Bacteriological agar

To assess how the fungal strains respond to cell wall stressors, 10 µL of a solution containing 10⁶ conidia/mL from the WT and Δepl2 strains of T. reesei were inoculated on the center of minimal media (MM) plates (1 g/L MgSO₄·7H₂O (Synth, Diadema, SP, Brazil), 10 g/L KH₂PO₄ (Synth, Brazil), 6 g/L (NH₄)₂SO₄ (Synth, Diadema, SP, Brazil), 3 g/L Na₃C₆H₅O₇ (Synth, Diadema, SP, Brazil), 20 mL/L trace elements, and 20 g/L bacteriological agar (Kasvi, Madrid, Spain)) with 2% glucose (Synth, Diadema, SP, Brazil) in the absence or presence of different concentrations of Calcofluor White (CFW) (Sigma-Aldrich, St. Louis, MO, USA) (20 and 40 µg/mL) or Congo Red (CR) (Sigma-Aldrich, St. Louis, MO, USA) (100 and 200 µg/mL). After incubation for 3 days, the mycelia diameter under the indicated conditions was recorded. Three replicates of each experiment were conducted. Statistical tests were performed using one-way analysis of variance (ANOVA) followed by Bonferroni’s test (available in Prism software v8.0) for comparing the growth of the parental and mutant strains. CFW and CR stains were used for chitin and β-1,3-glucan; the former interacts preferentially with chitin, and both interfere with cell wall assembly and integrity [34 (link)]. The use of these stressors allows the study of fungi strains with a role in cell wall maintenance.