Further experiments will focus on the upgrade of these protocols for the in planta detection of these bacteria as endophytes, encouraged by the results here obtained with the pathovar-specific TaqMan® probes. Moreover because of their multiplexing activity, these probes are already available to yield new important insights into the epidemiology of Psv, Psn and Psf and of the diseases they caused. Methods Bacterial strains this website and pathogenicity tests P. savastanoi strains used in this study are listed
in Table 1. P. savastanoi strains were routinely grown on King’s B agar (KB) [59], incubated at 26°C for 48 h. For liquid culture, bacteria were grown overnight on KB at 26°C on a rotary shaker (160 rpm). Bacterial suspensions were prepared from liquid cultures: after centrifugation (10 min at 7,000 g), the pellets were washed twice with sterile saline water (SSW, 0.85% NaCl in distilled water) and then resuspended in an appropriate volume of SSW to give the desired concentration [expressed as Colony Forming Units (CFU) per ml]. The concentration of each suspension was
verified by plating on KB agar plates 100 μl of SSW serial dilutions and counting single colonies after 2 days of incubation at 26°C. Bacterial epiphytes naturally occurring on P. savastanoi host plants (olive, oleander and ash) were also isolated and included in this study. To this Tipifarnib cell line purpose two chemically untreated plants for each species were sampled, randomly removing three below leaves per plant from one-year-old twigs. Each leaf was then resuspended in SSW (50 ml in a 100 cc Erlenmeyer flask) and incubated at 26°C on a rotatory shaker (200 rpm) for 18 hours. The leaves washings were then separately TPCA-1 cell line centrifuged (8,000 g, 15 min), each pellet resuspended in 200 μl of SSW, and then used for plating
on KB agar, containing cycloheximide (50 μg/ml) to avoid fungal growth. After an incubation of 2 days at 26°C, 50 individual and different bacterial colonies from each leaf washing were randomly isolated and submitted as unidentified pool to DNA extraction. For long term storage bacteria were maintained at -80°C on 20% (v/v) glycerol. In order to confirm their previous identification, almost-full-length 16S rRNA genes were amplified from all these isolates and amplifications were performed as described elsewhere [23]. The P. savastanoi strains used were also inoculated into 1-year-old olive, oleander and ash stems and tested for their pathogenicity and their virulence, as already described [21]. DNA extraction from bacteria and plants Genomic DNA was extracted and purified from 1 ml of bacterial titrated cultures (from 106 to 1010 CFU/ml), using Puregene® DNA Isolation Kit (Gentra System Inc., MN, USA), according to manufacturers’ instructions.