Dean Ban

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.

Preservation of plant genetic resources has become crucial for agricultural sustainability, particularly for vegetatively propagated crops like garlic. Modern agricultural practices, monoculture farming, environmental degradation, and climate change have led to genetic erosion, and there is growing interest in traditional garlic varieties, which may harbor superior traits. Despite being an asexually propagated crop, garlic exhibits a diverse range of ecotypes in phenotypic and agronomic characteristics. This study analyzed 77 garlic accessions from Istria and Kvarner, Dalmatia, Continental Croatia, and other countries, examining morphological traits alongside biochemical properties, namely allicin content, total phenolic content (TPC), and antioxidant capacity (with ferric reducing antioxidant power (FRAP) assay, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay). Morphological analysis categorized the accessions into two distinct morphotypes, which did not differ significantly in any biochemical parameter. In contrast, accession provenance was associated with differences in TPC and FRAP, with the highest values in accessions from Istria and Kvarner; in 2018/2019, these reached 820.8 mg GAE/100 g DW (TPC) and 354.6 µmol TE/100 g DW (FRAP), significantly exceeding Continental Croatia (494.1 and 180.4, respectively). This regional pattern varied between the two vegetation years. DPPH and allicin content instead differed between the two vegetation years, with DPPH increasing markedly from 2018/2019 to 2019/2020 (from 358.1 to 892.2 µmol TE/100 g DW in QlG1 and from 380.6 to 836.3 µmol TE/100 g DW in QlG2). These results indicate that, in this collection, morphotype was not a reliable predictor of biochemical quality in garlic and underscore the importance of combining morphological and biochemical assessment in germplasm conservation and breeding programs.

Fennel (Foeniculum vulgare Mill.) is an aromatic species of culinary and medicinal value, but the metabolic basis of its drought response and the extent of variation among wild populations remain poorly characterized. We investigated eleven wild fennel accessions from the Mediterranean region of Croatia under control and drought conditions, combining morphological measurements with targeted profiling of 78 primary and 55 secondary metabolites. Drought significantly reduced biomass, leaf area, length, and width, while leaf dry matter content increased. Primary metabolism shifted markedly: amino acids accumulated, with asparagine and arginine increasing by over 20-fold, whereas TCA-cycle (tricarboxylic acid cycle) organic acids declined. Pathway analysis identified Alanine, aspartate, and glutamate metabolism as most strongly affected by drought treatment. A decline in the GSH/GSSG ratio indicated oxidative stress under water shortage. Among secondary metabolites, free hydroxycinnamic acids and flavonoid aglycones accumulated while most glycosylated flavonoids declined, and eight compounds showed genotype-dependent responses in drought conditions. These results reveal accession-specific metabolic adjustments to drought and characterize Croatian wild fennel as a genetic resource with distinct phytochemical profiles relevant to future conservation and breeding.

This review summarizes current scientific knowledge on the use of protein hydrolyzate-based biostimulants in fruit production through evidence mapping, cross-species comparison, and evaluation of protocol-dependent responses within an agronomic framework, centered on foliar applications and their role in sustainable production systems. Research across a broad range of fruit species reports that protein hydrolyzates can significantly enhance yield, improve fruit quality, and mitigate the adverse effects of abiotic stresses such as drought and high temperatures. Treated plants often exhibit improved nutrient uptake, increased photosynthetic efficiency, and enhanced morphological traits, including better root development and vegetative growth. However, the effectiveness of these biostimulants varies depending on the fruit species, developmental stage, and application frequency, indicating the need for more tailored and crop-specific protocols. In conclusion, the literature confirms the functional role of protein hydrolyzates in enhancing resilience and productivity in fruit crops, while highlighting the need for further research to optimize their use under diverse agroecological conditions. Protocol harmonization and robust field validation will be essential for improving the reliability, interpretability, and practical relevance of future research on protein hydrolyzates in fruit production.

Selenium (Se) biofortification of vegetables can improve dietary Se intake; however, the dose-dependent balance between inorganic Se retention and organic Se assimilation following foliar selenate application remains insufficiently resolved across species. Five leafy vegetable species (garden rocket, wild rocket, dandelion, and two chicory cultivars) were grown under controlled greenhouse conditions and treated twice with foliar sodium selenate at increasing application rates (1 + 1, 2 + 2, 5 + 5, 10 + 0, 10 + 10, and 10 + 50 mg Se L⁻¹) across two experiments. Total Se and Se species were determined by HPLC-UV-HG-AFS following enzymatic extraction and cross-checked on selected extracts by HPLC-ICP-MS. Foliar selenate induced substantial Se accumulation in all species, reaching up to 102 µg g⁻¹ DW in garden rocket. At moderate application rates (notably 2 + 2 and 5 + 5 mg Se L⁻¹), a considerable proportion of extracted Se was converted into organic forms, with selenomethionine (SeMet) accounting for up to ~40% of total extracted Se. In contrast, at the highest application rate (10 + 50 mg Se L⁻¹), inorganic Se(VI) became predominant (often >40%), while SeMet proportion declined sharply to ~2–4%, indicating a saturation of metabolic assimilation capacity under high Se exposure. Leaf biomass was promoted at intermediate treatments (e.g., 5 + 5 and 10 + 0/10 + 10 mg Se L⁻¹), whereas the highest rate reduced growth. Overall, foliar selenate effectively biofortifies chicory, rocket, and dandelion leaves, but excessive application rates shift Se speciation toward inorganic storage and markedly suppress SeMet formation. These findings highlight the importance of dose optimization to maximize nutritional quality while avoiding metabolic overload.

Foliar application of fertilizers and bioactive compounds helps viticulture adapt to climate change, while biochar (BC) derived from grapevine pruning residues (GPRs) represents a versatile material that further contributes to climate change mitigation. In this study, the foliar application impact of seven different formulations on the chemical composition and quality of must and wine of Malvazija istarska (Vitis vinifera L.) was investigated. The suspensions contained various combinations of BC, urea, and amino acids. BC increased the pH of the solutions in which it was present due to its alkaline nature, thereby influencing the uptake of nutrients and other compounds. Treatments C (control) and A (amino acids) led to the highest amount of yeast-assimilable nitrogen (YAN) (170 and 172 mg N/L). The amino acid profile of the must differed from the typical composition, with glutamine identified as the predominant compound. The combination of BC with urea and amino acids was associated with a higher sugar concentration in the must compared to the application of BC alone, ranging from 208 to 223 g/L. Combining BC with other components led to wines that received superior sensory evaluation scores compared to both C and B. BC alone did not influence must or wine quality. However, its application in combination with other components makes it a suitable carrier for such compounds. Due to its benefits, easy and cheap production, foliar application of BC suspensions with fertilizers can become a standard operation in viticulture and contribute to sustainable fertilization.

Kale (Brassica oleracea L. var. acephala) has a long history of cultivation, and its importance as a source of mineral nutrients has received particular attention. This study aims to screen the variation in elemental leaf accumulation of 23 Croatian autochthonous kale accessions from the Adriatic basin. A two-factorial experiment with harvest time and kale accession as the main factors was conducted under field conditions. Further, the leaf elemental contents were measured and inter-elemental correlations computed. An analysis of the data revealed that IPT 418 accumulated the highest contents of Ca, Ni, and Li, while IPT 401 was most effective in Mg and Zn accumulation. The accessions IPT 411, IPT 384, and IPT 390 had the highest amounts of P, Mn, and Cr, respectively. Considering the range within the highest and lowest content, the accumulation varied 4-fold for Ca; 1.8-fold for Mg; 1.5-fold for P; 1.5-fold for Mn; 2.5-fold for Ni; 2-fold for Zn; 2.2-fold for Cr; and 1.7-fold for Li. According to the correlation matrix, positive correlations in elemental phytoaccumulation were found between macro- and micronutrients, while negative correlations were observed among macronutrients in most cases. In conclusion, the accessions most efficient in accumulating macro- and micronutrients may be potentially used to address nutrient malnutrition and produce more nutritional kale.

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 pomace (OP), an olive mill byproduct, poses environmental risks if mismanaged due to its high phenolic content, acidic pH, organic load, and electrical conductivity. This study evaluated the impact of olive pomace filtrate (OPF) at varying doses (OP-5, OP-10, OP-15) on broad bean (Vicia faba L.) growth, secondary metabolites, and nutrient accumulation. The highest OPF dose (OP-15) exhibited a clear negative, dose-dependent phytotoxic effect, causing stem discoloration, reduced root growth, necrosis, and chlorosis, while untreated controls showed vigorous growth. This significantly (p < 0.05) reduced leaf development, average number of leaves, and total leaf area, even at the lowest concentration (5%). Consequently, OP-15 reduced dry and fresh biomass by over 50% and shoot/root lengths by up to 61.55% compared to the control. Liquid chromatography mass spectrometry (LC-MS/MS) analysis revealed a positive dose-dependent effect of OPF on beneficial phenol and flavonoid accumulation, with significantly higher amounts of ferulic, isoferulic, caffeic, chlorogenic, and 4-hydroxybenzoic acids, as well as luteolin-4′-rutinoside and 4,7-dihydroxyflavone. OP application significantly (p < 0.05) decreased relative water content and increased electrolyte leakage and malondialdehyde, indicating stress. Furthermore, OP decreased the uptake of K, P, Fe, S, Zn, and Cu. Therefore, the intrinsic phytotoxicity of OPF suggests that mitigation measures are essential before considering environmental application to prevent potential adverse effects on sensitive crops and the wider ecosystem.

Olive pomace (OP) contains phytotoxic compounds that can impair plant growth and soil quality. Composting provides an effective method for detoxifying olive pomace (OP) and improving its suitability for agricultural use. Therefore, this study investigated the phytotoxic effects of raw olive pomace filtrate (OPF) on seed germination in radish (Raphanus sativus L.) and barley (Hordeum vulgare L.), as well as the impact of composted olive pomace (COP) on their growth. Seeds were exposed to OPF at concentrations of 0% (control), 1%, 3%, 5%, 10%, 20%, and 100%. Additionally, three composting treatments were evaluated: R1 (control: OP + barley straw), R2 (OP + barley straw + urea), and R3 (OP + barley straw + sheep litter). Results showed that OPF at concentrations of 10%, 20%, and 100% significantly reduced seed germination, with complete inhibition at concentrations > 10%. The COP treatments showed different physicochemical properties, such as R2 exhibiting better nutrient availability (C/N = 19, oil content = 0.04%). R3 had the highest concentrations of K (40,430.2 mg/kg) and P (6022.68 mg/kg). Results also indicated that R1 significantly reduced radish dry biomass production compared to barley, although R2 performed slightly better than R1 and R3. The findings highlight the need for proper compost stabilization to minimize the phytotoxicity and improve the agricultural potential of COP for improving soil health.