【作者】 郭会婧；

【导师】 蒋继志；

【作者基本信息】 河北大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 为了更有效地利用微生物源活性物质控制马铃薯晚疫病,本实验首先通过平板对峙拮抗试验从36种(株)微生物中筛选出了16种(株)对致病疫霉菌丝生长有显著抑制作用的微生物,其中B3282(细菌)、枯草芽孢杆菌和A32810(放线菌)这3种微生物的抑菌率均在70%以上,A4228、A32912、A5253和A32914等4种(株)放线菌的抑菌率均在60%以上。然后采用滤纸片法对36种(株)微生物单一发酵液进行抑菌作用的测定,结果表明：仅白菜黑斑病菌、梨黑斑病菌和A32914的发酵液对致病疫霉表现出较强的抑制作用,抑菌率分别为80.51%、70.20%和48.91%。在上述试验的基础上,开展了复合微生物源发酵液对致病疫霉抑制作用的测定,结果表明：供试的不同微生物的发酵液以等体积两两混配后,有1/4的组合抑菌效果介于两者单一使用效果之间,未表现出明显的增效作用；有1/4组合(其它微生物与梨黑斑病菌或白菜黑斑病菌发酵液的组合)表现出了明显的增效作用,特别是浓度为10mg/mL的梨黑斑病菌和白菜黑斑病菌的发酵液混配后抑制作用最显著(抑菌率达90.38%),明显高于单一发酵液的抑菌效果。此外,研究了单一及复合发酵液对致病疫霉的抑制机理。结果表明：单一和复合发酵液均可导致致病疫霉菌丝出现畸形和原生质浓缩等不正常现象；经处理后的致病疫霉菌丝体中可溶性蛋白含量低于对照,表明单一和复合发酵液均有可能抑制致病疫霉菌体中蛋白质的合成；单一和复合发酵液对致病疫霉游动孢子的释放和休止孢的萌发也均具有很强的抑制作用。最后,探讨了致病疫霉拮抗菌-白菜黑斑病菌和梨黑斑病菌产生的有效抗菌物质。结果显示：白菜黑斑病菌发酵液经121℃高压灭菌20min后,仍可保留72%的抑菌活性,说明发酵液中的抗菌物质具有较强的耐热性；白菜黑斑病菌发酵液经酸碱处理后,在pH2和pHH10下仍可分别保留76%和73%的抑菌活性,说明发酵液中的抗菌物质具有较好的耐强酸、强碱性；抗菌物质在乙酸乙酯和氯仿中的溶解性较大。该发酵液经100%浓度硫酸铵沉淀后所得到的沉淀物质对致病疫霉有明显抑制作用,上清液对致病疫霉无抑制作用；该发酵液的乙醇沉淀物质对致病疫霉无抑制作用,上清液对致病疫霉表现出较好的抑制作用；发酵液直接透析后的袋内物质未对致病疫霉表现出抑制作用。表明白菜黑斑病菌发酵液中的有效抗菌物质可能是蛋白质类物质。梨黑斑病菌发酵液的硫酸铵沉淀和乙醇沉淀对致病疫霉未表现出抑菌效果,两种处理的上清液均对致病疫霉有明显的抑菌作用；梨黑斑病菌菌丝体胞内提取物的饱和(NH4)2SO4沉淀和乙醇沉淀也未对致病疫霉表现出抑制作用,而两种处理得到的上清液对致病疫霉均表现出较好的抑制作用。表明梨黑斑病菌发酵液和菌丝体中的有效抗菌物质均为非蛋白类物质。更多还原

【Abstract】 In order to effectively control potato late blight using microbial material, different antagonistic microoganism strains were selected according to their inhibiton effects on growth of Phytophthora infestans (Mont.) de Bary by using dual-culture method. Among 36 species, 16 species showd the evident inhibition effects. The inhibition rates of B3282, Bacillus sp., and A32810 were more than 70%, and rates of A4228, A32912, A5253, and A32914 were more than 60%. However, there were only fermentations from 3 of 36 species, A. brassicae, A. kikuchiana, and A32914 displaying markable suppression. And their inhibition rates were 80.51%,70.20%, and 48.91%, respectively.The inhibitory effects of combination fermentations on the hypha growth of P.infestans were also investigated. The results showed that some combinations’inhibitory effects were between the single fermentations’ inhibition, which did not show significantly synergistic or cooperation inhibition to P. infestans; some of combination fermentations from two species microorganism’s fermentations showed significantly synergistic or cooperation inhibition to P.infestans. The treatmet of A. brassicae fermantation in combination with A. kikuchiana fermantation had the strongest inhibition, and the rate of which reached up to 90.38% higher than that of the single fermentation.We also studied the inhibition mechanisms from single A. brassicae fermantation, A.kikuchiana fermantation and their mixtures on P.infestans. The results showed that:all of the single fermentations and their mixtures had inhibition on protein synthesis of P. infestans’ mycelium, zoospore release and zoospore germination of P. infestans.The crude antagonistic substance which from A. brassicae fermentation was stable to pH and heat. The results also showed the antagonistic substance had strong polar. The ammonium sulfate precipitation from A. brassicae fermentation had strong inhibition to the hypha growth of P. infestans, but its ethanol precipitation did not show inhibition to the hypha growth of P.infestans. That showed the effective antagonistic substances in the A. brassicae fermentation may be protein substances. The ammonium sulfate precipitation and ethanol precipitation from A. kikuchiana fermentation and its hypha’s crude extracts did not show strong inhibition to the hypha growth of P.infestans. That showed the effective antagonistic substances in the A. kikuchiana fermentation and hypha were not protein substances.更多还原