Wheat releases compounds capable of inhibiting nitrification

Researchers at Aarhus University have identified compounds released by wheat roots with the potential to inhibit nitrification. The study points to benzoxazinoids as candidates for biological nitrification inhibitors, a process known by the acronym BNI. These compounds reduced the activity of nitrifying microorganisms in assays with Nitrosomonas europaea and appeared in higher concentrations in wheat lines with enhanced BNI characteristics.

Nitrification converts ammonium into nitrate through microbial action. This process favors nitrogen losses through leaching and nitrous oxide emissions. Biological inhibition of nitrification aims to retain more nitrogen in the soil in forms usable by plants. This strategy depends on the release of natural compounds by the roots.

The study evaluated 18 benzoxazinoids using a bioluminescence assay with Nitrosomonas europaea. Seven compounds showed strong inhibitory activity: MBOA, BOA, DIBOA, DIMBOA, APO, AAPO, and HPMA. The concentration values ​​required to inhibit fifty percent of nitrification ranged from eight to 47 micromolar.

Chemical forms

The results differentiate chemical forms with greater and lesser effect. MBOA and BOA, two benzoxazolinones aglycones, showed values ​​of eight and 13 micromolar, respectively. DIBOA and DIMBOA, two hydroxamic acids aglycones, showed values ​​of 11 and 20 micromolar, respectively. Glycosylated forms had low activity or no relevant inhibitory effect.

The team also compared three genotypes of Triticum aestivum: the parental lineage is Roelfs F2007, and there are two Roelfs BNI lineages. These two lineages carry a chromosomal fragment of Leymus racemosus, a wild grass associated with increased BNI trait. The plants were grown in a hydroponic system for three weeks.

The Roelfs BNI1 and Roelfs BNI2 lines released more active benzoxazinoids than the parental line. Their root exudates showed up to twice the inhibition compared to Roelfs F2007. This result was consistent with the higher concentration of these compounds in the exudates.

In exudates processed by rotary evaporation and collected in a medium with 0,5 millimolar ammonium sulfate, Roelfs BNI1 achieved 89,2 percent inhibition against ammonia-oxidizing bacteria. Roelfs BNI2 achieved 72,7 percent. The parental strain reached 36 percent. In an assay with ammonia-oxidizing archaea Candidatus Nitrosocosmicus franklandianus, Roelfs BNI1 had a performance of 29,5 percent, Roelfs BNI2 had a performance of 20 percent, and Roelfs F2007 had a performance of 3,7 percent.

Chemical analysis

Chemical analysis quantified nine benzoxazinoids in root exudates: BOA, DIBOA, DIMBOA, DIMBOA-Glc, HBOA, HBOA-Glc, HMBOA, HMBOA-Glc, and MBOA. DIMBOA appeared as the most abundant compound in exudates collected in ammonium sulfate, with values ​​between 89 and 252 micrograms per gram of root dry mass per day. HMBOA ranged from six to 14 micrograms per gram of root dry mass per day. MBOA ranged from five to seven micrograms per gram of root dry mass per day.

The Roelfs BNI1 and Roelfs BNI2 lines exuded DIMBOA in twice the amount of the parental line in the medium with ammonium sulfate. In the root tissue, the researchers quantified 15 benzoxazinoids. DIMBOA and DIMBOA-Glc were among the most abundant compounds.

Collection and preparation

The study also shows the importance of the method of collecting and preparing exudates. Acidification and the presence of salts interfered with the bioassay results. Without pH adjustment, the inhibition patterns remained inconsistent. After adjusting to pH 6,5, the difference between the BNI strains and the parental strain exceeded three times.

Researchers highlight an agronomic difference between biological inhibition and synthetic inhibitors. Synthetic inhibitors enter the system in concentrated doses. Compounds produced by the plant reach the rhizosphere gradually and locally. This dynamic can reduce non-target effects on microbial communities, although the CropSustain project is still evaluating impacts on soil microbiomes and non-target organisms.

This study positions benzoxazinoids as candidates for breeding programs focused on nitrogen use efficiency in wheat. Identifying the biosynthetic pathways involved may support the selection of cultivars with a greater capacity to exude nitrification-inhibiting compounds.

Further information at DOI:10.1016/j.plaphy.2026.111303