SCAR marker for Phytophthora nicotianae and a multiplex PCR assay for simultaneous detection of P. nicotianae and Candidatus Liberibacter asiaticus in citrus.
Identifieur interne : 000390 ( Main/Exploration ); précédent : 000389; suivant : 000391SCAR marker for Phytophthora nicotianae and a multiplex PCR assay for simultaneous detection of P. nicotianae and Candidatus Liberibacter asiaticus in citrus.
Auteurs : A K Das [Inde] ; S. Nerkar [Inde] ; N. Gawande [Inde] ; N. Thakre [Inde] ; A. Kumar [Inde]Source :
- Journal of applied microbiology [ 1365-2672 ] ; 2019.
Descripteurs français
- KwdFr :
- MESH :
- analyse : ADN.
- génétique : ADN, Marqueurs génétiques, Phytophthora, Rhizobiaceae.
- isolement et purification : Phytophthora, Rhizobiaceae.
- microbiologie : Citrus.
- méthodes : Réaction de polymérisation en chaine multiplex.
English descriptors
- KwdEn :
- MESH :
- chemical , analysis : DNA.
- chemical , genetics : DNA, Genetic Markers.
- genetics : Phytophthora, Rhizobiaceae.
- isolation & purification : Phytophthora, Rhizobiaceae.
- methods : Multiplex Polymerase Chain Reaction.
- microbiology : Citrus.
Abstract
AIMS
This study aimed to develop a random amplified polymorphic DNA (RAPD)-based sequence characterized amplified region (SCAR) marker for species-specific detection of Phytophthora nicotianae, a global plant pathogen. Another objective was to develop a multiplex PCR assay for simultaneous detection of P. nicotianae and huanglongbing-causing bacterium, Candidatus Liberibacter asiaticus (CaLas) in citrus roots using the developed SCAR marker and a previously published 16SrDNA-based CaLas-specific primer set.
METHODS AND RESULTS
The RAPD primer, OPA4, amplified a specific fragment of c. 400 bp only in P. nicotianae isolates. The fragment was eluted, purified, cloned and sequenced. One set of SCAR primers (SCAR4F/SCAR4R1), developed from the sequence information of the fragment, was found specific to P. nicotianae and produced an amplicon of 330 bp size, and was found non-specific to the five Phytophthora species (P. citrophthora, P. palmivora, P. lacustris, P. boehmeriae and P. insolita) and five other pathogens (Mycosphaerella citri, Alternaria alternata, Septobasidium pseudopedicillatum, Phytopythium vexans and Colletotrichum gloeosporioides) isolated from the citrus agroecosystem. The sensitivity of the primer pair was 5 pg µl
CONCLUSIONS
A rapid method using a RAPD-based SCAR marker for the detection of P. nicotianae was developed. Furthermore, a multiplex PCR assay was established for simultaneous detection of P. nicotianae and CaLas in citrus roots.
SIGNIFICANCE AND IMPACT OF THE STUDY
A RAPD-SCAR marker-based detection system and the one-step multiplex PCR method developed in this study can be applied to index citrus trees infected (individually or conjointly) with P. nicotianae and CaLas. The present technique developed would also be useful in monitoring disease epidemiology and phytosanitary surveillance.
DOI: 10.1111/jam.14392
PubMed: 31329353
Affiliations:
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Nerkar</name><affiliation wicri:level="1"><nlm:affiliation>ICAR-Central Citrus Research Institute, Nagpur, Maharashtra, India.</nlm:affiliation><country xml:lang="fr">Inde</country><wicri:regionArea>ICAR-Central Citrus Research Institute, Nagpur, Maharashtra</wicri:regionArea><wicri:noRegion>Maharashtra</wicri:noRegion></affiliation></author><author><name sortKey="Gawande, N" sort="Gawande, N" uniqKey="Gawande N" first="N" last="Gawande">N. Gawande</name><affiliation wicri:level="1"><nlm:affiliation>ICAR-Central Citrus Research Institute, Nagpur, Maharashtra, India.</nlm:affiliation><country xml:lang="fr">Inde</country><wicri:regionArea>ICAR-Central Citrus Research Institute, Nagpur, Maharashtra</wicri:regionArea><wicri:noRegion>Maharashtra</wicri:noRegion></affiliation></author><author><name sortKey="Thakre, N" sort="Thakre, N" uniqKey="Thakre N" first="N" last="Thakre">N. Thakre</name><affiliation wicri:level="1"><nlm:affiliation>ICAR-Central Citrus Research Institute, Nagpur, Maharashtra, India.</nlm:affiliation><country xml:lang="fr">Inde</country><wicri:regionArea>ICAR-Central Citrus Research Institute, Nagpur, Maharashtra</wicri:regionArea><wicri:noRegion>Maharashtra</wicri:noRegion></affiliation></author><author><name sortKey="Kumar, A" sort="Kumar, A" uniqKey="Kumar A" first="A" last="Kumar">A. Kumar</name><affiliation wicri:level="1"><nlm:affiliation>ICAR-Central Citrus Research Institute, Nagpur, Maharashtra, India.</nlm:affiliation><country xml:lang="fr">Inde</country><wicri:regionArea>ICAR-Central Citrus Research Institute, Nagpur, Maharashtra</wicri:regionArea><wicri:noRegion>Maharashtra</wicri:noRegion></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2019">2019</date><idno type="RBID">pubmed:31329353</idno><idno type="pmid">31329353</idno><idno type="doi">10.1111/jam.14392</idno><idno type="wicri:Area/Main/Corpus">000425</idno><idno type="wicri:explorRef" wicri:stream="Main" wicri:step="Corpus" wicri:corpus="PubMed">000425</idno><idno type="wicri:Area/Main/Curation">000425</idno><idno type="wicri:explorRef" wicri:stream="Main" wicri:step="Curation">000425</idno><idno type="wicri:Area/Main/Exploration">000425</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">SCAR marker for Phytophthora nicotianae and a multiplex PCR assay for simultaneous detection of P. nicotianae and Candidatus Liberibacter asiaticus in citrus.</title><author><name sortKey="Das, A K" sort="Das, A K" uniqKey="Das A" first="A K" last="Das">A K Das</name><affiliation wicri:level="1"><nlm:affiliation>ICAR-Central Citrus Research Institute, Nagpur, Maharashtra, India.</nlm:affiliation><country xml:lang="fr">Inde</country><wicri:regionArea>ICAR-Central Citrus Research Institute, Nagpur, Maharashtra</wicri:regionArea><wicri:noRegion>Maharashtra</wicri:noRegion></affiliation></author><author><name sortKey="Nerkar, S" sort="Nerkar, S" uniqKey="Nerkar S" first="S" last="Nerkar">S. Nerkar</name><affiliation wicri:level="1"><nlm:affiliation>ICAR-Central Citrus Research Institute, Nagpur, Maharashtra, India.</nlm:affiliation><country xml:lang="fr">Inde</country><wicri:regionArea>ICAR-Central Citrus Research Institute, Nagpur, Maharashtra</wicri:regionArea><wicri:noRegion>Maharashtra</wicri:noRegion></affiliation></author><author><name sortKey="Gawande, N" sort="Gawande, N" uniqKey="Gawande N" first="N" last="Gawande">N. Gawande</name><affiliation wicri:level="1"><nlm:affiliation>ICAR-Central Citrus Research Institute, Nagpur, Maharashtra, India.</nlm:affiliation><country xml:lang="fr">Inde</country><wicri:regionArea>ICAR-Central Citrus Research Institute, Nagpur, Maharashtra</wicri:regionArea><wicri:noRegion>Maharashtra</wicri:noRegion></affiliation></author><author><name sortKey="Thakre, N" sort="Thakre, N" uniqKey="Thakre N" first="N" last="Thakre">N. Thakre</name><affiliation wicri:level="1"><nlm:affiliation>ICAR-Central Citrus Research Institute, Nagpur, Maharashtra, India.</nlm:affiliation><country xml:lang="fr">Inde</country><wicri:regionArea>ICAR-Central Citrus Research Institute, Nagpur, Maharashtra</wicri:regionArea><wicri:noRegion>Maharashtra</wicri:noRegion></affiliation></author><author><name sortKey="Kumar, A" sort="Kumar, A" uniqKey="Kumar A" first="A" last="Kumar">A. Kumar</name><affiliation wicri:level="1"><nlm:affiliation>ICAR-Central Citrus Research Institute, Nagpur, Maharashtra, India.</nlm:affiliation><country xml:lang="fr">Inde</country><wicri:regionArea>ICAR-Central Citrus Research Institute, Nagpur, Maharashtra</wicri:regionArea><wicri:noRegion>Maharashtra</wicri:noRegion></affiliation></author></analytic><series><title level="j">Journal of applied microbiology</title><idno type="eISSN">1365-2672</idno><imprint><date when="2019" type="published">2019</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Citrus (microbiology)</term><term>DNA (analysis)</term><term>DNA (genetics)</term><term>Genetic Markers (genetics)</term><term>Multiplex Polymerase Chain Reaction (methods)</term><term>Phytophthora (genetics)</term><term>Phytophthora (isolation & purification)</term><term>Rhizobiaceae (genetics)</term><term>Rhizobiaceae (isolation & purification)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>ADN (analyse)</term><term>ADN (génétique)</term><term>Citrus (microbiologie)</term><term>Marqueurs génétiques (génétique)</term><term>Phytophthora (génétique)</term><term>Phytophthora (isolement et purification)</term><term>Rhizobiaceae (génétique)</term><term>Rhizobiaceae (isolement et purification)</term><term>Réaction de polymérisation en chaine multiplex (méthodes)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="analysis" xml:lang="en"><term>DNA</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>DNA</term><term>Genetic Markers</term></keywords><keywords scheme="MESH" qualifier="analyse" xml:lang="fr"><term>ADN</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Phytophthora</term><term>Rhizobiaceae</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>ADN</term><term>Marqueurs génétiques</term><term>Phytophthora</term><term>Rhizobiaceae</term></keywords><keywords scheme="MESH" qualifier="isolation & purification" xml:lang="en"><term>Phytophthora</term><term>Rhizobiaceae</term></keywords><keywords scheme="MESH" qualifier="isolement et purification" xml:lang="fr"><term>Phytophthora</term><term>Rhizobiaceae</term></keywords><keywords scheme="MESH" qualifier="methods" xml:lang="en"><term>Multiplex Polymerase Chain Reaction</term></keywords><keywords scheme="MESH" qualifier="microbiologie" xml:lang="fr"><term>Citrus</term></keywords><keywords scheme="MESH" qualifier="microbiology" xml:lang="en"><term>Citrus</term></keywords><keywords scheme="MESH" qualifier="méthodes" xml:lang="fr"><term>Réaction de polymérisation en chaine multiplex</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p><b>AIMS</b></p><p>This study aimed to develop a random amplified polymorphic DNA (RAPD)-based sequence characterized amplified region (SCAR) marker for species-specific detection of Phytophthora nicotianae, a global plant pathogen. Another objective was to develop a multiplex PCR assay for simultaneous detection of P. nicotianae and huanglongbing-causing bacterium, Candidatus Liberibacter asiaticus (CaLas) in citrus roots using the developed SCAR marker and a previously published 16SrDNA-based CaLas-specific primer set.</p></div><div type="abstract" xml:lang="en"><p><b>METHODS AND RESULTS</b></p><p>The RAPD primer, OPA4, amplified a specific fragment of c. 400 bp only in P. nicotianae isolates. The fragment was eluted, purified, cloned and sequenced. One set of SCAR primers (SCAR4F/SCAR4R1), developed from the sequence information of the fragment, was found specific to P. nicotianae and produced an amplicon of 330 bp size, and was found non-specific to the five Phytophthora species (P. citrophthora, P. palmivora, P. lacustris, P. boehmeriae and P. insolita) and five other pathogens (Mycosphaerella citri, Alternaria alternata, Septobasidium pseudopedicillatum, Phytopythium vexans and Colletotrichum gloeosporioides) isolated from the citrus agroecosystem. The sensitivity of the primer pair was 5 pg µl</p></div><div type="abstract" xml:lang="en"><p><b>CONCLUSIONS</b></p><p>A rapid method using a RAPD-based SCAR marker for the detection of P. nicotianae was developed. Furthermore, a multiplex PCR assay was established for simultaneous detection of P. nicotianae and CaLas in citrus roots.</p></div><div type="abstract" xml:lang="en"><p><b>SIGNIFICANCE AND IMPACT OF THE STUDY</b></p><p>A RAPD-SCAR marker-based detection system and the one-step multiplex PCR method developed in this study can be applied to index citrus trees infected (individually or conjointly) with P. nicotianae and CaLas. The present technique developed would also be useful in monitoring disease epidemiology and phytosanitary surveillance.</p></div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" IndexingMethod="Curated" Owner="NLM"><PMID Version="1">31329353</PMID><DateCompleted><Year>2019</Year><Month>12</Month><Day>06</Day></DateCompleted><DateRevised><Year>2020</Year><Month>01</Month><Day>08</Day></DateRevised><Article PubModel="Print-Electronic"><Journal><ISSN IssnType="Electronic">1365-2672</ISSN><JournalIssue CitedMedium="Internet"><Volume>127</Volume><Issue>4</Issue><PubDate><Year>2019</Year><Month>Oct</Month></PubDate></JournalIssue><Title>Journal of applied microbiology</Title><ISOAbbreviation>J Appl Microbiol</ISOAbbreviation></Journal><ArticleTitle>SCAR marker for Phytophthora nicotianae and a multiplex PCR assay for simultaneous detection of P. nicotianae and Candidatus Liberibacter asiaticus in citrus.</ArticleTitle><Pagination><MedlinePgn>1172-1183</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1111/jam.14392</ELocationID><Abstract><AbstractText Label="AIMS" NlmCategory="OBJECTIVE">This study aimed to develop a random amplified polymorphic DNA (RAPD)-based sequence characterized amplified region (SCAR) marker for species-specific detection of Phytophthora nicotianae, a global plant pathogen. Another objective was to develop a multiplex PCR assay for simultaneous detection of P. nicotianae and huanglongbing-causing bacterium, Candidatus Liberibacter asiaticus (CaLas) in citrus roots using the developed SCAR marker and a previously published 16SrDNA-based CaLas-specific primer set.</AbstractText><AbstractText Label="METHODS AND RESULTS" NlmCategory="RESULTS">The RAPD primer, OPA4, amplified a specific fragment of c. 400 bp only in P. nicotianae isolates. The fragment was eluted, purified, cloned and sequenced. One set of SCAR primers (SCAR4F/SCAR4R1), developed from the sequence information of the fragment, was found specific to P. nicotianae and produced an amplicon of 330 bp size, and was found non-specific to the five Phytophthora species (P. citrophthora, P. palmivora, P. lacustris, P. boehmeriae and P. insolita) and five other pathogens (Mycosphaerella citri, Alternaria alternata, Septobasidium pseudopedicillatum, Phytopythium vexans and Colletotrichum gloeosporioides) isolated from the citrus agroecosystem. The sensitivity of the primer pair was 5 pg µl<sup>-1</sup> of mycelial DNA. Furthermore, the specific SCAR primers coupled with a previously reported CaLas-specific primer set were used effectively in developing a multiplex PCR assay to detect P. nicotianae and CaLas simultaneously in root tissues of citrus plants.</AbstractText><AbstractText Label="CONCLUSIONS" NlmCategory="CONCLUSIONS">A rapid method using a RAPD-based SCAR marker for the detection of P. nicotianae was developed. Furthermore, a multiplex PCR assay was established for simultaneous detection of P. nicotianae and CaLas in citrus roots.</AbstractText><AbstractText Label="SIGNIFICANCE AND IMPACT OF THE STUDY" NlmCategory="CONCLUSIONS">A RAPD-SCAR marker-based detection system and the one-step multiplex PCR method developed in this study can be applied to index citrus trees infected (individually or conjointly) with P. nicotianae and CaLas. The present technique developed would also be useful in monitoring disease epidemiology and phytosanitary surveillance.</AbstractText><CopyrightInformation>© 2019 The Society for Applied Microbiology.</CopyrightInformation></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Das</LastName><ForeName>A K</ForeName><Initials>AK</Initials><Identifier Source="ORCID">https://orcid.org/0000-0002-5313-6118</Identifier><AffiliationInfo><Affiliation>ICAR-Central Citrus Research Institute, Nagpur, Maharashtra, India.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Nerkar</LastName><ForeName>S</ForeName><Initials>S</Initials><AffiliationInfo><Affiliation>ICAR-Central Citrus Research Institute, Nagpur, Maharashtra, India.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Gawande</LastName><ForeName>N</ForeName><Initials>N</Initials><AffiliationInfo><Affiliation>ICAR-Central Citrus Research Institute, Nagpur, Maharashtra, India.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Thakre</LastName><ForeName>N</ForeName><Initials>N</Initials><AffiliationInfo><Affiliation>ICAR-Central Citrus Research Institute, Nagpur, Maharashtra, India.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Kumar</LastName><ForeName>A</ForeName><Initials>A</Initials><AffiliationInfo><Affiliation>ICAR-Central Citrus Research Institute, Nagpur, Maharashtra, India.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><DataBankList CompleteYN="Y"><DataBank><DataBankName>GENBANK</DataBankName><AccessionNumberList><AccessionNumber>KY851111</AccessionNumber></AccessionNumberList></DataBank></DataBankList><GrantList CompleteYN="Y"><Grant><Agency>Indian Council of Agricultural Research</Agency><Country></Country></Grant></GrantList><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2019</Year><Month>08</Month><Day>09</Day></ArticleDate></Article><MedlineJournalInfo><Country>England</Country><MedlineTA>J Appl Microbiol</MedlineTA><NlmUniqueID>9706280</NlmUniqueID><ISSNLinking>1364-5072</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D005819">Genetic Markers</NameOfSubstance></Chemical><Chemical><RegistryNumber>9007-49-2</RegistryNumber><NameOfSubstance UI="D004247">DNA</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D002957" MajorTopicYN="N">Citrus</DescriptorName><QualifierName UI="Q000382" MajorTopicYN="Y">microbiology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D004247" MajorTopicYN="N">DNA</DescriptorName><QualifierName UI="Q000032" MajorTopicYN="N">analysis</QualifierName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D005819" MajorTopicYN="N">Genetic Markers</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D060885" MajorTopicYN="N">Multiplex Polymerase Chain Reaction</DescriptorName><QualifierName UI="Q000379" MajorTopicYN="Y">methods</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D010838" MajorTopicYN="N">Phytophthora</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName><QualifierName UI="Q000302" MajorTopicYN="N">isolation & purification</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D012230" MajorTopicYN="N">Rhizobiaceae</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName><QualifierName UI="Q000302" MajorTopicYN="N">isolation & purification</QualifierName></MeshHeading></MeshHeadingList><KeywordList Owner="NOTNLM"><Keyword MajorTopicYN="N">Phytophthora nicotianae
</Keyword><Keyword MajorTopicYN="N">Candidatus Liberibacter asiaticus</Keyword><Keyword MajorTopicYN="N">RAPD</Keyword><Keyword MajorTopicYN="N">SCAR</Keyword><Keyword MajorTopicYN="N">multiplex PCR</Keyword></KeywordList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="received"><Year>2018</Year><Month>12</Month><Day>19</Day></PubMedPubDate><PubMedPubDate PubStatus="revised"><Year>2019</Year><Month>06</Month><Day>21</Day></PubMedPubDate><PubMedPubDate PubStatus="accepted"><Year>2019</Year><Month>07</Month><Day>11</Day></PubMedPubDate><PubMedPubDate PubStatus="pubmed"><Year>2019</Year><Month>7</Month><Day>23</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2019</Year><Month>12</Month><Day>18</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2019</Year><Month>7</Month><Day>23</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">31329353</ArticleId><ArticleId IdType="doi">10.1111/jam.14392</ArticleId></ArticleIdList><ReferenceList><Title>References</Title><Reference><Citation>Anandaraj, M. 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