First Description of Non-Enzymatic Radical-Generating Mechanisms Adopted by Fomitiporia mediterranea: An Unexplored Pathway of the White Rot Agent of the Esca Complex of Diseases

Abstract

Fomitiporia mediterranea (Fmed) is the primary Basidiomycota species causing white rot in European vineyards affected by the Esca complex of diseases (ECD). In the last few years, an increasing number of studies have highlighted the importance of reconsidering the role of Fmed in ECD etiology, justifying an increase in research interest related to Fmed's biomolecular pathogenetic mechanisms. In the context of the current re-evaluation of the binary distinction (brown vs. white rot) between biomolecular decay pathways induced by Basidiomycota species, our research aims to investigate the potential for non-enzymatic mechanisms adopted by Fmed, which is typically described as a white rot fungus. Our results demonstrate how, in liquid culture reproducing nutrient restriction conditions often found in wood, Fmed can produce low molecular weight compounds, the hallmark of the non-enzymatic "chelator-mediated Fenton" (CMF) reaction, originally described for brown rot fungi. CMF reactions can redox cycle with ferric iron, generating hydrogen peroxide and ferrous iron, necessary reactants leading to hydroxyl radical (^•OH) production. These observations led to the conclusion that a non-enzymatic radical-generating CMF-like mechanism may be utilized by Fmed, potentially together with an enzymatic pool, to contribute to degrading wood constituents; moreover, indicating significant variability between strains.

Keywords: CMF; Fmed; GTDs; ferric iron; grapevine; phenolates; •OH.

Figure A1

MS/MS spectrum of compound (k) annotated as hypholomine B.

Figure 1

Dry weight of filtered liquid culture from 6 strains of Fmed (Fm1, Fm2, Fm3, Fm4, Fm5, and Fm6) grown at 27 °C under static conditions for 12 weeks in the dark. Values are the means ± standard deviation (n = 3). Results were analyzed by one-way ANOVA, followed by Fisher’s LSD multiple comparison test (p < 0.05). Different letters indicate significant differences according to multiple comparison test results.

Figure 2

Liquid media pH at the end of the experiment for the 6 strains of Fmed (Fm1, Fm2, Fm3, Fm4, Fm5, and Fm6) and in control (C_(-))flasks. Values are the means ± standard deviation (n = 3). Results were analyzed by Welch’s ANOVA (p < 0.05). Different letters indicate significant differences according to multiple comparison test results.

Figure 3

Total phenols from LMW methanolic extracts analyzed spectrophotometrically using a Folin–Ciocalteu assay. Values (expressed as mmol L⁻¹ gallic acid, GAE) are the means ± standard deviation (n = 3). Results from the negative control (C_(-)) and Fmed average value are shown on the left (nd = not detected). Results from different Fmed strains (Fm1, Fm2, Fm3, Fm4, Fm5, and Fm6) were analyzed by one-way ANOVA, followed by Fisher’s LSD multiple comparison test (p < 0.05). Different letters indicate significant differences according to multiple comparison test results.

Figure 4

Ferrous iron (expressed as µmol L⁻¹ of Fe²⁺) reduced from ferric iron (A) via the fungal secreted LMWCs and H₂O₂ (expressed as µmol L⁻¹) generated by redox cycling of LMWCs with Fe²⁺ and O₂ (B). Values are the means ± standard deviation (n = 3). Results from negative controls (C_(-)) and Fmed average values are shown on the left of each graph. Data from different Fmed strains (Fm1, Fm2, Fm3, Fm4, Fm5, and Fm6) were analyzed by the Kruskal–Wallis test, followed by Dunn’s multiple comparison tests (p < 0.05). Different letters indicate significant differences according to multiple comparison test results.

Figure 5

Heatmap (A) shows the LMW compounds’ average relative counts (Rc) analyzed by UHPLC-MS/MS. Chemical structure (B) of described compounds: (a) 4-hydroxybenzaldehyde, (b) benzoic acid, (c) 4-hydroxy-3-methoxybenzoic acid (vanillic acid), (d) 4-hydroxycinnamic acid (p-coumaric acid), (e) 2-hydroxybenzoic acid (salicylic acid), (f) 3-methoxybenzaldehyde, (g) N-acetyl-5-aminosalicylic acid, (h) 4-hydroxy-3-methoxybenzaldehyde (vanillin), (i) 5-hydroxyindole-3-acetic acid, (j) methyl-4-hydroxybenzoate, (k) hypholomine B (isomers a and b). Values of Rc in the heatmap are the means of four replicates (n = 4). Statistical analysis results for each compound are shown in Table S2.

Figure 6

Hydroxyl radical sample spectrum compared with theoretical simulation (A,B) and relative amplitude (C,D) generated from LMWCs redox cycle with iron, analyzed using spin-trapping in EPR. Values are the means ± standard deviation: for the reaction at pH 3.5 (C; n = 4), results from negative controls (C_(-)) and Fmed average values are shown on the left. Results from different Fmed strains (Fm1, Fm2, Fm3, Fm4, Fm5, and Fm6) were analyzed using the Kruskal–Wallis test, followed by Dunn’s multiple comparison tests (p < 0.05). Different letters indicate significant differences according to multiple comparison test results. For the reaction at pH 5.5 (D; n = 3), results were analyzed by the unpaired two-tailed t-test (p < 0.05). Asterisks indicate significance: ****, p < 0.0001. nd = not detected.

Figure 7

Increase in absorbance (measured spectrophotometrically at 540 nm) from the redox cycling of different LMWCs with (A) cellulose, (B) hemicellulose, and (C) Gewürztraminer wood sawdust. Values for each Fmed strain are the means ± standard deviation of 4 pooled replicates. Results from negative controls (C_(-)) and Fmed average values are shown on the left.