Soil sampling and the extraction and identification of nematodes
In January 2014 (after 10 years of N application), soil cores were collected from each plot (the treated central area) at 0-20 cm, 20-40 cm, and 40-60 cm soil depths. In each plot, five soil cores (3 cm in diameter) at each depth were randomly taken and combined to form one composite sample per depth per plot. The litter layer at the sample locations was removed before cores were collected. There were 36 soil samples (3 depths × 12 plots) in total.
After visible roots and stones were removed, the soil samples were passed through a 2-mm-mesh sieve and then stored at 4 °C for chemical analysis and nematode extraction. Soil water content (SWC, %, g of water per 100 g dry soil) was measured by comparing weights before and after oven-drying at 105 °C for 24 h, and soil pH was determined in a 1:2.5 (w/v) soil suspension. Concentrations of total N (TN, g/kg dry soil) and total phosphorus (TP, g/kg dry soil) were measured according to Zhou et al. (2019). Total C (TC, g/kg dry soil) was determined using an elemental analyzer (Perkin Elmer Instruments series II, USA). Contents of soil nitrate (NO3-N, mg/kg fresh soil) and ammonium nitrogen (NH4+-N, mg/kg fresh soil) were determined as previously described (Chen et al. 2015). Available P (AP, mg/kg dry soil) was extracted with a sodium bicarbonate solution (0.5 M, 30 min extraction), and the extracted phosphate was then quantified by the molybdenum blue procedure (Zhao et al.2018).
Nematodes were extracted from a 100-g subsample of fresh soil from each composite soil sample with Baermann funnels (Gray, 1984). The extracted nematodes were collected in a 4% formalin solution, and then counted and identified with a DIC microscope (ECLIPSE 80i, Nikon). The first 100 individuals encountered were identified to the genus or family level and classified into four trophic groups, including bacterial-feeding nematodes (BF), plant-feeding nematodes (PF), fungal-feeding nematodes (FF) and omnivorous-predatory nematodes (OP) (Bongers and Bongers, 1998; Yeates et al. 1993).