Potato dextrose agar (pda)

Leaf samples were processed for the isolation of fungal causal agents. The causal fungi were isolated from lesions using a single conidial isolation on 1.0% water agar containing 0.5 mg/l streptomycin under a stereo microscope according to the method described by Choi et al. [67 ]. The isolated plates were incubated at 25 °C for 24–48 h, and the germinated conidia were transferred onto potato dextrose agar (PDA; Conda, Madrid, Spain) containing 0.5 mg/l streptomycin. Pure fungal isolates were deposited in the Culture Collection of SDBR-CMU Laboratory, as previously mentioned.

The capacity of the collected fungal strains to grow in the presence of increasing concentrations of As(V) and Cr(VI) was evaluated on solid cultures. Into Petri dishes (12 cm in diameter), we poured potato dextrose agar (PDA; Condalab, Torrejón de Ardoz, Spain) supplemented with 2.5, 5.0, 7.5, 10.0, or 12.5 g of As⁵⁺ L⁻¹ from sodium arsenate (Na₂HAsO₄·7H₂O; Thermo Fisher Scientific, Kandel, Germany) and 0.1, 0.5, 1.0, 1.5, and 2.5 g of Cr⁶⁺ L⁻¹ from potassium dichromate (K₂Cr₂O₇; Scharlab ExpertQ^®, Sentmenat, Spain). Each BF strain was inoculated six times with an inoculation loop on each agar plate containing a defined concentration of As(V) or Cr(VI). The radial growth of each fungal colony was measured by averaging orthogonal diameters, and this value was then averaged again for all six colonies from every single plate. These measurements were repeated after 7, 15, 30, 45, and 60 days of incubation, which was performed at 25 °C under dark conditions. This prolonged timeframe was established to account for the potential long-term adaptation of slow-growing BF strains. Unamended control plates were also included. The tolerance index (TI) was calculated for every strain by dividing the measured growth when exposed to the metal in relation to the control plates.

Fungal isolations were performed from tissues (stem and crown) of healthy strawberry plants (Fragaria x ananassa) collected in April 2014 from various orchards located in Caserta province, Southern Italy. Tissue fragments (3–4 mm²) were excised with a surgical blade, dipped in 2% (vol/vol) NaClO for 30 s, rinsed twice in sterile distilled water and dried on sterile Whatman filter paper under a laminar flow hood. The samples were placed in 90 mm-diameter Petri dishes containing Potato Dextrose Agar (PDA, Conda, Madrid, Spain), amended with streptomycin sulphate (100 mg/L). The dishes were kept at 24 ± 2 °C in the dark. After 4–5 days of incubation, the fungal colonies emerging on the vegetable fragments were transferred on fresh PDA plates. Single-spore cultures were obtained by spreading 0.2 mL of a suspension containing 1 × 10³ conidia/mL, prepared from 7 days-old plates, on 1% water agar plates. Then, the germinated spores were transferred onto PDA dishes. The growing colonies were observed with an optical microscope to allow for a morphological characterization (colony diameter, colony color and degree of sporulation) (Olympus BH-2, Olympus America, Lake Success, NY, USA).