4.3.5. Conidia Viability *In Vitro*

For each set of experiments, conidial suspensions were obtained from 14-day-old colonies of *V. dahliae* isolate V180 growing on PDA, as described previously, and adjusted to <sup>8</sup> × 105 conidia mL−<sup>1</sup> using a haematocytometer. In parallel, OSE solutions were adjusted to 0, 1, 10, 20, 30, 40, 50, and 100% in sterile deionized distilled water (SDDW). Subsequently, a 5 μL drop of the conidial suspension was placed in the centre of a microscope coverslip (20 × 20 mm); then, a 5-μL drop of the OSE solution was mixed. Thus, the OSE was evaluated at the following final concentrations: 0, 0.5, 5, 10, 15, 20, 25, and 50%, with a concentration of 0% consisting of a 5 μL drop of the conidial suspension mixed with a 5 μL drop of sterile SDDW as a control. The coverslips were placed inside Petri dishes containing water agar, which were used as humid chambers, and were incubated at 23 ± 2 ◦C in the dark for 24 h. After the incubation period, a 5 μL drop of 0.01% acid fuchsine in lactoglycerol (1:2:1 lactic acid:glycerol:water) was added to each coverslip to stop conidial germination, and they were mounted on a slide. For each experiment (*I*, *II*, and *III*), there were three replicated coverslips per concentration of OSE obtained from each block and from each treatment or control (OSE at 0%, i.e., only SDDW). All the experiments were conducted twice.

In all cases, a total of 120 randomly selected conidia per replicated coverslip were observed at a ×400 magnification by means of a Nikon Eclipse 80i microscope (Nikon Corp., Tokyo, Japan), and the germinated and non-germinated conidia were counted. Conidia were considered germinated when the germ tube was at least one-half of the longitudinal axis of the conidia. Conidial viability was estimated as percentage (%) of conidial germination for each OSE concentration, and then, the inhibition of conidial germination (RGI; %) was estimated with respect to the control according to the following formula:

$$\text{RGI} \left( \% \right) = \left[ \text{(Ge}\_{\text{control}} \times \text{Ge}\_{\text{CSE solution}} \text{)} / \text{Ge}\_{\text{control}} \right]$$

where Gecontrol = percentage of germinated conidia after incubation in the SDDW, and GeOSEsolution = percentage of germinated conidia after incubation in the OSE solution from treated plants [36]. The RGI data were linearly regressed over the OSE concentration, and the predicted values of the effective OSE concentrations (μg mL−1) inhibiting 50% (EC50) of conidial germination were obtained from the fitted regressions.
