one step PCR-based method for the detection of economically important soft rot Erwinia species on micropropagated potato plants

A simple, rapid and sensitive PCR-based method was developed for the detection of all five subspecies of Erwinia carotovora, including subsp. carotovora and subsp. atroseptica, and all pathovars/biovars of Erwinia chrysanthemi, on plant tissue culture material. Primers SR3F and SR1cR, based on a conserved region of the 16S rRNA gene, amplified a DNA fragment of 119 bp from all 65 such strains tested. Detection limits of the method in vitro were 2(.)0 x 10(2)-3(.)4 x 10(3) cfu ml(-1) (equivalent to 1-17 cfu per PCR) and, following extraction of genomic DNA from plant extract, detection limits were 2(.)3 x 10(2)-1(.)9 x 10(4) cfu per microplant sample (equivalent to 5 cfu - 3(.)8 x 10(2) cfu per PCR). To improve the sensitivity of the method in planta, to obviate the need for complex and laborious DNA extractions, and to remove inhibitory substances present in the plant extract, an enrichment step was included prior to PCR. Following enrichment, the sensitivity of detection was < 10 cfu per microplant sample. This method provides the first sensitive means of detecting latent infection caused by several economically important soft rot erwinias simultaneously on potato tissue culture material.