4.3. In Planta Antifungal Activity

Cucumber plants cv. Bamboo in the second leaf stage were used to investigate the ability of geraniol and GNEs to inhibit B. cinerea B05. Plants were transplanted in 100 mL pots, irrigated regularly, and kept under greenhouse conditions at 20–25 °C with a 16/8 h photoperiod cycle and 60–70% RH, without any pesticide or fertilizer application. Eight (8) plants were used for each treatment, and the experiment was repeated thrice.

The pathogen was inoculated according to Elad et al. [68] with slight modifications using droplets (10 μL) of conidial suspension (2 × 10⁶ sp/mL) on the upper leaf part, and the application solution consisted of water: PDB (Potato Dextrose Broth) at 1:1 ratio and 0.1% Tween 20 as a surfactant.

Regarding the treatments of geraniol and GNEs, plants were spayed at concentrations causing 100% inhibition of B. cinerea, calculated based on the EC₅₀ values (470 and 210 μg/mL, respectively). The geraniol spraying solution was prepared by properly diluting the stock solution in water containing 0.1% DMSO and 0.1% Tween 20. Symptom evaluation was conducted daily for 96 h. GNEs were added as an oil-in-water solution.

Non-treated plants and plants treated with Luna Experience SC, at the highest recommended dose, were used as a control and a chemical control, respectively. In addition, plants sprayed with sole SDS at the same concentration used for the GNEs synthesis were tested for phytotoxicity.

The extent of the disease caused by B. cinerea on the leaves was evaluated according to a disease index (DI) based on symptom development as follows: 1: no symptom; 2: 1–12% of droplet area necrotic; 3: 13–25% necrosis; 4: 26–50% necrosis; 5: 51–100% necrosis; 6: necrotic area exceeding droplet diameter by up to 1 mm; 7: necrosis exceeding droplet diameter by 1–3 mm; 8: necrosis exceeding droplet diameter by more than 3 mm [68] (Figure 8).

Indicative symptoms demonstrating the extent of disease caused by B. cinerea on cucumber leaves according to disease index (DI): 1: no symptom; 2: 1–12% of droplet area necrotic; 3: 13–25% necrosis; 4: 26–50% necrosis; 5: 51–100% necrosis; 6: necrotic area exceeding droplet diameter by up to 1 mm; 7: necrosis exceeding droplet diameter by 1–3 mm; 8: necrosis exceeding droplet diameter by more than 3 mm [68].