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A multiplex PCR assay for Xanthomonas citri subsp. citri identification and pathotype determination

Vessela Mavrodieva: USDA APHIS PPQ S&T CPHST

<div>Asiatic citrus canker is a disease caused by the bacterial plant pathogen <em>Xanthomonas citri </em>subsp. <em>citri (Xanthomonas citri </em>pv. <em>citri; Xanthomonas axonopodis </em>pv.<em> citri</em> (<em>Xac))</em>. It causes distinctive necrotic raised lesions and severe infection may cause defoliation, twig dieback and general tree decline. This disease has both national and international trade implications. Three main citrus canker pathotypes have been identified namely A, A* and A<sup>w</sup>, which vary in their host range. Currently, there is no simple molecular diagnostic assay to differentiate them. To address this deficiency, a multiplex PCR assay was developed that confirms citrus canker and determines the pathotype of a sample. PCR primers were designed to target pathotype-specific genomic regions identified after aligning whole genome sequences of representative subspecies. Primers were designed in Geneious® and evaluated <em>in silico </em>using Primer-BLAST to check cross-reactivity with non-targets. <em>In silico</em> analysis to evaluate the likelihood of cross-reactions among the pathotype-specific primer sets as well as with the general <em>Xanthomonas citri</em> subsp. <em>citri</em> primers was also performed. The multiplex assay correctly identified the pathotype of 59 out of 62 isolated citrus canker strains from various hosts and geographic locations. This method allows for the confirmation of citrus canker and the identification of pathotype in a single reaction, which saves time and money in the event of an outbreak.</div>