Benzoxazinoids as candidate compounds for biological nitrification inhibition in wheat

Biological nitrification inhibition (BNI) is a sustainable plant-mediated approach to reduce nitrogen loss from soil through root exudation of nitrification inhibitors. Although BNI has been established in wheat, the key bioactive BNI compounds remain largely unexplored. We hypothesise that benzoxazinoids (BXs), specialised metabolites abundant in cereals and exuded by wheat roots, act as BNI compounds. We screened 18 BXs for their BNI efficiency using an ammonia-oxidising bacterium-based Nitrosomonas europaea bioluminescence assay. We also quantified the BNI-active BXs in the root exudates of two BNI-trait-translocated wheat lines and their parent line, Roelfs F2007, grown hydroponically for three weeks. Based on the bioluminescence assay, we identified seven BXs showing strong inhibitory activity: two aglucone benzoxazolinones (MBOA, BOA), two aglucone hydroxamic acids (DIBOA, DIMBOA), and three microbial transformation products (APO, AAPO, HPMA), with IC₅₀ values ranging from 8 to 47 μM. Root exudates of the BNI lines exhibited two-fold greater inhibition compared to their parent line, which correlated with higher concentrations of BXs in the root exudates. Overall, this study identifies BXs as strong candidate contributors to wheat BNI, suggesting that enhanced exudation of BXs can contribute to stronger inhibition of ammonia-oxidising bacteria and support more efficient and environmentally sustainable nitrogen use.