【作者】 胥丽娜；

【导师】 刘振宇；

【作者基本信息】 山东农业大学 ， 森林保护， 2009， 硕士

【摘要】 杨树水泡型溃疡病是由葡萄座腔菌(Botryosphaeria dothidea)侵染引起的重大枝干病害,该病普遍发生,危害严重,防治困难,严重影响着杨树的经济效益及生态效益。因而,寻求对杨树病害合理有效的防治措施已势在必行。生物防治具有安全而又环境友好的特点,越来越受到人们的关注,并展现出重大的发展潜力。有关杨树水泡溃疡病的生物防治研究,国内外报道较少。作为一类新型的生防因子,植物内生细菌是定植在健康植物的各种组织和器官中,与植物之间形成互惠共存、相互制约的一类微生物。从杨树体内分离筛选有益细菌并应用于杨树病害的生物防治实践中将具有重要的现实意义。本研究旨在从杨树体中分离内生细菌并对其进行多样性分析;筛选出具有较高拮抗活性的内生细菌;并对内生细菌PEBA20进行系统的分类学鉴定,测定了该内生细菌的发酵条件及抑菌活性;而对其代谢产物—蛋白质进行初步纯化。主要研究结果如下:1.于2007～2008年,3、6、9、12月4个月份,从泰安市城区选取多年生的三倍体毛白杨枝干共分离到126株内生细菌,并进行室内平板拮抗筛选测定,获得12株内生细菌对杨树水泡溃疡病病菌有较好的拮抗活性。通过4个月份的分离细菌,发现杨树体内含有大量的内生细菌,多样性较丰富。采用Shannon-Wiener分析其多样性,多样性指数(H)、丰度(D)、均匀度(J),发现在不同分离时间的杨树枝条中内生细菌的种类、数量和多样性指数明显不同。2.通过培养性状、染色反应、生理生化特征分析和16SrDNA序列分析等方法对PEBA20进行鉴定。结果表明,该菌为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。对峙培养发现PEBA20对杨树水泡溃疡病病菌产生较宽的抑菌圈,表现较好的抑菌作用。抑菌谱测定结果表明,PEBA20菌具有广谱抑菌活性,对杨腐烂病菌、茄子绵疫病菌、杨树炭疽病菌、合欢枯萎病菌等12种病原真菌均有较好的抑制作用。PEBA20菌株的活体和发酵滤液均表现广泛的拮抗谱,对杨树腐烂病菌抑菌效果最好,抑制率分别为:89.37%、82.62%;对杨树水泡溃疡该菌也有较好抑菌作用,抑制率分别为:69.95%、63.2%。这说明PEBA20对杨树病害的防治有较好的效果。该菌株也表现出良好的抗细菌活性,对核桃黑斑病菌、烟草青枯病菌、马铃薯环腐病菌、巨大芽孢杆菌等细菌的抑菌圈均在12mm以上。3.本研究初步探讨了不同培养条件下的发酵液对杨树水泡溃疡病菌的抑菌活性测定。测定不同培养条件的发酵液对杨树水泡溃疡病菌的抑菌效果,得出在NB培养基,培养120 h的PEBA20菌的发酵液,对杨树水泡溃疡病菌的抑菌效果最佳。通过二次正交设计优化发酵条件,计算得到该菌最适宜发酵条件为:T=28.5℃,rpm=140rpm,pH=5,通气量100mL,t=120h。4. PEBA20菌发酵培养后能够产生抗菌蛋白,不同培养条件下的蛋白粗提液对杨树水泡溃疡病病菌有着较好的拮抗效果。PEBA20菌发酵液的蛋白组分和0-60%饱和度的硫酸铵盐析粗蛋白,经CM-Sepharose Fast Flow阳离子交换柱层析,发现PEBA20菌发酵后的蛋白质中含有阳离子蛋白质,并能通过CM-Sepharose Fast Flow获得初步分离;经DEAE-Sepharose Fast Flow阴离子交换柱层析,形成明显的蛋白质吸收峰,各峰相对独立,峰形较尖锐,且没有拖尾。PEBA20菌的初步分离,为进一步分离纯化奠定基础。更多还原

【Abstract】 Poplar canker caused by Botryosphaeria dothidea, was a key stem disease which occurred widespreadly and was difficult to control.Biological control was an envirorment friendly method for the disease management with great potential development. There was little reports about biological control of Botryosphaeria dothidea. As a new type of biocontrol factors, plants endophytic bacteria were a class of microorganisms which colonied in a variety of tissues of healthy plants with mutually beneficial co-existence between plants. There was significance of controlling the diseases of poplars with endophytic bacteria isolated from the poplar body.In this study, we isolated endophytic bacteria from the poplar trees and analysed the bacteria diversity, selected the endophytic bacteria with high antagonistic activity, identificated the endophytic bacteria strain of PEBA20 with systematic taxonomy identification, determined its optimization of fermentation conditions and antibacterial activity, prelimary purified the proteins. The main findings showed as follows:1. 126 endophytic bacteria were isolated from branches of triploid Populus from Tai-an city in march, june, september and december, 2007 to 2008. We selected 12 poplar endophytic bacteria showed good antagonist activity to Botryosphaeria dothidea with flat antagonist screening method. We found a lot of poplar endophytic bacteria in poplars from these separated bacteria. Using Shannon-Wiener analysis with diversity index (H), abundance (D), evenness (J), the endophytic bacteria from poplar in different time showed significant difference.2. We identificated PEBA20 as Bacillus amyloliquefaciens through cultivation, staining reaction, physiological or biochemical characteristic analysis and 16SrDNA sequence analysis. PEBA20 showed good antimicrobial effect on Botryosphaeria dothidea. The results showed that the antibacterial spectrum of PEBA20 was broad, the antibacterial activity were high against Valsa sordida、Phytophthora capsiciLeon、Glomerella cingulata、 Fusarium oxysporum and other 12 kinds of pathogenic fungi, the inhibitory rate of the bacteria and fermentation filtrate against Valsa sordidaI was 79.53% and 67.32% respectively, while the inhibitory rate against Botryosphaeria dothidea was 53.27% and 40.48% respectively. This indicated that PEBA20 has good effect to control the disease of poplar trees. PEBA20 also showed good antibateria activity against Xanthomonas jugandis、Ralstonia solanacearum、Clavibacter sepedonicum、Bacillus megaterium , the inhibition zones to bacteria were more than 12mm.3. Antifungal activitives of different fermentation conditions of PEBA20 were determined. The results showed that culturing for 120h and cultured in NB medium were the favorable conditions for the antibiotic activity. We calculated the most suitable fermentation conditions for PEBA20 were T = 28.5℃, rpm = 140rpm, pH = 5, ventilation volume 100mL, t = 120h.4. Crude protein of PEBA20 under different culture conditions had better effects to Botryosphaeria dothidea,indicating the existence of th antibiotic proteins. The column chromatography with CM-Sepharose Fast Flow and DEAE-Sepharose Fast Flow demonstreated that the protein with antibiotic activity could be purified.更多还原