| Summary: | Leaf spot symptoms were spotted in two olive orchards in Istria and in Kvarner Gulf, Croatia. Fungal species from three representative isolates (P13 LECIII, R18 BI, JA20 NP) have been morphologically characterized based on the colony and conidial characteristics. Several techniques were performed for inducing the sporulation of the JA20 NP isolate. Only PDA + banana medium was successful. PCR was conducted for ITS, TUB, and EF1α gene regions. Phylogenetic analyses were performed using internal transcribed spacer, beta-tubulin, and translation elongation factor 1-alpha sequence data. Three types of tests were conducted: a pathogenicity test on detached leaves, on detached and scratched leaves, and on olive seedlings. Ultimately, from the morphological characterizations, DNA sequence analysis of ITS, TUB, and EF1α gene regions, and phylogenetic analysis, these species were identified as <i>Nigrospora gorlenkoana</i> Novobr., <i>Nigrospora osmanthi</i> Mei Wang & L. Cai, and <i>Nigrospora philosophiae-doctoris</i> M. Raza, Qian Chen & L. Cai. This is the first report of <i>Nigrospora</i> species causing leaf spot on olive trees and the first report of <i>Nigrospora philosophiae-doctoris</i> as a plant pathogen. Fungal leaf diseases in conditions that are favorable for infection and disease development can lead to a decrease in the yield and olive oil quality. Therefore, it is necessary to conduct further research and the monitoring of fungal leaf diseases.
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