**3. Discussion**

With a view to comparing the efficacy of the proposed nanocomposites verses other phenolic-based products against *Phytophthora* spp. discussed in the literature, it should be noted that Kim et al. [37] reported strong fungicidal activities of *Curcuma longa* L. rhizome-derived curcumin in ethyl acetate and hexane fractions against *P. infestans* with 100% and 84% control values at a concentration of 1000 <sup>μ</sup>g·mL−1. Apart from the higher concentrations used in those experiments as compared to the ones presented herein, it should be noted that a contribution of the pronounced cytotoxic activities of the solvents could not be excluded.

Pompimon et al. [38] assessed the anti-*P. capsici* activity of *C. longa* in acetone fraction, finding an inhibition of mycelial growth of ca. 90% at a concentration of 300 <sup>μ</sup>g·mL−1, higher than the EC90 of 184 <sup>μ</sup>g·mL−<sup>1</sup> of the curcumin-based treatment in DES media reported in this study.

The nanocomposites of the four polyphenols in DES medium would also be more active than, for instance, cuminic acid, which featured an EC50 value against mycelial growth of *P. capsici* of 19.7 μg· mL−<sup>1</sup> (which in turn was lower than the EC50 value of other benzoic acid derivatives in previous reports) [39]. Other natural compounds, such as furanocoumarins (e.g., psoralen or isopsoralen) would require concentrations of 500 <sup>μ</sup>g·mL−<sup>1</sup> to attain 82–84% disease control against *P. infestans* [40].

An EC50 value of amphopolycarboxyglycinate-stabilized AgNPs against *P. infestans* of 3.1 <sup>μ</sup>g·mL−<sup>1</sup> was reported by Krutyakov et al. [41], i.e., a 30 and 6 times higher concentration than those obtained for the AgNPs combined with gallic acid, silymarin and ferulic acid inclusion compounds in DES, respectively.

Banik and Pérez-de-Luque [42] found that the integration of copper nanoparticles (CuNPs) with non-nano copper like copper oxychloride, both at a 50 <sup>μ</sup>g·mL−<sup>1</sup> concentration, resulted in a 76% growth inhibition in vitro of the oomycete *P. cinnamomi* as compared to the control. Since in comparative assays between NPs the concentrations of AgNPs are usually 10 times higher than those CuNPs [43,44], the equivalent concentration of AgNPs to attain aforementioned effects should be 500 ca. <sup>μ</sup>g·mL−1, four times higher than that required by the DES treatments to attain a comparable mycelial growth inhibition (ca. 90%).

Chitosan has also been assayed against *P. infestans* [33], finding that concentrations of 500 μg· mL−<sup>1</sup> would be required to fully inhibit mycelial growth, similar to those of the COS-based nanocomposites with gallic acid, ferulic acid and curcumin in this study. On the other hand, *N*-(6-carboxyl cyclohex-3-ene carbonyl) chitosan with different degrees of substitution achieved an EC50 of 298 <sup>μ</sup>g·mL−<sup>1</sup> for *P. infestans* [45], better than the ones for the COS composites based on the three aforementioned polyphenols (in the 450–490 <sup>μ</sup>g·mL−<sup>1</sup> range).

The overall efficacy of the reported nanocomposites should be referred to the combination of the properties afforded by each of its constituents and their synergies.

According to Kim et al. [46], nanosilver may exert an antifungal activity by disrupting the structure of the cell membrane and inhibiting the normal budding process due to the destruction of the membrane integrity. Silver nanoparticles antifungal action may also result from the release of silver ions into the intracellular matrix of the pathogen [47]. Reports on the mechanism of inhibitory action of silver ions on microorganisms have shown that upon treatment with Ag+, DNA loses its ability to replicate, resulting in inactivated expression of ribosomal subunit proteins, as well as certain other cellular proteins and enzymes essential to ATP production. It has also been hypothesized that Ag+ would affect the function of membrane-bound enzymes, such as those in the respiratory chain [4].

Apropos of the role of the stevioside, the improvement in the solubility and bioavailability of the polyphenolic compounds should be ascribed to the formation of a nanocomposite structure comprising a transglycosylated compound, which includes the insoluble compounds. Transglycosylated materials have been reported to self-associate into particular micelle-like structures with a core-shell-like architecture, in which the hydrophobic skeleton is segregated from the aqueous exterior to form a novel drug-loading core, surrounded by a hydrophilic shell of sugar groups [48]. For instance, Kadota, Okamoto, Sato, Onoue, Otsu and Tozuka [24] found a 13000× increase in curcumin solubility when the tri-component system curcumin/*α*-glucosyl stevia/polyvinylpyrrolidone was used.

In relation to the phenolic compounds, they have been reported to have toxic activities against fungi involved in the deterioration of agricultural products by interfering with the development of mycelia [49]. They affect membrane functions such as electron transport, nutrition, enzyme activity, protein and nucleic acid synthesis, and they interact with membrane proteins, causing disruption of the structures and functionality. For instance, curcumin's efficacy would be influenced by its lipophilic nature, which leads to an adequate transmembrane permeability [16]. Its antifungal mechanism has been ascribed to the disruption of plasma membrane integrity, causing leakage of potassium ion from the cytosol and change in membrane potential [50]. On the other hand, gallic acid would exhibit both antioxidant as well as pro-oxidant characteristics, displaying a dual-edge sword behavior, which turns it into an efficient apoptosis inducing agen<sup>t</sup> [51].

Regarding the inhibition mode of chitosan, three mechanisms have been proposed [52]: (1) Its positive charge can interact with negatively charged phospholipid components of fungi membrane, increasing its permeability and causing the leakage of cellular contents, which subsequently leads to cell death; (2) it can act as a chelating agen<sup>t</sup> by binding to trace elements, causing the essential nutrients unavailable for normal growth of fungi; and (3) it may be able to penetrate the cell wall of fungi and bind to its DNA, inhibiting the synthesis of mRNA and, thus, affecting the production of essential proteins and enzymes.

As far as DES are concerned, they would act as a plasticizer, affecting the apparent viscosity of the solutions and enhancing water vapor permeability, water solubility and water sorption capability, as reported by Almeida, Magalh<sup>ã</sup>es, Souza and Gonçalves [27]. Nonetheless, it worth noting that, while choline chloride and urea show no inhibition as individual materials, their final product as ChCl:U DES has been reported to show inhibition towards *Candida cylindracea* [53]. This behavior could be due to the synergistic effect of forming DES [54] and can be used to prove that occasionally DES have a higher toxicological behavior than its original components.

The antimicrobial activity of DES is still not fully understood [55]. Some reports have noted that DES would increase the permeability of the lipid membrane of eukaryotic cells [56,57], as chitosan does. Since the mechanism for COS and DES would tentatively be similar, the differences in the performance of the composites presented herein in terms of fungal growth control should then be ascribed to differences in their ability to solubilize a wide range of solutes (e.g., components in the fungal cell membrane), pH, osmolality or chelation of membrane-bound divalent cations [58].

Since one of the possible mechanisms of action of silver requires that the silver ions enter the fungal cell for efficient killing, the enhancement of permeability driven by COS, DES and polyphenols would support that their interaction should be synergistic rather than simply additive.

From our work, the best results of mycelial growth inhibition at the lowest concentration (125 mg·mL−1) in DES (GI 91.5%), attained for the composite based on gallic acid, may be ascribed to the fact that gallic acid is extremely well absorbed, and very soluble in water as compared with other polyphenols [51]. Moreover, the introduction of the hydroxyl group on the cation in the chloride of choline salt has also been reported to significantly improve the extraction capacity of ionic liquids for gallic acid [59].

#### **4. Materials and Methods**
