Phenotypic and Molecular Characterization of Verticillium dahliae, the Causal Agent of Verticillium Wilt of Olive in Tunisia

Abstract

Aims: During the last two decades, verticillium wilt of olive has spread to young olive orchards were it causes severe yield losses and death of olive trees in southern and central regions of Tunisia. Therefore, identification of the causal agent as well as the study of its pathogenicity will be useful for design the appropriate management program. Place and Duration of Study: This work was performed in the Laboratory of Phytopathology at the Olive Tree Institute (Sfax, Tunisia) between July 2014 and June 2015. Methodology: This study was conducted using phenotypic and molecular methods to identify the causal agent of the decline and death of olive trees. The pathogen was recovered from infested tissues using a potato dextrose agar medium. Identity of the isolates was confirmed by ITS-RFLP and sequencing. Pathogenicity of the isolates was evaluated by infection bioassay on young olive plants. Results: All the fungal isolates were hyaline, flocculose and produced microsclerotia after 15 days of incubation at 25°C, which is in agreement with the identification key of Verticillium species. All the isolates were characterized by ITS-RFLP and sequencing of the internal transcribed spacer. All the isolates produced 470 bp using primers ITS1/ITS4. Digestion of the ITS product using EcoRI and HaeIII produced two fragment of 250 and 220 bp and three fragment of 300, 150, and 20 bp respectively. Pathogenicity was evaluated on two-year old olive plants using an artificial infection bioassay. After 15 days of inoculation, similar symptoms were produced as natural infection. Symptoms of wilt developed rapidly and caused the death of more than 50% of the inoculated plants. Conclusion: V. dahliae is present in almost all growing olive regions of Tunisia. However, local V. dahliae populations in Tunisia is predominated by a highly pathogenic clone which is able to overcome the resistance of the main olive cultivar.