Detection ofXanthomonas campestrispv.vesicatoriaAssociated with Pepper and Tomato Seed by DNA Amplification

Abstract

Detection of Xanthomonas campestris pv. vesicatoria associated with pepper and tomato seed was achieved by amplification of a DNA fragment of the bacterium by the polymerase chain reaction. Oligonucleotide primers specific for the hypersensitive reaction and pathogenicity (hrp) gene cluster were used in the reaction. The method includes extraction of total DNA from buffered seed washings to which sodium ascorbate and insoluble polyvinylpolypyrrolidone were added. The hrp fragments were amplified from the DNA preparations if cells of X. c. pv. vesicatoria were added to seed washes. Identification of an hrp fragment as that from X. c. pv. vesicatoria was attained by restriction enzyme analyses of the amplified fragment. The minimum number of cells that could be detected in washes from pepper or tomato seed was from 10 2 to 10 3 cfu per ml. This was about 1,000 times fewer than the minimum number of cells detected with an enzyme-linked immunosorbent assay. The pathogen was also detected in washes obtained from several lots of naturally contaminated pepper and tomato seed, one of which contained background bacterial microflora greater than 10 7 cfu per g of seed.