4.7. Evaluation of Effectiveness of T. asperellum against Fungal Pathogens in Tomato Plants

Roots of plants grown for 3 weeks in media with and without T. asperellum formulation were cut about 1 cm from the tips, and the roots were placed during 5 min in the F. oxysporum suspension (1 × 10⁵ conidia mL⁻¹). Control plants were mock-inoculated with sterile distilled water. Disease progression was followed through photograph records of symptoms 3 weeks post-inoculation (wpi). Plant fresh weight was recorded at 1 and 3 wpi.

Disease severity was calculated by the number of leaves showing different levels of wilt symptom and expressed as percentage in reference of total number of leaves per plant. Symptoms were recorded at 1 and 3 wpi. Phenotypic analysis, disease severity and loss weight assays were repeated three times with 10 replicates per treatment.

To explore disease progression, a colonization assay was performed in non- and pretreated plants with T. asperellum for 3 weeks and infected with F. oxysporum for 3 weeks.

Stem sections were collected from cotyledon node, first and second nodes. Sections were surface sterilized in 70% ethanol and rinsed in sterile distilled water and placed on PDA supplemented with 200 mg/mL streptomycin at 25 °C. Photographs were taken after 5 days of incubation on PDA. The experiment was repeated three times.

The B. cinerea strain was routinely cultivated in Petri dishes containing PDA. For inoculation, detached leaves from 5-week-old plants were laid on Petri dishes containing two blotting filter papers (Whatman) wetted with sterile water, then spotted with 5-mm-diameter agar plugs containing growing hyphae from B. cinerea [52]. Lesions were measured using the ImageJ software employing a calibration scale [50] at 1 and 2 wpi. Experiments were repeated three times with 10 replicates per treatment.