一株贝莱斯芽孢杆菌鉴定、全基因组学分析及其抗病促生特性

Identification, genomic analysis, and disease-resistant and plant growth-promoting characterization of a Bacillus velezensis

摘要:

植物病原菌侵染引起的作物病害给全球农业带来了巨大危害，而生物防治因其有效性和环境友好性等特点，已成为防控植物病害的研究热点。目的 从实验室保藏的菌种库中筛选拮抗植物病原菌的目标菌株，对其进行鉴定、全基因组学分析，并评价其抗病促生效果。方法 采用平板对峙法筛选拮抗菌；利用形态学观察、16S rRNA基因测序、基因组系统发育分析、基因组平均核苷酸相似性(average nucleotide identity, ANI)和数字DNA杂交(digital DNA-DNA hybridization, dDDH)值比较分析，以及生理生化特征等方法进行菌种分类学鉴定；对目标菌株进行全基因组序列分析，挖掘功能基因；通过测定溶磷、产铁载体、产蛋白酶和产纤维素酶活力来评价目标菌株的促生特性；采用二分隔培养皿检测目标菌株产生的挥发性气体对植物病原真菌抑制情况；通过盆栽试验探究目标菌株对番茄的促生防病效果。结果 获得1株对茄科罗尔斯通氏菌(简称青枯菌)及3种植物病原真菌具有明显抑菌活性的菌株MB1019，经鉴定其为贝莱斯芽孢杆菌(Bacillus velezensis)。生理生化特征显示，菌株MB1019的耐受温度为15-50 ℃，最大耐受NaCl浓度为10.0%，耐受pH值为5.0-9.0。全基因组分析显示，菌株MB1019基因组大小为3.8 Mb，G+C含量为46.4%，antiSMASH预测显示，菌株MB1019基因组含有17个次级代谢产物基因簇。dbCAN2预测显示，菌株MB1019含有52种、共108个CAZy家族基因，其中糖苷水解酶(glycoside hydrolases, GHs)家族种类和数量最多。菌株MB1019产生的挥发性气体能够显著抑制植物病原真菌的生长。盆栽试验结果显示，菌株MB1019对番茄具有明显促生防病效果。结论 贝莱斯芽孢杆菌MB1019具有拮抗青枯菌和植物病原真菌的能力，展现出耐高温、耐盐碱以及促生功能，其基因组中含有大量抑菌基因，并能产生挥发性气体有效抑制病原真菌的生长，MB1019菌液对番茄具有促生防病效果。综上所述，菌株MB1019作为微生物肥料和农药研发的优良菌种资源，展现出良好的开发和应用潜力。

Abstract:

Crop diseases caused by phytopathogens result in great harm to global agriculture. Biocontrol has garnered increasing attention in plant disease prevention and control because of its effectiveness and environmental friendliness.Objective To identify, analyze the genome, and evaluate the disease-resistant and plant growth-promoting effects of the strains with antagonistic activities that were screened from the culture collection.Methods Antagonistic strains were screened by plate confrontation method. and identified by morphological observation, phylogenetic analysis based on the 16S rRNA gene and genome, comparative analysis of average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values, and physiological and biochemical tests. The whole genome sequence of the target strain was analyzed for the mining of functional genes. The plant growth-promoting effect of the target strain was characterized in terms of the phosphorus-solubilizing, siderophore-producing, and proteinase- and cellulose-producing properties. The inhibition of volatile gas produced by the target strain on phytopathogenic fungi was examined in petri dishes with a septum. The effects of the target strain on tomato growth and diseases were studied by pot experiments.Results Strain MB1019 with obvious inhibitory effects on Ralstonia solanacearum and three phytopathogenic fungal strains was screened out and identified as Bacillus velezensis. The physiological and biochemical tests showed that strain MB1019 tolerated the temperature of 15-50 ℃, 10.0% NaCl, and pH 5.0-9.0. The genome of strain MB1019 was 3.8 Mb in length, with the G+C content of 46.4%. The prediction on antiSMASH suggested that the MB1019 genome had 17 synthetic gene clusters for secondary metabolites. The prediction on dbCAN2 suggested that MB1019 carried 108 genes belonging to 52 types of the CAZy family, among which glycoside hydrolases (GHs) were the richest and most abundant. The volatile gas produced by MB1019 significantly inhibited the growth of phytopathogenic fungi. The results of pot experiments showed that MB1019 promoted the growth and inhibited the diseases of tomato seedlings.Conclusion B. velezensis MB1019 capable of antagonizing R. solanacearum and phytopathogenic fungi has tolerance to high temperatures and saline-alkali and demonstrates plant growth-promoting effects. Its genome contains a large number of functional genes, and the volatile gas produced by this strain can inhibit the growth of pathogenic fungi. The fermentation broth of MB1019 has the effects of promoting the growth and preventing the diseases of tomato seedlings. In summary, B. velezensis MB1019 can be used as an elite strain in the research and development of microbial fertilizers and pesticides, demonstrating promising development and application prospects.