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Development of an Efficient Real-Time Quantitative PCR Protocol for Detection of Xanthomonas arboricola pv. pruni in Prunus Species ▿ †

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Development of an Efficient Real-Time Quantitative PCR Protocol for Detection of Xanthomonas arboricola pv. pruni in Prunus Species ▿ †

Auteurs : Ana Palacio-Bielsa ; Jaime Cubero ; Miguel A. Cambra ; Raquel Collados ; Isabel M. Berruete ; María M. L Pez

Source :

Applied and Environmental Microbiology [ 0099-2240 ] ; 2010.

RBID : PMC:3019718

Abstract

Xanthomonas arboricola pv. pruni, the causal agent of bacterial spot disease of stone fruit, is considered a quarantine organism by the European Union and the European and Mediterranean Plant Protection Organization (EPPO). The bacterium can undergo an epiphytic phase and/or be latent and can be transmitted by plant material, but currently, only visual inspections are used to certify plants as being X. arboricola pv. pruni free. A novel and highly sensitive real-time TaqMan PCR detection protocol was designed based on a sequence of a gene for a putative protein related to an ABC transporter ATP-binding system in X. arboricola pv. pruni. Pathogen detection can be completed within a few hours with a sensitivity of 10² CFU ml⁻¹, thus surpassing the sensitivity of the existing conventional PCR. Specificity was assessed for X. arboricola pv. pruni strains from different origins as well as for closely related Xanthomonas species, non-Xanthomonas species, saprophytic bacteria, and healthy Prunus samples. The efficiency of the developed protocol was evaluated with field samples of 14 Prunus species and rootstocks. For symptomatic leaf samples, the protocol was very efficient even when washed tissues of the leaves were directly amplified without any previous DNA extraction. For samples of 117 asymptomatic leaves and 285 buds, the protocol was more efficient after a simple DNA extraction, and X. arboricola pv. pruni was detected in 9.4% and 9.1% of the 402 samples analyzed, respectively, demonstrating its frequent epiphytic or endophytic phase. This newly developed real-time PCR protocol can be used as a quantitative assay, offers a reliable and sensitive test for X. arboricola pv. pruni, and is suitable as a screening test for symptomatic as well as asymptomatic plant material.

Url:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3019718

DOI: 10.1128/AEM.01593-10
PubMed: 21037298
PubMed Central: 3019718

Affiliations:

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PMC:3019718

Le document en format XML

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 pv. pruni in <italic>Prunus</italic>
 Species <xref ref-type="fn" rid="fn2">▿</xref>
 <xref ref-type="fn" rid="fn1">†</xref>
 </title>
<author><name sortKey="Palacio Bielsa, Ana" sort="Palacio Bielsa, Ana" uniqKey="Palacio Bielsa A" first="Ana" last="Palacio-Bielsa">Ana Palacio-Bielsa</name>
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<author><name sortKey="Cubero, Jaime" sort="Cubero, Jaime" uniqKey="Cubero J" first="Jaime" last="Cubero">Jaime Cubero</name>
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<author><name sortKey="Cambra, Miguel A" sort="Cambra, Miguel A" uniqKey="Cambra M" first="Miguel A." last="Cambra">Miguel A. Cambra</name>
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<author><name sortKey="Collados, Raquel" sort="Collados, Raquel" uniqKey="Collados R" first="Raquel" last="Collados">Raquel Collados</name>
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<author><name sortKey="Berruete, Isabel M" sort="Berruete, Isabel M" uniqKey="Berruete I" first="Isabel M." last="Berruete">Isabel M. Berruete</name>
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<author><name sortKey="L Pez, Maria M" sort="L Pez, Maria M" uniqKey="L Pez M" first="María M." last="L Pez">María M. L Pez</name>
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<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Development of an Efficient Real-Time Quantitative PCR Protocol for Detection of <italic>Xanthomonas arboricola</italic>
 pv. pruni in <italic>Prunus</italic>
 Species <xref ref-type="fn" rid="fn2">▿</xref>
 <xref ref-type="fn" rid="fn1">†</xref>
 </title>
<author><name sortKey="Palacio Bielsa, Ana" sort="Palacio Bielsa, Ana" uniqKey="Palacio Bielsa A" first="Ana" last="Palacio-Bielsa">Ana Palacio-Bielsa</name>
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<author><name sortKey="Cambra, Miguel A" sort="Cambra, Miguel A" uniqKey="Cambra M" first="Miguel A." last="Cambra">Miguel A. Cambra</name>
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<author><name sortKey="Berruete, Isabel M" sort="Berruete, Isabel M" uniqKey="Berruete I" first="Isabel M." last="Berruete">Isabel M. Berruete</name>
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<author><name sortKey="L Pez, Maria M" sort="L Pez, Maria M" uniqKey="L Pez M" first="María M." last="L Pez">María M. L Pez</name>
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<series><title level="j">Applied and Environmental Microbiology</title>
<idno type="ISSN">0099-2240</idno>
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<front><div type="abstract" xml:lang="en"><p><italic>Xanthomonas arboricola</italic>
 pv. pruni, the causal agent of bacterial spot disease of stone fruit, is considered a quarantine organism by the European Union and the European and Mediterranean Plant Protection Organization (EPPO). The bacterium can undergo an epiphytic phase and/or be latent and can be transmitted by plant material, but currently, only visual inspections are used to certify plants as being <italic>X. arboricola</italic>
 pv. pruni free. A novel and highly sensitive real-time TaqMan PCR detection protocol was designed based on a sequence of a gene for a putative protein related to an ABC transporter ATP-binding system in <italic>X. arboricola</italic>
 pv. pruni. Pathogen detection can be completed within a few hours with a sensitivity of 10<sup>2</sup>
 CFU ml<sup>−1</sup>
, thus surpassing the sensitivity of the existing conventional PCR. Specificity was assessed for <italic>X. arboricola</italic>
 pv. pruni strains from different origins as well as for closely related <italic>Xanthomonas</italic>
 species, non-<italic>Xanthomonas</italic>
 species, saprophytic bacteria, and healthy <italic>Prunus</italic>
 samples. The efficiency of the developed protocol was evaluated with field samples of 14 <italic>Prunus</italic>
 species and rootstocks. For symptomatic leaf samples, the protocol was very efficient even when washed tissues of the leaves were directly amplified without any previous DNA extraction. For samples of 117 asymptomatic leaves and 285 buds, the protocol was more efficient after a simple DNA extraction, and <italic>X. arboricola</italic>
 pv. pruni was detected in 9.4% and 9.1% of the 402 samples analyzed, respectively, demonstrating its frequent epiphytic or endophytic phase. This newly developed real-time PCR protocol can be used as a quantitative assay, offers a reliable and sensitive test for <italic>X. arboricola</italic>
 pv. pruni, and is suitable as a screening test for symptomatic as well as asymptomatic plant material.</p>
</div>
</front>
</TEI>
<pmc article-type="research-article"><pmc-comment>The publisher of this article does not allow downloading of the full text in XML form.</pmc-comment>
  <front><journal-meta><journal-id journal-id-type="nlm-ta">Appl Environ Microbiol</journal-id>
<journal-id journal-id-type="publisher-id">aem</journal-id>
<journal-title-group><journal-title>Applied and Environmental Microbiology</journal-title>
</journal-title-group>
<issn pub-type="ppub">0099-2240</issn>
<issn pub-type="epub">1098-5336</issn>
<publisher><publisher-name>American Society for Microbiology (ASM)</publisher-name>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">21037298</article-id>
<article-id pub-id-type="pmc">3019718</article-id>
<article-id pub-id-type="publisher-id">1593-10</article-id>
<article-id pub-id-type="doi">10.1128/AEM.01593-10</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Methods</subject>
</subj-group>
</article-categories>
<title-group><article-title>Development of an Efficient Real-Time Quantitative PCR Protocol for Detection of <italic>Xanthomonas arboricola</italic>
 pv. pruni in <italic>Prunus</italic>
 Species <xref ref-type="fn" rid="fn2">▿</xref>
 <xref ref-type="fn" rid="fn1">†</xref>
 </article-title>
</title-group>
<contrib-group><contrib contrib-type="author"><name><surname>Palacio-Bielsa</surname>
<given-names>Ana</given-names>
</name>
<xref ref-type="aff" rid="aff1">1</xref>
<xref ref-type="corresp" rid="cor1">*</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Cubero</surname>
<given-names>Jaime</given-names>
</name>
<xref ref-type="aff" rid="aff1">2</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Cambra</surname>
<given-names>Miguel A.</given-names>
</name>
<xref ref-type="aff" rid="aff1">3</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Collados</surname>
<given-names>Raquel</given-names>
</name>
<xref ref-type="aff" rid="aff1">3</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Berruete</surname>
<given-names>Isabel M.</given-names>
</name>
<xref ref-type="aff" rid="aff1">1</xref>
</contrib>
<contrib contrib-type="author"><name><surname>López</surname>
<given-names>María M.</given-names>
</name>
<xref ref-type="aff" rid="aff1">4</xref>
</contrib>
</contrib-group>
<aff id="aff1">Centro de Investigación y Tecnología Agroalimentaria de Aragón, Av. Montañana 930, 50059 Zaragoza, Spain,<label>1</label>
 Departamento de Protección Vegetal, INIA, Carretera de La Coruña km 7, 28040 Madrid, Spain,<label>2</label>
 Centro de Protección Vegetal, Av. Montañana 930, 50059 Zaragoza, Spain,<label>3</label>
 Instituto Valenciano de Investigaciones Agrarias, Carretera Moncada a Náquera km 4.5, 46113 Moncada, Valencia, Spain<label>4</label>
</aff>
<author-notes><corresp id="cor1"><label>*</label>
Corresponding author. Mailing address: Centro de Investigación y Tecnología Agroalimentaria de Aragón, Av. Montañana 930, 50059 Zaragoza, Spain. Phone: 34-976 71 63 75. Fax: 34-976 71 63 35. E-mail: <email>apalaciob@aragon.es</email>
</corresp>
</author-notes>
<pub-date pub-type="ppub"><month>1</month>
<year>2011</year>
</pub-date>
<pub-date pub-type="epub"><day>29</day>
<month>10</month>
<year>2010</year>
</pub-date>
<volume>77</volume>
<issue>1</issue>
<fpage>89</fpage>
<lpage>97</lpage>
<history><date date-type="received"><day>5</day>
<month>7</month>
<year>2010</year>
</date>
<date date-type="accepted"><day>21</day>
<month>10</month>
<year>2010</year>
</date>
</history>
<permissions><copyright-statement>Copyright © 2011, American Society for Microbiology</copyright-statement>
<copyright-year>2011</copyright-year>
</permissions>
<self-uri xlink:title="pdf" xlink:href="zam00111000089.pdf"></self-uri>
<abstract><p><italic>Xanthomonas arboricola</italic>
 pv. pruni, the causal agent of bacterial spot disease of stone fruit, is considered a quarantine organism by the European Union and the European and Mediterranean Plant Protection Organization (EPPO). The bacterium can undergo an epiphytic phase and/or be latent and can be transmitted by plant material, but currently, only visual inspections are used to certify plants as being <italic>X. arboricola</italic>
 pv. pruni free. A novel and highly sensitive real-time TaqMan PCR detection protocol was designed based on a sequence of a gene for a putative protein related to an ABC transporter ATP-binding system in <italic>X. arboricola</italic>
 pv. pruni. Pathogen detection can be completed within a few hours with a sensitivity of 10<sup>2</sup>
 CFU ml<sup>−1</sup>
, thus surpassing the sensitivity of the existing conventional PCR. Specificity was assessed for <italic>X. arboricola</italic>
 pv. pruni strains from different origins as well as for closely related <italic>Xanthomonas</italic>
 species, non-<italic>Xanthomonas</italic>
 species, saprophytic bacteria, and healthy <italic>Prunus</italic>
 samples. The efficiency of the developed protocol was evaluated with field samples of 14 <italic>Prunus</italic>
 species and rootstocks. For symptomatic leaf samples, the protocol was very efficient even when washed tissues of the leaves were directly amplified without any previous DNA extraction. For samples of 117 asymptomatic leaves and 285 buds, the protocol was more efficient after a simple DNA extraction, and <italic>X. arboricola</italic>
 pv. pruni was detected in 9.4% and 9.1% of the 402 samples analyzed, respectively, demonstrating its frequent epiphytic or endophytic phase. This newly developed real-time PCR protocol can be used as a quantitative assay, offers a reliable and sensitive test for <italic>X. arboricola</italic>
 pv. pruni, and is suitable as a screening test for symptomatic as well as asymptomatic plant material.</p>
</abstract>
</article-meta>
</front>
</pmc>
<affiliations><list></list>
<tree><noCountry><name sortKey="Berruete, Isabel M" sort="Berruete, Isabel M" uniqKey="Berruete I" first="Isabel M." last="Berruete">Isabel M. Berruete</name>
<name sortKey="Cambra, Miguel A" sort="Cambra, Miguel A" uniqKey="Cambra M" first="Miguel A." last="Cambra">Miguel A. Cambra</name>
<name sortKey="Collados, Raquel" sort="Collados, Raquel" uniqKey="Collados R" first="Raquel" last="Collados">Raquel Collados</name>
<name sortKey="Cubero, Jaime" sort="Cubero, Jaime" uniqKey="Cubero J" first="Jaime" last="Cubero">Jaime Cubero</name>
<name sortKey="L Pez, Maria M" sort="L Pez, Maria M" uniqKey="L Pez M" first="María M." last="L Pez">María M. L Pez</name>
<name sortKey="Palacio Bielsa, Ana" sort="Palacio Bielsa, Ana" uniqKey="Palacio Bielsa A" first="Ana" last="Palacio-Bielsa">Ana Palacio-Bielsa</name>
</noCountry>
</tree>
</affiliations>
</record>

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Development of an Efficient Real-Time Quantitative PCR Protocol for Detection of Xanthomonas arboricola pv. pruni in Prunus Species ▿ †

Development of an Efficient Real-Time Quantitative PCR Protocol for Detection of Xanthomonas arboricola pv. pruni in Prunus Species ▿ †

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Abstract

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