inhibitors (BNIs). The chemical structure was analyzed which inhibited Nitrosomonas by blocking AMO and HAO enzymatic pathways. The BNIs release required the presence of NH 4 + in the root environment and the stimulatory effect of NH 4 + 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 50 0.2 μM) than methyl 3-(4-hydroxyphenyl) propionate (MHPP) (ED 50 100 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.

Summary• Nitrification results in poor nitrogen (N) recovery and negative environmental impacts in most agricultural systems. Some plant species release secondary metabolites from their roots that inhibit nitrification, a phenomenon known as biological nitrification inhibition (BNI). Here, we attempt to characterize BNI in sorghum (Sorghum bicolor).• In solution culture, the effect of N nutrition and plant age was studied on BNI activity from roots. A bioluminescence assay using recombinant Nitrosomonas europaea was employed to determine the inhibitory effect of root exudates. One major active constituent was isolated by activity-guided HPLC fractionations. The structure was analysed using NMR and mass spectrometry. Properties and the 70% inhibitory concentration (IC 70 ) of this compound were determined by in vitro assay.• Sorghum had significant BNI capacity, releasing 20 allylthiourea units (ATU) g. Release of BNI compounds increased with growth stage and concentration of supply.-grown plants released several-fold higher BNI compounds than -grown plants. The active constituent was identified as methyl 3-(4-hydroxyphenyl) propionate.• BNI compound release from roots is a physiologically active process, stimulated by the presence of . Methyl 3-(4-hydroxyphenyl) propionate is the first compound purified from the root exudates of any species; this is an important step towards better understanding BNI in sorghum.