On a hydrophobic polypropylene surface, conidia germinated to 90%. Neither the single nor the double nor the triple hydrophobin mutants showed any difference in their germination behaviour when compared to the wild type (Figure 3A). To test the viability of the conidia under long term storage conditions, they were incubated for up to 12 weeks at 20°C and 32% humidity in the dark. Samples were taken at regular intervals,

and tested for germination of the conidia in full medium. No significant decrease in germination rates were observed for any of the mutant strains find more within this time period (data not shown), indicating that hydrophobin mutants of B. learn more cinerea do not display obvious defects in conidial viability. Figure 3 Phenotypic characterisation of hydrophobin mutants. A: Germination rates under different conditions, 24 h.p.i. I, II, II: Three transformants of hydrophobin triple mutant. Standard deviations are shown. B: Sclerotia formation on Gamborg agar plates. C: Germinated sclerotia with conidiophores and macroconidia (scale bar: 3 mm). D: Microconidia (hollow arrows) produced on phialides (filled arrows). Phialides were observed on branching hyphae and on macroconidia of B05.10 and the triple mutant (scale bar: 10 μm). E: Penetration into heat-killed onion epidermal cell layers

(16 h.p.i). Fungal structures at the epidermal surface were stained with trypan blue (scale bar: 25 μm). F: Lesion formation on detached tomato leaves. Standard deviations are shown. G: Wettability test with water and 0.2% SDS on non-sporulating mycelia. Pictures LY2835219 datasheet were taken after 3 h. H: Wettability test with SDS solutions on sporulating aerial mycelia. Pictures were taken after 7 h. The mutants Δbhp2, Δbhp3/bhp1 and Δbhp3/bhp2, were also tested in a radial growth assay on TMA and Gamborg glucose agar, in the presence of high temperature stress (28°C on TMA), and under salt stress (0.5 M NaCl in Gamborg

glucose agar). Again, no differences in growth rates of hydrophobin mutants and the wild type strain were observed (data not shown). In Verticillium dahliae, the class II hydrophobin VdhI has been described to be required for microsclerotia formation Sulfite dehydrogenase [17]. The increased expression of bhp2 in sclerotia indicated that it could play a role in sclerotia formation or function. To induce sclerotia formation in the wild type strain and the hydrophobin mutants, conidial suspensions were inoculated on Gamborg glucose agar and incubated for 28 days in the dark. As shown for the hydrophobin triple mutant in Figure 3B, all of the hydrophobin mutants produced sclerotia in similar size and number as the wild type. When water droplets were applied to wild type and mutant sclerotia, they remained on the surface, indicating a hydrophobic nature of the sclerotial surface (not shown).