Biostimulant-associated shifts in soil biochemical functioning and microbial community structure under field conditions: an exploratory field-scale assessment

The increasing adoption of agricultural biostimulants necessitates a clearer understanding of their ec ological impacts beyond plant performance, particularly regarding soil biochemical functioning and microbial community dynamics under field conditions. This study presents a field-scale exploratory assessment of a commercially available biostimulant applied within a maize ( Zea mays L.) production system in the Mopani region of Limpopo, South Africa. Soil samples were collected pre- and post-cropping season from treated and untreated plots managed under real-world agricultural conditions. To capture integrated field-scale signatures while accounting for spatial micro-heterogeneity, high-depth composite sampling was employed. The soil matrix was subjected to comprehensive physicochemical profiling and extracellular enzyme kinetic assays (sucrase, catalase, urease, and alkaline phosphatase). Total genomic DNA was extracted, and full-length high-throughput amplicon sequencing of the bacterial 16S and fungal ITS rRNA regions was executed utilizing the Pacific Biosciences platform. Resulting sequence data were processed using QIIME2 and VSEARCH pipelines. Soil nutrient dynamics varied in the control and treatment plots, but the control plots exhibited stability while the experimental plots varied, however, there was no significant difference caused by the soil type and sampling time. Furthermore, all but one of the soil parameters (NO 3 -N) declined in the biostimulant treated plot. Enzyme activities exhibited dynamic temporal variations, with apparent post-harvest declines in sucrase, urease, and alkaline phosphatase activities in the treated soil relative to the untreated control framework, contrasted by a robust persistence of soil catalase capacity. High-resolution sequencing revealed thorough microbial differences in the control and experimental plots. Bacterial alpha diversity indices demonstrated an upward trend over the cultivation period, driven by the expansion of fast-growing copiotrophic Pseudomonadota, while fungal diversity trended downward. Genus-level analyses highlighted a dual ecological response within the treated matrix, characterized by the positive improvement of plant-growth-promoting Streptomyces alongside the unexpected enrichment of opportunistic fungal lineages, including Humicola and the potential pathogen Fusarium . Functional predictions via FAPROTAX and FunFun corroborated these findings, indicating enriched bacterial nitrate reduction pathways alongside an expansion of fungal plant-pathogen metabolic clades. Collectively, these findings provide field-based, descriptive evidence that biostimulant application is associated with coordinated shifts in soil biochemical functioning and belowground community restructuring, establishing a valuable baseline for long-term agroecosystem monitoring.