Assessment of subtractive hybridization to select species and subspecies specific DNA fragments for the identification of Xylophilus ampelinus by polymerase chain reaction (PCR)

Eighteen Bsp143I digested DNA fragments specific to Xylophilus ampelinus we re cloned from a library enriched for X. ampelinus obtained after a subtrac tive hybridization step. It was also possible to clone specific DNA sequenc es directly after DNA digestion with Bsp143I probably because X. ampelinus is a unique bacterium. Nucleotidic sequences of four cloned specific fragme nts were determined. They did not share any homology with other DNA sequenc es in the EMBL/GeneBank database. Four primer sets were designed and tested for specificity to X. ampelinus. One primer set (Xamp 1.27) was a good can didate for a species-specific reagent in a procedure of identification of X . ampelinus using PCR. One primer set detected only Greek strains isolated from Vitis vinifera cv. Sultana. Genetic diversity within the X. ampelinus species can be used in further epidemiological studies on the bacterial nec rosis of grapevine.