Nitrification inhibitor chlorate and nitrogen substrates differentially affect comammox <i>Nitrospira</i> in a grassland soil

<b>Introduction</b>: Through the combined use of two nitrification inhibitors, Dicyandiamide (DCD) and chlorate with nitrogen amendment, this study aimed to investigate the contribution of comammox <i>Nitrospira</i> clade B, ammonia oxidizing bacteria (AOB) and archaea (AOA) to nitrification in a high fertility grassland soil, in a 90-day incubation study.<b>Methods</b>: The soil was treated with nitrogen (N) at three levels: 0 mg-N kg^-1 soil, 50 mg-N kg^-1 soil, and 700 mg-N kg^-1 soil, with or without the two nitrification inhibitors. The abundance of comammox <i>Nitrospira</i>, AOA, AOB, and nitrite oxidising bacteria (NOB) was measured using qPCR. The comammox <i>Nitrospira</i> community structure was assessed using Illumina sequencing.<b>Results and Discussion</b>: The results showed that the application of chlorate inhibited the oxidation of both NH₄⁺ and NO₂^- in all three nitrogen treatments. The application of chlorate significantly reduced the abundance of comammox <i>Nitrospira</i> <i>amo</i>A and <i>nx</i>rB genes across the 90-day experimental period. Chlorate also had a significant effect on the beta diversity (Bray-Curtis dissimilarity) of the comammox <i>Nitrospira</i> clade B community. Whilst AOB grew in response to the N substrate additions and were inhibited by both inhibitors, AOA showed litle or no response to either the N substrate or inhibitor treatments. In contrast, comammox <i>Nitrospira</i> clade B were inhibited by the high ammonium concentrations released from the urine substrates. These results demonstrate the differential and niche responses of the three ammonia oxidising communities to N substrate additions and nitrification inhibitor treatments. Further research is needed to investigate the specificity of the two inhibitors on the different ammonia oxidising communities.