Wood degradation by Fomitiporia mediterranea M. Fischer: Physiologic, metabolomic and proteomic approaches

Abstract

Fomitiporia mediterranea (Fmed) is one of the main fungal species found in grapevine wood rot, also called "amadou," one of the most typical symptoms of grapevine trunk disease Esca. This fungus is functionally classified as a white-rot, able to degrade all wood structure polymers, i.e., hemicelluloses, cellulose, and the most recalcitrant component, lignin. Specific enzymes are secreted by the fungus to degrade those components, namely carbohydrate active enzymes for hemicelluloses and cellulose, which can be highly specific for given polysaccharide, and peroxidases, which enable white-rot to degrade lignin, with specificities relating to lignin composition as well. Furthermore, besides polymers, a highly diverse set of metabolites often associated with antifungal activities is found in wood, this set differing among the various wood species. Wood decayers possess the ability to detoxify these specific extractives and this ability could reflect the adaptation of these fungi to their specific environment. The aim of this study is to better understand the molecular mechanisms used by Fmed to degrade wood structure, and in particular its potential adaptation to grapevine wood. To do so, Fmed was cultivated on sawdust from different origins: grapevine, beech, and spruce. Carbon mineralization rate, mass loss, wood structure polymers contents, targeted metabolites (extractives) and secreted proteins were measured. We used the well-known white-rot model Trametes versicolor for comparison. Whereas no significant degradation was observed with spruce, a higher mass loss was measured on Fmed grapevine culture compared to beech culture. Moreover, on both substrates, a simultaneous degradation pattern was demonstrated, and proteomic analysis identified a relative overproduction of oxidoreductases involved in lignin and extractive degradation on grapevine cultures, and only few differences in carbohydrate active enzymes. These results could explain at least partially the adaptation of Fmed to grapevine wood structural composition compared to other wood species, and suggest that other biotic and abiotic factors should be considered to fully understand the potential adaptation of Fmed to its ecological niche. Proteomics data are available via ProteomeXchange with identifier PXD036889.

Keywords: Esca; Fomitiporia mediterranea; adaptation; grapevine wood; white rot.

Figure 1

Experimental design.

Figure 2

Control, Fmed (F. mediterranea phco36) and Tver (T. versicolor BRFM1218) cultures on grapevine, beech and spruce at the end of the cultures. Grapevine and beech cultures were stopped at 3 months, and spruce cultures were stopped at 2 months. A yellow and wolly mycelium was observed for Fmed whereas Tver mycelium remains white and at the bottom of the flask. On spruce, a slight coloration of sawdust and white mycelium are observed with Fmed, and no mycelum is observed with Tver.

Figure 3

(A) Fmed and (B) Tver carbon mineralization rates during cultures and maximal respiration rates for grapevine, beech and spruce cultures. Statistics between mineralization rates during cultures are indicated for each point compared to the highest measured value in the same culture with Tukey test. Statistical differences between maximal mineralization rates were obtained with Tukey test. Significant differences are indicated by *** for p-values < 0.001, ** for p-values < 0.005 and * for p-values < 0.05.

Figure 4

Fmed and Tver cultures mass loss on spruce at 1 and 2  months, grapevine at 1, 2 and 3  months, and beech at 1 and 3  months. Significant differences obtained with Conover-Iman test of multiple comparisons are indicated by *** for p-values < 0.001, ** for p-values < 0.005 and * for p-values < 0.05.

Figure 5

Proportions in wood components in control, Fmed and Tver cultures on (A) grapevine, (B) spruce and (C) beech at 1  month (1 m), 2  months (2 m) or 3  months (3 m) of culture. Significant differences are given compared to the percentage in control according to Student–Newman–Keuls test, and are indicated by *** for p-values < 0.001, ** for p-values < 0.005 and * for p-values < 0.05.

Figure 6

Targeted metabolomic analysis of the wood degradation process. Log2 of significant metabolite fold changes for indicated pairwise comparisons are given by shades of red or blue colors according to the scale bar for (A) grapevine, (B) beech and (C) spruce cultures. Data represent the mean values of three biological replicates for each condition and time point. Statistical analyses were performed by Tukey’s honest significant difference method followed by false discovery rate (FDR) correction, with FDR < 0.05. For FDR ≥ 0.05, Log2 fold changes were set to 0. AA: amino acids; AL: aldehydes; F: flavonoid; PAc: phenolic acids; PAl: phenolic alcohols; St1 to St4: Stilbene monomers, dimers, trimers and tetramers; MXXX: m/z in positive mode. C: control.

Figure 7

Relative abundance of main classes of proteins in Fmed and Tver secretome on grapevine and beech cultures. Pept.: peptidases, GHs: glycoside hydrolases, PLs: polysaccharides lyases, CEs: carbohydrate esterases, Pect.: pectinases, AA9 = auxiliary enzymes family 9 (or LPMOs), MnPs: manganese peroxidases, Lacc.: laccases LiPs: lignin peroxidases, Oth. Ox.: other oxidoreductases. Classification was performed according to JGI database and percentages are given according to PAI relative abundancies for all proteins with emPAI higher than ten in at least one of the culture conditions.