Legume presence modulates soil bacterial diversity, composition, network complexity, and functions under drought in timothy-red clover mixed stands

Drought disrupts soil microbial communities by altering their diversity, composition, and network stability, impacting biogeochemical cycles that are critical for plant growth. While legume-based forage systems may enhance microbial resilience through nitrogen inputs, the response of soil bacterial communities to drought across different forage systems remains largely unexplored. This greenhouse study investigated soil bacterial community dynamics in timothy grass ( Phleum pratense L.) monocultures versus timothy-red clover ( Trifolium pratense L.) mixed stands under moderate (40% field capacity, FC), severe (20% FC), and no-drought (80% FC) conditions, followed by a post-drought recovery. Four weeks of drought significantly increased bacterial alpha and beta diversity in clover-timothy mixed stand soils, while grass monoculture soils showed no notable changes. Compositional shifts in mixed stand favors drought-tolerant Actinomycetota while reducing the desiccation-sensitive taxa. Co-occurrence network analysis revealed that mixed stands form larger, more complex, and more stable bacterial networks under moderate drought (during drought phase), and severe drought (during recovery phase) compared to grass monocultures. Notably, mixed stands exhibited enriched predicted nitrogen-cycling functions, potentially driven by nitrogen release from nodule senescence, with denitrification rates significantly exceeding those in monocultures, under both drought and recovery phases. Correspondingly, mixed stands outperformed the grass monocultures, exhibiting higher biomass, lower shoot C:N ratios, and increased total shoot nitrogen content, likely driven by legume rhizodeposition. These findings underscore the critical role of grass-legume mixtures in maintaining microbial network stability, nitrogen cycling, and forage productivity under drought and post-drought recovery, providing valuable insights for sustainable agriculture in climate-challenged environments. • Legume-grass mixtures maintain microbial diversity and stability under drought • Plant composition drives microbial drought responses beyond water limitation • Legume-derived nitrogen supports plant-microbe recovery from severe drought • Mixed stands enrich stress-tolerant taxa and nitrogen-cycling functions