2.2.Experimental design
There were three replicates per treatment for all experiments,and all of the soil samples were maintained in a controlled-environment incubatorat 25C ,which is equivalent to the approximate mean temperature of the irrigation period in the study region (from June to August).A total of 100 g of air-dried soil was added to a glass mason jar(730ml).The soil moisture was adjusted to 80% WFPS by carefully spraying 33.3 g of distilled water onto the soil to increase the soil moisture,and the soils were pre-incubated for 8 day store activate soil microorganisms(Harrison-Kirk et al.,2013).Next,all of the samples were maintained at or dried to their respective moisture contents under the same drying conditions for 12 days.The bulk density of the packed soil(1.26gcm3)and particle density(2.65gcm3)were used to calculate the WFPS,as described by Guo et al.(2014).
Three fertilization treatments were used:unfertilized(0N);7.4mg urea-N per 100 g dry soil,which is equivalent to 200 kg N ha1(200N);and 14.8 mg urea-N per 100 g dry soil,which is equivalent to 400 kg N ha1(400N),this application rate is a typical rate for cotton crop in this area.All of the urea-N fertilizers were dissolved in water.Two fertilization strategies were performed:a single application of urea(SI) at 0 day and a split urea application (SP) at 0,16,32 and 48 days(Fig.1).