Developing methods to evaluate phenotypic variability in biological nitrification inhibition ( BNI ) capacity of Brachiaria grasses

As part of the nitrogen (N) cycle in the soil, nitrification is an oxidation process mediated by microorganisms that transform the relatively immobile ammonium (NH4 + ) to the water soluble nitrate (NO3 ), producing nitrous oxide (N2O, a potent greenhouse gas) as a by-product (Canfield et al. 2010). Researchers at CIAT-Colombia, in collaboration with JIRCAS-Japan, reported that the tropical forage grass, Brachiaria humidicola, has the ability to inhibit the nitrification process by exuding chemical compounds from its roots to the soil. A major hydrophobic compound was discovered and named brachialactone (Subbarao et al. 2009). This capacity of Brachiaria grasses is known as biological nitrification inhibition (BNI) and could contribute to better N use efficiency in crop-livestock systems by improving recovery of applied N, while reducing NO3 leaching and N2O emissions. The current methodologies for quantifying the BNI trait need enhancement to accelerate the process of identifying differences between genotypes. In this paper, we aim to develop new (or improve the existing) phenotyping methods for this trait. Preliminary results were obtained using 3 different methods to quantify BNI: (1) a mass spectrometry method to quantify brachialactone; (2) a static chamber method to quantify N2O emissions from soils under greenhouse conditions; and (3) an improved molecular method to quantify microbial populations by real-time PCR (polymerase chain reaction). Using these 3 methods we expect to apply scores to a bi-parental hybrid population (n=134) of


Introduction
As part of the nitrogen (N) cycle in the soil, nitrification is an oxidation process mediated by microorganisms that transform the relatively immobile ammonium (NH 4 + ) to the water soluble nitrate (NO 3 -), producing nitrous oxide (N 2 O, a potent greenhouse gas) as a by-product (Canfield et al. 2010).Researchers at CIAT-Colombia, in collaboration with JIRCAS-Japan, reported that the tropical forage grass, Brachiaria humidicola, has the ability to inhibit the nitrification process by exuding chemical compounds from its roots to the soil.A major hydrophobic compound was discovered and named brachialactone (Subbarao et al. 2009).This capacity of Brachiaria grasses is known as biological nitrification inhibition (BNI) and could contribute to better N use efficiency in crop-livestock systems by improving recovery of applied N, while reducing NO 3 -leaching and N 2 O emissions.The current methodologies for quantifying the BNI trait need enhancement to accelerate the process of identifying differences between genotypes.
In this paper, we aim to develop new (or improve the existing) phenotyping methods for this trait.Preliminary results were obtained using 3 different methods to quantify BNI: (1) a mass spectrometry method to quantify brachialactone; (2) a static chamber method to quantify N 2 O emissions from soils under greenhouse conditions; and (3) an improved molecular method to quantify microbial populations by real-time PCR (polymerase chain reaction).Using these 3 methods we expect to apply scores to a bi-parental hybrid population (n=134) of ___________ Correspondence: Jacobo Arango, Centro Internacional de Agricultura Tropical (CIAT), A.A. 6713, Cali, Colombia.Email: j.arango@cgiar.org 2 B. humidicola accessions differing in their BNI capacity, CIAT 26146 (medium to low BNI) x CIAT 16888 (high BNI), in an attempt to identify QTLs (quantitative trait loci) associated with the BNI trait.

HPLC and GC-MS
For 24 hours, root exudates were collected from intact Brachiaria plants grown in a hydroponic system for 60 days after transplanting, using 0.5 L of aerated solutions of either NH 4 Cl (1 mM) or distilled H 2 O. BNI compounds were extracted by solvent partitioning using CH 2 Cl 2 .The organic fraction was collected and dried, while the residue was dissolved in CH 3 OH and separated by HPLC (Agilent 1200 with DAD detector) using a Zorbax Eclipse XDB-C18 column (4.6 x 150 mm, 5 µ).Detection was performed at 230, 240 and 280 nm.The HPLC fraction from the sample collected in NH 4 Cl at 35 min of retention time, was collected and mass spectra (MS) were recorded on a full scan mode using a GC (AT 6890 Series Plus), coupled to a MS (AT MSD5975 Inert XL).

Adaptation of a static chamber method for greenhouse gas (GHG) quantification
A method reported by Subbarao et al. (2009) for N 2 O emissions was adapted, by completely covering the pots, where individual Brachiaria accessions were growing (Figure 2A), allowing the collection of N 2 O gas manually with a syringe.For validation, 4 Brachiaria genotypes were evaluated for 5 weeks under greenhouse conditions with weekly measurements.In each measurement 4 gas samples were collected at 15 min intervals.

Improved molecular method to quantify microbial populations by real-time PCR
With the intention to diminish the error introduced by the differential soil DNA extraction efficiencies on individual samples for copy number quantification of amoA genes of ammonia-oxidizing bacteria and archaea through real-time PCR (Subbarao et al. 2009), a normalization method of soil DNA extraction reported by Park and Crowley (2005) was applied.Briefly, soil samples were spiked with known amounts of bacterial plasmid (pGEM-T easy ® promega) as an internal standard, and DNA extraction was performed using the FastDNA SPIN for soil kit (MP Biomedicals).

Results
The results for the identification of brachialactone by HPLC and GC-MS are shown in Figure 1. Figure 2 illustrates the setup of the experimental procedure and presents the N 2 O emissions, while Figure 3 shows the DNA quantification.

Discussion and Conclusions
The 3 phenotyping methods have shown distinct promise as a means of quantifying the BNI capacity of different species.
Positive identification of brachialactone (Figure 1) will allow rapid and precise estimation of the major BNI compound in Brachiaria grasses.Emissions of N 2 O from Brachiaria genotypes under confined conditions (Figure 2) measured with the covered pot method, have been successfully validated using data reported in field experiments by Subbarao et al. (2009).This will streamline the examination of more plant accessions to determine how they influence N 2 O emissions.Finally, the efficient normalization method (Figure 3) for quantification of DNA extracted from soil will overcome the problems associated with contaminants like humus, allowing more precise quantification of amoA genes in nitrifying microorganisms.

Figure 1 .
Figure 1.Identification of brachialactone from root exudates of Brachiaria humidicola by chromatography (HPLC) and mass spectrometry (GC-MS).A) Chromatogram of root exudates collected in aerated solutions of either NH 4 Cl (1 mM) or distilled H 2 O; putative brachialactone peak induced by NH 4 Cl is indicated.B) Positive mass spectrum identification of brachialactone and its chemical structure.

Figure 3 .
Figure 3. Normalization method for DNA extracted from soil to control different extraction efficiencies: A) pGEM-T easy map showing with the blue arrow the sequence used for normalization purposes; B) Quantification by real-time PCR of different amounts (100, 50 and 10 ng) of pGEM-T easy plasmid used as the internal standard in soil DNA extractions and the melting curve of the amplicons showing a specific amplification of a unique DNA sequence.