its highly active components was further studied at the cell level and physiological and biochemical level.The results are as follows:?1?The high activity strain SYS-5-2 was screened by plate confrontation and growth rate method,and it was classified as Schizophyllum commune according to morphological characteristics and phylogenetic analysis of ITS sequence.?2?After ethanol extraction,the yield of each extraction phase was N-butanol phase?18.03%?>ethyl acetate phase?11.06%?>petroleum ether phase?1.69%?,the inhibition rate of each extraction phase was ethyl acetate phase?65%?100%?>N-butanol phase?40.58%?100%?>petroleum ether phase?17.24%?28%?.In the gradient concentration antibacterial activity test,the ethyl acetate extract had an antifungal rate higher than 80%against the Thanatephorus cucumeris,and the IC₅₀was only 10.28 mg/m L.?3?The antifungal activity of the components extracted from ethyl acetate decreased gradually with the separation,which indicated that the high antifungal activity of the endophytic fungus SYS-5-2 was produced by the synergism of various substances.Oleanolic acid,a broad-spectrum antimicrobial terpenoid,was identified in the high active component by HPLC-MS,which was the first identification in the fermentation products of endophytic fungi of walnut.?4?After the treatment of high active components,the surface of the mycelium of the pathogen was shriveled and deformed under the scanning electron microscope;the conductivity value of the mycelium culture medium increased significantly with the increase of time;the soluble protein content in the mycelium of the pathogen remained stable basically;the total polysaccharide permeability increased gradually with the increase of the concentration of high active components;the activity of SOD,pod and PPO in the recipient plant capsicum increased significantly and it began to decline or stabilize after reaching the peak on the 4th day,and the effect of high active components was slightly higher than that of salicylic acid treatment.In conclusion,the high activity endophytic fungus SYS-5-2 was screened and identified as Schizophyllum commune,the ethyl acetate phase was highly active after ethanol extraction,after column chromatography,it can be seen that the high antifungal activity is produced by synergism of many substances,Oleanolic acid,a terpenoid substance with broad-spectrum bacteriostasis,was first identified in the high active component of endophytic fungus fermentation product of walnut,in the study of mechanism,it was proved that the high active components play the role of bacteriostasis by destroying the membrane permeability of pathogenic bacteria and enhancing the enzyme activity in plants.