Abstract
A bioluminescence assay using recombinant Nitrosomonas europaea was adopted to detect and quantify natural nitrification inhibitors in plant–soil systems. The recombinant strain of N. europaea produces a distinct two-peak luminescence due to the expression of luxAB genes, introduced from Vibrio harveyi, during nitrification. The bioluminescence produced in this assay is highly correlated with NO −2 production (r 2 = 0.94). Using the assay, we were able to detect significant amounts of a nitrification inhibitor produced by the roots of Brachiaria humidicola (Rendle) Schweick. We propose that the inhibitory activity produced/released from plants be termed ‘biological nitrification inhibition’ (BNI) to distinguish it from industrially produced inhibitors. The amount of BNI activity produced by roots was expressed in units defined in terms of the action of a standard inhibitor allylthiourea (AT). The inhibitory effect from 0.22 μM AT in an assay containing 18.9 mM of NH +4 is defined as one AT unit of activity. A substantial amount of BNI activity was released from the roots of B. humidicola (15–25 AT unit g−1 root dry wt day−1). The BNI activity released was a function of the growth stage and N content of the plant. Shoot N levels were positively correlated with the release of BNI activity from roots (r 2 = 0.76). The inhibitor/s released from B. humidicola roots suppressed soil nitrification. Additions of 20 units of BNI per gram of soil completely inhibited NO −3 formation in a 55-day study and remained functionally stable in the soil for 50 days. Both the ammonia monooxygenase and the hydroxylaminooxidoreductase enzymatic pathways in Nitrosomonas were effectively blocked by the BNI activity released from B. humidicola roots. The proposed bioluminescence assay can be used to characterize and determine the BNI activity of plant roots, thus it could become a powerful tool in genetically exploiting the BNI trait in crops and pastures.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Anon (1974) Technicon autoanalyzer II. Technicon Industrial Systems, Tarrytown
Bremner JM, McCarty GW (1993) Inhibition of nitrification in soil by allelochemicals derived from plants and plant residues. Soil Biochem 8:181–218
Fisher MJ, Rao IM, Ayarza MA, Lascano CE, Sanz JI, Thomas RJ, Vera RR (1994) Carbon storage by introduced deep-rooted grasses in the South American savannas. Nature 371:236–238
Giles J (2005) Nitrogen study fertilizes fears of pollution. Nature 433:791
Iizumi T, Nakamura K (1997) Cloning, nucleotide sequence, and regulatory analysis of the Nitrosomonas europaea dnaK gene. Appl Environ Microbiol 63:1777–1784
Iizumi T, Mizumoto M, Nakamura K (1998) A bioluminescence assay using Nitrosomonas europaea for rapid and sensitive detection of nitrification inhibitors. Appl Environ Microbiol 64:3656–3662
Ishikawa T, Subbarao GV, Ito O, Okada K (2003) Suppression of nitrification and nitrous oxide emission by the tropical grass Brachiaria humidicola. Plant Soil 255:413–419
Jarvis SC, Barraclough D (1991) Variation in mineral nitrogen content under grazed grassland swards. Plant Soil 138:177–188
Lata JC, Durand J, Lensi R, Abbadie L (1999) Stable coexistence of contrasted nitrification statuses in a wet tropical savanna system. Funct Ecol 13:762–763
Lata JC, Degrange V, Raynaud X, Maron PA, Lensi R, Abbadie L (2004) Grass populations control nitrification in savanna soils. Funct Ecol 13:762–763
Li Z, Peng DJ, Rae DJ, Zhou G (2001) Litter decomposition and nitrogen mineralization of soils in subtropical plantation forests of southern China, with special attention to comparisons between legumes and non-legumes. Plant Soil 229:105–116
Litchfield MD (1967) The automated analysis of nitrite and nitrate in blood. Analyst 92:132–136
Lodhi MAK (1982) Additional evidence of inhibition of nitrifiers and possible cycling of inhibitors produced by selected plants in a climax community. Bull Torrey Bot Club 109:199–204
McCarty GW (1999) Modes of action of nitrification inhibitors. Biol Fertil Soils 29:1–9
Meiklejohn J (1968) Number of nitrifying bacteria in some Rhodesian soils under natural grass and improved pastures. J Appl Ecol 5:291–300
Miranda CHB, Cadisch G, Urquiaga S, Miranda CHB, Boddey RM, Giller KE (1994) Mineral nitrogen in an oxisol from the Brazilian Cerrados in the presence of Brachiaria spp. Eur J Agron 3:333–337
Mosier AR, Duxbury JM, Freney JR, Heinemeyer O, Minami K (1996) Nitrous oxide emissions from agricultural fields: assessment, measurement, and mitigation. Plant Soil 181:95–108
Paavolainen L, Kitunen V, Smolander A (1998) Inhibition of nitrification in forest soil by monoterpenes. Plant Soil 205:147–154
Piccolo MC, Neill C, Cerri CC (1994) Net nitrogen mineralization and net nitrification along a tropical forest-to-pasture chronosequence. Plant Soil 162:61–70
Powell SJ, Prosser JI (1986) Effect of copper on inhibition by nitrapyrin of growth of Nitrosomonas europaea. Curr Microbiol 14:177–179
Prasad R, Power JF (1995) Nitrification inhibitors for agriculture, health, and the environment. Adv Agron 54:233–281
Raun WR, Johnson GV (1999) Improving nitrogen use efficiency for cereal production. Agron J 91:357–363
Rodgers GA (1986) Nitrification inhibitors in agriculture. J Environ Sci Health A 21:701–722
Stienstra AW, Klein Gunnewiek P, Laanbroek HJ (1994) Repression of nitrification in soils under a climax grassland vegetation. FEMS Microbiol Ecol 14:45–52
Subbarao GV, Ito O, Sahrawat KL, Berry WL, Nakahara K, Ishikawa T, Watanabe T, Suenaga K, Rondon M, Rao IM (2006) Scope and strategies for regulation of nitrification in agricultural systems—challenges and opportunities. Crit Rev Plant Sci 25:303–335
Sylvester-Bradley R, Mosquera D, Mendez JE (1988) Inhibition of nitrate accumulation in tropical grassland soils: effect of nitrogen fertilization and soil disturbance. J Soil Sci 39:407–416
Takagi S (1976) Naturally occurring iron-chelating compounds in oat-and-rice-root washing. 1. Activity, measurement and preliminary characterization. Soil Sci Plant Nutr 22:423–433
Varley JA (1966) Automatic method for the determination of nitrogen, phosphorus and potassium in plant material. Analyst 91:119–126
Vitousek PM, Gosz JR, Grier CC, Melillo JM, Reiners WA (1982) A comparative analysis of potential nitrification and nitrate mobility in forest ecosystems. Ecol Monogr 52:155–177
Ward BB, Courtney KJ, Langenheim JH (1997) Inhibition of Nitrosomonas europaea by monoterpenes from coastal redwood (Sequoia sempervirens) in whole-cell studies. J Chem Ecol 23:2583–2598
White CS (1988) Nitrification inhibition by monoterpenoids: theoretical mode of action based on molecular structures. Ecology 69:1631–1633
White CS (1991) The role of monoterpenes in soil nitrogen cycling in ponderosa pine. Biogeochemistry 12:43–68
Acknowledgments
We are grateful to Dr Taro Iizumi (Kurita Water Industries Limited) who has kindly provided us with the luminescent recombinant N. europaea strain for this research project work. Also, we acknowledge the participation and help from our colleagues at CIAT (Cali, Colombia) during this study and also provided the seed material used for various experiments described in this manuscript. We are also thankful to Dr K.L. Sahrawat (ICRISAT, India), who has gone through our manuscript critically and offered many suggestions to improve the clarity of our presentation and interpretation of the data.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Subbarao, G.V., Ishikawa, T., Ito, O. et al. A bioluminescence assay to detect nitrification inhibitors released from plant roots: a case study with Brachiaria humidicola . Plant Soil 288, 101–112 (2006). https://doi.org/10.1007/s11104-006-9094-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11104-006-9094-3