【作者】 张利辉；

【导师】 董金皋；

【作者基本信息】 河北农业大学 ， 植物病理学， 2009， 博士

【摘要】 瓜果腐霉（Pythium aphanidermatum（Eds）Fitzp）属于卵菌门,寄主范围较广,可引起多种植物的猝倒病和瓜果腐烂病。木实验室从黑麦草上分离到一株具有一定除草活性的瓜果腐霉菌株PA1,本试验以PA1以及分离自黄瓜的PAC菌株为出发菌株,通过发酵对菌株的除草活性物质的产生、分离及结构鉴定进行了系统研究。经对瓜果腐霉进行诱导、分离培养,得到了PAC、PAE等4个菌株,利用种子萌发法、生长抑制作用测定法对实验室保存的PA1及上述得到的4个菌株进行了除草活性测定,结果表明,来自黑麦草的PA1和来自黄瓜的PAC菌株分别对马唐和反枝苋表现出了较强的除草活性,其他3个菌株则活性不明显。对菌株PA1和PAC进行紫外诱变,分别得到了31株和33株诱变菌株,并对其除草活性进行了测定,结果表明,PA1菌株诱变后只有少数菌株的除草活性增强,如PA1-M1和PA1-M4菌株,多数菌株活性变化不明显;PAC菌株经紫外诱变不同时间处理后得到的菌株数量不等,除草活性结果表明,除草活性增强的菌株较多,其中PAC-M2对反枝苋的生长抑制作用较强。利用麦麸、小米、玉米等6种固体培养基质对瓜果腐霉的PA1-M1和PAC-M2菌株进行了固体发酵,经生物测定并结合TLC和HPLC,最终选定麦麸作为大量固体发酵的培养基质,在此基础上,又向麦麸培养基中加入适量的牛肉膏、蛋白胨和酵母膏,发现麦麸中加入蛋白胨可作为瓜果腐霉产生除草活性物质的最适培养基。利用PD培养基对PA1-M1菌株进行了液体发酵,比较了震荡和静置两种培养方式,以及培养滤液初始pH对菌丝干重和培养滤液除草活性的影响,结果表明,振荡方式、初始pH为5～7时,菌丝的产生量最大,培养滤液的除草活性也最强。对菌株PA1-M1和PAC-M2的培养滤液粗提物进行了除草活性测定,结果表明,PA1-M1培养滤液粗提物对马唐根芽的IC₅₀分别为0.36mg/mL和0.46mg/mL,PAC-M2培养滤液粗提物对反枝苋根和芽的IC₅₀分别为0.49mg/mL和0.36mg/mL。对PA1-M1菌株液体发酵后的培养滤液及粗提物进行了热稳定性、杀草谱的检测,发现该毒素对热稳定,对供试的马唐、反枝苋、藜等杂草均有较强的除草活性。两菌株菌丝的除草活性结果表明,PA1-M1菌丝对马唐根和芽的IC₅₀分别为7.36 mg/mL和4.09 mg/mL,PAC-M2菌丝粗提物对反枝苋根和芽和IC₅₀分别为3.74 mg/mL和4.76 mg/mL。利用溶剂提取、树脂柱层析、TLC、硅胶柱层析、HPLC等分离技术,对瓜果腐霉PA1-M1菌株的液体发酵代谢产物进行了系统的研究。从菌丝中分离出具有除草活性的化合物1和化合物2:确定了培养滤液粗提物的洗脱方案为50%乙醇、95%乙醇、乙酸乙酯、丙酮依次洗脱;对所得的乙酸乙酯洗脱液,95%乙醇洗脱液和50%乙醇洗脱液分别采用不同的方法进行分离,得到了化合物3、化合物4和化合物5、化合物6,根据标准样品及其保留时间判断化合物4为邻苯二甲酸二甲酯。对PA1-M1菌株的固体发酵产物利用乙酸乙酯提取,再经TLC、硅胶柱层析和HPLC分离,得到了化合物7、化合物8、化合物9、化合物10和化合物11。利用红外光谱（IR）、核磁共振波谱（NMR）、液质联用（LC-MS）技术对所得到的化合物1、化合物2,化合物5～化合物10进行了结构分析,确定了化合物7、化合物8、化合物9、化合物10的结构分别为阿魏酸、2-（1-H-吲哚-3-基）丁酸乙酯、阿魏酸正丁酯和2-（1-H-吲哚-3-基）己酸乙酯。化合物1为含有两个羟基的不饱和长链脂肪酸,其分子量为578,分子式为C₃₇H₇₀O₄。化合物2与化合物1属于同一类。化合物5的分子量为306。更多还原

【Abstract】 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.更多还原