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Find video protocols related to scientific articles indexed in Pubmed.
A novel plasmid pEA68 of Erwinia amylovora and the description of a new family of plasmids.
Arch. Microbiol.
PUBLISHED: 09-02-2014
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Recent genome analysis of Erwinia amylovora, the causal agent of fire blight disease on Rosaceae, has shown that the chromosome is highly conserved among strains and that plasmids are the principal source of genomic diversity. A new circular plasmid, pEA68, was found in E. amylovora strain 692 (LMG 28361), isolated in Poland from Sorbus (mountain ash) with fire blight symptoms. Annotation of the 68,763-bp IncFIIa-type plasmid revealed that it contains 79 predicted CDS, among which two operons (tra, pil) are associated with mobility. The plasmid is maintained stably in E. amylovora and does not possess genes associated with antibiotic resistance or known virulence genes. Curing E. amylovora strain 692 of pEA68 did not influence its virulence in apple shoots nor amylovoran synthesis. Of 488 strains of E. amylovora from seventeen countries, pEA68 was only found in two additional strains from Belgium. Although the spread of pEA68 is currently limited to Europe, pEA68 comprises, together with pEA72 and pEA78 both found in North America, a new plasmid family that spans two continents.
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Enteric pathogen survival varies substantially in irrigation water from Belgian lettuce producers.
Int J Environ Res Public Health
PUBLISHED: 07-02-2014
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It is accepted that irrigation water is a potential carrier of enteric pathogens, such as Salmonella and E. coli O157:H7 and, therefore, a source for contamination of fresh produce. We tested this by comparing irrigation water samples taken from five different greenhouses in Belgium. The water samples were inoculated with four zoonotic strains, two Salmonella and two E. coli O157:H7 strains, and pathogen survival and growth in the water were monitored up till 14 days. The influence of water temperature and chemical water quality was evaluated, and the survival tests were also performed in water samples from which the resident aquatic microbiota had previously been eliminated by filter sterilization. The pathogen's survival differed greatly in the different irrigation waters. Three water samples contained nutrients to support important growth of the pathogens, and another enabled weaker growth. However, for all, growth was only observed in the samples that did not contain the resident aquatic microbiota. In the original waters with their specific water biota, pathogen levels declined. The same survival tendencies existed in water of 4 °C and 20 °C, although always more expressed at 20 °C. Low water temperatures resulted in longer pathogen survival. Remarkably, the survival capacity of two E. coli 0157:H7 strains differed, while Salmonella Thompson and Salmonella Typhimurium behaved similarly. The pathogens were also transferred to detached lettuce leaves, while suspended in two of the water samples or in a buffer. The effect of the water sample on the pathogen's fitness was also reproduced on the leaves when stored at 100% relative humidity. Inoculation of the suspension in buffer or in one of the water samples enabled epiphytic growth and survival, while the pathogen level in the other water sample decreased once loaded on the leaves. Our results show that irrigation waters from different origin may have a different capacity to transmit enteric pathogens and an important impact on the fitness of the pathogens to sustain and even grow on the leaf surface.
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Comparative genome analysis of pathogenic and non-pathogenic Clavibacter strains reveals adaptations to their lifestyle.
BMC Genomics
PUBLISHED: 05-09-2014
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The genus Clavibacter harbors economically important plant pathogens infecting agricultural crops such as potato and tomato. Although the vast majority of Clavibacter strains are pathogenic, there is an increasing number of non-pathogenic isolates reported. Non-pathogenic Clavibacter strains isolated from tomato seeds are particularly problematic because they affect the current detection and identification tests for Clavibacter michiganensis subsp. michiganensis (Cmm), which is regulated with a zero tolerance in tomato seed. Their misidentification as pathogenic Cmm hampers a clear judgment on the seed quality and health.
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Evaluation of an attachment assay on lettuce leaves with temperature- and starvation-stressed Escherichia coli O157:H7 MB3885.
J. Food Prot.
PUBLISHED: 04-01-2014
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Attachment of enteric pathogens such as Escherichia coli O157:H7 to fresh produce is a crucial first step for contamination to occur, and irrigation water (IW) is considered a potentially important preharvest introduction route. In a natural situation, E. coli O157:H7 may be present in the irrigation water for some time and may, therefore, be starved. Most research, however, is performed with freshly cultured strains. The aim of this study was to examine the behavior of E. coli O157:H7 MB3885 under starvation stress in water used for overhead irrigation in the greenhouse and the consequence on its subsequent ability to attach to butterhead lettuce leaves. E. coli O157:H7 MB3885 was starvation stressed by introducing it at ±7.5 log CFU/ml into phosphate-buffered saline (PBS), sterile distilled water (SDW), or IW. The suspensions were stored at 4 or 20°C and were used after 0, 2, and 6 days for an attachment assay on butterhead lettuce. E. coli O157:H7 MB3885 levels were determined by plating method and live and dead quantitative PCR technique. A decrease in plate counts, an indicator of stress, was observed for most of the conditions, whereas a die-off, as revealed by the live and dead quantitative PCR data, was only observed in IW stored at 20°C. Overall, stress appeared to be highest in IW and lowest in PBS. The stressed cells were still able to recover, even at 4 °C, and to attach to the lettuce. Furthermore, our results show that standard laboratory solutions such as PBS and SDW may not be the best to simulate stressed cells in IW, in which the bacteria may behave significantly differently.
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Temperature-dependent development of the broad mite Polyphagotarsonemus latus (Acari: Tarsonemidae) on Rhododendron simsii.
Exp. Appl. Acarol.
PUBLISHED: 01-31-2014
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The broad mite, Polyphagotarsonemus latus (Banks), is one of the major pests causing severe economic damage in Rhododendron simsii Planch hybrid production in Belgium. In order to optimize biological control programs and to parameterize warning programs, we studied the effect of environmental temperature on the development of P. latus on R. simsii leaves. In combination with a photoperiod of 16:8 h (L:D) and a relative humidity of 80 ± 5 %, six constant temperatures (15, 17, 20, 25, 30 and 33 ± 1 °C), were studied. Total developmental times of 13.3, 10.5, 6.6, 4.2, 3.5 and 4.0 days were measured, respective to each of the aforementioned temperatures. Development of females took significantly longer than that of males at 15, 17, 20 and 30 °C. Survival rates observed between 17 and 30 °C varied between 43.5 and 96.9 %. Lower survival rates were found at 15 and 33 °C, i.e. 31.8 and 23.6 %, respectively. The lower, optimal and upper developmental threshold (t min , t opt and t max , respectively) and thermal constant (K) of the pest were estimated for each life stage by a linear and two non-linear models. Based on measurements of total development of P. latus thermal thresholds of 10.0, 30.1 and 36.0 °C were calculated for t min , t opt and t max , respectively. The number of degree-days needed to complete immature development when feeding on R. simsii was 66.7.
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Draft genome sequence of Xanthomonas fragariae reveals reductive evolution and distinct virulence-related gene content.
BMC Genomics
PUBLISHED: 11-20-2013
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Xanthomonas fragariae (Xf) is a bacterial strawberry pathogen and an A2 quarantine organism on strawberry planting stock in the EU. It is taxonomically and metabolically distinct within the genus Xanthomonas, and known for its host specificity. As part of a broader pathogenicity study, the genome of a Belgian, virulent Xf strain (LMG 25863) was assembled to draft status and examined for its pathogenicity related gene content.
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Draft Genome Sequences of Four Dickeya dianthicola and Four Dickeya solani Strains.
Genome Announc
PUBLISHED: 07-27-2013
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Dickeya dianthicola and "Dickeya solani" are currently the dominant bacterial pathogens of potatoes in Europe. Here, we present the draft genome sequences of four strains of each pathogen.
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Survival of enteric pathogens during butterhead lettuce growth: crop stage, leaf age, and irrigation.
Foodborne Pathog. Dis.
PUBLISHED: 04-24-2013
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The survival of Salmonella enterica serovar Thompson and Escherichia coli O157 was investigated on growing butterhead lettuce plants in the plant-growth chamber and greenhouse. All inoculation tests were made under conditions that approximate the greenhouse conditions for butterhead lettuce cultivation in Flanders (Belgium). The survival and proliferation of the pathogens on the leaves was determined at days 0, 4, and 8 after inoculation using standard plating techniques on selective medium. In the growth chamber, the extent to which both pathogens were able to multiply on the lettuce leaves was influenced by crop stage and leaf age. On young plants, the older leaves supported pathogen survival better. On nearly mature plants, pathogen population sizes were significantly higher on the old and young leaves compared with middle-aged leaves (p<0.001). In the greenhouse, the environmental regimen with high fluctuations in temperature and relative humidity was less conducive to the survival of E. coli O157, though its survival on nearly mature lettuce was enhanced by overhead irrigation. The moist conditions between the folded inner leaves are likely contributing to the survival of enteric pathogens in the lettuce head. Butterhead lettuce grown in greenhouses with a sprinkle irrigation system may present a potential health hazard when contaminated near harvest. Experimental design (growth chamber versus greenhouse) largely influences enteric pathogen behavior on growing lettuce plants.
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Genetic diversity of non-pathogenic Clavibacter strains isolated from tomato seeds.
Syst. Appl. Microbiol.
PUBLISHED: 02-20-2013
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Clavibacter michiganensis subsp. michiganensis (Cmm) is a seed-transmitted, quarantine pathogen which causes bacterial wilt and canker of tomato. Despite efforts to prevent seed contamination, new introductions are regularly detected, associated with new regions of tomato seed production. It seems as if the expanding diversity of Cmm also challenges the limited host range. Clavibacter-like isolates from tomato seed are phenotypically similar to Cmm in the common diagnostic semi-selective media and are identified as Cmm in the customary tests but are not pathogenic to tomato. In our first study four representatives formed a separate cluster in gyrB sequence analysis and in MALDI-TOF MS. Their presence on seed prevents clear judgment on the health status of tomato seeds. As their nature and function are unclear we aimed to investigate and compare them to Cmm. Twenty strains described as Clavibacter-like isolated from tomato seed and not pathogenic to tomato plantlets were selected. Leaf spots, wilting or cankers were not induced after local or systemic inoculation. Tomato stems were not colonized nor was there evidence of survival in tomato stems. Total DNA-DNA hybridization and sequence analysis of gyrB and dnaA proved that they belong to the Cm species but can be unambiguously separated from Cmm. Some of the genes encoding virulence determinants in Cmm strains were also detected in some of the non-pathogenic isolates. Moreover, Cmm strains formed a coherent group, while non-pathogenic Cm strains were heterogenic. The latter was confirmed by BOX-PCR. We speculate that tomato seeds likely represent a larger reservoir of unexplored Clavibacter diversity.
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Multilocus variable-number-tandem-repeats analysis (MLVA) distinguishes a clonal complex of Clavibacter michiganensis subsp. michiganensis strains isolated from recent outbreaks of bacterial wilt and canker in Belgium.
BMC Microbiol.
PUBLISHED: 02-18-2013
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Clavibacter michiganensis subsp. michiganensis (Cmm) causes bacterial wilt and canker in tomato. Cmm is present nearly in all European countries. During the last three years several local outbreaks were detected in Belgium. The lack of a convenient high-resolution strain-typing method has hampered the study of the routes of transmission of Cmm and epidemiology in tomato cultivation. In this study the genetic relatedness among a worldwide collection of Cmm strains and their relatives was approached by gyrB and dnaA gene sequencing. Further, we developed and applied a multilocus variable number of tandem repeats analysis (MLVA) scheme to discriminate among Cmm strains.
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Long-term survival of Escherichia coli O157:H7 and Salmonella enterica on butterhead lettuce seeds, and their subsequent survival and growth on the seedlings.
Int. J. Food Microbiol.
PUBLISHED: 01-02-2013
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The long-term survival of enteric pathogens on butterhead lettuce seeds, and their subsequent survival and growth on seedlings were investigated. Lettuce seeds were inoculated at a high level with two Salmonella enterica and two Escherichia coli O157:H7 strains each (±8 log?? CFU/g seed) and the survival of the pathogens was monitored over two years using standard plating techniques on selective medium. The Salmonella strains (serovars Typhimurium and Thompson) survived significantly better on the seeds than the E. coli O157:H7 strains (MB3885 and NCTC12900). When individual seeds were tested two years after inoculation, Salmonella was recovered from each individual seed, whereas E. coli O157:H7 only from 4% to 14% of the seeds, depending on the recovery method. When contaminated stored seeds were germinated and the seedlings examined for presence of the pathogens, it was clear that both pathogens were able to proliferate on the seedlings. Pathogen counts up to 5.92 log?? CFU and 4.41 log?? CFU per positive seedling were observed for Salmonella and E. coli O157:H7, respectively. Our study not only confirms the long-term survival of enteric pathogens on seeds but also shows that the pathogens maintain their ability to resuscitate and proliferate on the seedlings. Seeds or seedlings should be considered as contamination sources for the cultivation of leafy vegetables such as butterhead lettuce grown in greenhouses.
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Identification of new polymorphic microsatellite markers in the NA1 and NA2 lineages of Phytophthora ramorum.
Mycologia
PUBLISHED: 06-03-2011
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Phytophthora ramorum is a recently introduced pathogen in Europe and North America consisting of three clonal lineages. Due to the limited intralineage genetic variation, only a few polymorphic markers are available for use in studies involving the epidemiology and evolution of P. ramorum. A total of 159 primer pairs for candidate polymorphic SSR loci were tested with universal labeling. Four polymorphic microsatellite loci were identified within the NA1 lineage and one within the NA2 lineage, demonstrating the power and flexibility of the screening technique. The markers may significantly increase the number of genotypes that can be identified and as such can help better characterize the North American lineages of P. ramorum.
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Bacteriophages LIMElight and LIMEzero of Pantoea agglomerans, belonging to the "phiKMV-like viruses".
Appl. Environ. Microbiol.
PUBLISHED: 03-18-2011
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Pantoea agglomerans is a common soil bacterium used in the biocontrol of fungi and bacteria but is also an opportunistic human pathogen. It has been described extensively in this context, but knowledge of bacteriophages infecting this species is limited. Bacteriophages LIMEzero and LIMElight of P. agglomerans are lytic phages, isolated from soil samples, belonging to the Podoviridae and are the first Pantoea phages of this family to be described. The double-stranded DNA (dsDNA) genomes (43,032 bp and 44,546 bp, respectively) encode 57 and 55 open reading frames (ORFs). Based on the presence of an RNA polymerase in their genomes and their overall genome architecture, these phages should be classified in the subfamily of the Autographivirinae, within the genus of the "phiKMV-like viruses." Phylogenetic analysis of all the sequenced members of the Autographivirinae supports the classification of phages LIMElight and LIMEzero as members of the "phiKMV-like viruses" and corroborates the subdivision into the different genera. These data expand the knowledge of Pantoea phages and illustrate the wide host diversity of phages within the "phiKMV-like viruses."
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Aberrant genome size and instability of Phytophthora ramorum oospore progenies.
Fungal Genet. Biol.
PUBLISHED: 01-17-2011
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The functionality of the sexual cycle in the heterothallic pathogen Phytophthora ramorum, causal agent of Sudden Oak Death, has recently been demonstrated. Sexual reproduction could create genotypic variation and increase the pathogens ability to adapt to other host plants or changing environments. Genetic characterization using co-dominant microsatellite markers and flow cytometry of single-oospore progeny of crosses between a European A1 isolate and North American or European A2 isolates revealed a considerable number of non-Mendelian inheritance events. This includes inheritance of more than two alleles at a locus and non-inheritance of alleles from one parent at another locus. The progenies were mitotically unstable: zoospore and hyphal tip derivatives of the progenies showed genotypic rearrangements and phenotypic variation. Flow cytometry confirmed variation and instability in DNA content of the single-oospore progenies. This indicates that single-oospore progenies not only display aberrant genomic and phenotypic variation due to meiotic irregularities, but also extra variation as a result of post-meiotic genomic rearrangements.
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GyrB sequence analysis and MALDI-TOF MS as identification tools for plant pathogenic Clavibacter.
Syst. Appl. Microbiol.
PUBLISHED: 01-06-2011
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The bacterial genus Clavibacter has only one species, Clavibacter michiganensis, containing five subspecies. All five are plant pathogens, among which three are recognized as quarantine pests (mentioned on the EPPO A2 list). Prevention of their introduction and epidemic outbreaks requires a reliable and accurate identification. Currently, identification of these bacteria is time consuming and often problematic, mainly because of cross-reactions with other plant-associated bacteria in immunological tests and false-negative results in PCR detection methods. Furthermore, distinguishing closely related subspecies is not straightforward. This study aimed at evaluating the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and a fragment of the gyrB sequence for the reliable and fast identification of the Clavibacter subspecies. Amplification and sequencing of gyrB using a single primer set had sufficient resolution and specificity to identify each subspecies based on both sequence similarities in cluster analyses and specific signatures within the sequences. All five subspecies also generated distinct and reproducible MALDI-TOF MS profiles, with unique and specific ion peaks for each subspecies, which could be used as biomarkers for identification. Results from both methods were in agreement and were able to distinguish the five Clavibacter subspecies from each other and from representatives of closely related Rathayibacter, Leifsonia or Curtobacterium species. Our study suggests that proteomic analysis using MALDI-TOF MS and gyrB sequence are powerful diagnostic tools for the accurate identification of Clavibacter plant pathogens.
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Molecular detection of Puccinia horiana in Chrysanthemum x morifolium through conventional and real-time PCR.
J. Microbiol. Methods
PUBLISHED: 03-25-2009
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Puccinia horiana Henn. is a quarantine organism and one of the most important fungal pathogens of Chrysanthemum x morifolium cultivars grown for cut flower or potted plant production (florists chrysanthemum) in several regions of the world. Highly specific primer pairs were identified for conventional, nested, and real-time PCR detection of P. horiana based on the specific and sensitive PCR amplification of selected regions in the internal transcribed spacers (ITS1 and ITS2) of the nuclear ribosomal DNA (rDNA). Using these different PCR versions, 10 pg, 10 fg, and 5 fg genomic DNA could be detected, respectively. When using cloned target DNA as template, the detection limits were 5000, 50, and 5 target copies, respectively. These detection limits were not affected by a background of chrysanthemum plant DNA. The DNA extraction method was optimized to maximize the recoverability of the pathogen from infected plant tissue. A CTAB extraction protocol or a selection of commercial DNA extraction methods allowed the use of 10 ng total (plant+pathogen) DNA without interference of PCR inhibitors. Due to the specificity of the primers, SYBR Green I technology enabled reliable real time PCR signal detection. However, an efficient TaqMan probe is available. The lowest proportion of infected plant material that could still be detected when mixed with healthy plant material was 0.001%. The real-time PCR assay could detect as few as eight pure P. horiana basidiospores, demonstrating the potential of the technique for aerial detection of the pathogen. The amount of P. horiana DNA in plant tissue was determined at various time points after basidiospore inoculation. Using the real-time PCR protocol, it was possible to detect the pathogen immediately after the inoculation period, even though the accumulation of pathogen DNA was most pronounced near the end of the latent period. The detection system proved to be accurate and sensitive and could help not only in pathogen diagnosis but also in pathogen monitoring and disease forecasting systems.
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Phylogenetic relationships of Puccinia horiana and other rust pathogens of Chrysanthemum x morifolium based on rDNA ITS sequence analysis.
Mycol. Res.
PUBLISHED: 02-16-2009
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Isolates of the most important Puccinia species that have been reported on Chrysanthemum x morifolium were collected and the sequences of their ribosomal DNA internal transcribed spacers ITS1 and ITS2 were determined and used as phylogenetic markers. The focus of this study was on Puccinia horiana, due to its quarantine status and its impact in commercial chrysanthemum production. Three technical adjustments were needed to reliably obtain the nucleotide sequences starting from fresh or dried samples. The complete rDNA ITS nucleotide sequences of P. horiana, Puccinia chrysanthemi, and Puccinia tanaceti isolates of varying age and geographic origin were determined. We also identified an as yet undescribed Puccinia species on six old herbarium samples from chrysanthemum. This new species is morphologically similar to P. chrysanthemi and near identical to recent rust samples from Artemisia tridentata. P. tanaceti could not be confirmed as a pathogen of chrysanthemum. Different rDNA ITS sequences were present in P. horiana, with intra-isolate and inter-isolate variability in the length of three nucleotide repeat regions in the different rDNA tandem copies. We also identified three ITS types within P. horiana, with the rarer types displaying up to 67 bp nucleotide sequence differences. These rarer ITS types were detected at low copy number in all isolates. In general, very little rDNA ITS sequence variation was observed between P. horiana isolates from 1903 and 2003, and among isolates from different continents. Phylogenetic analyses using distance, Maximum Likelihood and Bayesian methods confirmed P. horiana, P. chrysanthemi, and the new Puccinia sp. as well-resolved groups, with P. horiana clustering in the clade where the economically important rust species of the Poaceae are located, and P. chrysanthemi and the new Puccinia sp. clustering in the clade where the majority of the rust fungi with hosts in the Asteraceae is located.
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Brenneria salicis, the bacterium causing watermark disease in willow, resides as an endophyte in wood.
Environ. Microbiol.
PUBLISHED: 02-10-2009
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Brenneria salicis has been studied in willow wood only in relation to watermark disease. In this pathogenic condition, the bacterium occurs at high concentrations. Pathogenicity of B. salicis is still uncontrollable and the disease unpredictable because the plant-bacteria interaction is not understood. Thanks to molecular techniques B. salicis can be detected at low concentrations, which are found in most non-pathogenic interactions. Brenneria salicis was identified and traced with a new specific three-primer polymerase chain reaction and its identity and relative concentration in biological samples confirmed through denaturing gradient gel electrophoresis profiling. Brenneria salicis was found in symptomless willows sampled randomly in Flanders agricultural areas, in young nursery willows, and also in poplar (Populus) and alder (Alnus). It harboured the nitrogenase reductase gene NifH and promoted growth and chlorophyll in willow. Inoculated luminescence-marked B. salicis circulated through the whole plant without inducing disease and exuded at the leaf margins. Other willow endophytes identified were Rahnella, Sphingomonas and Methylobacterium. In conclusion, because endophytic B. salicis is generally observed in willow, disease must not be dependent on infection. Leaf-to-leaf spread is proposed as an important mechanism for spread of B. salicis.
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Willow wood sap promotes the density-dependent pathogenesis of Brenneria salicis.
Environ. Microbiol.
PUBLISHED: 02-10-2009
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Brenneria salicis resides in symptomless willow (Salix spp.) and other tree species, but only willow trees develop watermark disease. To understand the conversion of B. salicis into a pathogen, its pathogenicity and differential growth in the various tree species are studied. Brenneria salicis was detected by plating and polymerase chain reaction-based techniques. Cell wall degradation and quorum sensing (QS) were assayed as possible pathogenicity mechanisms in wood. Differences in B. salicis growth capacities were tested in wood sap of the trees. Watermark diseased willow wood contained high concentrations of B. salicis with QS-induced cellulase activity. In the fall, wood sap of willow, and not of poplar and alder, promoted high density growth of B. salicis. In situ, B. salicis was the dominant bacterial type in willow wood during the fall and winter period. Willow sustains high densities of B. salicis at the time of leaf shedding. The cellulase in the immobilized wood sap has then a long-lasting contact with the xylem cell wall. Timing of dormancy and subsequent winter conditions might interfere with sap composition, B. salicis density, activity and survival, and be the reason, at least partly, for the variable occurrence of the disease.
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Pseudomonas cichorii as the causal agent of midrib rot, an emerging disease of greenhouse-grown butterhead lettuce in Flanders.
Syst. Appl. Microbiol.
PUBLISHED: 01-20-2009
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Bacterial midrib rot of greenhouse-grown butterhead lettuce (Lactuca sativa L. var. capitata) is an emerging disease in Flanders (Belgium) and fluorescent pseudomonads are suspected to play an important role in the disease. Isolations from infected lettuces, collected from 14 commercial greenhouses in Flanders, yielded 149 isolates that were characterized polyphasically, which included morphological characteristics, pigmentation, pathogenicity tests by both injection and spraying of lettuce, LOPAT characteristics, FAME analysis, BOX-PCR fingerprinting, 16S rRNA and rpoB gene sequencing, as well as DNA-DNA hybridization. Ninety-eight isolates (66%) exhibited a fluorescent pigmentation and were associated with the genus Pseudomonas. Fifty-five of them induced an HR+ (hypersensitive reaction in tobacco leaves) response. The other 43 fluorescent isolates were most probably saprophytic bacteria and about half of them were able to cause rot on potato tuber slices. BOX-PCR genomic fingerprinting was used to assess the genetic diversity of the Pseudomonas midrib rot isolates. The delineated BOX-PCR patterns matched quite well with Pseudomonas morphotypes defined on the basis of colony appearance and variation in fluorescent pigmentation. 16S rRNA and rpoB gene sequence analyses allowed most of the fluorescent isolates to be allocated to Pseudomonas, and they belonged to either the Pseudomonas fluorescens group, Pseudomonas putida group, or the Pseudomonas cichorii/syringae group. In particular, the isolates allocated to this latter group constituted the vast majority of HR+ isolates and were identified as P. cichorii by DNA-DNA hybridization. They were demonstrated by spray-inoculation tests on greenhouse-grown lettuce to induce the midrib rot disease and could be re-isolated from lesions of inoculated plants. Four HR+ non-fluorescent isolates associated with one sample that showed an atypical midrib rot were identified as Dickeya sp.
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CIM(®) monolithic anion-exchange chromatography as a useful alternative to CsCl gradient purification of bacteriophage particles.
Virology
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The use of anion-exchange chromatography was investigated as an alternative method to concentrate and purify bacterial viruses, and parameters for different bacteriophages were compared. Chromatography was performed with Convective Interactive Media(®) monoliths, with three different volumes and two matrix chemistries. Eleven morphologically distinct phages were tested, infecting five different bacterial species. For each of the phages tested, a protocol was optimized, including the choice of column chemistry, loading, buffer and elution conditions. The capacity and recovery of the phages on the columns varied considerably between phages. We conclude that anion-exchange chromatography with monoliths is a valid alternative to the more traditional CsCl purification, has upscaling advantages, but it requires more extensive optimization.
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A suggested new bacteriophage genus: "Viunalikevirus".
Arch. Virol.
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We suggest a bacteriophage genus, "Viunalikevirus", as a new genus within the family Myoviridae. To date, this genus includes seven sequenced members: Salmonella phages ViI, SFP10 and ?SH19; Escherichia phages CBA120 and PhaxI; Shigella phage phiSboM-AG3; and Dickeya phage LIMEstone1. Their shared myovirus morphology, with comparable head sizes and tail dimensions, and genome organization are considered distinguishing features. They appear to have conserved regulatory sequences, a horizontally acquired tRNA set and the probable substitution of an alternate base for thymine in the DNA. A close examination of the tail spike region in the DNA revealed four distinct tail spike proteins, an arrangement which might lead to the umbrella-like structures of the tails visible on electron micrographs. These properties set the suggested genus apart from the recently ratified subfamily Tevenvirinae, although a significant evolutionary relationship can be observed.
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Sequence diversity in the Dickeya fliC gene: phylogeny of the Dickeya genus and TaqMan® PCR for D. solani, new biovar 3 variant on potato in Europe.
PLoS ONE
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Worldwide, Dickeya (formerly Erwinia chrysanthemi) is causing soft rot diseases on a large diversity of crops and ornamental plants. Strains affecting potato are mainly found in D. dadantii, D. dianthicola and D. zeae, which appear to have a marked geographical distribution. Furthermore, a few Dickeya isolates from potato are attributed to D. chrysanthemi and D. dieffenbachiae. In Europe, isolates of Erwinia chrysanthemi biovar 1 and biovar 7 from potato are now classified in D. dianthicola. However, in the past few years, a new Dickeya biovar 3 variant, tentatively named Dickeya solani, has emerged as a common major threat, in particular in seed potatoes. Sequences of a fliC gene fragment were used to generate a phylogeny of Dickeya reference strains from culture collections and with this reference backbone, to classify pectinolytic isolates, i.e. Dickeya spp. from potato and ornamental plants. The reference strains of the currently recognized Dickeya species and D. solani were unambiguously delineated in the fliC phylogram. D. dadantii, D. dianthicola and D. solani displayed unbranched clades, while D. chrysanthemi, D. zeae and D. dieffenbachiae branched into subclades and lineages. Moreover, Dickeya isolates from diagnostic samples, in particular biovar 3 isolates from greenhouse ornamentals, formed several new lineages. Most of these isolates were positioned between the clade of D. solani and D. dadantii as transition variants. New lineages also appeared in D. dieffenbachiae and in D. zeae. The strains and isolates of D. dianthicola and D. solani were differentiated by a fliC sequence useful for barcode identification. A fliC TaqMan®real-time PCR was developed for D. solani and the assay was provisionally evaluated in direct analysis of diagnostic potato samples. This molecular tool can support the efforts to control this particular phytopathogen in seed potato certification.
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T4-related bacteriophage LIMEstone isolates for the control of soft rot on potato caused by Dickeya solani.
PLoS ONE
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The bacterium Dickeya solani, an aggressive biovar 3 variant of Dickeya dianthicola, causes rotting and blackleg in potato. To control this pathogen using bacteriophage therapy, we isolated and characterized two closely related and specific bacteriophages, vB_DsoM_LIMEstone1 and vB_DsoM_LIMEstone2. The LIMEstone phages have a T4-related genome organization and share DNA similarity with Salmonella phage ViI. Microbiological and molecular characterization of the phages deemed them suitable and promising for use in phage therapy. The phages reduced disease incidence and severity on potato tubers in laboratory assays. In addition, in a field trial of potato tubers, when infected with Dickeya solani, the experimental phage treatment resulted in a higher yield. These results form the basis for the development of a bacteriophage-based biocontrol of potato plants and tubers as an alternative for the use of antibiotics.
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