RAPID IDENTIFICATION OF A 2ND OUTBREAK OF ASIATIC CITRUS CANKER IN THE NORTHERN-TERRITORY USING THE POLYMERASE CHAIN-REACTION AND GENOMIC FINGERPRINTING

In May 1993, symptoms of asiatic citrus canker were found on West Indi an lime, lemon and grapefruit trees in a mixed citrus orchard at Lambe ll's Lagoon, near Darwin, Northern Territory, close to the site of a p revious (1991) canker outbreak. Symptoms occurred on the spring growth flush at the start of the wet season (October 1992). The time lag bet ween the appearance of symptoms and collection of material made it dif ficult to isolate the causal agent directly from the lesions, largely due to the overgrowth of secondary organisms. To overcome this difficu lty we used a method for detecting Xanthomonas campestris pv. citri ba sed 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 exud ates prepared from leaf or fruit lesions, from mixed cultures, from in oculated citrus leaves and from positive control DNAs prepared from re ference 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 preparat ion to detection of PCR products was less than 7 h. Colonies resemblin g X. citri were eventually recovered from only 2 of 11 symptomatic sam ples. A sensitive genomic fingerprinting technique provided strong evi dence that the 1991 outbreak was the source of the current infestation .