Fungal Degradation of Extractives Plays an Important Role in the Brown Rot Decay of Scots Pine Heartwood.

Scots pine heartwood is known to have resistance to wood decay due to the presence of extractives, namely stilbenes and resin acids. However, previous studies have indicated that these extractives are degradable by wood decaying fungi. This study aimed to investigate the relationship between extractive degradation and heartwood decay in detail and to gain insight into the mechanisms of extractive degradation. Mass losses recorded after a stacked-sample decay test with brown rot fungi showed that the heartwood had substantial decay resistance against Coniophora puteana but little resistance against Rhodonia placenta. Extracts obtained from the decayed heartwood samples revealed extensive degradation of stilbenes by R. placenta in the early stages of decay and a noticeable but statistically insignificant loss of resin acids. The extracts from R. placenta-degraded samples contained new compounds derived from the degraded extractives: hydroxylated stilbene derivatives appeared in the early decay stages and then disappeared, while compounds tentatively identified as hydroxylated derivatives of dehydroabietic acid accumulated in the later stages. The degradation of extractives was further analysed using simple degradation assays where an extract obtained from intact heartwood was incubated with fungal mycelium or extracellular culture fluid from liquid fungal cultures or with neat Fenton reagent. The assays showed that extractives can be eliminated by several fungal degradative systems and revealed differences between the degradative abilities of the two fungi. The results of the study indicate that extractive degradation plays an important role in heartwood decay and highlight the complexity of the fungal degradative systems.

Chemical Characterization and Visualization of Progressive Brown Rot Decay of Wood by Near Infrared Imaging and Multivariate Analysis.

Brown rot fungi cause a type of wood decay characterized by carbohydrate degradation and lignin modification. The chemical and physical changes caused by brown rot are usually studied using bulk analytical methods, but these methods fail to consider local variations within the wood material. In this study we applied hyperspectral near infrared imaging to Scots pine sapwood samples exposed to the brown rot fungi Coniophora puteana and Rhodonia placenta to obtain position-resolved chemical information on the fungal degradative process. A stacked-sample decay test was used to create a succession of decay stages within the samples. The results showed that the key chemical changes associated with decay were the degradation of amorphous and crystalline carbohydrates and an increase in aromatic and carbonyl functionality in lignin. The position-resolved spectral data revealed that the fungi initiated degradation in earlywood, and that earlywood remained more extensively degraded than latewood even in advanced decay stages. Apart from differences in mass losses, the two fungi produced similar spectral changes in a similar spatial pattern. The results show that near infrared imaging is a useful tool for analyzing brown rot decayed wood and may be used to advance our understanding of fungal degradative processes.

Using MALDI-FTICR-MS Imaging to Track Low-Molecular-Weight Aromatic Derivatives of Fungal Decayed Wood.

Low-molecular-weight (LMW) aromatics are crucial in meditating fungal processes for plant biomass decomposition. Some LMW compounds are employed as electron donors for oxidative degradation in brown rot (BR), an efficient wood-degrading strategy in fungi that selectively degrades carbohydrates but leaves modified lignins. Previous understandings of LMW aromatics were primarily based on "bulk extraction", an approach that cannot fully reflect their real-time functions during BR. Here, we applied an optimized molecular imaging method that combines matrix-assisted laser desorption ionization (MALDI) with Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS) to directly measure the temporal profiles of BR aromatics as Rhodonia placenta decayed a wood wafer. We found that some phenolics were pre-existing in wood, while some (e.g., catechin-methyl ether and dihydroxy-dimethoxyflavan) were generated immediately after fungal activity. These pinpointed aromatics might be recruited to drive early BR oxidative mechanisms by generating Fenton reagents, Fe2+ and H2O2. As BR progressed, ligninolytic products were accumulated and then modified into various aromatic derivatives, confirming that R. placenta depolymerizes lignin. Together, this work confirms aromatic patterns that have been implicated in BR fungi, and it demonstrates the use of MALDI-FTICR-MS imaging as a new approach to monitor the temporal changes of LMW aromatics during wood degradation.

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.