Premium
Co‐operational PCR Coupled with Dot Blot Hybridization for the Detection of Phaeomoniella chlamydospora on Infected Grapevine Wood
Author(s) -
Martos Soledad,
Torres Ester,
El Bakali Mohamed Abdessamad,
Raposo Rosa,
Gramaje David,
Armengol Josep,
Luque Jordi
Publication year - 2011
Publication title -
journal of phytopathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.53
H-Index - 60
eISSN - 1439-0434
pISSN - 0931-1785
DOI - 10.1111/j.1439-0434.2010.01758.x
Subject(s) - biology , amplicon , botryosphaeria , internal transcribed spacer , dot blot , microbiology and biotechnology , genomic dna , ribosomal dna , primer (cosmetics) , polymerase chain reaction , ribosomal rna , botany , dna , gene , genetics , phylogenetic tree , chemistry , organic chemistry
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 0.1 pg 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.