Identification and detection of Phoma tracheiphila, causal agent of citrus Mal secco disease, by real-time polymerase chain reaction

Phoma tracheiphila is the causal agent of a tracheomycotic disease of citrus called mal seccocausing the dieback of twigs and branches. This pathogen is of quarantine concern; therefore,fast and reliable protocols are required to detect it promptly. A specific primer pair and a duallabeledfluorogenic probe were used in a real-time polymerase chain reaction (PCR) with theCepheid Smart Cycler II System (Transportable Device TD configuration) to detect this fungusin citrus samples. Real-time PCR assay was compared to modified conventional PCR assay. Thesensitivity of the former was evaluated by testing P. tracheiphila DNA dilutions, and the minimumamount detectable was about 500 fg, whereas the linear quantification range was within100 ng to 1 pg. Conventional PCR sensitivity was 10 pg. Conventional and real-time PCR successfullydetected the fungus in woody samples of naturally infected lemon and artificially inoculatedsour orange seedlings. Nevertheless, real-time PCR was about 10- to 20-fold moresensitive than conventional PCR, and preliminary results indicate that the former techniqueachieves quantitative monitoring of the fungus in tissues. Simple and rapid procedures to obtainsuitable DNA samples from fungal cultures and citrus woody samples for PCR assays enablediagnosis to be completed in a short time.