拮抗菌AnH对植物病原真菌拮抗作用及樱桃树“黑疙瘩”病初探

发布时间：2019-04-18 12:07

【摘要】：植物病原菌引起的植物病害每年给作物产量及质量带来巨大的损失,生物防治途径对作物的植物病害具有很好的防治效果,越来越受到人们的关注。但是植物病害的生物防治剂的商业使用和应用一直很缓慢,获得广谱、高效的抑制植物病害的生防菌仍是一个挑战。本研究通过平板对峙法筛选出一株对12种植物病原真菌具有高效、广谱抗性的拮抗菌AnH,经生理生化试验以及16S rRNA测序鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens);拮抗菌AnH无菌发酵液中的抗菌物质耐高温和强酸强碱,经强光和紫外线照射后活性稳定,对蛋白酶K不敏感,且保存3个月对抗菌物质活性几乎没有影响,为以后田间实验打下基础;AnH同时具有纤维素酶和蛋白酶活性,对拮抗菌的发酵培养基进行了优化,得到最适宜的发酵条件为玉米粉15 g/L,酵母膏15 g/L,硫酸镁2 g/L,抑菌圈直径达18.1 mm;采用有机溶剂萃取法,硫酸铵沉淀法,盐酸沉淀法分离提取抗菌物质,结果表明其为水溶性物质,此外实验结果表明脂肽类粗提物能广谱抑制12种植物病原真菌的生长。采用凝胶过滤、LC-MS对其进行分离纯化分析得到分子量m/z1042.5、1056.5、1070.5三者分子量相差14 Da,正好是一个-CH2基团的大小,这符合脂肽类物质同系物的特征,这些结果暗示从拮抗菌AnH分离出来的抗菌物质是伊枯草菌素同系物。陕西白鹿原樱桃树出现了一种感染率高,蔓延快但发病机制尚不清楚的“黑疙瘩”病害,当地农民采用多种化学方法防治效果不佳。本研究采用微生物学分离技术对10棵患病樱桃树病组织进行分离培养,共分离得到135株细菌,进而通过形态、生理生化反应合并为不同的27株细菌,对其进行16S rRNA测序结果显示:23株为Enterobacteriaceae,其中 Pantoeasp.11 株,Enterobactersp.6 株,Salmonellasp.3 株,Escherichia sp.3株。病组织中的优势细菌属于Enterobacteriaceae;分离得到的8株真菌ITS测序结果显示:2株Wickerhamoms,2株Meyerozyma,1株Botryosphaeria dothidea,2 株 Fungal sp.,1 株 Gibberella。其中的真菌Botryosthea 是可引起多种果树患病的致病菌。用拮抗菌AnH的脂肽类及蛋白粗提物对病组织分离的细菌和真菌进行抑菌试验,结果显示拮抗菌AnH的脂肽类粗提物对其都有很好的抑菌作用。以上研究表明,拮抗菌AnH具有很好的应用潜能,为控制樱桃树“黑疙瘩”病及植物病害的发生提供了一定的科学依据和理论依据。
[Abstract]:Plant diseases caused by plant pathogens bring huge losses to crop yield and quality every year. Biological control methods have a good control effect on plant diseases of crops, and more attention has been paid to them. However, the commercial use and application of biological control agents for plant diseases have been slow. It is still a challenge to obtain a broad spectrum of biological control agents that can effectively control plant diseases. In this study, an antagonistic strain AnH, which was highly effective and broad-spectrum resistant to 12 plant pathogenic fungi, was screened out by plate confrontation method and identified as Bacillus amylase (Bacillus amyloliquefaciens); by physiological and biochemical tests and 16s rRNA sequencing. The antibacterial substance in aseptic fermentation broth of antagonistic strain AnH was high temperature resistant and strong acid and alkali. After irradiation with strong light and ultraviolet light, its activity was stable, it was not sensitive to protease K, and it had little effect on the activity of antibacterial substance after 3 months' storage. To lay the foundation for future field experiment; AnH had both cellulase and protease activity. The optimal fermentation conditions were as follows: corn meal 15 g / L, yeast extract 15 g / L, magnesium sulfate 2 g / L, and the optimal fermentation conditions were as follows: corn meal 15 g / L, yeast extract 15 g / L, magnesium sulfate 2 g / L. The diameter of bacteriostatic ring is up to 18.1 mm;. The antibacterial substances were separated and extracted by organic solvent extraction, ammonium sulfate precipitation and hydrochloric acid precipitation, and the results showed that they were water-soluble. In addition, the results showed that the lipopeptide crude extracts could inhibit the growth of 12 phytopathogenic fungi by broad spectrum. By gel filtration and LC-MS separation and purification, the molecular weight difference of molecular weight (MW) was 1042.5, 1056.5 and 1070.5, and the difference of molecular weight was just the size of a-CH2 group, which was consistent with the characteristic of lipopeptide homologue. These results suggest that the antibacterial substance isolated from antagonistic bacteria AnH is a homologue of eosinosporin. A kind of "black pimple" disease, which has a high infection rate and spread quickly but the pathogenesis is not clear, has appeared in the cherry tree of the White Deer Plantation in Shaanxi Province. Many chemical methods used by the local farmers are not effective in the prevention and control of black pimple. In this study, the microbiological isolation technique was used to isolate and culture 10 infected cherry tree disease tissues. 135 strains of bacteria were isolated, and then 27 different strains of bacteria were combined by morphological, physiological and biochemical reactions. The results of 16s rRNA sequencing showed that 23 Enterobacteriaceae, strains were Pantoeasp.11, Enterobactersp.6, Salmonellasp.3 and Escherichia sp.3. The dominant bacteria in the tissues belonged to 8 fungal ITS strains isolated from Enterobacteriaceae;. Sequencing results showed that two Wickerhamoms,2 strains, Meyerozyma,1 strain, Botryosphaeria dothidea,2 strain, Fungal sp.,1 strain, Gibberella., were isolated from the infected tissues. The fungus Botryosthea is one of the pathogenic bacteria which can cause many kinds of fruit tree diseases. The lipopeptide and crude protein extracts of antagonistic bacterium AnH were used to test the bacteriostatic activity of bacteria and fungi isolated from the disease tissue. The results showed that the crude lipopeptide extracts of antagonistic bacteria AnH had good bacteriostatic effect on the bacteria and fungi. The results showed that the antagonistic bacterium AnH had good application potential and provided scientific and theoretical basis for controlling the occurrence of "black pimple" disease and plant disease in cherry trees.
【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S476;S436.629

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