*2.4. Human Onychomycosis Assay*

Small pieces of nails (ca. 5 mm) without polish were washed with 1.25% (*v*/*v*) NaOCl containing 0.1% (*v*/*v*) Triton X-100 for 5 min. Nails were rinsed three times with sterile water and incubated for 3 days on PDA at 25 ± 2◦C. The non-infected nails were washed three times with sterile water to eliminate any adhered PDA fragments. Nails were placed in a wet chamber (Petri dishes) and the nail edge was infected with 100 μL of work conidia solution. Nails were observed for visible fungal growth from the fourth day onwards until the ninth day (Figure 2).

### *2.5. Fusarium Detection by Confocal Microscopy*

In order to visualize *Fusarium* spp. on the nails, samples were stained with WGA-Alexa Fluor® 488 conjugate (W11261, Life Technologies; CA USA), which binds to the chitin molecules on the fungal cell wall [26]. Samples were incubated for 30 min at room temperature in 1× PBS buffer (137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, pH 7.4) supplemented with 1 ng/μ<sup>L</sup> WGA. For visualization, stained nails were placed on a microscope slide and covered with a glass coverslip. Confocal microscopy (Leica TCS SP5 X) was used with the white laser for 499 nm excitation wavelength and emission ranges of 512–526 nm for WGA (green fluorescence) and 632–739 nm to ge<sup>t</sup> the autofluorescence signal (red fluorescence).

**Figure 1.** Diagrammatic representation of strategy to evaluate the infective capacity of fungi on plants.

**Figure 2.** Diagrammatic presentation of strategy to evaluate the infective capacity of fungi to cause onychomycosis.
