Application of ATR-FTIR Spectroscopy to Compare the Cell Materials of Wood Decay Fungi with Wood Mould Fungi

Wood fungi create vast damage among standing trees and all types of wood materials. The objectives of this study are to (a) characterize the cell materials of two major wood decay fungi (Basidiomycota), namely, Trametes versicolor and Postia placenta, and (b) compare the cell materials of decay fungi with four wood mould fungi (Ascomycota), namely, Aureobasidium pullulans, Alternaria alternata, Cladosporium cladosporioides, andUlocladium atrum. Fourier transform infrared (FTIR) spectroscopy is used to characterize the microbial cellular materials. The results showed that the IR bands for the fatty acid at ∼2900 cm were different for the two-decay-fungi genre. Postia placenta shows more absorbance peaks at the fatty acid region. Band ratio indices for amide I and amide II from protein amino acids were higher for the mould fungi (Ascomycota) than the decay fungi (Basidiomycota). Similarly, the band ratio index calculated for the protein end methyl group was found to be higher for the mould fungi than the decay fungi. Mould fungi along with the decay fungi demonstrated a positive correlation (R = 0.75) between amide I and amide II indices. The three-component multivariate, principal component analysis showed a strong correlation of amide and protein band indices.


Introduction
Wood fungi are broadly classified into two major categories, namely, decay fungi and mould fungi [1,2]. Decay or deterioration is performed predominantly by brown rot and white rot fungi, while staining or discolouration of wood is performed by mould fungi [3,4]. Decay and staining reduce the performance of wood material either structurally or aesthetically. Cell structure of fungi has been widely studied for taxonomy and physiology by using various methods other than spectroscopy [5][6][7][8][9][10][11][12][13]. The fatty acids that are produced in a fungal cell are species specific [14]. Fungal identification libraries of Phanerochaete chrysosporium, P. sordida, P. sanguinea, Trametes versicolor, T. hirsuta, and T. pubescens have been made by extraction and esterification of fungal cellular fatty acids that were characterized by gas chromatography [14]. However, analyzing the chromatographic result, the investigators observed wide variability in the maximum and minimum values. The variation in the amount of deoxyribonucleic acid (DNA) among fungi is often used to characterize species and strains. Attenuated total reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy is a fast surface characterizing tool that can provide chemical information about a substrate up to several micrometres deep. Fungal characterization by FTIR spectroscopy, though not new, is, therefore, an exciting and challenging task as vast information is made available from the microbial structure without the extraction of cell materials [6,[15][16][17]. The questions that originate from spectroscopic studies are as follows. (i) How to represent the spectroscopic findings? (ii) What information should be used for spectral analysis? (iii) How reliable are the spectroscopic data?
In this study, two naturally abundant wood rot fungi, Trametes versicolor and Postia placenta, and four common wood mould fungi, Aureobasidium pullulans, Alternaria alternata, Cladosporium cladosporioides, and Ulocladium atrum, were cultivated. The objectives of this study are to evaluate the applicability of ATR-FTIR spectroscopy to (1) identify cell materials of rot fungi (Basidiomycota); (2) identify cell materials of mould fungi (Ascomycota); (3) quantify spectral information of two rot fungi and four mould fungi.

Fungi
Characterization. Thermo Electron Nicolet 8700 FTIR spectrometer, fitted with a Smart Orbit attenuated total reflectance (ATR) accessory, was used to obtain infrared spectra. Mycelia of each fungal strain from agar plates ( Figure 1) were carefully picked by clean tweezers. Specimens were then placed on top of the diamond crystal of the ATR-FTIR spectroscopy accessory. Mechanical pressure from a rotating knob was applied to the specimens to acquire adequate contact with the diamond crystal. Vibrational spectra were collected at ambient conditions at room temperature and room humidity. Further spectral corrections were not performed. A minimum of three replicates of spectra were obtained from three specimens selected arbitrarily from the agar plates for each fungal species. The mid-IR region of 4000-400 cm −1 was evaluated to characterize the cell materials. Averages of 32 scans were obtained for a single spectrum.
Absorbance (log 10 (1/T)) Wavenumber (cm −1 ) OMNIC software was used for spectral analysis. Spectra with noise-free, sharp, and prominent peaks were selected to interpret the chemical structures, where absorbance ( ) of a pure specimen is governed by Beer-Lambert law as = log 10 (1/ ), with being the transmittance. Sigma Plot software (V. 11, Systat Software, Inc.) was used for plotting of graphs and for performing regression analysis. Scanning electron microscopic (SEM) back scatter electron (BSE) images, 15 kV, were obtained on a carbon black tape background.

Statistical Analysis.
Pearson's correlation coefficient ( ) was used to measure the strength of association between two variables. Coefficients of determination ( 2 ordinary and 2 adjusted for coefficients) were used to indicate the amount of variation in the response variable explained by the independent variable for the linear regression model. Larger 2 indicates more variability which is explained by the linear model. Chi-square distribution was used to ascertain the goodness of fit. Multivariate principal component analysis (PCA) was used to simplify the problem of variable groups by replacing a group of variables with a single new variable. Each principal component represents a linear combination of the original variables. The variance of this variable is the maximum among all possible choices of the axis. MINITAB software version 16.2.1 was used for statistical analysis. Three replicates from specimens of each fungal species were used for FTIR spectra representation and four replicates for each fungal species were used for regression analysis.

Results and Discussion
While fungi culture was performed for decay/rot fungi, the spectral findings of mould fungi were imported from a previous research work [18]. Several common IR bands were initially recognized in the FTIR vibrational spectra. (e)

pullulans
Wood decay fungi Wood mould fungi   The amount of hydroxyl radical from the nutrient substrate contributes significantly to the degradation mechanism by rot fungi [19,20]. Fatty acids show characteristic absorbance at the 2950-2845 cm −1 wavenumber region. The absence of high intensity peaks of fatty acids for Trametes versicolor at the fatty acid region is notable. Absorbances by amide and polysaccharide moieties were observed at wavenumbers ∼ 1650-1050 cm −1 . Amides show two absorption bands, amide I and amide II, at ∼1700-1500 cm −1 due to vibrational coupling between stretching frequencies of C=O ] str and N-H deformation. Amide I has high C=O ] str character from -sheet of protein, while amide II has lesser C=O character and demonstrates interactions between C=O ] str and N-H deformation coupling. Phospholipids and PO 2 show bands at 1237 cm −1 and 1082 cm −1 for asymmetric stretching and symmetric stretching vibrations, respectively [21]. Figure 3 for the two wood decay fungi and the four wood mould fungi shows the presence of aliphatic C-H ] str from esters or triglycerides at 2958 cm −1 , from fatty acids, phospholipids, glycerol, and triglycerides at 2921 cm −1 , 2915 cm −1 , and 2849 cm −1 , and from shoulders at 2872 cm −1 and 2852 cm −1 , respectively. Figure 4 shows that, at lower wavenumbers, mould fungi cell lipids show band at 1456 cm −1 for CH 2 bending mode and band at 1402 cm −1 for C=O symmetric stretching of COO groups as observed in previous research [18]. In general, aromatic and alkene C-H ] str bands appear slightly above ∼3000 cm −1 , while alkane C-H ] str appears below ∼3000 cm −1 . Glucans, chitins, amines, and acids are common fungal cell wall components [14,[22][23][24][25][26][27].

Decay
Fungi. Absorbance at ∼2200-1900 cm −1 is overshadowed by the interference from the diamond crystal absorption and spectral noise. Subsequently, there is no possibility to identify the presence of cumulative double bonds, antisymmetric C=C ] str , at 2000 cm −1 (Figure 4). However, there is a sharp peak at 2323 cm −1 for both decay and mould fungi that could possibly have originated from ketenes, O=C=O. The absence of any strong band at ∼2250 cm −1 eliminates the probability of presence of nitrile group, characterized by C≡N ] str absorption. Trametes versicolor shows higher intensities at the ∼3400 cm −1 region. Bonded amines, N-H ] str , display characteristic absorption at ∼3300 cm −1 region and ≡C-H ] str shows characteristic absorption band at ∼3320 cm −1 . The band at ∼2850 cm −1 is the characteristic absorption band for symmetric stretching of CH 2 . C-H ] str absorbance peaks are more prominent for Postia placenta compared to Trametes versicolor. The replicates of spectra from Trametes versicolor yield broad bands at 2921 cm −1 and 2852 cm −1 . The replicates of spectra from Postia placenta have strong absorption bands at 2958 cm −1 , 2915 cm −1 , 2872 cm −1 , and 2849 cm −1 . The absence of the band at 2872 cm −1 in the spectra of Trametes versicolor indicates that possibly Postia placenta has higher numbers of alkyl and alkane chain numbers in fatty acid configuration.
The fingerprint region for the wood rot decay fungi, Trametes versicolor and Postia placenta, is shown in Figure 4. The spectra for both fungi are largely similar in pattern except some more peaks for Postia placenta. Small shoulder at 1733 cm −1 of the spectrum originated from Trametes versicolor shows the presence of C=O ] str band for uronic acids or from saturated esters. The overtone of C=O ] str band is not distinguishable at 3400 cm −1 . A peak at 1717 cm −1 from both of the fungi and a small shoulder at 1700 cm −1 from Postia placenta suggest possible presence of carbonyl group in conjunction with a double bond or Glu (C=O) or Asp (C=O) amino acids [29]. The possibility of presence of the C=O group is further supported by a strong peak in the Postia placenta spectrum at 1683 cm −1 which could have resulted due to chelation between C=O group and a neighbouring O-H group. The presence of O-H along with C=O ] str is also evidenced by O-H deformation at 932 cm −1 found in the spectrum of both decay and mould fungi. Henceforth, these bands have originated from carboxyl groups present in the cell structures of the fungi. Two shoulders at 1654 cm −1 and 1652 cm −1 are probable peaks from characteristic absorptions of zwitterions -N + -H ] str and -CO 2 − ] str from asymmetric stretching of amino acids, Arg (CN 3 H + 5 ), and acetamides from chitin [28]. A strong absorption at ∼1556 cm −1 is due to amide II from -helix of protein, Glu (COO − ), and/or chitin. The band at 1505 cm −1 shows the presence of o-/msubstituted aromatic units, indole groups of amino acids (CN, CH, and NH), that is, Trp amino acid. The band at 1496 cm −1 is assigned to aromatic (CC) ring, Tyr (O − , CC, CH), or Trp (CC, CH) amino acid [29]. The small band at 1456 cm −1 for asymmetric CH 3 bending modes of end ethyl groups of protein and 1419 cm −1 could be due to absorption from carboxymethyl COO − units [29]. A strong band at 1375 cm −1 is due to chitin. The strong band at 1317 cm −1 can be assigned to amide III from proteins and chitins, 1248 cm −1 for stretching of non-hydrogen-bonded PO 2 group of phosphodiester groups of nucleic acid, 1202 cm −1 for P=O stretch, and 1148 cm −1 for -Glucans. Bands at 1146 cm −1 are due to COC/CC from -Glucan complexes, a shoulder at 1075 cm −1 is due to CO/CC from -Glucan complexes, the band at 1023 cm −1 is for -Glucan complexes or cellulose-OH, the band at 930 cm −1 is for aromatic rings, and finally the bands at 888 cm −1 and 848 cm −1 represent C-1-H of -Glucan and C-1-H of -Glucan units, respectively. Absorbance below 800 cm −1 could be due to the overtones of bands at higher frequencies.
There is an additional peak at 1683 cm −1 for Postia placenta. The observed differences between Trametes versicolor and Postia placenta in Figures 3 and 4 have resulted due to the presence and/or absence of different proportions of hydrocarbons and fatty acids. Presence of the acid groups would have resulted in sharp peaks, while absence would have resulted in no absorbance. Extracellular OH groups are present in Trametes versicolor [30]. Moreover, it is known that Postia placenta secretes oxalic acid into the sheath environment [19,31]. Hence, the sharp peak at 1683 cm −1 in Figure 4 is probably due to the IR detection of carbonyl groups.
Overall, as observed by previous researchers [6,32], it can be concluded that there is strong evidence of presence of chitin, Glucans, and glycoproteins that are covalently linked together in a dynamic process that could occur extracellularly or intercellularly. The probing depth of ATR-FTIR spectroscope was enough to sense bond-vibration signal of these active groups.

Quantitative Spectral Analysis for Six Fungal Species.
Based on the observations in Figures 2, 3, and 4, the ATR-FTIR band ratio indices at 1650 cm −1 , 1546 cm −1 , and 1456 cm −1 wavenumbers in the IR spectral region for the six fungal genre were calculated [18] in the following way: where is the band intensity at the observed wavenumber " " cm −1 and 2921 is the band intensity at wavenumber  2921 cm −1 . The band ratio index calculated at 1456 cm −1 and at 1650 cm −1 for the decay fungi and the mould fungi is not linear as is observed in Figure 5. Observing the pattern for the fungi belonging to Ascomycota and Basidiomycota, it may be concluded that the amide I and amide II indices do not overlap. Figure 5 demonstrates that the band ratio index for the protein ethyl group (1456 cm −1 ) and the amide II index (1546 cm −1 ) are higher for the four genera of wood mould fungi. The amide I index of one wood decay fungus (T. versicolor) is larger than the amide I index of three wood mould fungi, indicating that rot fungi have higher presence of amide I group over the lipids in the cellular structure. Additionally, the results from Figure 5 indicate that the increase in intensities of protein amide bands, amide I and amide II, will not be accompanied by an increase in intensity of the protein end ethyl group, since there is no significant visible relationship. Pearson's correlation coefficient, " ," measuring the degree of linear relationship between two variables obtained from statistical multivariate item analysis yields 0.806 (for indices at 1650 cm −1 and 1546 cm −1 ), 0.158 (for 1650 cm −1 and 1456 cm −1 ), and 0.307 (for 1546 cm −1 and 1456 cm −1 ). Therefore, from the correlation coefficient " ," it may be concluded that the IR band index pairs at higher wavenumbers (for 1650 cm −1 and 1546 cm −1 ) follow a high degree of linear trend, either increasing or decreasing, compared to the IR band index at the lower wavenumbers. Larger values of the band ratio indices are obtained for mould fungi and lower values are obtained for decay fungi from Figure 5. The investigated set of fungi shows presence of higher amount of protein materials for the mould fungi that may have resulted due to higher amount of nucleic acids. The other reason could be the fact that the mould fungi invade wood before the decay fungi attack sets in. Thus, the mould fungi had access to higher nitrogen rich nutrient sources and consequently their metabolic system shows higher presence of nitrogenous material.
In the linear regression model with 24 units of data from cumulative observations of all four replicates for the six fungi, it was observed that the statistical coefficient of determination, 2 = 0.75, for amide I accounts for 75% (adjusted 2 = 0.69) of the total variations in amide II. Henceforth, as seen in Figure 5, it may be concluded that the cell material of decay fungi is not similar to wood mould fungi resulting in 2 < 1.00. Previous research on bacterial cell component has reported similar positive correlation with the normalised intensities of amide II/C-H bands and the N/C elemental surface concentration ratio of the strains determined by X-ray photoelectron spectroscopy (XPS) [33]. The authors conferred that there is higher nitrogen content on the bacteria as compared to yeasts. Additionally, quantification of the peak areas at 1510 cm −1 , 1138 cm −1 , and 1758 cm −1 to follow degradation changes in wood substrate by ATR-FTIR spectroscopy showed increasing amount in lignin/carbohydrate ratio with degradation period [34][35][36]. Table 1 shows the descriptive statistics for each variable considered in Figure 5. Assuming normal distribution, Chisquare test was performed with hypothesized standard deviation = 0.5. From the value [probability ( > test statistic)] in Table 1, it can be concluded with 95% confidence limit that the band index calculated at 1456 / 2921 does not follow a multinomial distribution with specific proportion.
An IR spectrum contains more information than is needed. The entire spectrum is used to obtain absorbance values of a few peaks of interest. However, by using peak indices as variables ( 1650 / 2921 , 1546 / 2921 , and 1456 / 2921 ) in principal component analysis (PCA) technique, redundant data that are not useful can be reduced. A principal component analysis (PCA), assuming normal distribution, shows (   Figure 6 shows the scanning electron microscopic image of Basidiomycota fungal spores on a carbon black tape background. Researches have shown that there could be crystal depositions on the hypha depending on the wood substrate [19]. The small dimensions of the spores and hyphae from Figure 6 suggest that the ATR-FTIR signal from a single beam spot on fungal surface could not be singly due to the hypha; however, it could be from both the hyphae and amino acid rich spores.

Conclusions
Fourier transform infrared (FTIR) spectroscopy is used to compare cellular materials of wood decay and wood mould fungi. Infrared (IR) bands originating from six fungal species showed marked differences. Specifically, it was observed that (1) chitin and fatty acid were present in all six fungi; (2) spectra from Trametes versicolor (Basidiomycota) yield broad bands at 2921 cm −1 and 2852 cm −1 . Contrarily, the spectra from Postia placenta (Basidiomycota) have strong absorption bands at 2958 cm −1 , 2915 cm −1 , 2872 cm −1 , and 2849 cm −1 ; (3) Ascomycota and Basidiomycota amide I and amide II indices (for indices at 1650 cm −1 and 1546 cm −1 ) do not overlap. The calculated amide II index at 1546 cm −1 is less for the two decay (Basidiomycota) fungi than for the four mould (Ascomycota) fungi; (4) multivariate analysis provides Pearson's correlation coefficient, = 0.8, for amide I and amide II (for indices at 1650 cm −1 and 1546 cm −1 ) indicating a positive linear correlation; (5) additionally, 75% of the variation could be explained in the linear relationship between band indices of amide I at 1650 cm −1 and amide II at 1546 cm −1 ( 2 = 0.75); (6) the protein ethyl index at 1456 cm −1 shows a decreasing trend for the four mould fungi Cladosporium cladosporioides > Alternaria alternata > Aureobasidium pullulans > Ulocladium atrum; (7) principal component analysis was found to be useful to define the nature of correlation between the bands indices calculated from fingerprint regions. It was observed that the band index calculated at 1546 / 2921 has the largest percentage of the total data variation.
In conclusion, this study shows that ATR-FTIR spectroscopy provides information about fungal cellular structure that is specific to genus and species. Inter-and intraspecies comparison by FTIR spectroscopy shows the potential to generate future databanks to identify various fungi and pathogens.