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).