Development and evaluation of a real-time PCR assay targeting chromosomal DNA of Erwinia amylovora

Abstract

To develop and evaluate a new and reliable real-time PCR detection protocol on chromosomal DNA of the contagious plant pathogenic bacterium Erwinia amylovora, the causal agent of fire blight. A Taqman minor-groove-binder real-time PCR assay targeting a hypothetical protein coding gene of Erw. amylovora has been developed. Colony PCR of 113 bacterial strains from different taxa was performed to prove specificity. Serial decimal dilutions of Erw. amylovora showed a consistent detection sensitivity of 2 bacterial units per microl. All strains of Erw. amylovora could be identified, and there were no cross-reactions with matrices or other bacteria also testing naturally contaminated samples. Rapid, reliable and sensitive detection of Erw. amylovora is important to avoid the spread of the disease within orchards, and the distribution by contaminated plant material or vectors carrying the pathogen. The selected conserved target gene allows relative quantitative detection of Erw. amylovora from different sources and host taxa. The newly developed protocol also enables the detection of recently found natural strains that lack the species-specific plasmid pEA29, which was so far widely used as target for detection and identification of this plant pathogen by PCR. This study demonstrates that the newly developed and evaluated real-time assay can specifically be used for identifying all known strains of the EU quarantine plant pathogen Erw. amylovora. Low concentrations of the bacteria can be detected and relatively quantified using a different target area than other real-time PCRs designed so far.