2.3. Soil sampling and chemical and enzyme analysis
Soil samples were collected from 25 sites representing the five forest
states (Fig. 1). Sampling sites were located at least 500 m from one
another and are considered independent. Soils were sampled from each
site once in the middle of the dry season in October 2021 and once
towards the end of the rainy season in May 2022. Four samples were
extracted from the topsoil (0-10 cm) at each site and combined to
produce a composite sample. After sieving at 2 mm, samples were split
into two subsamples: the first was stored at 4 °C prior to biochemical
analysis and the second was air dried, crushed and sieved (<60
μm) for chemical analysis.
Soil pH was measured in a 1 M KCl extract (1:2.5, m/v) after shaking for
one hour (Hesse, 1971) using a pH meter (CRISON micro pH 2002).
Electrical conductivity (EC) was determined in water extract (1:5, m/v)
after shaking for one hour using a Conductivity Meter (CRISON micro CM
2201). Sample dry weights were used to calculate soil water content
(SWC) by the gravimetric method. Water-soluble carbon (WSC) content was
determined using a TOC-VE Shimadzu analyser after extraction with water
using a sample-to-extractant ratio of 1:10. Dehydrogenase activity (DHA)
was determined according to Trevors (1984) after soil incubation with
p-Iodonitrotetrazolium chloride
(2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride,
INT) and measurement of the Iodonitrotetrazolium formazan
(1-(4-Iodophenyl)-5-(4-nitrophenyl)-3-phenylformazan, INTF) absorbance
at 490 nm. Glucosidase activity (β-Glu) was measured according to Eivazi
and Tabatabai (1988) following soil incubation with
p-Nitrophenyl-β-D-glucopyranoside and measurement of the p-Nitrophenol
absorbance at 400 nm. Urease activity was determined according the
method proposed by Kandeler and Gerber (1988) and modified by Kandeler
et al. (1999).