基于基因组的一株土壤固氮菌分离菌株鉴定及其促生作用

Genome-based identification and plant growth promotion of a nitrogen-fixing strain isolated from soil

摘要:

[目的] 为获得高效固氮菌株，充分研究利用土壤固氮菌资源。[方法] 选取固氮能力较高的紫色土发育水稻土，采用富集纯化法分离固氮微生物菌株。通过16S rRNA基因系统发育分析和全基因组相关指数比较对新分离菌株进行物种鉴定。采用乙炔还原法和¹⁵N₂示踪法定量测定新分离菌株的固氮能力，通过培养特性和接种效果初步研究固氮菌株的促生作用。[结果] 从紫色土发育水稻土中分离得到1株可在无氮培养基上快速生长的菌株P208。基于16S rRNA基因和基因组92个核心基因的系统发育分析结果表明，新分离菌株P208与Azotobacter chroococcum IAM 12666^T（=ATCC 9043^T）系统发育距离最近（16S rRNA基因相似度为99.79%）。菌株P208与A.chroococcum ATCC 9043^T的基因组平均核苷酸一致性（ANI）、平均氨基酸一致性（AAI）和数字DNA-DNA杂交值（dDDH）高于物种分类阈值（ANI>；95%-96%，AAI>；95%-96%，dDDH>；70%），最大唯一匹配指数（MUMi）低于物种分类阈值（<；0.33），得出新分离菌株P208为褐球固氮菌（A.chroococcum）。A.chroococcum P208固氮活性为模式菌株A.chroococcum ATCC 9043^T的2.61倍。除固氮能力外，A.chroococcum P208具有IAA生成、溶磷活性和铁载体生成等促进植物生长潜力的培养特性，室内培养条件下接种A.chroococcum P208能够促进水稻、小麦幼苗根系的生长。[结论] 从固氮能力较强的水稻土中分离纯化得到1株具有较强固氮、促生潜力的固氮菌，具有潜在的开发应用价值，可为研究利用生物固氮提供微生物资源。

关键词:生物固氮;固氮菌;褐球固氮菌;促生作用;促生菌

Abstract:

[Objective] The aim of this study is to obtain efficient nitrogen-fixing strains and fully utilize soil nitrogen-fixing microbial resources.[Methods] The Entisol with superior nitrogen fixation capability was selected to isolate nitrogen-fixing strains via enrichment and purification procedure. The identification of newly isolated strain at species level was performed by means of 16S rRNA gene phylogenetic analysis and whole genome correlation index comparison. Quantitative determination of nitrogen fixation activity of newly isolated strain was performed by acetylene reduction assay and ¹⁵N₂ tracer method. The plant growth promotion potentials of the nitrogen-fixing strain were preliminarily determined through culture characteristics and inoculation effects.[Results] A strain, designated P208, which could grow rapidly in nitrogen-free medium, was isolated from Entisol. Phylogenetic analyses based on 16S rRNA gene and 92 core genes revealed that strain P208 was most phylogenetically closely related to Azotobacter chroococcum IAM 12666^T (=ATCC 9043^T) (99.79% 16S rRNA gene similarities). The average nucleotide identity (ANI), average amino acid identity (AAI) and digital DNA-DNA hybridization (dDDH) values based on genome sequences of strain P208 and A. chroococcum ATCC 9043^T were higher than the thresholds for species circumscription (ANI>95%-96%, AAI>95%-96%, dDDH>70%), and the maximal unique matches index (MUMi) value was lower than the species circumscription threshold (<0.33). Therefore, The newly isolated strain P208 could be identified as A. chroococcum. The nitrogen fixation activity of A. chroococcum P208 was 2.61 times that of the type strain A. chroococcum ATCC 9043^T. In addition to nitrogen fixation, A. chroococcum P208 had plant growth promotion potentials in cultures, such as IAA production, phosphate solubilization and siderophore production. Inoculation with A. chroococcum P208 could promote root growth of rice and wheat seedlings under controlled conditions.[Conclusion]] A strain with superior nitrogen fixation and plant growth promotion potential was isolated and purified from the paddy soil with superior nitrogen fixation capability. It has relatively good development and application potential value, and could provide microbial resource for the study and utilization of biological nitrogen fixation.

Key words:biological nitrogen fixation;nitrogen-fixing bacteria;Azotobacter chroococcum;plant growth promotion;plant growth-promoting bacteria