Plant–Endophyte Cross-Talk in Origanum heracleoticum L. In Vitro Axenic Culture: Endosphere-Driven Bacterial Interactions and Plant Metabolic Responses

: Origanum L. (Lamiaceae) is a commercially important medicinal and aromatic plant genus worldwide. Endophytic bacterial communities are recognized for promoting plant growth and physiology, although their interactions with host metabolism remain insufficiently understood. In this work, an in vitro model of axenic Origanum heracleoticum plants was established to investigate the relationship between endophytic bacteria and their tissue of origin. Specifically, we evaluated the adaptation of two strains, Bacillus sp. OHL2 and Pseudomonas sp. OHS18, and the potential role of Bacillus sp. OHL2 in modulating plant physiology and secondary metabolism. Bacterial inoculation and re-isolation highlighted niche-specific adaptation and possible co-evolution within the host, suggesting an active role of the plant in regulating bacterial colonization within the endosphere. Inoculation with Bacillus sp. OHL2 significantly enhanced photosynthetic rate, leaf area, dry weight, and chlorophyll content. No substantial overall changes in secondary metabolism were detected. Rosmarinic acid was the predominant phenolic, while monoterpenes dominated, with carvacrol dominant. A significant tissue-by-inoculation interaction was observed for α-humulene, which decreased in leaves of inoculated plants. Overall, the in vitro system provides a valuable platform to study plant-endophyte interactions and bacterial mechanisms underlying the stimulation of plant growth and metabolic responses.