Diversity and Pathogenicity of Botryosphaeriaceae Species Isolated from Olives in Istria, Croatia, and Evaluation of Varietal Resistance.

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 Buza, 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.

The Antibacterial Effect of Selected Essential Oils and Their Bioactive Constituents on Pseudomonas savastanoi pv. savastanoi: Phytotoxic Properties and Potential for Future Olive Disease Control.

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.

First Report of Olive Branch Dieback in Croatia Caused by Cytospora pruinosa Défago.

Olive (Olea europaea L.) is a very important crop grown in the Mediterranean part of Croatia. Olive branch and fruit dieback symptoms were observed in two olive orchards in Istria, Croatia. The samples from symptomatic trees were collected and brought to the laboratory for analysis. Based on their morphological characterization, isolated fungi were identified as Cytospora sp. Two representative isolates (one per orchard) were taken for molecular analysis, and based on DNA sequence data of the ITS and TUB gene regions, and phylogenetic analysis of the sequences, the isolates were identified as Cytospora pruinosa Défago. To determine pathogenicity, pathogenicity tests were conducted on detached olive branches and two-year-old olive trees in the greenhouse. This is the first report of C. pruinosa causing olive branch and fruit dieback in Croatia.

Identification and Characterisation of Pseudomonas savastanoi pv. savastanoi as the Causal Agent of Olive Knot Disease in Croatian, Slovenian and Portuguese Olive (Olea europaea L.) Orchards.

Strains of Pseudomonas savastanoi pv. savastanoi (Pss), isolated from infected olive trees (Olea europaea L.) in three European countries (Croatia, Slovenia and Portugal) were identified and characterised according to their colony morphology, physiological and biochemical features. According to the LOPAT scheme, 38.6% of Pss isolates were grouped in the Ib cluster. The Portuguese Pss strains were fully consistent with the typical LOPAT profile for this bacterium. Conversely, most Slovenian Pss strains showed delayed oxidase activity, whilst Croatian Pss strains did not produce any fluorescent pigment when grown in vitro. For Pss molecular identification, both end-point and real-time PCR were used, as well as MALDI-TOF, which was additionally used for proteomic analysis and the subsequent species identification of a number of strains that showed deviations from expected LOPAT results. Pss was confirmed as a causal agent of olive knot disease in 46.6% of olive orchards screened. Overall, these data suggests a possible correlation of certain Pss features with the geographical origin and the ecological niche of Pss isolates.

First Report of Phaeoacremonium iranianum Causing Olive Twig and Branch Dieback.

In an olive orchard on the western part of Istria, Croatia, twig and branch dieback was observed on several olive trees. In total, seven samples from symptomatic trees were collected. Samples were analyzed, and four fungal isolates showed morphological similarities to the species Phaeoacremonium. One isolate, chosen as a representative, was taken for molecular identification and pathogenicity tests. Based on the DNA sequence data of the ITS, TUB, and EF1α gene regions, the isolate was identified as P. iranianum. Pathogenicity tests were conducted on detached olive branches and olive trees in the greenhouse. To the best of our knowledge, this is the first report of twig and branch dieback on olive caused by Phaeoacremonium iranianum.

Inter-Varietal Diversity of Typical Volatile and Phenolic Profiles of Croatian Extra Virgin Olive Oils as Revealed by GC-IT-MS and UPLC-DAD Analysis.

Despite having an interesting native olive gene pool and a rapidly emerging olive oil industry, monovarietal extra virgin olive oils (EVOO) from Croatia are relatively unexplored. To investigate the inter-varietal diversity of typical volatile and phenolic profiles of Croatian EVOO, 93 samples from six olive (Olea europaea L.) varieties were subjected to gas chromatography-ion trap mass spectrometry (GC-IT-MS) and ultra-performance liquid chromatography with diode array detection (UPLC-DAD), respectively. Quantitative descriptive sensory analysis was also performed. Analysis of variance extracted many relevant exclusive or partial discriminators between monovarietal EVOOs among the identified volatile compounds and phenols. Successful differentiation model with a 100% correct classification was built by linear discriminant analysis, while the most typical volatiles for each monovarietal EVOO were confirmed by partial least squares discriminant analysis. Diverse typical sensory attributes among the EVOOs were tentatively ascribed to the variations in the composition of volatiles and phenols. It was proven that the approach that comprises GC-IT-MS and UPLC-DAD analysis may provide additional objective information about varietal origin and typicity which successfully complement those obtained by sensory analysis. The approach was characterized as universal in nature, with a significant potential to contribute in strengthening the varietal identities and position on the market of monovarietal and Protected Denomination of Origin (PDO) EVOO.

Towards understanding the varietal typicity of virgin olive oil by correlating sensory and compositional analysis data: a case study.

To investigate if the anecdotal varietal typicity concepts for two monovarietal virgin olive oils (VOO) can be scientifically supported and explained, VOO samples were sorted according to typicity grade and related to sensory and compositional data for volatiles and phenols. Clear inter-varietal, as well as intra-varietal boundaries between more and less typical samples were established by univariate and multivariate statistics. A variety-typical complex odor was related to a complex VOO volatile composition with relatively high amounts of lipoxygenase-derived compounds, accompanied by hydrocarbons, terpenes, and benzenoids. A simpler odor, described mostly as green and typical for the other monovarietal VOO, coincided with the lower amounts of the same volatiles. The intensity of bitterness and pungency was mostly in correlation with the amount of major phenols. Two monovarietal VOOs were clearly distinguished with respect to their variety-typical expression of pungency: the one described as gradually increasing was tentatively related to pinoresinol, while the other, described as immediately strong and burning, was assumed to originate mainly from p-HPEA-EDA. Intra-varietal typicity grades correlated with hedonic liking, as well as with general VOO positive sensory attributes related to key volatiles and phenols.