TY - JOUR
T1 - Co-operational PCR Coupled with Dot Blot Hybridization for the Detection of Phaeomoniella chlamydospora on Infected Grapevine Wood
AU - Martos, Soledad
AU - Torres, Ester
AU - El Bakali, Mohamed Abdessamad
AU - Raposo, Rosa
AU - Gramaje, David
AU - Armengol, Josep
AU - Luque, Jordi
PY - 2011/4/1
Y1 - 2011/4/1
N2 - The technique consisting of the co-operational PCR coupled with dot blot hybridization and posterior colorimetric visualization was developed for the detection of Phaeomoniella chlamydospora, one of the major pathogenic fungi involved in the Petri disease of grapevine. A partial region of the fungal rDNA including the internal transcribed spacer (ITS) region was amplified through co-operational PCR for P. chlamydospora and 17 additional grapevine-associated fungi included in the genera Botryosphaeria, Cryptovalsa, Cylindrocarpon, Dematophora, Diplodia, Dothiorella, Eutypa, Fomitiporia, Lasiodiplodia, Neofusicoccum, Phaeoacremonium, Phomopsis and Stereum, by using the primer pairs NSA3/NLC2 (external pair) and NSI1/NLB4 (inner pair). A specific probe (Pch2D) targeting the ITS2 region in the rDNA was developed for the detection of P. chlamydospora. Dot blot hybridizations carried out with the PCR products showed the specificity of the probe. Results indicated that Pch2D only hybridized with DNA amplicons of P. chlamydospora isolates, thus proving the specific detection of this fungus, while the 17 remaining species tested for the Pch2D probe resulted in negative results. Sensitivity of the technique was established below 0.1pg of genomic DNA. This technique was further validated using artificially inoculated grapevine cuttings with P. chlamydospora. The efficacy of detection was established at 80% after two independent blind assays. © 2010 Blackwell Verlag GmbH.
AB - The technique consisting of the co-operational PCR coupled with dot blot hybridization and posterior colorimetric visualization was developed for the detection of Phaeomoniella chlamydospora, one of the major pathogenic fungi involved in the Petri disease of grapevine. A partial region of the fungal rDNA including the internal transcribed spacer (ITS) region was amplified through co-operational PCR for P. chlamydospora and 17 additional grapevine-associated fungi included in the genera Botryosphaeria, Cryptovalsa, Cylindrocarpon, Dematophora, Diplodia, Dothiorella, Eutypa, Fomitiporia, Lasiodiplodia, Neofusicoccum, Phaeoacremonium, Phomopsis and Stereum, by using the primer pairs NSA3/NLC2 (external pair) and NSI1/NLB4 (inner pair). A specific probe (Pch2D) targeting the ITS2 region in the rDNA was developed for the detection of P. chlamydospora. Dot blot hybridizations carried out with the PCR products showed the specificity of the probe. Results indicated that Pch2D only hybridized with DNA amplicons of P. chlamydospora isolates, thus proving the specific detection of this fungus, while the 17 remaining species tested for the Pch2D probe resulted in negative results. Sensitivity of the technique was established below 0.1pg of genomic DNA. This technique was further validated using artificially inoculated grapevine cuttings with P. chlamydospora. The efficacy of detection was established at 80% after two independent blind assays. © 2010 Blackwell Verlag GmbH.
KW - Co-operational PCR
KW - Detection
KW - Petri disease
KW - Vitis vinifera
U2 - 10.1111/j.1439-0434.2010.01758.x
DO - 10.1111/j.1439-0434.2010.01758.x
M3 - Article
VL - 159
SP - 247
EP - 254
IS - 4
ER -