Characterisation of Phoma tracheiphila by RAPD-PCR, microsatellite-primed PCR and ITS rDNA sequencing and development of specific primers for in planta PCR detection Virgilio Balmas 1 , Barbara Scherm 2 , Stefano Ghignone 2 , Ali Ould Mohamed Salem 3 , Santa Olga Cacciola 4 and Quirico Migheli 1, * 1 Department of Plant Protection, Center for Biotechnology Development and Biodiversity Research, University of Sassari, Via E. De Nicola 9, 07100 Sassari, Italy; *Author for correspondence (Fax: +39 079 229316; E-mail: qmigheli@uniss.it); 2 Department of Plant Biology and IPP-CNR, University of Turin, V.le P.A. Mattioli 25, I-10125 Torino, Italy; 3 Department of Biology, University of Nouakchott, B.P. 5026 Nouakchott, Mauritania; 4 Department S.En.Fi.Mi.Zo., University of Palermo, Viale delle Scienze 2, 90128 Palermo, Italy Accepted 21 September 2004 Key words: Citrus, lemon, ‘‘mal secco’’ disease, molecular diagnostics, nuclear rRNA genes, population genetics, polymerase chain reaction Abstract Thirty six isolates of Phoma tracheiphila from Italy, the causal agent of the ‘‘mal secco’’ disease on Citrus species, were characterised by different molecular tools in comparison with representative isolates of other phytopathogenic Phoma species. These included analysis of the distribution of RAPD and microsatellite markers and sequencing of the internal transcribed spacer (ITS) region of the nuclear rRNA genes. The results obtained with 12 RAPD primers (92 markers) and 7 microsatellite primers (56 markers) suggest that Italian isolates of P. tracheiphila are genetically homogeneous, leading to identical patterns upon ampli- fication with 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 neighbor- joining analysis of ITS sequences of P. tracheiphila in comparison with those of other Phoma species, as well as with alignable sequences from anamorphic and teleomorphic taxa retrieved in BLAST searches, revealed a close relationship between P. tracheiphila and Leptosphaeria congesta. A pair of P. tracheiphila-specific primers was designed on the consensus sequence (555 residues) obtained from the alignment of the newly generated P. tracheiphila ITS sequences. A PCR-based specific assay coupled to electrophoretic separation of amplicons made it possible to detect P. tracheiphila in naturally infected Citrus wood tissue collected from both symptomatic and symptomless plants. The limit of detection was 10 pg of genomic DNA and 5 fg of the ITS target sequence. Introduction The mitosporic fungus Phoma traccheiphila (Petri) Kantschaveli et Gikachvili is the incitant of ‘‘mal secco’’, a serious disease of lemon (Citrus limon) and other Citrus species throughout the Mediter- ranean region, including the Black Sea area, (Punithalingam and Holliday, 1973; Perrotta and Graniti, 1988). The pathogen penetrates the host via wounds, and possibly through stomata (Perrotta and Graniti, 1988). The fungus invades the xylem and causes impairment of the water- transport system of the plant, leading to wilting of branches and eventually plant death. Typical symptoms consist of vein chlorosis, shedding of leaves and die-back of twigs and branches and a European Journal of Plant Pathology (2005) 111: 235–247 Ó 2005 Springer