**2. Results**

The antifungal activity of the different treatments in aforementioned two preparation media (COS in hydroalcoholic solution and DES) against *P. cinnamomi* (isolate MYC43) was studied in vitro by monitoring the radial growth of the mycelium (Figures 1 and 2).

**Figure 1.** *Phytophthora cinnamomi* growth inhibition assays with the nanocomposites based on the chitosan oligomers in hydroalcoholic solution preparation medium. *From left to right*: Control (no treatment) and treatments with AgNPs combined with gallic acid, silymarin, ferulic acid and curcumin inclusion compounds. *From top to bottom*: 125 <sup>μ</sup>g·mL−1, 250 <sup>μ</sup>g·mL−1, and 500 <sup>μ</sup>g·mL−<sup>1</sup> concentrations. Only one repetition per treatment is shown.

**Figure 2.** *P. cinnamomi* growth inhibition assays with the nanocomposites based on the deep eutectic solvent preparation medium. *From left to right*: Control (no treatment) and treatments with AgNPs combined with gallic acid, silymarin, ferulic acid and curcumin inclusion compounds. *From top to bottom*: 125 <sup>μ</sup>g·mL−1, 250 <sup>μ</sup>g·mL−<sup>1</sup> and 500 <sup>μ</sup>g·mL−<sup>1</sup> concentrations. Only one repetition per treatment is shown. The blue background in the control and curcumin-treated samples is due to the blue color of the paper on which the plates were on.

As shown in Figure 3, the increase in the concentration of the inclusion complexes from 125 <sup>μ</sup>g·mL−<sup>1</sup> to 500 <sup>μ</sup>g·mL−<sup>1</sup> resulted in a reduction in the radial growth of the mycelium in all cases. It may be observed that 100% mycelial growth inhibition occurred with the COS medium at the highest concentration of 500 <sup>μ</sup>g·mL−<sup>1</sup> for the composites with gallic acid, ferulic acid and curcumin (but not for silymarin). On the other hand, at lower concentrations (125 and 250 <sup>μ</sup>g·mL−1), silymarin and ferulic acid-based treatments were more effective than those based on gallic acid and curcumin.

As regards the nanocomposites with a DES preparation medium, total inhibitory activities were attained for the composites based on the four polyphenols under study concentrations of 250, and 500 <sup>μ</sup>g·mL−1. Further, at the lowest concentration of 125 <sup>μ</sup>g·mL−1, the antifungal performance of the composites in the DES medium was close to 90% for all polyphenols. Thus, the product efficacies were clearly higher than those of the composites based on the hydroalcoholic solution of COS.

Upon comparison with the treatments without phenolic inclusion compounds, it could be observed that the AgNPs-only treatment attained a lower inhibition than the composites for the COS medium, with mycelium growth even at the highest dose. On the other hand, the DES-based AgNPs-only treatment performed noticeably better than its COS counterpart, albeit it did not completely inhibit growth at a concentration of 250 mg·mL−<sup>1</sup> (whereas all the composites did). Therefore, an enhanced fungal growth control activity of the ternary mixtures was evidenced.

**Figure 3.** Radial growth values of *P. cinnamomi* in the presence of the composites, which consisted of different polyphenol inclusion compounds and silver nanoparticles at different concentrations, either in a chitosan hydroalcoholic solution (COS) or in a deep eutectic solvent (DES). A control (no treatment) and two treatments with AgNPs (in COS and DES) without the inclusion compounds are shown for comparison purposes. Error bars represent the standard deviation across three replicates.

The results from the sensitivity tests for *P. cinnamomi* may also be expressed with the help of EC50 and EC90 indicators (Table 1). The sensitivity of the isolate mainly varied as a function of the preparation media, but also according to the phenolic compound used. In line with the discussion presented above, *P. cinnamomi* (MYC43) was found to be remarkably more sensitive to the treatments prepared in DES, with EC50 values ranging from 0.1 to 8.9 <sup>μ</sup>g·mL−<sup>1</sup> and EC90 values between 77.9 and 184.3 <sup>μ</sup>g·mL−1. For comparison purposes, for the COS in hydroalcoholic solution medium treatments the EC50 values ranged from 171.6 to 279.9 <sup>μ</sup>g·mL−<sup>1</sup> and those of EC90 from 450.4 to 963.7 <sup>μ</sup>g·mL−1.


**Table 1.** Effective concentrations that inhibited mycelial growth by 50% and 90% (EC50 and EC90, respectively).

The highest sensitivity of the *P. cinnamomi* isolate (MYC43) corresponded to the inclusion compound with gallic acid in DES (EC50 = 0.1 <sup>μ</sup>g·mL−1), followed by the inclusion compounds with silymarin and ferulic acid, and finally by the one with curcumin, with EC50 values of 0.6, 0.6 and 8.9 <sup>μ</sup>g·mL−1, respectively.
