Identification and detection of rhizobium vitis casual agent of crown gall disease of grapevine (Vitis vinifera L.) by real-time PCR using LNA probe

In this study, a Real-Time PCR method using LNA (Locked Nucleic Acid) probe which was sensitive and selective were developed for identification and detection of Rhizobium vitis, a causal agent of Crown Gall Disease of grapevine from both bacterial cells and diseased plant material. The 475 bp of ocs (octopine synthase) gene belong to Rhizobium vitis strains catabolising octopine, and 394 bp of nos (nopaline synthase) gene belong to Rhizobium vitis strains catabolising nopaline were amplified by conventional PCR. The amplified PCR product were sequenced in order to develop primer and probe sets for Real-Time PCR. Spesific primer and probe sets for Rhizobium vitis strains catabolising vitopine were developed from the gene sequences of vis (vitopine synthase), vitopine iaaM (indole acetic acid synthase) and vitopine virD2 genes from the GenBank. Different Rhizobium vitis strains, other plant pathogenic bacteria from different genus and species, vine total genomic DNA and basal material and tumor tissue were tested in order to determine the sensitivity of primer sets and probes. Although the DNA fragment amplified from the Rhizobium vitis strains catabolising octopine and nopaline were 62 and 78 base pairs respectively, no amplified product was detected from the other bacterial pathogens and vine genomic DNA. The primer and probe sets developed in this Real-Time PCR method for the detection and identification of Rhizobium vitis strains catabolising octopine and nopaline were identified within 20-25 minutes. The sensitivity of detection limit by the developed Real-Time PCR methods for octopine and nopaline strains was 1 bacterial cell. The sensitivity limit of DNA level was 10 pg. The pathogenic bacterium obtained from bleeding sap of basal material and tumor tissues were identified and detected. In conclusion, Real-Time PCR method using primer and probe sets specific for each of Rhizobium vitis strains catabolising octopine and nopaline were sensitive and quick for identification and detection of Rhizobium vitis from bacterial cell, bleeding sap of grapevine, tumor tissues from grapevine and tomato