Metabolome fingerprinting reveals the presence of multiple nitrification inhibitors in biomass and root exudates of <i>Thinopyrum intermedium</i>

Biological Nitrification Inhibition (BNI) encompasses primarily NH₄ ⁺-induced release of secondary metabolites to impede the rhizospheric nitrifying microbes from performing nitrification. The intermediate wheatgrass <i>Thinopyrum intermedium</i> (Kernza®) is known for exuding several nitrification inhibition traits, but its BNI potential has not yet been identified. We hypothesized Kernza® to evince BNI potential through the presence and release of multiple BNI metabolites. The presence of BNI metabolites in the biomass of Kernza® and annual winter wheat (<i>Triticum aestivum</i>) and in the root exudates of hydroponically grown Kernza®, were fingerprinted using HPLC-DAD and GC-MS/MS analyses. Growth bioassays involving ammonia-oxidizing bacteria (AOB) and archaea (AOA) strains were conducted to assess the influence of the crude root metabolome of Kernza® and selected metabolites on nitrification. In most instances, significant concentrations of various metabolites with BNI potential were observed in the leaf and root biomass of Kernza® compared to annual winter wheat. Furthermore, NH₄ ⁺ nutrition triggered the exudation of various phenolic BNI metabolites. Crude root exudates of Kernza® inhibited multiple AOB strains and completely inhibited <i>N</i>. <i>viennensis</i>. Vanillic acid, caffeic acid, vanillin, and phenylalanine suppressed the growth of all AOB and AOA strains tested, and reduced soil nitrification, while syringic acid and 2,6-dihydroxybenzoic acid were ineffective. We demonstrated the considerable role of the Kernza® metabolome in suppressing nitrification through active exudation of multiple nitrification inhibitors.