甲基营养型芽孢杆菌BH21对葡萄灰霉病菌的拮抗作用  

Antagonism of Bacillus methylotrophicus Strain BH21 to Botrytis cinerea

作　　者：魏新燕[1] 黄媛媛[2] 黄亚丽[2] 杜克久[1] WEI XinYan1, HUANG YuanYuan, HUANG YaLi2, DU KeJiu1(1 College of Forestry, Hebei Agricultural University~National Engineering Research Center for Agriculture in Northern Mountainous Areas, Baoding 071001, Hebei; 2Institute of Biology, Hebei Academy of Sciences, Sho＇iazhuang 05005)

机构地区：[1]河北农业大学林学院/国家北方山区农业工程技术研究中心,河北保定071001 [2]河北省科学院生物研究所,石家庄050051

出　　处：《中国农业科学》2018年第5期883-892,共10页Scientia Agricultura Sinica

基　　金：国家重点研发计划(2016YFC0501303);河北省应用基础研究计划重点基础研究项目(15962904D)

摘　　要：【目的】甲基营养型芽孢杆菌(Bacillus methylotrophicus)BH21是一株对葡萄灰霉病菌(Botrytis cinerea)有较好拮抗作用的海洋源细菌,鉴定该菌株脂肽类抗菌物质合成基因,检测脂肽粗提物对葡萄灰霉病菌的拮抗作用,为应用该菌株防治葡萄灰霉病提供科学依据。【方法】通过特异引物的基因组PCR法检测甲基营养型芽孢杆菌BH21菌株合成脂肽的能力;盐酸沉淀和甲醇抽提法从无菌发酵液中提取脂肽粗提物;排油圈法检测脂肽粗提物的表面活性;采用菌丝生长速率法检测脂肽粗提物对灰霉病菌菌丝生长的抑制作用并计算有效中浓度EC50;利用高效液相色谱技术(HPLC)对脂肽粗提物洗脱分离,并采用菌丝生长速率法检测各组分对葡萄灰霉病菌的抑制能力;采用反相高效液相色谱(RP-HPLC)分析主要抑菌成分的脂肽类型。采用离体叶片接种法检测脂肽粗提物对葡萄灰霉病的防治效果。【结果】选取11对特异引物对菌株BH21基因组扩增,其中7对引物扩增出预期核酸片段;扩增产物经测序、BLAST比对分析,结果显示扩增产物与相关菌株脂肽基因相似度为96%—99%,扩增产物翻译的蛋白与相关菌株的脂肽合成蛋白相似度为96%—100%,表明甲基营养型芽孢杆菌BH21基因组中含有ituA、bamD、ituC、ituD、fenD、srfAB、yndJ,该菌株具有合成surfactin、iturin及fengycin等多种脂肽类抗菌物质的能力。盐酸沉淀和甲醇抽提从LB无菌发酵液中获得脂肽粗提物,得率为428 mg·L^(-1)。排油圈检测结果显示,脂肽粗提物使橄榄油膜形成排油圈,表明脂肽粗提物具有表面活性。脂肽粗提物对葡萄灰霉病菌菌丝生长具有显著的抑制作用,脂肽粗提物浓度为440μg·mL^(-1)时,对葡萄灰霉病菌菌丝生长的相对抑制率为82.8%。根据毒力方程计算,抑制葡萄灰霉病菌菌丝生长的脂肽粗提物EC50为144.39μg·mL^(-1)。HPLC分离纯化脂肽粗提物获得6个组分,�【Objective】Bacillus methylotrophicus strain BH21 is a marine derived bacterium that has a good antagonistic effect on Botrytis cinerea. The objective of this study is to identify the lipopeptide synthesis genes of the strain BH21 and investigate the antagonism of the crude extracts of lipopeptide to B. cinerea, and to provide a scientific basis for the prevention and control of B. cinerea. 【Method】 To determine the mechanisms of the antagonistic strain, PCR was used to screen strain BH21 for genes involved in biosynthesis of antimicrobial lipopeptide. Crude lipopeptide was extracted from the culture broth by hydrochloric acid precipitation and methanol extraction. Surface activity of the crude lipopeptide was determined by oil spreading method. The inhibition ability of the crude lipopeptide on mycelial growth of B. cinerea was investigated by mycelial growth rate method and EC50 was calculated. The crude lipopeptide was separated by liquid chromatography（HPLC） and the inhibition ability of each component to B. cinerea was detected by the mycelium growth rate method. Reversed phase high performance liquid chromatography（RP-HPLC） was used to analyze the types of the main antifungal components. The effect of the crude lipopeptide on the control of B. cinerea in grape was detected by tissue inoculation in vitro. 【Result】 A total of 11 pairs of specific primers were used for genomic amplification of strain BH21, and 7 gene fragments of the size expected to be correlated with biocontrol activities were efficiently amplified. After amplification, sequencing and BLAST analysis, the results showed that the similarity between the amplified products and the related lipopeptide genes was 96%-99%, the similarity between the protein produced by the nucleic acid fragment and the lipopeptide synthesized protein of the related strain was 96%-100%, which showed that the genome of strain BH21 contained ituA, bamD, ituC, ituD, fenD, srfAB and yndJ genes and the strain had the ability to synthesize antimicrobial

关 键 词：甲基营养型芽孢杆菌 葡萄灰霉病菌 抗真菌活性 脂肽 葡萄灰霉病 

分 类 号：S436.631[农业科学—农业昆虫与害虫防治] S476[农业科学—植物保护]