Abstract
Leaf scald disease (LSD) is caused by the Gram-negative bacterium, Xanthomonas albilineans. Genomic DNA from X. albilineans and Xanthomonas hyacinthi were analyzed by suppression subtractive hybridization (SSH) using X. albilineans as the tester from which unique sequences were sought and X. hyacinthi as the driver. Following the SSH procedure, amplification products within the size range of 100 - 600 bp were generated, purified, directly cloned with the Promega pGEM-T vector cloning kit, and transformed into ultracompetent Escherichia coli X L2-blue MRF’ cells (Stratagene, La Jolla, CA). Clones selected were sequenced (using a Perkin Elmer ABI PRISM Dye terminator cycle sequencing kit and ABI Model 377 DNA sequencer) in one direction with SP6 and T7 primers (Promega). Clone Xa 6 revealed very close homology with a probable bacterioferritin from Pseudomonas aeruginosa. Clone X. albilineans 12 showed 92% homology to the acetate repressor proteins and clone X. albilineans 18 displayed 85% homology to the plasmid pTOM9 from Alcaligenes xylosoxidans. Sequencing data also revealed homology to various hypothetical proteins.
Highlights
Leaf scald an insidious disease caused by the bacterium Xanthomonas albilineans colonizes the vascular system of sugarcane (Saccharum spp. hybrids) in either a chronic or acute phase
Genomic DNA from X. albilineans and X. hyacinthi were successfully digested by the restriction endonuclease Rsa1 (Figure 1)
It is appreciated that genomic variation within closely related groups of bacteria can be substantial, suggesting a need to define and study these differences (Sauerbaum and Achtman, 1999)
Summary
Leaf scald an insidious disease caused by the bacterium Xanthomonas albilineans colonizes the vascular system of sugarcane (Saccharum spp. hybrids) in either a chronic or acute phase. Numerous outbreaks have occurred throughout all regions of the world giving rise to concern that leaf scald could become a limiting economic factor in sugarcane production. This investigation focuses on the identification of sequences unique to X. albilineans using the SSH procedure. The availability of new genomic sequences in the last decade has created novel opportunities to analyze the organization of a genome’s regulatory machinery, the function of particular genes or gene clusters and the evolutionary relationships between different bacterial strains and species. The “pathogenicity islands” (PAIs) or multigene segments of virulent strains that tend to be absent from avirulent members of the same species and that help determine the nature and severity of disease are of special signific-
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