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Analysis of D-glucose metabolism of wood decay fungi using 13C-NMR and 13C-labeled substrates
2003 - IRG/WP 03-10475
D-Glucose metabolism is thought to be important during wood decay by fungi, not only for anabolic and catabolic purposes of central metabolism, but also as a potential source of peroxide required by extracellular peroxidases. There has been some confusion in the literature as to whether this peroxide-generating activity is of the glucose 1-oxidase or pyranose 2-oxidase (glucose 2-oxidase) type with various fungi or even within the same fungal species. Definitive classification requires accurate identification of the enzymatic products D-glucono-1,5-lactone and D-arabino-2-hexosulose (glucosone) with glucose 1-oxidase and pyranose 2-oxidase, respectively. We used 13C-NMR to distinguish these reactions starting with 13C-labeled glucose. The use of labeled substrates simplifies analysis and greatly increases detection sensitivity without requiring the isolation or derivatization of metabolites. We synthesized 13C-1-glucosone to study subsequent metabolism with crude enzyme preparations. Preliminary results with Phanerochaete chrysosporium are presented.
T H de Koker, M D Mozuch, P J Kersten

A discussion of current theories concerning CCA fixation
1983 - IRG/WP 3238
The understanding of the fixation mechanism of CCA and related preservatives in wood has been greatly improved by a significant series of recent scientific papers. In view of recent concerns in New Zealand regarding the long-term efficacy of CCA in high decay-hazard situations, it was considered appropriate to review this recent work and to contrast it with theories presented by previous workers.
D V Plackett

Characterisation of growth and stain of different groups of sapstain fungi on lodgepole pine
1999 - IRG/WP 99-10326
Canada is the world's largest exporter of softwood lumber. These softwood shipments are susceptible to a variety of wood-inhabiting fungi that can lead to sapstain discolouration, which in turn decrease the product value. Furthermore, the presence of these microorganisms may be unacceptable to the importing countries. The objective of this work is to assess the sapstaining capability and basic nutrition of thirty-four fungi isolates representing nine species that were isolated from sawmills across western Canada. The isolates were infected onto fresh lodgepole pine billets and assessed for staining ability, longitudinal growth, host-nutrient consumption, and host viability. The results indicated that the most aggressive saptain species on fresh logs was Ceratocystis coerulescens, followed consecutively by Leptographium spp, Ophiostoma minus, O. piliferum, O. piceae, Ophiostoma spp (D and E) and Aureobasidium pullulans. Preliminary HPLC analysis of soluble sugars indicated that mannose was the free monomer carbohydrate of choice for most of the staining fungi, followed by glucose. Arabinose and galactose were not well utilised. Gas chromatography of infected wood extracts that Leptographium sp. and C. coerulescens significantly reduced the triglyceride fraction.
C Fleet, C Breuil, A Uzunovic, A Byrne

The potential of 2-deoxy-D-glucose as an active ingredient in wood preservation
1999 - IRG/WP 99-30205
2-deoxy-D-glucose (2-DOG) is a potential active ingredient against wood decaying fungi. When dissolved in water, it can be used in pressure treatment of wood. Thereby the wood is protected from attack by wood decaying fungi. A concentration of 1.5% (mean retention 9.7 kg/cubic meter sap-wood) is adequate for brown rot fungi, and 3% (mean retention 19.5 kg/cubic meter sapwood) will also provide protection against white rot fungi. 2-DOG is easily soluble in water, and is therefore easily leached from the wood upon completion of the preservation process. Different types of fixa-tion methods have been tried and evaluated. It is possible to produce 2-DOG by the hydrolysis of chitin, a constituent of the exterior skeleton of shellfish and insects. There is a potential for exploita-tion of this waste product provided by the crab and shrimp industry. The yield of 1 kg of fresh shrimp is 75 g of 2-DOG.
O V Frederiksen, A P Koch

Conditions for basidiospore production in the brown rot fungus Gloeophyllum separium in axenic culture
1984 - IRG/WP 1232
Attempts to control and optimize the production of hymenial structures and basidiospore production in Gloeophyllum sepiarium in axenic culture resulted in the proposal of the following conditions as being suitable. The dikaryotic mycelia originally isolated from basidiocarps could consistently be induced to produce hymenial structures and pure basidiospore collects if illuminated by near ultraviolet light with emission maximum at 355 nm ("black light") at a temperature of 15°C on a chemically defined medium, where the concentration of the carbon and the nitrogen sources were shown to be of critical significance. The necessary conditions for basidiospore production in lignicolous fungi in general are is briefly discussed.
J Bjurman

CCA Chemistry
1983 - IRG/WP 3268
A Pizzi

Distribution of cellulases in the body of Coptotermes formosanus and the probability that the termite uses glucose as an energy and carbon sources
1997 - IRG/WP 97-10202
We assayed extracts of the digestive system and of the whole body of Coptotermes formosanus to determine where the various cellulases, glucose, and related substances were concentrated and to detect pyruvate dehydrogenase activity in the hindgut-removed body in order to verify its full cellulolytic system. About 20%, 18% and 36% of the total exo-1,4-ß-glucanase activity of C. formosanus were detected in the salivary glands, midgut, and hindgut, respectively. About a third of the total endo-1,4-ß-glucanase activity in the termite was detected in the salivary glands (34.5%) whereas the activities in the midgut and hindgut were 21.1% and 18.2%, respectively. About 75% of the total ß-D-glucosidase activity in the termite was detected in the midgut. Thus all the necessary cellulases for hydrolysis of natural cellulose to glucose were present in the region ranging from the salivary glands to the midgut in significant amounts. Most of the glucose and trehalose detected in the termite existed in the gutted-body. Most of the glucose detected in the gut existed in the midgut. Pyruvic acid was directly converted to acetyl-CoA in the presence of NAD+ by a crude extract of the gutted-body. These results suggest that natural cellulose ingested by the termite is hydrolyzed to oligosaccharides in the region of the foregut and midgut as well as in the hindgut, that oligosaccharides are hydrolyzed to glucose predominantly in the midgut, and that the resultant glucose is absorbed through the midgut wall into the tissues to be used as important energy and carbon sources.
S Itakura, H Tanaka, A Enoki

Physiologic response of Phanerochaete chrysosporium to exposure to triazole fungicides
1994 - IRG/WP 94-10066
Triazoles are increasingly important fungicides which are employed for a variety of applications included wood protection. Several recent studies suggest that white rot fungi are more tolerant of triazole compounds than other wood degrading fungi. Cultural studies using a white rot fungus, Phanerochaete chrysosporium, and 0.2 or 0.8 ppm of tebuconazole or propiconazole suggested that mycelial dry weight was most affected by the presence of triazoles. Extracellular carboxymethylcellulase, cellobiosidase and phenol oxidase activities were depressed but not inhibited by triazoles, while ß-glucosidase activity appeared to be slimulated by the presence of these biocides. The results suggest that white rot fungi may be less sensitive to triazoles and this diminished sensitivity may permit these fungi to become more important on wood treated with this biocide.
J J Morrell, R K Velicheti

Biosynthesis of ß-Glucan microfibrils by cellular fractions from brown-rot fungus Postia placenta (MAD-698 and ME-20) and white-rot fungus Schizophyllum commune (MAD-619)
1993 - IRG/WP 93-10025
In this study, we compared the brown rot fungus Postia placenta (MAD-698 and ME-20) with the white rot fungus Schizophyllum commune (MAD-619) to determine the location and distribution of glucan synthetase. We also measured the soluble protein content in subcellular fractions obtained by differential centrifugation MAD-698 is a degradative isolate, but ME-20 and MAD-619 do not produce significant weight loss in wood. The solubilized enzyme glucan synthetase catalyzes the UDP [U-l4C] glucose to synthesize an insoluble linear 1,3-ß-D-glucan polymer. Glucan is a component of basidiomycete cell walls and hyphal sheath. The wood-degrader MAD-698 showed the most glucan synthetase activity in the mixed membrane fractions, and the nondegradative isolates ME-20 and MAD-619 had the most activity in the cytoplasmic fractions. In fact, glucan synthetase activity was found in different proportions in different particulate fractions of MAD-698, ME-20, and MAD-619. Treatment with a mixture of the detergents octylglucoside and CHAPS ( 3 -[(3-Cholamidopropyl)-dimethylammonio]-1-propane-sulfonate) increased the glucan synthetase activity only in the cell wall fraction.
S C Croan, T L Highley

Controlling the sapstain fungus Ceratocystis coerulescens by metabolites obtained from Bjerkandera adusta and Talaromyces flavus
1993 - IRG/WP 93-10024
Sapstain causes severe damage to wood and wood products, posing a major economic problem for the wood industry. The purpose of this study was to determine if metabolites from Bjerkandera adusta and Talaromyces flavus would (1) decolorize stain in wood caused by Ceratocystis coerulescens and (2) prevent sapstain by Ceratocystis coerulescens. We studied the interaction of the sapstain fungus Ceratocystis coerulescens against the test fungi Bjerkandera adusta and Talaromyces flavus in dual cultures on agar medium. The metabolites obtained from test fungi were examined on pine veener disks stained by Ceratocystis coerulescens. Our results indicate that the test fungi were antagonistic to the sapstain fungus Ceratocystis coerulescens. The combination of metabolites from the antagonists decolorize the sapstained pine veener disks and killed the existing growth of Ceratocystis coerulescens.
S C Croan, T L Highley

Basisiospore production by Lentinus lepideus in vitro
1987 - IRG/WP 2276
Evaluation of fungicides active against the lower fungi by spore based bioassays is very common. Possibilities of using similar assays to evaluate preservatives against brown rot and white rot fungi, especially for use in above ground constructions, are limited by the lack of methods for production of basidiospores. Tested media containing glucose 10-30 g/l and ammonium tartrate concentrations 2-20 g/l supported fairly good spore production. Media buffered with the organic buffer 2 (N-morpholino) ethane sulfonate (MES) with phosphate concentrations of 12.5-50 mM supported good spore production.
J Bjurman

Fruitbody formation and basidiosporogenesis by the white rot fungus Pycnoporus cinnabarinus
1988 - IRG/WP 1348
Conditions for fruitbody formation by the white rot fungus Pycnoporus cinnabarinus in vitro were outlined. A marked difference in substrate requirements in comparison to previously tested brown rot fungi was revealed. Agar media containing Walseth cellulose and NH4 tartrate at 1 g/l permit profound production of basidiospores particularly at 15°C but only under light treatment. Higher NH4 tartrate concentrations and glucose addition slowed down or inhibited the fruitbody formation. Axenic basidiospores, to be used in tests for inhibitors contributing to natural durability, as well as in tests for fungicides could thus be produced.
J Bjurman

Wood and filter paper degradation, phenol oxidase and one-electron oxidation activities by the white rot fungus Ceriporiopsis subvermispora
2003 - IRG/WP 03-10486
The activities of one-electron oxidation and phenol oxidase during incubation of cultures of the white-rot basidiomycete Ceriporiopsis subvermispora containing either glucose or wood were periodically measured. Further, the degradation activities against wood and filter paper were examined during the course of cultivation. Weight losses of Japanese beech wood and Japanese cedar wood after 12 weeks were about 20% and 15%, respectively. Weight loss of filter paper was about 23% after 9 weeks. The one-electron oxidation and phenol oxidase activities in wood-containing cultures were higher than those in glucose-containing cultures. Extracellular low-molecular-weight substance has been isolated and has been characterized to compare with the substances from other wood degrading fungi that catalyze a redox reaction between O2 and electron donors to produce hydroxyl radicals. The mechanism on wood degradation caused by the white-rot fungus C. subvermispora is discussed.
H Tanaka, S Itakura, A Enoki

Enzymatic study of Ceratocystis sp., blue-stain fungi on Pinus nigra
1999 - IRG/WP 99-10315
One of the main problems that the forest exploitation industry has with Pinus nigra wood is the blue-stain fungi, whose causing agent is unknown. Therefore, the objective of this work has been to study, through enzymatic tests of the isolated cultures, if these fungi infect Pinus nigra in any specific way. After the incubations, isolates of Ceratocystis were obtained. These were cultured in a saline medium with sawdust of Pinus nigra and Pinus sylvestris, which were used as reference species. At different incubation times, carboxymethylcellulase, xylanase, cellobiohydrolase, laccase and manganese peroxidase were determinated. The results obtained show that the cellulolytic enzymes and laccase have higher activity on Pinus nigra sawdust than on Scots pine, while the Mn peroxidase showed higher values on the sawdust of the latter. Likewise, the cultures were developed in the same saline base medium with different monosacarides (glucose, galactose, mannose, xylose and arabinose), and in determining the residual sugar content, a marked prefrence for the consumption of pentoses in respect to the glucose was observed. The enzymatic activity tests carried out by APY ZYM also showed qualitative and semiquantitative differences between the isolated fungi and other Ceratocystis species tested, so, in addition to the above results, this could indicate a certain specificity of these fungi for this wood species.
M T De Troya, F Rubio, D Muñoz-Mingarro, F Llinares, C Rodríguez-Borrajo, M Yuste, M J Pozuelo, J I Fernández-Golfín

Fungal detoxification of organotin biocides
1985 - IRG/WP 1258
The ability of a range of wood decaying fungi to inactivate bis(tri-n-butyltin) oxide (TnBTO) in the extracellular growth medium, in stationary liquid culture was determined. A distinction between the ability to tolerate the fungicide and to inactivate it was made: the white-rot organism Coriolus versicolor being the most efficient inactivator. In an attempt to determine the extracellular agents responsible for any detoxification, Coriolus versicolor was shown to produce significantly greater levels of extracellular free radicals/peroxidase. Preliminary tests have shown the nature of the associated anion on the fungicide effects the susceptibility of tributyltin compounds to free radical attack in a chemical system. The ability of an free radical scavenger to reduce detoxification in such a system has also been demonstrated.
P S Belford, D J Dickinson

Characterization of wood decay enzymes by MALDI-MS for post-translational modification and gene identification
2002 - IRG/WP 02-10442
The recent sequencing of the Phanerochaete chrysosporium genome presents many opportunities, including the possibility of rapidly correlating specific wood decay proteins of the fungus with the corresponding gene sequences. Here we compare mass fragments of trypsin digests, determined by MALDI-MS (Matrix Assisted Laser Desorption Ionization-Mass Spectrometry), with predicted mass fragments derived from genome sequence. Glyoxal oxidase of P. chrysosporium is used for proof of concept because its genomic organization is known. Glyoxal oxidase was also chosen because it is a glycoprotein, as are many other fungal proteins, and post-translational sites are predicted by MALDI-MS.
T H de Koker, P J Kersten

Phenol oxidase activity and one-electron oxidation activity in wood degradation by soft-rot deuteromycetes
2007 - IRG/WP 07-10615
Wood degradation, one-electron oxidation activity as assayed by ethylene generation from 2-keto-4-thiomethylbutyric acid (KTBA), and phenol oxidase activity were measured in cultures of six deuteromyce fungi, with glucose or wood as the carbon source. The four fungi that degraded Japanese beech wood had higher one-electron oxidation activities in wood-containing cultures than in glucose-containing cultures. These four fungi also had measurable phenol oxidase activity in wood-containing cultures, but not in glucose-containing cultures. The two mould fungi that did not significantly degrade wood had no phenol oxidase activity in either wood- or glucose-containing cultures. The one-electron oxidation activity in intact cultures of the soft-rot deuteromycetes was roughly related with the rate of mass loss during wood degradation in those cultures. However, there was no clear relationship between phenol oxidase activity and either one-electron oxidation activity or the rate of wood mass loss, either over time, or in total. Most of the one-electron oxidation activity resulted from phenol oxidase and hydroxyl radical. Most of the phenol oxidase activity resulted from laccase. Furthermore, the mechanism of wood degradation by one of these deuteromycete fungi, Graphium sp., was investigated. Most of the phenol oxidase activity appeared to derive from laccase. Most of the ethylene generation from KTBA was attributed to hydroxyl radicals, produced by a low-molecular-mass substance in the extracellular media. This substance was composed of protein, carbohydrates, and Fe(II), and catalyzed redox reactions between O2 and unidentified electron donors, to produce hydroxyl radicals via H2O2. It is suggested that hydroxyl radicals may produce new phenolic substructures on the lignin polymer, making it susceptible to attack by laccase. Thus, one-electron oxidation acitivity and laccase activity are both important in wood degradation by Graphium sp.
H Tanaka, M Yamakawa, S Itakura, A Enoki

Oxidative stress and lignin peroxidase production in Phanerochaete chrysosporium
2008 - IRG/WP 08-10655
In Phanerochaete chrysosporium liquid cultures, the induction of lignin peroxidases is directly related to accumulation of reactive oxygen species at the mitochondrial level. In this study, we demonstrate that the expression of the mitochondrial thiol-related antioxidant system is not directly coupled to the LiPs expression. When the antioxidant systems are not able to cope with ROS accumulation, a major change in the respiratory pathway leading to a large modification of the oxidative phosphorylation occurs. The adaptative system revealed by this study plays probably a central role in the LiPs expression regulation and could allow identifying new targets for wood preservatives.
M Morel, L Diss, C Fourrey, M Chalot, M Droux, J P Jacquot, E Gelhaye

Effect of wood polymers degradation during heat treatment on extracellular enzymatic activities involved in beech degradation by Trametes versicolor
2008 - IRG/WP 08-40392
Effect of heat treatment on extracellular enzymes involved in wood degradation by Trametes versicolor was investigated. Heat-treated and untreated beech blocks were exposed to T. versicolor on malt agar medium and extracellular enzymatic activities investigated. A strong ABTS oxidizing activity has been detected during the first stage of colonization in both cases, while cellulase activities are mainly detected in the case of untreated beech wood. Further investigations carried out on holocellulose, isolated using sodium chlorite delignification procedure, either on untreated beech wood or heat treated one, indicate that commercially available cellulases are able to hydrolyse totally holocellulose from untreated sawdust, while hollocellulose from heat treated one was only partially hydrolysed. CP/MAS 13C NMR analysis of heat treated beech wood but also its lignin and holocellulose fractions obtained after acidic hydrolysis of polysaccharides or delignification with sodium chlorite indicates an important modification of hollocellulose showing degradation of hemicelluloses as generally described in the literature, but also formation of carbonaceous materials within the wood structure. All these data suggest that chemical modifications of wood components during heat treatment disturb enzymatic system involved in wood degradation.
S Lekounougou, G Nguila Inari, M Pétrissans, S Dumarçay, J P Jacquot, E Gelhaye, P Gérardin

Non-structural carbohydrates mobilization throughout the stem of Tectona grandis: A strategy for enhancing the wood natural durability
2010 - IRG/WP 10-10729
Non-structural carbohydrates (NSC) storage is an important feature of heartwood substances formation. Radial distributions of NSC before and after chemical (acid and basic) hydrolysis, were quantified using a spectrophotometric method after enzymatic reaction and the corresponding macromolecules of conjugated NSC analyzed by HPLC, were studied in teak stem with reference to wood in environmental condition. In sapwood, free NSC (starch, glucose, fructose and sucrose) content and that of conjugated NSC (glucose) decreased abruptly from sapwood to heartwood. In both sapwood and heartwood, NSC were bounded to two unidentified compounds HB1 and HB2 which were well-characterized by high performance liquid chromatography (HPLC)/diode array detector. Our results show that free (70%) and conjugated (30%) NCS mobilized in sapwood, underwent high catabolic activities in the transition zone leading to their drastic depletion in heartwood. In heartwood unmetabolized glucose was stored by glucosilation with HB1 and HB2 and probably with other molecules. These results indicate that NSC mobilization throughout the stem could be a strategy for long lasting species like teak with consequences on decay resistance of wood.
B F Niamké, N Amusant, D Stien, A Amissa Adima, C Jay-Allemand