Teliospores of smut fungi general aspects of teliospore walls and sporogenesis

Protoplasma (1998) 204:155-169 PROTOPLASMA �9 Springer-Verlag 1998 Printed in Austria

Teliospores of smut fungi General aspects of teliospore walls and sporogenesis §

M. Piepenbring*, R. Bauer, and F. Oberwinkler

Lehrstuhl Spezielle Botanik/Mykologie, Botanisches Institut, Universit~it Tiibingen, Tt~bingen

Received April 15, 1998 Accepted September 9, 1998

Summary. The concept and nomenclature for the elements of teliospore walls in smut fungi are presented and a survey of teliosporogenesis is given, as seen by light and transmission electron microscopy. Four developmental types are distinguished: the Ustila- go, Microbotryum, Tilletia, and Entorrhiza type. In the Ustilago type, sporogenons hyphae are completely segmented into teliospore initials which are embedded in a hyaline matrix formed by gelatinised hyphal walls (found in species of Anthracoidea, Cintractia, HeterotoIyposporium, Kuntzeomyces, Macalpinomyces, Melano- psichium, Sporisorium, Testicularia, Tolyposporium junci, Tri- chocintractia, and species of Ustilago infecting members of the family Poaceae). In the Microbotryum type, septate sporogenous hyphae are also completely segmented into teliospore initials, however, they are not surrounded by a hyaline matrix (Microbotryum, Sphacelothe- ca, Ustilago spp. infecting dicotyledons). A yeast-like budding of teliosporogenic cells is observed for some species of Microbotryum, Sphacelotheca, and Ustilago infecting dicotyledons. In the Tilletia type, teliospores differentiate locally in the sporogenous hyphae, in an apical or intercalary position, without a hyaline matrix (Con# diosporomyces, Doassinga, Entytoma, Erratomyces, Ingotdiomyces, Neovossia, Oberwinkleria, Rhamphospora, Tilletia). In all these types, the teliospore initials first develop a hyaline sheath under which the ornamentation, the exosporium, sometimes a middle layer, and the endosporium are successively deposited by the fungal cell. In the Entorrhiza type, the teliospores develop inside vital host cells with the wall of the sporogenous hypha included into the teliospore wall. The fungus develops a middle layer and an electron-transparent endosporium inside the hyphal wall while a layer forming the ornamentation is deposited onto the hyphal wall, probably by vesicles of dictyosomes of the host cell.

*Correspondence and reprints: Lehrstuhl Spezielle Botanik/Mykolo- gie, Botanisches Institut, Universit~it Tfibingen, Auf der Morgen- stelle 1, D-72076, TiJbingen, Federal Republic of Germany. +Part 164 in the series "Studies in Heterobasidiomycetes" from the Botanical Institute, University of Tfibingen

Keywords: Spores; Ultrastructure; Entorrhiza; Microbotryum; Tilletia; Ustilago.

Introduction

According to ultrastructural characteristics of septal pores, host-parasite interaction (Bauer et al. 1997), and nucleotide sequences of ribosomal DNA (Begerow et al. 1997) smut fungi (Microbotryales in the Urediniomycetes and most of the Ustilagino- mycetes, in the Basidiomycota, formerly the Ustilag- inales and Tilletiales) are biphyletic in origin. Conse- quently smut teliospores probably evolved at least twice during the phylogeny of basidiomycetes. This may be reflected in different ways teliospores develop (see the present paper); by different ultrastructure of mature teliospore walls and pathways of ornamentation development (Piepenbring et al. 1998a); and by special features of teliospores - like teliospore connections, appendages, and germ pores (Piepenbring et al. 1998b). Teliospore characteristics are discussed in relation to smut phylogeny and systematics by Piepenbring et al. (1998b). In order to evaluate gener- ic concepts in the present and the two following publications, most smut genera which are currently used (VAnky 1987 and more recent publications) are provi- sionally accepted.

Teliospores of smut fungi

The teliospores of smut fungi correspond to proba- sidia, which are usually thick-walled dispersal units

156 M. Piepenbring et al.: Teliosporogenesis in smut fungi

and dark coloured, ornamented resting spores. In humid conditions they germinate with basidia. They are also called brandspores, chlamydospores (inap- propriate because this term refers to asexual resting spores; see de Hoog and Boekhout 1982 and references therein), gemmae, melanospores, resting spores, teleutospores (term which should be restricted to rusts, Uredinales), and ustospores. Characteristics like the colour of the teliospore mass, the cellular arrangement of teliospores (single, in pairs, multicel- lular, in groups or balls), the presence and location of sterile cells, and the form, ornamentation, and size of the teliospores (ranging from 3.5 gm in Ustilago min- ima Arthur to 50 ~tm in Tilletia paradoxa Jaczewski) are the basis for smut identification and taxonomy (see, e.g., Zambettakis 1970, Vfinky 1991). Hitherto, the characteristics of the teliospores have not been used for systematic considerations at the supragener- ic level due to their apparent variability. The study of teliospore development may help to understand this morphological variability.

The walls of teliospores

According to de Bary (1884: p. 190) and many other authors, wails of typical mature smut teliospores are composed of the endospore (light-coloured inner layer), the epispore (episporium) or exospore (dark, ornamented outer layer), and sometimes a hyaline envelope (sheath or perispore), which can be distinguished by light microscopy (LM). The term "endospore", however, cannot be used for a spore wall layer in the English language because this also refers to endogenously developed spores. Therefore, the term "endosporium" and correspondingly "exosporium", "episporium", and "perisporium" are preferred (Singer 1962: p. 71, Payak 1964, Khanna and Payak 1968). In the present paper "exosporium" is preferred to "episporium" because in the latter the Greek syllable "epi" indicates covering a surface, while the respective layer is an essential part of the spore wall and is itself covered by the sheath. This nomenclature is applied in the present and some recent studies (e.g., Khanna and Payak 1972, Rober- son et al. 1990), but for teliospore walls of species of Tilletia and Entorrhiza, different nomenclature is used in literature (Piepenbring et al. 1998a). The following definitions of structural entities observed during teliospore wall development and in mature teliospores of smut fungi (Figs. 1 and 2) are part of the conclusions of the present study and Piepenbring et al. (1998a, b). They are presented

here, however, because they are of fundamental importance for the understanding of later descriptions.

Hyaline matrix: matrix embedding the teliospore initials, produced by the gelatinised walls of the sporogenous hyphae, hyaline as as seen by light microscopy (LM). Sheath: the first continuous envelope formed by a teliospore initial, completely surrounding it, separat- ing the protoplast from the gelatinised or disintegrat- ing wall of the sporogenous hypha; it is hyaline as seen by LM, often of medium to high electron density and fibrous as seen by transmission electron microscopy (TEM); it is more or less persistent in mature teliospores. Exosporium: the first continuous layer deposited underneath the sheath, dark as seen by LM and TEM, carrying ornamentation or forming the ornamentation by its sculptured surface, sometimes presenting a germ pore. Ornamentation: developing during a separate developmental phase preceding the deposition of the continuous layer of the exosporium (Fig. 1) or being part of the exosporium when the ornaments cannot be clearly distinguished from the continuous layer of the exosporium (Fig. 2). Middle layer: usually a thin additional layer between exosporium and endosporium, of different electron density, present only in certain taxa (not illustrated in Figs. 1 and 2); this layer is called "mesosporium" (Graham 1960: p. 116) or "partition layer" in teliospores of species of Tilletia and Entorrhiza. Endosporium: the innermost layer of the teliospore wall, light coloured as seen by LM and TEM, adjacent to the plasmalemma and the exosporium or the middle layer; it is the last layer deposited during teliosporogenesis.

Some authors distinguish a primary wall from a sec- ondary wall (e.g., Mires et al. 1992). These two terms are dispensable. The nomenclature proposed above is used for the structures of teliospore walls according to their relative position in the wall and their density as seen by LM and TEM. However, homology of simi- larly named layers in teliospores of far-related species is not always given.

Teliospore development

Teliospore development is initiated in or on a more or less specific organ of the host plant depending on the

M. Piepenbring et al.: Teliosporogenesis in smut fungi

respective species of the smut fungus and the host plant. Hypertrophied host tissue or sterile fungal cells can participate in the formation of the sorus. Usually at the base of the teliospore mass, sporogenous hyphae develop teliospore initials. In the past the development of smut teliospores has repeatedly been investigated by LM, mainly for members of the Usti- laginales and the Tilletiales. Several different ways of spore ontogeny are supposed to exist (e.g., Trione et al. 1989: p. 1678) but hitherto they have not been properly described and compared.

Ustilago type

The development of teliospores of species belonging to the Ustilaginales in the new sense (Bauer et al. 1997) is well documented in literature for species of the genera Anthracoidea, Cintractia, Macalpino- myces, Melanopsichium, Sporisorium, Trichocintrac- tia, and Ustilago infecting members of the family Poaceae (Tulasne and Tulasne 1847; Prillieux 1880; de Bary 1884; Seyfert 1927; Hutchins and Lutman 1938; Ainsworth and Sampson 1950; Kukkonen 1963; Kukkonen and Raudaskoski 1964; Kukkonen and Vaissalo 1964; Shinohara 1973; Langdon and Fullerton 1975, 1977; Trione 1980; Mims and Snetse- laar 1991; Mires etal. 1992; Piepenbring 1995, 1996). Parasitic dikaryotic hyphae in or between the host cells grow into spaces in the host tissue or onto its surface and disintegrate into hyphal segments, the teliospore initials, embedded in a hyaline matrix. The identity of individual hyphae may be retained for some time by the hyphal wall which is supposed to gelatinise forming the hyaline matrix. In the case of Ustilago maydis (DC.) Corda seen by TEM, the teliosporogenic cells contract their plasma, perform karyogamy, and probably multiply by mitotic divisions (Snetselaar and Mims 1994: p. 354). The sporogenous mycelium is generally used up completely in spore formation. Each teliospore initial first develops a hyaline sheath and later the usually dark- coloured wall. The hyaline matrix and the hyaline sheath of the young teliospores mostly disappear during the development of the spores, resulting in a dusty mass of mature teliospores. In the genus Sporisorium, more or less permanent teliospore balls develop from hyphae coiled in the hyaline matrix (as species of Sorosporium; Langdon and Fullerton 1975). Some species of this genus, like S. veracruzianum (Zundel & Dunlap) M. Piepenbr., however, form single teliospores from hyphae which are not coiled (Piepen- bring 1996: p. 103, fig. 5).

157

Microbotryum type

Short descriptions of teliosporogenesis of species of Microbotryum and Ustilago infecting dicotyledons are scattered in abundant literature (e.g., Fischer v. Waldheim 1869, Seyfert 1927). More detailed observations were published by Deml et al. (1980), Audran and Batcho (1980), and Deml and Oberwinkler (1982). Teliospores develop from sporogenous hyphae which are regularly septate and sometimes clamped. Dikaryotic teliospore initials are produced in series, completely using up the sporogenous hypha. Teliospore initials of variable form and size are covered by thin remains of the hyphal cell wall and develop a thick hyaline sheath as they become spherical. The different spore wall layers are deposited while the teliospore becomes diploid until maturity (Deml and Oberwinkler 1982: p. 351). A sporogenous zone without hyaline matrix, sporogenous hyphae completely used up for teliospore development, and young teliospores with thick hyaline sheaths were also observed for species of Sphacelotheca (Piepen- bring 1996) and in Aurantiosporium subnitens (Schr6ter & Henn.) M. Piepenbr. et al. (Piepenbring et al. 1996).

Tilletia type

During the teliosporogenesis of species belonging to the genera Neovossia and Tilletia a hyaline matrix is absent and, as seen by LM, teliospores develop indi- vidually by enlargement of terminal or sometimes intercalary parts of the hyphae (Tulasne and Tulasne 1847, Fischer v. Waldheim 1869, Prillieux 1880: p. 204, de Bary 1884, Fischer and Holton 1957, Karatygin 1981, Fuentes-Dfivila and Durgm 1986, Roberson and Luttrell 1987, Trione et al. 1989). In the case of Tilletia indica Mitra investigated by TEM, the dikaryotic plasma is concentrated in the terminal cell of the hypha with the subapical part of the hyphal cell being emptied and eventually segmented by retraction septa (Roberson and Luttrell 1987). Fusion of nuclei occurs during the early enlargement of the teliospore initial (Roberson and Luttrell 1987: p. 754). The position of the teliospore initials in relation to the sporogenous hypha is difficult to observe because the sporogenous hypha are coiled (Fuentes- Dfivila and Durfin 1986). Tulasne and Tulasne (1847) and Fischer v. Waldheim (1869), however, described and illustrated teliospore initials born on the tips of short lateral outgrowths of sporogenous hyphae for TilIetia caries (DC.) L.-R. & C. Tul. In culture, Fuentes-Dfivila and Durgm (1986) observed similar


s t ructures and add i t iona l ly " y " - s h a p e d septa at the

bases o f the short b ranches of T. indica. Hya l ine

appendages of t e l iospores o f Ingoldiomyces, Neovos- sia, Oberwinkleria, and Tilletia co r r e spond to rem-

nants o f the wal l o f the sporogenous hyphae (Piepen-

br ing et al. 1998b).

Sporogenous hyphae swel l l oca l ly in ap ica l or inter-

ca la ry parts to fo rm te l iospores in Erratomyces patelii (Pavgi & Thi rum.) M. Piepenbr . & R. Baue r (P iepen-

br ing and Bauer 1997). The te l iospore d e v e l o p m e n t

known for spec ies of the genera Entyloma (e.g., de

Bary 1884, S tempe l l 1934, Ka i se r 1936, He im 1962,

Ka ra tyg in 1981, P i epenbr ing 1996) and Rhampho- spora (e.g., Cunn ingham 1888) also co r re sponds to

the Ti l le t ia type because the te l iospores deve lop loca l -

ly in apical , subapica l , or in te rca la ry pos i t ion in

sporogenous hyphae .

Entor rh iza type

Tel iospores of species of Entorrhiza deve lop on the

t ips o f sp i ra l ly co i l ed hyphae ins ide hos t cel ls (e.g.,

F ineran 1980, Deml and O b e r w i n k l e r 1981). D e m l

and Obe rwink l e r (1981) i l lus t ra ted te l iospore ini t ia ls

of E. casparyana (Magnus) L a g e r h e i m as ap ica l ly

swol len parts o f hyphae de l imi t ed by a septum. There

is no ge la t inous fungal ma te r i a l a round the te l io-

spores (F ineran 1980: p. 269). The o rnamenta t ion

deve lops on the smoo th surface of the spore (Deml

and Obe rwink l e r 1981). F ine ran and F ine ran (1984:

p. 2530) conf i rm that " . . . the midd l e l aye r o f the wal l

[= their l ayer 2, exospo r ium of fu l ly deve loped

te l iospores] is the ini t ia l reg ion of the wal l upon

which the outer and inner layers are fo rmed" . The

mate r ia l fo rming the whole spore wal l is u sua l ly sup-

posed to be o f fungal or igin. F ineran and F ine ran

(1984: p. 2530) jus t men t ion that " . . . wal l ma te r i a l of

nonfunga l or ig in m a y somet imes sur round spores and

masks their surface ul t ras t ructure" .

Material and methods Material Material illustrated in the present paper is cited in the legends of the figures, while the general descriptions are based on observations on numerous different specimens cited in Piepenbring et al. (1998a, b). Specimens with H.U.V. numbers are deposited in the personal herbarium of K. Vfinky, those with MP (collection number) and H.U.P. numbers in the personal herbarium of the first author. Further collection numbers are AN (A. Nagler), FO (F. Oberwinkler), GD (G. Deml), and RB (R. Bauer) with the specimens deposited in the respective private herbaria.

Methods Teliospore development was studied with a Zeiss light microscope on hand-cut sections of young sori of recently collected material mounted in water. The ultrastructure of hyphae and teliospores was studied by TEM with a Zeiss EM 109 transmission electron microscope. Small pieces of fresh sori or of herbarium material were fixed in 2% glutaralde- hyde in 0.1 M Na-cacodylate (pH 7.2) for several days. After washing in 0.1 M Na-cacodylate (pH 7.2), the material was postfixed in 1% osmium tetroxide, washed with distilled water and stained in 1% uranyl acetate for 1 h in the dark. After washing in distilled water the material was dehydrated in an acetone series and embedded in Spurr's resin. On semi-thin cuts sporogenous areas were located and selected for ultrathin sections. These were placed on copper slot grids for TEM examination and poststained with lead citrate for 5 min. Semi-thin cuts were also used for observations by LM. Some material was freeze substituted. First, to remove air from intercellular spaces, samples were infiltrated with distilled water contain- ing 6% (v/v) (2.5 M) methanol for approximately 5 min at room temperature. Single samples were then placed in an aluminium holder and frozen immediately in the high-pressure freezer HPM 010 (Balz- ers Union, Balzers, Lichtenstein) as described in detail by Mendgen et al. (1991). Substitution medium (1.5 ml per specimen) consisted of 2% osmium tetroxide in acetone that had been dried over calcium chloride. Freeze substitution was performed at -90, -60, and -30 ~ 8 h for each step, using a Balzers Union FSU 010 freeze-substitution apparatus. The temperature then was raised to approximately 0 ~ over a 30 min period and samples were washed in dry acetone for another 30 rain. Infiltration with an Epon-Araldite mixture (Welter et al. 1988) was performed stepwise: 30% resin in acetone at 4 ~ for 7 h, 70 and 100% resin at 8 ~ for 20 h each, and 100% resin at 18 ~ for approximately 12 h. Samples were transferred to fresh resin and polymerised at 60 ~ for 10 h. Samples were processed like chemi- cally fixed samples described above except that the sections were additionally stained with 1% aqueous uranyl acetate for 1 h. The teliospores ofMycosyriroc cissi (DC.) Beck (MP 90), Rhamphospora nymphaeae Cunn. (RB 1076), Ustilagofiliformis (Schrank) Rostrup (RB 1083), and Ustilentyloma fluitans (Liro) K. V~inky (RB 900) were observed in material fixed by freeze substitution. This is speci- fied in the legends of the figures while conventionally fixed material is not designated.

Illustrations

Khanna and Payak (1968) proposed schematic illustrations for spores, called "sporograms" ("palynogram", Erdtman 1969). A sporogram shows half of a spore in optical section and the other half in superficial view. Schematic illustrations of the development of spines in a rust fungus published by Amerson and Van Dyke (1978) were also helpful for the elaboration of the schematic illustrations in this and the two following papers.

Results

Ustilago type

The paras i t ic hyphae of species be long ing to the Ust i -

lag ina les grow in inter- and in t race l lu la r spaces of the

hos t t issue. Dur ing te l iospore deve lopmen t they

b e c o m e segmen ted into t e l iospore ini t ials which are

M. Piepenbring et al.: Teliosporogenesis in smut fungi 159

\

plasma,

plasma- - - lemma

endosporium

exosporium

2

plasma

plasmalemma

J R -

en-

endospo tofthe

exospori .... exosporium

3 4

:..-.v.

�9 . . _ < . ..:...... . ;@;

' ', I, , I

10,urn 10jum

Figs. 1 and 2. Schematic illustrations of typical teliospore walls of smut fungi

Fig. 1. Ornamentation is distinguishable from the exosporium

Fig. 2. Ornamentation is part of the exosporium

Figs. 3 and 4. Sporogenous hyphae, teliospore initials, and young teliospores in a hyaline matrix (dots) seen by LM

Fig. 3. UstiIago rnaydis (H.U.P. 95). Thick black lines show walls of adjacent host cells

Fig. 4. Ustilago schroeteriana Hennings (MP 687)

embedded in a hyaline matrix (Figs. 3 and 4) in rather large intercellular spaces of the host tissue. Host cells rupture (Fig. 5) when sporogenous hyphae produce teliospore initials inside them or they are compressed and destroyed by the growing mass of teliospores. Seen by TEM, the parasitic hyphae are covered by a grey wall (Fig. 6). This wall gelatinises and surrounds the teliospore initials as a matrix, which is hyaline as seen by LM and of variable electron density with an often fibrous consistency as seen by TEM (Figs. 5-8). The gelatinous material causes the teliospore mass to be agglutinated at its base (especially in species of Anthracoidea and Cintractia) but it is lacking in pow- dery masses of mature teliospores of most of the Usti- laginales. In species of Melanopsichium, however, the hyaline matrix is persistent (Fig. 7) and surrounds the mature teliospores during their liberation. Each teliospore first develops the sheath, which is hyaline as seen by LM and, as seen by TEM, of low to medium electron density and sometimes fibrous (Figs. 7-9). This layer can either be lost in mature teliospores or persistent (Figs. 7 and 35). Underneath the

sheath, first the ornamentation and the exosporium, then the endosporium are deposited (Fig. 41). In addition to the genera mentioned in the literature (see Introduction), for which our results largely con- firm the Ustilago type of teliospore development (not shown), this developmental type has also been observed in Heterotolyposporium piluliforme (Berk.) K. Vgmky, HeterotoIyposporium lepidospermae K. Vinky, Kuntzeomyces ustilaginoideus (Henn.) Henn., Testicularia cyperi Klotzsch, and Tolyposporium junci (Schr6ter) Woronin. In species of Farysia, teliospores are developed in chains by septate hyphae which are completely consumed (Fig. 10). In these species, no conspicuous hyaline matrix could be observed. In Mycosyrinx cissi, the hyaline matrix is transparent as seen by TEM in conventionally fixed material (Fig. 11), while it is electron grey and fibrous when fixed by freeze substitution (Fig. 12). Parts of sporogenous hyphae which are not used for the teliospores are attached to developing teliospores (Figs. 11 and 12). The sheath of young teliospores of M. cissi is very thin (Figs. 11 and 12).

M. Piepenbring et al.: Tel iosporogenesis in smut fungi 161

13 T 14 18

16 ( ,um I

19

)

2O

Figs. 13--20. Teliospore initials surrounded by thick hyaline sheaths; LM

Figs. 13-16. Ustilago scabiosae (MP 2322). The anowed cells show yeast-like budding

Figs. 17-20. Ustilago stygia Liro (MP 2323). The lateraI cells in Figs. 17 and 19 are probably clamps

M i c r o b o t r y u m type

The paras i t i c h y p h a e of m e m b e r s of the M i c r o b o -

t rya les g row exc lus ive ly in in t e rce l lu l a r spaces .

Te l iospores deve lop w i t h o u t a h y a l i n e ma t r ix w h i c h

shou ld be seen by L M (Figs. 13 -20 ) or T E M (Figs.

2 1 - 2 4 ) . S p o r o g e n o u s hyphae , w h i c h are m o r e or less

b r a n c h e d and m a y carry c l amps (Figs. 17 and 19), are

septa ted at ra ther r egu la r in te rva l s (Figs . 13 -20) . The

te l iospore in i t ia l s are i n d i v i d u a l l y s u r r o u n d e d by th ick

Fig. 5. From left to right: host cells filled with parasitic hyphae, ruptured host cell walls, and teliospore initials (ti) in a hyaline matrix of Anthra- coideapannucea (Liro) K. Vfinky (MP 24). Bar: 5 gm

Fig. 6. Sporogenous hyphae (sh) of Cintractia taubertiana (Henn.) Clinton (MP 1801) with hyphal wall and teliospore initials (ti) in gelatinous matrix. Bar: 2 gm

Fig. 7. Part of a mature teliospore of Melanopsichium pennsylvanicum Hirschhorn (MP 801) surrounded by a persistent sheath and a hyaline matrix, en Endosporium, ex exosporium. Bar: 0.5 gm

Fig. 8. Part of a young teliospore of Ustilagofiliformis (freeze substituted; RB 1083) surrounded by the sheath and a hyaline matrix, p Plasma, pl plasma membrane. Bar: 0.2 gm

Fig. 9. Part of a young teliospore of Cintractia scleriae (DC.) Ling (MP 93) with an undulated plasma membrane and a thick sheath, p Plasma. Bar: 0.5 gm

Fig. 10. Young teliospores ofFarysia corniculata K. Vfinky (MP 964); TEM. Bar: 2 gm

Figs. 11 and 12. Teliospores of Mycosyrinx cissi

Fig. 11. A pair of young teliospores with a thin sheath and a remnant of the sporogenous hypha (conventionally fixed; H.U.V. 15194). Bar: 2 ~tm

Fig. 12. An almost mature pair of teliospores surrounded by a thin sheath and the hyaline matrix (freeze substituted; MP 90). Bar: 2 gm. Com- pare the appearance of the hyaline matrix with that in Fig. 11

162 M, Piepenbring et al.: Teliosporogenesis in smut fungi


sheaths which are hyaline as seen by LM and of variable electron density as seen by TEM (Figs. 21-24). Underneath the sheath the ornamentation, the exosporium, and the endosporium develop (Fig. 42). Repeatedly, teliospore initials have been observed forming yeast-like outgrowths. These outgrowths often protrude from the sheath of the mother cell and are covered by a sheath which is thinner than that of the mother cell. They are illustrated on the basis of LM for Ustilago scabiosae (Sowerby) Winter (Figs. 13, 15, and 16) and on TEM for Microbotryum violaceum (Pers.: Pers.) Deml & Oberw. (Figs. 21 and 22) and Ustilago tragopogonis-pratensis (Pers.) Roussel (Fig. 24). Three teliospore initials may be connected to one another (Fig. 22). Two separate teliospore initials may lie in a common sheath (Figs. 22 and 23). Similar yeast-like budding is illustrated as seen by LM of Sphacelotheca cf. hydropiperis (Schum.) de Bary by Piepenbring (1996: p. 91).

Tilletia type

Teliospore initials formed in apical position in short lateral outgrowths of sporogenous hyphae were observed for Tilletia caries (literature of the last cen- tury, see Introduction) and Tilletia contraversa Ktihn (Fig. 25). Hyphae with rows of adjacent young teliospores, as shown in Fig. 26, were often observed. Young teliospores frequently carry appendages formed mainly from remnants of the sporogenous hyphae (Figs. 27 and 28). The cytoplasm of a sporogenous hyphal cell is concentrated mostly in apical parts of the hyphae being withdrawn from other parts of the cell (Figs. 29, 31, and 32). The swelling, pyriform to spherical protoplast of the teliospore initial is delimited from the empty part of the sporogenous hypha by the sheath, which is hyaline as seen by LM, and fibrous and of medium electron density, as seen by TEM (Fig. 32). The hyphal wall covers most of the surface of the sheath as a thin layer which is fused to the sheath or lost in mature teliospores. The hyphal wall slightly gelatinises but does not form a matrix around the young teliospores (Figsl 29-32). Underneath the sheath the exosporium, with ornamented surface, and the endosporium are deposited (Fig. 43). This way of

163

I 5~um

Figs. 25-28. Sporogenous hyphae (Figs. 25 and 26) and young teliospores (Figs. 27 and 28) of Tilletia contraversa (MP 2083), as seen by LM

teliospore development is observed for species of Tilletia and Neovossia (see Introduction) as well as for Conidiosporomyces ayresii (Berk.) K. Vfinky (Figs. 31 and 32), Ingoldiomyces hyalosporus (Mas- see) K. Vfinky, and Oberwinkleria anulata K. & C. V~nky (not shown). As documented in the literature, teliospores in species of the genera Doassinga (Fig. 33), Entyloma (Fig. 34), and Rhamphospora (Fig. 35) develop as local, more or less terminal swellings of sometimes clamped hyphae.

Entorrhiza type

As suggested in the literature (see Introduction), the development of teliospore walls of E. casparyana

Figs. 21-24. Teliospore initials (ti) showing yeast-like development surrounded by thick hyaline sheaths (s); TEM

Figs. 21-23. Microbotryum violaceum (H.U.V. 31404). Bars: 1 Bin

Fig. 24. Ustilago tragopogonis-pratensis (GD 1867). Bar: l gm


starts at the persistent wall of the sporogenous hypha without any hyaline structure (Fig. 36). During the deposition of the middle layer and the endosporium from inside the teliospore initial by fungal activity, an ornamentation layer with an irregularly lamellate basal layer develops on the outer side of the hyphal wall (Figs. 37 and 38). Vesicles and Golgi apparatus in the host cell (Figs. 36-38) lie in the host plasma adjacent to the surface of the young teliospore which is covered by an irregularly folded plasma membrane. Based on this observation and the unusual sequence of layer deposition, we suggest that the thick ornamentation layer of medium to high electron density (Figs. 39-40) is of host origin (Fig. 44).

Discussion

General aspects of teliospore wall development

Teliospores of smut fungi may be called endogenous arthrospores (Madelin 1966) because they are formed inside the wall of sporogenous hyphae by segmenta- tion of the hyphal plasma. In contrast with endogenous ascospores, however, the plasma of one sporogenous cell is usually entirely used for one teliospore in the smut fungi. Sheath formation underneath the hyphal wall, formation of the exosporium, and formation of the endosporium are considered different phases of teliospore wall development. Formation of the ornamentation can be considered as a separate developmental phase, when the process of wall formation changes abruptly from local deposition of ornaments to the deposition of the exosporium on the entire surface of the cell. The young teliospore initial substantially increases in size during formation of the sheath, ornamentation, and the exosporium. The growth of the latter requires a coordinated balance between synthesis of new wall polymers and lysis of existing ones, as described by Bartnicki-Garcia (1973) for growth of hyphal cell walls in general. Vesicles or other structures in the

plasma related to the deposition of wall material could not be observed, in conventionally fixed or in freeze-substituted material (Fig. 8). The final size of the teliospore is not reached until the development of the exosporium is completed. In many smut species numerous young spores can be observed with fully developed exosporium but completely lacking endosporium. This suggests that there is a pause before the endosporium is deposited.

Types of teliospore development

The Ustilago type of teliospore development (Fig. 41) is characterised by teliospore initials surrounded by hyaline sheaths and embedded in a hyaline matrix and by sporogenous hyphae which are completely consumed by disintegration into teliospore initials. The failure to observe septate sporogenous hyphae suggests that the formation of septa is coupled with the separation of the teliospore initials by gelatinous material. The fact that sporogenous hyphae do not possess persistent septal pores (Bauer et al. 1997) is in accordance with this observation. The hyaline matrix is usually lost in mature sori but is somewhat persistent in species of Anthracoidea and Cintractia, resulting in an agglutinated spore mass at the base of the sori, and completely persistent in species of Melanopsichium. The Ustilago type of development has been found in species of the genera Anthracoidea, Cintractia, Heterotolyposporium, Kuntzeomyces, Macalpinomyces, Melanopsichium, Sporisorium, Tes- ticularia, in Tolyposporium junci, Trichocintractia, and also in species of Ustilago infecting members of the Poaceae. The teliospores of species of Farysia develop without a conspicuous hyaline matrix. How- ever, this is comparable to the Ustilago type of spore development, because the entire hyphae are used up in the formation of teliospore initials, there is no yeast-like development of teliospore initials, and the sheath is not as thick as in the Microbotryum type.

Figs. 29-34. Developmental stages of teliospores of the Tilletia type; TEM. hw Hyphal wall, s sheat, ti teliospore initial

Fig. 29. Pyriform teliospore initial of Tilletia olida (Riess) Schr6ter (H.U.P. 634). Bar: 1 pm

Fig. 30. Mature teliospore of T. oIida (H.U.P. 634). en Endosporium, o ornamentation. Bar: 5 gm

Fig. 31. Teliospore initials of Conidiosporomyces ayresii (MP 139). Bar: 3 pm

Fig. 32. Detail from Fig. 31 showing parts of three teliospore initials, one with an adjacent empty part of the sporogenous hypha, o Ornamenta- tion. Bar: 0.5 pm

Fig. 33. Sporogenous hypha with teliospore initial ofDoassinga callitrichis (Liro) K. Vfinky et al. (H.U.V. 2984). Bar: 2 gm

Fig. 34. Teliospore initial of Entyloma calendulae (Oudemans) de Bary (GD 736). Bar: 5 pm


41 ustilago type 4 2 Microbotryum type 4 3 Tilletia type 4 4 Entorrhiza type

o , ~': .-=-- plasma of �9 ~ . . . . host cell

~ ~ ' ~ o o : ' ~ ~ - ~ . ~ : ~ . ~ ~ .

l L hyaline ~' / matrix hyaline

~iii:;ii!.~.~ii!..i:.~.,.. sheath ~ , . ~ .

- s.eat. ~ OQ- 0

\ : ) i !00 ,ll o .~- v li.. ,~ : . : / / ; / ~ o ~ ~ , ~

~176 " ~ _--. .' " " ~ , , ~ ~x~, ~

Figs. 41-44. Schematic illustrations of teliospore development of the Ustilago type (Fig. 41), Microbotryum type (Fig. 42), Tilletia type (Fig. 43), and Entorrhiza type (Fig. 44) on the basis of literature (see Introduction) and present results. The haploid nuclei of dikaryotic hyphal cells fuse and form diploid nuclei in mature teliospores. Ornamentation (except in Fig. 44) and exact relative sizes are disregarded

Fig. 35. Fully grown teliospores with sporogenous hypha of Rhamphospora nymphaeae Cunn. (RB 862); TEM. Bar: 2 gm

Figs. 36-40�9 Teliosporogenesis of Entorrhiza casparyana; TEM (Figs. 36-39: RB 941; Fig. 40: MP 742)

Fig. 36. Part of a teliospore initial covered by the wall of the sporogenous hypha (hw) and the irregularly folded plasma membrane of the host cell (hpl). Note numerous Golgi apparatus (ga) in the plasma of the host. fpl Fungal plasma membrane. Bar: 0.5 gm

Fig. 37. Wall of a young teliospore with developing ornamentation (o) and adjacent Golgi apparatus (ga). Bar: 0.5 gm

Fig. 38. Young teliospore with spore wall layers of different electron density and developing ornamentation (o) adjacent to one Golgi apparatus (ga) and vesicles in the plasma of the host cell. hpl Host plasma membrane. Bar: 0.5 gm

Fig. 39. Wall of an almost mature teliospore, ml Middle layer. Bar: 0.5 gm

Fig. 40. Mature teliospore wall with the plasma broken off. en Endosporium, ex exosporium, ml middle layer, o ornamentation. Bar: 0.5 gm


The presence of a hyaline matrix suggests that teliospore development in species of Mycosyrinx is also closely related to the Ustilago type, although the sporogenous hypha are not completely used up. In contrast to the Ustilago type, in the Microbotryum type (Fig. 42) teliospores develop without a hyaline matrix from hyphae, which are clamped in some species. Corresponding to the Ustilago type, the sporogenous hyphae are completely used up for teliospore development and the development of individual teliospores starts with a hyaline sheath. The Microbotryum type has been observed for species of Microbotryum, Sphacelotheca, and Ustilago infecting dicotyledons. Cells with outgrowths covered by a thinner sheath suggest a multiplication of teliospore initials by budding in species of Microbotryum, Sphacelotheca, and Ustilago infecting dicotyledons. Snetselaar and Mires (1994) argue that diploid teliospore initials of U. maydis also multiply by mitotic divisions. Budding stages, like those observed in the Microbotryum type, have never been observed for members of the Ustilaginales, so the multiplication of teliospore initials of U. maydis may occur by cellular divisions without budding. In the Tilletia type of spore development (Fig. 43), only in certain parts of the sporogenous hyphae, the terminal, subterminal or intercalary position, do teliospores develop, which are delimited by a hyaline sheath. Empty parts of the hyphae may form appendages evident by LM. The Tilletia type of spore development occurs in species of Conidiosporo- myces, Ingoldiomyces, Neovossia, Oberwinkleria, and Tilletia with dusty teliospore mass, as well as in those of Doassinga, Entyloma, Erratomyces, and Rhamphospora with teliospores permanently embedded in host tissue. Teliospores of Entorrhiza type develop on the tips of coiled sporogenous hyphae in vital host cells, while host cells in which teliospores of species of the Usti- laginales develop are soon ruptured and killed. The exosporium of Entorrhiza casparyana corresponds to the wall of the sporogenous hypha which is covered by material probably deposited by the host cell forming the ornamentation without any hyaline structure (Fig. 44). This developmental type, therefore, is completely different from all the other types. Teliospore balls generally develop from coiled sporogenous hyphae in species of the genera Der- matosorus, genera in the Doassansia group, Doassan- siopsis, Moesziomyces, Sporisorium, Thecaphora,

Tolyposporium, Urocystis, Ustacystis, and others (Grayson and Lacy 1975, Nagler 1986 and references therein, V~nky 1987). The development of spore balls is not further considered here. For ultrastructural aspects of mature teliospore balls, see Piepenbring et al. (1998b).

Acknowledgements We thank A. Nagler, G. Deml, and especially K. V~nky for specimens, R. Berndt and R. M. Parton for critically reading the manu- script, and the Deutsche Forschungsgemeinschaft for financial sup- port.

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Teliospores of smut fungi general aspects of teliospore walls and sporogenesis

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