Assessing the interkingdom response of soil prokaryotes, fungi and protists to prairie restoration on retired agricultural lands

Belowground biodiversity responses to native ecosystem restoration remains uncertain, partly because factors regulating soil biota are complex and poorly understood. Here, we studied how prairie restoration of marginal croplands affected soil prokaryotes, fungi and protists on 5 farms from Ontario, Canada. Soil samples from crops and 5–9-year-old adjacent restored prairies were analyzed for microbial abundance (qPCR) and diversity (high-throughput sequencing). The response of microbial abundance and alpha diversity varied across groups and farms, yet restoration led to an average decrease in fungal abundance and protist richness. Prairie restoration also shaped microbial community composition, with high species turnover, although farm effects were generally stronger and modulated this response. Soils from both land uses were dominated by the same higher taxonomic ranks and shared 67–80% of genera, many of them generalists. Still, restoration altered the relative abundance of specific taxonomic and inferred functional groups, with increases in Glomeromycota and Acidobacteriota and decreases in protist phagotrophs. Fungal communities were overall the most sensitive, with more specialist taxa and shifts in inferred function, including increased symbionts and decreased saprotrophs and pathogens in restored prairies. Interkingdom networks revealed altered potential associations and assembly patterns, with only 8 associations in common between land uses. Our findings reveal a complex response of soil microbiota to prairie restoration, differing among microbial groups but also across farms, due to factors like soil properties, restoration practices, and legacy effects from previous management. This study highlights the importance of assessing soil biota restoration by exploring interkingdom responses while also disentangling the multiple factors that modulate them. • Restoration shifted microbial composition, with high species turnover. • On average, prairie soils had lower fungal abundance and protist richness. • Soil properties and other farm-specific factors shaped microbial responses. • Restoration-sensitive taxa and inferred functional groups were detected. • Restoration modified interkingdom association patterns and the taxa involved.