Interactions between Scots pine heartwood extractives and wood decaying fungi

The heartwoods of many wood species have natural resistance to degradation caused by wood decaying fungi. Many factors can contribute to the natural durability of heartwoods, but the most significant one is usually the presence of biologically active extractives in the wood material. This thesis investigated the interactions between heartwood extractives and wood decaying fungi in detail, using Scots pine as the wood material. The primary objectives of the thesis were to study the mechanisms of action of extractives and to explore the ways in which the extractives interact with fungi within the structure of wood. The mechanism of action studies focused on the antioxidant activity of extractives and on their ability to inhibit the enzymatic hydrolysis of wood polysaccharides. The antioxidant activity measurements showed that Scots pine heartwood extractives are active antioxidants, which means that they may be able to interfere with the radical-based degradative mechanisms used by wood decaying fungi, particularly brown rots. The extractives could also inhibit the action of hydrolytic enzymes, suggesting that they may be able to prevent the conversion of wood polysaccharides to digestible sugars. However, substantial hydrolase inhibition was only seen with a white rot enzyme preparation, which contained some enzyme(s) capable of modifying the heartwood extractives. The interaction studies used confocal Raman spectroscopy imaging to visualise the cellular level distributions of extractives and other chemical components in intact and decaying heartwood. The studies revealed that the phenolic pinosylvins were present throughout the heartwood tissues, suggesting that they have good ability to interact with fungi and their degradative agents during decay. However, the studies on decaying heartwood showed that pinosylvins were extensively degraded during incipient decay. The hydrophobic resin acids were only detected in the lumens of some tracheids and ray cells, but they were found to have higher resistance to degradation than the pinosylvins. Interestingly, the resin-rich extractives deposits found in tracheid lumens appeared to create local areas of reduced degradation in the decaying heartwood. The results of this thesis provide new information on the ways in which extractives can interact with fungi and contribute to natural durability. The results also increase our understanding of the origins of natural durability in Scots pine, the most abundant wood species in Finland

Männyn kuoren ja sisäoksien uuteaineiden kyllästepotentiaali

The purpose of this investigation was to assess the potential of Scots pine (Pinus sylvestris L.) bark and knot extractives to function as preservative chemicals. Scots pine was chosen to due to its importance in Finland; bark and knots were chosen due to their high extractives content and lack of industrial applications. The bark and knots of Scots pine were extracted with acetone and the extraction yields calculated. The chemical composition of the extracts was determined using gas chromatography-mass spectrometry, Fourier transform infrared spectroscopy, and UV Raman spectroscopy. Their antifungal activity was studied using a wood-free agar plate method. The bark and knots of Scots pine were found to contain large amounts of extractives, 16.8% and 24.6%, respectively. The bark extract consisted primarily of monosaccharides and resin acids; the knot extract of resin acids and pinosylvins. Both extracts also contained smaller amounts of fatty acids and other compounds. The bark extract was found to have no significant antifungal activity: at a low concentration the extract promoted the growth of fungi, while at a high concentration it had no statistically significant effect on growth. The lack of activity was concluded to be due to the chemical composition of the extract. None of the main compounds present in the bark extract are significantly antifungal, and the saccharides most likely acted as an additional food source, leading to increased growth at the lower concentration. The knot extract inhibited the growth of fungi at the higher concentration, and the activity of the extract was concluded to be due to the presence of large amounts of pinosylvins, which are known for their antifungal properties. The results of this investigation showed that Scots pine knot extractives may have potential as wood preservative chemicals. The knot extract showed significant antifungal character at a moderate concentration, and the extractives were obtainable in high yields. However, more study is needed before definitive conclusions can be drawn. Most importantly, the preservative potential of the extractives should be determined using tests that involve impregnation of wood. The use of extractives in wood impregnation is also subject to a number of general problems that should be studied before conclusions can be drawn.Tämän työn tarkoituksena oli tutkia männyn (Pinus sylvestris L.) kuoresta ja sisäoksista saatavien uuteaineiden potentiaalia toimia lahonsuoja-aineina. Mänty valittiin sen teollisen merkittävyyden johdosta; kuori ja oksat niiden korkean uuteainepitoisuuden ja teollisten sovellusten puutteen vuoksi. Lahonsuojapotentiaalin selvittämiseksi männyn kuorta ja oksia uutettiin asetonilla ja uuteaineiden saannot laskettiin. Uutteiden kemiallinen koostumus määritettiin kaasukromatografia-massaspektrometrian, Fourier-muunnos infrapunaspektroskopian, sekä UV Raman spektroskopian avulla. Uutteiden antifungaalisia ominaisuuksia tutkittiin puuvapaan agarmaljamenetelmän avulla. Männyn kuoren ja oksien havaittiin sisältävän merkittäviä määriä uuteaineita, 16.8% kuoressa ja 24.6% oksissa. Kuoriuute koostui pääosin monosakkarideista ja hartsihapoista, oksauute taas hartsihapoista ja pinosylviineistä. Molemmat uutteet sisälsivät myös pienempiä määriä rasvahappoja ja muita aineita. Kuoriuutteella ei havaittu olevan merkittäviä antifungaalisia ominaisuuksia: matalalla konsentraatiolla uute lisäsi sienten kasvua, korkealla konsentraatiolla uutteella ei taas ollut tilastollisesti merkittävää vaikutusta kasvuun. Antifungaalisten ominaisuuksien puutteen todettiin johtuvan kuoriuutteen kemiallisesta koostumuksesta. Mikään kuoriuutteen pääkomponenteista ei ole merkittävästi antifungaalinen, ja sakkaridit puolestaan saattoivat toimia ylimääräisenä ravinnonlähteenä, johtaen lisääntyneeseen kasvuun. Kuoriuute hidasti sienten kasvua merkittävästi korkealla konsentraatiolla, ja uutteen antifungaalisten ominaisuuksien todettiin olevan seurausta uutteessa suurissa määrin esiintyvistä pinosylviineistä. Pinosylviinien antifungaaliset ominaisuudet ovat hyvin tunnettuja. Tämän työn tulokset osoittavat, että männyn sisäoksien uuteaineilla saattaa olla potentiaalia toimia lahonsuoja-aineina. Oksauutteella havaittiin olevan merkittäviä antifungaalisia ominaisuuksia, ja lisäksi uuteaineiden saanto oksista oli korkea. Tutkimusta tulee kuitenkin jatkaa ennen kuin oksauutteiden lahonsuojapotentiaalista voidaan vetää varmoja johtopäätöksiä. Ensisijaisen tärkeää on määrittää uutteiden lahonsuojapotentiaali kokeissa, joissa tarkastellaan uutteilla kyllästettyä puuta. Uuteaineiden käyttöön kyllästyksessä myös liittyy lukuisia yleisen tason ongelmia joihin täytyy perehtyä ennen kuin johtopäätöksiä voidaan tehdä

Cellular level chemical changes in Scots pine heartwood during incipient brown rot decay

The heartwoods of many wood species have natural resistance to wood decay due to the accumulation of antifungal heartwood extractives. The natural durability of heartwoods has been extensively investigated, yet very little information is available on the initiation of heartwood decay. This experiment examined the onset of Rhodonia placenta brown rot decay in Scots pine heartwood in order to identify the key changes leading to heartwood decay. An imaging approach based on Raman imaging and multivariate image analysis revealed that the degradation of heartwood began in the innermost cell wall layers and then spread into the remaining cell walls and the middle lamella. Pinosylvins were extensively degraded in the cell walls, middle lamella and extractive deposits, while unidentified material most likely consisting of hemicelluloses and/or lipophilic extractives was removed from the inner cell wall layers. Changes similar to inner cell wall degradation were seen in the remaining cell walls in more advanced decay. The results indicate that the key change in incipient heartwood decay is the degradation of antifungal heartwood extractives. The inner cell wall degradation seen in this experiment may serve a nutritive purpose or facilitate the penetration of degradative agents into the cell walls and middle lamella

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.Peer reviewe

Relationship between decay resistance and moisture properties in wood modified with phenol formaldehyde and sorbitol-citric acid

Publisher Copyright: © 2023, The Author(s).Impregnation modifications improve the decay resistance of wood, but the mechanisms behind improved resistance are not yet fully understood. In this study, Scots pine sapwood samples were impregnation modified to investigate the relationship between moisture properties, decay resistance and chemical changes caused by decay. The samples were modified with phenol formaldehyde (PF) and sorbitol-citric acid (SCA) at different solids contents to study the effects of two different types of resins. The anti-swelling efficiency (ASE) and moisture exclusion efficiency (MEE) of the samples were measured, after which they were exposed to the brown rot fungi Coniophora puteana and Rhodonia placenta to determine their mass losses, moisture contents and chemical changes due to decay. The results showed that both modifications were able to increase ASE, MEE and decay resistance, and that neither modification was appreciably degraded by the fungi. However, no uniform relationship was found between mass loss and ASE or MEE for the two modifications, and there was a clear increase in the moisture contents of the decayed samples and sterile controls under decay test conditions with increasing modification degree. Overall, the results showed that modification with PF and SCA increases decay resistance, but the relationship between resistance and moisture properties requires further investigation.Peer reviewe

Decay Resistance of Surface Carbonized Wood

Surface carbonization, or charring, of wood is a one-sided modification method primarily intended for protection of exterior cladding boards. The heavily degraded surface acts as a barrier layer shielding the interior from environmental stresses, and as such acts as an organic coating. To test the durability of surfaces created in this manner, unmodified, contact charred, and flame charred spruce and birch samples were exposed to the brown rot fungus Coniophora puteana and white rot fungus Trametes versicolor for a period of nine weeks. All sides of the samples except the modified surfaces were sealed to investigate the protective effect of the surface. Mass losses were greatest for unmodified references (up to 60% and 56% for birch and spruce, respectively) and smallest for contact charred samples (up to 23% and 32%). The wood below the modified surfaces showed chemical changes typical of brown rot and simultaneous white rot. The measured glucosamine content revealed fungal biomass in both the modified surface as well as the layers beneath. According to the recorded values, the fungal biomass increased below the surface and was higher for flame charred samples in comparison to contact charred ones. This is likely due to the more intact, plasticized surface and the thicker thermally modified transition zone that restricts fungal growth more effectively in contact charred samples in comparison to the porous, cracked flame charred samples. Scanning electron microscope images verified the results by revealing fungal hyphae in all inspected wood types and species

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

Inhibitory effects of Scots pine heartwood extractives on enzymatic holocellulose hydrolysis by wood decaying fungi

The heartwood of Scots pine contains extractives that protect it against wood decaying fungi. Pine extractives such as pinosylvins are fungicidal compounds, but they may also have other mechanisms of action. This experiment investigated whether pinosylvins and other heartwood extractives can act as inhibitors of holocellulose hydrolysis, similarly to many other biomass-derived phenolic compounds. The inhibitory properties of extractives were studied against enzymes secreted by a brown rot (Coniophora puteana) and a white rot fungus (Trametes versicolor), as well as against a commercial Trichoderma reesei enzyme cocktail. The extractives were studied as wood-free extracts and extractives-containing wood powders. In all experiments, the behaviour of the white rot differed from that of the other two. The white rot hydrolases were strongly inhibited and deactivated by extractives, particularly pinosylvins, whereas the others showed only mild or moderate inhibition and no deactivation. The white rot enzymes seemedto modify the pinosylvins, with further studies suggesting that the modified pinosylvins may form complexes with enzymes and cause their deactivation. These results suggest that pine heartwood extractives have potential to contribute to decay resistance as hydrolase inhibitors but only when the fungus produces enzymes capable of modifying the extractives.Peer reviewe