【作者】 王莹；

【导师】 张艳菊；

【作者基本信息】 东北农业大学 ， 植物病理学， 2015， 硕士

【摘要】 黄瓜枯萎病是一种真菌土传病害,由尖孢镰孢菌黄瓜专化型引起,具有毁灭性,在全世界范围内发生,且在我国各地区都存在。黄瓜的产量会因为枯萎病而降低10%~30%,糟糕时甚至可达80%~90%,黄瓜生产中,最严重的就是枯萎病,能造成极大的经济损失。黄瓜种植区域连年增长,因此黄瓜枯萎病也越来越严重。所以,对黄瓜枯萎病的防治方法研究已成为生产中的重要问题。目前,药物控制是一个重要的方法防治枯萎病,然而因为缺少效率高、毒性小、没污染的杀菌剂,农药残留会威胁人类的身体健康。因为枯萎病是土传病害,土壤药剂处理面积过大,很难进行,耗资很高。现代社会不断发展,人们越来越重视非污染的绿色食品,并且越来越重视环境保护,生物防治已引起人们的关注。集价格低廉、抑制植物病虫害发生效果显著、同时增加作物产量、提高品质等多种积极作用于一身的蚯蚓粪进入了人们的视野。本研究从蚯蚓粪中筛选对黄瓜枯萎病菌具有拮抗作用的微生物,以期为今后生防菌在黄瓜枯萎病的防治奠定基础。采用稀释平板法,从新鲜蚯蚓粪中分离到374株细菌。筛选出对黄瓜枯萎病菌有拮抗活性的菌株28株,其中以D2的抑菌效果最好,抑菌直径达28.28 mm,且抑菌效果稳定。D2单菌落形态为圆形或近圆形,扁平状,中等大小,表面光滑,不透明。革兰氏染色为阴性。菌体杆状,不产生芽孢,(0.5~1.0)um×(1.5~4.0)um。在4℃不生长,41℃可以生长。在Na Cl浓度小于5%时可生长,大于7%时不生长。不产生荧光,可使明胶液化,可分解淀粉。D2的分子生物学鉴定中,得到目的片段大小为1462 bp,将得到的PCR产物测序结果和Genbank里的序列进行同源性比较,显示该菌株为洋葱伯克霍尔德菌(Burkholderia cenocepacia)。对D2的发酵培养基成分和培养条件进行了优化,筛选出最佳实验室发酵配方:酵母膏1%,蔗糖0.5%,Na Cl 0.3%,NH4NO3 0.3%;最佳培养条件:发酵温度30℃,培养基p H 6.0,接种量2.0%,装液量为100 m L的三角瓶中装液40 m L。D2发酵液优化条件下的对黄瓜治疗试验,D2稀释50倍时防效达70.48%,稀释100倍时防效达69.89%,稀释150倍时防效达69.58%,多菌灵防效达78.09%,D2稀释50倍防效仅次于多菌灵的防效。D2发酵液对黄瓜枯萎病预防试验,D2发酵液稀释50倍时,防治效果达到77.82%,稀释100倍时,防治效果为77.56%,稀释150倍时,防治效果为76.52%,多菌灵防效达70.59%,D2发酵液稀释50倍时防效比多菌灵的防效多7.23个百分点。更多还原

【Abstract】 Cucumber fusarium wilt disease caused by Fusarium oxysporum f. sp. cucumerinum is a worldwide destructive soil borne fungal diseases, occurred in all over china. Wilt can cause cucumber yield decreased by 10 % ~ 30 %, severe up to 80 % to 90 %, which is the most serious disease of cucumber, caused one of the biggest economic loss diseases. With the cucumber planting area has expanded each year, resulting in continuous cropping cucumber fusarium wilt disease cause is increasing. Therefore, in-depth study of the effective prevention and cure methods of cucumber fusarium wilt disease has become an important problem to be resolved at present. At present, chemical control is an important method to control cucumber fusarium wilt, but due to the lack of efficient, low toxicity, no pollution of fungicide, easy cause pesticide residues, harm people’s health. Due to cucumber fusarium wilt as soil-borne diseases, drug treatment is difficult to implement a large area of soil and the cost is higher. With the development of modern society, people pay more attention to the strong demand and protection of the environment for pollution-free food, biological control has aroused great attention. Set low price, suppress plant diseases and insect pests significant effect, while increasing crop yields many positive role in improving the quality of one vermicompost entered people’s field of vision. Screening antagonistic to plant disease microorganisms from vermicompost on plant growth, plays the role of plant diseases and insect pests prevention and control.A total number of 374 bacterial strains were isolated from fresh vermicompost by tube dilution assay. The result of plate test showed that twenty-eight strains had antagonistic activity against cucumber fusarium wilt. According to the results of the suppression assay, strain D2 was selected as one of the best effect. The inhibiting diameter was 28.8 mm and the inhibitory effect is stable.D2 single colonies were round or nearly round, flat, medium in size, smooth surface, opaque. Gram stain was negative. Cell function, it does not produce spores. Scanning electron microscope showed straight rod-shaped bacteria,(0.5~1.0) um×(1.5~4.0) um. Do not grow at 4 ℃, 41 ℃ can be grown. D2 growth in Na Cl was less than 5 %, D2 cannot grow in Na Cl was more than 7 %. D2 does not produce fluorescence, it can make the gelatin liquefaction, degradable starch.Identification of molecular biology of D2, obtain the target fragment size was 1462 bp, the PCR products were sequenced, sequence reported in Genbank sequencing and homology comparison, it was proved for the Burkholderia cenocepacia.The optimal fermentation medium components and the best optimal fermentation conditions for strain D2 were studied. The results were as follows: the medium of fermentation for D2 constituted of 1.5 % of yeast extract, 0.3 % of sucrose, 0.3 % of sodium chloride, 0.5 % of ammonium nitrate. The fermentation conditions were: cultural temperature 30 ℃, optimum initial p H 6.0, inoculation seed liquid 2.0 % and loading liquid 40 mL in 100 flasks.Optimizing conditions of cucumber fusarium wilt treatment trial fermentation broth of D2, D2 diluted 50 times, the preventive effect was 70.48 %, diluted 100 times, the preventive effect was 69.89 %, 150 fold dilution, the preventive effect was 69.58 % and carbendazim prevention effect was 78.09 %. The anti effect of D2 diluted 50 times was lower than carbendazim control effect.D2 fermentation liquid on cucumber fusarium wilt disease prevention trials, D2 fermentation broth diluted 50 times, the control effect reached 77.82 %, diluted 100 times, the control effect is 77.56 %, diluted 150 times, the control effect was 76.52 % and carbendazim prevention effect was 70.59 %. The preventive effect of D2 fermentation broth diluted 50 times was 7.23 percentage points higher than carbendazim control effect.更多还原