Biostimulant potential of triacontanol extracted from a legume-derived hydrolysate in Eruca sativa (L.)

Triacontanol (TRIA) is a long-chain aliphatic alcohol naturally occurring in plant epicuticular waxes and used as a biostimulant in agriculture to enhance plant health and yield. However, its effectiveness may vary depending on the source and extraction method. Thus, in this study, three sources of triacontanol were tested: a synthetic product (TRIA0), and two extracts (TRIA1 and TRIA2) obtained from a commercial legume-derived hydrolysate using supercritical CO₂ extraction at different conditions. Following chemical and spectroscopic characterization, the three TRIA samples were tested for their biostimulant effects on hydroponically grown rocket (Eruca sativa L.) plants under controlled conditions. A standardized concentration of TRIA (2 mg L^-1) was applied to assess the effects of the source matrix. The results demonstrated that extraction conditions strongly influenced TRIA efficacy. In particular, TRIA1 was the most effective in increasing the leaf and root fresh weight, SPAD values, nutrient accumulation (especially Ca, Fe, P, and S), and total phenols. By contrast, the higher extraction temperature used for TRIA2 appeared to partially compromise its activity, resulting in weaker physiological and metabolic responses, lower phenolic accumulation, and reduced stimulation of metabolic pathways. Metabolomic analysis evidenced treatment-dependent differences in metabolite profiles, particularly in pathways related to fatty acid, secondary metabolism, stress tolerance and growth regulation. These findings demonstrate the value of metabolomics in elucidating the molecular mechanisms underlying biostimulant action and in identifying key pathways activated by TRIA treatments and highlight the importance of extraction methods in determining TRIA performance and the potential of optimized legume-derived TRIA formulations to enhance crop responses linked to productivity.