First Report of Diplodia seriata as Causal Agent of Olive Dieback in Croatia.

In August 2010, a dieback of young olive (Olea europea L.) trees (cvs. Pendolino and Leccino) occurred in two orchards in Istria, Croatia. According to the producers, low temperatures during the winter severely damaged the plants and led to their decline. Distinctive symptoms, assumed fungal infection, were observed in internal tissue of stems and branches. Elongated brown necrosis, sometimes with black streaks, was visible under the bark, therefore Verticillium wilt was suspected. Of 1,086 trees in two orchards (4 ha), 165 (15%) showed symptoms. To isolate the causal agent, surface-sterilized wood chips of symptomatic tissue were placed on potato dextrose agar (PDA). Fungal colonies resembling Botryosphaeriaceae spp. grew from all wood fragments placed on PDA, and from these colonies, monohyphal isolates were obtained. For morphological identification, pycnidial formation was stimulated by growing the isolates on 2% water agar that included stems of plant species Foeniculum vulgare Mill. at room temperature under diffuse light. Pycnidia contained conidia that initially showed as hyaline, becoming light to dark brown as they matured, ovoid with truncated or rounded base and obtuse apex, aseptate, with wall moderately thick, externally smooth, roughened on the inner surface, and 22.8 to 23.5 × 9.6 to 10.5 μm. On the basis of these morphological characters, fungal species Diplodia seriata (teleomorph "Botryosphaeria" obtusa) was suspected (3). For molecular identification, four isolates (MN3, MN4, MN5, and MN6) were used for PCR to amplify the internal transcribed spacer (ITS) region and partial translation elongation factor 1-alpha (EF1-α) gene, using primers ITS4/ITS5 and EF1-728F/EF1-986R, respectively. Sequencing was performed with those amplified genes, then sequences were deposited in GenBank. Comparison of these sequences with GenBank sequences for referent D. seriata isolate CBS 112555 (AY259094 and AY573220) (3) showed 100% homology. On the basis of molecular data, the isolates were confirmed to be species D. seriata De Not. Pathogenicity tests were performed by inoculation of 2-year-old olive plants, six plants per tested cultivar (Pendolino and Leccino). For every cultivar, four plants were wounded and mycelium plugs from D. seriata cultures on PDA were placed on the wounds and sealed with Parafilm. Two control plants per tested cultivar were inoculated with sterile PDA plugs. After 2 months, six of eight inoculated plants wilted completely, and under the bark, brown necrosis was observed. D. seriata was constantly reisolated from the inoculated plants and fulfilled Koch's postulates and confirmed pathogenicity of D. seriata on olive as causal agent of olive dieback. Control plants showed no symptoms of the disease. This fungus has been recognized as the cause of fruit rot of olive (1) and branch canker or dieback in Spain (2). To our knowledge, this is the first report of D. seriata as a pathogen of olive in Croatia. Also, this is one of the first reports of D. seriata as the cause of olive dieback in the world, while Moral et al. (1,2) mostly reported it as the cause of olive fruit rot. Since the same symptoms of olive dieback were observed at other localities in Croatia, the disease could represent a serious threat, particularly for young olive orchards. References: (1) J. Moral et al. Plant Dis. 92:311, 2008. (2) J. Moral et al. Phytopathology 100:1340, 2010. (3) A. J. L. Phillips et al. Fungal Divers. 25:141, 2007.