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

Abstract

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, nitrification inhibitors (NIs) have been developed to suppress soil-nitrifier activity and decrease N losses. The NIs 3,4-dimethylpyrazole 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 effective to inhibit soil nitrification, their exact mode of action has not been confirmed. 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 nitrification in pure cultures of N. europaea. In the soil, we evidenced that DMPSA needs to be broken into DMP to achieve the inhibition of nitrification, which is mediated by a soil biological process that remains to be identified. Moreover, both DMPP and DMPSA are thought to inhibit nitrification due to their ability to chelate the Cu²⁺ cations that the ammonia monooxygenase enzyme (AMO) needs to carry on the first step of NH₄⁺ oxidation. However, the efficiency 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 nitrification in pure cultures together with the high efficiency of DMPP to inhibit nitrification 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.