Keystone rhizospheric diazotrophs may promote the pioneer plant growth in mine tailings

Nitrogen (N) shortage often hinders the bioremediation of mining-generated tailings. For pioneer plants living in mine tailings, biological N fixation (BNF) is an essential source of N. However, the diazotrophs in the rhizosphere of pioneer plants are rarely investigated. In this study, the pioneer plant Miscanthus sinensis samples were collected from four different tailings across Southwest China, and the 16S rRNA amplicon sequencing, multivariable statistical tools, ¹⁵N₂ DNA-SIP, metagenomic-binning, cultural screening methods, and pot experiment were applied to analyze the roles of diazotrophs in rhizosphere of M. sinensis. Firstly, genera Stenotrophomonas and Pseudomonas were identified as keystone taxa in the rhizosphere of M. sinensis cultivated in the tailings. Furthermore, ¹⁵N₂ DNA-SIP demonstrated that Stenotrophomonas and Pseudomonas were the rhizospheric diazotrophs. The metagenome-assembled genomes of Stenotrophomonas and Pseudomonas contained some genes involved in BNF, plant growth promotion, and metal(loid) resistance (As, Sb, Ni, Co, Zn, Mn, Cu, and Cd). Subsequent diazotrophic isolation was conducted, and two diazotrophic strains (Pseudomonas sp. R21 (nitrogenase activities, 1,161.9 nmol C₂H₄ mg⁻¹ protein h⁻¹) and Stenotrophomonas sp. R30 (1,890.2 nmol C₂H₄ mg⁻¹ protein h⁻¹)) were isolated from the rhizosphere soil of M. sinensis. Pot assay demonstrated that inoculation with Pseudomonas sp. R21 and Stenotrophomonas sp. R30 could promote plant growth, N content, and metal(loid) (arsenic and antimony) accumulation, which confirmed nitrogenase activity and plant growth-promoting ability of the two identified keystone taxa, highlighting the exciting opportunities of applying some keystone rhizospheric diazotrophs in the pioneer plants through microbiome manipulation for the bioremediation of tailings.