Abstract
The urgent need of research of new approaches to control bacterial disease on economical important crops, focuses our attention on bacteriophages as alternative biocontrol agents. Thus, the purpose of this paper is to present the isolation and initial characterization of three bacteriophages (SfXv124t/1, 2 and 3) isolated from rhizosphere soil of a healthy tomato plant in Bulgaria that are capable to lyse three phytopathogenic bacteria. The initial characterization includes determination of: their host range, plaque morphology, optimal storage temperature of pure phage lysates, their sensitivity to UV light, thermal inactivation, optimal multiplicity of infection (MOI) and virion morphology. The obtained results showed that one of the phage isolates was capable to lyse wild strains from three phytopathogenic bacterial species: Xanthomonas vesicatoria, Xanthomonas euvesicatoria and Xanthomonas gardneri, and the two remaining phages were active against X. vesicatoria and X. euvesicatoria. On X. vesicatoria lawn, the phages produced the same plaque types that differed only in their size. Storage at 4 °C for 26 days did not lead to decrease in phage titer as opposed to storage at 28 °C followed by decrease to varying degree for all three phages. The results obtained after exposure of the phage lysates to sunlight (UVA + B) and UVC light in separate experiments showed that UVC had a potent phagocidal effect as after 50 min of exposure there were no viable phages in the samples. UVA an UVB had lethal effect for two of the phage isolates and absolutely no lethal effect for the third one as after 50 min of exposure to sunlight there was no decrease in the initial phage titer. Phage isolates were tested for their thermal inactivation after incubation of pure phage lysates at three different temperatures: 55 °C, 75 °C and 95 °C for a period of 10 and 30 min. The most lethal temperature turned out to be 95 °C as after 10 min there were no viable phages in the samples. Phage isolate SfXv124t/1 was the most susceptible as its titer decreased by 1 lg after 10 min of incubation at 55 °C and by another 1 lg after 30 min. The most thermally resistant isolate was SfXv124t/3 as its titer remained stable after 30 min of incubation at 55 °C and decreased only by lg after incubation at 75 °C for 10 min. The optimal MOI for SfXv124t/3 was 0,01 (tested range 0,01-100) with maximal phage titer, reported at the 24(th) hour of incubation. TEM micrographs of the same isolates reveals that it belongs to family Podoviridae.