New insight regarding mode of action of brown rot decay of modified wood based on DNA and gene expression studies: A review

Abstract

Modified wood shows resistance against wood deteriorating fungi, but the mechanisms are still not fully understood. The aim of this paper was to summarise the molecular studies performed on modified wood with regard to brown rot decay fungi. The DNA data showed that fungi are present inside the laboratory wood test samples already after two weeks of inoculation. Generally the fungal DNA content reflects mass loss and wood moisture content. The oxidative gene expression seems to be higher in modified wood than in untreated wood and it tend to increase during incubation. Based on the gene expression data we suggest that the hypothesis of lack of substrate recognition by the fungus should be rejected. In the reviewed studies, total wood moisture content in the samples was generally not low enough to inhibit fungal colonisation. Hence, moisture distribution within the wood should be studied more closely.

Keywords

Acetylation Furfurylation DMDHEU Thermal modification Oxidative degradation Enzymatic degradation Moisture

Introduction

It is known that modified woods derived from non-durable wood species have an improved resistance against wood deteriorating fungi given sufficient treatment concentrations. Traditional wood preservation treatments achieve their resistance against fungi based on a toxic mode of action. Four established theories of mode of action of modified wood are prevalent:

(i)

fungal enzyme inefficiency due to non-recognition (Rowell 2005)

(ii)

fungal enzyme inefficiency due to lack of water at glycosidic bonds (Rowell 2005; Rowell et al. 2009)

(iii)

reduced flow of fungal molecules into the wood cell wall due to micropore blocking (Hill et al. 2005)

(iv)

inhibition of diffusion of fungal molecules due to insufficient amounts of moisture (Hill 2009; Boonstra and Tjeerdsma 2006).

Regarding moisture it has been discussed that the clue may not be the overall moisture content in the wood material but the moisture content and distribution within the wood cell wall (Papadopoulos and Hill 2002; Rapp et al. 2008; Ringman et al. 20114a) and whether there is sufficient moisture accessible around the hyphae to allow for fungal degradation. Alfredsen et al. (2013) found, based on statistical analysis of published data on acetic anhydride modified wood, that weight per cent gain could explain approximately 50% of the performance. Other parameters, like wood species or type of fungus, could reduce the variance in performance by additional 15%. Based on the surveyed literature the degree of cell wall bulking in combination with lowering of the equilibrium moisture content was suggested to be the primary mode of action.

Brown rot fungi degrade wood through oxidative and enzymatic action (Goodell et al. 1997; Arantes et al. 2012). First, the fungus induces production of hydroxyl radicals inside the wood cell wall through secreting reductants and hydrogen peroxide (Goodell et al. 1997). The reductants reduce ferric iron in the wood cell wall to ferrous iron (Goodell et al. 1997; 2006), which react with the hydrogen peroxide and produce hydroxyl radicals in the Fenton reaction (Fenton 1894). The hydroxyl radicals depolymerise hemicellulose and cellulose, modify lignin and generate sufficient rearrangements so that hydrolysing enzymes, which are too big to penetrate the intact wood cell wall, can diffuse into the cell wall and continue the degradation of the polysaccharides (Goodell et al. 1997; Baldrian and Valaskova 2008; Arantes et al. 2012).

A series of molecular studies have also elucidated some insight regarding fungal response mechanisms to modified wood materials. This paper reviews the work performed regarding DNA quantification and gene expression studies with focus on brown rot decay related to modified wood.

Method development

Eikenes et al. (2005) compared three different methods for quantification of Trametes versicolor colonisation of birch wood in lab; real-time PCR (qRT-PCR), kitin and ergosterol. Wood samples were harvested after 4, 8, 12, 16 and 20 weeks of incubation. The three indicators of fungal biomass were plotted against mass loss. They concluded that for early stages of fungal decay DNA quantification with real-time PCR (qRT-PCR) gave the best results. Pilgård et al. (2011) compared the same three methods with standard field evaluation ratings (EN 252 1990) in field stakes of furfurylated Scots pine, Scots pine heartwood and Cu-HDO treated Scots pine. They found that qRT-PCR in combination with microscopy provides relevant data about basidiomycete colonisation in wooden material.

Hietala et al. (2014) studied the influence of different temperature regimes on wood decay caused by P. placenta in sapwood and heartwood of Scots pine. The samples were incubated up to ten weeks at temperatures conducive or above optimal to wood decay. Mass loss and accompanying changes in wood composition, fungal DNA amount and transcript level profiles of fungal genes active during different phases decay was analysed. The differences between sapwood and heartwood were particularly pronounced for cultures incubated at 30°C. Unlike sapwood, heartwood showed no mass loss, poor substrate colonisation as indicated by low fungal DNA amount and marker gene transcript level profiles indicating a starvation situation. Hence, the data suggest that the suppressive effect of suboptimal temperature on wood decay was more pronounced in wood with increased durability than in wood with low decay resistance. This information should be investigated further also for modified wood.

Pilgård et al. (2010) quantified colonisation by the white rot fungus Trametes versicolor in furfurylated, acetylated and thermally modified Scots pine wood in lab. Cu-HDO and CCA was used as reference treatments. For all wood treatments the maximum fungal DNA level was recorded after an incubation period of 2 weeks. The results also indicated that the exploratory hyphal growth in Cu-HDO was limited owing to acute phytotoxicity. Later studies with extended incubation time has revealed that fungal DNA is found inside the modified wood sample up to 36 weeks even if no mass loss was detected (Schmöllerl et al. 2011; Pilgård et al. 2012; Alfredsen et al. 2013).

DNA and gene expression studies

Alfredsen and Fossdal (2010) studied Postia placenta gene expression during 2, 4 and 8 weeks of colonisation in furfurylated wood. The main finding was that genes related to oxidative metabolic activity generally was higher in furfurylated wood compared to untreated Scots pine. The expression of genes involved in enzymatic degradation 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 1,3β glucosidase the expression was higher in furfurylated wood. The wood moisture content in this study was 21% after eight weeks of incubation and thereby below the threshold of 25% given in EN 113 (CEN 1996). A challenge is that the threshold in EN 113 is based on a measure of the overall wood moisture content in the test samples. As mentioned above, whether this measure also reflects moisture available for the fungus in order to enable degradation needs to be looked into more closely for modified wood materials.

Schmöllerl et al. (2011) tested Scots pine with the following treatments: CCA, furfurylation, thermally modified and acetylation against Postia placenta after 2, 14 and 26 weeks of incubation. Sterile soil was used as substrate instead of malt agar in order to allow a long incubation time. The highest mass loss, wood moisture content and fungal DNA content were found in the control samples. Acetylated wood had the lowest mass loss and wood moisture content. The fungal DNA content was quite similar in the modified samples, while for the CCA the fungal DNA content was low until week 26. In the beginning of the incubation of modified 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 enzymatic degradation 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 putatively involved in lignin decomposition was detected. No water was added to the test system during the study, and at week 26 the soil was obviously drier than in the beginning of the test. Hence, lower moisture levels in the soil might have affected the fungal colonisation in samples harvested at 26 weeks. However, the wood moisture data from this test does not give any clear indications of such a drying effect. The overall wood moisture content was high enough to allow fungal colonisation according to EN 113 except for furufurylated samples week 2 and acetylated samples week 14 and 26.

Ringman et al. (2014b) studied DMDHEU-treated, acetylated and thermally modified Scots pine during 56 days of P. placenta incubation. The results indicate that the response of P. placenta upon the encounter of modified wood is to up-regulate the expression of the oxidative degradation machinery. The investigated P. placenta genes presumed to be involved in oxidative degradation (quinone reductase and alcohol oxidase) were expressed at higher levels in acetylated, DMDHEU-treated and thermally modified wood than in untreated wood. For the investigated genes involved in enzymatic degradation (endoglucanase and β-glucosidase), the levels of expression in modified woods were equal to or lower than those in untreated wood.

Pilgård et al. (2012) and Alfredsen and Pilgård (2013) examined three different levels of acetylated Southern yellow pine. All samples had above 25% wood moisture content when harvested. Pilgård et al. (2012) found no mass loss in the acetylated samples treated to a high treatment level (22·0±0·3% acetyl content) 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 alcohol oxidase (involved in production of H₂O₂), cytochrome P450 (related to breakdown of toxic compounds), and a NADH-quinone oxidoreductase (involved in generating biodegradative hydroxyl radicals via redox cycling) along the incubation time, growing on acetylated wood treated to a high treatment level.

Alfredsen and Pilgård (2013) studied how a standardised leaching procedure with water (EN 84 CEN 1996) affected the degradation of acetic anhydride modified samples by the brown rot fungi P. placenta compared to no leaching prior to incubation. The samples were harvested after 4 and 28 weeks. They compared changes in mass loss, wood moisture content, fungal DNA, and gene expression from five genes. If leaching changes the acetylated samples and makes them more susceptible for fungal deterioration the expected effect would be higher levels of these parameters. Generally, leaching resulted in a few differences between leached and non-leached samples at low levels of acetylation (11·7±0·3% acetyl content), while no changes were found for the highest acetylation level. No differences were found in gene expressions after 28 weeks. The possible protection of acetylated wood against oxidative fungal degradation was suggested to be interpreted in combination with the lowered wood moisture content.

Conclusion

Decay fungi are present inside laboratory test wood samples already two weeks after inoculation.

After 36 weeks of inoculation of acetylated wood samples fungal DNA was recorded but no mass loss was found.

Genes involved in oxidative degradation are up-regulated when the fungus is growing on modified wood compared to the control

Genes involved in enzymatic degradation are being expressed at similar levels as in the control.

The hypothesis of lack of substrate recognition by the fungus is not supported by the data and is suggested to be rejected.

The wood moisture content in the modified wood samples included in this review was generally high enough to allow for fungal colonisation. But how the moisture is distributed within the wood matrix and whether there is sufficient moisture accessible around the hyphae to allow for fungal degradation needs to be further investigated.

Footnotes

Acknowledgement

This paper is a part of COST Action FP1303 and the authors want to acknowledge the inspiring work performed by this COST Action.

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