Wood sample preparation

Sample disks were cut from Oriental beech trees (Fagus orientalis Lipsky.) at breast height. Sample blocks as 50(l)×20(t)×15(r) mm were then prepared from these disks to determine mass loss according to EN113 (1997). Dry mass and initial moisture contents of the blocks were determined prior to autoclaving (at 121ºC for 20 min). Following this, the blocks were placed in the decay test containers and inoculated with each test fungus.

Fungal identification and incubation

The fruiting bodies of P. ostreatus and T. versicolor were recovered from living beech trees (F. orientalis Lipsky.) at Alamdardeh forest (located in Sari, Iran). For isolation and identification of the fungi, small wood blocks were surface-sterilized with 95% ethanol for 10-15 sec followed by 1% sodium hypochlorite for 45-50 sec. Thereafter, the wood pieces were inoculated on Potato Dextrose Agar (PDA) medium. Cultures were incubated at 25°C for complete growth. A pure culture was obtained after successive transfer on MEA medium by hyphal tip purification, and preserved at 4°C. Molecular identification was done based on Polymerase Chain Reaction (PCR) in accordance with (^{Schmidt et al. 2012}, ^{Bari 2014}). The fungi were identified by DNA extraction and sequencing of the internal transcribed spacer region (ITS). The forward primer, ITS5, and reverse primer, ITS4, were used. PCR products were sent to Takapozist (Bioneer- Korea) for sequencing. Identification was done by sequence comparison with sequences deposited in the DNA databases using the NCBI’s BLAST tool.

For mass loss determination, Kolle-flasks containing Malt-extract-agar (MEA) (4,8%) were prepared and autoclaved at 121ºC for 20 min according to EN 113 (1997). Prepared malt extract was inoculated with actively growing fungal cultures and incubated until the surface of the medium was covered with mycelial growth. Autoclaved wood blocks were placed directly on the mycelia and incubated at 25°C and 65% relative humidity (RH) for 120 days. Ten of these blocks were harvested every 15 days for the duration of the experiment for mass loss determination. The remaining samples were used for microscopy and FT-IR spectroscopy.

Chemical analysis

Decayed and undecayed wood specimens from each sampling time point were ground to pass the powder through sieve (40-mesh) prior to analysis. Cellulose and lignin analysis was performed using the T-17 wd-97 (TAPPI 1997) and T-222 om-98 (TAPPI 1998) specifications, respectively. A modified T-249 cm-85 (TAPPI 1992) method for carbohydrate analysis (^{Davis 1998}) was used to determine total hemicellulose content. Average cellulose, lignin, and hemicellulose loss of decayed wood was calculated as a percent of the corresponding values for the unexposed samples.

Light and SEM microscopic imaging

Samples exposed to the fungi for 120 days were cut into small cubes (5×5×5 mm) and then sectioned with a sledge microtome (~ 10 µm). Since the moisture content of specimens was high and the tissue was soft, sectioning was carried out right after incubation, without any pretreatments. To distinguish the cell wall and hyphae, sections were double-stained with safranin O (0,5%) and Astrablue (0,3%) dissolved in distilled water followed by conventional microscopy including dehydration and mounting on microscopic slides (^{Gartner and Schweingruber 2013}, ^{Bari et al. 2015}c). The prepared slides were studied by BEL epifluorescence microscopy under bright field and ultraviolet light (330-400nm). Under bright field light, lignified cell walls appear red while hyphal walls and delignified xylem cells get blue color. UV fluorescence microscope, on the other hand, was used to qualitatively trace lignified versus delignified cells of xylem.

For scanning electron microscopy, the mini-blocks of decayed wood samples (5×5×5 mm) were clean cut using a razor blade and then chemically fixed according to ^{Mohebby (2003}). Sections were then mounted on SEM stubs, and sputter coated to a thickness of 30 nm with gold. A SEM microscope (VEGA\\TESCAN-LMU) was used for observation.

FT-IR spectroscopy

Dried wood samples were milled and sieved through a 40 mesh screen. The IR-spectra were obtained directly from wood powder using ATR technique by a Shimadzu 8400s FT-IR Spectrometer equipped with DLATGS detector. All samples were examined at a spectral resolution of 4 cm^-1 with 30 scans for each sample as well as the background. The background spectra were collected using an empty collector. A rubber band baseline correction method was also applied for all spectra. The CO₂ band was also removed from the spectra to make a suitable baseline correction (^{Mohebby 2005}).

Mass loss

The average mass loss of the wood samples at different periods of exposure to the white rot fungi, P. ostreatus and T. versicolor, are presented in Table 1. The data shows that both fungi attacked beech wood with similar decay capabilities resulting in an almost identical amount of mass loss. Both fungi decreased the wood mass about 15-18% at 30 days exposure and 26% after 120 days exposure. Both fungi exhibited similar capabilities to severely attack the wood within the first month (Table 1). Similar results were also previously reported by authors (^{Bravery 1978} and ^{Bari et al. 2015}d). Minor fluctuations in mass loss among blocks were also similar for both fungi.

Cell wall chemistry decomposition

The average cellulose, lignin, and total carbohydrate contents of unexposed beech wood were 39,6%; 22,2% and 67,6% of the dry wood weight, respectively. As shown in Table 2, both fungi were able to attack cellulose. Hemicellulose components were also attacked by both test fungi. The amount of cellulose and lignin remaining after fungal attack indicates similar capabilities of two fungi to degrade wood. Because these two white rot fungi are Basidiomycetes, they should degrade carbohydrates and lignin to a similar extent (^{Eriksson et al. 1990}). Cellulose is composed of two parts, crystalline and amorphous (^{Sjöström 1993}, ^{Kubicek 2013}). The amorphous region is much more susceptible than crystalline region for initial fungal metabolism (^{Eaton and Hale 1993}). Hence, a high rate of cellulose deconstruction at initial attack by the fungus would be normal. One of the major components in plant cell walls is the side by side chains of multiple hydroxyl groups in glucose (^{Klemm et al. 2004}, ^{O’Sullivan 1997}). To degrade the cellulose chains, fungi need specific enzymes such as endo- and exocellulases and β-glucosidase and most of the white-rot fungi are able to produce these enzymes (Eriksson et al. 1990, Eaton and Hal 1993, Kubicek 2013). It seems that nonselective white-rot fungi have these enzymatic systems. Since white-rot fungi use a hydrolysis system for the decomposition of cellulose chains (Kubicek 2013), they can cleave cellulosic chains by producing endoglucanase enzymes that can hydrolyze the ß-1,4-glycosidic bonds which breaks down crystalline cellulose to cellobiose (Eriksson et al. 1990, Kubicek 2013).

Table 2: Average of percent wood cell wall polymers losses in decayed Oriental beech wood samples by white-rot fungi. 

± values represent standard deviations of the means

^a Data in DP reported from ^{Bari et al. 2015}b.

The data in Table 2, shows that the average percent losses in hemicelluloses (total carbohydrate) at the end of the incubation were 6,73% and 7,32% for Pleurotus ostreatus and Trametes versicolor, respectively. Glucan, galactan, and arabinan were the most noticeable hemicellulose components depleted by both fungi. In contrast to cellulose, the structure of hemicelluloses contains heteropolysaccharides constituents (^{Sjöström 1993}, ^{Kubicek 2013}). One of the functions of hemicelluloses is protecting the cellulose microfibrils by providing physical barriers against hydrolytic enzymes. Hence, removal of the hemicellulose components from the cellulose microfibrils facilitates access of fungal hydrolytic enzymes to cellulose (^{Yang and Wyman 2004}). Since the arabinan and galactan are side chain elements of xylan and mannan; they are very susceptible to degradation (^{Timell 1967}). It has been suggested that they may be attacked before the main chain of the polymer (^{Curling et al. 2002}). Therefore, the full decomposition of hemicelluloses requires the activation of special enzymes such as endo-β-(1,4)-xylanase and β-xylosidase (^{Eriksson et al. 1990}, Kubicek 2013). Overall, hemicelluloses were the most degraded polymers in beech wood exposed to both fungi as shown in Table 2. Determination of the total carbohydrate showed similar reduction for beech exposed to both fungi as well as their ability to produce specialized enzymes for the decomposition of wood cell walls.

As presented in Table 2, the lignin content declined uniformly during the incubation. Moreover, this data indicates that both test fungi tended to consume lignin more than cellulose. This is in agreement with the literature (^{Eaton and Hale 1993}, ^{Schmidt 2006}), which indicates that both fungi are capable of degrading lignin more rapidly than carbohydrates. Lignin is a phenolic polymer that confers strength to plant cell walls, as well as serving as protection against pathogens (^{Eriksson et al. 1990}, Eaton and Hale 1993, Schmidt 2006). In general, obtaining nutrients through plant cell wall during the decomposition mechanisms is a fundamental strategy for success of many microorganisms. For example, saprophytic fungi inhabit dead organic materials like decaying wood. In order to acquire energy from these materials, both fungi need to produce enzymes in order to degrade a majority of plant cell wall polysaccharides present in the biomass.

Microscopic evaluations

The series of micrographs of decayed woods in Figure 2 showed that the decay patterns caused by the two test fungi are very similar. In addition, the diagnostic features of decay represent simultaneous white-rot for both fungi. The arrows denote fiber cell walls which have thinned due to fungal attack, indicating that all cell wall polymers are degraded by the fungi. This means that not only both fungi are capable of degrading lignin and hemicelluloses, but also they can attack cellulose polymer as well. The decomposition of S₂ and S₃ layers and the remainder of only a thin middle lamella (Figure 1, Figure 3A and Figure 3B) are the result of the fungi using all of the cell wall components, which has previously been reported (^{Liese 1970}, ^{Wilcox 1973}, ^{Schwarze et al. 2004}, ^{Schmidt 2006}, ^{Schwarze 2007}, ^{Kubicek 2013}). When decay develops throughout the xylem, previously tiny bore holes on the cell wall coalesce together and become ruptures in the cell walls (Figure 1C and Figure 1D; arrowheads). Erosion along with the reducing of the fiber's wall thickness are further evidences of similar patterns of decay by studied fungi. In simultaneous white-rot decay patterns, the decay occurs in vicinity of the hyphae, with subsequent formation of erosion troughs where the hyphae are growing on the cell walls (Wilcox 1973, ^{Anagnost 1998}, Schwarze et al. 2004, Schwarze 2007, Kubicek 2013, ^{Bari et al. 2015}c, ^{Kim et al. 2015}). In the case of samples decayed with Pleurotus ostreatus, the process and features of decay are similar for most components of the wood. However, in very confined areas of one sample, anatomical diagnosis of selective white-rot decay was also observed, e.g. separation of the cells, while the secondary walls remained almost intact (Figure 2). Complete decomposition of ray parenchyma cells (arrows) as well as degradation of the cell walls and vessels (arrowheads) occurred due to severe degradation of cell wall components (Figure 3C, Figure 3D). Separation of the ray cell walls along with vessel lumina (arrows) and serious decomposition of woody structure (arrowheads) took place causing massive consumption of cell wall components (Figure 3E and Figure 3F). It has been reported that some white-rot fungi are capable of optimizing their decomposition strategy by changing from simultaneous to selective decay or selectively digest lignin in one place of the sample and show symptoms of simultaneous decay at the other place of the same sample (^{Hartig 1878}, ^{Nagadesi et al. 2013}, Bari et al. 2015c). The switching in fungal behaviors, therefore, may causes changes in lingo-cellulolytic enzyme activity during the degradation of woody materials (^{Sunardi et al. 2016}).

Figure 1: Light microscopy from transverse sections showing decay patterns in Oriental beech wood samples by two white-rot fungi; A, C: Pleurotus ostreatus; B, D: Trametes versicolor. a and b: Eroding along with thinning (arrowheads) and ruptured cell walls as well as hyphal colonization of cell lumens (arrows). C and D: Decomposition and removing of cell walls (arrows) as well as eroding and thinning of cell walls (arrowheads), while the middle lamella remains intact even at advanced stages of decay. Scale bars in A, B: 30µm; C, D: 15µm. 

Figure 2: Oriental beech wood decayed by two white-rot fungi; a, b: Pleurotus ostreatus; c, d: Trametes versicolor under bright field (A-C) and UV light (D). Separation of secondary wall of a tracheid in cross section (A) and ray parenchyma cells in tangential section (B) of decayed wood resembling selective degradation. Under bright field, most of the tissue fibers are degraded while vessel walls show higher resistance with their red-stained color (C). This can be confirmed by viewing the same sample under UV light (D). Scale barsin A, B: 20µm; C, D: 50µm. 

Figure 3: Scanning electron micrographs of beech wood degradation after 120 days of exposure to the white-rot fungi; Pleurotus ostreatus (A,C&E); Trametes versicolor (B,D&F). A&B: cross sections: Severe eroded and thinned cell walls (arrows) as well as colonization of hyphae in cell lumina (arrowheads). C&D: radial sections: Complete decomposition of ray parenchyma (arrows) as well as deconstruction of cell walls and vessels (arrowheads) due to severe degradation of cell walls chemical materials. E&F: tangential section: separation of ray wall with vessels lumina (arrows) and serious decomposition of woody structure (arrowheads) due to massive consumption of cell wall chemicals. A&B: bar 20µm; C, D, E, F: bar 100µm. 

FTIR spectroscopy

The IR-spectra of white-rotted wood show changes of peaks in the fingerprint region (between 1800-600 cm⁻¹) for different stages of decay for both test fungi (Figure 4). As shown for the IR-spectra of the un-decayed beech wood, there is strong bond stretching by hydroxyl groups (O-H) at 3500-3300 cm⁻¹, prominent symmetric C-H bond stretching in aromatic methoxyl, methyl and methylene groups around 2885 cm ^{− 1}, and many well-defined peaks in the fingerprint region (between 1800-600 cm⁻¹). Detailed information on assigned peaks is shown in Table 3. Comparison between the peaks obtained from different stages of fungal decay with the un-decayed wood revealed disappearance of some peaks and appearance of new peaks after exposure to both fungi, which are prominent indications of new bonds generated from altered cell wall constituents and their cleaved structures (Figure 4).

Figure 4: IR-spectra for beech wood decayed by two white rot fungi, P. ostreatus and T. versicolor, during different periods of exposure. 

Table 3: IR assignment in decayed wood (^{Popescu et al. 2007}). 

S: syringyl; G: guaiacyl

Subtraction of the un-decayed peaks clearly shows the presence of newly formed peaks for the decayed wood (Figure 5, Figure 6, Figure 7, Figure 8, Figure 9). In Figure 5, Figure 6, Figure 7, Figure 8, Figure 9, the baselines (line zero) indicates changes in the decayed wood in comparison to the un-decayed wood. The subtracted peaks above the baseline showed a decrease in intensity of the present peaks and the peaks below the baseline indicate an increase of the present peaks as well as appearance of new bands due to fungal attack. When there is no difference between the peaks, the subtracted peak intensities are zero. In this situation, the subtracted peaks are located on baselines which indicate no changes in the wood during fungal attack. The spectra for the wood decayed by both fungi during the early stages of exposure revealed fewer alterations in wood chemistry which is probably due to reduced decay; while the wood attacked for 45 days revealed the appearance of new peaks (1800-400cm^-1); especially for hemicelluloses and lignin (Figure 6).

Figure 5: Subtracted IR-spectra for beech wood decayed by two white rot fungi, T. versicolor and P. ostreatus, during 15 days of fungal exposure. 

Figure 6: Subtracted IR-spectra for beech wood decayed by two white rot fungi, P. ostreatus and T. versicolor , during 45 days of fungal exposure 

Figure 7: Subtracted IR-spectra for beech wood decayed by two white rot fungi, P. ostreatus and T. versicolor, during 75 days of fungal exposure. 

Figure 8: Subtracted IR-spectra for beech wood decayed by two white rot fungi, P. ostreatus and T. versicolor, during 105 days of fungal exposure. 

Figure 9: Subtracted IR-spectra for beech wood decayed by two white rot fungi, P. ostreatus and T. versicolor, during 120 days of fungal exposure. 

The spectra for the decayed wood exposed to both white-rot fungi for 45, 75 and 105 days revealed appearance of new peaks at 1800-1650 cm^-1 and 1500-1300 cm^-1. Furthermore, new peaks appeared at 1800-1300cm^-1 for wood decayed by both white-rot fungi within 105 days. Those peaks represent fingerprint regions for hemicelluloses (especially xylan) and aromatic skeletal C-H as well as lignin derivatives. The new peaks indicate that new bands have developed due to breakdown of cell wall constituents, especially lignin and hemicelluloses. It is well known that white-rot fungi produce different types of hydrolyzing enzymes to dissolve lignin in wood cell walls as well as the middle lamella.

The new peaks disappeared for days 15, 75 and 120 (Figure 7, Figure 8, Figure 9). It is interesting to see considerable differences in spectra for decayed wood at days 105 and 120 (Figure 8, Figure 9). The new peaks appeared in wood exposed to the test fungi for 105 days (Figure 8) and disappeared at 120 days (Figure 9), indicating that both fungi degraded cell wall constituents until day 105 and then liberated them as small molecules. Afterwards, those molecules were assimilated as carbon sources by the fungi (Figure 9). Subsequently, fragments or non-degraded parts of the cell walls remained which are chemically similar to un-decayed wood. As shown in Figure 9, there are no substantial differences between spectra for decayed and un-decayed wood at this phase.

As ^{Mohebby (2005}) and ^{Karim et al. (2017}) stated that the white-rot fungus T. versicolor has the ability to remove most of the hemicelluloses adjacent to lignin and also partially remove the cellulose. According to the spectra assigned for the un-decayed wood (Table 3), the peaks related to hemicelluloses and lignin changed during fungal exposure, with partial changes in cellulose clearly indicating non-selective degradation of wood by both fungi. This phenomenon was also recently investigated and confirmed by microscopy and chemical studies for both fungi (^{Bari et al. 2015}b, ^{Bari et al. 2015}c). Mohebby (2005) as well as and ^{Pandey and Pittman (2003}) studied chemical changes in wood decayed by T. versicolor using FTIR spectroscopy and found decreased intensities of bands for lignin and carbohydrates. In contrast to the white-rot, during in the brown-rot decay the selective removal of structural carbohydrate components take place and causes increasing the higher lignin/carbohydrate ratio as decay progresses (^{Bouslimi et al. 2014}).

Comparison of the spectra of beech wood decayed by the two fungi used in this study after different periods of exposure showed considerable similarities, i.e., simultaneous appearance and disappearance of peaks. This suggests that both fungi use very similar mechanisms to attack wood. The authors conclude that these microscopic observations confirm the FTIR results.

Ratios of peak intensity at 1510 cm^-1 (C=C stretching in lignin aromatic skeletal structure) is presented in Figure 10 for both test fungi. The trends of the ratios vary similarly for both fungi during the exposure period with an increase at day 105. This increase indicates that the C=C band intensities in the decayed wood are higher than the un-decayed wood due to fungal attack, indicating that the lignin structure has been severely attacked by the test fungi at day 105 (Figure 4 and Figure 8).

Figure 10: Intensity ratio for wave number 1510 cm^-1 (C=C) of decayed to undecayed wood due to both fungi. 

The ratio of peaks at 1510/1158 cm^-1 (C=C stretching in lignin and C-O-C stretching in cellulose, respectively) is shown in Figure 11. The ratios varied for both test fungi with an increase at day 105 of fungal exposure. The increase shows that the C=C band intensities are higher than that of C-O-C at day 105, indicating that severe attack of the lignin has been occurred compared to attack of the cellulose.

Figure 11: Intensity ratio for wave number 1510 cm^-1 (C=C) to 1158 cm^-1 (C-O-C) in decayed wood due to both fungi. 

The ratio of peaks of the wave numbers 1510/895 cm^-1 is shown in Figure 12. The peak at wave number 895 cm^-1 is assigned for =C-H out of plane stretching which is related to lignin structure. There are similarities for both test fungi during exposure, with an increase occurring at day 105. However, there are some different fluctuations in the peak ratio for P. ostreatus. This indicates that both fungi attack the wood with similar patterns; however, they sometimes use different strategies to attack lignin. Any increase in peak ratio indicates that lignin has been broken down, many bonds are cleaved, and new bonds appeared in the lignin structure. In this case, an increase of =C-H bonds expressed as C=C bonds which have already been cleaved in lignin, or the intermolecular bonds in lignin monomers have already been broken down during fungal attack. The results presented here as well as the microscopic observations confirm the above mentioned results; especially Figure 4 and Figure 8. In general, it can be seen that the lignin has been severely attacked util2 day 105 and the liberated materials have been assimilated before day 120. Because there are quite similarities between days 120 and 0.

Figure 12: Intensity ratio for wave number 1510 cm^-1 (C=C) to 895 cm^-1 (out of plane =CH) in decayed wood due to both fungi.