Study Of Secondary Metabolites From Two Pythium Species

Pythium species are a kind of fungi that belong to Oomycetes with wide distribution.Most Pythium species are phytopathogenic fungi,which have a wide host range and they can cause damage to plants such as Cucurbitaceae,Leguminosae,Liliaceae and Gramineae etc.Pythium species can infect the roots,stems,leaves and other parts of plants,causing seedling damping-off,seedling rot,root rot,stem rot,and the decay of fruits and seeds.Pythium species can produce the beneficial secondary metabolites to human such as eicosapentaenoic acid(EPA),folic acid,ascorbic acid,growth hormone,they can also produce a series of harmful secondary metabolites such as phthalate esters,4-hydroxy-3-methoxycinnamic acid,etc.Few studies have been reported on secondary metabolites of Pythium species.In order to explore whether the Pythium species could produce the main mycotoxins or other secondary metabolites which would be harmful to human beings or plants,we studied the secondary metabolites from two Pythium strains isolated from giner and tomato.A large-scale fermentation was carried out on rice culture medium,and the secondary metabolites were extracted with ethyl acetate.The crude extracts were separated and purified by natural products separation technology.As a result,we obtained 18 compounds,among of them,14 compounds were identified by the methods of nuclear magnetic resonance(NMR),mass spectrometry(MS)and literature comparison.The specific research contents are as follows:1.The samples of infected ginger collected in Anqiu,Weifang,Shandong Province and the samples of infected tomato collected in Jiaozhou,Qingdao,Shandong Province.Then the collected samples were separated and purified,and two pathogenic fungus named SJ-1 and FQ-1 were obtained.The Pythium sylvaticum and Pythium ultimum were identified by observing the colony morphology,microscopic morphological characteristics and combining the ITS gene sequencing.2.We carried out large-scale fermentation of the two strains of Pythium species with rice medium.In the end,20 g crude extracts of P.sylvaticum and 12.5 g crude extracts of P.ultimum were obtained after ethyl acetate soak extraction and vacuum evaporation.3.The pathogenicity test of tomato seedlings was carried out by using P.ultimum crude extracts,the results showed that the crude extracts of P.ultimum had the effect of root rot and stem rot on tomato seedlings,and It can also cause the seedlings damping-off.The crude extracts of P.ultimum were tested by high performance liquid chromatogram-mass spectrometry(HPLC-MS)and compared with the database.No major mycotoxins were found in the crude extracts of P.ultimum.4.The crude extracts of P.sylvaticum and the crude extracts of P.ultimum were separated by silica gel column chromatography,and after TLC test,the similar components were combined.The combined components were separated by Sephadex LH-20 and HPLC.As a result,we obtained 18 compounds,among of them,14 compounds were identified by the methods of nuclear magnetic resonance(NMR),mass spectrometry(MS)and literature comparison.Eight compounds were isolated and identified from P.sylvaticum,thymidine,ind ole-3-ethanol,(R)-3-hydroxy-3-(2-hydroxyethyl)indolin-2-one,stigmasterol,?-sitoste rol,tyrosol,phenylacetic acid,dibutyl phthalate.Ten compounds were isolated from P.ultimum,among of them,six compounds were identified,phenazine-1-carboxylic acid,aerugine,aeruginol,13-methyltetradeca noic acid,phenazine-1-carboxamide,linoleic acid.The eight compounds were obtained from the cultured P.sylvaticum for the first time.The butyl phthalate and Phenylacetic acid isolated from the crude extracts of P.sylvaticum are harmful to human beings.Ten compounds were isolated from the crude extracts of P.ultimum,phenazine-1-carboxylic acid and phenazine-1-carboxamide were antibiotics,and we expanding the source of antibiotics.