Split Nitrogen Application Timing Steers Rhizosphere Nitrifiers and Nitrogen Utilization in Wheat

Split nitrogen (N) application is an important agronomic measure for improving wheat yield and quality, yet how rhizosphere nitrogen-transforming microbes respond to split N strategies and the underlying mechanisms remain unclear. This study investigated the effects of six N treatments, including control, basal application, jointing-stage soil topdressing, and foliar applications at booting, anthesis, and 10 days post-anthesis, on the community structure and diversity of key rhizospheric nitrogen cyclers (ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), and nitrite-oxidizing bacteria (NOB)) in wheat. Results showed that AOB and NOB alpha diversity were significantly modified by split N application. N application at anthesis enhanced AOB richness and diversity more than the later application, while concurrently decreasing NOB diversity. Booting-stage application enriched Nitrosospira and Nitrosomonas in the AOB community, whereas anthesis application increased Nitrososphaera sp. JG1 in AOA, but decreased Candidatus Nitrospira inopinata in NOB. Redundancy analysis identified soil pH, moisture, organic carbon, and key enzyme activities as the main drivers of microbial community assembly. Although no significant differences were observed in key agronomic traits among treatments, the 10 days post-anthesis treatment showed numerically superior yield and N uptake. Notably, AOB community evenness was significantly positively correlated with grain yield, protein yield, and N uptake, whereas NOB community diversity showed negative correlations. These findings demonstrate that split N application, particularly late foliar spray at 10 days post-anthesis, can modulate soil physico-chemical properties to selectively shape nitrogen-transforming microbial communities (notably AOB) in the wheat rhizosphere. This study provides a theoretical foundation for designing precise N management strategies rooted in rhizosphere ecology, with the goal of simultaneously improving yield, grain quality, and nitrogen use efficiency.