Potential use of soilborne lytic Podoviridae phage as a biocontrol agent against Ralstonia solanacearum

Abstract

A new podovirus RsPod1EGY Ralstonia phage (GenBank accession no MG711516) with a specific action against R. solanacearum phylotype IIa, sequevar I (race 3, biovar 2) was isolated from Egyptian soil. The potential efficacy of the isolated phage to be used as biocontrol agent was evaluated in vitro and under greenhouse conditions. The podovirus phage produced a plaque size of 3.0-4.0 mm in diameter and completed its infection cycle in 180 min after infection with a burst size of ∼27 virions per infected cell. On the basis of restriction endonuclease analysis, the genome size of the phage was about 41 kb of double-stranded DNA. In vitro studies showed that RsPod1EGY is stable at higher temperatures (up to 60 °C), and at a wide pH range (5-9). SDS-PAGE analysis indicated the major structural protein to be approximately 32 kDa. Bacteriolytic activity of RsPod1EGY against R. solanacearum was detected at different multiplicity of infection (MOI). RsPod1EGY proved to be effective in reduction and prevention of formation of surface polysaccharides of R. solanacearum, during the exponential growth phase of the latter. Interestingly, RsPod1EGY was effective in suppression of R. solanacearum under greenhouse conditions. All Phage-treated tomato plants showed no wilt symptoms or any latent infection during the experimental period, whereas all untreated plants have wilted by 10 days post-infection. The lytic stability of RsPod1EGY phage at higher temperature as well as its effective suppression of wilting symptoms under greenhouse conditions would contribute to biocontrol the bacterial wilt disease in Egypt under field conditions.

Keywords: biological control; disease severity; lytic bacteriophage; podoviridae; potato brown rot.