Rapid and accurate identification of Xanthomonas citri subspecies citri by fluorescence in situ hybridization

Citrus canker is an economically important disease caused by the bacterial pathogen Xanthomonas citri subsp. citri (Xcc). This organism targets a wide range of citrus plants, including sweet orange, grapefruit, lemon and lime. As Xcc is spread by environmental factors such as wind and rain, it is difficult to control its movement once the disease has established. In order to facilitate monitoring of citrus canker we sought to design a novel diagnostic protocol based on fluorescence in situ hybridization (FISH) for identification of bacterial cells directly from canker pustules without cultivation or DNA extraction. This method was validated for specificity against a range of Xanthomonas species and strains. We show that our assay is extremely rapid (typically requiring between 2 and 3 h), and possesses a similar specificity to existing PCR diagnostic tools. The sensitivity of the assay is comparable to that of an existing PCR-based technique and sufficient for identifying Xcc in symptomatic plant material. The method is easily transferable to diagnosticians without prior experience using FISH. SIGNIFICANCE AND IMPACT OF THE STUDY: Xanthomonas citri subsp. citri (Xcc) is an aggressive and hardy pathogen of citrus plants worldwide. Outbreaks are difficult and costly to contain and the establishment of citrus canker results in restricted trade. In order to extend the existing toolkit for identification of Xcc we developed a novel diagnostic approach based on fluorescence in situ hybridization. Our approach is of comparable specificity and sensitivity to existing methods but can be performed directly on infected tissue making it significantly faster than existing PCRs, and requiring fewer laboratory resources.