Development of a Polymerase Chain Reaction Protocol for Detection of <i>Xylella fastidiosa</i> in Plant Tissue

Abstract

A 7.4-kb <i>Eco</i>RI fragment of genomic DNA of <i>Xylella fastidiosa</i> strain PCE-RR (ATCC 35879) was used as a probe and was conserved in 18 strains of <i>Xylella</i>. The nucleotide sequence of a 1.0-kb internal <i>Eco</i>RV portion of the fragment was determined, and oligonucleotides were selected for primers that amplified genomic DNA specific to <i>X. fastidiosa</i> 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 <i>X. fastidiosa</i> 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 <i>Xylella</i> 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 <i>Rsa</i>I differentiated two pathotypes of <i>X. fastidiosa</i>.