Defoliation intensity drives post-recovery shifts in rhizosphere bacterial networks and assembly processes despite complete aboveground recovery in legume-grass mixtures

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Defoliation profoundly restructures rhizosphere microbial communities, yet the mechanistic basis of these shifts, particularly their post-recovery trajectories, remains poorly understood. We subjected red clover ( Trifolium pratense L.)-timothy ( Phleum pratense L.) mixtures to non-defoliated control, moderate, and severe defoliation treatments to investigate how stress intensity shapes rhizosphere bacterial community structure, assembly processes, and network organization following complete aboveground regrowth. While moderate defoliation triggered classic overcompensation with complete aboveground biomass recovery, severe defoliation revealed distinct post-recovery belowground legacies: significant root biomass decline, reduced N fixation, and fundamental microbial community restructuring. Defoliation shifted rhizosphere communities toward copiotrophic taxa, while decreased C:N ratios in companion grass shoots suggest increased N availability, consistent with N release from legume roots as previously documented. Despite stable alpha diversity, severe defoliation drove pronounced beta diversity shifts and recruited unique taxa absent from control communities. Network analysis revealed contrasting intensity-dependent responses: moderate defoliation caused the greatest structural disruption through loss of interactions and reduced connectivity, while severe defoliation triggered complete network reorganization with increased density but greater structural vulnerability. Lineage-based assembly profiling identified "switcher" bins that transitioned from stochastic drift to dispersal-limited assembly under severe defoliation, indicating emerging spatial constraints, with divergent trajectories within and across phyla revealing niche collapse in some lineages and competitive dominance in others. These findings demonstrate that belowground microbial legacies can still be detected beyond the point of aboveground recovery, revealing a critical temporal decoupling and providing fundamental insight into the ecological rules structuring rhizosphere communities under disturbance.

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