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 12 / 15 Conclusions In conclusion, the study demonstrated that a living mulch based on a mixture designed for establishing flower strips or to cover the soil in the interrow should not be sown into the apple tree rows during the orchard establishment, particu- larly when low vigour rootstocks (e.g., M9) are used, without additional interventions. Indeed, they competed for nutrient and water resources with the young apple trees, though they increased the soil N content and modified the bacterial community along the vegetative season. To limit the negative effects, the establishment of the living mulches could be complemented with either the application of organic or microbial biostimulants to the soil or foliar fertilization that can provide nutrients directly to the tree. Nevertheless, on a longer term, with the full development of the tree root system, the provision of organic matter and potential nitrogen fixation from both mixtures could partly balance the nutrient needs of the trees, particularly early season, allowing to reduce the needs of external inputs. This assumption is currently under evaluation as the limited time frame of the study can have potentially biased the observed overall effect of the living mulches. Nevertheless, the acquired knowledge could support the decision of farmers when implementing living mulches, are this practice is among those suitable for financial support within governmental programs meant to foster the adoption

of cultivation methods enhancing biodiversity in orchards. Showing contrasting effects on trees for the two mixtures (which were selected also to provide additional ecosystem services) can thus provide scientific evidence for the practical imple- mentation of the measure. The evaluation of the living mulches on yield and fruit quality, as well as on the containment of pests and weeds shall provide the conclusive information needed by operators. This assessment is currently ongoing. It is noteworthy that the mixture commonly used to establish flower strips outside the orchard showed to be also suit- able as a living mulch: its impact on beneficial fauna and reduction of apple pests in comparison to common flower strips is also currently under assessment. The selection of any of these kinds of mixtures for a commercial orchard could thus depend on the kind of ecosystem service that the farmer considers more useful for its cropping system, whether it shall support crop protection or increase soil health. Future work should thus consider the identification of the best mixture according to the pedo-climatic condi- tions and the evaluation of the mixture’s impact on functional microbiome, which would shade light on the complex rela- tionships established in the orchard soil. Supporting information S1 Table. Dynamic and concentration of soil nutrients and chemical parameters as affected by the introduction of two kinds of living mulches. Mean±SD, n = 3. Letters show statistically significant differences for p ≤ 0.05 (A-C for the

same treatment in different timepoints, a-b for different treatments in the same timepoint). (XLSX) S2 Table. Bacterial activity (AWCD) for six compound classes analysed with Biolog EcoPlate as affected by two kinds of living mulches mixtures during the vegetative season. Means±SD, n = 3. Different letters show statistically significant differences between sampling points within each compound class for p ≤ 0.05. (XLSX) S3 Table. Bacterial activity between soil samples collected from tested treatments in different parts of the 2022 vegetative season. Different letters show statistically significant differences between treatments within each sampling point for p ≤ 0.05. (XLSX) Author contributions Conceptualization: Ewa M. Furmanczyk, Eligio Malusà. Data curation: Ewa M. Furmanczyk.