A bioluminescence assay to detect nitrification inhibitors released from plant roots: a case study with Brachiaria humidicola

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 lM 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.

Reducing nitrous oxide emissions by changing N fertiliser use from calcium ammonium nitrate (CAN) to urea based formulations

“…Natural suppression of soil nitrification by plants has also been observed in some ecosystems, and is referred to as biological nitrification inhibition (Subbarao et al, 2006). This natural inhibition is most likely an evolutionary adaptation to ensure conservation and efficient use of N in natural systems 7 which have low-N availability (Lata et al, 2004).…”

Soil nitrogen dynamics after Brachiaria desiccation

“…Therefore, B. humidicola has been used as a model to characterize the suppressive ability of plants, which must have sufficient inhibitory activity to suppress soil nitrification and the emission of N 2 O (Subbarao et al, 2009). However, the presence or absence and intensity of this suppressive effect in soil depends on several factors, such as the soil moisture, temperature and type (Ipinmoroti et al, 2008), the N status of the plant (Subbarao et al, 2006a), the form (i.e., NH 4 + or NO 3 -) of N when applied and/or made available (Subbarao et al, 2007b,c), and especially the presence of NH 4 + in the rhizosphere, which is essential for the synthesis and release of biological nitrificationinhibiting compounds (BNIs) from the roots (Subbarao et al, 2007b,c;Subbarao et al, 2009). …”

Effect of soil-applied calcium carbide and plant derivatives on nitrification inhibition and plant growth promotion

“…Agricultural ecosystems depend heavily on large inputs of N fertilizer to sustain productivity, as naturally fixed N is seldom adequate for highproduction systems (Dinnes et al 2002;Subbarao et al 2006a;Abbasi and Khizar 2012). However, N is a highly dynamic and mobile element and significant N losses occur as a result of NO 3 -leaching, denitrification, runoff, NH 3 volatilization and gaseous emissions of N 2 O, and NO to the atmosphere (Zaman et al 2009).…”

“…is usually oxidized quite rapidly to NO 3 -, a highly mobile form of N, providing a much greater potential for N to be lostbeyond the rooting zone (leaching) and into the atmosphere as gaseous molecules (N 2 O, NO, and N 2 ) through denitrification (Subbarao et al 2006a). It is estimated that nearly 70 % of the applied N from managed ecosystems is lost through nitrification and subsequent processes (Raun and Johnson 1999;Glass 2003).…”

“…It is estimated that nearly 70 % of the applied N from managed ecosystems is lost through nitrification and subsequent processes (Raun and Johnson 1999;Glass 2003). These losses result in serious environmental consequences and inefficient use of both soil and applied N (Raun and Johnson 1999;Subbarao et al 2006a).…”