A simple assay for quantification of plant-associative bacterial nitrogen fixation

Accurate quantification of plant-associative bacterial nitrogen (N) fixation is crucial for selection and development of elite diazotrophic inoculants that could be used to supply cereal crops with nitrogen in a sustainable manner. Because a low oxygen environment that may not be conducive to plant growth is essential for optimal stability and function of the N-fixing catalyst nitrogenase, quantification of N fixation is routinely carried out on "free-living" bacteria grown in the absence of a host plant. Such experiments may not divulge the true extent of N fixation occurring in the rhizosphere where the availability and forms of nutrients such as carbon and N, which are key regulators of N fixation, may vary widely. Here, we present a modified in planta acetylene reduction assay, utilising the model cereal barley as a host, to quantify associative N fixation by diazotrophic bacteria. The assay is rapid, highly reproducible, applicable to a broad range of diazotrophs, and can be performed with simple equipment commonly found in most laboratories that investigate plant-microbe interactions. ImportanceExploiting "nitrogen-fixing" bacteria that reduce atmospheric dinitrogen into ammonia as inoculants of cereal crops has great potential to alleviate current inputs of environmentally deleterious fertiliser nitrogen and drive more sustainable crop production. Accurately quantifying plant-associative bacterial nitrogen fixation is central to the development of such inoculant bacteria, but most assays fail to adequately reproduce the conditions of plant root systems. In this work, we have validated and optimised a simple in planta assay to accurately quantify N fixation in bacteria occupying the root and surrounding soil of the model cereal barley. This assay represents a benchmark for quantification of plant-associative bacterial N fixation.