First report of Alternaria alternata causing bud and blossom blight on olive in Bosnia and Herzegovina

During 2020 and 2021, symptoms of withering on olive (Olea europaea) followed by necrosis on buds and flowers were observed in the Ljubuški and Mostar regions of western Herzegovina. These symptoms were observed in two of the ten orchards surveyed and on 83 olive (cv. Oblica) trees in the affected orchards (Figure 1), reaching a disease incidence of 60%. Small pieces from lesion margins were plated on potato dextrose agar and after five days' incubation at 25°C under a 12 hr light/dark regime, a single fungus consistently developed. The 10-day-old monoconidial cultures of two isolates (FAZ HZ and KRA HZ) were plated on potato carrot agar (PCA) and DRYES medium for morphological characterisation (Andersen et al., 2001; Simmons, 2007). Developed fungal colonies were olivaceous and cultures grown on PCA produced brown, branched and septate conidiophores with ellipsoid or ovoid conidia. Mean conidial size was 24.8 ±1.4 × 10.1 ±0.6 μm (Figure 2). Observed morphological features identified the fungus as Alternaria section alternata (Woudenberg et al., 2015). Fungal DNA from two isolates was amplified by PCR using primers for ITS (White et al., 1990), rpb2, endoPG, and Alt a 1 (Woudenberg et al., 2015). Amplicons of 540 bp (ITS), 904 bp (rpb2), 460 bp (endoPG), and 492 bp (Alt a 1) were produced from the FAZ HZ and KRA HZ isolates and sequenced (GenBank Accession Nos. OP010267 and OP010268 for ITS, OP038921 and OP038922 for rpb2, OP972863 and OP972864 for endoPG, and OP972865 and OP972866 for Alt a 1, respectively). A BLASTn analysis of these sequences showed 100% identity with the reference strain CBS 117.44 of A. alternata (Fries) Keissler in the ITS (KP124303), rpb2 (KP124772), endoPG (KP124001), and Alt a 1 (KP123854) regions. Phylogenetic analyses were done using the Maximum Likelihood method based on concatenated data set and reference CBS strains using MEGA 11 software (Tamura et al., 2021). Both olive isolates clustered in the same group as A. alternata and were clearly separated from closely related Alternaria species (Figure 3), thus confirming the morphological identification and BLASTn analyses. Thirty three-year-old seedlings of olive cv. Oblica were inoculated by suspension spraying (105 conidia/ml) in three different tests and maintained in a greenhouse for 10 days at 25- 28°C with a photoperiod of 12 hr light/12 hr dark. The same number of control plants were inoculated with sterilised distilled water. Necrotic flowers and buds appeared on infected seedlings, and A. alternata was consistently reisolated (Figure 4). No symptoms appeared on the control plants. This is the first report of A. alternata causing bud and blossom blight on olive in Bosnia and Herzegovina and, to the best of our knowledge, the first report of the fungus on any plant species in the country. Lagogianni et al. (2017) have previously reported A. alternata causing blight on olive in Greece. Since Mostar and Ljubuski are the major olive-producing areas and the increasing importance of olive in the country, this polyphagous pathogen could pose a serious threat to olive production and may spread to other crops. Extensive monitoring and early diagnosis will be of crucial importance for appropriate management of this disease. This research is funded by the Installation Research Project of the Croatian Science Foundation UIP-2020-02-7413 Natural bioactive compounds as a source of potential antimicrobial agents in the control of bacterial and other fungal pathogens of olives (Anti-Mikrobi-OL).