b-Carbonic Anhydrases in Fungi Figure 8. Germination rate of S. macrospora wild type and cas mutant strains. (A) Ascospores were isolated from strains grown in ambient air. Only strain Dcas1/2 grew at 5% CO2 (marked by an asterisk); because it develops mature perithecia with asci and ascospores only at elevated CO2 levels (Figure 7). 100 single spores from 10 different perithecia of wild type and mutant strains were isolated and plated on BMM medium with 0.5% sodium acetate. Ascospores were incubated in ambient air or at 5% CO2. Germinated spores were counted after 2–5 days of incubation. The percentage of germination efficiency was determined for each strain. (B) Mycelia from germinated spores of Dcas2, Dcas1/2 and Dcas2/3 are affected in growth velocity and hyphal density. Ten spores were plated from each strain and images were taken after two days of growth. (C) Germlings of Dcas2 mutants are highly vacuolated. Spores were plated on glass slides overlaid with a thin layer of BMM supplied with 0.5% sodium acetate and incubated for one day. Vacuoles in hyphae of Dcas2 are marked by white arrows Scale bar indicates 50 mm. doi:10.1371/journal.pone.0005177.g008 PLoS ONE | www.plosone.org 9 April 2009 | Volume 4 | Issue 4 | e5177