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Composition of extracted fungal cell walls as indicated by infrared spectroscopy
ARCHIVES
OF
BIOCHEMISTRY
Composition
AND
BIOPHYSICS
of Extracted Infrared A. J. MICHELL
C.S.I.R.O.,
Division
120, 628-637 (1967)
Fungal
Cell Walls
by
Spectroscopy G. SCURFIELD
AND
of Forest Products, Received
as Indicated
October
South Melbourne,
Australia
29, 1966
Materials have been removed from the hyphal walls of Polyporus myZZitae Cke. et Mass. by various chemical treatments. Infrared spectroscopv has been used in an attempt to determine the chemical composition of the residues. a-Chitin was shown to be the principal constituent possibly accompanied by fl-1,3-glucan. There was evidence also of the presence of carboxyl groups. Hyphae of a number of species of fungi from diverse taxonomic groups were extracted successively with potassium hydroxide and glacial acetic acid-hydrogen peroxide, and infrared spectra of the residues were determined. The fungi were divided into two groups on the basis of their spectra: Group I included species having cellulose and Group II those having a-chitin as a significant constituent. The residues of Epicoccum species possibly contained both cellulose and chitin. MATERIALS
Recent techniques for studying the cell wall composition of fungi have included X-ray diffraction to detect partly crystalline materials such as cellulose and chitin (l-5) and chromatographic analysis of hydrolyzates of mechanically isolated cell walls (5-8). Such hydrolyzates have been found to contain glucose, galactose, mannose, ribose, xylose, fucose, glucosamine, and galactosamine. Aronson (9), in a recent review, lists data previously obtained by the use of these and other methods. Infrared spectroscopy has been little used for studying the composition of fungal cell walls, although its value in polysaccharide studies has been demonstrated (10-13). In the present work residues obtained during a previous investigation (14) into the fine structure of the walls of Polyporus myllitae Cke. et Mass. were examined spectroscopically. An examination of the hyphal wall residues obtained by subjecting mycelia of various species of fungi to one such treatment was then undertaken with a view to determining the extent to which infrared spectra might have taxonomic value.
AND
METHODS
Aqueous suspensions of extracted hyphae were dried and the dry material was incorporated into potassium chloride discs (11). Infrared spectra were recorded on a Grubb-Parsons double-beam 54 spectrometer equipped with silica and sodium chloride prisms. Polyporus myllitae Cke. et Mass. (DFP 5029) was grown in liquid shake cultures and on solid media. The liquid medium had the following percentage composition: 0.15 KHIPOI + 0.05 MgSO1. 7 H20 + 2.0 dextrose + 0.08 peptone + 0.002 thiamine + tap water. The solid medium was 1.25yo malt agar. Subsequent treatments of the isolated mycelia were based upon those of Aronson and Preston (15), Fuller and Barshad (16), and Sturgeon (17), and have been detailed elsewhere (14). An additional treatment involved extraction of mycelia with boiling methanol for 3 hours and evaporation of the methanol extract in vacua. Inspection of extracted hyphae in the electron microscope indicated that extraction with hot 2yn potassium hydroxide (3 extractions each of W hour) followed by a 1:l (v/v) mixture of glacial acetic acid and 30% of hydrogen peroxide (1 hour at 70”) was adequate for revealing the microfibrillar texture of the cell walls (14). This extraction procedure was applied to mycelia of various species and infrared spectra used as a 628
COMPOSITION
OF EXTRACTED
means for comparing the composition of the residues obtained. The following species of fungi were examined: Mycelia Fungi
sterilia: imperfecti:
Phycomycetes:
* Cenococcum graniforme (Sow.) Fred. et Winge (DFP 10422) Alternaria solani (E. et M.) Sorauer * Diplodia pinea (Desm.) Kickx. (DFP 6096) Epicoccum sp. * HormodendTon resinae Lindau (DFP 10212) Pithomyces chartarum (Berk. et Curt.) M. B. Ellis Phytophthora cinnamomi Rands. Pythium
sp.
Ascomycetes:
Sclerotinia de Bary
Basidiomycetes:
* Armillaria * * *
*
sclerotiorum
(Lib.)
mellea (Vahl ex Fr.) Kummer (DFP 7132) Polyporus sulphureus (Bull.) Fr. (DFP 2400) Polyporus myllitae Cke. et Mass. (DFP 5029) Stereum chuillettii (Pers. ex Fr.) Fr. (DFP 10403) Corticium rolfsii Curzi (DFP 10601)
The Stereum chailettii culture was a Tasmanian isolate of the Sirex noctilio symbiont (18). Species marked (*) were grown on 1.25% malt Pithomyces Phytophthora cinnammi, agar; sclerotiorum were chartarum, and Sclerotinia grown on a standard potato agar + 0.1% Marmite; Alternaria solani, and the species of Pythium and Epicoccum, were grown on both media. In addition, the stipe and pileus of an Amanita species, the fruiting body of Coriolus zonatus (Nees ex Fr.) Quel., and the rhizomorph of Merulius lacrymans (Wulf) Fr., were examined. The species of Amanita and Coriolus were collected fresh in the field, while M. lacrymans rhizomorph was taken from the Division of Forest Products collection of fungi. Samples of 01-and B-chitins prepared by Hackman and Goldberg (19) from crab and cuttlefish, respectively, were kindly supplied by Dr. R. H. Hackman, C.S.I.R.O., Division of Entomology, Canberra; and the sample of yeast glucan by Dr. B. A. Stone, University of Melbourne. Chitosan was prepared from a commercial sample of crab chitin by the method of Fuller and Barshad (16). Cellulose I was isolated from wood, and cellulose II was prepared from this. Spectra of these substances were determined for comparative purposes.
FUNGAL
CELL WALLS
629
RESULTS SPECTRA
Reference spectra. Absorption maxima in the infrared spectra of polysaccharides are ill-defined because of the opportunities for coupling arising from the similar absorption frequencies of the predominant OH and CH groups (13), and of random interactions between the polymer chains (20). However, considerable progress has been made toward establishing band assignments useful for identifying these substances (13). Reference spectra of those likely to be important in any consideration of fungal cell wall composition are given in Fig. 1. The overall resemblance between the spectra is not surprising in view of the presence of pyranose rings in each polysaccharide. The spectra of cellulose I and II (Figs. 1.l and 1.2) can be distinguished most easily by differences near 1430, 1110, and 990 cm-’ (21-23). The most significant difference between the spectrum of yeast glucan (Fig. 1.3) and the spectra of the celluloses is probably the stronger band in the former near 2925 cm-l. The spectrum of glucomannan resembles that of cellulose II except for the presence of additional bands near 870 and 805 cm--‘, while galactans may be characterised by a band near 768 cm-’ (13). Bands near 3265, 3105, 1655, 1620, and 1550 cm-’ have been found (12, 24) to be characteristic of chitin spectra (Figs. 1.4 and 1.5). (Y- and p-chitin spectra, obtained by Hackman and Goldberg (19) using the pressed disc method, differ in the region 3700-2850 cm-‘, the bands in the spectrum of the a-material being much sharper and the band at 2960-l more intense. Present results indicate that, in addition, the band near 1640 cm-l is split in the a-chitin spectrum, and bands in the region 1160-890 cm+ appear to be sharper. The spectrum of chitosan (Fig. 1.6) resembles that of &chitin in the region 370&2850 cm-‘. It differs in other regions, especially in having weaker bands near 1640 and 1550 cm-’ and a new peak at 1590 cm-l. In protein spectra, the most distinctive bands arise from the amide group and occur near 1640 and 1550 cm-l (25). Thus, it is
630
MICHELL
AND
SCURFIELD
WAVE NUMBER (CM-‘) 1500 1250
I
I
I
I
2.75 3.00 3.25 FIG. 1. Infrared
1000
I
I
I
I
I
I
I
6
7
8
9
10
WAVELENGTH
(u )
spectra
difficult to distinguish spectroscopically between chitin and mixtures of proteins with, for example, glucans. However, some of the difficulty can be resolved by comparing spectra obtained before and after the substances have been treated with hot alkali in which chitin is much less soluble than protein. Spectra of the walls of Polyporus myllitae. Mycelia derived from cultures in a liquid medium gave spectra in which the bands were rather broad (Figs. 2.1-2.4). The most significant changes in the spectra resulting from chemical extraction of such mycelia occurred in the bands near 1730 and 1550 cm-‘. The former was removed by treatment with 2 %’ potassium hydroxide and the latter was intensified (cf. Figs. 2.1 and 2.2). On the other hand, treatment with either
of reference
900 I
800 I
7cm I
I
I
I
I
11
12
13
14
!
polysaccharides.
sulfuric acid or acetic acid-hydrogen peroxide increased the intensity of the 1730 cm-’ band and reduced that of the 1550 cm-’ band (Fig. 2.3). When potassium hydroxide was followed by sulfuric acid or glacial acetic acid-hydrogen peroxide or both successively, the band near 1730 cm-l reappeared weakly, but that near 1550 cm---’appeared unchanged (Fig. 2.4). The differences between the spectra in Figs. 2.1-2.4 in respect of the bands near 1730 cm-l and 1550 cm-’ would be explained if the water-washed material contained substances having both dissociated and undissociated carboxyl groups. The .latter absorb near 1730 cm-l, while the absorption of the dissociated groups would be expected to underlie the chitin band at 1550 cm-l. The greatly reduced intensity of the band near 1730 cm-1 in Fig. 2.4 indicated that such
COMPOSITION
3900
I
3400
I
OF EXTRACTED
3mo2950
I
I
2.75 3.00 3.25
FUNGAL
WAVE NUMBER (CM-‘1 1500 1250
I 6
I 7
I
CELL
moo900
I
8 9 WAVELENGTH t,!J )
631
WALLS
alo
700
I
I
I
I
I
10
11
12
13
14
FIG. 2. Infrared spectra of mycelia of Polyporus myllitae, before and after various chemical treatments. Spectra 14 refer to mycelia grown in liquid culture, and spectra 5-7 refer to those grown on a solid medium. Broken lines denote spectra obtained by incorporating increased concentrations of the materials into the discs. 1, Water-washed mycelia (liquid culture); 2, residue after treatment with 2% potassium hydroxide (3 half-hour treatments); 3, residue after treatment with 2% sulfuric acid or glacial acetic acid-30% hydrogen peroxide; 4, residue after treatment with 2% potassium hydroxide followed by glacial acetic acid-3ClR hydrogen peroxide and then by 2% sulfuric acid; 5, water-washed mycelia (solid culture); 6, residue after treatment with 10% trichloroacetic acid; 7, residue after treatment with 2y0 potassium hydroxide followed by glacial acetic acid-30% hydrogen peroxide and 2y0 sulfuric acid.
substances were largely removed by successive alkali and acid treatments. The general sharpening of bands in the overall spectrum, following acid and alkali treatments, indicated that amorphous polysaccharides of low degree of polymerization had been removed. Further evidence for the removal of such substances was provided b,y the reduction in intensity of bands in t,he region 900-800 cm-l, including those near 870 and 805 cm-’ (found in the
spectrum of mannans). These bands probably arose from vibrations associated with the backbones of linked pyranose rings (10). Figures 2.5-2.7 give the spectra, respectively, of water-washed mycelia derived from cultures on a solid medium and of two residues obtained by chemical extraction of these mycelia. Bands in the spectrum (Fig. 2.6) of the residue remaining after treatment with trichloroacetic acid, used by Sturgeon (17) as a solvent for phospho-
632
MICHELL
AND
glycoproteins, were generally sharper than in the spectrum of the water-washed mycelia (Fig. 2.5). The intensity of the band near 1730 cm+ was increased. Bands in the spectra were also sharpened after treatment of the mycelia with ethylene diamine alone, or trichloroacetic acid and then ethylene diamine, or with 5 % potassium hydroxide at 100” C for 12 hours, or successively with 2 % potassium hydroxide, 1:l acetic acidhydrogen peroxide, and 2 % sulfuric acid (Fig. 2.7). There was no discernible effect on the spectra when residues from the 5 % potassium hydroxide treatment were further treated with either acetic acid, or with acid and then cupriethylene diamine. However, in all such spectra the band near 1730 cm+ was absent. Comparison of the spectra in Fig. 2 with the reference spectra in Fig. 1 left no doubt that cu-chitin was a significant wall constituent. Treatment with anhydrous ethylene diamine would be expected to have produced changes in the bands near 1430, 1110, and 990 cm-1 if cellulose had been present. As mycelia before and after treatment with 5 % potassium hydroxide, potassium hydroxide followed by acetic acid, or potassium hydroxide followed by acetic acid and then cupriethylene diamine, gave similar spectra, it was unlikely that chitosan, or fl-1,4-linked polysaccharides, other than chitin, were present in significant amounts. A band at 2925 cm-l in the spectra could not be attributed to chitin. This band indicated another material which was resistant to all chemical treatments. It had a p-linked structure since absorption occurred near 890 cm-l rather than 850 cm-l (10). It may have been a p-1 ,&glucan since it was insoluble in cupriethylene diamine, in which p-1,4-linked polysaccharides dissolve (26). p-1 ,3-Glucans are known to be common constituents of fungal cell walls (9). Some indication of the effects of different cultural methods on wall composition could be obtained by comparing Fig. 2.1 with Fig. 2.5, and Fig. 2.4 with Fig. 2.7. The spectra in Figs. 2.5 and 2.7 were much sharper, especially in the regions 3500-3100 cm-’ and 1200-900 cm-‘, and resembled the spectrum of a-chitin (Fig. 1.4) much
SCURFIELD
more closely than those in Figs. 2.1 and 2.4. This suggested that the cY-chitin in the walls of the fungus grown on the solid medium was more highly crystalline. In addition, the band at 1730 cm-’ was much weaker in the spectrum in Fig. 2.5 compared wit’h that in Fig. 2.1. Possibly, therefore, the walls of the fungus grown on the solid medium were poorer in carboxyl-containing substances. A further point concerned the possible presence of lipids in cell walls. The infrared spectra of mycelia which had been washed with water and those which were extracted with boiling methanol gave almost identical spectra. However, the material obtained by evaporating the methanol extract to dryness gave a spectrum having some bands in common with those found in lipid spectra (27). Spectra of the wall residues of other species. The spectra of Phytophthora cinnamomi and the Pythium sp. (Figs. 3.1 and 3.2) resembled fairly closely the spectrum of cellulose I (Fig. 1.1). Small differences were observed in the region 1160-990 cm-‘, but these could have arisen from differences in crystallinity between fungal and wood cellulose. Extra bands were evident, however, in the spectrum of Pythium sp. at 2925 and 1730 cm-‘, so a little glucan and some carboxyl-containing substances may also have been present. The spectrum of Epicoccum sp. (Fig. 3.3) had several bands in common with those of the chitins, including bands near 1640 and 1550 cm-‘. However, the bands near 3450, 3265, and 3105 cm-’ were unresolved, as was also the band found near 953 cm-’ in the spectrum of cr-chitin. These results suggested that chitin was not the only wall constituent. The presence of a weak peak near 2925 cm-l indicated some /3-1,3-glucan, but the resemblance of the spectrum in the region 116&990 cm-l to those of Phytophthora and Pythium sp. suggested the possible presence of cellulose. The spectra of the remainder of the species examined were generally similar to one another (Fig. 4). The spectra of the following pairs of species were identical, and only those of the
COMPOSITION
3800 I
’
2.75
3400 I
300
3m I
3.25
OF EXTRACTED
FUNGAL
WAVE NUMBER (CM-‘) 1500 1250 1000 I I I
I
6
7 e 9 WAVELENGTH (cc,
10
633
CELL WALLS
800 I
900 I
11
12
700
13
FIG. 3. Infrared spectra of hyphal wall residues of fungi.
first named are reproduced: Polyporus myllitae (Fig. 2.7) and Corticium rolfsii, Pithomyces char&urn and Coriolus zonatus, Alternaria solani and Hormodendron resinae, Armillaria mellea and Stereum chaillettii. All spectra showed distinct bands near 3450, 3265, 2925, 2850, 1640, 1550, 1375, 1310, and 1155 cn-I, except the spectra of Armillaria, Stereum, and Cenoccoccum, in which bands near 3265 and 3105 cm-l were unresolved. Apart from the band at 2925 cm-1 such bands indicated the presence of chitin, almost certainly a-chitin, despite the fact that only in the spectrum of Amanita pileus was the band near 1640 cm-’ split. The distinctness of bands at 2960, 1450, and 1730 cm-’ varied from spectrum to spectrum. Thus the band at 2960 cm+ was detectable in the spectrum of Armillaria and Stereum, but not in that of Alternaria or Hormoden&on. The band at 1450 cm-l, most prominent in the spectrum of Cenococcum, and less prominent in the spectra of Sclerotinia, Diplodia, Alternaria, Hormodendron, Stereum, and Armillaria, was scarcely distinguishable in the spectra of the other species. These two bands could be attributed to C-H stretching and bending vibrations, respectively, but it was uncertain whether they arose from methyl or methylene groups.
The band at 1730 cm-‘, believed to signify the presence of carboxyl groups, was prominent in the spectra of Polyporus sulphureus and Diplodia, less prominent in those of Alternaria, Hormodendrcn, and Amanita, and absent in spectra of the other species. Bands at 953 and 894 cm-l, distinguishable in the spectra of Amanita, Polyporus sulphureus, Pithomyces, Coriolus, and Merulius, were absent from the spectra of Alternaria and Hormodendran, and were replaced by a broad band centred on 940 cm-1 in the spectra of Diplodia, Sclerotinia, Armillaria, Stereum, and Cenococcum. The bands at 800 and 780 cm-l, absent from the spectra of Diplodia, Sclerotinia, Armillaria, Stereum, Cenococcum, and Polyporus sulphureus, and barely distinguishable in the spectra of Pithomyces, Coriolus, Alternaria, Hormodendron, and Amanita, were prominent in the spectrum of Merulius. The spectrum of Merulius was also characterised by a band near 700 cm-l and that, of Cenococcum by the absence of a band near 1420 cm-l. From a diagnostic viewpoint the most promising bands, other than those already assigned, were the bands at 800 and 780 cm-l. Their frequency, and the absence of accompanying sharp higher frequency bands,
MICHELL
634
AND SCURFIELD
.
I
I
2.15
3.00
I
3.25
t
t
6
7
I
I
8 9 WAVELENGTH (/!L)
I
I
I
I
1
10
11
12
13
14
FIG. 4. Infrared spectra of hyphal wall residues of fungi.
suggested that they possibly arose from a polysaccharide linkage. However, they could also have arisen from inorganic constituents. In the spectra of Pithomyces, Coriolus, Merulius, Alternaria, and Hormodendron there was additional broad absorption underlying the chitin bands in the region 1250-1000 cm-‘, and in the spectra of Diplodia, Sclerotinia, Armillaria, Stereum, and Cmococcum walls, there was another broad band near 940 cm-‘. These bands
almost certainly arose from inorganic constituents. The spectra of mycelia of Alternaria, Pythium sp., and Epicoccum sp. grown on malt agar were similar to those grown on potato agar. DISCUSSION
Infrared spectroscopy is comparable with X-ray diffraction (1) as a technique for studying cell wall composition insofar as
COMPOSITION
OF EXTRACTED
both yield results which are more easily interpreted if the wall samples have been subjected t,o prior chemical treatment. They are comparable also in that both require similar amounts of material for an analysis, and both lead to the positive identification of crystalline organic substances, but are less definitive for mixtures and materials of lower crystallinity. The advantage of infrared spectroscopy is that it yields information concerning amorphous materials and the chemical groupings in the components of mixtures. Electron microscopic examination of the wall residues of Polyporus myllitae (14) has shown that the extent to which chemical treatments remove substances from the surfaces of hyphae varies somewhat with the type of t.reatment. Even the treatment with potassium hydroxide followed by glacial acetic acid-hydrogen peroxide is not entirely effective with every sort of fungal wall material. It is thus possible that some of the differences between spectra are due to the differential removal of such substances. This could account, for example, for the indications of the presence of inorganic materials in certain spectra. Frey (1) obtained X-ray diffraction photographs indicative of their presence in the walls or cells of various fungi. But apart from this, little appears to be known of the chemical nature of the materials removed. There is another possibility, namely, that substances of cytoplasmic origin are included in wall residues. The lipid material found in the methanol extract of P. myllitae walls, for example, might have come from the cell cytoplasm even though lipids have been reported to be wall components in other fungi (28, 29). The extent to which the various chemical treatments employed remove cytoplasm from the cells is uncertain, but alkali is believed to do so (16). The differences between the spectra of mycelia grown on solid and in liquid media could be due to the different methods of culture or to differences in the chemical composition of the media. The former possibility seems more likely in view of the near identity of the spectra of residues derived from mycelia grown on malt and on
FUNGAL
CELL
WALLS
635
potato agar. However, the question of the effect of growing conditions on wall composition needs closer examination, especially as the present conclusion is based on only one experiment. It is relevant to note that Bartnicki-Garcia and Nickerson (28) have shown that differences in cultural conditions, albeit more gross than those examined here, produce differences in the chemical composition of the walls of Mucor rouxii. The presence of carboxyl groups in fungal walls is of interest since they may possibly indicate the presence of uranic acids. A small quantity of uranic acid was found in a hot water-soluble fraction of the walls of Saprolegnia littoralis by Parker et al. (5). Crook and Johnston (S), however, failed to detect uranic acids in formic acid hydrolysates of the walls of several species of fungi. It has been pointed out above that the presence of proteins is not precluded, even in walls which, from their spectra, appear to consist of chitin. The possible importance of proteins can be judged from the fact that the walls of Pithomyces chartarum have been reported (17, 29) to contain 25 % protein, 40 % carbohydrate, and 10 % bound glucosamine. However, in view of the fact that hot 1 N potassium hydroxide has been found to dissolve nearly all the protein from the walls of Mucor rouxii (ZSj, it appears likely that the alkali treatment given here to wall residues eliminated any protein present in them. On the basis of the infrared spectra of their extracted hyphae, the fungi examined can evidently be divided into two groups: Group I includes those species having cellulose I, and Group II those having cr-chitin, as a significant wall constituent. The occurrence of cellulose I as a component in the hyphal walls of species of Phytophthora and Pythium confbms the result of Frey (1)) who used X-ray diff raction methods. Crook and Johnston (8), using chemical analysis, detected cellulose in the walls of Phytophthora cactorum. It has also been reported to be a component of the hyphal walls of species of Xaprolegnia, A chl ya, Lagenidium, and Brevilegnia (1, 5, 8), though the amounts present might
636
MICHELL
AND SCURFIELD
not exceed 15% of the unextracted wall weight (5).l On the other hand, chitin and not cellulose has been cited as a component of the walls of species of Chytridium, Rh~zo~h~d~~rn, AlEomyces, 3~~to~l~iella, Rh~z~u~~ ~~~~, and ~h~corn~~~ (1, 15,
28, 30), while the presence of both cellulose and chitin in the walls of Rhizidiomyces has been demonstrated by Fuller and Barshad 06).
Ascomycetes, other than yeasts, can generally be allocate to Group II (9). However, it has been claimed, mainly on the basis of X-ray diffraction data, that cellulose and chitin occur together in the conidiophore walls of Asporgillus (31) and the hyphal walls of Ceralocystis uZmi (32). Basidiomycetes~ without exception, can be allocated to Group II on the basis of the present data and the list given by Aronson (9). Infrared spectra indicate that many Fungi imperfecti can also be included in this group. The conclusion that both chitin and cellulose might be present in the walls of ~pi~o~~rn sp. needs further ~vestigation. Group II is evidently very large. The infrared spectra of the walls of species included in it indicate that it is also heterogeneous. The possibility of subdividing it on the basis of t,he characte~sti~s of such spectra might be worth pursuing with a wider range of speciesgrown under identical conditions. However, present data indicate that qualitative differences in wall composition between species in Group II are likely to be few. An obvious subgroup would indude those species whose walls have been sa.id to contain both chitin and cellulose. X-Ray diffraction and chemical analyses (1, 3, 6) have indicated that a Group III might be added to include those species of 1 hrote Added in Proof: Since this paper was written two new estimations of cellulose I in fungal cell walls have been published. BartnickiGarcia [J. Gen. Mierobiol. 42, 57 (1966)) has reported that, mechanically isolated walls of Phytophthora cinnamomi contain 25y0 cellulose I and 63% other fl-glucans. Aronson, Cooper and Fuller [Science 156, 332 (1967)J report that chemically isolated walls of P~~tophtho~~, A t~~~s~elZa,Achlya, and Pythium spp. contain 7, 31, 11, and 12% of cellulose I, respectiveIy, the rest of the walls being largely p-1,3- and p-1,6-glucans.
yeast shown to have a p-1,3-glucan rather than cellulose or chitin as a major wall component. The general conclusion from the above is that wall composition as deduced from infrared spectra has little taxonomic value at present, and that its phylogenetic value is Iimited to the phycomycetes. However, features in the spectra, such as the intense bands at 800 and 780 cm-’ in the spectrum of the rhizomorph walls of Merulius ZWYmans, do indicate fungi which might have wall compositions of special interest. ACKNOWLEDGMENTS The authors are grateful to Dr. Eileen E. Fisher, Victorian Plant Research Institute, Burnley; and Mr. E. W. B. Da Costa and Mr. N. E. M. Walt,ers, CSIRO, Division of Forest Products, for providing cultures of the fungi; and to Mrs. J. Bennett for laboratory assistance. REFERENCES 1. FREY, R., Ber. Schweiz.
BotGn. @es. 60, 199 (1950). 1, 1 (1954). 2. BLANK, F., Can. J. Microbial. 3. KREQER, D. R., Biochim. Biophys. Acta 13, 1 (1954)I 4. NICOLAI, E., AND PRESTON, R. D., Proc. Roy. Sot. (London), Ser. B 161, 244 (1959). 5. PARKER, B. C., PRESTON, R. D., AND FOGG, G. E., Proc. Roy. See. (London), Ser. B 158, 435 (1963). NORTHCOTE, D. H., AND HORNE, R. W., Biochem. J. 61,232 (1952). CUMMINS, C. S., AND HARRIS, H., J. Gen. MicrobioZ. 18, 173 (1962). CROOK, E. &I., AND JOHNSTON, I. R., Bioc~~. 3. 63,325 (1962). ARONSON, J. M. in “The Fungi” (G. C.
Ainsworth and A. S. Sussman, eds.), p. 49. Academic Press, New York (1965). 10. BARKER, S. A., BOURNE, E. J., STACEY, M., AND WHIFFEN, D. H., 3. C&m. Sot. 171 (1954). J. Chem. 10. 496 11. HIGGINS, H. G., Au&al. (1957)* 12. PEARSON, F. G., MBRCHESIAULT, R. H., AND LIANQ, C. Y., J. Polymer Sci. 43,101 (1960). 13. MARCHESSAULT, R. H., Pure Appl. Chem. 6, 107 (1962). 14. SCURFIEL~, G., .I. Linn. Sot, (Bob.) (Lon~o~). (in press) 15. ARONSON, J. M., AND PRESTON, R. D., J. Biophys. Biochem. Cytol. 6, 247 (1960).
COMPOSITION
OF EXTRACTED
16. FULLER, M. S., AND BARSHAD, I., Am. J. Botany 47, 105 (1960). 17. STURGEON, R. J., Biochem. J. 92,6OP (1964). 18. KINQ, J. M., Austral. J. Botany 14, 25 (1966). 19. HACKMAN, R. H., AND GOLDBERG, M., AustraZ. J. Biol. Sci. 18, 947 (1965). 20. LIANG, C. Y., KRIMM, S. H., AND SUTHERLAND, G.B.B.M., J. Chem. Phys. 26, 543 (1956). 21. LI~NG, C. Y., AND MARCHESSATJLT, R. H., J. Polymer Sci. 37, 385 (1959). 22. LIANG, C. Y., AND MARCHESSAULT, R. H., J. Polymer Sci. 89, 269 (1959). 23. MARCHESS.IULT, R. H., AND LIANG, C. Y., J. Polymer Sci. 43, 71 (1960). 24. RUDALL, K. M., Advan. Insect Physiol. 1, 257 (1963). 25. FRASER, R. D. B., in “A Laboratory Manual of Analytical Methods of Protein Chem-
FUNGAL
26. 27.
28. 29. 30. 31. 32.
CELL
WALLS
637
istry” (P. Alexander and R. J. Block, eds.), Vol. 2, p. 285. Pergamon, Oxford (1960). CLARKE, A. E., AND STONE, B. A., Rev. Pure Appl. Chem. 13, 134 (1963). FREEMAN, N. K., LINDGREN, F. T., Na, Y. C., AND NICHOLS, A. V., J. Biol. Chem. 203, 293 (1953). BARTNICKI-GARCIA, S., AND NICKERSON, W. J., Biochim. Biophys. Acta 68, 102 (1962). RUSSELL, D. W., STURGEON, R. J., AND WARD, V., J. Gen. MicrobioZ. 36, 289 (1964). ARONSON, J. M., AND PRESTON, R. D., Proc. Roy. Sot. (London), Ser. B 162,346 (1960). FARR, W. K., Trans. N.Y. Acad. Sci. 16, 209 (1954). ROSINSKI, M. A., AND CAMPANA, R. J., MycoZogia 66, 738 (1964).
OF
BIOCHEMISTRY
Composition
AND
BIOPHYSICS
of Extracted Infrared A. J. MICHELL
C.S.I.R.O.,
Division
120, 628-637 (1967)
Fungal
Cell Walls
by
Spectroscopy G. SCURFIELD
AND
of Forest Products, Received
as Indicated
October
South Melbourne,
Australia
29, 1966
Materials have been removed from the hyphal walls of Polyporus myZZitae Cke. et Mass. by various chemical treatments. Infrared spectroscopv has been used in an attempt to determine the chemical composition of the residues. a-Chitin was shown to be the principal constituent possibly accompanied by fl-1,3-glucan. There was evidence also of the presence of carboxyl groups. Hyphae of a number of species of fungi from diverse taxonomic groups were extracted successively with potassium hydroxide and glacial acetic acid-hydrogen peroxide, and infrared spectra of the residues were determined. The fungi were divided into two groups on the basis of their spectra: Group I included species having cellulose and Group II those having a-chitin as a significant constituent. The residues of Epicoccum species possibly contained both cellulose and chitin. MATERIALS
Recent techniques for studying the cell wall composition of fungi have included X-ray diffraction to detect partly crystalline materials such as cellulose and chitin (l-5) and chromatographic analysis of hydrolyzates of mechanically isolated cell walls (5-8). Such hydrolyzates have been found to contain glucose, galactose, mannose, ribose, xylose, fucose, glucosamine, and galactosamine. Aronson (9), in a recent review, lists data previously obtained by the use of these and other methods. Infrared spectroscopy has been little used for studying the composition of fungal cell walls, although its value in polysaccharide studies has been demonstrated (10-13). In the present work residues obtained during a previous investigation (14) into the fine structure of the walls of Polyporus myllitae Cke. et Mass. were examined spectroscopically. An examination of the hyphal wall residues obtained by subjecting mycelia of various species of fungi to one such treatment was then undertaken with a view to determining the extent to which infrared spectra might have taxonomic value.
AND
METHODS
Aqueous suspensions of extracted hyphae were dried and the dry material was incorporated into potassium chloride discs (11). Infrared spectra were recorded on a Grubb-Parsons double-beam 54 spectrometer equipped with silica and sodium chloride prisms. Polyporus myllitae Cke. et Mass. (DFP 5029) was grown in liquid shake cultures and on solid media. The liquid medium had the following percentage composition: 0.15 KHIPOI + 0.05 MgSO1. 7 H20 + 2.0 dextrose + 0.08 peptone + 0.002 thiamine + tap water. The solid medium was 1.25yo malt agar. Subsequent treatments of the isolated mycelia were based upon those of Aronson and Preston (15), Fuller and Barshad (16), and Sturgeon (17), and have been detailed elsewhere (14). An additional treatment involved extraction of mycelia with boiling methanol for 3 hours and evaporation of the methanol extract in vacua. Inspection of extracted hyphae in the electron microscope indicated that extraction with hot 2yn potassium hydroxide (3 extractions each of W hour) followed by a 1:l (v/v) mixture of glacial acetic acid and 30% of hydrogen peroxide (1 hour at 70”) was adequate for revealing the microfibrillar texture of the cell walls (14). This extraction procedure was applied to mycelia of various species and infrared spectra used as a 628
COMPOSITION
OF EXTRACTED
means for comparing the composition of the residues obtained. The following species of fungi were examined: Mycelia Fungi
sterilia: imperfecti:
Phycomycetes:
* Cenococcum graniforme (Sow.) Fred. et Winge (DFP 10422) Alternaria solani (E. et M.) Sorauer * Diplodia pinea (Desm.) Kickx. (DFP 6096) Epicoccum sp. * HormodendTon resinae Lindau (DFP 10212) Pithomyces chartarum (Berk. et Curt.) M. B. Ellis Phytophthora cinnamomi Rands. Pythium
sp.
Ascomycetes:
Sclerotinia de Bary
Basidiomycetes:
* Armillaria * * *
*
sclerotiorum
(Lib.)
mellea (Vahl ex Fr.) Kummer (DFP 7132) Polyporus sulphureus (Bull.) Fr. (DFP 2400) Polyporus myllitae Cke. et Mass. (DFP 5029) Stereum chuillettii (Pers. ex Fr.) Fr. (DFP 10403) Corticium rolfsii Curzi (DFP 10601)
The Stereum chailettii culture was a Tasmanian isolate of the Sirex noctilio symbiont (18). Species marked (*) were grown on 1.25% malt Pithomyces Phytophthora cinnammi, agar; sclerotiorum were chartarum, and Sclerotinia grown on a standard potato agar + 0.1% Marmite; Alternaria solani, and the species of Pythium and Epicoccum, were grown on both media. In addition, the stipe and pileus of an Amanita species, the fruiting body of Coriolus zonatus (Nees ex Fr.) Quel., and the rhizomorph of Merulius lacrymans (Wulf) Fr., were examined. The species of Amanita and Coriolus were collected fresh in the field, while M. lacrymans rhizomorph was taken from the Division of Forest Products collection of fungi. Samples of 01-and B-chitins prepared by Hackman and Goldberg (19) from crab and cuttlefish, respectively, were kindly supplied by Dr. R. H. Hackman, C.S.I.R.O., Division of Entomology, Canberra; and the sample of yeast glucan by Dr. B. A. Stone, University of Melbourne. Chitosan was prepared from a commercial sample of crab chitin by the method of Fuller and Barshad (16). Cellulose I was isolated from wood, and cellulose II was prepared from this. Spectra of these substances were determined for comparative purposes.
FUNGAL
CELL WALLS
629
RESULTS SPECTRA
Reference spectra. Absorption maxima in the infrared spectra of polysaccharides are ill-defined because of the opportunities for coupling arising from the similar absorption frequencies of the predominant OH and CH groups (13), and of random interactions between the polymer chains (20). However, considerable progress has been made toward establishing band assignments useful for identifying these substances (13). Reference spectra of those likely to be important in any consideration of fungal cell wall composition are given in Fig. 1. The overall resemblance between the spectra is not surprising in view of the presence of pyranose rings in each polysaccharide. The spectra of cellulose I and II (Figs. 1.l and 1.2) can be distinguished most easily by differences near 1430, 1110, and 990 cm-’ (21-23). The most significant difference between the spectrum of yeast glucan (Fig. 1.3) and the spectra of the celluloses is probably the stronger band in the former near 2925 cm-l. The spectrum of glucomannan resembles that of cellulose II except for the presence of additional bands near 870 and 805 cm--‘, while galactans may be characterised by a band near 768 cm-’ (13). Bands near 3265, 3105, 1655, 1620, and 1550 cm-’ have been found (12, 24) to be characteristic of chitin spectra (Figs. 1.4 and 1.5). (Y- and p-chitin spectra, obtained by Hackman and Goldberg (19) using the pressed disc method, differ in the region 3700-2850 cm-‘, the bands in the spectrum of the a-material being much sharper and the band at 2960-l more intense. Present results indicate that, in addition, the band near 1640 cm-l is split in the a-chitin spectrum, and bands in the region 1160-890 cm+ appear to be sharper. The spectrum of chitosan (Fig. 1.6) resembles that of &chitin in the region 370&2850 cm-‘. It differs in other regions, especially in having weaker bands near 1640 and 1550 cm-’ and a new peak at 1590 cm-l. In protein spectra, the most distinctive bands arise from the amide group and occur near 1640 and 1550 cm-l (25). Thus, it is
630
MICHELL
AND
SCURFIELD
WAVE NUMBER (CM-‘) 1500 1250
I
I
I
I
2.75 3.00 3.25 FIG. 1. Infrared
1000
I
I
I
I
I
I
I
6
7
8
9
10
WAVELENGTH
(u )
spectra
difficult to distinguish spectroscopically between chitin and mixtures of proteins with, for example, glucans. However, some of the difficulty can be resolved by comparing spectra obtained before and after the substances have been treated with hot alkali in which chitin is much less soluble than protein. Spectra of the walls of Polyporus myllitae. Mycelia derived from cultures in a liquid medium gave spectra in which the bands were rather broad (Figs. 2.1-2.4). The most significant changes in the spectra resulting from chemical extraction of such mycelia occurred in the bands near 1730 and 1550 cm-‘. The former was removed by treatment with 2 %’ potassium hydroxide and the latter was intensified (cf. Figs. 2.1 and 2.2). On the other hand, treatment with either
of reference
900 I
800 I
7cm I
I
I
I
I
11
12
13
14
!
polysaccharides.
sulfuric acid or acetic acid-hydrogen peroxide increased the intensity of the 1730 cm-’ band and reduced that of the 1550 cm-’ band (Fig. 2.3). When potassium hydroxide was followed by sulfuric acid or glacial acetic acid-hydrogen peroxide or both successively, the band near 1730 cm-l reappeared weakly, but that near 1550 cm---’appeared unchanged (Fig. 2.4). The differences between the spectra in Figs. 2.1-2.4 in respect of the bands near 1730 cm-l and 1550 cm-’ would be explained if the water-washed material contained substances having both dissociated and undissociated carboxyl groups. The .latter absorb near 1730 cm-l, while the absorption of the dissociated groups would be expected to underlie the chitin band at 1550 cm-l. The greatly reduced intensity of the band near 1730 cm-1 in Fig. 2.4 indicated that such
COMPOSITION
3900
I
3400
I
OF EXTRACTED
3mo2950
I
I
2.75 3.00 3.25
FUNGAL
WAVE NUMBER (CM-‘1 1500 1250
I 6
I 7
I
CELL
moo900
I
8 9 WAVELENGTH t,!J )
631
WALLS
alo
700
I
I
I
I
I
10
11
12
13
14
FIG. 2. Infrared spectra of mycelia of Polyporus myllitae, before and after various chemical treatments. Spectra 14 refer to mycelia grown in liquid culture, and spectra 5-7 refer to those grown on a solid medium. Broken lines denote spectra obtained by incorporating increased concentrations of the materials into the discs. 1, Water-washed mycelia (liquid culture); 2, residue after treatment with 2% potassium hydroxide (3 half-hour treatments); 3, residue after treatment with 2% sulfuric acid or glacial acetic acid-30% hydrogen peroxide; 4, residue after treatment with 2% potassium hydroxide followed by glacial acetic acid-3ClR hydrogen peroxide and then by 2% sulfuric acid; 5, water-washed mycelia (solid culture); 6, residue after treatment with 10% trichloroacetic acid; 7, residue after treatment with 2y0 potassium hydroxide followed by glacial acetic acid-30% hydrogen peroxide and 2y0 sulfuric acid.
substances were largely removed by successive alkali and acid treatments. The general sharpening of bands in the overall spectrum, following acid and alkali treatments, indicated that amorphous polysaccharides of low degree of polymerization had been removed. Further evidence for the removal of such substances was provided b,y the reduction in intensity of bands in t,he region 900-800 cm-l, including those near 870 and 805 cm-’ (found in the
spectrum of mannans). These bands probably arose from vibrations associated with the backbones of linked pyranose rings (10). Figures 2.5-2.7 give the spectra, respectively, of water-washed mycelia derived from cultures on a solid medium and of two residues obtained by chemical extraction of these mycelia. Bands in the spectrum (Fig. 2.6) of the residue remaining after treatment with trichloroacetic acid, used by Sturgeon (17) as a solvent for phospho-
632
MICHELL
AND
glycoproteins, were generally sharper than in the spectrum of the water-washed mycelia (Fig. 2.5). The intensity of the band near 1730 cm+ was increased. Bands in the spectra were also sharpened after treatment of the mycelia with ethylene diamine alone, or trichloroacetic acid and then ethylene diamine, or with 5 % potassium hydroxide at 100” C for 12 hours, or successively with 2 % potassium hydroxide, 1:l acetic acidhydrogen peroxide, and 2 % sulfuric acid (Fig. 2.7). There was no discernible effect on the spectra when residues from the 5 % potassium hydroxide treatment were further treated with either acetic acid, or with acid and then cupriethylene diamine. However, in all such spectra the band near 1730 cm+ was absent. Comparison of the spectra in Fig. 2 with the reference spectra in Fig. 1 left no doubt that cu-chitin was a significant wall constituent. Treatment with anhydrous ethylene diamine would be expected to have produced changes in the bands near 1430, 1110, and 990 cm-1 if cellulose had been present. As mycelia before and after treatment with 5 % potassium hydroxide, potassium hydroxide followed by acetic acid, or potassium hydroxide followed by acetic acid and then cupriethylene diamine, gave similar spectra, it was unlikely that chitosan, or fl-1,4-linked polysaccharides, other than chitin, were present in significant amounts. A band at 2925 cm-l in the spectra could not be attributed to chitin. This band indicated another material which was resistant to all chemical treatments. It had a p-linked structure since absorption occurred near 890 cm-l rather than 850 cm-l (10). It may have been a p-1 ,&glucan since it was insoluble in cupriethylene diamine, in which p-1,4-linked polysaccharides dissolve (26). p-1 ,3-Glucans are known to be common constituents of fungal cell walls (9). Some indication of the effects of different cultural methods on wall composition could be obtained by comparing Fig. 2.1 with Fig. 2.5, and Fig. 2.4 with Fig. 2.7. The spectra in Figs. 2.5 and 2.7 were much sharper, especially in the regions 3500-3100 cm-’ and 1200-900 cm-‘, and resembled the spectrum of a-chitin (Fig. 1.4) much
SCURFIELD
more closely than those in Figs. 2.1 and 2.4. This suggested that the cY-chitin in the walls of the fungus grown on the solid medium was more highly crystalline. In addition, the band at 1730 cm-’ was much weaker in the spectrum in Fig. 2.5 compared wit’h that in Fig. 2.1. Possibly, therefore, the walls of the fungus grown on the solid medium were poorer in carboxyl-containing substances. A further point concerned the possible presence of lipids in cell walls. The infrared spectra of mycelia which had been washed with water and those which were extracted with boiling methanol gave almost identical spectra. However, the material obtained by evaporating the methanol extract to dryness gave a spectrum having some bands in common with those found in lipid spectra (27). Spectra of the wall residues of other species. The spectra of Phytophthora cinnamomi and the Pythium sp. (Figs. 3.1 and 3.2) resembled fairly closely the spectrum of cellulose I (Fig. 1.1). Small differences were observed in the region 1160-990 cm-‘, but these could have arisen from differences in crystallinity between fungal and wood cellulose. Extra bands were evident, however, in the spectrum of Pythium sp. at 2925 and 1730 cm-‘, so a little glucan and some carboxyl-containing substances may also have been present. The spectrum of Epicoccum sp. (Fig. 3.3) had several bands in common with those of the chitins, including bands near 1640 and 1550 cm-‘. However, the bands near 3450, 3265, and 3105 cm-’ were unresolved, as was also the band found near 953 cm-’ in the spectrum of cr-chitin. These results suggested that chitin was not the only wall constituent. The presence of a weak peak near 2925 cm-l indicated some /3-1,3-glucan, but the resemblance of the spectrum in the region 116&990 cm-l to those of Phytophthora and Pythium sp. suggested the possible presence of cellulose. The spectra of the remainder of the species examined were generally similar to one another (Fig. 4). The spectra of the following pairs of species were identical, and only those of the
COMPOSITION
3800 I
’
2.75
3400 I
300
3m I
3.25
OF EXTRACTED
FUNGAL
WAVE NUMBER (CM-‘) 1500 1250 1000 I I I
I
6
7 e 9 WAVELENGTH (cc,
10
633
CELL WALLS
800 I
900 I
11
12
700
13
FIG. 3. Infrared spectra of hyphal wall residues of fungi.
first named are reproduced: Polyporus myllitae (Fig. 2.7) and Corticium rolfsii, Pithomyces char&urn and Coriolus zonatus, Alternaria solani and Hormodendron resinae, Armillaria mellea and Stereum chaillettii. All spectra showed distinct bands near 3450, 3265, 2925, 2850, 1640, 1550, 1375, 1310, and 1155 cn-I, except the spectra of Armillaria, Stereum, and Cenoccoccum, in which bands near 3265 and 3105 cm-l were unresolved. Apart from the band at 2925 cm-1 such bands indicated the presence of chitin, almost certainly a-chitin, despite the fact that only in the spectrum of Amanita pileus was the band near 1640 cm-’ split. The distinctness of bands at 2960, 1450, and 1730 cm-’ varied from spectrum to spectrum. Thus the band at 2960 cm+ was detectable in the spectrum of Armillaria and Stereum, but not in that of Alternaria or Hormoden&on. The band at 1450 cm-l, most prominent in the spectrum of Cenococcum, and less prominent in the spectra of Sclerotinia, Diplodia, Alternaria, Hormodendron, Stereum, and Armillaria, was scarcely distinguishable in the spectra of the other species. These two bands could be attributed to C-H stretching and bending vibrations, respectively, but it was uncertain whether they arose from methyl or methylene groups.
The band at 1730 cm-‘, believed to signify the presence of carboxyl groups, was prominent in the spectra of Polyporus sulphureus and Diplodia, less prominent in those of Alternaria, Hormodendrcn, and Amanita, and absent in spectra of the other species. Bands at 953 and 894 cm-l, distinguishable in the spectra of Amanita, Polyporus sulphureus, Pithomyces, Coriolus, and Merulius, were absent from the spectra of Alternaria and Hormodendran, and were replaced by a broad band centred on 940 cm-1 in the spectra of Diplodia, Sclerotinia, Armillaria, Stereum, and Cenococcum. The bands at 800 and 780 cm-l, absent from the spectra of Diplodia, Sclerotinia, Armillaria, Stereum, Cenococcum, and Polyporus sulphureus, and barely distinguishable in the spectra of Pithomyces, Coriolus, Alternaria, Hormodendron, and Amanita, were prominent in the spectrum of Merulius. The spectrum of Merulius was also characterised by a band near 700 cm-l and that, of Cenococcum by the absence of a band near 1420 cm-l. From a diagnostic viewpoint the most promising bands, other than those already assigned, were the bands at 800 and 780 cm-l. Their frequency, and the absence of accompanying sharp higher frequency bands,
MICHELL
634
AND SCURFIELD
.
I
I
2.15
3.00
I
3.25
t
t
6
7
I
I
8 9 WAVELENGTH (/!L)
I
I
I
I
1
10
11
12
13
14
FIG. 4. Infrared spectra of hyphal wall residues of fungi.
suggested that they possibly arose from a polysaccharide linkage. However, they could also have arisen from inorganic constituents. In the spectra of Pithomyces, Coriolus, Merulius, Alternaria, and Hormodendron there was additional broad absorption underlying the chitin bands in the region 1250-1000 cm-‘, and in the spectra of Diplodia, Sclerotinia, Armillaria, Stereum, and Cmococcum walls, there was another broad band near 940 cm-‘. These bands
almost certainly arose from inorganic constituents. The spectra of mycelia of Alternaria, Pythium sp., and Epicoccum sp. grown on malt agar were similar to those grown on potato agar. DISCUSSION
Infrared spectroscopy is comparable with X-ray diffraction (1) as a technique for studying cell wall composition insofar as
COMPOSITION
OF EXTRACTED
both yield results which are more easily interpreted if the wall samples have been subjected t,o prior chemical treatment. They are comparable also in that both require similar amounts of material for an analysis, and both lead to the positive identification of crystalline organic substances, but are less definitive for mixtures and materials of lower crystallinity. The advantage of infrared spectroscopy is that it yields information concerning amorphous materials and the chemical groupings in the components of mixtures. Electron microscopic examination of the wall residues of Polyporus myllitae (14) has shown that the extent to which chemical treatments remove substances from the surfaces of hyphae varies somewhat with the type of t.reatment. Even the treatment with potassium hydroxide followed by glacial acetic acid-hydrogen peroxide is not entirely effective with every sort of fungal wall material. It is thus possible that some of the differences between spectra are due to the differential removal of such substances. This could account, for example, for the indications of the presence of inorganic materials in certain spectra. Frey (1) obtained X-ray diffraction photographs indicative of their presence in the walls or cells of various fungi. But apart from this, little appears to be known of the chemical nature of the materials removed. There is another possibility, namely, that substances of cytoplasmic origin are included in wall residues. The lipid material found in the methanol extract of P. myllitae walls, for example, might have come from the cell cytoplasm even though lipids have been reported to be wall components in other fungi (28, 29). The extent to which the various chemical treatments employed remove cytoplasm from the cells is uncertain, but alkali is believed to do so (16). The differences between the spectra of mycelia grown on solid and in liquid media could be due to the different methods of culture or to differences in the chemical composition of the media. The former possibility seems more likely in view of the near identity of the spectra of residues derived from mycelia grown on malt and on
FUNGAL
CELL
WALLS
635
potato agar. However, the question of the effect of growing conditions on wall composition needs closer examination, especially as the present conclusion is based on only one experiment. It is relevant to note that Bartnicki-Garcia and Nickerson (28) have shown that differences in cultural conditions, albeit more gross than those examined here, produce differences in the chemical composition of the walls of Mucor rouxii. The presence of carboxyl groups in fungal walls is of interest since they may possibly indicate the presence of uranic acids. A small quantity of uranic acid was found in a hot water-soluble fraction of the walls of Saprolegnia littoralis by Parker et al. (5). Crook and Johnston (S), however, failed to detect uranic acids in formic acid hydrolysates of the walls of several species of fungi. It has been pointed out above that the presence of proteins is not precluded, even in walls which, from their spectra, appear to consist of chitin. The possible importance of proteins can be judged from the fact that the walls of Pithomyces chartarum have been reported (17, 29) to contain 25 % protein, 40 % carbohydrate, and 10 % bound glucosamine. However, in view of the fact that hot 1 N potassium hydroxide has been found to dissolve nearly all the protein from the walls of Mucor rouxii (ZSj, it appears likely that the alkali treatment given here to wall residues eliminated any protein present in them. On the basis of the infrared spectra of their extracted hyphae, the fungi examined can evidently be divided into two groups: Group I includes those species having cellulose I, and Group II those having cr-chitin, as a significant wall constituent. The occurrence of cellulose I as a component in the hyphal walls of species of Phytophthora and Pythium confbms the result of Frey (1)) who used X-ray diff raction methods. Crook and Johnston (8), using chemical analysis, detected cellulose in the walls of Phytophthora cactorum. It has also been reported to be a component of the hyphal walls of species of Xaprolegnia, A chl ya, Lagenidium, and Brevilegnia (1, 5, 8), though the amounts present might
636
MICHELL
AND SCURFIELD
not exceed 15% of the unextracted wall weight (5).l On the other hand, chitin and not cellulose has been cited as a component of the walls of species of Chytridium, Rh~zo~h~d~~rn, AlEomyces, 3~~to~l~iella, Rh~z~u~~ ~~~~, and ~h~corn~~~ (1, 15,
28, 30), while the presence of both cellulose and chitin in the walls of Rhizidiomyces has been demonstrated by Fuller and Barshad 06).
Ascomycetes, other than yeasts, can generally be allocate to Group II (9). However, it has been claimed, mainly on the basis of X-ray diffraction data, that cellulose and chitin occur together in the conidiophore walls of Asporgillus (31) and the hyphal walls of Ceralocystis uZmi (32). Basidiomycetes~ without exception, can be allocated to Group II on the basis of the present data and the list given by Aronson (9). Infrared spectra indicate that many Fungi imperfecti can also be included in this group. The conclusion that both chitin and cellulose might be present in the walls of ~pi~o~~rn sp. needs further ~vestigation. Group II is evidently very large. The infrared spectra of the walls of species included in it indicate that it is also heterogeneous. The possibility of subdividing it on the basis of t,he characte~sti~s of such spectra might be worth pursuing with a wider range of speciesgrown under identical conditions. However, present data indicate that qualitative differences in wall composition between species in Group II are likely to be few. An obvious subgroup would indude those species whose walls have been sa.id to contain both chitin and cellulose. X-Ray diffraction and chemical analyses (1, 3, 6) have indicated that a Group III might be added to include those species of 1 hrote Added in Proof: Since this paper was written two new estimations of cellulose I in fungal cell walls have been published. BartnickiGarcia [J. Gen. Mierobiol. 42, 57 (1966)) has reported that, mechanically isolated walls of Phytophthora cinnamomi contain 25y0 cellulose I and 63% other fl-glucans. Aronson, Cooper and Fuller [Science 156, 332 (1967)J report that chemically isolated walls of P~~tophtho~~, A t~~~s~elZa,Achlya, and Pythium spp. contain 7, 31, 11, and 12% of cellulose I, respectiveIy, the rest of the walls being largely p-1,3- and p-1,6-glucans.
yeast shown to have a p-1,3-glucan rather than cellulose or chitin as a major wall component. The general conclusion from the above is that wall composition as deduced from infrared spectra has little taxonomic value at present, and that its phylogenetic value is Iimited to the phycomycetes. However, features in the spectra, such as the intense bands at 800 and 780 cm-’ in the spectrum of the rhizomorph walls of Merulius ZWYmans, do indicate fungi which might have wall compositions of special interest. ACKNOWLEDGMENTS The authors are grateful to Dr. Eileen E. Fisher, Victorian Plant Research Institute, Burnley; and Mr. E. W. B. Da Costa and Mr. N. E. M. Walt,ers, CSIRO, Division of Forest Products, for providing cultures of the fungi; and to Mrs. J. Bennett for laboratory assistance. REFERENCES 1. FREY, R., Ber. Schweiz.
BotGn. @es. 60, 199 (1950). 1, 1 (1954). 2. BLANK, F., Can. J. Microbial. 3. KREQER, D. R., Biochim. Biophys. Acta 13, 1 (1954)I 4. NICOLAI, E., AND PRESTON, R. D., Proc. Roy. Sot. (London), Ser. B 161, 244 (1959). 5. PARKER, B. C., PRESTON, R. D., AND FOGG, G. E., Proc. Roy. See. (London), Ser. B 158, 435 (1963). NORTHCOTE, D. H., AND HORNE, R. W., Biochem. J. 61,232 (1952). CUMMINS, C. S., AND HARRIS, H., J. Gen. MicrobioZ. 18, 173 (1962). CROOK, E. &I., AND JOHNSTON, I. R., Bioc~~. 3. 63,325 (1962). ARONSON, J. M. in “The Fungi” (G. C.
Ainsworth and A. S. Sussman, eds.), p. 49. Academic Press, New York (1965). 10. BARKER, S. A., BOURNE, E. J., STACEY, M., AND WHIFFEN, D. H., 3. C&m. Sot. 171 (1954). J. Chem. 10. 496 11. HIGGINS, H. G., Au&al. (1957)* 12. PEARSON, F. G., MBRCHESIAULT, R. H., AND LIANQ, C. Y., J. Polymer Sci. 43,101 (1960). 13. MARCHESSAULT, R. H., Pure Appl. Chem. 6, 107 (1962). 14. SCURFIEL~, G., .I. Linn. Sot, (Bob.) (Lon~o~). (in press) 15. ARONSON, J. M., AND PRESTON, R. D., J. Biophys. Biochem. Cytol. 6, 247 (1960).
COMPOSITION
OF EXTRACTED
16. FULLER, M. S., AND BARSHAD, I., Am. J. Botany 47, 105 (1960). 17. STURGEON, R. J., Biochem. J. 92,6OP (1964). 18. KINQ, J. M., Austral. J. Botany 14, 25 (1966). 19. HACKMAN, R. H., AND GOLDBERG, M., AustraZ. J. Biol. Sci. 18, 947 (1965). 20. LIANG, C. Y., KRIMM, S. H., AND SUTHERLAND, G.B.B.M., J. Chem. Phys. 26, 543 (1956). 21. LI~NG, C. Y., AND MARCHESSATJLT, R. H., J. Polymer Sci. 37, 385 (1959). 22. LIANG, C. Y., AND MARCHESSAULT, R. H., J. Polymer Sci. 89, 269 (1959). 23. MARCHESS.IULT, R. H., AND LIANG, C. Y., J. Polymer Sci. 43, 71 (1960). 24. RUDALL, K. M., Advan. Insect Physiol. 1, 257 (1963). 25. FRASER, R. D. B., in “A Laboratory Manual of Analytical Methods of Protein Chem-
FUNGAL
26. 27.
28. 29. 30. 31. 32.
CELL
WALLS
637
istry” (P. Alexander and R. J. Block, eds.), Vol. 2, p. 285. Pergamon, Oxford (1960). CLARKE, A. E., AND STONE, B. A., Rev. Pure Appl. Chem. 13, 134 (1963). FREEMAN, N. K., LINDGREN, F. T., Na, Y. C., AND NICHOLS, A. V., J. Biol. Chem. 203, 293 (1953). BARTNICKI-GARCIA, S., AND NICKERSON, W. J., Biochim. Biophys. Acta 68, 102 (1962). RUSSELL, D. W., STURGEON, R. J., AND WARD, V., J. Gen. MicrobioZ. 36, 289 (1964). ARONSON, J. M., AND PRESTON, R. D., Proc. Roy. Sot. (London), Ser. B 162,346 (1960). FARR, W. K., Trans. N.Y. Acad. Sci. 16, 209 (1954). ROSINSKI, M. A., AND CAMPANA, R. J., MycoZogia 66, 738 (1964).