Cultivation and Maintenance of Pseudomonas savastanoi

The bacterial strains used in this study are listed in Table S1. Pseudomonas savastanoi pv. nerii strain Psn23 and its mutants were routinely grown at 26 °C on King’s B (KB) [26 (link)] or hrp-inducing minimal medium (MM) [27 (link)], while Escherichia coli strains TOP10 and ER2925 were grown on Luria-Bertani (LB) [28 ], as liquid or agarized cultures. Bacterial growth in liquid media was monitored by measuring optical density (OD) at 600 nm (OD₆₀₀) with a spectrophotometer (Infinite^® M200 PRO Multimode Reader, Tecan Group Ltd., Männedorf, Switzerland), while the concentration of viable bacteria was evaluated by plate counts and expressed as colony forming units per milliliter (CFU/mL). For long-term storage, bacteria were maintained at −80 °C on 40% (v/v) glycerol, and P. savastanoi cultures were periodically monitored by using specific PCR-based assays to exclude any bacterial contamination [29 (link),30 (link)]. Antibiotics were added to growth medium if needed, and used at the following final concentrations: 20 µg/mL streptomycin, 50 µg/mL nitrofurantoin, 10 µg/mL gentamicin, and 50 µg/mL kanamycin.