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Wood decay fungi from New Zealand leaky buildings – PCR identification (Part 2) and aerial spore trapping
2008 - IRG/WP 08-10649
Prior to this study, it was not know which species of decay fungi caused decay in New Zealand leaky buildings. Use of molecular biology methodology, polymerase chain reaction (PCR), and subsequent DNA sequencing, as well as classical mycological techniques based on morphology, has enabled identification of decay fungi and has provided insight into their relative importance based on isolation frequency. Fungi colonising Pinus radiata D. Don framing timber of leaky New Zealand buildings were isolated to produce pure cultures. Mycelia from these cultures on agar media were collected to extract DNA. To identify the fungi to the species level, PCR with primer pairs NSI1 + NLB4 and ITS1-F + ITS4 were performed followed by sequencing of the internal transcribed spacer (ITS) region. Identification was by BLAST (Basic Local Alignment Search Tool) search on sequences in GenBank. In total, 421 samples from leaky buildings were processed, mainly decayed timber, but also fibre cement boards and building paper. Sixty-eight fungal identifications were achieved of which 4 species are very common as follows: • Gloeophyllum sepiarium (Wulf.: Fr.) Karst. 13x • Oligoporus placenta (Fries 1865) Gilb. In Ryv.1985 11x • Antrodia sinuosa (Fr.) Karst. 8x • Gloeophyllum trabeum (Fr.) Murr 4x An aerial spore study of internal air, wall cavity air and exterior air of leaky buildings was carried out using a Merck MAS-100 instrument which collects spores directly onto various selective media plates. Also, decayed wood samples from the same leaky buildings enabled identification of G. sepiarium and A. sinuosa at the same test site. Viable fungal aerial spores were detected at every sampling location, with a highest mean of 3714 colony-forming units (CFU) per square meter found in water-damaged walls. The use of Carboxymethylcellulose medium further demonstrated the presence of cellulose degrading fungi within and around the location. Overall, the combination of these two approaches proved useful for detection of fungal species variation at a multi-unit building complex and it was possible to identify the brown rot decay fungal genus Antrodia with both methods.
D Stahlhut, R L Farrell, R Wakeling, M Hedley

The dry rot fungus and other fungi in houses. Part 2
1993 - IRG/WP 93-10001
J Bech-Andersen

An attempt to evaluate wood resistance against fungal decay in non-sterile conditions by measuring the variation of resistance to bending test
1988 - IRG/WP 2308
The main object of this work was to determine the variation of strength on large test specimens of wood (800 x 45 x 45 mm³) when exposed to accelerated fungal attacks close to natural conditions, out of test vessels. The modulus of elasticity (MOE) and the modulus of rupture (MOR) have been assessed. Thereby, the natural resistance of the wood species to fungal decay, the efficiency of preservative as well as the treatment applied are discussed. The wood tested is a guianese secondary species (Couma guianensis). The fungi tested are two guianese strains of brown and white rot. The exposure time is 12 weeks. No mould contamination has been recorded by use of a selective fungicide. The results obtained show that it is possible to infest in nonsterile conditions large wood specimens. Furthermore, modulus of rupture appears to be the most reliable criterion. The investigation, that requires limited equipment and staff could be performed in any tropical research station as it has been done at CTFT, French Guiana center.
L N Trong

Decay resistance of wood removed from poles treated with Trichoderma
1989 - IRG/WP 1386
Wood blocks removed from a distribution pole previously treated with a biological control product (Binab FYT pellets) were exposed in soil block tests to selected basidiomycetes. The blocks were removed from regions of poles where Trichoderma colonization had been confirmed by extensive sampling and computer mapping of microbial inhabitants. The results indicate that material from pole interiors colonized by Trichoderma is able to resist decay by Lentinus lepideus and Antrodia carbonica. Any decay prevention was lost however when the wood was steam sterilized prior to exposure to the basidiomycetes. The implications of the results for the use of biological control of internal decay in creosoted poles is briefly discussed.
A Bruce, B King, T L Highley

The dry rot fungus and other fungi in houses. Part 1
1992 - IRG/WP 92-2389
J Bech-Andersen

Monographic card on Antrodia serialis
1984 - IRG/WP 1145
G Seehan

Effect of asphyxiation on wood decay fungi treated with argon and nitrogen gas
2002 - IRG/WP 02-10452
The effects of low-oxygen conditions, achieved with either argon or nitrogen gas, on the viability of wood decay fungi Coniophora puteana, Antrodia vaillantii and Trametes versicolor, cultivated on PDA medium and infected wood samples, were examined. The fungal cultures were exposed to low oxygen concentration (below 10 ppm) for one to five weeks in hermetically sealed vessels. Anoxic treatment did not affect T. versicolor cultures in the time span of the experiment. Therefore treatment of only C. puteana and A. vaillantii mycelial cultures was extended to 10 and 16 weeks. After treatment, respiration and regeneration of mycelium were tested by measurements of CO2 production and resumed growth of hyphae onto fresh PDA growth medium. The effect of anoxic conditions on the mycelia of treated fungal species was expressed as an increased time needed for regeneration or as a complete absence of growth of inocula taken from the exposed cultures or wooden blocks reintroduced on new nutrient medium. The cultures that were retarded by the low oxygen concentration consequently produced less CO2. For C. puteana cultures, the effects of anoxic treatment became evident in the second week of the treatment. The number of affected cultures rose steadily with the prolongation of anoxic treatment. By the sixteenth week of the anoxic treatment, 80% of the inocula of C. puteana did not regenerate. A. vaillantii inocula regeneration was not affected until after the fourth week of treatment, and similarly for infected wood samples, after five weeks. The influence of anoxic treatment on the cultures of this species was more pronounced on the tenth and especially after the sixteenth week, when 67% of inocula did not regenerate. In general fungal species were differently sensitive to asphyxiation. T. versicolor cultures were not affected by anoxic conditions, caused by either argon or nitrogen gas, and A. vaillantii mycelial cultures proved to be less sensitive than those of C. puteana. In the test with infested wood blocks argon proved to be more effective, compared to nitrogen gas.
C Tavzes, F Pohleven, M Janisek, R J Koestler

About the relations between the natural durability of some tropical species and their extractives content
1983 - IRG/WP 1208
G R Y Déon

Experience with an industrial scale-up for the biological purification of CCA-treated wood waste
1997 - IRG/WP 97-50095
The biological purification of CCA-treated wood waste was tested in co-operation of the BFH and the Italian impregnation plant SoFoMe. Chipped poles were infested with the chromium and arsenic tolerant brown-rot fungus Antrodia vaillantii which can transform in the laboratory ca. 90% of the chromium and arsenic into watersoluble salts. These can be leached to 100-200 ppm residual metal content. The fermentation techniques tested will be described and the fermentation success as well as the possible use of the purified material will be discussed.
H Leithoff, R-D Peek

Bioprocessing preservative-treated waste wood
2000 - IRG/WP 00-50145
Disposal of preservative-treated waste wood is a growing problem worldwide. Bioprocessing the treated wood offers one approach to waste management under certain conditions. One goal is to use wood decay fungi to reduce the volume of waste with an easily managed system in a cost-effective manner. Wood decay fungi were obtained from culture collections in the Mycology Center and Biodeterioration research unit at the USDA-FS Forest Products Laboratory (FPL), Madison, Wisconsin, and from FPL field sites. The 95 isolates had originally been taken from at least 66 sites from around the United States. Isolates were screened in a bioassay (known as the 'choice test') for tolerance to CCA, ACQ, creosote and pentachlorophenol. A tolerant rating was based on fungal growth toward or on treated wood, with 17 tolerant to CCA, 21 to ACQ, 12 to creosote and 5 to pentachlorophenol. Decay capacity of the tolerant isolates was determined as percent weight loss by the ASTM D-1413-76 soil bottle method. We identified 8 isolates for experiments on preservative remediation. Isolates of Meruliporia incrassata and Antrodia radiculosa gave the highest percent degradation of ACQ and CCA-treated wood. Several A. radiculosa isolates and a Neolentinus lepideus isolate grew on creosote-treated wood, but had only a 4-5% weight loss. In this paper we discuss the potential use of decay fungi to degrade or remediate preservative-treated wood.
B Illman, V W Yang, L Ferge

Determination of absorption, accumulation and transport of copper in mycelium of some wood decay fungi
1999 - IRG/WP 99-10323
Copper compounds are common wood preservatives. However, tolerance of some wood decay fungi to copper compounds has been observed recently. Therefore, we tried to elucidate possible causes of this phenomenon. We investigated uptake, accumulation and secretion of copper in the mycelium of potentially copper tolerant fungi (Antrodia sp.) and non tolerant fungus Trametes versicolor. We observed that potentially tolerant fungi have lower uptake of copper to the mycelium than non tolerant species. They also do not transport copper into the medium. That means that copper tolerance of fungi is probably based on low uptake of copper to the mycelium and not on the active transport from the mycelium to the medium.
F Pohleven, S Breznikar, P Kalan, M Petric

Biochemical relationships between biodegradation of cellulose and formation of oxalic acid in brown-rot wood decay
1991 - IRG/WP 1472
Non-enzymic hydrolysis of cellulose with low concentrations of oxalic acid was examined. The incubation of pine wood pulp with 1% oxalic acid (pH 1.3) at 35°C for 4 weeks reduced the original viscosity to 60%. Reducing sugars were liberated from various cellulosic samples by the oxalic acid treatment. However, crystallinities of cellulose in those samples did not change before and after the treatments. Then, the enzymatic formation of oxalic acid was investigated in relation to cellulose biodegradation by brown-rot fungi. We succeeded in isolating oxaloacetase from the brown-rot fungus Tyromyces palustris in cell-free extracts which catalyze hydrolysis of oxaloacetate to produce oxalate and acetate. During the brown-rot wood decay process, oxaloacetase may play an important role in degradation of wood carbohydrate.
M Shimada, Y Akamatsu, A Ohta, M Takahashi

Microbial decomposition of salt treated wood
1993 - IRG/WP 93-50001-22
Specialized microorganisms which are able to convert fixed inorganic preservatives from treated wood into water soluble components are investigated. A number of brown rot fungi like Antrodia vaillantii have been isolated from cases of damage and examined under unsterile conditions with CCA-, CCB-, CCF- and CC-treated wood at retention levels of at least 50% higher than recommended for wood in ground contact. Depending on the kind of fungus, preservative retention, wood particle size, culture conditions and duration Cr and As can be almost completely leached from the treated wood. Cu reacts with oxalic acid to a compound of limited water solubility.
R-D Peek, I Stephan, H Leithoff

Evaluation of bacteria for biological control of wood decay
1990 - IRG/WP 1426
Laboratory soil-block and agar-block tests were carried out to evaluate the efficacy of bacteria as biological control agents against 5 brown-rot and 3 white-rot fungi. Pretreatment of Southern pine and sweetgum with a bacterial solution prevented decay in agar-block tests. However, the bacteria were generally ineffective in preventing decay in Southern pine, Douglas-fir, sweetgum and yellow poplar in soil-block tests. Wood blocks treated with an autoclaved bacterial solution were not decay resistant by either test method.
R Benko, T L Highley

Decay in window joinery in Sweden
1977 - IRG/WP 390
Systematic investigations of decay in window joinery in Sweden have not been undertaken until very recently. One might guess that one of the reasons for the minor interest is that this type of joinery did not rot strikingly faster than people expected. In the last 10-15 years, however, house owners, tenantowners, building societies and others have repeatedly argued that windows do rot more than in earlier days. As long as reliable figures supporting these complaints were not presented, only little attention was shown by the house-building industry and the authorities responsible for the building sector. Finally, 1974, Sveriges Allmännyttiga Bostadföretag (SABO), one of the major public building societies in Sweden, with financial support from the Building Research Council, started a special investigation of their dwellings with regard to rotting window joinery. When the results of this investigation was presented in September 1975, the magnitude of the problem became apparent and further investigations were immediately started.
B Henningsson

Intraspecific variability of durability of Wapa courbaril (Eperua grandiflora) against Antrodia sp. and Coriolus versicolor: effect of radial and height position in the stem
2004 - IRG/WP 04-10531
The variation of a lesser-used species, Eperua grandiflora attacked by brown and white rot has been examined. Trees of this specie showed differences in their behaviour against the type of rot used and also showed evidence of intra and inter tree variation. The “tree” effect is very significant concerning natural resistance. In the same way, there is variation of durability observed according to radial and vertical position in the stem. Although the vertical effect is less significant from practises point of view.
N Amusant, J Beauchêne, M Fournier

Growth of the copper tolerant brown rot fungus Antrodia vaillantii on different substrates
1995 - IRG/WP 95-10121
In recent years the copper tolerant brown rot fungus Antrodia vaillantii caused severe damages on impregnated wood in ground contact. The pattern of decay gave the impression that impregnated wood was even more severely attacked than unimpregnated. To investigate this question more closely laboratory tests were carried out. In a "choice test" Antrodia vaillantii grew preferably towards CC-impregnated wood. Furthermore, when adding nutrients to the specimens, the mass loss of impregnated wood significantly increased, while the nutrients did not affect the mass loss of the unimpregnated wood. In addition, the temperature tolerance of the fungi increased slightly when growing on impregnated wood. It could also be shown that Antrodia vaillantii reacts with an increased production of oxalic acid to the presence of a CC preservative.
H Leithoff, I Stephan, M-T Lenz, R-D Peek

Characterization of Poria indoor brown-rot fungi
1995 - IRG/WP 95-10094
The heterogeneous group of "Poria" fungi causing brown rot in buildings and also of wood in ground contact comprises Antrodia vaillantii, Antrodia serialis, Antrodia sinuosa, Antrodia xantha and Tyromyces placenta. These fungi have similar morphological appearance and biology. Their nomenclature has a confusing history and is still not uniform. As a consequence, misinterpretations may occur. SDS polyacrylamide gel electrophoresis showed a species-specific protein pattern for different cultures of Antrodia vaillantii separating the species from the other pore fungi as well as from Coniophora puteana and Serpula lacrymans. Electrophoresis also detected misidentifications. Investigations on growth rate, response to temperature, copper tolerance and wood decay revealed: Radial growth extension reached from 4 to 9 mm/d. Temperature optimum was 25 to 31°C. All withstood 1 hour at 60°C and some even 3 h at 65°C. Antrodia vaillantii was copper tolerant up to 0.05 M Cu. Wood weight loss after 20 weeks was higher by Tyromyces placenta (35%) and Antrodia sinuosa (33%) than by Antrodia xantha (21%), Antrodia serialis (16%) and Antrodia vaillantii (14%). Dual cultures revealed various inter- and intraspecific interactions and detected identity of differently coded cultures of a species. The former Poria vaporaria sensu Liese 'Normstamm II' for testing wood preservatives and the recent Poria placenta EN 113 strain FPRL 280 were shown to be either identical or at least sister monokaryons originating from the same individual.
O Schmidt

rDNA-ITS sequence of Serpula lacrymans and other important indoor rot fungi and taxon-specific priming PCR for their detection
1999 - IRG/WP 99-10298
Taxon-specific priming polymerase chain reaction (TSPP) is a powerful molecular tool for fungal diagnosis. For its application to indoor rot fungi, the internal transcribed spacer (ITS) of the ribosomal DNA (rDNA) of the main fungal species causing wood rot in European buildings was amplified with the polymerase chain reaction (PCR). The ITS region was sequenced. The complete sequences are presented. From base sequence divergency among the fungi, species-specific oligonucleotide primers were designed for TSPP. These marker molecules were suitable for the differential diagnosis of the dry rot fungus, Serpula lacrymans, the wild merulius, S. himantioides, the oak polypore, Donkioporia expansa, the brown cellar fungus, Coniophora puteana, the broad-spored white polypore, Antrodia vaillantii, the sap polypore, Tyromyces placenta, and the yellow-red gill polypore, Gloeophyllum sepiarium. Each specific marker identified isolates of its respective target species. Cross reaction with 'foreign' fungi was the exception. Species identification from unknown field samples from rot damage in buildings is also possible, because DNA from contaminating organisms does not response to the specific primers. Our variant of the technique is fast, because no preceding fungal pure cultures, no special DNA extraction/purification, and no restriction by endonucleases are necessary.
O Schmidt, U Moreth

Changes of EPR spectra of wood, impregnated with copper based preservatives, during exposure to Antrodia vaillantii
2000 - IRG/WP 00-10355
Spruce wood (Picea abies) samples were impregnated with two different aqueous solutions: copper(II) octanoate with ethanolamine or copper(II) sulfate (cCu = 1,0 x 10-2 mol/l). Impregnated and unimpregnated test pieces were exposed to wood rotting fungus Antrodia vaillantii. Some strains of this fungus are known as copper tolerant. After four weeks of exposure to A. vaillantii, we could not detect any Electron Paramagnetic Resonance (EPR) signal of Cu(II) in the spectrum of copper(II) sulfate treated wood. This means that Cu(II) was translocated or conversed into a form that cannot be detected by EPR. Instead of copper signal, manganese signal appeared. The same, manganese signal appeared also in untreated wood, after exposure to A. vaillantii. On the other hand, line shapes of the EPR spectra of Cu(II) in copper(II) octanoate/ethanolamine treated wood changed by rotting, from anisotropic to isotropic. We believe that these changes are mostly caused by oxalic acid, excreted by A. vaillantii, because we observed the same changes on EPR spectra of impregnated and unimpregnated wood, additionally treated with oxalic acid.
M Humar, M Petric, F Pohleven, M Šentjurc

Copper tolerance of various Antrodia vaillantii isolates
2001 - IRG/WP 01-10406
Copper based preservatives have been extensively used in the field of wood preservation. However, several brown-rot fungi to be tolerant to copper and consequently, efficacy of copper based wood preservatives may not be sufficient. Copper tolerance is especially by some fungi that are closely aligned to or included in genus Antrodia. The highest copper tolerance was found at some strains of wood rotting fungus A. vaillantii. In this research, we investigated the variation of tolerance to copper among 14 isolates of Antrodia using different screening tests. Screening test on potato dextrose agar wasused as nutrient medium. The concentration of copper(II) sulfate in growth medium varied between 1.0x10-3 mol/l and 5.0x10-2 mol/l. There was almost no growth detected at all observed isolates when nutrient media contained the highest concentration of copper. Nevertheless, six strains were still able to grow on solid media containing 2.5x10-2 mol/l of copper. Further tests confirmed copper tolerance atselected isolates. Thus we can conclude that various isolates performed different copper tolerance.
F Pohleven, A Malnaric, M Humar, C Tavzes

Fungal degradation of wood treated with metal-based preservatives. Part 1: Fungal tolerance
1996 - IRG/WP 96-10163
In recent years, concerns have arisen about the leaching of heavy metals from wood treated with chromated copper arsenate (CCA), particularly because of the large amount of CCA treated wood that will be discarded in the coming years. The long term objectives of this work are to determine the fate of copper, chromium and arsenic with the aging and potential decay of CCA-treated wood, and to develop strategies for recycling and remediation of disposed wood. In this study, we determined the ability of various decay fungi to decompose southern yellow pine wood treated with CCA or other metal-based preservatives. Isolates of Meruliporia incrassata and an isolate of Antrodia radiculosa caused the highest weight losses in CCA-treated southern yellow pine. One isolate of Meruliporia incrassata produced similar weight losses in CCA-treated and untreated southern pine after 10 weeks. Pine samples treated with very high levels of copper sulphate were decayed by Meruliporia incrassata, but the fungus was unable to decay wood treated with copper napthenate or copper-8-quinolinolate.
B Illman, T L Highley

Correlation between modulus of elasticity, mass losses and FTIR spectra of copper treated decayed wood
2006 - IRG/WP 06-10580
The composition of copper-based preservatives will change from copper-chromium to copper-ethanolamine, due to environmental demands. The most important drawback of copper-impregnated wood is the presence of tolerant fungal organisms that have developed an ability to degrade such preserved wood. In order to elucidate these processes, specimens (0.5×1.0×15 cm) made of Norway spruce (Picea abies) were vacuum-impregnated with copper-, chromium-, and copper-ethanolamine-based aqueous solutions (cCu=0.5%), and afterwards exposed to copper-sensitive Gloeophyllum trabeum and copper-tolerant Antrodia vaillantii for various times, between one and eight weeks. After incubation, specimens were isolated, and modulus of elasticity (MOE) losses determined using a nondestructive technique. Mass losses, FTIR spectra, and color changes were measured as well. The results showed that there is significant difference between brown rot decay caused by G. trabeum and A. vaillantii. Decay caused by A. vaillantii is more selective than that caused by G. trabeum. Additionally, it was proven that copper effectively protected spruce from G. trabeum, but not completely against A. vaillantii. Decay of copper-impregnated wood by copper-tolerant fungi is similar to decay of control, unimpregnated wood. Whereas decay of copper-impregnated specimens by G. trabeum, was effectively stopped in its initial stage.
M Humar, B Bucar, F Pohleven

Effect of Cinnamomum kanehirae Extractives on the Compositions of the Fermentation Broth of Antrodia cinnamomea
2007 - IRG/WP 07-10632
The fruiting body of Antrodia cinnamomea, called niu-chang-chih, is well known and highly prized in Taiwan as a folk medicine. This fungus is endemic to Taiwan and the growth of which is restricted to the inner heartwood cavities of another endemic tree species, Cinnamomum kanehirae. The fungal basidiomes have been used for treating food and drug poisoning, diarrhea, abdominal pain, hypertension, itchy skin and cancer. The purpose of this study was to investigate the effect of C. kanekirae extractives on the fungal mycelium production and the spore germination. The heartwood was extracted using 70% ethanol, and then liquid-liquid partitions using n-hexane, ethyl acetate, n-butanol and distilled water were carried out respectively. The effects of treating the fermentation broths with C. kanehirae extractives on the fungal biomass, antioxidant scavenging activity using 1, 1-diphyenyl-2-picrylhydrazyl (DPPH) were examined, and the compositions of the fermentation broth were monitored using high-performance liquid chromatography analysis. The results indicate that C. kanehirae extractives treated fungal broths had higher growth promotional effect on their biomass then that of the control (0.0313 g). The n-hexane soluable fraction treated mycelia showed the greatest dry mass (0.0480 g), followed by ethyl acetate soluable fraction (0.0333 g). The addition of crude extract increased the spore germination activity from 8.1 x 106 cfu/ml to 1.1 x 107 cfu/ml. The n-butanol soluble fraction showed the best spore germination activity of 1.3 x 107 cfu/ml. The active fungal ingredient, dehydroeburicoic acid, was found to increase with the addition of C. kanehirae extractive fractions according to the HPLC analysis. The C. kanehirae extractives were effective DPPH free radical scavengers which masked the antioxidant effect of the fungal broth.
Ruo-Yun Yeh, Yuan-Min Shen , Shang-Tzen Chang

Antifungal activity of essential oils against common wood degrading/decaying fungi
2008 - IRG/WP 08-30465
Despite the wide use of essential oils in pharmaceutical and food industry as antimicrobial agents, their use as wood preservatives has not been fully explored. In this study, 12 essential oils were screened in nutrient medium for their antifungal activity against 8 common mould, sapstain or decay fungi. Subsequently, one essential oil, eugenol was evaluated for decay resistance in an agar/wood block tests using both unleached and leached cycles with radiata pine sapwood blocks. During the initial in-vitro screening trial, variability in the tolerance of the tested fungi towards the selected essential oil was apparent. Some of the essential oils such as geranium, cinnamon leaf and eugenol completely inhibited the growth of all test fungi at 0.5% w/v on nutrient medium, whereas, three essential oils; eucalyptus, olive leaf and kolorex® were unable to restrict the growth of any test fungi even at 1% w/v concentration. Durability test results on radiata pine confirmed the antifungal activity of eugenol but highlighted the leachibility of this compound from wood. Blocks treated with 3% w/v eugenol without a leaching cycle had less than 1% weight loss when exposed to all three tested wood decaying fungi, Oligoporus placenta, Coniophora puteana and Antrodia Xantha. However, blocks which were leached showed weight losses in the range of 13.40 to 23.12%. This study identified eugenol as a potential benign wood preservative for treatment of timber not exposed to severe leaching, e.g. New Zealand Hazard Class H1.2. However, to be used for higher decay hazard situations, further work for in-situ polymerization of eugenol to fix active(s) in wood will be required.
T Singh, C Chittenden

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