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 Brachiaria humidicola (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.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

Fig. 1: Mechanism of nitrification in soils and the associated nitrogen losses from NO3 leaching and N2O emissions

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.

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

Compound no:1 Compound no:2

Fig. 6: Fractionation of NI activity in the open column

Fig. 7: Further fractionation of the 60% MeOH fraction

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

Fig. 4: Physical map of recombinant Nitrosomonas, that was used in the assay(Source: Iizumi, et al., 1998)

Fig. 5: Electron transfer pathways andluciferase 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.