Identification and detection of Pseudomonas savastanoi pv. savastanoi ,the causal agent of olive knot disease and it's epiphytic population by quantitative real-time membran bio-PCR

In this study, a sensitive and selective Real-Time PCR method using LNA (Locked Nucleic Acid) probe was developed for identification and detection of Pseudomonas savastanoi pv. savastanoi (Pss) which causes olive knot disease and results in significant loss of quality and product in economic aspects of olive farming. For Real-Time PCR method, Indoleacetate-lysine ligase (iaaL) gene sequence of Pseudomonas savastanoi pv. savastanoi was used in order to develop a specific primer set and probe. Pss isolates, different plant pathogenic bacteria genus and total genomic DNA of olives were tested for determination of the specificity of the designed primer and probe sets. Results showed no amplification for the genomes of different plant pathogenic bacteria and olive genomic DNA. On the other hand all local and foreign Pss isolates showed amplification at 69 bp. In this study, cell sensitivity limit was 2 cells and the sensitivity limit on DNA level was determined as 11,8 pg. Quantitative Real-Time Membrane Bio-PCR method was used to detect only alive Pseudomonas savastanoi pv. savastanoi in the epiphytic population and quantitatively determine the population density. By this method, the epiphytic population rate of Pss on olive trees could be determined. The method allowed the detection of the rate of change in the epiphytic populations by applying in different regions or in the same region at different times. In conclusion, Quantitative Real-Time Membrane Bio-PCR method using LNA probes developed in this study, can be used for rapid (24 min), sensitive and quantitatively identification and detection of Pseudomonas savastanoi pv. savastanoi strains from direct bacterial cell and diseased olive plant and also can be used for determination of epiphytic population and only alive bacteria within this population.