Recombinase Polymerase Amplification Assay for in Field Detection of Tomato Bacterial Spot (Xanthomonas euvesicatoria, X. gardneri, and X. perforans)
Amanda Strayer: University of Florida, Department of Plant Pathology
<div>Bacterial spot, one of the most detrimental diseases of tomato that occurs worldwide, is caused by <em>Xanthomonas </em><em>euvesicatoria</em> (Xe), <em>X. gardneri </em>(Xg), <em>X. perforans</em> (Xp), and <em>X. vesicatoria </em>(Xv). Currently, there is no in-field diagnostic assay available for bacterial spot. In this study, we developed two recombinase polymerase amplification (RPA) assays that can detect Xe, Xg, and Xp in crude plant samples in the field using a portable detection instrument. Three RPA exo probes and two primer sets were designed to amplify regions of the <em>hrpB</em> gene of Xe, Xg, and Xp. The Xg RPA assay amplified DNA extracted from 18 pure cultures of Xg and 24 crude plant samples infected with Xg. The Xe RPA assay amplified DNA extracted from 36 pure cultures of Xe and Xp, and 63 crude plant samples infected with either Xe or Xp. The Xp RPA assay only amplified DNA extracted from 18 pure cultures of Xp. Thus, the Xe RPA assay can be used to diagnose bacterial spot caused by either Xe or Xp in the field, which can be differentiated using the Xp RPA assay following isolation in the laboratory. Although an additional RPA exo probe and primer set was designed to detect Xv, it was unable to amplify DNA extracted from pure cultures of Xv. Since Xv is becoming less prevalent worldwide, it could be considered as the least important bacterial spot pathogen. Since the Xe and Xg RPA assays detected Xe, Xg, and Xp in crude plant samples, it holds great potential as an in-field diagnostic tool.</div>
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