Sampling Period Contributes More than Cultivar to Shaping the Olive (Olea europaea L.) Leaf Metabolome: A Full-Season LC-MS/MS Survey

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.