R 210 215 evaporator

The bacterial strains used in this study are listed in Supplementary Table S1 and were grown for twenty-one days in modified basic propionate (BAP) media described previously (Ngom et al., 2016 (link)) according to conditions listed in Supplementary Table S1. Bacterial cultures were exposed to plant root exudates (RE) for five days as described previously (Beauchemin et al., 2012 (link); Clavijo et al., 2015 (link); Chabaud et al., 2016 (link)). Cell-free supernatant fluids were purified from cultures showing an absorbance of 0.3 at 595 nm. Cultures were collected by centrifugation at 4,000 g for 5 min and the supernatant fluids were filtered through a 0.22 μm filter as described in Chabaud et al., 2016 (link). Unless otherwise indicated, experiments were performed with the supernatant fluids of a F. casuarinae culture induced with RE from its host plant C. glauca and referred to as FCS for Frankiacasuarinaesupernatant. FCS were concentrated fifty times (FCS 50X) using an R-210/215 evaporator (BÜCHI Labortechnik AG, Switzerland). Nodulation experiments with the different Frankia strains were performed as described previously (Alloisio et al., 2010 (link); Svistoonoff et al., 2010b (link); Imanishi et al., 2011 (link)).

The extracts with the use of a pressure vessel were prepared in accordance with the method proposed in the previous study [16 (link)].
Briefly fresh chicory roots after cleaning and grinding (1300 g) were mixed with the solvent in the amount of 2100 mL and extracted with the method proposed by Budryn et al. [17 (link)] with some modifications, in a pressure vessel type PS-5692 (Vienna, Austria). As a solvent, a water–methanol mixture (70/30 or 50/50, v/v) was used at 80 °C for 20 min. The pressure in the vessel during extraction was 0.2 MPa. The obtained suspensions were filtered using the KNF 18 035.3 N vacuum pump (Neuberger, NJ, USA) and paper filters with a density of 84 g/m² Poch (Gliwice, Poland). The methanol was then evaporated at 446 mbar in a Rotavapor R-210/215 evaporator (Büchi, Switzerland). The obtained extracts, devoid of organic solvent, were frozen at −80 °C for 24 h and freeze-dried (Delta 1-24 LSC lyophilizer, Martin Christ GmbH, Osterode am Harz, Germany). The obtained lyophilized preparations were stored at −25 °C before further analysis.