Plasmid-Based Hybridization Probes for Detection and Identification of Xanthomonas campestris pv. citri. John S. Hartung, U.S. Department of Agriculture, Agricultural Research Service, Plant Science Institute, Fruit Lab, Beltsville, MD 20705-2350. . Plant Dis. 76:889-893. Accepted for publication 24 February 1992. This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. The American Phytopathological Society, 1992. DOI: 10.1094/PD-76-0889.

Two hybridization probes were developed from plasmid DNA of pathotype A strain XC62 of Xanthomonas campestris pv. citri. Plasmid pFL62.42 was constructed by cloning a 4.2-kb BamHI fragment of an indigenous plasmid of strain XC62 in the vector pUC9. Plasmid pFL1 contains an internal 700-bp EcoRI fragment from pFL62.42; this fragment was also cloned in the vector pUC9. The plasmid probes were highly specific for X. c. citri. Both plasmids hybridized with DNA purified from 44 pathotype A strains of X. c. citri, which had been isolated from diseased Citrus spp. in 15 countries. A dot blot format with chemiluminescent detection by biotinylated probes was used for these experiments. Probe pFL62.42 was also used to detect 13 of 15 pathotype B, C, and D strains of X. c. citri, and probe pFL1 was used to detect 11 of 15 pathotype B, C, and D strains of X. c. citri. The limit of detection for pathotype A was between 2 and 7 ng of DNA per dot, and the limit of detection for pathotypes B, C, and D was about two- to fourfold higher. Neither plasmid hybridized with DNA from 56 strains of X. campestris associated with citrus bacterial spot disease. The probes also did not hybridize with DNA from four epiphytic strains of X. campestris isolated from Citrus foliage or with DNA from Flavobacterium balustinum, Enterobacter cloacae, Escherichia coli, Erwinia carotovora, E. amylovora, and Pseudomonas putida. When 16 other pathovars of X. campestris were screened by DNA dot blotting, limited cross reaction was observed with plasmid pFL62.42. Specificity of detection was improved with plasmid pFL1. Only DNA from X. c. vignicola and from a single strain of X. c. bilvae hybridized with plasmid pFL1. Probe pFL1 also specifically detected pathotype A of X. c. citri in extracts of leaf lesions. These probes will be useful in detecting X. c. citri.