Once upon a Time, There Was a Piece of Wood: Present Knowledge and Future Perspectives in Fungal Deterioration of Wooden Cultural Heritage in Terrestrial Ecosystems and Diagnostic Tools.

Wooden Cultural Heritage (WCH) represents a significant portion of the world's historical and artistic heritage, consisting of immovable and movable artefacts. Despite the expertise developed since ancient times to enhance its durability, wooden artefacts are inevitably prone to degradation. Fungi play a pivotal role in the deterioration of WCH in terrestrial ecosystems, accelerating its decay and leading to alterations in color and strength. Reviewing the literature of the last 25 years, we aimed to provide a comprehensive overview of fungal diversity affecting WCH, the biochemical processes involved in wood decay, and the diagnostic tools available for fungal identification and damage evaluation. Climatic conditions influence the occurrence of fungal species in threatened WCH, characterized by a prevalence of wood-rot fungi (e.g., Serpula lacrymans, Coniophora puteana) in architectural heritage in temperate and continental climates and Ascomycota in indoor and harsh environments. More efforts are needed to address the knowledge fragmentation concerning biodiversity, the biology of the fungi involved, and succession in the degradative process, which is frequently centered solely on the main actors. Multidisciplinary collaboration among engineers, restorers, and life sciences scientists is vital for tackling the challenges posed by climate change with increased awareness. Traditional microbiology and culture collections are fundamental in laying solid foundations for a more comprehensive interpretation of big data.

Co-culture Wood Block Decay Test with Bacteria and Wood Rotting Fungi to Analyse Synergism/Antagonism during Wood Degradation.

Mixed communities of fungi and bacteria have been shown to be more efficient in degrading wood than fungi alone. Some standardised protocols for quantification of the wood decay ability of fungi have been developed (e.g., DIN V ENV 12038:2002 as the legal standard to test for the resistance of wood against wood-destroying basidiomycetes in Germany). Here, we describe a step-by-step protocol developed from the official standard DIN V ENV12038 to test combinations of bacteria and fungi for their combined wood degradation ability. Equally sized wood blocks are inoculated with wood decay fungi and bacterial strains. Axenic controls allow the analysis of varying degradation rates via comparison of the wood dry weights at the end of the experiments. This protocol provides new opportunities in exploration of inter- and intra-kingdom interactions in the wood-related environment and forms the basis for microcosm experiments. Key features • Quantification of wood decay ability of mixed cultures. • Allows testing if fungi are more efficient in degrading wood when bacteria are present.

Changes in Chemical Structure of Thermally Modified Spruce Wood Due to Decaying Fungi.

Fungi play a critical role in the decomposition of wood and wood-based products in use. The ability of decaying fungi to cause degradation of polysaccharides and lignin in the thermally modified Norway spruce (Picea abies L. Karst.) wood was examined with pure culture decomposition tests in laboratory conditions using the brown-rot fungus Serpula lacrymans (Schumacher ex Fries) S.F. Gray and white-rot fungus Trametes versicolor (Linnaeus ex Fries) Pilat. Spruce wood samples were primary thermally treated under atmospheric pressure at the temperatures of 100, 150, 200, 220, 240 and 260 °C during 1, 3 and 5 h, whereby larger losses in their mass, holocellulose, mannose and xylose were achieved at harder thermal regimes. Meanwhile, the holocellulose percent content reduced considerably, and the percent content of lignin increased sharply. Spruce wood thermally modified at and above 200 °C better resisted to brown-rot fungus S. lacrymans than the white-rot fungus T. versicolor. Due to the decay processes, the mass fractions of holocellulose, cellulose and hemicelluloses were lower in those spruce wood samples in which thermal degradation was more intensive, with achieving the highest mass loss values after thermal treatments, after which the decay attacks were poorer or even none with the minimal mass loss values due to action by the brown-rot fungus S. lacrymans and the white-rot fungus T. versicolor. The mannose and glucose percent content in thermally-fungally attacked spruce wood was intensive reduced, e.g., by 17% to 98% in wood after thermal treatments at temperature equal and above 200 °C.

Cytochrome P450 Complement May Contribute to Niche Adaptation in Serpula Wood-Decay Fungi.

Serpula wood-decay fungi occupy a diverse range of natural and man-made ecological niches. Serpula himantioides is a forest-floor generalist with global coverage and strong antagonistic ability, while closely related species Serpula lacrymans contains specialist sister strains with widely differing ecologies. Serpula lacrymans var. shastensis is a forest-floor specialist in terms of resource preference and geographic coverage, while Serpula lacrymans var. lacrymans has successfully invaded the built environment and occupies a building-timber niche. To increase understanding of the cellular machinery required for niche adaptation, a detailed study of the P450 complement of these three strains was undertaken. Cytochrome P450 monooxygenases are present in all fungi and typically seen in high numbers in wood decay species, with putative roles in breakdown of plant extractives and lignocellulose metabolism. Investigating the genomes of these related yet ecologically diverse fungi revealed a high level of concordance in P450 complement, but with key differences in P450 family representation and expression during growth on wood, suggesting P450 proteins may play a role in niche adaptation. Gene expansion of certain key P450 families was noted, further supporting an important role for these proteins during wood decay. The generalist species S. himantioides was found to have the most P450 genes with the greatest family diversity and the highest number of P450 protein families expressed during wood decay.

Microbiota Associated with Different Developmental Stages of the Dry Rot Fungus Serpula lacrymans.

The dry rot fungus Serpula lacrymans causes significant structural damage by decaying construction timber, resulting in costly restoration procedures. Dry rot fungi decompose cellulose and hemicellulose and are often accompanied by a succession of bacteria and other fungi. Bacterial-fungal interactions (BFI) have a considerable impact on all the partners, ranging from antagonistic to beneficial relationships. Using a cultivation-based approach, we show that S. lacrymans has many co-existing, mainly Gram-positive, bacteria and demonstrate differences in the communities associated with distinct fungal parts. Bacteria isolated from the fruiting bodies and mycelia were dominated by Firmicutes, while bacteria isolated from rhizomorphs were dominated by Proteobacteria. Actinobacteria and Bacteroidetes were less abundant. Fluorescence in situ hybridization (FISH) analysis revealed that bacteria were not present biofilm-like, but occurred as independent cells scattered across and within tissues, sometimes also attached to fungal spores. In co-culture, some bacterial isolates caused growth inhibition of S. lacrymans, and vice versa, and some induced fungal pigment production. It was found that 25% of the isolates could degrade pectin, 43% xylan, 17% carboxymethylcellulose, and 66% were able to depolymerize starch. Our results provide first insights for a better understanding of the holobiont S. lacrymans and give hints that bacteria influence the behavior of S. lacrymans in culture.

Extraction of Vanillin Following Bioconversion of Rice Straw and Its Optimization by Response Surface Methodology.

Value-added chemicals, including phenolic compounds, can be generated through lignocellulosic biomass conversion via either biological or chemical pretreatment. Currently vanillin is one of the most valuable of these products that has been shown to be extractable on an industrial scale. This study demonstrates the potential of using rice straw inoculated with Serpula lacrymans, which produced a mixture of high value bio-based compounds including vanillin. Key extraction conditions were identified to be the volume of solvent used and extraction time, which were optimized using response surface methodology (RSM). The vanillin compounds extracted from rice straw solid state fermentation (SSF) was confirmed through LC-ESI MS/MS in selective ion mode. The optimum concentration and yield differed depending on the solvent, which was predicted using 60 mL ethyl acetate for 160 min were 0.408% and 3.957 µg g-1 respectively. In comparison, when ethanol was used, the highest concentration and yields of vanillin were 0.165% and 2.596 µg g-1. These were achieved using 40 mL of solvent, and extraction time increased to 248 min. The results confirm that fungal conversion of rice straw to vanillin could consequently offer a cost-effect alternative to other modes of production.

Biochemical characterization of Serpula lacrymans iron-reductase enzymes in lignocellulose breakdown.

Putative iron-reductase (IR) genes from Serpula lacrymans with similarity to the conserved iron-binding domains of cellobiose dehydrogenase (CDH) enzymes have been identified. These genes were cloned and expressed to functionally characterize their activity and role in the decomposition of lignocellulose. The results show that IR1 and IR2 recombinant enzymes have the ability to depolymerize both lignin and cellulose, are capable of the reduction of ferric iron to the ferrous form, and are capable of the degradation of nitrated lignin. Expression of these genes during wheat straw solid-state fermentation was shown to correlate with the release of compounds associated with lignin decomposition. The results suggest that both IR enzymes mediate a non-enzymatic depolymerisation of lignocellulose and highlight the potential of chelator-mediated Fenton systems in the industrial pre-treatment of biomass.

Distinct Growth and Secretome Strategies for Two Taxonomically Divergent Brown Rot Fungi.

Brown rot fungi are wood-degrading fungi that employ both oxidative and hydrolytic mechanisms to degrade wood. Hydroxyl radicals that facilitate the oxidative component are powerful nonselective oxidants and are incompatible with hydrolytic enzymes unless they are spatially segregated in wood. Differential gene expression has been implicated in the segregation of these reactions in Postia placenta, but it is unclear if this two-step mechanism varies in other brown rot fungi with different traits and life history strategies that occupy different niches in nature. We employed proteomics to analyze a progression of wood decay on thin wafers, using brown rot fungi with significant taxonomic and niche distances: Serpula lacrymans (Boletales; "dry rot" lumber decay) and Gloeophyllum trabeum (order Gloeophyllales; slash, downed wood). Both fungi produced greater oxidoreductase diversity upon wood colonization and greater glycoside hydrolase activity later, consistent with a two-step mechanism. The two fungi invested very differently, however, in terms of growth (infrastructure) versus protein secretion (resource capture), with the ergosterol/extracted protein ratio being 7-fold higher with S. lacrymans than with G. trabeum In line with the native substrate associations of these fungi, hemicellulase-specific activities were dominated by mannanase in S. lacrymans and by xylanase in G. trabeum Consistent with previous observations, S. lacrymans did not produce glycoside hydrolase 6 (GH6) cellobiohydrolases (CBHs) in this study, despite taxonomically belonging to the order Boletales, which is distinguished among brown rot fungi by having CBH genes. This work suggests that distantly related brown rot fungi employ staggered mechanisms to degrade wood, but the underlying strategies vary among taxa.IMPORTANCE Wood-degrading fungi are important in forest nutrient cycling and offer promise in biotechnological applications. Brown rot fungi are unique among these fungi in that they use a nonenzymatic oxidative pretreatment before enzymatic carbohydrate hydrolysis, enabling selective removal of carbohydrates from lignin. This capacity has independently evolved multiple times, but it is unclear if different mechanisms underpin similar outcomes. Here, we grew fungi directionally on wood wafers and we found similar two-step mechanisms in taxonomically divergent brown rot fungi. The results, however, revealed strikingly different growth strategies, with S. lacrymans investing more in biomass production than secretion of proteins and G. trabeum showing the opposite pattern, with a high diversity of uncharacterized proteins. The "simplified" S. lacrymans secretomic system could help narrow gene targets central to oxidative brown rot pretreatments, and a comparison of its distinctions with G. trabeum and other brown rot fungi (e.g., Postia placenta) might offer similar traction in noncatabolic genes.

Fungal variegatic acid and extracellular polysaccharides promote the site-specific generation of reactive oxygen species.

This study aims to clarify the role of variegatic acid (VA) in fungal attack by Serpula lacrymans, and also the generation and scavenging of reactive oxygen species (ROS) by the fungus. VA promotes a mediated Fenton reaction to generated ROS after oxalate solubilizes oxidized forms of iron. The fungal extracellular matrix (ECM) ß-glucan scavenged ROS, and we propose this as a mechanism to protect the fungal hyphae while ROS generation is promoted to deconstruct the lignocellulose cell wall. A relatively high pH (4.4) also favored Fe(III) transfer from oxalate to VA as opposed to a lower pH (2.2) conditions, suggesting a pH-dependent Fe(III) transfer to VA employed by S. lacrymans. This permits ROS generation within the higher pH of the cell wall, while limiting ROS production near the fungal hyphae, while ß-glucan from the fungal ECM scavenges ROS in the more acidic environments surrounding the fungal hyphae.