Isolation and characterization of bacteriophages specific for Rhizobium leguminosarum biovar phaseoli

Abstract

Two lytic phages, designated as H3V and R2V, specific for Rhizobium leguminosarum biovar phaseoli, were isolated and characterized. Phage H3V was active against four indigenous isolates (HURR-3, HURR-21, HURR-35, and HURR-56) and two standard strains (RCR-3605 and USDA-2669) whereas R2V was specific to one indigenous (Raj-2) and one standard (USDA-2676) strain; there was no cross infectivity. Both phages had distinct morphology; phage H3V had an oblate polyhedral head (58 × 76 nm) and a flexible noncontractile tail (120 × 10 nm), while phage R2V had a hexagonal head (56 nm wide) and a very short tail (11 × 10 nm). The lytic cycle of phage R2V requires Ca2+ ions (1 mM), which considerably reduce its latent period and burst size. Adsorption and one-step growth experiments of phages revealed that H3V had a slower adsorption rate (0.56 × 10−9 cm3/min), a longer latent period (255 min), and a higher burst size (240 plaque-forming units/cell) than R2V, which had an adsorption rate of 0.94 × 10−9 cm3/min, a 210-min latent period, and a burst size of 200 plaque-forming units/cell. Inactivation of these phages by heat, osmotic shock, and uv irradiation showed that phage H3V was comparatively more sensitive than R2V. These phages were frequently detected in healthy nodules of French beans (Phaseolus vulgaris) at two different field locations and no correlation between phage titer and nodule size or colour was observed. Phage titer varied from 2.8 × 102 to 1.2 × 106 plaque-forming units/nodule.Key words: Rhizobium, phages, morphology.