Nitrate (NO₃^-) leaching is a serious health and ecological concern in global agroecosystems, particularly those under the application of agricultural-managed aquifer recharge (Ag-MAR); however, there is an absence of information on microbial controls affecting NO₃^- leaching outcomes. We combine natural dual isotopes of NO₃^- (¹⁵N/¹⁴N and ¹⁸O/¹⁶O) with metagenomics, quantitative polymerase chain reaction (PCR), and a threshold indicator taxa analysis (TITAN) to investigate the activities, taxon profiles, and environmental controls of soil microbiome associated with NO₃^- leaching at different depths from Californian vineyards under Ag-MAR application. The isotopic signatures demonstrated a significant priming effect (P < 0.01) of Ag-MAR on denitrification activities in the topsoil (0-10 cm), with a 12-25-fold increase of ¹⁵N-NO₃^- and ¹⁸O-NO₃^- after the first 24 h of flooding, followed by a sharp decrease in the enrichment of both isotopes with ∼80% decline in denitrification activities thereafter. In contrast, deeper soils (60-100 cm) showed minimal or no denitrification activities over the course of Ag-MAR application, thus resulting in 10-20-fold of residual NO₃^- being leached. Metagenomic profiling and laboratory microcosm demonstrated that both nitrifying and denitrifying groups, responsible for controlling NO₃^- leaching, decreased in abundance and potential activity rates with soil depth. TITAN suggested that Nitrosocosmicus and Bradyrhizobium, as the major nitrifier and denitrifier, had the highest and lowest tipping points with regard to the NO₃^- changes (P < 0.05), respectively. Overall, our study provides new insight into specific depth limitations of microbial controls on soil NO₃^- leaching in agroecosystems.

Agricultural managed aquifer recharge (Ag-MAR, on-farm recharge), where farmland is flooded with excess surface water to intentionally recharge groundwater, has received increasing attention by policy makers and researchers in recent years. However, there remain concerns about the potential for Ag-MAR to exacerbate nitrate (NO₃^-) contamination of groundwater, and additional risks, such as greenhouse gas emissions and crop tolerance to prolonged flooding. Here, we conducted a large-scale, replicated winter groundwater recharge experiment to quantify the effect of Ag-MAR on soil N biogeochemical transformations, potential NO₃^- leaching to groundwater, soil physico-chemical conditions, and crop yield. The field experiment was conducted in two grapevine vineyards in the Central Valley of California, which were each flooded for 2 weeks and 4 weeks, respectively, with 1.31 and 1.32 m³ m^-2 of water. Hydrologic, geochemical, and microbial results indicate that NO₃^- leaching from the first 1 m of the vadose zone was the dominant N loss pathway during flooding. Based on pore water sample and N₂O emission data, denitrification played a lesser role in decreasing NO₃^- in the root zone but prolonged anoxic conditions resulted in a significant 29 % yield decrease in the 4-week flooded vineyard. The results from this research, combined with data from previous studies, are summarized in a new conceptual model for integrated water-N dynamics under Ag-MAR. The proposed model can be used to determine best Ag-MAR management practices.