Pectobacterium parmentieri is a Gram-negative plant-pathogenic bacterium able to infect potato (Solanum tuberosum L.). Little is known about lytic bacteriophages infecting P. parmentieri and how phage-resistance influences the environmental fitness and virulence of this species. A lytic phage vB_Ppp_A38 (ϕA38) has been previously isolated and characterized as a potential biological control agent for the management of P. parmentieri. In this study, seven P. parmentieri SCC 3193 Tn5 mutants were identified that exhibited resistance to infection caused by vB_Ppp_A38 (ϕA38). The genes disrupted in these seven mutants encoded proteins involved in the assembly of O-antigen, sugar metabolism, and the production of bacterial capsule exopolysaccharides. The potential of A38-resistant P. parmentieri mutants for plant colonization and pathogenicity as well as other phenotypes expected to contribute to the ecological fitness of P. parmentieri, including growth rate, use of carbon and nitrogen sources, production of pectinolytic enzymes, proteases, cellulases, and siderophores, swimming and swarming motility, presence of capsule and flagella as well as the ability to form biofilm were assessed. Compared to the wild-type P. parmentieri strain, all phage-resistant mutants exhibited a reduced ability to colonize and to cause symptoms in growing potato (S. tuberosum L.) plants. The implications of bacteriophage resistance on the ecological fitness of P. parmentieri are discussed.

Resistance to bacteriophage infections protects bacteria in phage-replete environments, enabling them to survive and multiply in the presence of their viral predators. However, such resistance may confer costs for strains, reducing their ecological fitness as expressed as competitiveness for resources or virulence or both. There is limited knowledge about such costs paid by phage-resistant plant pathogenic bacteria in their natural habitats. This study analyzed the costs of phage resistance paid by the phytopathogenic pectinolytic bacterium Dickeya solani both in vitro and in potato (Solanum tuberosum L.) plants. Thirteen Tn5 mutants of D. solani IPO 2222 were identified that exhibited resistance to infection by lytic bacteriophage vB_Dsol_D5 (ΦD5). The genes disrupted in these mutants encoded proteins involved in the synthesis of bacterial envelope components (viz. LPS, EPS and capsule). Although phage resistance did not affect most of the phenotypes of ΦD5-resistant D. solani such as growth rate, production of effectors, swimming and swarming motility, use of various carbon and nitrogen sources and biofilm formation evaluated in vitro, all phage resistant mutants were significantly compromised in their ability to survive on leaf surfaces as well as to grow within and cause disease symptoms in potato plants.

We present a simple, fast and inexpensive screening assay to preselect candidate Pectobacterium spp. and Dickeya spp. Tn5 mutants, which carry transposon insertions in genes putatively encoding proteins used by lytic bacteriophages to interact with host cells, for the follow-up studies. The proposed method is fast and costeffective and it does not need any specialized laboratory equipment and/or technical support. The Tn5 mutants are generated using random transposon mutagenesis with the mini-Tn5 transposon. The obtained bacterial mutants are incubated in the presence of viable lytic bacteriophage particles in liquid bacterial growth medium supplemented with resazurin for 12 h at 28°C in a 96-well microtiter plate assay. During the cultivation, the Tn5 mutants that are susceptible to phage infection are lysed. The mutants that are resistant to a viral infection (not lysed after contact with bacteriophages) irreversibly reduce the resazurin violet dye to pink/yellowish-colored resorufin indicating active bacterial metabolism (a positive reaction). The change of the culture color can be observed by eye. The Tn5 mutants that are positive in the screen are selected for sequencing of the Tn5 insertion site directly from bacterial genome. The proposed assay allows generation of a number of immediately-available Tn5 mutants expressing phage-resistant phenotypes that can be later selected for further examinations. As a proof-of-concept, we used this method to evaluate resistance to viral infection of Tn5 mutants of Dickeya solani strain IPO2222 and Pectobacterium parmentieri strain SCC3193 using lytic bacteriophages ɸD5 and ɸA38, respectively.

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.

Dickeya solani is an emerging plant pathogenic bacterium, causing disease symptoms in a variety of agriculturally relevant crop species worldwide. To date a number of D. solani genomes have been sequenced and characterized, the great majority of these genomes have however come from D. solani strains isolated from potato (Solanum tuberosum L.) and not from other plant hosts. Herewith, we present the first complete, high-quality genome of D. solani strain IPO 2019 (LMG 25990) isolated from ornamental plant Hyacinthus orientalis. The genome of D. solani strain IPO 2019 consists of one chromosome of 4,919,542 bp., with a GC content of 56.2% and no plasmids. The genome contains 4502 annotated features, 22 rRNA genes, 73 tRNA genes and 1 CRISPRS. We believe that the information of this high-quality, complete, closed genome of D. solani strain isolated from host plant different than potato (i.e. hyacinth) will provide resources for comparative genomic studies as well as for analyses targeting adaptation and ecological fitness mechanisms present in Dickeya solani species.