Deciphering the Olive Fruit Volatilome: A Multivariate Approach to Assess Cultivar Variation and Biotic Stress Response in a Changing Agroclimatic Context

Understanding olive tree metabolism and its interactions with biotic and abiotic factors is crucial for the sustainability and resilience of olive cultivation in a changing agroclimatic context. In response to biotic stress, plants activate complex signaling pathways that trigger the production of specialized metabolites, particularly volatile organic compounds (VOCs). This study investigates the volatolomic profile naturally emitted by whole olive fruits using an integrated metabolomic strategy that combines design of experiments (DoE), targeted and untargeted analyses, and multivariate statistics. Optimal headspace solid-phase microextraction (HS-SPME) conditions were established using 30 g of sample, a 50 °C extraction temperature, a 50 min extraction time, and a 3 min injection at 250 °C, identifying extraction time and temperature as the most critical factors influencing VOC recovery. The data demonstrated significant cultivar-dependent variation in the volatile emissions from healthy olive fruit among six representative varieties. Furthermore, robust partial least squares-discriminant analysis (PLS-DA) and random forest models provided a clear separation between healthy and damaged olive fruits, achieving high predictive accuracy (90%) and identifying key volatile biomarkers derived from the lipoxygenase (LOX) pathway. This novel multivariate optimization approach (SPME–GC/MS) represents a powerful tool for establishing a reliable chemical fingerprint of the olive fruit “volatilome” under evolving agroclimatic challenges.