Molecular Diagnostic Tools for the Detection and Characterization of Phoma tracheiphila

In the recent years PCR-based techniques for the identification and detection of Phoma tracheiphila, the causal agent of citrus mal secco disease, have been evaluated aiming to provide tools for biological and epidemiological studies. A wide collection of P. tracheiphila strains was used to evaluate and validate diagnostic protocols and a fAFLP method for fungal characterization. Conventional and real-time PCR protocols were successfully tested for the specific identification of P. tracheiphila and its detection in planta. A further improvement of the real-time PCR protocol and the DNA extraction methods allowed the quantification of the fungus both from naturally infected and artificially inoculated citrus species as well from soil. The protocol was proved diagnostic flexible, rapid and more sensitive than any other (0.1 pg fungal DNA). Real-time PCR from leaf and stem samples taken at various time after inoculation allowed quantitative monitoring of P. tracheiphila spread in planta. Variable DNA concentrations were detected and quantified by real-time in naturally contaminated soil sampled beneath infected lemon trees in four different citrus groves from spring to autumn. In other experiments, representative P. tracheiphila isolates were used to develop a fluorescent-AFLP protocol to detect intraspecific variability. fAFLP were able to differentiate fungal isolates but no correlation with the geographic origin, isolate morphology or virulence was observed. To our knowledge, the protocol makes available a tool to discriminate isolates of P. tracheiphila, useful for epidemiological and population studies.