Bacteria from the genera Dickeya (formerly Erwinia chrysanthemi) and Pectobacterium (formerly E. carotovora) are the agents of blackleg and soft rot on many important crops. In 2005, Dickeya solani was isolated for the first time in Poland from a symptomatic potato plant. To establish the presence and diversity of Dickeya spp. in Poland, we surveyed potato fields and water sources, including surface waters near potato fields and water from potato-processing facilities and sewage plants. Only D. dianthicola and D. solani were isolated from symptomatic potato, and only D. zeae and D. chrysanthemi were isolated from water sources. The Dickeya spp. isolated from potato formed a relatively homogenous group, while those from water sources were more diverse. To our knowledge, this is the first comprehensive characterization of Dickeya spp. isolated during several years from regions with a temperate climate in Central Europe.

Pectobacterium parmentieri (formerly Pectobacterium wasabiae) is a newly established species of pectinolytic plant-pathogenic bacteria responsible for the symptoms of soft rot and blackleg on potato. In this work, we describe biodiversity and the population structure of P. parmentieri strains isolated during two consecutive growing seasons from the seed potato fields in Poland. About 450 samples of diseased potato tubers, potato plants, or accompanying weeds were collected throughout the country and tested for the presence of P. parmentieri by molecular identification methods. We found that P. parmentieri strains commonly occur in almost all regions of Poland. Furthermore, these isolates constituted significant fraction of pectinolytic bacteria from seed potato fields because 16% (2013) and 13% (2014) of the analyzed plant samples were infected with P. parmentieri. Subsequently, a detailed characterization of the obtained strains was conducted basing on repetitive sequences profiling, recA-gene-based phylogeny, and phenotypic features. By applying repetitive extragenic palindromic sequence-based polymerase chain reaction (REP-PCR), we revealed the presence of five distinct genomic profiles among P. parmentieri strains, with profile I being the most abundant (approximately 44%). The performed recA gene-based phylogenetic analysis divided P. parmentieri isolates into two distinct clades, although the strains originating from different years did not group separately. Evaluation of the phenotypic traits playing crucial roles for the virulence of pectinolytic bacteria (namely, pectinase, cellulase and protease activities, and siderophore production, in addition to potato tissue maceration, swimming, and swarming motility) indicated some differences among the characterized strains. To the best of our knowledge, this is the first study that describes biodiversity and the population structure of P. parmentieri isolated in two growing seasons under temperate climate conditions and, hence, illustrates high heterogeneity within this species.

BackgroundPectobacterium parmentieri is a newly established species within the plant pathogenic family Pectobacteriaceae. Bacteria belonging to this species are causative agents of diseases in economically important crops (e.g. potato) in a wide range of different environmental conditions, encountered in Europe, North America, Africa, and New Zealand. Severe disease symptoms result from the activity of P. parmentieri virulence factors, such as plant cell wall degrading enzymes. Interestingly, we observe significant phenotypic differences among P. parmentieri isolates regarding virulence factors production and the abilities to macerate plants. To establish the possible genomic basis of these differences, we sequenced 12 genomes of P. parmentieri strains (10 isolated in Poland, 2 in Belgium) with the combined use of Illumina and PacBio approaches. De novo genome assembly was performed with the use of SPAdes software, while annotation was conducted by NCBI Prokaryotic Genome Annotation Pipeline.ResultsThe pan-genome study was performed on 15 genomes (12 de novo assembled and three reference strains: P. parmentieri CFBP 8475T, P. parmentieri SCC3193, P. parmentieri WPP163). The pan-genome includes 3706 core genes, a high number of accessory (1468) genes, and numerous unique (1847) genes. We identified the presence of well-known genes encoding virulence factors in the core genome fraction, but some of them were located in the dispensable genome. A significant fraction of horizontally transferred genes, virulence-related gene duplications, as well as different CRISPR arrays were found, which can explain the observed phenotypic differences. Finally, we found also, for the first time, the presence of a plasmid in one of the tested P. parmentieri strains isolated in Poland.ConclusionsWe can hypothesize that a large number of the genes in the dispensable genome and significant genomic variation among P. parmentieri strains could be the basis of the potential wide host range and widespread diffusion of P. parmentieri. The obtained data on the structure and gene content of P. parmentieri strains enabled us to speculate on the importance of high genomic plasticity for P. parmentieri adaptation to different environments.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-5140-9) contains supplementary material, which is available to authorized users.

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The objective of the present study was to evaluate the antibacterial properties of a plant secondary metabolite - caffeine. Caffeine is present in over 100 plant species. Antibacterial activity of caffeine was examined against the following plant-pathogenic bacteria: Ralstonia solanacearum (Rsol), Clavibacter michiganesis subsp. sepedonicus (Cms), Dickeya solani (Dsol), Pectobacterium atrosepticum (Pba), Pectobacterium carotovorum subsp. carotovorum (Pcc), Pseudomonas syringae pv. tomato (Pst), and Xanthomonas campestris subsp. campestris (Xcc). MIC and MBC values ranged from 5 to 20 mM and from 43 to 100 mM, respectively. Caffeine increased the bacterial generation time of all tested species and caused changes in cell morphology. The influence of caffeine on the synthesis of DNA, RNA and proteins was investigated in cultures of plant pathogenic bacteria with labelled precursors: [(3)H]thymidine, [(3)H]uridine or (14)C leucine, respectively. RNA biosynthesis was more affected than DNA or protein biosynthesis in bacterial cells treated with caffeine. Treatment of Pba with caffeine for 336 h did not induce resistance to this compound. Caffeine application reduced disease symptoms caused by Dsol on chicory leaves, potato slices, and whole potato tubers. The data presented indicate caffeine as a potential tool for the control of diseases caused by plant-pathogenic bacteria, especially under storage conditions.

A multiplex polymerase chain reaction (PCR) assay for simultaneous, fast and reliable detection of the main soft rot and blackleg potato pathogens in Europe has been developed. It utilises three pairs of primers and enables detection of three groups of pectinolytic bacteria frequently found in potato, namely: Pectobacterium atrosepticum, Pectobacterium carotovorum subsp. carotovorum together with Pectobacterium wasabiae and Dickeya spp. in a multiplex PCR assay. In studies with axenic cultures of bacteria, the multiplex assay was specific as it gave positive results only with strains of the target species and negative results with 18 non-target species of bacteria that can possibly coexist with pectinolytic bacteria in a potato ecosystem. The developed assay could detect as little as 0.01 ng µL–1 of Dickeya sp. genomic DNA, and down to 0.1 ng µL–1 of P. atrosepticum and P. carotovorum subsp. carotovorum genomic DNA in vitro. In the presence of competitor genomic DNA, isolated from Pseudomonas fluorescens cells, the sensitivity of the multiplex PCR decreased tenfold for P. atrosepticum and Dickeya sp., while no change was observed for P. carotovorum subsp. carotovorum and P. wasabiae. In spiked potato haulm and tuber samples, the threshold level for target bacteria was 101 cfu mL–1 plant extract (102 cfu g–1 plant tissue), 102 cfu mL–1 plant extract (103 cfu g–1 plant tissue), 103 cfu mL–1 plant extract (104 cfu g–1 plant tissue), for Dickeya spp., P. atrosepticum and P. carotovorum subsp. carotovorum/P. wasabiae, respectively. Most of all, this assay allowed reliable detection and identification of soft rot and blackleg pathogens in naturally infected symptomatic and asymptomatic potato stem and progeny tuber samples collected from potato fields all over Poland.

Background: Dickeya solani is an important plant pathogenic bacterium causing severe losses in European potato production. This species draws a lot of attention due to its remarkable virulence, great devastating potential and easier spread in contrast to other Dickeya spp. In view of a high need for extensive studies on economically important soft rot Pectobacteriaceae, we performed a comparative genomics analysis on D. solani strains to search for genetic foundations that would explain the differences in the observed virulence levels within the D. solani population. Results: High quality assemblies of 8 de novo sequenced D. solani genomes have been obtained. Wholesequence comparison, ANIb, ANIm, Tetra and pangenome-oriented analyses performed on these genomes and the sequences of 14 additional strains revealed an exceptionally high level of homogeneity among the studied genetic material of D. solani strains. With the use of 22 genomes, the pangenome of D. solani, comprising 84.7% core, 7.2% accessory and 8.1% unique genes, has been almost completely determined, suggesting the presence of a nearly closed pangenome structure. Attribution of the genes included in the D. solani pangenome fractions to functional COG categories showed that higher percentages of accessory and unique pangenome parts in contrast to the core section are encountered in phage/mobile elements-and transcription-associated groups with the genome of RNS 05.1.2A strain having the most significant impact. Also, the first D. solani large-scale genome-wide phylogeny computed on concatenated core gene alignments is herein reported. Conclusions: The almost closed status of D. solani pangenome achieved in this work points to the fact that the unique gene pool of this species should no longer expand. Such a feature is characteristic of taxa whose