【作者】 顾爱国；

【导师】 吴文君；

【作者基本信息】 西北农林科技大学 ， 农药学， 2004， 硕士

【摘要】 本论文以一株编号为 2B 的苦皮藤内生真菌为出发菌株，应用形态学的方法鉴定了其种属地位；研究了其代谢产物的农用生物活性，并对目标活性物质进行分离和结构解析，采取诱变育种手段选育了高产菌株，并对高产菌株的发酵条件进行了优化。通过试验结果的分析和讨论，取得了以下结论： 1 根据真菌的分类及鉴定方法，用光学显微镜观察 2B 菌株气生菌丝、大型分生孢子、小型分生孢子及菌落性状，对比相关分类系统，将 2B 菌株鉴定为层出镰刀菌(Fusarium proliferatum)。 2 在对 2B 菌株发酵产物生物活性研究中，发现 2B 菌株发酵产物甲醇提取物有明显的抑菌活性，在 1000 μg·mL-1浓度下对番茄早疫病菌（Alternaria solani）、烟草赤星病菌（Alternaria alternata）、小麦根腐病菌（Bipolaris sorokiniana）、玉米大斑病菌（Exserohilum turcicum）4 种供试菌的菌丝生长和孢子萌发抑制率均大于 80%；在盆栽试验中，对黄瓜霜霉病菌（Pseudoperonospora cubensis）的治疗和保护作用均在 70%以上。 3 采用常压柱层析、HPLC 制备等方法，在生物活性追踪指引下，从 2B 菌株发酵产物分离出 4 个化合物 2B-1，2B-2，2B-3，2B-4。经波谱技术( IR，1H NMR，13C NMR，COSY，MS)，结合化学检验方法，并与相关文献对比，将分离得到的化合物分别鉴定为 enniatin B，enniatin B1，enniatinA1，ergosterin。化合物 2B-1，2B-2，2B-3 在 100μg·mL-1浓度下，对玉米大斑病原菌菌丝生长抑制率分别为 68.18%，87.49%，90.91%。 4 建立了恩镰孢菌素（enniatins）的 HPLC 检测方法。检测条件为色谱柱 (C18 柱，0.9×30cm,10μm)；流动相(V∶V)甲醇∶水(88∶12)；流速 1 mL·min-1；检测波长UV229nm；室温下 5μL 进样，该方法相对标准偏差 RSD< 1%，平均回收率在 98%以上。 5 采用紫外线（UV）,甲基磺酸乙酯（EMS），超声波+UV + EMS 3 种诱变方式进行复合诱变，同时结合形态变异与产量之间的相关关系，挑选形态差异明显的单菌落，然后通过摇瓶发酵筛选 enniatins 高产菌株，通过 3 轮诱变筛选大幅度提高了菌种的生产能力，最后得到的突变株 UE152 代谢 enniatinns 总产量为 59.72 mg·100mL-1，是出发菌株摇瓶效价 2.9 倍，且该突变株的高产遗传特性稳定。 6 通过单因子试验和多因子正交试验筛选获得 UE152 菌株最佳的摇瓶培养基配方和发酵条件为葡萄糖 2%、土豆 20%、蛋白胨 0.5%、NH4Cl 0.3%、MgS040.1%和 CaCO30.1%，培养温度 26℃，接种量 10 块菌饼（每 50 mL 发酵液），初始 pH 值为 6.0~7.0，装液量 50 mL（每 250 mL 三角瓶），转速 160rpm，发酵时间 120h。在上述实验条件下enniatinns 总产量可达 88.68 mg·100mL-1，从正交试验结果分析当中看出，如果适当增加通气量，产量可望更高。更多还原

【Abstract】 Endophytic fungus of strain 2B isolated from Celastrus angulatus has been studied on the taxonomy,agricultural bioactivities, isolation and identification of active components, breeding of high yield strains,and optimization of fermentation conditions in this paper. The main results are as follows:1 By means of observation of aerial mycelium, macroconidia, microconidia and colony morphologycharacteristics of strain 2B using optics microscope, comparised with related classify system, strain 2B wasidentified as Fusarium proliferatum.2 The agricultural bioactivities of the strain 2B fermentation products were tested. The results showedthat the methanol extracts of mycelium had evident fungicidal activity. The extracts was tested againstAlternaria solani, Alternaria longipes, Bipolarissorokiniana shoem, Exserohilum turcicum, the resultsindicated that the inhibiting rates against mycelium growth and spore germination were both more than80% at the concentration of 1000 μg·mL-1. The results of pot test indicated that the methanol extracts ofmycelium exhibited more than 70% for both protective efficacy and therapeutic efficacy againstPseudoperonospora cubensis disease at the same concentration.3 By means of open column chromatography on silica gel and preparative HPLC, four compounds,2B-1, 2B-2, 2B-3, 2B-4, were isolated from the methanol extracts of fermentation products of strain 2B.On the basis of spectral technology (MS, FT-IR, H NMR, 1 13C NMR, COSY), chemical methods andcomparison with related reports of literatures, their structures were identified as enniatinB, enniatinB1,enniatinA1 and ergosterin, respectively. At the concentration of 100 μg·mL-1, the inhibiting rates ofcompound 2B-1, 2B-2, 2B-3 against the mycelium growth of Exserohilum turcicum were 68.18%, 87.49%,90.91%, respectively.4 The analytical method of HPLC for enniatins was set up. The operating conditions were chromatogramcolumn Hypersil ODS (C18, 300mm × 9mm i.d.，10 μm), inject volume 5μL at room temperature,methanol-water (88:12, V/V) as mobile phase with a flow rate of 1.0mL·min-1and UV detection at 229nm.The relative standard deviation (RSD) was less than 1%, and the average recovery was over 98%.5 Using Endophytic fungus of strain 2B as starting strain, mutagenized with ultraviolet (UV) , EMS andultrasonic+UV + EMS in sequence, at the same time ,based on the relationship between the variation ofmodality and the capcity of the mutants’ yield, the mutant strains which had great variation of modalitywere selected. Through three times mutagenized and the shake flask fermentation test, a mutant UE-152high-producing enniatins was obtained, which had much higher productive qualitied than its original strain.Its enniatins yield reached 59.72 mg·100mL-1 as 2.9 times high as that of original strain, and the heredity<WP=11>character of the high productivity was stable.6 The optimum composition of culture medium and fermentation conditions of the strain UE152 weredemonstrated by the test of uni-factor and orthogonal design, respectively. The optimum culture mediumconsisted of glucose 2%, potato 20%, peptone 0.5% , NH4Cl 0.3%, MgS04 0.1%, CaCO3 0.1% and originalpH6.0-7.0. Inoculum volume was 10 entries per 50mL medium. A 250mL flask containing 50mL mediumwas cultivated at 26℃ for 120h and the rotatory velocity was 160 rpm. Under the best culture conditions,the general yield of Enniatins was 88.68 mg·100 mL-1. The results of orthogonal test showed if increasedthe aeration level, the yield could be higher.更多还原