Metabolome fingerprinting reveals the presence of multiple nitrification inhibitors in biomass and root exudates of Thinopyrum intermedium.

Biological Nitrification Inhibition (BNI) encompasses primarily NH₄ ⁺-induced release of secondary metabolites to impede the rhizospheric nitrifying microbes from performing nitrification. The intermediate wheatgrass Thinopyrum intermedium (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 (Triticum aestivum) 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 N. viennensis. 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.