On a quiet weekend morning in a greenhouse in AU Flakkebjerg, rows of wheat plants stand growing with their roots submerged in clear water. There is no soil, no buffer, no automation to take over the work. Every day, weekends and holidays included, Postdoc Purna Kumar Khatri comes by to check them. He adjusts the pH drop by drop. If he does not, the roots will suffer. And the experiment will fail.
“It’s physically demanding,” he says, “but also mentally. You have to be precise. Every single day.”
This meticulous routine is part of a much larger story: one that begins beneath the soil surface, where plants quietly negotiate with microbes over one of agriculture’s most precious resources: nitrogen.
Modern agriculture runs on nitrogen. Yet less than half of the nitrogen applied as fertiliser is actually used by crops. The rest is lost through leaching or gaseous emissions into the atmosphere as nitrous oxide.
For decades, researchers and policymakers have tried to manage this problem with regulations and fertiliser limits. Another way to limit the problem is synthetic nitrification inhibitors. These chemicals slow down the microbial conversion of ammonium to nitrate, which can reduce nitrous oxide emissions by limiting nitrification and subsequent denitrification, but they are costly, must be reapplied, and may affect non-target soil organisms.
This is the core idea behind biological nitrification inhibition (BNI): a process where plant roots release natural compounds that suppress the activity of nitrifying microbes in the soil. The result: more nitrogen remains in a form plants can use, less is lost to the environment, and fertiliser efficiency improves.
“Plants are not passive,” Purna Kumar Khatri explains. “They have strategies. They defend themselves. And they try to secure nutrients in the soil. We are just beginning to understand how sophisticated those strategies are.”
At the centre of Purna Kumar Khatri´s research are benzoxazinoids: a group of naturally occurring compounds well known in cereals like wheat, maize, and rye. For decades, these chemicals have been studied mainly for their role in plant defence: deterring insects, suppressing weeds, and inhibiting nematodes.
What Purna Kumar Khatri and his colleagues have now shown is that several of these compounds also act as powerful nitrification inhibitors.
In a newly published study, they screened 18 different benzoxazinoids using a sensitive bioluminescence assay with Nitrosomonas europaea, a model nitrifying bacterium. Seven compounds stood out, including BOA, MBOA, DIBOA and DIMBOA, all capable of strongly suppressing nitrification at relatively low concentrations.
Crucially, these compounds are not synthetic additives. They are produced by the plant itself and released from the roots into the surrounding soil.
“It was good news,” Purna Kumar Khatri says. “Not because the compounds were completely unknown, but because now we understand them better. They were already known as defence chemicals. Now we see that they may also be key players in nitrogen management.”
The study compared three wheat lines: a conventional parent line and two so-called BNI wheat lines, which carry a chromosome fragment from a wild grass (Leymus racemosus) known to enhance BNI traits.
When grown hydroponically, the BNI wheat lines released significantly higher amounts of active benzoxazinoids than the parent line. Their root exudates inhibited nitrification up to two-fold more strongly, correlating directly with the higher concentrations of these compounds.
This matters because the implications go far beyond the lab.
“If you can increase nitrogen-use efficiency by even ten percent in real field conditions,” Purna Kumar Khatri says, “the absolute savings in fertiliser and emissions are enormous.”
Some modelling studies suggest that BNI-enabled crops could reduce nitrogen losses by 20–30 percent. And importantly, early field experiments show no yield penalty: farmers can apply less fertiliser and harvest the same amount of grain.
One of the strengths of plant-mediated nitrification inhibition is its timing and precision. Synthetic inhibitors are applied in large doses, all at once. Plant-produced compounds, by contrast, are released gradually and locally exactly where and when they are needed.
“The plant produces very small amounts,” Purna Kumar Khatri explains. “But it does so continuously. And these chemicals have been part of natural ecosystems forever. They are not something new we introduce from outside.”
This also reduces the risk of unintended side effects. While synthetic inhibitors may disrupt soil microbial communities, naturally exuded compounds tend to have more targeted effects. In the large international CropSustain project, parallel research groups are now studying how BNI traits influence soil microbiomes and non-target organisms over time.
For Purna Kumar Khatri, the long-term goal is clear: turn chemical insight into breeding knowledge.
If researchers can identify which biosynthetic pathways produce the most effective nitrification-inhibiting compounds, breeders can select for wheat varieties that express these traits more strongly. The result would be crops that are inherently more climate-friendly without changing farmers’ practices or requiring new inputs.
“This is where chemistry meets genetics,” he says. “Once we know the compounds, breeders can work on enhancing the plant’s capacity to produce them.”
In the meantime, his days remain anchored in the greenhouse. Bottles of collected root exudate fill freezers. Samples wait to be analysed. The work is slow, repetitive, and often invisible.
But beneath the surface, something remarkable is taking shape.
“If you break a cycle naturally,” Purna Kumar Khatri says, “not with chemicals but with biology, then everyone benefits: the plant, the farmer, and the environment.”
Collaborators: Department of Agroecology at Aarhus University, Department of Plant and Environmental Sciences at Copenhagen University and School of Biological Sciences at University of Aberdeen.
Funding: This work is supported by the Novo Nordisk Foundation (NNF) under the Biological Nitrification Inhibition (BioNI) project (Grant No. NNF22SA0078638), and in collaboration with CIMMYT and supported by NNF under the CropSustaiN/AU-AGRO project (Sub-grant Agreement No. GWP-2024-115 with Aarhus University). CGR was supported by a Royal Society University Research Fellowship (URF150571). CIMMYT provided the seeds for Roelf parent and Roelf BNI wheat lines used in this study.
Conflict of interest: None
Read more: The publication “ Benzoxazinoids as candidate compounds for biological nitrification inhibition in wheat ” is published in Plant Physiology and Biochemistry. It is written by: Purna Kumar Khatri, Jasmeet Kaur-Bhambra, Kenneth Madriz-Ordenana, Bente Birgitte Laursen, Hans Thordal-Christensen, Xiaoping Fan, Jonathan Sølve, Cecile Gubry-Rangin, Jawameer Hama, Kristian Koefoed Brandt, and Inge S. Fomsgaard.
Contact: Postdoc Purna Kumar Khatri , Department of Agroecology, Aarhus University. Mail: purna.khatri@agro.au.dk
Communications Adviser Camilla Brodam Galacho , Department of Agroecology, Aarhus University. Tel.: +45 9352 2136 or mail: brodam@agro.au.dk
Plant Physiology and Biochemistry
Benzoxazinoids as candidate compounds for biological nitrification inhibition in wheat
1-May-2026
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.