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Selective detection of Pseudomonas syringae pv. tomato using dot blot hybridization and real‐time PCR
Author(s) -
Fanelli V.,
Cariddi C.,
FinettiSialer M.
Publication year - 2007
Publication title -
plant pathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.928
H-Index - 85
eISSN - 1365-3059
pISSN - 0032-0862
DOI - 10.1111/j.1365-3059.2007.01612.x
Subject(s) - pseudomonas syringae , pathovar , biology , dot blot , southern blot , dna , polymerase chain reaction , real time polymerase chain reaction , hybridization probe , rapd , microbiology and biotechnology , pseudomonadaceae , genomic dna , pseudomonas , bacteria , genetics , gene , population , demography , sociology , genetic diversity
Two rapid detection methods based on dot blot hybridization with a nonradioactive DNA probe and molecular beacon‐PCR were developed for the specific detection of Pseudomonas syringae pv . tomato , the causal agent of bacterial speck of tomato. A 1378 bp DNA fragment (Acc. No. AM039892), obtained from the extension of a 255 bp fragment generated by a RAPD protocol, was used to find a suitable combination of primers specific for the tomato pathovar. A 138 bp fragment from the genome of P. syringae pv. tomato DC 3000 was used as DNA probe. In dot blots of DNA extracted from either pure cultures or artificially contaminated seeds washes, the probe recognized specifically the tomato pathovar. A molecular beacon was designed from the same region for the specific detection and quantification of P. syringae pv . tomato by real‐time PCR. A highly significant correlation was observed between the amount of target DNA and the cycle threshold (Ct). Using a fast protocol for DNA extraction, from pure cultures and from washes of artificially contaminated seeds, the limit of detection was about 1 × 10 2 CFU. The diagnostic tools developed proved highly specific for P. syringae pv. tomato and simple to use. They can therefore be applied to large‐scale testing of tomato seeds and seedlings for the assessment of their phytosanitary condition in nurseries.