Isolation,screening And Fermentation Optimization Of Endophytic Fungi Produing Anti-TMV Active Substances From Fructus Bruceae

In this paper,Endophytic fungi were isolated from different organs of the traditional Chinese medicine plant Brucea javanica.The second metabolites which were obtained by liquid shake-flask fermentation against TMV activity was measured to screen strains which could inhibit TMV infection and replication.Then,the strains were identified and fermentation conditions of which were optimized.Last,active substances against TMV were separated and purified.The main results are as follows:138 endophytic fungi were isolated from different parts of Brucea javanica such as the roots,branches,leaves and flowers,45 strains isolated from roots,45 from branches,45 from leaves,10 from flowers.Shaking flask fermentation at 25 ?and ratating speed of 120 r/min,after 7 days,the antiviral activity of the crude extracts of the fermentation broth against TMV were determined by leaf discs assay.The results showed that the 39 endophytic fungi's crude extracts at a concentration of 1 mg/mL were able to inhibit the TMV replication with the inhibition rate over 50%.Especially inhibition rate of the strain Rll at a concentration of 1 mg/mL reached 75.35%.The 39 endophytic fungi's crude extracts at a concentration of 1 mg/mL were used to examine their inhibitory activities Anti-TMV infection by half-leave method.Each strain's inhibition rate was tested 5 times.The result showed that the inhibition rate of 25 strains was higher than 80%at a concentration of 1 mg/mL,The best inhibition rate of Anti-TMV infection were 100%?Combined with the results of morphological identification and ITS molecular identification,the taxonomic status of the R11 was determined,which could inhibit TMV infection and proliferation.Rll was identified as,Aspergillus,aspergillus niger.Through the screening of fermentation medium,ShaShi medium was selected as a foundation for fermentation medium optimization.The optimal culture medium was determined by one factor experimental design and orthogonal experimental design.The results showed that the best culture medium of R11 produing anti-TMV active substances was 3%sucrose,1.5%yeast extrat,0.15%Cacl2,28?,15 days,125 r/min,the initial pH 6.0,15%initial inoculation.The inhibition rate of Anti-TMV infection of Rll's crude extracts at a concentration of 1 mg/mL of optimal culture broth was 98.98%,increased 2.1%compared with that of basic culture broth and the inhibition rate of Anti-TMV replication was 82.91%,increased 7.56%compared with that of basic culture broth.The active monomer was isolated and purified from fermentation broth of R11 with the column chromatography methods.The activity test showed that IC50 of Anti-TMV infection and replication of the monomer was 23.7 ?g/mL and 46.5 ?g/mL respectively.