Effects of reduced nitrogen on the nifH-harboring soil microbiome in a soybean-maize strip intercropping system

Introduction Nitrogen (N) is a core limiting factor for crop growth, with approximately 50% of global food production relying on chemical N fertilizer inputs. However, excessive N application results in N use efficiency below 40%, and unabsorbed N triggers environmental problems. Maize-soybean relay strip intercropping (MSSI) enhances vertical resource partitioning, increases land productivity, and optimizes N utilization, but its effects on nifH -marked N-fixing microbiota under reduced N input remain unclear. This study aimed to investigate the abundance and diversity of N-fixing microbiota in response to the MSSI system with reduced N application. Methods A 2-year field experiment was conducted in two soil textures (sandy loam in Wuji and medium loam in Gaocheng) including three cropping systems: monocropping maize, monocropping soybean, and MSSI. To further explore the underlying mechanism, an N gradient experiment with four fertilizer rates was established in Wuji. At the maturation stage, rhizosphere soil samples were collected, and q-PCR, enzyme activity assays, and high-throughput sequencing were used to analyze N cycle-related marker genes, enzyme activities, and nifH gene abundance and diversity. Results The MSSI system maintained maize yields comparable to monocropped maize, while soybean yields reached 60.1–69.6% of monocropped levels. MSSI significantly increased nifH gene abundance in soybean rhizosphere soil, but reduced the Chao1, Shannon, Simpson, and observed species indices of N-fixing microbiota. Specifically, MSSI decreased N-fixer diversity (Shannon: −18.2%) and richness (Chao1: −12.5%), whereas the 25% reduced N input treatment (ISN 25 ) enhanced diversity (Shannon: +15.7%) by improving community evenness without altering species richness. Discussion Our results demonstrate that the MSSI system significantly alters soil N fertility and the community structure of nifH -marked N-fixing bacteria. The reduced N input combined with MSSI can optimize N utilization by regulating N-fixing microbial communities, providing a theoretical basis for sustainable agricultural practices that balance food security and ecological protection.