Improving the nutritional quality of food through advanced and sustainable agricultural practices has become a key objective of modern vegetable crop production. Emphasis is placed on increasing the content of health-promoting bioactive compounds, such as vitamins and polyphenols, particularly flavonoids whose accumulation is strongly affected by various biotic and abiotic stress factors. To mitigate stress-induced limitations and enhance plant performance, biostimulants are increasingly applied. Among them, Trichoderma spp. are widely recognized for their ability to promote plant growth and resilience, primarily through enzymatic activity and the production of bioactive metabolites. The aim of this study was to evaluate the potential of the native Trichoderma sp. strain STP8 to enhance the production of bioactive compounds through seed and soil applications at planting and 26 days after planting (DAP), applied individually or in combination. A spore suspension (4 × 106 spores mL−1) was used. The experiment was arranged in a randomized complete block design with five replicates. At harvest (43 DAP), dry matter, ascorbic acid, chlorophyll, and carotenoid contents were determined. Additionally, flavonoids and non-flavonoids, total phenolics, individual phenolic compounds, and antioxidant capacity were analyzed. Achieved results demonstrate that the effects of the native Trichoderma sp. strain STP8 on lettuce secondary metabolism and antioxidant properties are strongly dependent on the developmental stage at which inoculation is performed, providing further insight into the stage-specific interactions between plans and Trichoderma. Practically, a single application at planting proved to be the most effective strategy for enhancing the accumulation of bioactive compounds, indicating that optimized application timing may improve the efficacy of Trichoderma-based biostimulants, while avoiding unnecessary repeated applications. These findings support the potential use of native Trichoderma strains as sustainable tools for improving the nutritional and functional quality of lettuce. Further research integrating physiological, biochemical, and molecular analyses is required to elucidate the mechanisms by which the native Trichoderma sp. strain STP8 regulates the biosynthesis of bioactive compounds in lettuce.
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