Identification and sensitive endophytic detection of the fire blight pathogen Erwinia amylovora with 23S ribosomal DNA sequences and the polymerase chain reaction.
Two short sequences, situated in the bacterial 23S rDNA gene, were used as primers for the PCR detection of E. amylovora. All 34 E. amylovora strains tested, from different geographical and host plant origins and of different virulence, produced a 565-bp PCR fragment. E. amylovora was distinct from other phytobacteria, with which no PCR product was observed. Only Escherichia coli was cross-recognized by the production of a weaker PCR band of similar size to E. amylovora. In a fast PCR protocol, where 2 temp. were cycled, E. amylovora in pure culture was detected on gel at concn as low as 3×102 c.f.u/ml. This corresponded to a detection limit of 1.5 bacteria/PCR. Reliable PCR detection in woody host plant tissue was only obtained with PVP/PVPP-treated sample extracts. Using E. amylovora-spiked plant extracts and extracts of fruit tree shoots artificially infected with E. amylovora, the PCR detection sensitivity was 6.6×102 c.f.u/ml of extract. Starting from the plant samples, the PCR detection results were visualized on gels within 5 h.