Validation of qPCR assays for the detection of citrus canker

Citrus canker, a serious bacterial disease affecting the citrus industry worldwide, is caused by Xanthomonas citri subsp. citri (Xcc) pathotypes A, A* and Aw, and to a lesser extent by X. fuscans subsp. aurantifolii (Xfa). The recent citrus canker outbreak in Australia has emphasised the need to re-evaluate the efficiency of molecular assays used for detecting citrus canker bacteria. Two published probe-based qPCR assays targeting the pth and lrp genes were tested for Xcc, whereas a SYBR Greenbased qPCR assay was tested for Xfa. The Xcc pth gene and Xfa qPCR assays were shown to be specific towards all pathotypes of Xcc and Xfa, respectively. The detection limit for both assays were 1 pg of genomic DNA or 103 CFU in bacteria-spiked leaf sample. The Xcc lrp gene qPCR assay was able to discriminate Xcc pathotypes with a detection limit of 1 ng of genomic DNA or 106 CFU in bacteriaspiked leaf sample, but this assay showed cross-reaction with Xfa. To allow rapid high-throughput detection of all Xcc pathotypes, a duplex probe-based qPCR assay was developed by incorporating COX primers as an internal control for plant DNA into the pth gene qPCR assay.