Molecular Diagnostics of Biscogniauxia mediterranea, the Causal Agent of Charcoal Rot Disease on Oak Using Species-Specific Primers

Biscogniauxia mediterranea, the cause of charcoal rot disease on oak, is known as one of the main agents involved in oak decline in Iran. However, very little is known on the extent of distribution, host range and mechanisms of dispersal of the pathogen in infected areas as well as incidence of the disease in other regions of country. The present study was aimed to develop a diagnostic tool for molecular identification and detection of the causal agent of charcoal rot disease based on species-specific primer set using polymerase chain reaction. To this aim, samples were collected from oak trees with typical charcoal rot disease from Ilam province. The causal agent was isolated and purified using routine plant pathology methods. Biscogniauxia isolates were identified using a combination of morphological and sequence data from ITSrDNA region. Blast search analysis of the sequence data obtained in this study against the sequence data in GenBank confirmed the identity of the isolates as B. mediterranea.
Seqeunce data of ITS-rDNA region for all of Biscogniauxia were obtained from GenBank and aligned together with seqeunce data gnerated in this study. A pair of species-specific primer (BmF/BmR) with expcted amplicon size of 400 bp was designed for B. mediterranea. Efficacy of this primer set was tested and verified on DNA extracted from B. mediterranea and other fungal species isolated from oak tree tissues.The results of amplication profile showed that a 400 bp band was amplified only from B. mediterranea isolates. The species-specific primer set designed in this study can be used in monitoring programmes for the detection of the causal agent, the extent of distribution and mechanisms of dispersal of the pathogen.