【作者】 张武岗；

【导师】 张兴；

【作者基本信息】 西北农林科技大学 ， 农药学， 2009， 博士

【摘要】 本研究以分离自三江源生态环境中的一株放线菌(编号:19G-317)为研究对象,对其农用抑菌活性、生长特性、分类地位、发酵条件、抑菌活性成分的分离与鉴定方面进行了较为系统的研究,得出以下主要研究结果:1、采用菌丝生长速率法、活体组织法和盆栽试验测定了放线菌19G-317菌株发酵产物的农用抑菌活性。试验结果表明:放线菌19G-317菌株发酵产物(发酵液、菌体提取液和发酵液提取物)不但对22种供试植物病原真菌具有较强的抑菌活性,而且对4种细菌也有一定的抑制作用。菌丝生长速率法试验结果表明,在1 mg/mL浓度下,菌体提取液对8种供试植物病原真菌的菌丝生长抑制率大于90 %;发酵液提取物对11种供试植物病原真菌的菌丝生长抑制率大于90 %。孢子萌发试验结果表明,在5 mg/mL浓度下,发酵产物对番茄灰霉病菌(Botrytis cinerea)、水稻稻瘟病菌(Pyricularia oryzae)、番茄叶霉病菌(Fulvia fulva)孢子萌发的抑制率均大于80 %,发酵液、菌体提取液和发酵液提取物对番茄灰霉病菌孢子萌发的抑制率分别为97.30 %、98.99 %和100%。组织法试验结果表明,在10 mg/mL浓度下,发酵液、菌体提取液和发酵液提取物对番茄灰霉病的药效分别为72.17 %、76.95 %和88.63 %,高于对照药剂(50%速克灵);菌体提取液和发酵液提取物对油菜菌核病(Sclerotinia sclerotiorum)的药效均为100 %。盆栽试验表明,发酵产物对辣椒疫霉病(Phytophthora capsici)、番茄灰霉病、小麦白粉病(Blumeria graminis)、小麦全蚀病(Gaeumannomyces graminis)和小麦纹枯病(Rhizoctonia cerealis)均表现出一定的防治效果。在10 mg/mL浓度下,菌体提取液和发酵液提取物对小麦纹枯病的治疗效果分别为62.40 %和74.08 %;发酵液、菌体提取液和发酵液提取物对小麦白粉病的保护效果分别为63.35 %、59.47 %和71.77 %;发酵液提取物对辣椒疫霉病的保护效果和治疗效果为54.18 %和56.46 %。2、对放线菌19G-317菌株的生长特性和传代稳定性进行了测定。试验结果表明,该菌株的生长对氧气的需求比较高,属于专性好气型;菌落在pH6.8～8.4的生长环境下生长良好;菌落生长对渗透压的适应范围比较宽,在NaCl质量浓度为0.5～20 g/L的生长环境中均能生长,且生长情况基本一样;菌落生长对紫外线比较敏感,在紫外线照射1 h后,菌落生长明显减弱;菌株的遗传性状在试验范围内保持稳定。3、根据放线菌19G-317菌株的形态特征、培养特征、生理生化特征以及16S rDNA序列研究结果,初步鉴定该菌株为链霉菌属黄色类群链霉菌。将其16S rDNA序列已注册于GeneBank,注册号为EU872020。4、综合单因素试验、Plackett–Burman设计和响应面等方法对放线菌19G-317菌株发酵工艺进行了优化。放线菌19G-317菌株优化后的发酵培养基组成为:麦芽糖50.3 g/L、胰蛋白胨9.6 g/L、NaCl 2.5 g/L、CaCO3 2.0 g/L;优化后的揺瓶发酵条件为:装液量125 mL/250mL、转速195 r/min、温度28℃、接种量7 %、初始pH 7。5、对放线菌19G-317菌株的发酵产物稳定性和发酵培养特性进行了测定。结果表明,该菌株发酵产物对热(20-100℃)比较稳定,对酸碱和紫外线的稳定性较差。发酵液随着处理温度的升高和处理时间的延长对番茄灰霉病菌和枯草芽孢杆菌的抑制率略有下降,下降幅度不明显;发酵液在偏酸或偏碱条件下对番茄灰霉病菌和枯草芽孢杆菌的抑制率均明显低于中性条件(pH 7);紫外线照射45 min后,发酵液对番茄灰霉病菌和枯草芽孢杆菌的抑菌活性与照射5 min的抑菌活性相比,抑制率分别下降30.83 %、8.7 %。发酵培养特性研究结果表明菌株的生长符合微生物生长的一般规律,在0～48 h为生长延缓期,在48～72 h为对数生长期,培养72 h后菌株生长达到稳定期,168 h后进入衰亡期。6、从放线菌19G-317菌株发酵产物中分离纯化出14个化合物,最终确定出8个化合物结构,分别为1-甲基-9-羟甲基吩嗪(1-methyl-9-hydroxymethylphenazin)(A),β-谷甾醇(β-Sitosterol) (C),硬脂酸(Stearic acid,D),十六酸乙酯(Ethyl-hexadecanoate,H),4-烯-1,11-(1,5-戊二醇)-癸内酯(J),5-烯-1,18-十八烷二酸二甲酯(K),3,4,6-三乙酸-1-环己烯(L),环(丙氨酰-苯丙氨酸) [cyclo-(Ala-Phe),M]。试验结果表明,菌株代谢产物的抑菌活性成分主要属于中等极性或高极性物质,对发酵产物分离纯化得到三个活性较好的化合物。这3个化合物(M、N、O)对番茄灰霉病菌和枯草芽孢杆菌均有较好的抑制作用。在1 mg/mL浓度下,3个化合物对枯草芽孢杆菌的抑菌圈直径分别为11 mm、13 mm和19 mm,对番茄灰霉病菌的菌丝生长抑制率分别为59.67%、84.43%和97.88 %,对番茄灰霉病菌的孢子萌发抑制率分别为77.9 %、100.0 %和100.0 %。更多还原

【Abstract】 In this thesis, a strain of actinomycete (No. 19G-317) isolated from the soil of natural conservation area of Sanjiangyuan Nature Reserve was chose as the main research object, the antifungal activity, the biology characteristics, taxonomic status, fermentation condition optimization, isolation and identified of bioactivity compounds from strain 19G-317 metabolites were studied. The main results and conclusions were outlined as follows:(1) The fungicidal activities of the fermentation product of actinomycete strain 19G-317 were evaluated by mycelium growth rate, in vivo inhibition and potted plant experiment against plant pathothic fungi and bacteria.The bioassay results showed that both of fermentation broth, extract product of mycelium and fermentation broth not only have stronger bioactivity against 22 tested plant pathogenic fungi, but also have certain inhibition activity against 4 tested plant pathogenic bacteria. The mycelium growth inhibition rate of extract product of actinomycete (No. 19G-317) mycelium were over 90 % to 8 tested plant pathogenic fungi; meanwhile, the mycelium growth inhibition rate of extract product of actinomycete (No. 19G-317) fermentation broth were over 90 % to 11 tested plant pathogenic fungi under the concentration of 1 mg/mL; The results of spore germination test showed that the inhibition rate of the fermentation broth and the extract product of mycelium and the fermentation extraction were 97.30 %、98.99 % and 100.00 % to Botrytis cinerea under the concentration of 5 mg/mL. Tissue experiment showed that the efficacy of the fermentation broth, the extract product of mycelium and the fermentation extraction were 72.17 %、76.95 % and 88.63 % against Botrytis cinerea under the concentration of 10 mg/mL. Furthermore, the control efficacy of the extract product of mycelium and the fermentation extraction were all 100% against Sclerotinia sclerotiorum under the concentration of 10 mg/mL. Potted plant experiment showed that the fermentation products exhibited a protective efficacy of 23.80 %、38.60 %、63.35 % and a therapeutic efficacy of 34.24 %、35.17 %、43.80 % against Phytophthora capsici, Botrytis cinerea and Blumeria graminis, the extract product of mycelium were 43.80 %、47.59 %、59.43 % and 42.74 %、44.57 %、45.09%; The efficacy of the fermentation broth、the extract product of mycelium were 30.60 % and 49.19 %, 45.08 % and 60.42 % against Gaeumannomyces graminis and Rhizoctonia cerealis.(2) The biology characteristics and passage stability of the strain 19G-317 were determined. The results showed that the growth of the strain 19G-317 need more oxygen, that is, it was aerobic microorganism; the favorite PH value for the strain growth ranged from 6.8 to 8.4, and the optimum PH was 7.6; The osmotic pressure for colony growth of the strain was much broader, it can grow constantly on the concentration of NaCl ranged from 0.5～20 g/L. However, the strain was sensitive to ultraviolet radiation, the colony growth become weaker after 1h ultraviolet radiation; Furthermore, the stability result showed that heredity character of the strain 19G-317 was stable.(3) Based on morphological characteristics, growth characteristics, biochemical and physiological characteristics and 16S rDNA sequence of the strain 19G-317, it was identified as Streptomyces flavus sp. The 16s rDNA sequences have been deposited in GenBank with the accession numbers EU872020.(4) Statistical methods such as Single factor, Plackett–Burman design and response surface methodology were employed synthetically to optimize the fermentation medium and conditions of actinomycete strain 19G-317. The optimum composition of the fermentation medium (g/L): Maltose 50.8 g/L、Tryptone 10.5 g/L、NaCl 2.5 g/L、CaCO3 2.0 g/L;and the optimum fermentation condition: medium volume 125mL/250mL、rotary speed 195 r/min、temperature 28℃、inoculation volume 7 %、initial PH 7。After optimization, the antibiotic activity of strain 19G-317 improved by 22.67 % and 38.30 % against Botrytis cinerea and Bacillus subtilis, respectively.(5) Stability test of fermentation products of strain 19G-317 had been carried out to acid, alkali, temperature and ultraviolet radiation. The fermentation products of strain 19G-317 were stable to temperature, but it was not stable to acid, alkali and ultraviolet radiation. The inhibition rate of fermentation products of strain 19G-317 against Botrytis cinerea and Bacillus subtilis continued to adjust downwards, in line with the rising temperature and extended handling time. The inhibition rate of fermentation products of strain 19G-317 against Botrytis cinerea and Bacillus subtilis under acidic or alkali conditions were lower obviously than under neutral conditions. After 45 min ultraviolet radiation, the inhibition rate of fermentation products of strain 19G-317 against Botrytis cinerea and Bacillus subtilis decreased by 30.83 %、8.7 % than that 5 min ultraviolet radiation. Furthermore, study on the culture characteristics of strain 19G-317showed that the strains growth was in accordance with the general discipline of microorganism growth.(6) 14 compounds were isolated from fermentation products of strain 19G-317 and the chemical structures of 8 compounds were identified. Those chemicals include 1-methyl-9-hydroxymethylphenazin (A),β-Sitosterol (C), stearic acid(D) ,Ethyl-hexadecanoate(H), 4- alkene -1,11-(1,5-pentadiol)- Decalactone(J), 5- alkene -1,18- Octadecane diacid, dimethyl ester(K), 3,4,6- triacetic acid -1- cyclohexene(L), cyclo-(Ala-Phe)(M).The bioassay results of the chemicals for the actinomycete strain 19G-317 showed that the main bioactivity components belongs to middle or high polarity compounds, the further bioassay indicated that three compounds(M、N、O) have stronger inhibitory effect on Botrytis cinerea and Bacillus subtilis, the inhibition zone diameter of three compounds were over 11mm against Bacillus subtilis, the inhabition rate were over 59.67 % against mycelium growth of Botrytis cinerea, and over 77.9 % against spore germination of Botrytis cinerea.更多还原