<![CDATA[This study reports the first confirmed occurrence of Didymosphaeria variabile associated with leaf spot disease of Eucalyptus globulus in urban plantations in Addis Ababa, Ethiopia. A systematic field survey conducted from March to May 2024 across 10 plantation sites spanning elevations of 2,138–2,961 m a.s.l. revealed characteristic light-brown necrotic leaf lesions bearing scattered pycnidia. Across all surveyed sites, the overall mean disease incidence was 92.9 ± 8.03%, while mean disease severity was 81.87 ± 6.75%. A total of 250 symptomatic leaves were collected for pathogen isolation, characterization, and identification. Morphological examination revealed pycnidia and ovoid, septate conidia consistent with D. variabile, with conidia measuring 2.9–4.5 × 1.2–2.6 µm and a mean colony radial growth rate of 2.1 ± 0.3 mm day⁻¹. Molecular identification based on the internal transcribed spacer (ITS) region showed 100% sequence identity with reference sequences of D. variabile. Maximum-likelihood phylogenetic analysis placed the Ethiopian isolates within a well-supported clade containing D. variabile reference sequences (bootstrap support: 100%). Pathogenicity assays on detached E. globulus leaves reproduced field-like symptoms within 10–14 days, and the pathogen was successfully reisolated, thereby fulfilling Koch’s postulates. Inoculated leaves developed a mean lesion diameter of 8.3 ± 1.2 mm, whereas no lesions developed in the controls. Statistical analysis confirmed significant differences among treatments (F₂, ₂₇: 187.4, P < 0.001; Tukey’s HSD, P < 0.05). This study provides the first confirmed report of D. variabile associated with leaf spot disease of E. globulus in Ethiopia and highlights the need for strengthened disease surveillance, urban forestry management, and long-term monitoring of Eucalyptus plantations in East Africa.]]>
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