Identification And Metabolites Of Three Fungal Strains From Artemisia Annua Linn.

Three endophytic fungi,coded SPS-02,03,04,were isolated from healthy stems of Artemisia annua Linn.On the basis of morphorgical character,culturing property and phylogenetic tree,three strains had been respectively classified as Aspergillus fumigatus,Fusarium oxysporum and Alternaria alternata.A total of 12 metabolites were isolated and 10 compounds were unambiguously identified by a combination of spectroscopic methods.Bioassay indicated that 8 compounds had inhibitory effect on the human pathogenic fungus Canidia albicans.The results were described in detail as follows:Firstly,strains SPS-02,03,04 appeared different features in physiology and biochemistry.SPS-02 colony was light green and exsited the micromorphological structures of conidiophore planted in the matrix,septate hypha and spore spherical.SPS-03 appeared on white colony and the structures of oval conidia,penicillate-capitate and branched filamentous hypha.SPS-04 showed on black colony,radial grooves and smooth,rod diaphragm with inner bud spore.Secondly,physiological study indicated that strain SPS-02 had the strongest vitality,which can grow at high concention(≥200 μg/mL)of ampicillin,penicillin and streptomycin,jinggangmycin,amphotericin and sodium hypochlorite.SPS-03,04.SPS-02 could utilize different kind of carbon and nitrogen source for rapid growth.In contrast,the ability of utilizing carbon of SPS-03 was weak and SPS-04 could not grow normally in the ammonium salts or amide compounds.The antimicrobial evaluation indicated that strain SPS-02,03,04 had inhibitory effects on Escherichia coli and Staphylococcus aureus.Especially SPS-02 could strogly inhibited phytopathogenic fungi such as Alternaria alternate.The phylogenetic trees of SPS-02,03,04 were generated by 18 S rDNA sequences analyses using Clustal X and MEGA.SPS-02 were showed to be 99% in the homology and clustered in the same branch of the strain A.fumigatus(KF322139.1).SPS-03 was clustered in the same branch of the strain F.oxysporum(JN546125.1).And SPS-04 was close to the branch of the strain Alternaria sp.(GQ253348.1).Thirdly,after scale-up fermentation and extraction,the yields of crude extract of fermentation broth of strains SPS-02,03,04 were respectively 0.244 g/L,0.395 g/L,0.642 g/L.12 metabolites were isolated by p-HPLC and UV-LC.Up to now,10 compounds had been identified to be N-(2,4-dihydroxybenzoyl)-L-serine(1),2,4-dihydroxybenzoic acid(2),2H-1-benzopyran-2,5(6H)-dione(3),bisdethiobis(methylthio)gliotoxin(4),bis(2-ethylhexyl)-phthalate(5),5-butyl-2-pyridinecarboxylic acid(6),oleic acid(7),dodecanoic acid(8),methyl 2,6-dihydroxy-4-methylbenzoate(9),methyl undecanoate(10).Compound 1 was firstly isolated from an endophtic A.fumigatus.Lastly,among these compounds,compounds 4 and 5 were shown to strongly inhibit C.albicans with MIC values of 0.899,0.821 μM,respectively.Compound 2,3,6,7 and 10 also had antimicrobial activity against C.albicans with the range of 1.79~8.84 μM.However,these compounds(1-12)exhibited weak inhibitory effects on E.coli and S.aureus(MIC>60 μM).These findings provided the experimental basis for systematic investigation on biodiversity and chemodiversity of endophytic microbes from A.annua.