Invasive Species Compendium

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Abstract

Identification of the fire blight pathogen, Erwinia amylovora, by PCR assays with chromosomal DNA.

Abstract

E. amylovora, the causal agent of fire blight, was identified independently from the common plasmid pEA29 by 3 different PCR assays with chromosomal DNA. PCR with 2 primers was performed with isolated 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 exopolysaccharide synthesis. The amplified fragment of 1.6 kb was analysed and the sequence was found to be identical for 2 E. amylovora strains. The identity of the PCR products was further confirmed by restriction analysis. The 1.6 kb signal was also used for detection of E. amylovora in the presence of other plant-associated bacteria and 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 restriction enzyme HaeIII. The pattern obtained for E. amylovora was different from that of other bacteria. The sequence of the 16S rRNA gene was determined from a cloned fragment and was closely related to the sequences of Escherichia coli and other Erwinia species. Arbitrarily primed PCR with a 17-mer oligonucleotide derived from the sequence of transposon Tn5 produced a unique banding pattern for all E. amylovora strains investigated. These methods expand identification methods for E. amylovora, which include DNA hybridization and a PCR technique based on plasmid pEA29.