【作者】 高伟；

【导师】 田黎；

【作者基本信息】 青岛科技大学 ， 生物化工， 2009， 硕士

【摘要】 当前农业病害的防治以化学农药为主,但化学农药制剂较高的残留,且生产过程对环境污染严重,生物农药具有低毒、低残留、不易产生抗药性等优点,其研发备受广大科研工作者青睐。生物农药的研发很大程度上依赖杀菌剂防治机制的研究,通过研究发现海洋芽孢杆菌B-9987对植物病原菌尖刀镰孢菌、茄链格孢菌、灰葡萄孢菌、立枯丝核菌有较好的抑菌活性,其发酵液对茄链格孢菌和灰葡萄菌的生物防治效果分别为85.1%和88.4%,并且对生物防治机制做了系统研究,主要研究内容如下:(1)分别用B-9987活菌原液、无细胞滤液和粗提物稀释液处理番茄,研究了处理后番茄植株抗病代谢相关酶系的影响,结果表明,各处理番茄植株五种防御酶活性均显著增加,其中活菌原液处理诱导效果最好。对五种酶诱导作用在第3d天开始表现,并且诱导作用持续时间较长。通过比较测定了在温室和田间两种环境条件下,两种提取液(活菌原液和无细胞滤液)对番茄防御酶诱导作用的时间变化趋势。其研究结果表明,除超氧化物歧化酶(SOD)外,室内环境较室外更利于B-9987菌株处理液对番茄防御酶系的诱导作用发挥。B-9987菌株对番茄防御酶系有较好的诱导作用,但诱导作用的发挥受环境条件影响明显。(2)分别测定了活菌原液和无细胞滤液不同稀释度及4倍稀释度下不同诱导时间对番茄挥发性物质的影响,研究表明,较低浓度的B-9987提取液对挥发性物质有明显的促进作用,4倍和8倍稀释度对挥发性物质的诱导作用明显优于2倍稀释度。在对诱导时间对挥发性物质的试验中,总挥发性物质、氧合脂类、萜类化合物的变化趋势相近,均在第9-11d大量释放。芳香类化合物往往出现两个活性峰,且在诱导第1-3d就有明显增加,不同的变化趋势可能与植物抗病代谢相关联从而达到增加系统抗性的目的。(3)通过研究B-9987菌株抗真菌代谢产物其对茄链格孢菌的抑菌活性及其对细胞形态、菌体渗透性、细胞壁、细胞膜成分的影响。研究结果表明,代谢产物对茄链格孢菌的最小抑菌浓度和最低杀菌浓度分别为0.35mg/mL和5.0mg/mL。茄链格孢菌经B-9987代谢产物处理后引起菌体蛋白质、核酸等大分子物质的外流;细胞壁葡聚糖和细胞膜成份分析表明,B-9987粗提物对茄链格孢菌能显著影响其孢子萌发,迅速改变菌丝体细胞的通透性,其主要影响细胞壁葡聚糖合成,对细胞膜甾醇成分影响较小。研究结果表明海洋芽孢杆菌B-9987对植物病原真菌具有优良的抑菌活性,并且对番茄早疫病和灰霉病具有很好的田间生物防治效果,其生物防治机制包括诱导抗性、拮抗作用并且能够诱导番茄抗菌挥发性物质的增加。更多还原

【Abstract】 The geoponic disease was prevented by chemical fungicide usually, but it produced the environmental problem and also contaminated the agricultural products. The biological fungicide was presumed to be no harms to the environment and humankind. The application of biological fungicide depended on the research of the bacterio-mechanism to some extant. The fungistatic activity of Bacillus marinus B-9987 was studied in this paper. The metabolite of B-9987 have an perfect antifungal effect on Fusarium oxysporum, Alternaria solani, Bortrytis cinerea and Rhizoctonia solani. The biocontrol effect on tomato was 85.1% to early blight, 88.4% to grey mold. The biocontrol mechanism elicited by Bacillus marinus B-9987 was systematically investigated as follows:(1)The inducement of defense enzymes by different extracts of B-9987 was investigated. The result showed that the activities of five defense enzymes in each treatment were significantly improved compared to the control. In comparison with other treatment, the fermentation broth presented the highest inducing effect. According to the trends of the enzymes` activities, the inducement can be observed in the third day after sprayed with different extracts, and insisted to the seventh day mostly. The comparative study on the activities of defense enzymes was also obtained under different environmental conditions. According to the result, the B-9987 extracts had an inducing effect on the 5 defense enzymes in tomato leaves. The indoor activities of the enzymes (except superoxide dismutase SOD) was higher than the outdoor enzymes activities, and the fermentation broth show better inducement in the same environmental condition. It can be concluded from the result that the B-9987 extracts had an inducing effect on the defense enzymes in tomato. The inducing effect depended on environmental condition.(2)In the defensive responses of plants towards pathogens, volatiles may play a potential role in plant chemical defense strategies. In our work, the leaves of tomato were treated with fermentation broth and cell free filtration in different dilutions and times. The volatiles in the leaves were analyzed by GC/MS. The results demonstrated that the B-9987 extracts with high dilution increased the amount of total antipathogenic volatiles significantly, 4 and 8 times dilution present higher inducing effect than 2 times dilution. Total volatile compounds, phytoxylipin, terpenoids compounds had an releasing burst between the 9th to 11th in 13 days trend, and the trends were similar to each other. The aromatic compounds enhanced in the 1st to 3rd day, the different trend can be inferred the aromatic compounds may have an relationship with disease resistance metabolism.(3) The fungistatic activities and the effect on the cell morphology, permeability, the composition of cell wall and membrane of Alternaria solani of the metabolites of B-9987 was studied. The results showed that the metabolites have strong antifungal activity with MIC and MFC value being 0.35mg/mL and 5.0mg/mL respectively. Detected the protein and nucleotide in extracellular fluid showed the morphology was affected. According to the analysis result of membrane and cell wall, the metabolites affected the synthesis of glucan but had little influence on ergosterol.All the results show that the Bacillus marinus B-9987 has strong inhibition on plant pathogen, and perfect biocontrol effect on tomato early blight and grey mold. The biocontrol mechanism of B-9987 includes inducing resistant, antagonism and volatile compounds releasing promotion. The Bacillus marinus B-9987 has a bright prospect on the application of biocontrol.更多还原