Selection Of Highly Active Endophytic Fungi From Juglans Regia L. And Its Metabolites Antifungal Mechanism(?)

Endophytic fungi from Juglans regia L.have rich biodiversity.In order to make better use of this microbial resources,this study selected the active endophytic fungi from walnut,optimized fermentation conditions,separated and purified metabolites,and studed inhibition mechanism.The main results are as follows:?1?Using the growth rate method,6 common plant pathogens were used as indicator fungi to select 16 endophytic fungi of walnut.The results showed that fermentation product of endophytic fungus G3 derived from the roots of improved walnut trees showed a certain antifungal activity against the 6 pathogens.And the inhibition rate of Valsa ceratosperma reached 100%,which completely inhibited the growth of pathogenic mycelium;the inhibition rate of Thanatephoru-s cucumeris?Frank?Donk.and Gloeosporium fructigenum were more than 75%.The G3 strain was identified as Fusarium camptoceras by observation of morphological features combined with molecular biology identification.?2?The inoculation amount,fermentation temperature and fermentation days were taken as the influencing factors,and the inhibition rate of G3 fermentation product on Valsa ceratosperma was evaluated.The fermentation conditions of walnut endophytic fungus G3were optimized by single factor experiment and orthogonal test.The results showed that the optimal process of G3 fermentation was the inoculation amount is 10%,the fermentation temperature is 28?,and the fermentation time is 30 d;under this condition,the inhibition rate of G3 fermentation product against Valsa ceratosperma was 85.17%.The influence of the influencing factors on the inhibition rate was as follows:fermentation temperature>fermentation days>inoculation amount.?3?The ethanol extract of the endophytic fungus G3 solid fermentation product was extracted with petroleum ether,ethyl acetate and n-butanol respectively by liquid-liquid distribution method.The results showed that the IC₅₀ values of n-butanol phase and ethyl acetate phase against Valsa ceratosperma were 3.3 mg/mL and 4.3 mg/mL,respectively.Combined with the quality of each extract phase and its inhibitory effect on Valsa ceratosperma,the n-butanol phase and the ethyl acetate phase were chosen as the active extract phase for further study.?4?By pre-testing the chemical components of each extract phase,the results show that the n-butanol phase mainly contains organic acids,and the ethyl acetate phase is mainly Phenols.It is speculated that the active compounds of G3 fermentation products may be organic acids and phenols.The extracting phase of ethyl acetate was separated by column chromatography to obtain the highly active fraction Fry2-2.When the concentration was 1mg/mL,the inhibition rate of Valsa ceratosperma reach 89.36%.A total of 15 compounds were detected by GC-MS analysis and they are mainly amines,lipids,phenols and alkanes.After further separation of Fry2-2,Compound A was precipitated in the Fry2-2-2 component.After recrystallization purification and nuclear magnetic resonance detection,compound A was identified as 2,2'-methylenebis[6-?1,1-dimethylethyl?]-4-methyl-phenol and the IC₅₀value of inhibiting Valsa ceratosperma is 0.11 mg/mL.?5?Under the conditions of in vitro and in vivo,the mechanism of G3 highly active fraction inhibiting Valsa ceratosperma was studied.The results showed that under the treatment of highly active fraction,the mycelium of the pathogen was damaged or torn,or the spindle is formed at the tip,which caused abnormal growth and decreased mycelial volume;the conductivity of the pathogenic fungal liquid increases,the ergosterol content decreases,and the chitinase activity increases.The activity of defense enzymes SOD,PPO and POD in susceptible apples increased significantly.The defensive enzyme activity showed a similar trend of increasing first and then decreasing under the treatment by high-activity components and 5 mmol/L salicylic acid.And the high-activity components can significantly inhibit the expansion of Valsa ceratosperma lesions,the maximum inhibition rate is 77.61%.