Isolation,Purification And Structural Identification Of The Antifungal Substance From Fermentation Broth Of Trichoderma Longibr-achiatum T05

Trichoderma is a kind of biocontrol fungi existing widely in the nature,which has a variety of biocontrol mechanisms.Trichoderma longibrachiatum T05 was collected in the northeast area and was tolerant of the low temperature,so it was of great importance to do research about its prevention mechanism and secondary metabolites to control plant diseases of cold area in northern.In this paper,a series of studies on the T05 including antagonistic mechanism,optimization of antimicrobial capacity of fermentation broth,stability of fermentation broth,isolation,purification and structure identification of secondary metabolites were carried out.Findings as follows:When T05 and pathogenic fungi were cultured alone,T05 grew fastest and showed its strong ability to occupy space and nutrients.The volatile substances produced by T05 had some antifungal activity against the Botrytis cinerea the Botrytis cinerea,which made the hyphae of the pathogen become twisted,dry and thin.Testing juncture hyphae under microscope discovered that,strain T05 compete with pathogenic fungi for space and nutrients by winding and coverage and some hyphae of T05 also invade to absorb nutrients in the hyphae of pathogenic fungi.Ethyl acetate extract of T05 fermentation liquid showed obviously antifungal activity against 5 kinds of pathogenic fungi.Research showed that the inhibition rate of the extract of fermentation broth was different by using different culture methods.By screening,the best fermentation combination of the highest inhibition rate was obtained:oscillating culture,culturing for 6 days,temperature 20?,initial pH=6,and the initial inoculum volume 0.5mL in 500mL conical flask bottled 250mL medium.The results of the stability of fermentation broth of T05 showed that strong acids showed greatly effect to the fermentation broth of T05.Temperature(less than 100?)had little effect on the antimicrobial activity of the fermentation broth,but high temperature and pressure had a certain effect on the antifungal activity of the fermentation broth.The ultraviolet radiation has little effect on the antibacterial activity of the fermentation broth,and the short time of the inhibition rate is unchanged.In the light of 10d,the inhibitory rate of fermentation broth was relatively stable,which was decreased obviously after 10d.With the increase of the concentration of oxidant and reducing agent,the inhibitory rate of fermentation broth decreased gradually.The Na+?K+?Fe2+?Fe3+?Mg2+and Cu2+showed less effect on the antifungal activity of the fermentation broth and the Zn2+ had certain effect on the antifungal activity of the fermentation broth.The fermentation broth is stable in normal temperature environment and can be stored for a long time without loss of activity.Isolation,purification and structure identification of the secondary metabolites with antifungal activity in the fermentation broth of T05 strain was researched by using TLC,Silica Gel Column Chromatography,Sephadex LH-20 Column Chromatography,UV,LC-MS and NMR by using active tracking method.The compound was bislongiquinolide and its molecular formula was C28H32O8 by UV,LC-MS and NMR analysis.