A simple assay for quantification of plant-associative bacterial nitrogen fixation
A simple assay for quantification of plant-associative bacterial nitrogen fixation
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DOI：
10.1101/2021.03.31.437999
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发表时间：
2021-04
期刊：
影响因子：
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通讯作者：
Timothy L. Haskett;P. Poole
中科院分区：
文献类型：
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作者：
Timothy L. Haskett;P. Poole
植物联合细菌固氮的准确定量对于选择和开发可用于以可持续方式为谷类作物提供氮的优良固氮接种剂至关重要。因为可能不利于植物生长的低氧环境对于固氮催化剂固氮酶的最佳稳定性和功能是必不可少的，所以常规地对在没有宿主植物的情况下生长的“自由生活”细菌进行固氮的定量。这样的实验可能无法揭示发生在根际的固氮的真实程度，在根际，作为固氮关键调节因子的营养物质（如碳和氮）的可用性和形式可能差异很大。在这里，我们提出了一个修改后的植物乙炔还原试验，利用模型谷物大麦作为主机，量化联合固氮菌固氮。该方法快速，重现性好，适用于广泛的固氮生物，并且可以用大多数研究植物-微生物相互作用的实验室中常见的简单设备进行。重要性利用能够将大气中的二氮还原为氨的“固氮”细菌作为谷类作物的接种剂，对于缓解目前对环境有害的肥料氮的投入并推动更可持续的作物生产具有巨大的潜力。准确定量植物相关细菌固氮是这种接种细菌的发展的核心，但大多数测定未能充分再现植物根系的条件。在这项工作中，我们已经验证和优化了一个简单的植物测定，以准确地量化固氮细菌占据的根和周围的土壤模型谷物大麦。该测定代表了用于定量植物缔合细菌固氮的基准。
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. Importance Exploiting “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.