Olive (Olea europaea L.) leaf phenolics are increasingly valued across various industrial sectors. This study evaluated whether foliar application of copper (Cu), zinc (Zn), and manganese (Mn) can influence phenolic accumulation in olive leaves. One-year-old olive plantlets grown under greenhouse conditions were sprayed with these micronutrients. Samples of treated mature leaves were collected at 30, 60, and 90 days after treatment (DAT), whereas young leaves that developed after application were sampled at 90 DAT. Foliar fertilization significantly influenced the phenolic profile of olive leaves depending on the micronutrient applied, the sampling time, and the age of the leaves. The highest mean oleuropein concentration was recorded at 30 DAT (209.43 mg/100 g DW), driven mainly by the Cu × 30 combination (302.36 mg/100 g DW), whereas 90 DAT showed a significant decrease in most phenolic classes. In young leaves, oleuropein concentrations were an order of magnitude higher (2661–5105 mg/100 g DW) than those measured in mature leaves (14.5–302 mg/100 g DW). Furthermore, correlation analysis revealed nutrient-specific modulation of phenolic metabolism: Cu was positively associated with secoiridoids in mature leaves, Zn was positively associated with secoiridoids at 90 DAT, and Mn was negatively associated with flavonoids and with Cu and Zn at 90 DAT in young leaves. These findings suggest that foliar fertilization affects phenolic pathways differently depending on the applied micronutrient and the leaf developmental stage, providing new insights into the temporal regulation of olive leaf phenolic metabolism and identifying suitable harvest periods for the targeted phenolic compounds.

Olive leaf biomass, generated as a residue of olive cultivation, represents a readily available source of phenolic compounds with recognized nutraceutical potential. However, the composition of these bioactive compounds is influenced by both genetic and environmental factors. Therefore, this study aimed to evaluate the effects of cultivar and sampling period on the biophenolic composition and mineral nutrient content of olive leaves from six cultivars maintained under comparable environmental conditions in central Dalmatia, Croatia. Leaves were collected during the harvest and pruning periods. Phenolic compounds were determined by HPLC and mineral nutrients by ICP-MS, FAAS, and UV/Vis spectrophotometry. Significant cultivar-dependent differences were observed for most analyzed phenolic compounds and mineral nutrients. Cultivar ‘Leccino’ exhibited the highest concentrations of several phenolic compounds and total phenols during the pruning period, while distinct accumulation patterns were recorded among cultivars. Ten of the thirteen individual phenolic compounds showed a significant sampling-period effect; nine were higher during the pruning period, whereas apigenin was higher at harvest. Seven of the ten mineral nutrients were significantly affected by sampling period, all of them decreasing towards pruning. Multivariate analysis identified cultivar as the dominant source of variation in both fractions, and the phenolic and mineral profiles were congruent among cultivars within each sampling period. Higher phenolic accumulation during the pruning period differed in magnitude among cultivars for 15 of the 24 variables. These findings support the potential valorization of olive leaves as a source of bioactive compounds and highlight cultivar and sampling period as important considerations for their targeted utilization.

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.