Brown-rot Fungus Fomitopsis Palustris Research Articles

Pectin decomposition at the early stage of brown-rot decay by Fomitopsis palustris.

The sapwood of Japanese cedar (Cryptomeria japonica D. Don) was decayed by the brown-rot fungus Fomitopsis palustris under bright and dark conditions. Scanning electron microscopy revealed the presence of mycelia inside the wood even after 1 week from the start of fungal exposure. Moreover, holes were observed in the torus after fungal exposure. Ruthenium red staining revealed that the pectin in pits was largely absent for 3 weeks. These events occurred before the mass loss of wood samples was confirmed at the early stage. Moreover, FpPG28A was more highly expressed at the hyphal front on a pectin-containing medium under dark conditions compared with bright conditions. This up-regulation under dark conditions indicated that the pectin decomposition ability was promoted inside the wood where light could not reach. In conclusion, we suggest that the brown-rot fungus completed its hyphal expansion within the wood via pectin decomposition in pits before holocellulose decomposition.

Starch-degrading enzymes from the brown-rot fungus Fomitopsis palustris

Brown-rot fungi preferentially degrade softwood and cause severe breakdown of wooden structures. At the initial stage of the brown-rot decay, penetrating hyphae of the fungi are observed in ray parenchyma. Since starch grains are known to be present in the ray parenchyma of sapwood, investigation of the functions and roles of the starch-degrading enzymes is important to understand the initial stage of brown-rot decay. We purified and characterized two starch-degrading enzymes, an α-amylase (FpAmy13A) and a glucoamylase (FpGLA15A), from the brown-rot fungus, Fomitopsis palustris, and cloned the corresponding genes. The optimal temperature for both enzymes was 60 °C. FpAmy13A showed higher activity at a broad range of pH from 2.0 to 5.0, whereas FpGLA15A was most active at pH 5.0–6.0. Notable thermal stability was found for FpGLA15A. Approximately 25% of the activity remained even after treatment at 100 °C for 30 min in sodium phosphate buffer at pH 7.0. These different characteristics imply the different roles of these enzymes in the starch degradation of wood.

A GH family 28 endo-polygalacturonase from the brown-rot fungus Fomitopsis palustris: Purification, gene cloning, enzymatic characterization and effects of oxalate

Brown-rot fungi are the wood-decay basidiomycetes and have ability to break down plant cell wall carbohydrates. It has been suggested that degradation of pectin is important for the initial stages of brown rot. We purified an endo-polygalacturonase (FpPG28A) from the brown-rot fungus Fomitopsis palustris, analysis of the predicted amino acid sequence indicated that FpPG28A belongs to GH family 28. The highest activity of purified FpPG28A was observed at 60 °C in 50 mM sodium acetate buffer (pH 5.0); this activity was highly specific for polygalacturonic acid chains. However, calcium polygalacturonate gel was not degraded by FpPG28A under those optimal conditions. We observed that calcium polygalacturonate gel was readily degraded by the enzyme in the oxalate buffer. Furthermore, the thermostability of FpPG28A was elevated in oxalate buffer at pH 3.0. These results indicated that oxalate has an important role in the degradation of woody pectin by FpPG28A.

Functional characterization of a thermostable endoglucanase belonging to glycoside hydrolase family 45 from Fomitopsis palustris

A gene encoding an endoglucanase belonging to subfamily C of glycoside hydrolase family 45 (GH45) was identified in the brown rot fungus Fomitopsis palustris and functionally expressed in Pichia pastoris. The recombinant protein displayed hydrolytic activities toward various substrates such as carboxymethyl cellulose, phosphoric acid swollen cellulose, glucomannan, lichenan, and β-glucan. In particular, the enzyme had a unique catalytic efficiency on β-1,4-glucans rather than mixed β-1,3/1,4-glucans as compared to other GH45 endoglucanases. The fungal enzyme was relatively thermostable, retaining more than 91.4% activity at 80°C for 1h. Site-directed mutagenesis studies revealed that the mutants N95D and D117N had significantly reduced enzymatic activities, indicating that both residues are essential for the catalytic reaction. Our study expands knowledge and understanding of the catalytic mechanism of GH45 subfamily C enzymes and also suggests that this thermostable endoglucanase from F. palustris has great potential in industrial applications.

Wood decaying properties of the termite mushroom Termitomyces eurrhizus

Four strains of the termite mushroom Termitomyces eurrhizus collected in Japan were surveyed for their wood decaying properties in three softwood and two hardwood species, in comparison with the white-rot fungus Trametes versicolor and the brown-rot fungus Fomitopsis palustris. All strains of T. eurrhizus degraded only the surfaces of the wood samples, and differences in mass-loss rates between heartwood and sapwood were generally not significant. Higher mass-loss rates were generally obtained in softwood than in hardwood. The results of chemical analyses of decayed wood samples indicated that T. eurrhizus does not have high lignin-degradation ability, even though it is categorized as a white-rot fungus. These results clearly suggest the unique physiological characteristics of T. eurrhizus.

Analysis of microbial volatile organic compounds produced by wood-decay fungi.

Microbial volatile organic compounds (MVOCs) produced by the brown-rot fungus Fomitopsis palustris and white-rot fungus Trametes versicolor grown on wood chip and potato dextrose agar were analyzed by GC-MS. In total, 110 organic compounds were identified as MVOCs. Among them, only 23 were MVOCs commonly observed in both types of fungi, indicating that the fungi have differential MVOC expression profiles. In addition, F. palustris and T. versicolor produced 38 and 22 MVOCs, respectively, which were detected only after cultivation on wood chip. This suggests that the fungi specifically released these MVOCs when degrading the cell-wall structure of the wood. Time course analysis of MVOC emission showed that both types of fungi produced the majority of MVOCs during the active phase of wood degradation. As both fungi produced specific MVOCs in the course of wood degradation indicates the possibility of the application of MVOCs as detection markers for wood-decay fungus existing in woody materials.

Antifungal and antitermitic activities of wood vinegar from Vitex pubescens Vahl

Antifungal and antitermitic activities of wood vinegar produced from Vitex pubescens were evaluated. Three kinds of wood vinegar were produced at three different pyrolysis temperatures, i.e. at 350, 400 and 450 °C. A PDA dilution method was employed to assay antifungal activity of the vinegars with a white-rot fungus Trametes versicolor and a brown-rot fungus Fomitopsis palustris. Termiticidal activity and repellent effect were evaluated by a no-choice test and a choice test with Reticulitermes speratus and Coptotermes formosanus. All wood vinegars exhibited antifungal activity against both fungi. Wood vinegar of 450 °C had the higher activity than those of 400 and 350 °C. It was assumed that acid component contributed to the increase in controlling the growth of fungal. The wood vinegar exhibited antitermite activity to both R. speratus and C. formosanus workers in the no-choice experiment. However, it needed relatively high concentration to obtain the perfect mortality. For instance, the wood vinegar of 10 % concentration was needed to achieve 100 % mortality against C. formosanus, whereas for R. speratus only 3 % of wood vinegar was required. In the direct-choice experiment, wood vinegar had a significantly repellent effect to both termites at the lowest treating concentration of 10 %.

Characterization of two acidic β-glucosidases and ethanol fermentation in the brown rot fungus Fomitopsis palustris

Two acidic β-glucosidases (βGI and βGII) from the brown rot fungus Fomitopsis palustris were purified to homogeneity by several chromatographic steps. βGI and βGII had molecular weights of 130 and 213 kDa, respectively, and exhibited optimum activity at pH 2.5 and 55 °C. The K m values of βGI and βGII for p-nitrophenyl-β- d-glucopyranoside were 0.706 and 0.971 mM, respectively. Although the effect of metal ions and inhibitors differed between the two enzymes, both β-glucosidases exhibited preferential glucose release during hydrolysis of cello-oligosaccharides, indicating that βGI and βGII possess effective exo-type activities. Notably, F. palustris was able to produce ethanol when cultured on medium containing 20 g/l of glucose, mannose, cellobiose, and maltose, in which the maximum ethanol concentrations measured were 9.2, 8.7, 9.0, and 8.9 g/l, corresponding to 90.2%, 85.3%, 88.2%, and 87.3% of the theoretical yield, respectively. These findings suggest that F. palustris has the ability not only to secrete β-glucosidase enzymes effective at low pH, but also to function as a biocatalyst, which may be suitable for the conversion of lignocellulosic materials into ethanol.

Laboratory evaluation of wood-based composites treated with alkaline copper quat against fungal and termite attacks

Vacuum-impregnation with alkaline copper quat (ACQ) was applied as a post-treatment to five commercially-available wood-based composites (approximately 12-mm thick), including softwood plywood (SWP), hardwood plywood (HWP), medium density fiberboard (MDF), oriented strand board (OSB) and particleboard (PB). In general, ACQ-treated wood-based composites were not as resistant to biological attack as treated Cryptomeria japonica sapwood blocks, possibly due to the uneven distribution of preservative in the composites. Untreated and treated composites were tested for their resistance to two decay fungi (brown-rot fungus Fomitopsis palustris and white-rot fungus Trametes versicolor) and the subterranean termite Coptotermes formosanus by Japanese standardized laboratory test methods. Untreated MDF was the most resistant to both biological attacks, followed by PB, which was less resistant to C. formosanus. ACQ did not adequately protect SWP from F. palustris or termite, or OSB from F. palustris or T. versicolor even at the highest test retention of 5.2 kg/m 3, whereas the biological resistance of HWP was reasonably improved by ACQ. Since cut-end-coated composites treated at higher retentions of both ACQ and copper azole performed much better than uncoated materials, cut-ends were considered to provide decay fungi and termites with easy access or penetration. These findings support the importance of remedial treatment of processed building components at construction sites.

Heat Modification of Wood: Chemical Properties and Resistance to Mold and Decay Fungi

The resistance of heat-modified sugi (Cryptomeria japonica D.) sapwood against mold and decay fungi was evaluated under laboratory conditions. Wood specimens were subjected to heat treatment at 180°C for 2 and 4 hours and at 220°C for 2 hours. Changes in lignin and polysaccharide weight percent composition, solubility, and pH of heat-modified specimens were determined. Treatment at 220°C for 2 hours decreased wood polysaccharide content. Heat treatment slightly increased resistance against Rhizopus javanicus and Gliocladium (Trichoderma) virens, but not against Aspergillus niger. The highest temperature used increased resistance to decay by the white-rot fungus Trametes versicolor, but mass losses with the brown-rot fungus Fomitopsis palustris were higher than those with T. versicolor.

Biological performance of copper azole-treated wood and wood-based composites

Abstract Vacuum-impregnation with copper azole was applied as a post-treatment to five commercially available wood-based composites with thicknesses of approximately 12 mm, such as softwood plywood (SWP), hardwood plywood (HWP), medium density fiberboard (MDF), oriented strand board (OSB), and particleboard (PB). Untreated and treated composites were tested for their resistance to decay fungi (brown rot fungus Fomitopsis palustris and white rot fungus Trametes versicolor) and the subterranean termite Coptotermes formosanus by Japanese standardized laboratory test methods. Untreated MDF was highly resistant to both biological attacks and seemed to require no preservative treatment under less hazardous conditions, i.e., under protected and above-ground applications, with possible occasional wetting. PB was ranked second, and needed further protection only against C. formosanus. Copper azole did not adequately protect SWP from F. palustris and termite. OSB was not protected either against F. palustris and T. versicolor even at a concentration level of 1 kg copper azole per m3 in contrary to blocks of Cryptomeria japonica sapwood, which was protected at these concentration levels. The biological resistance of HWP was reasonably improved by copper azole. The performance of wood-based composites treated with copper azole, which was inferior to the biological resistance of treated C. japonica sapwood blocks, might depend on the thickness or layer profile, density as a result of porosity, uneven distribution of the preservative in the composites, and the susceptibility of the component raw materials.

Involvement of FpTRP26, a thioredoxin-related protein, in oxalic acid-resistance of the brown-rot fungus Fomitopsis palustris

Brown-rot fungus Fomitopsis palustris grows vigorously at high concentrations of oxalic acid (OA), which is fungal metabolite during wood decay. We isolated a cDNA FpTRP26 from F. palustris by functional screening of yeast transformants with cDNAs grown on plates containing OA. FpTRP26 conferred a specific resistance to OA on the transformant. OA-content in transformants grown with 2 mM OA decreased by 65% compared to that of the control. The amount of FpTRP26 transcript in F. palustris amplified with increasing OA-accumulation, and was maintained at high levels even in the stationary phase. Its transcription in F. palustris was inducible in response to exogenously added OA. These results suggest that FpTRP26 is involved in the OA-resistance in F. palustris.

Combined effect of boron compounds and heat treatments on wood properties: Boron release and decay and termite resistance

Abstract The decay and termite resistance of boric acid (BA)- and di-sodium octoborate tetrahydrate (DOT)-treated sugi sapwood was tested in the context of additional heat treatments at two temperature levels. Heat treatments had no effect on boron release and almost all boron was leached from specimens during a 10-day weathering period. Decay tests with the brown-rot fungus Fomitopsis palustris and the white-rot fungus Trametes versicolor and a 3-week termite resistance test with the subterranean termite Coptotermes formosanus were performed. Heat treatments did not increase the decay resistance of either BA- or DOT-treated specimens against the brown-rot fungus. However, the decay resistance of BA-treated specimens against the same fungus increased after heat treatment at 220°C for 2 h. Heat treatments at 180°C for 4 h and 220°C for 2 h also resulted in increased decay resistance of DOT-treated specimens against T. versi-color. Increased resistance against termite attack was observed only in DOT-treated specimens heated at 180°C for 4 h or at 220°C for 2 h. Accordingly, a synergistic effect between heat and DOT treatments was observed for resistance against white-rot decay and termites.

The effect of substituent distribution on the decay resistance of chemically modified wood

Fungal durability of chemically modified wood was investigated with respect to the macroscopic distribution of substituents. Sugi (Cryptomeria japonica D. Don) sapwood samples were treated with modifying chemicals, which involved acid anhydrides (benzoic and n-hexanoic anhydrides) and acid chlorides (benzoyl and n-hexanoyl chlorides), to prepare benzoylated and n-hexanoylated wood. Neutron radiography technique was applied to visualize and evaluate the distribution of the substituents in those modified wood samples. Results revealed that the reaction with acid chlorides led to the heterogeneous distribution, high content of the substituents near the cross sections but relatively low content inside, while the modification with acid anhydrides resulted in the almost homogeneous distribution. The effect of such different distribution on the decay resistance was estimated by using the brown-rot fungus Fomitopsis palustris. It was found that wood samples modified with benzoyl and n-hexanoyl anhydrides exhibited clearly higher resistance against the brown-rot decay than those modified with corresponding acid chlorides, indicating the homogeneous distribution of the substituents is more effective. These results demonstrate that the substituent distribution is one of the important factors for the decay resistance of chemically modified wood.

Blockboard with boron-treated veneers: laboratory decay and termite resistance tests

This study is evaluated by measuring the weight loss, and the ability of white- and brown-rot fungi and termites to attack untreated and boron-treated blockboard manufactured using untreated fir (Abies bornmulleriana M.) strips sandwiched between Ekaba (Tetraberlinia bifoliolata Harms.) veneers at final assembly. The veneers were treated with either boric acid or disodium octoborate tetrahydrate, or mixtures of these chemicals, and blockboard specimens were subjected to fungal decay resistance tests performed according to the Japanese Industrial Standard (JIS) A-9201 method using the brown-rot fungus Fomitopsis palustris (Berkeley et Curtis) Murrill and the white-rot fungus, Trametes versicolor (L. ex Fr.) Quel. Blockboard specimens were also tested against the subterranean termite Coptotermes formosanus Shiraki to determine termite resistance. Blockboard specimens with boron-treated veneers demonstrated increased durability against decay fungi and termite attack. However, sealing of untreated fir strips before decay resistance tests helped reduce significantly the weight losses in blockboard with either untreated or boron-treated veneers. Although blockboard is usually used in indoor applications, incorporation of boron-based biocides may be required for increasing resistance to fungal decay and termite attack and giving fire retardancy.

Decay and termite resistance of boron-treated and chemically modified wood by in situ co-polymerization of allyl glycidyl ether (AGE) with methyl methacrylate (MMA)

Chemical modification was carried out on disodium octoborate tetrahydrate (DOT)-treated wood using allyl glycidyl ether (AGE) in combination with methyl methacrylate (MMA) to limit boron leaching. Co-polymerization of the monomers in DOT-treated sugi ( Cryptomeria japonica D. Don) wood was completed by catalyst heat treatment. Besides boron leachability, the dimensional stability and decay, and termite resistance of chemically modified wood were also determined. Dimensional stability in terms of anti-swelling efficiency and water absorption was found to improve in chemically modified wood specimens. Co-polymerization of AGE with MMA in DOT-treated wood resulted in less boron leaching compared with DOT-only-treated wood. Chemically modified wood containing boron proved to be more resistant to the brown-rot fungus Fomitopsis palustris (Berkeley et Curtis) Murrill (FFPRI 0507) and the white-rot fungus Trametes versicolor (L. ex Fr.) Quel. (FFPRI 1030) in laboratory decay resistance tests after severe leaching for 10 days. Termite resistance tests also showed that wood specimens treated with DOT and MMA plus AGE monomers were effective against subterranean termites Coptotermes formosanus Shiraki even after severe leaching cycles.

Physical and Chemical Properties of an Extracellular Low-Molecular-Weight Substance from the Brown-Rot Basidiomycete Fomitopsis palustris

A low-molecular-weight, extracellular substance with one-electron-oxidizing activity was isolated from wood-degrading cultures of the brown-rot basidiomycetous fungus Fomitopsis palustris. The substance was partially purified by acetone precipitation, gel-filtration chromatography on Sephadex G-50 and G-25, and DEAE-Sepharose ion-exchange chromatography. The partially-purified material was a glycopeptide composed of (54%, w/w) protein and (42%, w/w) neutral carbohydrate, containing (0.06%, w/w) Fe (II) by weight. The molecular weight as estimated by Tricine-SDS-PAGE was 7, 200-10, 000; whereas the molecular weight as determined by size-exclusion or gel-filtration chromatography was 1, 000-5, 000. The glycopeptide reduced O2to H2O2 and Fe (III) to Fe (II), and thus could generate hydroxyl radical via a Fenton reaction. One mg of the partially-purified glycopeptide reduced 1.7μ mol of Fe (III) to Fe (II) and contained at least 0.6μ mol of α-hydroxyketone or endiol groups. Most of the α-hydroxyketone groups were 1-amino-2-ketoses produced by the condensation of side-chain amino groups and carbohydrate aldehydes. The function of the glycopeptide in brown-rot decay is discussed.

Preservative treatment of wood-based composites with 3-iodo-2-propynyl butylcarbamate using supercritical carbon dioxide impregnation

Five structural-use wood-based composites [medium density fiberboard (MDF), hardwood plywood, softwood plywood, particleboard, oriented strand board (OSB)] were treated with 3-iodo-2-propynyl butylcarbamate (IPBC) using supercritical carbon dioxide (SC-CO2) as a carrier solvent. Treatment was conducted at 35°C/7.85 MPa (80 kgf/cm2), 35°C/9.81 MPa (100 kgf/cm2), 35°C/11.77 MPa (120 kgf/cm2), 45°C/7.85 MPa, 45°C/9.81 MPa, 45°C/11.77 MPa, 55°C/7.85 MPa, 55°C/9.81 MPa, and 55°C/11.77 MPa. A decay test was carried out in the laboratory according to the modified Japanese standard method in which untreated and treated specimens were exposed for 12 weeks to a monoculture of the white-rot fungus Trametes versicolor (L.: Fr.) Pilat or the brown-rot fungus Fomitopsis palustris (Berk. et Curt.) Gilbn. and Ryv. IPBC/SC-CO2 treatment protected the treated materials from decay by the two fungi, although the relative efficiency against decay varied with the treatment conditions and the type of wood-based composite. Better performance for MDF, hardwood plywood, and particleboard was generally obtained at high temperatures and pressures, whereas softwood plywood and OSB were most protected at low temperatures under any of the pressure levels tested.

Low molecular weight silicic acid - inorganic compound complex as wood preservative

A new wood preservative containing low molecular weight and low-toxicity silicic acid (LWSA) was investigated. To prevent environmental pollution with the wood preservative, a silicic acid monomer aqueous solution (SAMS) or colloidal silicic acid solution (CSAS) was combined with various metal compounds or boric acid. Agents where SAMS or CSAS was combined with boric acid gave good protection against decay caused by the brown-rot fungus Fomitopsis palustris, the treated wood (Cryptomera japonica D. Don) specimens after the leaching test maintained a high resistance to decay. The leaching and decay tests revealed high quantities of chemicals leaching from wood treated with SAMS-metal agents. However, when wood was treated with SAMS-boric acid, there was little leaching of agent in either test. The mechanism of resistance of wood, which was treated with boric acid mixed with CSAS or SAMS, to the brown-rot fungus F. palustris were investigated. When the concentration of boric acid was high, mycelial growth was inhibited completely and no protein production was detected. When the amount of boric acid was low, the xylanase, mannase and cellulase activities were lower than with control wood powder. When powdery boric acid was combined with CSAS, it was considered that the treated woods have higher anti-weather properties than when boric acid-methanol solution was mixed with CSAS. The agent-preparation method adopted should be considered carefully after taking the treatment process and the intended use of the preservative-treated wood into account.

A metabolic role of the glyoxylate and tricarboxylic acid cycles for development of the copper-tolerant brown-rot fungus Fomitopsis palustris.

Fruit bodies of the copper-tolerant brown-rot fungus Fomitopsis palustris were produced in liquid medium for the first time. To induce fruit body formation of this fungus, it was important to inoculate the liquid medium with mycelia grown on potato dextrose agar plates and also to adjust the initial pH of the medium to 5.0. The metabolic role of the glyoxylate and tricarboxylic acid cycles during fungal development in the liquid culture was investigated in relation to oxalate biosynthesis. The enzymes for the glyoxylate cycle and oxalate biosynthesis in mycelium showed greater activities at the vegetative growth stage than at the fruiting stage. The ratios of isocitrate dehydrogenase activity to isocitrate lyase activity in mycelium were 0.3 and 4.0 at the vegetative and fruiting stage, respectively. Thus, isocitrate lyase of the glyoxylate cycle played a more important role in oxalate synthesis at the earlier stage of the cultivation, whereas isocitrate dehydrogenase played a major role in glutamate synthesis during fruit body formation.