Study On The Secondary Metabolities From Two Fungi

Biodiversity of microbial resources determines structural diversity and variety of biological activities of their metabolites. As an important member of the microflora, fungi have immense number of species, extremely wide distribution, as well as miscellaneous biological activities for their secondary metabolites, such as antimicrobial, antitumor, regulating plant growth and so on.In this study, the secondary metabolites of two fungi were investigated. Strain 443 preserved in our lab, which had been isolated from the rhizosphere soil of tomato in Suqian, Jiangsu Province, China. Strain 443 was identified as Irpex sp. according to colony morphology and molecular biological methods. The other strain named By254 was endophytic fungus Phomopsis sp.. The soid fermentation products of the two fungi were isolated and purified, with a view to finding compounds which had efficient bioactivities.According to antagonistic test in vitro, the petroleum ether extracts of fermentation products of strain 443 exhibited favorable activities against Sclerotinia sclerotiorum, Botrytis cinerea, Phytophthora capsici, Pythium ultimum, Rhizoctonia cerealis and had obvious inhibitory effects against Botrytis cinerea and Phytophthora capsici, particularly. We further validated the potential value of strain 443 in terms of biological control efficacy against Phytophthora blight and growth-promoting for pepper in the greenhouse. Moreover, according to colony morphology and homology analysis of ITS sequence, strain 443 was identified as Irpex sp. Accession number in GenBank of NCBI is JF737033.Solid-state fermentation of strain 443 was carried out on wheat bran media, fermented materials were extracted by mixture of chloroform/methanol (1:1, v/v) and then evaporated the solvent in vacuo to give crude extract, resuspended in water, and then extracted with petroleum ether. After that,10 g of the petroleum ether extract was separated by silica gel column (200 g,200-300 mesh) eluated by gradient of acetone:petroleum ether with grandient of 0:100→1:100→5:100→10:100→20:100→40:100→80:100→100:0.Eight fractions were obtained, then each fraction was separated by column chromatography, thin layer chromatography (TLC), high-performance liquid chromatography (HPLC), recrystallization, etc, obtaining six compounds (compound 1-6). The configuration of compounds was determined by mass spectrometry (HR-ESI-MS, ESI-MS), magnetic resonance (1H-NMR,13C-NMR, DEPT,13C-1H-HSQC, 13C-1H-HMBC,1H-1H COSY) etc, and finally we have determined them as sterols or cerebrosides.Compound 1-5, which were separated from Irpex sp.443, are ergosterol, chol -esterol,3β,5α,6β-trihydroxyergosta- 7,22 -diene (cereviosterol), (2S,2'R,3R,3'E,4E,8E)-1-O-D-glucopyranosyl-2-N-(2'-hydroxy-3'-octadecenoyl)-3-hydroxy-9-methyl-4,8-sphingadienine (cerebrosides C) and (2S,2'R,3R,3'E,4E,8E)-1-O-β-D-gluc -opyranosyl-2-N-(2'-hydroxy-octadecylselyl)-3-hydroxy-9-methyl-4,8-sphingadien -ine (cerebrosides D), respectively. Data of 1H-NMR of compound 6, which was isolated from Phomopsis sp., By254 was coincidence with cereviosterol, and was also detected and analyzed by HPLC. Finally, it was determined as cereviosterol.Ergosterol, cholesterol, cereviosterol, cerebrosides C, cerebrosides D were firstly isolated from Irpex sp. Cereviosterol was isolated from Phomopsis sp. for the first time. Although cerebroside C and cerebroside D only had feeble antagonistic activities against pathogenic bacteria, they could act as an elictor to activate plant defense response to confer resistance to plant pathogens and therefore relieve occurrence of plant disease in some extent. These bioactive compounds lay the foundation for further exploitation and development of biological pesticides.