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Antibiotics 2018, 7, 76 8 of 12 y el Carbón Vegetal, Spain. The isolate was maintained in potato-dextrose-agar (PDA) slant tubes, supplied by Merck Millipore (Darmstadt, Germany), stored at 4 ◦C. 4.8. Efficacy of the Nanocomposites for the Control of Phytophthora cinnamomi Agar disks (8 mm in diameter) were cut from the margin of a 7-day-old colony growing on PDA and were transferred to a PDA medium supplemented with the nanocomposites at final concentrations of 125 μg·mL−1, 250 μg·mL−1 and 500 μg·mL−1. Three replicates were performed for each treatment. For each active ingredient and concentration, inhibition of radial mycelial growth (mm) compared with the untreated control was evaluated after 7 days of incubation at 24 ◦C, in the dark. The relative growth inhibition (%) of each treatment compared to untreated control was calculated as follows: Growth inhibition (%) = [(dc − dt)/dc] × 100, where dc stands for the average diameter of the fungal colony in the control and dt is the average diameter of the treated colony [63]. Results were expressed as effective concentrations EC50 and EC90 (i.e., the concentrations which reduced growth inhibition by 50% and 90%) by regressing the inhibition of radial mycelial growth values (% control) against the values of the antifungal nanocomposite concentrations. 5. Conclusions Composites consisting of silver nanoparticles and polyphenol inclusion compounds were synthesized using two preparation media, one based on chitosan oligomers in an hydroalcoholic solution and the other based on a deep eutectic solvent. Both types of composites showed an increase in the water solubility of the polyphenols and in the in vitro antifungal activity against Phytophthora cinnamomi (MYC43 isolate). Nonetheless, the DES-based samples efficacy was remarkably higher than that of their counterparts with a chitosan oligomers-based matrix: Complete inhibition of mycelial growth was attained at concentrations of 250 and 500 μg·mL−1, and even at the lowest dose of 125 μg·mL−1, they resulted in a 90% growth inhibition. As regards the impact of the choice of the different polyphenols, for the DES-based treatments, the highest sensitivity of P. cinnamomi corresponded to the composite with gallic acid (EC50 = 0.1 μg·mL−1), followed by those with silymarin and ferulic acid, and finally by the one with curcumin, with EC50 values of 0.6, 0.6 and 8.9 μg·mL−1, respectively. This may be ascribed to the fact that gallic acid is extremely well absorbed, and very soluble in water as compared with other polyphenols. The reported activities for the composites were remarkably higher than those reported for Phytophthora spp. using AgNPs, chitosan or polyphenols separately. This points to the possibility of a successful application of these nanocomposites in agriculture, with the aim of reducing the use of toxic and expensive conventional systemic fungicides. Further research on the ability of the prepared nanocomposites to inhibit growth of P. cinnamomi in the context of a plant infection model and on the basic mechanism involved, fungistatic or fungicidal, is underway. 6. Patents The work reported in this manuscript is related to Spanish patent P201731489. Author Contributions: P.M.-R., M.T.B.-A., E.P.-L. and J.M.-G. designed the experiments; J.M.-G. contributed reagents and conducted the synthesis; P.M.M. and B.M.I. performed the experiments; P.M.M., J.M.-G., M.T.B.-A. and P.M.-R. analyzed the data and wrote the paper. Funding: This research was partly funded by Sistemas Biotecnología y Recursos Naturales. Acknowledgments: P.M.-R. would like to thank Santander Universidades for its financial support through the “Becas Iberoamérica Jóvenes Profesores e Investigadores, España” scholarship program. Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.PDF Image | Silver Nano Polyphenol Inclusion Compounds Composites
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