A fluorometric assay for high-throughput phosphite quantitation in biological and environmental matrices

Phosphite, the anion of phosphorus acid, is increasingly recognized as an important metabolite in the global biogeochemical phosphorus cycle and as a fungicide and fertilizer in agriculture. As such, methods for detecting phosphite quantitatively and selectively are critical to evidencing phosphorus redox chemistry. Here, we present a fluorescence-based assay for phosphite, based on the NAD+-dependent oxidation of phosphite by phosphite dehydrogenase and the subsequent reduction of resazurin to resorufin. With the application of a thermostable phosphite dehydrogenase, a medium-invariant analytical approach, and novel sample preparation methods, the assay is capable of rapid and accurate phosphite quantification with a 3 mm limit of detection in a wide array of biologically- and environmentally-relevant matrices, including bacterial and archaeal cell lysate, seawater, anaerobic digester sludge, and plant tissue. We demonstrate the utility of the assay by quantitating phosphite uptake in a model crop plant in the presence or absence of a phosphite-oxidizing strain of Pseudomonas stutzeri as a soil additive, establishing this bacterium as an efficient phosphite converting biofertilizer.