Abstract
Assessment 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. Although diazotrophic bacteria possess diverse oxygen tolerance mechanisms, most require a sub 21% oxygen environment to achieve optimal stability and function of the N-fixing catalyst nitrogenase. Consequently, assessment of N fixation is routinely carried out on “free-living” bacteria grown in the absence of a host plant and such experiments may not accurately divulge activity 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 situ acetylene reduction assay (ARA), utilizing the model cereal barley as a host to comparatively assess nitrogenase activity in 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. Thus, the assay could serve as a first point of order for high-throughput identification of elite plant-associative diazotrophs.
Highlights
Exploiting diazotrophic bacteria that reduce atmospheric dinitrogen (N2) into ammonia (NH3+) as inoculants of cereal crops has great potential to alleviate current inputs of environmentally deleterious fertilizer nitrogen (N) in agricultural systems to establish more sustainable crop production (Santos et al, 2019)
We have demonstrated that our simple in situ acetylene reduction assays (ARA) is highly reproducible, rapid and is applicable to a diverse range of diazotrophic bacteria
The simple standardization involved in our assay workflow is one of its most beneficial features, offering a reduced workload compared to other potential approaches
Summary
Exploiting diazotrophic bacteria that reduce atmospheric dinitrogen (N2) into ammonia (NH3+) as inoculants of cereal crops has great potential to alleviate current inputs of environmentally deleterious fertilizer nitrogen (N) in agricultural systems to establish more sustainable crop production (Santos et al, 2019). Assessment of plant-associative bacterial N fixation is central to the selection and development of elite inoculant strains and is typically carried out using 15N incorporation assays (Chalk, 2016; Doty et al, 2016; Herridge and Giller, 2016; Van Deynze et al, 2018). While these assays can be highly accurate, they are laborious and must be performed on both symbiotic partners if measurements of total N-fixed are required. Monitoring this alternative reaction provides a rapid strategy to measure total nitrogenase activity independently of the fate of fixed N and can serve as a proxy for measurements of N fixation
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