2.3. Inoculation Methodology

Four methods were used to inoculate seedlings with T. paradoxa: drip, infiltration, cutting, and direct contact with cultured agar blocks. The seedlings were maintained for 15 days under control conditions, monitoring temperature and humidity using a data logger thermohygrometer. Humidity was held at 80–100%, and the temperature was maintained at 26–28 °C, from the moment of the inoculation. (i) Inoculation with Mycelium blocks: agar blocks containing mycelium were placed at the base of the third leaf, which had previously been given a 2 mm diameter round surface wound with a pipette tip. The agar blocks were covered with parafilm to guarantee humid conditions. Sterile agar blocks were used as controls. (ii) Local Infiltration: a hypodermic syringe was used for infiltrating 0.1 milliliters of endoconidial suspension at the base of the third leaf. The infiltration buffer was used as the control. (iii) Cutting of the Leaf: the apex of the third leaf was cut using scissors previously soaked in the endoconidial suspension. For control, scissors were soaked in a buffer solution. (iv) Drip Inoculation: one milliliter of endoconidial suspension was drip-applied at the base of the third leaf, which had previously been given a superficial wound. A buffer drip was used as a control.

In the dose-response trial, the disease was assessed 15 days after fungal inoculation at 10⁴, 10⁵, and 10⁶ endoconidia mL⁻¹. Each seedling was considered a replicate, with three replicates per treatment. The complete experiment was repeated three times.

In the time-disease trial, symptom development was assessed at 0, 24, 48, 72, 96, 120, 240, and 360 h post-infection (hpi).