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

Balmas, Virgilio and Scherm, Barbara and Ghignone, Stefano and Salem, Ali Ould Mohamed and Cacciola, Santa Olga and Migheli, Quirico (2005) Characterisation of Phoma tracheiphila by RAPD-PCR, microsatellite-primed PCR and ITS rDNA sequencing and development of specific primers for in planta PCR detection. European Journal of Plant Pathology, Vol. 111 (3), p. 235-247. eISSN 1573-8469. Article.

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DOI: 10.1007/s10658-004-4173-x

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 amplification 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.

Item Type:Article
ID Code:498
Status:Published
Refereed:Yes
Uncontrolled Keywords:Citrus, lemon, mal secco disease, molecular diagnostics, nuclear rRNA genes, population genetics, polymerase chain reaction
Subjects:Area 07 - Scienze agrarie e veterinarie > AGR/12 Patologia vegetale
Divisions:001 Università di Sassari > 02 Centri > Centro di eccellenza interdisc. sviluppo ricerca biotecnologica e studio biodiversità della Sardegna e dell'area mediterranea
001 Università di Sassari > 01 Dipartimenti > Protezione delle piante
Publisher:Springer Netherlands
eISSN:1573-8469
Deposited On:18 Aug 2009 10:02

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