【作者】 张晓云；

【导师】 马平；

【作者基本信息】 河北农业大学 ， 植物病理学， 2011， 硕士

【摘要】 枯草芽孢杆菌菌株CAB-1是从黄瓜根际土壤中分离筛选到的一株生防细菌,对黄瓜白粉病、番茄灰霉病及番茄叶霉病等蔬菜病害均具有良好的防治效果。本研究对菌株CAB-1产生的三种抑菌物质:蛋白、脂肽及挥发性物质进行了分离、鉴定和分析。枯草芽孢杆菌菌株CAB-1的抑菌蛋白:采用40%饱和度的硫酸铵对菌株CAB-1的发酵上清液进行盐析,得到蛋白粗提物。该蛋白粗提物能明显降低黄瓜白粉菌分生孢子的萌发率,并造成菌丝顶端膨大成球状无法继续生长。子叶喷雾法测得其对黄瓜白粉病的保护作用和治疗作用的防效分别为73.33%和76.85%。单因素实验表明该蛋白粗提物的最佳产生条件为:3号培养基,2%接种量,培养温度30℃,转速180 r/min,装液量100 mL/250 mL,培养时间48 h。该粗蛋白对胰蛋白酶及蛋白酶K不敏感,胃蛋白酶处理使其活性降低14%;抑菌蛋白在100℃以下处理30 min活性变化差异不显著,而121℃处理30 min其抑菌活性为对照的72%;抑菌蛋白对酸碱的耐受范围广,在pH值3-12的范围内抑菌活性均能保持在75%以上;经甲醇、氯仿、乙酸乙酯及丙酮处理后该粗蛋白的抑菌活性变化不大。采用HiTrap DEAE-Sepharose Fast Flow阴离子交换层析柱对蛋白粗提物进行分离纯化后获得五个吸收峰,其中峰Ⅲ具有较大抑菌活性。峰Ⅲ收集液经SDS-PGAE检测后为单一蛋白条带,其分子量约为66 KDa,经MALDI-TOF-MS鉴定分析该蛋白与putative phage-related pre-neck appendage protein匹配率最高,分值为135(P<0.05)。枯草芽孢杆菌菌株CAB-1的脂肽类抑菌物质:采用盐酸沉淀法提取得到脂肽类物质,子叶喷雾法测得该物质对黄瓜白粉病的防效为76.17%。MALDI-TOF-MS分析结果显示该脂肽类物质的[M+H]+值分别呈现1447.725,1461.813,1477.109和1500.041,1513.839,1527.566两类信号。其中1447.725,1461.813,1477.109推测为Fengycin A(C15-C17);而在目前的报道中并没有检索到与1500.041,1513.839和1527.566分子量相似的脂肽类物质,推测可能是一种新的物质。枯草芽孢杆菌菌株CAB-1的挥发性抑菌物质:通过平板熏蒸法测定了菌株CAB-1所产挥发性抑菌物质(VCs)对黄瓜白粉菌的抑制作用,结果表明VCs对黄瓜白粉病的防效为85.44%。经GC-MS分析鉴定,菌株CAB-1产生的挥发性抑菌物质由多种成分组成,包括胺类、醇类、烷烃类、酚类、酰胺类等27种物质。VCs防治黄瓜白粉病的最佳作用条件为:在0.012 m~3的密闭空间内,2皿CAB-1平板培养熏蒸96 h,防效达到94.60%。更多还原

【Abstract】 Bacillus Subtilis strain CAB-1 was isolated from rhizosphere soil of cucumber and showed strong inhibition activity against many vegetable diseases such as cucumber powdery mildew, tomato gray mold, tomato leaf mold etc. The objective of this study was to isolate and identify the antifungal protein,lipopeptides and volatile compounds produced by B. subtilis strain CAB-1 and to analyze their inhibitory activities.Antifungal protein was isolated from the cultural supernatant of B. subtilis strain CAB-1 using 40% Ammonium sulfate precipitation. The crude protein effectively restrained the conidia germination and the growth of mycelia of Sphaerotheca fulginea. The crude protein reduced the disease index of cucumber by cotyledon-spraying method, the protective and curative effect were 73.33% and 76.85 respectively. The optimum culture condition for antifungal protein production was 2% inoculums volume to NO.3 medium, 100 mL loading amount in a 250 mL flask, at 30℃for 48 h on a rotary orbital shaker with 180 r/min speed. The crude protein produced by strain CAB-1 was not sensitive to Trypsin and Proteinase K, while the antifungal activity was reduced 14% by Pepsin. The crude protein showed stable activity at 100℃, and retained 72% antifungal activity at 121℃for 30 min. The antifungal activity of crude protein kept stable over a wide pH value. The antifungal activity maintained in organic solvents such as methanol, chloroform, ethyl acetate and acetone. The antifungal protein was purified by DEAE-Sepharose Fast Flow chromatography, and five fractions were harvested. FractionⅢshowed higer antifungal activity and a single band with a molecular mass of 66 kDa was collected following the SDS-PAGE method. According to the analysis of MALDI-TOF-MS, the similarity of amino acid sequence between the the purified protein and putative phage-related pre-neck appendage protein was very high.Lipopeptides were isolated from the cultural supernatant of strain CAB-1 by the methods of hydrochloric acid precipitation. The protective value of lipopeptides-treated plot was 76.17%. According to MALDI-TOF-MS analysis, the lipopeptides revealed two groups [M+H]+ ion peaks at 1447.725,1461.813,1477.109 and 1500.041,1513.839,1527.566 respectively. And the first group was determined as Fengycin A (C15-C17), however, the other group of homologues was undetermined.We also assayed the fumigating effect of the volatiles compounds(VCs) from strain CAB-1 to S. fulginea . VCs fumigation reduced the disease index of cucumber and the protective value was 85.44%. The VCs produced by CAB-1were identified by using GC-MS method, yielded 27 compounds, which included a range of alcohols, phenols, amines, alkanes, amides etc. Comparing with different doses and different times treatments, 2 plates fumigating for 4 days in 0.012 m~3 airtight space was our choice in the controlled greenhouse.更多还原