Nitrification inhibition activity, a novel trait in root exudates of rice

AoB PLANTS http://aobplants.oxfordjournals.org/ Open access – Research article Nitrification inhibition activity, a novel trait in root exudates of rice Juan Pariasca Tanaka, Pierfrancesco Nardi and Matthias Wissuwa* Japan International Research Center for Agricultural Sciences (JIRCAS) Crop Production and Environment Division, 1-1 Ohwashi, Tsukuba, Ibaraki 305-8686, Japan Received: 20 May 2010; Returned for revision: 30 July 2010; Accepted: 13 September 2010; Published: 17 September 2010 Citation details: Pariasca Tanaka J, Nardi P, Wissuwa M. 2010. Nitrification inhibition activity, a novel trait in root exudates of rice. AoB PLANTS 2010: plq014, doi:10.1093/aobpla/plq014 Abstract Background and aims Nitrification is an important process in soil–plant systems for providing plant-available nitrate (NO32). However, NO32 is less stable in soils compared with ammonium (NH4+) and is more easily lost through leaching, runoff or denitrification. This study tested whether biological nitrification inhibition (BNI) activity is present in the root exudates of rice (Oryza sativa) and also the extent of variation between different genotypes. Methodology The BNI activity of root exudates was estimated by a bioluminescence assay using a recombinant Nitrosomonas europaea strain. Afterwards, the effect of a single application of concentrated root exudates and that of exudates deposited in the rhizosphere soil was tested on BNI using soil incubation. Soil was added with (NH4)2SO4 and water to reach 60 % of the waterholding capacity and incubated at 30 8C for different periods. Amounts of NH4+ and NO32 were determined using a continuous-flow auto-analyser. Principal results In an initial screening experiment, BNI activity in the exudates of 36 different rice genotypes was evaluated using a bioassay based on a recombinant Nitrosomonas strain. Significant genotypic variation was detected with the upland cultivar IAC25 demonstrating consistently high BNI activity, while modern lowland varieties like Nipponbare or IR64 exhibited lower activity. Subsequent experiments ruled out the possibility that BNI activity is simply due to non-specific (solute) leakage from roots. Soil incubation studies with concentrated root exudates of IAC25 showed significant reductions in NO32 formation. This effect was confirmed by detecting lower NO32 levels in incubation experiments using rhizosphere soil obtained from IAC25. Conclusions Our results provide first evidence that root exudates of rice can reduce nitrification rates in soil. Having shown this for a model crop, rice, offers possibilities for further exploitation of this phenomenon through molecular and genetic tools. Introduction The process of nitrification, in which ammonia (NH3) is converted to nitrate (NO32), is a key soil process that provides plant roots with the nitrogen (N) form that is preferentially taken up. However, when nitrification occurs rapidly, NO32 supply may exceed plant demand. Such excess soil NO32 is easily lost because NO32 is less tightly bound to the soil compared with NH4+. Losses of the highly mobile NO32 due to leaching, * Corresponding author’s e-mail address: AoB PLANTS Vol. 2010, plq014, doi:10.1093/aobpla/plq014, available online at www.aobplants.oxfordjournals.org & The Authors 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5/uk/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. AoB PLANTS Vol. 2010, plq014, doi:10.1093/aobpla/plq014 & The Authors 2010 1 Pariasca Tanaka et al. — Nitrification inhibition activity in root exudates of rice runoff or denitrification can amount to 60 % of applied fertilizer N in coarse-textured soils (Gaines and Gaines, 1994). Such a reduction in N use efficiency represents a large economic cost, estimated to be around US$ 15 billion annually (Subbarao et al., 2006). In addition, off-site movement of NO32 can also cause environmental problems such as groundwater contamination and eutrophication of surface water. Nitrogen lost by gaseous emission, as the powerful greenhouse gas N2O, contributes to the climate change phenomenon. In general, the regulation of nitrification to a level in which nitrification rates are in synchrony with NO3 uptake by plants may alleviate these problems and help achieve a more sustainable modern agriculture. Nitrification can be generalized as a two-step process wherein the initial oxidation of NH3 to NO22, considered as the rate-limiting step, is followed by the oxidation of NO22 to NO32. Historically, the former step was believed to be carried out solely by chemolithoautotrophic ammonia-oxidizing bacteria (AOB), represented by the genus Nitrosomonas spp., and the latter step by chemolithoautotrophic nitrite-oxidizing bacteria, represented by the genus Nitrobacter. However, recent evidence, reviewed by Hayatsu et al. (2008), suggests that the process is much more complex, involving autotrophic Archaea nitrifiers, heterotrophic nitrifiers and anammox bacteria, which convert ammonium and nitrite into N gas under anaerobic conditions (Mulder et al., 1995). Adding to this complexity is the potential for plants to stimulate or inhibit each of these groups specifically or as a whole. Numerous compounds that interfere with nitrification have been identified, but very few of these have been used successfully as synthetic nitrification inhibitors in agriculture and horticulture (Slangen and Kerkhoff, 1984; Zerulla et al., 2001). The additional cost and variability in the effectiveness of synthetic inhibitors because of site-specific factors, such as soil type and weather conditions (Goertz, 1994), have limited their wide adoption. Recent research suggests that plant root exudates can inhibit nitrification in soil, as shown for the tropical grass Brachiaria humidicola (Subbarao et al., 2009). This opens possibilities for using biological nitrification inhibition (BNI) as a low-cost in situ biological alternative. The BNI potential of root exudates has often been estimated by using a recombinant Nitrosomonas europaea strain (harbouring luxAB genes from Vibrio harveyi) in a bioluminescence assay (Iizumi et al., 1998; Ishikawa et al., 2003). This methodology has been used extensively in characterizations of BNI-related processes, including the identification of putative specific inhibitor compounds in roots of B. humidicola and sorghum (Sorghum bicolor) (Gopalakrishnan et al., 2007; Zakir et al., 2008; Subbarao et al., 2009). 2 Biological nitrification inhibition has first been described in detail in B. humidicola; however, this is a rather minor crop that is typically grown with little or no application of N fertilizers. It is therefore highly desirable to identify BNI in a crop that (i) is of major imp (...truncated)