Identification and detection by real-time pcr using lna probe of Dickeya chrysanthemi, Pectobacterium carotovorum subsp. carotovorum, Pseudomonas cichorii, Pseudomonas corrugata, Pseudomonas fluorescens, Pseudomonas mediterranea and Pseudomonas viridiflava, casual agents of tomato (Solanum lycopersicum L.) pith necrosis disease

In this study, Dickeya chrysanthemi, Pectobacterium carotovorum subsp. carotovorum, Pseudomonas cichorii, Pseudomonas corrugata, Pseudomonas fluorescens, Pseudomonas mediterranea and Pseudomonas viridiflava, causal agents of tomato pith necrosis disease causing serious economical yield losses on tomato, were sensitively and selectively identified and detected in short time by Real-Time PCR methods using LNA (Locked Nucleic Acid)probes. The sensitivity of primer and probe sets developed for Dickeya chrysanthemi, Pectobacterium carotovorum subsp. carotovorum, Pseudomonas cichorii, Pseudomonas corrugata, Pseudomonas fluorescens, Pseudomonas mediterranea and Pseudomonas viridiflava. Primer and probe sets developed in this study were tested against different Dickeya chrysanthemi, Pectobacterium carotovorum subsp. carotovorum, Pseudomonas cichorii, Pseudomonas corrugata, Pseudomonas fluorescens, Pseudomonas mediterranea and Pseudomonas viridiflava strains, other plant pathogenic bacteria from different genus and species. Although no amplication product was detected from genome of different species of plant pathogenic bacteria, 65 bp-amplification product of Dickeya chrysanthemi, 63 bp-amplification product of the Pectobacterium carotovorum subsp. carotovorum, 73 bp-amplification product of Pseudomonas cichorii, 70 bp-amplification product were detected from different strains of Pseudomonas corrugata, 70 bp-amplification product of Pseudomonas fluorescens, 73 bp-amplification product of Pseudomonas mediterranea and 77 bp-amplification product of Pseudomonas viridiflava. In this study, the detection of bacterial pathogen causing pith necrosis by Real- Time PCR was carried out from bacterial cell suspension, genomic DNA and plant material. The detection from cell suspension was carried out with precision limits of 2 cell for Dickeya chrysanthemi, 4 cell for Pectobacterium carotovorum subsp. carotovorum, 4 cell for Pseudomonas cichorii, 4 cell for Pseudomonas corrugata, 5 cell for Pseudomonas fluorescens, 5 cell for Pseudomonas mediterranea and 2 cell for Pseudomonas viridiflava. The detection of genomic DNA in pg (picogram) of the bacterial genome was also found to be 12 pg for Dickeya chrysanthemi, 11 pg for Pectobacterium carotovorum subsp. carotovorum, 14 pg for Pseudomonas cichorii, 14 pg for Pseudomonas corrugata, 14 pg for Pseudomonas fluorescens and 13 pg for Pseudomonas mediterranea 12 pg for Pseudomonas viridiflava. In conclusion, Real-Time PCR method using primer and probe sets specific for each of 7 different tomato bacterial pith necrosis pathogens including Dickeya chrysanthemi, Pectobacterium carotovorum subsp. carotovorum, Pseudomonas cichorii, Pseudomonas corrugata, Pseudomonas fluorescens, Pseudomonas mediterranea and Pseudomonas viridiflava were sensitive and quick (17-27 min.) for identification of the bacteria from the cells and for detection of the pathogens from the tomato diseased plant tissue.