• ISSN 1008-505X
  • CN 11-3996/S
高晓丹, 马鸣超, 曹凤明, 朱倬漪, 李俊, 姜昕. 基于生物化学和基因组学方法的Bacillus velezensis19573-3生防潜力评价[J]. 植物营养与肥料学报, 2021, 27(6): 1068-1079. DOI: 10.11674/zwyf.2021108
引用本文: 高晓丹, 马鸣超, 曹凤明, 朱倬漪, 李俊, 姜昕. 基于生物化学和基因组学方法的Bacillus velezensis19573-3生防潜力评价[J]. 植物营养与肥料学报, 2021, 27(6): 1068-1079. DOI: 10.11674/zwyf.2021108
GAO Xiao-dan, MA Ming-chao, CAO Feng-ming, ZHU Zhuo-yi, LI Jun, JIANG Xin. Biocontrol potential of Bacillus velezensis 19573-3 identified using biochemistry and genomics methods[J]. Journal of Plant Nutrition and Fertilizers, 2021, 27(6): 1068-1079. DOI: 10.11674/zwyf.2021108
Citation: GAO Xiao-dan, MA Ming-chao, CAO Feng-ming, ZHU Zhuo-yi, LI Jun, JIANG Xin. Biocontrol potential of Bacillus velezensis 19573-3 identified using biochemistry and genomics methods[J]. Journal of Plant Nutrition and Fertilizers, 2021, 27(6): 1068-1079. DOI: 10.11674/zwyf.2021108

基于生物化学和基因组学方法的Bacillus velezensis19573-3生防潜力评价

Biocontrol potential of Bacillus velezensis 19573-3 identified using biochemistry and genomics methods

  • 摘要:
    目的 筛选具有防病促生功能的芽孢杆菌优良菌株,为植物病原菌生物防治提供生态绿色、环境友好的菌种资源。
    方法 采用菌丝生长速率抑制法和离体叶片防效筛选拮抗多种病原菌的高效菌株,通过HPLC法检测抑菌活性物质,借助基础生物学及UPLC-MS法,测定其产生植物激素等代谢物能力水平,并初步解析其促生防病相关特性。通过Biolog系统鉴定、多序列系统进化和全基因组序列分析,明确菌株19573-3的分类地位,并对其可能产生的相关次级代谢产物基因簇进行注释分析。
    结果 菌株19573-3对于灰霉菌、腐霉菌、疫霉菌、镰刀菌等多种病原真菌具有良好的拮抗能力,具有广谱抑菌活性,且该菌株对番茄灰霉病有显著的防效,经HPLC检测发现该菌株能够产生抑菌活性物质丰原素 (fengycin)。此外,该菌株兼具产生纤维素酶、蛋白酶和铁载体的能力,还可产生水杨酸、细胞分裂素和生长激素等与植物促生密切相关的物质。通过生理生化指标、16S rRNA与gyrA基因序列系统进化分析以及基于基因组水平的ANI分析鉴定,菌株19573-3属于贝莱斯芽孢杆菌 (Bacillus velezensis)。B. velezensis 19573-3基因组全长3990203 bp,G+C含量为46.56%,共编码基因4164个,编码区总长度占全基因组的比例90.03%,染色体上存在86个tRNA,27个rRNA和8个sRNA。利用antiSMASH预测菌株B. velezensis 19573-3基因组中可能存在合成丰原素 (fengycin)、表面活性素 (surfactin)、铁载体 (bacillibactin) 等与拮抗和促生相关的13个次级代谢产物合成基因簇。
    结论 细菌19573-3属于Bacillus velezensis,对引起植物病害的灰霉菌、腐霉菌和疫霉菌等多种病原真菌均具有良好的拮抗能力,菌株代谢物显示了根际促生和生物防治功能。19573-3菌株由一个环状染色体构成,不含质粒,基因组全长3990203 bp,G+C含量为46.56%。在B. velezensis 19573-3基因组序列之中,注释到了一个长度为37669 bp的基因簇,包含fenA、fenB、fenC、fenD、fenE、fenF等基因,负责产生丰原素 (Fengycin),注释到了合成铁载体 (bacillibactin) 的基因簇以及与生长激素合成相关的基因ysnS和yhcX,从基因角度证实了B. velezensis 19573-3具备产生激素的能力。

     

    Abstract:
    Objectives Bacillus spp. strain was screened and identified for eco-friendly biological control of plant pathogens, disease prevention and growth promotion.
    Methods The antagonistic ability of the strain was determined by mycelial growth rate and detached leaves method, antimicrobial metabolites were determined by HPLC method, while plant hormones were assessed based on the basic biology and UPLC-MS. Based on the results of biolog system, multi-sequence phylogenic analysis and genome mining, the strain 19573-3 was identified, and the secondary metabolite gene clusters were analyzed.
    Results Strain 19573-3 showed broad-spectrum antagonistic activity against Botrytis spp., Pythium spp., Phytophthora spp., Fusarium spp., etc. and had significant control efficiency on Bortrytis cinerea. Fengycin, salicylic acid, cytokinin and IAA were identified and quantitatively determined in the secondary metabolism of the strain. The production of cellulase, protease and siderophores were proved by selective medium plate. According to physiological and biochemical characteristics, the phylogenetic analysis of 16S rRNA and gyrA, and average nucleotide identity, strain 19573-3 was identified as Bacillus velezensis. B. velezensis 19573-3 had a single circular chromosome of 3,990,203 bp with a 46.56% G+C content, including 86 tRNA, 27 rRNA, and 8 sRNA. The bacterial genome contained 4146 genes that occupied 90.03% of the genome. Meanwhile, 13 secondary metabolite synthesis gene clusters were related to antagonism and growth promotion, which might encode the fengycin, surfactin, and bacillibactin.
    Conclusions Strain 19573-3 is identified as B. velezensis. Good antagonistic effect was proved biologically and chemically as the determination of fengycin and plant hormones in the secondary metabolism extracts. Strain 19573-3 was composed of a circular chromosome of 3,990,203 bp in size, and the code clusters for producing fengycin, surfactin, and bacillibactin were predicted on it.

     

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