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Observations on the fine structure of the hemiascomycete Cephaloascus fragrans Hanawa, with emphasis… Higham, Michael Thomas 1971

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i OBSERVATIONS ON THE FINE STRUCTURE OF THE HEMIASCOMYCETE CEPHALOASCUS  FRAGRANS HANAWA, WITH EMPHASIS ON CELL WALLS, SEPTA, AND ASCOSPORES. by ;MICHAEL THOMAS HIGHAM B.Sc^, University of B r i t i s h Columbia, 1967 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the 'Department of BOTANY We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA JULY 1971 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permi ssi'on for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his. representatives. It is'understood that copying or publication of"this thesis for financial gain shal1 not be allowed without my written permission. Michael Thomas Higham Department of BOTANY The University of British Columbia Vancouver 8, Canada Date JULY, 1971 ABSTRACT The presence of multiperforate septa i n vegetative hyphae of Cephaloascus fragrans i s demonstrated by t h i n -s e ctioning. The number of micropores per septum i s much greater than that reported i n other m u l t i p e r f o r a t e l y septate fungi. Brown encrustations of the ascophores, previously observed by light-microscopy, are resolved as granular depositions between the ascophore wall and an outer i n v e s t i n g membrane. Similar depositions are observed between the ascal walls and t h e i r i n v e s t i n g membranes. The morphology of these b l i s t e r - l i k e depositions i s studied by t h i n - s e c t i o n i n g , freeze-etching and scanning electron microscopy. Several stages of ascosporogenesis are observed which may i n d i c a t e a process d i f f e r i n g from that shown i n other Ascomycetes. The observed structure of ascospores i s i n agreement with that of other cucula.te ascospores studied by elec t r o n microscopy. i i i TABLE OF CONTENTS Abstract i i Table of contents .. i i i L i s t of plates i v Acknowledgment v Introduction • • 1 Materials and Methods . ...y j 5 Observations 9 Discussion ..................•.•••'« 18 Summary • • • \ ,• ••• 30 Plates f..-? •; •; -.J...r. 31 Bibliography > i . . . •• 44 Appendix •.,. • » •'•••jv? 51 i v LIST OF PLATES Plate 1. Electron micrographs of the plasma membrane of vegetative hyphae. Plates 2 & 3. Electron micrographs of uniperforate and multiperforate septa. Plates 4 & 5. Light and electron micrographs of the ascophore and i t s "encrustations". Plate 6. Light and electron micrographs of the ascus and ascus septum. Plates 7 - 9 . Electron micrographs of stages of asco-sporogenesis. Plate 10. Electron micrographs of ascus "encrustations" and the ascus-investing membranes. Plate 11. Electron micrographs of mature ascospores held i n mucous droplets at the ascophore t i p s . Plate 12. Electron micrographs of i n t e r n a l structure of mature ascospores. Plate 13. Results of vitamin additions to growth media. V ACKNOWLEDGMENT The author wishes to thank the following people for their assistance during the research reported in this thesis, and during preparation of the thesis it s e l f o Dr. R.J. Bandoni, Professor, to whom, as supervisor of this thesis, I am most indebted. Dr. T. Bisalputra, Associate Professor, who generously made available his laboratory and electron microscope f a c i l i t i e s , and provided financial support for this research from his N.R.C. Grant A-2288. Dr. G.C. Hughes III and Dr. K.M. Cole, Associate Professors, who, upon reading the original manuscripts made many useful suggestions* Marguerite Long and Shirley Reid who typed this thesis. And to the Faculty, S t a f f and Students of ,the Botany Department who have a s s i s t e d the author during t h i s work. 1. INTRODUCTION The fine-structure of Hemiascomycetes has been discussed i n reviews of fungal cytology by Hawker (1965), Moore (1965), and Bracker (1967), and by Matile et a l (1969) i n a review restricted to cytology of yeasts. More recent studies include those on Saccharomyces (Lynn and Magee, 1970; Strunk, 1970; Zickler, 1970; Ghosh, 1971; Moens, 1971); Pityrosporum (Keddie and Barajan, 1969); and Wickerhamia (Bauer, 1970). Despite the number of electron microscopic studies of the unicellular and pseudomycelial yeasts,<there are few reports on the fine structure of mycelial yeasts. Endomycopsis was f i r s t studied by Kawakami and Nehira (1958b), who observed sporulation and germination. The septa in this genus were studied by Takada et j i l (1965) who reported plasmodesmata providing cytoplasmic continuity through the cross-walls. Kreger-van Rij and Veenhuis (1969) confirmed the presence of plasmodesmata and also demonstrated swollen septa resembling the basidiomycetous dolipore septum. An ascospore of Endomycopsis was also shown i n thin-section by Kreger-van R i j (1969). Endomyces was studied by Kreger-van Rij and Veenhuis (1961) and i t also possesses swollen "dolipore" type septa. The structure of c e l l walls of Endomyces was shown in thin-section by Hagedorn (1964), and after freeze-etching, by Streiblova (1968). 2. The f i n e structure of the mycelial stage of Blastomyces has been studied by Edwards and Edwards (1960), Carbonell and Rodriguez (1968), and Garrison et a l (1970). The l a t t e r authors also observed the mycelial phase of Histoplasma, which was l a t e r studied with the scanning e l e c t r o n microscope (Garrison and Lane, 1971). The asporogenous yeast, Geotrichum, has been observed by Hashimoto et a l (1964), Wilsenach and Kessel (1965a), and Kirk and S i n c l a i r (1966), a l l of whom demonstrated the presence of micropores or plasmodesmata i n the septa. Only a s i n g l e micrograph, (of the ascospore), of Nematospora has been published (Kreger-van R i j , 1969). One of the most i n t e r e s t i n g fungi i n the Endomycetaceae i s Cephaloascus fragrans Hanawa. In t h i s genus, sexual reproduction involves the production of large, erect, brown-encrusted ascophores. From the apex of each ascophore, whorls of a s c i i n branched chains are produced, (see F i g . 20). The a s c i mature b a s i p e t a l l y , each r e l e a s i n g four cuculate ascospores i n t o a mucous droplet which i s retained at the ascophore apex. Cephaloascus fragrans was f i r s t i s o l a t e d from the external ear of a st,udent by Hanawa (1920), and was not rediscovered u n t i l D.E. Wells (1954) obtained i s o l a t e s from cut lumber. Unaware of 3. the e a r l i e r publication by Hanawa, Wells proposed the name Ascocybe grovesii for this organism. Following the c l a s s i f i c a t i o n of Zender (1925), she placed this monotypic genus i n the Endomycetaceae, based on the production of cuculate ascospores and the presence of d i s t i n c t ascophores. Dixon (1959) studied the relationships between the ascal and conidial stages of Ascocybeand showed the occurrence of an alternation of heteromorphic generations: d i p l o i d ascophores and a s c i , and haploid vegetative mycelium, conidia, and ascospores. The nuclear behaviour of Ascocybe was observed by "Wilson (1961) using orcein and hematoxylin preparations. The diplophase was seen to be i n i t i a t e d by fusion of two nuclei i n the c e l l which becomes the base of the ascophore. No nuclear fusion occurs i n the ascus, and meiosis was seen to take place i n basipetal order i n the chains of a s c i . The synonomy of Ascocybe grovesii Wells and Cephaloascus  fragrans Hanawa was indicated by Ainsworth (1961), and the name Ascocybe grovesii was rejected by Schippers-Lammertse and Heyting (1962) after comparing isolates of both type cultures. These authors, using the c l a s s i f i c a t i o n of Gaumann (1949), concurred with Wells i n placing Cephaloascus i n the Endomycetaceae, and proposed the erection of a new subfamily, the Cephaloascoideae, because of the unique structure of the f r u c t i f i c a t i o n . The habitat and n u t r i t i o n of Cephaloascus was studied by Batra (1963) who isolated this fungus from ambrosia beetle tunnels i n recently f e l l e d pine trees. He noted that ascophores produced i n these tunnels were devoid of brown pigmentation. The f i r s t report of the ultrastructure of Cephaloascus was by Besson (1967), who observed the ascophore septa and mature ascospores. She demonstrated three wall layers of the spores, and a centrally uniperforate septum i n the ascophore. The only other published electron micrographs of Cephaloascus are two of septa of vegetative hyphae (Kreger-van R i j and Veenhuis, 1969). These septa are also centrally uniperforate, and one has a number of small granules associated with the pore aperture. This report presents a wide range of u l t r a s t r u c t u r a l observations of Cephaloascus fragrans, obtained by several techniques of electron microscopy. 5. MATERIALS AND METHODS 1. Cultures,, The c u l t u r e of Cephaloascus fragrans Hanawa used i n t h i s work was provided by Dr. R.J. Bandoni, and bore the U.B.C. Culture C o l l e c t i o n number 204. The cult u r e o r i g i n a l l y came from the Government of Canada Forest Products Laboratory, Vancouver, B.C. Preliminary growth t r i a l s showed that a standard mycolo^ical medium, Malt-Yeast-Peptone agar, M.Y.P. (Appendix), while supporting good vegetative growth, severely r e s t r i c t e d production of the perfect stage. A s i m i l a r condition was encountered by Schippers-Lammertse and Heyting (1962) with an o l d larboratory c u l t u r e of t h i s fungus. Attempts to increase ascophore production by a d d i t i o n of vitamins were successful: pyridoxine, (0.1 m g / l i t r e ) , stimulated ascophore productions while s l i g h t l y reducing the vegetative growth r a t e , and both b i o t i n (5 u g / l i t r e ) and i n o s i t o l (5 mg/litre) stimulated sexual reproduction and vegetative growth. The r e s u l t s of these growth t r i a l s are presented v i s u a l l y i n Plate 13. Cultures for e l e c t r o n microscopy were grown at 20 - 25° C. on M.Y.P. agar containing a four-vitamin s o l u t i o n (Appendix), which provided for e x c e l l e n t ascophore production. Sampling was done a t 15-20 days when colony diameter was 3-4 cm., and v e g e t a t i v e and sexual stages formed a dense m y c e l i a l mat. 2. L i g h t Microscopy. Whole mounts of the fungus were prepared by removing p o r t i o n s of the m y c e l i a l mat w i t h f i n e f o r c e p s , and tea s i n g apart the hyphae and ascophores. Water-mount s l i d e s were observed and photographed w i t h b r i g h t - f i e l d or phase-contrast o p t i c s using a Zeiss Photomicroscope. 3. Scanning E l e c t r o n Microscopy. (S.E.M.) Small areas of the hyphal mat were gently removed from the agar surface and were immediately q u i c k - f r o z e n i n Freon 22 (cooled i n l i q u i d n i t r o g e n ) , followed by immersion i n l i q u i d n i t r o g e n . The frozen samples were then placed on the precooled stage of a Pearse Speedivac Tissue Dryer (Model 1) and freeze-d r i e d f o r 24 hours at -40° C. Fo l l o w i n g f r e e z e - d r y i n g , the samples were mounted onto S.E.M. specimen stubs u s i n g DAG 416, an adhesive c o n s i s t i n g of a s i l v e r suspension, (Atcheson C o l l o i d s Co.), which serves to ground the specimen and prevent e l e c t r o n - c h a r g i n g . Samples were then rotary-shadowed w i t h gold i n a Mikros VE-10 vacuum evaporator, and examined w i t h a Cambridge Stereoscan e l e c t r o n microscope operated at 20 kV. 7. 4 . Thin-sectioning. Entire culture dishes with 1 5 - 2 0 day colonies were slowly flooded with water agar ( 0 . 8 7 » ) , which was allowed to s o l i d i f y . This procedure was found to confer greatest protection to asci and ascophores during subsequent chemical f i x a t i o n procedures, and also served to dissipate the clusters of ascospores held i n mucous drops around the ascal whorls. Each agar-embedded colony was cut, using an acetone-cleaned razor blade, irito 1 mm. thick s l i c e s ; which were immediately submerged i n f i x a t i v e . Fixation was carried out for 1 - 2 hours at room temperature, since t r i a l s at 4 ° C. showed no improvement i n tissue preservation. The single f i x a t i v e used consisted of 2 . 5 7 o glutaraldehyde i n 0 . 0 5 M. sodium cacodylate buffer (pH 6 . 8 ) combined with equal volumes with 27o osmium tetroxide prepared i n d i s t i l l e d water. This f i x a t i v e produced the best results i n t r i a l s with single and double fi x a t i o n s at various pH's, and with several buffer concentrations. Dehydration was accomplished by a graded ethanolj series, followed by embedding i n low-viscosity epoxy resin (Spurr, 1 9 6 9 ) or i n Maraglas (Bisalputra and Weier, 1 9 6 3 ) v i a a propylene oxide series. After heat polymerization i n a vacuum oven, the epoxy-embedded specimens were trimmed and sectioned on a Sorvall Porter-Blum MT-1 ultramiqrotome, using a duPont diamond 8. knife. Sections were collected on Formvar-coated copper grids, stained with uranyl acetate (saturated solution i n 707o methanol) followed by lead c i t r a t e (Reynolds, 1963), and were observed with a Zeiss E.M.-9A electron microscope. 5. Freeze-Etching. Portions of the hyphal mat were removed with fine forceps and placed i n gold support cups, which were then submerged i n Freon 22 (cooled i n l i q u i d nitrogen) followed by submersion i n l i q u i d nitrogen. The quick-frozen mycelium and cup were quickly transferred to the precooled specimen stage of a Balzers BA-360M apparatus. The specimens were subjected to the freeze-etching method of Moor et a l (1961), using a cutting temperature of -110° C. and sublimation times of 30-90 seconds at 3 x 10 ^  Torr pressure. The etched surfaces were shadowed with platinum/ carbon, then, strengthened by evaporation of carbon. The re s u l t -ing replicas were treated with sulphuric acid and sodium hypochlorite to remove c e l l u l a r debris (Kalley and Bisalputra, 1970) , and were collected on Formvar-coated copper grids and observed i n a Zeiss E.M.-9A electron microscope. 9. OBSERVATIONS Hyphae In freeze-etched samples, the exposed plasma membranes of the hyphae are seen to possess grooves measuring 100-200 mu in length, 25-35 mp in width and 20-30 mu i n depth. When observed from the exterior of the c e l l , (Fig. 1), the exposed membrane fracture face i s covered with granules 75-95 A in diameter. This face represents the interior of the plasma membrane, the outer half of which has been removed by the fracture. Several indications of i • f i b r i l l a r elements can be seen i n the grooves, (Fig. 1, arrows). In Fig. 2 the membrane is seen from the cel\ interior, and a lack of granules i s evident on this face, suggesting that i t i s the true cytoplasmic face of the membrane. The interior aspects of grooves show no f i b r i l l a r structures or granules. F i b r i l s of 35-50 A diameter occur i n the grooves and a l l along the fractured wall-membrane interface (Fig. 3). These f i b r i l s appear to connect grooves to wall material (Fig. 3, arrows). The hyphal wall, 80-100 mu in thickness, shows no detectable layering in freeze-etched preparations. It appears granular rather than f i b r i l l a r in composition, although this may be caused by the plane of fracturing. In thin-section, the vegetative hyphae are found to ibe divided by t y p i c a l ascomycetous septa, 70-80 mu thick, with a single central pore, 50-60 mu i n diameter (Fig.4). Several dark granules are located on either side*qf the septal pore, but none* are seen blocking pore .apertures. The;se septate J hyphae are approximately lu i n ^ diameter and occur i n the surface mat and below the agar surface. A larger hyphal type (4-6u i n diameter), also occurs which i s r e s t r i c t e d to the surface mat. These large hyphae are divided by multiperforate septa (Fig. 5), 100-200 rryj thick. As shown i n Figure 6, there are approximately 35 micropores i n the septum^cross-section, each separated from the next by 100-350 mu. From th i s data, one can calculateithat in^a discoid septum 2 of the diameter i l l u s t r a t e d (5.7 ju), with an arek of 24 ^  , there could be from 200 to 1000 perforations (determined from maximum and minimum pore'spacing). This high number of perforations i s supported by Fi g . 7 which shows the porjes of one1 .septum i n both longitudinal and cross section. There i s no evident organization of pore spacing and pores separated by as l i t t l e as 20 mju are observed ( F i g . 8). When observed at high magnification (Figs. 8 and 9) the pores are seen to be membrane-lined by a continuation 4 of the plasma membrane. In Figure 8 the pore-aperture diameter i s approximately 30 mu (excluding the 85-95 A membrane). In longitudinal section, (Fig. 9), the pores taper toward the middle of the septum, where, the diameter i s reduced to 18-20 mu. In Figures 6 and 9 the pores appear to be plugged by a dark-staining (osmiophylic) material; i n Figures 7 and 8, the pores contain no apparent plug m a t e r i a l . The Ascophore Under low power bright f i e l d ( F i g . 10) and phase contrast i l l u m i n a t i o n ( F i g . 11), the ascophores appear to be encrusted with* p a r t i c l e s from a short distance below the apex down to the l e v e l of the mycelial mat. At higher magnification, however, the encrustations are seen to be smooth rather than angular, i n d i c a t i n g a n o n - c r y s t a l l i n e nature ( F i g . 12). In the scanning electron microscope, the exterior encrustations are resolved as 'blisters'. In one' ascophore' ( F i g . 13) , which apparently l o s t i t s cytoplasm during freeze-drying, the - i n t e r i o r of the ascophore wall also appears i r r e g u l a r . These i n t e r i o r i r r e g u l a r i t i e s may Represent cytoplasmic remnants. Figure 14 shows the surface topography of an ascophore; i t s e n t i r e surface i s covered"with b l i s t e r s of varying s i z e s . The b l i s t e r s have very smooth surfaces, suggesting a thi n wall or membranous covering. In order to ensure that the b l i s t e r morphology i s not an a r t i f a c t of S.E.M. preparation techniques, several ascophores have been freeze-etched. The r e s u l t s of freeze-etching generally confirm the S.E.M. ascophore topography ( F i g . 15). The " b l i s t e r " surface i s smooth and other areas of the ascophore possess lesser undulations. The i n t e r i o r of the b l i s t e r , exposed by 12. cross-fracture, does not demonstrate a d i s t i n c t boundary, and the contents appear continuous with the ascophore w a l l . The explanation for the smooth b l i s t e r surface i s apparent i n thin section. Figure 16, o'f an ascophore i n cross-section, shows that the c e l l wall (0.3-0.6u thick) i s bounded on the inside by the plasma membrane and on the outside by an investing membrane. At higher magnification, ( F i g . 17), i t i s evident that the b l i s t e r s are formed by deposition of granular material between the wall and i t s investing membrane. The l a t t e r i s 110-130 A thick; and the plasma membrane measures 75-90 A i n thickness. The investing membrane also d i f f e r s i n appearing as a single s o l i d l i n e , whereas the plasma membrane shows a more ty p i c a l trilaminar structure. The ascophore wall i s bilaminar, with a granular outer layer (approx. 240 nyi thick) and an inner amorphous layer (approx. 130 ima t h i c k ) . In longitudinal section, however, the" wall shows a fibrous pattern (Figs. 18 and 19), suggesting a primarily longitudinal f i b r e orientation. The b l i s t e r contents are resolved as granules 150-180 A i n diameter;: the same type granules also comprise a 30-50 mu layer over the entire ascophore surface. The Ascus The asci are produced i n whorls from c e l l s at the ascophore apqx and, normally, contain four cuculate spores, (Fig. 20). Figure 21 shows a basal c e l l with an ascus attached, and the remains of a second ascus attachment s i t e i s also present. The attached ascus has borne two asci ^ as evidenced by attachment scars at the ascus apex. These scars are closed by septa. The ascus contains mature spores, only three of which are seen i n most sectioiis because of their special arrangement. There are cytoplasmic membranes, s u p e r f i c i a l l y resembling endoplasmic reticulum, at the base of the ascus, ( F i g . 22), and an incomplete seiptum occurs between the ascus and basal c e l l . At high magnification, ( F i g . 23), the septal pore apparatus appears to consist of a short tube-like structure, approximately 50 rmujin diameter, and extending 50-70 mu on either side of the 100 mu thick septuiru" "The.walls of the tube are 30 mu th-Lck, and may be closed at the basal c e l l side. In a maturing ascus, as evidenced bys incompletely formed spores, ( F i g . 24), the ascospores are surrounded by numerous cytoplasmic membrane fragments, and the ascus i s enclosed by onje, and i n places by two (Fig. 24, arrows), investing membranes. No membranes are seen on the outside of the spore walls, although spore plasma membranes are evident. In Figure 25, the cytoplasmic membranes of the ascus are resolved as trilaminar structures 60-70 A thick, while the spore plasma membranes measure 70-90 A thick. The small granules on the ascal cytoplasmic membranes are 30-60 A i n diameter, and therefore are far too small to be ribosomes. In several places, (Fig. 25, arrows), the cytoplasmic membranes are 1 4 . closely appressed to the immature outer spore w a l l . These1membranes ar el'probably derived from a breakdown of endoplasmic reticulum or ascal plasma membrane. The outline of the outer spore w a l l , (Fig. 26, arrows) suggests that thite area w i l l become part of the "brim" of the cuculate1- spore. Again, no spore-investing membran^ i s present to form the outer boundary of the spore w a l l . Figure 27 shows a large fragment of cytoplasmic membrane p a r t i a l l y surrounding the spore. This piece of membrane i s the- same thickness (70-80 A), as the spore plasma membrane, and i s devoid of the'i granules characteristic of the majority of ascal cytoplasmic membranes. The staining of the-spore plasma membrane i s very s l i g h t , while the |ascal cytoplasmic membrane fragment i s densely stained, whicb. may indicate chemical/, differences or merely beibecause of- protection of the spore plasma membrane \by the spore w a l l . In the mature lascus, ( F i g . 28) an outer investing merhbrane i s v i s i b l e on the spores. There^ are* fewer cytoplasmic membrane fragments, and the ascus wall and i t s investing membranes appear to be degenerating (Fi g . 28, arrows). Higher magnification, ( F i g . 29), shows the location of spore wall layers and membranes, the- remains of cytoplasmic membranes, and the' absence of ascus-investing membranes. The^ascus plasma membrane i s not apparent i n Figures 28 and 29 and was never observed i n mature a s c i . The remnants of this membrane probably are represented by the cytoplasmic membranes, either as a resul t of natural breakdown or as a f i x a t i o n a r t i f a c t . 15. The ascus boundary i s interesting i n that b l i s t e r s , (Figs. 30-32) ,! similar to those of the ascophore, (Figs. 16-19), are /formed below the ascus/-investing membrane. However, the ascal b l i s t e r s d i f f e r i n often having two investing membranes (Fig. 32), and the granular contents do not form a continuous thin layer outside bliister areas as they do i n the^ ascophore, (Figs. 30 and 31). Both investing membranes stain much moire densely than cytoplasmic and plasma membranes, and usually appear as single l i n e s , the inner membrane being 75-95 A thick, while; the ouster i s t y p i c a l l y thinnery approximately 60 A thick,. At only one s i t e can a trilaminar pattern be seen, ( F i g . 32, arrows) and here both investing membranes appear to be of the same thickness, 50-60 A , indicating that additional dimension i s a r e s u l t pf staining of material located on or near the! inner membrane. The Mature Ascospore • Mature ascospores are released from the ascus by dissolutioyi of the ascus wal\ and investing membrane, and are retained i n a drop of mucous at the ascophore apex. Theimucous presumably i s derived from the ascus cytoplasm. In colonies, movemept of the ascophores causes spore^droplets to adheye-to one \.another , (Fig. 33). This produces masses of sporep (Fig. 34), numbering i n the'thousands, at the tops of clusters of over one hundred ascophores. In Figure 35, an isolated ascophore can be^ seen, with some 'of i t s basal c e l l s s t i l l i n t a c t , supporting the mucous balj. 16. of hundreds of ascospores produced by this single - f r u c t i f i c a t i o n . At higher magnification ( F i g . 36), strands of dried mucous can be seen connecting smooth-surfaced and wrinkled ascospores. Thei. rough surfaces of spores may be caused i n part by thel freezer-drying preparatory technique jused, although a lesser degree of irregulkri^ty i s also seen i n freeze-etched spores? ( F i g . 37). Partj of the invent-ing membrane has been removed, exposing the f i n e l y granular outer ' sporei.wall. In cross-fracture, ( F i g . 38), the;wall material of the brim i s exposed, and i t appears to contain pockets where large » granules were?'removed by fracturing, or where no wall material was deposited during spore maturation. UnlikeJthe hyphal plasma membranes, the spore investing membranes are devoid of grooves. The absence of a granular fracture faqe indicates that the exposed surface { i s a true outer membrane face. This type of fracturing may r e f l e c t membrane chemistry differences, sinc^ i t also occurs on the ascophore- investing membrane. In thin-section, (Fig. 39), the mature spore wall can be distinguished as three layers, the inner and outer layers being of the same low electron density and of thei. same-, thickenjss, 60-80 mu. These layers are separated by an electron transparent zone-, 25 mji wide* The spore cytoplasm contains several mitochondria, and a large vacuole, a feature' found i n approximately one third* of ascospores examined i n thin section. I d e n t i f i c a t i o n of nuclei could not be made with certainty, duetto poor staining of internal membranesi. The* fibrous nature of the inner wall* ,layer i s demonstra-ted i n Figure 40, and the continuity of the brim with the outer wall layer i s evident, as i s the Ntrilaminar structure of the investing membrane, (Fig. 40, double•arrows), where brim wall material i s not closely appressed to the membrane. This was a common observation (see also Figures 29 and 39) and may be a result of addition of this membrane after wall formation has ceased, since i f wall shrinkage was the cause, orie would expect both edges of the brims to have excess membrane, which was not the case i n most mature spores J observed following^ chemical f i x a t i o n procedures. 18. DISCUSSION Hyphae .Invaginations of the fungal plasma membrane, (Figs. 1-3) were f i r s t demonstrated i n freeze-etched preparations of Saccharomyces cerevisiae by Moor and Muhlethaler (1963). They have since been observed i n a wide variety of fungal c e l l s and spores: P e n i c i l l i u m , (Sassen _et al_, 1967); Pyrenochaeta, (Hess, 1968); Wickerhamia (Bauer, 1970); Hypholoma, (Bonchat and Bemoulin, 1970); Coniothyrium, (Jones and Johnson, 1970); Psilocybe, (Stocks and Hess, 1970); T i l l e t i a , (Allen _et a l , 1971); and Paneolus, ( G r i f f i t h s , 1971). However, there are few reports of such grooves i n the plasma membranes of hyphal c e l l s , (Matile _et _al, 1965), such as i s shown i n this report. The physical, structure of these invaginations i s similar i n a l l fungi so far studied. They consist of a groove 300-400 & wide, 500 A deep, and vary i n length up to 1 j». In chemically-fixed and sectioned material, preservation of the membrane has rarely been good enough to show such invaginations; only those-micrographs by Darling et a l (1969) and Ghosh (1971) have closely approximated the frozen-etched morphology. Moor and Muhlethaler (1963) indicated that the number of grooves increases with increasing c e l l age, but this i s more d i f f i c u l t to determine i n mycelial fungi and was not attempted with Cephaloascus. There are several reports of anastomosis of grooves (Nec*as et. a_l, 1969; 19. Ghosh, 1971) but i n Cephaloascus a l l grooves were isolated (Figs. 1-3). The p a r t i c l e s on the exposed fracture surface'of the plasma membrane (Figs. 1 and 3), observed i n most other freeze-etching studies, were believed, by Sassen et a l (1967), to be multienzyme complexes. Their conclusion was based on similar studies of algal plasma membranes by Muhlethaler _et a l (1965). However, these, p a r t i c l e s were subsequently isolated and characterized as glyco-protein by Matile _et _al_, (1967). The absence of such p a r t i c l e s from the invaginations has been p a r t i a l l y explained by N^cas (.et a l (1969), who presented micrographs of the reverse side of the fracture face. This face bears a groove-like pattern of p a r t i c l e s on an otherwise smooth surface. This interpretation supports the contention of Branton (1966) that the freeze-etching technique fractures within membranes, rather than along "either-*.outer face. The l a t t e r viewpoint i s supported by the observation of f i b r i l s ( F i g . 3) extending from wall material to the fracture surface. Similar f i b r i l s have been observed by Streiblova (1968) and G r i f f i t h s (1971) and are generally considered to represent glucan (Matilef -et all, 1969) , although Jones and Johnson (1970) suggest that, i n Coniothyrium, they are c h i t i n f i b r i l s . Hyphal.' c e l l s of Ascomycetes t y p i c a l l y are septate; the- septa have a centrally located pore, and often have densely staining Woronin bodies associated with them. The Woronin bodies areVbelieved 20. to function as pore valves to control cytoplasmic flow (Bracker, 1967). In Cephaloascus, this type of septum (Fig. 4) repeatedly was observed i n vegetative hyphae of the mycelial mat and i n those penetrating the substrate. The septa never were observed with ( associated bodies large enough to block pore-appertures, but groups of smaller granules, ( F i g . 4, arrows) were common. Similar septa with granules were seen by Kreger-van R i j and Veenhuis (1969) i n this fungus. Besson (1967) also found such septa i n the ascophore of Cephaloascus. No complex pore structures were observed such as those described by Furtado (1971) i n Sordaria, or by Moore (1963) and C a r r o l l (1967) i n Ascodesmis. I t i s Evident that the' vegetative 1 hyphae of Cetphaloascus are functionally coenocytic since the sizq of the poije apperture, ja, has been shown to allow organelle passage i n otheir fungi. In colonies of C_. fragrans, there i s a t r a n s i t i o n from fine hyphae 1 )i diameter, to large 6-10 p. ascophores. This t r a n s i t i o n apparently i s accomplished by intermediate-sized hyphae, 3-6 ju i n diameter, which occur only i n the^hyphal mat and are never seen i n the* substrate or i n the a e r i a l mycelium above the colony. I t was i n this hyphal type that multiperforate septa were found (Figs. 5-9). The occurrence |of multiple perforations i n fungal septa was f i r s t reported by Hashimoto j|t a l , (1964) i n Geotrichum. This observation was repeated-by Wilsenach and Kessel (1965a) and by Kirk and S i n c l a i r (1966). Hawker et a l (1966) described plasmode^smata-like1 periforations i n Rhizopus and G i l b e r t e l l a . Micropores similar to ^ those > 21.' shown i n Cephaloascus ( F i g s . 5 and 6) were demonstrated by Takada jet a l (1965) and by Kreger-van R i j and Veenhuis (1969) i n 3 species of the, c l o s e l y r e l a t e d genus, Endomycopsis. The most obvious d i f f e r e n c e i n m u l t i p e r f o r a t e septa of Cephaloascus and Endomycopsis i s the number of pores per septum. Endomycopsis has l e s s than 100, but Cephaloascus has several hundred to over 1000 pores. The t o t a l area of pore apertures i n such septa can be c a l c u l a t e d to represent 0.5-2.07. of the t o t a l area of the cross w a l l , and i s the same as that percentage c a l c u l a t e d f o r the u n i p e r f o r a t e septum of the f i n e vegetative hyphae. The f u n c t i o n of t h i s septum i s , t h e r e f o r e , not to exclude cytoplasmic communication, but to prevent o r g a n e l l e movement by v i r t u e of the s i z e (10-30 rryu) of the micropores. This creates an e f f e c t i v e i s o l a t i o n of the reproductive structures from an i n f l u x of o r g a n e l l e s , while maintaining n u t r i t i o n a l c o n t i n u i t y with the remainder of the colony. I t should be pointed out that the m u l t i p e r f o r a t e septum described i n Fusarium ( R e i c h l e , 1965) i s not ( comparable i n f u n c t i o n to those of Cephaloascus, Endomycopsis, and others with micropores. The apertures (approximately 10) i n the Fusarium septum are of the same diameter (0.1 u ) , as those of a t y p i c a l u n i p e r f o r a t e , ascomycetous septum, and thus would not exclude o r g a n e l l e passage. The Ascophore Well6 (1954), i n d e s c r i b i n g the ascophore of Cephaloascus (= Ascocybe g r o v e s i i ) stated that: "The w a l l becomes ye l l o w i s h and then deepens to a medium 22. brown, at the s a m e time becoming roughened and encrusted with irregularly-shaped brown p a r t i c l e s . " Despite^ the uniqueness o f this structure, l i t t l e more informa-tion on i t s chemical or physical structure i s available. Attempts to isoJjate and characterize £he brown pigment have .thus far been unsuccessful (T. C s e r j e s i , B.C. Forest Products Laboratory, personal communication). The* only published micrograph of the Cephaloascus ascophore, (Besson, 1967) , provides the ..information that the c e l l walls are bounded by two membranes; an inner plasma membrane, and an outer investing membrane; and that the ascophore cross walls are of the centrally uni|>erforate type! Using l o W | magnification l i g h t microscopy, (Figs. 10 and 11), the ascophore closely f i t s the description by Wells (1954). However, under high power, phase> contrast optics, (Fig. 12), the so-called encrustations appear as smooth^ rounded " b l i s t e r s " rather than irregularly-shaped p a r t i c l e s . U t i l i z i n g the higher magnification and greater dep^ tbX of f i e l d of the scanning electron microscope, (Figs. 13 and 14), the. I'particles" are seen to be G r o u n d e d b l i s t e r -l i k e structures that appear continuous with the surface of the-ascophore and thus with one another. This suggests that the "encrustations" are actually part of the c e l l w a l l , and are therefore covered by the inventing mjembrane i l l u s t r a t e d by Besson (1967). The i n t e r i o r of a blisters, rqvealed by freeze-etching ( F i g . 15) shows apparent continuity with the ascophore w a l l . 23. In thin sections, however, the b l i s t e r contents are, revealed as granular depositions s t r u c t u r a l l y d i s t i n c t from the ascophor-e wall (Figs. 16-19). Thel presence 'of a bounding membrane may explain d i f f i c u l t i e s encountered i n i s o l a t i n g the granular pigment, and a knowledge of thfe structures involved w i l l hopefully be of somej help. The^function, i f any, of pigment granules outside, the ascophoreiwall i s not obvious. L i t t l e increase/, i n strength i s indicated, but the pigment may offerIprot^ction to d i p l o i d n u c l e i i from high l i g h t i n t e n s i t i e s . I t w i l l be/of intenest to see i f future investigations demonstrate similar fine structure i n "encrusted" hyphae of other fungi. The Ascus The septum between the ascus and basal c e l l ( F i g . 21-23), of Cephaloascus i s centrally perforate and there i s a s t r u c t u r a l l y simple pore"apparatus (Fig . 23). Since- septa between a s c i , or where asci had broken away, are complete crosswalls, i t i s l i k e l y that the basipetal maturation of asci results i n closure of the central pores as each successive ascus matures, thus preventing loss of cytoplasm from the immature ascus immediately below. Nov complex septal structures of the types reported by C a r r o l l (1967b) i n Ascodesmis and Saccobolus were found i n Cephaloascus. The wall.-of the ascus consists of a single layer and i s bounded on the outside by a darkly staining membrane, the ascus investing membrane (Fig. 24 and 30-32). Similar structures have appeared i n the micrographs by several authors in the asci of a wide variety of Ascomycetes (Carroll, 1967b; Reeves, 1967, Kreger-van R i j , 1969; K. Wells, 1970) but the investing membrane has not been previously described as such, since the trilaminar structure (Fig. 32) was not demonstrated. In the present study, the ascus investing membranes often occur i n pairs (Fig. 24 and 32), but only for part of the ascus surface. The explanation for this could be that the outer membrane i s more easily destroyed during processing for thin sectioning; that this membrane i s i n the process of natural breakdown during ascus maturation, or that this outer membrane i s an art i f a c t produced by adherance of liberated cytoplasmic membrane fragments from mature ,asci to: the single investing membrane of maturing asci. The observed differences i n staining properties of the outer ascus investing membrane and the ascal cytoplasmic membrane fragments, indicate that these fragments are not the origin of double investing membranes. The only other source of such membrane fragments would be the investing membranes of other asci removed during processing. The natural origin of such a membrane (or membranes) outside the c e l l wall i s as hard to explain for asci as i t i s for ascophores, since such an investing membrane i s not found in hyphae. Therefore such membrane synthesis must occur through the plasma membrane and c e l l wall, a phenomenon which warrants further investigation. Spore release i n Cephaloascus i s effected by disintegration ) 25. ( F i g . 28) of the ascus apex (Dixon, 1959). Therefore no modifica-t i o n s of the ascus w a l l , such as havje been shown by Greenhalgh and Evans (1967) and Reeves (1971) i n Euascomycetes, were observed* The occurrence of b l i s t e r s of granular m a t e r i a l on the a s c i ( F i g . 24 and 30-32) i s reported here f o r the f i r s t time, as no brown encrustations on the a s c i are d i s c e r n i b l e with the l i g h t microscope. The granular contents are seemingly i d e n t i c a l to those i n ascophore b l i s t e r s , but are l i m i t e d to the b l i s t e r areas. Without exact knowledge of the chemical nature of t h i s granular m a t e r i a l , one cannot advance theories as to whether i t i s f u n c t i o n a l i n the w a l l area or i s merely an excretory product of the cytoplasm. The ascus cytoplasm contains few i d e n t i f i a b l e o r g a n e l l e s other than endoplasmic r e t i c u l u m - l i k e sheets of membranes, the quantity of which apparently decreases as spores mature ( F i g . 21, 24 and 28). However^ the lack of organelles may be a f i x a t i o n a r t e f a c t . Although t h i s i s not intended as a study of ascosporogenesis, because o f the many a s c i examined, some of the l a t e r stages i n spore formation are observed. From these observations the development of spores of Cephaloascus may be u n l i k e that reported for other Ascomycetes. Mature spores of Cephaloascus are enclosed i n a spore-investing membrane, and spore release i s by d i s s o l u t i o n of the ascus w a l l and i t s i n v e s t i n g membrane, ( F i g . 28). Therefore the lack of a spore i n v e s t i n g membrane, combined with the presence of a complete ascus wall and ascus i n v e s t i n g membrane i s considered 2 6 . indicative of an immature ascus (Fig. 24 and 30). Thus, i n Cephaloascus, the deposition of the spore wall may occur outside the spore plasma membrane but without the presence of an outer investing membrane. The above observation differs from a l l previous electron microscopic reports i n which there are undeniably two membranes present around each spore nucleus, between which the wall i s deposited (eg. Beckett, 1968; Conti and Naylor, 1960; Delay, 1966; Greenhalgh and Evans, 1968; Greenhalgh and G r i f f i t h s , 1970; Lynn and Magee, 1970; Madelin, 1966; Moore, 1963; Moore and McAlear, 1962; Wells, 1970). These authors were not able to determine the 1 origin of the double-membranes of the spore, however, several authors have speculated on such origins. Bandoni et a l (1967), G r i f f i t h s (1968), and Bracker (1967) suggest the ascus plasma membrane as an origin. Bracker and Williams (1966), Reeves (1967), Schrantz (1966a,b), and Wilsenach and Kessel (1965b) support the idjea of endoplasmic reticulum forming these membranes. Carroll (1967a) the v"blebbing" of the nuclear envelope i n combination with elements of endoplasmic reticulum. This i s followed by formation of the spore membranes from, an "unwinding*" of the above four-membraned "blebs" or vesicles. In his studies of Saccharomyces, Moens (1971) described the formation, of the "prospore" (double-membraned wall progenitor) in close association with centriolar plaques on the nuclear membrane. The prospore i s 27. extended around each spore nucleus as i t is differentiated from i the parent nucleus. This is in contrast to earlier reports on ascosporogenesis in Saccharomyces by Hashimoto et all (1960) who reported only one membrane (the spore plasma membrane) and Marquardt (1963) who reported formation of the spore investing membrane from deposition from the ascus cytoplasm onto an already formed spore coat. The centriolar; plaques observed by Moens have also been reported in Ascobolus and Podospora (Zickler, 1970) but were not noticeably involved in "prospore" formation, since these membranes were observed to result from fusion of vesicles at the ascus periphery. In Cephaloascus, no spores were observed without a plasma membrane, and a l l stages of spore maturation observed were after the inner wall (endospore) was essentially complete. While the outer spore wall layer i s forming, i t i s closely appressed by short lengths of cytoplasmic membranes (Fig„ 24 and 25). The "brim" of the cuculate spore i s formed from this outer layer (Fig. 26) without tl^e benefit of an outer membrane to delimit i t s boundaries. However, this i s not inconceivable, since the spore wall of Hansenula (Bandoni et 1967) is formed normally in areas not closely bounded by membranes. This occurs between the "brim" of one spore and the^"crown" of the next. Also, both spore membranes are often convoluted while the walls between them are smooth and regular, as noted by Greenhalgh and Evnas (1968). Several spores of Cephaloascus were observed with relatively large 28. sheets of membrane i n close proximity to the maturing outer spqre wall (Fig. 27). The o r i g i n of such membranes was not determined, but a reduction i n amount of ascus. cytoplasmic membranes during spore maturation may indicate that they are the o r i g i n of the spore investing membrane. This situation i s not unlike that described by Marquardt (1963) i n Saccharomyces. Very few asci containing mature spores were observed, presumably since release ojccurs very soon after spores are completed. Therefore t h i s stage i s i l l u s t r a t e d with lower quality micrographs (Fig . 28 and 29) than are e a r l i e r stages. The{Mature Ascospore. The structure of other cucula.te ascospores has been reported (Bandoni, et a l , 1967; Besson, 1966, 1967; Kreger-van R i j , 1969). Those of Cephaloascus are essent i a l l y the same; consisting of a wall of two layers: an inner electron-transparent endospore,j and an ouiter, more electron-dense, epispore which also forms the brim (Fig. 38-40). Theispore wall i s bounded on the outside by a densely staining, 60-70A thick, membrane (Fig. 40), the iinvesting membrane, (=spore membrane or perispore)./ Though not shown i n previous observations of ascosporjes, a vacuole was sometimes present (Fig. 39 and the-spore cytoplasm contains several mitochondria ( F i g . 39 and 40). Other, non-cuculate ascospores have a similar ultrastructure' 1, with variations mainly occurring i n the number of wall layers (Besson, 1966; Hashimoto _et a l , 1958; Kawakami and Nehira, 1958a; Lowry and Sussman, 1958), although the electron-transparent inner 29. wall layer (endospore) i s common to a l l , and i s the layjer destined to give) r i s e tto the hyphal wall upon spore germination (Lowry and Sussman, 1968) j. The. scanning electron microscope i s currently being used i - n the ( S t u d y of, ascospores for taxonomic purposes (eg. Hawker, 1968^. The surface of a spore\(i.e. the investing membrane or pe^rispore) i s l i k e l y to be most dependent on preparatory techniques, as evidenced by the_ rough and smooth surfaces on Cephaloascus spores (Fig . 36). Both types are•produced i n the same f r u c t i f i c a t i o n ( F i g . 35). Spore surfaces of freeze-etched spores, presumably the best preserved, show only s l i g h t i r r e g u l a r i t i e s (Fig. 37). The u l t r a s t r u c t u r a l s i m i l a r i t i e s of cuculate ascospores, while seemingly supporting the contention of Zender (1925), that a l l cuculate-spored genera should be placed i n a single family, i s apparently contradictied by the lack of agreement on their modes of ascosporogenesis. This taxonomic problem cannot be s a t i s f a c t o r i l y solved u n t i l a l l such genera have been f u l l y studied by electron microscopy. S U M M A R Y The plasma membrane of vegetative hyphae of Cephaloascus  fragrans consists of an i n t e r n a l p a r t i c u l a t e f r a c t i o n and has a grooved surface. i Vegetative hyphae possess both u n i p e r f o r a t e and m u l t i -perforate c r o s s - w a l l s . The m u l t i - p e r f o r a t e septa possess several hundred plasmodesmata-like micropores. The ascophore and a s c i have depositions of granular, pigmented m a t e r i a l between an outer i n v e s t i n g membrane and the c e l l w a l l . Late stages of ascosporogenesis are observed without an outer spore-investing membrane. This may be due/to l o s s of t h i s membrane during f i x a t i o n , or may represent the method of spore w a l l formation i n Cephaloascus. Despite p o s s i b l e d i f f e r e n c e s i n ascospore formation, the f i n e s t r u c t u r e of mature ascospores i s s i m i l a r to that reported f o r other cuculate-spored genera. 31. Plate I Fig. 1. Internal fracture face of plasma membrane, showing p a r t i c l e s , grooves and f i b r i l s (arrows) X 68,400 (Freeze-etched). F i g . 2. Cytoplasmic face of plasma membrane showing smooth, pa r t i c l e - f r e e surface. X 76,000 (Freeze-etched). F i g . 3. Internal fracture face of plasma membrane (pm), showing tonoplast ( t ) , cytoplasm ( c ) , c e l l wall (cw), and f i b r i l a r wall components (arrows). X 62,700 (Freeze-etched).) 32. Plate I I Fig . 4 . Vegetative hypha with a centrally uniperforate septum and septum-associated cytoplasmic granules (arrows)J X 38,000 (Glutaraldehyde-osmium) .\ F i g . 5. Large diameter vegetative hyphae with multiperforate septum. X 9,000 (Glutaraldehyde-osmium). Fi g . 6. Large diameter hypha with multiperforate septum ( s ) , endoplasmic reticulum ( e r ) , a plasma membrane (pm), with an invagination (mi), lamellate body (lb) and glycogen (g). X 19,200 (Glutaraldehyde-osmium). 33. Plate I I I Fig. 7 . A multiperforate septum (s) showing the micropores i n long and cross-section and the septum connection to the c e l l wall (cw). X 28,800 (Glutaraldehyde-osmium). Fig. 8. Micropores i n cross-section, showing plasma membrane (pm) l i n i n g the aper.fures through the septum ( s ) . X 108,400 (Glutaraldehyde-osmium) ,> Fig. 9. Micropores i n long-section with plasma membranes (pm) within the pores of the bilaminar septum.1] X 108,400 (Glutaraldehyde-osmium). 34. Plate IV Fig. IO.J Ascophore "encrustations". X 250 ( B r i g h t - f i e l d ) . Fig. 11. Ascophore "encrustations". X 320 (Phase-contrast).! Fig. 12J Ascophore b l i s t e r s . X 1100 (Phase-contrast). Fig. 13.J Internal (ib) and external ascophore b l i s t e r s . X 5000 (S.E.M.) Fi g . 14. External topography of an ascophore (as) showing smooth b l i s t e r s (b). X 6000 (S.E.M.) Fi g . 15. External topography of an ascophore (as), showing smooth b l i s t e r s (b), and granular b l i s t e r contents (be). X 20,000 (Freeze-etched) Plate V Cross-section of an ascophore with plasma membrane (pm) and an outer investing membrane (im). X 18,000 (Glut.-osmium). Cross-section of ascophore showing granular (g) and amorphous (a) c e l l wall layers, plasma (pm) and investing (im) membranes^ X 38,000 (Glut.-osmium) Longitudinal section of ascophore showing fibrous c e l l w a l l , and a surface b l i s t e r . X 12,000 (Glut.-osmium) High magnification of a b l i s t e r i n longitudinal section, showing arrangement of the c e l l wall (cw), granular b l i s t e r contents (be) , and the investing membrane. X 38,000 (Glut.'-osmium) 36. Plate VI Fig. 20. Whole mount of the f r u c t i f i c a t i o n o^ Cephaloascus fragrans, showing the arrangement of ascospores and asci.| X 1300 ( B r i g h t - f i e l d ) Fi g . 21. An ascus and basal c e l l separated by a uniperforate septum ( s ) . Complete septa (cs) seal the site s of attachment of the secondary-whorl a s c i . X 13,200 (Glu):.-osmium) Fig . 22. Ascal cytoplasmic membranes (er) i n a mature ascus, as indicated by the presence of. membrane-enclosed spore walls (sw). X 38,000 (Glut.-osmium) Fig. 23. High magnification of incomplete septum betweeni ascus and basal c e l l (be), showing septal pore (sp) and pore apparatus (pa).>j X 80,000 (Gluti-osmium) 37. Plate VII Fig. 24. Immature ascus containing many cytoplasmic membranes, and bounded, i n several places by two investing membranes (arrows), j X: 30,800 (Glut.-osmium) Fi g . 25. The vimmature spore wall consisting of an inner ( electron-transparent layerj(iw) and an outer more electron-dense layer (ow). I t i s bounded i n many areas (arrows), by cytoplasmic membrane fragments of the ascus cytoplasm ( e r ) . X 80,500 (Glut.-osmium) 38. Plate VIII Fig. 26. Immature -.ascospore with inner (iw) and outer (ow) wall layers, and the def i n i t e absence vof an outer inventing membrane^ (arrows). A plasma membrane (pm) i s presen|:. X 95,000 (Glut.-osmium) Fig. 27. A large fragment of cytoplasmic membrane (er) i n close proximity to the wall of a maturing spore,'; This membrane i s of the same ditrjensions as both the spore plasma membrane (pm) and the investing membrane of mature spores. X 120,000 (Glut.-osmium) 39. Plate .IX Fig. 28. A mature ascus with disintegrating c e l l wall (cw), remnants of the disintegrated ascus-investing membrane, (arrows), and a small amoujit of endoplasmic reticulum ( e r ) . Mature spores are characterized by the presence of a plasma membrane'^ (pm) and an outer investing membrane (im)^ X 32,400 (Glut.-osmium) F i g . 29. A mature ascospore with plasma (pm) and investing (im) membranes. The ascus wall (aw) i s no^longer cl e a r l y defined, and the ascus cytoplasm (ac) contains mostly vesicular elements of endoplasmic reticulum-like membrane/s (er).j X 57,500 (Glut.-osmium); 40. Plate X Fig. 30. An ascus, with blisters (b) similar to thosp of the ascophore, consisting of granular depositions between the ascus wall (aw) and the ascus-investing membnane (im). X 35,700 (Glut.-osmium) Fig. 31. An ascus blister (b) at higher magnification; ascus wall (aw), investing membrane (im). X 66,500 (Glut.-osmium) Fig. 32. A blister, bounded by two investing/membranes which can be yresolved as trilaminar structures (arrows) ^  Dissimilarity of the^ ascus wall (aw) and a spore \ wall (sw) are apparent'. X 150,000 (Glut.;rosmium) © a w s w 41. Plate, XI F i g . 33. Morphology of an o l d colony i n which ascophores (as) are .held together by mucous d r o p l e t s of spores ( s d ) . X 1^ 0 (S.E .Mp F i g . 34. Surface of a spore d r o p l e t . X 7,000 (S.E.M.) F i g . 35. j An ascophore (as) w i t h i n t a c t basal c e l l s (be) , together w i t h the spore d r o p l e t i t has produced. X 2,000 (S.E.M.) F i g . 36. Rough surfaced ( r s ) and smooth surfaced (ss) cuculate ascospores, held together by strands of d r i e d mucous m a t e r i a l (m). X 10,000 (S.E.JM.) F i g . 37. Mature ascospore w i t h exposed surfaces of i n v e s t i n g membrane (i m ) , outer w a l l l a y e r (ow) and brim ( b r ) . X 30,500 (Freeze-etched) Figj. 38. A f r a c t u r e d ascospore showing i n t e r n a l s t r u c t u r e of the brim (br) and the i n t e r n a l surface of the spore i n v e s t i n g membrane (im).} X 67,300 (Freeze-etched) 42. Plate XII F i g . 39. A mature ascospore with vacuole ( v ) , tonoplast ( t ) , plasma membrane (pm), investing membrane (im), brim (br) , and inner (iw) and outer (ow) wall laye r s . X 48,100 (Glut;.-osmium) F i g . 40. D e t a i l of the brim ( b r ) , and investing membrane (im), which can be resolved as a tri l a m i n a r structure (double arrows). The plasma membrane (pm) its convoluted. A mitochondrion (mi) i s indicated i n the cytoplasm. X 120,000 (Glut.-osmium) 43. Pljate XIII Fig. 41. 21 day growth of C. fragrans on M.Y.P. agar. Ascophore production i s sparse and colonies are white, except bordering a contaminant colony (cc) where ascophore production was stimulated and the Cephaloascus colony was brown (arrows). Fig. 42. 21 day growth on M.Y.P. + pyridoxine. Growth rate i s slower but ascophore production i s greatly increased and colonies are brown. Fig . 43. 21 day growth on M.Y.P. + i n o s i t o l . Growth rate and ascophore production are increased over M.Y.P. re s u l t s . Fig. 44. 21 day growth on M.Y.P. + b i o t i n . Best growth and ascophore production achieved. 44. BIBLIOGRAPHY Ainsworth, G.C. 1961. Dictionary of the fungi. (5th e d i t i o n ) . Commonwealth Mycological I n s t i t u t e , Kew, Surrey. A l l e n , J.V., Hess, W.M., and Weber, D.J. 1971J U l t r a s t r u c t u r a l i nvestigations of dormant T i l l e t i a caries t e l i o s p o r e s . Mycologia 63:144-156. Bandoni', R.J. ,. B i s a l p u t r a , A.A. , and Bis a l p u t r a , T. 1967. Ascospore development i n Hansenula anomala. Can. J.Bot. 45:361-366. Batra, L.R. 1963. Habitat and n u t r i t i o n of Dipodascus and Cephaloascus. Mycologia 55:508-520. Bauer, H.1 1970. A freeze—etch study of membranes i n the yeast Wickerhamia florescens.' 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