Evaluation of a real-time PCR and a loop-mediated isothermal amplification for detection of Xanthomonas arboricola pv. pruni in plant tissue samples

Palacio-Bielsa, Ana; López-Soriano, Pablo; Bühlmann, Andreas; Doorn, J. van; Pham, K.T.K.; Cambra, M. A.; Berruete, Isabel M.; Pothier, Joël F.; Duffy, Brion; Olmos, Antonio; López, Marı́a M.

doi:10.1016/j.mimet.2015.03.005

Cited by 12 publications

(18 citation statements)

References 11 publications

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“…and phenotypically similar to X. arboricola were initially identified as Xap by using a real-time PCR protocol (Palacio-Bielsa et al, 2011, 2015). Additionally, the plasmid pXap41, which is considered a specific feature of Xap , was not detected by PCR amplification of the genes repA1, repA2 and mobC (Pothier et al, 2011b) from strains CITA 14, CITA 42, CITA 49, CITA 51, CITA 124, and CITA 149 and, therefore, they were not considered as Xap but Xap -look-a-like strains.…”

Section: Resultsmentioning

confidence: 99%

“…For an initial Xap classification, a real-time PCR reaction in the gene ftsX of an ABC transporter (Palacio-Bielsa et al, 2011, 2015), and a multiplex PCR for plasmid pXap41 (Pothier et al, 2011b) were performed. Those strains that showed a positive result only for the real-time assay were considered as Xap -look-a-like strains, and were further identified according to a multilocus sequence typing scheme (MLSA) based in the partial sequences of the housekeeping genes dnaK, fyuA, gyrB and rpoD (Young et al, 2008).…”

Section: Methodsmentioning

confidence: 99%

“…After 21 dpi, the infiltrated leaves were macerated on sterile distilled water and tenfold dilution of the macerated were plated on YPGA (0.5% yeast extract, 0.5% bactopeptone, 1.0% glucose, 2.0% agar) supplemented with 250 mg/l of cycloheximide. Colonies showing a Xanthomonas -like phenotype were confirmed as Xap -look-a-like using a real time PCR protocol indicated above (Palacio-Bielsa et al, 2011, 2015). Additionally, for P. persica rootstock GF-305, colonies were counted in order to determine the bacterial concentration in the inoculated tissue at the end of the assay (Ah-You et al, 2007).…”

Section: Methodsmentioning

confidence: 99%

“…Sterile distilled water was used as negative control. Additionally, a real-time PCR protocol previously described (Palacio-Bielsa et al, 2011, 2015) was also applied on all the 99 bacterial strains tested for xopE3 gene.…”

Section: Methodsmentioning

confidence: 99%

“…Statistical analyses were performed by using Statgraphics Plus v.5.1 software. The amplification efficiency of the protocol for each kind of sample was calculated as described previously (Palacio-Bielsa et al, 2011, 2015). Linear regression curves representing the C ts of each reaction were plotted against the logarithmic values of bacterial or DNA concentration.…”

Section: Methodsmentioning

confidence: 99%

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Pan-Genomic Analysis Permits Differentiation of Virulent and Non-virulent Strains of Xanthomonas arboricola That Cohabit Prunus spp. and Elucidate Bacterial Virulence Factors

et al. 2017

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Xanthomonas arboricola is a plant-associated bacterial species that causes diseases on several plant hosts. One of the most virulent pathovars within this species is X. arboricola pv. pruni (Xap), the causal agent of bacterial spot disease of stone fruit trees and almond. Recently, a non-virulent Xap-look-a-like strain isolated from Prunus was characterized and its genome compared to pathogenic strains of Xap, revealing differences in the profile of virulence factors, such as the genes related to the type III secretion system (T3SS) and type III effectors (T3Es). The existence of this atypical strain arouses several questions associated with the abundance, the pathogenicity, and the evolutionary context of X. arboricola on Prunus hosts. After an initial characterization of a collection of Xanthomonas strains isolated from Prunus bacterial spot outbreaks in Spain during the past decade, six Xap-look-a-like strains, that did not clustered with the pathogenic strains of Xap according to a multi locus sequence analysis, were identified. Pathogenicity of these strains was analyzed and the genome sequences of two Xap-look-a-like strains, CITA 14 and CITA 124, non-virulent to Prunus spp., were obtained and compared to those available genomes of X. arboricola associated with this host plant. Differences were found among the genomes of the virulent and the Prunus non-virulent strains in several characters related to the pathogenesis process. Additionally, a pan-genomic analysis that included the available genomes of X. arboricola, revealed that the atypical strains associated with Prunus were related to a group of non-virulent or low virulent strains isolated from a wide host range. The repertoire of the genes related to T3SS and T3Es varied among the strains of this cluster and those strains related to the most virulent pathovars of the species, corylina, juglandis, and pruni. This variability provides information about the potential evolutionary process associated to the acquisition of pathogenicity and host specificity in X. arboricola. Finally, based in the genomic differences observed between the virulent and the non-virulent strains isolated from Prunus, a sensitive and specific real-time PCR protocol was designed to detect and identify Xap strains. This method avoids miss-identifications due to atypical strains of X. arboricola that can cohabit Prunus.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Pan-Genomic Analysis Permits Differentiation of Virulent and Non-virulent Strains of Xanthomonas arboricola That Cohabit Prunus spp. and Elucidate Bacterial Virulence Factors

et al. 2017

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Xanthomonas arboricola pv. pruni (bacterial canker of stone fruit)

Osdaghi¹

2022

CABI Compendium

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Rapid Detection of Xanthomonas citri pv. fuscans and Xanthomonas phaseoli pv. phaseoli in Common Bean by Loop-Mediated Isothermal Amplification

Paiva

Wendland²,

Teixeira³

et al. 2020

Plant Disease

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A single loop-mediated isothermal amplification (LAMP) assay was developed for specific detection of both pathogens that cause bacterial blight in common bean, Xanthomonas phaseoli pv. phaseoli (Xpp) and Xanthomonas citri pv. fuscans (Xcf). The objective was to provide a simple, easy-to-use, specific, and sensitive method to investigate the presence of one or both pathogens in plant material and seeds for routine diagnosis. The detection limits for both pathogens were 10 CFU/ml for cell suspensions and 1 fg of DNA, whereas in conventional PCR, the primers detected up to 105 CFU/ml and 1 ng of DNA. Specificity was confirmed by testing DNA from bean leaves, other Xanthomonas species, common fungal and bacterial bean pathogens, and bacteria from the leaf microbiota. The method was tested with bean leaves inoculated with Xpp, and the pathogen could be detected from 4 h up to 15 days postinoculation, even before disease symptoms were visible. When the method was applied to bacterium detection (Xpp or Xcf) in seed lots from infected plants, the bacterium detection rate was 100% (24 of 24). The pathogens were detected in seeds incubated for just 1 h in saline solution (0.85%), reducing the time needed for bacterium detection. The LAMP assay could be useful as a tool in bean bacterial blight management. Rapid and sensitive detection of bacteria in bean seed lots would reduce the risks of planting highly contaminated seeds in environments favorable to blight multiplication.

show abstract

Evaluation of a real-time PCR and a loop-mediated isothermal amplification for detection of Xanthomonas arboricola pv. pruni in plant tissue samples

Cited by 12 publications

References 11 publications

Pan-Genomic Analysis Permits Differentiation of Virulent and Non-virulent Strains of Xanthomonas arboricola That Cohabit Prunus spp. and Elucidate Bacterial Virulence Factors

Pan-Genomic Analysis Permits Differentiation of Virulent and Non-virulent Strains of Xanthomonas arboricola That Cohabit Prunus spp. and Elucidate Bacterial Virulence Factors

Xanthomonas arboricola pv. pruni (bacterial canker of stone fruit)

Rapid Detection of Xanthomonas citri pv. fuscans and Xanthomonas phaseoli pv. phaseoli in Common Bean by Loop-Mediated Isothermal Amplification

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