2.1 Site Description and Experimental Design
The experiment was conducted in Baicao Garden Teaching and Research base of Shenyang Agricultural University, Shenyang City Liaoning Province, northeast China (123°25’E, 41°46’N). The region belonged to a temperate semi-humid continental climate zone, with an annual average temperature of 6.5 ℃, annual precipitation of 700 mm, and brown soil type (Typic Eutrochrepts in the US soil taxonomy). The soil properties were as follows: an organic carbon content of 7.33 g·kg-1, a total nitrogen content of 0.93 g·kg-1, an ammoniacal nitrogen content of 3.21 mg·kg-1, a nitrate nitrogen content of 25.99 mg·kg-1, available P 12.36 mg·kg-1, and available K 98.96 mg·kg-1, pH of 7.0. The lawn was planted in the experimental site on May 7, 2016 and was dominated by Kentucky bluegrass (Poa pratensis L.) of the variety Merit.
The experiment was started by applying N fertilizer in 2016. Four gradients were set according to the standard of lawn management and fertilization in north China. The gradients decreased from 300 kg kg·ha·yr-1 to 225 kg·ha·yr-1, 150 kg·ha·yr-1 and control treatments. They were referred to as the N300, N225, N150, and N0 treatments, respectively. In total, 12 plots of 1 × 1 m size were fully randomized throughout the study site, and each treatment was repeated three times. The annual urea equivalent was divided into two parts; fertilization was carried out on May 15 and August 15, continuous fertilization for two years. Sprinkler irrigation was carried out immediately after fertilization.
Five soil cores were randomly taken from each plot from a depth of 0-20 cm and then mixed to form one composite sample on the day after the last fertilization. All soil samples were transported to the laboratory and sieved through a 2 mm mesh within 24 h. The soils were divided into two parts. One part of the soil was used to measure physicochemical properties. The remainder of each sample was stored at -80 ℃ for subsequent laboratory tests and total DNA extraction of soil microorganisms.