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.