EVALUATION OF A DIGOXYGENIN-LABELED PHASEOLOTOXIN GENE FRAGMENT AS A DNA-PROBE FOR DETECTION OF PSEUDOMONAS-SYRINGAE PV PHASEOLICOLA FROM BEAN-SEEDS

Several EcoR1 fragments in the DNA region of Pseudomonas syringae pv. phaseolicola controlling phaseolotoxin production were cloned into the bacteriophage M13. One of them, pGi63, was labelled by incorporating digoxygenin dUTP and was tested for its ability to serve as a diagnost ic probe. This probe showed high specificity for the 27 strains of P. s. pv. phaseolicola tested. All 23 other non-target strains gave no si gnal. By dot blot hybridization the non-radioactive probe resulted in sensitivity equal to that of a P-32-labelled probe. Two substrates for alkaline phophatase were tested. The luminescent substrat AMPPD(R) ga ve a more intense signal than the colorimetric complex (X-phosphate/NB T) and was the only one able to directly detect the pathogen in seed s oak liquid. However, this technique is not sensitive enough to be used in routine seed assays. The detection limit was 5 x 10(5) cells per d ot when bacteria were suspended in water and seed soak liquid of bean cv. 'Michelet'. The sensitivity was ten fold less (5 x 10(6)) for bact eria suspended in seed soak liquid of pigmented cultivars 'Vernel', 'C oco Rubico', 'Primel', 'Delinel'. However, the digoxygonin-labelled pr obe was reliable for confirmining the identification of colonies after plating assays.