Effects of Reduced Nitrogen Application on the nifH-Harboring Microbiota in Soybean Rhizosphere under Maize-Soybean Relay Strip Intercropping

Abstract

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. Thus, optimizing N fertilizer application has become a critical concern for balancing food security and ecological protection. Maize-soybean relay strip intercropping (MSSI) enhances vertical resource partitioning by stacking tall maize with low soybean, thereby increasing land productivity and optimizing N utilization, through rational planting ratio allocation to facilitate N sharing and improve overall N use efficiency. This study investigated the abundance and diversity of nifH-marked N-fixing microbiota in response to the MSSI system under reduced N input levels. A 2-year field experiment was conducted in three cropping systems, two different soil textures (sandy loam soil in Wuji and medium loam soil in Gaocheng): monocropping maize, monocropping soybean, and MSSI. The results indicate that the MSSI system can ensure maize yields comparable to those of monocropped maize, whereas soybean yields can reach 60.1%-69.6% of MS levels. Analysis of nifH gene abundance in experimental fields revealed that MSSI significantly elevated nifH levels in soybean rhizosphere soil. To further analyze the efficient mechanism of the MSSI model, we set up a N gradient field experiment at Wuji. Rhizosphere soil samples were from four different rates of N fertilizer application were collected at the maturation stage. We quantified bacterial marker genes associated with the N cycle enzymes via q-PCR, detected the activity of enzymes involved in the N cycle, and analyzed the abundance and diversity of the nifH gene via high-throughput sequencing technology. The analysis of nifH and rhizosphere soil microbial diversity revealed that MSSI systems significantly increased nifH and reduced the Chao1, Shannon, Simpson, and observed species indices. The results indicated that the MSSI system significantly altered soil N fertility and the community structure of soil N-fixing bacteria marked by nifH. MSSI decreased N-fixer diversity (Shannon: - 18.2%) and richness (Chao1: -12.5%), while ISN25 enhanced diversity (Shannon: +15.7%) by improving community evenness without changing species richness.