Xanthomonas citri subsp citri is the causal agent of citrus canker, a serious disease of citrus found in over 30 countries that is currently exotic to Australia. Several incursions have been recorded in Australia, the most recent in 2004, with the eradication campaign costing ~$17.6 million. Early and accurate detection of the pathogen increases the likelihood of a successful eradication.
The symptoms of mature citrus canker are quite distinct, though in the early stages they can be easily confused with the fungal disease citrus scab. It is at this stage that an effective molecular diagnostic tool is most effective. Within the Australian context, there are a number of endemic Xanthomonads, including the closely related Xanthomonas citri subsp. malvacearum which also carries the pthA region and are capable of generating false positive results with the Cubero & Graham 2002 protocol. Previously, a false positive occurred when these primers yielded a band for a Xanthomonas citri subsp malvacearum present on a citrus leaf which was tested due to the presence of an early scab lesion.
Whole-genome sequencing technology has been used to sequence numerous Xanthomonads (176 for this analysis); including 29 Xanthomonas citri subsp. citri from previous incursions and interceptions, 13 X. citri subsp. maniferaeindicae, 67 X. citri subsp. malvacearum and 67 endemic Xanthomonads. Comparative genomic analyses of 176 Xanthomonads identified unique regions of X. citri subps. citri for diagnostic targeting. There was approximately 2Mbp of a 5-6Mbp genome total difference between the X. citri subsp. citri and X. citri subsp. malvacearum. This comprised of 58 unique regions, which were greater than 50bp, for which 38 primer sets were designed and 16 were successful at identifying X. citri subsp. citri from the other Xanthomonads. These same regions have now been used for LAMP (loop-mediated isothermal amplification) assay development.