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 3 / 15 beneficial arthropods. The working hypothesis was to verify whether the increase of above-ground biodiversity, con- sidering the plants coenosis, could improve the soil microbial biodiversity and functions as well, thus providing addi- tional ecosystem services to the cropping system. Therefore, the living mulches effect on the soil nutrient content and bacterial microbiome, as well as on the development of the apple trees was assessed in the first years of orchard establishment. Materials and methods Experimental site and management practices The experiment was conducted in a commercial apple orchard located in Leczeszyce (51°46’18” N, 20°47’49” E, central Poland). The location is characterized by the average annual temperature of 10°C and an average annual precipitation of 650 mm. The orchard was established on 28th April 2021 by planting 2-year old saplings cv. Golden Delicious on M9 rootstock, spaced at 3.0 m x 0.9 m (3700 trees/ha). Plant protection was carried out according to the integrated production guidelines [25,26], according to the needs. Two plant mixtures were tested as living mulches: one consisting of Trifolium repens (20%) and Festuca ovina (80%) and second being a mixture of 10 different species designed for flowering strips establishment composed of: Trifolium repens (10%), Festuca ovina (80%), Plantago lanceolata (0.5%), Sanguisorba minor (4%), Silene vulgaris (1.5%), Achillea millefolium (1.0%), Dianthus deltoides (1.0%), Leontodon hispidus (0.5%),

Thymus pulegioides (0.5%) and Prunella vulgaris (1.0%). These mixtures were commercially available and recommended for either covering the soil to reduce erosion and weeds control (Mix1) or to support pollinators and beneficial arthropods as flowering strip (Mix2) consisting of perennial species commonly found in Poland and Europe. The plant mixtures were sown along the tree row on 26th May 2021, four weeks after trees planting (10 g/m2). The control was set as natural cover with herbicide treatment during the season: two hand weeding during first season and two chemical treatments with Gal- lup 360 SL (glyphosate 360 g/l) and Kileo 400 SL (240 g/l glyphosate and 160 g/l 2,4-dichlorophenoxyacetic acid) during second season, on 28th May and 26th July 2022 respectively. Each treatment consisted of three replicates (5 trees each). The field research was carried out in a private orchard. No animals or whole plants were collected from the environment. The study involved only soil and leaf sampling, which did not require any official permits. Plant growth and yield potential assessment Plant growth was assessed by determining the increase of the average trunk growth based on two perpendicular mea- surements performed about 20 cm above the grafting union, and counting the number of one-year-old shoots and their length at the end of each vegetative period (BBCH 95) on each tree of the replicates (30th Sep 2021, 15th Nov 2022 respectively). The blooming intensity and fruitlets number were considered as proxy of the yield potential. The number of flowers and

fruitlets was counted per each tree during the second vegetative season (on 12th May – BBCH 65 and 11th July 2022 – BBCH 72, respectively). A hail event made not possible to estimate the fruit yield in that season. Leaf nutrient analysis Leaf samples were collected randomly from each replicate in July 2022, gathering 60 mature leaves from current-year shoots from the central section of the tree. The leaves were washed with distilled water, dried at the temperature of 60°C in a forced-air oven, then ground in a Wiley stainless-steel mill. The samples were microwave digested in HNO3, using closed Teflon vessels. Inductively coupled plasma spectrometry (ICP Model OPTIMA 2000DV, Perkin Elmer, USA) was used to determine P, K, Na, Ca, and Mg [27]. The N content was determined using the Kjeldahl method (Vapodest, Gerhardt, Germany) after mineralization in concentrated sulfuric acid in the presence of copper-potassium catalyst [27].