Development of a Polymerase Chain Reaction Protocol for Detection of Xylella fastidiosa in Plant Tissue

A 7.4-kb EcoRI fragment of genomic DNA of Xylella fastidiosa strain PCE-RR (ATCC 35879) was used as a probe and was conserved in 18 strains of Xylella. The nucleotide sequence of a 1.0-kb internal EcoRV portion of the fragment was determined, and oligonucleotides were selected for primers that amplified genomic DNA specific to X. fastidiosa in 33 strains tested by the polymerase chain reaction (PCR). Plant extracts for PCR and enzyme-linked immunosorbent assay (ELISA) were obtained by maceration of grape petioles and by vacuum extraction of citrus stems. Known cell numbers of X. fastidiosa were added to the plant extracts contained in a succinate-citrate-phosphate buffer prior to assay. Amplification of DNA by PCR was inhibited in the presence of plant extracts unless sodium ascorbate and acid-washed polyvinylpyrrolidone were added to the extraction buffer. Detection of Xylella by PCR was 100-fold more sensitive than by ELISA; the limits of detection were 1 × 10² cfu/ml for PCR and 2 × 10⁴ cfu/ml for ELISA. Restriction endonuclease digestion of PCR amplification products with RsaI differentiated two pathotypes of X. fastidiosa.