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Rapid strain classication and taxa delimitation within the edible mushroom genus Pleurotus through the use of diffuse reectance infrared Fourier transform (DRIFT) spectroscopy
Georgios I. ZERVAKISa,*, Georgios BEKIARISa, Petros . TARANTILISb, Christos S. PAPPASb
Agricultural University of Athens, Department of Agricultural Biotechnology, Laboratory of General and Agricultural Microbiology, Iera Odos 75, 11855 Athens, Greece b Agricultural University of Athens, Department of Science, Laboratory of Chemistry, Iera Odos 75, 11855 Athens, Greece
a

article info
Article history: Received 23 January 2012 Received in revised form 28 March 2012 Accepted 7 April 2012 Available online 19 April 2012 Corresponding Editor: Kentaro Hosaka Keywords: Filamentous fungi FT-IR spectroscopy Mushroom identication Oyster mushroom Pleurotus taxonomy

abstract
Fourier transform infrared (FT-IR) spectroscopy has been successfully applied for the identication of bacteria and yeasts, but only to a limited extent for discriminating specic groups of lamentous fungi. In the frame of this study, 73 strains e from different associated hosts/substrates and geographic regions e representing 16 taxa of the edible mushroom genus Pleurotus (Basidiomycota, Agaricales) were examined through the use of diffuse reectance infrared Fourier transform (DRIFT) spectroscopy. A binary matrix, elaborated on the basis of presence/absence of specic absorbance peaks combined with cluster analysis, demonstrated that the spectral region 1800e600 cm1 permitted clear delimitation of individual strains into Pleurotus species. In addition, closely related species (e.g., Pleurotus ostreatus and Pleurotus pulmonarius) or taxa of the subgenus Coremiopleurotus demonstrated high similarity in their absorbance patterns, whereas genetically distinct entities such as Pleurotus dryinus, Pleurotus djamor, and Pleurotus eryngii provided spectra with noteworthy differences. When specic regions (1800e1700, 1360e1285, 1125e1068, and 950e650 cm1) were evaluated in respect to the absorbance values demonstrated by individual strains, it was evidenced that this methodology could be eventually exploited for the identication of unknown Pleurotus specimens with a stepwise process and with the aid of a dichotomous key developed for this purpose. Moreover, it was shown that the nature of original fungal material examined (mycelium, basidiomata, and basidiospores) had an effect on the outcome of such analyses, and so did the use of different mycelium growth substrates. In conclusion, application of FT-IR spectroscopy provided a fast, reliable, and cost-efcient solution for the classication of pure cultures from closely related mushroom species. 2012 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction
The genus Pleurotus (Fr.) P. Kumm. (Basidiomycota, Agaricales) comprises species which are widely exploited for bioconverting lignocellulosic byproducts into edible mushrooms of high

nutritional and medicinal value. Commercial mushroom production of Pleurotus spp. corresponds to ca. 30 % of the respective total; the latter annually exceeds 15 million tons and yields a turnover of more than 50 billion US dollars (Chang 2008).

* Corresponding author. Tel.: 30 21052894341; fax: 30 2105294354. E-mail address: zervakis@aua.gr 1878-6146/$ e see front matter 2012 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.funbio.2012.04.006

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Precise identication of wild mushroom isolates/taxa and elucidation of their relationships are essential prerequisites for the development of biotechnological applications through the use of the respective genetic resources. The genus Pleurotus is one of the most taxonomically challenging groups of macrofungi comprising several species and subspecic entities with complex afnities, whose discrimination/delimitation is in many cases problematic (Zervakis 2004). Traditional taxonomic approaches such as morphological studies are often inadequate in distinguishing among closely related Pleurotus spp. (mainly due to the signicant inuence exerted by environmental factors on macroscopic characters). Furthermore, mating compatibility tests are laborious and in some cases inconclusive, while molecular techniques are known to be expensive and require a rather high level of expertise (Vilgalys & Sun 1994; Zervakis & Balis 1996; Petersen & Hughes 2003; Zervakis et al. 2004; Ravash et al. 2010). Vibrational (e.g., Fourier transform infrared e FT-IR) spectroscopy measures bending, contracting, and stretching vibrations of molecules that are excited by an infrared beam. When infrared light interacts with a chemical functional group, the latter tends to adsorb infrared radiation and vibrates by producing bands in well dened regions, which are characteristic for particular classes of compounds. Microorganisms have specic biochemical composition and are thus known to produce unique ngerprint spectra over the midinfrared region (4000e600 cm1). Hence, this technique has been used for studying the molecular composition as well as for identifying biological samples (Naumann et al. 1991; Naumann et al. 1996; Movasaghi et al. 2008). The approach is fast, reagent-free, noninvasive, highly specic, and it requires limited amounts of sample to be examined. An alternative FT-IR spectroscopy method for the analysis of microbial cells using diffuse reectance absorbance (DRIFT) was developed by Goodacre et al. (1996) for acquiring infrared spectra of powders and of materials with rough surfaces. DRIFT offers among others the additional advantages of simpler sample preparation and the capacity to analyze nontransparent materials. FT-IR spectroscopy has been successfully applied in a wide range of studies, including identication/differentiation of various types of biological materials (Pappas et al. 2008; Tarantilis et al. 2008) as well as in the discrimination and metabolic responses of several bacteria and yeast species (Lamprell et al. 2006; Toubas et al. 2007; Kamnev 2008). As regards lamentous fungi in particular, FT-IR spectroscopy was used for examining a few groups of signicant importance, e.g., causes of fungal infections in humans (Erukhimovitch et al. 2005), mycotoxins producing agents  nchez et al. 2008), and plant pathogens (Salman (Galvis-Sa et al. 2010). On the other hand, and with the exception of some wood-rot macrofungi (Naumann 2009), mushroom species were only marginally addressed through FT-IR spectroscopy in investigations principally aiming at determining specic compounds present in basidiomata and basidiospores ek-Gro (Mohac sev et al. 2001; De Gussem et al. 2005). The present work aimed at evaluating the use of DRIFT spectroscopy for taxonomic purposes in the edible mushroom genus Pleurotus, and at assessing its wider applicability for pertinent studies with relevant biological material. Of

particular interest was to evidence its suitability for the delimitation of Pleurotus species by studying mycelium samples through the use of absorbance patterns and cluster analysis. In addition, a dichotomous key was elaborated on the basis of FT-IR data for potential identication and classication of individual Pleurotus specimens. Finally, the effects of different types of fungal material and growth media on the resulting spectra were examined.

Materials and methods

Organisms
In the frame of this study, 73 strains originally assigned to 16 taxa of the genus Pleurotus with a world-wide distribution were evaluated: Pleurotus abalonus Y.H. Han, K.M. Chen, & S. Cheng, Pleurotus abieticola R.H. Petersen & K.W. Hughes, Pleurotus australis Sacc., Pleurotus calyptratus (Lindblad ex Fr.) Sacc., Pleurotus citrinopileatus Singer, Pleurotus columbinus  l., Pleurotus cornucopiae (Paulet) Rolland, Pleurotus cystidioQue sus O.K. Mill., Pleurotus djamor (Rumph. ex Fr.) Boedijn, Pleurol., tus dryinus (Pers.) P. Kumm., Pleurotus eryngii (DC.) Que Pleurotus fuscosquamulosus D.A. Reid & Eicker, Pleurotus nebrol., Pleurotus ostreatus (Jacq.) P. Kumm., Pleudensis (Inzenga) Que l., and Pleurotus sapidus Que let rotus pulmonarius (Fr.) Que (Table 1). Stock cultures of all the strains examined are maintained in the fungal culture collection of the Laboratory of General and Agricultural Microbiology, Agricultural University of Athens (AUA), Athens, Greece.

Preparation of fungal samples

Fungal strains were routinely maintained on potato dextrose agar (PDA, Corda). This medium also served for mycelium growth to be used for the main part of FT-IR measurements. Precultures developing on PDA were used for inoculating the main cultures to be subsequently grown on the same medium in Petri dishes. After 5e10 d (depending on the linear growth rates of individual taxa), and during their active-growth phase, mycelia were harvested by carefully scraping-off the aerial hyphae from the surface of the medium. Then, they were frozen at 80  C, lyophilized, and pulverized in an agate mortar. For assessing the effect that another nutrient substrate could have on the FT-IR spectra, several selected strains from all taxa (Table 1) were grown on a cellulose-based medium (CM) prepared as previously described (Zervakis & Balis 1996). For each one of the two media (PDA and CM), three replicates were used for each strain examined. Furthermore, for studying possible differences in the FT-IR spectra obtained from biological material other than mycelium, i.e., dried basidiomata (maintained as herbarium specimens at AUA, Laboratory of General and Agricultural Microbiology) basidiospores from selected Pleurotus ostreatus strains were also examined (Table 1). For the former type of material, a small quantity (only context, ca. 2 g) was cut off the pileus of the respective specimen. Then it was frozen at 80  C, lyophilized, and pulverized as described above.

DRIFT spectroscopy for Pleurotus strains classication and taxa delimitation

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Table 1 e Details of the 73 strains from 16 Pleurotus taxa studied by FT-IR spectroscopy. Analysis of mycelium grown on PDA was performed for all strains; additional examinations were also conducted for the strains indicated by superscript letters (a,b,c), as specied at the end of the Table. Most of the strains presented here were previously examined through the application of other approaches as well.d a/a
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Taxon
P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. abalonus abalonus abalonus abalonus abalonus abalonus abalonus abieticola australis calyptratus citrinopileatus columbinus cornucopiae cystidiosus cystidiosus cystidiosus djamor djamor dryinus dryinus dryinus dryinus dryinus dryinus eryngii eryngii eryngii eryngii eryngii var. eryngii eryngii var. eryngii eryngii var. eryngii eryngii var. eryngii eryngii var. ferulae eryngii var. ferulae eryngii var. ferulae eryngii var. ferulae eryngii var. ferulae eryngii var. ferulae eryngii var. ferulae eryngii var. ferulae eryngii var. thapsiae eryngii var. thapsiae fuscosquamulosus fuscosquamulosus fuscosquamulosus nebrodensis nebrodensis nebrodensis nebrodensis nebrodensis nebrodensis nebrodensis nebrodensis ostreatus ostreatus ostreatus ostreatus ostreatus ostreatus ostreatus

Geographic origin
Japan Japan Japan China Thailand Philippines Japan Australia exCzechoslovakia Malaysia Italy Iran USA South Africa USA

Collection code no.

LGMACC 39 LGMACC-PO37 ASIK 2 CBS 80391 DSM 5335 FCUP 661 IFO 31074 MUCL44554 D 2245.11 MUCL 28909 MUCL 28684 CBS 37351 S 660 ATCC 28597 ATCC 28598 CFMR 6474 CAS Y55 CAS Y60 CBS 44977 CBS 72483 CBS 80485 LGAM P114 LGAM P157 LGAM P159 ATCC 36047 LGMACC 81 LGMACC 831102 CAS Y607 LGAM P63 LGAM P64 UPA 10 UPA 12 LGAM P66 LGAM P102 LGAM P124 LGAM P125 LGAM P156 LGAM P160 LGAM P169 LGMACC 841043 UPA 5 UPA 27 LGAM P50 LGAM P100 LGAM P164 LGAM P147 LGAM P163 LGAM P177 UPA 8 UPA 28 UPA 32 CAS Y456 CAS Y596 CBS 29147 CCBAS 443 LGAM P67 LGAM P104 LGAM P105 LGAM P112 LGAM P123

Abbreviation used
P.aba L39 P.aba PO37 P.aba A2 P.aba 80391 P.aba 5335 P.aba 661 P.aba 31074 P.abi 44554 P.aus 2245.11a P.cal 28909a P.cit 28684a P.col 37351a P.cor 660 P.cys 28597 P.cys 28598 P.cys 6474a P.dja Y5a P.dja Y60a P.dry 44977 P.dry 71483 P.dry 80485 P.dry P114 P.dry P157 P.dry P159 P.ery 36047 P.ery L81 P.ery 831102 P.ery Y607 P.ery P63a P.ery P64 P.ery U10 P.ery U12 P.ery P66 P.ery P102 P.ery P124 P.ery P125 P.ery P156 P.ery P160 P.ery P169 P.ery 841043 P.ery U5 P.ery U27 P.fus P50a P.fus P100 P.fus P164 P.neb P147 P.neb P163 P.neb P177 P.neb U8 P.neb U28a P.neb U32 P.neb Y456 P.neb Y596 P.ost 29147a P.ost 443 P.ost P67 P.ost P104b,c P.ost P105 P.ost P112b,c P.os P123b,c (continued on next page)

exCzechoslovakia Netherlands Netherlands Greece Greece Greece France France Greece Greece Italy Italy Greece Greece Greece Greece Greece Greece Greece Italy Italy Italy Greece Greece Greece China Greece Greece Italy Italy Italy China China Italy Greece Greece Greece Greece Greece

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Table 1 e (continued ) a/a

61 62 63 64 65 66 67 68 69 70 71 72 73

Taxon
P. P. P. P. P. P. P. P. P. P. P. P. P. ostreatus ostreatus pulmonarius pulmonarius pulmonarius pulmonarius pulmonarius pulmonarius pulmonarius pulmonarius pulmonarius pulmonarius sapidus

Geographic origin
Greece Greece Greece Greece Greece Greece Greece Hungary India Malaysia Hong Kong USA

Collection code no.

LGAM P146 LGAM P149 LGAM P12 LGAM P16 LGAM P47 LGAM P111 LGAM P133 LGMACC 850403 LGMACC 37 MUCL 28683 MUCL 29757 CCBAS 666 ATCC 24986

Abbreviation used
P.ost P146b,c P.ost P149 P.pul P12 P.pul P16 P.pul P47 P.pul P111 P.pul P133 P.pul 850403a P.pul L37a P.pul 28683a P.pul 29757 P.pul 666 P.sap 24986

a Mycelia developed on CM were also included in the analysis. b Basidiospores were also included in the analysis. c Basidiomata were also included in the analysis. d Strains (a/a) 1, 3e7, 15, 16, and 43 were studied by Zervakis et al. (2004); 25e27, 29e33, 41, 42, and 49e51 by Zervakis et al. (2001); 1e7, 9, 14e16, and 43 by Zervakis (1998); 10e12, 19e21, 25e27, 29, 30, 33, 40, 43, 54, 56, 64, 65, 68, 69, and 73 by Zervakis & Balis (1996); 1, 12, 25, 29, 33, 40, 43, 68, 69, and 73 by Irac abal et al. (1995); 1, 12, 14, 19, 21, 25e27, 29, 30, 33, 40, 43, 54, 63, 64, 68, 69, and 73 by Zervakis et al. (1994).

Basidiospores were obtained from spore-prints (maintained on the surface of microscope glass-slides), and they were frozen at 80  C, lyophilized, and pulverized as described above. All samples were examined in triplicate.

Then the baseline was corrected by the automatic baseline correct function (default setting) that automatically corrects the tilted baselines of the selected spectra with the baseline points selected by the software.

Spectroscopic measurements
In the DRIFT spectroscopy apparatus, an infrared beam emitted from a glowing ember source enters the interferometer compartment of the spectrophotometer where spectral encoding takes place, and an interferogram signal is produced. Then the infrared beam is focused onto the surface of the solid sample, diffuse reectance (which penetrates into the sample and then scatters in all directions) is collected and refocused by special reection accessories. Finally, the beam enters the detector for nal measurement, the resulting signal is digitalized and sent back to the computer. There, Fourier transformation takes place and thus an infrared spectrum is produced. For the purposes of this study, 2 mg from each sample in the form of a dry nely-ground powder were placed in a Micro sampling cup (Spectra-Tech Inc., USA). Then, the sample was mounted onto the DRIFT accessory sample-holder of a ThermoScientic Nicolet 6700 FT-IR spectrophotometer (Nicolet Instrument Corp., Madison, WI) equipped with a deuterated triglycine sulfate (DTGS) detector (Nichrome source with a potassium bromide (KBr) beamsplitter). Measurements were recorded in the range of 4000e600 cm1 with an interval of 4 cm1 against a KBr background. The nal spectrum of each sample was obtained by averaging 100 scans. Spectral processing was performed using the OMNIC ver. 7.3 software (Thermo Electron Inc., San Jose, CA). All spectra were smoothed using the automatic smooth function of the software, which uses the SavitskyeGolay algorithm (95-point moving second-degree polynomial). This function (default setting) is automatically smoothing the high-frequency component of the sample data, which is useful for improving the appearance of peaks obscured by noise.

Data analysis
Following smoothing and baseline-correction of obtained spectra, FT-IR spectroscopy data deriving from selected spectral regions for each strain examined were exported as Excel les. The absorption values in these les were further processed through the SPSS Statistics ver. 19 (IBM) software package by applying Ward/Euclidean distance methods. FT-IR spectroscopy measurements and relevant calculations (means and standard deviations) were conducted entirely through the use of the OMNIC software. This software calculates one average spectrum per strain by taking into account the pertinent data deriving from the three replicates of the strain. For each average spectrum produced within the 1800e600 cm1 range, 624 mean absorbance values were calculated, and they were subsequently used for producing the resulting graph. At the same time, OMNIC generated the respective 624 standard deviation values as well. Hence, the study of all 73 Pleurotus strains yielded a total number of 91 104 (624 73 2) mean and standard deviation values, which are available upon request from the corresponding author. For delimiting individual Pleurotus strains into distinct taxa, the existence or not of absorption peaks at specic wavenumbers was monitored and scored as present (1) or absent (0) respectively (they were automatically determined by OMNIC, pertinent sensitivity setting was adjusted at 100 %). The binary matrix thus created was used as input in the SPSS software for the generation of pertinent dendrograms. In the case where all strains were individually examined for assessing the use of the dichotomous key, then their respective spectra absorbance details (after being elaborated through OMNIC) were used for direct input into the SPSS software.

DRIFT spectroscopy for Pleurotus strains classication and taxa delimitation

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Results and discussion

Samples originating from pure cultures of lamentous fungi (i.e., use of mycelium instead of single cells) were only recently examined through FT-IR (Linker & Tsror 2008; Naumann 2009; Salman et al. 2010) since they are more demanding in their analysis requirements. For the purposes of the present study, all DRIFT spectroscopic measurements were repeated twice for ensuring results reproducibility. In addition, for avoiding any possible negative effects of the KBr matrix on the spectroscopic images of polar functional groups involved in H-bonding (e.g., polyester or protein Oe or Ne containing moieties; Kamnev et al. 2008), only pure dried fungal biomass was used in spectroscopy measurements (without mixing it with KBr, which used to be a common practice in FT-IR applications). The majority of the Pleurotus strains/taxa included in the present study were previously examined (Table 1) as regards their taxonomic identity and their intra and intertaxon relationships through a combination of approaches, including morphological, ecophysiological, mating compatibility, biochemical, and molecular studies (Zervakis et al. 1994, 2001, 2004; Irac abal et al. 1995; Zervakis & Balis 1996; Zervakis 1998). For a given spectrum examined through FT-IR, observed peaks signify that a specic compound (or mixture of compounds) presents absorbance in this particular wavelength. The nature of the functional group(s) is identied on the basis of the absorbance presented by known (reference) compounds, which produce identical absorption patterns at the same wavelength. The spectral range 1800e600 cm1 was selected for the delimitation of Pleurotus taxa since it is informative of the fungal cell-wall and cell-membrane associated compounds. This same entire spectrum (or individual spectral regions within this particular range) has been successfully used in several studies involving lamentous fungi (Table 2). The higher wavenumbers region corresponds mainly to the water absorption bands (e.g., 3350 cm1), whereas peaks at 3000e2800 cm1 are attributed to absorbance by fatty acids (Sivakesava et al. 2004); however, these proved to be of no taxonomic signicance for Pleurotus species. Hence, further analysis focused in the absorbance regions which appear in Table 3 together with their respective functional groups and/ ek-Gro or macromolecules (Mohac sev et al. 2001; Sivakesava et al. 2004; Erukhimovitch et al. 2005). Peaks below 1030 cm1 form part of the so-called ngerprint area corresponding mainly to mannans or CeH deformations of a- and b- anomers ek-Gro of glucans (Mohac sev et al. 2001). Indicative average spectra of four strains belonging in different Pleurotus species are presented in Fig 1 (in addition, the standard deviation spectrum is provided for each one of these strains, under Supplementary data). Noteworthy was that there were clear differences in comparisons among spectra obtained from genetically distant species (e.g., Pleurotus dryinus vs. Pleurotus cystidiosus), whereas similar patterns were detected for related taxa (e.g., Pleurotus ostreatus vs. Pleurotus pulmonarius). For assembling FT-IR spectroscopy data from all Pleurotus species examined, cluster analysis with Wards algorithm was employed. Initially, the entire selected region (1800e600 cm1) served for discriminating among individual

taxa. For this purpose, a dendrogram (Fig 2) was constructed on the basis of data deriving from a binary matrix (presence/ absence of absorbance peaks at specic spectra wavelengths, see Table 3), and not by using the entire range of absorbance values. For this clustering process mean absorbance values were calculated for all taxa. Nevertheless, the use of either mean values for strains or of individual values for all replicates did not alter the taxa delimitation efciency of the method (a dendrogram based on individual values of strain replicates is provided under Supplementary data). The use of this particular dendrogram (Fig 2) permitted the separation of all discrete species, and more importantly grouped in the same clusters closely related taxa, (i.e., P. ostreatus, P. pulmonarius, and Pleurotus sapidus, and P. cystidiosus, Pleurotus fuscosquamulosus, and Pleurotus abalonus), conrming thus previous mating compatibility and molecular phylogeny studies (Vilgalys & Sun 1994; Zervakis & Balis 1996; Petersen et al. 1997; Zervakis 1998; Zervakis et al. 2004). In addition, species that are well separated through the application of other taxonomic approaches demonstrated high levels of spectral differences as well, e.g., P. dryinus, Pleurotus abieticola, Pleurotus djamor, and Pleurotus calyptratus (Vilgalys & Sun 1994; Zervakis & Balis 1996; Petersen & Hughes 1997). Of interest was also the relative placement of Pleurotus australis versus the rest of the Coremiopleurotus taxa (i.e., P. abalonus, P. cystidiosus, P. fuscosquamulosus) verifying previous investigations employing mating and molecular methodologies (Zervakis 1998; Zervakis et al. 2004). Furthermore, the distant positioning of Pleurotus eryngii versus Pleurotus nebrodensis is in accordance with the outcome of recent studies (Zervakis et al. 2001; Rodriguez-Estrada et al. 2010), whereas intraspecic taxa of the P. eryngii species-complex were not discriminated by the application of this approach. In general, cluster analysis based on FT-IR spectroscopy data does not necessarily reect hierarchic positioning of taxa and it might not always represent the real taxonomic relationships among them (Naumann 2009). Differences in metabolic products could inuence spectra of closely related taxa and consequently affect the subsequent clustering process. Such observations were previously made for classication down to the genus level for microbial species (Naumann 2000). More particularly, the examination of the individual spectrum of each Pleurotus taxon permitted its own assessment and it facilitated comparative evaluation with other members of the genus. Pleurotus abalonus, P. cystidiosus, and P. fuscosquamulosus strains produced absorbance peaks in 12 out of a total of 19 regions detected for Pleurotus species (Table 3). Of particular importance was the peak noted at 1744 cm1 (esters of phospholipids) and the absence of absorbance at 1055 cm1 (CeO stretching, carbohydrates). In contrast, P. australis did not present peaks at the 1744e1736 cm1 and 802e796 cm1 regions, and this distinctly differentiates this taxon from the previous three with which it shares the common feature of producing asexual synnematoid fructications (Zervakis 1998; Zervakis et al. 2004). Pleurotus columbinus is a taxon with high genetic afnity to P. ostreatus (Irac abal et al. 1995); results of the present study demonstrated that P. columbinus possessed all ten absorbance peaks of P. ostreatus plus an additional one at 1110e1105 cm1.

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Table 2 e Applications of FT-IR spectroscopy in lamentous fungi. Organism(s)

Basidiomycota and Ascomycota (82 spp.) Fusarium graminearum Coniophora puteana, Trametes versicolor and Phanerochaete chrysosporium Lactarius (four spp.) Penicillium, Memnoniella and Fusarium Trametes versicolor and Schizophyllum commune Airborne lamentous fungi Bipolaris sorokiniana

Studys objective(s) e outcome

Identication of various glucan types in sporocarps and identication of fungi to genus level Mycotoxin detection in corn Determination of modications in wood chemistry by wood-decay fungi Analyses of basidiospores content in specic compounds Identication of fungal infections in humans Localization and identication of white-rot fungi in wood Identication and intraspecies characterization Quantitative analysis of total mycotoxins in fungal metabolic extracts Identication of plant pathogenic strains Evaluation of biodegradation of ligninocellulosics Detection of ochratoxin A in dried fruits Determination of biochemical composition of hyphae/spores Discrimination at genus or strain level Identication of fungal strains causing wood decay Characterization of fungal degraded wood Detection and identication of plant pathogens at genus level Fungal degradation of leaf-litter

Spectra used (cm1)

950e750 and 1200e1000 respectively 3300e900 3400e800

Reference
ek-Gro Mohac sev et al. (2001)

Kos et al. (2003) Pandey & Pitman (2003)

1800e200 1500e1300 1800e600 1765e715 (four distinct regions) 3000e1450

De Gussem et al. (2005) Erukhimovitch et al. (2005) Naumann et al. (2005) Fischer et al. (2006) Marder et al. (2006)

Fusarium (ve spp.) Echinodontium taxodii and Trametes versicolor Penicillium, Aspergillus Neurospora, Rhizopus Soil-borne fungi (ve spp.) Basidiomycota and Ascomycota (24 spp.) Chaetomium globosum Rhizoctonia, Colletotrichum, Verticillium and Fusarium Ciliochorella spp. (Ascomycota)

3000e500 1734, 1510, 1378, 1163, 898 1800e600 4000e800 3000e2800 and 1550e900 3700e600 (six distinct regions) 3900e2700, 1800e900 3020e2800, 1780e1680, 1200e950 1800e900

Nie et al. (2007) Zhang et al. (2007)  nchez et al. (2008) Galvis-Sa Jilkine et al. (2008) Linker & Tsror (2008) Naumann (2009) Popescu et al. (2010) Salman et al. (2010) Saparrat et al. (2010)

On the other hand, P. ostreatus, P. pulmonarius, and P. sapidus were three of the ve Pleurotus taxa that had in common ten absorbance regions within the spectrum range examined. All three showed high similarity, which could be regarded as indicative of their high genetic relatedness (Zervakis et al. 1994; Irac abal et al. 1995). Pleurotus nebrodensis is the fourth taxon with the same absorbance pattern; however, it presented minor differences in the exact wavenumber of some peaks. More importantly, it was clearly distinguished from the closely related P. eryngii (Zervakis et al. 2001; Ravash et al. 2010; Rodriguez-Estrada et al. 2010) by lacking two absorbance peaks at 1742 cm1 and 1105 cm1. Pleurotus citrinopileatus (the fth taxon with identical absorbance pattern, again with slight different peaks within the same absorbance regions) and Pleurotus cornucopiae are closely related taxa (Ohira 1990; Zervakis 2004); their FT-IR spectra demonstrated high similarity (ten out of 11 peaks were common), and only a peak at the amide III region exhibited by P. cornucopiae differentiated them. Furthermore, P. djamor FT-IR spectrum yielded 13 absorbance regions, three of which appeared rarely or never in other taxa: 706e702, 1003, and 1110e1105 cm1. For P. abieticola, noteworthy were the distinct peak at 1056 cm1 and the two peaks in the

760e700 cm1 regions. Pleurotus calyptratus presented a peak at the amide III region (1320 cm1), while no peaks were detected at the 1744e1736 cm1 and 1056e1055 cm1 regions. Pleurotus dryinus was one of the best separated species based on the results of this methodology; it exhibited a unique peak at 919 cm1 and another one at 1055 cm1 presented only by P. abieticola too; noteworthy were also the peaks at 763 cm1 and 1736 cm1. At a next stage, Pleurotus strains were individually examined through cluster analysis of their absorbance values for determining whether DRIFT spectroscopy could yield results permitting classication of strains into different species. For this purpose, specic spectra regions were selected for all Pleurotus taxa represented by more than one strain (with the sole exception of P. sapidus since this taxon was considered as closely related to P. pulmonarius). Initially, the 1744e1736 cm1 region was chosen since its use permitted the separation of ten Pleurotus taxa into two large groups according to the production of an absorbance peak at this particular wavelength. Group A included strains of P. dryinus, P. eryngii, P. abalonus, P. cystidiosus, and P. fuscosquamulosus, which were further subdivided into two large clusters. Group

DRIFT spectroscopy for Pleurotus strains classication and taxa delimitation

Table 3 e Absorbance peaks (cmL1) demonstrated by 73 strains of 16 Pleurotus taxa when subjected to DRIFT spectroscopy analysis, and their corresponding functional groups and macromolecules. Taxa Wavenumbers (cm )
706e702 763e756 802e796 856e846 919 939e934 1003 1056e1055 1091e1085 1110e1105 1155e1150 1251e1244 1328e1318 1409e1404 1453e1447 1550e1547 1665e1659 1744e1736 706 760 798 849 934 1056 1090 702 756 850 935 796 854 934 801 848 939 802 849 938 797 849 937 798 850 936 802 849 939 1003 1090 1110 1151 1247 1055 1088 705 763 856 919 801 848 937 798 849 937 798 850 936 800 850 937 800 850 938 800 852 934
1

P.aba

P.abi

P.aus

P.cal

P.cit

P.col

P.cor

P.cys

P.dja

P.dry

P.ery

P.fus

P.neb

P.ost

P.pul

P.sap Interpretationa
Fingerprint area Fingerprint area Fingerprint area Fingerprint area Fingerprint area Fingerprint area Fingerprint area CeO stretching PO2 symmetrical stretching CeC and CeO stretching and CeH deformation CeO stretching PO2 antisymmetrical stretching Protein amide III CeOeH bending Asymmetric CH3 bending in proteins Protein amide II Protein amide I C]O bonds of phospholipids

798 848 938

1089

1090

1089

1086

1091 1110 1152 1246

1086

1086

1090 1105 1152 1246

1087

1089

1087

1087

1087

1152 1244 1322 1407 1453 1550 1663 1744

1155 1246

1152 1244 1326 1408 1452 1548 1664

1152 1248 1328 1408 1450 1550 1665

1151 1248

1151 1245 1318 1404 1451 1548 1664

1151 1245 1321 1407 1453 1548 1662 1741

1152 1251

1152 1244 1321 1407 1453 1549 1662 1743

1152 1246

1151 1246

1150 1248

1151 1246

1408 1452 1547 1659 1736

1406 1452 1550 1665

1409 1448 1550 1663

1407 1450 1550 1665

1408 1453 1547 1664 1736

1409 1451 1548 1658 1742

1408 1451 1548 1661

1407 1450 1549 1661

1407 1448 1549 1663

1407 1448 1549 1663

ek-Gro a According to Erukhimovitch et al. (2005), Mohac sev et al. (2001), and Sivakesava et al. (2004).

721

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G. I. Zervakis et al.

Fig 1 e Indicative FT-IR spectra (1800e600 cmL1 region) obtained from the following four Pleurotus strains (from top to bottom): P. cystidiosus (ATCC 28597), P. pulmonarius (LGMACC 850403), P. ostreatus (CBS 29147), and P. dryinus (CBS 80485). DRIFT analysis was performed with mycelium samples grown on PDA, and mean spectra from three replicates per strain were calculated through the use of the OMNIC ver. 7.3 software. The main functional groups within this spectral region are also illustrated (see also Table 3).

Fig 2 e Dendrogram illustrating the grouping of 16 Pleurotus taxa after Ward linkage analysis and rescaled distance clustering of the pertinent DRIFT spectroscopy data in the 1800e600 cmL1 region. For the clustering process, mean absorbance values were calculated for all taxa through the OMNIC ver. 7.3 software, and the dendrogram was produced on the basis of data deriving from a binary matrix (presence/absence of absorbance peaks at specic spectra wavelengths, see Table 3).

DRIFT spectroscopy for Pleurotus strains classication and taxa delimitation

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Fig 3 e Dendrogram illustrating the separation of P. nebrodensis (Group C) from P. djamor, P. ostreatus, P. pulmonarius, and P. sapidus (Group D) after Ward linkage analysis and rescaled distance clustering of the pertinent DRIFT spectroscopy data in the 1800e600 cmL1 region. For the clustering process, absorbance values from individual strains were directly used for the generation of the dendrogram.

B was composed of strains belonging to P. nebrodensis, P. ostreatus, P. pulmonarius, P. sapidus, and P. djamor. Intergroup heterogeneity values ranged from 12 to 25, while intragroup heterogeneity did not exceed the value 4 for Group A and the value 3 for Group B. From this point, numerical analysis continued for each Group separately. For Group A, examination of the 770e750 cm1 region permitted the clear separation of P. dryinus from the other ve taxa. Then, analysis of the protein amides region (1608e1125 cm1) separated P. eryngii from the Coremiopleurotus taxa; the latter cluster included P. abalonus, P. cystidiosus, and P. fuscosquamulosus, which grouped together as anticipated due to their close phylogenetic relationships (Zervakis et al. 2004). On the other hand, the entire spectrum 1800e600 cm1 was examined for taxa of Group B. In this way, P. nebrodensis strains were positioned into a distinct cluster (Group C) permitting their separation from the other six taxa which formed Group D (Fig 3). At a further step, the 710e695 cm1 region separated P. pulmonarius (incl. P. sapidus, Group E) from the remaining two taxa (Fig 4). Its subsequent exclusion and the

use of the entire ngerprint region (950e650 cm1) led to the grouping of all but one P. ostreatus strains (Group G) into a distinct cluster well separated from P. djamor strains (Fig 5). Alternatively, when the objective is the delimitation of an unknown Pleurotus specimen, an identication process could be elaborated in the form of a dichotomous key, which is primarily based on mean absorbance values of Pleurotus taxa (Table 3) combined where necessary with cluster analyses:

1 Absorbance at the 1744e1736 cm1 region No absorbance at the 1744e1736 cm1 region 2(1) Absorbance at the 763e756 cm1 region No absorbance at the 763e756 cm1 region 3(2) Absorbance at 919 cm1 No absorbance at 919 cm1 4(2) Absorbance at the 1110e1105 cm1 region

2 (Group A) 5 (Group B) 3 4 dryinus abieticola eryngii

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G. I. Zervakis et al.

No absorbance at the 1110e1105 cm1 region 5(1) Absorbance at the 1322e1318 cm1 region No absorbance at the 1322e1318 cm1 region 6(5) Absorbance at the 1328e1324 cm1 region No absorbance at the 1328e1324 cm1 region 7(6) Absorbance at the 763e756 cm1 region No absorbance at the 763e756 cm1 region 8(6) Absorbance at the 1110e1105 cm1 region No absorbance at the 1110e1105 cm1 region 9(8) Absorbance at the 706e702 cm1 region No absorbance at the 706e702 cm1 region 10(8) Absorbance at 1089 cm1 No absorbance at 1087 cm1 11(10) Absorbance at 1665 cm1 No absorbance at 1665 cm1 12(11) Absorbance at 1450 cm1 No absorbance at 1450 cm1

Coremiopleurotus taxa cornucopiae 6 7 8 australis calyptratus 9 10 djamor columbinus nebrodensis 11 citrinopileatus 12 ostreatus pulmonarius, sapidus

It should be noted that the above key is robust for taxa demonstrating distinct absorbance regions/peaks as it is evident from the data presented in Table 3. Classication of Pleurotus strains falling under the last three steps of the key (from

steps 10 to 12) could be exercised with greater difculty since the corresponding taxa presented minute differences in their spectra. However, cluster analysis performed for these particular strains/taxa resulted in very good discrimination of the strains examined (Figs 3e5). In total, accurate identication of Pleurotus strains through cluster analysis exceeded 87 %, and for many taxa this percentage was 100 %. These values resulted by estimating the percentage ratio of the strain(s) number, which was positioned out of the respective taxons cluster (e.g., strain P.pul P133; Fig 4) over the number of strains which were correctly classied. Furthermore, the effectiveness of the dichotomous key was successfully evaluated by examining a different subset of spectra from the one used for its establishment; the latter included strains that were studied in the past through more than one methodologies, see Table 1. Instead, the validation subset consisted of strains which had been initially identied through the application of morphological criteria, i.e., P.aba PO37, P.cys 28597, P.dja Y60, P. dry P114, P.ery Y607, P.fus P164, P.neb P177, P.ost P105, P.pul P111. The biological material used for all types of analyses conducted above was mycelium derived from pure cultures grown on solidied potato dextrose (PDA) laboratory medium. For determining the possible effect that the nature of the growth substrate might have on the spectra derived, another medium (cellulose-based medium) was used for mycelium production from 14 selected Pleurotus strains (Table 1). Results demonstrated high absorbance in the amide III region (1328e1318 cm1) for most strains examined (Fig 6A). This

Fig 4 e Dendrogram illustrating the separation of P. pulmonarius and P. sapidus (Group E) from P. ostreatus and P. djamor (Group F) after Ward linkage analysis and rescaled distance clustering of the pertinent DRIFT spectroscopy data in the 710e695 cmL1 region. For the clustering process, absorbance values from individual strains were directly used for the generation of the dendrogram.

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Fig 5 e Dendrogram illustrating the separation of P. ostreatus (Group G) from P. djamor after Ward linkage analysis and rescaled distance clustering of the pertinent DRIFT spectroscopy data in the 950e650 cmL1 region. For the clustering process, absorbance values from individual strains were directly used for the generation of the dendrogram.

observation was in contrast to what was the case in PDA media, where weak or no absorbance was noted at this particular wavelength. In addition, the use of CM resulted in much weaker or no absorbance at all in the 1744e1736 cm1 and/ or 1110e1105 cm1 regions (Fig 6A). Such differences in absorbance intensities were often accompanied with a marked shift in the peak wavelength, which occurred more often within the ngerprint region. Another issue that was studied was the effect of the type of the biological material examined on the resulting FT-IR spectra. In addition to the mycelium samples, tissue from the pileus (basidioma) of dried mushroom (exsiccata maintained in the herbarium collection of AUA) and basidiospores from four selected P. ostreatus strains were analyzed (Table 1). Of signicant interest was that all basidiospore samples demonstrated high absorbance at the phospholipids, the end eCH3 group of proteins and the amide III regions (1744e1736 cm1, 1453e1447 cm1, and 1328e1314 cm1 respectively) in contrast to the respective mycelium spectra which did not produce pertinent peaks (Fig 6B). As regards the spectra obtained from basidiomata, a double peak was noted at 1207 cm1 (characteristic for this type of material), whereas no peak was observed at the 1453e1447 cm1 region, which was the case in the other two types of materials (Fig 6B). These results are in accordance with a previous report deriving from examination of numerous wild mushroom species stating that the spectra of pileus, stipe, and basidiospores from the same basidioma presented signicant differences, therefore indicating high variability in the chemical composition of ek-Gro mushroom parts (Mohac sev et al. 2001). Despite very limited in number, previous FT-IR spectroscopy studies successfully identied several lamentous fungi

to genus and/or species level. For example, the use of different spectra (3020e2800, 1780e1680, and 1200e950 cm1), which were carefully-chosen and successively used in a stepwise process resulted in the discrimination of the genera Fusarium, Rhizoctonia, Colletotrichum, and Verticillium (Salman et al. 2010). On the other hand, the combined use of four spectra regions (1765e1590 cm1, 1470e1275 cm1, 1170e1000 cm1, and 930e715 cm1) succeeded at delimiting Aspergillus and Penicillium species (Fischer et al. 2006). Similarly, Naumann (2009) employed together six different spectra regions (four of them within the range 1800e600 cm1) to discriminate 26 strains of wood-decaying macrofungi belonging to 24 species. Data obtained from this DRIFT spectroscopy analysis were subsequently used for the construction of reference libraries containing spectra of examined Pleurotus material. As anticipated, the value of such databases increases by the number of evaluated strains per taxon it contains; however, it is of paramount importance to ascertain that the initial battery of specimens used for founding the library is accurately identied through (preferably) a combination of different methodologies (e.g., morphology, mating compatibility where suitable, and molecular analysis). Furthermore, despite the fact that mycelium samples deriving from pure cultures present practical advantages over the use of single-cell material such as basidiospores, they require a carefullyelaborated preparation protocol. The latter is associated with standardization of the nature and composition of the growth medium, cultivation conditions, harvesting, and processing of the mycelium prior to FT-IR spectroscopy. By strictly adhering to it, the much-sought after reproducibility can be achieved, making it possible to correctly identify unknown strains after the comparison of their spectra versus

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Fig 6 e (A). FT-IR spectra in the 1800e600 cmL1 region of the P. eryngii LGAM P63 strain obtained from the analysis of mycelium grown on PDA (solid line) and on CM (dashed line). (B). FT-IR spectra in the 1800e600 cmL1 region of the P. ostreatus LGAM P123 strain obtained from the analysis of mycelium (dashed line), basidiomata (dotted and dashed line), and basidiospores (solid line).

those of reference material maintained in the constructed library.

Conclusion
Application of FT-IR spectroscopy produced characteristic spectra for most of the Pleurotus taxa examined. A binary matrix, elaborated on the basis of presence/absence of such specic peaks, combined with cluster analysis demonstrated that the region 1800e600 cm1 clearly separated

among Pleurotus species. The pertinent results are in accordance with the outcome of previous studies employing several other well-established methodologies for the discrimination among Pleurotus taxa. Alternatively, absorbance values for all Pleurotus strains taken at specic spectrum regions indicated that this approach could be eventually exploited for identication of unknown Pleurotus specimens, either directly (through the use of carefullyelaborated reference libraries) or with the aid of a suitably-developed dichotomous key. Hence, DRIFT spectroscopy provides a solid and reliable tool for screening large

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number of fungal specimens and for conducting fast classication analyses with minimal cost and technical requirements.

Acknowledgements
We would like to thank D.M. Dimou, who kindly provided Pleurotus basidiospore samples and herbarium material. The constructive comments by two anonymous reviewers on the submitted manuscript are greatly appreciated.

Supplementary material
Supplementary data related to this article can be found online at doi:10.1016/j.funbio.2012.04.006.

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