ISSN:
1365-3059
Source:
Blackwell Publishing Journal Backfiles 1879-2005
Topics:
Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
Notes:
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 µL. The same number of X. ampelinus CFU could be detected in the presence of 1·5 × 105 CFU µL−1 of Erwinia herbicola cells using the n-PCR procedure.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1046/j.1365-3059.2001.00568.x
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