RAPID AND SENSITIVE COLORIMETRIC DETECTION OF XANTHOMONAS-AXONOPODIS PV CITRI BY IMMUNOCAPTURE AND A NESTED-POLYMERASE CHAIN-REACTION ASSAY

We have developed a sensitive and specific assay for Xanthomonas axono podis pv. citri, the causal agent of citrus bacterial canker. The assa y is based on sequential nested amplification by polymerase chain reac tion (PCR) of a region of plasmid DNA that is very highly conserved in X. axonopodis pv. citri. Specific amplification products were observe d in reactions containing three or fewer target molecules, an improvem ent of 50- to 100-fold over single-stage PCR, and similar results were observed when beginning with purified DNA or living bacterial cells. Colorimetric detection of amplification products was performed with th e DIANA (detection of immobilized amplified nucleic acids) method, whi ch uses labeled primers to allow amplification product capture and det ection in a microtiter plate. Predicted amplification products were pr oduced from all strains of X. axonopodis pv. cirri and from four of si x strains of X. axonopodis pv. aurantifolii but not from other xanthom onads, including citrus epiphytes, except for X. axonopodis pv. vignic ola and one strain isolated from Feronia elephantiacum, consistent wit h previous hybridization results. No amplification products were obser ved from strains of X. axonopodis pv. citrumelo that incite citrus bac terial spot disease in Florida citrus nurseries. Amplification was com pletely inhibited by copper hydroxide when present in the reaction mix at 13.6 mu g/ml. Concentrated leaf extracts from tangelo and mandarin orange, but not similar extracts from other citrus varieties, also in hibited amplification. Immunomagnetic separation of target bacteria pr ior to amplification was used to concentrate and recover X. axonopodis pv. citri from samples containing compounds that inhibit amplificatio n (i.e., copper and concentrated citrus extracts). Immunocapture, by c oncentrating target bacteria from dilute plant extracts, improved the sensitivity of the assay by 100-fold over nested-PCR alone. The combin ation of sensitivity, specificity, and speed of the assay could make t his a widely used assay both in plant quarantine and in areas where X. axonopodis pv. citri is endemic and clean planting stock programs are to be initiated.