Evaluation of the physical properties of the conidial surface The conidial cell surface electrostatic charge was assessed by microelectrophoresis with a Zetasizer and the cell surface hydrophobiCity (CSH) was assessed by two-phase partitioning with hexadecane as the hydrocarbon phase or using a two-aqueous phase system. Results showed that the electronegative charge of the conidial surface for mutant isolates was much lower than that of the wild-type strains (Table 5). Likewise, two-phase partitioning showed a decrease in CSH for conidia of pigmentless or brownish isolates. This decreased hydrophobiCity

is consistent with the increased wettability observed during the preparation of conidial suspensions. Table 5 Physical properties of the conidial surface Strain or isolate number Zeta potential (mV) Water/hexadecane (%)1 PEG/dextran2 Reference strains          CBS 113.26

Inhibitor Library mw – 43.8 10 2.37    IHEM 18963 – 39.1 11 2.8 Mutant isolates          IHEM 2508 – 21.5 2 2.04    IHEM 9860 – 26 0.05 1.14    IHEM 15998 – 25.6 2.2 1.8 1 Results are expressed as the percentage of conidia that were excluded from the aqueous phase. 2 Results are expressed as the ratio between the absorbance of the upper phase (rich in PEG and hydrophobic) and that of the lower phase (rich in dextran and hydrophilic) Ultrastructure of the conidial wall visualised by transmission electron microscopy The conidial wall of reference strains was composed of several superimposed layers, with a thick electron transparent inner layer and two

thin electron dense outer layers, the outermost layer being responsible for the ornamentations of the cell Belnacasan research buy wall (Figure 5). However, conidia of mutant isolates, as well as those from reference strains cultivated in the presence of pyroquilon, showed a thinner cell wall devoid of the outermost layer which could sometimes be seen free in the surrounding medium. Figure 5 Ultrastructure learn more of the conidial wall as visualised by transmission electron microscopy. Conidia from reference strains CBS 113.26 (A) and IHEM 18963 (B and C) cultivated in the presence (C) or not (A and B) of pyroquilon 20 μg/mL, or of mutant isolates (D and E: pigmentless isolates IHEM 2508 and 9860; F: brownish isolate IHEM 15998) were processed for ultrastructural examination of their cell wall. Note the smooth surface of the conidia of reference strains cultivated in the presence (C) of pyroquilon and mutant isolates (D, E, F) and the lack of the outermost cell wall layer (arrowheads) which sometimes appears free in the surrounding medium (arrows). Bars correspond to 500 nm. Visualisation of the hydrophobic rodlet layer by atomic force microscopy We also investigated the presence of a hydrophobic rodlet layer on the conidial surface, to provide support for our hypothesis. This protein film is usually composed of about 10-nm thick rodlets of varying length organized into bundles or fascicles, in which individual rodlets lie parallel within a single fascicle.