Secondary Metabolites From Endophytic Fungi Of Artemisia Argyi Origin And Their Activity Against Marine-derived Aquatic Pathogens

Aquaculture industry has made great contribution to food security and is one of the fastest growing industries in large-scale agriculture.However,with the expansion of the scale of aquaculture and the increase of intensification,the frequency and degree of disease occurrence are also increasing.Especially in recent years,the diseases caused by aquatic pathogenic bacteria are also continuously erupting on a large scale.Therefore,it is urgent to study and discover new compounds of antimicrobial activity against aquatic pathogens.The natural products have ideal inhibitory effect on marinederived pathogens and have research value in the field of aquaculture.The chemical structures of natural product from fungal kingdom are novel and variable,and with significant pharmacological values as well.Microbial secondary metabolites play an important role in the communication of those within microbial species,microbial interspecies,and cross-border species.It is likely easier to find active substances for anti-marine pathogens from the species that genetically far from marine organisms.Endophytic fungi have been an essential resource of the novel and bioactivity natural products,and the organic extract of the fungal culture exhibited antimicrobial activity against several marine-derived pathogens in our primary screening,but the chemical constituents of endophytic fungi from A.argyi have not yet been reported.Therefore,we studied the secondary metabolites of endophytic fungi isolated from A.argyi,with the aim to discover new leading compounds of antimicrobial activity against aquatic pathogens.A total of 12 endophytic fungal strains were isolated from the medicinal plant Artemisia argyi.The 12 strains were initially fermented in PDA,MH2 and rice solid medium for screening,and the crude extracts were evaluated for the activity against marine aquatic pathogenic bacteria,and HPLC and TLC analysis were also performed.As a result,two fungal strains,Trichoderma koningiopsis QA-3 and T.virens QA-8 were selected for further chemical investigation.In the antibacterial activity tests,the crude extracts of QA-3 and QA-8 exhibited antimicrobial activities against marine aquatic pathogens Vibrio alginolyticus,Aeromonas hydrophilia and V.parahaemolyticus.In addition,the crude extracts of QA-3 also showed some inhibition against Edwardsiella tarda and V.harveyi.Chemical study of the fungal strain T.koningiopsis QA-3 resulted in the isolation of a total of thirty-eight secondary metabolites,with eighteen of them are new compounds,including fifteen new polyketides(TKO1–TKO15),one new α-pyrone derivatives(TKO16)and two new terpenoids(TKO17 and TKO18).The activity of TKO1 against V.vulnificus was stronger than that of the positive control chloramphenicol(MIC,4.0 μg/mL vs 8.0 μg/mL).TKO6 showed strongest activity against V.alginolyticus(MIC=1.0 μg/mL),while TKO8 against V.harveyi(MIC=4.0 μg/mL),TKO11 against Ed.tarda(MIC=2.0 μg/mL),and TKO29 against M.luteus(MIC=1.0 μg/mL),which are comparable to that of the positive control chloramphenicol.Chemical investigation of the fungal strain T.virens QA-8 resulted in the isolation of a total of forty-three secondary metabolites with different planar structures,with twenty of them are new compounds,including twelve new cadinane sesquiterpenes(TV1–TV12,three of which are halogenated compounds)and eight new carotene-type terpenoids(TV13–TV20).Compound TV6 had activity against Ed.tarda and V.anguillarum with MIC values 1.0 and 2.0 μg/mL,respectively,which is comparable to that of the positive control(chloramphenicol,with MIC values 0.5 and 1.0 μg/mL,respectively).Compounds TV7 and TV8 had inhibitory activity against all tested pathogenic strains with MIC values ranging from 0.5–64 μg/mL,while compounds TV11 and TV12 have activity against all other seven tested strains,except for M.luteus,with MIC values ranging from 0.5–16 μg/mL.Compounds TV14–TV18 and TV23–TV25 have inhibitory activity against M.luteus with MIC values ranging from 0.5–32 μg/mL,and the activity of compounds TV14 and TV16 were similar to or stronger than that of the positive control.In conclusion,a total of 81 metabolites with diverse scaffolds were isolated and identified from the extracts of the above fungal strains,and 38 of them were new compounds.The evaluation of antimicrobial bioactivity showed that several compounds had potential to be developed as anti-aquatic bacterial agents.Our study not only enriched the structure types of natural products,but also provided some reference value for the follow-up research on discovering new types of anti-aquatic bacterial compounds.