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Rapid strain classification and taxa delimitation within the

edible mushroom genus Pleurotus through the use of diffuse

reflectance infrared Fourier transform (DRIFT) spectroscopy

Georgios I. ZERVAKISa,*, Georgios BEKIARISa, Petros Α. TARANTILISb, Christos S. PAPPASb

aAgricultural University of Athens, Department of Agricultural Biotechnology, Laboratory of General and Agricultural Microbiology,

Iera Odos 75, 11855 Athens, GreecebAgricultural University of Athens, Department of Science, Laboratory of Chemistry, Iera Odos 75, 11855 Athens, Greece

a r t i c l e i n f o

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 identification

Oyster mushroom

Pleurotus taxonomy

a b s t r a c t

Fourier transform infrared (FT-IR) spectroscopy has been successfully applied for the iden-

tification of bacteria and yeasts, but only to a limited extent for discriminating specific

groups of filamentous fungi. In the frame of this study, 73 strains e from different associ-

ated hosts/substrates and geographic regions e representing 16 taxa of the edible mush-

room genus Pleurotus (Basidiomycota, Agaricales) were examined through the use of diffuse

reflectance infrared Fourier transform (DRIFT) spectroscopy. A binary matrix, elaborated

on the basis of presence/absence of specific absorbance peaks combined with cluster anal-

ysis, demonstrated that the spectral region 1800e600 cmÀ1 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 demonstratedhigh similarity in their absorbance patterns, whereas genetically distinct entities such

as Pleurotus dryinus, Pleurotus djamor, and Pleurotus eryngii provided spectra with noteworthy

differences. When specific regions (1800e1700, 1360e1285, 1125e1068, and 950e650 cmÀ1)

were evaluated in respect to the absorbance values demonstrated by individual strains, it

was evidenced that this methodology could be eventually exploited for the identification 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 out-

come of such analyses, and so did the use of different mycelium growth substrates. In con-

clusion, application of FT-IR spectroscopy provided a fast, reliable, and cost-efficient

solution for the classification of pure cultures from closely related mushroom species.

ª 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 bioconvert-

ing lignocellulosic byproducts into edible mushrooms of high

nutritional and medicinal value. Commercial mushroom pro-

duction of Pleurotus spp. corresponds to ca. 30 % of the respec-

tive total; the latter annually exceeds 15 million tons and

yields a turnover of more than 50 billion US dollars

(Chang 2008).

* Corresponding author. Tel.: þ30 21052894341; fax: þ30 2105294354.E-mail address: [email protected]

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / f u n b i o

f u n g a l b i o l o g y 1 1 6 ( 2 0 1 2 ) 7 1 5 e7 2 8

1878-6146/$ e see front matter ª 2012 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

doi:10.1016/j.funbio.2012.04.006

mailto:[email protected]

http://www.elsevier.com/locate/funbio

http://dx.doi.org/10.1016/j.funbio.2012.04.006






http://www.elsevier.com/locate/funbio

mailto:[email protected]

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Precise identification 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 Pleuro-

tus is one of the most taxonomically challenging groups of

macrofungi comprising several species and subspecific enti-

ties with complex affinities, whose discrimination/delimita-

tion 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 significant influence

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) spec-

troscopy measures bending, contracting, and stretching vibra-

tions of molecules that are excited by an infrared beam. Wheninfrared light interacts with a chemical functional group, the

latter tends to adsorb infrared radiation and vibrates by pro-

ducing bands in well defined regions, which are characteristic

for particular classes of compounds. Microorganisms have

specific biochemical composition and are thus known to pro-

duce unique ‘fingerprint’ spectra over the midinfrared region

(4000e600 cmÀ1). 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, non-

invasive, highly specific, and it requires limited amounts of

sample to be examined. An alternative FT-IR spectroscopy

method for the analysis of microbial cells using diffuse reflec-tance absorbance (DRIFT) was developed by Goodacre et al.

(1996) for acquiring infrared spectra of powders and of mate-

rials with rough surfaces. DRIFT offers among others the addi-

tional 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 identification/differentiation of

various types of biological materials (Pappas et al. 2008;

Tarantilis et al. 2008) as well as in the discrimination and met-

abolic responses of several bacteria and yeast species

(Lamprell et al. 2006; Toubas et al. 2007; Kamnev 2008). As

regards filamentous fungi in particular, FT-IR spectroscopy

was used for examining a few groups of significant impor-tance, e.g., causes of fungal infections in humans

(Erukhimovitch et al. 2005), mycotoxins producing agents

(Galvis-Sanchez et al. 2008), and plant pathogens (Salman

et al. 2010). On the other hand, and with the exception of

some wood-rot macrofungi (Naumann 2009), mushroom spe-

cies were only marginally addressed through FT-IR spectros-

copy in investigations principally aiming at determining

specific compounds present in basidiomata and basidiospores

(Mohacek-Grosev 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 delim-

itation 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 identification and classification 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

Qu

el., Pleurotus cornucopiae (Paulet) Rolland, Pleurotus cystidio-sus O.K. Mill., Pleurotus djamor (Rumph. ex Fr.) Boedijn, Pleuro-

tus dryinus (Pers.) P. Kumm., Pleurotus eryngii (DC.) Quel.,

Pleurotus fuscosquamulosus D.A. Reid & Eicker, Pleurotus nebro-

densis (Inzenga) Quel., Pleurotus ostreatus (Jacq.) P. Kumm., Pleu-

rotus pulmonarius (Fr.) Quel., and Pleurotus sapidus Quelet

(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 À80 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 strainsfrom all taxa (Table 1) were grown on a cellulose-based me-

dium (CM) prepared as previously described (Zervakis &

Balis 1996).For each one ofthe two media (PDAand 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 myce-

lium, i.e., dried basidiomata (maintained as herbarium speci-

mens 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 À80 C, lyophilized, and pulverized as described above.

716 G. I. Zervakis et al.

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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 onPDA was performed for all strains; additional examinations were also conducted for the strains indicated by superscript letters ( a,b,c ), as specified at the end of the Table. Most of the strains presented here were previously examined through theapplication of other approaches as well.d

a/a Taxon Geographic origin Collection code no. Abbreviation used

1 P. abalonus Japan LGMACC 39 P.aba L39

2 P. abalonus Japan LGMACC-PO37 P.aba PO373 P. abalonus Japan ASIK 2 P.aba A2

4 P. abalonus China CBS 80391 P.aba 80391

5 P. abalonus Thailand DSM 5335 P.aba 5335

6 P. abalonus Philippines FCUP 661 P.aba 661

7 P. abalonus Japan IFO 31074 P.aba 31074

8 P. abieticola MUCL44554 P.abi 44554

9 P. australis Australia D 2245.11 P.aus 2245.11a

10 P. calyptratus exCzechoslovakia MUCL 28909 P.cal 28909a

11 P. citrinopileatus Malaysia MUCL 28684 P.cit 28684a

12 P. columbinus Italy CBS 37351 P.col 37351a

13 P. cornucopiae Iran S 660 P.cor 660

14 P. cystidiosus USA ATCC 28597 P.cys 28597

15 P. cystidiosus South Africa ATCC 28598 P.cys 28598

16 P. cystidiosus USA CFMR 6474 P.cys 6474a

17 P. djamor CAS Y55 P.dja Y5a

18 P. djamor CAS Y60 P.dja Y60a

19 P. dryinus exCzechoslovakia CBS 44977 P.dry 44977

20 P. dryinus Netherlands CBS 72483 P.dry 71483

21 P. dryinus Netherlands CBS 80485 P.dry 80485

22 P. dryinus Greece LGAM P114 P.dry P114



25 P. eryngii ATCC 36047 P.ery 36047

26 P. eryngii France LGMACC 81 P.ery L81

27 P. eryngii France LGMACC 831102 P.ery 831102

28 P. eryngii CAS Y607 P.ery Y607

29 P. eryngii var. eryngii Greece LGAM P63 P.ery P63a

30 P. eryngii var. eryngii Greece LGAM P64 P.ery P64

31 P. eryngii var. eryngii Italy UPA 10 P.ery U10

32 P. eryngii var. eryngii Italy UPA 12 P.ery U1233 P. eryngii var. ferulae Greece LGAM P66 P.ery P66

34 P. eryngii var. ferulae Greece LGAM P102 P.ery P102






40 P. eryngii var. ferulae Italy LGMACC 841043 P.ery 841043

41 P. eryngii var. thapsiae Italy UPA 5 P.ery U5

42 P. eryngii var. thapsiae Italy UPA 27 P.ery U27

43 P. fuscosquamulosus Greece LGAM P50 P.fus P50a

44 P. fuscosquamulosus Greece LGAM P100 P.fus P100

45 P. fuscosquamulosus Greece LGAM P164 P.fus P164

46 P. nebrodensis China LGAM P147 P.neb P147

47 P. nebrodensis Greece LGAM P163 P.neb P16348 P. nebrodensis Greece LGAM P177 P.neb P177

49 P. nebrodensis Italy UPA 8 P.neb U8

50 P. nebrodensis Italy UPA 28 P.neb U28a

51 P. nebrodensis Italy UPA 32 P.neb U32

52 P. nebrodensis China CAS Y456 P.neb Y456

53 P. nebrodensis China CAS Y596 P.neb Y596

54 P. ostreatus Italy CBS 29147 P.ost 29147a

55 P. ostreatus CCBAS 443 P.ost 443

56 P. ostreatus Greece LGAM P67 P.ost P67

57 P. ostreatus Greece LGAM P104 P.ost P104b,c



60 P. ostreatus Greece LGAM P123 P.os P123b,c

(continued on next page)

DRIFT spectroscopy for Pleurotus strains classification and taxa delimitation 717

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Basidiospores were obtained from ‘spore-prints’ (maintained

on the surface of microscope glass-slides), and they were fro-

zen at À80 C, lyophilized, and pulverized as described above.

All samples were examined in triplicate.

Spectroscopic measurements

In the DRIFT spectroscopy apparatus, an infrared beam emit-

ted from a glowing ember source enters the interferometer

compartment of the spectrophotometer where ‘spectral

encoding’ takes place, and an interferogram signal is pro-

duced. Then the infrared beam is focused onto the surfaceof the solid sample, diffuse reflectance (which penetrates

into the sample and then scatters in all directions) is collected

and refocused by special reflection accessories. Finally, the

beam enters the detector for final measurement, the resulting

signal is digitalized and sent back to the computer. There,

Fourier transformation takes place and thus an infrared spec-

trum is produced.

For the purposes of this study, 2 mg from each sample in

the form of a dry finely-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 Ther-

moScientific Nicolet 6700 FT-IR spectrophotometer (Nicolet

Instrument Corp., Madison, WI) equipped with a deuteratedtriglycine sulfate (DTGS) detector (Nichrome source with a po-

tassium bromide (KBr) beamsplitter).

Measurements were recorded in the range of

4000e600 cmÀ1 with an interval of 4 cmÀ1 against a KBr back-

ground. The final 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 algo-

rithm (95-point moving second-degree polynomial). This

function (default setting) is automatically smoothing the

high-frequency component of the sample data, which is use-

ful for improving the appearance of peaks obscured by noise.

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.

Data analysis

Following smoothing and baseline-correction of obtained

spectra, FT-IR spectroscopy data deriving from selected spec-

tral regions for each strain examined were exported as Excel

files. The absorption values in these files were further pro-

cessed through the SPSS Statistics ver. 19 (IBM) software pack-age by applying Ward/Euclidean distance methods.

FT-IR spectroscopy measurements and relevant calcula-

tions (means and standard deviations) were conducted en-

tirely through the use of the OMNIC software. This software

calculates one average spectrum per strain by taking into ac-

count the pertinent data deriving from the three replicates

of the strain. For each average spectrum produced within

the 1800e600 cmÀ1 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

91104 (¼624 Â 73 Â 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 specific wave-

numbers was monitored and scored as present (1) or absent (0)

respectively (they were automatically determined by OMNIC,

pertinent sensitivity setting wasadjusted at 100 %). The binary

matrix thus createdwas used as input in theSPSSsoftware 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 absor-

bance details (after being elaborated through OMNIC) were

used for direct input into the SPSS software.

Table 1 e ( continued )

a/a Taxon Geographic origin Collection code no. Abbreviation used



63 P. pulmonarius Greece LGAM P12 P.pul P12


65 P. pulmonarius Greece LGAM P47 P.pul P4766 P. pulmonarius Greece LGAM P111 P.pul P111


68 P. pulmonarius Hungary LGMACC 850403 P.pul 850403a

69 P. pulmonarius India LGMACC 37 P.pul L37a

70 P. pulmonarius Malaysia MUCL 28683 P.pul 28683a

71 P. pulmonarius Hong Kong MUCL 29757 P.pul 29757

72 P. pulmonarius CCBAS 666 P.pul 666

73 P. sapidus USA ATCC 24986 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 Iracabal 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).


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Results and discussion

Samples originating from pure cultures of filamentous fungi

(i.e., use of mycelium instead of single cells) were only re-

cently examined through FT-IR (Linker & Tsror 2008;

Naumann 2009; Salman et al. 2010) since they are more de-

manding 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 measure-

ments (without mixing it with KBr, which used to be a com-

mon 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 rela-

tionships through a combination of approaches, including morphological, ecophysiological, mating compatibility, bio-

chemical, and molecular studies (Zervakis et al. 1994, 2001,

2004; Iracabal et al. 1995; Zervakis & Balis 1996; Zervakis 1998).

For a given spectrum examined through FT-IR, observed

peaks signify that a specific compound (or mixture of com-

pounds) presents absorbance in this particular wavelength.

The nature of the functional group(s) is identified on the basis

of the absorbance presented by known (reference) com-

pounds, which produce identical absorption patterns at the

same wavelength. The spectral range 1800e600 cmÀ1 was se-

lected for the delimitation of Pleurotus taxa since it is informa-

tive of the fungal cell-wall and cell-membrane associated

compounds. This same entire spectrum (or individual spectralregions within this particular range) has been successfully

used in several studies involving filamentous fungi (Table 2).

The higher wavenumbers region corresponds mainly to the

water absorption bands (e.g., 3350 cmÀ1), whereas peaks at

3000e2800 cmÀ1 are attributed to absorbance by fatty acids

(Sivakesava et al. 2004); however, these proved to be of no tax-

onomic significance for Pleurotus species. Hence, further anal-

ysis focused in the absorbance regions which appear in

Table 3 together with their respective functional groups and/

or macromolecules (Mohacek-Grosev et al. 2001; Sivakesava

et al. 2004; Erukhimovitch et al. 2005). Peaks below 1030 cmÀ1

form part of the so-called fingerprint area corresponding

mainly to mannans or CeH deformations of a-and b- anomersof glucans (Mohacek-Grosev 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 ge-

netically distant species (e.g., Pleurotus dryinus vs. Pleurotus cys-

tidiosus), 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 Ward’s algorithm

was employed. Initially, the entire selected region

(1800e600 cmÀ1) 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 specific 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 repli-

cates did not alter the taxa delimitation efficiency of themethod (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. cysti-

diosus, Pleurotus fuscosquamulosus, and Pleurotus abalonus), con-

firming 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 appli-

cation of other taxonomic approaches demonstrated high

levels of spectral differences as well, e.g., P. dryinus, Pleurotusabieticola, Pleurotus djamor, and Pleurotus calyptratus (Vilgalys

& Sun 1994; Zervakis & Balis 1996; Petersen & Hughes 1997).

Of interest was also the relative placement of Pleurotus aus-

tralis versus the rest of the Coremiopleurotus taxa (i.e., P. abalo-

nus, P. cystidiosus, P. fuscosquamulosus) verifying previous

investigations employing mating and molecular methodolo-

gies (Zervakis 1998; Zervakis et al. 2004). Furthermore, the dis-

tant 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 intraspecific

taxa of the P. eryngii species-complex were not discriminated

by the application of thisapproach. In general, cluster analysis

based on FT-IR spectroscopy data does not necessarily reflecthierarchic positioning of taxa and it might not always repre-

sent the real taxonomic relationships among them

(Naumann 2009). Differences in metabolic products could in-

fluence spectra of closely related taxa and consequently affect

the subsequent clustering process. Such observations were

previously made for classification 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 assess-

ment 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 cmÀ1 (esters of phospholipids) and the absence of ab-

sorbance at 1055 cmÀ1 (CeO stretching, carbohydrates). In

contrast, P. australis did not present peaks at the

1744e1736 cmÀ1 and 802e796 cmÀ1 regions, and this dis-

tinctly differentiates this taxon from the previous three

with which it shares the common feature of producing asex-

ual synnematoid fructifications (Zervakis 1998; Zervakis

et al. 2004). Pleurotus columbinus is a taxon with high genetic

affinity to P. ostreatus (Iracabal 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 cmÀ1.


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On the other hand, P. ostreatus, P. pulmonarius, and P. sapidus

werethree ofthe five Pleurotus taxathat had in common ten ab-

sorbanceregionswithinthespectrumrangeexamined.Allthree

showed highsimilarity, which could be regardedas indicative of

their high genetic relatedness (Zervakis et al. 1994; Iracabal et al.

1995). Pleurotus nebrodensis is thefourth taxon with thesame ab-

sorbance 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 cmÀ1 and

1105 cmÀ1. Pleurotus citrinopileatus (the fifth taxon with identical

absorbance pattern,again with slight different peaks within the

same absorbance regions) and Pleurotus cornucopiae are closely

relatedtaxa (Ohira 1990; Zervakis 2004); their FT-IR spectradem-

onstrated high similarity (ten out of 11 peaks were common),

andonly a peak at theamide IIIregionexhibitedby P. cornucopiae

differentiated them.

Furthermore, P.djamorFT-IRspectrumyielded13absorbance

regions, three of which appeared rarely or never in other taxa:

706e702,1003,and1110e1105cmÀ1.For P. abieticola, noteworthy

were the distinct peak at 1056 cmÀ1 and the two peaks in the

760e700 cmÀ1 regions. Pleurotus calyptratus presented a peak at

the amide III region (1320 cmÀ1), while no peaks were detected

at the 1744e1736 cmÀ1 and 1056e1055 cmÀ1 regions. Pleurotus

dryinus was one of the best separated species based on the

results of this methodology; it exhibited a unique peak at

919cmÀ1andanotheroneat1055cmÀ1presented onlyby P.abie-

ticola too; noteworthy were also the peaks at 763 cmÀ1 and

1736 cmÀ1.

At a next stage, Pleurotus strains were individually exam-ined through cluster analysis of their absorbance values for

determining whether DRIFT spectroscopy could yield results

permitting classification of strains into different species. For

this purpose, specific spectra regions were selected for all Pleu-

rotus 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 cmÀ1 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 par-

ticular 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

Table 2 e Applications of FT-IR spectroscopy in filamentous fungi.

Organism(s) Study’s objective(s) e outcome Spectra used (cmÀ1) Reference

Basidiomycota and

Ascomycota (82 spp.)

Identification of various glucan

types in sporocarps and

identification of fungi to genus level

950e750 and 1200e1000

respectively

Mohacek-Grosev et al. (2001)

Fusarium graminearum Mycotoxin detection in corn 3300e900 Kos et al. (2003)

Coniophora puteana,Trametes versicolor

and Phanerochaete

chrysosporium

Determination of modifications in wood

chemistry by wood-decay fungi

3400e

800 Pandey & Pitman (2003)

Lactarius (four spp.) Analyses of basidiospores

content in specific compounds

1800e200 De Gussem et al. (2005)

Penicillium, Memnoniella

and Fusarium

Identification of fungal

infections in humans

1500e1300 Erukhimovitch et al. (2005)

Trametes versicolor and

Schizophyllum commune

Localization and identification

of white-rot fungi in wood

1800e600 Naumann et al. (2005)

Airborne filamentous fungi Identification and intraspecies

characterization

1765e715 (four distinct regions) Fischer et al. (2006)

Bipolaris sorokiniana Quantitative analysis of total

mycotoxins in

fungal metabolic extracts

3000e1450 Marder et al. (2006)

Fusarium (five spp.) Identification of plantpathogenic strains

3000e

500 Nie et al. (2007)

Echinodontium taxodii

and Trametes versicolor

Evaluation of biodegradation

of ligninocellulosics

1734, 1510, 1378, 1163, 898 Zhang et al. (2007)

Penicillium, Aspergillus Detection of ochratoxin

A in dried fruits

1800e600 Galvis-Sanchez et al. (2008)

Neurospora, Rhizopus Determination of biochemical

composition of hyphae/spores

4000e800 Jilkine et al. (2008)

Soil-borne fungi (five spp.) Discrimination at genus

or strain level

3000e2800 and 1550e900 Linker & Tsror (2008)

Basidiomycota and

Ascomycota (24 spp.)

Identification of fungal

strains causing wood decay

3700e600 (six distinct regions) Naumann (2009)

Chaetomium globosum Characterization of

fungal degraded wood

3900e2700, 1800e900 Popescu et al. (2010)

Rhizoctonia, Colletotrichum,

Verticillium and Fusarium

Detection and identification of

plant pathogens at genus level

3020e2800, 1780e1680, 1200e950 Salman et al. (2010)

Ciliochorella spp. (Ascomycota) Fungal degradation of leaf-litter 1800e900 Saparrat et al. (2010)


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Table 3 eAbsorbance peaks (cmL1 ) demonstrated by 73 strains of 16 Pleurotus taxa when subjected to DRIFT spectroscopy analysisgroups and macromolecules.

Taxa 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.p

Wavenumbers (cmÀ1)

706e702 706 702 705

763e756 760 756 763

802e796 798 798 796 801 802 797 798 802 801 798 798 800 8

856e846 848 849 850 854 848 849 849 850 849 856 848 849 850 850 8

919 919

939e934 938 934 935 934 939 938 937 936 939 937 937 936 937 9

1003 1003

1056e1055 1056 1055

1091e

1085 1089 1090 1090 1089 1086 1091 1086 1086 1090 1088 1090 1087 1089 1087 101110e1105 1110 1110 1105

1155e1150 1152 1155 1152 1152 1151 1152 1151 1151 1151 1152 1152 1152 1152 1151 11

1251e1244 1244 1246 1244 1248 1248 1246 1245 1245 1247 1251 1246 1244 1246 1246 12

1328e1318 1322 1326 1328 1318 1321 1321

1409e1404 1407 1408 1408 1408 1406 1409 1404 1407 1407 1408 1409 1407 1408 1407 14

1453e1447 1453 1452 1452 1450 1452 1448 1451 1453 1450 1453 1451 1453 1451 1450 14

1550e1547 1550 1547 1548 1550 1550 1550 1548 1548 1550 1547 1548 1549 1548 1549 15

1665e1659 1663 1659 1664 1665 1665 1663 1664 1662 1665 1664 1658 1662 1661 1661 16

1744e1736 1744 1736 1741 1736 1742 1743

a According to Erukhimovitch et al. (2005), Mohacek-Grosev et al. (2001), and Sivakesava et al. (2004).

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Fig 1 e Indicative FT-IR spectra (1800e600 cmL1 region) obtained from the following four Pleurotus strains (from top to bot-

tom): 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 2eDendrogram 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 specific spectra wavelengths, see Table 3 ).


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B wascomposed of strains belonging to P. nebrodensis, P. ostrea-

tus, P. pulmonarius, P. sapidus, and P. djamor. Intergroup hetero-

geneity 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 cmÀ1

region permitted the clear separation of P. dry-inus from theother five taxa. Then, analysis of theprotein am-

ides region (1608e1125 cmÀ1) 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, theentire spectrum 1800e600 cmÀ1 was

examined for taxa of Group B. In this way, P. nebrodensis

strains were positioned into a distinct cluster (Group C) per-

mitting their separation from the other six taxa which formed

Group D (Fig3).At a further step, the 710e695 cmÀ1 region sep-

arated P. pulmonarius (incl. P. sapidus, Group E) from the

remaining two taxa (Fig 4). Its subsequent exclusion and the

use of the entire fingerprint region (950e650 cmÀ1) led to the

grouping of all but one P. ostreatus strains (Group G) into a dis-

tinct cluster well separated from P. djamor strains (Fig 5).

Alternatively, when the objective is the delimitation of an

unknown Pleurotus specimen, an identification process could

be elaborated in the form of a dichotomous key, which is pri-

marily based on mean absorbance values of Pleurotus taxa

(Table 3) combined where necessary with cluster analyses:

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.

1 Absorbance at the 1744e1736 cmÀ1

region

2 (Group A)

No absorbance at the 1744e1736 cmÀ1

region

5 (Group B)

2(1) Absorbance at the 763e756 cmÀ1

region

3

No absorbance at the 763e756cmÀ1 region 4

3(2) Absorbance at 919 cmÀ1 dryinus

No absorbance at 919 cmÀ1 abieticola


region

eryngii


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It should be noted that the above key is robust for taxa

demonstrating distinct absorbance regions/peaks as it is evi-

dent from the data presented in Table 3. Classification of Pleu-

rotus strains falling under the last three steps of the key (from

steps 10 to 12) could be exercised with greater difficulty since

the corresponding taxa presented minute differences in their

spectra. However, cluster analysis performed for these partic-

ular strains/taxa resulted in very good discrimination of the

strains examined (Figs 3e5). In total, accurate identification

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 taxon’s

cluster(e.g., strain P.pul P133; Fig 4) over thenumber of strains

which were correctly classified.

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 identified

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 con-ducted above was mycelium derived from pure cultures

grown on solidified 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 cmÀ1) for most strains examined (Fig 6A). This


region

Coremiopleurotus taxa


region

cornucopiae


region

6


region

7


region

8


region

australis

No absorbance at the 763e756 cmÀ1 region calyptratus


region

9


region

10


region

djamor

No absorbance at the 706e702 cmÀ1 region columbinus

10(8) Absorbance at 1089 cmÀ1 nebrodensis

No absorbance at 1087 cmÀ1 11

11(10) Absorbance at 1665 cm

À1

citrinopileatusNo absorbance at 1665 cmÀ1 12

12(11) Absorbance at 1450 cmÀ1 ostreatus

No absorbance at 1450 cmÀ1 pulmonarius, sapidus

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.


7/27/2019 hongos IR


observation was in contrast to what was the case in PDA me-

dia, 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 cmÀ1 and/

or 1110e1105 cmÀ1 regions (Fig 6A). Such differences in absor-

bance intensities were often accompaniedwith a marked shiftin the peak wavelength, which occurred more often within the

fingerprint region.

Anotherissue that was studied was theeffect of the type of

the biological material examined on the resulting FT-IR spec-

tra. In addition to the mycelium samples, tissue from the pi-

leus (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

significant interest was that all basidiospore samples demon-

strated high absorbance at the phospholipids, the end eCH3

group of proteins and the amide III regions (1744e1736 cmÀ1,

1453e1447 cmÀ1, and 1328e1314 cmÀ1 respectively) in con-

trast to the respective mycelium spectra which did not pro-duce pertinent peaks (Fig 6B). As regards the spectra

obtained from basidiomata, a double peak was noted at

1207 cmÀ1 (characteristic for this type of material), whereas

no peak was observed at the 1453e1447 cmÀ1 region, which

was thecase in the other two typesof materials (Fig6B). 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 significant differences, therefore

indicating high variability in the chemical composition of

mushroom parts (Mohacek-Grosev et al. 2001).

Despite very limited in number, previous FT-IR spectros-

copy studies successfully identified several filamentous fungi

to genus and/or species level. For example, the use of different

spectra (3020e2800, 1780e1680, and 1200e950 cmÀ1), 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 cmÀ1, 1470e1275 cmÀ1, 1170e1000 cmÀ1, and

930e715 cmÀ1) succeeded at delimiting Aspergillus and Penicil-

lium species (Fischer et al. 2006). Similarly, Naumann (2009)

employed together six different spectra regions (four of

them within the range 1800e600 cmÀ1) 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 antici-

pated, 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 iden-tified through (preferably) a combination of different meth-

odologies (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 mate-

rial such as basidiospores, they require a carefully-

elaborated preparation protocol. The latter is associated

with standardization of the nature and composition of the

growth medium, cultivation conditions, harvesting, and pro-

cessing 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 un-

known strains after the comparison of their spectra versus

Fig 5 e Dendrogram illustrating the separation of P. ostreatus (Group G) from P. djamor after Ward linkage analysis and re-

scaled 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.


7/27/2019 hongos IR


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 specific peaks, combined with cluster analysis dem-

onstrated that the region 1800e600 cmÀ1 clearly separated

among Pleurotus species. The pertinent results are in accor-

dance with the outcome of previous studies employing sev-

eral other well-established methodologies for the

discrimination among Pleurotus taxa. Alternatively, absor-

bance values for all Pleurotus strains taken at specific spec-

trum regions indicated that this approach could be

eventually exploited for identification of unknown Pleurotus

specimens, either directly (through the use of carefully-

elaborated reference libraries) or with the aid of a suit-

ably-developed dichotomous key. Hence, DRIFT spectros-

copy provides a solid and reliable tool for screening large

Fig 6 e (A). FT-IR spectra in the 1800e600 cmL1 region of the P. eryngii LGAM P63 strain obtained from the analysis of my-

celium 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 basid-

iospores (solid line).


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number of fungal specimens and for conducting fast classi-

fication analyses with minimal cost and technical

requirements.

Acknowledgements

We would like to thank D.M. Dimou, who kindlyprovided Pleu-rotus basidiospore samples and herbarium material. The con-

structive 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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