Pm1 and pm2a microplates

Root nodules obtained from Coria-ria japonica23 (link) growing in Japan (Tosa district) were used to inoculate Coriaria myrtifolia seedlings grown on sterile sand and maintained in a greenhouse. Root nodules were harvested, surface disinfected (Na hypochlorite 1.05% for 30 min) and washed several times with sterile distilled water. Preparation of vesicle clusters was performed as described previously24 (link). Lobes were crushed in 0.05 M sodium phosphate buffer, pH 7.2 using a Broeck tissue homogenizer and incubated with the following scavengers for hydrogen peroxide (10 mM N,N’-dimethylthiourea) hydroxyl radical (0.28 M dimethylsulfoxide), for singlet oxygen (0.02 M L-histidine), for superoxide anion (10 mM 4,5-dihydroxybenzene-1,3-disulfonate), for peroxyl radical (100 μM α-tocopherol) and for peroxynitrite anion (100 μM uric acid). Released microsymbiont vesicle clusters were retained by gravity filtration on the 52-μm nylon screen and used to assess cells viability using PM1 and PM2A microplates (Biolog) for carbon sources, as well as PM9 and PM10 microplates (Biolog) for pH and NaCl tolerance, respectively. Viability of endophytic cell clusters was weekly assessed in a subset of PM1, PM2A, PM9 and PM10 microplates using only the live/dead assay BacLight Bacterial Viability Kit for microscopy (Life Technologies, Paisley, UK).