Sara Godena

Olive leaves represent a metabolically active tissue that plays an important role in plant responses to biotic stress. The present study comprised two independent experiments investigating biochemical responses of olive leaves to bacterial and fungal challenge under controlled conditions. Changes in primary metabolites (sugars, organic acids and free amino acids), phenolic compounds and lipid peroxidation were analyzed using chromatographic and spectrophotometric methods. In the bacterial experiment, pronounced differences were observed in primary metabolism. Tryptophan concentrations ranged from approximately 50 mg kg⁻¹ DW to more than 360 mg kg⁻¹ DW in ‘Istarska bjelica’, while sucrose concentrations reached up to 87 g kg⁻¹ DW, demonstrating cultivar-dependent differences in carbohydrate metabolism. Phenolic profiling showed that secoiridoids were the dominant phenolic class, with oleuropein concentrations exceeding 27 g kg⁻¹ DW across bacterial treatments. In the fungal experiment, amino acids showed greater variability than sugars and phenolic compounds, whereas MDA concentrations ranged from approximately 190 to 300 nmol g⁻¹ DW but did not differ significantly among pathogen treatments. Overall, the two experiments showed distinct patterns of metabolite variation associated with bacterial and fungal challenge. These findings contribute to a better understanding of cultivar-dependent metabolic responses and provide a basis for future studies of olive–microbe interactions.

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.

The disposal of olive mill wastewater (OMWW) poses significant environmental challenges due to its high content of phytotoxic and pollutant compounds. This study aims to explore the chemical composition of OMWW derived from various olive varieties (Buža, Buža puntoža, Istarska bjelica, Leccino, and Rosinjola) and assess its antifungal potential against phytopathogenic fungi from the Botryosphaeriaceae family. OMWW samples were analyzed for their physicochemical properties, phenolic composition via LC-MS/MS, and antifungal activity against Botryosphaeria dothidea (Moug. ex Fr.) Ces. & De Not., Diplodia mutila (Fr.) Fr., D. seriata De Not., Dothiorella iberica A.J.L. Phillips, J. Luque & A. Alves, Do. sarmentorum (Fr.) A.J.L. Phillips, Alves & Luque, and Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips. Antifungal efficacy was tested at varying concentrations, alongside the phenolic compounds hydroxytyrosol and vanillic acid. Antifungal activity varied across fungal species and OMWW concentrations. Lower OMWW concentrations inhibited mycelial growth in some pathogens, while higher concentrations often had a stimulatory effect. Among the OMWW treatments, Leccino and Buža showed the most significant antifungal activity against species from the Botryosphaeriaceae family. The results demonstrated significant variability in OMWW composition, with Istarska bjelica exhibiting the highest concentrations of phenolic compounds, sugars, dry matter, and carbon and nitrogen content. The results also highlight the impact of acidification on the phenolic profile of OMWW. Treatment with HCl significantly altered the concentration of individual phenolic compounds, either enhancing their release or contributing to their degradation. Among the two compounds, vanillic acid showed greater efficacy than hydroxytyrosol. In addition, microorganisms isolated from OMWW, including Bacillus velezensis Ruiz-Garcia et al., Rhodotorula mucilaginosa (A. Jörg.) F.C. Harrison, Nakazawaea molendiniolei (N. Cadez, B. Turchetti & G. Peter) C. P. Kurtzman & C. J. Robnett, and Penicillium crustosum Thom, demonstrated antagonistic potential against fungal pathogens, with B. velezensis showing the strongest inhibitory effect. The greatest antagonistic effect against fungi was observed with the species Do. Iberica. The findings highlight the potential of OMWW as a sustainable alternative to chemical fungicides, simultaneously contributing to the management of waste and protection of plants through circular economy principles.

One of the most persistent and damaging diseases in olive trees is olive knot disease. This disease is caused by an infection by the Gram-negative phytopathogenic bacterium Pseudomonas savastanoi pv. savastanoi that is notoriously difficult to control. The increasing demand for eco-friendly and sustainable agricultural solutions has driven research into plant-based agents. This study investigated the antibacterial properties of essential oils (EOs) and their constituents, olive mill wastewater (OMWW), the phenolic compound hydroxytyrosol (HTyr), and algae and garlic extracts, as well as copper-based and plant-stimulating commercial products against P. savastanoi pv. savastanoi, a significant olive tree pathogen. Antibacterial activity was determined using the Kirby–Bauer disc diffusion and broth microdilution methods. The EOs derived from Thymus vulgaris (thyme) and Origanum compactum (oregano), and their key components thymol and carvacrol, exhibited the strongest antibacterial efficacy. Conversely, the OMWW, plant-stimulating products, and algae and garlic extracts showed limited to no antibacterial activity in vitro, with their antibacterial properties determined using the disc diffusion method. While the EOs were highly effective in vitro, regardless of the testing method, their efficacy in bacterial growth inhibition was strain- and concentration-dependent, possibly highlighting some metabolic or genetic variability in the target pathogen, even though the MIC values against all tested strains of P. savastanoi pv. savastanoi were equal. Bacterial membrane disruption and the consequent leakage of metabolites were determined as the modes of action of carvacrol and oregano EO. Carvacrol also promoted plant growth in lettuce without significant phytotoxic effects, although minor necrotic lesions were observed in young olive leaves at higher concentrations, presenting these agents as potential next-generation green bactericides.

The antifungal effectiveness of essential oils (EOs) and their predominant components were tested on 14 phytopathogenic fungi isolated from olive trees. Commercial EOs from holy basil (Ocimum tenuiflorum L.), Chinese cinnamon (Cinnamomum aromaticum Ness), lemon (Citrus × limon), peppermint (Mentha × piperita L.), oregano (Origanum compactum Benth), and thyme (Thymus vulgaris L.) and components eugenol, e-cinnamaldehyde, limonene, menthol, carvacrol, and thymol were used. Antifungal efficacy was tested on six species from the Botryosphaeriaceae family: Botryosphaeria dothidea (Moug. ex Fr.) Ces. & De Not.; Diplodia mutila (Fr.) Fr.; D. seriata De Not.; Dothiorella iberica A.J.L. Phillips, J. Luque & A. Alves; Do. sarmentorum (Fr.) A.J.L. Phillips, Alves & Luque; and Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips. Other tested species included Biscogniauxia mediterranea (De Not.) Kuntze, B. nummularia (Bull.) Kuntze; Cytospora pruinosa Défago; Nigrospora gorlenkoana Novobr.; N. osmanthi Mei Wang & L. Cai; N. philosophiae-doctoris M. Raza, Qian Chen & L. Cai; Phaeoacremonium iranianum L. Mostert, Grafenhan, W. Gams & Crous; and Sordaria fimicola (Roberge ex Desm.) Ces. & De Not. The results show that Chinese cinnamon and oregano EOs, along with their components, completely inhibited the growth of all tested fungi, indicating their potential as biological control agents in sustainable agriculture. In contrast, the least effective treatments were the EOs derived from lemon and peppermint, as well as the components limonene, menthol, and thymol. Notably, the fungi Do. iberica and N. gorlenkoana were among the most sensitive to all the treatments applied.

During 2021 and 2022, a field investigation was conducted in Istria, Croatia, searching for trees exhibiting signs of Botryosphaeria dieback. Samples of symptomatic trees were collected from 26 different locations and analysed. Isolates that morphologically corresponded to species from the Botryosphaeriaceae family were selected, and detailed morphological characterisation and molecular identification of the isolates were conducted. Based on morphological characteristics and phylogenetic analysis using the internal transcribed spacer (ITS), beta-tubulin (TUB2), and translation elongation factor 1-alpha (TEF1-α) regions, six species of fungi from the Botryosphaeriaceae family were identified: Botryosphaeria dothidea (Moug. ex Fr.) Ces. & De Not.; Diplodia mutila (Fr.) Fr.; Diplodia seriata De Not.; Dothiorella iberica A.J.L. Phillips, J. Luque & A. Alves; Dothiorella sarmentorum (Fr.) A.J.L. Phillips, Alves & Luque; and Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips. This is the first report of D. mutila, Do. sarmentorum, and Do. iberica causing Botryosphaeria dieback on olive trees in Croatia, and the first study investigating the resistance of Croatian olive varieties to species from the Botryosphaeriaceae family. Pathogenicity testing of selected isolates and assessment of variety resistance were conducted on four different olive varieties, namely Buža, Istarska bjelica, Leccino, and Rosinjola, using representative isolates of the mentioned species. The most aggressive species was found to be N. parvum. Olive varieties exhibited differences in susceptibility depending on the fungus they were infected with.

Olive knot disease, caused by the bacterium Pseudomonas savastanoi pv. savastanoi, causes great damage in olive orchards. While control measures of P. savastanoi pv. savastanoi in olive orchards primarily rely on pruning and copper-based treatments, the use of antibiotics as bactericidal preparations in agriculture is limited and highly restricted. However, plants are naturally endowed with protective molecules, such as phenolic compounds, which defend them against herbivores, insects, and microorganisms. This research aimed to test the virulence of five strains of P. savastanoi pv. savastanoi isolated from different growing regions and olive varieties, and to examine whether there is a difference in plant susceptibility based on the variety. An additional goal was to test the antimicrobial activity of olive mill wastewater, known for its high content of phenolic compounds, and aqueous garlic hydrolysate, as well as to compare them with a commercial copper-based product, pure hydroxytyrosol, and a standard antibiotic as references. Analysis of knot characteristics showed variations in the virulence of the P. savastanoi pv. savastanoi strains, with the highest virulence being observed for the strain I7L and the lowest virulence for the strain B45C-PR. The olive cultivar Rosinjola displayed higher susceptibility compared to Frantoio, Buža, and Leccino, while cv. Istarska bjelica exhibited the least susceptibility compared to the other investigated olive cultivars. In an attempt to explore alternative solutions for disease control, in vitro tests revealed that the phenol HTyr, GE, and the wastewater with the highest total phenolic content (cv. Istarska bjelica) possess the highest antibacterial activity. This supports the role of polyphenols in host defense, aligning with previous field observations of lower susceptibility of cv. Istarska bjelica to olive knot disease. These findings highlight the complex nature of olive knot interactions with bacterial strains and olive cultivars, simultaneously accentuating and underscoring the importance of considering the host’s defenses as well as bacterial virulence in disease management strategies.

A field investigation of olive trees in Istria, Croatia, revealed branch dieback and cracked bark. Samples of diseased branches were collected from eight different locations and analysed. Additionally, meteorological data from two locations were analysed to determine if there was a connection between climatic changes and the appearance of pathogens in the region. Pathogenicity tests were conducted on olive seedlings. This study provides a description of Biscogniauxia and Sordaria species’ morphology and elucidates their phylogeny based on the internal transcribed spacer (ITS), beta-tubulin (TUB2) and translation elongation factor 1-alpha (TEF1- α) regions. This research represents the first documented occurrence of Biscogniauxia mediterranea causing charcoal disease in olive trees in Croatia. Additionally, it is the first report of Biscogniauxia nummularia (Bull.) Kuntze and Sordaria fimicola causing diseases in olive trees anywhere in the world. Furthermore, this study marks one of the initial forays into molecular investigations of these species isolated from olive trees. Considering the potential threat posed by the inherent aggressiveness of Biscogniauxia species, further research is deemed necessary to curb the development of charcoal disease.

Plant pathogenic bacteria pose a significant threat to olive cultivation, leading to substantial economic losses and reduced yield. The efficacy of antimicrobial agents against these pathogens is of great interest for sustainable disease management strategies. As such, the management of olive knot disease is one of the major challenges in olive protection. In the presented study, through a series of in vitro assays, we investigated the antimicrobial effect of six essential oils (EOs) and their most concentrated constituents against causative agent of olive knot disease—Pseudomonas savastanoi pv. savastanoi, highlighting the high potential of Origanum compactum EO and its constituent carvacrol. Carvacrol exhibited the highest potential for practical application, demonstrating membrane disruption as its mechanism of action even at the lowest concentration. The bactericidal effect of antimicrobials was confirmed in a time–kill assay, where concentrations of MIC, 2× MIC, and 4× MIC were evaluated. Some of the applied treatments resulted in inhibition equal or higher than copper-based treatment. Additionally, we assessed the phytotoxicity of carvacrol by foliar application on olive cv. Leccino. The appearance of phytotoxic injuries majorly occurred on the young leaves of olive plants, with the highest proportion of damaged canopy observed when the 2× MIC concentration was applied. Due to its great efficiency against P. savastanoi pv. savastanoi in vitro, these findings highlight the potential of carvacrol as a molecule of interest for the development of environmentally friendly biopesticides. This study also contributes to the advancement of disease management practices in olive cultivation, leading to enhanced crop protection.