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Wood Decay Research Using Molecular Procedures, What Can It Tell Us?
2008 - IRG/WP 08-10678
There are many unanswered questions in wood decay and protection research, and no single research technique will ever answer all of these questions. The process of wood decay is a very complex series of biochemical and chemical reactions that are heavily influenced by the hundreds of bacterial and fungal species found on environmental samples of wood. There are a variety of molecular techniques being used that can allow researchers to probe these wood decay organisms and their activities further. This presentation will highlight the types of questions that different molecular techniques can help us explore as well as consider the limitations of this line of research. Several molecular techniques and their potential uses will be discussed in simple non-technical terms, including (1) sequencing and cloning for species identifications, (2) real-time PCR for detection of decay enzymes, (3) proteomics for exploring gene activation /inactivation during decay or biocide breakdown, and (4) microbial community analysis. A critical gap in our knowledge, and consequently in the ability to maximize use of these techniques, is the lack of annotated, whole-genome sequences for important wood decay fungi.
S V Diehl, M. L Prewitt, Young-Min Kang, L Mangum, J D Tang


Postia placenta gene expression of oxidative and carbohydrate metabolism related genes during growth in furfurylated wood
2009 - IRG/WP 09-10701
A range of studies the last decade have shown that modified wood can provide excellent protection against a range of wood deteriorating organisms, including decay fungi. However, we still lack information about why the modified wood is protected from microbial attack. Several hypotheses have been put forward e.g. inhibition of action of specific enzymes, but they still need testing. An understanding of the mechanisms utilized by decay fungi when exposed to modified wood is important for further optimisation of new modified wood products. In this study gene expression of the brown rot fungus Postia placenta has been monitored after 2, 4 and 8 weeks of colonization in furfurylated Scots pine and control samples. Preliminary results are given. The main finding was that genes related to oxidative metabolic activity was higher in furfurylated wood compared to untreated Scots pine, and that carbohydrate metabolism related expression was lower in furfurylated wood compared to untreated control.
G Alfredsen, C G Fossdal


Gene expression of selected decay enzymes produced during biodeterioration of three wood types
2009 - IRG/WP 09-10702
Comparative studies were conducted on the expression of several decay enzyme genes during the decay of pine, cedar, and ACQ treated pine over 10 months. Measurements of MOE, decay rating, and moisture control were monitored for physical properties. Identification of decay fungi and the detection of decay enzymes were carried out before gene expression levels were measured. The MOE of wood stakes decreased more in pine than in cedar and ACQ treated pine, indicating a greater loss of strength. Visual decay rating data paralleled MOE data at each sampling time. Moisture contents of all three wood types in each container varied between 40% and 100% over the 10 months. Basidiomycetes were found on all samples as determined by PCR amplification of the basidiomycete 18s rRNA gene after 4 months. For identification of decay fungi at 4 months, PCR products of basidiomycete 18s rRNA gene were cloned and sequenced. Four species of basidiomycetes on cedar, six on ACQ treated pine, and twelve on pine were identified. The white rot fungus, Phlebia radiata was identified on all wood stakes. P. radiata species specific primers were then designed to track the expression of three decay enzyme genes: lignin peroxidase (Lip), manganese peroxidase (Mnp), and laccase (Lcc) on wood samples. Gene expression data as measured by Real-time PCR indicated that the Mnp expression level on pine at 4 months was very similar to the expression level on ACQ-pine but was not detected on cedar. The Lcc expression level on pine samples at 6 months was 30% less than the expression level on ACQ-treated pine while no Lcc was detected on cedar. The results clearly show that the naturally durable properties of cedar reduce the wood decay community and its activities in comparison to untreated pine and ACQ-treated pine. P.radiata attempted to decay the ACQ-treated wood by producing a large amount of Lcc than was produced on pine but the MOE data showed this attack was not successful. Thus, it appears ACQ-treated wood does not stop the production of the decay enzymes but does inhibit the effectiveness of the enzymes. Results from the study indicate that different resistant woods have different effects on the microbial communities and its enzymatic activities during decay.
Young-Min Kang, L Prewitt, S Diehl


Postia placenta gene expression during growth in furfurylated wood
2010 - IRG/WP 10-10734
Modified wood can provide protection against a range of wood deteriorating organisms. But we still lack information about why the modified wood is protected from microbial attack. Several hypotheses have been put forward for the mode of action against wood decaying fungi, including inhibition of action of specific enzymes, but they still need further testing. In this study gene expression of the brown rot fungus Postia placenta FPRL 280 has been monitored after 2, 4 and 8 weeks of colonization in furfurylated Scots pine (Pinus sylvestris L.) and in untreated control samples. Preliminary results are given. The main finding was that genes related to oxidative metabolic activity generally was higher in furfurylated wood compared to untreated Scots pine. Carbohydrate metabolism related expression varied. For one endo-glucanase and two β -glucosidases the expression was lower in furfurylated wood compared to untreated control, while for one glucoamylase and one glucan 1,3b glucosidase the expression was higher in furfurylated wood. The four cytochrome P450 tested, involved in breakdown of toxic compounds, gave inconsistent results between furfurylated and untreated control samples. Phenylalanine ammonia lyase and cytosolic oxaloacetase gave higher expression in control than in furfurylated samples.
G Alfredsen, C G Fossdal


Towards Understanding the Biology of Wood Decay
2010 - IRG/WP 10-10739
Our previous research has focused primarily on ways to identify the wood decay fungi and microbial community. We continue to explore this complex and dynamic community and its interactions through microbial community ecology studies, gene expression interactions and proteomics. However, in order to better understand the mechanisms of fungal decay, we have sequenced the genome of a copper tolerant brown rot fungus, Antrodia radiculosa. To advance our goals, we will be using structural and comparative genomics to identify novel genes and functional genomics and transcriptomics to systematically discover what genes are activated during wood decay under different environmental conditions.
J Tang, K Jenkins, L Parker, 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


The effects of acetylation level on the growth of Postia placenta
2011 - IRG/WP 11-10751
To understand the defence mechanisms utilized by decay fungi when exposed to different wood protection systems the study of gene expression can give us some answers. When the DNA sequences are known, primers can be designed to detect transcripts of genes with gene products related to basic cellular processes and hyphal growth. The characteristic gene products induced in different fungi by different wood protection systems can be identified. Studies on the expression of fungal genes will give us a better understanding of the fungal degradation of wood and we can optimize wood protection systems. Hence, no single technique will give us the answer to all questions about the decay of wood we need to gather small pieces of the puzzle using different approaches. The aim of the present study was to investigate the effects of acetylation level on the growth of Postia placenta with regard to amount of total DNA and gene expression targeting 7 different genes. This paper presents preliminary results after 4 weeks of incubation. The results presented in this paper are parts of a larger project which reaches over a period of 36 weeks with sampling times after 12, 20, 28 and 36 weeks. We found no mass loss in the acetylated samples after 4 weeks of incubation in a modified soil-block test. The presence of P. placenta DNA and the absence of mass loss could indicate on an inability of the mycelia to establish a wood exploitation phase. Two genes related to carbohydrate metabolism were expressed in a higher amount in P. placenta during growth on untreated wood than during growth on acetylated wood. However, for a third gene, also related to carbohydrate metabolism, the relationship was the opposite. Two genes related to oxidative metabolism were expressed in a higher amount in P. placenta during growth on acetylated wood than during growth on untreated wood and another two genes related to oxidative metabolism showed inconsistent results.
A Pilgård, G Alfredsen, C G Fossdal, C J Long II


Molecular investigation of Postia placenta growing in modified wood
2011 - IRG/WP 11-10756
Brown rot is the most common and destructive type of fungal decay for wood in service. These fungi depolymerize preferentially the structural carbohydrates, cellulose and hemicellulose in the cell wall leaving oxidized lignin behind. Modified wood can provide protection against a variety of wood deteriorating organisms, including decay fungi. However, there is still little known about the mode of function of the different wood modifications concerning the decay resistance. The biochemical mechanisms and gene products induced in brown rot during growth in modified wood are poorly understood. In this paper the data collected from mass loss studies and qPCR and qRT-PCR were used for profiling growth dynamics and gene expression of the brown rot fungus Postia placenta in different wood substrates through different stages of decay. Pinus sylvestris (L.) sapwood was used for the following treatments and modifications: chromated copper arsenate CCA (0.67%), furfurylation (WPG 37), thermal modification (D212) and acetylation (WPG 23). Untreated Pinus sylvestris (L.) sapwood was used as control. Samples were taken at different time intervals from 2 to 26 weeks. The highest mass loss and the highest fungal DNA content were found in the control samples while acetylated wood had the lowest mass loss and fungal DNA content. These results reflect a close relation of mass loss and fungal DNA content, both reflecting the amount of Postia placenta decaying the samples. Generally, expression of the investigated genes was highest in CCA treated wood. In the beginning of the incubation of all treated wood samples, the genes coding for oxidative metabolic activity had higher expression levels than the untreated control. In the end of the incubation most of these genes were less expressed than in the untreated control. The genes used for carbohydrate metabolism and the alcohol oxidase showed a significant decrease after 14 weeks of incubation. At the same time an increase in gene expression of an enzyme putative involved in lignin decomposition was detected.
B Schmöllerl, G Alfredsen, C G Fossdal, M Westin, A Steitz


Variation in two Postia placenta strains, MAD-698-R and FPRL 280 – mass loss, DNA content and gene
2012 - IRG/WP 12-10781
Brown-rot fungi such as Postia placenta are common inhabitants of forest ecosystems and brown rot fungi are also largely responsible for the destructive decay of wooden structures. The aim of this study was to compare two commonly used strains of Postia placenta – MAD-698-R and FPRL 280. Scots pine sapwood samples were exposed for two and eight weeks to both fungal strains. The following was investigated: mass loss, fungal gDNA content and gene expression. A significant difference was found in mass loss after eight weeks between the P. placenta strains MAD-698-R and FPRL 280. MAD-698-R gave higher mass loss than FPRL 280. However, MAD-698-R seems to have a slightly slower growth rate than FPRL 280, reflected in lower gDNA content after two weeks. After eight weeks of exposure the gDNA content dropped and no significant difference was found between MAD-698-R and FPRL 280. We observed differences in mass loss, colonization-rate and gene expression between the two Postia strains. Results suggest significant differences in the regulation of key lignocellulose degrading enzymes between MAD-698-R and FPRL 280.
N Thaler, G Alfredsen, C G Fossdal


The effects of acetylation level on the growth of Postia placenta over 36 weeks
2012 - IRG/WP 12-40589
Genomic sequencing gives us a tool to systematically and rapidly discover novel genes, how their products function in the cell, and explore their interactions. When the DNA sequences are known, primers can be designed to detect transcripts of genes with gene products related to basic cellular processes and hyphal growth. The characteristic gene products induced in different fungi by different wood protection systems during decay can be identified. This knowledge will give us a better understanding of the fungal degradation of wood and we can optimize wood protection systems. Hence, no single technique will give us the answer to all questions about the decay of wood we need to gather small pieces of the puzzle using different approaches. The aim of the present study was to investigate the effects of acetylation level on the growth of Postia placenta with regard to amount of total DNA and gene expression targeting six different genes. This paper presents preliminary results after 36 weeks of incubation. We found no mass loss in the acetylated samples treated to a high treatment level after 36 weeks of incubation in a modified monoculture soil-block test. The presence of P. placenta DNA and the absence of mass loss could indicate on an inability of the mycelia to establish a wood exploitation phase. The results also showed that P. placenta increased the expression of AlO (involved in production of H2O2), cytochrome P450 (related to breakdown of toxic compounds), and QRD (involved in generating biodegradative hydroxyl radicals via redox cycling) along the incubation time, growing on acetylated wood treated to a high treatment level.
A Pilgård, G Alfredsen, C G Fossdal, C J Long II


Postia placenta cellulase gene expression in modified wood during incipient decay
2013 - IRG/WP 13-40626
In optimization of modified wood, it is important to understand the mode of action of the wood modification and how the fungi response to it. The aim of this study was to investigate the expression of cellulases during the first two weeks of Postia placenta exposure in acetylated, DMDHEU-treated and thermally modified as well as in untreated wood. Using real-time PCR, the gene expression patterns of the P. placenta endoglucanase Ppl103675 and β-glucosidase Ppl112501 during 56 days of decay was analyzed. Preliminary data indicate that both genes are expressed at higher values in untreated wood at 56 days of exposure (56% mass loss) than at 14 days. We also saw high values for both genes at ten days of exposure for both untreated (11% mass loss) and modified woods (all 0% mass loss). We conclude that the high values at 10 and 56 days in untreated wood may be due to that monosaccharides and by-products from the cellulose degradation process, known to induce cellulase expression, have been released from the wood cell wall. Furthermore, the high values at ten days in the modified woods may be the result of an unknown regulatory mechanism. In addition, we found that the expression patterns for the P. placenta endoglucanase Ppl103675 and β-glucosidase Ppl112501 are very similar in the three modified woods investigated, but different from the patterns in untreated wood. This might be an indication that all the herein studied wood modifications affect fungi in the same way, i.e. they have the same mode of action.
R Ringman, A Pilgård, K Richter


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


Isolation of a gene from the melanin pathway of the sapstaining fungi Ophiostoma piceae using PCR
1997 - IRG/WP 97-10219
To prevent sapstaining fungi from discoloring wood, it is necessary to determine what factors affect the biosynthesis and characteristics of the pigment(s) and to identify the genes involved in the pathway. Using inhibitors and heterologous DNA probes from Alternaria alternata, we suggest that melanin, the pigment of Ophiostoma piceae, is produced by the dihydroxynaphthalene (DHN) pathway. Recently, sequences were published for one of the enzymes in the DHN pathway of Colletotrichtum lagenarium, a cucumber pathogen, and Magnaporthe grisea, a pathogen of rice. From this information we synthesized degenerate oligonucleotides to the conserved regions of the trihydroxynaphthalene (THR1) and tetrahydroxynaphthalene reductase (ThnR) genes. Using these primers and genomic O. piceae as template DNA, we obtained a 365 nucleotide PCR product. The deduced amino acid sequence of the product had 85% homology to the Thr1 of C. lagenarium and 80% homology to the ThnR of M. grisea. This PCR product will be used to screen a genomic library of O. piceae in order to isolate the entire gene sequence in the melanin pathway. Complete characterization of the genes involved should facilitate more direct development of anti-stain strategies.
R Eagen, J Kronstad, C Breuil


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


Effect of Heavy Metals on the Expression of Manganese Dependent Peroxidases of Phanerochaete chrysosporium
2010 - IRG/WP 10-10722
This work is part of an ongoing investigation that studies the application of a solution rich in Zn and Mn, obtained from a recycling process, as a preservative for wood. The effects of Zn2+ and Mn2+ on the enzyme-expression level of manganese-dependent peroxidase (MnP) from Phanerochaete chrysosporium was studied by real-time PCR. The glyceraldehyde 3-phosphate dehydrogenase gene (gpd) was used as reference for the relative quantification of the expression of the three isoenzymes. The expression kinetics was studied for different media and culture conditions. The conditions and culture time leading to maximum gene expression and enzyme activity were determined for each isoenzyme. The effects of medium composition (replete, ligninolytic: carbon-limited, nitrogen-limited), temperature (28ºC and 37ºC), and shaking (static or agitated at 125 rpm) were studied. Incubation in a static carbon-limited medium at 37ºC showed the highest expression of the mnp genes. In these conditions, the concentration of Zn2+ and/or Mn2+ that stimulate or inhibit the expression of mnps was determined. The selection of suitable culture conditions and the sensitivity of real-time PCR enabled a differentiation in the expression pattern of the three studied MnP isoenzymes in the Zn2+ and/or Mn2+-containing media. Zn2+ concentrations lower than 0.5 mM led to variable expression levels for the different isoenzymes, but were found to have a clear inducing effect when Zn2+ was used in combination with 100 µM Mn2+. Results of the above analysis were compared with enzymatic activity data obtained spectrophotometrically.
C Ibáñez, M Rabinovich, M Barraco, G Gecchetto, M Cerdeiras


Isolation and evaluation of Lactobacillus brevis from chilli waste for potential use as a wood preservative
2011 - IRG/WP 11-10749
Lactic acid bacteria were isolated from chilli waste and evaluated for their ability to arrest wood rotting basidiomycetes. In previous work a quick screening method using 96 well plates and measuring absorbance to determine fungal growth was developed specifically to investigate the efficacy of isolated bacteria against wood decay fungi. Using this method, one bacterium (isolate C11) was identified from three bacterial isolates as having significant antifungal properties against Oligoporus placenta. This isolate was identified as Lactobacillus brevis by 16S rRNA gene sequencing and BLAST analysis of the NCBI database. To determine antifungal activity in wood, Pinus radiata blocks were impregnated with L. brevis strain C11 cell free supernatant (CFS) and exposed to brown rot fungi O. placenta, Antrodia xantha, and Coniophora puteana. The CFS treated timber demonstrated resistance to degradation from all fungi especially when L. brevis was incubated for one week before filtering the culture to retrieve the supernatant. To determine the nature of the bacterial metabolites affecting fungal growth, the affect of pH, temperature and proteinaceous enzymes on the CFS was assessed using the 96 well quick screening method. The antifungal metabolites were heat stable and not affected by proteinase K, but were affected by neutralisation with NaOH suggesting the metabolites were of an acidic nature.
D O’Callahan, T Singh, I R McDonald


Identification of soft-rot fungi existed in the samples from the galley excavated at Yenikapi
2014 - IRG/WP 14-10833
The shipwrecks of the Middle Byzantine period were excavated during the construction of the Marmaray railway and metro station in Yenikapı between 2004 and 2012. This has become the largest investigations because of the size and the number of the shipwrecks and its associated artifacts. In the previous report, the electron micrographs of the samples revealed that a wide range in the degree of deterioration by erosional bacteria and soft-rot fungus obtained from one shipwreck (Köse and Taylor 2013). In this study, therefore, genetical approaches using rDNA ITS region was conducted to identify fungal species existed in the samples. Six ascomycete strains were detected, and one of them was Penicillium paneum, which is known as a typical soft-rot fungus of wood. We are identifying other fungal strains and erosion bacteria to clarify the mechanism of degradation for remediation and restoration of the shipwrecks.
T Wada, C Köse, K Igarashi