Fine structure of a new mycophagous amoeba and its feeding on Cochliobolus sativus

Soil Biol. Biochem. Vol. 17, No. 5, pp. 64S655, 1985 Printed in Great Britain. All rights reserved

0038-0717/85 $3.00 + 0.00 Copyright 0 1985 Pergamon Press Ltd

FINE STRUCTURE OF A NEW MYCOPHAGOUS AMOEBA AND ITS FEEDING ON COCHLIOBOLUS SATIVUS K. M. OLD, S. CHARRAB~RTYand R. GIBBS C.S.I.R.O.

Division

of Forest

Research, (Accepted

Summary-An

P.O. Box 4008, Canberra,

A.C.T.

2600, Australia

12 March 1985)

unidentified mycophagous soil amoeba is described. The pigmented soil-borne fungus

Cochliobolus satiuus and four other fungal species, both pigmented and hyaline, were utilized as food.

Spores were ingested and lysed within digestive vacuoles by general wall erosion. This contrasts with the wall perforation mechanism described for other mycophagous amoebae. Ultrastructural studies of trophozoites showed that large quantities of electron dense granules were released into the digestive vacuoles during fungal cell lysis. These were incorporated into the amoeba1 protoplast. Bacteria were commonly present in the amoeba1 protoplasts and within digestive vacuoles. Their possible role as endosymbionts is discussed.

INTRODUCTION There are at least 13 genera of soil amoebae known to feed on fungal propagules. These include Aruchnulu (Old and Darbyshire, 1978), Vurnpyrellu (Anderson and Patrick, 1978), Theratromyxu (Old and Oros, 1980; Anderson and Patrick, 1980), Arceliu and Geococcus (Dreschler, 1936), Gephyrumoebu, Mayorellu and Succamoebu (Chakraborty et al., 1983), a Ripidomyxu (Chakraborty and Pussard, 1985), Thecamoeba (Pussard et al., 1979), Hurtmunella (C. Palzer, personal communication), Thecumoeba (Esser et al., 1975), Cashia (Pussard et al., 1980) and Acunthamoebu (unpublished information). Continuing interest in this phenomenon is likely to result in the extension of this list. Chakraborty et al. (1983) summarized the steps in fungal feeding by soil amoebae. Three stages were identified; attachment of trophozoites to cell walls, engulfment of propagules and finally, digestion of wall material and protoplasts. Digestion may be initiated by penetration of fungal cell walls at discrete points followed by invasion of the cell lumina. This perforation-lysis has been recorded for A. imputiens (Old, 1977), Vampyrellu spp (Anderson and Patrick, 1980) a Leptomyxida isolate (Chakraborty and Old, 1982), Thecumoebu grunijka ssp. minor, (Pussard et al., 1980), Succumoebu sp. and Gephyrumoeba sp. (Chakraborty et al., 1983). Chakraborty and Old (1982) also noted that digestion of fungal propagules could occur in food vacuoles within trophozoites and result in a more general lysis of the cells. The Leptomyxida isolate completely lysed chlamydospores of Phytophthoru cinnumomi Rands leaving an amorphous residue. Conidia of Cochfiobolus sativus (Ito et Kurib). Dreschs.

ex Dastur

within

food vacuoles

foration, and are especially useful when combined with scanning electron microscopy (SEM). The latter provides information on the type of perforation caused by the amoebae and differentiates between those initiated as annular depressions (Old, 1977; Anderson and Patrick, 1978) by A. imputiens and the small perforations caused by other amoebae. Transmission electron microscopy (TEM) was used by Old (1978) to investigate the mechanism of perforation of C. sutivus spores by A. imputiens but has not been utilized in other studies. We describe the morphology and feeding behaviour of a newly-isolated mycophagous amoeba as observed with light and electron microscopy.

of Muyor-

ellu

sp. suffered rupture of internal septa and protoplast disorganization, although no perforated conidia were found in the feeding trials. Descriptions of mycophagous mechanisms of amoebae have largely been based on observations with the light microscope. Such studies are essential to confirm the occurrence of spore lysis or per645

MATERIALSAND METHODS Isolates of the amoeba were obtained from a sandy loam wheatfield soil from the Eyre Peninsula South Australia and a red basaltic loam soil from a mixed eucalypt-rainforest community south of Bumie, NW Tasmania. Soil samples were collected from the “A” horizon and stored in plastic bags at room temperature before use. Amoebae were recovered from the soils using a method (Old, 1977) which employs conidia of C. sutivus sandwiched between pairs of Nuclepore Filter membranes (Nuclepore Corporation, California) and buried in soil moistened to near field capacity. Single-cell cultures of the isolated amoebae and their associated bacteria were made by spreading suspensions of amoebae and spores on Prescott and James’ (1955) solution solidified with 1.0% Bacto agar. Small blocks of agar bearing single amoeba were transferred to fresh suspensions of conidia in sterile Neff’s amoeba saline solution (AS) in 9 cm Petri dishes (Page, 1976). Cultures were held at room temperature (15-25°C). Subcultures were necessary every 4-6 weeks to keep the cultures viable and active. All observations of feeding were made by means of a Zeiss inverted microscope. Stock cultures of C. sativus were maintained on Czapeks Dox Agar at 25°C and conidia obtained by flooding plates with sterile AS. Conidial suspensions

K. M. OLD et al.

646

cytological structures distinguishable by light microscopical examination are the single posteriorlylocated contractile vacuole and many phase-bright particles and crystals giving the endoplasm a granular appearance. The nucleus is rendered inconspicuous due to the abundance of the phase-bright particles, however the uninucleate nature of the trophozoites is evident from stained light microscopic (Fig. 3) and TEM observations (Fig. 11). In stained preparations, nuclei measure 8-10 pm in diameter. Trophozoites do not readily adhere to glass surface. Although a distinctive floating form has not been observed, suspended cells often appear to have more tubular pseudopodia. Locomotion is predominantly monopodial but can be polypodial at times (Figs 1 and 2). Pseudopodia of the type lobosa are produced by the eruptive action of the ectoplasm into which the granular endoplasm may flow. Commonly the amoeba extends more than one pseudopodium from the advancing margin. Only one of these becomes the major pseudopodium and determines the direction of movement but other pseudopodia form concurrently and may take over this role at any time. In active cultures pseudopodial advance has been measured at approximately 2pm s-‘. Spherical cysts, 3@-40 pm in dia are formed singly or in clusters in older cultures (Fig. 4) and have in their walls two distinctly visible layers which are separated by a space of several microns. Excystment has not been observed. In aged cultures, rounded forms of the amoeba, dia 17-35 pm, are the most commonly found structures. These may represent moribund cells or some stage in cyst formation.

were filtered through sterile cheese cloth to remove mycelium. Feeding trials were conducted with conidia of several other fungi including Cylindrocarpon didymum (Hartig) Wollenw., Fusarium oxysporum f.sp pini (Hartig) Snyd & Hansen, Epicoccum nigrum Link ex Link and Endothia gyrosa (Schw. Fr.) Fries. Preparation for examinatidn with the TEM, (JEOL, 100 C) was by fixing suspensions of amoebae, which were feeding actively on conidia, with 3% glutaraldehyde in phosphate buffer pH 7. After 24 h at 4°C the suspensions were rinsed with fresh buffer, centrifuged in a refrigerated centrifuge for 8 min at 6000 rev min-’ and the supernatant was discarded. The cells were then resuspended in the centrifuge tube in a few ml of 2% distilled water agar at 50°C and immediately centrifuged for 5 min at 6000 rev min-‘. This resulted in a dense suspension of cells embedded in agar. Blocks of agar, of a few cubic mm volume were cut out and fixed for 2 h in 3% acrolein in phosphate buffer pH 7, rinsed with fresh buffer and fixed again with 1% 0~0, for 2 h in phosphate buffer. The material was dehydrated in an ethanol series to absolute ethanol and embedded in Spurr resin @purr, 1969). Ultra-thin sections were cut with glass knives on a Reichert Ultracut microtome and stained with a saturated solution of uranyl acetate in 50% ethanol and aqueous lead citrate. Spores were prepared for SEM examination by the method of Old and Darbyshire (1978). RESULTS

Morphology

of the amoeba

Trophozoites of this amoeba are more or less palmate to elongate in shape and measure 6&120 pm in length and 15-57 pm in breadth. The cell cytoplasm consists of a granular endoplasm and hyaline ectoplasm. In mobile amoebae, the ectoplasmic layer forms hyaline caps to the pseudopodia. The other

Trophozoite fine structure

The fine structure of a trophozoite is shown in Fig. 10. The cell is approx. 40 pm in diameter and shows differentiation of ectoplasm and endoplasm. The greater width of ectoplasm at one side of the cell

Plate 1. Phase contrast light micrographs. Fig. 1. Trophozoites of the amoeba showing polypodial locomotion, note the hyaline advancing edge of a pseudopod (P). Fig. 2. Trophozoite Fig. 3. Fixed and stained

showing

trophozoite

predominantly

showing

nucleus conidia.

Fig. 4. Cyst showing Figs 5-9. Successive

ectocyst

stages

Fig. 7. Conidium

visible, cytoplasm

Fig. 8. Conidium

is contained

Fig. 9. Conidium Plates

lysed

of a conidium.

and has swollen within

a separate

has been ejected

226. Transmission

(CV) and 5 partly

has septa intact. has contracted

lacks septa and cytoplasm

locomotion.

vacuole

and endocyst.

in the digestion

Fig. 5. Conidium Fig. 6. Septa are no longer

monopodial

(n) contractile

electron

to form three discrete

masses.

to a more spheroidal

shape.

digestive

vacuole.

from the amoeba. micrographs.

Plate 2. Fig. 10. Ultra-thin section of a trophozoite showing differentiation of ectoplasm (ect) and endoplasm. Cytoplasmic organelles include vacuoles, mitochondria (m). Food vacuoles contain an unidentified microorganism (urn) and the cytoplasm contains bacteria (b). Fig. 11. Nucleus

and cytoplasm showing the nuclear membrane (NM), chromatin, diffuse nucleoplasm within an outer more dense zone traversed by concentric fibrils (f).

Fig. 12. Cytoplasm showing golgi bodies (g) and bacteria (b) contained within vacuoles around which vesicles (vs) are aggregated. Note the absence of electron-dense granules present in Figs 13-16.

Fine structure of fungal feeding by an amoeba

Plate 1.

647

648

K. M.

OLD

Plate 2.

et al.

649

Fine structure of fungal feeding by an amoeba probably corresponds to the anterior portion of the amoeba. Despite the tendency of amoebae to round up during fixation several of the sectioned amoebae showed a cluster of protuberances at what may be the posterior of the cell. This may correspond to the more complex uroid structures present in some Amoebidae and function as a simple holdfast or pivot during locomotion. Mitochondria with vesicular cristae are common (Figs 11 and 14) and the endoplasm is extensively traversed by endoplasmic reticulum (Fig. 13). Organelles include golgi bodies (Figs 12 and 13) small electron transparent vacuoles and other vacuoles containing amorphous residues and microorganisms which may be small protozoa (Fig. 10) in various stages of lysis. The cytoplasm also contains bacteria which are not contained within cytoplasmic membranes suggesting that they are not merely a source of food for the amoeba (Figs 10 and 12). Vesicles possibly originating from the golgi bodies have aggregated around these bacteria in some sectioned amoebae (Fig. 12). Nuclei have been sectioned occasionally and are characterized by a central body of chromatin or endosome surrounded by a diffuse area of nucleoplasm and a more dense peripheral zone. The latter is traversed by concentric fibrils or strands (Fig. 11). The nuclear membrane is composed of two unit membranes and in some sections has a distinctly beaded appearance, corresponding to the locations of nuclear pores. Mycophagy

by the amoeba

Trophozoites of the amoeba have been cultured for more than a year on suspensions of conidia of C. sativus. Conidia are ingested and contained within the amoeba1 cell. As many as five conidia have been observed within a single trophozoite. Segments of hypae can also be engulfed. The digestion process takes at least 8 h. During this time the amoeba remains motile although cells engorged with several conidia often remain relatively stationary. When fresh subcultures are set up the amoeba1 population develops a degree of synchrony and after l-2 weeks the majority of cells will be feeding on conidia. Evidence of spore lysis is the disappearance of septa after several hours (Figs 5, 6 and 7), deformation of conidium shape and the development of vacuoles within which individual conidia are contained (Fig. 8). Lysed conidia are egested along with debris from the food vacuole (Fig. 9). Although lysed spores often have irregular breaches in their walls SEM examination has failed to show discrete regular perforations in smooth spore surfaces as found with many other mycophagous amoebae (Old and Oros, 1980; Chakraborty et al., 1983). No attempt was made to conduct feeding trails with a broad range of taxa as carried out with Arachnula impatiens (Old and Darbyshire, 1978), however E. nigrum, C. didymum, E. gyrosa and F. oxysporum f.sp. pini were all utilized as food by the amoeba. Fine structure

of mycophagy

electron-dense granules approximately 3&50 nm dia (Fig. 13). Each granule appears to be composed of 3-5 sub units (Fig. 15). So far the granules have not been found in amoebae not containing conidia (Figs l&12). Figure 17 is a section through an amoeba which contains three conidia in various stages of digestion. The cell walls and protoplast of conidium No. 1 are intact. Penetration of chemicals and resin into this spore have been poor, as is common with control conidia. The control conidium shown in Fig. 21 is well embedded but the protoplast shows a negative image with electron transparent membranes. By contrast the protoplast of conidium No. 2 is well fixed and stained. Normally stained mitochondria can be seen. The better fixation is due to the extensive erosion of the melanized outer layer of the spore wall (Fig. 16) which in control conidia prevents adequate penetration of chemicals. Conidium 3 is completely lysed and is an empty shell with a thin layer of cell wall remaining. By detailed examination of these spores, and the digestive vacuoles within which they are contained, information can be derived on the mechanism of lysis. Figures 14 and 15 show the interface between the amoeba1 protoplast and the melanized outer wall of the conidium. The vacuolar membrane shows irregular invaginations and a space, about 100 nm wide is present between the amoeba1 membrane and the spore. This space is filled with an amorphous electron dense substance. In contrast, conidia 2 and 3 are contained within large food vacuoles. The membrane of the vacuole containing conidium No. 3 is lined with arrays of the electron dense granules described above as being present within the cytoplasm of the trophozoite (Figs 18 and 19). Remarkably these granules were not found associated with the membrane of the vacuole containing conidium No. 2. The fine structure of amoeba1 feeding on C. didymum was also studied. Conidia are thin walled compared to those of C. satiuus and are not melanized. The electron dense granules were found in the amoeba1 cytoplasm and in very dense arrays within food vacuoles containing partially lysed conidia (Fig. 22). Lysis of the conidia was very extensive, some food vacuoles containing folded layers of cell wall remnants (Fig. 23). In some preparations of amoeba1 feeding on either fungus vesicles were found arrayed nearby the vacuolar membrane (Fig. 19). It was not possible to distinguish whether these resulted from phagocytosis of vacuolar contents by the cell membrane or were membrane-bound products of cytoplasm about to discharge into the vacuolar lumen. A significant feature of the cytoplasm of amoebae feeding on C. didymum was the accumulation of lipid bodies (Fig. 23). These structures were also common in intact cells of the fungus and were apparently a major source of nutrition for the amoeba. Food vacuoles contained bacteria some of which were embedded in the outer wall of conidia (Fig. 20).

by the amoeba

The cytoplasm of amoebae containing condia of C. sativus is distinctive in that areas of the protoplast throughout the trophozoite contain clusters of

DISCUSSION

The amoeba studied and this will require

here remains to be identified more detailed study of its

650

K. M. OLD et al.

morphology by light and electron microscopy. A culture has been deposited with the Culture Centre of Algae and Protozoa, Institute of Terrestrial Ecology, University of Cambridge. As with earlier studies, this amoeba has been shown to digest melanized fungal conidia of C. sativus. However, instead of the perforation-lysis mechanism shown for A. impatiens (Old, 1978), which leaves the outer wall otherwise intact, the amoeba digests spore walls within food vacuoles by general erosion of the outer melanized layer followed by lysis of the protoplast. Study of the fine structure of amoebae feeding on conidia in conjunction with light microscopical examination provides an insight into the mechanism involved. The section shown in Fig. 17 is of particular value in this regard as the three conidia are clearly in successive stages of lysis. Supporting information was derived from observation of sections through individual amoeba and amoebae sectioned randomly as dense suspensions of embedded cells. Ingestion of the spore is apparently followed by close association between the amoeba1 plasmalemma and the spore surface (Figs 14 and 15). The irregular invaginations of the cell membrane may represent an increase in the surface area for passage of lytic enzymes from the amoeba1 protoplast to the spore wall. Golgi activity in the amoeba1 cell was particularly intense as shown by the frequency of these bodies in the trophozoite. Phase contrast microscopy suggests that the period before a distinct vacuole forms may be several hours although such obser-

vations are limited by the resolution of the microscope. The second stage of digestion represented by conidium No. 2 is characterized by food vacuole formation. These were also found containing lysing conidia of C. didymum (Fig. 23). Distinctive electron dense granules were present in the cytoplasm and food vacuoles of the amoebae. It is suggested that these are products of digestion of conidia. The presence of these particles in amoebae feeding on melanized and hyaline conidia indicates that they are not a product of melanin degradation. Their uniform size, shape and surface properties have resulted in aggregation into arrays in the vacuolar sap and their attraction to surfaces, either the vacuolar membrane or the remains of fungal cell wall. This suggests that they have their origins in a relatively common substrate within the conidia, possibly a major cell wall component and in Fig. 16 distinct granules appear to have formed within a macerated portion of a spore wall. It is not clear how the granules pass into the amoebae cytoplasm, or how they are egested from the cell, although breaks in the vacuolar membrane have been found (Fig. 22). Digestion of conidia results ultimately, for C. sutivus, in an empty shell composed largely of the remains of the outer melanized wall layer (Fig. 17). Conidia of C. didymum on the other hand are commonly reduced to a formless accumulation of cell wall residues. Bacteria were present freely in the cytoplasm of all dissected amoebae and in food vacuoles containing conidia. The role of these bacteria is uncertain. Many

Plate 3. Fig. 13. Detail of the cytoplasm of the amoeba in Fig. 17 showing endoplasmic reticulum, electron dense granules, a golgi body (g) and a storage granule possibly lipid (1). Fig. 14. Interface between amoeba1 protoplast and spore 1 (Fig. 17) showing the close association between the amoeba1 membrane and the conidium wall. Fig. 15.Detail of Fig. 14 showing the deeply invaginated amoeba1 membrane, the electron dense material between the membrane and the spore wall (arrows) and the electron dense granules (ed). Fig. 16. Interface between the amoeba1 protoplast and spore No. 2 (Fig. 17) showing a macerated portion of the outer spore-wall. Electron-dense granules (arrows) can be seen in the lysing wail. Plate 4. Fig. 17. Amoeba1 trophozoite containing three conidia numbered 1, 2 and 3 in successive stages of digestion. Note the presence of electron-dense granules throughout the cytoplasm and bacteria (b) within the lumina of digestive vacuoles. Arrows indicate arrays of granules in the vacuole containing conidium No. 3. Plate 5. Fig. 18. The remaining shell of spore No. 3 is shown. The digestive vacuole separates the spore wall from the amoeba1 membrane and a dense array of electron dense granules is aggregated at the amoeba1 membrane bordering the vacuole. Fig. 19. Vesicle bordering the digestive vacuole containing conidium No. 3. Fig. 20. Conidium contained within an amoeba1 food vacuole. The wall of the spore is colonized by bacteria. Fig. 21. Control conidium of C. suriuu.s. Note the multilayered spore wall with an outer melanized layer (arrow) and an inner non-melanized fibrillar matrix. The protoplast shows poor fixation due to the impervious nature of the intact conidium wall. Plate 6. Fig. 22. Electron dense granules (ed) in the amoeba1 cytoplasm and vacuole resulting from lysis of C. didymum spores. Note the break in the vacuolar membrane (arrow). Fig. 23. Amoeba containing cells of C. didymum in various stages of digestion. Note the large numbers of lipid bodies (1) in the cytoplasm and one lipid body within the intact fungal spore.

Fine structure of fungal feeding by an amoeba

Plate 3.

651

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K. M. OLD

et al.

Plate 4.

Fine structure of fungal feeding by an amoeba

Plate 5.

653

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K. M. OLD et al.

Plate 6.

Fine structure of fungal feeding by an amoeba amoebae, including members of the family Amoebidae and the genus Saccamoeba contain bacteria freely in their cytoplasm rather than enclosed in digestive vacuoles. In some instances these are endosymbionts essential for continued reproduction and cell viability (Jeon, 1980; Jeon and Jeon, 1976). The function of the vesicles clustered around the bacterial cells in some amoebae is not known. It is possible that these vesicles contained enzymes originating from either the amoeba1 cytoplasm or the bacterial cells which function in the lysis of the fungal cell wall. Spore lysis would then occur to the benefit of amoebae and bacteria alike. Soil bacteria have not so far been found to lyse intact conidia of C. satiuus (Old and Robertson, 1970) which are protected from digestion by the outer melanized layer. The protected niche of the amoeba1 cell may provide a habitat for melanin-lysing bacteria to function. Alternatively the presence of bacteria in the lumina of digestive vacuoles may be purely fortuitous as the trials were conducted in non-axenic cultures. In this case the melanized layer must be first eroded by enzymes of amoeba1 origin, exposing substrates which are susceptible to bacterial digestion. The above description and discussion of mycophagy by the amoeba is based on the interpretation of electron micrographs and further work with these and other combinations of amoebae and fungi are needed. The chemical nature of the electron dense granules and their origin need to be unequivocally established. The significance of bacteria within the amoeba1 protoplast and digestive vacuole with relation to lysis of fungal cells is also not clear. Unlike the majority of mycophagous amoebae studied so far, the feeding mechanism is not perforation Iysis but by general cell wall erosion. Acknowledgements-We thank C.S.I.R.O. Division of Entomology for EM facilities; J. M. Oros and P. Hay for preparing photographic plates; V. Mosmondor for composing montages for Figs 10 and 17; and Drs N. Malajczuk and J. F. Darbyshire for critical reading of the manuscript. REFERENCES

Anderson T. R. and Patrick Z. A. (1978) Mycophagous amoeboid organisms from soil that perforate Thielaviopsis basicola and Cochliobolus sativus. Phytopathology 68, 16181626. Anderson T. R. and Patrick Z. A. (1980) Soil vampyrellid amoebae that cause small perforations in conidia of Cochliobolus sativus. Soil Biology & Biochemistry 12, 1599167.

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Chakraborty S. and Old K. M. (1982) Mycophagous soil amoebae: interactions with three plant pathogenic fungi. Soil Biology & Biochemistry 14, 247-255. Chakrabortv S. and Pussard M. (1985) Rioidomvxa australiensis ng., nsp. A new mycophagous amoeba from Australian soil. Prorisfologica 21, 133-140. Chakraborty S., Old K. M. and Warcup J. H. (1983) Amoebae from a take-all suppressive soil which feed on Gaeumannomyces graminis tritici and other soil fungi. Soil Biology & Biochemistry 15, 17-24. Dreschler C. (1936) A Fusarium-like species of Dactylella, caoturina and consuming testaceous rhizopods. Journnl of the Washington Academy qf Science 26, 397404. ” Esser R. P., Ridings W. H. and Sobers E. K. (1975) Ingestion of fungus spores by protozoa. Proceedings of the Soil and Crop Science Society of Florida 34, 206208. Jeon K. W. (1980) Symbiosis of bacteria with amoebae. In Cellular Interactions in Symbiotic and Parasitic Relationships (C. B. Cook. P. Pappas and E. Rudolph, Eds), pp. 245-262. Ohio State Universitv Press, Columbus. Jean K. W. and Jeon M. S. (1976) Endosymbiosis in amoebae: recently established endosymbionts have become required cytoplasmic components. Journal of Cell Physiology 89, 337-347. Old K. M.1977) Giant soil amoebae cause perforation and lysis of spores of Cochliobolus sativus. Transactions of the British Mycological Society 68, 277-28 1. Old K. M. (1978) Fine structure of perforation of Cochliobolus sativus conidia bv giant amoebae. Soil Bioloav -. & Biochemistry 10, 509-516.Old K. M. and Darbyshire J. F. (1978) Soil fungi as food for giant amoebae. Soil Biology & Biochemistry 10, 93-100. Old K. M. and Oros J. M. (1980) Mycophagous amoebae in Australian forest soils. Soil Biology _. & Biochemistry 12, 169-175. Old K. M. and Robertson W. M. (1970) Effects of lytic enzymes and natural soil on the fine structure of conidia of Cochliobolus sativus. Transactions of the British Mycological Society 54, 343-350. Page F. C. (1976) An Illustrated Key to Freshwater and Soil Amoebae. Freshwater Biological Association. Scientific publication No. 34. Prescott D. M. and James T. W. (1955) Culturing of Amoeba proteus on Tetrahymena. Experimental Cell Research 8. 256-258. Pussard M.. Allabouvctte C. and Pons R. (1979) Etude preliminaire d’une amibe mycophage Thecamoeba grant&a ssp. minor (Thecamoebidae, Amoebida) Protistologica 15, 139-149. Pussard M., Allabouvette C., Lemaitre I. and Pons R. (1980) Une nouvelle amibe mycophage endogee Cashia mycophaga n. sp. (Hartmannellidae, Amoebida) Protistologica 16, 44345 1. Spurr A. R. (1969) A low viscosity epoxy resin embedding medium for electron microscopy. Journal of Ultrastructural Research 26, 3143.