PCR-BASED DETECTION OF ARTIFICIAL LATENT INFECTIONS OF GERANIUM BY XANTHOMONAS-CAMPESTRIS PV. PELARGONII

A procedure entailing biological enrichment and PCR amplification was developed to detect small populations of Xanthomonas campestris pv. pe largonii (X.c. pv. pelargonii) in tissues of geranium. Known numbers o f colony forming units (CFU) of X.c. pv. pelargonii were introduced in to 'Red Elite' geraniums through wounding of petioles and stems. Immed iately after inoculation, sections of the petioles and stems were harv ested and incubated for 24 or 48 h in nutrient broth (biological enric hment). After enrichment, bacterial cells were collected by centrifuga tion, followed by rapid extraction of total nucleic acid from the cell s with GeneReleaser(TM) PCR amplification of DNA with pathovar-specifi c primers, and ethidium bromide-stained agarose gel electrophoretic an alysis of the PCR products. After 48 h biological enrichment, it was p ossible to detect as few as 1 CFU of X.c. pv. pelargonii in stems and petioles collected immediately after inoculation, with the detection l imit ranging between 1 and 120 CFU during multiple experiments. It als o was possible to detect systemic movement of the bacterium in intact plants sampled 24 h after inoculation with a minimal inoculum (4 CFU). This procedure may have application in geranium certification program s concerned with the detection of latent infections associated with lo w levels of X.c. pv. pelargonii.