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Detection of the dry rot fungus Serpula lacrymans by amplified ribosomal DNA restriction analysis
1998 - IRG/WP 98-10245
Isolates of the dry rot fungus Serpula lacrymans and the wild merulius S. himantioides were investigated by amplified ribosomal DNA restriction analyses (ARDRA) of the internal transcribed spacers (ITS). The technique uses the polymerase chain reaction (PCR) to amplify the variable region of the ITS between the conserved 18S and 28S genes of the nuclear ribosomal DNA (rDNA). The ITS region of all isolates of Serpula lacrymans and S. himantioides was successfully amplified. The length of the amplified product was about 630 bp. Subsequent double digest of the ITS with the restriction endonucleases Haelli and Taql separated the closely related fungi by species-specific fragments. Thus, ARDRA-ITS proved to be suited for the detection of the dry rot fungus.
O Schmidt, U Moreth

Characterization of tropical wood-decaying fungi by RFLP analysis of PCR amplified rDNA
1998 - IRG/WP 98-10251
Five brown-rot fungi (Tyromyces palustris, Gloeophyllum trabeum, Antrodia sp., Poria placenta, Coniophora puteana) and three white-rot fungi (Coriolus versicolor, Pycnoporus sanguineus, Lentinus squarrosulus) registred as representative wood-decaying fungi for international standards of wood durability tests have been characterized using molecular tools (PCR/RFLP). The Internal Transcribed Spacer between 18S and 25S rDNA has been amplified using ITS1 and ITS4 primers. The amplified fragments were digested using nine restriction enzymes (RFLP). The analysis of RFLP gel electrophoresis allowed to clearly discriminate every strain from each others. The 150 wood-decaying fungi strains currently in collection in the laboratory will be studied to create a data bank of reference profiles.
A Zaremski, M Ducousso, Y Prin, D Fouquet

16S rRNA Analysis of the Bacteria Associated with Biocide Degradation
2004 - IRG/WP 04-10543
The bacterially mediated degradation of the new anti-fungal biocide, bethoxazin, was studied in vitro, by means of 16S rRNA PCR-amplification and cloning techniques. Woodblocks impregnated with a subtoxic concentration of bethoxazin were incubated in compost, and the micro-organisms associated with the wood after 6 and 12 weeks was studied. It was found that after 6 weeks the wood contained a large number of proteobacteria, including Pseudomonas fluorescens, Bordetella petrii, Asticcacaulis biprosthecium and Alcaligenes spp. These organisms appear to be the dominant organisms involved in bethoxazin detoxification. After 12 weeks, the population of bacteria was found to have altered considerably, with the dominant bacteria being identified as Afipia, Rhizobium and Verrucomicrobium. This population is thought to have developed after the biocide had been detoxified and represents a succession in the wood, outcompeting the previous detoxifying population.
D F Wallace, D J Dickinson

Effects of chlorothalonil (CTN) and butylated hydroxytoluene (BHT) on microbial communities involved in the deterioration of wood using T-RFLP II: Results from field studies
2007 - IRG/WP 07-30429
The effects of Chlorothalonil (CTN) and Butylated Hydroxytoluene (BHT) on microbial species diversity in wood and the surrounding soil are being assessed by Terminal Restriction Fragment Length Polymorphism (T-RFLP). CTN was selected as a trial organic wood preservative, and the non-biocidal BHT was evaluated for its synergistic effects with CTN. ACQ-C was a positive control and untreated SYP stakes were negative controls. Tests were installed at two separate field sites in MS that represent two different AWPA hazard zones. Samples were taken every 3 months over a 15 month period and visually evaluated for termite attack and decay. Samples were processed and whole genomic DNA was extracted for molecular analyses. Upon initial amplification of DNA using both specific and general primers, the presence or absence of target fungi was confirmed using gel electrophoresis. We are currently using T-RFLP to analyze the patterns of microbial colonization over time and in response to external stimuli (i.e., wood preservative treatment) to identify potential shifts in microbial community. Preliminary results indicate that the presence of non-basidiomycete fungi (i.e. molds, stains, and soft rots) are uniformly distributed throughout the samples regardless of treatment, while basidiomycetes are less common and severe decreases in overall basidiomycete populations occurred during periods of drought at both test sites.
G T Kirker, M L Prewitt, S V Diehl

Microbial Community Analysis Using Terminal Restriction Fragment Length Polymorphism (T-RFLP) Analysis: Field Study Results
2008 - IRG/WP 08-20377
The effects of chlorothalonil (CTN) and butylated hydroxytoluene (BHT) on the fungal community in southern yellow pine (SYP) were assessed using terminal restriction fragment length polymorphism (T-RFLP) analysis. Field stakes treated with 0.25% and 0.37% ammoniacal copper quat (ACQ-C), 0.1 and 0.25% CTN, and 0.1 and 0.25% CTN combined with 2% BHT were installed with untreated controls in field sites in Mississippi. Stakes were sampled at 90 days intervals and rated for decay damage. Microbial DNA was extracted from wood and amplified using non-specific (bacteria and fungi) and specific (basidiomycete) primers. Alpha diversity (richness and diversity), species composition, and beta diversity, were all calculated using T-RFLP data. Results indicate the presence of wood preservatives containing biocides: (1) increases initial colonization by bacteria that decreases over time, (2) slows the initial colonization of field stakes by fungi resulting in lower richness and diversity that increased over time, and (3) increased richness and diversity of basidiomycetes. Preservative treatment also changed the community composition of bacteria, fungi, and basidiomycetes found in wood, that became more similar over time to untreated controls for fungi and basidiomycetes, but not bacteria. The beta diversity of treated samples was less similar in early stages of exposure (3-9 months), but coalesced over time into stable populations that were similar to fungal and basidiomycete communities in untreated controls, but still bacteria remained different. Correlations were found between depletion of 0.1% CTN and increasing bacterial and fungal diversity.
G T Kirker, S V Diehl, M L Prewitt, W J Diehl

Analysis on dynamics of basidiomycetes in decayed wood by a method using non-specific amplification of DNA
2009 - IRG/WP 09-20426
A combination of non-specific amplification of DNA by Phi29 DNA polymerase and specific amplification of the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) by polymerase chain reaction (PCR) was applied to elucidate the ratio of basidiomycete species inhabiting decayed wood from an outdoor bench. After more than 8 hours incubation with Phi29 DNA polymerase, concentration of DNA were reached to 50 ng/μl, which was approximately 1,000 times higher than that of DNA in the original sample. It was revealed that the increase of PCR cycles biased basidiomycete species from original ratio, whereas longer incubation time of the non-specific amplification less affected on it, suggesting the advantage of amplification by Phi29 DNA polymerase to analysis on dynamics of basidiomycetes in decayed wood.
T Wada, K Igarashi, M Samejima

Bacterial Degradation of Pentachlorophenol Indicated in Groundwater Undergoing Long Term In-Situ Remediation
2016 - IRG/WP 16-50318
Pentachlorophenol (PCP, Penta) is a worldwide soil and water contaminant. The pervasive use of PCP as a fungicide, herbicide, pesticide, and as preservative for wood products has led to its global distribution. It is a highly effective preventative measure for most invasive insects, bacteria, fungi, and molds, as well as an excellent wood protection treatment. This study examined a known PCP contaminated site that has been undergoing remediation treatment since 2000 to determine if PCP degrading bacteria could be identified using molecular techniques. Bacteria were identified by sequencing the 16s rDNA fragment from DNA extracted from groundwater cultures and comparing those sequences to a database using a basic local alignment search tool, BLAST. The PCP-degraders Burkholderia cepacia and Flavobacterium (Sphingobium) chlorophenolicum were identified in multiple wells, as were the 4-chlorophenol degrader Herbaspirillum chlorophenolicum, and the common soil bacteria Pseudomonas sp., Aquaspirillum sp., and Rhodocista sp., among others. Numerous bacterial samples also appeared in the results as “uncultured”. Diversity measures including species richness, Simpson’s and Shannon’s indices, and species evenness were calculated from operational taxonomic unit results for each well at each sampling point in order to better understand changes in the bacterial community. Species richness tended to be higher at wells further away from the biosparging line, while diversity and evenness varied throughout the area. Correlations between PCP concentration, operational taxonomic units, and distance from biosparging wells were determined by Pearson’s product-moment correlation and Spearman’s rank correlation. Positive correlations were found between distance from biosparging wells and PCP concentration, species richness and distance, and to a smaller degree, diversity and distance. Biosparging remediation has a significant impact on the types of PCP-degrading bacteria within the groundwater matrix, and installations of this type of treatment should be applied to maximize the use of the native bacteria to assist in degradation of the contaminant.
C E Stokes, H Borazjani, L Prewitt

Analysis of tebuconazole in wood treated with Tanalith™ E
1999 - IRG/WP 99-20158
A simple gas chromatographic method for determining tebuconazole in Tanalith™ E treated wood is described. A two step sequential extraction procedure with methanol was used. Sample extracts were analysed without cleanup or concentration using capillary column GC with thermionic specific detection. The performance of the method was assessed using radiata pine (Pinus radiata) sapwood, radiata pine heartwood, and spotted gum (Corymbia citriodora) sapwood as substrates. Recoveries from fortified samples ranged from 97% to 103%. The precision of the method was assessed by analysing a number of actual treated wood samples over a range of retention levels, which produced relative standard deviations in the range of 3% to 8%.
D E Ferlazzo

The applicability of life cyle analysis and alternative methods in the wood preservation industry
1994 - IRG/WP 94-50023
In the Netherlands, several case studies have been performed using the life cycle analysis method (LCA). This type of research is aimed at an inventory and classification (sometimes including also evaluation) of the environmental impacts of a product, from the raw material to waste stage ("cradle to grave" approach). In a LCA each environmental impact is assessed in terms of, for example, mass of raw material use (kg), energy consumption (MJ), emissions (COx, NOX, SOx, etc.) and final waste (in kg). The critical point in an LCA is the definition of comparable "functional units" for similar products made of different materials with different service lifes. As the LCA method has often proved to be very complex, lime-consuming, expensive and difficult to interpret and translate into practically usefull results, alternative methods are developed. Three methods are described and compared on the basis of various examples. It is hoped that this may be of use as a starting point for further discussion on the suitability of applying the LCA on (preservative treated) timber products.
P Esser, J Cramer

Utilization of curcumin for detection of presence of boron in wood
1982 - IRG/WP 3191
It has been shown that curcumin is not a reliable reagent for detecting boron in wood that has been attacked by fungi
M-L Edlund

Results of chemical analyses in the field of wood preservation in the Bundesanstalt für Materialprüfung
1973 - IRG/WP 321
The results of qualitative and quantitative chemical analyses of wood preservatives are often the basis for evaluating the various works in the field of wood preservation. In the past 10 to 15 years a number of such works was carried out in the Bundesanstalt fur Materialprüfung, Berlin-Dahlem, dealing with the identification and effectiveness of wood preservatives and with methods of wood preservation. Fundamental realisations were made which will be summarised below. It seems advisable to differentiate between inorganic and organic chemical wood preservatives and methods of analyses. These are two distinct fields which differ also with regard to the analytical techniques applied.
H J Petrowitz

Modelling of PCP migration in the environment: Feeding the models with laboratory data
1993 - IRG/WP 93-50001-08
In 1989, Hydro-Québec began a study program on pentachlorophenol (PCP) to ensure safe use of the product at all stages. One of the aspects of the study is the creation of a predictive system for evaluating the behavior of PCP and oil migration from wood poles to the environment. This system comprises four mathematical models for predicting PCP and oil migration in and on the surface of the pole, in soil and in groundwater, and for predicting runoff. Laboratory experiments aimed at quantifying and supplying the input for each model have been designed. A method of analyzing both PCP and oil in water. wood and soil has been developed. The radial and longitudinal distributions of PCP and oil concentrations have been established for several combinations of wood species and treatments. Laboratory setups and preliminary results are presented.
A Besner, P Tétreault, R Gilbert

Penetration analysis of two common bamboo species - borak and jawa of Bangladesh
2002 - IRG/WP 02-40247
Preservative treatment of two bamboo species, namely borak (Bambusa balcooa Roxb.) and jawa (Bambusa salarkhanii Alam) was carried out with chromated copper boron (CCB) preservative by dipping method. The variation in preservative penetration between the two different species was determined. It was found that preservative penetrates into borak quicker than into jawa and easier into air-dried bamboo than into green one.
M O Hannan, A K Lahiry, N M Islam

Finite element analysis of boron diffusion in wooden poles
2003 - IRG/WP 03-40263
The problem of describing the migration of dissolved boron in wood is treated with special reference to the commonly used remedial treatment of wooden poles. The governing equations are derived and discussed together with some of the material parameters required. The equations are solved by the finite element method and finally, results showing the effect of different treatment strategies are presented.
K Krabbenhøft, P Hoffmeyer, C G Bechgaard, L Damkilde

Detection of defects in standing poles by X-ray techniques
1980 - IRG/WP 2132
The application of X-ray techniques to wood pole inspection procedures has been evaluated. Internal defects were accurately identified using X-ray inspection methods. These methods and their possible influence on the safety and economic aspects of wood pale utilization are discussed.
W D Gardner, R S Johnstone, W Pitt

Conforming to european standards for preservative-treated timber: Specifying with confidence
2000 - IRG/WP 00-20194
A four-year collaborative study between four industrial partners and BRE has assessed timber treated by current UK industrial practices in the light of current European Standards. Data were collected for CCA and creosote treated timber components, and compared with the requirements laid out in EN351-1 and -2. A number of difficulties were encountered that have been described in previous IRG papers (98-20150, 99-20156), such as the poor reproducibility of chemical analyses and variable timber density. This paper describes the conclusions of our collaboration, focusing on the application of the findings and how to overcome any difficulties encountered. The data collected allowed the calculation of figures that have been submitted for inclusion into the UK's proposed national code for preservative-treated timber (DD239). An example is the recommendation of new minimum retention figures for creosote-treated commodities. This paper describes the factors that will enable UK specifiers to use the European Standards with confidence and greater understanding of how they map onto traditional methods of specification. In addition valuable lessons have been learnt applicable to the industry world wide.
E D Suttie, R J Orsler

Quantitative determination of Chromium: A comparison of three instruments
1995 - IRG/WP 95-50053
A comparison is made between three instruments for measuring levels of chromium in the leachate of copper-chrome-arsenic (CCA) treated timber. These include an atomic absorption spectrophotometer, an inductively coupled plasma spectrometer and the RQflexâ. The RQflexâ is a new hand-held instrument which measures chromium by dipping a ReflectoquantÒ strip into the leachate and inserting the strip into the RQflexâ. Readings are determined reflectometrically following a reaction between chrome and the Reflectoquantâ strip. It provides an inexpensive and quick technique which is used in this paper to determine the degree of preservative fixation prior to the removal of CCA treated timber from drip pads of wood treatment sites. The results indicate that RQflexâ is a suitable instrument for the rapid assessment of chromium levels in rain wash off from treated timber. A comparison is given of the economics and accuracy of the instruments for measuring chromium.
S Walley, P R S Cobham, P Vinden

Electron microscopic detection and chemical analysis of three-lamellar structures in wood-destroying fungi
1984 - IRG/WP 1240
In the course of transmission electron microscopical investigations of pine wood decay by various brown- and white-rot fungi extracellular three-lamellar structures (TLS) formed by the fungi were found in specimens stained with ruthenium red. These structures occured in the lumen of the wood cell surrounding the hypha at the outermost layer of the fungal cell wall. In the course of the investigations these structures were also detected in fungi cultivated with glucose on a rotary shaker, where they showed forms similar to tubuli and vesicles. The three-lamellar structures formed by the white-rot fungus Sporotrichum pulverulentum, which were contained in the outermost cell wall layer, were isolated by disintegration of the fungal pellet and subsequent digestion of the fungal cell wall by snail enzyme. It was found that these structures are resistant to the enzymatic digestion and are composed of 80 to 90% carbohydrates, mainly consisting of glucose monomeres, 5 to 10% proteins, containing 5 fractions with molecular weights between 30000 and 200000, and finally 5 to 10% lipids which do not contain any phospholipid.
R Foisner, K Messner, H Stachelberger, M Röhr

Observations on the failure of anti-sapstain treated timber under non-drying conditions
1990 - IRG/WP 1437
A range of bacteria and yeasts were isolated from antisapstain treated timber and fresh sawdust. Solution samples containing 100 ppm of TCMTB in a nutrient medium were inoculated with these organisms and incubated at 25°C for 5 days. The levels of TCMTB remaining in solution were determined by HPLC analysis after this time. Results indicated high losses of active ingredient for a range of organisms. These results suggest that active biodetoxification of organic biocides could occur in a short period of time during storage of antisapstain treated timber under favourable conditions. The implications of these results are discussed.
G R Williams

Collaborative field experiment: Analysis of copper and chromium in stakes of the four reference timbers
1982 - IRG/WP 3213
The stakes analysed belong to the Swedish set of the four reference timbers of the collaborative field experiment (Document No: IRG/WP/367). Before the field exposure 60 mm were cut off the end of each stake. This was done in order to be able to carry out chemical analyses as well as different types of testing. Some of the material has been used in softrot tests (Document No: IRG/WP/1151).
T Nilsson

Collaborative soft rot tests: 2nd interim report
1975 - IRG/WP 258
J G Savory, J K Carey

A bibliography of organic solvent-based wood preservatives
1973 - IRG/WP 322
H Alliot

X-ray analysis of selected anatomical structures in copper/chrome/arsenic treated wood
1973 - IRG/WP 320
Application of analytical electron microscopy to problems in wood preservation has been very limited. Indeed, less than ten workers appear to have published their results using the technique, and of these' only two papers deal with energy dispersion procedures in the scanning electron microscope; the others employ the more familiar wavelength dispersive methods of the electron probe.
H Greaves

Comparative studies on the distribution of lignin and CCA elements in birch using electron microscopic X-ray microanalysis
1987 - IRG/WP 1328
The microdistribution of metal preservatives in treated wood has received considerable research over the last two decades. Despite this, little effort seems to have been made to try and correlate the distribution of CCA elements with respect to naturally occurring wood cell wall components in wood. In the present preliminary study an attempt is made to relate the distribution of lignin in-situ with that of CCA elements. For the study matched samples of Betula verrucosa were first either mercurized to specifically label the lignin or vacuum impregnated with a commercial 2% K33 CCA preservative solution. Thereafter using SEM-EDXA, the relative distribution of labelled lignin and CCA elements were compared for both different cell types and cell wall regions. Results showed the relative microdistribution of CCA to follow closely that of the lignin distribution. Regions showing high lignin levels showed high CCA levels and vice-versa. Highest CCA and lignin levels were recorded in the vessel, fibre and ray middle lamella cell corners regions while the lowest levels were detected in the fibre (S2) secondary walls. Both the low lignin level (and syringyl type) and CCA uptake in fibre S2 walls would seem in close agreement with the known high susceptibility of these elements to soft rot attack in both treated and untreated birch. Comparisons made between the lignin content of the S2 layer for birch fibres and other known soft rot resistant species (e.g. Alstonia scholaris) showed great differences, with the latter showing much higher lignin (ca 3x) levels.
G F Daniel, T Nilsson

Ultrastructural and TEM-EDAX studies on the degradation of CCA treated radiata pine by tunnelling bacteria
1985 - IRG/WP 1260
An ultrastructural study was carried out on bacterial attacked Pinus radiata stakes treated with a high level (24.7 kg/m³) of Tanalith NCA preservative. The purpose of the investigation was to determine whether the organism possessed intracellular and/or extracellular detoxification mechanisms in order to overcome the high levels of copper, chrome and arsenic present within the wood fibre walls. Correlated T.E.M. and T.E.M.-EDAX studies showed most of the preservative elements to remain outside the bacteria associated with bacterial tunnel wall and cross-wall extracellular secretions. T.E.M.-EDAX showed the levels of preservative elements present in the tunnel walls and cross walls to often greatly exceel that recorded in neighbouring S2 cell wall regions, while studies on the bacteria showed that only copper and very low levels of chromium and arsenic had entered the cells. Observations suggested that the metals found within the cells were associated with electron-dense deposits or inclusions within the nuclear region or cell cytoplasm, the deposits often containing high levels of phosphorus and calcium together with lesser levels of other cations. The study also provided evidence for the direct visualisation of CCA preservative elements within fibre walls using T.E.M. without any form of secondary chemical enhancing, and in addition, considerable new information on the nature and structure of the single celled, Gram-negative, motile bacteria involved in the decay.
G F Daniel, T Nilsson

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