Trans. Br. mycol, Soc. 72 (2) 207-212 (1979)

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Printed in Great Britain.

ULTRASTRUCTURE OF CONIDIUM ONTOGENY OFPSEUDOBASIDIOSPORA CAROLINIANA

By R. CAMPBELL,

Department of Botany, Bristol University, Bristol BS8 1 UG

The conidia of Pseudobasidiospora caroliniana are formed eccentrically on the conidio-genous cell by holoblastic extension of the. wall. The unequal development of theconidium base is related to the distribution of endoplasmic reticulum and the vesiclesoriginating from it.

Conidium ontogeny in the hyphomycetes hasnow been extensively studied at both the lightand the electron microscope level and there is someagreement on the main types of conidia and theirontogeny (Kendrick, 1971). There has been muchless work on the coelomycetes, particularly withthe electron microscope, though it seems that theycontain similar ontogeny types to the hyphomy-cetes. Most of the conidiogenous cells in coelo-mycetes are very small and difficult to see withthe light microscope. Sutton (1977) has resolved agreat deal of the nomenclatural confusion, butmuch more information is needed on conidiumontogeny if the coelomycetes are to be describedand classified in the same terms as the hypho-mycetes.

Dyko & Sutton (1978) described Pseudobasidio-spora caroliniana (Deuteromycotina, Coelomycetes)as having holoblastic conidia which were unusual inthat they were positioned eccentrically at the apexof a filiform extension of the conidigenous cell.This paper reports the ultrastructure of the form-ation of these conidia and suggests the mechanismwhich determines this conidial shape.

MATERIALS AND METHODS

P. caroliniana Dyko & Sutton (IMI 208458) wasgrown on 2 % (wIv) malt agar at room temperaturein daylight. Material was fixed by flooding theculture with glutaraldehyde-formaldehyde fixative(2% (v/v) and 1 % (w/v) respectively in 0'1 M

sodium cacodylate buffer pH 7'2) and then re-moving pycnidia to fresh fixative for half an hour.The material was then washed in the samebuffer, post-fixed in osmium tetroxide (1 %, w/v)in the buffer, washed in water, stained in aqueousuranyl acetate and dehydrated in an ethanol-water series. After embedding in Spurr's resinit was sectioned on a LKB ultramicrotome,stained in Reynold's lead citrate and observedin an AEI EM6G microscope.

Specimens were also prepared for fluorescence

microscopy by mounting fresh material in 0'05 %(wIv) Calcofluor White M2R New (AmericanCyanimid Co.). These were viewed in a Zeissphotomicroscope with epifluorescence illuminationand Zeiss exciter filters BP450-490 and 455,chromatic beam splitter F510 and barrier filterBP520-560. Photographs were recorded on IlfordHP5film.

RESUL TS

The conidia are formed at the tip of the filiformprocess of the conidiogenous cell (Fig. 1). Theconidiogenous cell usually has a doliiform base,and occasionally there may appear to be two cellsin this swollen portion (Fig. 1), but this is causedby oblique sectioning of the cluster of conidio-genous cells. The wall of the base of the conidio-genous cell is continuous with the filamentouspart (Fig. 2). The mature conidium is eccen-trically placed above a slight constriction of thefiliform process and the wall of the conidium andthe conidiogenous cell are continuous (Fig. 3).The conidium is holoblastic and only one isproduced on each conidiogenous cell.

The plasmalemma along the sides of the develop-ing conidium is much convoluted (e.g. Fig. 3)and has many invaginations and paramural bodies(Fig. 4). In contrast to this, the plasmalemmaadjacent to the eccentrically placed conidiumbase is continuous and relatively smooth and thereis little vesicle activity in this region (Figs 3, 6).Frequently the plasmalemma in this regionappears more electron dense than in the restof the conidium (Fig. 6), because it is sectioneduniformly at right angles to its surfaces, ratherthan in glancing sections of circular or irregularprofiles where it is convoluted and associatedwith vesicles as in the rest of the conidium. Thereare a large number of vesicles, apparently origina-ting from the endoplasmic reticulum (Fig. 4)which is extensively developed as conidiumgrowth occurs (Fig, 5).

0007-1536/79/2828-4730 $01.00 © 1979 The British Mycological Society

208 Conidium ontogeny of Pseudobasidiospora

Figs. 1-3. For description see p. 210.

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R. Campbell 209

Figs. 4-10. For description see page 210.

MYC 72

210 Conidium ontogeny of Pseudobasidiospora

A B c o E F

Fig. 11. Diagram, based on electron, visible and u.v, microscopy, of the proposed means of conidiumformation; arrows indicate the direction of growth. The developing, filamentous part of the conidio-genous cell grows apically (A). The tip expands (B) and the wall then grows on one side only, bending thetip over to form the eccentric conidium base (C). The side walls now extend uniformly (D) to give thecharacteristic shape (E) with the mucilagenous wall, septum and eccentric apiculus of the mature coni-dium (F). Not to scale.

EXPLANATION OF FIGURES 1-10

Fig. 1. Montage of a developing conidium and its conidiogenous cell with the swollen base and the fili-form process. The conidium has been sectioned so that the eccentric base is not shown. x 8300.

Fig. 2. Swollen base of the conidiogenous cell with the wall continuous with the filiform process. Thewell-developed endoplasmic reticulum may be associated with the wall or mucilage production. Fibrousmucilage fills the cavity of the pycnidium. x 17000.

Fig. 3. A developing conidium eccentrically placed on the filiform process of the conidiogenous cell.Note the convoluted plasmalemma along the sides of the conidium. x 18200.

Fig. 4. Vesicles, formed from golgi cisternae, fusing with the plasmalemma during the elongation of thesides of the conidium. x 26800.

Fig. 5. Similar stage to Fig. 4: the extending wall is approximately parallel to the rough endoplasmicreticulum profiles. x 38900.

Fig. 6. The base of the developing conidium where the plasmalemma is smooth and there is little vesicleactivity except on the sides of the conidium (right of the photograph). x 21300.

Fig. 7. A nearly mature conidium with the thin wall which is slightly electron dense and the muchbroader, electron transparent, mucilaginous wall which merges into the fibrous mucilage filling thepycnidium. The vesicles and plasmalemmasomes found at this stage may be associated with the muci-lagenous wall production. x 38200.

Figs 8-10. Fresh material mounted in CaJcofluor. All x 700.

Fig. 8. Basal cells with the apices of the filiform processes fluorescing more brightly, presumablyindicating tip growth.

Fig. 9. Young expanding conidia fluorescing brightly except in the position next to the junction with thefiliform process (arrows). The basal swellings also fluoresce.

Fig. 10. Mature conidia faintly fluorescing except at the septa (arrows) and the basal cells.

R. Campbell 211

An electron transparent layer forms aroundeach conidium, while the general contents of thepycnidium appear more electron dense andfibrous (Fig. 7). This is interpreted as the mucilagesurrounding each conidium (Dyko & Sutton,1978). The electron transparent layer does notcover the conidiogenous cell wall. When theconidium is mature a septum, in which no porehas been seen, forms across the top of the filiformprocess of the conidiogenous cell, leaving anapiculus on the conidium when it is released.

When mounted in Calcofluor the basal swellingsof the conidiogenous cells almost always fluoresce,regardless of their age (Figs 8, 9, 10). As thefiliform process of the conidiogenous cell isproduced, it fluoresces at its tip (Fig. 8), and theconidium fluoresces as it matures, as judged by itsshape and size and the formation of the septum(Fig. 9). In some developing conidia there isless intense fluorescence in the basal region.Mature conidia and filiform processes show littlefluorescence (Fig. 10), except where the septaare formed.

DISCUSSION

Calcofluor binds to p-linked hexopyranosyl poly-saccharides in the cell walls (Maeda & Ishida, 1967)and has been used to demonstrate areas of walldifferentiation such as hyphal tips, septa andbranch points (Gull & Trinci, 1974). Meristematicregions of conidiogenous cells (Hawes & Beckett,1977) also fluoresce in Calcofluor. In older wallsthe p-linked hexopyranosyl polysaccharides maybe masked by other polymers, so that the wallsdo not fluoresce, and even in known meristematicregions there may be no fluorescence (e.g. in thebasauxic conidiophores of Arthrinium, unpubl.).Fluorescence in Calcofluor solutions is thereforeuseful corroborative evidence for meristems andgrowth points, but the presence or absence offluorescence is not definitive. With these reser-vations it is suggested that the filiform process ofthe conidiogenous cell grows apically then expandsholoblastically to form the conidium. The wallgrowth of the conidium cannot however be com-pletely uniform, for the eccentric attachmentmust involve uneven wall growth. The shape ofthe conidium is very similar to that of manybasidiospores: McLaughlin (1977) has attributedthe shape of Coprinus basidiospores to differentdegrees of plasticity and setting of the wall atdifferent stages in development. An electrondense region, the hilar appendix body, was associ-ated with the hilar appendix and may also beinvolved in determining spore shape. There is noevidence in the present study for anything equi-

valent to the hilar appendix body, and the shapeof the conidia of Pseudobasidiospora is thoughtto be determined solely by different rates of wallgrowth. Wall growth is thought to result fromthe fusion, with the plasmalemma, of vesiclesderived from Golgi cisternae or cisternal ringswhich may contain either lytic enzymes or wallprecursors (Beckett, Heath & McLaughlin, 1974).In Pseudobasidiospora there is evidence of extensivevesicle fusion with the plasmalemma for all partsof the conidium except the basal swelling whichcauses the eccentric position on the conidiogenouscell. Over this region the plasmalemma showslittle sign of vesicle fusion so the wall productionover the area is possibly less than that over therest of the conidium. The wall is therefore bentover as the conidium initial is formed, and sub-sequent rapid growth along the sides of the develo-ping conidium results in the typical shape (Fig. 11).A lack of new wall growth in the basal part ofdeveloping conidia is suggested by the reductionin fluorescence in this region (Fig. 9).

Dyko & Sutton (1978) drew attention to theaffinity of Pseudobasidiospora with Tiarosporellaand Neottiospora. There is also a similarity toColeophoma, recently described by Nag Raj (1978)as having holoblastic conidia originating fromampulliform conidiogenous cells. The generaBasilocula, Ceuthosira and Xenodomus are reducedto synonyms of Coleophoma. None of thesegenera have the filiform extension of the conidio-genous cell and the eccentric positioning of theconidium which are characteristic of Pseudo-basidiospora.

REFERENCES

BECKETT, A., HEATH, 1. B. & McLAUGHLIN, D. J.(1974). An Atlas of Fungal Ultrastructure. London:Longmans. 221 pp.

DYKO, B. J. & SUTTON, B. C. (1978). Two genera ofwater-borne Coelomyeetes from submerged leaflitter. Nova Hedwigia 29, 167-178.

GULL, K. & TRINCI, A. J. P. (1974). Detection of areasof wall differentiation in fungi using fluorescentstaining. Archives of Microbiology 96, 53-57.

HAWES, C. R. & BECKETT, A. (1977). Conidium onto-geny in the Chalara state of Ceratocystis adiposa. 1.Light microscopy. Transactions of the BritishMycological Society 68, 259-265.

KENDRICK, W. B. (1971). Taxonomy of the FungiImperfecti. Toronto: University of Toronto Press3°9 pp.

McLAUGHLIN, D. J. (1977). Basidiospore initiation andearly development in Coprinus cinereus. AmericanJournal of Botany 64, 1-16.

MAEDA, H. & ISHIDA N. (1967). Specificity of bindingof hexopyranosyl polysaccharides with fluorescentbrightener. Journal of Biochemistry 62, 276-278.

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212 Conidium ontogeny of Pseudobasidiospora

NAG RAJ, T. R. (1978). Genera Coelomycetum. XIV.Allelochaeta, Basilocula, Ceuthosira, Microgloeum,Neobarclaya, Polynema, Pycnidiochaeta and Xenodo-mus. Canadian Journal of Botany 56, 686-707.

SUTTON, B. C. (1977). Coelomycetes. VI. Nomencla-ture of generic names proposed for Coelomycetes.Mycological Papers, 141. Commonwealth Mycologi-cal Institute, Kew, England. 253 pp.

(Accepted for publication 12 June 1978)