Soil biochemistry analysis in Shorea robusta forests

We hypothesized that: 1) disturbance regimes would negatively affect soil microbial biomass and enzyme stoichiometry by increasing the substrate stress (organic matter) input from under-canopy vegetation (shrubs and herbs) and decline soil properties compared to non-disturbed; 2) moderate disturbance (MD) would reduce the microbial C and P limitation in severely P-limited soil by improving soil nutrients status and key physicochemical properties. The study followed a randomized complete stands design, incorporating 24 treatment combinations based on four disturbance regimes (ND, LD, MD, and HD), three seasons (summer, monsoon, and winter), and two soil depths (0-15 and 15-30 cm). As per Shankar & Garkoti (2023), 45 permanent forest stands (≥ 0.1 ha) were categorized into four ecological zones—ND (8), LD (25), MD (9), and HD (3)—based on disturbance intensity (DI, %) and canopy coverage (CC, %) as illustrated in Fig. 1b. These zones are defined as follows: No Disturbance (ND) with DI ≤ 5% and CC > 70%, Low Disturbance (LD) with 5% < DI ≤ 20% and CC > 50%, Moderate Disturbance (MD) with 20% < DI ≤ 50% and CC < 50%, and High Disturbance (HD) with DI > 50% and CC < 30%. Using a soil corer (15 cm × 5 cm × 5 cm), five soil cores were randomly collected at 1 to 1.5 meters from the base of tree stumps of dominant species, at soil depths of 0-15 cm and 15-30 cm. The soil from each predefined stand at the same depth was combined into one composite sample. This process yielded 48 composite soil samples for further analysis (four disturbance regimes × two replicates × two soil depths × three seasons). The freshly collected soil samples were placed in bags and stored at 4ºC before transportation to the laboratory. The moist samples were sieved through a ≤ 2 mm mesh to remove visible stones, pebbles, plant roots, and debris, and then homogenized. Each homogenized composite sample was divided into two portions: one was stored at 4ºC for soil microbial biomass and enzyme activity analysis, while the other was air-dried for assessing soil physicochemical properties. In each ecological zone, five random quadrats of 5 × 5 m² were established for shrubs, and 1 × 1 m² quadrats (with four points at the corners and one at the diagonal intersection) were used for herbs to assess cumulative understory diversity.