First Report of <i>Neocosmospora solani</i> Causing Wilt of <i>Olea europaea</i> L. in Bosnia and Herzegovina

In Bosnia and Herzegovina, olive trees are cultivated in the karstic, warm, and dry southern areas of the country. New olive groves have recently been established on anthropogenically modified sites, being converted into intensive olive plantations. In September 2023, 3-year-old olive trees (cvs. Leccino and Oblica) exhibiting a high incidence of dried leaves, dead branches, necrotic areas in the trunk cross-section and plant mortality were observed at the Studenci–Ljubuški site (43.154698N; 17.617926E) in West Herzegovina Canton, with an estimated disease incidence of approximately 50%. Symptomatic plant material was collected and surface-disinfected using 2% sodium hypochlorite and 70% ethanol. Branch samples from approximately 8 trees were cut into small pieces, plated onto potato dextrose agar (PDA), and incubated for 5-7 days at 25°C. Morphologically similar slow-growing fungal colonies developed from nearly all wood fragments. Culture were purified through successive transfers of hyphal tips from the edges of slow-growing colonies onto fresh PDA. Microscopic observations revealed septate mycelium, microconidia (single-celled or bicellular, with various shapes), fusiform multicellular macroconidia and chlamydospores produced singly or in pairs. The macroconidia were typically 3-5 septate, slightly curved, and within the size range (approximately 25-50 × 3–5 µm) described for members of the Fusarium solani species complex, FSSC (Leslie and Summerell 2006). Based on morphological (macroscopic and microscopic) characteristics, the fungus was identified as Neocosmospora solani (Summerbell and Schroers 2002). N. solani belongs to the genus Neocosmospora (OʼDonnell et al. 2008). Fungal DNA was extracted using a DNA extraction kit (Qiagen) and used for amplification of the translational elongation factor 1-alpha (TEF) gene with universal TEF (EF-1/EF-2) primers and the RNA polymerase II second largest subunit (RPB2) gene with 5F2/7cR primers (OʼDonnell et al. 1998). The PCR products of two isolates (OLEHZ1 and OLEHZ2) were sequenced by Macrogen Europe (Netherlands), yielding TEF sequences of 645 bp (GenBank accession numbers PV600648 and PV600649) and RPB2 sequences of 842 bp (GenBank accession numbers PX584566 and PX584567). These sequences were compared with GenBank available sequences, showing 99.69-100% nucleotide identity with the N. solani sequences for both genes. A phylogenetic tree was constructed using the maximum likelihood method and both isolates were clustered with N. solani isolates, confirming their identity. Pathogenicity tests were conducted on olive cv. Oblica using N. solani isolates obtained from symptomatic plants. One-year-old rooted olive seedlings (8 plants) were washed under tap water and then left at room temperature for 24 hours before testing. The seedlings were surface-disinfected with a 0.5% sodium hypochlorite for 10–15 minutes, rinsed with sterile water and left for 2 hours before inoculation. A spore suspension (10⁶ spores mL⁻¹), prepared from a 5-day-old culture grown on PDA, was sprayed on the leaves under high-humidity conditions (approximately 90–95% relative humidity), which was maintained by covering the plants with a plastic enclosure to reduce transpiration and evaporation. The plants were incubated at 20°C under a 14-hour photoperiod. The assay was carried out twice, using two control plants. After 40 days, inoculated plants exhibited brown stem lesions and leaf chlorosis. The fungus was reisolated from symptomatic tissues and was found to be identical morphologically with original isolates. Non-inoculated control plants remained healthy. To the best of our knowledge, this is the first report of N. solani causing wilt and mortality of young olive trees (O. europaea L.) in Bosnia and Herzegovina, highlighting its potential threat to olive cultivation in the region.