甲基营养型芽孢杆菌BH21对葡萄灰霉病菌的拮抗作用

doi:10.3864/j.issn.0578-1752.2018.05.007

摘要/Abstract

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440 μg·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144.39 μg·mL^-1。HPLC分离纯化脂肽粗提物获得6个组分，只有组分BH21-2和BH21-3抑制葡萄灰霉病菌的生长，RP-HPLC色谱图分析表明组分BH21-2和BH21-3属于fengycin家族脂肽。葡萄灰霉病离体叶片试验结果表明，脂肽粗提物浓度为400 μg·mL^-1时，对葡萄叶片灰霉病防病效果为100%；脂肽粗提物浓度为220 μg·mL^-1时，对葡萄叶片病斑扩展相对抑制率为94.4%。【结论】甲基营养型芽孢杆菌菌株BH21具有合成surfactin、iturin及fengycin等多种脂肽类抗菌物质的基因，该菌株脂肽粗提物对葡萄灰霉病菌具有较强的拮抗作用，在葡萄灰霉病生物防治中具有应用潜力。

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

Abstract: 【Objective】Bacillusmethylotrophicusstrain 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 lipopeptide such as surfactins, iturins and fengycins. Antifungal lipopeptide produced by BH21 was extracted by hydrochloric acid precipitation and methanol extraction, and the yield was 428 mg·L^-1. The results of the oil spreading test showed that the crude lipopeptide had surface activity. The crude lipopeptide significantly inhibited mycelial growth of B. cinerea when the concentration was 440 μg·mL^-1, the relative inhibition rate of mycelial growth of B. cinerea was 82.8%, and the effective medium concentration EC₅₀ was 144.39 μg·mL^-1. Six fractions were collected with elution time through HPLC, only BH21-2 and BH21-3 inhibited the growth of B. cinerea. RP-HPLC chromatogram analysis showed that the components BH21-2 and BH21-3 belong to the fengycin family. Grape leaf in vitro test results showed that when the concentration of crude lipopeptide was 440 μg·mL^-1,the control effect againstgrape gray leaf spot was 100%, while the concentration was 220 μg·mL^-1, the relative inhibitory rate of grape leaf lesion was 94.4%.【Conclusion】 The strain BH21 has the genes for synthesizing antimicrobial lipopeptide such as surfactins, iturins and fengycins, and the lipopeptide extracted from this strain has strong antagonism to B. cinerea, so it has potential application in the biological control of B. cinerea.

Key words: Bacillus methylotrophicus, Botrytis cinerea, antifungal activity, lipopeptide, grape gray mold

魏新燕，黄媛媛，黄亚丽，杜克久. 甲基营养型芽孢杆菌BH21对葡萄灰霉病菌的拮抗作用[J]. 中国农业科学, 2018, 51(5): 883-892.

WEI XinYan, HUANG YuanYuan, HUANG YaLi, DU KeJiu. Antagonism of Bacillus methylotrophicus Strain BH21 to Botrytis cinerea[J]. Scientia Agricultura Sinica, 2018, 51(5): 883-892.

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