Šime Marcelić

Olive (Olea europaea L.) leaf composition depends on both collection date and cultivar, yet these two sources of variation are rarely separated within one orchard over a full year. Leaves of the indigenous Istrian cultivars ‘Istarska bjelica’ and ‘Rošinjola’ and the introduced ‘Leccino’ were sampled in one orchard every two months from April 2025 to February 2026. Of 82 metabolites quantified by LC-MS/MS, 64 were detected in every cultivar and period and analysed. The sampling period significantly affected 56 metabolites, while cultivar influenced 35. Their interaction was significant for 23 metabolites, indicating that seasonal changes are cultivar-dependent. Effect sizes confirmed this ordering: PERMANOVA attributed 45.3% of the total metabolome variance to sampling period against 17.3% to cultivar. At the whole-metabolome level, sampling period affected all three cultivars, whereas cultivar differentiation was absent in one period. By class, sampling period affected all six classes, whereas cultivar was significant for phenolic acids, flavonoids and B-group vitamins, with no effect on the total metabolite pool (p = 0.189). Unsupervised PCA grouped samples by season, and supervised PLS-DA detected cultivar differences (96.3% cross-validated accuracy, permutation p = 0.012) although they were not resolved by the first two principal components; this accuracy describes separability within this dataset of nine experimental units rather than predictive performance. Cultivar information is therefore spread across many metabolites, and fixed sampling dates are essential when comparing cultivars or assessing leaves as raw material.

Olive leaves are a by-product of olive farming and have a high phenolic content. However, the effect of farming system, cultivar, and harvest time on their content is poorly studied. In June, August, and October 2022 (108 samples), leaves of trees ‘Istarska bjelica’, ‘Leccino’, and ‘Oblica’ within an organic and integrated grove were sampled. From these samples, the seven main phenolic compounds and twelve mineral elements were quantified. Data were analysed by three-way ANOVA with tree as a random effect. All 19 compounds and elements showed at least one significant interaction and 14 elements and compounds showed a significant three-way interaction; thus, no factor could be interpreted in isolation. Eight of the ten elements with a three-way interaction showed a reversal of system difference with cultivar–date combinations. Five of the six phenolics with a farming system effect were 26% to 65% higher with organic management; in contrast, apigenin-7-O-glucoside was 37% lower. Sampling date predominantly affected the secoiridoids: oleuropein and oleacein increased 5.9- and 15.6-fold, respectively. Phenolic compounds and mineral elements were correlated: boron correlated negatively with oleuropein (r = −0.76), whereas silicon correlated positively with oleacein (r = 0.46). To standardise olive leaves as a bioactive source, cultivar, harvest date, and farming system must be specified.

Trešnja je popularna vrsta koštičavog voća, posebice za svježu potrošnju. Podloge SL 64, MaxMa 14 i Gisela 5 pokazuju dobru prilagodljivost za različita područja uzgoja. Rizobakterije u tlu potiču rast biljaka te izravno ili neizravno utječu na razvoj biljke. Vrsta Azospirillum brasilense Tarrand, Krieg i Döbereiner, 1978., pozitivno pridonosi rastu biljke. Utjecaj A. brasilense na kontejnerski uzgojene podloge koštičavih vrsta do sada je nedovoljno istražen. Ciljevi rada su utvrditi vegetativni rast, indeks sadržaja klorofila (CCI) i makrohraniva u lišću podloga trešnje kao rezultat zalijevanja sadnica podloge rizobakterijom. Ispitan je utjecaj mikrobiološkog gnojiva AZOS (A. brasilense, 10⁶ CFU g⁻¹) na vegetativni rast, indeks sadržaja klorofila (CCI) i sadržaj makrohraniva u lišću sadnica podloga trešnje SL 64, MaxMa 14 i Gisela 5 u kontejnerskom uzgoju. Poljski pokus u slučajnom bloknom rasporedu (n=3) uključivao je tretmane: T1 (mjesečno zalijevanje, 1,12 g L⁻¹), T2 (dvomjesečno) i kontrolu T0. Uzorci listova su prikupljeni i analizirani u laboratoriju. Podaci su obrađeni dvosmjernom analizom varijance (ANOVA), dok je značajnost razlika između srednjih vrijednosti utvrđena Duncanovim testom (p<0,05). Na temelju dobivenih rezultata vidljivo je da su T1 i T2 značajno povećali poprečni presjek debla (103,8 i 101,5 mm²), sadržaj N (1,83 % i 1,72 %), uz značajno smanjenje sadržaja Ca (1,26 % i 1,21 %) u odnosu na kontrolu. Podloga SL 64 ostvarila najveći TCSA (124,5 mm²), a MaxMa 14 najviši N (1,86 %). Ovo je prvo istraživanje utjecaja A. brasilense na podloge trešnje u poljskim uvjetima koje otkriva specifične interakcije genotipa i tretmana, potvrđujući potencijal PGPRa za smanjenje mineralnih gnojiva u kontejnerskom uzgoju.

Regenerative agriculture is oriented around restoring soil health through natural processes. In this context, soil biota plays a central role, and bioinoculation represents a potentially effective approach for targeted modification of microbial communities. Among beneficial microorganisms, Trichoderma atroviride is prominent for its biocontrol agent (BCA) activity against plant-parasitic nematodes (PPNs), whereas its effects on free-living nematodes (FLNs) under in vivo conditions remain insufficiently explored. The aim of this study was to assess the response of nematode communities to bioinoculation with T. atroviride as an indicator of soil functional status. A three-year field study was conducted in organic olive orchards at Vodnjan and Nadin on four autochthonous olive cultivars, applying two inoculum doses of T. atroviride: 1 × 10⁶ spores mL⁻¹ (LD) and 1 × 10⁸ spores mL⁻¹ (HD). Bioinoculation increased the diversity of the soil nematode communities at both locations. However, the responses differed between the two inoculum doses. Both doses were associated with an increased abundance of FLNs and a reduced abundance of herbivorous nematodes relative to the control, with LD showing a more consistent and ecologically favourable effect. In combination with biotic and abiotic factors, the LD dose was associated with greater trophic diversity and a more structured soil food web, whereas increasing the inoculum concentration (HD) did not result in additional functional improvement.

This research focuses on the additional valorization of olive leaves, a by-product of regular olive pruning, by increasing their secondary metabolite content through the combined application of biochar and a phenolic extract from olive leaves. A suspension of biochar, obtained by the pyrolysis of grapevine pruning residues, was prepared by mixing it in demineralized water (1.5 g; 5 L; 24 h). The phenolic extract was obtained by extracting lyophilized and ground olive leaves in demineralized water (50 g; 5 L; 24 h), while the combined preparation was obtained in an analogous manner (1.5 g biochar; 50 g olive leaf powder; 5 L water; 24 h). Treatments were applied at the beginning of July, 50 days after anthesis (May 16th) and included the following: (i) control treatment (demineralized water), (ii) biochar solution, (iii) phenolic extract solution, and (iv) a combined aqueous preparation of biochar and phenolic extract, all with the addition of a wetting agent. Trees of the olive cultivars Leccino and Istarska bjelica were sprayed with the corresponding preparation until runoff. Olive leaves were sampled three weeks after treatment (July 26th) and, after washing and drying, and were prepared for LC-MSMS analysis. Both biochar-based treatments induced the most potent effects, although responses differed between cultivars. In particular, apigenin derivatives, hydroxytyrosol, luteolin-7-rutinoside, and the secoiridoid oleacein showed apparent differences between biochar treatments and the control. Overall, higher concentrations of the sum of detected secoiridoids were observed in the leaf samples of ‘Istarska bjelica’ under BCH and BCH+PH treatments, whereas no such differences were found for ‘Leccino’ cultivar. Further research is needed to clarify the cultivar-dependent response of secondary metabolism in these olive cultivars and the mechanisms by which biochar foliar application modulates metabolite profiles.

Olive (Olea europaea L.) cultivars often exhibit genotype-specific responses to micronutrient management. In this study, we investigated the metabolic leaf fingerprinting of three cultivars ‘Rošinjola’, ‘Leccino’, and ‘Istarska bjelica’ at two sampling periods (SP-I = 64 days after treatment (DAT) and SP-II = 118 DAT), following boron foliar fertilisation (+B = 41.62 mM B; −B = 0 mM B) applied 50 days after anthesis. To our knowledge, this is the first study to provide such a detailed evaluation of boron-induced shifts in phenolic metabolism in olive leaves. At harvest (SP-II), all three cultivars showed higher concentrations of total identified phenolic compounds in +B plants compared with the −B controls. Notably, the concentration of verbascoside at harvest was higher in +B plants of ‘Istarska bjelica’ and ‘Leccino’, but not in ‘Rošinjola’. Oleuropein content increased in +B plants at harvest to a level higher than 4870 mg/100 g DW, irrespective of cultivar. Conversely, apigenin-7-glucoside declined from SP-I to SP-II in ‘Leccino’ regardless of treatment, whereas in ‘Istarska bjelica’, this decrease occurred only in control plants, with boron preventing the seasonal decline. These findings confirm the prolonged effect of boron foliar fertilisation on phenolic metabolism in olive leaves and highlight cultivar-specific differences in metabolic responses. Further research is needed to clarify how these metabolic shifts relate to primary plant metabolism and how they influence olive oil quality traits among cultivars grown under Croatian conditions.

Research background. Anthocyanins, the most abundant pigments in red wine, play an important role in the visual aspect of wine sensory properties. However, due to their unstable nature, their ability to polymerise with tannins is important for colour stability. Their content varies with grapevine variety, growing conditions, viticultural and winemaking practices. Leaf removal, a common viticultural practice, enhances anthocyanin accumulation in red grapevines, and partial fruit zone leaf removal at different phenological stages can significantly influence the anthocyanin content of grapes and wine. This two-year study examined how two different times of fruit zone leaf removal at different phenological stages affect the initial anthocyanin content in wine and their stability during ageing in Merlot, Syrah and Cabernet Sauvignon wines grown in a Mediterranean climate. Experimental approach. Partial leaf removal was applied during flowering and during vérasion and compared with untreated control. The wines obtained from all treatments and varieties were bottled two months after the end of fermentation, and then stored and matured under the cellar conditions for one year. To determine the influence of different times of leaf removal on the concentration of anthocyanins and their stability in the wine, the wines were analysed immediately after bottling and again after 6 and 12 months of storage. For the determination of all phenolic compounds, high-performance liquid chromatography (HPLC) was used. Results and conclusions. Leaf removal treatments increased the concentration of anthocyanins in all three cultivars. The obtained results showed that malvidin-3-O-glucoside (Mal-3-Glc) was the most abundant individual anthocyanin, while the most unstable anthocyanin was petunidin-3-O-coumaroyl glucoside (Pet-3-Coum-Glc). Initial concentration of total anthocyanins in all wine samples was significantly affected by different conditions in the two years of study, but with a significant effect of the defoliation treatments. Anthocyanin concentration decreased during the ageing of the wine, and the degradation of anthocyanins ranged from 36 to 90 %. The stability of anthocyanins in wine was most influenced by ageing time, while year and treatment had no influence. The concentration of total phenolic acids increased during wine ageing, while the concentration of total flavonol glycosides (TFG) decreased in all wine samples except Merlot from 2016. Novelty and scientific contribution. The results of this study contribute to a better understanding of the stability of increased concentrations of anthocyanins in wines during ageing obtained by the practice of grapevine leaf removal in the vineyard.

All olive (Olea europaea L.) plant tissues have a high phenolic content. However, the effects of the cultivar and sampling period on the tissue phenolic content remain almost unknown; in addition, the interactions between nutrient uptake and leaf phenol concentrations have not been clarified. This study sampled olive leaves to explore how the cultivar, sampling period, and their interaction affect leaf phenol and nutrient concentrations. Leaves were collected from six cultivars during three seasonal periods: harvest (October; SP1), dormancy (January; SP2), and pruning (March; SP3). Five were Istrian cultivars (‘Bova’, ‘Buža muška’, ‘Buža puntoža’, ‘Istarska bjelica’, ‘Rošinjola’), and one was the Italian cultivar ‘Leccino’. Phenolic profiles in olive leaves were correlated with potassium (K), phosphorus (P), and copper (Cu) concentrations. However, significant correlations between these nutrients and oleuropein, verbascoside, and total phenolic content (TPC) were determined only for ‘Rošinjola’. Oleuropein was the most abundant phenolic compound, while among genotypes, ‘Buža muška’ showed the highest oleuropein levels across all sampling periods, indicating its potential source of oleuropein in olive leaves. Seasonal variations in olive leaf phenolic compounds appear to be strongly influenced by phenological phase, nutrient dynamics, and weather conditions, as confirmed by multivariate analysis across sampling periods and cultivars. The findings emphasise the importance of selecting both an appropriate cultivar and sampling period to maximise the accumulation of olive leaf phenolic compounds. Nevertheless, long-term experimentation on cultivars with a high leaf phenolic potential, like ‘Buža muška’ and ‘Rošinjola’, is necessary in order to develop appropriate farming strategies for maximising phenolic compounds with human or plant health benefits.

Secondary metabolites in olive (Olea europaea L.) leaves constitute a complex framework wherein phenylpropanoids, terpenoids, and secoiridoids in particular, serve as major contributors to olive plant resilience. Silicon (Si) stands as a mediator of defense mechanisms in plants, enhancing their protective responses and adaptability. A field trial on one-year-old plantlets of two metabolically distinct olive genotypes was conducted to investigate the effects of foliar-applied Si on the phytochemical profiles of locally treated leaves. Silicon’s systemic effects in juvenile leaves were also appraised. We accounted for intervarietal differences in nutrient uptake and conducted in situ measurements of physiological indices. The peak of the summer season and the onset of autumn were chosen as the two sampling time points. Intense summer conditions prompted metabolic adjustments that resulted in phytochemical profiles unique to each cultivar. These profiles were further significantly altered by Si while remaining genotype-specific, with substantial increases in prominent compounds like oleuropein (105% and 252%) and verbascoside (62% and 126%), depending on the genotype. As the pressure from environmental factors eased, the differences in Si-mediated phytochemical responses emerged. Silicon had a limited effect on the phytochemical profile of the resilient cultivar which acquired a metabolic steady-state, while it significantly altered the profile of its metabolically more versatile counterpart, resulting with a progressive increase in its oleuropein (37%) and verbascoside (26%) levels. These effects extended to untreated, juvenile leaves as well. While effective in altering and improving the phytochemical composition of olive leaves, Si acted in a manner that adhered to each genotype’s metabolic foundation. The intensity of environmental constraints, along with each cultivar’s inherent sensitivity to them, seems to be tied to silicon’s capacity to mediate significant phytochemical alterations. The extent of silicon’s prophylactic function may therefore be dependent on a genotype’s metabolic foundation and overall sensitivity, and as such it seems inseparable from stress and its intensity.

Sweet cherry (Prunus avium L.) is a valuable fruit crop for fresh consumption. Due to its early availability in season, it achieves relatively high prices on the market. Self-fertile cultivar Lapins is one of the world’s leading sweet cherry varieties. Intensive cherry production seeks for new technologies such as using more adaptable rootstocks and microbiological products that could help plants adopt more sustainable growth in different soils/climates. The aim of this work is to determine the substrate properties, vegetative growth, leaf chlorophyll and mineral content of maiden trees grafted on three different rootstocks due to the application of growth-promoting rhizobacteria. A pot experiment was carried out on one-year-old maiden trees of cv. Lapins grafted on SL 64, MaxMa 14 and Gisela 5 and grown in 12 L plant pots filled with commercial substrate. Plant growth-promoting rhizobacteria Azospirillum brasilense was added by watering the plants with 1.12 g L⁻¹ per pot once a month (T₁) or every two months (T₂) from March to September with seven treatments in T₁ and four treatments in T₂. At the same time, control (C) plants were watered with rainwater. Plant height, trunk circumference and leaf chlorophyll content index (CCI) were measured. In addition, shoot growth and internode number were measured in three development stages (BBCH 34, 39 and 91). The substrate and leaf samples were collected and analyzed in the laboratory in accordance with established procedures. Data were processed by ANOVA and the Tukey test. Results have showed that rootstock affected substrate electrical conductivity (EC); nitrate (NO₃⁻), phosphorous (P₂O₅), calcium (Ca) and magnesium (Mg) content, including mineral nitrogen (N) content; tree height, circumference, shoot length and internode number; the leaf chlorophyll content index (CCI); and leaf potassium (K), Ca and Mg content. Furthermore, treatment significantly affected the CCI, average internode length, ammonia (NH₄⁺) and Ca content in the substrate and leaf N, Ca and Mg content. Rhizobacteria A. brasilense can be used as an additional biofertilizer in sustainable agricultural practices for obtaining healthier sweet cherry maiden trees, but microbial biotechnology rules must be respected.