Site description and soil characteristics
The field research was conducted at the Miyaluo Experimental Forests on the eastern QTP in Lixian County, Sichuan Province, China (31° 35′N, 102° 35′E, and 3150 m a.s.l.). The mean annual temperature is 8.9°C; July is the warmest month (12.6 °C) and January is the coldest month (-8°C). The mean annual precipitation ranges from 600 to 1100 mm; the highest precipitation frequency and amount occur from May to September. According to the local Forestry Bureau, large areas of natural forests were deforested for agriculture from the 1950s to 1980s. Subsequently, plantations were established and currently cover one million hectares in western Sichuan Province, China, accounting for approximately 50% of the forest area in this region. The current study was conducted in a 70-year-old planted forest dominated by dragon spruce (Picea. asperata; hereafter called “the plantation”) and in a 200-year-old natural forest dominated by spruce (Picea. asperata) and fir (Abies faxoniana) (hereafter called “the natural forest”). The understory of the spruce plantation is less dense and is dominated by the herbaceous plants Festuca ovina, Deyeuxia arundinacea and Carex capillifoemia. The understory of the spruce-fir forest is dominated by Acer mono, Lonicera spp., and Betula albosinensis with occurrences of the herbaceous plants Anemone rivularis and Carex capilliformis. The soils at both sites are Cambic Umbrisols according to the IUSS Working Group (2007).
Soil sampling was conducted at the height of the warm and rainy season (July) in 2017. From each forest type, soil samples were collected from three grids (approximately 4 m × 4 m) that were randomly placed in a representative 100 m × 100 m plot. In each grid, the O horizon was removed, and five subsamples were then taken close to the targeted species (P. asperata) using a large diameter soil core (15 cm diameter, 0-15 cm depth) to obtain as many fine roots of P. asperata as possible. This depth was chosen because it had the largest fine root biomass at these sites (Liu et al., 2008). In the field, five subsamples were pooled together and the living roots of P. asperata were empirically identified by features such as morphology, color, and elasticity. Rhizosphere soil was operationally defined as soil adhering to the roots of P. asperata and was carefully collected using forceps and brushes, while the remaining soil was considered bulk soil (sensu Phillip & Fahey, 2006). Soil samples (rhizosphere and bulk soil) in each grid were sieved (2 mm), and homogenized in situ and then split into three parts. One part was used immediately after sieving to determine gross N cycling in situ. The second part was immediately placed in an ice box, transported to the laboratory and stored at -4°C for analysis of microbial biomass and enzymatic activity. The third part was air dried for analysis of soil properties.
The measurement of gross N production and retention
Potential rates of gross N mineralization and nitrification were measured using the 15N pool dilution techniques (Hart et al., 1994). For the 15N labeling experiments, 50 g of the rhizosphere or bulk soil was weighted into a black cylindrical plastic jar with a wide mouth (4.7 cm diameter × 10.2 cm height). A total of four jars per soil fraction (rhizosphere or bulk soil) for each grid were prepared. Two were injected with (15NH4+)2SO4 solution, and the other two were injected with K15NO3 solution. The concentrations of the labeled solution were determined based on measurement of the background concentrations of NH4+ and NO3- in the soils. Specifically, each jar received five 0.5 mL injections of the solutions (50 atom% 15N) that contained 90 μg NH4+ mL-1 or 45 μg NO3- mL-1, equivalent to an average rate of 4.5 μg NH4+ g-1 soil or 2.25 μg NO3- g-1 soil. The injected NH4+ was 40-62% (rhizosphere and bulk soils in the plantation) and 29-38% (rhizosphere and bulk soils in the plantation) of the initial soil NH4+ concentration. The injected NO3- was 40-50% (rhizosphere and bulk soils in the plantation) and 31-36% (rhizosphere and bulk soils in the plantation) of the initial NO3- concentration. One jar from each labeled pair was collected and the soil was mixed in a plastic bag and subsamples (15 g) were extracted with 2 M KCl solution (solution:soil = 5:1) 10 min after 15N injection (T0) (Silver et al., 2005; Henneron et al., 2020). The other jar of the labeled pair was incubated for 1 day (T1) in situ and subsamples (15 g) were extracted with 2 M KCl solution (solution:soil = 5:1). Simultaneously, we also used the 15NH4+- and 15NO3--labeled samples to assess microbial NH4+ immobilization (IA) and microbial NO3- immobilization rates (IN), respectively (as measures of microbial N retention). Approximately 10 g of the T115NH4+- and 15NO3--labeled samples was fumigated with CHCl3 for 48h, and the corresponding subsamples were incubated without CHCl3 fumigation and then extracted with 0.5 M K2SO4 (approximately a 5:1 ratio of solution to soil) (Davison et al., 1991). For the extracts of T1 samples, extractable organic N and 15N enrichment were determined using persulfate digestion as described by Corre et al. (2007). The concentrations of NH4+ and NO3- were determined on a continuous-flow analyzer (Skalar, Breda, Netherlands). Part of the extracts was frozen immediately and kept frozen during transport by air to Nanjing for 15N analyses at the School of Geography Sciences, Nanjing Normal University. For isotope analysis, NH4+ and NO3- were separated following the same 15N diffusion procedures described by Zhang et al. (2013). The isotopic compositions of NH4+ and NO3- were determined using an automated C/N analyzer coupled to an isotope ratio mass spectrometer (Europa Scientific Integra, UK).
The gross rates of N mineralization, nitrification, and microbial NH4+ and NO3- immobilization were calculated to characterize the production and retention of NH4+ and NO3- following the equations given by Kirkham and Bartholomew (1954) and Davidson et al. (1991). Rates of DNRA, also a measure of microbial NO3- retention, were calculated from the 15NO3--labeled samples following the same calculations used by Silver et al. (2001, 2005). The formulas are listed in the Appendix A.
To allow an overall comparison between two soil compartments and to study to what extent mineral N immobilization plays an important role in N retention, different ratios were calculated as described by Vervaet et al. (2004). The ratio of microbial NH4+ immobilization to gross mineralization (IA:GM) or microbial NO3- immobilization to gross nitrification (IN:GN) was calculated to evaluate the extent to which microbes contribute to retaining the newly produced NH4+ or NO3- (Geisseler et al., 2010). The ratio of gross nitrification to gross mineralization (i.e., relative nitrification, GN:GM) was calculated to indicate which type of N form (NH4+ or NO3-) dominated the N cycling (Gilliam et al., 2001). High rates of relative nitrification suggest that N cycling in soils is mostly dominated by NO3- rather than NH4+ and vice versa.
Microbial biomass C and N and microbial nutrient limitation
Soil microbial biomass C and N were determined from the unlabeled soil samples using the CHCl3 fumigation and 0.5 M K2SO4 solution extraction method (Brookes et al., 1985; Vance et al., 1989). Organic C and total N in the extracts were determined simultaneously on a TOC/TN analyzer (Multi-N/C 2100, Analytik Jena AG, Germany). Microbial biomass C and N were calculated based on the differences in extractable organic C and total N between the fumigated and nonfumigated samples, correcting for unrecovered biomass using a factor of 0.45 (Jenkinson, 2004). The stoichiometric imbalance ratios between microbes and their resources in rhizosphere and in the bulk soils were calculated as the ratio of SOC:N to microbial biomass C:N, which reflect the relative extent to microbial nutrient limitation of rhizosphere compared to the bulk soil (Mooshammer et al., 2014). An increasing stoichiometric imbalance indicates increasing microbial N limitation, and vice versa, a decreasing stoichiometric imbalance indicates increasing microbial C limitation.
Enzyme activity assays
The potential activity of six enzymes involved in soil N cycling in the rhizosphere and bulk soil, including proteases, leucine aminopeptidase (LAP), β-1,4-N-acetylglucosaminidase (NAG), phenol oxidase (POX), peroxidase(PER) and nitrate reductase (NIR), was assayed. The specific information for each enzyme is shown in Table A.1. Simply speaking, the activities of NAG and LAP enzyme were measured with 4-methyl-umbelliferyl substrate conjugate using microplate fluorometic assay with 365 nm excitation and 450 nm emission filters on Varioskan Flash multiplate reader (Thermo Scientific, USA), and the activities of PPO and POX enzyme were measured spectrophotometrically as absorbance at 460 nm using L-3,4-dihydroxyphenylalanine (L-DOPA) as the substrate (Saiya-Cork et al., 2002). We assayed potential proteases activity using trichloroacetic acid solution to terminate the activity following the method modified from Brzostek et al. (2012). The concentration of amino acids in the incubated and initial subsamples was quantified using the o-phthaldialdehyde and β-mercaptoethanol (OPAME) method with glycine as the standard (Jones et al., 2002). Protease activity was calculated from the difference in amino acid concentration between the incubated and initial samples. NIR activity was measured the sulphamic acid-naphthalamine colorimetric method with the absorbance reading at 543 nm after incubating in dark and anaerobic conditions for 24 h using KNO3 as the substrate (Schinner et al., 1996). The detailed processes of enzyme activity analysis are shown in the Appendix A.
Other supporting parameters
In this study, a series of soil physicochemical characteristics (including the concentration of soil organic C (SOC), total N (TN), dissolved organic C (DOC), NH4+, NO3-, pH, CEC, and base saturation (BS)) were measured to analyze whether these factors control the gross rates of mineral N production and retention. Subsamples of rhizosphere and bulk soils were ground to pass through a 0.15 μm sieve, and then the SOC and TN concentrations were analyzed using a CN analyzer (Vario MACRO, Elementar Analysensysteme GmbH, Hanau, Germany). The soil pH was measured with a pH meter (Meltler-Toledo Instruments Co., Ltd., Shanghai, China) using a 2.5:1 soil to H2O extract. The CEC was determined from air-dried soils (passed through a 2 mm sieve) by percolating with unbuffered 1 M NH4Cl, followed by analysis of the percolates for exchangeable element concentrations (Ca, Mg, K, Na, Fe, Al and Mn) using inductively coupled plasma optical emission spectroscopy (ICP-OES; Optima 5300 DV, Perkin Elmer). BS was calculated as the percentage of exchangeable base cations relative to the CEC. The concentrations of different N forms in the rhizosphere and bulk soils were also measured to evaluate the differences in soil N availability between the two soil fractions. The NH4+ and NO3- were extracted with 2 M KCl solution (solution:soil = 5:1) and then determined on a continuous-flow analyzer (Skalar, Breda, Netherlands). The soil organic N (SON) was calculated as the difference between the total N concentration and inorganic N content (SON = TN – ([NH4+] + [NO3−])) (Staelens et al., 2011; Masses et al., 2016).
The normality and equality of variance were tested using the Kolmogorov-Smirnov D and the Levene statistic for each parameter in the rhizosphere and bulk soils of the plantation and the natural forest. Log transformation was conducted when the parameters showed heterogeneous variance. Considering that many previous studies have compared difference in soil N cycling between forest types, we focused on illuminating how plants regulate rhizosphere gross N transformations to improve soil N availability in alpine forest ecosystems with regardless of the differences between plantation and natural forest in this study. Therefore, paired Student’s t-tests were conducted to examine the differences in soil physicochemical characteristics, N-cycling rates (GM, GN, IA, IM and DNRA) and microbial properties (microbial biomass and N-cycling enzyme activities) between the rhizosphere and the bulk soil at each site. The RE were calculated as the percentage difference of the N-cycling rates (GM, GN, IA, IM and DNRA) and N-pool sizes between the rhizosphere and bulk soil to assess whether rhizosphere and bulk soils differed in N supply capacities (Phillips and Fahey, 2006). To assess the influence of soil physicochemical and microbial properties on soil N cycling rates in the rhizosphere and bulk soil, Pearson correlation tests were conducted to identify relationships between gross mineral N transformation rates, soil variables, and microbial variables. Means and standard errors (SE) of the four replicates for each soil compartment in each forest type are reported as measures of central tendency and dispersion. Differences are considered statistically significant when p-values <0.05, unless otherwise stated. All analyses were conducted using SAS 8.0 for Windows (SAS Institute Inc., Cary NC, USA). Graphic illustrations were generated using Origin 8.0 software (Origin Lab Corporation, Northampton, MA, USA).