Vario el

Physicochemical analyses of soil pH, conductivity, water content, and concentrations of soil organic carbon (SOC) and total nitrogen (TN) were performed as previously described [38 (link)]. In brief, the soil moisture was determined as the differences in mass of fresh soil dried at 105°C for 24 h. Air-dried soils were passed through a 100-mesh screen, then concentrations of SOC and TN were determined using the CHNS-analyzer system (Elementar Vario EL, Elementar Analysensysteme GmbH, Hanau, Germany) with the burning method at 450 and 1250°C, respectively. Soil pH was measured using 1M KCl (5 g soil in a 25mL solution), and soil conductivity was determined in 1:2 soil/deionized water slurry.

Within each plot, three soil samples at 0–30 cm depth were obtained at sowing, heading and maturity stages over the two growing seasons. To evaluate the differences of soil nutrient dynamics between treatments, each soil sample was collected by mixing the soil cores from three representative positions around each plant (Fig. 9). Each soil sample was air-dried at the time of the measurement and any visible extraneous material (e.g., residues, roots, and stones) was removed before the sample was ground, passed through a 2-mm sieve, and weighed. Soil organic carbon (SOC) and total nitrogen (STN) were measured using the dry combustion (450 °C) method with a CHNS analyzer (Elementar Vario El, Elementar Analysen Systeme GmbH, Hanau, Germany). Soil available phosphorus (SAP) was determined by extracting with 0.5 M NaHCO₃ and analyzed using a UV-vis spectrophotometer^{37 (link)}. The C/N ratio was then calculated as the ratio of SOC to STN for each sample. Soil pH was determined by a pH electrode in a 5: 1 water:soil ratio slurry.

All chemicals were purchased from commercial sources and used without further purification. Solvents were dried and distilled using conventional methods. Melting points were measured on Heiztisch Mikroskop—Polytherm A (Wagner & Munz, Germany). NMR spectra were performed on Agilent 400-MR DDR2 (¹H: 400 MHz, ¹³C: 100 MHz). Deuterated solvents used are indicated in each case. Chemical shifts (δ) are expressed in ppm and refer to the residual peak of the solvent or TMS as an internal standard; coupling constants (J) are in Hz. The mass analyses were performed using the ESI technique on a Q–TOF (Micromass) spectrometer. Elemental analyses were carried out on Perkin–Elmer 240, Elementar vario EL (Elementar, Germany) or Mitsubishi TOX–100 instruments. All samples were dried in the desiccator over P₂O₅ under vacuum (1 Torr) at 80 °C for 8 h. The IR spectra were measured on an FT–IR spectrometer Nicolet 740 or Bruker IFS66 spectrometers equipped with a heated Golden Gate Diamante ATR–Unit (SPECAC) in KBr. A total of 100 Scans for one spectrum were co–added at a spectral resolution of 4 cm⁻¹. The courses of the reactions were monitored using TLC aluminium sheets with Silica gel 60 F254 (Merck). The column chromatography was performed on Silica gel 60 (Merck). HPLC was performed on Büchi Pure 850 FlashPrep chromatography instrument using Prontosil, 150 × 20 mm, 5 μm column.