Exploring diversity of Erwinia amylovora population in Serbia by conventional and automated techniques and detection of new PFGE patterns

Ivanović, Milan; Obradović, Aleksa; Gašić, Katarina; Minsavage, Gerald V.; Dickstein, E. R.; Jones, Jeffrey B.

doi:10.1007/s10658-012-9950-3

Cited by 10 publications

(9 citation statements)

References 19 publications

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“…Although all studied strains formed levantype colonies, differences were observed in their size, convexity and edges, which grouped the strains into three types. Similar variations in the shape of colonies were also reported by Momol & Aldwinckle (2000), especially between strains isolated from Maloideae and Rubus spp., and later observed also by Ivanović et al, (2012). The results of our research did not indicate a correlation between the morphology of colonies on KB and NSA media and the pathogenicity of the strains, nor their host species or geographical origin.…”

Section: Discussioncontrasting

confidence: 62%

“…The PFGE has been used for differentiation of E. amylovora strains, as well as for monitoring of introductions and spread of the pathogen (Jock et al, 2002;Donat et al, 2007). This method revealed at least two possible directions of E. amylovora introduction into Serbia (Ivanović et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

“…Although this technique was useful in differentiation of E. amylovora strains from Ireland (Brennan et al, 2002), it revealed high homogeneity among E. amylovora strains from Israel (Manulis-Sasson et al, 1998), Australia (Taylor & Hale, 1998), Austria and Hungary (Keck et al, 2002) and Poland (Pulawska et al, 2006). So far, the diversity of E. amylovora population from Serbia has been studied by PFGE and automated techniques, such as Biolog TM and fatty acid analysis (Ivanović et al, 2012). The objective of this research was to study the population with other molecular methods, such as REP, ERIC and BOX-PCR and by using six primers in RAPD-PCR.…”

Section: Introductionmentioning

confidence: 99%

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Characterization and population diversity of Erwinia amylovora strains originating from pome fruits in Serbia

et al. 2018

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The diversity of 30 Erwinia amylovora strains, isolated from quince, pear and apple trees on 14 localities in Serbia, was studied using bacteriological and molecular methods. In pathogenicity tests, all strains caused necrosis and oozing of bacterial exudate on inoculated immature pear, cherry and plum fruits, and induced hypersensitive reaction in tobacco leaves. The studied strains were Gram and oxidase negative, non-fluorescent, levan and catalase positive and facultatively anaerobic. The strains did not reduce nitrates, but utilized citrate and produced acid from sorbitol, hydrolyzed gelatine, produced reducing substances from sucrose and grew in the presence of 5% NaCl, but not at 36ºC. Identity of the strains was confirmed by conventional and nested PCR methods. Rep-PCR with REP, ERIC and BOX primers resulted in amplification of several DNA fragments respectively, but showed no variation within the strains. However, different genetic profiles were obtained with RAPD-PCR by using six primers which enabled differentiation of the strains into four groups. Genetic differences between the studied strains did not correlate with the host plants, geographical origin or year of isolation.

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Section: Discussioncontrasting

confidence: 62%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Characterization and population diversity of Erwinia amylovora strains originating from pome fruits in Serbia

et al. 2018

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“…The strains were isolated from different host plants, at different localities and in different years in Serbia and had various PFGE patterns in their genome, representing distant populations of the pathogen. The strains were previously identified based on biochemical properties, immature pear fruit assay, Biolog TM MicroPlate System and Fatty acid analysis (IVANOVIĆ et al, 2012). Type strain of E. amylovora NCPPB 595 was used as a positive control.…”

Section: Bacterial Strains Growth Conditions and Specificity Of Pcr mentioning

confidence: 99%

Specificity and sensitivity of three PCR-based methods for detection of Erwinia amylovora in pure culture and plant material

et al. 2019

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2019):Specificity and sensitivity of three PCR-based methods for detection of Erwinia amylovora in pure culture and plant material. -Genetika, Vol 51, No.3, 1039-1052 Three PCR methods, referred in this study as "conventional", "nested" and "chromosomal" PCR and suggested for routine detection of Erwinia amylovora in pure culture and plant material, were evaluated according to their specificity and sensitivity. Specificity of PCR methods was analyzed by using 42 strains of E. amylovora, originating from different locations and plant species, with diverse PFGE profiles, representing distant populations of the pathogen. Sensitivity of PCR protocols in pure culture was studied by using nine different concentrations of E. amylovora in sterile ultrapure water as a template in PCR reactions. In order to study inhibitory effect of plant DNA and other inhibitors on sensitivity of the three PCR methods bacterial dilutions were mixed with plant macerate of pear, apple and quince prior to the PCR reaction. In specificity assays, tested PCR protocols were able to detect all E. amylovora strains regardless of the host of the strain, its origin or PFGE group, indicating primer specificity. On the other hand, sensitivity among tested methods varied, depending on bacterial 1040 GENETIKA, Vol. 51, No3, 1039-1052, 2019 concentration and selected plant material used in the PCR. When working with pure cultures nested PCR showed the greatest sensitivity by detecting 1.9 bacterial cells per PCR reaction, followed by detection limit of 9.5 cells per PCR reaction with conventional PCR and 1.910 5 cells/PCR reaction with chromosomal PCR. In spiked samples plant inhibitors either did not affect or they decreased the sensitivity of the PCR reaction, depending on the protocol and/or type of plant macerate. In our experiments, inhibitors from pear and quince macerates did not affect sensitivity of nested PCR, while apple macerate reduced its sensitivity by a factor of 10. Conventional PCR protocol was able to detect 95 cells/PCR reaction in pear and apple macerate, but only 9.510 3 cells/PCR in quince macerate. Greatest decrease in sensitivity of the PCR method was observed in spiked samples with chromosomal PCR since bacterial DNA was not detected in each of the spiked samples. Our research shows that all three PCR protocols are specific for detection of E. amylovora, but nested PCR proved to be most sensitive when working with pure cultures and plant material.

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“…Recently, in 2012, E. amylovora was reported from different host plants and locations in Serbia and Montenegro (Ivanovic et al, 2012) and it is also present in other countries like Tunisia, first reported in 2014 in pear (Rhouma et al, 2014) or Algeria, that had its first characterization of isolates in 2012 (Laala et al, 2012).…”

Section: Global Distributionmentioning

confidence: 99%

Elucidating the molecular basis of copper stress in Erwinia amylovora

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March 2017 3.9. Validation of the differentially expressed genes by quantitative real-time PCR 3.10. Mutant construction and complementation 3.11. Experiments with mutants of E. amylovora to evaluate the role of different genes 3.11.1. The possible role of rpoS gene in the survival of strain CFBP 1430 3.11.2. Assays to evaluate the possible role of copA, soxS, arcB, yjcE, ygcF, yhhQ, galF and EAM_3469 genes of strain Ea1189 after copper shock 3.11.2.1. Copper tolerance in vitro 3.11.2.2. Effect of introduction of target genes in the mutants 3.12. The copA case 3.12.1. Expression curve of copA in E. amylovora after copper-shock induction 3.12.2. Copper tolerance in planta 4. RESULTS 4.1. Minimal inhibitory concentration for copper 4.2. Short-term assay for E. amylovora survival in AB medium supplemented with different copper concentrations 4.3. Efficiency of RNA extraction 4.4. Sensitivity of rpoS, katG and dsbC primers 4.5. Real-time PCR standard curve for rpoS gene 4.6. Long-term assay for E. amylovora survival in AB medium supplementd with copper sulfate and expression of rpoS gene through time 4.7. General characteristics of the E. amylovora Ea1189 strain transcriptome in response to a copper shock 4.8. Evaluation of differentially expressed genes under copper conditions by quantitative real-time PCR 4.9. Mutants of selected genes of E. amylovora 4.9.1. Assays to evaluate the possible role of rpoS gene 4.9.2. Assays to evaluate the possible role of copA, soxS,

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Exploring diversity of Erwinia amylovora population in Serbia by conventional and automated techniques and detection of new PFGE patterns

Cited by 10 publications

References 19 publications

Characterization and population diversity of Erwinia amylovora strains originating from pome fruits in Serbia

Characterization and population diversity of Erwinia amylovora strains originating from pome fruits in Serbia

Specificity and sensitivity of three PCR-based methods for detection of Erwinia amylovora in pure culture and plant material

Elucidating the molecular basis of copper stress in Erwinia amylovora

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