Novel Tetraplex Quantitative PCR Assays for Simultaneous Detection and Identification of Xylella fastidiosa Subspecies in Plant Tissues.
Identifieur interne : 000282 ( PubMed/Corpus ); précédent : 000281; suivant : 000283Novel Tetraplex Quantitative PCR Assays for Simultaneous Detection and Identification of Xylella fastidiosa Subspecies in Plant Tissues.
Auteurs : Enora Dupas ; Martial Briand ; Marie-Agnès Jacques ; Sophie CesbronSource :
- Frontiers in plant science [ 1664-462X ] ; 2019.
Abstract
Xylella fastidiosa (Xf) is an insect-borne bacterium confined to the xylem vessels of plants. This plant pathogen has a broad host range estimated to 560 plant species. Five subspecies of the pathogen with different but overlapping host ranges have been described, but only three subspecies are widely accepted, namely subspecies fastidiosa, multiplex, and pauca. Initially limited to the Americas, Xf has been detected in Europe since 2013. As management of X. fastidiosa outbreaks in Europe depends on the identification of the subspecies, accurate determination of the subspecies in infected plants as early as possible is of major interest. Thus, we developed various tetraplex and triplex quantitative PCR (qPCR) assays for X. fastidiosa detection and subspecies identification in planta in a single reaction. We designed primers and probes using SkIf, a bioinformatics tool based on k-mers, to detect specific signatures of the species and subspecies from a data set of 58 genome sequences representative of X. fastidiosa diversity. We tested the qPCR assays on 39 target and 30 non-target strains, as well as on 13 different plant species spiked with strains of the different subspecies of X. fastidiosa, and on samples from various environmental and inoculated host plants. Sensitivity of simplex assays was equal or slightly better than the reference protocol on purified DNA. Tetraplex qPCR assays had the same sensitivity than the reference protocol and allowed X. fastidiosa detection in all spiked matrices up to 103 cells.ml-1. Moreover, mix infections of two to three subspecies could be detected in the same sample with tetraplex assays. In environmental plant samples, the tetraplex qPCR assays allowed subspecies identification when the current method based on multilocus sequence typing failed. The qPCR assays described here are robust and modular tools that are efficient for differentiating X. fastidiosa subspecies directly in plant samples.
DOI: 10.3389/fpls.2019.01732
PubMed: 31956326
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Novel Tetraplex Quantitative PCR Assays for Simultaneous Detection and Identification of <i>Xylella fastidiosa</i> Subspecies in Plant Tissues.</title><author><name sortKey="Dupas, Enora" sort="Dupas, Enora" uniqKey="Dupas E" first="Enora" last="Dupas">Enora Dupas</name><affiliation><nlm:affiliation>IRHS, Agrocampus-Ouest, INRA, University of Angers, SFR 4207 QuaSaV, Beaucouzé, France.</nlm:affiliation></affiliation></author><author><name sortKey="Briand, Martial" sort="Briand, Martial" uniqKey="Briand M" first="Martial" last="Briand">Martial Briand</name><affiliation><nlm:affiliation>IRHS, Agrocampus-Ouest, INRA, University of Angers, SFR 4207 QuaSaV, Beaucouzé, France.</nlm:affiliation></affiliation></author><author><name sortKey="Jacques, Marie Agnes" sort="Jacques, Marie Agnes" uniqKey="Jacques M" first="Marie-Agnès" last="Jacques">Marie-Agnès Jacques</name><affiliation><nlm:affiliation>IRHS, Agrocampus-Ouest, INRA, University of Angers, SFR 4207 QuaSaV, Beaucouzé, France.</nlm:affiliation></affiliation></author><author><name sortKey="Cesbron, Sophie" sort="Cesbron, Sophie" uniqKey="Cesbron S" first="Sophie" last="Cesbron">Sophie Cesbron</name><affiliation><nlm:affiliation>IRHS, Agrocampus-Ouest, INRA, University of Angers, SFR 4207 QuaSaV, Beaucouzé, France.</nlm:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2019">2019</date><idno type="RBID">pubmed:31956326</idno><idno type="pmid">31956326</idno><idno type="doi">10.3389/fpls.2019.01732</idno><idno type="wicri:Area/PubMed/Corpus">000282</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">000282</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">Novel Tetraplex Quantitative PCR Assays for Simultaneous Detection and Identification of <i>Xylella fastidiosa</i> Subspecies in Plant Tissues.</title><author><name sortKey="Dupas, Enora" sort="Dupas, Enora" uniqKey="Dupas E" first="Enora" last="Dupas">Enora Dupas</name><affiliation><nlm:affiliation>IRHS, Agrocampus-Ouest, INRA, University of Angers, SFR 4207 QuaSaV, Beaucouzé, France.</nlm:affiliation></affiliation></author><author><name sortKey="Briand, Martial" sort="Briand, Martial" uniqKey="Briand M" first="Martial" last="Briand">Martial Briand</name><affiliation><nlm:affiliation>IRHS, Agrocampus-Ouest, INRA, University of Angers, SFR 4207 QuaSaV, Beaucouzé, France.</nlm:affiliation></affiliation></author><author><name sortKey="Jacques, Marie Agnes" sort="Jacques, Marie Agnes" uniqKey="Jacques M" first="Marie-Agnès" last="Jacques">Marie-Agnès Jacques</name><affiliation><nlm:affiliation>IRHS, Agrocampus-Ouest, INRA, University of Angers, SFR 4207 QuaSaV, Beaucouzé, France.</nlm:affiliation></affiliation></author><author><name sortKey="Cesbron, Sophie" sort="Cesbron, Sophie" uniqKey="Cesbron S" first="Sophie" last="Cesbron">Sophie Cesbron</name><affiliation><nlm:affiliation>IRHS, Agrocampus-Ouest, INRA, University of Angers, SFR 4207 QuaSaV, Beaucouzé, France.</nlm:affiliation></affiliation></author></analytic><series><title level="j">Frontiers in plant science</title><idno type="ISSN">1664-462X</idno><imprint><date when="2019" type="published">2019</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><i>Xylella fastidiosa</i> (<i>Xf</i>) is an insect-borne bacterium confined to the xylem vessels of plants. This plant pathogen has a broad host range estimated to 560 plant species. Five subspecies of the pathogen with different but overlapping host ranges have been described, but only three subspecies are widely accepted, namely subspecies <i>fastidiosa</i>, <i>multiplex</i>, and <i>pauca</i>. Initially limited to the Americas, <i>Xf</i> has been detected in Europe since 2013. As management of <i>X. fastidiosa</i> outbreaks in Europe depends on the identification of the subspecies, accurate determination of the subspecies in infected plants as early as possible is of major interest. Thus, we developed various tetraplex and triplex quantitative PCR (qPCR) assays for <i>X. fastidiosa</i> detection and subspecies identification <i>in planta</i> in a single reaction. We designed primers and probes using SkIf, a bioinformatics tool based on <i>k</i>-mers, to detect specific signatures of the species and subspecies from a data set of 58 genome sequences representative of <i>X. fastidiosa</i> diversity. We tested the qPCR assays on 39 target and 30 non-target strains, as well as on 13 different plant species spiked with strains of the different subspecies of <i>X. fastidiosa</i>, and on samples from various environmental and inoculated host plants. Sensitivity of simplex assays was equal or slightly better than the reference protocol on purified DNA. Tetraplex qPCR assays had the same sensitivity than the reference protocol and allowed <i>X. fastidiosa</i> detection in all spiked matrices up to 10<sup>3</sup> cells.ml<sup>-1</sup>. Moreover, mix infections of two to three subspecies could be detected in the same sample with tetraplex assays. In environmental plant samples, the tetraplex qPCR assays allowed subspecies identification when the current method based on multilocus sequence typing failed. The qPCR assays described here are robust and modular tools that are efficient for differentiating <i>X. fastidiosa</i> subspecies directly in plant samples.</div></front></TEI><pubmed><MedlineCitation Status="PubMed-not-MEDLINE" Owner="NLM"><PMID Version="1">31956326</PMID><DateRevised><Year>2020</Year><Month>01</Month><Day>23</Day></DateRevised><Article PubModel="Electronic-eCollection"><Journal><ISSN IssnType="Print">1664-462X</ISSN><JournalIssue CitedMedium="Print"><Volume>10</Volume><PubDate><Year>2019</Year></PubDate></JournalIssue><Title>Frontiers in plant science</Title><ISOAbbreviation>Front Plant Sci</ISOAbbreviation></Journal><ArticleTitle>Novel Tetraplex Quantitative PCR Assays for Simultaneous Detection and Identification of <i>Xylella fastidiosa</i> Subspecies in Plant Tissues.</ArticleTitle><Pagination><MedlinePgn>1732</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.3389/fpls.2019.01732</ELocationID><Abstract><AbstractText><i>Xylella fastidiosa</i> (<i>Xf</i>) is an insect-borne bacterium confined to the xylem vessels of plants. This plant pathogen has a broad host range estimated to 560 plant species. Five subspecies of the pathogen with different but overlapping host ranges have been described, but only three subspecies are widely accepted, namely subspecies <i>fastidiosa</i>, <i>multiplex</i>, and <i>pauca</i>. Initially limited to the Americas, <i>Xf</i> has been detected in Europe since 2013. As management of <i>X. fastidiosa</i> outbreaks in Europe depends on the identification of the subspecies, accurate determination of the subspecies in infected plants as early as possible is of major interest. Thus, we developed various tetraplex and triplex quantitative PCR (qPCR) assays for <i>X. fastidiosa</i> detection and subspecies identification <i>in planta</i> in a single reaction. We designed primers and probes using SkIf, a bioinformatics tool based on <i>k</i>-mers, to detect specific signatures of the species and subspecies from a data set of 58 genome sequences representative of <i>X. fastidiosa</i> diversity. We tested the qPCR assays on 39 target and 30 non-target strains, as well as on 13 different plant species spiked with strains of the different subspecies of <i>X. fastidiosa</i>, and on samples from various environmental and inoculated host plants. Sensitivity of simplex assays was equal or slightly better than the reference protocol on purified DNA. Tetraplex qPCR assays had the same sensitivity than the reference protocol and allowed <i>X. fastidiosa</i> detection in all spiked matrices up to 10<sup>3</sup> cells.ml<sup>-1</sup>. Moreover, mix infections of two to three subspecies could be detected in the same sample with tetraplex assays. In environmental plant samples, the tetraplex qPCR assays allowed subspecies identification when the current method based on multilocus sequence typing failed. The qPCR assays described here are robust and modular tools that are efficient for differentiating <i>X. fastidiosa</i> subspecies directly in plant samples.</AbstractText><CopyrightInformation>Copyright © 2019 Dupas, Briand, Jacques and Cesbron.</CopyrightInformation></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Dupas</LastName><ForeName>Enora</ForeName><Initials>E</Initials><AffiliationInfo><Affiliation>IRHS, Agrocampus-Ouest, INRA, University of Angers, SFR 4207 QuaSaV, Beaucouzé, France.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>French Agency for Food, Environmental and Occupational Health & Safety, Plant Health Laboratory, Angers, France.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Briand</LastName><ForeName>Martial</ForeName><Initials>M</Initials><AffiliationInfo><Affiliation>IRHS, Agrocampus-Ouest, INRA, University of Angers, SFR 4207 QuaSaV, Beaucouzé, France.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Jacques</LastName><ForeName>Marie-Agnès</ForeName><Initials>MA</Initials><AffiliationInfo><Affiliation>IRHS, Agrocampus-Ouest, INRA, University of Angers, SFR 4207 QuaSaV, Beaucouzé, France.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Cesbron</LastName><ForeName>Sophie</ForeName><Initials>S</Initials><AffiliationInfo><Affiliation>IRHS, Agrocampus-Ouest, INRA, University of Angers, SFR 4207 QuaSaV, Beaucouzé, France.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2019</Year><Month>12</Month><Day>27</Day></ArticleDate></Article><MedlineJournalInfo><Country>Switzerland</Country><MedlineTA>Front Plant Sci</MedlineTA><NlmUniqueID>101568200</NlmUniqueID><ISSNLinking>1664-462X</ISSNLinking></MedlineJournalInfo><KeywordList Owner="NOTNLM"><Keyword MajorTopicYN="N">SkIf</Keyword><Keyword MajorTopicYN="N">TaqMan</Keyword><Keyword MajorTopicYN="N">diagnostic</Keyword><Keyword MajorTopicYN="N">multiplexing</Keyword><Keyword MajorTopicYN="N">real-time PCR</Keyword><Keyword MajorTopicYN="N">subspecies differentiation</Keyword></KeywordList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="received"><Year>2019</Year><Month>07</Month><Day>19</Day></PubMedPubDate><PubMedPubDate PubStatus="accepted"><Year>2019</Year><Month>12</Month><Day>09</Day></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2020</Year><Month>1</Month><Day>21</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="pubmed"><Year>2020</Year><Month>1</Month><Day>21</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2020</Year><Month>1</Month><Day>21</Day><Hour>6</Hour><Minute>1</Minute></PubMedPubDate></History><PublicationStatus>epublish</PublicationStatus><ArticleIdList><ArticleId 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