Comprehensive Soil Physicochemical Analysis

Soil pH was determined using a fresh soil to water ratio of 1:5 using a pH monitor (Thermo 0rion-868, MA, USA). Soil moisture was measured gravimetrically after a 16-h desiccation at 105 °C. Soil samples for C and N analyses were air dried (2 mm mesh), handpicked to remove plant litter and fine roots, and ground. Total soil C and N content for each plot were determined by combustion (2400 II CHNS/0 Elemental l Analyzer, Perkin-Elmer, Boston, MA, USA). Dissolved organic carbon (DOC) and dissolved total nitrogen (DTN) were extracted by adding 50 ml of 0.5 MK₂SO₄ to 10 g fresh soil, shaking for 1 h, and vacuum filtering through a G4 glass fiber filter with a pore space of 1.2 μm (Fisher). DOC and DTN were determined using a total organic carbon-total nitrogen (TOC-TN) analyzer (Shimadzu, Kyoto, Japan). Ammonium (NH₄⁺) and nitrate (NO₃⁻) concentrations in extracts were assessed colorimetrically by automated segmented flow analysis (AAIII; Bran and Luebbe, Germany) using the salicylate/dichloroisocyanuric acid and cadmium column/sulfanilamide reduction methods, respectively. Through HF and HClO₄ digestion, total potassium (TK) was determined by flame photometry (FP640, INASA, China), while total phosphorus (TP) was determined using the molybdenum blue method. Available potassium (AK) was determined in 1 M ammonium acetate extracts by flame photometry (FP640, INASA, China). Soil available phosphorus (AP) was extracted by 0.5 M NaHCO₃ and determined using the molybdenum blue method. Organic carbon was determined according to potassium dichromate oxidation titration. Soil electric conductivity was determined by a conductivity monitor using a dry soil to water ratio of 1:5 (Thermo 0rion-868, MA, USA). Soil samples were air dried and homogenized by grinding in an agate mortar and then passed through a 0.149 mm sieve to analyze the elements. These samples (~ 0.4–0.5 ± 0.0001 g) were digested with nitric acid (HNO₃), hydrofluoric acid (HF), and perchloric acid (HClO₄) (5 mL: 10 mL: 5 mL) on a hot plate. Soil total Mg, Ca, K, and Fe were measured with an ICP-AES Optima 8000 (Perkin-Elmer, Waltham, MA, USA), while total Cd, chromium(Cr), Mn, copper(Cu), Zn, plumbum (Pb), and As were measured with an HPLC-ICP-MS (7700X, Agilent, USA). A certified soil reference material (GBW07408, National Research Center for Certified Reference Materaials, China) were used to ensure that the accuracy of the analytical data and the accuracy ranged from 93.9 to 107.4%. All soil variables are described in Additional file 2: Table S9.