Effect of compound 3 on the production of laccases by B. cinerea 50 ml Erlenmeyer flasks containing 5 ml of minimum medium in the presence of 1% (w/v) of glucose were inoculated with conidia (16106 conidia ml21) and incubated for three days at 22uC in static conditions. After this incubation time, culture media were discarded and fresh minimum medium without glucose containing of 100 mg ml21 of compound 3 or methanol-DMSO, at the same concentration as treatments, was added to mycelium. Cultures were incubated at 22uC and laccase activity was determined in the supernatants. To evaluate laccase activity, syringaldazine was used as the substrate [46]. Enzyme activity was determined spectrophotometrically by monitoring the absorbance at 530 nm. The reaction mixture (1 ml) contained 30 ml of culture filtrates and 0.1 mmol l21 syringaldazine in 50 mmol l21 sodium phosphate buffer (pH 6.0) and was incubated at 22uC for 15 min. One unit per ml of laccase (U ml21) corresponds to the amount of enzyme that increases the absorbance in 0.1 in 1 min. Protein concentration was determined as described [47]. Determination of the effect of compound 3 on the membrane integrity of B. cinerea This was determined using the SYTOX Green uptake assay [48]. B. cinerea conidia at a final concentration of 16105 conidia ml21 were inoculated in 24-well plates (lined with 12-mm glass coverslips) containing 1 ml of liquid minimum medium. Cultures were incubated at 22uC for 15 h to permit the germination of the conidia. After this time, liquid medium was removed and same medium with 70% (v/v) ethanol (positive control), 8% (v/v) methanol-DMSO (negative control), or 20, 40 and 100 mg ml21 of compound 3 was added to each well. The mixtures were incubated at 22uC for one, four and six hours in the case of compound 3 and methanol-DMSO or for 10 min when ethanol was used. B. cinerea hyphae adhered to glass coverslips were washed three times with liquid minimum medium and were stained with 50 nmol l21 SYTOX Green (Molecular Probes, Eugene, OR, USA). After 10 min of incubation, the hyphae were washed with minimum medium and glass coverslips containing hyphae were mounted in slides. For the assembly of the samples in the slides, 15 ml of DABCO (1,4-diazabicyclo[2.2.2]octane) was used. The fluorescence of B. cinerea hyphae stained with SYTOX Green was observed under a confocal microscope (Carl Zeiss LSM 510) at an excitation wavelength of 488 nm and an emission wavelength of 540 nm. These experiments were done at least in triplicate. Determination of the effect of compound 3 on the oxygen consumption of B. cinerea conidia Oxygen consumption was determined polarographically at 25uC with a Hansatech oxygen electrode by using germinating conidia in a total volume of 1 ml. To obtain conidia in suspension, Murashige and Skoog’s basal medium at 4.4 g l21 (Phytotechnol- ogy Laboratories, Lenexa, KS, USA) was added to Petri dishes containing conidia. The conidia were harvested by scraping with a sterile spatula. To eliminate mycelium, the suspension was filtered through glass wool. The conidia concentration was adjusted to 16107 conidia ml21 with minimum liquid media, in the presence of 2% (w/v) glucose. Conidia were incubated for 2 hours at 22uC. The measurement of basal oxygen consumption was carried out for 2 min in the same minimum liquid medium. After this time, 0.05 M carbonyl cyanide m-chlorophenylhydra- zone (CCCP), 10 mmol l21 KCN or the compound 3, dissolved in methanol-DMSO (24:1) at a final concentration of 40 or 100 mg ml21 were added. Control contained methanol-DMSO Antifungal Resveratrol Derivatives at the same concentration as treatments. Oxygen consumption was determined for eight more minutes. These experiments were done at least in triplicate. Statistical analyses Data presented in Figures 2, 4 and 6 are expressed as mean 6 SD from at least three independent results. Significant differences were determined using a one way analysis of variance (Microsoft Office Excel 2007). Mean values were separated using the least significant difference test (P,0.05). Theoretical calculations Lipophilicity values were taken from SciFinder database, originally calculated using Advanced Chemistry Development (ACD/Labs) Software V9.04 (1994–2010 ACD/Labs). Geometry optimization for the 3-furyl resveratrol derivatives was performed with theoretical methods using the density functional theory (DFT) program DMol3, implemented in Materials Studio 4.4 (PC platform) from Accelrys (San Diego, USA). The density setting was the general gradient approximation (GGA) [49] and the Becke exchange (BP) functional [50]. A double numeric basis set with polarization functions (DNP) for an all electron calculation was used [51]. The same conditions were employed to calculate frequencies, useful to determine geometries in a minimum of energy, and population charges. In this ab-initio study the approximate co- planarity of the molecules was assumed because resveratrol and its derivatives prefer this resonance stabilized conformation. In this study we do not investigate the mechanism of hydrolysis, which may be demethoxylation [19–20] or demethylation. Therefore, by using the word demethoxylation we mean hydroxyl formation, and do not exclude the demethylation mechanism. Procedure information about figures Figure 2. Compounds were dissolved in methanol-DMSO (24:1). The slides were inoculated with dry conidia, placed in a humid chamber, and incubated at 22uC. Percentages of germination relative to control were determined after 7 h of incubation. Each bar represents the mean of at least three independent experiments 6 standard deviation. Figure 4. Each bar represents the mean of at three independent experiments 6 standard deviation. The final methanol-DMSO concentration was identical in control and treatment assays. Figure 5. The fluorescence of B. cinerea hyphae stained with SYTOX Green was observed under a confocal microscope. These photographs are representatives of five independent experiments. Figure 6. 16107 Conidia ml21 were suspended in minimum liquid media, in the presence of 2% (w/v) glucose. Oxygen concentration was determined in the presence of methanol-DMSO at the same concentration as treatments, compound 3 at 40 or 100 mg ml21, 10 mM KCN or 0.05 M carbonyl cyanide m-chlorophenylhydrazone (CCCP). Percentage of oxygen consumption was determined relative to control in the absence of methanol-DMSO. Supporting Information Figure S1 (TIF) Figure S2 (TIF) Figure S3 (TIF) Figure S4 (TIF) Scheme synthesis of compound 1. Scheme synthesis of compound 2 and 3. H-NMR of compound 1. H-NMR of compound 2. PLoS ONE | www.plosone.org 8 October 2011 | Volume 6 | Issue 10 | e25421