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The adaptive proliferation of bacteria, or cell division in the absence of an exogenous substrate, is controlled by density-dependent mechanisms with the participation of AHL- and AI-2-dependent quorum sensing systems. Along with the signaling molecules of these bacterial communication systems, bacterial metabolites that are permanently released during microbial metabolism, for example, CO2, can also participate in the regulation and can serve as biomarkers of cell density. It has been found that carbon dioxide is necessary to the adaptive proliferation initiation, and the elevated atmospheric CO2 content causes this process to terminate prematurely. Thus, CO2 is able to regulate bacterial adaptive reactions and is probably one of the signals involved in the initiation and termination of the process of adaptive proliferation. It was shown that CO2 in the form of the bicarbonate ion can activate the cAMP-dependent signaling cascade and is also included in the bacterial cell mass. [ABSTRACT FROM AUTHOR]

Potato (Solanum tuberosum L.) is considered one of the main food crops in many countries worldwide. The present work was planned to manage potato black-leg disease using organic treatments as well as mineral-nitrogenous treatments, in addition to examining their effects on the pathogenic bacterium (Pectobacterium atrosepticum) population under artificial inoculation conditions. The obtained data indicated that applications of cabbage, onion and peppermint dry ground leaves as organic matters, and applications of ammonium super-phosphate, potassium sulfate and urea as mineral-nitrogenous fertilizers reduced P. atrosepticum population and black-leg disease of potato compared with the control treatment. In addition, these treatments led to increasing potato yield, while the beneficial effects increased with increasing their doses compared with the control treatment. Meanwhile, the organic matter appeared highly effective compared with mineral-nitrogenous fertilizers in disease reduction. However, mineral-nitrogenous fertilizers appeared highly effective in increasing potato yield. Meanwhile, onion dry ground leaves (organic matter) and urea (nitrogenous fertilizer) were the most effective on the pathogenic bacterium population and severity of the disease compared with other treatments

Potato (Solanum tuberosum L.) is considered one of the main food crops in many countries worldwide. The present work was planned to manage potato black-leg disease using organic treatments as well as mineralnitrogenous treatments, in addition to examining their effects on the pathogenic bacterium (Pectobacterium atrosepticum) population under artificial inoculation conditions. The obtained data indicated that applications of cabbage, onion and peppermint dry ground leaves as organic matters, and applications of ammonium super-phosphate, potassium sulfate and urea as mineral-nitrogenous fertilizers reduced P. atrosepticum population and black-leg disease of potato compared with the control treatment. In addition, these treatments led to increasing potato yield, while the beneficial effects increased with increasing their doses compared with the control treatment. Meanwhile, the organic matters appeared highly effective compared with mineral-nitrogenous fertilizers in disease reduction. However, mineral-nitrogenous fertilizers appeared highly effective in increasing potato yield. Meanwhile, onion dry ground leaves (organic matter) and urea (nitrogenous fertilizer) were the most effective on the pathogenic bacterium population and severity of the disease compared with other treatments. [ABSTRACT FROM AUTHOR]

Potato farming is a vital component of food security and the economic stability especially in the under developing countries but it faces many challenges in production, blackleg disease caused by Pectobacterium atrosepticum (Pa) is one of the main reason for damaging crop yield of the potato. Effective management strategies are essential to control these losses and to get sustainable potato crop yield. This study was focused on characterizing the Pa and the investigating new chemical options for its management. The research was involved a systematic survey across the three district of Punjab, Pakistan (Khanewal, Okara, and Multan) to collect samples exhibiting the black leg symptoms. These samples were analyzed in the laboratory where gram-negative bacteria were isolated and identified through biochemical and pathogenicity tests for Pa. DNA sequencing further confirmed these isolates of Pa strains. Six different chemicals were tested to control blackleg problem in both vitro and vivo at different concentrations. In vitro experiment, Cordate demonstrated the highest efficacy with a maximum inhibition zones of 17.139 mm, followed by Air One (13.778 mm), Profiler (10.167 mm), Blue Copper (7.7778 mm), Spot Fix (7.6689 mm), and Strider (7.0667 mm). In vivo, Cordate maintained its effectiveness with the lowest disease incidence of 14.76%, followed by Blue Copper (17.49%), Air One (16.98%), Spot Fix (20.67%), Profiler (21.45%), Strider (24.99%), and the control group (43.00%). The results highlight Cordate's potential as a most effective chemical against Pa, offering promising role for managing blackleg disease in potato and to improve overall productivity. [ABSTRACT FROM AUTHOR]

Pectobacterium spp. cause blackleg and soft rot on potato worldwide. Potato is a staple crop in China, and blackleg and soft rot diseases caused by Pectobacterium spp. have increased significantly as potato cultivation expanded rapidly in the last decade. This study was conducted to isolate and characterize the lytic bacteriophages infecting the major pathogen causing potato blackleg and soft rot, Pectobacterium atrosepticum (P. a.), in sewage water and bulk soil samples collected in the cities Wuhan and Datong in China. Three lytic bacteriophages were obtained and named Pectobacterium Phages 1, 2, and 7 (PP1, 2 and 7). Transmission electron microscopic analysis revealed that PP1 and PP7 were similar in size with a head diameter (ca. 70–80 nm, n = 10), and PP2 had a smaller head size (ca. 50 nm, n = 10). PP2 and PP7 had a short tail, and no tail was observed for PP1. The adsorption of phages PP1, PP2, and PP7 to P. a. after 18, 27, and 18 min was 98, 99 and 98%, respectively. The latent periods for PP1, PP2, and PP7 were 20, 40, and 50 min, respectively. In vitro bacterial challenge assays showed that these three phages were able to infect P. a. 1 h after mixing. The potential of these bacteriophages to protect potato seedlings was tested. After 27 days of inoculation, seedlings inoculated with water and those sprayed with phages alone exhibited healthy growth with no symptoms. Conversely, seedlings solely inoculated with P. a. displayed a significantly lower survival rate compared to the water-inoculated seedlings. However, seedlings co-inoculated with P. a. and single or mixed phages showed a substantial improvement in survival rates by 33% and 30%, respectively, compared to the group without phage treatment. [ABSTRACT FROM AUTHOR]

Potato farming is a vital component of food security and the economic stability especially in the under developing countries but it faces many challenges in production, blackleg disease caused by Pectobacterium atrosepticum (Pa) is one of the main reason for damaging crop yield of the potato. Effective management strategies are essential to control these losses and to get sustainable potato crop yield. This study was focused on characterizing the Pa and the investigating new chemical options for its management. The research was involved a systematic survey across the three district of Punjab, Pakistan (Khanewal, Okara, and Multan) to collect samples exhibiting the black leg symptoms. These samples were analyzed in the laboratory where gram-negative bacteria were isolated and identified through biochemical and pathogenicity tests for Pa. DNA sequencing further confirmed these isolates of Pa strains. Six different chemicals were tested to control blackleg problem in both vitro and vivo at different concentrations. In vitro experiment, Cordate demonstrated the highest efficacy with a maximum inhibition zones of 17.139 mm, followed by Air One (13.778 mm), Profiler (10.167 mm), Blue Copper (7.7778 mm), Spot Fix (7.6689 mm), and Strider (7.0667 mm). In vivo, Cordate maintained its effectiveness with the lowest disease incidence of 14.76%, followed by Blue Copper (17.49%), Air One (16.98%), Spot Fix (20.67%), Profiler (21.45%), Strider (24.99%), and the control group (43.00%). The results highlight Cordate’s potential as a most effective chemical against Pa, offering promising role for managing blackleg disease in potato and to improve overall productivity.

The alternative sigma factor RpoS is considered to be one of the major regulators providing stress resistance and cross-protection in bacteria. In phytopathogenic bacteria, the effects of RpoS have not been analyzed with regard to cross-protection, and genes whose expression is directly or indirectly controlled by RpoS have not been determined at the whole-transcriptome level. Our study aimed to determine RpoS-regulated genes and phenotypes in the phytopathogenic bacterium Pectobacterium atrosepticum. Knockout of the rpoS gene in P. atrosepticum affected the long-term starvation response, cross-protection, and virulence toward plants with enhanced immune status. The whole-transcriptome profiles of the wild-type P. atrosepticum strain and its ΔrpoS mutant were compared under different experimental conditions, and functional gene groups whose expression was affected by RpoS were determined. The RpoS promoter motif was inferred within the promoter regions of the genes affected by rpoS deletion, and the P. atrosepticum RpoS regulon was predicted. Based on RpoS-controlled phenotypes, transcriptome profiles, and RpoS regulon composition, the regulatory role of RpoS in P. atrosepticum is discussed. [ABSTRACT FROM AUTHOR]

Potato is one of the most important food crops in the world. Cultivation of potatoes is an important segment of the agriculture and economy as a whole in Ukraine. Bacterial diseases such as ring rot caused by Clavibacter sepedonicus, as well as soft rot and blackleg caused by Pectobacterium atrosepticum evoke tremendous losses of the potato crop. Stored seed tubers are the main source of accumulating pathogens in latent form. In addition, potato crop yield and quality are significantly affected by weather conditions. Along with affecting potatoes directly, meteorological factors such as air temperature and humidity can also impact tuber infestation with bacterial pathogens. The aim of the study was to monitor the prevalence of bacterial ring rot pathogen C. sepedonicus and soft rot pathogen P. atrosepticum in potato tubers on the territory of Ukraine in 2020 and 2021 years and assess its association with different weather conditions. Methods. Potato seed lots from newly harvested crops in eight regions of Ukraine (Odesa, Kyiv, Donetsk, Kherson, Cherkasy, Mykolaiv, Dnipropetrovsk, and Zhytomyr regions) without visual symptoms of diseases were used for the experiments. The detection of C. sepedonicus and P. atrosepticum was conducted by the immunochemical method DAS ELISA. Basal meteorological data (the sum of precipitation (mm/month) and average air temperature (°C) were obtained from the local meteorological stations. The amplitude of air temperature and the sum of precipitation during potato planting, growing, and harvesting seasons, as well as Sielianinov's hydrothermal coefficient (K) were additionally calculated. The Spearman correlation coefficient was used for the analysis of the correlation between pathogen prevalence and weather parameters. Results. Both ring rot pathogen C. sepedonicus and soft rot pathogen P. atrosepticum were detected in potato lots from all surveyed regions, but the prevalence of latent infestation varied between 2020 and 2021. In 2020, the highest soft rot pathogen prevalence values were registered for Mykolaiv, Kyiv, and Cherkasy regions (21.7, 10.5, and 10% respectively), where high rainfall levels in May and June were observed. In 2021, potato planting, growing, and harvesting seasons were marked by significantly higher precipitation levels, as compared to 2020, in all regions. Average occurrence values for P. atrosepticum in potato lots were 1.7 times higher than that in 2020. The highest prevalence (40%) was observed in the Donetsk region wiThextremely high air humidity during the potato growing period. Strong positive correlation (r = 0.721) was found between soft rot pathogen prevalence and K coefficients during the potato growing season, as well as between P. atrosepticum prevalence and the sum of precipitation during planting and early growing seasons. Prevalence of ring rot pathogen didn't differ significantly in 2020 and 2021. The highest C. sepedonicus prevalence was observed in 2020 in the Kyiv region (18.9%), and in 2021 -- in the Donetsk region (20%). A moderate positive correlation (r = 0.591) was found between pathogen prevalence and air temperature during planting and early growing season. Conclusions. Weather factors such as air temperature and humidity over the planting and early growing potato season can influence progeny tuber infestation with P. atrosepticum and C. sepedonicus. Knowledge of the effect of the changing weather conditions on the susceptibility of potato varieties with different genetic features to infestation with soft rot and ring rot pathogens warrants future investigation since it is crucially important for developing measures for disease control by potato producers. [ABSTRACT FROM AUTHOR]

An intragene involves the rearrangement of gene configurations based on switching coding sequences and regulatory regions within a species. The overexpression of specific GSL (Gibberellin Stimulated-Like) genes is known to confer enhanced disease resistance. A potato intragenic expression cassette, with the potato GSL1 gene placed under the regulatory control of the light-inducible Lhca3 gene from potato, was designed and constructed. The resulting intragene was transformed using Agrobacterium-mediated gene transfer into potato cultivar Iwa to investigate whether overexpression of the potato GSL1 intragene can confer disease resistance. The transgenic status of the resulting plants was confirmed by PCR. The majority of the transgenic potato lines were determined to overexpress the GSL1 gene at the mRNA level using quantitative RT–PCR analysis. From these results, seven lines were selected for further characterisation and thoroughly evaluated in bioassays for resistance to Pectobacterium atrosepticum which causes blackleg disease in potato. These pathogenicity bioassays demonstrated that high transcriptional overexpression of GSL1 confers high resistance to blackleg disease. This confirms a functional role of the GSL1 gene in plant defence against pathogens and provides an intragenic approach for disease resistance in potatoes. [ABSTRACT FROM AUTHOR]

Bacterial adaptation is regulated at the population level with the involvement of intercellular communication (quorum sensing). When the population density is insufficient for adaptation under starvation, bacteria can adjust it to a quorum level through cell divisions at the expense of endogenous resources. This phenomenon has been described for the phytopathogenic bacterium Pectobacterium atrosepticum (Pba), and it is called, in our study, adaptive proliferation. An important attribute of adaptive proliferation is its timely termination, which is necessary to prevent the waste of endogenous resources when the required level of population density is achieved. However, metabolites that provide the termination of adaptive proliferation remained unidentified. We tested the hypothesis of whether quorum sensing-related autoinducers prime the termination of adaptive proliferation and assessed whether adaptive proliferation is a common phenomenon in the bacterial world. We showed that both known Pba quorum sensing-related autoinducers act synergistically and mutually compensatory to provide the timely termination of adaptive proliferation and formation of cross-protection. We also demonstrated that adaptive proliferation is implemented by bacteria of many genera and that bacteria with similar quorum sensing-related autoinducers have similar signaling backgrounds that prime the termination of adaptive proliferation, enabling the collaborative regulation of this adaptive program in multispecies communities. [ABSTRACT FROM AUTHOR]

Bacterial attachment on root surfaces is an important step preceding the colonization or internalization and subsequent infection of plants by pathogens. Unfortunately, bacterial attachment is not well understood because the phenomenon is difficult to observe. Here we assessed whether this limitation could be overcome using optical trapping approaches. We have developed a system based on counter-propagating beams and studied its ability to guide Pectobacterium atrosepticum (Pba) cells to different root cell types within the interstices of transparent soils. Bacterial cells were successfully trapped and guided to root hair cells, epidermal cells, border cells, and tissues damaged by laser ablation. Finally, we used the system to quantify the bacterial cell detachment rate of Pba cells on root surfaces following reversible attachment. Optical trapping techniques could greatly enhance our ability to deterministically characterize mechanisms linked to attachment and formation of biofilms in the rhizosphere. [ABSTRACT FROM AUTHOR]

Cyclamen (Cyclamen persicum Mill.) is an ornamental plant affected by various phytosanitary issues including bacterial soft rots, which cause crop losses of 20% worldwide. In Colombia, however, the symptoms of bacterial soft rots are not well described and the causal agents are unknown. This study aimed to characterize the bacterial soft rot disease according to its symptoms, and identify its causal agents at the species level. For this purpose, we visited nurseries in the municipality of San Antonio del Tequendama, Colombia, and selected diseased plants to describe symptoms, and to isolate and characterize pathogens using morphological, biochemical, and molecular methods. Soft rotting activity in potato tubers and pathogenicity in cyclamen plants also was evaluated. Six symptoms were found to be associated with the disease, namely: wilt, soft corm, bacterial exudates on corms, root rot, soft rot and necrotic soft rot on petioles and peduncles. Of 47 isolates, seven were pathogenic: one from the soft corms; two from petioles with soft rot; two from petioles with necrotic soft rot; and two from root rot. A multiple correspondence analysis clustered isolates in the genera Pseudomonas, Dickeya and Pectobacterium based on the morphological, biochemical, and pathogenic characteristics. A multi-locus analysis (MLSA) based on sequencing of the 16S rDNA region and housekeeping genes (gapA, icdA, mdh, pgi, rpoB and rpoD) confirmed the bacterial species Pseudomonas asplenii, Dickeya solani and Pectobacterium atrosepticum as the causal agents of cyclamen bacterial soft rot in Colombia. [ABSTRACT FROM AUTHOR]

ABSTRACT Acetylcholine is a central biological signal molecule present in all kingdoms of life. In humans, acetylcholine is the primary neurotransmitter of the peripheral nervous system; it mediates signal transmission at neuromuscular junctions. Here, we show that the opportunistic human pathogen Pseudomonas aeruginosa exhibits chemoattraction toward acetylcholine over a concentration range of 1 μM to 100 mM. The maximal magnitude of the response was superior to that of many other P. aeruginosa chemoeffectors. We demonstrate that this chemoattraction is mediated by the PctD (PA4633) chemoreceptor. Using microcalorimetry, we show that the PctD ligand-binding domain (LBD) binds acetylcholine with a equilibrium dissociation constant (KD) of 23 μM. It also binds choline and with lower affinity betaine. Highly sensitive responses to acetylcholine and choline, and less sensitive responses to betaine and l-carnitine, were observed in Escherichia coli expressing a chimeric receptor comprising the PctD-LBD fused to the Tar chemoreceptor signaling domain. We also identified the PacA (ECA_RS10935) chemoreceptor of the phytopathogen Pectobacterium atrosepticum, which binds choline and betaine but fails to recognize acetylcholine. To identify the molecular determinants for acetylcholine recognition, we report high-resolution structures of PctD-LBD (with bound acetylcholine and choline) and PacA-LBD (with bound betaine). We identified an amino acid motif in PctD-LBD that interacts with the acetylcholine tail. This motif is absent in PacA-LBD. Significant acetylcholine chemotaxis was also detected in the plant pathogens Agrobacterium tumefaciens and Dickeya solani. To the best of our knowledge, this is the first report of acetylcholine chemotaxis and extends the range of host signals perceived by bacterial chemoreceptors. IMPORTANCE P. aeruginosa causes a significant number of deaths annually worldwide. For many pathogens, chemotaxis plays an import role in the initial stages of infection, and deciphering the key chomoeffectors and their cognate chemoreceptors may permit the development of strategies to inhibit this process. Genome analyses have shown that many bacteria possess a large number of chemoreceptors. The chemoeffectors recognized by the large majority of chemoreceptors are unknown. However, identifying these chemoeffectors is crucial for deciphering the evolutionary forces that have shaped chemosensory signaling mechanisms in bacteria with different lifestyles. Our current understanding of the relationship between bacterial lifestyle and chemoreceptor repertoire is limited, and this work contributes to closing this gap in our knowledge. By expanding the list of known chemoeffectors and chemoreceptors, progress is made toward identifying functional receptor homologs in other bacteria.

PCR and qPCR are important methods for plant disease diagnosis and quantification of pathogen populations in host tissues or soil. For most plant diseases, DNA extracted from infected tissue or soil is a prerequisite for PCR or qPCR. The efficiency of DNA extraction should have a direct impact on the sensitivity of PCR and qPCR systems. In this study, plant pathogen DNA extraction efficiencies were evaluated based on three plant disease systems and three DNA extraction methods. DNA of bacterial (Pectobacterium atrosepticum), protist (Plasmodiophora brassicae) and fungal (Botrytis cinerea) pathogens was extracted and aliquots were prepared. Aliquots of the DNA were mixed with healthy tissues of the corresponding host plant and DNA was extracted again from the mixture. The resultant mixed DNA, as well as the original pathogen DNA, were analysed by qPCR to assess the quantities of the pathogen DNA. Extraction efficiency was calculated based on the qPCR Cq values of the mixed DNA and the original pathogen DNA for each extraction method on each pathogen. Our results indicated the pathogen DNA extraction efficiencies were generally less than, or near, 50%. This result called attention to the importance of: 1) including the DNA extraction efficiency in the evaluation and announcement of new qPCR diagnostic systems, and 2) developing and using high efficient DNA extraction methods in plant disease diagnosis and pathogen quantification. [ABSTRACT FROM AUTHOR]

Potato soft rot Pectobacteriaceae (SRP) cause large yield losses and are persistent in seed lots once established. In Norway, different Pectobacterium species are the predominant cause of soft rot and blackleg disease. This work aimed to evaluate the potential of real-time PCR for quantification of SRP in seed tubers, as well as investigating the status of potato seed health with respect to SRP in Norway. A total of 34 seed potato lots, including certified seeds, was grown and monitored over three consecutive years. All seed lots contained a quantifiable amount of SRP after enrichment, with very few subsamples being free of the pathogens. A high SRP prevalence based on a qPCR assay, as well as a high symptom incidence in certified seeds were observed, suggesting that current criteria for seed certification are insufficient to determine tuber health and predict field outcomes. Pectobacterium atrosepticum was the most abundant species in the examined seed lots and present in all lots. Consistently good performance of first generation seed lots with respect to blackleg and soft rot incidence, as well as low quantity of SRP in these seed lots demonstrated the importance of clean seed potatoes. Weather conditions during the growing season seemed to govern disease incidence and SRP prevalence more than seed grade. The impact of temperature, potato cultivar and Pectobacterium species on tuber soft rot development were further examined in tuber infection experiments, which showed that temperature was the most important factor in nearly all cultivars. Large-scale quantification of latent infection and predictive models that include contributing factors like weather, infecting bacterial species and cultivar are needed to reduce soft rot and blackleg. [ABSTRACT FROM AUTHOR]

Pectobacterium atrosepticum is a species of plant pathogenic bacteria responsible for significant losses in potato production worldwide. Pectobacterium atrosepticum can cause blackleg disease on potato stems as well as the tuber disease termed potato soft rot. Methods for the effective control of these diseases are limited and are primarily based on good agricultural practices. Bacteriophages, viruses of bacteria, could be used as an alternative, environmentally friendly, control measure. Here, we describe the isolation and characterization of 29 phages virulent to P. atrosepticum. The phages belong to 12 different species based on a 95% sequence identity cut-off. Furthermore, based on sequence diversity and propagation results, we selected six of these phages to form a phage cocktail. The phages in the cocktail was tested on a number of P. atrosepticum strains in order to determine their host range. The phages was found to lyse 93% of the tested strains. The cocktail was subsequently tested for its effectiveness in combatting potato soft rot under simulated storage conditions. Use of the phage cocktail reduced both disease incidence and disease severity by 61% and 64%, respectively, strongly indicating that phage biocontrol has the potential to reduce the economic impact of soft rot in potato production. A phage cocktail consisting of six bacteriophages can significantly reduce soft rot symptoms on potato tubers infected with Pectobacterium atrosepticum under simulated storage conditions. [ABSTRACT FROM AUTHOR]

The recent taxonomic diversification of bacterial genera Pectobacterium and Dickeya, which cause soft rot in plants, focuses attention on the need for improvement of existing methods for the detection and differentiation of these phytopathogens. This research presents a whole genome-based approach to the selection of marker sequences unique to particular species of Pectobacterium. The quantitative real-time PCR assay developed is selective in the context of all tested Pectobacterium atrosepticum strains and is able to detect fewer than 102 copies of target DNA per reaction. The presence of plant DNA extract did not affect the sensitivity of the assay.

Soft rot caused by Pectobacterium species is a devastating plant disease poorly characterized in terms of host plant responses. In this study, changes in the transcriptome of tobacco plants after infection with Pectobacterium atrosepticum (Pba) were analyzed using RNA-Seq. To draw a comprehensive and nontrivially itemized picture of physiological events in Pba-infected plants and to reveal novel potential molecular “players” in plant–Pba interactions, an original functional gene classification was performed. The classifications present in various databases were merged, enriched by “missed” genes, and divided into subcategories. Particular changes in plant cell wall-related processes, perturbations in hormonal and other regulatory systems, and alterations in primary, secondary, and redox metabolism were elucidated in terms of gene expression. Special attention was paid to the prediction of transcription factors (TFs) involved in the disease’s development. Herewith, gene expression was analyzed within the predicted TF regulons assembled at the whole-genome level based on the presence of particular cis-regulatory elements (CREs) in gene promoters. Several TFs, whose regulons were enriched by differentially expressed genes, were considered to be potential master regulators of Pba-induced plant responses. Differential regulation of genes belonging to a particular multigene family and encoding cognate proteins was explained by the presence/absence of the particular CRE in gene promoters.

To date, Certrevirus is one of two genera of bacteriophage (phage), with phages infecting Pectobacterium atrosepticum, an economically important phytopathogen that causes potato blackleg and soft rot disease. This study provides a detailed description of Pectobacterium phage CB7 (vB_PatM_CB7), which specifically infects P. atrosepticum. Host range, morphology, latent period, burst size and stability at different conditions of temperature and pH were examined. Analysis of its genome (142.8 kbp) shows that the phage forms a new species of Certrevirus, sharing sequence similarity with other members, highlighting conservation within the genus. Conserved elements include a putative early promoter like that of the Escherichia coli sigma70 promoter, which was found to be shared with other genus members. A number of dissimilarities were observed, relating to DNA methylation and nucleotide metabolism. Some members do not have homologues of a cytosine methylase and anaerobic nucleotide reductase subunits NrdD and NrdG, respectively. Furthermore, the genome of CB7 contains one of the largest numbers of homing endonucleases described in a single phage genome in the literature to date, with a total of 23 belonging to the HNH and LAGLIDADG families. Analysis by RT-PCR of the HNH homing endonuclease residing within introns of genes for the large terminase, DNA polymerase, ribonucleotide reductase subunits NrdA and NrdB show that they are splicing competent. Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) was also performed on the virion of CB7, allowing the identification of 26 structural proteins—20 of which were found to be shared with the type phages of the genera of Vequintavirus and Seunavirus. The results of this study provide greater insights into the phages of the Certrevirus genus as well as the subfamily Vequintavirinae.

Pectinolytic bacteria were isolated from 48 potato plants showing the symptoms of blackleg and collected in different fields of commercial potato production areas at Samsun, Amasya, Corum and Yozgat provinces in Turkey in 2015. The survey resulted in the isolation of 26 pectinolytic strains that belonged to P. atrosepticum, P. carotovorum subsp. brasiliense, P. carotovorum subsp. carotovorum and P. parmentieri species based on molecular identification with species-specific PCR and phenotypic characterization. The identified strains indicated typical biochemical characteristics of the assigned species. For 16 representative Pectobacterium isolates 10 unique rep-PCR band patterns were obtained. The 16S rRNA and recA and gapA gene fragment sequencing confirmed the species identity of the isolates. The phenotypic characterization of the strains revealed that for all assays but one (cellulase, protease activity, swimming but not swarming), the tested Pectobacterium species were significantly different from each other proving the diversity of the strains belonging to these genera. Recent outbreaks of blackleg and/or soft rot in potato production areas in Turkey may pose a threat on other crops, as tomato, pepper, cucumber, onion, cabbage, broccoli and sugar beet are cultivated in the same provinces. [ABSTRACT FROM AUTHOR]

The quorum quenching (QQ) activity of endophytic bacteria associated with medicinal plants was explored. Extracts of the Gram-negative Enterobacter sp. CS66 possessed potent N-acylhomoserine lactone (AHL) hydrolytic activity in vitro. Using degenerate primers, we PCR-amplified an open reading frame (denoted aiiE) from CS66 that was 96% identical to the well-characterised AHL-lactonase AiiA from Bacillus thuringiensis, but only 30% was identical to AHL-lactonases from other Gram-negative species. This confirms that close AiiA homologs can be found in both Gram-positive and Gram-negative bacteria. Purified AiiE exhibited potent AHL-lactonase activity against a broad range of AHLs. Furthermore, aiiE was able to reduce the production of secreted plant cell wall-degrading hydrolytic enzymes when expressed in trans in the economically important plant pathogen, Pectobacterium atrosepticum. Our results indicate the presence of a novel AHL-lactonase in Enterobacter sp. CS66 with significant potential as a biocontrol agent. [ABSTRACT FROM AUTHOR]

In the present study, we attempted to elucidate if the harmful phytopathogenic bacteria of Pectobacterium genus ( P. atrosepticum) possess the enzymes for oxidation of phenolic compounds. Polyphenol oxidase (laccase) activity was revealed in P. atrosepticum cell lysates. Using bioinformatic analysis, an ORF encoding a putative copper-containing polyphenol oxidase of 241 amino acids with a predicted molecular mass of 25.9 kDa was found. This protein (named Pal1) shares significant level of identity with laccases of a new type described for several bacterial species. Cloning and expression of the pal1 gene and the analysis of corresponding recombinant protein confirmed that Pal1 possessed laccase activity. The recombinant Pal1 protein was characterized in terms of substrate specificity, kinetic parameters, pH and temperature optimum, sensitivity to inhibitors and metal content. Pal1 demonstrated alkali- and thermo-tolerance. The kinetic parameters Km and kcat for 2,6-dimethoxyphenol were 0.353 ± 0.062 mM and 98.79 ± 4.9 s−1, respectively. The protein displayed high tolerance to sodium azide, sodium fluoride, NaCl, SDS and cinnamic acid. The transcript level of the pal1 gene in P. atrosepticum was shown to be induced by plant-derived phenolic compound (ferulic acid) and copper sulfate. [ABSTRACT FROM AUTHOR]

In western Europe, Pectobacterium carotovorum subsp. brasiliense is emerging as a causal agent of blackleg disease. In field experiments in the Netherlands, the virulence of this pathogen was compared with strains of other Dickeya and Pectobacterium species. In 2013 and 2014, seed potato tubers were vacuum infiltrated with high densities of bacteria (106 CFU mL-1) and planted in clay soil. Inoculation with P. carotovorum subsp. brasiliense and P. atrosepticum resulted in high disease incidences (75-95%), inoculation with D. solani and P. wasabiae led to incidences between 5% and 25%, but no significant disease development was observed in treatments with P. carotovorum subsp. carotovorum, D. dianthicola or the water control. Co-inoculations of seed potatoes with P. carotovorum subsp. brasiliense and D. solani gave a similar disease incidence to inoculation with only P. carotovorum subsp. brasiliense. However, coinoculation of P. carotovorum subsp. brasiliense with P. wasabiae resulted in a decrease in disease incidence compared to inoculation with only P. carotovorum subsp. brasiliense. In 2015, seed potatoes were inoculated with increasing densities of P. carotovorum subsp. brasiliense, D. solani or P. atrosepticum (103-106 CFU mL-1). After vacuum infiltration, even a low inoculum density resulted in high disease incidence. However, immersion without vacuum caused disease only at high bacterial densities. Specific TaqMan assays were evaluated and developed for detection of P. carotovorum subsp. brasiliense, P. wasabiae and P. atrosepticum and confirmed the presence of these pathogens in progeny tubers of plants derived from vacuum-infiltrated seed tubers. [ABSTRACT FROM AUTHOR]

Some bacteria, when infected by their viral parasites (bacteriophages), undergo a suicidal response that also terminates productive viral replication (abortive infection [Abi]). This response can be viewed as an altruistic act protecting the uninfected bacterial clonal population. Abortive infection can occur through the action of type III protein-RNA toxin-antitoxin (TA) systems, such as ToxINPa from the phytopathogen Pectobacterium atrosepticum. Rare spontaneous mutants evolved in the generalized transducing phage &3934;M1, which escaped ToxINPa-mediated abortive infection in P. atrosepticum. &3934;M1 is a member of the Podoviridae and a member of the "KMV-like" viruses, a subset of the T7 supergroup. Genomic sequencing of &3934;M1 escape mutants revealed single-base changes which clustered in a single open reading frame. The "escape" gene product, M1-23, was highly toxic to the host bacterium when overexpressed, but mutations in M1-23 that enabled an escape phenotype caused M1-23 to be less toxic. M1-23 is encoded within the DNA metabolism modular section of the phage genome, and when it was overexpressed, it copurified with the host nucleotide excision repair protein UvrA. While the M1-23 protein interacted with UvrA in coimmunoprecipitation assays, a UvrA mutant strain still aborted &3934;M1, suggesting that the interaction is not critical for the type III TA Abi activity. Additionally, &3934;M1 escaped a heterologous type III TA system (TenpINPl) from Photorhabdus luminescens (reconstituted in P. atrosepticum) through mutations in the same protein, M1-23. The mechanistic action of M1-23 is currently unknown, but further analysis of this protein may provide insights into the mode of activation of both systems. IMPORTANCE: Bacteriophages, the viral predators of bacteria, are the most abundant biological entities and are important factors in driving bacterial evolution. In order to survive infection by these viruses, bacteria have evolved numerous antiphage mechanisms. Many of the studies involved in understanding these interactions have led to the discovery of biotechnological and gene-editing tools, most notably restriction enzymes and more recently the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems. Abortive infection is another such antiphage mechanism that warrants further investigation. It is unique in that activation of the system leads to the premature death of the infected cells. As bacteria infected with the virus are destined to die, undergoing precocious suicide prevents the release of progeny phage and protects the rest of the bacterial population. This altruistic suicide can be caused by type III toxin-antitoxin systems, and understanding the activation mechanisms involved will provide deeper insight into the abortive infection process. [ABSTRACT FROM AUTHOR]

Pectobacterium atrosepticum is a pectolytic enterobacterium that causes blackleg in plants. Bacteriophage Peat2 was isolated from P. atrosepticum infected potato tissue and its genome sequenced. Analysis of the 48,659 bp genome revealed that Peat2 resembles the Myoviridae family Klebsiella pneumoniae bacteriophage JD001 and includes a CRISPR Cas 4 RecB-like nuclease. A lytic bacteriophage, Peat2 details provide important information for application as a biocontrol agent of disease caused by phytopathogenic P. atrosepticum. [ABSTRACT FROM AUTHOR]

Pectobacterium atrosepticum is a phytopathogen of economic importance as it is the causative agent of potato blackleg and soft rot. Here we describe the Pectobacterium phage vB_PatP_CB5 (abbreviated as CB5), which specifically infects the bacterium. The bacteriophage is characterized in detail and TEM micrographs indicate that it belongs to the Podoviridae family. CB5 shares significant pairwise nucleotide identity (≥80%) with P. atrosepticum phages φM1, Peat1, and PP90 and also shares common genome organization. Phylograms constructed using conserved proteins and whole-genome comparison-based amino acid sequences show that these phages form a distinct clade within the Autographivirinae. They also possess conserved RNA polymerase recognition and specificity loop sequences. Their lysis cassette resembles that of KP34virus, containing in sequential order a U-spanin, a holin, and a signal–arrest–release (SAR) endolysin. However, they share low pairwise nucleotide identity with the type phage of the KP34virus genus, Klebsiella phage KP34. In addition, phage KP34 does not possess several conserved proteins associated with these P. atrosepticum phages. As such, we propose the allocation of phages CB5, Peat1, φM1, and PP90 to a separate new genus designated Phimunavirus.

This study reports the development of a real-time, loop-mediated isothermal amplification (RealAmp) assay for the detection of Pectobacterium atrosepticum ( P. atrosepticum). A phylogenetic tree was constructed based on the gyrB gene of P. atrosepticum and related species. Pectobacterium atrosepticum from different sources can be clustered in the same branch with 100% support rate. The RealAmp primers targeting the gyrB gene of P. atrosepticum worked most efficiently at 61·0°C. Compared with 55 related bacterial strains, the eight P. atrosepticum strains displayed positive reaction in the RealAmp assay. The melting temperature (Tm) of P. atrosepticum amplified products was about 85·0°C. The detection limit of the RealAmp assay for the detection of P. atrosepticum in pure culture was approx. 3 CFU reaction−1. The detection limit of the RealAmp assay for the detection of P. atrosepticum in artificially contaminated samples was 22 CFU reaction−1. The detection rate of the RealAmp assay for the detection of potato tubers was 28·5-32·0% higher than that of the conventional PCR. In summary, a specific, sensitive and rapid RealAmp assay based on the gyrB gene of P. atrosepticum, which can be easily performed and real-time monitored, was established. Significance and Impact of the Study Potato blackleg caused by Pectobacterium atrosepticum ( P. atrosepticum) which is mainly transmitted through the seed potato leads to the decline in potato production. To reduce yield loss, rapid detection of P. atrosepticum in seed potato remains essential. Based on the gyrB gene of P. atrosepticum, species-specific primers were designed. A real-time, loop-mediated isothermal amplification (RealAmp) assay was established for the detection of P. atrosepticum. The RealAmp assay is a specific, rapid and sensitive method for P. atrosepticum detection. Therefore, it provides an effective diagnosis of potato blackleg in both the growing and stored potato. [ABSTRACT FROM AUTHOR]

* Background and aims Potato (Solanum tuberosum) is an important food crop and is grown worldwide. It is, however, significantly sensitive to a number of soil-borne pathogens that affect roots and tubers, causing considerable economic losses. So far, most research on potato has been dedicated to tubers and hence little attention has been paid to root structure and function. * Methods In the present study we characterized root border cells using histochemical staining, immunofluorescence labelling of cell wall polysaccharides epitopes and observation using laser confocal microscopy. The monosaccharide composition of the secreted exudates was determined by gas chromatography of trimethylsilyl methylglycoside derivatives. The effects of root exudates and secreted arabinogalactan proteins on bacterial growth were investigated using in vitro bioassays. * Key Results Root exudate from S. tuberosum was highly enriched in galactose-containing molecules including arabinogalactan proteins as major components. Treatment of the root with an elicitor derived from Pectobacterium atrosepticum, a soil-borne pathogen of potato, altered the composition of the exudates and arabinogalactan proteins. We found that the growth of the bacterium in vitro was differentially affected by exudates from elicited and nonelicited roots (i.e. inhibition versus stimulation). * Conclusions Taken together, these findings indicate that galactose-containing polymers of potato root exudates play a central role in root-microbe interactions. [ABSTRACT FROM AUTHOR]

Representatives of Pectobacterium genus are some of the most harmful phytopathogens in the world. In the present study, we have elucidated novel aspects of plant- Pectobacterium atrosepticum interactions. This bacterium was recently demonstrated to form specific 'multicellular' structures - bacterial emboli in the xylem vessels of infected plants. In our work, we showed that the process of formation of these structures includes the pathogen-induced reactions of the plant. The colonisation of the plant by P. atrosepticum is coupled with the release of a pectic polysaccharide, rhamnogalacturonan I, into the vessel lumen from the plant cell wall. This polysaccharide gives rise to a gel that serves as a matrix for bacterial emboli. P. atrosepticum-caused infection involves an increase of reactive oxygen species (ROS) levels in the vessels, creating the conditions for the scission of polysaccharides and modification of plant cell wall composition. Both the release of rhamnogalacturonan I and the increase in ROS precede colonisation of the vessels by bacteria and occur only in the primary xylem vessels, the same as the subsequent formation of bacterial emboli. Since the appearance of rhamnogalacturonan I and increase in ROS levels do not hamper the bacterial cells and form a basis for the assembly of bacterial emboli, these reactions may be regarded as part of the susceptible response of the plant. Bacterial emboli thus represent the products of host-pathogen integration, since the formation of these structures requires the action of both partners. [ABSTRACT FROM AUTHOR]