【作者】 董法宝；

【导师】 牟志美；

【作者基本信息】 山东农业大学 ， 特种经济动物饲养， 2010， 硕士

【摘要】 家蚕细菌病和真菌病每年都给蚕桑生产造成严重的经济损失,现有药物虽有较好的防治效果,但也存在一些缺点,如长期使用能够使病原菌抗药性增强,污染环境,有些药物甚至对人有害等。为克服现有药物的诸多限制性问题,开发新型微生物药剂作为蚕药是一个重要的研究方向。本实验室从桑树健康叶片中分离到一株内生菌Burkholderia cepacia Lu10-1,该菌株对桑炭疽病菌(Colletotrichum morifolium)、桑粘格孢菌(Septogloeum mori)、杨树水泡溃疡病菌(Dothiorella gregaria)、金黄色葡萄球菌(Staphylococcus aureus)以及苏芸金芽孢杆菌(Bacillus thuringis)等多种动植物病原菌具有较强的拮抗作用,且能够进入桑树体内稳定定殖,呈现出良好的开发前景。本研究通过形态观察法和药理实验法两种方法探讨Burkholderia cepacia Lu10-1菌株对家蚕黑胸败血菌(Bacillus bombysepticus)和白僵菌(Beauveria bassiana)的抑菌机制,研究该菌株发酵上清液对家蚕黑胸败血病和白僵病的防治效果,并探讨其对家蚕生理及茧质的影响。主要研究结果如下:1测定了Lu10-1菌株抗菌粗提物对家蚕黑胸败血菌的拮抗效果和最小抑杀菌浓度。结果表明:抗菌粗提物对黑胸败血菌具有较强的拮抗作用,其抑菌圈直径为18.20 mm;最小抑菌浓度(MIC)和杀菌浓度(MBC)分别为1.56 mg/mL和3.13 mg/mL。2采用分光光度法、电导率法和SDS-PAGE分析等方法测定了Lu10-1菌株抗菌粗提物对黑胸败血菌生长、胞外溶液电导率、胞内外总糖、胞内外蛋白质的影响。结果表明:经抗菌粗提物处理后,黑胸败血菌未出现对数生长期、细胞膜的渗透性发生改变、胞内糖和蛋白质发生渗漏、胞内分子量较大的蛋白出现降解现象。3采用扫描电镜和透射电镜分别观察了Lu10-1菌株抗菌粗提物对黑胸败血菌菌体形态和结构的破坏情况。结果表明:抗菌粗提物能够使黑胸败血菌菌体破裂,细胞内容物流出,形成空腔,最后消融。表明抗菌粗提物的抑菌机制为破坏黑胸败血菌的细胞壁或细胞膜。4测定了Lu10-1菌株抗菌粗提物对家蚕白僵菌的拮抗效果、对孢子萌发和菌丝的抑制作用。结果表明:抗菌粗提物对白僵菌具有较强的拮抗作用,其抑菌圈直径为17.00 mm;抗菌粗提物可以有效的抑制白僵菌孢子的萌发和菌丝的生长。5采用光学显微镜、扫描电镜和透射电镜分别观察了Lu10-1菌株抗菌粗提物对白僵菌孢子、菌丝的形态和结构的破坏情况。光学显微镜观察结果表明:抗菌粗提物作用于孢子后,孢子出现聚集、内容物分布不均、变形、空腔及胞壁断裂等异常,作用于菌丝后,菌丝出现变粗、扭曲、皱褶、膨大、消融等现象;电镜观察结果表明:抗菌粗提物能够使白僵菌孢子和菌丝的细胞质浓缩,质壁发生分离,细胞壁变薄、破裂,内容物流出,形成空腔。说明抗菌粗提物的抑菌机制为破坏白僵菌的细胞壁或细胞膜。6测定了Lu10-1菌株发酵上清液对家蚕黑胸败血病和白僵病的防治效果、及对家蚕生长发育和茧质的影响。结果表明:发酵上清液对黑胸败血病和白僵病具有较好的防治效果,对黑胸败血病的预防和治疗有效率分别为41.19%和24.00%,对白僵病的预防和治疗有效率分别为55.88%和34.00%;发酵上清液对家蚕的生长发育和茧质无不良影响。更多还原

【Abstract】 The bacteriosis and fungous disease of silkworm caused heavy economic losses every year. The drugs in hand have better control efficiency, but they have some disadvantages, for instance long-term using could reinforced drug resistance of pathogenic bacteria; impacted on the environment; some drugs had harmful to human body. To overcome the limiting problems of the drugs, developing new type microbial preparation as silkworm drugs is a key research direction.Burkholderia cepacia Lu10-1 is an antagonistic bacteria, isolated from the mulberry leaves, it was had strong antagonistic activity to the pathogenic bacteria of many animals and plants, for instance Colletotrichum morifolium, Septogloeum mori, Dothiorella gregaria, Staphylococcus aureus and Bacillus thuringis, and the strain Lu10-1 could stably colonize in the mulberry body, has a bright future.This research through modality observation and pharmacological experimental methods, analyzed its antagonistic activity to the Bacillus bombysepticus and Beauveria bassiana of silkworm, and the control efficiency of the supernatant of strain Lu10-1 zymotic fluid to septicemia and white muscardine, and investigated the effect of the physiological functions and the cocoon quality of silkworm by the supernatant. The main results were showed as the followings.1 We measured the inhibitory effect, minimal inhibitory concentration (MIC), and minimal bacteriocidal concentration(MBC) of antibacterial crude extract to the Bacillus bombysepticus. The results indicated that the antibacterial crude extract had strong antagonistic activity against the Bacillus bombysepticus. Its diameter of inhibition zone reached 18.20 mm. The MIC and MBC of antibacterial crude extract were 1.56 mg/mL and 3.13 mg/mL.2 Using spectrophotometric method, conductimetric method, and SDS- PAGE method measured the effect of the antibacterial crude extract to the growth, extracellular conductivity, intracellular and extracellular saccharide and proteins of the Bacillus bombysepticus. The results showed that after being treated by antibacterial crude extract, the Bacillus bombysepticus never appeared logarithmic phase, cell membrane permeability had changed, intracellular saccharide and protein leaked from the cell, macromolecular protein of intracellular degraded.3 The modality failure of the Bacillus bombysepticus cell was observed by electron microscope. The results showed that the antibacterial crude extract of strain Lu10-1 made the modality of the Bacillus bombysepticus damaged, inner substance outflowed, formed cavity and ablation of the cells. That just shows the antibacterial crude extract of strain Lu10-1 has apparent inhibitory effect to the Bacillus bombysepticus by destroying its cell wall and membrane.4 We measured the inhibitory effect of the antibacterial crude extract of strain Lu10-1 to the spore germination and growth of Beauveria bassiana. The results showed that the antibacterial crude extract had strong antagonistic activity against the Beauveria bassiana. Its diameter of inhibition zone reached 17.00 mm; and its also inhibit the growth and spore germination of Beauveria bassiana.5 The modality failure of the Beauveria bassiana cell was observed by electron microscope. The observation of optical microscope indicated after being treated with the antibacterial crude extract, the spore of Beauveria bassiana became collective and had such abnormalities as uneven distribution of cellular contents, deformation of spores, disappear of cellular contents and disrupted cellwall; and the mycelia of Beauveria bassiana became thicker, retorted, corrugated, and inflated with inner substance outflowed and ablation of the cells. The observation of electron microscope indicated the antibacterial crude extract could make the spore and mycelia of cytoplasm concentrated, plasmolysis, cellwall became attenuation, damaged, inner substance outflowed and formed cavity. That just shows the antibacterial crude extract has apparent inhibitory effect to the Beauveria bassiana by destroying its cell wall and membrane.6 We measured the control effect of the supernatant of strain Lu10-1 zymotic fluid to the septicemia and white muscardine of silkworm, and the effect of the physiological functions and the cocoon quality of silkworm. The results showed the supernatant of strain Lu10-1 had better control effect to the septicemia and white muscardine of silkworm, the supernatant of strain Lu10-1 achieved 41.19% control efficiency and 24.00% prophylactic effect on silkworm septicemia respectively; the prophylactic and treatment effective rate of supernatant to white muscardine respectively reached 55.88% and 34.00%; while no harmful effect was observed on the physiological functions and cocoon quality of the silkworm.更多还原