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Ultrastructural studies of the patterns of conidiogenesis in Dacrymyces stillatus nees: Fries (basidiomycete)
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Ultrastructural studies of the patterns of conidiogenesis in Dacrymyces sti&ztus Nees: F ries (Basidiomycete) Dominique C. MOSSEBO*, Amougou AKOA & R. Atangana ETEME University of Yaounde’1, Mycological Laboratory, B.l? 1456 Yaoundk, Cameroon
New patterns of conidial formation on basidiospores and vegetative hyphae are identified, illustrated and described.The mechanismsof conidial ontogeny so far unsatisfactorilly studied are elucidated and now better understood. Several types of conidiogenous cells are describedand their ontogeny better illustrated and explained.Arthroconidia of the fungi are for the first time studied at ultrastructural level and their mode of formation and secessionas well better understood. Likewise, chlamydospores of D. stillutus are for the first time investigated at ultrastructural level, what enablesa better observationof their structureand morphology and as well a better understanding of their origin and various modes of formation. 0 2001 Adac / l?ditions scientifiques et m&&ales Elsevier SAS Abstract -
Conidia I microconidia chlamydospores
/ conidiogenous cells I denticles / yeast-like budding I arthroconidia
1
RCsumC - De nouveaux modes de formation de conidies auparavant non d&rites sur les basidiosporeset les hyphes vCgttatives sont identifiCs, puis illust.r& et d&its. L’ontogtnie des conidiesjusqu’alors d&rite de faGoninsuffisanteest Clucid6eet maintenantmieux comprise.Aussi, plusieurs types de cellules conidioghnessont d&rites et leur ontogenieillustie et mieux comprise ?I l’ultrastructure. Les arthroconidies de l’espbce sont pour la premi&e fois BtudiCesau niveau ultrastmcturel et leurs modes de formation et de secession mieux compris. De msme, les chlamydosporesde D. stillatus sont pour la premii%efois examineesau niveau ultrastructurel, ce qui permet une meilleure observationde leur structure et leur morphologie ainsi qu’une meilleure comprehension de leur origine et leurs diffkrents modes de formation. 0 2001 Adac /Editions scientifiques et mCdicalesElsevier SAS Conidies / microconidies / cellules conidiog&nes / denticules / bourgeonnement levure / arthroconidies / chlamydospores
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INTRODUCTION The conidial formation on basidiospores and vegetative hyphae of Dacrymyces stillatus was already in the past summarily mentioned by authors like Kennedy (1958a, b) and M C Nabb (1972) and somewhat examined by other authors like Brefeld (18%X), Yen (1949), Bulat (1953), Magasi (1965), GGttel(l983) and Mossebo (1995); but mostly done just by means of light microscopy (it’s only in the works of the last above mentioned author that a study of the conidial structures of D. stillatus was started at ultrastructural * Correspondenceand reprints: [email protected]
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level) and that superficially in the framework of other main research topics, the results of these investigations didn’t so far enable to elucidate some important aspects of the mechanisms of conidiogenesis of this wood-destroying fungus, and particularly the modes of formation and secession of conidia from conidiogenous cells, so as the patterns of development, structure and morphology of these conidiogenous cells bearing microconidia. On account of these shortcomings in the knowledge of the conidial ontogeny in Dacrymyces stillatus, it appeared necessary for a better understanding of these patterns of development to proceed to deep investigations at ultrastructural level.
MATERIAL
AND METHODS
Collection of basidiospores and artbrospores in their original form and for conidial production Basidiospores collected on water-agar medium (Agat-Agar (Merck): 15 g, distilled water: 1 1) from teleomorph fungal fruitbodies (plate I) were incubated just for 24-48 h at 20-25 “C when they were desired in their original form or nearly so for ultrastructural examination. The time of incubation was extended to 5-7 days or up to 2-3 weeks and sometimes more, either that conidiogenous structures growing directly on the surface of basidiospore or rather on mycelial hyphae (needed 2 to 3 weeks incubation or more to grow) were desired. It was noticed in the course of the experiments carried out that any medium with other nutritious components added to agar boosted a very rapid growth of the mycelium, what appeared to delay or negatively affect the growth of the above mentioned conidiogenous structures which therefore looked very less differentiated or produced in an incomplete manner and sometimes rather rare to observe, compared to the structures obtained on water-agar medium which are usually well developed. In order to study the ultrastructure of arthrospores (arthroconidia) produced by the anamorph fruit bodies (plate I) of the fungus, these fruit bodies were selected from the collection and still on their substrat, they were properly cleaned on running tap water and afterwards with sterile water. Thereafter in a clean bench, their gelatinous part (visible part of the anamorph fruit body attached to the sub&at) was gently removed using a sterile inoculating needle and transfered onto fresh plates of the medium by forming streaks on the surface. Since the artbrospores were needed in their original form (shape at the moment they are produced by the fruit body), the inoculated plates were incubated just for about 24 h at 20-25 “C in order to avoid germination. Sample preparation for scanning electron microscope Agar squares of about 3 mm3 cut out of the mycelial cultures of basidiospores and arthrospores at different stages of growth as mentioned earlier were fixed in a solution of 2 % glutaraldehyde in 0.1 M cacodylatbuffer (pH 7.2) for about 1 h at 4 “C. The samples were thereafter washed twice (10 min for each wash) in 0.2 M cacodylatbuffer and dehydrated in ethanol using the following series: 30 %, 50 %, 70 %, 95 %, 100 % and 15 min for each concentration. After dehydration, the samples were critical-point-dried in a critical point drier using CO, as the carrier gas. Samples were then mounted on scanning
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Plate I. Different types of fructifications of Dacrymyces stillatus in their natural habitat and mycelial cultures from basidiospores. a. Teleomorph fructifications. b. Anamorph fructifications. c. Mycelial culture of normal growth obtained from monospore cultures. d, e. slow growing mycelium with a rough to verrucous surface. The chlamydospores (see plate VII) of the fungus were obtained only on this type of mycelium. f. A polysperm culture of Dacrymyces stillatus. Samples were taken from mycelium of type c, d, e, f (about 8 weeks old cultures) for electron microscope examinations. (Fig. c-f in 9 cm Petri dishes).
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electron microscope ‘grids’ and sputter-coated with gold-palladium. They were examined using a Stereoscam MK 250 scanning electron microscope.
Sample preparation for transmission electron m icroscope Samples were taken from cultures and fixed in the same manner as described above. They were then washed 6 times (10 n-tin each) in 0.1 M cacodylatbuffer and fixed a second time in 1 % osmiumtetroxyde in 0.1 m cacodylatbuffer for 1 h in the darkness at room temperature. Thereafter, osmiumtetroxyde was washed in distilled water at the rate of 6 washes of 10 m m each. The samples were then contrasted with uranylacetate for 1 h in the darkness at room temperature. After contrasting, uranylacetate was washed with distilled water, 3 washes of 10 min each. The material was then dehydrated in the following series of ethanol: 10 %, 25 %, 50 %, 70 %, 95 %, 3 x 100 % and 10 min for each step of dehydration. The samples were afterwards infiltrated with resin (Epoxydharz = ERL) in the following series: 1) ERL-Aceton 1: 1 for 30 min, 2) ERLAceton 2:l for 30 min and 3) pure ERL overnight in the exicator. Following infiltration, the samples were embedded with fresh ERL (resin) in silicon blocs and placed in a special oven at 70 “C for 12 h for polymerisation. Ultra-thin films of 75 to 100 nm were cut from resin blocs (L x W x H = 12 x 5 x 5 mm) bearing samples at their end, this by using a microtome ULTRACUT/E. The films were individually placed on microscope grids and contrasted with bleicitrate combined with sodium hydroxyde (NaOH) pellets. They were then examined using a Zeiss EM 109 electron microscope.
RESULTS Patterns of conidial development in Ducrymyces stiZlutzu M icroconidia formation on basidiospores(plate II) As those produced on vegetative hyphae, microconidia that are directly produced on basidiospores equally play a prominent role in the reproduction and sexual life-cycle of Dacrymyces stilkztus. As illustrated by Figs a, b, c of plate II, two main patterns of conidial development on basidiospores could be distinguished: 1) Microconidia (Figs a2, aa) formed on numerous tiny outgrowths (denticles) born on short conidiogenous cells (Figs a,, a,, as) growing directly on the surface of basidiospores and, 2) Microconidia born on tiny denticles formed at the apex of germ tubes which develop on basidiospores (Figs b,, b,, c,).
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M icroconidia formed on conidiogenouscells growing on basidiospores
Conidigenous cells generally short and thicker at their apex (Figs a,,a,, a, and c,) develop directly on the surface of basidiospores from the cell walls outwards and give rise to numerous little outgrowths at their apex on which microconidia (Figs a,, as) form. These conidiogenous cells are of various sizes and forms (Figs a,, a,, a5,.c2, d,,d,,d,,e,,e,). They usually produce microconidia in clusters, but also solitary termmal comdla at their tip (Fig. el). These solitary microconidia at the tip of conidiogenous cells of D. stillutus were formerly classified by Kendrick & Watling (1979) as well in the group ‘solitary blastic conidia on basidiospores (Group 1 B)’ as in that of ‘conidial ballistospores (Group 2)‘.
Ultrastructural studies of the patterns of conidiogenesis
Plate II. Microconidia formation on basidiospores
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The authors who based their classification on various data from literature didn’t however give any indication on the other patterns of development of conidigenous cells as illustrated on the figures of plate II. Figs 4 and d, show that conidiogenous cells continue to grow during the differentiation of the first formed microconidia, either by producing a germ-tube-like hypha (Fig. da) growing at their apex or by continuing to lengthen (Fig. d2) with the multiplication of denticles bearing microconidia. These two figures (da and ds) very likely illustrate a proliferous development of the conidigenous cells. It’s worth mentioning here that structures similar to conidiogenous cells were observed by Yen (1949) on basidiospores of D. stillutus by means of light microscopy and these structures were schematically represented by the author. In other studies, Kobayasi & Tubaki (1965) found the formation of microconidia in clusters or solitary on basidiospores so typical among the Dacrymycetaceae in general that they characterized it as <>.
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Microconidia formed on conidiogenouscells at the apex of germ-tubes
Microconidia formed on conidiogenous cells at the apex of germ-tubes (plate II / Figs b,, b,, cl) were rarely observed and above all difficult to obtain in culture on synthetic medium. The type of medium used and the proper adjustment of the incubation time (Section II.1.) was very determining to raise the above mentioned structures.Only the other modes of formation previously described (Figs a, d,, da, e) were so far mentioned in literature. As shown on figure f (arrows) small germ-tubes sometimes branched and measuring about 5 to 6 pm long first grow on basidiospores and later on produce conidiogenous cells bearing microconidia on their apex (Figs b,, b,, c,). Compared to the previously described structures, these conidiogenous cells are usually smaller and less branched than those growing directly on the surface of basidiospores and the two modes of formation of microconidia could simultaneously occur on the same basidiospore (Figs ci and c,).
Conidia formation on conidiogenouscells growing on vegetative hyphae (plate III(l), III(Z), IV(l) and IV(2)) These conidiogenous cells are polymorphic and of extremely variable size. They are sometimes reduced to a single denticle (Figs a,, b,, ci) bearing a solitary microconidia at its end. Some outgrowths present a dichotomous branching (Figs a,, b,), others are more branched (Figs I+, c,), sometimes in form of clusters (Fig. d) and some others are club-shaped (Figs c,, cgr e) or ampulliform (Fig. c,). The conidia appear more or less scatteredalong the vegetative hypha (Figs a, b: plate III(l)) or close together in higher density (Fig. f, plate III(2)). The denticles bearing microconidia appear like little conical projections (Figs b,, cl, CJ or tooth-like structures (Figs a2, cd, cs, e) growing on conidiogenous cells or vegetative hyphae on which they form at variable density. They sometimes present irregular forms (Figs a, and as,). Beside the previously described proliferous development of conidiogenous cells (plate II / Figs d,, ds), determinate type of conidiogknous cells (plate III( 1)/d) were also observedon vegetative hyphae. Their main characteristic is that they cease development during or just after the differentiation of the conidia they produce. In addition to the different modes of conidial arrangement on conidiogenous cells formerly described, the botryose development of conidia was also identified. It’s
Ultrastructural studies of the patterns of conidiogenesis
Plate III (1). Conidia formation on conidiogenous cells growing on vegetative hyphae
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Plate III (2). Conidia formation on conidiogenous cells growing on vegetative hyphae (continuation)
Ultrastmctural studies of the patterns of conidiogenesis
Plate IV (1). Conidiogenous cells formation on vegetative hyphae
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Plate IV (2). Conidiogenous cells formation on vegetative hyphae (continuation)
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essentially characterized by a more or less considerable swelling of the apcx of conidiogenous cells on which conidia are synchronously or asynchronously formed (plate III(l) / Figs C, and d). Sympodially proliferating conidiogenous (plate 111(2)/g,, g2) were also obtained in some cultures. They are characterized by a proliferous and alternate development of conidiogenous cells on two opposite sides of a rachis. Conidia formed on sympodially proliferating conidiogenous cells were characterized by Kendrick & Watling (1979) as <,a group in which about 60 species among which many Dacrymycetdes were classified by these authors using literature from various sources. Other types of conidiogenous cells on vegetative hyphae with characteristics different from those previously described are presented on Figs h, i, j, k, 1, m, n, o, p, q of plates IV( 1) and IV(2). They are also polymorphic and above all larger than the others, but with generally few denticles bearing conidia on their external surface. They are either born laterally on hyphae (Figs h, i, j, k, 1) or issued from morphological and structural transformations of these hyphae (Figs m, n, o, p, q). - In the lateral development, the starting point is a little outgrowth (Fig. hi) on the vegetative hypha, which instead of giving rise to a conidium (Fig. h,: conidium came-off here already) continue to grow in length and thickness (Figs h,, ii, ia) to form conidiogenous cells. At maturity, these conidiogenous cells (Figs j, k, 1) generally produce very few conidia with the main characteristics that they are born only at the apex (Figs j i, k(arrows)) of conidiogenous cells and not as in some previously studied cases on their entire external surface. Some conidiogenous cells continue to enlarge (Fig. 12) during or before the differentiation of apical conidia (Figs k(arrows), Ii), others rather branch out (Figs ja, k,) during this process. These two patterns of development constitute an additional evidence of the proliferous development of conidiogenous cells in Dacrymyces stillutus. - About the development of conidiogenous cells by morphological and structural transformations of vegetative hyphae (plate IV(2)/m, n, o, p, q), three main patterns of formation could be distinguished here: 1) 2 to 4 terminal cells (Figs m, n) of a vegetative hypha enlarge and form a succession of conidiogenous cells whose ends (see arrows) are defined by the cross walls (septa) of the generating hypha. The conidiogenous cells formed are of different size, sometimes with branchings (Figs n,, na) and they usually produce very few conidia. 2) An entire vegetative hypha (Fig. n) changes morphologically and structurally into a conidiogenous cell with the development on its external surface of more or less numerous denticles (see arrows) bearing conidia. 3) The morphological and structural transformations of vegetative hyphae sometimes result in the formation of conidiogenous cells of irrregular shape (Figs p, q) with more or less denticles bearing microconidia on their external surface.
Microconidia
formation by germination of other microconidia aud by yeast-like budding
These two modes of germination have already been mentioned very shortly in the past in the framework of other works done by authors like Brefeld (1888), Yen (1949) and Oberwinkler (personal communications), though neither Brefeld, nor Yen didn’t use the term ‘yeast-like budding’ in their respective schematic representations. The ultrastructural studies carried out here enabled to throw more light on this pattern of development in D. stillutus. So, it can be noticed on Figs a, and a,. of plate III(l) that conidia still on
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vegetative hypha can also multiply by budding as it occurs for instance in Candida albicans as described by Cole (1975a) and Cole & Samson (1979). Germination of microconidia with germ-tubes (plate II/g) was also obtained. Shape aud nuclear state of conidia About the shape of microconidia obtained by different modes of production previously described, they are mostly round (1.5-2 pm diam) and rarely cylindrical (plate III(l)/d and III(2)/f). Their external surface is usually smooth. It nevertheless sometimes shows ornamentations (plate III(l)/d and 111(2)/g, and gZ). The nucleus state of conidia in relation to their origin was also examined using the ‘Dapi’ (4,6-Diamidino 2-phenylindol dihydrochloridhydrat, 98 %) staining method after Bnmk et al. (1979), Hooley et al. (1982) and Raju M.B. (1982). This study revealed that conidia produced on haploid hyphae are always uninucleate (plate 111(2): Fig. 1N Mic) whereas those formed on dicaryotic hyphae are as their generating hyphae always binucleate (plate 111(2):Fig. 2N Mic). Cross-walls between conidia and conidiogenoti cells and mode of conidial secession from conidiogenous cells (plate V) The process of conidial formation in Dacrymyces stillatus begins by a non-polar swelling (plate V, Figs a, b) of the initial cell (IC) during which the cell walls uniformly thicken (Figs a, b: arrows). It’s only after this non-polar swelling that the conidium formed is delimited by a cross-wall (Fig. c: arrows). Some conidia are exceptionally delimited by two cross walls (Fig. d: arrows). Figs a, b, c and d show that the internal as well as the external layers of the cell wall are involved in the formation of conidia. This represents a holoblastic development of conidia. The mode of conidial secession from conidiogenous cells was also examined. It’s a centripetal separation of the two layers of the septum as shown on Figs e and f. This represents a shizolitic mode of conidial secession in Dacrymyces stillatus as described by Cole & Samson (1979). Mode of formation
and characteristics of arthroconidia (plate VI)
in Dacrymyces stilhztus
The formation of arthroconidia in Dacrymyces stillutus occurs by fragmentation and disintegration of vegetative hyphae (plate VI/a,b). This fragmentation always occurs at septal level (Figs b and c: arrows right) by a centripetal separation of the two layers of the cross walls, what confers a shizolitic mode of secession of these arthroconidia. The conidia formed in this process are usually bicellular (Figs a,, ba). However, unicellular conidia (Figs a,, b,) could also be formed during the fragmentation of vegetative hyphae. The arthroconidia of Dacrymyces stillatus are usually more or less cylindrical (Figs b,, b,, c), however some of sausage-shape (Fig. f) were rarely observed. They measure 2-3 pm (diam) x 10-12 pm (L) when they are bicellular and 2-3 pm (diam) x 4-6 l.un (L) when unicellular. Their ultrastructure reveals as in the case of Geotrichum candidum link ex. pers. (Cole 1975b; Cole & Samson 1979) that the pattern of conidiogenesis comprises a phase of lateral swelling (Figs b/c: arrows left and Fig. d,)
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Plate V. Cell-walls between conidia and conidiogenous cells and mode of conidial secession. IC: Initial cell; N: Nucleus; L: Lipid drops
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Plate VI. Mode of formation and secession of arthroconidia
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of arthroconidia which is contrary to that of Geotrichum candidum less pronounced without a concomitant swelling of the artbroconidium apex. This lateral swelling of arthroconicia is very likely linked to the centripetal separation of the cross walls and the tearing apart of the internal and external layers (Fig. c, arrow right) of the cell walls. Figs d, and d, respectively show the remains of the tom cell wall and the scar of the dolipore septum after the shizolitic separation of arthroconidia. All layers of the hypha cell walls are involved in the formation of arthroconidia as shown on Figs c and d. This characterizes a holoarthric mode of development and as the arthroconidia are formed in chains (Fig. a), they can be qualified as thallic-arthric arthroconidia. As basidiospores,’ the arthroconidia of Dacrymyces stillatus germinate by producing germ-tubes (Fig. e: arrow) on which conidia later on develop.
Chlamydosporesand their mode of development in Dacrymycesstilkiztus (plate VII) In addition to the morphological and structural transformations of vegetative hyphae previously described (plates IV(l) and IV(2)), other types of hyphal transformations were observed as presented on plate VII. Although sometimes resembling to the above mentioned ones, these structures were not considered as conidiogenous cells, but rather as chlamydospores for 3 main reasons: 1) Their mode of development, their structure and external appearance are quite different from those of conidiogenous cells. 2) The hyphae undergoing these morphological transformations never produce denticles on which conidia form as it’s usually the case with conidiogenous cells. 3) The hyphal transformations as presented on plate VII are essentially characterized by a swelling of one or more apical hyphal cells during which the cell wall of the swollen segments thickens (Fig. a: arrows). Moreover, the above described features considered together better match the characteristics of chlamydospores as defined by Griffiths (1974): “A viable, asexually produced, accessory spore resulting from the structural modification of a vegetative hyphal segment(s) possessing an inner secondary wall, usually impregnated with hydrophobic material, and whose function is primarily perennation and not dissemination.” Chlamydospores are reserves organs which generally endure all natural climatic conditions and therefore ensure the perennety of the species in the nature given that they are always capable of germination when the climatic conditions become favourable. According to these definitions, Figs a, b, c, d, e and f of plate VII undoubtedly represent the different types of chlamydospores in Dacrymyces stillatus. About their shape, they are round (Figs a, b, d) to ovoid (Figs e, f) with a rough to verrucous surface and measure 3-5 pm diameter. They are formed at hyphal apex by the swelling of terminal segments either on both sides of the cross-wall (Fig. f, arrows) or in the cellular room between successive cross-walls of the hypha (Fig. e, arrows). Both patterns of development give rise to chlamydospores in chains similar to those of “Asterophoratype” (Group 11D”) in the classification of Kendrick & Watling (1979). Figure c represents a chain of chlamydospores still in differentiation. It’s worth mentioning that in a previous study of the cultural characteristics of spores of Dacrymyces ellisii, Bulat (1953) already reported the presence of terminal or intercellary, ovoid to spherical structures with thick walls that he characterized as chlamydospores and Yen (1949) also reported such structures in old mycelial parts of
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Plate VII. Chlamydospores and their mode of development
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mono- and polysperm cultures of Dacryomyces deliquescens (now Dacrymyces stillatus) that he rather designated as ‘allocystes’. The author didn’t however give any schematic representation of these structures. It’s also important to mention that all types of chlamydospores observed in Dacrymyces stillatus were exclusively obtained in cultures with a somewhat slow mycelial growth and a rough to verrucous mycelial surface as presented on Figs d and e of Plate I.
DISCUSSION The choice of a relevant culture medium, the proper adjustment of the duration of samples culture according to the type of investigated material and above all the utilization of the scanning and transmission electron microscope enabled us to clarify many until now unknown or badly known aspects of the mechanisms of conidiogenesis in Dacrymyces stillatus, thereby enabling also a better understanding of the conidial ontogeny in this wood-fungus. Compared to the features of several other species previously described by Cole (1975 a,b; 1981), Cole & Samson (1979), Hughes (1953), Kendrick (1971) and Kendrick & Watling (1979), it is clear that D. stillatus shows a far greater diversity in the patterns of conidial formation. In addition to the great variability in the ontogeny of conidiogenous cells (diagrams: ‘summary of the patterns of conidiogenesis in Dacrymyces stillatus’ on the following two pages), their characteristics as well as those of the conidia they produce make of D. stillatus one of the rare so far known fungus, which displays for itself alone so many different patterns in the formation of conidia. Moreover, the yeast-like budding which was so far known only on detached basidiospores and on conidia in culture in liquid medium was also observed on vegetative hypha, what confirms this great diversity in the conidial ontogeny in D. stilhtus. The experiments carried out here should in future be extended to other members of the Dacrymycetaceae and even to the Dacrymycetales in general in order to find out whether they display the same or different features in the patterns of con&al formation. Acknowledgements. The practical part of this work was carried out at the Botanic Institute (Departmentof Mycology) of the University of Ttibingen in Germany.I herebyextendmy best thanksto Prof. Dr. Franz Oberwinkler,the Director of this Institute for his permanentassistance and technical advice during the execution of the work and also for the laboratory material he put at our disposal to this end. I do also thank the German organization ‘Deutscher Akademischer Austauschdienst’(DAAD) that financially supportedour researchstay in Germany.
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Summary of the patterns of conidiogenesis in Dacrymycesstillatus Holoblastic development
CONIDIOGENOUS
CELL 1) lateral growth on hypha 2) Swelling of terminal cells (2 to 4) on
1) direct growth on basidiospore 2) growth on short germ-tubes
3) Morphological and structural transformations og hypha
VEGETATIVE
. BASIDIOSPORE
* Prohferous non-sympodiat conidiogenous cells can either be botryose or extend their growth in other forms before or after the differentiation of conidia, that is : - either by growing in length with the multiplication of denticles bearing microconidia, - or still in length with the development of other conidiogenous cells which are usually bigger and more or less elongated wtth very few denticles bearing microconidia. - or finally by developing germ tubes.
Ultrastructural studies of the patterns of conidiogenesis
Patterns of development of thallic-arthric
conidia
CONIDIA -uni and bicellular
I
Vegetative hypha
-always binucleate
I
The arthroconidia of Dacrymyces stillatus are formed by fragmentation and disintegration of vegetative hyphae. This disintegration occurs in a shizoiitic manner and a holoarthric development. The arthroconidia formed are cylindrical, uni- and bicellular and always binucleate. They usually develop in chains (catenulate development) and can therefore be characterized as thallic-arthric conidia.
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Untersuchongen aus dem Gesamtgebiet der Mykologie. VII. Heft: II. Protobasidiomyceten. Leipzig. BRLJNKC. F., JONES K. C. & JAMES T. W., 1979 - Assay for nanogram quantities of DNA in cellular homogenates.Analytical Biochemistry 92 : 497-500. BULAT T. J., 1953 - Cultural studies of Dacrymyces Ellisii. Mycologia 45 : 40-45. COLE G. T., l975a - A preparatoq technique for examination of imperfect fungi by scanning electron microscopy. cytobios12: 115-121. COLE G. T., l975b - The Thallic mode of conidiogenese in the fungi imperfecti. Canadian Basidiomyceten
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GOTTEL G., 1983 -
Untersuchung zur Systematik der Gattung Dacrymyces Nees per Fr: (Basidiomycetes). DisseflatiOn, Fakultat fiir Biologie, Universitat Ttibingen. GRIFEITBS D. A.. 1974 -The origin, structure and function of chlamydospores in fungi. Nova Hedwigm 25 : 503-547. HOOLEY P. A., gym A. M., EVOLA M. & SHAW D. S., 1982 - Duplication cycle in Nuclei of germinating zoospores of Phytophtora drechsleri LJS revealed by Dapi staining. Transactions of the British Mycological Society 79 : 563-566. HUGHESS. J., 1953 - Conidiophores, Conidia and Classification. Canadian Journal of Botany 31 : 577-659. KENDRICK B., 1971 -Taxonomy of Fungi Imperfecti. In Proceedings of the$rst International specialists Workshop Conference on Criteria and Terminology in the Classification of Fungi Impe$ecti. B. Kendtick University of Toronto press. KEmRICK B. & WATLING R., 1979 - Mitospores in Basidiomycetes. In EDITORS (I?&), The Whole fungus, Vol. 2, Ottawa Canada, National Museum of Natural Sciences, Kendrick B./P&i, pp. 473-545. KENNEDY L. L., l958a - The Genera Of the Dacrymycetaceae. Mycologia 50 : 874-895. KENNEDY L. L., 1958b - The Genus Dacrymyces. Mycologia 50 : 896899. ~~~~Y. ~. &_. TUBAKI K. K., 1965 - Studies on cultural characters and asexuel reproduction KOBAYASSI of Heterobasidiomycete I. Transactions of the Mycological Society of Japan 6 : 29-36. MAGASI L. p., 1965 - Conidial discharge in Dacrymyces. Mycologia 57 : 136-138. Mc NABB R. E. R., 1972 - Taxonoinic Studies in the Dacrymycetaceae VIII. Dacrymyces Nees ex Pies New Zealand Jout?d of Botany 11 : 461-524 . MOSSEBOD. c., 1995 - Untersuchung zur sexuellen Fortpjanzung von Dacrymyces stillatus Nees ex Pries (Basidiomycete) and Ultrastruktur der Konidiogenese. Dissertation (Ph.D Thesis), Universitgt Tiibingen. Deutschland. RAJU N. B., 1982 - Easy methods for fluorescent Staining of Neurospora nuclei. Neurospora Newsletter 29 : 24-26. YEN H. C., 1949 - Contribution a 1’etude de 1a sexualite et du mycelium des Basidiomycetes saprophytes. Annales de l’universite de Lyon, Secteur C: Sciences Nat. 6 : 5-127.
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L”“I,
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,A,.
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0 2001 Ada&&ions scientifiques et mkdicales Elsevier SAS. TOW clroits r&e&s S0181158401010661/FLA
Ultrastructural studies of the patterns of conidiogenesis in Dacrymyces sti&ztus Nees: F ries (Basidiomycete) Dominique C. MOSSEBO*, Amougou AKOA & R. Atangana ETEME University of Yaounde’1, Mycological Laboratory, B.l? 1456 Yaoundk, Cameroon
New patterns of conidial formation on basidiospores and vegetative hyphae are identified, illustrated and described.The mechanismsof conidial ontogeny so far unsatisfactorilly studied are elucidated and now better understood. Several types of conidiogenous cells are describedand their ontogeny better illustrated and explained.Arthroconidia of the fungi are for the first time studied at ultrastructural level and their mode of formation and secessionas well better understood. Likewise, chlamydospores of D. stillutus are for the first time investigated at ultrastructural level, what enablesa better observationof their structureand morphology and as well a better understanding of their origin and various modes of formation. 0 2001 Adac / l?ditions scientifiques et m&&ales Elsevier SAS Abstract -
Conidia I microconidia chlamydospores
/ conidiogenous cells I denticles / yeast-like budding I arthroconidia
1
RCsumC - De nouveaux modes de formation de conidies auparavant non d&rites sur les basidiosporeset les hyphes vCgttatives sont identifiCs, puis illust.r& et d&its. L’ontogtnie des conidiesjusqu’alors d&rite de faGoninsuffisanteest Clucid6eet maintenantmieux comprise.Aussi, plusieurs types de cellules conidioghnessont d&rites et leur ontogenieillustie et mieux comprise ?I l’ultrastructure. Les arthroconidies de l’espbce sont pour la premi&e fois BtudiCesau niveau ultrastmcturel et leurs modes de formation et de secession mieux compris. De msme, les chlamydosporesde D. stillatus sont pour la premii%efois examineesau niveau ultrastructurel, ce qui permet une meilleure observationde leur structure et leur morphologie ainsi qu’une meilleure comprehension de leur origine et leurs diffkrents modes de formation. 0 2001 Adac /Editions scientifiques et mCdicalesElsevier SAS Conidies / microconidies / cellules conidiog&nes / denticules / bourgeonnement levure / arthroconidies / chlamydospores
sow forme de
INTRODUCTION The conidial formation on basidiospores and vegetative hyphae of Dacrymyces stillatus was already in the past summarily mentioned by authors like Kennedy (1958a, b) and M C Nabb (1972) and somewhat examined by other authors like Brefeld (18%X), Yen (1949), Bulat (1953), Magasi (1965), GGttel(l983) and Mossebo (1995); but mostly done just by means of light microscopy (it’s only in the works of the last above mentioned author that a study of the conidial structures of D. stillatus was started at ultrastructural * Correspondenceand reprints: [email protected]
120
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level) and that superficially in the framework of other main research topics, the results of these investigations didn’t so far enable to elucidate some important aspects of the mechanisms of conidiogenesis of this wood-destroying fungus, and particularly the modes of formation and secession of conidia from conidiogenous cells, so as the patterns of development, structure and morphology of these conidiogenous cells bearing microconidia. On account of these shortcomings in the knowledge of the conidial ontogeny in Dacrymyces stillatus, it appeared necessary for a better understanding of these patterns of development to proceed to deep investigations at ultrastructural level.
MATERIAL
AND METHODS
Collection of basidiospores and artbrospores in their original form and for conidial production Basidiospores collected on water-agar medium (Agat-Agar (Merck): 15 g, distilled water: 1 1) from teleomorph fungal fruitbodies (plate I) were incubated just for 24-48 h at 20-25 “C when they were desired in their original form or nearly so for ultrastructural examination. The time of incubation was extended to 5-7 days or up to 2-3 weeks and sometimes more, either that conidiogenous structures growing directly on the surface of basidiospore or rather on mycelial hyphae (needed 2 to 3 weeks incubation or more to grow) were desired. It was noticed in the course of the experiments carried out that any medium with other nutritious components added to agar boosted a very rapid growth of the mycelium, what appeared to delay or negatively affect the growth of the above mentioned conidiogenous structures which therefore looked very less differentiated or produced in an incomplete manner and sometimes rather rare to observe, compared to the structures obtained on water-agar medium which are usually well developed. In order to study the ultrastructure of arthrospores (arthroconidia) produced by the anamorph fruit bodies (plate I) of the fungus, these fruit bodies were selected from the collection and still on their substrat, they were properly cleaned on running tap water and afterwards with sterile water. Thereafter in a clean bench, their gelatinous part (visible part of the anamorph fruit body attached to the sub&at) was gently removed using a sterile inoculating needle and transfered onto fresh plates of the medium by forming streaks on the surface. Since the artbrospores were needed in their original form (shape at the moment they are produced by the fruit body), the inoculated plates were incubated just for about 24 h at 20-25 “C in order to avoid germination. Sample preparation for scanning electron microscope Agar squares of about 3 mm3 cut out of the mycelial cultures of basidiospores and arthrospores at different stages of growth as mentioned earlier were fixed in a solution of 2 % glutaraldehyde in 0.1 M cacodylatbuffer (pH 7.2) for about 1 h at 4 “C. The samples were thereafter washed twice (10 min for each wash) in 0.2 M cacodylatbuffer and dehydrated in ethanol using the following series: 30 %, 50 %, 70 %, 95 %, 100 % and 15 min for each concentration. After dehydration, the samples were critical-point-dried in a critical point drier using CO, as the carrier gas. Samples were then mounted on scanning
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Plate I. Different types of fructifications of Dacrymyces stillatus in their natural habitat and mycelial cultures from basidiospores. a. Teleomorph fructifications. b. Anamorph fructifications. c. Mycelial culture of normal growth obtained from monospore cultures. d, e. slow growing mycelium with a rough to verrucous surface. The chlamydospores (see plate VII) of the fungus were obtained only on this type of mycelium. f. A polysperm culture of Dacrymyces stillatus. Samples were taken from mycelium of type c, d, e, f (about 8 weeks old cultures) for electron microscope examinations. (Fig. c-f in 9 cm Petri dishes).
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electron microscope ‘grids’ and sputter-coated with gold-palladium. They were examined using a Stereoscam MK 250 scanning electron microscope.
Sample preparation for transmission electron m icroscope Samples were taken from cultures and fixed in the same manner as described above. They were then washed 6 times (10 n-tin each) in 0.1 M cacodylatbuffer and fixed a second time in 1 % osmiumtetroxyde in 0.1 m cacodylatbuffer for 1 h in the darkness at room temperature. Thereafter, osmiumtetroxyde was washed in distilled water at the rate of 6 washes of 10 m m each. The samples were then contrasted with uranylacetate for 1 h in the darkness at room temperature. After contrasting, uranylacetate was washed with distilled water, 3 washes of 10 min each. The material was then dehydrated in the following series of ethanol: 10 %, 25 %, 50 %, 70 %, 95 %, 3 x 100 % and 10 min for each step of dehydration. The samples were afterwards infiltrated with resin (Epoxydharz = ERL) in the following series: 1) ERL-Aceton 1: 1 for 30 min, 2) ERLAceton 2:l for 30 min and 3) pure ERL overnight in the exicator. Following infiltration, the samples were embedded with fresh ERL (resin) in silicon blocs and placed in a special oven at 70 “C for 12 h for polymerisation. Ultra-thin films of 75 to 100 nm were cut from resin blocs (L x W x H = 12 x 5 x 5 mm) bearing samples at their end, this by using a microtome ULTRACUT/E. The films were individually placed on microscope grids and contrasted with bleicitrate combined with sodium hydroxyde (NaOH) pellets. They were then examined using a Zeiss EM 109 electron microscope.
RESULTS Patterns of conidial development in Ducrymyces stiZlutzu M icroconidia formation on basidiospores(plate II) As those produced on vegetative hyphae, microconidia that are directly produced on basidiospores equally play a prominent role in the reproduction and sexual life-cycle of Dacrymyces stilkztus. As illustrated by Figs a, b, c of plate II, two main patterns of conidial development on basidiospores could be distinguished: 1) Microconidia (Figs a2, aa) formed on numerous tiny outgrowths (denticles) born on short conidiogenous cells (Figs a,, a,, as) growing directly on the surface of basidiospores and, 2) Microconidia born on tiny denticles formed at the apex of germ tubes which develop on basidiospores (Figs b,, b,, c,).
-
M icroconidia formed on conidiogenouscells growing on basidiospores
Conidigenous cells generally short and thicker at their apex (Figs a,,a,, a, and c,) develop directly on the surface of basidiospores from the cell walls outwards and give rise to numerous little outgrowths at their apex on which microconidia (Figs a,, as) form. These conidiogenous cells are of various sizes and forms (Figs a,, a,, a5,.c2, d,,d,,d,,e,,e,). They usually produce microconidia in clusters, but also solitary termmal comdla at their tip (Fig. el). These solitary microconidia at the tip of conidiogenous cells of D. stillutus were formerly classified by Kendrick & Watling (1979) as well in the group ‘solitary blastic conidia on basidiospores (Group 1 B)’ as in that of ‘conidial ballistospores (Group 2)‘.
Ultrastructural studies of the patterns of conidiogenesis
Plate II. Microconidia formation on basidiospores
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The authors who based their classification on various data from literature didn’t however give any indication on the other patterns of development of conidigenous cells as illustrated on the figures of plate II. Figs 4 and d, show that conidiogenous cells continue to grow during the differentiation of the first formed microconidia, either by producing a germ-tube-like hypha (Fig. da) growing at their apex or by continuing to lengthen (Fig. d2) with the multiplication of denticles bearing microconidia. These two figures (da and ds) very likely illustrate a proliferous development of the conidigenous cells. It’s worth mentioning here that structures similar to conidiogenous cells were observed by Yen (1949) on basidiospores of D. stillutus by means of light microscopy and these structures were schematically represented by the author. In other studies, Kobayasi & Tubaki (1965) found the formation of microconidia in clusters or solitary on basidiospores so typical among the Dacrymycetaceae in general that they characterized it as <
-
Microconidia formed on conidiogenouscells at the apex of germ-tubes
Microconidia formed on conidiogenous cells at the apex of germ-tubes (plate II / Figs b,, b,, cl) were rarely observed and above all difficult to obtain in culture on synthetic medium. The type of medium used and the proper adjustment of the incubation time (Section II.1.) was very determining to raise the above mentioned structures.Only the other modes of formation previously described (Figs a, d,, da, e) were so far mentioned in literature. As shown on figure f (arrows) small germ-tubes sometimes branched and measuring about 5 to 6 pm long first grow on basidiospores and later on produce conidiogenous cells bearing microconidia on their apex (Figs b,, b,, c,). Compared to the previously described structures, these conidiogenous cells are usually smaller and less branched than those growing directly on the surface of basidiospores and the two modes of formation of microconidia could simultaneously occur on the same basidiospore (Figs ci and c,).
Conidia formation on conidiogenouscells growing on vegetative hyphae (plate III(l), III(Z), IV(l) and IV(2)) These conidiogenous cells are polymorphic and of extremely variable size. They are sometimes reduced to a single denticle (Figs a,, b,, ci) bearing a solitary microconidia at its end. Some outgrowths present a dichotomous branching (Figs a,, b,), others are more branched (Figs I+, c,), sometimes in form of clusters (Fig. d) and some others are club-shaped (Figs c,, cgr e) or ampulliform (Fig. c,). The conidia appear more or less scatteredalong the vegetative hypha (Figs a, b: plate III(l)) or close together in higher density (Fig. f, plate III(2)). The denticles bearing microconidia appear like little conical projections (Figs b,, cl, CJ or tooth-like structures (Figs a2, cd, cs, e) growing on conidiogenous cells or vegetative hyphae on which they form at variable density. They sometimes present irregular forms (Figs a, and as,). Beside the previously described proliferous development of conidiogenous cells (plate II / Figs d,, ds), determinate type of conidiogknous cells (plate III( 1)/d) were also observedon vegetative hyphae. Their main characteristic is that they cease development during or just after the differentiation of the conidia they produce. In addition to the different modes of conidial arrangement on conidiogenous cells formerly described, the botryose development of conidia was also identified. It’s
Ultrastructural studies of the patterns of conidiogenesis
Plate III (1). Conidia formation on conidiogenous cells growing on vegetative hyphae
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Plate III (2). Conidia formation on conidiogenous cells growing on vegetative hyphae (continuation)
Ultrastmctural studies of the patterns of conidiogenesis
Plate IV (1). Conidiogenous cells formation on vegetative hyphae
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Plate IV (2). Conidiogenous cells formation on vegetative hyphae (continuation)
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essentially characterized by a more or less considerable swelling of the apcx of conidiogenous cells on which conidia are synchronously or asynchronously formed (plate III(l) / Figs C, and d). Sympodially proliferating conidiogenous (plate 111(2)/g,, g2) were also obtained in some cultures. They are characterized by a proliferous and alternate development of conidiogenous cells on two opposite sides of a rachis. Conidia formed on sympodially proliferating conidiogenous cells were characterized by Kendrick & Watling (1979) as <
Microconidia
formation by germination of other microconidia aud by yeast-like budding
These two modes of germination have already been mentioned very shortly in the past in the framework of other works done by authors like Brefeld (1888), Yen (1949) and Oberwinkler (personal communications), though neither Brefeld, nor Yen didn’t use the term ‘yeast-like budding’ in their respective schematic representations. The ultrastructural studies carried out here enabled to throw more light on this pattern of development in D. stillutus. So, it can be noticed on Figs a, and a,. of plate III(l) that conidia still on
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vegetative hypha can also multiply by budding as it occurs for instance in Candida albicans as described by Cole (1975a) and Cole & Samson (1979). Germination of microconidia with germ-tubes (plate II/g) was also obtained. Shape aud nuclear state of conidia About the shape of microconidia obtained by different modes of production previously described, they are mostly round (1.5-2 pm diam) and rarely cylindrical (plate III(l)/d and III(2)/f). Their external surface is usually smooth. It nevertheless sometimes shows ornamentations (plate III(l)/d and 111(2)/g, and gZ). The nucleus state of conidia in relation to their origin was also examined using the ‘Dapi’ (4,6-Diamidino 2-phenylindol dihydrochloridhydrat, 98 %) staining method after Bnmk et al. (1979), Hooley et al. (1982) and Raju M.B. (1982). This study revealed that conidia produced on haploid hyphae are always uninucleate (plate 111(2): Fig. 1N Mic) whereas those formed on dicaryotic hyphae are as their generating hyphae always binucleate (plate 111(2):Fig. 2N Mic). Cross-walls between conidia and conidiogenoti cells and mode of conidial secession from conidiogenous cells (plate V) The process of conidial formation in Dacrymyces stillatus begins by a non-polar swelling (plate V, Figs a, b) of the initial cell (IC) during which the cell walls uniformly thicken (Figs a, b: arrows). It’s only after this non-polar swelling that the conidium formed is delimited by a cross-wall (Fig. c: arrows). Some conidia are exceptionally delimited by two cross walls (Fig. d: arrows). Figs a, b, c and d show that the internal as well as the external layers of the cell wall are involved in the formation of conidia. This represents a holoblastic development of conidia. The mode of conidial secession from conidiogenous cells was also examined. It’s a centripetal separation of the two layers of the septum as shown on Figs e and f. This represents a shizolitic mode of conidial secession in Dacrymyces stillatus as described by Cole & Samson (1979). Mode of formation
and characteristics of arthroconidia (plate VI)
in Dacrymyces stilhztus
The formation of arthroconidia in Dacrymyces stillutus occurs by fragmentation and disintegration of vegetative hyphae (plate VI/a,b). This fragmentation always occurs at septal level (Figs b and c: arrows right) by a centripetal separation of the two layers of the cross walls, what confers a shizolitic mode of secession of these arthroconidia. The conidia formed in this process are usually bicellular (Figs a,, ba). However, unicellular conidia (Figs a,, b,) could also be formed during the fragmentation of vegetative hyphae. The arthroconidia of Dacrymyces stillatus are usually more or less cylindrical (Figs b,, b,, c), however some of sausage-shape (Fig. f) were rarely observed. They measure 2-3 pm (diam) x 10-12 pm (L) when they are bicellular and 2-3 pm (diam) x 4-6 l.un (L) when unicellular. Their ultrastructure reveals as in the case of Geotrichum candidum link ex. pers. (Cole 1975b; Cole & Samson 1979) that the pattern of conidiogenesis comprises a phase of lateral swelling (Figs b/c: arrows left and Fig. d,)
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Plate V. Cell-walls between conidia and conidiogenous cells and mode of conidial secession. IC: Initial cell; N: Nucleus; L: Lipid drops
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Plate VI. Mode of formation and secession of arthroconidia
Ultrastructural studies of the patterns of conidiogenesis
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of arthroconidia which is contrary to that of Geotrichum candidum less pronounced without a concomitant swelling of the artbroconidium apex. This lateral swelling of arthroconicia is very likely linked to the centripetal separation of the cross walls and the tearing apart of the internal and external layers (Fig. c, arrow right) of the cell walls. Figs d, and d, respectively show the remains of the tom cell wall and the scar of the dolipore septum after the shizolitic separation of arthroconidia. All layers of the hypha cell walls are involved in the formation of arthroconidia as shown on Figs c and d. This characterizes a holoarthric mode of development and as the arthroconidia are formed in chains (Fig. a), they can be qualified as thallic-arthric arthroconidia. As basidiospores,’ the arthroconidia of Dacrymyces stillatus germinate by producing germ-tubes (Fig. e: arrow) on which conidia later on develop.
Chlamydosporesand their mode of development in Dacrymycesstilkiztus (plate VII) In addition to the morphological and structural transformations of vegetative hyphae previously described (plates IV(l) and IV(2)), other types of hyphal transformations were observed as presented on plate VII. Although sometimes resembling to the above mentioned ones, these structures were not considered as conidiogenous cells, but rather as chlamydospores for 3 main reasons: 1) Their mode of development, their structure and external appearance are quite different from those of conidiogenous cells. 2) The hyphae undergoing these morphological transformations never produce denticles on which conidia form as it’s usually the case with conidiogenous cells. 3) The hyphal transformations as presented on plate VII are essentially characterized by a swelling of one or more apical hyphal cells during which the cell wall of the swollen segments thickens (Fig. a: arrows). Moreover, the above described features considered together better match the characteristics of chlamydospores as defined by Griffiths (1974): “A viable, asexually produced, accessory spore resulting from the structural modification of a vegetative hyphal segment(s) possessing an inner secondary wall, usually impregnated with hydrophobic material, and whose function is primarily perennation and not dissemination.” Chlamydospores are reserves organs which generally endure all natural climatic conditions and therefore ensure the perennety of the species in the nature given that they are always capable of germination when the climatic conditions become favourable. According to these definitions, Figs a, b, c, d, e and f of plate VII undoubtedly represent the different types of chlamydospores in Dacrymyces stillatus. About their shape, they are round (Figs a, b, d) to ovoid (Figs e, f) with a rough to verrucous surface and measure 3-5 pm diameter. They are formed at hyphal apex by the swelling of terminal segments either on both sides of the cross-wall (Fig. f, arrows) or in the cellular room between successive cross-walls of the hypha (Fig. e, arrows). Both patterns of development give rise to chlamydospores in chains similar to those of “Asterophoratype” (Group 11D”) in the classification of Kendrick & Watling (1979). Figure c represents a chain of chlamydospores still in differentiation. It’s worth mentioning that in a previous study of the cultural characteristics of spores of Dacrymyces ellisii, Bulat (1953) already reported the presence of terminal or intercellary, ovoid to spherical structures with thick walls that he characterized as chlamydospores and Yen (1949) also reported such structures in old mycelial parts of
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Plate VII. Chlamydospores and their mode of development
Ultrastructural studies of the patternsof conidiogenesis
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mono- and polysperm cultures of Dacryomyces deliquescens (now Dacrymyces stillatus) that he rather designated as ‘allocystes’. The author didn’t however give any schematic representation of these structures. It’s also important to mention that all types of chlamydospores observed in Dacrymyces stillatus were exclusively obtained in cultures with a somewhat slow mycelial growth and a rough to verrucous mycelial surface as presented on Figs d and e of Plate I.
DISCUSSION The choice of a relevant culture medium, the proper adjustment of the duration of samples culture according to the type of investigated material and above all the utilization of the scanning and transmission electron microscope enabled us to clarify many until now unknown or badly known aspects of the mechanisms of conidiogenesis in Dacrymyces stillatus, thereby enabling also a better understanding of the conidial ontogeny in this wood-fungus. Compared to the features of several other species previously described by Cole (1975 a,b; 1981), Cole & Samson (1979), Hughes (1953), Kendrick (1971) and Kendrick & Watling (1979), it is clear that D. stillatus shows a far greater diversity in the patterns of conidial formation. In addition to the great variability in the ontogeny of conidiogenous cells (diagrams: ‘summary of the patterns of conidiogenesis in Dacrymyces stillatus’ on the following two pages), their characteristics as well as those of the conidia they produce make of D. stillatus one of the rare so far known fungus, which displays for itself alone so many different patterns in the formation of conidia. Moreover, the yeast-like budding which was so far known only on detached basidiospores and on conidia in culture in liquid medium was also observed on vegetative hypha, what confirms this great diversity in the conidial ontogeny in D. stilhtus. The experiments carried out here should in future be extended to other members of the Dacrymycetaceae and even to the Dacrymycetales in general in order to find out whether they display the same or different features in the patterns of con&al formation. Acknowledgements. The practical part of this work was carried out at the Botanic Institute (Departmentof Mycology) of the University of Ttibingen in Germany.I herebyextendmy best thanksto Prof. Dr. Franz Oberwinkler,the Director of this Institute for his permanentassistance and technical advice during the execution of the work and also for the laboratory material he put at our disposal to this end. I do also thank the German organization ‘Deutscher Akademischer Austauschdienst’(DAAD) that financially supportedour researchstay in Germany.
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Summary of the patterns of conidiogenesis in Dacrymycesstillatus Holoblastic development
CONIDIOGENOUS
CELL 1) lateral growth on hypha 2) Swelling of terminal cells (2 to 4) on
1) direct growth on basidiospore 2) growth on short germ-tubes
3) Morphological and structural transformations og hypha
VEGETATIVE
. BASIDIOSPORE
* Prohferous non-sympodiat conidiogenous cells can either be botryose or extend their growth in other forms before or after the differentiation of conidia, that is : - either by growing in length with the multiplication of denticles bearing microconidia, - or still in length with the development of other conidiogenous cells which are usually bigger and more or less elongated wtth very few denticles bearing microconidia. - or finally by developing germ tubes.
Ultrastructural studies of the patterns of conidiogenesis
Patterns of development of thallic-arthric
conidia
CONIDIA -uni and bicellular
I
Vegetative hypha
-always binucleate
I
The arthroconidia of Dacrymyces stillatus are formed by fragmentation and disintegration of vegetative hyphae. This disintegration occurs in a shizoiitic manner and a holoarthric development. The arthroconidia formed are cylindrical, uni- and bicellular and always binucleate. They usually develop in chains (catenulate development) and can therefore be characterized as thallic-arthric conidia.
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Untersuchongen aus dem Gesamtgebiet der Mykologie. VII. Heft: II. Protobasidiomyceten. Leipzig. BRLJNKC. F., JONES K. C. & JAMES T. W., 1979 - Assay for nanogram quantities of DNA in cellular homogenates.Analytical Biochemistry 92 : 497-500. BULAT T. J., 1953 - Cultural studies of Dacrymyces Ellisii. Mycologia 45 : 40-45. COLE G. T., l975a - A preparatoq technique for examination of imperfect fungi by scanning electron microscopy. cytobios12: 115-121. COLE G. T., l975b - The Thallic mode of conidiogenese in the fungi imperfecti. Canadian Basidiomyceten
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