Development of a TaqMan PCR assay for specific detection and quantification of Pectobacterium brasiliense in potato tubers and soil
Pectobacterium brasiliense (Pb) is one of the causal agents of soft rot and blackleg diseases and has become a pathogen of economic importance in many potato production regions worldwide. Accurate, sensitive and timely identification of Pb is critical for improved management of the pathogen to mitig...
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| Veröffentlicht in: | European journal of plant pathology 2020-10, Vol.158 (2), p.521-532 |
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| creator | Muzhinji, N. Dube, J. P. de Haan, E. G. Woodhall, J. W. van der Waals, J. E. |
| description | Pectobacterium brasiliense
(Pb) is one of the causal agents of soft rot and blackleg diseases and has become a pathogen of economic importance in many potato production regions worldwide. Accurate, sensitive and timely identification of Pb is critical for improved management of the pathogen to mitigate yield losses. This study describes the development and validation of a TaqMan probe-based quantitative real-time PCR assay for rapid and specific detection of Pb in plant material and soil. A primer-pair that amplifies a 125-bp fragment was designed from the 16-23 s intergenic spacer region ribosomal RNA and the tRNA-Glu gene region. The specificity of the assay was evaluated with 24 isolates representative of nine different
Pectobacterium
and
Dickeya
species associated with soft rot and blackleg of potatoes. The designed Pb species-specific primers and FAM-labelled TaqMan probe were specific for detection of Pb in all the assays performed and it did not detect other
Pectobacterium
and
Dickeya
species. The TaqMan PCR assay could detect Pb DNA quantities as low as 10 fg/µl and DNA from a concentration of cells as low as 10
3
colony forming units/ml. The assay was capable of identifying and quantifying Pb in potato tubers and in field soils without the interference of inhibitors. The developed TaqMan PCR assay can be used for routine Pb diagnostics, surveillance and epidemiological studies. |
| doi_str_mv | 10.1007/s10658-020-02097-4 |
| format | Article |
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(Pb) is one of the causal agents of soft rot and blackleg diseases and has become a pathogen of economic importance in many potato production regions worldwide. Accurate, sensitive and timely identification of Pb is critical for improved management of the pathogen to mitigate yield losses. This study describes the development and validation of a TaqMan probe-based quantitative real-time PCR assay for rapid and specific detection of Pb in plant material and soil. A primer-pair that amplifies a 125-bp fragment was designed from the 16-23 s intergenic spacer region ribosomal RNA and the tRNA-Glu gene region. The specificity of the assay was evaluated with 24 isolates representative of nine different
Pectobacterium
and
Dickeya
species associated with soft rot and blackleg of potatoes. The designed Pb species-specific primers and FAM-labelled TaqMan probe were specific for detection of Pb in all the assays performed and it did not detect other
Pectobacterium
and
Dickeya
species. The TaqMan PCR assay could detect Pb DNA quantities as low as 10 fg/µl and DNA from a concentration of cells as low as 10
3
colony forming units/ml. The assay was capable of identifying and quantifying Pb in potato tubers and in field soils without the interference of inhibitors. The developed TaqMan PCR assay can be used for routine Pb diagnostics, surveillance and epidemiological studies.</description><identifier>ISSN: 0929-1873</identifier><identifier>EISSN: 1573-8469</identifier><identifier>DOI: 10.1007/s10658-020-02097-4</identifier><language>eng</language><publisher>Dordrecht: Springer Netherlands</publisher><subject>Agriculture ; Assaying ; Biomedical and Life Sciences ; Blackleg ; Deoxyribonucleic acid ; DNA ; Ecology ; Economic importance ; Epidemiology ; Glu gene ; Life Sciences ; Pathogens ; Pectobacterium ; Plant Pathology ; Plant Sciences ; Polymerase chain reaction ; Potatoes ; rRNA ; Soft rot ; Soils ; Spacer region ; Species ; tRNA Glu ; Tubers ; Vegetables</subject><ispartof>European journal of plant pathology, 2020-10, Vol.158 (2), p.521-532</ispartof><rights>Koninklijke Nederlandse Planteziektenkundige Vereniging 2020</rights><rights>Koninklijke Nederlandse Planteziektenkundige Vereniging 2020.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c2344-57254cd5a8a957cfdc9798d1922037dd2453f49a3c5b5882f693d4002c8166713</citedby><cites>FETCH-LOGICAL-c2344-57254cd5a8a957cfdc9798d1922037dd2453f49a3c5b5882f693d4002c8166713</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s10658-020-02097-4$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s10658-020-02097-4$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,780,784,27923,27924,41487,42556,51318</link.rule.ids></links><search><creatorcontrib>Muzhinji, N.</creatorcontrib><creatorcontrib>Dube, J. P.</creatorcontrib><creatorcontrib>de Haan, E. G.</creatorcontrib><creatorcontrib>Woodhall, J. W.</creatorcontrib><creatorcontrib>van der Waals, J. E.</creatorcontrib><title>Development of a TaqMan PCR assay for specific detection and quantification of Pectobacterium brasiliense in potato tubers and soil</title><title>European journal of plant pathology</title><addtitle>Eur J Plant Pathol</addtitle><description>Pectobacterium brasiliense
(Pb) is one of the causal agents of soft rot and blackleg diseases and has become a pathogen of economic importance in many potato production regions worldwide. Accurate, sensitive and timely identification of Pb is critical for improved management of the pathogen to mitigate yield losses. This study describes the development and validation of a TaqMan probe-based quantitative real-time PCR assay for rapid and specific detection of Pb in plant material and soil. A primer-pair that amplifies a 125-bp fragment was designed from the 16-23 s intergenic spacer region ribosomal RNA and the tRNA-Glu gene region. The specificity of the assay was evaluated with 24 isolates representative of nine different
Pectobacterium
and
Dickeya
species associated with soft rot and blackleg of potatoes. The designed Pb species-specific primers and FAM-labelled TaqMan probe were specific for detection of Pb in all the assays performed and it did not detect other
Pectobacterium
and
Dickeya
species. The TaqMan PCR assay could detect Pb DNA quantities as low as 10 fg/µl and DNA from a concentration of cells as low as 10
3
colony forming units/ml. The assay was capable of identifying and quantifying Pb in potato tubers and in field soils without the interference of inhibitors. The developed TaqMan PCR assay can be used for routine Pb diagnostics, surveillance and epidemiological studies.</description><subject>Agriculture</subject><subject>Assaying</subject><subject>Biomedical and Life Sciences</subject><subject>Blackleg</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>Ecology</subject><subject>Economic importance</subject><subject>Epidemiology</subject><subject>Glu gene</subject><subject>Life Sciences</subject><subject>Pathogens</subject><subject>Pectobacterium</subject><subject>Plant Pathology</subject><subject>Plant Sciences</subject><subject>Polymerase chain reaction</subject><subject>Potatoes</subject><subject>rRNA</subject><subject>Soft rot</subject><subject>Soils</subject><subject>Spacer region</subject><subject>Species</subject><subject>tRNA Glu</subject><subject>Tubers</subject><subject>Vegetables</subject><issn>0929-1873</issn><issn>1573-8469</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2020</creationdate><recordtype>article</recordtype><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><recordid>eNp9kEtLxDAUhYMoOI7-AVcB19U8m2Yp4xNGHGRchzRNJUMn6SSp4No_bmcquHNxuXDu-c6FA8AlRtcYIXGTMCp5VSCC9iNFwY7ADHNBi4qV8hjMkCSywJWgp-AspQ0aISnJDHzf2U_bhX5rfYahhRqu9e5Fe7havEGdkv6CbYgw9da41hnY2GxNdsFD7Ru4G7TPe10fpJFfjddQa5NtdMMW1lEn1znrk4XOwz5knQPMQ21jOiSk4LpzcNLqLtmL3z0H7w_368VTsXx9fF7cLgtDKGMFF4Qz03BdacmFaRsjhawaLAlBVDQNYZy2TGpqeM2rirSlpA1DiJgKl6XAdA6uptw-ht1gU1abMEQ_vlSEMVxyyhAfXWRymRhSirZVfXRbHb8URmpftprKVmPR6lC2YiNEJyiNZv9h41_0P9QP26eCkQ</recordid><startdate>20201001</startdate><enddate>20201001</enddate><creator>Muzhinji, N.</creator><creator>Dube, J. 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P. ; de Haan, E. G. ; Woodhall, J. W. ; van der Waals, J. E.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c2344-57254cd5a8a957cfdc9798d1922037dd2453f49a3c5b5882f693d4002c8166713</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2020</creationdate><topic>Agriculture</topic><topic>Assaying</topic><topic>Biomedical and Life Sciences</topic><topic>Blackleg</topic><topic>Deoxyribonucleic acid</topic><topic>DNA</topic><topic>Ecology</topic><topic>Economic importance</topic><topic>Epidemiology</topic><topic>Glu gene</topic><topic>Life Sciences</topic><topic>Pathogens</topic><topic>Pectobacterium</topic><topic>Plant Pathology</topic><topic>Plant Sciences</topic><topic>Polymerase chain reaction</topic><topic>Potatoes</topic><topic>rRNA</topic><topic>Soft rot</topic><topic>Soils</topic><topic>Spacer region</topic><topic>Species</topic><topic>tRNA Glu</topic><topic>Tubers</topic><topic>Vegetables</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Muzhinji, N.</creatorcontrib><creatorcontrib>Dube, J. P.</creatorcontrib><creatorcontrib>de Haan, E. G.</creatorcontrib><creatorcontrib>Woodhall, J. W.</creatorcontrib><creatorcontrib>van der Waals, J. 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P.</au><au>de Haan, E. G.</au><au>Woodhall, J. W.</au><au>van der Waals, J. E.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Development of a TaqMan PCR assay for specific detection and quantification of Pectobacterium brasiliense in potato tubers and soil</atitle><jtitle>European journal of plant pathology</jtitle><stitle>Eur J Plant Pathol</stitle><date>2020-10-01</date><risdate>2020</risdate><volume>158</volume><issue>2</issue><spage>521</spage><epage>532</epage><pages>521-532</pages><issn>0929-1873</issn><eissn>1573-8469</eissn><abstract>Pectobacterium brasiliense
(Pb) is one of the causal agents of soft rot and blackleg diseases and has become a pathogen of economic importance in many potato production regions worldwide. Accurate, sensitive and timely identification of Pb is critical for improved management of the pathogen to mitigate yield losses. This study describes the development and validation of a TaqMan probe-based quantitative real-time PCR assay for rapid and specific detection of Pb in plant material and soil. A primer-pair that amplifies a 125-bp fragment was designed from the 16-23 s intergenic spacer region ribosomal RNA and the tRNA-Glu gene region. The specificity of the assay was evaluated with 24 isolates representative of nine different
Pectobacterium
and
Dickeya
species associated with soft rot and blackleg of potatoes. The designed Pb species-specific primers and FAM-labelled TaqMan probe were specific for detection of Pb in all the assays performed and it did not detect other
Pectobacterium
and
Dickeya
species. The TaqMan PCR assay could detect Pb DNA quantities as low as 10 fg/µl and DNA from a concentration of cells as low as 10
3
colony forming units/ml. The assay was capable of identifying and quantifying Pb in potato tubers and in field soils without the interference of inhibitors. The developed TaqMan PCR assay can be used for routine Pb diagnostics, surveillance and epidemiological studies.</abstract><cop>Dordrecht</cop><pub>Springer Netherlands</pub><doi>10.1007/s10658-020-02097-4</doi><tpages>12</tpages></addata></record> |
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| ispartof | European journal of plant pathology, 2020-10, Vol.158 (2), p.521-532 |
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| source | SpringerLink Journals - AutoHoldings |
| subjects | Agriculture Assaying Biomedical and Life Sciences Blackleg Deoxyribonucleic acid DNA Ecology Economic importance Epidemiology Glu gene Life Sciences Pathogens Pectobacterium Plant Pathology Plant Sciences Polymerase chain reaction Potatoes rRNA Soft rot Soils Spacer region Species tRNA Glu Tubers Vegetables |
| title | Development of a TaqMan PCR assay for specific detection and quantification of Pectobacterium brasiliense in potato tubers and soil |
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