Evidences towards deciphering the mode of action of dimethylpyrazole-based nitrification inhibitors in soil and pure cultures of Nitrosomonas europaea

Background: Agriculture relies on the intensive use of synthetic nitrogen (N) fertilizers to maximize crop yields, which has led to the transformation of agricultural soils into high-nitrifying environments. Nevertheless, nitrifcation inhibitors (NIs) have been developed to suppress soil-nitrifer activity and decrease N losses. The NIs 3,4-dimethyl‑ pyrazole phosphate (DMPP) and 2-(3,4-dimethyl-1H-pyrazol-1-yl) succinic acid isomeric mixture (DMPSA) are able to reduce N2O emissions and maintain soil NH4

• for a longer time. Although both NIs have been proven to be efective to inhibit soil nitrifcation, their exact mode of action has not been confrmed. We aimed to provide novel insights to further understand the mode of action of DMP-based NIs. We evaluated the performance of DMPP and DMPSA in soil and pure cultures of nitrifying bacteria Nitrosomonas europaea. Results: DMPSA did not inhibit nitrifcation in pure cultures of N. europaea. In the soil, we evidenced that DMPSA needs to be broken into DMP to achieve the inhibition of nitrifcation, which is mediated by a soil biological process that remains to be identifed. Moreover, both DMPP and DMPSA are thought to inhibit nitrifcation due to their ability to chelate the Cu2+ cations that the ammonia monooxygenase enzyme (AMO) needs to carry on the frst step of NH4
• oxidation. However, the efciency of DMPP was not altered regardless the Cu2+ concentration in the medium. In addition, we also showed that DMPP targets AMO but not hydroxylamine oxidoreductase enzyme (HAO). Conclusions: The inability of DMPSA to inhibit nitrifcation in pure cultures together with the high efciency of DMPP to inhibit nitrifcation even in presence of toxic Cu2+ concentration in the medium, suggest that the mode of action of DMP-based NIs does not rely on their capacity as metal chelators.