In January 2023, symptoms of disease were detected on Cupressus sempervirens trees (36°23′39″ N; 10°22′38″ E; alt. 137m) in a nursery located in Nabeul, northeast Tunisia. Thirty five % of trees in the nursery (n = 100) had branch cankers associated with gummy exudates and numerous pycnidia emerged through the bark (Figure 1).

        Small fragments (3 × 3 mm) of infected branches were cut, surface-sterilised in 70% ethanol for two minutes and rinsed three times in sterilised water, before being placed in Petri dishes containing potato dextrose agar (PDA) and incubated in darkness at 25°C for three days. Pure cultures were obtained by removing a small fragment of mycelium from the margin of each colony and incubating under the same conditions. To enhance sporulation, sterile pine needles were placed over actively growing cultures and incubating under light at 25°C (Hlaiem et al., 2021). Colonies were first fluffy white, becoming dark grey with abundant mycelia. After seven days’ incubation, black pycnidia were noticed on the sterile pine needles (Figure 2). Conidiogenous cells were cylindrical, hyaline, thin-walled and smooth, and produced a single conidium at the tip. Conidia were first hyaline becoming dark brown, before discharge from pycnidia. They were aseptate, ovoid, apex obtuse, base truncate with a thick melanised cell wall, widest in the middle, and 19 to 26.8 × 9.5 to 14.2 μm in size (Figure 3). Based on the morphological features of colonies, the fungus was identified as Diplodia seriata De Not. (Phillips et al., 2007). Molecular characterisation was achieved by sequencing the internal transcribed spacer (ITS)-rDNA and part of the translation elongation factor 1-α (EF1-α) region. BLAST searches of the ITS (GenBank Accession No. OR186981) and elongation factor EF-1-α (OR497211) showed that the sequences had 100% identity with reference sequences of Diplodia seriata isolate UCR-DS13 (MW393566 and MW496404, respectively).

        The pathogenicity assay was conducted according to Hlaiem et al. (2021) by inoculating the fungus into a shallow wound (3 mm) made by a sterile scalpel on five one-year-old C. sempervirens seedlings. As a control, sterile PDA plugs were placed into similar wounds on five healthy seedlings of C. sempervirens. After four weeks, all seedlings inoculated with D. seriata had necrotic lesions 4.55 ±0.25 cm in length. The control seedlings remained asymptomatic. The fungus was successfully reisolated, fulfilling Koch's postulates.

        To our knowledge, this is the first report of Diplodia seriata causing branch canker disease on Cupressus sempervirens in Tunisia and globally. Diplodia seriata has been reported to cause canker on hawthorn trees in Turkey (Kurt et al., 2022) and has been recognised causing dieback disease of apple trees in Portugal (Phillips et al., 2012) and grapevine in Iran (Mohammadi et al., 2013).

        Source of information:https://bsppjournals.onlinelibrary.wiley.com/doi/full/10.1002/ndr2.12234