Table 1. Effect of compounds on in vitro mycelial growth of B. cinerea. Compounds Compound 1 Compound 2 Compound 3 Resveratrol Pterostilbene LogPa 4.0 4.4 4.3 3.0 4.1 ED50b±SD(mg ml21) 71.664.5 42.065.4 16.663.1 35066.9 10063.7 aEstimated lipophilicity values and sigma, calculated using Advanced Chemistry Development (ACD/Labs) Software V9.04 (1994–2010 ACD/Labs), as stored in SciFinder. bDetermination of median effective doses (ED50), based on colony diameter measurements after 96 h of incubation. doi:10.1371/journal.pone.0025421.t001 Effect of the compound 3 on the production of laccases by B. cinerea The effect of compound 3 on the production of laccases by B. Cinerea was determined by inoculating pre-grown mycelium in minimum media containing compound 3 (Figure 4A); results showed increased production of laccases compared to the control group inoculated with just the solvent at the same concentration as treatment. Since the addition of compound 3 to the culture showed a difference in the mycelium growth, the enzymatic activity after six days of incubation was also expressed as U mg21 of protein (Figure 4B). The enzymatic activity, in the presence of compound 3, was twice that produced by the control. Effect of compound 3 on the cytoplasmatic membrane and on respiratory chain of B. cinerea Since reports that some stilbenes can interact with fungal membranes are known [10], the effect of compound 3 on the plasma membrane of B. cinerea was analyzed using Sytox Green staining (Figure 5). In the negative control, (methanol-DMSO), nuclei exhibit no fluorescence (Figure 5A), while when hyphae were treated with ethanol (positive control) fluorescent nuclei are observed, indicating alteration of the membrane integrity (Figure 5B). Treatment with Antifungal Resveratrol Derivatives compound 3 produced alteration of the B. cinerea plasma membrane after 6 h of incubation (Figures 5C–5E); after 4 h of incubation, an unspecific stain was observed, which did not correspond to nuclei fluorescence. In addition, resveratrol is known to inhibit the respiratory chain [8,12] by affecting the rotary mechanism of F1-ATPase [21]. For this reason, the effect of compound 3 on the oxygen consumption of germinating conidia of this fungus was also analyzed (Figure 6). KCN, an inhibitor of the respiratory chain, and carbonyl cyanide m-chlorophenylhydrazone (CCCP), an uncoupler of the oxidative phosphorylation, were used as controls. In the presence of KCN, oxygen consumption decreased to 50%; KCN does not completely inhibit the oxygen consumption of B. cinerea conidia because this fungus contains a constitutive alternative oxidase [22]. The uncoupling compound CCCP increased oxygen consumption up to 250% while compound 3 at 40 mg ml21 did not affect the oxygen consumption, although, at 100 mg ml21 a slight increase of oxygen consumption was observed. In this study, three 2-furyl derivatives of resveratrol with one, two and three methoxy groups were tested on B. cinerea isolate T50 and the results were compared to that obtained with resveratrol and the closely related dimethoxylated-resveratrol species, pterostilbene (3,5-dimethoxy-49-hydroxy-trans-stilbene). Earlier studies show that pterostilbene is more active against B. cinerea than resveratrol on the mycelia growth of B. Cinerea [5]. Besides confirming these results, our study also demonstrates that compound 3 with two meta methoxy groups shows higher antifungal activity than mono- or tri- methoxylated furyl derivatives. The two dimethoxylated stilbene derivatives had different effects on B. cinerea: compound 3 was more active on mycelial growth, whereas pterostilbene was a more effective inhibitor of conidia germination. Therefore, the presence of two meta methoxy groups in stilbene seems to be an important factor for antifungal activity against B. cinerea while the presence of furyl or phenolic groups determines the target of action of these compounds. The high antifungal activity of compound 3 against B. cinerea can be explained through a fungal-mediated demethoxylation reaction to generate the aromatic hydroxyl groups that are considered to be important structural features in stilbenes for free radical scavenging and prooxidant activity [23]. Our theoretical DFT calculations seem to support the production of these reactive hydroxyl groups Figure 2. Effect of the compounds on conidial germination of B. cinerea. From left to right: Resveratrol, pterostilbene, compound 1, compound 2 and compound 3, see also ‘‘Materials and methods section k Procedure information about figures’’. Different letters indicate that the means are significantly different at P,0.05. doi:10.1371/journal.pone.0025421.g002 PLoS ONE | www.plosone.org 3 October 2011 | Volume 6 | Issue 10 | e25421