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An Oligonucleotide Array for the Identification and Differentiation of Bacteria Pathogenic on Potato

First and third authors: Eastern Cereal and Oilseed Research Centre, Research Branch, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, Ontario K1A 0C6; and second author: Canadian Food Inspection Agency, Centre for Animal and Plant Health, 93 Mount Edward Road, Charlottetown, Prince Edward Island C1A 5T1

Oligonucleotides, 16 to 24 bases long, were selected from the 3′ end of the 16S gene and the 16S--23S intergenic spacer regions of bacteria pathogenic on potato, including Clavibacter michiganensis subsp. sepedonicus, Ralstonia solanacearum, and the pectolytic erwinias, including Erwinia carotovora subsp. atroseptica and carotovora and E. chrysanthemi. Oligonucleotides were designed and formatted into an array by pin spotting on nylon membranes. Genomic DNA from bacterial cultures was amplified by polymerase chain reaction using conserved ribosomal primers and labeled simultaneously with digoxigenin-dUTP. Hybridization of amplicons to the array and subsequent serological detection of digoxigenin label revealed different hybridization patterns that were distinct for each species and subspecies tested. Hybridization of amplicons generally was restricted to appropriate homologous oligonucleotides and cross-hybridization with heterologous oligonucleotides was rare. Hybridization patterns were recorded as separate gray values for each hybridized spot and revealed a consistent pattern for multiple strains of each species or subspecies isolated from diverse geographical regions. In preliminary tests, bacteria could be correctly identified and detected by hybridizing to the array amplicons from mixed cultures and inoculated potato tissue.

Additional keywords: blackleg, brown rot, macroarray, microarray, reverse dot blot, ring rot, soft rot.