Studies Of Antimicrobial Secondary Metabolites From 29 Freshwater Fungi

In recent years,due to the excessive use of antibiotics,the resistance of various pathogenic microorganisms to various drugs has shown an upward trend.The search for new antibiotics with broad-spectrum antibacterial effects has always been a hot spot of researchers.Secondary metabolites of fungi are the best source of new drug discovery.In this study,the screening,composition and mechanism of the secondary metabolites of freshwater fungi were investigated.The submerged specimens were collected from freshwater habitats in Thailand.A total of 29 fungal taxa are recorded,in which three taxa are described as new species.They are Chloridium aquaticum,Chloridium aseptatum and Dictyochaeta aquatic in Chaetosphaeriaceae,Sordariomycetes.Chloridium aquaticum is the only species having proliferating conidiophores and holoblastic conidiogenous cells in Chloridium.A detailed description and illustrations of the taxa is provided and phylogenetic relationship between the new taxa and their relatives are compared and discussed.The Et OAc extracts of rice fermentation culture of 29 freshwater fungi are assayed in vitro against three gram-negative bacteria,three gram-positive bacteria and three pathogenic fungi strains using the oxford-cup method.Antibacterial activity test shows that seven samples have antibacterial activity against all six bacteria.The proportion is 20.7%.They belong to seven genera including Clohesyomyces,Dictyochaeta and Dictyosporella.Gram-positive bacteria are inhibited by 15 samples(51.7%)and Gram-negative bacteria are inhibited by 10 samples(34.5%).Antifungal activity tests shows that Annulatascus velatisporus has the strongest antifungal activity against all three pathogenic fungi.Another 16 samples also have antifungal activity against 1-3 pathogenic fungi.After conducting chemical fingerprinting,the strongest antibiotic activities are recorded in Dictyochaeta aquatic and Distoseptispora sp..Nine polarity section A-I were isolated from Et OAc extract of Dictyochaeta aquatic E and F were found have the strongest antibiotic activities.To study the antibacterial mechanism of E and F on E.coli and Staphylococcus aureus.Ion leakage measurement,scanning electron micrographs,large molecules leakage measurement and AKP leakage measurement to determine the effect of E and F on cell membrane.Agarose gel electrophoresis was carried out to analyze the effect of E and F on DNA.SDS-PAGE patterns of proteins was carried out to analyze the effect of E and F on proteins.The result was shown that E and F could change cell membrane permeability.DNA of E.coli treated with F was extracted and compared with blank control group,the band of DNA of experiment group emerged dimmed and trailing.In others,E and F can act on proteins of E.coli and Staphylococcus aureus.Three pure compounds are separated from Et OAc extract of Dictyochaeta aquatica by silica gel column chromatography and recrystallization.The structure was identified by NMR and literature,including two steroids: ergosta-5,7,22-triene-3?-ol(1)and 5?,8?-epidioxyergosta-6,22-dien-3?-ol(2);a compound: TAN-1496 B(3).In addition,the separation components of Dictyochaeta aquatic were analyzed,and 31 methyl esterification components were identified;18 separation components of Distoseptispora sp.were analyzed.Among them,octanoic acid,hydroxydecanoate acid,palmitic acid,lauric acid,myristic acid,palmitoleic acid,oleic acid,linoleic acid,stearic acid and arachidonic acid are all fatty acids with antibacterial activity.In this study,the mechanism of the secondary metabolites of freshwater fungi with antibacterial activity and the preliminary identification of active components were established,which laid a foundation for the effective utilization of freshwater fungi resources.