Detection and quantification of Phytophthora ramorum, P. kernoviae, P. citricola and P. quercina in symptomatic leaves by multiplex real‐time PCR
Identifieur interne : 000408 ( Istex/Corpus ); précédent : 000407; suivant : 000409Detection and quantification of Phytophthora ramorum, P. kernoviae, P. citricola and P. quercina in symptomatic leaves by multiplex real‐time PCR
Auteurs : Leonardo Schena ; Kelvin J. D. Hughes ; David E. L. CookeSource :
- Molecular Plant Pathology [ 1464-6722 ] ; 2006-09.
Abstract
New species of Phytophthora such as Phytophthora ramorum, P. kernoviae and P. quercina together with P. citricola are plant pathogens which impact on forest health, natural ecosystem stability and international trade. A real‐time multiplex PCR approach based on TaqMan PCR was developed to simultaneously identify and detect these four Phytophthora species. Specific primers and probes labelled with FAM (P. ramorum), Yakima Yellow (P. kernoviae), Rox (P. citricola) and Cy5 (P. quercina) were designed in different regions of the ras‐related protein (Ypt1) gene. A new set of Black Hole Quenchers (BHQ), which dissipate energy as heat rather than fluorescence, were utilized. The method proved to be highly specific in tests with target DNA from 72 Phytophthora isolates (35 species). For all pathogens, the detection limit was 100 fg of target DNA and was not improved utilizing a nested approach to provide a first round of amplification with Phytophthora spp.‐specific primers. Cycle threshold (Ct) values were linearly correlated with the concentration of the target DNA (correlation coefficients ranged from 0.947 to 0.996) and were not affected by the presence of plant extracts, indicating the appropriateness of the method for qualitative and quantitative analyses. Two universal primers and a TaqMan probe were also developed to evaluate the quality and quantity of extracted DNA and to avoid false negatives. The reliability of the entire procedure was assessed using both artificially and naturally infected leaves of a range of plant species. The method, combined with a rapid procedure for DNA extraction, proved to be rapid, reliable, sensitive and cost effective as multiple pathogens were detected within the same plant extract by using different primer/probe combinations.
Url:
DOI: 10.1111/j.1364-3703.2006.00345.x
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Cooke</name><affiliation><mods:affiliation>Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j">Molecular Plant Pathology</title><idno type="ISSN">1464-6722</idno><idno type="eISSN">1364-3703</idno><imprint><publisher>Blackwell Publishing Ltd</publisher><pubPlace>Oxford, UK</pubPlace><date type="published" when="2006-09">2006-09</date><biblScope unit="volume">7</biblScope><biblScope unit="issue">5</biblScope><biblScope unit="page" from="365">365</biblScope><biblScope unit="page" to="379">379</biblScope></imprint><idno type="ISSN">1464-6722</idno></series><idno type="istex">BD3C09110BC1A13FFD561F31BD73BE3BF1ED76CD</idno><idno type="DOI">10.1111/j.1364-3703.2006.00345.x</idno><idno type="ArticleID">MPP345</idno></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">1464-6722</idno></seriesStmt></fileDesc><profileDesc><textClass></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">New species of Phytophthora such as Phytophthora ramorum, P. kernoviae and P. quercina together with P. citricola are plant pathogens which impact on forest health, natural ecosystem stability and international trade. A real‐time multiplex PCR approach based on TaqMan PCR was developed to simultaneously identify and detect these four Phytophthora species. Specific primers and probes labelled with FAM (P. ramorum), Yakima Yellow (P. kernoviae), Rox (P. citricola) and Cy5 (P. quercina) were designed in different regions of the ras‐related protein (Ypt1) gene. A new set of Black Hole Quenchers (BHQ), which dissipate energy as heat rather than fluorescence, were utilized. The method proved to be highly specific in tests with target DNA from 72 Phytophthora isolates (35 species). For all pathogens, the detection limit was 100 fg of target DNA and was not improved utilizing a nested approach to provide a first round of amplification with Phytophthora spp.‐specific primers. Cycle threshold (Ct) values were linearly correlated with the concentration of the target DNA (correlation coefficients ranged from 0.947 to 0.996) and were not affected by the presence of plant extracts, indicating the appropriateness of the method for qualitative and quantitative analyses. Two universal primers and a TaqMan probe were also developed to evaluate the quality and quantity of extracted DNA and to avoid false negatives. The reliability of the entire procedure was assessed using both artificially and naturally infected leaves of a range of plant species. The method, combined with a rapid procedure for DNA extraction, proved to be rapid, reliable, sensitive and cost effective as multiple pathogens were detected within the same plant extract by using different primer/probe combinations.</div></front></TEI><istex><corpusName>wiley</corpusName><author><json:item><name>LEONARDO SCHENA</name><affiliations><json:string>Department of Plant Protection and Applied Microbiology, Via Amendola 165/A, 70126, Bari, Italy</json:string><json:string>E-mail: leonardo.schena@agr.uniba.it</json:string></affiliations></json:item><json:item><name>KELVIN J. D. HUGHES</name><affiliations><json:string>Central Science Laboratory, Sand Hutton, York, North Yorkshire YO41 1LZ, UK</json:string></affiliations></json:item><json:item><name>DAVID E. L. COOKE</name><affiliations><json:string>Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK</json:string></affiliations></json:item></author><articleId><json:string>MPP345</json:string></articleId><language><json:string>eng</json:string></language><originalGenre><json:string>article</json:string></originalGenre><abstract>New species of Phytophthora such as Phytophthora ramorum, P. kernoviae and P. quercina together with P. citricola are plant pathogens which impact on forest health, natural ecosystem stability and international trade. A real‐time multiplex PCR approach based on TaqMan PCR was developed to simultaneously identify and detect these four Phytophthora species. Specific primers and probes labelled with FAM (P. ramorum), Yakima Yellow (P. kernoviae), Rox (P. citricola) and Cy5 (P. quercina) were designed in different regions of the ras‐related protein (Ypt1) gene. A new set of Black Hole Quenchers (BHQ), which dissipate energy as heat rather than fluorescence, were utilized. The method proved to be highly specific in tests with target DNA from 72 Phytophthora isolates (35 species). For all pathogens, the detection limit was 100 fg of target DNA and was not improved utilizing a nested approach to provide a first round of amplification with Phytophthora spp.‐specific primers. Cycle threshold (Ct) values were linearly correlated with the concentration of the target DNA (correlation coefficients ranged from 0.947 to 0.996) and were not affected by the presence of plant extracts, indicating the appropriateness of the method for qualitative and quantitative analyses. Two universal primers and a TaqMan probe were also developed to evaluate the quality and quantity of extracted DNA and to avoid false negatives. The reliability of the entire procedure was assessed using both artificially and naturally infected leaves of a range of plant species. The method, combined with a rapid procedure for DNA extraction, proved to be rapid, reliable, sensitive and cost effective as multiple pathogens were detected within the same plant extract by using different primer/probe combinations.</abstract><qualityIndicators><score>8</score><pdfVersion>1.3</pdfVersion><pdfPageSize>595 x 782 pts</pdfPageSize><refBibsNative>true</refBibsNative><abstractCharCount>1790</abstractCharCount><pdfWordCount>9211</pdfWordCount><pdfCharCount>59398</pdfCharCount><pdfPageCount>15</pdfPageCount><abstractWordCount>262</abstractWordCount></qualityIndicators><title>Detection and quantification of Phytophthora ramorum, P. kernoviae, P. citricola and P. quercina in symptomatic leaves by multiplex real‐time PCR</title><refBibs><json:item><host><author></author><title>Commission Decision (2002/757/EC) of 19 September 2002 on provisional emergency phytosanitary measures to prevent the introduction into and the spread within the Community of Phytophthora ramorum Werres, De Cock & Man in't Veld sp. novOfficial Journal of the European Communities</title></host></json:item><json:item><author><json:item><name>Y. Balci</name></json:item><json:item><name>E. Halmschlager</name></json:item></author><host><volume>86</volume><pages><first>442</first></pages><author></author><title>Plant Dis</title></host><title>First confirmation of Phytophthora quercina on oaks in Asia</title></json:item><json:item><author><json:item><name>Y. Balci</name></json:item><json:item><name>E. Halmschlager</name></json:item></author><host><volume>52</volume><pages><last>702</last><first>694</first></pages><author></author><title>Plant Pathol</title></host><title>Phytophthora species in oak forests in Turkey and their association with declining oak trees</title></json:item><json:item><author><json:item><name>Y. Balci</name></json:item><json:item><name>E. Halmschlager</name></json:item></author><host><volume>33</volume><pages><last>174</last><first>157</first></pages><author></author><title>For. Pathol</title></host><title>Incidence of Phytophthora species in oak forests in Austria and their possible involvement in oak decline</title></json:item><json:item><author><json:item><name>P.A. Beales</name></json:item><json:item><name>T. Brokenshire</name></json:item><json:item><name>A.V. Barnes</name></json:item><json:item><name>V.C. Barton</name></json:item><json:item><name>K.J.D. Hughes</name></json:item></author><host><volume>53</volume><pages><first>524</first></pages><author></author><title>Plant Pathol</title></host><title>First report of ramorum leaf blight and dieback (Phytophthora ramorum) on Camellia spp. in the UK</title></json:item><json:item><author><json:item><name>C.M. Brasier</name></json:item><json:item><name>P.A. Beales</name></json:item><json:item><name>S.A. Kirk</name></json:item><json:item><name>S. Denman</name></json:item><json:item><name>J. Rose</name></json:item></author><host><volume>109</volume><pages><last>859</last><first>853</first></pages><author></author><title>Mycol. Res</title></host><title>Phytophthora kernoviae sp. nov., an invasive pathogen causing bleeding stem lesions on forest trees and foliar necrosis of ornamentals in the UK</title></json:item><json:item><author><json:item><name>C. Brasier</name></json:item><json:item><name>S. Denman</name></json:item><json:item><name>A. Brown</name></json:item><json:item><name>J. Webber</name></json:item></author><host><volume>108</volume><pages><last>1110</last><first>1107</first></pages><author></author><title>Mycol. Res</title></host><title>Sudden oak death (Phytophthora ramorum) discovered on trees in Europe</title></json:item><json:item><author><json:item><name>C.M. Brasier</name></json:item><json:item><name>S.A. Kirk</name></json:item><json:item><name>J. Delcan</name></json:item><json:item><name>D.E.L. Cooke</name></json:item><json:item><name>T. Jung</name></json:item><json:item><name>W.A. Man In't Veld</name></json:item></author><host><volume>108</volume><pages><last>1184</last><first>1172</first></pages><author></author><title>Mycol. Res</title></host><title>Phytophthora alni sp. nov. & its variants: designation of emerging heteroploid hybrid pathogens spreading on Alnus trees</title></json:item><json:item><author><json:item><name>C.M. Brasier</name></json:item><json:item><name>E. Sanchez‐Hernandez</name></json:item><json:item><name>S.A. Kirk</name></json:item></author><host><volume>107</volume><pages><last>484</last><first>477</first></pages><author></author><title>Mycol. Res</title></host><title>Phytophthora inundata sp. nov., a part heterothallic of trees and shrubs in wet or flooded soils</title></json:item><json:item><author><json:item><name>P. Bridge</name></json:item><json:item><name>B. Spooner</name></json:item></author><host><volume>232</volume><pages><last>154</last><first>147</first></pages><author></author><title>Plant Soil</title></host><title>Soil fungi: diversity and detection</title></json:item><json:item><author><json:item><name>Y. Chen</name></json:item><json:item><name>R. Roxby</name></json:item></author><host><volume>181</volume><pages><last>94</last><first>89</first></pages><author></author><title>Gene</title></host><title>Characterization of a Phytophthora infestans gene involved in the vesicle transport</title></json:item><json:item><author><json:item><name>D.E.L. Cooke</name></json:item><json:item><name>A. Drenth</name></json:item><json:item><name>J.M. Duncan</name></json:item><json:item><name>G. Wagels</name></json:item><json:item><name>C.M. Brasier</name></json:item></author><host><volume>30</volume><pages><last>32</last><first>17</first></pages><author></author><title>Fungal. Genet. Biol</title></host><title>A molecular phylogeny of Phytophthora and related Oomycetes</title></json:item><json:item><author><json:item><name>D.E.L. Cooke</name></json:item><json:item><name>T. Jung</name></json:item><json:item><name>N.A. Williams</name></json:item><json:item><name>R. Schubert</name></json:item><json:item><name>G. Bahnweg</name></json:item><json:item><name>W. Oβwald</name></json:item><json:item><name>J.M. Duncan</name></json:item></author><host><volume>103</volume><pages><last>804</last><first>799</first></pages><author></author><title>Mycol. Res</title></host><title>Molecular evidence supports Phytophthora quercina as distinct species</title></json:item><json:item><author><json:item><name>D.E.L. Cooke</name></json:item><json:item><name>T. Jung</name></json:item><json:item><name>N.A. Williams</name></json:item><json:item><name>R. Schubert</name></json:item><json:item><name>W. Osswald</name></json:item><json:item><name>J.M. Duncan</name></json:item></author><host><volume>35</volume><pages><last>70</last><first>57</first></pages><author></author><title>For. Pathol</title></host><title>Genetic diversity of European populations of the oak fine‐root pathogen Phytophthora quercina</title></json:item><json:item><author><json:item><name>F. Corpet</name></json:item></author><host><volume>16</volume><pages><last>10890</last><first>10881</first></pages><author></author><title>Nucleic Acids Res</title></host><title>Multiple sequence alignment with hierarchical clustering</title></json:item><json:item><author><json:item><name>D.W. Cullen</name></json:item><json:item><name>P.R. Hirsch</name></json:item></author><host><volume>30</volume><pages><last>993</last><first>983</first></pages><author></author><title>Soil Biol. Biochem</title></host><title>Simple and rapid method for direct extraction of microbial DNA from soil for PCR</title></json:item><json:item><author><json:item><name>D.W. Cullen</name></json:item><json:item><name>A.K. Lees</name></json:item><json:item><name>I.K. Toth</name></json:item><json:item><name>J.M. Duncan</name></json:item></author><host><volume>107</volume><pages><last>398</last><first>387</first></pages><author></author><title>Eur. J. Plant Pathol</title></host><title>Conventional PCR and real‐time quantitative PCR detection of Helminthosporium solani in soil and potato tubers</title></json:item><json:item><author><json:item><name>E.M. Hansen</name></json:item><json:item><name>J.L. Parke</name></json:item><json:item><name>W. Sutton</name></json:item></author><host><volume>89</volume><pages><last>70</last><first>63</first></pages><author></author><title>Plant Dis</title></host><title>Susceptibility of Oregon forest trees and shrubs to Phytophthora ramorum: a comparison of artificial inoculation and natural infection</title></json:item><json:item><author><json:item><name>E.M. Hansen</name></json:item><json:item><name>P.W. Reeser</name></json:item><json:item><name>W. Sutton</name></json:item><json:item><name>L.M. Winton</name></json:item><json:item><name>N. Osterbauer</name></json:item></author><host><volume>87</volume><pages><first>1267</first></pages><author></author><title>Plant Dis</title></host><title>First report of A1 mating type of Phytophthora ramorum in North America</title></json:item><json:item><author><json:item><name>K.J. Hayden</name></json:item><json:item><name>D. Rizzo</name></json:item><json:item><name>J. Tse</name></json:item><json:item><name>M. Garbelotto</name></json:item></author><host><volume>94</volume><pages><last>1083</last><first>1075</first></pages><author></author><title>Phytopathology</title></host><title>Detection and quantification of Phytophthora ramorum from California forests using a real‐time polymerase chain reaction assay</title></json:item><json:item><author><json:item><name>B. Henricot</name></json:item><json:item><name>C. Prior</name></json:item></author><host><volume>18</volume><pages><last>156</last><first>151</first></pages><author></author><title>Mycologist</title></host><title>Phytophthora ramorum, the cause of sudden oak death or ramorum leaf blight and dieback</title></json:item><json:item><author><json:item><name>K.J.D. Hughes</name></json:item><json:item><name>J.A. Tomlinson</name></json:item><json:item><name>R.L. Griffin</name></json:item><json:item><name>N. Boonham</name></json:item><json:item><name>A.J. Inman</name></json:item><json:item><name>C.R. Lane</name></json:item></author><host><volume>in press</volume><author></author><title>Phytopathology</title></host><title>Development of a one‐step real‐time PCR assay for diagnosis of Phytophthora ramorum</title></json:item><json:item><author><json:item><name>A. Ippolito</name></json:item><json:item><name>L. Schena</name></json:item><json:item><name>V. Soleti Ligorio</name></json:item><json:item><name>T. Yaseen</name></json:item><json:item><name>F. Nigro</name></json:item></author><host><volume>110</volume><pages><last>843</last><first>833</first></pages><author></author><title>Eur. J. Plant Pathol</title></host><title>Real time detection of Phytophthora nicotianae and P. citrophthora in citrus roots and soils</title></json:item><json:item><author><json:item><name>U. Jönsson</name></json:item><json:item><name>T. Jung</name></json:item><json:item><name>U. Rosengren</name></json:item><json:item><name>B. Nihlgard</name></json:item><json:item><name>K. Sonesson</name></json:item></author><host><volume>158</volume><pages><last>364</last><first>355</first></pages><author></author><title>New Phytol</title></host><title>Pathogenicity of Swedish isolates of Phytophthora quercina to Quercus robur in two different soils</title></json:item><json:item><author><json:item><name>H.S. Judelson</name></json:item><json:item><name>T.A. Randall</name></json:item></author><host><volume>41</volume><pages><last>615</last><first>605</first></pages><author></author><title>Genome</title></host><title>Families of repeated DNA in the oomycete Phytophthora infestans and their distribution within the genus</title></json:item><json:item><author><json:item><name>T. Jung</name></json:item><json:item><name>H. Blaschke</name></json:item><json:item><name>P. Neumann</name></json:item></author><host><volume>26</volume><pages><last>272</last><first>253</first></pages><author></author><title>For. Pathol</title></host><title>Isolation, identification and pathogenicity of Phytophthora species from declining oak stands</title></json:item><json:item><author><json:item><name>T. Jung</name></json:item><json:item><name>H. Blaschke</name></json:item><json:item><name>W. Osswald</name></json:item></author><host><volume>49</volume><pages><last>718</last><first>706</first></pages><author></author><title>Plant Pathol</title></host><title>Involvement of soilborne Phytophthora species in Central European oak decline and the effect of site factors on the disease</title></json:item><json:item><author><json:item><name>T. Jung</name></json:item><json:item><name>D.E.L. Cooke</name></json:item><json:item><name>H. Blaschke</name></json:item><json:item><name>J.M. Duncan</name></json:item><json:item><name>W. Oβwald</name></json:item></author><host><volume>103</volume><pages><last>798</last><first>785</first></pages><author></author><title>Mycol. Res</title></host><title>Phytophthora quercina sp. nov., causing root rot of European oaks</title></json:item><json:item><author><json:item><name>T. Jung</name></json:item><json:item><name>E.M. Hansen</name></json:item><json:item><name>L. Winton</name></json:item><json:item><name>W. Oβwald</name></json:item><json:item><name>C. Delatour</name></json:item></author><host><volume>106</volume><pages><last>411</last><first>397</first></pages><author></author><title>Mycol. Res</title></host><title>Three new species of Phytophthora from European oak forests</title></json:item><json:item><author><json:item><name>T. Jung</name></json:item><json:item><name>G.W. Hudler</name></json:item><json:item><name>S.L. Jensen‐Tracy</name></json:item><json:item><name>H.M. Griffiths</name></json:item><json:item><name>F. Fleischmann</name></json:item><json:item><name>W. Osswald</name></json:item></author><host><volume>19</volume><pages><last>166</last><first>159</first></pages><author></author><title>Mycologist</title></host><title>Involvement of Phytophthora species in the decline of European beech in Europe and the USA</title></json:item><json:item><author><json:item><name>T. Jung</name></json:item><json:item><name>J. Nechwatal</name></json:item><json:item><name>D.E.L. Cooke</name></json:item><json:item><name>G. Hartmann</name></json:item><json:item><name>M. Blaschke</name></json:item><json:item><name>W.F. Oβwald</name></json:item><json:item><name>J.M. Duncan</name></json:item><json:item><name>C. Delatour</name></json:item></author><host><volume>107</volume><pages><last>789</last><first>772</first></pages><author></author><title>Mycol. Res</title></host><title>Phytophthora pseudosyringae sp. nov., a new species causing root and collar rot of deciduous tree species in Europe</title></json:item><json:item><author><json:item><name>P. Kong</name></json:item><json:item><name>C.X. Hong</name></json:item><json:item><name>S.N. Jeffers</name></json:item><json:item><name>P.A. Richardson</name></json:item></author><host><volume>93</volume><pages><last>831</last><first>822</first></pages><author></author><title>Phytopathology</title></host><title>A Species‐specific polymerase chain reaction assay for rapid detection of Phytophthora nicotianae in irrigation water</title></json:item><json:item><author><json:item><name>P. Kong</name></json:item><json:item><name>C.X. Hong</name></json:item><json:item><name>P.A. Richardson</name></json:item></author><host><volume>52</volume><pages><last>693</last><first>681</first></pages><author></author><title>Plant Pathol</title></host><title>Rapid detection of Phytophthora cinnamomi using PCR with primers derived from the Lpv putative storage protein genes</title></json:item><json:item><author><json:item><name>P. Kong</name></json:item><json:item><name>C.X. Hong</name></json:item><json:item><name>P.W. Tooley</name></json:item><json:item><name>K. Ivors</name></json:item><json:item><name>M. Garbelotto</name></json:item><json:item><name>P.A. Richardson</name></json:item></author><host><volume>38</volume><pages><last>439</last><first>433</first></pages><author></author><title>Lett. Appl. Microbiol</title></host><title>Rapid identification of Phytophthora ramorum using PCR‐SSCP analysis of ribosomal DNA ITS‐1</title></json:item><json:item><author><json:item><name>L.P.N.M. Kroon</name></json:item><json:item><name>F.T. Bakker</name></json:item><json:item><name>G.B.M. Van Den Bosch</name></json:item><json:item><name>P.J.M. Bonants</name></json:item><json:item><name>W.G. Flier</name></json:item></author><host><volume>41</volume><pages><last>782</last><first>766</first></pages><author></author><title>Fungal Genet. Biol</title></host><title>Phylogenetic analysis of Phytophthora species based on mitochondrial and nuclear DNA sequences</title></json:item><json:item><author><json:item><name>C.R. Lane</name></json:item><json:item><name>P.A. Beales</name></json:item><json:item><name>K.J.D. Hughes</name></json:item><json:item><name>R.L. Griffin</name></json:item><json:item><name>R.L. Munro</name></json:item><json:item><name>C.M. Brasier</name></json:item><json:item><name>J.F. Webber</name></json:item></author><host><volume>52</volume><pages><first>414</first></pages><author></author><title>Plant Pathol</title></host><title>First outbreak of Phytophthora ramorum in England, on Viburnum tinus</title></json:item><json:item><author><json:item><name>C.R. Lane</name></json:item><json:item><name>P.A. Beales</name></json:item><json:item><name>K.J.D. Hughes</name></json:item><json:item><name>J.A. Tomlinson</name></json:item><json:item><name>A.J. Inman</name></json:item><json:item><name>K. Warwick</name></json:item></author><host><volume>53</volume><pages><first>522</first></pages><author></author><title>Plant Pathol</title></host><title>First report of ramorum dieback (Phytophthora ramorum) on container‐grown English yew (Taxus baccata) in England</title></json:item><json:item><author><json:item><name>A.K. Lees</name></json:item><json:item><name>D.W. Cullen</name></json:item><json:item><name>L. Sullivan</name></json:item><json:item><name>M.J. Nicolson</name></json:item></author><host><volume>51</volume><pages><last>302</last><first>293</first></pages><author></author><title>Plant Pathol</title></host><title>Development of conventional and quantitative real‐time PCR assays for the detection and identification of Rhizoctonia solani AG‐3 in potato and soil</title></json:item><json:item><author><json:item><name>C.A. Lévesque</name></json:item><json:item><name>A.W.A.M. De Cock</name></json:item></author><host><volume>108</volume><pages><last>1383</last><first>1363</first></pages><author></author><title>Mycol. Res</title></host><title>Molecular phylogeny and taxonomy of the genus Pythium</title></json:item><json:item><author><json:item><name>B. Lievens</name></json:item><json:item><name>M. Brouwer</name></json:item><json:item><name>A.C.R.C. Vanachter</name></json:item><json:item><name>C.A. Lévesque</name></json:item><json:item><name>B.P.A. Cammue</name></json:item><json:item><name>B.P.H.J. Thomma</name></json:item></author><host><volume>223</volume><pages><last>122</last><first>113</first></pages><author></author><title>FEMS Microbiol. Lett</title></host><title>Design and development of a DNA array for rapid detection and identification of multiple tomato vascular wilt pathogens</title></json:item><json:item><author><json:item><name>I.M. Mackay</name></json:item><json:item><name>K.E. Arden</name></json:item><json:item><name>A. Nitsche</name></json:item></author><host><volume>30</volume><pages><last>1305</last><first>1292</first></pages><author></author><title>Nucleic Acids Res</title></host><title>Real‐time PCR in virology</title></json:item><json:item><author><json:item><name>F.N. Martin</name></json:item><json:item><name>P.W. Tooley</name></json:item></author><host><volume>95</volume><pages><last>284</last><first>269</first></pages><author></author><title>Mycologia</title></host><title>Phylogenetic relationships among Phytophthora species inferred from sequence analysis of mitochondrially encoded cytochrome oxidase I and II genes</title></json:item><json:item><author><json:item><name>F.N. Martin</name></json:item><json:item><name>P.W. Tooley</name></json:item></author><host><volume>107</volume><pages><last>1391</last><first>1379</first></pages><author></author><title>Mycol. Res</title></host><title>Phylogenetic relationships of Phytophthora ramorum, P. nemorosa, and P. pseudosyringae, three species recovered from areas in California with sudden oak death</title></json:item><json:item><author><json:item><name>F.N. Martin</name></json:item><json:item><name>P.W. Tooley</name></json:item></author><host><volume>94</volume><pages><last>991</last><first>983</first></pages><author></author><title>Phytopathology</title></host><title>Identification of Phytophthora isolates to species level using restriction fragment length polymorphism analysis of a polymerase chain reaction‐amplified region of mitochondrial DNA</title></json:item><json:item><author><json:item><name>F.N. Martin</name></json:item><json:item><name>P.W. Tooley</name></json:item><json:item><name>C. Blomquist</name></json:item></author><host><volume>94</volume><pages><last>631</last><first>621</first></pages><author></author><title>Phytopathology</title></host><title>Molecular detection of Phytophthora ramorum, the causal agent of sudden oak death in California, and two additional species commonly recovered from diseased plant material</title></json:item><json:item><author><json:item><name>G.W. Moorman</name></json:item><json:item><name>S. Kang</name></json:item><json:item><name>D.M. Geiser</name></json:item><json:item><name>S.H. Kim</name></json:item></author><host><volume>86</volume><pages><last>1231</last><first>1227</first></pages><author></author><title>Plant Dis</title></host><title>Identification and characterization of Pythium species associated with greenhouse floral crops in Pennsylvania</title></json:item><json:item><author><json:item><name>R.A. Mumford</name></json:item><json:item><name>K. Walsh</name></json:item><json:item><name>I. Barker</name></json:item><json:item><name>N. Boonham</name></json:item></author><host><volume>90</volume><pages><last>453</last><first>448</first></pages><author></author><title>Phytopathology</title></host><title>Detection of potato mop top virus and tobacco rattle virus using a multiplex real‐time fluorescent reverse‐transcription polymerase chain reaction assay</title></json:item><json:item><author><json:item><name>J. Nechwatal</name></json:item><json:item><name>W.F. Oβwald</name></json:item></author><host><volume>31</volume><pages><last>273</last><first>257</first></pages><author></author><title>For. Pathol</title></host><title>Comparative studies on the fine root status of healthy and declining spruce and beech trees in the Bavarian Alps and occurrence of Phytophthora and Pythium species</title></json:item><json:item><author><json:item><name>J. Nechwatal</name></json:item><json:item><name>A. Schlenzing</name></json:item><json:item><name>T. Jung</name></json:item><json:item><name>D.E.L. Cooke</name></json:item><json:item><name>J.M. Duncan</name></json:item><json:item><name>W.F. Oβwald</name></json:item></author><host><volume>31</volume><pages><last>97</last><first>85</first></pages><author></author><title>For. Pathol</title></host><title>A combination of baiting and PCR techniques for the detection of Phytophthora quercina and P. citricola in soil samples from oak stands</title></json:item><json:item><author><json:item><name>J.L. Parke</name></json:item><json:item><name>R.G. Linderman</name></json:item><json:item><name>N.K. Osterbauer</name></json:item><json:item><name>J.A. Griesbach</name></json:item></author><host><volume>88</volume><pages><first>87</first></pages><author></author><title>Plant Dis</title></host><title>Detection of Phytophthora ramorum blight in Oregon nurseries and completion of Koch's postulates on Pieris, Rhododendron, Viburnum, and Camellia</title></json:item><json:item><author><json:item><name>D.M. Rizzo</name></json:item><json:item><name>M. Garbelotto</name></json:item></author><host><volume>1</volume><pages><last>204</last><first>197</first></pages><author></author><title>Front. Ecol. Environ</title></host><title>Sudden oak death: endangering California and Oregon forest ecosystems</title></json:item><json:item><host><author></author><title>Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.</title></host></json:item><json:item><host><author></author><title>Assessing the potential of regions of the nuclear and mitochondrial genome to develop a ‘molecular tool box’ for the detection and characterization of Phytophthora speciesJ. Microbiol. Meth.</title></host></json:item><json:item><author><json:item><name>L. Schena</name></json:item><json:item><name>A. Ippolito</name></json:item></author><host><volume>85</volume><pages><last>25</last><first>15</first></pages><author></author><title>J. Plant Pathol</title></host><title>Rapid and sensitive detection of Rosellinia necatrix in roots and soils by real time Scorpion‐PCR</title></json:item><json:item><author><json:item><name>L. Schena</name></json:item><json:item><name>F. Nigro</name></json:item><json:item><name>A. Ippolito</name></json:item></author><host><volume>43</volume><pages><last>280</last><first>273</first></pages><author></author><title>Phytopathol. Med</title></host><title>Real‐time PCR detection and quantification of soilborne fungal pathogens: the case of Rosellinia necatrix, Phytophthora nicotianae, P. citrophthora, and Verticillium dahliae</title></json:item><json:item><author><json:item><name>L. Schena</name></json:item><json:item><name>F. Nigro</name></json:item><json:item><name>A. Ippolito</name></json:item><json:item><name>D. Gallitelli</name></json:item></author><host><volume>110</volume><pages><last>908</last><first>893</first></pages><author></author><title>Eur. J. Plant Pathol</title></host><title>Real‐time quantitative PCR: a new technology to detect and study phytopathogenic and antagonistic fungi</title></json:item><json:item><author><json:item><name>R. Schubert</name></json:item><json:item><name>G. Bahnweg</name></json:item><json:item><name>J. Nechwatal</name></json:item><json:item><name>T. Jung</name></json:item><json:item><name>D.E.L. Cooke</name></json:item><json:item><name>J.M. Duncan</name></json:item><json:item><name>G. Müller‐Starck</name></json:item><json:item><name>C. Langebartels</name></json:item><json:item><name>H. Sandermann Jr</name></json:item><json:item><name>W.F. Oβwald</name></json:item></author><host><volume>29</volume><pages><last>188</last><first>169</first></pages><author></author><title>For. Pathol</title></host><title>Detection and quantification of Phytophthora species which are associated with root‐rot diseases in European deciduous forests by species‐specific polymerase chain reaction</title></json:item><json:item><author><json:item><name>B.L. Shearer</name></json:item><json:item><name>C.E. Crane</name></json:item><json:item><name>A. Cochrane</name></json:item></author><host><volume>52</volume><pages><last>443</last><first>435</first></pages><author></author><title>Aust. J. Bot</title></host><title>Quantification of the susceptibility of the native flora of the south‐west botanical province, Western Australia, to Phytophthora cinnamomi</title></json:item><json:item><author><json:item><name>E. Stokstad</name></json:item></author><host><volume>303</volume><pages><first>1959</first></pages><author></author><title>Science</title></host><title>Nurseries may have shipped sudden oak death pathogen nationwide</title></json:item><json:item><author><json:item><name>K. Tanaka</name></json:item><json:item><name>I.B. Heath</name></json:item><json:item><name>P.B. Moens</name></json:item></author><host><volume>24</volume><pages><last>396</last><first>385</first></pages><author></author><title>Can. J. Genet. Cytol</title></host><title>Karyotipe, synaptonemal complexes and possible recombination nodules of the Oomycete fungus Saprolegnia</title></json:item><json:item><author><json:item><name>N. Thelwell</name></json:item><json:item><name>S. Millington</name></json:item><json:item><name>A. Solinas</name></json:item><json:item><name>J. Booth</name></json:item><json:item><name>T. Brown</name></json:item></author><host><volume>28</volume><pages><last>3761</last><first>3752</first></pages><author></author><title>Nucleic Acids Res</title></host><title>Mode of action and application of Scorpion primers to mutation detection</title></json:item><json:item><author><json:item><name>F.M. Thomas</name></json:item><json:item><name>R. Blank</name></json:item><json:item><name>G. Hartmann</name></json:item></author><host><volume>32</volume><pages><last>307</last><first>277</first></pages><author></author><title>For. Pathol</title></host><title>Abiotic and biotic factors and their interactions as causes of oak decline in Central Europe</title></json:item><json:item><author><json:item><name>J.A. Tomlinson</name></json:item><json:item><name>N. Boonham</name></json:item><json:item><name>K.J.D. Hughes</name></json:item><json:item><name>R.L. Griffin</name></json:item><json:item><name>I. Barker</name></json:item></author><host><volume>71</volume><pages><last>6710</last><first>6702</first></pages><author></author><title>Appl. Environ. Microbiol</title></host><title>On‐site DNA extraction and real‐time PCR for detection of Phytophthora ramorum in the field</title></json:item><json:item><author><json:item><name>P.W. Tooley</name></json:item><json:item><name>K.L. Kyde</name></json:item><json:item><name>L. Englander</name></json:item></author><host><volume>88</volume><pages><last>999</last><first>993</first></pages><author></author><title>Plant Dis</title></host><title>Susceptibility of selected ericaceous ornamental host species to Phytophthora ramorum</title></json:item><json:item><author><json:item><name>P.W. Tooley</name></json:item><json:item><name>F.N. Martin</name></json:item><json:item><name>M.M. Carras</name></json:item><json:item><name>R.D. Frederick</name></json:item></author><host><volume>96</volume><pages><last>345</last><first>336</first></pages><author></author><title>Phytopathology</title></host><title>Real‐time fluorescent polymerase chain reaction detection of Phytophthora ramorum and Phytophthora pseudosyringae using mitochondrial gene regions</title></json:item><json:item><author><json:item><name>A.M. Vettraino</name></json:item><json:item><name>G.P. Barzanti</name></json:item><json:item><name>M.C. Bianco</name></json:item><json:item><name>A. Ragazzi</name></json:item><json:item><name>P. Capretti</name></json:item><json:item><name>E. Paoletti</name></json:item><json:item><name>N. Luisi</name></json:item><json:item><name>N. Anselmi</name></json:item><json:item><name>A. Tannini</name></json:item></author><host><volume>32</volume><pages><last>28</last><first>19</first></pages><author></author><title>For. Pathol</title></host><title>Occurrence of Phytophthora species in oak stands in Italy and their association with declining oak trees</title></json:item><json:item><author><json:item><name>A.M. Vettraino</name></json:item><json:item><name>O. Morel</name></json:item><json:item><name>C. Perlerou</name></json:item><json:item><name>C. Robin</name></json:item><json:item><name>S. Diamandis</name></json:item><json:item><name>A. Vannini</name></json:item></author><host><volume>111</volume><pages><last>180</last><first>169</first></pages><author></author><title>Eur. J. Plant Pathol</title></host><title>Occurrence and distribution of Phytophthora species in European chestnut stands, and their association with Ink Disease and crown decline</title></json:item><json:item><author><json:item><name>H. Voglmayr</name></json:item><json:item><name>J. Greilhuber</name></json:item></author><host><volume>25</volume><pages><last>195</last><first>181</first></pages><author></author><title>Fungal Genet. Biol</title></host><title>Genome size determination in Peronosporales (Oomycota) by feulgen image analysis</title></json:item><json:item><author><json:item><name>Z.Y. Wang</name></json:item><json:item><name>A. Göttlein</name></json:item><json:item><name>H. Rodenkirchen</name></json:item><json:item><name>F. Fleischmann</name></json:item><json:item><name>W. Oβwald</name></json:item></author><host><volume>151</volume><pages><last>368</last><first>365</first></pages><author></author><title>J. Phytopathol</title></host><title>The Influence of Phytophthora citricola on rhizosphere soil solution chemistry and the nutritional status of European beech seedlings</title></json:item><json:item><author><json:item><name>R.A.M. Wattier</name></json:item><json:item><name>L.L. Gathercole</name></json:item><json:item><name>S.J. Assinder</name></json:item><json:item><name>C.J. Gliddon</name></json:item><json:item><name>K.L. Deahl</name></json:item><json:item><name>D.S. Shaw</name></json:item><json:item><name>D.I. Mills</name></json:item></author><host><volume>3</volume><pages><last>138</last><first>136</first></pages><author></author><title>Mol. Ecol. Notes</title></host><title>Sequence variation of intergenic mitochondrial DNA spacers (mtDNA‐IGS) of Phytophthora infestans (Oomycetes) and related species</title></json:item><json:item><author><json:item><name>S. Werres</name></json:item></author><host><volume>25</volume><pages><last>390</last><first>381</first></pages><author></author><title>For. Pathol</title></host><title>Influence of the Phytophthora isolate and the seed source on the development of beech (Fagus sylvatica) seedling blight</title></json:item><json:item><author><json:item><name>S. Werres</name></json:item><json:item><name>R. Marwitz</name></json:item><json:item><name>W.A. Man In't Veld</name></json:item><json:item><name>A.W.A.M. De Cock</name></json:item><json:item><name>P.J.M. Bonants</name></json:item><json:item><name>M. De Weerdt</name></json:item><json:item><name>K. Themann</name></json:item><json:item><name>E. Ilieva</name></json:item><json:item><name>R.P. Baayen</name></json:item></author><host><volume>105</volume><pages><last>1165</last><first>1155</first></pages><author></author><title>Mycol. Res</title></host><title>Phytophthora ramorum sp. nov., a new pathogen on Rhododendron and Viburnum</title></json:item><json:item><author><json:item><name>L.M. Winton</name></json:item><json:item><name>J.K. Stone</name></json:item><json:item><name>L.S. Watrud</name></json:item><json:item><name>E.M. Hansen</name></json:item></author><host><volume>92</volume><pages><last>116</last><first>112</first></pages><author></author><title>Phytopathology</title></host><title>Simultaneous one‐tube quantification of host and pathogen DNA with real‐time polymerase chain reaction</title></json:item><json:item><author><json:item><name>M. Zerjav</name></json:item><json:item><name>A. Munda</name></json:item><json:item><name>C.R. Lane</name></json:item><json:item><name>A.V. Barnes</name></json:item><json:item><name>K.J.D. Hughes</name></json:item></author><host><volume>53</volume><pages><first>523</first></pages><author></author><title>Plant Pathol</title></host><title>First report of Phytophthora ramorum on container‐grown plants of rhododendron and viburnum in Slovenia</title></json:item></refBibs><genre><json:string>article</json:string></genre><host><volume>7</volume><publisherId><json:string>MPP</json:string></publisherId><pages><total>15</total><last>379</last><first>365</first></pages><issn><json:string>1464-6722</json:string></issn><issue>5</issue><genre><json:string>journal</json:string></genre><language><json:string>unknown</json:string></language><eissn><json:string>1364-3703</json:string></eissn><title>Molecular Plant Pathology</title><doi><json:string>10.1111/(ISSN)1364-3703</json:string></doi></host><categories><wos><json:string>science</json:string><json:string>plant sciences</json:string></wos><scienceMetrix><json:string>natural sciences</json:string><json:string>biology</json:string><json:string>plant biology & botany</json:string></scienceMetrix></categories><publicationDate>2006</publicationDate><copyrightDate>2006</copyrightDate><doi><json:string>10.1111/j.1364-3703.2006.00345.x</json:string></doi><id>BD3C09110BC1A13FFD561F31BD73BE3BF1ED76CD</id><score>1</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/document/BD3C09110BC1A13FFD561F31BD73BE3BF1ED76CD/fulltext/pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/document/BD3C09110BC1A13FFD561F31BD73BE3BF1ED76CD/fulltext/zip</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/document/BD3C09110BC1A13FFD561F31BD73BE3BF1ED76CD/fulltext/tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main" xml:lang="en">Detection and quantification of Phytophthora ramorum, P. kernoviae, P. citricola and P. quercina in symptomatic leaves by multiplex real‐time PCR</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher>Blackwell Publishing Ltd</publisher><pubPlace>Oxford, UK</pubPlace><availability><p>WILEY</p></availability><date>2006</date></publicationStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a" type="main" xml:lang="en">Detection and quantification of Phytophthora ramorum, P. kernoviae, P. citricola and P. quercina in symptomatic leaves by multiplex real‐time PCR</title><author xml:id="author-1"><persName><forename type="first">LEONARDO</forename><surname>SCHENA</surname></persName><email>leonardo.schena@agr.uniba.it</email><affiliation>Department of Plant Protection and Applied Microbiology, Via Amendola 165/A, 70126, Bari, Italy</affiliation></author><author xml:id="author-2"><persName><forename type="first">KELVIN J. D.</forename><surname>HUGHES</surname></persName><affiliation>Central Science Laboratory, Sand Hutton, York, North Yorkshire YO41 1LZ, UK</affiliation></author><author xml:id="author-3"><persName><forename type="first">DAVID E. L.</forename><surname>COOKE</surname></persName><affiliation>Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK</affiliation></author></analytic><monogr><title level="j">Molecular Plant Pathology</title><idno type="pISSN">1464-6722</idno><idno type="eISSN">1364-3703</idno><idno type="DOI">10.1111/(ISSN)1364-3703</idno><imprint><publisher>Blackwell Publishing Ltd</publisher><pubPlace>Oxford, UK</pubPlace><date type="published" when="2006-09"></date><biblScope unit="volume">7</biblScope><biblScope unit="issue">5</biblScope><biblScope unit="page" from="365">365</biblScope><biblScope unit="page" to="379">379</biblScope></imprint></monogr><idno type="istex">BD3C09110BC1A13FFD561F31BD73BE3BF1ED76CD</idno><idno type="DOI">10.1111/j.1364-3703.2006.00345.x</idno><idno type="ArticleID">MPP345</idno></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>2006</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>New species of Phytophthora such as Phytophthora ramorum, P. kernoviae and P. quercina together with P. citricola are plant pathogens which impact on forest health, natural ecosystem stability and international trade. A real‐time multiplex PCR approach based on TaqMan PCR was developed to simultaneously identify and detect these four Phytophthora species. Specific primers and probes labelled with FAM (P. ramorum), Yakima Yellow (P. kernoviae), Rox (P. citricola) and Cy5 (P. quercina) were designed in different regions of the ras‐related protein (Ypt1) gene. A new set of Black Hole Quenchers (BHQ), which dissipate energy as heat rather than fluorescence, were utilized. The method proved to be highly specific in tests with target DNA from 72 Phytophthora isolates (35 species). For all pathogens, the detection limit was 100 fg of target DNA and was not improved utilizing a nested approach to provide a first round of amplification with Phytophthora spp.‐specific primers. Cycle threshold (Ct) values were linearly correlated with the concentration of the target DNA (correlation coefficients ranged from 0.947 to 0.996) and were not affected by the presence of plant extracts, indicating the appropriateness of the method for qualitative and quantitative analyses. Two universal primers and a TaqMan probe were also developed to evaluate the quality and quantity of extracted DNA and to avoid false negatives. The reliability of the entire procedure was assessed using both artificially and naturally infected leaves of a range of plant species. The method, combined with a rapid procedure for DNA extraction, proved to be rapid, reliable, sensitive and cost effective as multiple pathogens were detected within the same plant extract by using different primer/probe combinations.</p></abstract></profileDesc><revisionDesc><change when="2006-09">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/document/BD3C09110BC1A13FFD561F31BD73BE3BF1ED76CD/fulltext/txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="Wiley, elements deleted: body"><istex:xmlDeclaration>version="1.0" encoding="UTF-8" standalone="yes"</istex:xmlDeclaration><istex:document><component version="2.0" type="serialArticle" xml:lang="en"><header><publicationMeta level="product"><publisherInfo><publisherName>Blackwell Publishing Ltd</publisherName><publisherLoc>Oxford, UK</publisherLoc></publisherInfo><doi origin="wiley" registered="yes">10.1111/(ISSN)1364-3703</doi><issn type="print">1464-6722</issn><issn type="electronic">1364-3703</issn><idGroup><id type="product" value="MPP"></id><id type="publisherDivision" value="ST"></id></idGroup><titleGroup><title type="main" sort="MOLECULAR PLANT PATHOLOGY">Molecular Plant Pathology</title></titleGroup></publicationMeta><publicationMeta level="part" position="09005"><doi origin="wiley">10.1111/mpp.2006.7.issue-5</doi><numberingGroup><numbering type="journalVolume" number="7">7</numbering><numbering type="journalIssue" number="5">5</numbering></numberingGroup><coverDate startDate="2006-09">September 2006</coverDate></publicationMeta><publicationMeta level="unit" type="article" position="5" status="forIssue"><doi origin="wiley">10.1111/j.1364-3703.2006.00345.x</doi><idGroup><id type="unit" value="MPP345"></id></idGroup><countGroup><count type="pageTotal" number="15"></count></countGroup><titleGroup><title type="tocHeading1">Original articles</title></titleGroup><eventGroup><event type="firstOnline" date="2006-07-17"></event><event type="publishedOnlineFinalForm" date="2006-07-17"></event><event type="xmlConverted" agent="Converter:BPG_TO_WML3G version:2.3.2 mode:FullText source:FullText result:FullText" date="2010-03-09"></event><event type="xmlConverted" agent="Converter:WILEY_ML3G_TO_WILEY_ML3GV2 version:3.8.8" date="2014-02-03"></event><event type="xmlConverted" agent="Converter:WML3G_To_WML3G version:4.1.7 mode:FullText,remove_FC" date="2014-10-31"></event></eventGroup><numberingGroup><numbering type="pageFirst" number="365">365</numbering><numbering type="pageLast" number="379">379</numbering></numberingGroup><correspondenceTo> * <i>Correspondence</i>: <email>leonardo.schena@agr.uniba.it</email></correspondenceTo><linkGroup><link type="toTypesetVersion" href="file:MPP.MPP345.pdf"></link></linkGroup></publicationMeta><contentMeta><countGroup><count type="figureTotal" number="4"></count><count type="tableTotal" number="4"></count><count type="formulaTotal" number="0"></count><count type="referenceTotal" number="74"></count><count type="wordTotal" number="7930"></count></countGroup><titleGroup><title type="main">Detection and quantification of <i>Phytophthora ramorum</i>, <i>P. kernoviae</i>, <i>P. citricola</i> and <i>P. quercina</i> in symptomatic leaves by multiplex real‐time PCR</title><title type="shortAuthors">L. SCHENA <i>et al.</i></title><title type="short">Multiplex detection of forest phytophthoras</title></titleGroup><creators><creator creatorRole="author" xml:id="cr1" affiliationRef="#a1" corresponding="yes"><personName><givenNames>LEONARDO</givenNames><familyName>SCHENA</familyName></personName></creator><creator creatorRole="author" xml:id="cr2" affiliationRef="#a2"><personName><givenNames>KELVIN J. D.</givenNames><familyName>HUGHES</familyName></personName></creator><creator creatorRole="author" xml:id="cr3" affiliationRef="#a3"><personName><givenNames>DAVID E. L.</givenNames><familyName>COOKE</familyName></personName></creator></creators><affiliationGroup><affiliation xml:id="a1" countryCode="IT"><unparsedAffiliation>Department of Plant Protection and Applied Microbiology, Via Amendola 165/A, 70126, Bari, Italy</unparsedAffiliation></affiliation><affiliation xml:id="a2" countryCode="GB"><unparsedAffiliation>Central Science Laboratory, Sand Hutton, York, North Yorkshire YO41 1LZ, UK</unparsedAffiliation></affiliation><affiliation xml:id="a3" countryCode="GB"><unparsedAffiliation>Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK</unparsedAffiliation></affiliation></affiliationGroup><abstractGroup><abstract type="main" xml:lang="en"><title type="main">SUMMARY</title><p>New species of <i>Phytophthora</i> such as <i>Phytophthora ramorum, P. kernoviae</i> and <i>P. quercina</i> together with <i>P. citricola</i> are plant pathogens which impact on forest health, natural ecosystem stability and international trade. A real‐time multiplex PCR approach based on TaqMan PCR was developed to simultaneously identify and detect these four <i>Phytophthora</i> species. Specific primers and probes labelled with FAM (<i>P. ramorum</i>), Yakima Yellow (<i>P. kernoviae</i>), Rox (<i>P. citricola</i>) and Cy5 (<i>P. quercina</i>) were designed in different regions of the ras‐related protein (<i>Ypt</i>1) gene. A new set of Black Hole Quenchers (BHQ), which dissipate energy as heat rather than fluorescence, were utilized. The method proved to be highly specific in tests with target DNA from 72 <i>Phytophthora</i> isolates (35 species). For all pathogens, the detection limit was 100 fg of target DNA and was not improved utilizing a nested approach to provide a first round of amplification with <i>Phytophthora</i> spp.‐specific primers. Cycle threshold (Ct) values were linearly correlated with the concentration of the target DNA (correlation coefficients ranged from 0.947 to 0.996) and were not affected by the presence of plant extracts, indicating the appropriateness of the method for qualitative and quantitative analyses. Two universal primers and a TaqMan probe were also developed to evaluate the quality and quantity of extracted DNA and to avoid false negatives. The reliability of the entire procedure was assessed using both artificially and naturally infected leaves of a range of plant species. The method, combined with a rapid procedure for DNA extraction, proved to be rapid, reliable, sensitive and cost effective as multiple pathogens were detected within the same plant extract by using different primer/probe combinations.</p></abstract></abstractGroup></contentMeta></header></component></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>Detection and quantification of Phytophthora ramorum, P. kernoviae, P. citricola and P. quercina in symptomatic leaves by multiplex real‐time PCR</title></titleInfo><titleInfo type="abbreviated" lang="en"><title>Multiplex detection of forest phytophthoras</title></titleInfo><titleInfo type="alternative" contentType="CDATA" lang="en"><title>Detection and quantification of Phytophthora ramorum, P. kernoviae, P. citricola and P. quercina in symptomatic leaves by multiplex real‐time PCR</title></titleInfo><name type="personal"><namePart type="given">LEONARDO</namePart><namePart type="family">SCHENA</namePart><affiliation>Department of Plant Protection and Applied Microbiology, Via Amendola 165/A, 70126, Bari, Italy</affiliation><affiliation>E-mail: leonardo.schena@agr.uniba.it</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">KELVIN J. D.</namePart><namePart type="family">HUGHES</namePart><affiliation>Central Science Laboratory, Sand Hutton, York, North Yorkshire YO41 1LZ, UK</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">DAVID E. L.</namePart><namePart type="family">COOKE</namePart><affiliation>Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="article" displayLabel="article"></genre><originInfo><publisher>Blackwell Publishing Ltd</publisher><place><placeTerm type="text">Oxford, UK</placeTerm></place><dateIssued encoding="w3cdtf">2006-09</dateIssued><copyrightDate encoding="w3cdtf">2006</copyrightDate></originInfo><language><languageTerm type="code" authority="rfc3066">en</languageTerm><languageTerm type="code" authority="iso639-2b">eng</languageTerm></language><physicalDescription><internetMediaType>text/html</internetMediaType><extent unit="figures">4</extent><extent unit="tables">4</extent><extent unit="references">74</extent><extent unit="words">7930</extent></physicalDescription><abstract lang="en">New species of Phytophthora such as Phytophthora ramorum, P. kernoviae and P. quercina together with P. citricola are plant pathogens which impact on forest health, natural ecosystem stability and international trade. A real‐time multiplex PCR approach based on TaqMan PCR was developed to simultaneously identify and detect these four Phytophthora species. Specific primers and probes labelled with FAM (P. ramorum), Yakima Yellow (P. kernoviae), Rox (P. citricola) and Cy5 (P. quercina) were designed in different regions of the ras‐related protein (Ypt1) gene. A new set of Black Hole Quenchers (BHQ), which dissipate energy as heat rather than fluorescence, were utilized. The method proved to be highly specific in tests with target DNA from 72 Phytophthora isolates (35 species). For all pathogens, the detection limit was 100 fg of target DNA and was not improved utilizing a nested approach to provide a first round of amplification with Phytophthora spp.‐specific primers. Cycle threshold (Ct) values were linearly correlated with the concentration of the target DNA (correlation coefficients ranged from 0.947 to 0.996) and were not affected by the presence of plant extracts, indicating the appropriateness of the method for qualitative and quantitative analyses. Two universal primers and a TaqMan probe were also developed to evaluate the quality and quantity of extracted DNA and to avoid false negatives. The reliability of the entire procedure was assessed using both artificially and naturally infected leaves of a range of plant species. The method, combined with a rapid procedure for DNA extraction, proved to be rapid, reliable, sensitive and cost effective as multiple pathogens were detected within the same plant extract by using different primer/probe combinations.</abstract><relatedItem type="host"><titleInfo><title>Molecular Plant Pathology</title></titleInfo><genre type="journal">journal</genre><identifier type="ISSN">1464-6722</identifier><identifier type="eISSN">1364-3703</identifier><identifier type="DOI">10.1111/(ISSN)1364-3703</identifier><identifier type="PublisherID">MPP</identifier><part><date>2006</date><detail type="volume"><caption>vol.</caption><number>7</number></detail><detail type="issue"><caption>no.</caption><number>5</number></detail><extent unit="pages"><start>365</start><end>379</end><total>15</total></extent></part></relatedItem><identifier type="istex">BD3C09110BC1A13FFD561F31BD73BE3BF1ED76CD</identifier><identifier type="DOI">10.1111/j.1364-3703.2006.00345.x</identifier><identifier type="ArticleID">MPP345</identifier><recordInfo><recordContentSource>WILEY</recordContentSource><recordOrigin>Blackwell Publishing Ltd</recordOrigin></recordInfo></mods></metadata><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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