Study On The Secondary Metabolites Of Two Strains Of Marine-Derived Fungi And Their Antibacterial Activities

Phytopathogens are one of the main factors that cause plant diseases and lead to significant economic losses to agriculture annually.The control agents of these two diseases rely on chemical pesticides for a long time,which led to pathogen resistance,pesticide residues and environmental pollutions.In recent years,bio-pesticides that are environmentally friendly,efficient,low-toxic,and resistant biopesticides have become hot spots for research and development.As marine fungi live in a unique marine environment,they can produce many secondary metabolites with novel structures and significant biological activities,which have become important sources for lead compounds of biopesticides.Among them,Aspergillus sp.and Trichoderma sp.are important sources of marine fungi-derived compounds.In this thesis,we obtained lead compounds of biopesticides with anti-phytopathogenic activity from a seaweed-derived Aspergillus sp.D40 and a mangrove-derived Trichoderma harzianum D13.It mainly includes the following research contents.1.Screening and identification of target strains.Fourty strains of marine-derived fungi preserved in the laboratory were screenedfor their anti-phytopathogenic activity against Phytophtora parasitica var.nicotianae and Ralstonia solanacearum.The agar diffusion method and mycelium growth rate method were used to test the antibacterial activity of the extracts.A total of eight fungal extracts including D4,D10,D12,D20,D33,D35,D38 and D40 showed antibacterial effects against P.parasitica var.nicotianae.The extracts of D12,D13,D14 and D40 exhibited antibacterial activity against R.solanacearum.Aspergillus sp.D40 and T.harzianum D13 were selected to be the target strains according to chemical and biological screening,and were identified by molecular and morphological methods.2.Separation and identification of compounds.Aspergillus sp.D40 and T.harzianum D13 were subjected to large-scale fermentation to obtain their extracts.Silica gel,reverse silica gel and Sephadex LH-20 gel column chromatography,as well as semi-preparative high-performance liquid chromatography?HPLC?were applied for compounds isolation and purification.UV,NMR,MS,ECD and other spectroscopic methods were used to identify the structures of compounds,including relative and absolute configurations.A total of 11 compounds?1–11?were isolated and identified from D40,including 10polyketones and one alkaloid,among which asperfuranone A–C?1–3?were new ones.Interestingly,compounds 1–4 and 9 exist as inseparable mixtures of a pair of epimers,and the relative configuration of compound 9b was first reported.Six compounds?12–17?were isolated and identified from D13,including two nafuredin compounds,one isocoumarin compound,and three dipeptide compounds.The absolute configuraiton of the new compound nafuredin C?12?and trichodermamide G?14?were determined by ECD calculation.Notably,trichodermamide G?14?had a novel structure of 4,7 thiobridged benzoxazine which was first reported.3.Determination of antibacterial activity of compounds and optimization of Aspergillus sp.D40fermentation.All of the isolated compounds were subjected to their antibacterial activity against R.solanacearum,Xanthomonas campestris,Acidovorax avenae,Xanthomonas citri,Clavibacter michiganensis,Erutima carafavora,and antifungal activity against P.parasitica var.nicotianae,Alteraria alternata,Magnaporthe grisea,A.brassicicola,Pestalotiopsis theae.Penicillic acid?7?displayed significant antibacterial activity against six plant pathogenic bacteria with IC₅₀ values ranging from 11.6to 58.2?g/mL.The new compound 12 showed anti-phytopathogenic activity against four plant pathogens,and compounds 12 and 13 exhitied significant antifungal activity against M.grisea,with MIC of 3.13 and6.25?g/mL,respectively.As penicillic acid is toxic to plants,animals,and humans,we used one strain many compounds?OSMAC?fermentation optimization to further explore the antibacterial potential of D40 by changing the culture media.It revealed that the broth extract of malt extract seawater medium?BMEW?exhibited the strongest antibacterial activity against R.solanacearum,whereas a mycelial extract from the malt extract seawater medium?MMEW?was inactive.BMEW and MMEW were compared using HPLC-HRMS-based metabolomics to study into their composition and to guide future isolation of bioactive compounds.Phenylpropanoids and small molecular phenolic derivatives were the main constituents of the 109 up-regulated compounds and may be related to the antibacterial activities of BMEW,and provided a basis for the subsequent isolation and identification of antibacterial compounds from this fungus.