Changes in melanin amount in conidia as the age of culture progresses.

<p>Melanin amount in the conidial wall changed with the age of culture as revealed by UV-visible spectrophotometry (<b>A</b>) or electron paramagnetic resonance (<b>B</b>). (<b>A</b>) The mean rank of the quantity of melanin extracted from conidia was significantly different among cultures of different age (<i>p</i><0.05). (<b>B</b>) EPR spectra of <i>P. boydii</i> conidia taken from cultures of different age (5, 9 and 14 days). The spectra were recorded at a microwave power of 30 db. Spectra are representative of three independent experiments.</p

Changes in the physical properties of the conidial surface induced by melanin inhibition.

<p>The surface electronegativity of conidia recovered from cultures grown with pyroquilon was markedly reduced with respect to conidia recovered from control cultures or cultures grown with glyphosate (<i>p = </i>0.0010).</p

Primers for sequencing genes involved in the melanin synthesis pathway.

<p>*Primers carrying the same number represent forward and reverse primers included in the same amplification reaction. Tm of each amplification reaction is indicated in the same row next to the forward primers.</p><p>**IUPAC-IUB symbols for nucleotide nomenclature: M = A or C; Y = C or T; W = A or T; K = G or T; and H = A, C or T.</p

Transmission electron microscopy examination of conidia from cultures grown with or without melanin inhibitors.

<p>Control conidia (<b>A</b>) or conidia recovered from cultures grown with the melanin inhibitors glyphosate (<b>B</b>) or pyroquilon (C) examined by transmission electron microscopy (scale bar = 200 nm).</p

Atomic force microscopy image of the surface of <i>P. boydii</i> conidia.

<p>Conidia recovered from 9-day-old cultures examined by AFM in the tapping mode showed a smooth surface without any rodlet layer of hydrophobins (10 µm×10 µm image).</p

UV-visible spectrophotometry of melanin extracts.

<p>Melanin extracts from control conidia or conidia recovered from cultures grown in the presence of DOPA-melanin (kojic acid or glyphosate) or DHN-melanin (pyroquilon, tricyclazol or carpropamid) inhibitors were examined under UV-visible spectrophotometry. A spectrum similar to synthetic melanin (0.05 mg/ml) was obtained only for extracts from control conidia or conidia produced with DOPA-melanin inhibitors.</p

Analysis of <i>PKSI</i> and <i>4HNR</i> gene sequences and predicted protein sequences.

<p>Alignment of protein and nucleotide sequences for polyketide synthase type I of the fungi (Accession number): <i>Pseudallescheria boydii</i> (KC440182, this work), <i>Colletotrichum lagenaria</i> (BAA18956.1), <i>Verticillium dahliae</i> (EGY13508.1), <i>Magnaporthe oryzae</i> (ELQ39536.1), <i>Ophiostoma piceae</i> (ABD47522.2). Alignment of protein and nucleotide sequences for tetrahydroxynaphtalene reductase of the fungi (Accession number): <i>Pseudallescheria boydii</i> (KJ549637, this work), <i>Colletotrichum orbiculare</i> (AB661336), <i>Ophiostoma floccosum</i> (AF285781), <i>Magnaporthe grisea</i> (AY846877), and <i>Scedosporium prolificans</i> strain 3.1 (JX861395). Protein functional domains were predicted by conserved domain search in NCBI.</p

Fluorescence labeling of cell wall mannan groups in <i>P. boydii</i> conidia.

<p>Conidia recovered from 5-day-old cultures were incubated with concanavalin A conjugated to fluorescein isothiocyanate (Con A-FITC), and examined under fluorescence microscopy. Whereas a few conidia were highly fluorescent (asterisks) and some others were faintly labeled (arrows), the majority exhibited labeling exclusively on their release scar (<b>A</b>) as demonstrated by examination of the same field by phase-contrast microscopy (<b>B</b>). In (<b>C</b>) and (<b>D</b>), conidia incubated with a large excess of the inhibitor methyl α-D-mannopyranoside prior labeling with FITC-Con A showed no fluorescence, thus attesting the specificity of the labeling.</p

Repercussion of increased melanin synthesis on the surface labelling of conidia with concanavalin A.

<p>(<b>A</b>) Trasmission electron microscopy showing conidia labeled with gold-conjugated concanavalin A (Con A; 5-nm gold particle). Scale bar = 200 nm. (<b>B, </b><b>C</b>) Quantitative analysis by flow cytometry of fluorescence at the surface of conidia treated with fluorescein isothiocyanate (FITC)-ConA. (B) Comparison of mean specific fluorescence intensity of conidia recovered from 5-, 9-, or 14-day- old cultures grown with or without glyphosate or pyroquilon. Contrary to conidia with glyphosate treatment, conidia with pyroquilon treatment showed a significant increase in labelling with respect to their counterparts from 9- or 14-day-old control cultures grown without any melanin inhibitor (<i>p</i><0.001). Specific fluorescence was obtained after normalizing against the fluorescence in conidia untreated with FITC-ConA or conidia treated with methyl α-D-mannopyranoside prior to the addition of FITC-Con A. (<b>C</b>) Fluorescence frequency distribution histograms: the blue-lined histogram represents the relative signal of conidia from cultures grown without any melanin inhibitor, whereas the red- or green-lined histograms represent the relative signal of conidia treated with pyroquilon or glyphosate, respectively (number of fungal cells in the y-axis versus relative fluorescence intensity in the x-axis expressed as arbitrary units on a logarithmic scale).</p

Influence of the age of the cultures on the physical surface properties of conidia.

<p>Surface electrostatic charge (<b>A</b>) and cell surface hydrophobicity (<b>B</b>) of conidia recovered from 5-, 9-, and 14-day-old cultures were significantly different among the three groups with a <i>p</i><0.01 for (A) and <i>p</i><0.001 for (B).</p