Separation And Identification Of The Herbicidal Fractions Produced By Pythium Aphanidermatum

Pythium aphanidermatum is a kind of pathogen belongs to Oomycete,which can cause many diseases such as bed rot of many kinds of crops,melons and fruits. The previous results showed that PA1 of P.aphanidermatum,which was isolated from the infected Perennial ryegrass had herbicidal activity.In this study,PA1 and the other 4 isolates that isolated from different hosts were researched detailedly,such as the production,separation and structure identification of the herbicidal substances.Different melons and fruits were used to isolate the pathogen P. aphanidermatum,and 4 isolates were obtained.Bioassy of the crude toxins produced by the 4 isolates and PA1 was tested by the methods of seed germination and inhibition on weed growth.The results showed that PA1 had the stronger herbicidal activity to Digitaria sanguinalis,and the isolate PAC had stronger activity to Amaranthus retroflexus.31 and 33 mutant-isolates were obtained by irradiating the mycelia of PA1 and PAC under UV illumination and their herbicidal activity were researched.The results showed that only fewness mutant isolates as PA1-M1and PA1-M4 from PA1 had a increased herbicidal activity,and the activity of other isolates was nealy the same as PA1 or decreased.Different numbers of isolates were obtained from PAC when irradiated by different time,and the herbicidal activity of most of the mutant isolates was increased,and that of the isolate PAC-M2 was stronger to A.retroflexus.The wheat bran,millet,rice,corn,oat and soybean meal were used as solid media to ferment PA1-M1 and PAC-M2,and the crude toxins were bioassyed and separated using the method of TLC and HPLC.The results showed that the herbicidal activity of the crude toxins from wheat bran was better than the others. HPLC analysis was done to the crude toxins from wheat bran after adding different nutration such as beef extracts,peptone,yeast extracts,and the results showed that the toxin production of that of peptone was highest,so the wheat bran,in which added some peptone was selected as the optimal solid culture for the PA1-M1 and PAC-M2. PD media was used to ferment PA1 and PAC-M2,and the affection of culturing modes such as shaking and stillness,and pH of PD on the dry weight of mycelia and herbicidal activity of the filtrate were studied.The results showed that the more suitable condition for the producing of mycelia and filtrate were shaking culturing at intial pH5-7.Herbicidal activity of filrate extracts and mycelia extracts produced by PA1-M1 and PAC-M2 was tested.The results showed that the IC₅₀ of filtrate extracts from PA1-M1 to the root and bud of Digitaria sanguinalis was 0.36mg/mL and 0.46mg/mL respectively,while that from PAC-M2 were 0.49mg/mL and 0.36mg/mL respectively.The filtrate extracts were steady to heat and had herbicidal activity to the weeds tested.The IC(50) of mycelia extracts from PA1-M1 to the root and bud of Amaranthus retroflexus was 7.36 mg/mL and 4.09 mg/mL,and that from PAC-M2 was 3.74 mg/mL and 4.76 mg/mL respectively.The metabolites produced by PA1 in liquid media was researched in detail using the method of solvent extacting,adsorbing by colophony,TLC,Silica Gel Column, Chromatography and HPLC.Compound 1 and compound 2 was obtained from the mycelia extracts.The eluting mode was determined as 50%ethanol,95%ethanol, ethyl acetate and acetone in turn,and the eluting fractions were separated with different methods.In this way,compound 3,compound 4,compound 5 and compound 6 were obtained respectively.The results of HPLC showed that compound 4 was determined as dimethyl 4-nitrophthalate.Compound 7,compound 8,compound 9 and compound 10 were obtained by using ethyl acetate extrating,TLC,Silica Gel Column Chromatography and HPLC from the solid fermented extration.IR,NMR,LC-MS were used to identify and analysis the chemical structure of the compounds obtained.The structure of compound 7,8,9 and 10 was ferulic acid, 2-(1H-indol-3-yl)ethyl butyrate,butyl-3-(3-methoxy-4-methylphenyl)acrylate,and 2-(1H-indol-3-yl)ethyl hexanoate,respectively.Compound 1 was an unsaturated fatty acid with 2 hydroxyls and the molecular weight and molecular formula was 578 and C₃₇H₇₀O₄ respectively.Compound 2 was concluded to be the same type with componud 1,and MS spectrum indicated the molecular weight of compound 5 was 306.