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Aims: The ability to suppress soil nitrification through the release of nitrification inhibitors from plant roots is termed 'biological nitrification inhibition' (BNI). Here, we aimed at the quantification and characterization of the BNI function in sorghum that includes inhibitor production, their chemical identity, functionality and factors regulating their release. Methods: Sorghum was grown in solution culture and root exudate was collected using aerated NHCl solutions. A bioluminescence assay using recombinant Nitrosomonas europaea was employed to determine the BNI activity. Activity-guided chromatographic fractionation was used to isolate biological nitrification inhibitors (BNIs). The chemical structure was analyzed using NMR and mass spectrometry; pH-stat systems were deployed to analyze the role of rhizosphere pH on BNIs release. Results: Sorghum roots released two categories of BNIs: hydrophilic- and hydrophobic-BNIs. The release rates for hydrophilic- and hydrophobic- BNIs ranged from 10 to 25 ATU g root dwt. d. Addition of hydrophilic BNIs (10 ATU g soil) significantly inhibited soil nitrification (40 % inhibition) during a 30-d incubation test. Two BNI compounds isolated are: sakuranetin (ED 0.6 μM; isolated from hydrophilic-BNIs fraction) and sorgoleone (ED 13.0 μM; isolated from hydrophobic-BNIs fraction), which inhibited Nitrosomonas by blocking AMO and HAO enzymatic pathways. The BNIs release required the presence of NH in the root environment and the stimulatory effect of NH lasted 24 h. Unlike the hydrophobic-BNIs, the release of hydrophilic-BNIs declined at a rhizosphere pH >5.0; nearly 80 % of hydrophilic-BNI release was suppressed at pH ≥7.0. The released hydrophilic-BNIs were functionally stable within a pH range of 5.0 to 9.0. Sakuranetin showed a stronger inhibitory activity (ED 0.2 μM) than methyl 3-(4-hydroxyphenyl) propionate (MHPP) (ED 100 μM) (isolated from hydrophilic-BNIs fraction) in the in vitro culture-bioassay, but the activity was non-functional and ineffective in the soil-assay. Conclusions: There is an urgent need to identify sorghum genetic stocks with high potential to release functional-BNIs for suppressing nitrification and to improve nitrogen use efficiency in sorghum-based production systems. [ABSTRACT FROM AUTHOR]