Rapid Identification of a Second Outbreak of Asiatic Citrus Canker in the Northern Territory Using the Polymerase Chain Reaction and Genomic Fingerprinting.

Abstract

In May 1993, symptoms of asiatic citrus canker were found on West Indian lime, lemon and grapefruit trees in a mixed citrus orchard at Lambell’s Lagoon, near Darwin, Northern Territory, close to the site of a previous (1991) canker outbreak. Symptoms occurred on the spring growth flush at the start of the wet season (October 1992). The time lag between the appearance of symptoms and collection of material made it difficult to isolate the causal agent directly from the lesions, largely due to the overgrowth of secondary organisms. To overcome this difficulty we used a method for detecting Xanthomonas campestris pv. citri based on the polymerase chain reaction. A primer pair, known to amplify only DNA from group A of X. c. citri, directed the amplification of a DNA fragment of the expected size (222 base pairs, bp) from crude exudates prepared from leaf or fruit lesions, from mixed cultures, from inoculated citrus leaves and from positive control DNAs prepared from reference cultures of X. c. citri. A second primer pair and a duplex PCR were then tested, these also generated products of the expected sizes, and hence a presumptive diagnosis of asiatic citrus canker was made. We believe that this is the first use of PCR technology to diagnose a field outbreak of citrus canker. The total time from specimen preparation to detection of PCR products was less than 7 h. Colonies resembling X. citri were eventually recovered from only 2 of 11 symptomatic samples. A sensitive genomic fingerprinting technique provided strong evidence that the 1991 outbreak was the source of the current infestation.