Comparative Transcriptomics During Brown Rot Decay in Three Fungi Reveals Strain-Specific Degradative Strategies and Responses to Wood Acetylation.

Brown rot fungi degrade wood in a two-step process in which enzymatic hydrolysis is preceded by an oxidative degradation phase. While a detailed understanding of the molecular processes during brown rot decay is mandatory for being able to better protect wooden products from this type of degradation, the underlying mechanisms are still not fully understood. This is particularly true for wood that has been treated to increase its resistance against rot. In the present study, the two degradation phases were separated to study the impact of wood acetylation on the behavior of three brown rot fungi commonly used in wood durability testing. Transcriptomic data from two strains of Rhodonia placenta (FPRL280 and MAD-698) and Gloeophyllum trabeum were recorded to elucidate differences between the respective decay strategies. Clear differences were found between the two decay stages in all fungi. Moreover, strategies varied not only between species but also between the two strains of the same species. The responses to wood acetylation showed that decay is generally delayed and that parts of the process are attenuated. By hierarchical clustering, we could localize several transcription factors within gene clusters that were heavily affected by acetylation, especially in G. trabeum. The results suggest that regulatory circuits evolve rapidly and are probably the major cause behind the different decay strategies as observed even between the two strains of R. placenta. Identifying key genes in these processes can help in decay detection and identification of the fungi by biomarker selection, and also be informative for other fields, such as fiber modification by biocatalysts and the generation of biochemical platform chemicals for biorefinery applications.

Degradative Capacity of Two Strains of Rhodonia placenta: From Phenotype to Genotype.

Brown rot fungi, such as Rhodonia placenta (previously Postia placenta), occur naturally in northern coniferous forest ecosystems and are known to be the most destructive group of decay fungi, degrading wood faster and more effectively than other wood-degrading organisms. It has been shown that brown rot fungi not only rely on enzymatic degradation of lignocellulose, but also use low molecular weight oxidative agents in a non-enzymatic degradation step prior to the enzymatic degradation. R. placenta is used in standardized decay tests in both Europe and North America. However, two different strains are employed (FPRL280 and MAD-698, respectively) for which differences in colonization-rate, mass loss, as well as in gene expression have been observed, limiting the comparability of results. To elucidate the divergence between both strains, we investigated the phenotypes in more detail and compared their genomes. Significant phenotypic differences were found between the two strains, and no fusion was possible. MAD-698 degraded scots pine more aggressively, had a more constant growth rate and produced mycelia faster than FPRL280. After sequencing the genome of FPRL280 and comparing it with the published MAD-698 genome we found 660,566 SNPs, resulting in 98.4% genome identity. Specific analysis of the carbohydrate-active enzymes, encoded by the genome (CAZome) identified differences in many families related to plant biomass degradation, including SNPs, indels, gaps or insertions within structural domains. Four genes belonging to the AA3_2 family could not be found in or amplified from FPRL280 gDNA, suggesting the absence of these genes. Differences in other CAZy encoding genes that could potentially affect the lignocellulolytic activity of the strains were also predicted by comparison of genome assemblies (e.g., GH2, GH3, GH5, GH10, GH16, GH78, GT2, GT15, and CBM13). Overall, these mutations help to explain the phenotypic differences observed between both strains as they could interfere with the enzymatic activities, substrate binding ability or protein folding. The investigation of the molecular reasons that make these two strains distinct contributes to the understanding of the development of this important brown rot reference species and will help to put the data obtained from standardized decay tests across the globe into a better biological context.

Hydrophobic and Hydrophilic Extractives in Norway Spruce and Kurile Larch and Their Role in Brown-Rot Degradation.

Extractives found in the heartwood of a moderately durable conifer (Larix gmelinii var. japonica) were compared with those found in a non-durable one (Picea abies). We identified and quantified heartwood extractives by extraction with solvents of different polarities and gas chromatography with mass spectral detection (GC-MS). Among the extracted compounds, there was a much higher amount of hydrophilic phenolics in larch (flavonoids) than in spruce (lignans). Both species had similar resin acid and fatty acid contents. The hydrophobic resin components are considered fungitoxic and the more hydrophilic components are known for their antioxidant activity. To ascertain the importance of the different classes of extractives, samples were partially extracted prior to subjection to the brown-rot fungus Rhodonia placenta for 2-8 weeks. Results indicated that the most important (but rather inefficient) defense in spruce came from the fungitoxic resin, while large amounts of flavonoids played a key role in larch defense. Possible moisture exclusion effects of larch extractives were quantified via the equilibrium moisture content of partially extracted samples, but were found to be too small to play any significant role in the defense against incipient brow-rot attack.

Toxic hazard of leachates from furfurylated wood: comparison between two different aquatic organisms.

Environmental concern regarding the use of toxic preservatives such as chromated copper arsenate (CCA) has been put forward. In the European Union, United States, and Japan, CCA has been phased out for residential and water-contact applications. Ecotoxicological studies of wood treated with conventional preservatives were carried out in the late 1990s, and it was concluded that the main impact is to water and aquatic organisms. Today, alternatives to conventional preservation methods, marketed as "environmentally friendly" or "nontoxic," are emerging. Examples of such alternatives are modified wood, e.g., thermally modified, furfurylated, and acetylated wood. To date, not enough hazard characterization has been performed. In the present study, the Microtox assay with the marine bacterium Vibrio fischeri and the Daphtox procedure with the crustacean Daphnia magna were used as screening methods in an effect assessment. Both organisms were exposed to water leachates from furfurylated wood using two different leaching procedures. The results indicate that Microtox is more sensitive to the toxic components from furfurylated wood than Daphtox. Furthermore, the toxicity of treated Pinus radiata was higher than that of treated Pinus sylvestris. The toxicity did not diminish over the test period, as is the case for preservative-treated wood. The present study found that treatment conditions can influence the toxicity considerably, so toxicity studies should be included in the development of new treatment process. The present study also shows that using an intermediate vacuum-drying step, leading to a more efficient curing/polymerization, results in slightly less hydrophobic oligomers in the product, such that the leachates become less toxic to bacteria.

Toxic hazard and chemical analysis of leachates from furfurylated wood.

The furfurylation process is an extensively investigated wood modification process. Furfuryl alcohol molecules penetrate into the wood cell wall and polymerize in situ. This results in a permanent swelling of the wood cell walls. It is unclear whether or not chemical bonds exist between the furfuryl alcohol polymer and the wood. In the present study, five different wood species were used, both hardwoods and softwoods. They were treated with three different furfurylation procedures and leached according to three different leaching methods. The present study shows that, in general, the leachates from furfurylated wood have low toxicity. It also shows that the choice of leaching method is decisive for the outcome of the toxicity results. Earlier studies have shown that leachates from wood treated with furfuryl alcohol prepolymers have higher toxicity to Vibrio fischeri than leachates from wood treated with furfuryl alcohol monomers. This is probably attributable to differences in leaching of chemical compounds. The present study shows that this difference in the toxicity most likely cannot be attributed to maleic acid, furan, furfural, furfuryl alcohol, or 2-furoic acid. However, the difference might be caused by the two substances 5-hydroxymethylfurfural and 2,5-furandimethanol. The present study found no difference in the amount of leached furfuryl alcohol between leachates from furfurylated softwood and furfurylated hardwood species. Earlier studies have indicated differences in grafting of furfuryl alcohol to lignin. However, nothing was found in the present study that could support this. The leachates of furfurylated wood still need to be