Study Site
The study site of this experiment was located in the Xilin River Basin,
a temperate steppe (116°14′E, 43°13′N) near the Inner Mongolia Grassland
Ecosystem Research Station (IMGERS) in the Inner Mongolia Autonomous
Region, China. The experimental area is about 100 ha natural grassland
with an elevation up to1250 m, which has been fenced since 1999 to
exclude large animal grazing. Based on a long-term observational dataset
(1980-2013), the mean annual temperature here was 0.9 °C, with mean
monthly temperatures ranging from −21.4 °C in January to 19.7 °C in
July, and the mean annual precipitation was about 350.00 mm,
approximately 70%-80% of which fell between May and August. The soil
at the site is either a Haplic Calcisol or Calcic-Orthic Aridisol, as
classified by the FAO and the US soil classification system,
respectively. The dominant plant species in the community areStipa grandis and Leymus chinensis , which together account
for more than 60% of the total peak aboveground biomass, the other 40%
of vegetation are forbs.
Experimental design
Eighty experimental plots were established, with a
randomized complete block design in
September 2008 (Y. Zhang et al., 2016). Each plot was measured 10 m × 10
m, and there were 2-m walkways between adjacent plots. Each plot was
treated with one of five different levels of N addition, i.e., 0, 2, 10,
20 and 50 g N m-2 yr-1 (designated
as N-0, N-2, N-10, N-20, and N-50, respectively), at one of two N
addition frequencies (twice a year vs. monthly), and with one of two
management regimes (un-mowing vs. mowing). Hence, there were 20
treatments (5 × 2 × 2) in total, each with 4 replicates. The form of N
added to the plots was purified NH4NO3(> 99%). N additions started on 1 November and were
continued on the first day of June of the same year and November of the
next year for the low frequency treatments (2 N addition
yr-1), or started on 1 November and continued on the
first day of each month for the high frequency treatment (monthly, or 12
N addition yr-1). Fertilizer was applied referring to
the method of Zhang et al. (2016). Mowing treatments were carried out
two times a year, with 10 cm
stubble
height
left after mowing, on 25 June to 30 June and 25 August to 30 August in
every year from 2009 to 2012.
Soil and plant sampling and measurements
Plant and soil samples were collected in mid-August 2012. To investigate
vegetation biomass and collect plant and soil samples, a 2 m × 0.5 m
quadrat was randomly placed in each plot, but at least 50 cm inside the
border of each plot to avoid edge effects. The aboveground biomass (AGB;
g m-2) was measured by clipping all plants above the
soil surface, oven-drying the combined clippings at 65 °C for 48 h,
recording the sample’s dry weight, and then finely crushing the sample
in a mill to determine the C and N content of plants
(Cplant and Nplant; mg
kg-1). Three root cores were collected using a 7 cm
diameter root auger at 10 cm intervals in the plots to a soil depth of
30 cm. These core samples were then mixed and soaked in water to remove
the mineral components of the soil, then treated as described above to
measure the belowground biomass (BGB; g m-2). Soil
samples were collected with a soil augur (3 cm in diameter), and in each
plot three cores were taken with 0-10 cm depth and
at least 50 cm apart. Soil samples
were then mixed and sieved through 2-mm mesh, stored at 4 °C for
laboratory analysis of soil ammonium
(NH4+-N; mg kg-1),
nitrate
(NO3--N;
mg kg-1), and moisture (SM %) content, as well as
microbial biomass carbon and N (MBC and MBN; mg kg-1).
Soil subsamples were air-dried for analysis of soil pH, and soil C and N
(Csoil and Nsoil; mg
kg-1). The Cplant,
Croot, and Csoil were determined by a
H2SO4-K2Cr2O7oxidation method (X. T. Lü, Lü, Zhou, Han, & Han, 2012). The
Nplant, Nroot, and Nsoilwere determined by using the Kjeldahl acid-digestion method with an
Alpkem auto-analyzer (Kjektec System
1026 distilling unit, Sweden). Soil
NH4+-N
and NO3--N were analysed using a flow
injection auto analyzer (FLAstar 5000 Analyzer; Foss Tecator, Hillerød,
Denmark), in which these substances were extracted from 10 g of fresh
soil sample with 50 ml of 0.5 M K2SO4solution. MBC and MBN were analysed with a fumigation extraction method
(Vance, Brookes, & Jenkinson, 1987). Soil pH was determined by
measuring pH of a soil-water suspension (soil: water = 1: 2.5) with a pH
meter (FE20-FiveEasy).
Gross ammonification and nitrification rates
The 15N pool dilution technique was used to determine
the gross ammonification rate (GA; mg
kg-1 SDW d-1) and gross
nitrification rate (GN; mg kg-1 SDW
d-1) in intact soil cores with one from each
experimental plot. Soil in the cores were labelled with15N-(NH4)2SO4or 15N-KNO3 (30 atom%15N enrichment and 3 mL 100 g-1 dry
soil equivalent) for quantification of gross N ammonification or
nitrification. A custom-made multi-injector, which consisted of 10
simultaneously operated syringes with custom-made side-port cannulas,
was used to ensure homogenous labelling. Each injection was equivalent
to 0.1 mL, 100 g-1 soil, and then was labelled with 3
mL of 15N-enriched solution that corresponded to 2 mg
N kg-1 dry soil; 3 replicate labels were made from
each core. When soil was sampled, the fresh soil core was labelled
immediately with the apparatus above mentioned (3 times at 0.3 cm, 1.5
cm, and 2.5 cm depth). About one hours later (t1), the first soil core
was collected and extracted by 0.5 M
K2SO4 solution with a soil: solution
ratio of 1:2 as described by
Dannenmann et al. (2009). Forty
hours after labelling (t2), the second soil core was extracted using the
same procedure as at t1.
Soil extracts were frozen for further processing. The diffusion
technique described by Dannenmann et al. (2006) was used to collect
NH4+and NO3- on acid filter traps, which
were then analysed for 15N enrichment with an isotope
ratio mass spectrometer (Delta Plus XP; Thermo) at the Center of Stable
Isotopes of KIT-IMK-IFU. Subsamples of the soil extracts were analysed
to determine their NH4+ and
NO3- concentrations by a commercial
laboratory in order to avoid bias. Gross ammonification and
nitrification rates were calculated with the equation provided by
Kirkham & Bartholomew (Kirkham & Bartholomew, 1954)(1954).
Statistical analyses
All results are presented in figures as mean ± SE (standard error).
Repeated measures analysis of variance (ANOVA) for a randomized complete
block design was performed, and Tukey’s HSD post hoc test was used to
test for differences in the response variables between different
treatments if the ANOVA revealed an overall significant difference. IfP < 0.05, differences were concluded to be
statistically significant. Linear regression was also performed to
examine the relationships of GA and GN rates with soil pH and
NH4+-N +
NO3--N. All statistical tests were
performed with SAS 9.0, except for regression analyses, which were
performed in the SIGMAPLOT software (SIGMAPLOT 12.5 for windows; Systat
Software Inc., San Jose, CA, USA) that was also used to produce graphs.
Structural equation modelling (SEM) was carried out with the software
package IBM SPSS Amos 21.0.