Data_Sheet_1_A Simple in situ Assay to Assess Plant-Associative Bacterial Nitrogenase Activity.xlsx

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.

植物共生固氮细菌氮（N）固定能力的评估对于筛选和开发能够以可持续方式为谷物作物提供氮素的优良固氮菌剂至关重要。尽管固氮细菌拥有多样的氧耐受机制，但大多数细菌需要低于21%的氧气环境才能实现固氮催化剂氮化酶的最佳稳定性和功能。因此，氮固定能力的评估通常在无宿主植物的“自由生活”细菌上进行，此类实验可能无法准确揭示根际中的活性，而在根际中，如碳和氮等关键调控氮固定的营养物质的可用性和形态可能存在广泛差异。在此，我们提出了一种改进的现场乙炔还原法（ARA），利用模型谷物大麦作为宿主，以比较评估固氮细菌的氮化酶活性。该测试方法快速、高度重现性，适用于广泛的固氮菌，并且可以使用大多数研究植物-微生物相互作用的实验室中常见的简单设备进行。因此，该测试可以作为高吞吐量筛选优良植物共生固氮菌的首选方法。