Spontaneous mutations in hlyD and tuf genes result in resistance of Dickeya solani IPO 2222 to phage ϕD5 but cause decreased bacterial fitness and virulence in planta.

Lytic bacteriophages able to infect and kill Dickeya spp. can be readily isolated from virtually all Dickeya spp. containing environments, yet little is known about the selective pressure those viruses exert on their hosts. Two spontaneous D. solani IPO 2222 mutants (0.8% of all obtained mutants), DsR34 and DsR207, resistant to infection caused by lytic phage vB_Dsol_D5 (ΦD5) were identified in this study that expressed a reduced ability to macerate potato tuber tissues compared to the wild-type, phage-susceptible D. solani IPO 2222 strain. Genome sequencing revealed that genes encoding: secretion protein HlyD (in mutant DsR34) and elongation factor Tu (EF-Tu) (in mutant DsR207) were altered in these strains. These mutations impacted the DsR34 and DsR207 proteomes. Features essential for the ecological success of these mutants in a plant environment, including their ability to use various carbon and nitrogen sources, production of plant cell wall degrading enzymes, ability to form biofilms, siderophore production, swimming and swarming motility and virulence in planta were assessed. Compared to the wild-type strain, D. solani IPO 2222, mutants DsR34 and DsR207 had a reduced ability to macerate chicory leaves and to colonize and cause symptoms in growing potato plants.

The viability of lytic bacteriophage ΦD5 in potato-associated environments and its effect on Dickeya solani in potato (Solanum tuberosum L.) plants.

Dickeya solani is one of the most important pectinolytic phytopathogens responsible for high losses in potato, especially in seed potato production in Europe. Lytic bacteriophages can affect the structure of the host population and may influence spread, survival and virulence of the pathogen and in consequence, infection of the plant. In this study, we aimed to acquire information on the viability of the broad host lytic bacteriophage ΦD5 on potato, as well as to apprehend the specific effect of this bacteriophage on its host D. solani type-strain in different settings, as a preliminary step to target co-adaptation of phages and host bacteria in plant environment. Viability of the ΦD5 phage in tuber extract, on tuber surface, in potting compost, in rainwater and on the leaf surface, as well as the effect of copper sulfate, were examined under laboratory conditions. Also, the interaction of ΦD5 with the target host D. solani in vitro and in compost-grown potato plants was evaluated. ΦD5 remained infectious in potato tuber extract and rain water for up to 72 h but was inactivated in solutions containing 50 mM of copper. The phage population was stable for up to 28 days on potato tuber surface and in potting compost. In both, tissue culture and compost-grown potato plants, ΦD5 reduced infection by D. solani by more than 50%. The implications of these findings are discussed.

Genomic, proteomic and morphological characterization of two novel broad host lytic bacteriophages ΦPD10.3 and ΦPD23.1 infecting pectinolytic Pectobacterium spp. and Dickeya spp.

Pectinolytic Pectobacterium spp. and Dickeya spp. are necrotrophic bacterial pathogens of many important crops, including potato, worldwide. This study reports on the isolation and characterization of broad host lytic bacteriophages able to infect the dominant Pectobacterium spp. and Dickeya spp. affecting potato in Europe viz. Pectobacterium carotovorum subsp. carotovorum (Pcc), P. wasabiae (Pwa) and Dickeya solani (Dso) with the objective to assess their potential as biological disease control agents. Two lytic bacteriophages infecting stains of Pcc, Pwa and Dso were isolated from potato samples collected from two potato fields in central Poland. The ΦPD10.3 and ΦPD23.1 phages have morphology similar to other members of the Myoviridae family and the Caudovirales order, with a head diameter of 85 and 86 nm and length of tails of 117 and 121 nm, respectively. They were characterized for optimal multiplicity of infection, the rate of adsorption to the Pcc, Pwa and Dso cells, the latent period and the burst size. The phages were genotypically characterized with RAPD-PCR and RFLP techniques. The structural proteomes of both phages were obtained by fractionation of phage proteins by SDS-PAGE. Phage protein identification was performed by liquid chromatography-mass spectrometry (LC-MS) analysis. Pulsed-field gel electrophoresis (PFGE), genome sequencing and comparative genome analysis were used to gain knowledge of the length, organization and function of the ΦPD10.3 and ΦPD23.1 genomes. The potential use of ΦPD10.3 and ΦPD23.1 phages for the biocontrol of Pectobacterium spp. and Dickeya spp. infections in potato is discussed.