【作者】 徐宁；

【导师】 王秀峰； 魏珉；

【作者基本信息】 山东农业大学 ， 蔬菜学， 2012， 博士

【摘要】 黄瓜是设施主栽蔬菜,连作障碍已成为制约其高产高效和可持续发展的重要因素,防控技术研究备受关注。已有研究表明,大葱轮作可显著改善温室黄瓜连作土壤理化和生物学性状,提高微生物多样性,增加细菌和放线菌数量,减少真菌数量,抑制枯萎病原菌繁殖,促进生长,提高产量,但其内在机制尚不明确,大葱根系分泌物及其化感作用可能是重要原因。为此,本课题在研究大葱根系分泌物对温室黄瓜和枯萎病原菌化感效应的基础上,分离鉴定其中主要化感物质并进行功能验证,以期从化感作用角度揭示大葱轮作防控温室黄瓜连作障碍的原因,为制定合理栽培制度提供理论依据。主要研究结果如下：1.轮作大葱根际土壤EC值显著降低,pH值升高,增加了细菌和放线菌数量,减少了真菌和镰刀菌数量,提高了转化酶、脲酶、过氧化氢酶和多酚氧化酶活性。与黄瓜根际土壤相比,大葱根际土壤中邻苯二甲酸二乙酯和二苯并呋喃含量较高,且含有6种硫代化合物,其中5种属于亚硫酸酯类。2.利用连续根系分泌物收集系统(CRETS)收集到的根系分泌物种类多于浸根法,增加了烃类和胺类化合物。树脂+萃取法比直接萃取法分离获得的物质更多,增加了醇类、苯甲酸衍生物等多种根系分泌物成分。采用较低的浓缩温度(35℃)有利于保持根系分泌物的稳定性。采用连续根系分泌物收集系统(CRETS),在较低的浓缩温度(35℃)下更有利于大葱根系分泌物的提取。3.采用静态顶空(SHS)法提取大葱根系挥发性成分,并用气相色谱-质谱联用(GC-MS)技术分析其构成。结果表明,大莓根系挥发性物质包括醚类(硫醚)、醇类(硫醇)、醛类、烃类、酯类、酸类、杂环化合物等,其中大部分属于硫代化合物,硫代物以甲硫醇、丙硫醇为主,二硫代物以二甲基二硫醚、甲基丙基二硫醚、二丙基二硫醚为主,三硫代物以二甲基三硫醚和二丙基三硫醚为主。4.‘元藏大葱,根系的挥发性物质比‘章丘大葱’多。幼苗期和成株期大葱的挥发性物质构成差异不大。大葱根系挥发性物质提取过程中,剪切获得的硫醇和酸类物质相对含量较高；研磨获得的挥发性物质种类更多,以烃类和醛类为主。5.大葱根系分泌物对黄瓜种子萌发和芽苗生长均表现低浓度促进、高浓度抑制的作用,‘元藏大葱,比‘章丘大葱’的化感作用更强；随着根系分泌物浓度升高,对枯萎病原菌菌丝生长和孢子萌发的抑制作用逐渐增强,‘元藏大葱’根系分泌物的抑制程度大于‘章丘大葱’6.大葱根系分泌物中含有烃类、醇醚类、酚类、萘类、醛酮类、酸类、酯类、酰胺类以及杂环化合物等物质,其中,杂环化合物、酯类和酰胺类化合物所占的比例较大；邻苯二甲酸酯类和苯并噻唑类物质相对含量较高,2,2’-亚甲基双(4-甲基-6-叔丁基苯酚)、苯甲酸苄酯、苯甲酸-4-甲基苯酯、2-甲硫基异硫氰酸苯酯等相对含量较低,上述物质在‘元藏大葱’根系分泌物中的相对含量高于‘章丘大葱’7.大葱根系分泌物不同pH提取组分对黄瓜芽苗生长均表现为促进作用,对枯萎病原菌菌丝生长表现为抑制作用。以碱性组分的化感作用最强,酸性组分次之,水溶性组分最弱。碱性组分中相对含量较高的为噻唑类杂环化合物和酯类化合物,较酸性和水溶性组分增加了相对含量为21.88%的硫代化合物和8.22%的酰胺类化合物。8.利用四种不同极性有机溶剂(乙酸乙酯、乙醚、三氯甲烷、正丁醇)提取大葱根系分泌物,各提取组分对黄瓜芽苗生长均表现为促进作用,对枯萎病原菌菌丝生长表现为抑制作用,以乙酸乙酯提取组分(A)的化感作用最强。进一步分离化感优势组分(A),以80%乙酸乙酯+20%乙醚分离物(A-1)的化感作用最强。大葱根系分泌物A-1组分的物质构成包括邻苯二甲酸二丁酯、邻苯二甲酸二异辛酯、邻苯二甲酸二乙酯、2-甲硫基苯并噻唑、苯并噻唑、苯甲酸-4-甲基苯酯和环莠隆等。9.邻苯二甲酸二丁酯和苯甲酸-4-甲基苯酯对黄瓜芽苗生长表现促进作用,对枯萎病原菌菌丝生长表现抑制作用,但随着浓度升高,前者的化感作用先增强后减弱,后者的化感作用逐渐增强；苯并噻唑对黄瓜种子萌发、芽苗生长表现为“低促高抑”对枯萎病原菌菌丝生长具有抑制作用,且随浓度升高先增强后减弱。黄瓜子叶淀粉酶活性变化与芽苗生长趋势一致,下胚轴吲哚乙酸氧化酶活性变化与之相反。更多还原

【Abstract】 Cucumber(Cucumis sativus L.) is one of the main vegetables under protected cultivation, and continuous cropping obstacles have become important factors restricting high production and sustainable development, the control of continuous cropping obstacles has been paid more and more attention. Previous studies have shown that rotation with Welsh onion can significantly ameliorate the physicochemical and biological properties of continuous cropped cucumber soil in greenhouse and improve the diversity of microorganisms, the number of bacteria and actinomycetes were increased and fungi was reduced, the reproduction of Fusarium oxysporum f.sp. cucumerinum was inhibited, and the growth and yield of cucumber were promoted, but the internal mechanism is not clear. Welsh onion root exudates and its allelopathy may be the important reasons. Therefore, this paper has studied on the Welsh onion(Allium fistulosum L.) root exudates and their allelopathy on cucumber and Fusarium oxysporum f.sp. cucumerinum, the main allelochemicals were isolated, identificated and verified, and execpected explaining the reasons through allelopathy of rotation with Welsh onion control the continuous cropping obstacles of cucumber in greenhouse and providing theoretical basis for the development of culture system reasonably. The main results were as follows:1. After Welsh onion rotation, the EC of rhizosphere soil was decreased significantly while the pH increased. The number of bacteria and actinomycetes in Welsh onion rhizosphere soil were increased, the number of fungi and Fusarium oxysporum were decreased, and the activities of invertase, urease, catalase, polyphenol oxidase were increased. Relative content of diethyl phthalate and dibenzofuran were higher in Welsh onion rhizosphere soil than cucumber rhizosphere soil, and containing six sulfur compounds, five compounds of them belong to the sulfite esters.2. More compounds were collected by the continuous root exudates trapping system (CRETS) than the root soaking method (RS) and an increase in the hydrocarbon and amine compounds was observed in the solution prepared by the CRETS. More chemical compositions were detected by the resin plus extraction method than the direct extraction method and an increase in the alcohol and benzozic acid derivatives was observed in the solution prepared by the resin plus extraction method. The stability of root exudates can be maintained at a relatively lower temperature (35℃). It have more advantages in the analysis of the compounds in the Welsh onion root exudates when collect the Welsh onion root exudates by the CRETS and at a relatively lower temperature (35℃). 3. Volatile compounds in Welsh onion roots were extracted by static headspace (SHS) and identified by gas chromatography-mass spectrometry (GC-MS). The ether (thioether), alcohol (mercaptan), aldehyde, hydrocarbon, ester, acid, heterocyclic were detected in Welsh onion roots volatile compounds, most of these were organo-sulfur compounds. The major mono-thiosulphinates was methanethiol and propanethiol. The major di-thiosulphinates was disulfide dimethyl, disulfide methyl propyl and disulfide dipropyl. The major tri-thiosulphinates was dimethyl trisulfide and trisulfide dipropyl.4. The roots of’Yuanzang’have more volatile compounds than’Zhangqiu’, but there is little difference between the seedling stage and adult stage. During the extraction process of volatile compounds from Welsh onion roots, higher relative content of mercaptan and acids were got by cuting processing while more material were got by grinding treatment, in which the relative content of hydrocarbon and aldehyde compounds was higher.5. The seed germination and sprout growth of cucumber were promoted by root exudates at low concentration while suppressed at high concentration, and the allelopathy of’Yuanzang’ was stronger than’Zhangqiu’. Inhibitory effects on hypha growth and spore germination of Fusarium oxysporum f.sp. cucumerinum enhanced with increasing concentration of root exudates, and’Yuanzang’showed stronger effects than’Zhangqiu’.6. Hydrocarbon, alcohol, ether, phenol, naphthalene, aldehyde, ketone, acid, ester, amide, heterocyclic compounds were detected in Welsh onion root exudates, of which the heterocyclic, ester and amide compounds occupied higher proportion. The phthalate esters and benzothiazole derivatives have a higher relative content while p-cresol2,2’-methylenebis[6-tert-butyl-, benzyl benzoate, benzoic acid p-tolyl ester,2-(methylthio) phenyl isothiocyanate have a lower relative content, which were higher in’Yuanzang’than in ’Zhangqiu’.7. The sprout growth of cucumber were promoted by different pH extract fractions from Welsh onion root exudates while hypha growth of Fusarium oxysporum f.sp. cucumerinum were inhibited. The allelopathy of alkali fraction was strong, followed by acidic fraction and water-soluble fraction. The higher relative content compositions of alkali fraction were thiazole heterocyclic and ester compounds, and there were increased the relative content of21.88%of sulfur compounds and8.22%of amides as compared with acidic fraction and water-soluble fraction.8. Four different polarity organic solvents (ethyl acetate, ethyl ether, chloroform, butyl alcohol) were used to extracte Welsh onion root exudates. The sprout growth of cucumber were promoted by different organic solvents extract components while hypha growth of Fusarium oxysporum f.sp. cucumerinum were inhibited. The allelopathy of ethyl acetate extract component (A) was the strongest, which is the allelopathic advantage component. Further separate the allelopathic advantage fraction (A), the allelopathy of component separated by the80%ethyl acetate+20%ether (A-1) was the strongest, the A-1component identified by GC-MS have seven compounds of which were dibutyl phthalate, diisooctyl phthalate, diethyl phthalate,2-(methylthio) benzothiazole, benzothiazole, benzoic acid p-tolyl ester, cycluron.9. The sprout growth of cucumber were promoted by dibutyl phthalate and benzoic acid p-tolyl ester while hypha growth of Fusarium oxysporum f.sp. cucumerinum were inhibited. The allelopathy of dibutyl phthalate increased first and then decreased with the increasing of concentration, and the allelopathy of benzoic acid p-tolyl ester gradually increased. The seed germination and sprout growth of cucumber were promoted by benzothiazole at low concentration while suppressed at high concentration, the hypha growth of Fusarium oxysporum f.sp. cucumerinum were inhibited by benzothiazole, the inhibition increased first and then decreased. Change of amylase activity in cucumber cotyledon was in agreement with the tendency of sprout growth, the tendency of IAA oxidase activity in cucumber hypocotyl in reverse.更多还原