Antifungal Activity against Botryosphaeriaceae Fungi of the Hydro-Methanolic Extract of Silybum marianum Capitula Conjugated with Stevioside

Abstract

Silybum marianum (L.) Gaertn, viz. milk thistle, has been the focus of research efforts in the past few years, albeit almost exclusively restricted to the medicinal properties of its fruits (achenes). Given that other milk thistle plant organs and tissues have been scarcely investigated for the presence of bioactive compounds, in this study, we present a phytochemical analysis of the extracts of S. marianum capitula during the flowering phenological stage (stage 67). Gas chromatography-mass spectroscopy results evidenced the presence of high contents of coniferyl alcohol (47.4%), and secondarily of ferulic acid ester, opening a new valorization strategy of this plant based on the former high-added-value component. Moreover, the application of the hydro-methanolic extracts as an antifungal agent has been also explored. Specifically, their activity against three fungal species responsible for the so-called Botryosphaeria dieback of grapevine (Neofusicoccum parvum, Dothiorella viticola and Diplodia seriata) has been assayed both in vitro and in vivo. From the mycelial growth inhibition assays, the best results (EC₉₀ values of 303, 366, and 355 μg·mL^-1 for N. parvum, D. viticola, and D. seriata, respectively) were not obtained for the hydroalcoholic extract alone, but after its conjugation with stevioside, which resulted in a strong synergistic behavior. Greenhouse experiments confirmed the efficacy of the conjugated complexes, pointing to the potential of the combination of milk thistle extracts with stevioside as a promising plant protection product in organic Viticulture.

Keywords: coniferyl alcohol; ferulic acid; grapevine trunk diseases; milk thistle; stevioside.

Figure 1

Colony growth measures of (a) N. parvum, (b) D. viticola, and (c) D. seriata strains when cultured in PDA plates containing the various control products (viz. stevioside, S. marianum hydromethanolic extract, coniferyl alcohol, ferulic acid, stevioside–S. marianum, stevioside–coniferyl alcohol and stevioside–ferulic acid conjugate complexes) at concentrations in the 62.5–1500 μg·mL⁻¹ range. The same letters above concentrations indicate that they are not significantly different at p < 0.05. Error bars represent standard deviations.

Figure 2

(a) Highly efficient enzymatic cascade engineered for biotransformation of eugenol to silybin and isosilybin. Adapted from [44]. (b) Schematic representation of the non-β-oxidative pathway for conversion of ferulic acid into vanillin, according to [51].