First report of cytospora punicae causing trunk canker of pomegranate (punica granatum) in Cyprus

Organized cultivation of pomegranate (Punica granatum L.) in Cyprus is relatively recent and accelerating, with approximately 420 acres currently under cultivation. In August 2013, a pomegranate orchard cv. Ermioni with severe trunk malformations, located in the mountainous region of Pitsilia (elevation 1100 m) in the province of Limassol, Cyprus, was brought to our attention. Trees were 2 years old and almost 30% of trees exhibited on their trunk longitudinal sunken cankers, ranging from 5 to 30 cm long. At the canker site, the bark was torn open, rendering exposed sapwood reddish-brown. The foliage was yellowish, while in a few cases the trunk was girdled, causing tree death, while no canker formation was found on the tree branches. Fungal isolations were performed by plating plant tissue from discolored sapwood on potato dextrose agar (PDA) amended with streptomycin sulfate at 100 μg/ml, and representative isolates were subcultured onto PDA at 25°C. Developed thalli were comprised of septate hyphae that were whitish and progressively turned olive green to dark brown with maturity. Dark-brown pycnidia (n = 25) were obvious after 2 weeks and measured 248.6 to 580.4 μm in diameter (mean 389.4 μm; SD ± 90.9 μm). Conidia (n = 50) were hyaline, aseptate, and allantoid, ranging from 4.5 to 5.4 μm long (mean = 4.8 μM; SD ± 0.4 μm) × 0.9 to 1.9 μm wide (mean = 1.4 μm; SD ± 0.3 μm). The internal transcribed spacer region (ITS1-5.8S-ITS2) of rDNA and the translation elongation factor 1-alpha gene (EF1-α) were amplified and sequenced. Consensus sequences were deposited in GenBank (Accession Nos. KR020716 and KR020717) and BLAST alignment in the NCBI database revealed high homology (99 to 100%) to other previously reported for Cytospora punicae Sacc. (Saccardo 1884) (Accession Nos. KJ621688, KJ621689, KJ621684, and JX438568). In October 2013, 10 one-year-old branches of P. granatum cv. Ermioni were inoculated by placing mycelium plugs from actively growing colonies on PDA into wounds made by removing the bark with a cork borer, while 5 control branches were inoculated with sterile PDA plugs. Inoculations sites were covered with Vaseline and wrapped with parafilm to prevent desiccation. Six months later, branches were examined for canker development. C. punicae-inoculated branches developed longer cankers (25.8 cm) compared with the control branches (0.6 cm). The fungus was reisolated only from inoculated branches, whereas isolations from nonexpanding wounds were negative, indicating that the fungal mycelium placed in the wounds was responsible for canker formation. Koch’s postulates were fulfilled. C. punicae has been previously described as a stem, twig, and branch pathogen causing canker and dieback on pomegranate trees (Palou et al. 2013; Peduto Hand et al. 2014). The opportunistic nature of Cytospora spp. fungi is well described and canker development has been reported to begin at trees predisposed to inciting factors causing acute stress, including drought, flood, insect damage, and frost (Guyon et al. 1996). Thus, further research to determine the predisposing factors for trunk canker development in pomegranates by C. punicae may be considered in the near future. To our knowledge, this is the first report of the aforementioned pathosystem in Cyprus