The Pistoia Mountains exhibit a great variety of flora, particularly rich in aromatic plants, such as juniper, mint, savory, helichrysum, and thyme. Thyme is especially notable for its essential oil, typically displaying high thymol and carvacrol content. While the chemotype of thyme determined by its specific terpene composition is genetically controlled, environmental factors, plant age, and seasonality can influence terpene production. This article investigates the morpho-chemical variability of wild thyme plants collected from two different regions of the Pistoia Mountains, identifying five distinct chemotypes. The gas chromatography–mass spectrometry (GC-MS) technique was used to characterize the chemical profiles and determine the seasonal variation in terpene production, identifying spring and summer as the balsamic period, the optimal time for essential oil collection. Furthermore, high-value thyme clones were preserved through in vitro micropropagation, ensuring chemotype stability. These findings contribute to a deeper understanding of wild thyme biodiversity and provide a foundation for practical applications, including the development of value-added products like herb-infused cheeses, plant and animal disease treatments, and integrated pest management strategies in agricultural systems.

The Biolog FF MicroPlates are a powerful tool for assessing fungal metabolic activity and for exploring the metabolic impact of fungicidal or fungistatic agents. Terpenes, key constituents of essential oils, have recently attracted considerable interest as natural antimicrobial compounds. However, two intrinsic properties of terpenes strongly limit their use: high volatility and low aqueous solubility. In this study, we developed a proof-of-concept approach to investigate the indirect action of terpenes on fungal metabolism using Biolog FF MicroPlates. We evaluated the metabolic response of selected fungi to vapor-phase exposure of pure terpene and terpene-based cocrystal, a crystalline solid formed by a terpene and another organic molecule, which slows down terpene release. Pure terpene induced an early but transient inhibition of fungal growth, with effect diminishing at later stages. In contrast, cocrystal maintains a prolonged terpene release and action, allowing robust and reproducible assessment of terpene-induced metabolic changes under indirect contact conditions. Principal Component Analysis revealed that while terpene-treated samples initially diverged from controls, their metabolic trajectories partially reconverge over time, whereas cocrystal-treated samples maintained a consistent separation, indicating prolonged metabolic disruption. Quantitative analyses confirmed that terpene exerted stronger inhibitory effects at early time points, whereas the cocrystal became increasingly effective over time, showing statistically significant inhibition across most carbon source classes. Notably, the cocrystal exhibited enhanced suppression of glucoside and nitrogen-containing substrate utilization, suggesting selective interference with key metabolic pathways. This strategy offers a novel platform for studying volatile bioactive compounds on fungal metabolism and increases the terpene applications in different areas of investigation.