赤霉病菌拮抗菌Bacillus subtilis AF0907抗菌物质研究  

Study of Antagonistic Substance from Bacillus subtilis AF0907 against Fusarium graminearum

作　　者：胡晓丹[1,2] 王建伟[2] 李孝敬 祭芳[2] 史建荣[2,3] 徐剑宏 

机构地区：[1]南京农业大学生命科学学院/农业部农业环境微生物工程重点开放实验室,南京210095 [2]江苏省农业科学院食品质量安全与检测研究所/江苏省食品质量安全重点实验室/省部共建国家重点实验室培育基地/农业部农产品质量安全控制技术与标准重点实验室/江苏省转基因安全评价公共服务中心,南京210014 [3]江苏省现代粮食流通与安全协同创新中心,南京210014

出　　处：《中国生物防治学报》2015年第3期378-385,共8页Chinese Journal of Biological Control

基　　金：公益性行业(农业)科研专项(201303088);国家自然科学基金项目(31471662);江苏省农业科技创新基金(CX(13)3092);农业部风险评估项目(GJFP201500702)

摘　　要：菌株AF0907是从土壤中分离到的1株对小麦赤霉病菌具有显著拮抗作用的枯草芽孢杆菌,该菌株不仅对小麦赤霉病菌具有很好的拮抗活性,对其他多种植物病原菌都具有很好的拮抗效果。为了明确菌株AF0907对赤霉病菌的抑菌机理,本研究首先使用平板对持法对拮抗物质进行定位,结果表明该菌分泌的拮抗物质主要位于细胞上清液中。采用不同饱和度的硫酸铵沉淀细胞上清液,确定了60%的硫酸铵饱和度是沉淀该拮抗物质的最佳条件;分别研究了温度、p H值、蛋白酶K、氯仿对该拮抗物质活性的影响,结果表明该拮抗物质在低温下比较稳定,在60℃以上的高温下,活性迅速降低;在酸性或碱性条件下不稳定;该拮抗物质分别加入蛋白酶K和氯仿作用后拮抗活性分别下降了60.68%、80.07%,因此,初步判断该拮抗物质为蛋白质。利用DEAE-52离子交换柱层析法分离纯化了该拮抗蛋白并进行了SDS-PAGE电泳,结果显示该拮抗蛋白的分子量为48 k Da;利用PPSQ-31A蛋白自动测序仪测定拮抗蛋白N-末端15个氨基酸序列为:His-Glu-Phe-Pro-Thr-Tyr-Lys-His-Met-Tyr-Gln-Val-Met-His-Leu。Bacillus subtilis AF0907 with strong inhibitory activity against Fusarium graminearum was isolated from soil. In order to clarify the mechanism of inhibition activity of stain AF0907, the distribution of antagonistic substances was studied by confrontation culture on PDA plates and found to be mainly distributed in cell supernatant. The cell supernatant of strain AF0907 was precipitated by ammonium sulfate at different concentration, and the result showed that 60% ammonium sulfate saturation is the most suitable condition for the antagonistic substance precipitation. The effects of temperature, p H, proteinase K, and chloroform on the inhibition activity of antagonistic substance in AF0907 were then studied. The results showed that antagonistic substance was stable in temperature lower than 60 ℃, but the inhibition activity decreased immediately in temperature higher than 70 ℃. The antagonistic substance was also not stable under acidic or basic conditions. The inhibition activity of the antagonistic substance in AF0907 declined by 60.68%、80.07% after treated with proteinase K and chloroform for one hour in 37 ℃ respectively. According to the antagonistic characterization, the antagonistic substance in AF0907 was identified as protein consequently. The antagonistic protein was then isolated and purified by DEAE-52 column after precipitating by ammonium sulfate and its molecular weight was about 48 k Da detected by SDS-PAGE electrophoresis. The amino acid at N terminal amino acid of the antagonistic protein was sequenced as His-Glu-Phe-Pro-Thr-Tyr-Lys-His-Met-Tyr-Gln-Val-Met-His-Leu by PPSQ-31 A protein automated sequencer.

关 键 词：小麦赤霉病菌 枯草芽孢杆菌 拮抗菌 拮抗蛋白 

分 类 号：S476[农业科学—农业昆虫与害虫防治] S435.121.45[农业科学—植物保护]