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.