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Synthetic nitrification inhibitors (SNI) and biological nitrification inhibitors (BNI) are promising tools to limit nitrogen (N) pollution derived from agriculture. Modern wheat cultivars lack sufficient capacity to exude BNIs, but, fortunately, the chromosome region (Lr#n-SA) controlling BNI production in Leymus racemosus, a wild relative of wheat, was introduced into two elite wheat cultivars, ROELFS and MUNAL. Using BNI-isogenic-lines could become a cost-effective, farmer-friendly, and globally scalable technology that incentivizes more sustainable and environmentally friendly agronomic practices. We studied how BNI-trait improves N-uptake, and N-use, both with ammonium and nitrate fertilization, analysing representative indicators of soil nitrification inhibition, and plant metabolism. Synthesizing BNI molecules did not mean a metabolic cost since Control and BNI-isogenic-lines from ROELFS and MUNAL presented similar agronomic performance and plant development. In the soil, ROELFS-BNI and MUNAL-BNI plants decreased ammonia-oxidizing bacteria (AOB) abundance by 60% and 45% respectively, delaying ammonium oxidation without reducing the total abundance of bacteria or archaea. Interestingly, BNI-trait presented a synergistic effect with SNIs since made it also possible to decrease the AOA abundance. ROELFS-BNI and MUNAL-BNI plants showed a reduced leaf nitrate reductase (NR) activity as a consequence of lower soil NO3- formation and a higher amino acid content compared to BNI-trait lacking lines, indicating that the transfer of Lr#-SA was able to induce a higher capacity to assimilate ammonium. Moreover, the impact of the BNI-trait in wheat cultivars was also noticeable for nitrate fertilization, with improved N absorption, and therefore, reducing soil nitrate content.

Active nitrifiers and rapid nitrification are major contributing factors to nitrogen losses in global wheat production. Suppressing nitrifier activity is an effective strategy to limit N losses from agriculture. Production and release of nitrification inhibitors from plant roots is termed "biological nitrification inhibition" (BNI). Here, we report the discovery of a chromosome region that controls BNI production in "wheat grass" Leymus racemosus (Lam.) Tzvelev, located on the short arm of the "Lr#3Ns(b)" (Lr#n), which can be transferred to wheat as T3BL.3Ns(b)S (denoted Lr#n-SA), where 3BS arm of chromosome 3B of wheat was replaced by 3Ns(b)S of L. racemosus. We successfully introduced T3BL.3Ns(b)S into the wheat cultivar "Chinese Spring" (CS-Lr#n-SA, referred to as "BNI-CS"), which resulted in the doubling of its BNI capacity. T3BL.3Ns(b)S from BNI-CS was then transferred to several elite high-yielding hexaploid wheat cultivars, leading to near doubling of BNI production in "BNI-MUNAL" and "BNI-ROELFS." Laboratory incubation studies with root-zone soil from field-grown BNI-MUNAL confirmed BNI trait expression, evident from suppression of soil nitrifier activity, reduced nitrification potential, and N2O emissions. Changes in N metabolism included reductions in both leaf nitrate, nitrate reductase activity, and enhanced glutamine synthetase activity, indicating a shift toward ammonium nutrition. Nitrogen uptake from soil organic matter mineralization improved under low N conditions. Biomass production, grain yields, and N uptake were significantly higher in BNI-MUNAL across N treatments. Grain protein levels and breadmaking attributes were not negatively impacted. Wide use of BNI functions in wheat breeding may combat nitrification in high N input-intensive farming but also can improve adaptation to low N input marginal areas.