贝莱斯芽孢杆菌(Bacillus velezensis) JJYY防控土传病害效果评价及其全基因组测序分析和抗菌成分鉴定

Bacillus velezensis JJYY against soil-borne diseases: biocontrol effect, whole genome sequencing, and antimicrobial components

摘要:

【背景】土传病害是世界农业可持续发展的关键限制因子，在我国蔬菜和中药材上发生日趋严重，引起严重的连作障碍。生物防治对环境和农产品安全，是目前研究的热点和重点。【目的】明确对土传病原菌具有广谱抗菌活性菌株JJYY的种类及其防控效果和主要抗菌产物，为新型生物农药开发奠定基础。【方法】结合扫描电镜观察、生理生化分析和16S rRNA基因扩增测序技术鉴定菌株JJYY。分别利用比浊法和菌丝生长抑制法测定该菌提取物对4种土传病原细菌和5种土传病原真菌的EC₅₀，利用盆栽试验评价对番茄青枯病等病害的防控效果。利用二代Illumina NovaSeq与三代PacBio Sequel相结合测序技术对菌株JJYY进行全基因组测序，使用PGAP等软件进行基因注释等分析，利用反相制备液相色谱和质谱初步分离鉴定抗菌物质。【结果】菌株JJYY是一株贝莱斯芽孢杆菌（Bacillus velezensis），该菌提取物对4种土传细菌和5种土传真菌的EC₅₀分别为0.940-1.092 mg/mL和2.733-3.678 mg/mL。对番茄青枯病、菊花根腐病和辣椒枯萎病的最高防效在80.00%-87.74%之间，与化学药剂链霉素或噁霉灵无显著差异（P>0.05）。该菌基因组大小为3 929 792 bp，编码3 895个基因，与B.velezensis JS25R和B.subtilis 168具有3 445个和2 997个同源编码基因。预测该基因组共有12个次级代谢产物合成功能基因簇。从该菌株提取物中共分离出9种抗菌组分，其中2个初步判定为已知大环内酯类抗生素macrolactin D和7-O-malonyl-macrolactin A。【结论】菌株JJYY是一株对土传病原细菌和真菌病害均有较高防效的贝莱斯芽孢杆菌，该菌基因组与已知贝莱斯芽孢杆菌不同，并产生多种已知和未知的抗菌物质，继续深入分析鉴定其抗菌物质和抗菌机制将为开发新型高效的生物农药奠定良好基础。

Abstract:

[Background] Soil-borne diseases are a key factor limiting the agriculture sustainable development in the world and become increasingly serious in vegetables and Chinese medicinal herbs in China, leading to critically continuous-cropping obstacles. Biocontrol of soil-borne diseases, without causing safety problems to the environment and agricultural products, is a hot research topic. [Objective] To identify the strain JJYY with a broad antimicrobial spectrum to soil-borne pathogens, evaluate its control effects on major soil-borne diseases, and identify its major antimicrobial metabolic products. [Methods] This strain was identified by a combination of morphological observation with scanning electron microscopy, physiological and biochemical tests, and 16S rRNA gene sequencing. Turbidimetry and mycelial growth inhibition assay were employed to determine the EC₅₀ of this strain against four soil-borne bacterial pathogens and five fungal pathogens, respectively. Pot experiments were carried out to evaluate the control effects of the strain on tomato bacterial wilt and other diseases. Illumina NovaSeq and PacBio Sequel were used for whole genome sequencing of this strain, followed by functional annotation in PGAP. Preparative reversed-phase liquid chromatography (prep-RPLC) and mass spectrometry (MS) were employed to isolate and identify the metabolites of this strain. [Results] The strain JJYY was identified as Bacillus velezensis. The EC₅₀ values of the extract of this strain against four soil-borne bacterial pathogens and five soil-borne fungal pathogens were 0.940-1.092 mg/mL and 2.733-3.678 mg/mL, respectively. The strain showed the highest control effects of 80.00%-87.74% on tomato bacterial wilt, Chrysanthemum morifolium root rot, and capsicum Fusarium wilt, with no significant difference compared with streptomycin or hymexazol (P>0.05). The whole genome of this strain was 3 929 792 bp in length, carrying 3 895 genes. The genome of this strain shared 3 445 and 2 997 homologous genes with B. velezensis JS25R and B. subtilis 168, respectively. Twelve gene clusters were predicted to be associated with the synthesis of secondary metabolites. A total of 9 antimicrobial components were separated from the extract of this strain, of which two were preliminarily identified as macroactin D and 7-O-malonyl-macroactin A. [Conclusion] B. velezensis JJYY demonstrates definite control effects on both soil-borne bacterial and fungal diseases, with the genome distinct from reported B. velezensis strains. It produces diverse known and possible novel antibiotics. Identifying the antimicrobial substances and deciphering the antimicrobial mechanisms of this strain would facilitate the development of novel effective biocontrol agents.