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The effects of copper proximity on oxalate production in Fibroporia radiculosa
2014 - IRG/WP 14-10823
Copper remains a key component used in wood preservatives available today. However, the observed tolerance of several critical wood rotting organisms continues to be problematic. Tolerance to copper has been linked to the production and accumulation of oxalate, which precipitates copper into insoluble copper-oxalate crystals, thus inactivating copper ions. The purpose of this study was to assess differences in oxalate production and decay capacity of four wood decay fungi (three copper-tolerant and one copper-sensitive) exposed to various applications of copper. Three Fibroporia radiculosa isolates and one Gloeophyllum trabeum isolate were subjected to one formulation of copper citrate presented to the test fungi by four different treatments in Southern pine wood blocks for an eight week period. Samples were evaluated for oxalate production and weight loss every two weeks. Two of the copper-tolerant isolates evaded the inhibitory effects of all four copper treatments by week eight. The copper-sensitive organism exhibited some limitations to actively decay blocks in two of the four copper treatments. These findings suggest that proximity to copper citrate, available in any form (i.e. impregnation, direct contact, free liquid or close proximity) generally, had no negative effect on fungal growth, oxalate production, and decay capacity of the copper-tolerant organisms. Results also suggested that the copper-sensitive fungus was restricted in its ability to effectively decay wood when copper was pressure treated or directly added to the surface of wood blocks. This study also suggested that close proximity to copper alone (i.e. not pressure treated) did not completely inhibit decay of the copper-tolerant or copper-sensitive test fungi.
K M Jenkins, C A Clausen, F Green III

Selection of Wood-Rotting Basidiomycetes for Inoculation of an Accelerated Soil Bed Test
2014 - IRG/WP 14-20543
There is a need for a test method that guarantees exposure of treated wood to soil and preservative-tolerant wood-rotting basidiomycetes as standard field tests do not do this reliably. The ability of a range of selected wood-rotting basidiomycetes to grow through unsterile forest soil was investigated in a Mason jar test assembly. None of the white-rot fungi in test grew through this soil. Four of the brown-rot fungi grew through the soil, and decayed water-treated control mini-stakes to the point of failure during 12 weeks of incubation. Based on the results of this study, consideration of prevalence in North America, preservative tolerance, and pattern of growth, one isolate each of two brown-rot fungi was selected to be used in future accelerated soil bed test experiments to ensure the presence of wood-rotting basidiomycetes in the soil . These were L. pinastri Findlay 141 and F. radiculosa L-7878-Sp. In addition, this unsterile soil-jar test method should be developed into a laboratory screening test for ground contact wood preservatives where biodegradation and basidiomycete exposure are assured.
P I Morris, A Uzunovic, J Ingram

The copper-transporting ATPase pump and its potential role in copper-tolerance
2016 - IRG/WP 16-10859
Copper-tolerant brown-rot decay fungi exploit intricate mechanisms to neutralize the efficacy of copper-containing preservative formulations. The production and accumulation of oxalate is the most widely recognized theory regarding the mechanism of copper-tolerance in these fungi. The role of oxalate, however, may be only one part of a series of necessary components required for this complex mechanism. Annotation of the Fibroporia radiculosa genes involved in copper-tolerance characterized a subset of proteins, three copper-transporting ATPase pumps, which regulate copper concentrations inside the fungal cell by exporting excess copper ions. The goal of this study was to determine the relevance of copper-transporting ATPase pumps in the mechanism of F. radiculosa copper-tolerance. Southern pine test blocks were pressure-treated with 0.6%, 1.2%, and 2.4% ammoniacal copper citrate and subjected to a copper-tolerant strain of F. radiculosa and a copper-sensitive strain of Gloeophyllum trabeum in decay tests over a four week period. Untreated Southern pine test blocks subjected to both test fungi served as controls. Expression levels of three copper-transporting ATPase pumps were evaluated each week by qRT-PCR. F. radiculosa showed up-regulation of all three ATPase pumps when exposed to the copper treatments over the course of this study. G. trabeum showed down-regulation of ATPase1 and ATPase2 and no expression of ATPase3 when exposed to the copper treatments over the course of this study. Up-regulation of the three ATPase pumps can be correlated to the ability of F. radiculosa to decay copper-treated wood (12% weight loss at week 4). Down-regulation of ATPase1 and ATPase2 and lack of ATPase3 expression can be correlated to the inability of G. trabeum to decay copper-treated wood (1% weight loss at week 4). Preliminary results indicate these three ATPase pumps function as an essential component of the complex mechanism of copper-tolerance utilized by F. radiculosa.
K M Ohno, C A Clausen, F Green III, G Stanosz

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

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

Biological detoxification of wood treated with salt preservatives
1992 - IRG/WP 92-3717
The use of microorganisms that are capable to convert chemically fixed inorganic preservative complexes from impregnated wood waste into watersoluble components is investigated. A number of fungi were isolated from deteriorated and initially well-treated wood. They revealed an exceptionally high production of organic acids (pH 2). The fungi were identified and used together with others of the same genus for experiments under non-sterile conditions on a laboratory scale with CCA-, CCB and CC-treated wood at retention levels of at least 50% higher than recommended for wood in ground contact. As a result Cr and As were leached to more than 90% depending on culture conditions, whereas Cu reacts with oxalic acid to a complex with limited water solubility.
I Stephan, R-D Peek

Silicon tetrachloride: A potential wood preservative
1980 - IRG/WP 3133
In its present form, this paper is a preliminary draft of a paper that will eventually be submitted for publication in the Forest Products Journal. We are continuing the work on the effects of silicon tetrachloride on wood and expect to have additional data for the meeting in May 1980. Specifically we are measuring the penetration rate and depth of SiCl4, and we are chemically analyzing the treated wood with respect of phenolic and other extractives. Our results continue to appear quite favourable for the future use of SiCl4 as a preservative.
C W Owens, W T Shortle, A L Shigo

The influence of fungal species and the level of decay on the mortality and feeding activity of adult Euophryum confine (Broun)
2002 - IRG/WP 02-10448
The mortality and feeding activity of adult Euophryum confine (Broun) (Coleoptera: Curculionidae) are used to establish the substrate conditions most suitable for their rearing. Weevils were allowed to feed on Pinus nigra (Arnold) sapwood blocks, either undecayed or decayed to 10% weight loss ±5% by two brown rots Coniophora puteana (Schum.:Fr.) Karst. and Serpula lacrymans (Wulf.:Fr.) Schroeter, or the white rot Fibroporia vaillantii (DC.:Fr.) Parmasto in a non-choice test for seven days at 20°C (±1°C) and 92% relative humidity ±5%. Weevils were also allowed to feed on blocks acid pre-treated to 10% (±1%) weight loss. Weevil mortality and block weight losses resulting from feeding were recorded. Those pre-treatments resulting in low mortality and high feeding activity were considered most suitable for rearing adult weevils.
M Green, A J Pitman

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

A genomic sequencing approach to study wood decay and copper tolerance in the brown rot fungus, Antrodia radiculosa
2010 - IRG/WP 10-10720
We used Illumina paired-end short read sequencing (76 nt, 300 bp insert size) to produce a de novo assembly of the genome of Antrodia radiculosa, a copper-tolerant brown rot fungus that is capable of aggressive wood decay. Quality analysis of the base calls in the dataset (8.95 Gb) showed that the majority of the nucleotide sequence was of the highest quality with 5% in the lowest quality group. Low quality scores were cumulative as position in the read increased, indicating that once a read went bad, the rest of the read was also likely to be bad. Analysis of the DNA sequence showed that 1.4% of the bases called was ambiguous. These N's occurred as homopolymers up to 76 nt long. To assess how poor scores and N homopolymers affected the assembly, reads were filtered at increasingly stringent thresholds. Each dataset was then assembled at varying kmer lengths (k) using Velvet 0.7.55. The N50 metric, which describes the size distribution of contigs in the assembly, was then plotted against kmer. Results showed that each dataset was characterized by a max N50 value, but the lower the quality of the dataset, the more dramatic the fluctuation in N50. A second observation was that the larger dataset had greater max N50, despite the presence of the low quality data. The max N50 value of the semi-clean dataset (k = 45) was more than 2x greater than the max N50 value for the very clean dataset (k = 37). Using Genemark-ES v2, we predicted 8000 and 5700 genes from contigs >= 20 kb for these two max N50 assemblies, respectively. Average gene length, percent GC, intron size, number of introns, exon size, and CDS length were very similar for the two assemblies, suggesting that Velvet successfully removed error from the poor scores and the N homopolymers without sacrificing accuracy of the assembly. This work demonstrates that gene prediction from short read sequencing data of fungi is technically feasible and represents a significant step towards accelerating a genome-wide understanding of how brown rot fungi decay wood and tolerate high levels of copper.
J D Tang, T Sonstegard, S Burgess, S V Diehl

Gene expression analysis of a copper-tolerant brown rot fungus on MCQ-treated wood
2011 - IRG/WP 11-10748
Most brown rot fungi are copper-tolerant, which makes them difficult to control with copper-based wood preservatives like MCQ. To better understand what biological processes are regulated, we used our model species, Antrodia radiculosa, to examine expression of genes on MCQ-treated wood. Our hypothesis was genes that decreased copper bioavailability would be up-regulated early, when wood showed no strength loss, while genes that degraded the structural polysaccharides would be up-regulated late, when wood exhibited high strength loss. We used a global profiling strategy called RNA-Seq to record all the genes that were actively being expressed at the two time points. We found 544 differentially expressed gene models. 52 of these gene models had putative functions directly related to oxalate production and polysaccharide degradation. Increased oxalate production at the early time point was caused by up-regulated expression of the following gene models: two pyruvate decarboxylases (3x), one citrate synthase (4x), one isocitrate lyase (8x), one oxaloacetate hydrolase (4x), and four mitochondrial carrier proteins (up to 9x). Up-regulation of oxalate is consistent with the theory that fungi remove copper toxicity by forming insoluble copper oxalate crystals. With respect to the late time point, we found sixteen gene models from at least six different glycoside hydrolase families (GH5, GH10, GH12, GH3, GH61, and GH53) that were highly up-regulated (as much as 23x), along with many sugar transporter genes. Function of the glycoside hydrolases involved cleavage of the -bonds typical of the hemicelluloses and cellulose, which explained the 52% strength loss observed at the late time point. Interestingly, two different sets of gene models for pectin hydrolysis were up-regulated at both early and late time points, suggesting that the pectic substances they targeted were slightly different. These results are significant for wood protection because we have identified the genes that are regulated to uptake sugars, control oxalate levels, and to enzymatically degrade pectin, cellulose, and the hemicelluloses. By knowing these control points, we can rationally develop the next generation of environmentally safe wood preservatives. We also hope to exploit novel brown rot biochemistries for new industries, like biological pretreatment of wood for cellulosic ethanol production.
J D Tang, A Perkins, S V Diehl