Use of Species-Specific Satellite DNA from<i>Bursaphelenchus xylophilus</i>as a Diagnostic Probe

Tarès, Sophie; Lemontey, J.-M.; Guiran, G. de; Abad, Pierre

doi:10.1094/phyto-84-294

Cited by 31 publications

(10 citation statements)

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“…Total DNA, extracted from wood, bark and nematode suspensions of pine wood samples and from M. galloprovincialis, was successfully used as template for PCR detection of PWN by amplification of the species specific satDNA, generating a PCR specific pattern of monomer and multimers of the 160 monomer unit, as previously described (Tarés et al 1994;Castagnone et al 2005). The results obtained show that these DNA extraction methods are suitable for obtaining DNA free from PCR inhibitors, which are commonly present in wood and insects (Wilson 1997).…”

Section: Discussionmentioning

confidence: 99%

“…However, few studies on the use of these methodologies for direct detection of PWN in pine wood and insect vector, without the preliminary steps of nematode extraction, have been conducted (François et al 2007;Takeuchi and Futai 2007;Kikuchi et al 2009;Takeuchi and Futai 2009;Kanetani et al 2011;Wang et al 2010;Hu et al 2011;Wang et al 2011). In the present study, a new methodology was developed for the direct detection of PWN, performed by PCR amplification of the species specific MspI satDNA, leading to a pattern of monomer and multimers of the 160 bp monomer (Tarés et al 1994;Castagnone et al 2005), as recommended by the European and Mediterranean Plant Protection Organization (EPPO) (2009), using total DNA extracted from P. pinaster wood and bark samples and from the insect vector, M. galloprovincialis.…”

Section: Introductionmentioning

confidence: 99%

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Direct molecular detection of the pinewood nematode, Bursaphelenchus xylophilus, from pine wood, bark and insect vector

2011

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The pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease. The international economic impact of the introduction of the PWN into new areas has highlighted the need for the development of accurate and reliable detection methods of B. xylophilus, which are essential to define aspects of its control and management. In the present study, a methodology was developed for the direct detection of PWN by conventional PCR assay, with a species specific set of primers based on PWN satellite DNA, using total DNA extracted directly from maritime pine, Pinus pinaster, wood and bark samples, and from the insect vector, Monochamus galloprovincialis. This methodology involves homogenisation of wood, bark and insects using liquid nitrogen, DNA extraction and one or two PCR amplification steps, which permit the rapid and direct detection of one single nematode present in 100 mg of wood and bark and in one entire insect without the preliminary steps of nematode extraction.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Direct molecular detection of the pinewood nematode, Bursaphelenchus xylophilus, from pine wood, bark and insect vector

2011

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“…Interspecies forms have also been found, making it even more difficult to distinguish between these two closely related species (15). To distinguish B. xylophilus from B. mucronatus, several DNA-based methods have been developed, such as genomic DNA hybridization (16), PCR-restriction fragment length polymorphism (17)(18)(19) and species-specific PCR (ssPCR) (20). More recently, a real-time PCR protocol using the 5S rRNA as a probe was developed (21).…”

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confidence: 99%

A Combination of Biochemical and Proteomic Analyses Reveals Bx-LEC-1 as an Antigenic Target for the Monoclonal Antibody 3-2A7-2H5-D9-F10 Specific to the Pine Wood Nematode

Lee

Seo

Nam

et al. 2011

Molecular & Cellular Proteomics

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Pine wilt disease (PWD) is one of the most devastating forest diseases in Asia and Europe. The pine wood nematode, Bursaphelenchus xylophilus, has been identified as the pathogen underlying PWD, although the pathology is not completely understood. At present, diagnosis and confirmation of PWD are time consuming tasks that require nematode extraction and microscopic examination. To develop a more efficient detection method for B. xylophilus, we first generated monoclonal antibodies (MAbs) specific to B. xylophilus. Among 2304 hybridoma fusions screened, a hybridoma clone named 3-2A7-2H5 recognized a single protein from B. xylophilus specifically, but not those from other closely related nematodes. We finally selected the MAb clone 3-2A7-2H5-D9-F10 (D9-F10) for further studies. To identify the antigenic target of MAb-D9-F10, we analyzed proteins in spots, fractions, or bands isolated from SDS-PAGE, two-dimensional electrophoresis, anion exchange chromatography, and immunoprecipitation via nano liquid chromatography electrospray ionization quadrupole ion trap mass spectrometry (nano- The pinewood nematode, Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle (1), is a serious pathogen of the forest, particularly of Pinus species, causing pine wilt disease (PWD) 1 . Even though PWD was first reported in Japan in 1905 (2), the relationship between B. xylophilus and PWD was not elucidated until 1971 (3). Currently, PWD has spread to Far East Asian countries including Korea (4), China (5), and Taiwan (6) as well as to Portugal in the European Union (7). The distribution of B. xylophilus is currently extensive, explaining the seriousness of PWD. LC-ESI-Q-IT-MS). Peptides of galactose-bindingDistribution and development of PWD within pine trees have been described (8 -10). The Japanese pine sawyer beetle, Monochamus alternatus, is the known vector for B. xylo-

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“…However, diagnosis is often hindered by the presence of other Bursaphelenchus spp., including B. mucronatus (Mamiya & Enda, 1979). Bursaphelenchus mucronatus is a closely related pine wood nematode species that is morphologically similar to B. xylophilus yet is non-pathogenic under field conditions (Mamiya & Enda, 1979;Kiyohara & Bolla, 1990;Tares et al, 1994;Kanzaki & Futai, 2006), although it is often detected with B. xylophilus in dead pine wood (Nagashima et al, 1975;Kishi, 1995). Bursaphelenchus mucronatus has also been found in many regions of Korea, suggesting its broad distribution (I.S.…”

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confidence: 99%

Rapid and accurate prediction of the frequencies of Bursaphelenchus xylophilus and B. mucronatus in mixed nematode samples using real-time species-specific PCR

Kang

Moon

Lee

et al. 2009

Nematol

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Accurate detection of Bursaphelenchus xylophilus and prediction of its frequency in crude nematode samples is often hindered by the coexistence of related nematode species, such as B. mucronatus, that are morphologically similar but non-pathogenic. To establish a detection system enabling determination of the relative frequencies of B. xylophilus and B. mucronatus from field nematode samples, we developed a real-time species-specific PCR (rtssPCR) protocol which targets the substantial sequence differences in the 5S rRNA marker gene between the two nematode species. Using standard DNA mixtures of B. xylophilus and B. mucronatus in various ratios, plots of percent species proportion vs cycle threshold value (Ct value) were generated for the prediction of species frequency. The rtssPCR protocol enables the detection of target nematode frequencies as low as 0.16% at the 95% confidence level. When nematode DNA samples were extracted from the mixed specimens of B. xylophilus and B. mucronatus in various ratios and analysed by rtssPCR, the semi-log plot was nearly identical to the plot generated from standard mixed DNA samples, demonstrating that field populations of the nematodes can be directly used for rtssPCR analysis. The rapid and accurate determination of B. xylophilus or B. mucronatus frequencies by this rtssPCR protocol makes it ideal for routine monitoring and quarantine of B. xylophilus in the field.

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Use of Species-Specific Satellite DNA fromBursaphelenchus xylophilusas a Diagnostic Probe

Cited by 31 publications

References 0 publications

Direct molecular detection of the pinewood nematode, Bursaphelenchus xylophilus, from pine wood, bark and insect vector

Direct molecular detection of the pinewood nematode, Bursaphelenchus xylophilus, from pine wood, bark and insect vector

A Combination of Biochemical and Proteomic Analyses Reveals Bx-LEC-1 as an Antigenic Target for the Monoclonal Antibody 3-2A7-2H5-D9-F10 Specific to the Pine Wood Nematode

Rapid and accurate prediction of the frequencies of Bursaphelenchus xylophilus and B. mucronatus in mixed nematode samples using real-time species-specific PCR

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