Figure 7. Fruiting-body developmental of wild type, single and double knock-out strains.

(A) Strains were grown on BMM medium for 7 days under natural environmental conditions (low CO₂) or at high CO₂ concentrations (5%). No morphological changes were observed in single and double Δcas1/3 and Δcas2/3 knock-out strains, respectively. Mature perithecia of Δcas1/2 strain in ambient air were not detectable after 7 days. Elevated CO₂ concentrations complemented this phenotype. Scale bar indicates 500 µm (B) CO₂/HCO₃ ⁻ accumulates in the fungal cell. Growth defect of Δcas2 is severely increased when mycelium is grown from germinated single spores (Δcas2_ssi: single spore isolate). BMM plate supplied with 0.5% sodium acetate was either inoculated with Δcas2 mutant strain or with an isolated single ascospore of Δcas2. Plates were incubated with or without CO₂ for three days and images were taken from young mycelia. After prolonged incubation, Δcas2_ssi develops less fruiting-bodies under ambient air than a consecutively transferred mutant strain after nine days of growth. The defect is complemented at 5% CO₂. Scale bar indicates 1 mm (C) Double knock-out mutant strain Δcas1/2 produced only few perithecia after 21 days of growth. Fruiting-bodies exhibit a wild-type phenotype, but are accumulated at several places on the plate. No mature ascospores were discharged from perithecia developed from Δcas1/2 strain.