Spatial heterogeneity in the rhizosphere microbiome and root nitrogen assimilation gene expression in soil-based and soilless cucumber and tomato organic greenhouse systems

Aims The rhizosphere microbiome plays a central role in plant nutrition, yet it is often treated as a spatially homogeneous zone of plant influence. In organic greenhouse systems, where nutrients are supplied through surface-applied organic fertilizers, resource gradients may generate spatially heterogeneous rhizospheres. This study investigated how rhizosphere selection and sampling depth shape microbial communities, root nitrogen (N) assimilation gene expression, and their relationships with crop N offtake. Methods A split-plot experiment was conducted over two crop cycles (cucumber and tomato) in soil-based and soilless greenhouse systems under four organic fertilization regimes. Rhizosphere and bulk samples were collected at 0–5 and 10–15 cm, and microbial community composition, functional profiles, and root expression of N-related genes were analyzed. Results Rhizosphere selection interacted with sampling depth to shape prokaryotic and fungal community composition in both systems. Prokaryotic communities in the 0–5 cm rhizosphere were enriched in putative N-cycling functions relative to bulk soil and the 10–15 cm rhizosphere, particularly in the soilless system. Saprotrophic fungal communities were more evenly distributed across depths. Glutamine synthetase type 1 expression was consistently higher in roots from the 0–5 cm layer and was positively associated with numerous microbial taxa. Sampling depth also influenced the relationships among rhizosphere community composition, root N-assimilation gene expression, and crop N offtake. Conclusions These findings demonstrate that the rhizosphere is spatially heterogeneous and that accounting for this heterogeneity can improve understanding and management of root-soil-microbiome interactions in agroecosystems.