Detection of Xylophilus ampelinus in grapevine cuttings using a nested polymerase chain reaction

A sensitive and specific assay was developed to detect bacterial blight of grapevine caused by Xylophilus ampelinus (Panagopoulos, 1969) comb. nov. in grapevine cuttings. The 16S?23S rDNA intergenic spacer region of X. ampelinus was sequenced and pathogen-specific primers were designed from a region in the spacer between the tRNA (Ala) and the 23S genes. A nested PCR (n-PCR) reaction was applied with a first-stage PCR using universal primers within the ends of the 16S and 23S genes, followed by a second-stage PCR with nested primers specific to the X. ampelinus spacer region. A 277-bp fragment was amplified from 38 Xylophilus strains tested, but not from saprophytes associated with grapevine or phylogenetically related phytobacteria. The 277-bp product was shown to be derived from the X. ampelinus spacer region by restriction with Dra I, Sau 3AI, Taq I and Msp I, Southern hybridization and genomic DNA dot blots. When the (n-PCR) procedure was applied in the absence of nontarget DNA, the limit of detection was less than 10 colony-forming units (CFU) per µ L. The same number of  X. ampelinus CFU could be detected in the presence of 1·5 × 10⁵ CFU  µ L^?1 of Erwinia herbicola cells using the n-PCR procedure.