Acid phosphates activity and mycorrhizal fungi
We used colorimetric methods to analyze acid phosphates activity (Tabatabai, 1994). Specifically, four grams of moist soil or 0.2 grams of litter was added with 60 ml of 50 mM acetate buffer (pH 5) in a blender for 1 min. A subsample (0.75 ml) of the slurry was then added to 0.75-ml substrate (5 mM). Two controls (buffer + substrate and buffer + slurry subsample) were also analyzed. Each analysis was repeated three times. After 1h incubation at 20 °C in the dark, each sample was centrifuged at 2091×g, then 0.2 ml of supernatant was transferred into a 96-well plate. Finally, 0.03 ml ultrapure water and 0.02 ml 1 M NaOH were added to each well. Absorbance of each well was measured at 405 nm by Multiskan EX (Thermo Scientific, Waltham, MA). A p-nitrophenol (p -NP) calibration curve was performed, and results were expressed in μmol of p -NP released h−1 g−1 of dry soil.
PLFA assays were performed using established methods to estimate the abundance of arbuscular mycorrhizal fungi (16:1ω5c) and ectomycorrhizal fungi (18:2ω6)(65 ). In brief, lipids were extracted from 8 g of lyophilized soil, fractionated and methylated. The resulting fatty acid methyl esters (FAMEs) were separated using a GC gas chromatograph (Agilent GC7890A; Agilent Technologies Inc.). Peaks were identified using the Sherlock System (v. 6.1; MIDI Inc.).