Development and Application of qPCR and RPA Genus- and Species-Specific Detection of Phytophthora sojae and P. sansomeana Root Rot Pathogens of Soybean.
Identifieur interne : 000A32 ( Main/Exploration ); précédent : 000A31; suivant : 000A33Development and Application of qPCR and RPA Genus- and Species-Specific Detection of Phytophthora sojae and P. sansomeana Root Rot Pathogens of Soybean.
Auteurs : J Alejandro Rojas [États-Unis] ; Timothy D. Miles ; Michael D. Coffey ; Frank N. Martin [États-Unis] ; Martin I. Chilvers [États-Unis]Source :
- Plant disease [ 0191-2917 ] ; 2017.
Abstract
Phytophthora root rot of soybean, caused by Phytophthora sojae, is one of the most important diseases in the Midwestern United States, and is estimated to cause losses of up to 1.2 million metric tons per year. Disease may also be caused by P. sansomeana; however, the prevalence and damage caused by this species is not well known, partly due to limitations of current diagnostic tools. Efficient, accurate, and sensitive detection of pathogens is crucial for management. Thus, multiplex qPCR and isothermal RPA (recombinase polymerase amplification) assays were developed using a hierarchical approach to detect these Phytophthora spp. The assays consist of a genus-specific probe and two species-specific probes that target the atp9-nad9 region of the mitochondrial genome that is highly specific for the genus Phytophthora. The qPCR approach multiplexes the three probes and a plant internal control. The RPA assays run each probe independently with a plant internal control multiplexed in one amplification, obtaining a result in as little as 20 mins. The multicopy mitochondrial genome provides sensitivity with sufficient variability to discern among different Phytophthora spp. The assays were highly specific when tested against a panel of 100 Phytophthora taxa and range of Pythium spp. The consistent detection level of the assay was 100 fg for the qPCR assay and 10 pg for the RPA assay. The assays were validated on symptomatic plants collected from Michigan (U.S.) and Ontario (Canada) during the 2013 field season, showing correlation with isolation. In 2014, the assays were validated with samples from nine soybean producing states in the U.S. The assays are valuable diagnostic tools for detection of Phytophthora spp. affecting soybean.
DOI: 10.1094/PDIS-09-16-1225-RE
PubMed: 30682964
Affiliations:
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Miles</name><affiliation><nlm:affiliation>School of Natural Sciences, California State University, Monterey Bay, Seaside, 93955.</nlm:affiliation><wicri:noCountry code="subField">93955</wicri:noCountry></affiliation></author><author><name sortKey="Coffey, Michael D" sort="Coffey, Michael D" uniqKey="Coffey M" first="Michael D" last="Coffey">Michael D. Coffey</name><affiliation><nlm:affiliation>Department of Plant Pathology and Microbiology, University of California, Riverside, 92521.</nlm:affiliation><wicri:noCountry code="subField">92521</wicri:noCountry></affiliation></author><author><name sortKey="Martin, Frank N" sort="Martin, Frank N" uniqKey="Martin F" first="Frank N" last="Martin">Frank N. Martin</name><affiliation wicri:level="2"><nlm:affiliation>United States Department of Agriculture-Agricultural Research Service, Crop Improvement and Protection Research Unit, Salinas, CA 93905.</nlm:affiliation><country xml:lang="fr">États-Unis</country><placeName><region type="state">Californie</region></placeName><wicri:cityArea>United States Department of Agriculture-Agricultural Research Service, Crop Improvement and Protection Research Unit, Salinas</wicri:cityArea></affiliation></author><author><name sortKey="Chilvers, Martin I" sort="Chilvers, Martin I" uniqKey="Chilvers M" first="Martin I" last="Chilvers">Martin I. Chilvers</name><affiliation wicri:level="4"><nlm:affiliation>Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing, 48824; and Program in Ecology, Evolutionary Biology and Behavior, Michigan State University, East Lansing, 48824.</nlm:affiliation><orgName type="university">Université d'État du Michigan</orgName><country>États-Unis</country><placeName><settlement type="city">East Lansing</settlement><region type="state">Michigan</region></placeName></affiliation></author></analytic><series><title level="j">Plant disease</title><idno type="ISSN">0191-2917</idno><imprint><date when="2017" type="published">2017</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Phytophthora root rot of soybean, caused by Phytophthora sojae, is one of the most important diseases in the Midwestern United States, and is estimated to cause losses of up to 1.2 million metric tons per year. Disease may also be caused by P. sansomeana; however, the prevalence and damage caused by this species is not well known, partly due to limitations of current diagnostic tools. Efficient, accurate, and sensitive detection of pathogens is crucial for management. Thus, multiplex qPCR and isothermal RPA (recombinase polymerase amplification) assays were developed using a hierarchical approach to detect these Phytophthora spp. The assays consist of a genus-specific probe and two species-specific probes that target the atp9-nad9 region of the mitochondrial genome that is highly specific for the genus Phytophthora. The qPCR approach multiplexes the three probes and a plant internal control. The RPA assays run each probe independently with a plant internal control multiplexed in one amplification, obtaining a result in as little as 20 mins. The multicopy mitochondrial genome provides sensitivity with sufficient variability to discern among different Phytophthora spp. The assays were highly specific when tested against a panel of 100 Phytophthora taxa and range of Pythium spp. The consistent detection level of the assay was 100 fg for the qPCR assay and 10 pg for the RPA assay. The assays were validated on symptomatic plants collected from Michigan (U.S.) and Ontario (Canada) during the 2013 field season, showing correlation with isolation. In 2014, the assays were validated with samples from nine soybean producing states in the U.S. The assays are valuable diagnostic tools for detection of Phytophthora spp. affecting soybean.</div></front></TEI><pubmed><MedlineCitation Status="PubMed-not-MEDLINE" Owner="NLM"><PMID Version="1">30682964</PMID><DateRevised><Year>2019</Year><Month>11</Month><Day>20</Day></DateRevised><Article PubModel="Print-Electronic"><Journal><ISSN IssnType="Print">0191-2917</ISSN><JournalIssue CitedMedium="Print"><Volume>101</Volume><Issue>7</Issue><PubDate><Year>2017</Year><Month>Jul</Month></PubDate></JournalIssue><Title>Plant disease</Title><ISOAbbreviation>Plant Dis</ISOAbbreviation></Journal><ArticleTitle>Development and Application of qPCR and RPA Genus- and Species-Specific Detection of Phytophthora sojae and P. sansomeana Root Rot Pathogens of Soybean.</ArticleTitle><Pagination><MedlinePgn>1171-1181</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1094/PDIS-09-16-1225-RE</ELocationID><Abstract><AbstractText>Phytophthora root rot of soybean, caused by Phytophthora sojae, is one of the most important diseases in the Midwestern United States, and is estimated to cause losses of up to 1.2 million metric tons per year. Disease may also be caused by P. sansomeana; however, the prevalence and damage caused by this species is not well known, partly due to limitations of current diagnostic tools. Efficient, accurate, and sensitive detection of pathogens is crucial for management. Thus, multiplex qPCR and isothermal RPA (recombinase polymerase amplification) assays were developed using a hierarchical approach to detect these Phytophthora spp. The assays consist of a genus-specific probe and two species-specific probes that target the atp9-nad9 region of the mitochondrial genome that is highly specific for the genus Phytophthora. The qPCR approach multiplexes the three probes and a plant internal control. The RPA assays run each probe independently with a plant internal control multiplexed in one amplification, obtaining a result in as little as 20 mins. The multicopy mitochondrial genome provides sensitivity with sufficient variability to discern among different Phytophthora spp. The assays were highly specific when tested against a panel of 100 Phytophthora taxa and range of Pythium spp. The consistent detection level of the assay was 100 fg for the qPCR assay and 10 pg for the RPA assay. The assays were validated on symptomatic plants collected from Michigan (U.S.) and Ontario (Canada) during the 2013 field season, showing correlation with isolation. In 2014, the assays were validated with samples from nine soybean producing states in the U.S. The assays are valuable diagnostic tools for detection of Phytophthora spp. affecting soybean.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Rojas</LastName><ForeName>J Alejandro</ForeName><Initials>JA</Initials><Identifier Source="ORCID">http://orcid.org/0000-0001-5492-5963</Identifier><AffiliationInfo><Affiliation>Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing, 48824; and Program in Ecology, Evolutionary Biology and Behavior, Michigan State University, East Lansing, 48824.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Miles</LastName><ForeName>Timothy D</ForeName><Initials>TD</Initials><Identifier Source="ORCID">http://orcid.org/0000-0002-7484-9360</Identifier><AffiliationInfo><Affiliation>School of Natural Sciences, California State University, Monterey Bay, Seaside, 93955.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Coffey</LastName><ForeName>Michael D</ForeName><Initials>MD</Initials><AffiliationInfo><Affiliation>Department of Plant Pathology and Microbiology, University of California, Riverside, 92521.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Martin</LastName><ForeName>Frank N</ForeName><Initials>FN</Initials><AffiliationInfo><Affiliation>United States Department of Agriculture-Agricultural Research Service, Crop Improvement and Protection Research Unit, Salinas, CA 93905.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Chilvers</LastName><ForeName>Martin I</ForeName><Initials>MI</Initials><AffiliationInfo><Affiliation>Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing, 48824; and Program in Ecology, Evolutionary Biology and Behavior, Michigan State University, East Lansing, 48824.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2017</Year><Month>05</Month><Day>15</Day></ArticleDate></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>Plant Dis</MedlineTA><NlmUniqueID>9882809</NlmUniqueID><ISSNLinking>0191-2917</ISSNLinking></MedlineJournalInfo></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="entrez"><Year>2019</Year><Month>1</Month><Day>27</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="pubmed"><Year>2017</Year><Month>7</Month><Day>1</Day><Hour>0</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2017</Year><Month>7</Month><Day>1</Day><Hour>0</Hour><Minute>1</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">30682964</ArticleId><ArticleId IdType="doi">10.1094/PDIS-09-16-1225-RE</ArticleId></ArticleIdList></PubmedData></pubmed><affiliations><list><country><li>États-Unis</li></country><region><li>Californie</li><li>Michigan</li></region><settlement><li>East Lansing</li></settlement><orgName><li>Université d'État du Michigan</li></orgName></list><tree><noCountry><name sortKey="Coffey, Michael D" sort="Coffey, Michael D" uniqKey="Coffey M" first="Michael D" last="Coffey">Michael D. Coffey</name><name sortKey="Miles, Timothy D" sort="Miles, Timothy D" uniqKey="Miles T" first="Timothy D" last="Miles">Timothy D. Miles</name></noCountry><country name="États-Unis"><region name="Michigan"><name sortKey="Rojas, J Alejandro" sort="Rojas, J Alejandro" uniqKey="Rojas J" first="J Alejandro" last="Rojas">J Alejandro Rojas</name></region><name sortKey="Chilvers, Martin I" sort="Chilvers, Martin I" uniqKey="Chilvers M" first="Martin I" last="Chilvers">Martin I. Chilvers</name><name sortKey="Martin, Frank N" sort="Martin, Frank N" uniqKey="Martin F" first="Frank N" last="Martin">Frank N. Martin</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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