Alfalfa fields with different stand ages alters soil inorganic nitrogen dynamics through shifts in nitrogen-cycling functional genes across growth stages

Abstract

Introduction Alfalfa cultivation is associated with soil nitrogen availability and microbial nitrogen-cycling functional potential. However, how soil inorganic nitrogen and nitrogen-cycling functional genes vary with alfalfa stand age across growth stages remains unclear. Methods Using a chronosequence approach, we investigated soil inorganic nitrogen and nitrogen-cycling functional genes in a maize-field control (CK) and 2-, 3-, 4-, and 7-year-old alfalfa fields (T1–T4) during the regreening, flowering, and maturity-harvest stages. Soil NO3--N and NH4+-N concentrations and TPM-normalized abundances of nitrogen-cycling functional genes were determined. Correlation analysis, PERMANOVA, and structural equation modeling (SEM) were used to examine their relationships. Results Soil NO3--N and NO4+-N concentrations differed significantly among stand ages and generally exhibited unimodal patterns, with the highest observed values in the 4-year field (T3) and lower values in the 7-year field (T4). The abundances of genes involved in nitrification, denitrification, and nitrate reduction also varied with stand age and showed growth-stage-specific patterns. Growth stage explained a greater proportion of variation in nitrogen-cycling gene profiles than stand age, although stand age was also significantly associated with these profiles. The relationships between functional genes and soil inorganic nitrogen varied among growth stages. SEM analysis suggested that growth stage was the primary factor associated with variation in nitrogen-cycling gene groups. Nitrification-related genes were positively correlated with soil inorganic nitrogen, whereas nitrate reduction-related genes showed negative correlations. Conclusion Overall, the 4-year alfalfa field exhibited the highest observed soil inorganic nitrogen concentrations, while this pattern was less evident in the 7-year field. These findings indicate that soil nitrogen status and nitrogen-cycling functional genes vary jointly with alfalfa stand age and growth stage. Nitrogen-cycling functional genes may serve as potential indicators of soil nitrogen status, although further experimental studies are needed to verify these relationships.