Antifungal Activity of Resveratrol against Botrytis cinerea Is Improved Using 2-Furyl Derivatives Francesco Caruso1*, Leonora Mendoza2, Paulo Castro2, Milena Cotoras2, Maria Aguirre2, Betty Matsuhiro2, Mauricio Isaacs3, Miriam Rossi4*, Angela Viglianti5, Roberto Antonioletti1 1 Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, University of Rome, Istituto Chimico, Rome, Italy, 2 Facultad de Qu´ımica y Biolog´ıa, Universidad de Santiago de Chile, Santiago, Chile, 3 Facultad de Ciencias, Departamento de Qu´ımica, Universidad de Chile, Santiago, Chile, 4 Vassar College, Department of Chemistry, Poughkeepsie, New York, United States of America, 5 University of Rome, Istituto Chimico, Rome, Italy Abstract The antifungal effect of three furyl compounds closely related to resveratrol, (E)-3,4,5-trimethoxy-b-(2-furyl)-styrene (1), (E)- 4-methoxy-b-(2-furyl)-styrene (2) and (E)-3,5-dimethoxy-b-(2-furyl)-styrene (3) against Botrytis cinerea was analyzed. The inhibitory effect, at 100 mg ml21 of compounds 1, 2, 3 and resveratrol on conidia germination, was determined to be about 70%, while at the same concentration pterostilbene (a dimethoxyl derivative of resveratrol) produced complete inhibition. The title compounds were more fungitoxic towards in vitro mycelial growth than resveratrol and pterostilbene. Compound 3 was the most active and a potential explanation of this feature is given using density functional theory (DFT) calculations on the demethoxylation/demethylation process. Compound 3 was further evaluated for its effects on laccase production, oxygen consumption and membrane integrity of B. cinerea. An increase of the laccase activity was observed in the presence of compound 3 and, using Sytox Green nucleic acid stain, it was demonstrated that this compound altered B. cinerea membrane. Finally, compound 3 partially affected conidia respiration. Citation: Caruso F, Mendoza L, Castro P, Cotoras M, Aguirre M, et al. (2011) Antifungal Activity of Resveratrol against Botrytis cinerea Is Improved Using 2-Furyl Derivatives. PLoS ONE 6(10): e25421. doi:10.1371/journal.pone.0025421 Editor: Matthew H. Todd, University of Sydney, Australia Received January 28, 2011; Accepted September 5, 2011; Published October 11, 2011 Copyright: 2011 Caruso et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Consiglio Nazionale delle Ricerche, Rome, Italy; Departamento de Investigaciones Cient´ıficas y Tecnolo´ gicas (DICYT) at Universidad de Santiago de Chile; the International Foundation for Science Grants Nu F/3115-1 (LM) and C/2807-2 (MC), and FONDECYT N 1090723 (LM, MC and FC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: caruso@vassar.edu (FC); rossi@vassar.edu (MR) Introduction Botrytis cinerea is a facultative phytopathogenic fungus that attacks flowers, fruits, leaves, and stems of more than 200 plant species causing several pre- and post-harvest diseases [1]. The continuous use of commercial fungicides such as dicarboximide and benzimidazole has caused the appearance of highly resistant strains of B. cinerea and the contamination of soil and water [2]. However, some natural products isolated from plants exert antifungal activity and could be good alternatives to commercial fungicides [3]. Some plant secondary metabolites, stilbenes, have received considerable interest in this area also because of their role in human health [4]. The resistance of Vitis vinifera grape to infection by B. cinerea is due, in part, to the plant response through production of several stilbenes including resveratrol, pterostilbene and e-viniferin, a resveratrol dehydrodimer [5]. These compounds have been tested in vitro against B. cinerea and results show that pterostilbene inhibits conidia germination and in vitro mycelia growth of B. cinerea more efficiently than resveratrol, indicating that methylation of hydroxyphenyl groups is important to the antifungal activity [5]. Schouten et al. demonstrated that resveratrol, while not toxic to B. cinerea, is converted into a fungitoxic compound by a specific laccase of this fungus that then causes self-intoxication [6]. Laccases are copper-containing polyphenol oxidases that catalyze the oxidation of phenolic compounds and the reduction of molecular oxygen into water [7]. The mechanism of action of these stilbenes against B.cinerea is not well understood. It has been suggested that resveratrol inhibits the respiration of fungal cells, probably by acting as an uncoupling agent [8]. Another explanation for the mode of action of hydroxystilbenes may involve membrane peroxidation [9]. Pterosilbene causes destruction of the endoplasmic reticulum and the nuclear and mitochondrial membranes in B. cinerea dormant conidia [10]. A positive correlation among antifungal activity of natural and synthetic stilbenes and their hydrophobicity was found [11] suggesting that pterostilbene is more active than the less hydrophobic resveratrol, due to its increased diffusion through the cytoplasmic membrane. In vitro evidence suggests that pathogenicity of B. cinerea strains in grapevines is associated with their ability to degrade pterostilbene and resveratrol [12]; those that metabolize stilbenes by a laccase- like stilbene oxidase [12–13] were more pathogenic to the grapevine than those that do not. Of three different B. cinerea laccase genes that have been characterized, Bclcc1, Bclcc2 and Bclcc3, resveratrol induces only Bclcc2 expression in liquid culture, suggesting that this gene participates in the metabolic oxidation of stilbene derivatives [6]. Since co-planarity between rings and the connecting double bond in stilbene based compounds, like resveratrol, seems to be a central feature for other biological activities [14–15], we were interested in exploring whether this feature contributed to the antifungal mechanism by choosing three coplanar furyl derivatives PLoS ONE | www.plosone.org 1 October 2011 | Volume 6 | Issue 10 | e25421