Structures And Bioactivities Of Secondary Metabolites From Four Marine-Alga-Epiphytic Trichoderma Strains

The structurally diverse and active secondary metabolites of marine Trichoderma spp.are important sources of small molecule natural products and drug development.By the end of 2021,more than 250 species of Trichoderma strains have been publicly reported,and no more than sixteen species of marine Trichoderma have been studied for their secondary metabolites,including no more than nine species of marine algicolous Trichoderma.Most of the current research on marine algicolous Trichoderma strains has focused on alga-endophytic Trichoderma fungi,and less on alga-epiphytic Trichoderma fungi.Herein,the chemical structure and biological activity of secondary metabolites from marine alga-epiphytic Trichoderma fungi were investigated.In July 2018,four hundred and seventeen strains of algae epiphytic fungi were isolated from the surface of alga obtained from the sea near Zhoushan,Zhejiang Province,which constructed a library of algae epiphytic fungi strains.After that,four algae epiphytic fungi strains including Trichoderma hamatum Z36-7,T.virens Z8-7,T.reesei Z56-8,and T.aethiopicum Z58-7 were selected as the research object through bioactive and chemical screening.In total,one hundred and nineteen secondary metabolites were isolated and identified,including 7 new compounds.The structural types included cyclonerane sesquiterpenes,bisobolane sesquiterpenes,acorane sesquiterpenes,cadinane sesquiterpenes,carotane sesquiterpenes,diterpenes,alkaloids,diketopiperazines,benzene derivatives,and sterols.Some isolates were evaluated for their anti-microalgal and anti-bacterial activities and toxic effects on Artemia salina and Brachionus plicatilis.Reversed-phase column chromatography,Sephadex LH-20 gel column chromatography,preparative thin-layer chromatography,and semi-preparative high performance liquid phase were used to obtain the monomeric compounds.Modern spectroscopic techniques such as 1D/2D NMR,UV,MS,IR,and comparison with literature data were used to identify the structures of the monomeric compounds.A total of forty-four compounds were isolated from the fermentation product of T.hamatum Z36-7,including five new compounds(TH1*-TH5*).The new isolates comprised two cyclonerane sesquiterpenes as well as two polyketides,and one new natural product(TH3*)containing halogen.A total of twenty-six compounds were isolated and identified from the fermentation of T.virens Z8-7,including one new cadinane sesquiterpene(AV1*).Thirty compounds were isolated and identified from the culture of T.reesei Z56-8,including one new vertinolide compound(TR1*),and nineteen compounds were isolated from the culture of T.aethiopicum Z58-7.Some of the isolated secondary metabolites were evaluated for their biological activities against Colletotrichum lagenarium,C.gloesoporioides,Fusarium oxysporium f.sp.cucumerinum,F.oxysporum f.sp.cubense,Glomerella cingulate,Rhizoctonia solani and antimicrobial activity against Vibrio harveyi,V.anguillarum,V.splengidus,V.parahaemolyticus as well as brine shrimp lethality against A.salina and lethal activity against B.plicatilis as well as the microalgal inhibition of Heterocapsa circularisquama,Prorocentrum lima,Heterosigma akashiwo,P.minimum,P.micans,P.donghaiense,Amphidinium carterae,Chattonella marina.The results showed that the polyketides compounds TH28 and TH30 exhibited highly significant inhibitory activity against Chattonella marina with IC50 less than0.1 μg/m L.The compound TR1 from T.reesei Z56-8 exhibited significant inhibitory activity against P.donghaiense with IC50 2.8 μg/m L.TH20 and TR20 displayed good inhibitory effects on all four species of microalgae and may have broad-spectrum antimicroalgal activity.The results of the antifungal activity showed that the sorbicillin compound TR20 exhibited significant inhibitory activity with a minimum inhibitory concentration of 3.9 μg/m L against G.cingulata.Compounds TA9,TH20,TH32,TR22,and TV22 showed moderate inhibitory activity against G.cingulata with MIC values ranging from 15.6 to 62.5 μg/m L.Compounds TR20 and TR22 indicated moderate inhibitory activity against F.oxysporium f.sp.cucumerinum with MIC values of 15.6 and 62.5 μg/m L,respectively.The results of lethal activity against B.plicatilis showed that compounds TA14,TH21,TR11-12,TR20,TR22,TV4 and TV16 had high toxicity with IC50 values of 1.0 to 7.8 μg/m L.In this dissertation,the study on the secondary metabolites of T.aethiopicumZ58-7 and the marine origin of T.hamatum Z36-7 were the first time.More than thirty compounds were found to have significant anti-microalgal and antibacterial activities and Brachionus plicatilis toxicity.The research not only enriched the study of natural products from marine Trichoderma strains,but provided theoretical foundation for development and utilization of marine Trichoderma.