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Soft rot disease in potato is a major problem in fields and warehouses all over the world. Although it is known that bacteria from the genera Pectobacterium and Dickeya are the main causative agents of soft rot diseases, recent studies indicate the involvement of pectinolytic Bacillus and Paenibacillus in this disease. In the present research, samples of potato with soft rot symptoms were collected from eight governorates of Tunisia. Two hundred and seventy bacterial isolates were acquired from tubers. Twenty of the isolated strains indicted pectinolytic activity by forming deep cavities on crystal violet pectate medium. All pectinolytic isolates were able to macerate potato tuber tissue. Phenotypic characterisation showed that these isolates were Gram-positive bacilli, exhibiting pectinolytic, cellulolytic, proteolytic and amylolytic activity. The majority of the isolates indicated swimming and swarming motility. The application of API test, MALDI-TOF MS and 16S rDNA sequencing allowed for the assignment of nineteen of the tested isolates to the species Bacillus pumilus and one to the species Paenibacillus amyloliticus. To the best of our knowledge, this is the first report of soft rot in potato caused by pectinolytic B. pumilus in Tunisia.

Pectobacterium atrosepticum (Pba) are phytopathogenic, pectinolytic, non-sporulating, gram-negative rods causing blackleg and soft rot diseases in potato. They are ubiquitously present under temperate climate conditions and contribute to significant economic losses. Currently, there are no efficient control methods against soft rot Pectobacteriaceae (SRP). As limitation of the spread of SRP is solely prevention-based, we undertook broad characterization on both genotypic and phenotypic levels of the P. atrosepticum isolates acquired during monitoring of potato fields in Poland in 2013, 2014 and 2016. Of REP, ERIC and BOX-based genomic fingerprinting, the BOX-based approach turned out to be the most informative and allowed for classification of 118 Pba strains into six groups. The present study indicated that IV and I BOX profiles dominated among the investigated population. Finally, 23 Pba strains representing all BOX groups and originating from various growing seasons were selected for genotypic, phylogenetic and phenotypic studies. rpoS-based phylogeny revealed intraspecies variation (16 SNP sites) among the studied Pba strains, in contrast to the analyses relying on gyrA (1 SNP) and recA (0 SNP) sequences. Pba strains showed higher potency to macerate potato tissue at 20 °C than 28 °C. These isolates exhibited rather uniform production of plant cell wall degrading enzymes (pectinases, cellulases and proteases), lipases, siderophores and biofilm, however in the majority of cases inferior in comparison to Dickeya solani and Pectobacterium carotovorum strains. In conclusion, this study revealed genotypic and phenotypic uniformity in addition to high virulence potential of Pba strains isolated from potato fields during several growing seasons in Poland.

Soft rot and blackleg diseases, caused by pectinolytic bacteria from the numerous species of Dickeya and Pectobacterium, pose a serious threat to the world potato production. Besides, infections triggered by these pectinolytic bacteria lead to huge economic losses in the cultivation of other crops, vegetables, and ornamentals. Strains belonging to the genus Pectobacterium tend to be isolated from various environments such as rotten or asymptomatic plants, weeds, soil or water. The main virulence factors of these phytopathogenic bacteria involve plant cell wall degrading enzymes (PCWDEs) i.e. pectinases, cellulases and proteases. Among accessory virulence factors, there is often lipopolysaccharide (LPS) listed. This constituent of the external part of bacterial cell wall contains lipid A, inner and outer core in addition to O-polysaccharide (OPS). LPS plays an important role in plant-microbe interactions, in particular during the first step of pathogen recognition. In this study we present the chemical structure of OPS of the first Pectobacterium aquaticum strain (IFB5637) isolated from water in Poland. The OPS consists of two common hexoses, such as mannose and glucose, as well as an abequose (3,6-dideoxy-d-xylo-hexose), the first 3,6-dideoxyhexose identified among the Pectobacteriaceae family: According to our best knowledge this is the first determined structure of the OPS of P. aquaticum.

Bacteria from the genera Dickeya and Pectobacterium, the causative agents of soft rot and blackleg, trigger significant economic losses in potato production worldwide. Efficient struggle with these phytopathogens is highly challenging taking into consideration the lack of available control procedures. As only preventive measures are accessible, we decided to provide insight into the soft rot Pectobacteriaceae (SRP) present in Poland. During the growing seasons of 2013 and 2014, altogether 531 potato plants were collected from 138 seed potato fields and 23 storage facilities. Plant origin of the isolated bacteria, frequencies of coinfections with different species, the affected potato cultivars in addition to seasonal variation in the occurrence of SRP were studied. It was shown that bacteria from the Pectobacterium genus were abundant and outnumbered the ones classified to Dickeya spp. The vast majority of strains was isolated from the plant samples collected in July 2013 or in June–July 2014. The presence of all taxa of interest: Pectobacterium atrosepticum, Pectobacterium carotovorum, Pectobacterium parmentieri, Dickeya dianthicola and Dickeya solani were confirmed in July each year. We were able to isolate bacteria from the genus Dickeya and Pectobacterium from 35 out of 58 potato cultivars tested. The majority of SRP was isolated from potato stems, not from potato tubers. In four cases, coinfections of potato samples with even three diverse species of SRP, i.e. P. atrosepticum, P. carotovorum and P. parmentieri, were noted. It seems that since the first documented appearance of Dickeya solani in Poland in 2005, this pathogen has not played a dominating role in our country. The reported data describing the appearance and distribution of SRP in Poland might allow for prediction of the risks associated with infections initiated by these bacteria.

Doxycycline (DOX), an antibiotic commonly used in medicine and veterinary, is frequently detected in natural waterways. Exposition of bacteria to DOX residuals poses a selective pressure leading to a common occurrence of DOX-resistance genetic determinants among microorganisms, including virulent human pathogens. In view of diminishment of the available therapeutic options, we developed a continuous-flow reaction-discharge system generating pulse-modulated radio-frequency atmospheric pressure glow discharge (pm-rf-APGD) intended for DOX removal from liquid solutions. A Design of Experiment and a Response Surface Methodology were implemented in the optimisation procedure. The removal efficiency of DOX equalling 79 ± 4.5% and the resultant degradation products were identified by High-Performance Liquid Chromatography–Diode Array Detection, Liquid Chromatography Quadruple Time of Flight Mass Spectrometry, Ultraperformance Liquid Chromatography–Tandem Mass Spectrometry, total organic carbon, total nitrogen, Attenuated Total Reflectance Furrier Transform–Infrared, and UV/Vis-based methods. The pm-rf-APGD-treated DOX solution due to the generated Reactive Oxygen and Nitrogen Species either lost its antimicrobial properties towards Escherichia coli ATCC25922 or significantly decreased biocidal activities by 37% and 29% in relation to Staphylococcus haemolyticus ATCC29970 and Staphylococcus aureus ATCC25904, respectively. Future implementation of this efficient and eco-friendly antibiotic-degradation technology into wastewater purification systems is predicted.

Acquisition of high-quality bacterial genomes is fundamental, while having in mind investigation of subtitle intraspecies variation in addition to development of sensitive species-specific tools for detection and identification of the pathogens. In this view, Pacific Biosciences technology seems highly tempting taking into consideration over 10,000 bp length of the generated reads. In this work, we describe a bacterial genome assembly pipeline based on open-source software that might be handled also by non-bioinformaticians interested in transformation of sequencing data into reliable biological information. With the use of this method, we successfully closed six Dickeya solani genomes, while the assembly process was run just on a slightly improved desktop computer.

High availability of fast, cheap, and high-throughput next generation sequencing techniques resulted in acquisition of numerous de novo sequenced and assembled bacterial genomes. It rapidly became clear that digging out useful biological information from such a huge amount of data presents a considerable challenge. In this chapter we share our experience with utilization of several handy open source comparative genomic tools. All of them were applied in the studies focused on revealing inter- and intraspecies variation in pectinolytic plant pathogenic bacteria classified to Dickeya solani and Pectobacterium parmentieri. As the described software performed well on the species within the Pectobacteriaceae family, it presumably may be readily utilized on some closely related taxa from the Enterobacteriaceae family. First of all, implementation of various annotation software is discussed and compared. Then, tools computing whole genome comparisons including generation of circular juxtapositions of multiple sequences, revealing the order of synteny blocks or calculation of ANI or Tetra values are presented. Besides, web servers intended either for functional annotation of the genes of interest or for detection of genomic islands, plasmids, prophages, CRISPR/Cas are described. Last but not least, utilization of the software designed for pangenome studies and the further downstream analyses is explained. The presented work not only summarizes broad possibilities assured by the comparative genomic approach but also provides a user-friendly guide that might be easily followed by nonbioinformaticians interested in undertaking similar studies.

To the present day, no efficient plant protection method against economically important bacterial phytopathogens from the Pectobacteriaceae family has been implemented into agricultural practice. In this view, we have performed a multivariate optimization of the operating parameters of the reaction-discharge system, employing direct current atmospheric pressure glow discharge, generated in contact with a flowing liquid cathode (FLC-dc-APGD), for the production of a plasma-activated liquid (PAL) of defined physicochemical and anti-phytopathogenic properties. As a result, the effect of the operating parameters on the conductivity of PAL acquired under these conditions was assessed. The revealed optimal operating conditions, under which the PAL of the highest conductivity was obtained, were as follows: flow rate of the solution equaled 2.0 mL min−1, the discharge current was 30 mA, and the inorganic salt concentration (ammonium nitrate, NH4NO3) in the solution turned out to be 0.50% (m/w). The developed PAL exhibited bacteriostatic and bactericidal properties toward Dickeya solani IFB0099 and Pectobacterium atrosepticum IFB5103 strains, with minimal inhibitory and minimal bactericidal concentrations equaling 25%. After 24 h exposure to 25% PAL, 100% (1−2 × 106) of D. solani and P. atrosepticum cells lost viability. We attributed the antibacterial properties of PAL to the presence of deeply penetrating, reactive oxygen and nitrogen species (RONS), which were, in this case, OH, O, O3, H2O2, HO2, NH, N2, N2+, NO2−, NO3−, and NH4+. Putatively, the generated low-cost, eco-friendly, easy-to-store, and transport PAL, exhibiting the required antibacterial and physicochemical properties, may find numerous applications in the plant protection sector.

Pectinolytic bacteria from the genus Pectobacterium cause high economic losses in various crops, vegetables, and ornamentals including potato. Thus far, these strains have been isolated from distinct environments such as rotten or asymptomatic plants, soil, and waterways. The prevalence of soft rot Pectobacteriaceae in different depths of Pomeranian lakes was performed by a qualified scuba diver over 2 years of monitoring. It allowed for the isolation and broad characterization of a strain from the newly established species Pectobacterium aquaticum. Phylogenetic analysis on the sequences of dnaX and recA genes revealed the highest similarity of this strain to P. aquaticum CFBP 8637T. In addition to the determination of analytical profile index (API 20E), we discovered that this strain possesses a smooth form of a lipopolysaccharide with O-polysaccharide consisting of mannose, glucose, and abequose. Moreover, the characterized strain, described as P. aquaticum IFB5637, produced plant-cell–wall-degrading enzymes, such as pectinases, cellulases, proteases, and was capable of macerating potato and chicory tissues under laboratory conditions. In view of more frequent irrigation of seed potato fields resulting from the ongoing climate warming, it is important to monitor the occurrence of potential disease-causing agents in natural waterways.

Gold nanoparticles (AuNPs) are of scientific and industrial significance; however, the traditional synthesis methods employ toxic compounds. Hence, non-toxic and environmentally friendly AuNPs synthesis methods are of special interest. Here, AuNPs were produced using four solutions of fermented grape juices. UV/Vis absorption spectrophotometry and transmission electron microscopy indicated that AuNPs synthesized with a solution based on semi-sweet red grapes were mostly spherical with narrow size distribution (average diameter of 82.1 ± 36.2 nm). AuNPs of similar spherical morphology but smaller size were obtained using a solution based on semi-dry red grapes (57.1 ± 16.4 nm). A large variety of AuNPs shapes and broader size distribution were produced when solutions based on semi-sweet or dry white grapes were applied. In this case, the average sizes of the AuNPs were 271.6 ± 130.2 nm and 76.0 ± 47.2 nm, respectively. Using energy dispersive X-ray spectroscopy, Au, C, and O were detected, confirming formation of biogenic AuNPs in all cases. Mie theory calculations for AuNPs synthesized with the aid of solutions based on red grapes suggest that their optical properties are different and best suited for distinct downstream applications. Attenuated total reflectance Fourier transform infrared spectroscopy, the Folin-Ciocalteu assay, and the Bertrand’s method were used to examine bioactive compounds present in the solutions applied for synthesis. Phenolics, and to a lesser extent reducing sugars, were identified as likely playing a significant role in reduction and stabilization of the AuNPs. These results display the great potential of these solutions for green synthesis of size defined AuNPs, and illustrate that different grape varieties may be used to obtain AuNPs with unique properties. Keywords: Nanostructures, Bioreduction process, Phenolics, Reducing sugars, Mie scattering

Soft rot Pectobacteriaceae (SRP) are ubiquitous on earth as there are records of findings from all continents where host plants are grown. This chapter describes information on soft rot diseases on these continents. For some countries, detailed information is provided by local experts on the SRP present, their economic damage, and the management strategies applied for their control. The focus of the chapter is mainly on SRP as causative agents of potato blackleg, although in specific cases details are provided on SRP in other host plants. In Europe, the SRP cause important economic losses mainly on potato, with most species described in the literature being found. In Latin America significant losses are also reported due to potato diseases caused by various Dickeya and Pectobacterium species, while in Australia and Oceania, recent outbreaks of D. dianthicola in potato have resulted in high economic losses. In Asia, however, SRP cause economic losses mainly in vegetable crops other than potato, while in North America SRP cause diseases on a wide range of crops (including potato and ornamental plants) in both field and storage. In Africa SRP are only known to occur in 17 of the 54 African countries but where it is known, potato is the most affected crop.

Acquisition of high-quality bacterial genomes is fundamental, while having in mind investigation of subtitle intraspecies variation in addition to development of sensitive species-specific tools for detection and identification of the pathogens. In this view, Pacific Biosciences technology seems highly tempting taking into consideration over 10,000 bp length of the generated reads. In this work, we describe a bacterial genome assembly pipeline based on open-source software that might be handled also by non-bioinformaticians interested in transformation of sequencing data into reliable biological information. With the use of this method, we successfully closed six Dickeya solani genomes, while the assembly process was run just on a slightly improved desktop computer.

High availability of fast, cheap, and high-throughput next generation sequencing techniques resulted in acquisition of numerous de novo sequenced and assembled bacterial genomes. It rapidly became clear that digging out useful biological information from such a huge amount of data presents a considerable challenge. In this chapter we share our experience with utilization of several handy open source comparative genomic tools. All of them were applied in the studies focused on revealing inter- and intraspecies variation in pectinolytic plant pathogenic bacteria classified to Dickeya solani and Pectobacterium parmentieri. As the described software performed well on the species within the Pectobacteriaceae family, it presumably may be readily utilized on some closely related taxa from the Enterobacteriaceae family. First of all, implementation of various annotation software is discussed and compared. Then, tools computing whole genome comparisons including generation of circular juxtapositions of multiple sequences, revealing the order of synteny blocks or calculation of ANI or Tetra values are presented. Besides, web servers intended either for functional annotation of the genes of interest or for detection of genomic islands, plasmids, prophages, CRISPR/Cas are described. Last but not least, utilization of the software designed for pangenome studies and the further downstream analyses is explained. The presented work not only summarizes broad possibilities assured by the comparative genomic approach but also provides a user-friendly guide that might be easily followed by nonbioinformaticians interested in undertaking similar studies.

We present an optimized non-thermal atmospheric plasma (NTAP)-based reaction-discharge system that was applied for a continuous-flow treatment of apple juice (AJ). To optimize this system for a high-throughput production of AJ with ameliorated properties, the effect of several parameters was studied using design of experiments approach followed by the response surface methodology. Additionally, nutritional, physicochemical, microbiological and cytotoxic properties of resulting AJ were assessed. It was established that NTAP treatment of AJ led to rise in concentration of Ca, Fe, K, Mg, Na and Sr by 8–10% as well as Al, B, Ba, Cu, Mn and Zn by 11–15%. Additionally, the increased total phenolic content by ~ 11% in addition to the prolonged by up to 12 days shelf life of the product were observed. Moreover, it was found that the NTAP-treatment of AJ did not change the structure of organic compounds present in AJ, in addition to its °Brix value, color and ferric ion reducing antioxidant power. Furthermore, AJ subjected to NTAP did not show any cytotoxic activity towards non-malignant human intestinal epithelial cells but exhibited induced cell cytotoxicity in human colorectal adenocarcinoma cells. Our study provided arguments for future introduction of these types of preparations to the global market.

The species Dickeya aquatica was established in 2014 after the genomic characterization of the pectinolytic bacteria isolated from water. It was demonstrated that D. aquatica was able to cause symptoms of soft rot on the fruit of tomato and cucumber. According to earlier works, lipopolysaccharides are regarded as an important virulence factor of Pectobacteriaceae. An O-specific polysaccharide containing d -Fuc and l -Rha was obtained by mild acid hydrolysis of the lipopolysaccharide of D. aquatica IFB0154 (strain Dw044 isolated in Finland). By means of compositional analyses and NMR spectroscopy, the chemical repeating unit of the polymer was identified as a linear disaccharide of the structure shown below. The rhamnose residue was partially acetylated at O-2 or O-3. OAc (~40%) ↓2 →3)-α- d -Fucp-(1 → 4)-α- l -Rhap-(1→ ↑3 OAc (~30%) The O-polysaccharides isolated from Dickeya dianthicola IFB0485 and Dickeya zeae IPO946 have a different structure, identical to that previously described for several strains of Dickeya solani and Dickeya dadantii 3937.

Background: Dickeya solani was pointed as a significant trait to potato production in Europe and drew much of scientific attention due to remarkable virulence, great devastating potential and easier spread in contrast to other Dickeya spp. In a view of a high need for extensive studies on economically important soft rot Pectobacteriaceae, we performed a nearly conclusive pangenome 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. Whole-sequence comparison, ANIb, ANIm, Tetra and pangenome-oriented analyses performed on these genomes sequences and the sequences of 14 additional strains revealed 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 revealed 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 for taxa, whose representatives either occupy isolated ecological niches or lack efficient mechanisms for gene exchange and recombination, which seems rational concerning a strictly pathogenic species with clonal spread and population structure. Finally, no obvious correlations between the geographical origin of D. solani strains and their phylogeny was found, which might reflect the specificity of the international seed potato market.

Background Pectobacterium 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. Results The 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. Conclusions We 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 material The online version of this article (10.1186/s12864-018-5140-9) contains supplementary material, which is available to authorized users.

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.

The hazardous and endocrine disturbing compounds (EDCs), present in the wastewaters, notably impair reproduction efficacy of fish and amphibians. Disruption of hormonal homeostasis in humans is also postulated. For this reason, development of an efficient method for purification of wastewaters from these compounds is of the highest environmental concern. Therefore, we employed cold atmospheric pressure plasmas (CAPPs), operated in high-throughput mode, for removal of hazardous and EDCs from liquid disposals. It was found that the studied CAPPs led to effective degradation of all the analyzed EDCs, including Bisphenol A (BPA), Bisphenol S (BPS), 17-alpha-ethinylestradiol (EE2), Dexamethasone (DXM), Benzophenone (BPH), Dapsone (DPS), and 2-Nitrophenol (2-NIP), as assessed by high-performance liquid chromatography with diode array detection (HPLC-DAD). Degradation products of the studied EDCs were identified by applying ultra performance liquid chromatography – tandem mass spectrometry (UPLC-MS/MS). To reveal the CAPP-liquid interactions, the concentrations of total organic carbon (TOC) and total nitrogen (TN) in the analyzed EDCs samples were estimated. Then, an attenuated total reflectance Furrier transform – infrared (ATR FT-IR) spectroscopy and UV/Vis absorption spectrophotometry were employed to decipher the contribution of the studied CAPPs to decomposition of EDCs. The collected results suggest that the analysed CAPPs might be applied in the future in wastewater treatment plants.

Plant pathogenic bacteria cause significant economic losses in the global food production sector. To secure an adequate amount of high-quality nutrition for the growing human population, novel approaches need to be undertaken to combat plant disease-causing agents. As the currently available methods to eliminate bacterial phytopathogens are scarce, we evaluated the effectiveness and mechanism of action of a non-thermal atmospheric pressure plasma (NTAPP). It was ignited from a dielectric barrier discharge (DBD) operation in a plasma pencil, and applied for the first time for eradication of Dickeya and Pectobacterium spp., inoculated either on glass spheres or mung bean seeds. Furthermore, the impact of the DBD exposure on mung bean seeds germination and seedlings growth was estimated. The observed bacterial inactivation rates exceeded 3.07 logs. The two-minute DBD exposure stimulated by 3–4% the germination rate of mung bean seeds and by 13.4% subsequent early growth of the seedlings. On the contrary, a detrimental action of the four-minute DBD subjection on seed germination and early growth of the sprouts was noted shortly after the treatment. However, this effect was no longer observed or reduced to 9.7% after the 96 h incubation period. Due to the application of optical emission spectrometry (OES), transmission electron microscopy (TEM), and confocal laser scanning microscopy (CLSM), we found that the generated reactive oxygen and nitrogen species (RONS), i.e., N2, N2+, NO, OH, NH, and O, probably led to the denaturation and aggregation of DNA, proteins, and ribosomes. Furthermore, the cellular membrane disrupted, leading to an outflow of the cytoplasm from the DBD-exposed cells. This study suggests the potential applicability of NTAPPs as eco-friendly and innovative plant protection methods.

Dickeya spp. and Pectobacterium spp. are etiological agents of soft rot on crops, vegetables, and ornamentals. They also cause blackleg on potato. These pectinolytic phytopathogens are responsible for significant economic losses, mostly within the potato production sector. Importantly, there are no methods to eradicate these microorganisms once they have infected plant material. Solely preventive measures remain, including early detection and identification of the pathogens, monitoring of their spread in addition to planting certified seed material tested for latent infections. As proper identification of the causative agent allows for efficient limitation of disease spread, numerous detection and differentiation methods have been developed. Most commonly followed procedures involve: isolation of viable bacterial cells (alternatively post-enrichment) on semi-selective media, identification to species level by PCR (single, multiplex, Real time), serology or fatty acids profiling. Differentiation of the isolates is often accomplished by sequencing the housekeeping genes or molecular fingerprinting. In view of lowering total costs of next-generation sequencing (NGS), a huge amount of generated data reveals subtle differences between strains that have proven to be potentially useful for the establishment of specific novel detection pipelines. Successful implementation of molecular diagnostic methods is exemplified by 20-year studies on the populations of pectinolytic bacteria on potatoes in Poland. The presented work aims to gather the characteristics of Dickeya spp. and Pectobacterium spp. important for the identification process in addition to providing an overview of modern and newly developed specific, rapid, high-throughput and cost-effective screening methods for the detection and identification of these phytopathogens.

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.

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.

Development of efficient plant protection methods against bacterial phytopathogens subjected to compulsory control procedures under international legislation is of the highest concern having in mind expensiveness of enforced quarantine measures and threat of the infection spread in disease-free regions. In this study, fructose-stabilized silver nanoparticles (FRU-AgNPs) were produced using direct current atmospheric pressure glow discharge (dc-APGD) generated between the surface of a flowing liquid anode (FLA) solution and a pin-type tungsten cathode in a continuous flow reaction-discharge system. Resultant spherical and stable in time FRU-AgNPs exhibited average sizes of 14.9 &plusmn, 7.9 nm and 15.7 &plusmn, 2.0 nm, as assessed by transmission electron microscopy (TEM) and dynamic light scattering (DLS), respectively. Energy dispersive X-ray spectroscopy (EDX) analysis revealed that the obtained nanomaterial was composed of Ag while selected area electron diffraction (SAED) indicated that FRU-AgNPs had the face-centered cubic crystalline structure. The fabricated FRU-AgNPs show antibacterial properties against Erwinia amylovora, Clavibacter michiganensis, Ralstonia solanacearum, Xanthomonas campestris pv. campestris and Dickeya solani strains with minimal inhibitory concentrations (MICs) of 1.64 to 13.1 mg L&minus, 1 and minimal bactericidal concentrations (MBCs) from 3.29 to 26.3 mg L&minus, 1. Application of FRU-AgNPs might increase the repertoire of available control procedures against most devastating phytopathogens and as a result successfully limit their agricultural impact.

International audience; Bacteria belonging to the genera Dickeya and Pectobacterium are responsible for significant economic losses in a wide variety of crops and ornamentals. During last years, increasing losses in potato production have been attributed to the appearance of Dickeya solani. The D. solani strains investigated so far share genetic homogeneity, although different virulence levels were observed among strains of various origins. The purpose of this study was to investigate the genetic traits possibly related to the diverse virulence levels by means of comparative genomics. First, we developed a new genome assembly pipeline which allowed us to complete the D. solani genomes. Four de novo sequenced and ten publicly available genomes were used to identify the structure of the D. solani pangenome, in which 74.8 and 25.2% of genes were grouped into the core and dispensable genome, respectively. For D. solani panregulon analysis, we performed a binding site prediction for four transcription factors, namely CRP, KdgR, PecS and Fur, to detect the regulons of these virulence regulators. Most of the D. solani potential virulence factors were predicted to belong to the accessory regulons of CRP, KdgR, and PecS. Thus, some differences in gene expression could exist between D. solani strains. The comparison between a highly and a low virulent strain, IFB0099 and IFB0223, respectively, disclosed only small differences between their genomes but significant differences in the production of virulence factors like pectinases, cellulases and proteases, and in their mobility. The D. solani strains also diverge in the number and size of prophages present in their genomes. Another relevant difference is the disruption of the adhesin gene fhaB2 in the highly virulent strain. Strain IFB0223, which has a complete adhesin gene, is less mobile and less aggressive than IFB0099. This suggests that in this case, mobility rather than adherence is needed in order to trigger disease symptoms. This study highlights the utility of comparative genomics in predicting D. solani traits involved in the aggressiveness of this emerging plant pathogen.

Bacteria belonging to the genera

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.

Diseases caused by phytopathogenic bacteria are responsible for significant economic losses, and these bacteria spread through diverse pathways including waterways and industrial wastes. It is therefore of high interest to develop potent methods for their eradication. Here, antibacterial properties of direct current atmospheric pressure glow discharge (dc-APGD) generated in contact with flowing bacterial suspensions were examined against five species of phytopathogens. Complete eradication of Clavibacter michiganensis subsp. sepedonicus, Dickeya solani, and Xanthomonas campestris pv. campestris from suspensions of OD600 ≈ 0.1 was observed, while there was at least 3.43 logarithmic reduction in population densities of Pectobacterium atrosepticum and Pectobacterium carotovorum subsp. carotovorum. Analysis of plasma-chemical parameters of the dc-APGD system revealed its high rotational temperatures of 2,300 ± 100 K and 4,200 ± 200 K, as measured from N2 and OH molecular bands, respectively, electron temperature of 6,050 ± 400 K, vibrational temperature of 4000 ± 300 K, and high electron number density of 1.1 × 1015 cm-1 . In addition, plasma treatment led to formation of numerous reactive species and states in the treated liquid, including reactive nitrogen and oxygen species such as NOx , NH, H2 O2 , O2 , O, and OH. Further examination revealed that bactericidal activity of dc-APGD was primarily due to presence of these reactive species as well as to UVA, UVB, and UVC irradiation generated by the dc-APGD source. Plasma treatment also resulted in an increase in temperature (from 24.2 to 40.2 °C) and pH (from 6.0 to 10.8) of bacterial suspensions, although these changes had minor effects on cell viability. All results suggest that the newly developed dc-APGD-based system can be successfully implemented as a simple, rapid, efficient, and cost-effective disinfection method for liquids originating from different industrial and agricultural settings.

O-polysaccharides were isolated from lipopolysaccharides obtained from four different strains of plant pathogenic bacteria belonging to the species Dickeya solani : two of them were isolated in Poland (IFB0099 and IFB0158), the third in Germany (IFB0223) and the last one, D. solani Type Strain IPO2222, originated from the Netherlands. In addition, the O-polysaccharide of a closely related species D. dadantii strain 3937 was isolated. The purified polysaccharides of the five strains were analyzed using NMR spectroscopy and chemical methods. Sugar and methylation analyses, including absolute configuration assignment, together with NMR data revealed that all O-polysaccharides tested are homopolymers of 6-deoxy- d -altrose ( d -6dAlt) the following structure: →2)-β- d -6dAlt p -(1→

Animal manures are routinely applied to agricultural lands to improve crop yield, but the possibility to spread bacterial phytopathogens through field fertilization has not been considered yet. We monitored 49 cattle, horse, swine, sheep or chicken manure samples collected in 14 Polish voivodeships for the most important plant pathogenic bacteria - Ralstonia solanacearum (Rsol), Xanthomonas campestris pv. campestris (Xcc), Pectobacterium carotovorum subsp. carotovorum (Pcc), Pectobacterium atrosepticum (Pba), Erwinia amylovora (Eam), Clavibacter michiganensis subsp. sepedonicus (Cms) and Dickeya sp. (Dsp). All of the tested animal fertilizers were free of these pathogens. Subsequently, the growth dynamics of Pba, Pcc, Rsol, and Xcc in cattle, horse, swine, sheep and chicken manures sterilized either by autoclaving or filtration was evaluated. The investigated phytopathogens did not exhibit any growth in the poultry manure. However, the manure filtrates originating from other animals were suitable for microbial growth, which resulted in the optical density change of 0.03-0.22 reached within 26 h (48 h Rsol, 120 h Xcc), depending on bacterial species and the manure source. Pcc and Pba multiplied most efficiently in the cattle manure filtrate. These bacteria grew faster than Rsol and Xcc in all the tested manure samples, both the filtrates and the autoclaved semi-solid ones. Though the growth dynamics of investigated strains in different animal fertilizers was unequal, all of the tested bacterial plant pathogens were proven to use cattle, horse, swine and sheep manures as the sources of nutrients. These findings may contribute to further research on the alternative routes of spread of bacterial phytopathogens, especially because of the fact that the control of pectionolytic bacteria is only based on preventive methods.

O-Polysaccharides were isolated from the lipopolysaccharides of two strains of plant pathogenic bacteria Pectobacterium wasabiae isolated in Poland in 2013 (IFB5408 and IFB5427). The purified polysaccharides were analyzed using 1D and 2D NMR spectroscopy ((1)H, DQF-COSY, TOCSY, ROESY, HSQC, HSQC-TOCSY, and HMBC) and the chemical methods. Sugar and methylation analyses of native polysaccharides, absolute configuration assignment of constituent monosaccharides together with NMR spectroscopy data revealed that the chemical structures of both O-polysaccharides are the same.