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Pectobacterium parmentieri (former Pectobacterium wasabiae) is an emerging pectinolytic bacterial pathogen causing losses in potato (Solanum tuberosum L.) worldwide. Until now, there are no effective measures to protect crops from infections caused by P. parmentieri. Likewise, little is known about lytic bacteriophages that can infect and kill P. parmentieri, and that could be used in the (bio)control of this pathogen in agricultural applications. A novel lytic bacteriophage vB_Ppp_A38 (ϕA38), belonging to the order Caudovirales, was isolated and characterised in our previous studies. This report provides information about its complete genome sequence and accompanying phylogenomics. The genome of vB_Ppp_A38 consists of 75,764-bp-length, linear, double-stranded DNA with an average GC content of 48.7% and is predicted to have 97 open reading frames (ORFs), with an average length of 732 nucleotides. The open reading frames were classified into functional groups, including structural units, packing, DNA metabolism, regulation and additional (miscellaneous) functions. Comparative genomic analyses based on the whole-genome sequence indicated that the phage vB_Ppp_A38 is most similar to N4-like viruses of the family Schitoviridae, genus Cbunavirus, sharing more than 90% identity with the phages vB_PatP_CB1, vB_PatP_CB3 and vB_PatP_CB4 infecting P. atrosepticum. The bacteriophage vB_Ppp_A38 is the first Cbunavirus bacteriophage infecting other Pectobacterium spp. hosts than P. atrosepticum. As vB_Ppp_A38 is a lytic virus able to kill plant-pathogenic P. parmentieri, it can be used in the biological control of this pathogen in agricultural applications.

A simple and fast procedure has been developed to generate soft rot Pectobacteriaceae (SRP: Pectobacterium spp. and Dickeya spp.) Tn5 mutants in genes encoding receptors used by bacteriophages to interact with their hosts, for the follow-up studies. The procedure is inexpensive and does not require any specialized tools and/or dedicated technical support. The neomycin-resistant SRP Tn5 mutants are generated via conjugation with a transposon donor Escherichia coli ST18 strain (requiring 5-aminolevulinic acid (5-ALA) to survive) carrying pFAJ1819-mini-Tn5-neoR. The conjugation is done on solid medium supplemented with 5-ALA. After conjugation bacterial cells are collected, suspended in liquid bacterial medium, added to the suspension containing lytic bacteriophages and incubated for the additional 30 min with shaking (120 rpm). During this stage, the transposon recipients (Pectobacterium spp. and/or Dickeya spp. Tn5 mutants), susceptible to bacteriophage infection are lysed. Likewise, due to the lack of 5-ALA in the growth medium, E. coli ST18 (transposon donor) cells die at this stage. Finally, after incubation, the bacterial mutants with the Tn5 insertions, resistant to phage infection are selected on solid growth medium supplemented with neomycin. The Tn5 insertion sites are sequenced to acquire knowledge about the Tn5-distrupted genes and their putative function in phage-host interactions. The proposed assay allows generation of a number of immediately-available Tn5 mutants expressing phage-resistant phenotypes in a short time (ca. 48 h) that can be later characterized for various other phenotypic features. In this study, as a proof-of-concept, this method has been used to generate Dickeya solani IPO2222 Tn5 mutants resistant to infection caused by the lytic bacteriophage ɸD5.

As a part of a blackleg and soft rot survey on potato plants cultivated in the agricultural region of Southern Greenland during the growing season of 2019 diseased potato plants and tubers were collected at six localities. Samples of plants and tubers with typical blackleg and soft rot symptoms were cut into parts weighing ca. 10–15 g each and incubated in potato enrichment broth (PEB) under anaerobic conditions for 4–7 days at ca. 20–22 °C (conditions during shipment of the samples from Greenland to Poland). After enrichment plant extracts were plated on crystal violet pectate (CVP) medium to isolate individual cavity-forming bacterial colonies. The inoculated plates were incubated at 22, 25 and 28 °C to maximize the chance of finding pectinolytic bacteria with different growth temperature optima. Cavity-forming isolates were collected and purified to pure cultures on tryptone soya agar (TSA). Ten cavity-forming isolates, named Green1 – Green10, representing isolates from symptomatic plants from different locations, were selected for analyses. The isolates gave a 434 bp. product in Pectobacterium spp.-specific PCR and a 439 bp. product in P. atrosepticum (Pba) -specific PCR. These isolates were Gram (−) rods, facultative anaerobic, catalase positive, oxidase and indole negative, grew in TSB + 5% NaCl, produced acid from lactose, maltose and raffinose. BlastN analyses of the ca. 1300 bp. 16S rDNA sequences of all 10 strains indicated a 99–100% similarity to the 16S rDNA of Pectobacterium atrosepticum. All 10 isolates caused soft rot of potato tuber slices after 72 h at 28 °C. Phylogenetic analysis based on the recA gene sequence grouped the isolates together with P. atrosepticum strains CFBP1526T and SCRI1043. This is the first report on isolation of P. atrosepticum from blackleg-diseased potato plants in Greenland. The presence of P. atrosepticum and its possible impact on potato cultivation in Greenland is discussed.

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 cost-effective 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.

Possibilities to protect potato tubers from rotting caused by Soft Rot Pectobacteriaceae (SRP) under disease favoring conditions were investigated using compatible mixtures of bacterial antagonists and tested with a newly developed stepwise efficacy-based screening protocol. Twenty-two bacterial antagonists were evaluated against a combination of five Pectobacterium and Dickeya strains representing species and subspecies most often associated with potato soft rot in Europe. To enable potential synergistic activity, the antagonists were initially tested against the combination of pathogens in 15 random mixtures containing up to 5 antagonists each. Three mixtures (M2, M4, and M14) out of 15 tested reduced tuber tissue maceration due to soft rot. The individual antagonists derived from M2, M4, and M14 mixtures were tested on potato slices and whole tuber injection assays. These five strains (S. plymuthica strain A294, E. amnigenus strain A167, R. aquatilis strain H145, S. rubidaea strain H440, and S. rubidaea strain H469) were combined to develop a tailored biological control mixture against potato soft rot. The new mixture, designated the Great Five (GF), was tested on seed potato tubers vacuum infiltrated with antagonists and subsequently with the combination of five SRP pathogens. In these experiments, the GF mixture provided stable protection of inoculated potato tubers, reducing soft rot by 46% (P = 0.0016) under high disease pressure conditions. The A294, A167, H145, H440, and H469 antagonists were characterized for features important for viable commercial applications including growth at different temperatures, resistance to antibiotics, and potential toxicity toward Caenorhabditis elegans. The implications for control of soft rot caused by SRP with the use of the GF mixture of antagonists are discussed.

Dickeya solani is a Gram-negative bacterium able to cause disease symptoms on a variety of crop and ornamental plants worldwide. Weeds including Solanum dulcamara (bittersweet nightshade) growing near agricultural fields have been reported to support populations of soft rot bacteria in natural settings. However, little is known about the specific interaction of D. solani with such weed plants that may contribute to its success as an agricultural pathogen. The aim of this work was to assess the interaction of D. solani with its crop plant (Solanum tuberosum) and an alternative (S. dulcamara) host plant. From a collection of 10,000 Tn5 transposon mutants of D. solani IPO2222 carrying an inducible, promotorless gusA reporter gene, 210 were identified that exhibited plant tissue-dependent expression of the gene/operon into which the Tn5 insertion had occurred. Thirteen Tn5 mutants exhibiting the greatest plant tissue induction of such transcriptional units in S. tuberosum or S. dulcamara as measured by qRT-PCR were assessed for plant host colonization, virulence, and ability to macerate plant tissue, as well as phenotypes likely to contribute to the ecological fitness of D. solani, including growth rate, carbon and nitrogen source utilization, motility, chemotaxis toward plant extracts, biofilm formation, growth under anaerobic conditions and quorum sensing. These 13 transcriptional units encode proteins involved in bacterial interactions with plants, with functions linked to cell envelope structure, chemotaxis and carbon metabolism. The selected 13 genes/operons were differentially expressed in, and thus contributed preferentially to D. solani fitness in potato and/or S. dulcamara stem, leaf, and root tissues.

Two bacteriophages, ϕA38 and ϕA41, infecting Pectobacterium parmentieri strain SCC 3193 (former Pectobacterium wasabiae strain SCC 3193) were isolated from arable soil samples collected in different regions of Poland. ϕA38 and ϕA41 have a typical morphology of the members of the family Podoviride and order Caudovirales, with a head diameter of ca. 60 nm and tail length of ca. 20 nm. Phages ϕA38 and ϕA41 exhibited a similar RFLP pattern with Csp6I restriction endonuclease. They were stable in a range of pHs, temperatures and osmolarities but were rapidly inactivated by UV light. During the first 20 min., 74 and 69% of ϕA38 and ϕA41 phages, respectively, were adsorbed to SCC 3193 cells. In one-step growth experiments, ϕA38 and ϕA41 showed latent period of ca. 20–30 min and burst size of 102 and 141 phages, respectively. The optimal multiplicity of infection (MOI) was calculated to be 0.01 for both bacteriophages. In the host range experiments, both phages were able to infect six from 21 of the tested P. parmentieri isolates but the phages were unable to infect other members of the Pectobacterium spp. or Dickeya spp. In the proof-of-concept experiments, ϕA38 and ϕA41 were able to inhibit the growth of P. parmentieri strain SCC 3193 and to protect potato tuber tissue maceration caused by the bacterium. The potential use of ϕA38 and ϕA41 bacteriophages for the biocontrol of P. parmentieri in potato is discussed.

Here, we present a simple and fast screening method to preselect candidate Dickeya solani Tn5 mutants which carry transposon insertions in plant tissue-induced genes for the follow-up studies. The described method is faster and cost-effective in comparison to standard IVET, expression microarrays and RNAseq, and it does not require any specialized laboratory equipment and/or technical assistance. The Tn5 mutants are generated using mini-Tn5 transposon carrying inducible reporter gene (promoterless gusA coding for β-glucuronidase) and are screened for the β-glucuronidase positive phenotypes under non-inductive conditions with X-gluc as a glucuronidase substrate. The bacterial mutants negative in the first screen are then screened for β-glucuronidase phenotypes in the presence of plant tissues, viz. roots, stems and leaves, suspended in liquid bacterial growth medium supplemented with X-gluc in a 48-well microtiter plate assay. The mutants that are positive in the screen with plant tissues are selected for sequencing of the Tn5 insertion sites directly from bacterial genome. This method allows generation of a number of ready-to-use Tn5 mutants showing up-regulation by plant tissue which can be later selected for further studies. We used this method to evaluate interaction of D. solani IPO2222 (type strain) with bittersweet nightshade (Solanum dulcamara L.) and potato (Solanum tuberosum L.) tissues.

Soft rot and blackleg of potato caused by pectinolytic bacteria lead to severe economic losses in potato production worldwide. To investigate the species composition of bacteria causing soft rot and black leg of potato in Norway and Poland, bacteria were isolated from potato tubers and stems. Forty-one Norwegian strains and 42 Polish strains that formed cavities on pectate medium were selected for potato tuber maceration assays and sequencing of three housekeeping genes (dnaX, icdA and mdh) for species identification and phylogenetic analysis. The distribution of the species causing soft rot and blackleg in Norway and Poland differed: we have demonstrated that mainly P. atrosepticum and P. c. subsp. carotovorum are the causal agents of soft rot and blackleg of potatoes in Norway, while P. wasabiae was identified as one of the most important soft rot pathogens in Poland. In contrast to the other European countries, D. solani seem not to be a major pathogen of potato in Norway and Poland. The Norwegian and Polish P. c. subsp. carotovorum and P. wasabiae strains did not cluster with type strains of the respective species in the phylogenetic analysis, which underlines the taxonomic complexity of the genus Pectobacterium. No correlation between the country of origin and clustering of the strains was observed. All strains tested in this study were able to macerate potato tissue. The ability to macerate potato tissue was significantly greater for the P. c. subsp. carotovorum and Dickeya spp., compared to P. atrosepticum and P. wasabiae.

Colonization of Solanum dulcamara (bittersweet nightshade) plants by a GFP-tagged Dickeya solani type strain IPO2222 (IPO2254) was investigated by selective plating and epifluorescence stereomicroscopy (ESM), using in vitro plants and plants grown in compost soil. Replicated experiments were carried out in a growth chamber and the progress of infection and disease symptoms on tissue of the cultured plants, following leaf- and stem-base inoculations with bacteria, was evaluated. Microscopy observations were confirmed by spread-plating dilutions of plant extracts onto agar medium directly after the harvest. In experiments where the stem base of in vitro plants inoculated with a range of inocula of D. solani (104 to 108 colony forming units [cfu] ml−1) was examined at 14 days post infection (dpi), blackleg-like symptoms developed in more than 80% plants together with a reduction of the plant fitness (disease symptoms, weight, height, and appearance). In leaf-inoculated plants at 14 dpi, 15% of the plants exhibited severe blackleg-like symptoms. In detached S. dulcamara leaf assays, IPO2254 survived on the adaxial surface for 14 days at populations of 106 cfu per leaf. Thirty days after stem inoculation of plants grown in compost soil in pots, up to 104 cfu g−1 of GFP-tagged D. solani were found inside the stems. D. solani were detected inside the vascular tissue (xylem vessels) of stems, in the pith tissue in roots, and on the internal surface of the stem hollow. The implications of S. dulcamara infection by D. solani for the long-distance dispersal of the bacterial inoculum are discussed.

Dickeya solani is a Gram-negative necrotrophic, plant pathogenic bacterium able to cause symptoms in a variety of plant species worldwide. As a facultative anaerobe, D. solani is able to infect hosts under a broad range of oxygen concentrations found in plant environments. However, little is known about oxygen-dependent gene expression in Dickeya spp. that might contribute to its success as a pathogen. Using a Tn5 transposon, harboring a promoterless gusA reporter gene, 146 mutants of D. solani IPO2222 were identified that exhibited oxygen-regulated expression of the gene into which the insertion had occurred. Of these mutants 114 exhibited higher expression under normal oxygen conditions than hypoxic conditions while 32 were more highly expressed under hypoxic conditions. The plant host colonization potential and pathogenicity as well as phenotypes likely to contribute to the ecological fitness of D. solani, including growth rate, carbon and nitrogen source utilization, production of pectinolytic enzymes, proteases, cellulases and siderophores, swimming and swarming motility and the ability to form biofilm were assessed for 37 strains exhibiting the greatest oxygen-dependent change in gene expression. Eight mutants expressed decreased ability to cause disease symptoms when inoculated into potato tubers or chicory leaves and three of these also exhibited delayed colonization of potato plants and exhibited tissue specific differences in gene expression in these various host tissues. The genes interrupted in these eight mutants encoded proteins involved in fundamental bacterial metabolism, virulence, bacteriocin and proline transport, while three encoded hypothetical or unknown proteins. The implications of environmental oxygen concentration on the ability of D. solani to cause disease symptoms in potato are discussed.

Dickeya solani is a Gram-negative necrotrophic, plant pathogenic bacterium able to cause symptoms in a variety of plant species worldwide. As a facultative anaerobe, D. solani is able to infect hosts under a broad range of oxygen concentrations found in plant environments. However, little is known about oxygen-dependent gene expression in Dickeya spp. that might contribute to its success as a pathogen. Using a Tn5 transposon, harboring a promoterless gusA reporter gene, 146 mutants of D. solani IPO2222 were identified that exhibited oxygen-regulated expression of the gene into which the insertion had occurred. Of these mutants 114 exhibited higher expression under normal oxygen conditions than hypoxic conditions while 32 were more highly expressed under hypoxic conditions. The plant host colonization potential and pathogenicity as well as phenotypes likely to contribute to the ecological fitness of D. solani, including growth rate, carbon and nitrogen source utilization, production of pectinolytic enzymes, proteases, cellulases and siderophores, swimming and swarming motility and the ability to form biofilm were assessed for 37 strains exhibiting the greatest oxygen-dependent change in gene expression. Eight mutants expressed decreased ability to cause disease symptoms when inoculated into potato tubers or chicory leaves and three of these also exhibited delayed colonization of potato plants and exhibited tissue specific differences in gene expression in these various host tissues. The genes interrupted in these eight mutants encoded proteins involved in fundamental bacterial metabolism, virulence, bacteriocin and proline transport, while three encoded hypothetical or unknown proteins. The implications of environmental oxygen concentration on the ability of D. solani to cause disease symptoms in potato are discussed.

The potential for control of Dickeya solani infections in potato by elicitation of in vitro grown potato plants with salicylic acid (SA) was investigated by selective plating and confocal laser scanning macroscopy (CLSM). In replicated experiments potato plants grown on medium supplemented with 25 or 50 μM of SA were evaluated for the phytotoxic effects. Potato plants grown on medium supplemented with SA and inoculated with GFP-tagged D. solani were investigated for blackleg development and colonization of potato plants by the bacteria. Three days after inoculation, colonization of roots by D. solani was observed in 100 % control plants grown on medium without SA but not in plants grown on medium supplemented with 50 μM SA. After 14 days, 100 % of control plants showed severe disease symptoms, whereas plants grown on medium supplemented with 50 μM SA and inoculated with bacteria did not express any symptoms. After 14 days bacteria were found inside 100 % stems of control plants in densities of ca. 103–104 cfu g−1 and inside ca. 10–15 % stems of plants treated with 50 μM SA in densities similar to these in the control plants. The GFP-tagged bacteria were macroscopically detected on the surface of the roots of control plants but not on the surface of the plants treated with 50 μM SA 14 days after inoculation. The implications of SA treatments on plant fitness and disease development are discussed.

In this study, colonization and disease development of axenically-grown tomato plants by Clavibacter michiganensis subsp. michiganensis (Cmm), the causative agent of bacterial wilt and canker, was investigated. For this, a spontaneous rifampicin resistant strain of Cmm was tagged with a marker that expressed a green fluorescent protein (GFP) in a stable way and which possessed a similar virulence to the parental strain. In vitro plants were drop-inoculated at the stem base and the population dynamics was determined by dilution pour-plating in a selective medium. At 3 h after inoculation, Cmm was already present in low densities in roots, stems and leaves. At 16 dpi, Cmm was found throughout the entire plant in high densities of ca. 1010 cfu g−1. Symptoms developed in the in vitro plants typical for Cmm, such as canker, wilting and growth reduction. The presence of Cmm in vascular and parenchymatic tissue of in vitro tomato plants was confirmed by epifluorescence stereo- and confocal laser scanning microscopy. This study showed that in vitro tomato plants can be effectively used for detailed studies on interactions between Cmm and its host, in particular if a GFP-tagged strain of the pathogen is used.

Nine bacteriophages infecting Dickeya spp. biovar 3 (‘Dickeya solani’) were isolated from soil samples collected in different regions in Poland. The phages have a typical morphology of the members of the order Caudovirales, family Myoviridae, with a head diameter of c. 90–100 nm and tail length of c. 120–140 nm. In host range experiments, phage ϕD5 expressed the broadest host range, infecting members of all Dickeya spp., and phage ϕD7 showed the narrowest host range, infecting isolates of Dickeya dadantii and ‘D. solani’ only. None of the phages was able to infect Pectobacterium spp. isolates. All phages were prone to inactivation by pH 2, temperature of 85°C and by UV illumination for 10 min (50 mJ cm−2). Additionally, phages ϕD1, ϕD10 and ϕD11 were inactivated by 5 m NaCl and phage ϕD2 was inactivated by chloroform. Phages ϕD1, ϕD5, ϕD7 and ϕD10 were characterized for optimal multiplicity of infection and the rate of adsorption to the bacterial cells. The latent period was 30 min for ϕD1, 40 min for ϕD5, 20–30 min for ϕD7 and 40 min for ϕD10. The estimated burst size was c. 100 plaque-forming units per infected cell. The bacteriophages were able to completely stop the growth of ‘D. solani’ in vitro and to protect potato tuber tissue from maceration caused by the bacteria. The potential use of bacteriophages for the biocontrol of biovar 3 Dickeya spp. in potato is discussed.

Xanthomonas fragariae is the causative agent of angular leaf spot disease of strawberry. Greenhouse experiments were conducted using a X. fragariae isolate tagged with a green fluorescent protein (GFP) for detailed population dynamic studies in and on leaves after spray-inoculation. The GFP-tagged bacteria were monitored with dilution plating of leaf washings and leaf extracts, and analysis of intact leaves using a non-invasive monitoring system called PathoScreen, based on laser radiation of fluorescent cells in plant tissues and signal recording with a sensitive camera. PathoScreen was also used to monitor bacteria grown on an agar medium after leaf printing. During the first 3 days after inoculation, bacterial populations washed off leaves rapidly decreased by at least a factor of 1000, after which populations remained stable until 14 days post-inoculation (dpi), when symptoms first started to appear. Thereafter, populations increased to a level of 1012 colony-forming units (CFU) g−1 of leaf material or higher. Similarly, densities in leaf extracts were low during the first 3 days after inoculation, at a level of 100–1000 CFU g−1 of leaf tissue. Gradually populations increased to a level of 109–1012 CFU g−1 at 28 dpi. Higher densities of epiphytic populations were found on the abaxial side than on the adaxial leaf side during the first 2 weeks after inoculation. After spray-inoculation of leaves, bacterial populations released from infected plants remained low until symptoms appeared, after which plants became highly infectious, in particular under high humidity.

Treatments of tubers with chemical disinfectants (70 % ethanol, 1 % sodium hypochlorite, 2 % copper sulphate, 5 % peracetic acid, 10 % hydrogen peroxide, 1 % MennoClean (benzoic acid), 5 % trisodium phosphate and 0.2 % caffeine) were evaluated for control of blackleg caused by ‘D. solani’. All disinfectants effectively killed bacteria in axenic cultures within 5 min and all, except hydrogen peroxide and trisodium phosphate, were able to kill ‘D. solani’ in spiked potato extracts. Treatments with all disinfectants except trisodium phosphate, reduced the pathogen populations on the periderm of tubers previously inoculated by dipping in a suspension of ‘D. solani’, when the disinfectant was applied for at least 15 min. However, in replicated greenhouse experiments, treatments with all disinfectants except hydrogen peroxide and caffeine resulted in phytotoxicity, reducing sprouting of tubers with 10 – 100 %. Sodium hypochlorite and MennoClean were selected for further studies. Treatments of inoculated tubers with these disinfectants prevented developing of soft rot symptoms on tubers when subjected to favourable rotting conditions. In replicated greenhouse experiments, inoculated tubers treated with sodium hypochlorite and MennoClean reduced the blackleg disease incidences from 50 % to 0 % and to 5 %, respectively, and symptomless infection of potato stems from 93 % to 0 % and to 5 %, respectively. A combination of both compounds was highly phytotoxic. The potential use of sodium hypochlorite and MennoClean in an integrated management strategy for potato blackleg is discussed.

Studies were conducted to explain the relative success of ‘Dickeya solani’, a genetic clade of Dickeya biovar 3 and a blackleg-causing organism that, after recent introduction, has spread rapidly in seed potato production in Europe to the extent that it is now more frequently detected than D. dianthicola. In vitro experiments showed that both species were motile, had comparable siderophore production and pectinolytic activity, and that there was no antagonism between them when growing. Both ‘D. solani’ and biovar 1 and biovar 7 of D. dianthicola rotted tuber tissue when inoculated at a low density of 103 CFU mL-1. In an agar overlay assay, D. dianthicola was susceptible to 80% of saprophytic bacteria isolated from tuber extracts, whereas ‘D. solani’ was susceptible to only 31%, suggesting that ‘D. solani’ could be a stronger competitor in the potato ecosystem. In greenhouse experiments at high temperatures (28°C), roots were more rapidly colonized by ‘D. solani’ than by biovar 1 or 7 of D. dianthicola and at 30 days after inoculation higher densities of ‘D. solani’ were found in stolons and progeny tubers. In co-inoculated plants, fluorescent protein (GFP or DsRed)-tagged ‘D. solani’ outcompeted D. dianthicola in plants grown from vacuum-infiltrated tubers. In 3 years of field studies in the Netherlands with D. dianthicola and ‘D. solani’, disease incidence varied greatly annually and with strain. In summary, ‘D. solani’ possesses features which allow more efficient plant colonization than D. dianthicola at high temperatures. In temperate climates, however, tuber infections with ‘D. solani’ will not necessarily result in a higher disease incidence than infections with D. dianthicola, but latent seed infection could be more prevalent

Interactions between Serratia plymuthica A30 and a blackleg-causing biovar 3 Dickeya sp. were examined. In a potato slice assay, S. plymuthica A30 inhibited tissue maceration caused by Dickeya sp. IPO2222 when co-inoculated at a density at least 10 times greater than that of the pathogen. In glasshouse experiments, population dynamics of the antagonist and of the pathogen in planta were studied by dilution plating and confocal laser scanning microscopy (CLSM) using fluorescent protein-tagged strains. Pathogen-free minitubers were vacuum-infiltrated with DsRed-tagged Dickeya sp. IPO2222 and superficially treated during planting with a water suspension containing GFP-tagged S. plymuthica A30. A30 reduced the blackleg incidence from 55% to 0%. Both the pathogen and the antagonist colonized the seed potato tubers internally within 1 day post-inoculation (dpi). Between 1 and 7 dpi, the population of A30 in tubers increased from 101 to c. 103 CFU g-1 and subsequently remained stable until the end of the experiment (28 dpi). Populations of A30 in stems and roots increased from c. 102 to c. 104 CFU g-1 between 7 and 28 dpi. Dilution plating and CLSM studies showed that A30 decreased the density of Dickeya sp. populations in plants. Dilution plating combined with microscopy allowed the enumeration of strain A30 and its visualization in the vascular tissues of stem and roots and in the pith of roots, as well as its adherence to and colonization of the root surface. The implications of these finding for the use of S. plymuthica A30 as a biocontrol agent are discussed.

Possibilities for biocontrol of biovar 3 Dickeya sp. in potato were investigated, using bacteria from rotting potato tissue isolated by dilution plating on nonselective agar media. In a plate assay, 649 isolates were screened for antibiosis against Dickeya sp. IPO2222 and for the production of siderophores. Forty-one isolates (6·4%) produced antibiotics and 112 isolates (17·3%) produced siderophores. A selection of 41 antibiotic-producing isolates and 41 siderophore-producing isolates were tested in a potato slice assay for control of the Dickeya sp. Isolates able to reduce rotting of potato tuber tissue by at least 50% of the control were selected. Isolates were characterized by 16S rDNA analysis as Bacillus, Pseudomonas, Rhodococcus, Serratia, Obesumbacterium and Lysinibacillus genera. Twenty-three isolates belonging to different species and genera, 13 producing antibiotics and 10 producing siderophores, were further characterized by testing acyl-homoserine lactone (AHL) production, quorum quenching, motility, biosurfactant production, growth at low (4·0) and high (10·0) pH, growth at 10°C under aerobic and anaerobic conditions and auxin production. In replicated greenhouse experiments, four selected antagonists based on the in vitro tests were tested in planta using wounded or intact minitubers of cv. Kondor subsequently inoculated by vacuum infiltration with an antagonist and a GFP (green fluorescent protein)-tagged biovar 3 Dickeya sp. strain. A potato endophyte A30, characterized as S. plymuthica, protected potato plants by reducing blackleg development by 100% and colonization of stems by Dickeya sp. by 97%. The potential use of S. plymuthica A30 for the biocontrol of Dickeya sp. is discussed.

This paper briefly reviews research on the causative agents of blackleg and soft rot diseases of potato, namely Pectobacterium and Dickeya species, and the disease syndrome, including epidemiological and aetiological aspects. It critically evaluates control methods used in practice based on the avoidance of the contamination of plants, in particular the use of seed testing programmes and the application of hygienic procedures during crop production. It considers the perspective of breeding and genetic modification to introduce resistance. It also evaluates the application of physical and chemical tuber treatments to reduce inoculum load and examines the possibility of biocontrol using antagonistic bacteria and bacteriophages.

Translocation of a green fluorescent protein (GFP)-tagged Dickeya sp. from stems or from leaves to underground parts of potato plants was studied in greenhouse experiments. Thirty days after stem inoculation, 90% of plants expressed symptoms at the stem base and 95% of plants showed browning of internal stem tissue. The GFP-tagged Dickeya sp. was detected by dilution plating in extracts of the stem interiors (100%), stem bases (90%), roots (80%), stolons (55%), and progeny tubers (24%). In roots, the GFP-tagged Dickeya sp. was found inside and between parenchyma cells whereas, in stems and stolons, the GFP-tagged Dickeya sp. was found in the xylem vessels and protoxylem cells. In progeny tubers, this strain was detected in the stolon end. Thirty days after leaf inoculation, the GFP-tagged Dickeya sp. was detected in extracts of 75% of the leaves, 88% of the petioles, 63% of the axils, and inside 25% of the stems taken 15 cm above the ground level. UV microscopy confirmed the presence of the GFP-tagged Dickeya sp. inside petioles and in the main leaf veins. No blackleg or aerial stem rot and no translocation of the GFP-tagged Dickeya sp. to underground plant parts was observed. The implications for contamination of progeny tubers are discussed.

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.

In Western and Northern Europe, Dickeya species are causing increasingly severe economic losses in potato (Solanum tuberosum) crops. The costs of seed potato production resulting from Dickeya spp. infection are high due to rejection and declassification of seed tubers (Slawiak et al., 2009). Potato blackleg caused by Dickeya spp. is primarily a seed tuber-borne disease (Tsror et al., 2009). Symptoms including blackening of the stem base, wilting of plants and rotten seed tubers are observed. Recently, outbreaks of potato blackleg were recorded in Georgia on three cultivars, in Samtskhe-Javakheti region, Akhalkalaki district, in an area in excess of 100 hectares

Colonization of potato plants by soilborne, green fluorescent protein (GFP)-tagged Dickeya sp. IPO2254 was investigated by selective plating, epifluorescence stereo microscopy (ESM), and confocal laser scanning microscopy (CLSM). Replicated experiments were carried out in a greenhouse using plants with an intact root system and plants from which ca. 30% of the lateral roots was removed. One day after soil inoculation, adherence of the pathogen on the roots and the internal colonization of the plants were detected using ESM and CLSM of plant parts embedded in an agar medium. Fifteen days post-soil inoculation, Dickeya sp. was found on average inside 42% of the roots, 13% of the stems, and 13% of the stolons in plants with undamaged roots. At the same time-point, in plants with damaged roots, Dickeya sp. was found inside 50% of the roots, 25% of the stems, and 25% of the stolons. Thirty days postinoculation, some plants showed true blackleg symptoms. In roots, Dickeya sp. was detected in parenchyma cells of the cortex, both inter- and intracellularly. In stems, bacteria were found in xylem vessels and in protoxylem cells. Microscopical observations were confirmed by dilution spread-plating the plant extracts onto agar medium directly after harvest. The implications of infection from soilborne inoculum are discussed.

Detailed studies were conducted on the distribution of Pectobacterium carotovorum subsp. carotovorum and Dickeya spp. in two potato seed lots of different cultivars harvested from blackleg-diseased crops. Composite samples of six different tuber sections (peel, stolon end, and peeled potato tissue 0.5, 1.0, 2.0 and 4.0 cm from the stolon end) were analysed by enrichment PCR, and CVP plating followed by colony PCR on the resulting cavity-forming bacteria. Seed lots were contaminated with Dickeya spp. and P. carotovorum subsp. carotovorum (Pcc), but not with P. atrosepticum. Dickeya spp. and Pcc were found at high concentrations in the stolon ends, whereas relatively low densities were found in the peel and in deeper located potato tissue. Rep-PCR, 16S rDNA sequence analysis and biochemical assays, grouped all the Dickeya spp. isolates from the two potato seed lots as biovar 3. The implications of the results for the control of Pectobacterium and Dickeya spp., and sampling strategies in relation to seed testing, are discussed.

Sixty-five potato strains of the soft rot-causing plant pathogenic bacterium Dickeya spp., and two strains from hyacinth, were characterised using biochemical assays, REP-PCR genomic finger printing, 16S rDNA and dnaX sequence analysis. These methods were compared with nineteen strains representing six Dickeya species which included the type strains. A group of twenty-two potato strains isolated between 2005-2007 in the Netherlands, Poland, Finland and Israel were characterised as belonging to biovar 3. They were 100% identical in REP-PCR, dnaX and 16S rDNA sequence analysis. In a polyphasic analysis they formed a new clade different from the six Dickeya species previously described, and may therefore constitute a new species. The strains were very similar to a Dutch strain from hyacinth. On the basis of dnaX sequences and biochemical assays, all other potato strains isolated in Europe between 1979 and 1994 were identified as D. dianthicola (biovar 1 and 7), with the exception of two German strains classified as D. dieffenbachia (biovar 2) and D. dadantii (biovar 3), respectively. Potato strains from Peru were classified as D. dadantii, from Australia as D. zeae and from Taiwan as D. chrysanthemi bv. parthenii, indicating that different Dickeya species are found in association with potato.