Detection of Phytophthora capsici in Irrigation Water using Loop-Mediated Isothermal Amplification.
Identifieur interne : 000248 ( Main/Exploration ); précédent : 000247; suivant : 000249Detection of Phytophthora capsici in Irrigation Water using Loop-Mediated Isothermal Amplification.
Auteurs : Owen Hudson ; Sumyya Waliullah ; Justin Hand ; Romina Gazis-Seregina ; Fulya Baysal-Gurel ; Md Emran Ali [États-Unis]Source :
- Journal of visualized experiments : JoVE [ 1940-087X ] ; 2020.
Descripteurs français
- KwdFr :
- MESH :
- génétique : ADN, Phytophthora.
- isolement et purification : Phytophthora.
- méthodes : Techniques d'amplification d'acides nucléiques.
- parasitologie : Eau, Sol.
- Irrigation agricole.
English descriptors
- KwdEn :
- MESH :
- chemical , genetics : DNA.
- genetics : Phytophthora.
- isolation & purification : Phytophthora.
- methods : Nucleic Acid Amplification Techniques.
- chemical , parasitology : Soil, Water.
- Agricultural Irrigation.
Abstract
Phytophthora capsici is a devastating oomycete pathogen that affects many important solanaceous and cucurbit crops causing significant economic losses in vegetable production annually. Phytophthora capsici is soil-borne and a persistent problem in vegetable fields due to its long-lived survival structures (oospores and chlamydospores) that resist weathering and degradation. The main method of dispersal is through the production of zoospores, which are single-celled, flagellated spores that can swim through thin films of water present on surfaces or in water-filled soil pores and can accumulate in puddles and ponds. Therefore, irrigation ponds can be a source of the pathogen and initial points of disease outbreaks. Detection of P. capsici in irrigation water is difficult using traditional culture-based methods because other microorganisms present in the environment, such as Pythium spp., usually overgrow P. capsici making it undetectable. To determine the presence of P. capsici spores in water sources (irrigation water, runoff, etc.), we developed a hand pump-based filter paper (8-10 µm) method that captures the pathogen's spores (zoospores) and is later used to amplify the pathogen's DNA through a novel loop-mediated isothermal amplification (LAMP) assay designed for the specific amplification of P. capsici. This method can amplify and detect DNA from a concentration as low as 1.2 x 102 zoospores/mL, which is 40 times more sensitive than conventional PCR. No cross-amplification was obtained when testing closely related species. LAMP was also performed using a colorimetric LAMP master mix dye, displaying results that could be read with the naked eye for on-site rapid detection. This protocol could be adapted to other pathogens that reside, accumulate, or are dispersed via contaminated irrigation systems.
DOI: 10.3791/61478
PubMed: 32658194
Affiliations:
Links toward previous steps (curation, corpus...)
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Floyd Nursery Research Center, Tennessee State University.</nlm:affiliation><wicri:noCountry code="subField">Tennessee State University</wicri:noCountry></affiliation></author><author><name sortKey="Ali, Md Emran" sort="Ali, Md Emran" uniqKey="Ali M" first="Md Emran" last="Ali">Md Emran Ali</name><affiliation wicri:level="1"><nlm:affiliation>Department of Plant Pathology, Coastal Plain Experiment Station, University of Georgia; emran.ali@uga.edu.</nlm:affiliation><country wicri:rule="url">États-Unis</country><wicri:regionArea>Department of Plant Pathology, Coastal Plain Experiment Station</wicri:regionArea><wicri:noRegion>Coastal Plain Experiment Station</wicri:noRegion></affiliation></author></analytic><series><title level="j">Journal of visualized experiments : JoVE</title><idno type="eISSN">1940-087X</idno><imprint><date when="2020" type="published">2020</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Agricultural Irrigation (MeSH)</term><term>DNA (genetics)</term><term>Nucleic Acid Amplification Techniques (methods)</term><term>Phytophthora (genetics)</term><term>Phytophthora (isolation & purification)</term><term>Soil (parasitology)</term><term>Water (parasitology)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>ADN (génétique)</term><term>Eau (parasitologie)</term><term>Irrigation agricole (MeSH)</term><term>Phytophthora (génétique)</term><term>Phytophthora (isolement et purification)</term><term>Sol (parasitologie)</term><term>Techniques d'amplification d'acides nucléiques (méthodes)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>DNA</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Phytophthora</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>ADN</term><term>Phytophthora</term></keywords><keywords scheme="MESH" qualifier="isolation & purification" xml:lang="en"><term>Phytophthora</term></keywords><keywords scheme="MESH" qualifier="isolement et purification" xml:lang="fr"><term>Phytophthora</term></keywords><keywords scheme="MESH" qualifier="methods" xml:lang="en"><term>Nucleic Acid Amplification Techniques</term></keywords><keywords scheme="MESH" qualifier="méthodes" xml:lang="fr"><term>Techniques d'amplification d'acides nucléiques</term></keywords><keywords scheme="MESH" qualifier="parasitologie" xml:lang="fr"><term>Eau</term><term>Sol</term></keywords><keywords scheme="MESH" type="chemical" qualifier="parasitology" xml:lang="en"><term>Soil</term><term>Water</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Agricultural Irrigation</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Irrigation agricole</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Phytophthora capsici is a devastating oomycete pathogen that affects many important solanaceous and cucurbit crops causing significant economic losses in vegetable production annually. Phytophthora capsici is soil-borne and a persistent problem in vegetable fields due to its long-lived survival structures (oospores and chlamydospores) that resist weathering and degradation. The main method of dispersal is through the production of zoospores, which are single-celled, flagellated spores that can swim through thin films of water present on surfaces or in water-filled soil pores and can accumulate in puddles and ponds. Therefore, irrigation ponds can be a source of the pathogen and initial points of disease outbreaks. Detection of P. capsici in irrigation water is difficult using traditional culture-based methods because other microorganisms present in the environment, such as Pythium spp., usually overgrow P. capsici making it undetectable. To determine the presence of P. capsici spores in water sources (irrigation water, runoff, etc.), we developed a hand pump-based filter paper (8-10 µm) method that captures the pathogen's spores (zoospores) and is later used to amplify the pathogen's DNA through a novel loop-mediated isothermal amplification (LAMP) assay designed for the specific amplification of P. capsici. This method can amplify and detect DNA from a concentration as low as 1.2 x 10<sup>2</sup> zoospores/mL, which is 40 times more sensitive than conventional PCR. No cross-amplification was obtained when testing closely related species. LAMP was also performed using a colorimetric LAMP master mix dye, displaying results that could be read with the naked eye for on-site rapid detection. This protocol could be adapted to other pathogens that reside, accumulate, or are dispersed via contaminated irrigation systems.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" IndexingMethod="Curated" Owner="NLM"><PMID Version="1">32658194</PMID><DateCompleted><Year>2020</Year><Month>10</Month><Day>14</Day></DateCompleted><DateRevised><Year>2020</Year><Month>10</Month><Day>14</Day></DateRevised><Article PubModel="Electronic"><Journal><ISSN IssnType="Electronic">1940-087X</ISSN><JournalIssue CitedMedium="Internet"><Issue>160</Issue><PubDate><Year>2020</Year><Month>06</Month><Day>25</Day></PubDate></JournalIssue><Title>Journal of visualized experiments : JoVE</Title><ISOAbbreviation>J Vis Exp</ISOAbbreviation></Journal><ArticleTitle>Detection of Phytophthora capsici in Irrigation Water using Loop-Mediated Isothermal Amplification.</ArticleTitle><ELocationID EIdType="doi" ValidYN="Y">10.3791/61478</ELocationID><Abstract><AbstractText>Phytophthora capsici is a devastating oomycete pathogen that affects many important solanaceous and cucurbit crops causing significant economic losses in vegetable production annually. Phytophthora capsici is soil-borne and a persistent problem in vegetable fields due to its long-lived survival structures (oospores and chlamydospores) that resist weathering and degradation. The main method of dispersal is through the production of zoospores, which are single-celled, flagellated spores that can swim through thin films of water present on surfaces or in water-filled soil pores and can accumulate in puddles and ponds. Therefore, irrigation ponds can be a source of the pathogen and initial points of disease outbreaks. Detection of P. capsici in irrigation water is difficult using traditional culture-based methods because other microorganisms present in the environment, such as Pythium spp., usually overgrow P. capsici making it undetectable. To determine the presence of P. capsici spores in water sources (irrigation water, runoff, etc.), we developed a hand pump-based filter paper (8-10 µm) method that captures the pathogen's spores (zoospores) and is later used to amplify the pathogen's DNA through a novel loop-mediated isothermal amplification (LAMP) assay designed for the specific amplification of P. capsici. This method can amplify and detect DNA from a concentration as low as 1.2 x 10<sup>2</sup> zoospores/mL, which is 40 times more sensitive than conventional PCR. No cross-amplification was obtained when testing closely related species. LAMP was also performed using a colorimetric LAMP master mix dye, displaying results that could be read with the naked eye for on-site rapid detection. This protocol could be adapted to other pathogens that reside, accumulate, or are dispersed via contaminated irrigation systems.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Hudson</LastName><ForeName>Owen</ForeName><Initials>O</Initials><AffiliationInfo><Affiliation>Department of Plant Pathology, Coastal Plain Experiment Station, University of Georgia.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Waliullah</LastName><ForeName>Sumyya</ForeName><Initials>S</Initials><AffiliationInfo><Affiliation>Department of Plant Pathology, Coastal Plain Experiment Station, University of Georgia.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Hand</LastName><ForeName>Justin</ForeName><Initials>J</Initials><AffiliationInfo><Affiliation>University of Georgia Cooperative Extension.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Gazis-Seregina</LastName><ForeName>Romina</ForeName><Initials>R</Initials><AffiliationInfo><Affiliation>Department of Plant Pathology, Tropical Research & Education Center, University of Florida.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Baysal-Gurel</LastName><ForeName>Fulya</ForeName><Initials>F</Initials><AffiliationInfo><Affiliation>Department of Agricultural and Environmental Sciences, Otis L. Floyd Nursery Research Center, Tennessee State University.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Ali</LastName><ForeName>Md Emran</ForeName><Initials>ME</Initials><AffiliationInfo><Affiliation>Department of Plant Pathology, Coastal Plain Experiment Station, University of Georgia; emran.ali@uga.edu.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType><PublicationType UI="D059040">Video-Audio Media</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2020</Year><Month>06</Month><Day>25</Day></ArticleDate></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>J Vis Exp</MedlineTA><NlmUniqueID>101313252</NlmUniqueID><ISSNLinking>1940-087X</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D012987">Soil</NameOfSubstance></Chemical><Chemical><RegistryNumber>059QF0KO0R</RegistryNumber><NameOfSubstance UI="D014867">Water</NameOfSubstance></Chemical><Chemical><RegistryNumber>9007-49-2</RegistryNumber><NameOfSubstance UI="D004247">DNA</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D057914" MajorTopicYN="Y">Agricultural Irrigation</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D004247" MajorTopicYN="N">DNA</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D021141" MajorTopicYN="N">Nucleic Acid Amplification Techniques</DescriptorName><QualifierName UI="Q000379" MajorTopicYN="Y">methods</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D010838" MajorTopicYN="N">Phytophthora</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName><QualifierName UI="Q000302" MajorTopicYN="Y">isolation & purification</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D012987" MajorTopicYN="N">Soil</DescriptorName><QualifierName UI="Q000469" MajorTopicYN="N">parasitology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D014867" MajorTopicYN="N">Water</DescriptorName><QualifierName UI="Q000469" MajorTopicYN="Y">parasitology</QualifierName></MeshHeading></MeshHeadingList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="entrez"><Year>2020</Year><Month>7</Month><Day>14</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="pubmed"><Year>2020</Year><Month>7</Month><Day>14</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2020</Year><Month>10</Month><Day>21</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>epublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">32658194</ArticleId><ArticleId IdType="doi">10.3791/61478</ArticleId></ArticleIdList></PubmedData></pubmed><affiliations><list><country><li>États-Unis</li></country></list><tree><noCountry><name sortKey="Baysal Gurel, Fulya" sort="Baysal Gurel, Fulya" uniqKey="Baysal Gurel F" first="Fulya" last="Baysal-Gurel">Fulya Baysal-Gurel</name><name sortKey="Gazis Seregina, Romina" sort="Gazis Seregina, Romina" uniqKey="Gazis Seregina R" first="Romina" last="Gazis-Seregina">Romina Gazis-Seregina</name><name sortKey="Hand, Justin" sort="Hand, Justin" uniqKey="Hand J" first="Justin" last="Hand">Justin Hand</name><name sortKey="Hudson, Owen" sort="Hudson, Owen" uniqKey="Hudson O" first="Owen" last="Hudson">Owen Hudson</name><name sortKey="Waliullah, Sumyya" sort="Waliullah, Sumyya" uniqKey="Waliullah S" first="Sumyya" last="Waliullah">Sumyya Waliullah</name></noCountry><country name="États-Unis"><noRegion><name sortKey="Ali, Md Emran" sort="Ali, Md Emran" uniqKey="Ali M" first="Md Emran" last="Ali">Md Emran Ali</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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