2.2. Experimental design and sampling method
We selected three predominant land use patterns discussed above (alfalfa artificial grassland: AG, wheat-maize rotation field: WM, and native grassland: PC). Each land use pattern was located in three representative sample sites with relative flat surface and had the same physiographical units and slope gradients, and established nine 20 × 20 m2 plots in each land use type in spring 2011. In total, we determined 3 representative sample sites and 9 plots as true replicated in each land use. The native grassland with more than 30-45 m width was located beside the reclamation field, respectively, and the distance between each plot exceeded the spatial correlation of most soil physicochemical and microbial characteristics (< 13.5 m) (Jiao et al., 2019). The cultivation history of three land use types and soil basic physiochemical properties in 0-20 cm depth when plots were established in spring 2011 were shown in Table 1 and Table 2, respectively.
A field survey was conducted in mid-June 2020 (after the winter wheat was harvested). We selected 5 quadrats of 1m×1m in each plot for soil sample collection according to the “S” shape sampling method, and five soil sampling drills were located at the center and the four diagonal corners of each 1m×1m quadrat at three depths (0-20cm, 20-40cm, 40-60cm) by using a drill (8 cm-diameter). All the soil samples at the same layer of each quadrat were mixed to create a representative soil sample. Overall, 81 composite soil samples were collected, representing three land uses, three depths, and nine replicates. The mixed soil samples were taken back to the laboratory. After removing roots and stones, the samples were divided into two parts. One part was stored in the refrigerator at -80℃ for the enzymatic activity, ammonium nitrogen (NH4+-N), and nitrate nitrogen (NO3--N). The other part of soil samples was screened with 0.15 mm and 2 mm sieves respectively after dried and crushed for soil properties determined. Soil bulk density (BD) was collected at the intermediate position of 0-20, 20-40, and 40-60cm soil layer of each quadrat using a cutting ring with 5.0 cm inner diameter and 100 cm3 volume. The undisturbed soil was collected in 0-20, 20-40, and 40-60cm soil layer of each quadrat with aluminum box for the content of water stable aggregate (WSA).