Symbiotic Interaction Analysis of Bradyrhizobium and A. americana

The symbiotic interaction of wild-type Bradyrhizobium DOA9 and derivatives with A. americana was analyzed as described by Wongdee et al. (2016 (link), 2018) (link). At 20 days post-inoculation (dpi), the numbers of nodules on the roots were determined, and the nitrogenase enzyme activity of the plants was measured using the acetylene reduction assay (ARA). For cytological analysis, fresh nodules were sliced into 30-micron sections using a vibratome (Leica VT1000S Vibrating blade microtome, United States). Nodule sections of plants inoculated with DOA9-PmrpoN strains were stained with X-gluc (Bonaldi et al., 2010 (link)). After staining, the sections were mounted on microscope slides and observed by light microscopy. Nodule section of plants inoculated with rpoN mutants were stained with Live-Dead staining reagent (Invitrogen), after which they were analyzed using confocal microscopy as described by Okazaki et al. (2016) (link).

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Wongdee J., Piromyou P., Songwattana P., Greetatorn T., Teaumroong N., Boonkerd N., Giraud E., Nouwen N, & Tittabutr P. (2023). Role of two RpoN in Bradyrhizobium sp. strain DOA9 in symbiosis and free-living growth. Frontiers in Microbiology, 14, 1131860.

Publication 2023

VT1000S Vibrating blade microtome Live-Dead staining reagent

Acetylene Assay Bradyrhizobium Confocal microscopy Derivatives Enzyme activity Inoculation Light microscopy Microscope Microtome Nitrogenase Plants Roots Strains Symbiotic interaction

Corresponding Organization :

Other organizations : Suranaree University of Technology, Centre Occitanie-Montpellier, Centre de Coopération Internationale en Recherche Agronomique pour le Développement

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Variable analysis

independent variables

Bradyrhizobium DOA9 wild-type and derivatives

dependent variables

Number of nodules on the roots at 20 days post-inoculation (dpi)
Nitrogenase enzyme activity of the plants measured using the acetylene reduction assay (ARA)

control variables

Time point of measurement (20 dpi)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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