Isolation,identification Of Endophytic Fungi From Loranthus Tanakae And Study On Secondary Metabolites Of Alternaria Alternata

Objective:Endophytic fungi were isolated and purified from a medicinal plant,Loranthus tanakae.Strains were identified based on morphological analysis combined with molecular biological method.High activity strains with anti-oxidant and bacteriostatic activities were screened.The ethyl acetate extracts were obtained by large-scale fermentation of the active strains.The secondary metabolites of the active strains were isolated,identified,and analyzed for their activity.This project provides a reference for the development and utilization of endophytic fungal resources in L.tanakae.Methods:(1)Set different disinfection time and different concentrations of sodium hypochlorite for surface disinfection of L.tanakae leaves,Endophytic fungi of L.tanakae were isolated by tissue inoculation culture,and purified by hypha tip picking method.Identification of endophytic fungi using morphology combined with ITS sequence alignment.(2)Small-scale liquid fermentation of L.tanakae endophytic fungi,Extraction of endophytic fungus fermentation broth with ethyl acetate,concentrating under reduced pressure to obtain an extract,and measuring its DPPH radical scavenging activity and ABTS~+radical scavenging activity.(3)Further screening of strains with antibacterial activity,the filter paper agar diffusion method was used for the preliminary screening of bacteriostatic activity,and the minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of the bactericidal active strain after the initial screening were determined by 96-well plate method.(4)The large-scale liquid fermentation experiment was performed on the active strains,and the secondary metabolites were separated by column chromate-graphy and other methods.The structure of the monomeric compounds was identified by spectroscopy,and the antioxidant and antibacterial activities of the monomeric compounds were determined.Results:(1)The L.tanakae leaves disinfection conditions were determined as follows:5%ethanol immersion and disinfection for 1 minute,and sterilized water was washed 3times;5%sodium hypochlorite solution was immersed and disinfected for 5 minutes,and sterilized water was washed 3 times;and finally,immersion disinfection with 75%ethanol 30 seconds,rinse 5 times with sterile water.A total of 29 endophytic fungi of L.tanakae were isolated and purified,belonging to 17 genera,including Botryo-sphaeria,Phoma,Fusarium,Didymella,Alternaria,Penicillium,Neonectria,Clados-porium,Didymella,Epicoccum,Dothideomycetes,Dothiorella,Cytospora,Peyronellaea,Leptosphaerulina,Colletotrichum,Diaporthe.Among them,the dominant floras are Alternaria,Penicillium and Colletotrichum.(2)When the sample concentration is 0.02~0.2 mg/mL,the fermen-tation products of the endophytic fungi BS06,BS07 and BS27 of L.tanakae have better scavenging ability to DPPH free radicals,and slightly weaker ABTS~+free radical scavenging activity.The fermentation product of strain BS27 has the highest scavenging rate for DPPH free radicals,when the sample concentration is 0.2 mg/mL,the scavenging rate reaching95.17%.(3)When the sample concentration is 50 mg/mL,the fermentation products of endophytic fungi BS06,BS07 and BS27 have inhibitory ability on Staphylococcus aureus and Bacillus subtilis,but have no obvious inhibitory effect on Escherichia coli and Proteus species.Among them,the endophytic fungus BS27 has the strongest bacteriostasis ability.The inhibition zone for S.aureus reaches 15.60 mm,the MIC value is 12.25 mg/mL,and the MBC value is 25 mg/mL.The inhibition zone for B.subtilis reaches 14.35 mm,the MIC value was 6.25 mg/mL,and the MBC value was 12.5mg/mL.(4)Three compounds were isolated from the second metabolites product of BS27and identified as alternariol-5-O-methyl ether(1),alternariol(2),cis,cis-9,12-octadecadienoic acid(3),respectively.Compounds 1 and 2 have weak scavenging ability to ABTS~+free radicals,at a concentration of 0.2 mg/mL,the scavenging rate of ABTS~+radical by Compound 1 was 16.15%,and the scavenging rate of ABTS~+radical by Compound 2 was 21.49%.Compounds 1 and 3 have certain antibacterial activity.Compound 1 showed antibacterial activity against E.coli,P.species,S.aureus,and B.subtilis,which the MIC of 250?g/mL and the MBC of 500?g/mL.The MIC of compound 3 to E.coli was 500?g/mL,MBC was 1 000?g/mL,MIC to S.aureus and B.subtilis was 250?g/mL,and MBC was 500?g/mL.Conclusion:Endophytic fungi are abundant in L.tanakae.29 endophytic fungi isolated and purified for the first time,which were identified as 17 genera by morphological and ITS sequence comparison.Alternaria,Penicillium and Colletotrichum are the dominant floras.Among them,the fermentation product of Alternaria endophytic fungus BS27 has good antioxidant activity and antibacterial activity.The secondary metabolites of the active strain BS27 were studied,and three compounds were isolated.The preliminary exploration of the endophytic fungal resources of L.tanakae provides reference basis for the further research of endophytic fungi resources of L.tanakae.