Fiastar 5000 analyzer

Total soil organic carbon and nitrogen (TOC and TN) at the depths of 0–10 and 10–20 cm were measured by a TOC-5000A analyzer (Shimadzu Corp, Kyoto, Japan) and a Vario EL III Elemental Analyzer (Elementar, Hanau, Germany). The supernatant of soil solution was used for NH₄⁺-N and NO₃⁻-N measurement through a FIAstar 5000 Analyzer (FOSS, Hillerd, Denmark) as previously described (Yang et al. 2013 ).
Aboveground vegetation properties were measured by indices of species number, abundance, diversity, aboveground biomass, and average height based on common protocols (Yang et al. 2013 ). Among these, the species number and abundance were counted during field sampling. Plant biomass was weighed after mowing. The diversity was calculated by the Shannon–Weaver index (Liu et al. 2014 ). To measure the average height and coverage of the vegetation canopy, the 1 × 1 m quadrat was divided into 100 0.1 × 0.1 m small squares, then touched species were measured by 0.1 cm marks along a vertical ruler held behind the pin. The canopy height of shrub in 1 × 1 m quadrat was calculated by the average of all species in the zone.

All of the nine replicate soil samples collected from each grazing zone were analyzed for physical and chemical properties. Soil pH was determined using a 1:2.5 soil/water mixture. After the mass was stabilized by drying at 105°C for 24 h, the water content of the soil was calculated. Water and inorganic carbon were removed by reacting the samples with phosphoric acid (Schumacher, 2002 ), after which soil organic carbon and total nitrogen were measured using the NC analyzer dry combustion method (Sumigraph NC-900; Sumika Chemical Analysis Service, Tokyo, Japan). For chemical analyses, soil was digested in H₂SO₄ for total nitrogen and in H₂SO₄/HCl for total phosphorus, extracted with KCl for nitrate and ammonium, and analyzed using an automated flow analysis instrument (FIAstar 5000 Analyzer; Foss Tecator, Hillerød, Denmark).

The soil samples were collected from 0 to 10 cm soil layer in each plot using an auger in mid-August every year. The soil available N content (NH₄⁺–N and NO₃^––N) was extracted with a 2 mol L^–1 KCl solution and determined with a continuous flow spectrophotometer (FIAstar 5000 Analyzer; Foss Tecator, Hillerød, Denmark). The soil microbial biomass C (MBC) and N (MBN) were measured using the chloroform fumigation extraction method (Brookes et al., 1985 (link); Fu et al., 2012 (link)). Fumigated and unfumigated soil samples were extracted with 100 ml 0.5 mol L^–1 K₂SO₄ and filtered with a 0.45-μm membrane. The extractable organic carbon and total N were determined by an automatic carbon analyzer (Phoenix 8000) and flow injection nitrogen analyzer (FIAStar5000, FOSS Inc), respectively. Extractable carbon and N were converted to MBC and MBN using a conversion coefficient of 0.45 for both measurements (Fu et al., 2012 (link)).

The leaf-blade area of all leaves of the sample branches was measured with an LI-3100C optical area meter (LI-COR Biosciences, Lincoln, NE, USA). To calculate a tree’s total foliage area, a regression model was used (Table 2). Leaf area ratio (LAR) was considered as the total leaf area per unit tree aboveground dry mass.
Leaf N concentrations were determined by block digestion and steam distillation methods (Kjeltec Auto 1030 Analyzer, FOSS Tecator AB, Höganäs, Sweden). Phosphorus (P) concentrations of plant material were determined spectrophotometrically from Kjeldahl digests using a FIAstar 5000 Analyzer (FOSS Tecator AB). Chemical analyses were performed at the Laboratory of Biochemistry, Estonian University of Life Sciences.

Soil moisture content was measured gravimetrically after drying at 105°C for 24 h. Soil pH was measured in 1 M KCl (1∶5 w/v) using a pH electrode. Soil organic carbon and total N were analyzed by the CHNS-analysis system (Elementar Analysen Systeme, GmbH, Hanau, Germany) with the burning method at 450°C and 1250°C, respectively. The concentration of soil available N (NO₃-N+NH₄-N) was measured using a FIAstar 5000 Analyzer (FOSS, Hillerød, Denmark) after extraction with 2 M KCl (1∶5 w/v). Soil available P was extracted following the Mehlich-3 method and measured using the molybdate-blue colorimetrical method [43] . Plant N content was analyzed by the San⁺⁺ Continuous Flow Analyzer (Skalar Analytical B.V., Breda, Netherlands) using the semi-micro Kjeldahl method. Plant P content was measured using the molybdate-blue colorimetric method after digestion with sulfuric acid [44] .

Dry soil aliquots (humus layer and mineral soil), as well as fine and coarse root samples were milled to a fine powder (Retsch MN 400, Haan, Germany). The soil pH was determined using a glass electrode (pH meter, WTW, Weilheim, Germany) in a 1:2.5 soil:water solution (w/v). The total carbon (TC) and total nitrogen (TN) were measured using an Elemental Analyzer (Elementar, Vario ELIII, Elementar Analysen Systeme GmbH, Germany). Nitrate (NO₃⁻) and ammonium (NH₄⁺) concentrations were determined via flow injection analysis (FIAstar 5000 Analyzer, Foss Tecator, Hilleroed, Denmark) following the extraction of the soil samples in 2 mo1/L KCL solution. The NO₃⁻ was determined through the Dual Wavelength Spectrophotometry method and NH₄⁺ by the indophenol blue colorimetric technique [35 ]. The total phosphorus (TP) was measured via Inductively Coupled Plasma Optical Emission Spectrometry (ICP OES) (iCAP 6300 Series, Thermo Fischer Scientific, Dreieich, Germany), subsequent to extraction by 65% HNO₃ at 160 °C for 12 h. The available phosphorus (AP) was extracted using 0.5 M NaHCO₃ (pH 8.5) and then quantified using the molybdenum blue method. The gravimetric soil water content was determined by weighing the soil samples prior to and following drying at 105 °C for 24 h [36 (link)].

Concentrations of NH₄⁺, NO₂⁻, and NO₃⁻ were measured according to the US EPA procedures [30 ] using a fully automated flow-injection system (FIA-star 5000 analyzer, Foss Tecator, Höganäs, Sweden) in water samples.