Real-time PCR for detection and quantification of Xanthomonas axonopodis pv. dieffenbachiae on Anthurium

Effective control of Xanthomonas axonopodis pv. dieffenbachiae, the causal agent of anthurium bacterial blight, requires efficient screening of quarantine plants against this pathogen. A reproducible real-time PCR method that targets a portion of a gene from the LPS cluster, encoding a putative ABC transporter, was developed to detect X. axonopodis pv. dieffenbachiae in anthurium samples. The specificity of the assay was confirmed with a panel of strains belonging to X. axonopodis pv dieffenbachiae and other pathovars and species. The assay was conducted with serial 10-fold dilutions of purified bacterial DNA and pure cultures (12 strains tested). It was possible to construct standard curves with a high correlation coefficient (r2 = 0.99) in the range of 15 ng to 1.5 pg for purified DNA and 107 to 102 CFU/ml for bacterial cultures. DNA extraction methods were compared for maximum DNA yield from anthurium plants. Homogenization of tissues with a Homex grinder (Bioreba) followed by purification using the DNeasy blood and tissue kit (Qiagen) was the most efficient method. The assay was successfully used to monitor the in vivo infection of anthurium experimentally inoculated with X. axonopodis pv. dieffenbachiae. This Real-Time PCR assay provides a rapid method for the early detection of the bacterium in symptomless anthurium tissues. It should therefore be a very useful diagnostic tool for indexing propagation material in nurseries and for surveillance of international movement of X. axonopodis pv. dieffenbachiae on anthurium. (Texte intégral