Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

Development of High-Throughput Methods to Assist Measure of Biological Nitrification Inhibition in Populus Soils


John F. Cahill* (, Dana L. Carper


Oak Ridge National Laboratory


This project seeks to discover and validate gene functions and pathways in Populus trichocarpa that lead to generation of biological nitrification inhibitors and to understand their role affecting nitrogen use efficiency.


Plants have evolved to mediate nitrification processes by exuding biological nitrification inhibitors (BNIs) in soil to alter the functions of the plant-associated microbiome. BNIs have emerged as one route to mitigate loss of nitrogen (N) and improve nitrogen use efficiency (NUE). Little is known of the genes, gene families, and associated pathways related to BNI production and function. Thus, experimental data that comprehensively link host traits, genetics, and exudation of BNI compounds are needed. Such data are critical to creating systems network models that can deconvolute complex host-microbiome interactions, system feedbacks on the microbiome, and impact on biogeochemical cycles. This project seeks to discover and validate gene functions and pathways in P. trichocarpa that lead to generation of BNIs and understand their role affecting NUE. To begin, methods and protocols are needed to measure nitrification traits in soil obtained from a P. trichocarpa genome-wide association study (GWAS) population. To accurately characterize such processes, measure of gross nitrification rate (GNR) using 15N-isotope dilution techniques is needed. Unfortunately, current assays can be costly and difficult to implement and are relatively low throughput. Linking plant genomic function to nitrification inhibition activity requires measure of GNR phenotypes across a diverse population comprising hundreds and thousands of samples, hence the need for alternate methods. Here, chemical derivatization protocols are adapted to enable isotope dilution measurements with high throughput and sensitivity.

Methods were developed to capitalize on the high-throughput capabilities of an immediate drop-on-demand (I.DOT) system coupled with open port sampling interface–mass spectrometry (OPSI-MS). Derivatization products were analyzed by dispensing 10 nanoliters of diluted (water) samples into a flow of solvent which is subsequently characterized by mass spectrometry. Reactions were optimized for reaction time, derivative concentration, and other variables. Soil samples were kept refrigerated until supplemented with 15N-rich ammonium sulfate or potassium nitrate, incubated for 24 hours, and extracted with 1 molar potassium chloride. Measure of nitrate was achieved by modifying derivatization protocols of 2,3-diaminonaphthalene. Using the I.DOT/OPSI-MS system, the reaction could be monitored over time using various 2,3-diaminonaphthalene concentrations. Without a concentration step, a limit of detection (LOD) of low micrometer was achieved for 14N-2,3-naphthotriazole (NAT) and even lower for 15N-NAT. O-phthaldialdehyde (OPA), dansyl chloride (DAN), and 9-fluorenylmethoxycarbonyl chloride (FMOC-Cl) derivatization reactions were explored for detection of ammonia. Of these, OPA resulted in the highest sensitivity with the simplest derivatization procedure. An LOD of low µM was achieved using OPA. Both DAN and OPA methods had <10% coefficient of variation. DAN and OPA methods were used to demonstrate measures of GNR and gross mineralization rate in soil samples collected near to a P. trichocarpa with varying concentrations of N (milligram 15N per kilogram of soil). Notably the DAN and OPA methods were able to quantitively relate 15N/14N isotope ratios using high-resolution mass spectrometry or selected multiple reaction monitoring with 3 s/sample throughput and no sample cleanup. Together, these methods represent a simple, flexible, and fast approach for measure of nitrate and ammonia in soil extracts.

Funding Information

This work is supported by the U.S. DOE, Office of Science, BER program, through an Early Career Award.