Earthworms and phytate drive phosphorus hotspots in the drilosphere: Enzymatic, microbial, and spatial insights from in situ imaging

Soil organic phosphorus (P) represents a critical component of terrestrial ecosystems, yet its mineralization dynamics in biologically-active zones like earthworm drilosphere remain poorly understood. This study combined microcosm experiments with in situ imaging to elucidate the mechanisms of phytate mineralization in earthworm drilosphere, 2-mm soil layer surrounding the burrows. Under phytate application, endogeic earthworms increased the available P content by 6.3% in the drilosphere and elevated phytase activity by 38%. In situ imaging using diffusive gradients in thin-films and soil zymography revealed that phytate application increased the maximum labile P flux by 54% and maximum acid phosphatase activity by 67%. Hotspot analysis showed phytate application expanded labile P hotspots from 3.1 to 4.2% in soil while reducing acid phosphatase hotspots from 4.8 to 4.6%, indicating elevated baseline enzyme activity in the drilosphere. Microbial community analysis revealed that earthworms restructured drilosphere communities, with Gammaproteobacteria and Sordariomycetes emerging as dominant functional taxa. Functional gene expression analysis showed selective upregulation of phoD and phyA phytase genes and downregulation of P-starvation response genes phoU and phoR , boosting microbial P acquisition capacity. These findings demonstrate that earthworms drive phytate mineralization through integrated mechanisms: enzymatic effects, gene regulations, and microbial community restructuring. This work helps to understand the spatially-heterogeneous P dynamics, highlighting soil fauna as ecosystem engineers by creating self-reinforcing systems to maximize organic P utilization. • Earthworms increased available P by 6.3% and phytase activity by 38% in soil; • Phytate application expanded labile P hotspots from 3.1 to 4.2% in soil; • Gammaproteobacteria and Sordariomycetes dominated P-cycling microbial taxa; • Gene expression showed upregulated phytase phoD and phyA under phytate application; • Integrated imaging revealed spatial coupling of enzyme activity and P availability.