Structure-activity relationship study in soil identifies an effective broad-spectrum inhibitor of archaeal nitrification

Ammonia-oxidizing archaea (AOA) can contribute substantially to nitrification in agricultural soils, yet the portfolio of AOA-targeting nitrification inhibitors (NIs) with demonstrated efficacy under soil conditions remains limited. Moreover, the performance of candidate inhibitors in soil is often difficult to predict from pure-culture assays because their activity is strongly influenced by soil-dependent factors. In this study, we employed a recently developed high-throughput miniaturized soil incubation assay to perform a soil-based structure-activity relationship (SAR) analysis of SIAS (Specific Inhibitor of AOA in Soil), a pyrazolo-pyridine derivative previously identified as an AOA-targeting compound with strong additive nitrification inhibition effects when combined with the AOB-targeting inhibitor DMP (3,4-dimethyl-1H-pyrazole). To identify structural features associated with improved and more robust soil efficacy, a library of 27 SIAS-derived pyrazolo-pyridine analogues was evaluated directly in soil. The SAR analysis showed that inhibitory activity was governed by a specific combination of structural features and led to the discovery of Optimized SIAS (Opt. SIAS), a simplified analogue that consistently outperformed the original lead compound across diverse agricultural soils. Correlation analysis further revealed that soil pH and, particularly, silt content were major determinants of Opt. SIAS efficacy. The strong additive effects obtained through combined application of Opt. SIAS and DMP highlight the contribution of AOA to residual nitrification following AOB inhibition and demonstrate the potential of simultaneously targeting both groups. Together, these results expand the limited portfolio of effective AOA-targeting nitrification inhibitors and provide a foundation for the development of next-generation nitrification inhibition strategies aimed at improving nitrogen-use efficiency and reducing nitrogen losses from agricultural soils.