Identification and detection by quantitative real-time PCR of erwinia amylovora, causal agent of the fire blight disease

Erwinia amylovora, causal agent of Fire Blight Disease which causes significant losses in pomaceous plants, was sensitively identified and detected in short time by Quantitative Real-Time PCR method using LNA probe. Probe and primers for identification of the bacterium by Real-Time PCR were designed from internal sequence of chromosomal 1.6 kb-ams gene which is responsible for the polysaccharide production of the pathogenic bacteria. 63 bp PCR product from genome of Erwinia amylovora was amplified by the primer and probe as designed. Different Erwinia amylovora strains and bacterial species were used to determine the specificity of the probe and primers. Although no amplication was detected from genome of different species of pathogenic bacteria, the 63 bp-amplifications were detected from different strains of E. amylovora. While sensitivity limit of the prob and primers from direct bacterial suspension was found to be 6 cfu/2 ? , the genomic DNA detection limit was 10 pg. Quantitative Real-Time PCR method using probe and primers developed in this study gave an oppurtunity for detection of epiphytic population of E. amylovora on diseased and healthy plant materials including leaf, shoot and flower in early stage.