Nitrogen fertilization outweighs plant species loss in shaping bacterial belowground diversity in an alpine meadow on the central Tibetan Plateau.

Plant species loss and nitrogen fertilization affect grassland biodiversity. However, their interactive effects on plant communities, soil properties, and the soil microbiome remain insufficiently understood. We analyzed how the removal of plant species, with and without urea addition, influenced plant diversity, soil properties, and soil bacterial communities in a Tibetan Plateau grassland. Continuous plant species removal and urea addition over seven years modified plant beta-diversity equally strong, while urea exerted a stronger negative effect on plant alpha-diversity. Both, plant species removal and urea addition caused soil acidification and an increase in NO₂⁻/NO₃⁻, while dynamics in TOC and TON were mainly driven by the growing season. Structural equation modeling identified soil acidification via urea addition as the most important indirect driver that negatively affected bacterial alpha-diversity and shifted bacterial beta-diversity. Urea addition also exerted direct negative effects on bacterial alpha- and beta-diversity, causing repression of oligotrophic (Acidobacteriota, Chloroflexota, Planctomycetota, Gemmatimonadota) and stimulation of copiotrophic (Bacillota, Bacteroidota, Pseudomonadota) bacterial taxa. Plant species removal caused slight increases in bacterial alpha-diversity, paralleled by less diverse but more even plant communities. We show that soil acidification by urea fertilization outweighs plant species loss in its negative effect on bacterial soil biodiversity in Tibetan grasslands.