Excessive nitrogen (N) fertilization harms the diversity, structure, and function of the soil microbiome. Yet, whether such adverse effects can be repaired through reducing the subsequent N fertilization rate remains not completely clear so far. Here, using a long-term N-overfertilized wheat-maize cropping field, we assessed the effect of reducing various proportions of the subsequent N fertilization rate over six years on crop productivity, soil physicochemical and biochemical properties, and microbiome. Five treatments were employed in our field experiment: the farmers’ conventional N fertilization rate (zero reduction, as a control) and the reduction in the farmers’ N rate by 20%, 40%, 60%, and 100%. The results showed that moderate N reduction (20–40%) enhanced crop productivity and soil fertility but did not affect soil enzyme activity. Soil bacterial and fungal community diversity were insensitive to N fertilization reduction, whereas their community structures changed significantly, with more prominent alteration in the fungal community. Functional prediction indicated that average relative abundance of arbuscular mycorrhizal fungi increased with N fertilization reduction but that of ectomycorrhizal fungi decreased. Moderate N reduction (20–40%) enhanced species interactions and, thus, provided a more complex cross-kingdom microbial co-occurrence network. Both bacterial and fungal community assembly were governed by stochastic processes, and this was not altered by N fertilization reduction. Overall, the response of the soil microbiome to N fertilization reduction was greatly dependent on the reduced N proportion. The findings obtained here shed light on the importance of optimal N fertilization rate in the intensively cultivated, high-input grain production system.

The application of phosphate fertilizers significantly influences soil microbial communities and nutrient cycling. Soil enzymes, which are sensitive to nutrient levels, play a critical role in microbial metabolism. However, the impact of phosphate fertilizers on nutrient limitations within the microbial metabolism of agricultural soils remains poorly understood. In this study, soil samples were collected from a depth of 0–20 cm from a wheat crop subjected to a three-year field experiment with six different phosphorus (P) application rates. Soil β-glucosidase (BG) and leucine aminopeptidase (LAP) activities were highest under the P3 (60 kg P₂O₅ ha⁻¹) treatment over the three-year study period. The responses of soil N-acetyl-β-glucosaminidase (NAG) and alkaline phosphatase (AKP) to increasing P additions varied across different years. The EES C:N, C:P, and vector length were significantly greater than 1. Soil nutrient characteristics accounted for 70.71% of the variation in soil enzyme stoichiometry. The vector length and angle of soil enzymes explained by soil nutrient characteristics were 0.65 and 0.73, respectively. Among these factors, ROC exhibited the largest direct and total effect on the soil enzyme vector length and angle. These research findings offer valuable insights for the management of agricultural fertilizers. Consequently, it is recommended to enhance soil carbon levels to alleviate carbon limitations and improve P utilization efficiency.

Partial substitution of mineral N fertilizer with manure (organic substitution) is considered as an effective way to reduce N input in intensive agroecosystems. Here, based on a 3-year field experiment, we assessed the influence of different organic substitution ratios (15%, 30%, 45%, and 60%, composted chicken manure applied) on vegetable productivity and soil physicochemical and biochemical properties as well as microbiome (metagenomic sequencing) in an intensive greenhouse production system (cucumber-tomato rotation). Organic substitution ratio in 30% got a balance between stable vegetable productivity and maximum N reduction. However, higher substitution ratios decreased annual vegetable yield by 23.29–32.81%. Organic substitution (15–45%) improved soil fertility (12.18–19.94% increase in soil total organic carbon content) and such improvement was not obtained by higher substitution ratio. Soil mean enzyme activity was stable to organic substitution despite the activities of some selected enzymes changed (catalase, urease, sucrase, and alkaline phosphatase). Organic substitution changed the species and functional structures rather than diversity of soil microbiome, and enriched the genes related to soil denitrification (including nirK, nirS, and nosZ). Besides, the 30% of organic substitution obviously enhanced soil microbial network complexity and this enhancement was mainly associated with altered soil pH. At the level tested herein, organic substitution ratio in 30% was suitable for greenhouse vegetable production locally. Long-term influence of different organic substitution ratios on vegetable productivity and soil properties in intensive greenhouse system needs to be monitored.