In-depth insight into the short-term effect of floor management practice on young apple trees development and soil microbial biodiversity and activity

PLOS One | https://doi.org/10.1371/journal.pone.0329979 August 7, 2025 4 / 15 Soil sample collection Soil samples for chemical and biological analyses were collected three times during the 2022 growing season within well-established living mulches (12th May, 11th July and 14th September). Sampling was performed using Egner’s auger (2.5 cm diameter) collecting at least 10 subsamples at 0–20 cm depth in the volume of soil near the tree root system (30 cm around the trunk). The subsamples were pooled and mixed to form the analytical sample. Visible animals, organic parts (plant residues, roots) as well as small stones were removed at this stage. The samples were stored in 4°C for up to one week or proceeded immediately for soil chemical analysis, and microbial biodiversity and activity determination. Soil chemical analysis The soil samples were analyzed in terms of pH, salinity, and the levels of macronutrients: N-NO3, N-NH4, P, K, Ca and Mg. After drying at a temp. of 65°C, the soil was mineralized in concentrated nitric acid in a Candela Mars 5 microwave digestion oven. Nitrogen was determined colorimetrically with a Skalar SanPlus automated flow analyzer [28], and phos- phorus and potassium were analyzed with a Perkin Elmer OPTIMA 2000 DV plasma spectrometer [29]. The pH of the soil samples was determined on KCl extract: 10 g of homogenized and air-dried soil was mixed with 25 mL of 1 M KCl, and the measurement was carried out on the solution after 24 h using a pH meter (Fisher Scientific, Warsaw, Poland). Soil microbial biodiversity and activity assessment

Soil microbial biodiversity and activity were determined using the BIOLOG system and EcoPlates plates. A suspension was obtained from 1 g of soil using sterile distilled water (1:9 w:v) and serially diluted three times. Plates were inoculated with 100 μl of appropriate soil suspension (10−3) on each well and then incubated in the dark at 26°C. After 72h the absor- bance at 590 nm (OD) was determined. The activity of microorganisms was estimated on the basis of Average Well Colour Development (AWCD) [30] and the overall activity index or activity index for each compound class were calculated according to the formula: AWCD = ∑ OD i/n (1) where ODi is the optical density of the individual wells and n is the number of substrates included in specific category (31 for overall AWCD, 10 for carbohydrates, 7 for carboxylic acids, 6 for amino acids, 4 for polymers, 2 for phenolic com- pounds and 2 for amines). The microbial biodiversity index was estimated using the Shannon-Weaver coefficient (H): H = – ∑ pi(ln p i) (2) where pi is the level of microbial activity in each well (ODi) divided by the activity in all the wells (Ʃ ODi) [31]. When cal- culating the level of activity of microorganisms for the H index and the amount of metabolized substrates, the threshold value OD = ODi – OD(control well) was used. Substrate richness (S), which is the number of utilized carbon substrates, was calculated using an OD590 value of 0.500 as threshold for positive response. The results were used for comparison and

visualization of the potential metabolic profiles of the soil microbial communities. Statistical analysis Statistical analysis of data was performed using the R software version 4.1.3 [32]. The Shapiro-Wilk test was used to verify if the data followed a normal distribution and the Levene’s test was used to verify the homogeneity of variances. The data were thus analysed by ANOVA and means differences tested with Tukey’s test at p ≤ 0.05 with HSD test function