tolaasii, which are naturally resistant to phage infection (Munsch & Olivier, 1995; Yoon et al., 2011). The aim of this study was to isolate bacteriophages that are effective against P. tolaasii and some other pathogenic pseudomonads. The isolation, purification, and host range of these bacteriophages, as well
as the morphology and the complete genome sequence analysis of the Bf7 bacteriophage – having one of the widest host ranges of them – are described. Bacterial strains used for the host range determination of bacteriophages selleck derived from the Belgian Co-ordinated Collections of Micro-organisms (BCCM/LMG, Gent, Belgium), from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig, Germany), and from decaying sporocarps of oyster mushroom, isolated previously from a Hungarian mushroom farm (Sajben et al.,
2011) (Table 1). Pseudomonas tolaasii MLN2238 manufacturer causes yellowing of the oyster mushroom sporocarp during cultivation; therefore, we used necrotic caps to isolate bacteriophages against the pathogen. Infected mushrooms (5 g of each) were smashed, diluted in 10 mL SM buffer (100 mM NaCl, 8 mM MgSO4, 50 mM Tris–HCl, pH 7.5, and 0.01% gelatin in distilled water), and incubated overnight at 25 °C with gentle agitation. The mushroom particles and bacterial cells were removed by centrifugation at 4000 g for 20 min at 4 °C, then the supernatant was centrifuged at 20 000 g for 60 min at 4 °C to collect the phages. Chloroform was added after the centrifugation to eliminate the residual bacterial cells. 150 μL from this mixture was added to 50 μL of P. tolaasii LMG 2342T culture (OD620 nm = 1) incubated previously at 25 °C for 18 h. The mixture was diluted in 6 mL of soft Triptic Soy Base (TSB) agar (0.7%), overlaid on 2% agar plates and allowed to solidify. The phage plaques were detected after 18 h of incubation at 25 °C. To select for phages with increased host range, these plaques were diluted in SM buffer, and the previously described method was
repeated with a culture of Pseudomonas putida DSM 9278. After this step, the resulting plaques derive from bacteriophages that are able to infect both previously applied pseudomonads. Single plaques were collected, and then phage stocks were prepared using P. putida as an indicator strain and stored at 4 °C. Phage titers were determined Coproporphyrinogen III oxidase by the double agar layer method (Adams, 1959) with minor modifications. Soft TSB with 0.7% agar was used for the top layer. Ten-fold serial dilutions were prepared from the phage lysates and added to the host bacteria. The mixture was poured onto the bottom agar layer consisting of LB medium. Number of plaques was scored after 18–24 h incubation at 25 °C. To evaluate the host range of the isolated phages, a collection of Pseudomonas strains (Table 1) were tested for sensitivity by the spot lysis assay (Day & Marchesi, 1996) performed with minor modifications. Bottom agar layers were prepared with 2% agar without nutrient source.