Abstract
The chemical and morphological changes in heartwood specimens of Liquidambar orientalis Mill. caused by the white-rot fungus Trametes versicolor and the brown-rot fungi Tyromyces palustris and Gloeophyllum trabeum were studied by wet chemistry, FT-IR, GC-MS analyses, and photo-microscopy. According to GC-MS results, 26 extracts identified in the ethanol/toluene extraction and 17 in the ethanol extraction were found. Heartwood specimens of L. orientalis were highly susceptible to the fungi tested. While 1% NaOH solubility increased 35% in the specimen decayed by T. palustris, only an 8% increase was seen in the specimen exposed to T. versicolor when compared to the control specimen. Decayed wood by T. palustris showed a 5.5% increase in the Klason lignin content when compared to control specimens; however, the Klason lignin content decreased after a T. versicolor attack for 12 weeks. A T. versicolor attack in the cell walls was seen both from the lumina and from the cell corners, and the attack from the cell corners was mainly clear in ray parenchyma cells. An excessive destruction was detected in the wood structure attacked by T. palustris. The cell collapse was caused by a distortion in the plane of the wood cells. This extensive degradation was seen in all types of cell walls. Cracks in the cell walls were also detected in the specimens.
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Evaluation of Fungal Deterioration in Liquidambar orientalis Mill. Heartwood by FT-IR and Light Microscopy
Nural Yilgor,a,* Dilek Dogu,b Roderquita Moore,c Evren Terzi,b and S. Nami Kartal b
The chemical and morphological changes in heartwood specimens of Liquidambar orientalis Mill. caused by the white-rot fungus Trametes versicolor and the brown-rot fungi Tyromyces palustris and Gloeophyllum trabeum were studied by wet chemistry, FT-IR, GC-MS analyses, and photo-microscopy. According to GC-MS results, 26 extracts identified in the ethanol/toluene extraction and 17 in the ethanol extraction were found. Heartwood specimens of L. orientalis were highly susceptible to the fungi tested. While 1% NaOH solubility increased 35% in the specimen decayed by T. palustris, only an 8% increase was seen in the specimen exposed to T. versicolor when compared to the control specimen. Decayed wood by T. palustris showed a 5.5% increase in the Klason lignin content when compared to control specimens; however, the Klason lignin content decreased after a T. versicolorattack for 12 weeks. A T. versicolor attack in the cell walls was seen both from the lumina and from the cell corners, and the attack from the cell corners was mainly clear in ray parenchyma cells. An excessive destruction was detected in the wood structure attacked by T. palustris. The cell collapse was caused by a distortion in the plane of the wood cells. This extensive degradation was seen in all types of cell walls. Cracks in the cell walls were also detected in the specimens.
Keywords: Fungal degradation; GC-MS; Extractives; Morphology of wood decay; FT-IR; Liquidambar orientalis
Contact information: a: Department of Forest Products Chemistry and Technology, Forestry Faculty, Istanbul University, 34473, Istanbul, TURKEY; b: Department of Forest Biology and Wood Protection Technology, Forestry Faculty, Istanbul University, 34473, Istanbul, TURKEY; c: USDA Forest Service, Forest Products Laboratory, Madison, WI, 53726, USA; *Corresponding author: yilgorn@istanbul.edu.tr
The use of trade or firm names in this publication is for reader information and does not imply endorsement by the U.S. Department of Agriculture of any product or service. The Forest Products Laboratory is maintained in cooperation with the University of Wisconsin. This article was written and prepared by U.S. Government employees on official time, and it is therefore in the public domain and not subject to copyright.
INTRODUCTION
Wood is a natural, renewable, and valuable construction material. Since being thought of as naturally resistant to wood-degrading organisms, wood has often been used as a shelter, and has many outdoor applications throughout human history. However, the durability of wood varies depending on its tree species, chemical composition, and the environmental conditions the wood is exposed to. Wood biodegradation occurs in different ways such as fungal, bacterial, and insect attack. Fungal decay is the most widespread type of wood degradation, and wood decay fungi are usually classified in three main groups: white, soft, and brown-rot (Eriksson et al. 1990; Blanchette 1995). Of these, only white-rot fungi are capable of degrading the hemicelluloses, cellulose, and lignin, whereas soft-rot fungi can degrade cellulose and hemicellulose and partially digest lignin. Brown-rot fungi are not able to digest lignin while they decompose the hemicelluloses and cellulose (Erikkson et al. 1990; Blanchette 1995).
The development of new techniques has provided a great deal of understanding about the mechanism of fungal degradation of wood polymers, but there are still many questions that remain unanswered regarding wood biodegradation by fungi (Hatakka and Hammel 2010).
In this study, Liquidambar orientalis, one of the most important endemic hard-wood species in Turkish flora, was investigated with respect to some of its chemical and anatomical properties after being exposed to the white- and brown-rot fungi. In recent years, various projects regarding protection of L. orientalis trees, development of new plantations, and realization of new management plans for balsam production have been started in Turkey due to the usage of its wood for indoor building and roofing material as well as balsam production (Terzi et al. 2012). The influence of fungal degradation on both gymnosperm and angiosperm wood species in terms of anatomical and chemical changes have been studied. Flournoy et al. (1991) examined the mechanism of cellulose depolymerization in sweetgum (L. styraciflua L.) wood specimens exposed to brown-rot fungus, Postia placenta. Highley and Murmanis (1987) studied micro-morphological changes in sweetgum and western hemlock sawdust caused by white-rot fungus, Trametes versicolor. However, many of the wood species and the degradation caused by fungi have become an interest for many researchers after the development of new instrumental techniques such as FT-IR, GC-MS, NMR, etc. Of these, FT-IR is one of the most useful and valuable tools for investigating fungal decay and has been used to observe the effects of fungi in wood structures. It is also useful to characterize the chemical changes in the molecular structure of woods exposed to biological attacks such as fungal and bacterial. Faix et al. (1991) examined beech wood attacked by white-rot fungus by FT-IR spectroscopy and analytical pyrolysis; results indicated that lignin was modified more than polysaccharides. Pandey and Pitman (2003) studied Pinus sylvestris L. sapwood and Fagus sylvatica L. wood specimens by FT-IR after different exposure periods by brown and white rot fungi. Another study by Pandey and Pitman (2004) examined the lignin content of both softwood and hardwood after decay by brown rot fungus with FT-IR. Naumann et al. (2005) demonstrated that FT-IR technology has an ability to identify wood fungi, and it is possible to detect fungi in wood by FT-IR microscopy. Rumana et al. (2010) used FT-IR spectroscopy to characterize the lignin structure, and five tropical timber wood species of the family of Dipterocarpaceae were investigated with regard to chemical and histochemical properties. FT-IR spectroscopy, on the other hand, requires the preparation of small pellets with KBr which might be time consuming and tedious. In recent years, a new technique called “Attenuated Total Reflectance” (ATR) has been developed and used in conjunction with infrared spectroscopy. Mohebby (2005) studied beech wood that had been exposed to a white rot fungus for a period 84 days with ATR spectroscopy by means of chemical alteration of the wood structure. The study claimed that ATR spectroscopy was a very feasible and rapid method for examining biodegradation caused by fungi in wood.
The goal of this study was to identify some of the chemical components of L. orientalis heartwood by GC-MS and monitor the changes in wood structure after brown and white fungi exposure by means of FT-IR ATR spectroscopy and light microscopy.
EXPERIMENTAL
Decayed Wood Specimens
Details regarding heartwood specimens and decay tests are given in our previous study (Terzi et al.2012). Soil block decay resistance tests were performed based on the AWPA E10-09 standard method by using 12 replicate heartwood specimens of L. orientalis (19 by 19 by 19 mm) for each test fungus. Two brown rot and one white rot fungi (Tyromyces (Fomitopsis) palustris (Berkeley and Curtis). Murrill (FFPRI 0507) and Gloeophyllum trabeum (Pers:Fries) Murrill, MAD 617 and Trametes versicolor (L.: Fr.) Quel. MAD 697 were employed as test fungi (AWPA 2010).
Chemical Analyses
Before chemical analyses, the heartwood and sapwood of L. orientalis were separated, and then the air-dry wood specimens were ground and screened through a 40 to 80-mesh sieve according to TAPPI T-257 cm-85 (TAPPI 1992). The ash content of the wood specimens, hot water, and dilute alkali solubility and extractives soluble in solvents were determined according to TAPPI standards T 211 om-93, T 207 om-93, T 212 om-98, and T 204 om- 88, respectively (TAPPI 1999 and 1987).
The Klason lignin content of heartwood was performed based on Runkel and Wilke (1951). The Klason lignin values were corrected for the ash content gravimet-rically following the incubation of lignin at 575 °C for less than 3 h.
The carbohydrates content of the hydrolysates was determined by anion exchange high performance liquid chromatography (HPLC) using pulsed amperometric detection (Davis 1998). Wood sugars were quantitated using an internal standard method, and the results were reported in terms of a percentage of the original specimen mass.
In addition, in decayed wood specimens, the Klason lignin content and 1% NaOH solubility were determined using the above standards.
Heartwood Extracts Preparation
To determine the heartwood extractives, ethanol/toluene and ethanol extraction steps were performed according to TAPPI method T 204 om-88 (TAPPI 1987) with slight modifications. The ground wood samples were magnetically stirred with solvents for 48 h. In the first 24 h (2:1) toluene and 95% ethanol were used, and the solvent and extracts were filtered. In the second 24 h, 95% ethanol was used and filtered. The wood extractions had a yellow tint in the solvent. The solvents were dried in the rotary evaporator and the extractives were collected for further investigation by GC-MS.
Gas Chromatography-Mass Spectrometry (GC-MS) Analyses
The extracts were analyzed on a Shimadzu GC-MS-QP2010 instrument (Kyoto, Japan) equipped with split-splitless inlets, a mass spectrometer, and an auto injector. SHRXI-5ms (30m X 0.25 mm I.D., 0.25 μm film thickness) capillary column was used and the carrier gas was helium. The temperature injection was 250 oC. The oven was temperature-programmed from 50 oC (1 min) to 320 oC (10 min) at 10 oC/min. The National Institute of Standards and Technology (NIST) library was used for identifying components.
FT-IR ATR Spectral Analyses
The IR absorption data were obtained by using a Perkin Elmer 100 FT-IR Spectrometer combined with an ATR unit (Universal ATR Diamond Zn/Se) at a resolution of 4 cm-1 for 32 scans in the spectral range 600 to 4000 cm-1. Dried specimens were milled and passed through a mesh 80 sieve, and the analyses were performed on undecayed specimens and specimens exposed to the fungi. The spectra were baseline corrected and normalized to the highest peak. FT-IR spectroscopy was improved by an attenuated total reflectance (ATR) unit that provided direct interaction of the measuring beam with the sample and reflected the attenuated radiation to the spectrometer and increased the sensitivity of FT-IR-based analyses.
Anatomical Examinations
Degraded heartwood specimens were cut directly into thin sections by using a sliding microtome. The sections were then stained with safranin and picro-aniline-blue. A specimen obtained from the heartwood of sound wood served as the control. That sample was kept under vacuum in the presence of alcohol, glycerin, and water at room temperature to soften it before it was sectioned. The sections were only stained by safranin. Evaluations were performed using an Olympus BX51 Light Microscope, and images were taken using analySIS FIVE Software; a DP71 Digital Camera was installed and adapted on the microscope. All of the microscopic investigations and evaluations were realized on cross, radial, and tangential sections.
RESULTS AND DISCUSSION
Chemical Analyses
Table 1 shows the amount of Klason lignin and the monomer sugars in undecayed heartwood specimens of L. orientalis. The solubility values by ethanol/toluene, ethanol, hot water, and 1% NaOH are given in Table 2. The ash content of heartwood specimen was determined as 0.42%. Table 3 represents the amount of Klason lignin and hot alkali solubility of the specimens exposed to the brown rot and white rot fungi tested; percentage changes in these values after fungal attack were based on the undecayed specimens. The ethanol/toluene extractable content of the wood consisted of the waxes, fats, resins, oils, and tannin. The hot water extraction removed a part of the extraneous components, such as inorganic compounds, tannins, gums, sugars, coloring matter, and starches. Hot alkali soluble extracts contain low molecular weight carbohydrates and degraded cellulose. Besides undecayed specimens, Klason lignin content and 1% NaOH solubility were determined in the specimen exposed to either Tyromyces (Fomitopsis) palustris or Trametes versicolor. While alkali solubility was found as 18.7% in the control specimen, it was 25.3% and 20.2% in the specimens exposed to T. palustris and T. versicolor, respectively. As expected, 1% NaOH solubility remarkably increased in the specimen decayed by T. palustris since the brown-rot fungi degraded wood carbohydrates selectively. Even though the high solubility of 1% NaOH indicates the degradation of the hemicelluloses and degraded cellulose in wood, it was 20.2% in the specimen exposed to T. versicolor in our study. While 1% NaOH solubility showed a 35.3% increase in the specimen decayed by T. palustris, only an 8% increase in the specimen exposed to T. versicolor was seen when compared to the control specimen. An increase in alkali solubility is an efficient sign of carbohydrate decomposition, and T. palustriscaused a remarkable decomposition in the carbohydrates compared to T. versicolor due to high alkali solubility in the study (Table 3). The decayed wood specimens by T. palustris showed a 5.5% increase in the Klason lignin content when compared to the control specimen; however, the Klason lignin content decreased after the T. versicolor attack for 12 weeks. This might be an indication that the fungus, T. versicolor, degraded the lignin part of the wood preferentially.
Table 1. Chemical Properties of Undecayed Heartwood of L. orientalis
All values are average of duplicate analyses.
Table 2. Solubility of Undecayed Heartwood of L. orientalis
All values are average of duplicate analyses.
Table 3. Klason Lignin Content, 1% NaOH Solubility, and Percentage Changes in Control and Decayed Wood Specimens
* Based on undecayed control specimens. All values are average of duplicate analyses.
The present test results were in good agreement with a study by Winandy and Morrell (1993), who studied the relationship between decay and the chemical composi-tion of Douglas-Fir heartwood, and performed decay tests with A. placenta and G. trabeum as the brown-rot fungi and T. versicolor and B. adusta as the white-rot fungi. Their results showed that the brown rot fungi caused more substantial changes in the chemical composition. The alkali solubility of the specimens colonized by the brown-rot fungi gradually increased with longer incubation periods. The Klason lignin content of the specimens colonized by either of the two brown-rot fungi remarkably increased during the 177-day-incubation period. Furthermore, it was suggested that both of the white-rot fungi caused a minimal effect on the chemical properties of the wood, as shown by the alkali solubility.
Mass Loss in Decayed Wood Specimens
Heartwood specimens of L. orientalis were susceptible to fungal decay by the two brown-rot fungi and one white-rot fungus tested. Mass losses have been reported for such specimens in decay resistance tests (Terzi et al. 2012). T. palustris degradation caused around a 50% mass loss in the specimens. Mass losses in the specimens varied between 37% and 43% for the G. trabeum attack.
The white-rot fungus T. versicolor caused mass losses that varied from 34% to 41%. According to the ASTM D 2017 classification (ASTM 2010), the heartwood specimens of L. orientalis were “non-resistant” against the T. palustris; however, the specimens were moderately resistant against G. trabeum and T. versicolor based on the average mass losses (Terzi et al. 2012).
GC-MS Analyses
Results from the GC-MS analyses are shown in Figs. 1a and 1b. Compounds having a peak area of 1% or more are given in Table 4. According to the GC-MS results, there were 26 extracts identified in the ethanol/toluene extraction and 17 in the ethanol extraction. Thirty-five was the total number of compounds identified from both extrac-tions. Three replicates of the samples were injected into the GC-MS.
As can be seen in Fig. 1a, 26 compounds were identified by the NIST library in the first extraction step with the solvents ethanol/toluene. Some of the compounds’ concentrations were in trace amounts. Compounds 9, 10, 11, and 12 did not have baseline separation; however, they were identified by the NIST library as 2-furancarboxaldehyde, 5-(hydroxymethyl) (9), hydrocinnamic alcohol (10), 2-butanone, 4-phenyl- (11), and benzene, 2-ethenyl-1,3,5-trimethyl-(12). The most abundant compound is benzaldehyde, representing peak 1 in the ethanol/toluene extraction step.
Fig. 1. a) Ethanol/toluene extracts fractionation chromatogram (26 compounds identified by the NIST); b) Ethanol extracts fractionation chromatogram (17 compounds identified by the NIST)
Figure 1b shows the compounds identified by the NIST library in the second extraction step with ethanol. There were some compounds in trace amounts for this extraction as well. Peaks 3, 4, and 5 were not completely separated; however, they were conclusively identified as 2-furancarboxaldehyde, 5-(hydroxymethyl)-, hydrocinnamic alcohol, and 1-phenoxypropan-2-ol, respectively. In the ethanol extraction, benzyl alcohol was more abundant than benzaldehyde. Peak 2 represents benzyl alcohol and peak 1 represents benzaldehyde. Peaks 13, 14, and 15 are naphthalene peaks. Naphthalene has been reported to be a chemical component of storax (Hafizoglu et al. 1996). Further investigations are needed to determine whether the naphthalene peaks are natural components of these extractives or contaminates. Benzaldehyde, benzyl alcohol, hydro-cinnamic alcohol, benzyl benzoate, tetradecanoic acid, salicylic acid, and tritetracontane were identified in both extractions. Some of the chemicals found in both of the extractions have been documented as having contributing factors in biological activities.
Lee et al. (2009) investigated benzaldehyde, hydrocinnamic alcohol, and benzyl alcohol for anti-fungal activity. The inhibition rates of benzaldehyde and hydrocinnamic alcohol as individual compounds were 100% and 50.3% at 28×10-3 mg/mL air concentration, respectively against P. cactorum. Benzyl alcohol showed no fungal activity. This study found 4 out of 8 chemicals in L. orientalis heartwood having antifungal activity. Benzyl benzoate is known for showing a high toxicity against insects. Terzi et al. (2012) demonstrated in the termite and larvae resistance tests that L. orientalis heartwood is resistant against these organisms.
IR Spectroscopy Analyses
Undecayed wood (control)
The IR spectra of undecayed L. orientalis heartwood are shown in Fig. 2, and the explanations of the wood peaks can be seen in Table 5. The peaks are defined with reference to Harrington et al. 1964; Faix 1991; Pandey and Pitman 2003; and Naumann et al. 2005. A strong hydrogen bond (O-H) stretching absorption was seen at 3336 cm-1 (1), and the following C-H stretching absorption band was around 2917cm-1 (2) (Fig. 2).
The region between 1800 and 600 cm-1 is called the “fingerprint region” and many significant and well-defined peaks due to the various functional groups present in wood structure can be observed in this region (Pandey and Pitman 1999; Moheby 2005). It is known that the holocellulose content of hardwood is higher than softwood, and even though it is suggested that the position of the carbonyl peak is in a higher wave number for hardwoods (≥1740) (Pandey 1999), there are different findings which indicate lower wave numbers for carbonyl peaks in hardwoods, such as 1730 cm-1 in Eucalyptus regnans (Harrington et al. 1964), 1724 cm-1 in Fagus sylvatica (Mohebby 2005), and 1738 cm-1 in F. sylvatica L. (Pandey and Pitman 2004). However, in our study, strong bands at 1734 cm-1(3) and 1235 cm-1 (11) were observed, indicating an unconjugated C=O stretching in xylan. Besides the 1734 cm-1 band, the 1235 cm-1 band also represented acetyl and carboxyl vibrations in the xylan (Harrington et al. 1964). According to Harrington, the other wood fractions’ contribution to this band is the lowest when compared to the other bands that represent xylan, such as 1260 cm-1, 1055 cm-1, 1030 cm-1, 990 cm-1, and 895 cm-1 (Harrington et al. 1964). The peak at 1157cm-1 (12) is assigned to the C-O-C vibration in cellulose and hemicellulose.
The peaks observed at 1593 cm-1 (5) and 1504 cm-1 (6) are characteristic peaks for lignin as seen in Fig. 2. Other than 1734 cm-1 (3), absorption bands at 1157 cm-1 (12) and 896 cm-1 (16), also from the carbohydrates, are accepted as reference peaks for polysaccharides (Pandey and Pitman 2003). It is suggested that while the guaiacyl type of lignin, which is characteristic of softwood lignin, absorbs near 1266-1270 cm-1 and 1230 cm-1 (Faix 1991; Pandey1999), the syringyl type of lignin, which is characteristic for hardwood lignin, absorbs only at 1230 cm-1 (Pandey 1999). Similarly, the band at 1235 cm-1 (11) was seen at L. orientalis heartwood, but the band at 1265 cm-1 representing guaiacyl lignin was not detected in L. orientalis heartwood. Nevertheless, hardwood lignin contains guaiacyl moieties but the strong absorption band at 1235 cm-1(11) might have suppressed the band at 1265 cm-1and prevented its appearance (Pandey and Pitman 2003). The peaks in the absorption band at 1372 cm-1 (9) and 1324 cm-1 (10) are assigned to the C-H deformation in cellulose, the hemicelluloses, and the C-H vibration in cellulose and C1-O vibration in syringyl derivatives, respectively (Pandey and Pitman 2003; Faix 1991). The band near 830 cm-1 (17) observed in L. orientalis heartwood is referred to as syringyl linkage which represents the hardwood lignin (Higgins et al. 1961; Harrington et al. 1964).
Fig. 2. IR spectrum bands of L. orientalis heartwood (control)
Table 5. Assignment of IR Bands
Some of the bands of the spectra in this work slightly differ from literature values. Numbers in parenthesis show the references.
1: Pandey and Pitman 2003; 2: Harrington et al. 1964; 3: Faix 1991; 4: Naumann et al. 2005
Wood Decayed by T. palustris and G. trabeum
The IR spectra of L. orientalis heartwood exposed to either T. palustris or G. trabeum for 12 weeks compared to the control specimen are shown in Fig. 3a. It is obvious that the IR spectrum bands of these two brown rot fungi overlap each other except for the spectrum band at 1106 cm-1 (13) (Fig. 3a). Actually, the band at 1106 cm-1 (13) at the control specimen shifted to 1117 cm-1 and 1110 cm-1 after deterioration by T. palustris and G. trabeum, respectively. The shifting of T. palustris-spectrums was greater than that of G. trabeum compared to the control specimen spectrum (Fig. 3a). According to Harrington et al. (1964), the band near 1110 cm-1 is assigned to an associated OH bending band on the basis of its response to deuteration in cellulose (Higgins et al. 1961; Harrington et al. 1964). It is clear that both fungi modified the cellulose, since the spectrum at 1106 cm-1 (13) showed an important change when compared to the control specimen (Fig. 3a). Since the spectrums of both brown-rot fungi showed a big similarity and the IR spectrum peaks overlapped, except the band at 1106 cm-1 (13), only the spectral peaks of the fingerprint region for T. palustris were evaluated along with the control specimen for the fingerprint region (Fig. 3b). As can be seen in Fig. 3b, the absorption peak at 1734 cm-1 (3) assigned to a carbonyl group in xylan decreased in the specimen degraded by T. palustriswhen compared to the control specimen. Besides the absorption band at 1734 cm-1 (3), a decrease in the absorbance spectrum at 1372 cm-1 (9) for the C-H deformation in cellulose and hemicellulose was observed, causing more intensity in the following band at 1324 cm-1 (10) from lignin. Even though a 1324 cm-1 absorption band was also assigned to the C-H vibration in cellulose, the increase in the intensity of this band indicated that syringyl units were not degraded, and as expected T. palustris degradation in the carbohydrates took place selectively. A decrease in the intensity in the spectrum band at 1648 cm-1 (4) in the specimen exposed to T. palustris was observed, causing an increase in the intensity of the spectrum band at 1593 cm-1 (5). The peak at 1648 cm-1 (4) was assigned to the H-O-H deformation vibration of absorbed water, and also the region of 1650 to 1600 cm-1 could be assigned to conjugated/aromatic carbonyl groups and aromatic rings (Harrington et al. 1964; Nuopponen et al. 2006). Besides the strong absorption band observed at 1235 cm-1 (11) in the control specimen, a shoulder at 1265 cm-1 was also detected; this is typical for guaiacyl lignin in the specimen degraded by T. palustris (Fig. 3b). Even though the test wood is a hardwood specimen; the shoulder observed at 1265 cm-1 in the specimens degraded by the brown rot fungi may be caused by the content of guaiacyl moieties which remain unaffected while the decrease in the intensities of 1235 cm-1 (11) band is due to the degradation of xylan (Pandey and Pitman 2003). The intensity at 1157 cm-1 (12) representing the carbo-hydrates also decreased in the specimens degraded by brown-rot fungi, as can be seen in Fig. 3b. As expected, the intensity of the carbohydrate band at 896 cm-1 (16) was nearly lost when compared to the control specimen. These results were in good agreement with the results by Faix et al. 1991 and Pandey and Pitman 2003.
According to ATR spectroscopy results, after 12 weeks of exposure to the brown rot fungi, a significant decrease in the intensity at 1734 cm-1 (3), 1372 cm-1 (9), 1157 cm-1 (12), and 896 cm-1 (16) from the polysaccharides occurred. Besides the severe decrease in the intensity of polysaccharide bands, an increase in the intensity of absorption bands at 1593 cm-1 (5) and 1504 cm-1 (6) was observed, indicating the presence of lignin. It was clear that both brown rot fungi, T. palustris and G. trabeum, caused a selective degradation in the polysaccharide composition of the wood structure, and this was indicated in a relative increase in the intensities of the lignin bands. Additionally, some of the peaks shifted in the specimen degraded by T. palustris. The band at 1235 cm-1 (11) shifted to 1221 cm-1 and the band at 1106 cm-1 (13) shifted to 1117 cm-1 in the degraded specimen when compared to the control specimen (Fig. 3b). Moreover, while the band at 1106 cm-1 (13) was a shoulder in the control specimen, it became a remarkable peak after the brown rot fungi degradation, as is clearly apparent in Fig. 3b. Another interesting result concerned the band at 1235 cm-1 (11), which shifted and decreased in intensity. This indicated the degradation of xylan and the modification of lignin because this band showed both C-O vibration of acetyl groups in xylan and also the presence of the syringyl type of lignin (Pandey and Pitman 2003). These findings were compatible with the Klason lignin and hot alkali solubility results obtained from the specimen exposed to the brown rot fungi (Table 3).
Fig. 3a. IR spectra of specimen decayed by brown-rot fungi (T. palustris and G. trabeum) and control specimen
Fig. 3b. Comparison of T. palustris and control specimens IR bands in the range of 1800 and 750 cm-1
Wood Decayed by T. versicolor
Figure 4a shows FT-IR spectra of the control specimen and the specimen exposed to T. versicolor. Unlike the brown-rot fungi (T. palustris and G. trabeum), no significant change was observed in the specimens degraded by T. versicolor in the carbonyl absorption peak at 1734 cm-1 (3), which characterizes unconjugated C=O stretching in xylan. Significant decreases were observed in the intensities of the bands at 1593 cm-1 (5) and 1504 cm-1 (6) which originate from conjugated carbonyl groups in lignin (Fig. 4b).
Fig. 4a. IR spectra of specimen decayed by T. versicolor and control specimen
Fig. 4b. IR bands of T. versicolor and control specimens in the range of 1800 to 750 cm-1
A remarkable increase in intensity that was observed at the 1648 cm-1 (4) band, originating from lignin, can be referred to both the decrease in intensity at the 1593 cm-1 (5) band and the structural change in lignin during T. versicolor degradation. These findings were compatible with the results from a study by Faix et al. (1991). According to their results, beech wood degraded by white-rot fungi displayed an increased intensity of the 1646 cm-1 band while the band at 1596 and 1506 cm-1 showed a decreased intensity (Faix et al. 1991). Furthermore, the band at 830 cm-1 (17) from syringyl lignin was nearly absent in the specimen degraded by T. versicolor. The spectra of the degraded specimen by T. versicolor displayed lower absorbance compared to the control specimen in the peak at the wave number 1456 cm-1 (7) related to the polysaccharides. Even though the mass losses were relatively low in the specimen degraded by T. versicolor compared to T. palustris and G. trabeum (Terzi et al. 2012), small changes were observed in polysaccharides’ band intensities compared to T. palustris and G. trabeum. Thus, it is clear that T. versicolor degraded mostly lignin and also a small amount of carbohydrates, while T. palustris and G. trabeum selectively degraded the polysaccharides of the wood. As expected, smaller changes were observed in the polysaccharide band intensities of the decayed specimen by T. versicolor compared to T. palustris and G. trabeum degradation. This result is also compatible with the relatively low mass losses in the specimen (Terzi et al. 2012). Remarkable differences occurred in the degradation of the lignin in the specimen degraded by T. versicolor. According to the ATR spectroscopy results, decreases and modifications in the lignin bands were more significant compared to polysaccharides’ absorption bands.
Anatomical Observations
Microscopic investigations performed on the deteriorated heartwood samples of L. orientalis by white-rot and brown-rot fungi were evaluated separately. Images of the control sample are shown in Fig. 5 for comparisons.
Fig. 5. Light micrographs of control: a) Cross section, b) Radial section, c) Tangential section.
The heartwood is basically reddish brown to brown. The sapwood color is distinct from the heartwood color and it has a distinctive sapwood border. Since the growth rings are marked by a few rows of radially flattened fibers, the growth ring boundaries are distinct. The wood is diffuse-porous and the vessels are mostly solitary, rarely in short (2-3 vessels) radial rows. The shapes of the solitary vessels are slightly angular in their outline. The number of vessels/mm² are 49 to 65. The mean tangential vessel diameter is 66 to 56 µm. The mean vessel element length is 775 µm. Inter-vessel pits are scalariform. Vessel-ray pits with much reduced borders are apparently simple: pits vary from horizontal (scalariform, gash-like) to vertical (palisade). Rays are 1-3(-6)-seriate, and body ray cells are procumbent with mostly 1 to 4 rows of upright and / or square marginal cells. The traumatic axial canals are sometimes present in long tangential lines (Efe 1987; Bozkurt et al. 1989; Wheeler et al.2010).
Anatomical changes in the decayed wood by the white-rot fungus, Trametes versicolor
Figure 6 (in multiple parts) shows samples exposed to fungal decay.
Fig. 6. (T. versicolor attack). a) Fungal attack from the lumina (arrows 1) and from the cell corners of fibers (arrows 2), b) Fungal attack from the cell corners of ray parenchyma (arrows 1), bore holes composed due to the progressing of pit erosion (arrows 2), and the separations in the ray parenchyma cells indicating selective degradation (arrows 3), c) Hyphae boring the cell walls (arrows 1), dentate structure in fiber cell walls indicating irregular degradation (arrows 2), and splits in the fiber cell walls (arrows 3), d) General thinning in the secondary walls of fiber cells (arrows 1), dentate structure in fiber cell walls (arrows 3), and minute cracks in the secondary walls of fiber cells (arrows 2)
Fig. 6. (continued). e) Local degradation resulting in the localized removal of the fiber cell walls and middle lamella (arrows 1), separations between fiber-ray parenchyma cells in less quantity (arrows 2), f) Local degradation resulting in the localized removal of the fiber cell walls and middle lamella (arrows)
After 12 weeks of an incubation period, selective and simultaneous white-rot decay was found in the wood specimens. The attack of T. versicolor in the cell walls was observed both from the lumina and from the cell corners (Fig. 6a and Fig. 6b). Fungal attack from the cell corners was detected particularly in the ray parenchyma cells (Fig. 6b). Hyphae boring the cell walls were also seen (Fig. 6c). This morphological alteration has been attributed in the literature to the interference of extractives (Lee et al. 2009). That type of fungal attack resulted in cell wall thinning from the lumen outwards.
Two types of cell wall thinning were observed in the microscopic sections; one was characterized by the general erosion of the cell walls adjacent to the hyphae growing in the lumen surface (Fig. 6d), and the other one was a local degradation resulting from the localized removal of the cell walls and middle lamella (Fig. 6e and Fig. 6f). General thinning in the fiber cell walls showed a dentate structure, indicating irregular degradation (Fig. 6c and Fig. 6d). Although, the fiber cell wall thinning was extensive in some parts of sections, the middle lamellae and the cell corners were much more resistant to decomposition. Minute cracks in large quantities were detected in the secondary cell walls of the fibers (Fig. 6d). However, splits in the cell walls were rarely seen (Fig. 6c). Erosion of the simple and bordered pits caused round or oval openings (Fig. 6g). Bore holes composed due to the progressing of pit erosion were detected (Fig. 6b and Fig. 6g). In the longitudinal sections, erosion channels appeared with U-shaped notches (Fig. 6h). All of the changes in the anatomical structure of the wood mentioned above were interpreted as simultaneous degradation. It has been reported that the presence of cell wall thinning, minute cracks in the cell walls, rounded or oval pit erosions, bore holes, and erosion channels indicate simultaneous degradation (Wilcox 1968; Liese 1970; Highley and Murmanis 1987; Schwarze 1995; Anagnost 1998; Luna et al. 2004).
Fig. 6 (continued). g) Round or oval openings in pits (arrows 1), bore holes composed due to the progressing of pit erosion (arrows 2), h) Erosion channel with U-shaped notches (arrows), i) Separations in the ray parenchyma cells indicating selective degradation (arrows), j) Lighter color areas in the middle lamella (arrows 1) and in the secondary cell walls (arrows 2) reflect the occurrence of selective delignification.
Some clues supporting selective degradation were also found. Cell separations were evaluated as one of the most important pieces of evidence for selective degradation. Those separations in connection with the degradation of the middle lamella were clear in ray parenchyma cells (Fig. 6b and Fig. 6i). On the other hand, separations were observed in less quantity both between the fiber cells and the fiber-ray parenchyma cells (Fig. 6a
and Fig. 6e). The middle lamella of the wood cells stained with safranin showed lighter areas reflecting the occurrence of selective delignification (Fig. 6j). Those areas were also observed in the secondary walls of the wood cells.
Schwarze (2007) noticed that lignin was first removed from the secondary wall nearest the lumen and then throughout the secondary wall toward the middle lamella in selective degradation. The middle lamella and cell corners were the last areas to be degraded. Schwarze (2007) also reported that parenchyma cells were often preferentially degraded by white-rot fungi causing a selective delignification. Although observations were not performed on different stages of decomposition, the results of the current study can be considered to be compatible with the results of Schwarze (2007).
Obst et al. (1994) studied the degraded sapwood blocks of seven hardwood species including sweetgum (L. styraciflua L.) by T. versicolor. Their results showed that after 6 weeks of decay, the fiber cell walls were degraded preferentially to the cell corners, middle lamellae, vessels, and ray parenchyma cells. Vessels and ray parenchyma cells were rapidly colonized by fungal hyphae but were resistant to decay in a high degree compared to the fiber cells.
Fig. 7. (T. palustris attack). a) Destruction in the wood structure, hyphae on the surface of S3 layer in the lumen side (arrows), b-c) Destruction in the wood structure, d) Hyphae on the surface of S3 layer in the lumen side (arrow)
Since in the current study, the microscopic investigations were not realized in the specimens decayed at weekly intervals, as had been done in most of the previous studies, it is difficult to state which types of wood cells were degraded preferentially. On the other hand, the cell corners and middle lamella of the fiber cells can be considered much more resistant to decay when compared with the ray parenchyma cells.
Highley and Murmanis (1987) reported that T. versicolor severely degraded the middle lamella and the cell corners of sweetgum (L. styraciflua L.) without significant degradation of the adjacent cell walls. Therefore, T. versicolor was considered as a nonselective white-rot fungus in that study. There are also some studies that reported that T. versicolor is a nonselective white rotter, since it degrades carbohydrates and lignin more or less uniformly (Cowling 1961; Liese 1970; Anagnost 1998). Since the degraded middle lamella and cell corners of the ray parenchyma cells showed significant degrada-tion together with the adjacent cell walls in the current study, it was considered selective degradation.
Fig. 7. (continued). e) Cracks in the cell walls (arrows), loose structure in the all cell walls, f) Cracks in the cell walls (arrows), g) Loose structure in the all cell walls
Anatomical changes in decayed wood by the brown-rot fungus, Tyromyces palustris
A great amount of destruction was observed in the wood structure. It is thought that the cell collapse caused destruction because there was a distortion in the plane of wood cells (Fig. 7a,b,c). Extensive degradation in all types of cell walls was also observed. Most of the hyphae were seen on the surface of the S3 layers in the lumen side (Fig. 7a and Fig. 7d). Cracks in the cell walls were detected, whilst no erosion channels and distinct boreholes were observed (Fig. 7e,f). Almost all of the cell walls showed a loose structure (Fig. 7e,g), and they were in an expanded form. There is an assumption about the penetration of cellulose degrading enzymes into the cell walls stating that the enzymes cannot penetrate without any loosening of the cell wall matrix (Cowling and Kirk 1976; Hill and Papadopoulos 2001).
Some studies have been reported that parenchyma cells are resistant to brown-rot decay (Worrall et al. 1997; Schwarze et al. 2000; Schwarze et al. 2003). However, the results of the current study showed almost the same degree of decomposition in all of the types of cell walls.
CONCLUSIONS
- Twenty-six extracts were identified in the ethanol/toluene extraction and 17 extracts were identified from the ethanol extraction. There were 13 main extractives listed in the table (peak area above 1%) for this study. The extractives identified in the heartwood of L. orientalis are known for contributing to biological activity. Individual chemicals found in L. orientalisheartwood extracts have been shown in the literature to have more of an impact on biological activity.
- There was a morphological alteration occurring where the hyphae were boring the cell wall during the white-rot degredation. According to the literature, this observation is due to an interference of the extractives. More investigations are needed to determine whether the extractives play a role in this observation.
- The microscopy examinations demonstrated that T. versicolor primarily degraded lignin and carbohydrates simultaneously, but it was also observed that in some sections the lignin was degraded preferentially. As expected, less changes were observed in the polysaccharide FT-IR band intensities of the decayed specimen by T. versicolor compared to T. palustris and G. trabeum degradation, which was compatible with relatively low mass losses in the specimen.
- ATR spectroscopy examinations also supported that the degree of lignin degradation was higher than hemicellulose degradation, while cellulose showed no deterioration after 12 weeks of T. versicolor degradation.
- A great amount of destruction and extensive degradation in all types of cell walls were observed in the wood structure during the brown rot decay. During the brown rot decay, polysaccharides were extensively depolymerized and removed, and although the lignin may also have been modified, its residue largely remained behind. This was verified by both the Klason lignin determination and FT-IR ATR spectroscopy observations.
- The remarkable increase in the hot alkali solubility value in the specimen exposed to T. palustrisindicated the decomposition and removal of polysaccharides. This was also compatible with the FT-IR ATR spectroscopy results, indicating a significant decrease in the intensities of the bands at 1734 cm-1, 1372 cm-1, 1157 cm-1, and 896 cm-1. It was clear that T. palustris and G. trabeumcaused a remarkable degradation of polysaccharide structure of wood and also caused some modifications in the lignin structure.
ACKNOWLEDGEMENTS
Decayed test specimens were obtained from our previous study supported by the Coordination Unit for Scientific Research Projects, Istanbul University, Turkey (Project No: 4436).
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Article submitted: March 13, 2013; Peer review completed: April 9, 2013; Revised version received and accepted: April 18, 2013; Published: April 23, 2013.