Validation of a molecular method for the diagnosis of malsecco disease of citrus

Mal secco disease, caused by the mitosporic fungus Phoma tracheiphila, is a destructive vascular disease of lemon (Citrus limon) in the Mediterranean region. P. tracheiphila is a quarantine pathogen on the list of most regional plant protection organizations including the European and Mediterranean Plant Protection Organization (EPPO). According to the standard diagnostic protocol of EPPO, P. tracheiphila can be identified by both conventional and molecular methods. In 2005, a PCR-based assay for P. tracheiphila was published and a pair of specific primers (Pt-FOR2 and Pl-REV2) were designed on the consensus sequence obtained from the alignment of the internal transcribed spacer (ITS) region of the nuclear rRNA genes. This PCR-based specific assay made it possible to detect the fungus in infected tissues of both symptomatic and symptomless citrus plants. In the last three years, this molecular method has been used to test the susceptibility of various lemon cultivars and hybrids to artificial infections with P. tracheiphila. The susceptibility to malsecco of stem-inoculated lemon accessions as evaluated on the basis of symptom severity correlated with the colonization rate of the xylem by the pathogen and the results of PCR assay were consistent with conventional isolations on artificial media. However, the molecular method proved faster and sensitive than conventional isolation. In summer, symptomatic plants recovered and the pathogen was not detected in new vegetation flushes by both diagnostic methods, confirming that high temperature inhibits xylem colonization by the fungus.