Natural variation of the wheat root exudate metabolome and its influence on biological nitrification inhibition activity.

Excessive nitrogen use and low nitrogen use efficiency (NUE) in current agroecosystems are disrupting the global nitrogen cycle. Chemical inhibitors offer only temporary relief, while plant-derived biological nitrification inhibitors (BNIs) remain safer but underexplored. Identifying biological nitrification inhibition (BNI) traits in nitrogen-demanding crops like wheat is key to improving sustainability. In this study, a combined GC- and LC-MS platform was used to determine the metabolome of the root exudates of 44 diverse wheat genotypes originating from India and Austria. With more than 6000 metabolic features, a pronounced genotype-specific variation, a clear geographic pattern and an unexpected complexity of the root exudate metabolome were observed. A novel high-throughput assay utilizing diverse ammonia-oxidizing bacteria (AOB) and archaea (AOA) was developed for rapid BNI testing, highlighting distinct inhibition and even growth stimulation capacities between genotypes. Network analysis and advanced machine and deep learning analysis identified combinations of 32 metabolites linked to high BNI activity, including phenylpropanoids sinapinic acid, syringic acid and others, as well as glycosylated flavones isoschaftoside and others. This indicates that the concurrent presence of specific metabolites, rather than a single compound, drives nitrification inhibition in the rhizosphere. Variation in BNI activity among wheat genotypes, classified as either spring or winter types, suggests that root architecture modulates the dynamics of root exudation and the potential for nitrification inhibition. The unique combination of high-throughput metabolomics analysis and the BNI fast-track assay allows for screening of large germplasm collections as an essential requirement to introduce BNI and related NUE traits into modern breeding programmes.