1. In grassland ecosystems, large herbivorous animal grazing activity and increasing nitrogen deposition strongly alters microbial community structure and function. Understanding the effects of grazing and nitrogen addition on the spatial heterogeneity in soil microbial community structure, enzymatic activities and the underlying mechanisms are crucial for making better predictions of soil organic matter dynamics and nutrient cycling. 

2. We examined the spatial heterogeneity of soil microbial community structure and enzymatic activity associated with changes in soil microclimate, soil characteristics, plant biomass and soil nutrient responses to grazing and nitrogen addition using a manipulative experiment with control (CK), grazing (G), nitrogen addition (N) and grazing plus nitrogen addition (NG) treatments in a Leymus chinensis meadow steppe, in northeastern China. 

3. The results demonstrated that soil microbial community structure and enzymatic activities showed a high level of spatial dependence [C/(C + C0)≥0.9] in the CK plot. G, N and NG treatments not only reduced the spatial variability ofsoil microbial community structure and enzymatic activities, but also reshaped the spatial links between enzymes activities and microbial community structure. Litter biomass, soil temperature and soil nutrients (soil dissolved inorganic nitrogen or soil dissolved organic carbon) explained 21-27% of the spatial variability of soil microbial community structure in the CK treatment and pH was the strongest driver for the spatial variability of soil enzymatic activities. Meanwhile, the homogenization in soil water content induced by the N addition treatment was a determinant of the reduction in spatial heterogeneity of the microbial community structure. The combination of soil physicochemical properties (bulk density, soil pH and soil dissolved inorganic nitrogen), soil temperature and root biomass explained 32-43% of the spatial variability of the microbial community structure in the G treatment, and N and G treatments had additive effects on the spatial heterogeneity of total PLFAs by homogenizing root biomass. Plant biomass and microbial community structure were the major drivers for the spatial heterogeneity of enzymatic activities under G, N and NG. In NG, the change in spatial variability of enzymatic activities was dominated by N addition. Regardless of grazing, N addition facilitated the spatial correlation between microbial community structure and enzyme activities. 

4. Overall, our results revealed the drivers of soil microbial community structure and enzymatic activities spatial pattern shift due to grazing and N addition, highlighting the role that spatial variability in soil microbial community structure and enzymatic activities has on the L. chinensis meadow steppe.

To examine how spatial heterogeneity of soil microbial community structure and enzymatic activities respond to grazing and nitrogen addition at a small scale of 15 ×15 m, we established a field experiment with control (CK), grazing (G), nitrogen addition (N) and grazing plus nitrogen addition (NG) treatments in a Leymus chinensis meadow steppe, in northeastern China. We explored the mechanisms of shifting spatial variability in microbial community structure and enzyme activities by measuring associated changes in soil microclimate and characteristics (soil temperature, soil water content, bulk density and pH), plant biomass (aboveground biomass, AB; root biomass, RB; litter biomass, LB) and soil nutrients (soil dissolved inorganic nitrogen, TIN; soil dissolved organic carbon, DOC; soil organic carbon, SOC; soil total nitrogen, TN) of 75 samples within each treatment. We replaced blank rows with null.