Electron microscopic X-ray microprobe and cytochemical study of isolated cell walls of mucoralean fungi

Notes and brief articles

536 REFERENCES

BRANDT, W. H . & REESE,]. E. (1964). Morphogenesis in Vert icillium : a self-produced diffusible morphogenetic factor . Amer ican Journal of Botany 51, 922--927. BRANDT, W. H . & ROTH, ] . N. (1965). Loss of melanincontaining structures by Verticillium cultures. Phytopathology 55, 1200-1202. BURNETT, ]. H. (1978). Aspect s of the structure and growth of hyphal walls. In Fungal Walls and Hyphal Growth (ed . ]. H . Burnett & A. P. ] . Trinci), pp . 1-25. Cambridge University Press. CoHEN, ]., KATZ, D. & ROZENBERGER, R. F. (1969). Temperature sensitive mutant of Asp ergillus nidulans lacking amino sugars in its cell wall. Nature, London 229,713-7 15. GULL, K. & TRINCI, A. P. ]. (1974). Detection of areas of

wall different iation in fungi using fluorescent staining. Archives of Microbiology 96, 53-57. HEALE,] . B. & ISAAC, I. (1965). Environmental factors in the production of dark resting structures in Verticillium albo-atrum , V . dahliae and V. tricorpus. Transactions of the British Mycological Society 48, 39-50. SMITH, H . c. (1965). The morphology of Verticillium albo-atrum, V. dahliae and V. tricorpus. N ew Zealand Journal of Agricultural Research 8, 450-478. TRINCI, A. P.]. (1978). Wall and hyphal growth. Science Progress, Oxford 65, 75--99. VALENTINE, B. P. & BAINBRIDGE, B. W. (1978). The relevance of a study of a temperature-sensitive ballooning mutant of Aspergillus nidulans defective in manno se metabolism to our understanding of mannose as a wall component and carbon/energy source. J ournal of General Microbiology 109, 155-166.

ELECTRON MICROSCOPIC X-RAY MICROPROBE AND CYTOCHEMICAL STUDY OF ISOLATED CELL WALLS OF MUCORALEAN FUNGI BY G. M. CAMPOS-TAKAKI

Departmeno de Antibioticos, Universidade Federal de Pernambuco, Recife, Pernambuco, Brazil G. W . BEAKES

Department of Plant Biology, University of Newcastle upon Tyne , Newcastle upon Tyne , NEI 7R U, U.K. AND S. M. C. DIETRICH

Institute de Botanica, C.P. 4005, Sao Paulo, Brazil The diverse chemical composition of fungal cell walls has been used to characterize the main classes of fungi and found to be in good agreement with classical phylogeny based on morphological criteria (see review by Barmicki-Garcia, 1968). Following the earlier scanning electron microscope X-ray microprobe study of the elemental composition of isolated cell walls of Cunninghamella echinulata (Jones, McHardy, Farmer & Jones, 1977) it was decided to evaluate the use of the more sensitive transmission electron microscope (TEM) based X-ray microanalysis in determining the elemental composition of isolated cell walls of a wider range of mainly mucoralean zygomycetes. It was hoped that this might provide a quick and simple way of revealing qualitative differences in the chemical composition of fungal cell walls for use in chemotaxonomic studies. In addition zygomycete fungi are unusual in that their cell walls contain large amounts of phosphorous ( > 10 %, Datema, Van Den Ende & Wessels, 1977; Jones et al ., 1977) and therefore, a cytochemical technique previously used to identify polyphosphate granules in bluegreen algal cells (Jensen, 1968) was also applied to Trans . Br . mycol. Soc. 80 (3) (1983)

this material to see if the spatial localization of phosphate with the walls could be resolved. The fungi examined are listed in Tables 1 and 2 and were obtained from the culture collection of the Instituto de Botanica, Sao Paulo, Brazil (SP C). The fungi were maintained on potato dextrose agar and large scale culturing was carried out for 6 days at 25°C in 1 I flasks containing 400 ml synthetic liquid medium (H esseltine & Anderson, 1957). Vegetative mycelium was harvested using sintered glass filters and the cell walls were isolated and purified as described by Letourneau, Deven & Manocha (1976). Samples of isolated cell wall suspensions were dried onto formvar coated grids (200 mesh) and either shadowed with Au/Pd for observation of fine-structure or coated with spectroscopic carbon for microanalysis. Microfibrillar skeletons of some wall samples were also prepared by chemical hydrolysis as described by Hawes (1980) and processed as above. The elemental composition of whole (WW) and microfibrillar (M F) wall fractions was determined in a CORA TEM. Selected wall fragments were analysed (at 8 K magnification) for

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Notes and brief articles Table

1.

537

Summary of elemental composition of whole and microfibrillar f ractions of isolated Zygomy cete walls Elements in whole wall (and microfibrillar) wall fractions

Species Choanephora cucurbitarum (Berk, & Rav.) Thaxter Cun ninghamella elegans

Lendner Linderina pennispora Rape r & Fennell Mortie rella alpin a Peyronel A bsidia blakesleeana Lendner Abs idia cylindrospora Hagem Absidia orchidis (Vuill.) Hagem Absidia pseudocylindrospora Hess eltine & Ellis Cir cinella anagarensis (Schost.) Zycha Ci rcinella mucoroides Saito Gongronella butleri (Lendn er) Peyronel & Dal Vesco M . griseocyanus Hagem f. gr iseocyanus M . griseocyanus Hag em f. j anssenii (Lendn er) Schipper Mucor mucedo Fresen. Rhiz opus arrhizus Fischer Syncephalastrum racemosum Cohn ex Schroter

SPC no .

Mg

P

S

Cl

K

Ca

373

± (-)

+ (- )

+* (+ )

+ (±)

+ (±)

+ (+)

70

+ (-)

+ (- )

± (± )

- ( -)

-

( -)

+ (+)

368

- ( -)

+ (-)

+* (+ )

- (±)

- ( -)

+ ( +)

+

+

+

+

+

+ (- )

± ( +)

- ( -)

± (±)

+ (+)

+ +

+* +

+

+

±

+

+

342 341

+ (-)

316 340 317

+

+

348

- (+)

+ (+)

+ (+)

- (±)

-

( -)

+ (+ )

332 343

+ + (-)

+ + (- )

±* ± * (+ )

- (± )

- (- )

+ + (+)

310

+ (-)

+ (± )

- ( ±)

(- )

+ (- )

+ (+)

322

+

+

+

+

344 361 365

- ( +) + (-)

+ + (±) + (-)

- ( ±) + (-)

+ - ( +) + ( +)

-

+ + + ( +) + (+)

- ( d;)

+ (- )

+ , present 7 out of 10 samples; ±, pre sent 5 out of 10 samples ; *, pr esent in some background spectra. Where no bracketed data given microfibrillar fractions not analysed . 120 s using a probe size of ca 5 pm and fixed condenser setting. For each sample at least 10 replicate spectra were recorded, together with at least one background spectrum from the supporting formvar. Finally, some wall samples were also proces sed for ultrathin sectioning using the histochemical procedure described by Jensen (1968) to visualize cytoplasmic polyphosphates. Controls were prepared by extracting walls with 10 % TCA prior to fixing. The typical ultrastructural app earance of metalshadowed WW and MF mucoralean wall fragments as represented by Syncephalastrum racemosum, are shown in Figs l (a) and l (b) respectively. The whole wall fragments were relatively electronopaque with both the outer and inner faces of the flattened cylindrical walls appearing uniformly smooth (Fig. 1 a). The chemically cleaned MF wall fract ion is more electron transparent than WW fract ion and showed very littl e contamination with amorphous matrix material (F ig. lb ). The microfibrils form a dense randomly intermeshing netT ran s. Br. my col. S oc. 80 (3) (1983)

work in all the walls examined. The elements present in the spectra in the WW and MF wall fragments analysed in this investigation are sum marized in Table 1 and representative spectra for S. racemosum given in Figs 2 (a), (b). Unfortunately , elements with low atomic numbers « 10) are not detected efficiently with this system . The main elements of biological interest within the fungal wall were Mg, P, S, CI, K and Ca (T able 1). There were three main background peaks, Al from the specimen holder, Fe from the microscope column and Cu from the support grid. In some samples a small S peak was also detected in the formvar support film (T able 1). Phosphorus was the only element present in all the walls although Ca was detected in all but Rhizopus arrhizus and Absidia orchidis (T able 1). The number of elements detect ed in th e isolated wall fragments ranged from a minimum of two in A . orchidis to five or six in Choanephora cucurbitarum, Mucor ja van icus and S. racemosum (T able 1). Unfortunately, no obvious pattern emerged amongst the gener a examined with

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Notes and brief articles Table

2.

Summary of staining reaction of isolated mucoralean walls with phosphate localization technique of Jensen (1968)

Phosphate localization Granules associated with wall surface

Species

++ + + ++ +++ +++ ++ +++ ++ ++ +++ +++ + +++ +++

Cunninghamella elegans Linderina pennispora Absidia blakesleeana Absidia orchidis Absidia pseudocylindrospora Circinella angarensis Circinella mucoroides Gongronella butleri M. griseocyanus f. griseocyanus M. griseocyanus f. janssenii Mucor javanicus Mucor lamprosporus Mucor mucedo Mucor plumbeus Syncephalastrum racemosum Saprolegnia ferax

++

Bacteria (unidentified) ND: not determined;

Staining within wall

TCA extracted controls

++ ++ + ++ +

+ ++ ++ +

+ + ++ +++

+

++

ND ND

+ < + + + , relative intensity of lead staining; -, unstained.

regard to the elemental composition of their walls. The possibility was considered that some of the elements, such as Cl and K, may be derived from fragments of contaminant cytoplasm and so selected wall samples were washed and given additional sonication before re-examination. The elemental spectra however, remained unchanged, indicating all elements detected were bound firmly to the wall fraction. The more electron transparent MF fractions (Fig. 1 b), not surprisingly, had much lower overall count rates during the 2 min analysis. The most striking feature of most of the MF spectra was the absence or marked reduction in P peaks (Table 1; Fig. zb). Most MF spectra contained fewer

elements than WW fractions (Table 1) but most had prominent S peaks (Fig. zb ; Table 1). The wall elements detected by TEM microprobe analysis with the exception of K, are identical to those reported in Cunninghamella echinulate using SEM probe and confirmed by chemical analysis (Jones et al., 1977). However the problem remains of distinguishing those elements that are true wall constituents and those that are firmly bound contaminants adsorbed during preparation. Cell walls of these zygomycete fungi are known to contain exceptionally large amounts of phosphate, often in excess of 10 % (Campos-Takaki, unpubl.). This is confirmed by the prominent phosphorus peaks in all the elemental spectra of

Fig. 1. Whole mount preparations of isolated cell walls of Syncephalastrum racemosum. (a) Whole wall fraction x 30000; (b) Microfibrillar fraction x 31000. Fig. 2. Representative X-ray spectra showing elemental composition between 0·8 and 4·oKeV (ca Na-Ca) for (a) Whole walls of S. racemosum; (b) Microfibrillar fraction of S. racemosum. Spectra were obtained under as near possible identical conditions, for 120 s count time. The figure in brackets is maximum (full scale) count number at which spectrum plotted. Figs 3-5. Selection of isolated walls stained with Pb(N0 3)./(NH4).S to localize phosphate. Fig. 3(a) TCA-extracted control showing absence of lead deposits x 60000. (b) Phosphate-localized wall showing electron dense surface deposits of lead and layered intra-wall staining x 65000. Fig. 4. Wall of Syncephalastrum racemosum stained for phosphate showing typical pattern of surface granules and no intra-wall staining x 74000. Fig. 5. Wall (between arrows) of Saprolegnia ferax stained for phosphate showing complete absence of staining. Note electron dense deposits associated with walls of contaminating bacterial cells x 60000. Trans. Br. mycol. Soc. 80 (3) (1983)

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Notes and brief articles

p

539

Micr o fibrils (500)

Whole wall ( 1000)

s K

AI

2 (0 )

5

3(b)

Trans. Br . mycol. Soc. 80 (3) ( 1983)

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540

Notes and brief articles

isolated whole walls. Members ofthe Mucorales are known to accumulate large quantities of inorganic polyphosphates in their vegetative mycelium Games & Casida, 1964; Harold, 1966; Dietrich, 1976). Harold (1962) has reported that added inorganic polyphosphates will bind strongly to glycan sites at the surface of Neurospora walls. Datema et al. (1977) also have shown that the polyphosphates from Mucor mucedo walls are bound to glucuronan fractions. Some of the wall phosphorus in the Mucorales may therefore be derived from the adsorption of water soluble polyphosphates (Dietrich, 1976) from the cytoplasmic pool during isolation. The results of the cytochemical localization of phosphate using the lead nitrate/ammonium sulphide staining technique described by Jensen (1968) have been summarized in Table 2 and Figs 3-5. This staining technique gave rather variable results and not all of the TCA-extracted controls were negative (Table 2). It is unclear whether this reflects a lack of specificity in the staining procedure or the incomplete removal of phosphate in the TCA-treated controls. The most commonly encountered staining pattern, shown clearly in Mucor griseocyanus f. janssenii (Fig. 3 b) and Syncephalastrum racemosum (Fig. 4) was the deposition of electron-dense globules on the surfaces of the walls (Table 1). These were shown by X-ray microanalysis to be composed oflead. The extent of the deposition varied considerably from a dense coating as in Fig. 3(b), to more scattered granules over the surface (Fig. 4). Although this staining pattern suggests a non-specific adsorption of stain to the wall, it was not observed in most of the controls treated with TCA (Fig. 3 a). In M. griseocyanus f.janssenii electron dense deposits also occurred in localized 'lamellar' bands within the wall (Fig. 3b). In contrast, the walls of Saprolegnia ferax which lack polyphosphate in their walls (Novaes-Lidieu, Jimenez-Martinez & Villanueva, 1967; Dietrich, 1975) and cytoplasm (Dietrich, 1976) remained completely unstained, but the walls of contaminating bacteria stained densely (Fig. 5). Therefore, in general, this cytochernical localization technique gave positive results in walls known to contain phosphate. However, the rather varied staining pattern of walls with this technique suggests it is not of sufficiently good resolution to reveal unequivocally the precise spatial localization of phosphate within the walls. Nevertheless, the presence of high levels of phosphorus in all whole mucoralean walls and the staining of wall layers with the phosphate-localization technique supports the view of Jones et al. (1977) that phosphates are genuine wall constituents in zygomycete fungi, rather than just being cytoplasmic contaminants as Trans. Br. mycol. Soc. 80 (3) (1983)

previously suggested (Harold, 1962; Datema et al., 1977). Although the use of TEM electron probe X-ray microanalysis on bulk samples of isolated fungal walls has given rather variable and equivocable results, in future, analysis of fixed and embedded wall preparations may make possible a more precise analysis of elements present. In addition, by calibrating with standards as recently described by Harvey, Flowers, Hall & Spurr (1980) the data may be quantified. Although a simple qualitative comparison of elemental composition of isolated zygomycete walls appears to be of limited use in chemotaxonomy it may still prove useful as an indicator of gross differences in the chemical composition of fungal walls.

G. M. C- T. gratefully acknowledges the receipt of a CAPES overseas fellowship during her period in Newcastle. We would like to thank Bob Hewitt for EM technical support and Sandra and Sue Clothier for secretarial assistance. REFERENCES

BARTNICKI-GARCIA, S. (1968). Cell wall chemistry, morphogenesis and taxonomy of fungi. Annual Review of Microbiology ZZ, 87-108. DATEMA, R., VAN DEN ENDE, H. & WESSELS, J. G. H. (1977). The hyphal wall of Mucor mucedo. 1. Polyanionic polymers. Europeanfournal of Biochemistry 80, 611--619. DIETRICH, S. M. C. (1975). Comparative study of hyphaI wall components of oomycetes: Saprolegnia and Pythiaceae. Annals Academie Brasiliensis Ciencia 47, 155-162. DIETRICH, S. M. C. (1976). Presence of polyphosphate of low molecular weight in Zygomycetes. Journal of Bacteriology 1Z7, 14°8-1413. HAROLD, F. M. (1962). Binding of inorganic polyphosphate to the cell wall of Neurospora crassa. Biochemica et Biophysica Acta 57, 5g--66. HAROLD, F. M. (1966). Inorganic polyphosphates in biology: structure, metabolism, and function. Bacteriological Reviews 30, 772-794. HARVEY, D. M. R, FLOWERS, T. J., HALL, J. L. & SPURR, A. R (1980). The preparation of calibration standards for sodium, potassium and chlorine analysis by analytical electron microscopy. Journal of Microscopy 118, 143-152. HAWES, C. R (1980). Conidium germination in Chalara state of Ceratocystis adiposa: a light and electron microscope study. Transactions of the British Mycological Society 74, 321-328. HESSELTINE, C. W. & ANDERSON, R. F. (1957). Microbiological production of carotenoids 1. Zygospores and carotene produced by intraspecific crosses of Choanephoraceae in liquid medium. Mycologia 49, 449-552. JAMES, A. W. & CASIDA, L. E. (1964). Accumulation of phosphorus compounds by Mucor racemosus.Journal of Bacteriology 87, 150-155.

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Notes and brief articles T. E. (1968). Electron microscopy of polyphosphate bodies in a blue-green alga, Nostoc pruniforme. Archives fur Mikrobiologie 62, 144-152. JONES, D., McHARDY, W. T., FARMER, V. C. & JONES, Y. (1977). Electron probe microanalysis of cell walls of Cunninghamella echinulata. Transactions of the British Mycological Society 69, 71-75. LETOURNEAU, D. R., DEVEN, J. H. & MANOCHA, S. M. JENSEN,

54 1

(1976). Structure and composition of the cell wall of Choanephora cucurbitarum. Canadian Journal of Microbiology, 202, 486-494. NOVAES-LIDIEU, M., JIMENEZ-MARTINEZ, A. & VILLANUEVA, J. R. (1967). Chemical composition of hyphal wall of phycomycetes. Journal of General Microbiology 47, 237-245.

SCANNING ELECTRON MICROSCOPY OF THE SOIL FUNGUS STILBELLA BULBICOLA BY D. JONES

Department of Microbiology, The Macaulay Institute for Soil Research, Craigiebuckler, Aberdeen, AB9 2QJ, Scotland

During studies on the ecology and physiology of phenolic acid-decomposing fungi from soil around the root region of various agricultural crops, an infrequently isolated species was Stilbella bulbicola P. Henn., first described as a new species by Hennings (1905). It is of special interest because of its antagonism towards soil fungi when grown on certain agars. To date no active antagonistic (antibiotic) substance has been isolated from cultures and preliminary studies have indicated that this antagonism is possibly due to pH changes. This note describes scanning electron miscroscope observations on the asexual fruiting structures of the fungus which will supplement the original description by Hennings. Blocks of agar (Oxoid Czapek Dox), approximately 2 mm square, were cut from cultures of the fungus and prepared for critical point drying by methods described previously (Jones, 1978). Dried specimens were sputter coated with gold before being examined in a Cambridge Instruments S4 scanning electron microscope. Because the mass of phialospores that accumulate in mucoid heads on the hyaline synnemata obscure the detail of the phialides, some cultures were flushed with distilled water to remove most of them prior to critical point drying. The hyaline aseptate phialospores gather in

Trans. Br. mycol. Soc. 80 (3) (1983) 18

cream to pale-yellow coloured mucoid heads, one of which is illustrated in Fig. 1. The phialospores are shown in more detail in Figs 2 and 3. It will be seen from Fig. 3 that the ovate phialospore generally terminates in a short papilla or apiculus. Figure 4 is of a washed specimen which reveals the conidiophores aggregated into a synnema and diverging at the tip to terminate in phialides. Figure 5 shows a phialospore emerging from the phialide, through a cylindrical collarette (arrowed) and Fig. 6 illustrates three fully emerged phialospores (a, b, c). A culture of this fungus has been deposited at the Culture Collection, Commonwealth Mycological Institute, Kew, Surrey, U.K. and given the number IM1 259 852. I thank Dr Kitty Brady of the Commonwealth Mycological institute for identifying the fungus under study. REFERENCES

P. (1905). Einige schadliche parasitische Pilze auf exotischen Orchideen unserer Gewachshauser. Hedwigia 44, 168-178. JONES, D. (1978). Scanning electron microscopy of cystosori of Spongospora subterranea. Transactions of the British Mycological Society 70, 292-293. HENNINGS,

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