【作者】 王远宏；

【导师】 李洪连； 袁虹霞；

【作者基本信息】 河南农业大学 ， 植物病理学， 2002， 硕士

【摘要】 本文针对防治小麦纹枯病的上百种生防放线菌进行筛选后，选择出目标菌株024，随后对它的菌种的初步鉴定、初始发酵条件与最适发酵条件进行了探索，并对024放线菌所产抗生素的纯化、理化性质、发酵液的防治效果等内容进行了研究。 对024放线菌进行初步鉴定后认为，该菌是小链孢菌属（Microstreptospora）的一个新种或变种。抑菌谱测定表明024放线菌的发酵液对多种真菌如：苹果炭疽病菌（Colletotrichum gloeosporioides）、小麦赤霉病菌（Fusarium gramiaearum）、小麦长孺孢（Helminthosporium sativum）、泡桐溃疡病菌（Botryosphaeria dothidea）、番茄早疫病菌（Alternaria solani）、板栗疫病菌（Endothia parasitica）等有良好的抑制效果。 研究表明，摇床发酵初始培养基组成为：黄豆饼10g/L，葡萄糖10g/L，蛋白胨3g/L，NaCl 2.5g/L，CaCO₃，2g/L，初始pH值7`0；发酵条件为：装液量100ml/500ml，转速110r/min，温度28℃，培养时间10d；改良后的最适培养基成分为：黄豆饼10g/L，葡萄糖10g/L，蛋白胨3g/L，NaGl 2.5g/L，CaCO₃ 2g/L，MgSO₄ 5mmol/L，KH₂PO₄ 5mmol/L，FeCl₃ 1mmol/L，初始pH值7.0；发酵液培养条件为：装液量50ml/250ml，转速170r/min，温度28℃—39℃（24h变温），培养时间2d。改良后的发酵液产生的抗生素的抑菌能力提高到79.2％，较原来有较大提高。初始培养基颜色为红褐色，改良后的培养基颜色为淡黄色，且改良后发酵液过滤速度快，蛋白含量和色素含量少。对发酵液透析、超滤、金属离子沉淀、加热等处理研究后结论如下：024放线菌发酵液中的抗生物质为非蛋白质类，分子量较大，具有较强的热稳定性；出苗试验与防效对比试验表明024 放线菌发酵液在低浓度与原液浓度条件下能提高小麦种子发芽率，促进 苗的生长，并对小麦纹枯病有着良好的防治效果，但高浓度下使出苗率 与防效都降低。 024放线菌发酵物质－抗生素纯化研究结果表明，通过冷冻干燥，甲 醇浸取以及丙酮、氯仿和甲醇的连续粗提后，上氧化铝和硅胶柱层析可‘以有效地对024放线菌所产抗生素进行纯化。粗提制品的理化性质研究 表明，024放线菌所产抗生素为无色晶体，易溶于水，可溶于甲醇和乙 醇，且在甲醇中的溶解度较大，不溶或难溶于丙酮、氯仿、正丁”醇、乙 酸乙酯与苯。在有机溶剂中稳定性很强，在PH6E范围内有较强的抑菌 能力，在这一范围之外，活性急剧下降。紫外吸收峰在234urn处。更多还原

【Abstract】 This paper studied the screening and identification of a new Actinomycetes ( 024 ) for controling the disease caused by Rhizoclonici cerealis on wheat, its initial and optimal fermentation condition of production of antifungal substance, and the methods of purification, the physical and chemical character.etc..The actinomyces was idicntified as a new species of the Microsircplosporu Its antifungal spectrum 024-antibiotic includes many plantpathogenic fungi, such as Glomerella cingulata, Fusarium gramiaearum, Helmmthosporiuin sativitnt, /iotrvosphticriu dothidea, Alternaria xolani, Endothia parasitica etc..The results show that the composition of initial fermentation medium is soybean cake 10g/L, glucose 10g/L, peptone3g/L, NaCl 2.5g/I,, CaCO3 2g/L, The condition is the initial pH of medium 7.0, 50ml liquid medium in 250ml flask, fermentation temperature 28℃. HOrpm for 10days; the composition of optimal fermentation medium is soybean cake 10g/L, glucose 10g/L, peptone.1g/L. NaCl 2.5g/L, CaCO3 2g/L, MgSO4 5mmol/L, KH2PO4 5mmol/L, FeCl3 1mmol/L. The optimal condition is the initial pH of medium 7.0, 50ml liquid medium in 250ml flask, fermentation temperature 28℃ -39℃(changing the temperature at 24th h), 1 70rpm for 2days. After the initial medium was improved, the activity of 024-antibiotic was increased obviously ( 79.2% ), the colour of fermentation broth was changed from henna into buff and the filter rate was speeded. Through investigating the character of the fermentation broth, the following conclusions can be drawn that the antifungal substance is thermostable and not protein: it has better control effect against wheat sharp eye-spot under mid-concentrationjiowever decrease the effect under high- concentration ; the molecular weight is bigger; it can improve the wheat seed germination rate and promote its growth.Different extracting and purifing methods were taken, such as organic solvent extraction, soak, alumina column chromatography and silica gel column chromatography etc.. At last, the effective approach was found that the 024-antibiotic can be purified by freez-dry, methyl alcohol soaking, acetone, chloroform, and methyl alcohol continuously washing, alumina and silica gel column chromatography.The 024-antibiotic is achromatic, diffluent in water, and can be dissolved in methyl alcohol and ethanol.it is difficult for 024-antibiotic to be dissolved in acetone, chloroform, n-butanol, acetic anhydride and benzene. It is stable in organic solvent. In the range pH6-8, the 024-antibiotic has strong inhibilitory activity, and out of the range the activity will drop rapidly.lt can be absorbed at 234nm tempestuously.更多还原