P status in plant, litter, soil and leachate
The P concentrations of plant and litter were measured photometrically after digestion with H2SO4-H2O2(Anderson & Ingram, 1989). Total plant P uptake was calculated by the sums of tissue biomass multiplied by corresponding P concentrations of the four species. Soil microbial biomass P were analyzed using the fumigation-extraction method (Vance et al., 1987): a subsample was extracted using 0.03 M NH4F and 0.025 M HCl to determine extracted P, and another subsample was fumigated for 24 h with chloroform and extracted with the same extract. The extracted P concentrations were corrected for the sorption of P to soil particles during extraction based on the difference between samples spiked and not spiked with P (25 µg of inorganic P added as KH2PO4). The microbial biomass P values were calculated as the differences between the values of fumigated and un-fumigated subsamples corrected by the conversion factor (i.e., 0.4).
Soil P fractionation was measured according to the method described by Tiessen & Moir (1993) and McDowell & Condron (2000). The P in soil was sequentially extracted by 1.0 M NH4Cl (soluble P), 0.1 M NH4F (aluminum phosphate, Al-P), 0.1 M NaOH (ferric phosphate, Fe-P). The non-extracted P in soils after sequential extraction was residual P (occluded P). In brief, 3.0 g of finely ground air-dried soil was extracted by 30 mL of each extractant solution on a horizontal shaker. The shaking time was 30 min for NH4Cl, 16 h for NH4F and NaOH. The solution after shaking was centrifuged at 10,000 r/min for 10 min to collect supernatant for inorganic and total P analysis. Phosphorus extracted by NH4Cl was classified as easily-available P, while P in NH4F and NaOH extracts was classified as moderately-available P. The organic P concentration in NH4F and NaOH extracts were calculated as the difference between total P analyzed after persulphate digestion and inorganic P. The moderately-available organic P was the sums of organic P extracted by NH4F and NaOH. All soil extracted P was determined using an inductively coupled plasma optical emission spectrometer (ICP-OES; Optima 2000 DV, Perkin Elmer, USA).
Leachate P concentrations were measured colorimetrically. Total leachate P was calculated by summing both surface and underground soil solutions, after multiplying P concentrations by the leachate volume, in each month. Rainfall was similar among treatments throughout the experiment due to similar slope aspects and close distance across the three sites.