IDENTIFICATION OF THE FIRE BLIGHT PATHOGEN, ERWINIA-AMYLOVORA, BY PCRASSAYS WITH CHROMOSOMAL DNA

Erwinia amylovora, the causative agent of fire blight, was identified independently from the common plasmid pEA29 by three different PCR ass ays with chromosomal DNA. PCR with two primers was performed with isol ated DNA and with whole cells, which were directly added to the assay mixture. The oligonucleotide primers were derived from the ams region, and the PCR product comprised the amsB gene, which is involved in exo polysaccharide synthesis. The amplified fragment of 1.6 kb was analyze d, and the sequence was found to be identical for two E. amylovora str ains. The identity of the PCR products was further confirmed by restri ction analysis. The 1.6-kb signal was also used for detection of the f ire blight pathogen in the presence of other plant-associated bacteria acid in infected plant tissue. For further identification of isolated strains, the 16S rRNA gene of E. amylovora and other plant-associated bacteria was amplified and the products were digested with the restri ction enzyme HaeIII. The pattern obtained for E. amylovora was differe nt from that of other bacteria. The sequence of the 16S rRNA gene was determined from a cloned fragment and was found to be closely related to the sequences of Escherichia coli and other Erwinia species. Finall y, arbitrarily primed PCR with a 17-mer oligonucleotide derived from t he sequence of transposon Tn5 produced a unique banding pattern for al l E. amylovora strains investigated. These methods expand identificati on methods for E. amylovora, which include DNA hybridization and a PCR technique based on plasmid pEA29.