贝莱斯芽孢杆菌L-1对梨灰霉和青霉病菌的抑制作用评价及全基因组分析

Complete genome analysis of Bacillus velezensis L-1 and its inhibitory effect on pear gray and blue mold

摘要:

[目的]明确贝莱斯芽孢杆菌（Bacillus velezensis）L-1对梨灰霉和青霉病菌的抑制作用，明确菌株L-1无菌发酵液拮抗活性的稳定性及可能的拮抗机制。[方法]通过离体测定、活体测定和病原菌菌丝形态观察，评价菌株L-1对梨灰霉和青霉病菌的拮抗活性。以梨灰霉病菌为供试病原菌，利用牛津杯法测定菌株L-1无菌发酵液拮抗活性的稳定性。利用Pacbio RSⅡ三代测序技术测定L-1的全基因序列，将全基因序列与基因蛋白质序列数据库进行BLAST比对分析，预测菌株L-1可能产生的次生代谢产物及潜在的作用机制。[结果]菌株L-1对梨灰霉和青霉病菌的活体抑制率分别为92.88%和77.47%，能引起病原菌菌丝膨大、畸形。菌株L-1在含10% NaCl的培养液中仍能正常生长，其无菌发酵液耐高温、酸、碱、紫外照射和蛋白酶降解，对病原菌具有稳定的拮抗活性。全基因序列分析结果显示菌株L-1有112个基因参与了多种碳源的代谢，可以利用多种碳源进行生长；含有参与亚精胺、海藻糖等与菌株抗逆性相关化合物合成的基因；次生代谢产物预测结果显示：L-1含有合成surfactin、fengycin、bacillibactin、bacillaene、macolactin、difficidin、bacilysin等多种肽聚糖和聚酮糖类抗性化合物的基因簇，以及能够降解病原菌细胞壁的β-1，3-葡聚糖酶和几丁质酶相关的基因；此外菌株L-1含有生成乙偶姻等能够诱导植物抗性的基因。[结论]菌株L-1能有效拮抗多种梨果采后病害，抗逆性强，拮抗活性稳定，预测菌株L-1能够通过产生多种拮抗活性化合物和细胞壁水解酶类以及诱导植物抗性实现防病效果，具有很大的应用潜力。

关键词:梨灰霉病;梨青霉病;生物防治;贝莱斯芽孢杆菌;全基因组序列

Abstract:

[Objective] This work aimed to uncover the inhibitory effects of Bacillus velezensis L-1 on pear gray and blue mold, the inhibitory stability of its cell-free supernatant, and understand the molecular mechanism underlying the biocontrol processes.[Methods] We analyzed the inhibitory effects of strain L-1 on pear gray and blue mold both in vitro and in vivo, and observed the influences of strain L-1 on the mycelium growth of pathogens by microscopes. We determined the inhibitory stability of strain L-1 on Botrytis cinerea by oxford cup method and performed the complete genome sequencing of strain L-1 by Pacbio RSⅡ platform. We also annotated the obtained sequences through different protein databases to predict the mechanisms involved in the biocontrol processes.[Results] The control efficacy of strain L-1 against pear gray and blue mold was 92.88% and 77.47% respectively, and strain L-1 caused the swallow and abnormal growth of the pathogens' mycelium. Strain L-1 could grow normally in the broth containing 10% (W/V) NaCl, and its cell-free supernatant showed stable inhibitory effects against pear gray mold under acid, alkali, UV light, heat and protease treatments. Complete genome sequence analysis showed that there were 112 genes involved in the metabolism of different carbons, indicating its ability to grow on different carbon sources. Strain L-1 contained genes encoding the alimine and trehalose, that are related to stress resistance. Strain L-1 contained gene clusters related to the biosynthesis of a variety of polypeptide and polyketide compounds, such as surfactin, fengycin, bacillibactin, bacillaene, macolactin, difficidin, and bacilysin, and the genes encoding enzymes like β-1,3-glucase and chitinase, that could hydrolyze the pathogen cell wall. In addition, strain L-1 contained genes related to the biosynthesis of actoin, which could induce host resistance.[Conclusion] B. velezensis L-1 has the potential as an effective biocontrol agent.

Key words:pear gray mold;pear blue mold;biological control;Bacillus velezensis;complete genome sequence