A simplified protocol of subtractive hybridization based on the technique of L. M. Kunkel, A. P. Monaco, W. Middlesworth, H. D. Ochs & S. A. Latt (1985, , 82, 4778-4782) was used to obtain DNA sequences specific to isolated from diseased citrus plants. As a driver, DNA extracted from bacteria showing different degrees of relatedness was used: 788 isolated from another host (plum), pv. and strains. A DNA fragment, f14, showing no hybridization to the driver DNA, was used as a probe specific to from citrus and oleander. This fragment was sequenced and the predicted protein showed 40% similarity to the central region of flagellin of serotypes H1 and H12. A pair of internal primers (f14-1 and f14-2) was designed for amplification of DNA. These primers detected strains isolated from citrus and oleander and yielded an amplification product of about 600 bp. They were also able to detect the bacteria in extracts from citrus plants with or without symptoms of disease. No amplification reaction was obtained using DNA extracted from other species and pathovars of and phytopathogens of citrus ( pv. ) and coffee ( pv. ). The isolation of a DNA fragment specific to from citrus showed that the simplified protocol of subtractive hybridization used in this work is potentially applicable to other microorganisms.


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