Fifty-four Dickeya solani thermoregulated genes were identified using Tn5 transposon mutagenesis with an inducible gusA reporter system; 45 genes were up-regulated at 37°C, whereas nine were up-regulated at 18°C. The relative level of gene up-regulation ranged from 2-1200 and 5-650 U/mg total proteins at 18 and 37°C, respectively. Among the temperature-regulated loci, genes coding for proteins involved in fundamental bacterial metabolism, membrane-related proteins and pathogenicity-corresponding factors and several hypothetical unknown proteins were found. The mutants were tested for their pathogenicity in planta and for features known to be important for D. solani virulence viz. production of pectinolytic enzymes, cellulases, proteases, siderophores and auxins as well as for motility and the ability to form a biofilm. Eight Tn5 mutants, four up-regulated at high and four up-regulated at low temperature, expressed visible phenotypes including the decreased ability to cause symptoms on potato tubers and chicory leaves, impairment in phospholipase production and/or deficiency in biofilm formation. The implications of environmental temperature 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. 10 3 -10 4 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.

Pectobacterium parmentieri is a pectinolytic plant pathogenic bacterium causing high economic losses of cultivated plants. The highly devastating potential of this phytopathogen results from the efficient production of plant cell wall-degrading enzymes, i.e., pectinases, cellulases and proteases, in addition to the impact of accessory virulence factors such as motility, siderophores, biofilm and lipopolysaccharide (LPS). LPS belongs to pathogen-associated molecular patterns (PAMPs) and plays an important role in plant colonization and interaction with the defense systems of the host. Therefore, we decided to investigate the heterogeneity of O-polysaccharides (OPS) of LPS of different strains of P. parmentieri, in search of an association between the selected genomic and phenotypic features of the strains that share an identical structure of the OPS molecule. In the current study, OPS were isolated from the LPS of two P. parmentieri strains obtained either in Finland in the 1980s (SCC3193) or in Poland in 2013 (IFB5432). The purified polysaccharides were analyzed by utilizing 1D and 2D NMR spectroscopy (1H, DQF-COSY, TOCSY, ROESY, HSQC, HSQC-TOCSY and HMBC) in addition to chemical methods. Sugar and methylation analyses of native polysaccharides, absolute configuration assignment of constituent monosaccharides and NMR spectroscopy data revealed that these two P. parmentieri strains isolated in different countries possess the same structure of OPS with a very rare residue of 5,7-diamino-3,5,7,9-tetradeoxy-l-glycero-l-manno-non-2-ulosonic acid (pseudaminic acid) substituted in the position C-8: →3)-β-d-Galf-(1→3)-α-d-Galp-(1→8)-β-Pse4Ac5Ac7Ac-(2→6)-α-d-Glcp-(1→6)-β-d-Glcp-(1→. The previous study indicated that three other P. parmentieri strains, namely IFB5427, IFB5408 and IFB5443, exhibit a different OPS molecule than SCC3193 and IFB5432. The conducted biodiversity-oriented assays revealed that the P. parmentieri IFB5427 and IFB5408 strains possessing the same OPS structure yielded the highest genome-wide similarity, according to average nucleotide identity analyses, in addition to the greatest ability to macerate chicory tissue among the studied P. parmentieri strains. The current research demonstrated a novel OPS structure, characteristic of at least two P. parmentieri strains (SCC3193 and IFB5432), and discussed the observed heterogenicity in the OPS of P. parmentieri in a broad genomic and phenotype-related context.

Temperature is one of the critical factors affecting gene expression in bacteria. Despite the general interest in the link between bacterial phenotypes and environmental temperature, little is known about temperature-dependent gene expression in plant pathogenic Pectobacterium atrosepticum, a causative agent of potato blackleg and tuber soft rot worldwide. In this study, twenty-nine P. atrosepticum SCRI1043 thermoregulated genes were identified using Tn5-based transposon mutagenesis coupled with an inducible promotorless gusA gene as a reporter. From the pool of 29 genes, 14 were up-regulated at 18 °C, whereas 15 other genes were up-regulated at 28 °C. Among the thermoregulated loci, genes involved in primary bacterial metabolism, membrane-related proteins, fitness-corresponding factors, and several hypothetical proteins were found. The Tn5 mutants were tested for their pathogenicity in planta and for features that are likely to remain important for the pathogen to succeed in the (plant) environment. Five Tn5 mutants expressed visible phenotypes differentiating these mutants from the phenotype of the SCRI1043 wild-type strain. The gene disruptions in the Tn5 transposon mutants caused alterations in bacterial generation time, ability to form a biofilm, production of lipopolysaccharides, and virulence on potato tuber slices. The consequences of environmental temperature on the ability of P. atrosepticum to cause disease symptoms in potato are discussed.

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