Soil Bacteriome Shifts along a Cultivation Gradient in Southwestern Spanish Wetlands

Understanding how long-term agricultural practices affect soil bacteriome is essential for sustainable land management. In the Guadalquivir Marshes of southwestern Spain, which encompass both Doñana National Park and one of Europe's most productive rice cultivation areas, decades of rice farming have transformed natural wetlands into artificial agroecosystems. Although bacterial degradation in cultivated soils has been previously suggested, comparative analyses between rice paddies and adjacent natural wetlands remain scarce.Here, we characterized the soil bacteriome across a cultivation gradient by comparing undisturbed natural marshes, within Doñana National Park, with rice fields cultivated for 25 years (Cantarita) and 80 years (Mínima 2). Using full 16S rRNA gene via long-read metabarcoding and standardized soil physicochemical assays, we analysed taxonomic composition, environmental associations, and predicted functional profiles.Our results reveal a progressive restructuring of bacterial communities with increased cultivation time, notably a significant enrichment of Chloroflexota (especially Anaerolineae) and a decline in Actinomycetota and Planctomycetota in paddy soils. Functional predictions indicated a higher potential for denitrification in cultivated soils-likely involving Chloroflexota taxa-compared to more diverse nitrogen pathways in natural sites. These shifts were strongly associated with changes in pH, electrical conductivity, calcium carbonate, and nitrate levels. Remarkably, most bacterial differences were already evident within the first 25 years of cultivation, underscoring the rapid ecological impact of intensive rice cultivation.Notably, we identified specific bacterial groups (Anaerolineae and Nocardioides in paddy soils; Euzebya, Rubrobacter, and Planctomycetota in natural wetlands), whose enrichment was associated with soil type. This approach highlights the value of integrating bacterial-based assessments into sustainable wetland management strategies.