Nitrification Inhibition (NI) compounds from Brachiaria humidicola , a tropical grass

1. Nitrification Inhibition (NI) compounds from Brachiaria humidicola, a tropical grass Gopalakrishnan, S.*, Subbarao, G.V., Nakahara, K. and Ito, O. Japan International Research Center for Agricultural Sciences (JIRCAS), 1-1 Ohwashi, Tsukuba (Ibaraki), Japan 305-8686 • Introduction: • Though nitrification is an essential biological process it also results in loss of 50 to 70% of the fertilizer-N and contribute to global warming by destruction of the ozone layer in stratosphere through N2O emissions and serious NO3 pollution of surface- and groundwater bodies. • If nitrification process is inhibited/slowed, then plants will have adequate time to uptake the fertilizer-N, thus will substantially improve N-recovery and uptake, NO3 pollution problems and also global warming will be reduced. • JIRCAS in collaboration with Center for Tropical Agriculture (CIAT), Columbia showed that root exudates of Brachiariahumidicola (BH) inhibited nitrification. • However, the compound(s) responsible for NI activity are yet to be understood. So the present research was aimed at isolating the NI compound(s) from root tissues of BH. Fig. 1: Mechanism of nitrification in soils and the associated nitrogen losses from NO3 leaching and N2O emissions • Methods: • Several chromatographic tools were used that includes: Solvent Partitioning, SPE, TLC, Open Column and HPLC. NMR and Mass Spectrometer tools were used for identification of the compound(s). • Each and every stages of the purification was monitored by an assay employing Nitrosomonas cells. Fig. 2: B. humidicola grown in the field and a whole plant cultivated in hydroponic culture Fig. 3: Nitrosomonas europea • Bioassay protocol: • The basic methodology was adopted from Iizumi, et al., 1998 (Applied and Environmental Microbiology 64(10): 3656-3662). • Recombinant Nitrosomonas europaea cells were employed in the assay that produces bioluminescence (due to the expression of luxAB genes) and the intensity of light emission was measured in a luminometer. • Results and discussion: • Reversed phase open column (C18) separated the NI activity, isolated from the root tissues. • More than 75% of the total recovered NI activity was found in three MeOH fractions namely 20%, 60% and 80% fractions. • Two NI compounds were purified from 60% MeOH fraction of the open column. • These two NI compounds were identified and confirmed for activity. Fig. 4: Physical map of recombinant Nitrosomonas, that was used in the assay (Source: Iizumi, et al., 1998) Fig. 5: Electron transfer pathways and luciferase reaction in N. europaea that was used in the assay (Source: Iizumi et al., 1998) • Conclusion: • Two of the NI compounds were identified from the root tissues of BH. • Reference: • Iizumi, T., Mizumoto, M. and Nakamura, K. (1998). A bioluminescence assay using Nitrosomonas europaea for rapid and sensitive detection of nitrification inhibitors. Applied and Environmental Microbiology, 64(10): 3656-3662. Fig. 7: Further fractionation of the 60% MeOH fraction Fig. 6: Fractionation of NI activity in the open column Compound no:1 Compound no:2 Fig.8: Purity check of the 1st compound in HPLC (C18 column) Fig. 9: Purity check of the 2nd compound in HPLC (C18 column) * Corresponding author: gopal@jircas.affrc.go.jp This poster was prepared for presentation at 30th International Symposium on High performance Liquid Phase Separations and Related Techniques (HPLC 2006), June 17-23, 2006, San Francisco, USA