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

Abstract

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

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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