Diversification of corn-based cropping systems with wheat increases plant growth-promotion potential of soil microbial communities

Diversifying corn-based crop rotations by including wheat can enhance soil health and increase corn yield resilience, but long-term effects on soil microbial communities and microbial plant growth-promoting (PGP) functions are unclear. Moreover, responses of soil microbiota to rotational changes are influenced by other interacting agricultural practices. We analyzed soil samples from a 25-year field trial in southern Ontario, Canada, comparing four corn-based crop rotations in combination with conventional and reduced tillage practices and presence and absence of nitrogen fertilization. Microbial community composition and PGP potential were assessed via amplicon sequencing of 16S rRNA genes and ITS regions and qPCR quantification of PGP functional genes. Tillage had strongest impacts on microbial composition, followed by crop rotation, then fertilization. Reducing tillage exerted stronger effects on prokaryotic than fungal composition, whereas increasing rotational diversity had stronger effects on fungal than prokaryotic composition. A strong rotation × tillage interaction indicated that diversification effects were more pronounced under reduced tillage, promoting distinct microbial taxa across tillage systems. Diversifying rotations with wheat enriched potential PGP genera such as Marquandomyces , Clonostachys , and Edaphobaculum, and increased gene abundances related to phosphate mineralization, nitrification, sulfur oxidation, and plant stress tolerance. Functional shifts were most evident under conventional tillage, suggesting enhanced microbial capacity for nutrient availability and stress mitigation in intensive systems. Overall, our findings demonstrate that crop rotation-induced shifts in soil microbiota are strongly shaped by tillage, and integrating wheat into corn-based rotations can enhance microbial plant growth-promotion potential, particularly under conventional tillage. • Tillage and crop rotation had stronger impacts on soil microbiota than fertilization. • Impacts of crop rotation on community composition varied by tillage intensity. • Diversifying corn-based crop rotations with wheat promoted PGP taxa and functions. • Wheat benefits for PGP microbes were strongest under conventional tillage. • Rotation diversification may improve sustainability of conventional tillage systems.