Real-Time PCR and LAMP Assays for the Detection of Spores of Alternaria solani and Sporangia of Phytophthora infestans to Inform Disease Risk Forecasting.
Identifieur interne : 000399 ( Main/Exploration ); précédent : 000398; suivant : 000400Real-Time PCR and LAMP Assays for the Detection of Spores of Alternaria solani and Sporangia of Phytophthora infestans to Inform Disease Risk Forecasting.
Auteurs : A K Lees [Royaume-Uni] ; D M Roberts [Royaume-Uni] ; J. Lynott [Royaume-Uni] ; L. Sullivan [Royaume-Uni] ; J L Brierley [Royaume-Uni]Source :
- Plant disease [ 0191-2917 ] ; 2019.
Descripteurs français
- KwdFr :
- Agriculture (méthodes), Alternaria (génétique), Appréciation des risques (méthodes), Phytophthora infestans (génétique), Réaction de polymérisation en chaine en temps réel (MeSH), Solanum tuberosum (parasitologie), Sporanges (génétique), Spores de protozoaire (génétique), Spores de protozoaire (isolement et purification), Techniques d'amplification d'acides nucléiques (MeSH).
- MESH :
- génétique : Alternaria, Phytophthora infestans, Sporanges, Spores de protozoaire.
- isolement et purification : Spores de protozoaire.
- méthodes : Agriculture, Appréciation des risques.
- parasitologie : Solanum tuberosum.
- Réaction de polymérisation en chaine en temps réel, Techniques d'amplification d'acides nucléiques.
English descriptors
- KwdEn :
- Agriculture (methods), Alternaria (genetics), Nucleic Acid Amplification Techniques (MeSH), Phytophthora infestans (genetics), Real-Time Polymerase Chain Reaction (MeSH), Risk Assessment (methods), Solanum tuberosum (parasitology), Sporangia (genetics), Spores, Protozoan (genetics), Spores, Protozoan (isolation & purification).
- MESH :
- genetics : Alternaria, Phytophthora infestans, Sporangia, Spores, Protozoan.
- isolation & purification : Spores, Protozoan.
- methods : Agriculture, Risk Assessment.
- parasitology : Solanum tuberosum.
- Nucleic Acid Amplification Techniques, Real-Time Polymerase Chain Reaction.
Abstract
Real-time loop-mediated isothermal amplification (LAMP) assays for the detection of sporangia of the causal pathogen of late blight, Phytophthora infestans, and spores of the main causal pathogen of early blight, Alternaria solani, were developed to facilitate the in-field detection of airborne inoculum to improve disease forecasting. These assays were compared with an existing real-time PCR assay for P. infestans and a newly developed real-time PCR assay for A. solani. Primers were designed for real-time LAMP of P. infestans and A. solani. The specificity of the P. infestans real-time LAMP assay was similar to that of an existing real-time PCR assay: DNA of P. infestans was consistently amplified as was DNA of the taxonomically closely related species Phytophthora mirabilis, Phytophthora phaseoli, and Phytophthora ipomoea; no amplification of DNA from the potato pathogens Phytophthora erythroseptica or Phytophthora nicotianae occurred. Real-time LAMP and PCR assays were developed for A. solani, and the specificity was compared with an existing conventional PCR assay. Importantly, the A. solani real-time LAMP and PCR assays did not amplify the species Alternaria alternata. However, cross-reactivity with Alternaria dauci was observed with the real-time PCR assay and Alternaria brassicae with the real-time LAMP assay. The sensitivity of all assays for the detection of DNA extracted from sporangia/spores of the target pathogens was evaluated. The P. infestans real-time LAMP assay reliably detected 5 pg of DNA, equivalent to ∼1 sporangia per reaction. By comparison, 20 fg of DNA was detectable with the existing real-time PCR assay. In the case of A. solani, real-time LAMP detected 4.4 pg of DNA, equivalent to ∼1 spore per reaction, and real-time PCR detected 200 fg of DNA. In-field air samplers were deployed in two trial plots planted with potato: one infected with P. infestans, and the other infected with A. solani. Four additional samplers were located in commercial potato fields. Air samples were taken through the season, and detection of airborne inoculum of P. infestans and A. solani with both real-time PCR and LAMP was assessed.
DOI: 10.1094/PDIS-04-19-0765-RE
PubMed: 31657996
Affiliations:
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Lynott</name><affiliation wicri:level="1"><nlm:affiliation>The James Hutton Institute, Invergowrie, Dundee DD2 5DA, United Kingdom.</nlm:affiliation><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>The James Hutton Institute, Invergowrie, Dundee DD2 5DA</wicri:regionArea><wicri:noRegion>Dundee DD2 5DA</wicri:noRegion></affiliation></author><author><name sortKey="Sullivan, L" sort="Sullivan, L" uniqKey="Sullivan L" first="L" last="Sullivan">L. 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Lynott</name><affiliation wicri:level="1"><nlm:affiliation>The James Hutton Institute, Invergowrie, Dundee DD2 5DA, United Kingdom.</nlm:affiliation><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>The James Hutton Institute, Invergowrie, Dundee DD2 5DA</wicri:regionArea><wicri:noRegion>Dundee DD2 5DA</wicri:noRegion></affiliation></author><author><name sortKey="Sullivan, L" sort="Sullivan, L" uniqKey="Sullivan L" first="L" last="Sullivan">L. Sullivan</name><affiliation wicri:level="1"><nlm:affiliation>The James Hutton Institute, Invergowrie, Dundee DD2 5DA, United Kingdom.</nlm:affiliation><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>The James Hutton Institute, Invergowrie, Dundee DD2 5DA</wicri:regionArea><wicri:noRegion>Dundee DD2 5DA</wicri:noRegion></affiliation></author><author><name sortKey="Brierley, J L" sort="Brierley, J L" uniqKey="Brierley J" first="J L" last="Brierley">J L Brierley</name><affiliation wicri:level="1"><nlm:affiliation>The James Hutton Institute, Invergowrie, Dundee DD2 5DA, United Kingdom.</nlm:affiliation><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>The James Hutton Institute, Invergowrie, Dundee DD2 5DA</wicri:regionArea><wicri:noRegion>Dundee DD2 5DA</wicri:noRegion></affiliation></author></analytic><series><title level="j">Plant disease</title><idno type="ISSN">0191-2917</idno><imprint><date when="2019" type="published">2019</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Agriculture (methods)</term><term>Alternaria (genetics)</term><term>Nucleic Acid Amplification Techniques (MeSH)</term><term>Phytophthora infestans (genetics)</term><term>Real-Time Polymerase Chain Reaction (MeSH)</term><term>Risk Assessment (methods)</term><term>Solanum tuberosum (parasitology)</term><term>Sporangia (genetics)</term><term>Spores, Protozoan (genetics)</term><term>Spores, Protozoan (isolation & purification)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Agriculture (méthodes)</term><term>Alternaria (génétique)</term><term>Appréciation des risques (méthodes)</term><term>Phytophthora infestans (génétique)</term><term>Réaction de polymérisation en chaine en temps réel (MeSH)</term><term>Solanum tuberosum (parasitologie)</term><term>Sporanges (génétique)</term><term>Spores de protozoaire (génétique)</term><term>Spores de protozoaire (isolement et purification)</term><term>Techniques d'amplification d'acides nucléiques (MeSH)</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Alternaria</term><term>Phytophthora infestans</term><term>Sporangia</term><term>Spores, Protozoan</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Alternaria</term><term>Phytophthora infestans</term><term>Sporanges</term><term>Spores de protozoaire</term></keywords><keywords scheme="MESH" qualifier="isolation & purification" xml:lang="en"><term>Spores, Protozoan</term></keywords><keywords scheme="MESH" qualifier="isolement et purification" xml:lang="fr"><term>Spores de protozoaire</term></keywords><keywords scheme="MESH" qualifier="methods" xml:lang="en"><term>Agriculture</term><term>Risk Assessment</term></keywords><keywords scheme="MESH" qualifier="méthodes" xml:lang="fr"><term>Agriculture</term><term>Appréciation des risques</term></keywords><keywords scheme="MESH" qualifier="parasitologie" xml:lang="fr"><term>Solanum tuberosum</term></keywords><keywords scheme="MESH" qualifier="parasitology" xml:lang="en"><term>Solanum tuberosum</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Nucleic Acid Amplification Techniques</term><term>Real-Time Polymerase Chain Reaction</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Réaction de polymérisation en chaine en temps réel</term><term>Techniques d'amplification d'acides nucléiques</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Real-time loop-mediated isothermal amplification (LAMP) assays for the detection of sporangia of the causal pathogen of late blight, <i>Phytophthora infestans</i>, and spores of the main causal pathogen of early blight, <i>Alternaria solani</i>, were developed to facilitate the in-field detection of airborne inoculum to improve disease forecasting. These assays were compared with an existing real-time PCR assay for <i>P. infestans</i> and a newly developed real-time PCR assay for <i>A. solani</i>. Primers were designed for real-time LAMP of <i>P. infestans</i> and <i>A. solani</i>. The specificity of the <i>P. infestans</i> real-time LAMP assay was similar to that of an existing real-time PCR assay: DNA of <i>P. infestans</i> was consistently amplified as was DNA of the taxonomically closely related species <i>Phytophthora mirabilis</i>, <i>Phytophthora phaseoli</i>, and <i>Phytophthora ipomoea</i>; no amplification of DNA from the potato pathogens <i>Phytophthora erythroseptica</i> or <i>Phytophthora nicotianae</i> occurred. Real-time LAMP and PCR assays were developed for <i>A. solani</i>, and the specificity was compared with an existing conventional PCR assay. Importantly, the <i>A. solani</i> real-time LAMP and PCR assays did not amplify the species <i>Alternaria alternata</i>. However, cross-reactivity with <i>Alternaria dauci</i> was observed with the real-time PCR assay and <i>Alternaria brassicae</i> with the real-time LAMP assay. The sensitivity of all assays for the detection of DNA extracted from sporangia/spores of the target pathogens was evaluated. The <i>P. infestans</i> real-time LAMP assay reliably detected 5 pg of DNA, equivalent to ∼1 sporangia per reaction. By comparison, 20 fg of DNA was detectable with the existing real-time PCR assay. In the case of <i>A. solani</i>, real-time LAMP detected 4.4 pg of DNA, equivalent to ∼1 spore per reaction, and real-time PCR detected 200 fg of DNA. In-field air samplers were deployed in two trial plots planted with potato: one infected with <i>P. infestans</i>, and the other infected with <i>A. solani</i>. Four additional samplers were located in commercial potato fields. Air samples were taken through the season, and detection of airborne inoculum of <i>P. infestans</i> and <i>A. solani</i> with both real-time PCR and LAMP was assessed.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" IndexingMethod="Curated" Owner="NLM"><PMID Version="1">31657996</PMID><DateCompleted><Year>2019</Year><Month>11</Month><Day>27</Day></DateCompleted><DateRevised><Year>2019</Year><Month>11</Month><Day>27</Day></DateRevised><Article PubModel="Print-Electronic"><Journal><ISSN IssnType="Print">0191-2917</ISSN><JournalIssue CitedMedium="Print"><Volume>103</Volume><Issue>12</Issue><PubDate><Year>2019</Year><Month>Dec</Month></PubDate></JournalIssue><Title>Plant disease</Title><ISOAbbreviation>Plant Dis</ISOAbbreviation></Journal><ArticleTitle>Real-Time PCR and LAMP Assays for the Detection of Spores of <i>Alternaria solani</i> and Sporangia of <i>Phytophthora infestans</i> to Inform Disease Risk Forecasting.</ArticleTitle><Pagination><MedlinePgn>3172-3180</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1094/PDIS-04-19-0765-RE</ELocationID><Abstract><AbstractText>Real-time loop-mediated isothermal amplification (LAMP) assays for the detection of sporangia of the causal pathogen of late blight, <i>Phytophthora infestans</i>, and spores of the main causal pathogen of early blight, <i>Alternaria solani</i>, were developed to facilitate the in-field detection of airborne inoculum to improve disease forecasting. These assays were compared with an existing real-time PCR assay for <i>P. infestans</i> and a newly developed real-time PCR assay for <i>A. solani</i>. Primers were designed for real-time LAMP of <i>P. infestans</i> and <i>A. solani</i>. The specificity of the <i>P. infestans</i> real-time LAMP assay was similar to that of an existing real-time PCR assay: DNA of <i>P. infestans</i> was consistently amplified as was DNA of the taxonomically closely related species <i>Phytophthora mirabilis</i>, <i>Phytophthora phaseoli</i>, and <i>Phytophthora ipomoea</i>; no amplification of DNA from the potato pathogens <i>Phytophthora erythroseptica</i> or <i>Phytophthora nicotianae</i> occurred. Real-time LAMP and PCR assays were developed for <i>A. solani</i>, and the specificity was compared with an existing conventional PCR assay. Importantly, the <i>A. solani</i> real-time LAMP and PCR assays did not amplify the species <i>Alternaria alternata</i>. However, cross-reactivity with <i>Alternaria dauci</i> was observed with the real-time PCR assay and <i>Alternaria brassicae</i> with the real-time LAMP assay. The sensitivity of all assays for the detection of DNA extracted from sporangia/spores of the target pathogens was evaluated. The <i>P. infestans</i> real-time LAMP assay reliably detected 5 pg of DNA, equivalent to ∼1 sporangia per reaction. By comparison, 20 fg of DNA was detectable with the existing real-time PCR assay. In the case of <i>A. solani</i>, real-time LAMP detected 4.4 pg of DNA, equivalent to ∼1 spore per reaction, and real-time PCR detected 200 fg of DNA. In-field air samplers were deployed in two trial plots planted with potato: one infected with <i>P. infestans</i>, and the other infected with <i>A. solani</i>. Four additional samplers were located in commercial potato fields. Air samples were taken through the season, and detection of airborne inoculum of <i>P. infestans</i> and <i>A. solani</i> with both real-time PCR and LAMP was assessed.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Lees</LastName><ForeName>A K</ForeName><Initials>AK</Initials><Identifier Source="ORCID">http://orcid.org/0000-0003-4034-2059</Identifier><AffiliationInfo><Affiliation>The James Hutton Institute, Invergowrie, Dundee DD2 5DA, United Kingdom.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Roberts</LastName><ForeName>D M</ForeName><Initials>DM</Initials><AffiliationInfo><Affiliation>The James Hutton Institute, Invergowrie, Dundee DD2 5DA, United Kingdom.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Lynott</LastName><ForeName>J</ForeName><Initials>J</Initials><AffiliationInfo><Affiliation>The James Hutton Institute, Invergowrie, Dundee DD2 5DA, United Kingdom.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Sullivan</LastName><ForeName>L</ForeName><Initials>L</Initials><AffiliationInfo><Affiliation>The James Hutton Institute, Invergowrie, Dundee DD2 5DA, United Kingdom.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Brierley</LastName><ForeName>J L</ForeName><Initials>JL</Initials><AffiliationInfo><Affiliation>The James Hutton Institute, Invergowrie, Dundee DD2 5DA, United Kingdom.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2019</Year><Month>10</Month><Day>28</Day></ArticleDate></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>Plant Dis</MedlineTA><NlmUniqueID>9882809</NlmUniqueID><ISSNLinking>0191-2917</ISSNLinking></MedlineJournalInfo><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D000383" MajorTopicYN="N">Agriculture</DescriptorName><QualifierName UI="Q000379" MajorTopicYN="N">methods</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D000528" MajorTopicYN="Y">Alternaria</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D021141" MajorTopicYN="Y">Nucleic Acid Amplification Techniques</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D055750" MajorTopicYN="Y">Phytophthora infestans</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D060888" MajorTopicYN="Y">Real-Time Polymerase Chain Reaction</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D018570" MajorTopicYN="Y">Risk Assessment</DescriptorName><QualifierName UI="Q000379" MajorTopicYN="N">methods</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D011198" MajorTopicYN="N">Solanum tuberosum</DescriptorName><QualifierName UI="Q000469" MajorTopicYN="N">parasitology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D058432" MajorTopicYN="N">Sporangia</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D033761" MajorTopicYN="N">Spores, Protozoan</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName><QualifierName UI="Q000302" MajorTopicYN="N">isolation & purification</QualifierName></MeshHeading></MeshHeadingList><KeywordList Owner="NOTNLM"><Keyword MajorTopicYN="N">IPM</Keyword><Keyword MajorTopicYN="N">air sampling</Keyword><Keyword MajorTopicYN="N">fungi</Keyword><Keyword MajorTopicYN="N">oomycetes</Keyword><Keyword MajorTopicYN="N">pathogen detection</Keyword><Keyword MajorTopicYN="N">rotating air samplers</Keyword></KeywordList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="pubmed"><Year>2019</Year><Month>10</Month><Day>29</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2019</Year><Month>11</Month><Day>28</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2019</Year><Month>10</Month><Day>29</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">31657996</ArticleId><ArticleId IdType="doi">10.1094/PDIS-04-19-0765-RE</ArticleId></ArticleIdList></PubmedData></pubmed><affiliations><list><country><li>Royaume-Uni</li></country></list><tree><country name="Royaume-Uni"><noRegion><name sortKey="Lees, A K" sort="Lees, A K" uniqKey="Lees A" first="A K" last="Lees">A K Lees</name></noRegion><name sortKey="Brierley, J L" sort="Brierley, J L" uniqKey="Brierley J" first="J L" last="Brierley">J L Brierley</name><name sortKey="Lynott, J" sort="Lynott, J" uniqKey="Lynott J" first="J" last="Lynott">J. Lynott</name><name sortKey="Roberts, D M" sort="Roberts, D M" uniqKey="Roberts D" first="D M" last="Roberts">D M Roberts</name><name sortKey="Sullivan, L" sort="Sullivan, L" uniqKey="Sullivan L" first="L" last="Sullivan">L. Sullivan</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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