2.5. Sporulation and infection tests

The fungal isolates were grown on potato dextrose agar (PDA, AppliChem) and synthetic minimal medium (MM) containing 2% sucrose, 0.1% KH₂PO₄, 0.3% NaNO₃, 0.05% KCl, 0.05% MgSO₄•7H₂O, pH 5.0 (Plaza et al 2013). The spore production were measured from cultures on PDA, each strain was seeded with 0.5 mm circular section of PDA agar with mycelium of the fungus; the isolate was incubated in PDA for 2 weeks at 20ºC under a 24 h photoperiod (12 h light/ 12 h darkness). The spores were collected and filtered with miracloth (Merck, USA) a resuspended in 10 mL water sterile and counted with Neubauer camera. For sclerotia formation, each isolate was seeded with 0.5 mm circular section of PDA agar with mycelium of the fungus in MM for 2 weeks at 17ºC in darkness.

Infection tests of apple fruits were performed as described by Doehlemann et al. (2006). Prior to inoculation, the fruit tissues were wounded with a pinprick of a 21G syringe and surface-sterilized by immersion in 75% ethanol for 1 min. Inoculation of the fruits was performed with 5 μL droplets of 2.5×10⁵ conidia/mL conidial suspensions for 4 days at 20ºC in Percival incubators (Percival, USA). The B05.10 wild-type strain was used as a control strain due its virulence on apple, tomato or grapevine as well as others fruit or plant (Nafisi et al., 2014; Plaza et al., 2015; Zhang et al., 2016; Plaza et al., 2018; Liu et al., 2019). The pathogenicity of B. cinerea isolates in this study was compared to the B05.10 strain.