Characterisation of Phoma tracheiphila by RAPD-PCR, microsatellite-primed PCR and ITS rDNA sequencing and development of specific primers for in planta PCR detection

Thirty six isolates of Phoma tracheiphila from Italy, the causal agent of the ‘‘mal secco’’ disease on Citrusspecies, were characterised by different molecular tools in comparison with representative isolates of otherphytopathogenic Phoma species. These included analysis of the distribution of RAPD and microsatellitemarkers and sequencing of the internal transcribed spacer (ITS) region of the nuclear rRNA genes. Theresults obtained with 12 RAPD primers (92 markers) and 7 microsatellite primers (56 markers) suggest thatItalian isolates of P. tracheiphila are genetically homogeneous, leading to identical patterns upon amplificationwith all the tested primers. Accordingly, ITSI-5.8S-ITS2 sequences were highly conserved(98–100% identity along a 544-characters alignment) among all the isolates of P. tracheiphila. A neighborjoininganalysis of ITS sequences of P. tracheiphila in comparison with those of other Phoma species, as wellas with alignable sequences from anamorphic and teleomorphic taxa retrieved in BLAST searches, revealeda close relationship between P. tracheiphila and Leptosphaeria congesta. A pair of P. tracheiphila-specificprimers was designed on the consensus sequence (555 residues) obtained from the alignment of the newlygenerated P. tracheiphila ITS sequences. A PCR-based specific assay coupled to electrophoretic separationof amplicons made it possible to detect P. tracheiphila in naturally infected Citrus wood tissue collectedfrom both symptomatic and symptomless plants. The limit of detection was 10 pg of genomic DNA and5 fg of the ITS target sequence