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

Promoter-probe vectors carrying fluorescent protein-reporter genes are powerful tools used to study microbial ecology, epidemiology, and etiology. In addition, they provide direct visual evidence of molecular interactions related to cell physiology and metabolism. Knowledge and advances carried out thanks to the construction of soft-rot Pectobacteriaceae biosensors, often inoculated in potato Solanum tuberosum, are discussed in this review. Under epifluorescence and confocal laser scanning microscopies, Dickeya and Pectobacterium-tagged strains managed to monitor in situ bacterial viability, microcolony and biofilm formation, and colonization of infected plant organs, as well as disease symptoms, such as cell-wall lysis and their suppression by biocontrol antagonists. The use of dual-colored reporters encoding the first fluorophore expressed from a constitutive promoter as a cell tag, while a second was used as a regulator-based reporter system, was also used to simultaneously visualize bacterial spread and activity. This revealed the chronology of events leading to tuber maceration and quorum-sensing communication, in addition to the disruption of the latter by biocontrol agents. The promising potential of these fluorescent biosensors should make it possible to apprehend other activities, such as subcellular localization of key proteins involved in bacterial virulence in planta, in the near future.

Soft Rot Pectobacteriaceae (SRP; Pectobacterium spp. and Dickeya spp., formerly known as pectinolytic Erwinia spp.) are necrotrophic bacterial pathogens infecting a large number of plant species worldwide, including agriculturally-important crops. Despite the SRP importance in agriculture, little is known about the bacteriophages infecting them, and even less about the prophages present in their genomes. Prophages are recognized as factors underlying bacterial virulence, genomic diversification and ecological fitness that contribute to the novel phenotypic properties of bacterial hosts. Likewise, they are recognized as a driving force of bacterial evolution. In this study, 57 complete genomes of Pectobacterium spp. and Dickeya spp. deposited in NCBI GenBank, were analyzed for the presence of prophage-like elements. Viral sequences were discovered in 95% of bacterial genomes analyzed with the use of PHASTER, PhiSpy, and manual curation of the candidate sequences using NCBI BLAST. In total 37 seemingly intact and 48 putatively defective prophages were found. The 37 seemingly intact prophages (27 sequences in Dickeya spp. genomes and 10 sequences in Pectobacterium spp. genomes) were annotated using RAST. Analysis of the prophage genes encoding viral structural proteins allowed classification of these prophages into different families of the order Caudovirales (tailed bacteriophages) with the SRP prophages of the Myoviridae family (81% of found prophages) being the most abundant. The phylogenetic relationships between prophages were analyzed using amino acid sequences of terminase large subunit (gene terL), integrase (gene int), holin (gene hol), and lysin (gene lys). None of these markers however proved fully useful for clear phylogenetic separation of prophages of SRP into distinct clades. Comparative analyses of prophage proteomes revealed six clusters: five present in Dickeya spp. and one within Pectobacterium spp. When screened for the presence of bacterial genes in the genomes of intact prophages, only one prophage did not contain any ORFs of bacterial origin, the other prophages contained up to 23 genes acquired from bacterial hosts. The bacterial genes present in prophages could possibly affect fitness and virulence of their hosts. The implication of prophage presence in the genomes of Pectobacterium spp. and Dickeya spp. is discussed.

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

This is the first report describing precipitation of bacteriophage particles with zinc chloride as a method of choice to isolate infectious lytic bacteriophages against Pectobacterium spp. and Dickeya spp. from environmental samples. The isolated bacteriophages are ready to use to study various (ecological) aspects of bacteria-bacteriophage interactions. The method comprises the well-known precipitation of phages from aqueous extracts of the test material by addition of ZnCl2, resuscitation of bacteriophage particles in Ringer’s buffer to remove the ZnCl2 excess and a soft agar overlay assay with the host bacterium to isolate infectious individual phage plaques. The method requires neither an enrichment step nor other steps (e. g., PEG precipitation, ultrafiltration, or ultracentrifugation) commonly used in other procedures and results in isolation of active viable bacteriophage particles.

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

The soft rot Enterobacteriaceae (SRE) Pectobacterium and Dickeya species (formerly classified as pectinolytic Erwinia spp.) cause important diseases on potato and other arable and horticultural crops. They may affect the growing potato plant causing blackleg and are responsible for tuber soft rot in storage thereby reducing yield and quality. Efficient and cost-effective detection and identification methods are essential to investigate the ecology and pathogenesis of the SRE as well as in seed certification programmes. The aim of this review was to collect all existing information on methods available for SRE detection. The review reports on the sampling and preparation of plant material for testing and on over thirty methods to detect, identify and differentiate the soft rot and blackleg causing bacteria to species and subspecies level. These include methods based on biochemical characters, serology, molecular techniques which rely on DNA sequence amplification as well as several less-investigated ones.

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

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

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

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

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

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

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

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

The soil isolate Ochrobactrum sp. A44 inactivates N-acyl homoserine lactone (AHL) quorum sensing signal molecules and is capable of quenching the AHL-dependent virulence of Pectobacterium carotovorum in planta. To characterize this AHL inactivating activity, Ochrobactrum cell extracts were prepared and their capacity to degrade a broad range of AHLs was determined. AHLs with acyl chains ranging from C4 to C14 with or without 3-oxo or 3-hydroxy substituents were all inactivated to varying extents; long chain AHLs were generally more susceptible than short chain compounds irrespective of the three position substituent. HPLC and LC-tandem mass spectrometry of the AHL degradation products revealed that the AHL inactivating activity present in the Ochrobactrum cell extract cleaved the AHL amide bond. To identify the gene(s) responsible for AHL degradation, Ochrobactrum sp. A44 was subjected to random transposon (Tn) mutagenesis and the resulting mutants screened for the loss of AHL acylase activity. The Tn insertion in mutant A6731 was mapped to a gene termed aiiO, the translated product of which belongs to the α/β hydrolase superfamily which constitutes a novel type of AHL acylase.

Little is known about lytic bacteriophages infecting plant-pathogenic Dickeya spp. These bacteria cause economically significant losses in arable crops and ornamental plant production worldwide. At present, there is no effective control of diseases caused by Dickeya spp. A novel bacteriophage, ϕD5, belonging to the family Myoviridae, order Caudovirales, that could be used to control these bacteria was isolated previously. This report provides information on its characterization. The ϕD5 genome consists of 155,346-bp-length double-stranded DNA with a GC content of 49.7 % and is predicted to have 196 open reading frames (ORFs) with an average length of 711 nucleotides each. The ORFs were classified into functional groups, including phage structure, packaging, DNA metabolism, regulation, and additional functions. The phage lifestyle predicted from PHACTS indicated that ϕD5 may be a lytic phage and therefore can efficiently kill plant-pathogenic Dickeya spp. Electronic supplementary material The online version of this article (doi:10.1007/s00705-014-2170-8) contains supplementary material, which is available to authorized users.

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

Rispail, Nicolas et al., Mitogen-activated protein kinase (MAPK) cascades and the calcium–calcineurin pathway control fundamental aspects of fungal growth, development and reproduction. Core elements of these signalling pathways are required for virulence in a wide array of fungal pathogens of plants and mammals. In this review, we have used the available genome databases to explore the structural conservation of three MAPK cascades and the calcium–calcineurin pathway in ten different fungal species, including model organisms, plant pathogens and human pathogens. While most known pathway components from the model yeast Saccharomyces cerevisiae appear to be widely conserved among taxonomically and biologically diverse fungi, some of them were found to be restricted to the Saccharomycotina. The presence of multiple paralogues in certain species such as the zygomycete Rhizopus oryzae and the incorporation of new functional domains that are lacking in S. cerevisiae signalling proteins, most likely reflect functional diversification or adaptation as filamentous fungi have evolved to occupy distinct ecological niches., This analysis was carried out by members of the SIGNALPATH Marie Curie training network (MRTN-CT-2005-019277), which provided financial support for N.R., C.A., R.C., A.G., R.H., E.P.N., A.P., E.S., V.V. and M.Y.

A scalable open systems and standards derived software ecosystem is described for computer vision analytics (CVA) assisted exploitation of full motion video (FMV). The ecosystem, referred to as the Advanced Video Activity Analytics (AVAA), has two instantiations, one for size, weight, and power (SWAP) constrained conditions, and the other for large to massive cloud based configurations. The architecture is designed to meet operational analyst requirements to increase their productivity and accuracy for exploiting FMV using local cluster or scalable cloudbased computing resources. CVAs are encapsulated within a software plug-in architecture and FMV processing pipelines are constructed by combining these plug-ins to accomplish analytical tasks and manage provenance of processing history. An example pipeline for real-time motion detection and moving object characterization using the flux tensor approach is presented. An example video ingest experiment is described. Quantitative and qualitative methods for human factors engineering (HFE) assessment to evaluate cognitive loads for alternative work flow design choices are discussed. This HFE process is used for validating that an AVAA system instantiation with candidate workflow pipelines meets CVA assisted FMV exploitation operational goals for specific analyst workflows. AVAA offers a new framework for video understanding at scale for large enterprise applications in the government and commercial sectors.

Soft rot Enterobacteriaceae (Pectobacterium spp. and Dickeya spp., formerly pectinolytic Erwinia spp.) are ubiquitous necrotrophic bacterial pathogens that infect a large number of different plant species worldwide, including economically important crops. Despite the fact that these bacteria have been studied for more than 50 years, little is known of their corresponding predators: bacteriophages, both lytic and lysogenic. The aim of this minireview is to critically summarize recent ecological, biological and molecular research on bacteriophages infecting Pectobacterium spp. and Dickeya spp. with the main focus on current and future perspectives in that field.

Plant necrotrophic Dickeya spp. are among the top ten most devastating bacterial plant pathogens able to infect a number of different plant species worldwide including economically important crops. Little is known of the lytic bacteriophages infecting Dickeya spp. A broad host lytic bacteriophage ϕD3 belonging to the family Myoviridae and order Caudovirales has been isolated in our previous study. This report provides detailed information of its annotated genome, structural proteome and phylogenetic relationships with known lytic bacteriophages infecting species of the Enterobacteriaceae family.

Quorum sensing plays a role in the regulation of soft rot diseases caused by the plant pathogenic bacterium Pectobacterium carotovorum subsp. carotovorum. The signal molecules involved in quorum sensing in P. carotovorum subsp. carotovorum belong to the group of N-acyl homoserine lactones (AHLs). In our study, we screened bacteria isolated from the potato rhizosphere for the ability to degrade AHLs produced by P. carotovorum subsp. carotovorum. Six isolates able to degrade AHLs were selected for further studies. According to 16S rDNA sequence analysis and fatty acid methyl ester profiling, the isolates belonged to the genera Ochrobactrum, Rhodococcus, Pseudomonas, Bacillus, and Delftia. For the genera Ochrobactrum and Delftia, for the first time AHL-degrading isolates were found. Data presented in this study revealed for the first time that Ochrobactrum sp. strain A44 showed the capacity to inactivate various synthetic AHL molecules; the substituted AHLs were inactivated with a lower efficiency than the unsubstituted AHLs. Compared with the other isolates, A44 was very effective in the degradation of AHLs produced by P. carotovorum subsp. carotovorum. It was verified by polymerase chain reaction, DNA–DNA hybridization, and a lactone ring reconstruction assay that Ochrobactrum sp. strain A44 did not possess AHL lactonase activity. AHL degradation in Ochrobactrum sp. strain A44 occurred intracellularly; it was not found in the culture supernatant. AHL-degrading activity of A44 was thermo sensitive. Experiments in planta revealed that Ochrobactrum sp. strain A44 significantly inhibited the maceration of potato tuber tissue. Since A44 did not produce antibiotics, the attenuation of the decay might be due to the quenching of quorum- sensing-regulated production of pectinolytic enzymes. The strain can potentially serve to control P. carotovorum subsp. carotovorum in potato.Key words: AHL degradation, Ochrobactrum sp., Pectobacterium carotovorum.

Dickeya solani is one of the most important pectinolytic phytopathogens responsible for high losses in potato, especially in seed potato production in Europe. Lytic bacteriophages can affect the structure of the host population and may influence spread, survival and virulence of the pathogen and in consequence, infection of the plant. In this study, we aimed to acquire information on the viability of the broad host lytic bacteriophage ΦD5 on potato, as well as to apprehend the specific effect of this bacteriophage on its host D. solani type-strain in different settings, as a preliminary step to target co-adaptation of phages and host bacteria in plant environment. Viability of the ΦD5 phage in tuber extract, on tuber surface, in potting compost, in rainwater and on the leaf surface, as well as the effect of copper sulfate, were examined under laboratory conditions. Also, the interaction of ΦD5 with the target host D. solani in vitro and in compost-grown potato plants was evaluated. ΦD5 remained infectious in potato tuber extract and rain water for up to 72 h but was inactivated in solutions containing 50 mM of copper. The phage population was stable for up to 28 days on potato tuber surface and in potting compost. In both, tissue culture and compost-grown potato plants, ΦD5 reduced infection by D. solani by more than 50%. The implications of these findings are discussed.

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

The soil isolate Ochrobactrum sp. A44 inactivates N-acyl homoserine lactone (AHL) quorum sensing signal molecules and is capable of quenching the AHL-dependent virulence of Pectobacterium carotovorum in planta. To characterize this AHL inactivating activity, Ochrobactrum cell extracts were prepared and their capacity to degrade a broad range of AHLs was determined. AHLs with acyl chains ranging from C4 to C14 with or without 3-oxo or 3-hydroxy substituents were all inactivated to varying extents; long chain AHLs were generally more susceptible than short chain compounds irrespective of the three position substituent. HPLC and LC-tandem mass spectrometry of the AHL degradation products revealed that the AHL inactivating activity present in the Ochrobactrum cell extract cleaved the AHL amide bond. To identify the gene(s) responsible for AHL degradation, Ochrobactrum sp. A44 was subjected to random transposon (Tn) mutagenesis and the resulting mutants screened for the loss of AHL acylase activity. The Tn insertion in mutant A6731 was mapped to a gene termed aiiO, the translated product of which belongs to the α/β hydrolase superfamily which constitutes a novel type of AHL acylase.

A Serratia plymuthica-specific TaqManA (R) assay was designed based on the consensus nucleotide sequence from the 3'- end of the luxS gene present in all S. plymuthica strains tested. The specificity of the assay was demonstrated by testing 21 Serratia spp. strains and 30 isolates belonging to various species that can potentially coexist with S. plymuthica in the same environment. Positive reactions in the TaqManA (R) assay were observed only for S. plymuthica isolates and not for other bacteria. The TaqManA (R) assay could detect down to 1.95 ng of S. plymuthica DNA, down to 5 bacterial cells per reaction (100 cfu ml(-1)) in vitro, down to 50 bacterial cells per reaction (1,000 cfu ml(-1)) in spiked potato root extracts and down to 5 bacterial cells per reaction (100 cfu ml(-1)) in spiked potato haulm extracts. We used this assay to quantify S. plymuthica A30 cells in potato and tomato haulms and roots grown from S. plymuthica A30-inoculated potato seed tubers and tomato seeds. The results were comparable with the spread-plating of plant extracts on a newly developed S. plymuthica A30 selective medium (CVTR2Arif). The TaqManA (R) assay can be used to quantify S. plymuthica isolates in different ecosystems and in complex substrates.

Serratia plymuthica A30 is a Gram-negative bacterium expressing antagonistic activity toward blackleg- and soft rot-causing Dickeya sp. biovar 3 (“ Dickeya solani ”). Here, we present the draft genome sequence of strain A30, which has been isolated from rotten potato tuber tissue.

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

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

In Ustilago maydis the pheromone signal is transmitted via a mitogen-activated protein kinase (MAP kinase) module to the transcription factor Prf1. Prf1 activates transcription of a and b mating type genes by binding to pheromone response elements (PREs) located in regulatory regions of these genes. Here we show that the CCAAT-box binding protein Hap2 from U. maydis regulates prf1 expression. Hap2 was initially identified as a potential interaction partner of the MAP kinase Kpp6 in yeast two-hybrid screens and was subsequently also shown to interact with the MAPK Kpp2. Deletion of hap2 in haploid cells abolished mating, resulting from a defect in pheromone-induced gene expression. Crosses of haploid hap2 deletion strains were completely impaired in pathogenicity. Constitutive expression of prf1 complemented the pheromone response defect in Δhap2 strains. Chromatin immunoprecipitation assays indicated that Hap2 binds directly to CCAAT motifs in the prf1 promoter. Point mutations in two putative MAPK phosphorylation sites in Hap2 attenuated the pheromone response. In a solopathogenic strain hap2 deletion affected filamentation and the mutants showed reduced pathogenicity symptoms. These data suggest that Hap2 is a novel regulator of prf1 with additional functions after cell fusion.

Many Gram-positive and Gram-negative bacteria communicate using small diffusible signal molecules called autoinducers. This process, known as quorum sensing (QS), links cell density to the expression of genes as diverse as those associated with virulence factors production of plant and animal pathogens, bioluminescence, antibiotic production, sporulation or biofilm formation. In Gram-negative bacteria, this communication is mainly mediated by N-acyl-homoserine lactones (AHLs). It has been proven that inactivation of the signal molecules attenuates many of the processes controlled by QS. Enzymatic degradation of the signal molecules has been amply described. Two main classes of AHL-inactivating enzymes were identified: AHL lactonases which hydrolyse the lactone ring in AHLs, and AHL acylases (syn. AHL amidases) which liberate a free homoserine lactone and a fatty acid. Recently, AHL oxidoreductase, a novel type of AHL inactivating enzyme, was described. The activity of these enzymes results in silencing the QS-regulated processes, as degradation products cannot act as signal molecules. The ability to inactivate AHL (quorum quenching, QQ) might be useful in controlling virulence of many pathogenic bacteria.

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

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

Pectinolytic Pectobacterium spp. and Dickeya spp. are necrotrophic bacterial pathogens of many important crops, including potato, worldwide. This study reports on the isolation and characterization of broad host lytic bacteriophages able to infect the dominant Pectobacterium spp. and Dickeya spp. affecting potato in Europe viz. Pectobacterium carotovorum subsp. carotovorum (Pcc), P. wasabiae (Pwa) and Dickeya solani (Dso) with the objective to assess their potential as biological disease control agents. Two lytic bacteriophages infecting stains of Pcc, Pwa and Dso were isolated from potato samples collected from two potato fields in central Poland. The ΦPD10.3 and ΦPD23.1 phages have morphology similar to other members of the Myoviridae family and the Caudovirales order, with a head diameter of 85 and 86 nm and length of tails of 117 and 121 nm, respectively. They were characterized for optimal multiplicity of infection, the rate of adsorption to the Pcc, Pwa and Dso cells, the latent period and the burst size. The phages were genotypically characterized with RAPD-PCR and RFLP techniques. The structural proteomes of both phages were obtained by fractionation of phage proteins by SDS-PAGE. Phage protein identification was performed by liquid chromatography-mass spectrometry (LC-MS) analysis. Pulsed-field gel electrophoresis (PFGE), genome sequencing and comparative genome analysis were used to gain knowledge of the length, organization and function of the ΦPD10.3 and ΦPD23.1 genomes. The potential use of ΦPD10.3 and ΦPD23.1 phages for the biocontrol of Pectobacterium spp. and Dickeya spp. infections in potato is discussed.