Abundant culturable diazotrophs within Actinomycetia rather than rare taxa are underlying inoculants for nitrogen promotion in desert soil

Desert soil may harbor various culturable diazotrophs that can endure multiple environmental stresses and promote ecological restoration. To explore nitrogen-fixing microorganisms that can be potential inoculants for nutrition improvement in nitrogen-limited ecosystems and disclose whether rare taxa contribute to the nitrogen incorporation process, we collected soil from the Mu Us Desert and combined 16S rRNA high-throughput sequencing, functional gene arrays (GeoChip) method, and culture-dependent approaches to observe the composition and key groups of culturable diazotrophs. Proteobacteria was the most dominant phylum, accounting for 73.07 % of the 823 operational taxonomic units (OTUs), grown in nitrogen-free media. Among all the valid assigned putative nitrogen-fixing OTUs, 53.21 % were identified as rare taxa in the soil. Among all the rare OTUs that could be effectively enriched as abundant taxa in the medium, over 58 % were also affiliated with Proteobacteria. Despite the predominance of Proteobacteria, the ordinary least squares regression model implied that less abundant Actinomycetia was significantly related to the nifH gene signal intensity generated by GeoChip, while rare culturable diazotrophic categories had no relationship with the nifH gene. The random forest analyses further confirmed that the most important indicator of the nitrogen-fixing process was Actinomycetia. The two stably generated pure cultures, classified as Streptomyces sp. (Actinomycetia) and Bacillus pumilus (Firmicutes) by Sanger sequencing and BLAST were inoculated into sterilized desert soil. Available nitrogen, total dissolved nitrogen (TDN), and total nitrogen (TN) were measured, and the two strains significantly increased the soil ammonium content, nitrate content, and TDN content. Our findings provide genomic and solid evidence that rare taxa within Proteobacteria maintain the biodiversity of culturable nitrogen fixers, whereas abundant Actinomycetia possess potential value as nitrogen-fixing inoculants in drylands.