DEVELOPMENT OF A MULTIPLEX PCR ASSAY FOR THE SIMULTANEOUS DETECTION OF CLAVIBACTER MICHIGANENSIS subsp MICHIGANENSIS, PSEUDOMONAS SYRINGAE pv. TOMATO AND XANTHOMONAS AXONOPODIS pv. VESICATORIA USING PURE CULTURES

A multiplex PCR assay for the simultaneous detection of three bacterial seed-borne pathogens of tomato was developed. Published primers: (i) CMM-5-CMM-6 for Clavibacter michiganensis pv. michiganensis; (ii) primer 1-primer 2 for Pseudomonas syringae pv. tomato; (iii) RST2-RST3 for Xanthomonas axonopodis pv. vesicatoria, were used in the assay. Annealing temperatures were determined by gradient PCR individually for each pathogen and primer concentration ratios were investigated. Sensitivity assays were carried out and compared with single PCR. Temperature of 59 +/- 1 degrees C was optimal for annealing. Optimal primer concentrations were determined as 0.36 mu Mol l(-1) for C. in. michiganensis, 0.30 mu Mol l(-1) for X. a. vesicatoria, and 0.12 mu Mol l(-1) for P s. tomato. Sensitivity assays showed that 3 CFU in 50 mu l sterile distilled water, derived from pure cultures of C. in. michiganensis, P s. tomato and X. a. vesicatoria could reliably be detected by multiplex PCR when applied to pure cultures. The detection limit was determined to be ca. 10 times lower than that of single PCR. Multiplex PCR provided less labor and rapid results for the detection of bacterial pathogens of tomato, but the sensitivity of detection was reduced. Thus, the sensitivity of this technique should be assayed prior to its use in place of single PCR.