PMAxx-Based Viability Staining for Plant Microbiome

The viability staining was performed with an improved version of PMA, PMAxx (Biotium Inc., Fremont, CA, USA), according to Santander et al. [5 (link)]. Briefly, 0.4 mL aliquots of plant macerates were mixed with 0.1 mL of 5 × PMA Enhancer for Gram-negative bacteria (Biotium Inc., Fremont, CA, USA) and 2.5 μL of 20 mM PMAxx in 2 mL tubes. The tubes were then incubated for 5 min under dark conditions, at room temperature, in a shaker at 150 rpm to allow the dye to penetrate the dead cells. For PMAxx photo-activation, the tubes were placed horizontally and half-dipped on ice in a shallow tray located 20 cm distance under two halogen lamps (500 W each) for 10 min, with the researcher gently mixing the tubes’ content every 5 min. After treatment, samples were pelleted via centrifugation at 14,500× g for 10 min, the supernatants were discarded, and the pelleted materials were stored at −80 °C or directly used for DNA extraction, according to PMAxx manufacturer’s instructions.
Unless stated differently, the DNA extractions of pelleted PMAxx-treated and non-PMAxx-treated samples in this study were carried out with the Plant DNeasy Mini Kit (Qiagen, Frederick, MD, USA), following the manufacturer’s instructions.