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.本研究提出如下假说：1）相较于未受干扰（non-disturbed）样地，干扰制度（disturbance regime）会通过增加冠下植被（under-canopy vegetation，灌丛与草本植物）输入的底物压力（substrate stress，有机质），并降低土壤属性，对土壤微生物生物量（soil microbial biomass）与酶化学计量（enzyme stoichiometry）产生负面影响；2）中度干扰（MD）可通过改善土壤养分状况与关键理化性质，缓解严重磷限制土壤（severely P-limited soil）中的微生物碳、磷限制。本研究采用随机完全林分设计（randomized complete stands design），基于4种干扰制度（未受干扰ND、低干扰LD、中度干扰MD、高干扰HD）、3个季节（夏季、季风季、冬季）以及2个土壤深度（0~15 cm与15~30 cm），共设置24种处理组合。参照Shankar与Garkoti（2023）的方法，将45个永久性林分（面积≥0.1 ha）依据干扰强度（DI，%）与冠层覆盖率（CC，%）划分为4个生态区——未受干扰区（ND，8个林分）、低干扰区（LD，25个林分）、中度干扰区（MD，9个林分）、高干扰区（HD，3个林分），具体划分方式如图1b所示。各生态区定义如下：未受干扰（ND）：干扰强度≤5%且冠层覆盖率>70%；低干扰（LD）：5%<干扰强度≤20%且冠层覆盖率>50%；中度干扰（MD）：20%<干扰强度≤50%且冠层覆盖率<50%；高干扰（HD）：干扰强度>50%且冠层覆盖率<30%。使用规格为15 cm ×5 cm ×5 cm的土壤钻（soil corer），在优势树种树桩基部1~1.5 m处，分别于0~15 cm和15~30 cm土层随机采集5个土芯。将同一预设林分相同土层的土壤混合为1个复合样品（composite sample）。该采样流程共获得48个复合土壤样品用于后续分析（4种干扰制度×2个重复×2个土壤深度×3个季节）。新鲜采集的土壤样品装入密封袋中，于4℃冷藏保存后运输至实验室。将湿样通过孔径≤2 mm的筛网过筛，以去除可见石块、砾石、植物根系及碎屑，随后进行均质化处理。将每份均质后的复合样品分为两份：一份置于4℃冷藏，用于土壤微生物生物量与酶活性分析；另一份风干，用于测定土壤理化性质。在每个生态区内，设置5个5×5 m²的样方用于灌丛调查，同时设置1×1 m²的样方（四角及对角线交点共设置5个采样点）用于草本植物调查，以评估林下植被的累积多样性。