Soil sampling and laboratory analysis
Three soil sub-plots (2 m × 2 m) were randomly selected in each land use type from four blocks for four times including October, 2016; January, 2017; April, 2017; July, 2017. Five soil cores (diameter = 5cm) from each sub-plot were randomly collected from two soil depths including 0-10 cm and 10-30 cm. A total of 32 soil samples (three subplots in four plots with two soil layers) were collected to represent each land use type one season, with a grand total of 96 samples across all four land use types one season and with a grand total of 384 samples across all seasons. All the fresh soil samples were sieved with a 2 mm sieve after the visible plant material and stones were removed with forceps. Then, each soil sample was divided into two subsamples. One subsample was stored at -20 ℃ for the measurement of enzyme activities and PLFAs, the other subsample was air-dried for the soil physicochemical properties analyses.
Soil moisture was measured by oven-drying fresh soil at 105 ℃ to a constant weight. Soil pH was determined from soil water suspension (1:2.5 v: v) with a digital pH meter. The C and N concentrations was determined by the methods used by Rovira and Vallejo (2002). We used a two-step acid hydrolysis procedure to extractant to measure the labile and recalcitrant SOC, SON of bulk soil. Briefly, a portion of air-dried soil (approximately 2000 mg) was treated with 5 mL 1 mol/L HCl for 24 h to remove inorganic C for the measurements of soil organic carbon and nitrogen (SOC and SON). Then, soil recalcitrant N (RN) was obtained by acid hydrolysis. The SOC, SON and RN were analyzed by using an isotope ratio mass spectrometer (Thermo Finnigen, Delta- Plus, Flash, EA, 1112 Series, USA). Soil labile N (LN) is made by the difference between SON and RN. Soil N recalcitrance index (RIN) was calculated as follows (Cheng et al., 2013; Rovira and Vallejo, 2002):
RIN (%) = RN/ SON * 100%
Soil hydrolase activities, including N-acetyl-β-glucosaminidase (NAG), leucine aminopeptidase (LAP), β-glucosidase (BG) and phosphatase enzyme (AP) were determined by a fluorimetric microplate method (Deforest, 2009). The substrates used for enzyme assay were 4- MUB-N-acetyl-β-D-glucosaminide, ι-Leucine-7-amido-4-methylcoumarin and 4-methylumbelliferyl (MUB)-β-D-glucopyranoside for NAG and LAP, BG, respectively (Deforest, 2009). The BG, NAG and AP enzyme activities were measured using a modified fluorescent-linked substrate (4-methylumbelliferone (MUB) and the LAP enzyme activities were measure by fluorescent-linked substrate 7-Amino-4-methylcoumarin Chromophore (MUC) microplate protocol in situ pH conditions and temperature (Smith et al., 2015). Briefly, 1 g fresh soil was homogenized in 90 mL sodium tris buffer using a magnetic stirrer. Hydrolytic enzyme activities were measured fluorometrically in black polystyrene 96-well, 300-ml microplates (Whatman Inc., Florham Park, NJ). The dispensing of tris buffer, soil suspension, standard solution (10 μM 4-MUB) and fluorescent substrate solution (200 μM) specific to each enzyme followed a strict order on the well plate. The microplates were incubated in the dark at 25 ℃ for 3 hours. Following incubation, 10μl of 1mol/L NaOH was added to stop the reaction and then the plates were read on a Beckman-Coulter DTX880 fluorescent microplate reader with excitation of 360 nm and emission of 450 nm (Beckman-Coulter, Fullerton, CA, USA). Enzyme activities were expressed as nmol fluorescene g−1 dry fraction h−1. Specific enzyme activities (i.e. enzyme activities per unit of SON) were calculated by dividing enzyme activities over the SON (Raiesi and Beheshti, 2014).
We used phospholipid fatty acids (PLFAs) to determine total PLFAs and ratios of fungal to bacterial biomass (F: B ratios) (Bossio and Scow, 1998). Briefly, total lipids were extracted using 23 mL extraction mixture [chloroform: methanol: phosphate buffer (1:2:0.8 v/v/v)] from 8 g of lyophilized soil. Samples containing nonadecanoic acid methyl ester (19:0) as an internal standard were analyzed with an Agilent 6890 Gas Chromatograph equipped with an Ultra 2-methylpolysiloxane column.