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.