Basidium development in some species of Ustilago

Basidium development in some species of Ustilago

Mycol. Res. 93 (4): 405-412 (1989) 405 Prinled in Greal Brilain Basidium development in some species of Ustilago C. T. INGOLD 11 Buckner's Close, ...

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Mycol. Res. 93 (4): 405-412 (1989)

405

Prinled in Greal Brilain

Basidium development in some species of Ustilago

C. T. INGOLD 11 Buckner's Close, Benson, Oxford OX9 6LR, UK

Basidium development in some species of Ustilago. Mycological Research 93 (4): 405-412 (1989). Teliospore germination in four species of Ustilago is described. In U. kuehneana a long 4-celled metabasidium is formed, each cell initially producing a single basidiospore. In U. aschersoniarrtl on malt agar a branched metabasidium is formed with one 2-celled and two I-celled branches, each cell forming sporidia. On water agar, germination of a different kind occurs involving conjugation: sporidia are rarely produced. In U. scrobiculata no sporidia are formed, conjugation occurring between cells of the metabasidium. The hyphae resulting from conjugation give compact colonies of limited growth in which the hyphae eventually break up forming arthrospores. In U. commelinae the metabasidium is 2-celled, each cell prodUCing a long, unbranched hypha; no sporidia are formed. The whole range of basidial apparatus, resulting from teliospore germination, in Ustilago and related genera is discussed and illustrated by a comprehensive diagram. Key words: Ustilago, Basidium, Teliospore, Arthrospore, Conjugation.

In recent publications (Ingold, 1983, 1984, 1985, 1986, 1987, 1988) I have attempted to interpret the immediate product of germination of the teliospore in Ustilago and closely related genera in terms of a basidium. The essential feature of this attempt has been the study of the course of germination of individual teliospores. This paper reports teliospore germination in four further species and provides an opportunity to survey the various modifications of the basic pattern of the basidium that I have illustrated during the past few years.

MATERIALS AND METHODS The following smuts were studied: Ustilago kuehneana Wolff on Rumex acetosa L. collected at Scabbacombe Head, near Kingswear, Devon, U.K. in May 1988 and sent to me by Prof. J. Webster; U. aschersoniana Fischer v. Waldheim on Cutandia memphitica (Sprengel) K. Richter collected in Azerbajdzhan, U.s.S.R. in May 1986; U. scrobiculala Liro on Calamagroslis purpurea (Trin.) Trin. collected in Tome, Lappmark, Sweden in Aug. 1986; and U. comme/inae (Komarov) Zundel on Commelina communis L. collected in Hirosaki, Bunchyo-cho, Japan in Sept. 1986. The last three smuts were kindly sent to me as exsiccala by Dr K. Vanky. For germination, teliospores were spread thinly on agar in Petri dishes. The medium was normally 0'2 % malt agar (MA), but occasionally water agar (WA) was also used. A square of agar (ca 10 mm side) with a suitable scatter of teliospores was cut out, transferred to a slide and, without added water, covered with a square cover-glass (11 mm side). Teliospores that had just started to germinate were located under the

microscope, and their subsequent growth was recorded by camera lucida drawings at appropriate intervals.

RESULTS Ustilago kuehneana From the point of view of basidial structure, U. kuehneana is of special interest because of the illustrations of Brefeld (1893). He showed whorls of sporidia, apparently all at the same stage of development, at the septa of the metabasidium. It is difficult to envisage how this appearance could be reconciled with the basic idea of a basidium initially with four basidiospores. A few days after collection teliospores were spread on MA. They germinated 14-36 h later, each forming a straight, 4celled metabasidium which was relatively long (40-60 x 3-4 \.1m), the lowermost cell sometimes accounting for over half of its length (Fig. 1). An unusual feature was basal vacuolation which drove the visible protoplasm into the germtube. In all other species of Ustilago and its close relatives that I have studied, vacuole formation of this nature did not occur, some granular cytoplasm remaining within the parent teliospore. Initially, each cell of the metabasidium produced' a single sporidium (basidiospore) which usually arose close to a septum, although rarely one was formed at the apex of the metabasidium. Often sporidia developed on either side of a septum and crossed one another as the upper grew backwards and the lower forwards. After the production of the first sporidium from a cell of the meta basidium, another usually developed close to, but not at the same level as, the first. 27-2

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Fig. 1. Us/ilago kuehneana. A, Four germinated teliospores at the stage when the hrst sporidium (basidiospore) has been formed from each cell of the metabasidium; B. basidium development; at zero h (when the teliospore had been on MA for 2 d) the agar was covered with a coverglass; subsequent drawings at indicated times in h and min; temp. ca 20°C; C. liberated sporidia, some budding; D, conjugation between sporidia.

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However, the number of sporidia arising in this way was small. In the material I studied, the pattern of teliospore germination was consistent and may be regarded as the standard pattern for the genus, namely with the four initial sporidia being produced by the external part of the metabasidium which itself remains intact. There was no indication of the remarkable whorls of sporidia figures by Brefeld (1883). Liberated sporidia increased by budding and often joined pairwise by conjugation tubes. Ustilago aschersoniana

The dried material from which the teliospores were obtained had been collected two years previously. On 0'2% MA at 18-20 °C they germinated after 1-2 d, and by the end of the third day the germination was about 20 %. Initially a straight hypha (I5-30 X 3-4 ~m), not constricted at its emergence

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from the teliospore, was formed. At an early stage it appeared to become divided into two cells by a transverse septum which, however, was visible only with optimum illumination and careful focusing. It is suggested that this hypha (or germtube) corresponds to half the metabasidium, the rest remaining within the teliospore (Fig. 2 A. B). Shortly before the first germ-tube had aHained its full length, a second began to develop. Mostly this made its appearance alongside the first. but occasionally it grew through the wall of the teliospore in a position often remote from the first (Fig. 2 G, H). Early in its development the first germ-tube usually produced a sporidium from both of its cells, and often these arose close together on either side of the transverse wall (Fig. 2 B). Some hours after the second germ-tube had arisen, a third developed giving a tight cluster where the three hyphae emerged from the spore. Later the situation often became further complicated by the development of sporidia in this region. Perhaps the second and third germ-tubes, which seem to remain for a considerable time without septa, have

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Fig. 2. Ustilago aschersoniana. A, B, Two genninating teliospores; at zero h each had been on MA for 1-2 d and was then covered with a coverglass; times in h and min; temp. 18-20°; C-H, teliospores that have genninated on MA. Cell contents not shown: pigmentation of teliospores indicated diagrammatically by diagonal shading.

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developed from the two quarters of the metabasidium which, it is supposed, remained within the teliospore wall after the emergence of the first germ-tube. On water agar (WA) the pattern of germination was strikingly different. After 2-3 d a single germ-tube was formed which soon became divided by a cross-wall into two

cells which only rarely produced sporidia (Fig. 3). Instead they conjugated after the manner well-known in Ustilago trifici (Pers.) Rostrup (Fig, 3 A-E). When, as on MA, a further germtube appeared, normally by the side of the first, it grew forward to give a long, unbranched hypha which did not form sporidia. Hyphae of this kind reached a length of up to

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Fig. 3. Ustilago aschersoniana. Teliospores germinating on water agar. A, Stages in germination; covered with coverglass at zero h;

times in h and min; B-E, conjugation between the two cells of the first-formed germ-tube; F, conjugation between an attached sporidium and the upper cell of the first-formed germ-tube; G, only one germ-tube has formed and conjugation occurs as in F; H L no conjugation occurs, both cells of the first-formed germ-tube are producing sporidia; J, (at a lower scale) the second-formed germ-tube has become long with retraction-septa in the oldest part. Pigmentation of teliospores indicated diagrammatically by diagonal shading.

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Fig. 4. Ustilago scrobiculata. A-D, Teliospores germinating after 6-9 d on MA; in D the same one is shown at zero hand 7 h later.

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500 !-lm with the living apical part limited to about 100 !-lm, the 'empty' basal part having retraction-septa at intervals (Fig. 3 J). Occasionally only the first germ-tube was produced, and very rarely a third developed. Although conjugation of the two cells of the first-formed germ-tube normally produced a 'knee-type' junction, occasionally the lower cell formed a sporidium which was contacted by a short hypha from the upper cell, followed by fusion (Fig. 3 F, G). At no time was a hypha seen developing from a fused pair of cells.

Ustilago scrobicuIata Nearly two years after collection, teliospores from dried infected Calamagrostis purpurea were streaked on 0'2 % MA. A week later at room temperature a few « 1 %) germinated. A prostrate 4-celled metabasidium was formed usually with the lowermost of its three cross-walls visible just beyond the outline of the teliospore. However, sometimes only two septa could be seen. No sporidia were formed, but the cells of the metabasidium conjugated in pairs as in Ustilago tritiei (Fig. 4). From each conjugated pair a hypha grew out which might have been dikaryotic. On MA these hyphae from germinating teliospores gave rise to very compact surface colonies of branched hyphae. These colonies soon ceased to grow, rarely exceeding 500 !-lm. After about a week the constituent hyphae became converted into branched chains of long, cylindrical, unicellular spores widely separated from one another as the result of protoplasmic contraction. It is reasonable to regard them as arthrospores. When one of these colonies was cut out, inverted and dragged over the surface of MA, a streak of liberated arthrospores was produced. After two days many had germinated, normally by a subterminal germ-tube (Fig. 5 B). Each gave rise to a colony just like its parent. The production of arthrospores by a smut appears to be unusual.

Ustilago commelinae Teliospores were spread on 0'2 % MA and kept at room temperature (18-20°). After 10 d < 0'1 % had germinated. Thereafter more teliospores germinated daily and after a further 10 d the germination rate had risen to about 5 %. The course of germination was very consistent (Fig. 6). A single straight hypha (25-40 x 4-5 !-lm), lying on the agar, was produced, constituting the metabasidium, and became divided into two cells by a median septum. Then, just below this, a narrow hypha (2!-lm diam) arose. Almost simultaneously, another grew from the upper cell either in an apical position or just above the septum in the metabasidium, but never in an intermediate position. The hyphae grew to a length of several hundred micrometres, but no branching occurred. Further growth ceased and no sporidia were produced. This species was originally placed in Tilletia because its teliospores had conspicuous surface reticulations, and was retained in that gen~s by Duran & Fischer (1961) who

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remarked, however, that 'if the species turns out to be a Ustilago, a generic transfer will be necessary'. Teliospore germination was, indeed, achieved by McLain (1960) who demonstrated the 2-celled nature of the metabasidium and the production of a single hypha from each of its cells. Later Duran & Safeeulla (1968) published a photograph of a teliospore with a metabasidium. They noted that 'the promycelium with its 2 cells which neither fuse nor produce sporidia ... is unique among smut fungi'.

DISCUSSION In addition to studying teliospore germination in the species considered above, I have examined the process in many other smuts belonging to Ustilago and closely related genera (Ingold, 1983, 1984, 1985, 1986, 1987, 1988). It seems appropriate now to attempt a comparative review of the products of germination which can usually be interpreted in terms of a transversely septate basidium. This account is illustrated by a diagram (Fig. 7) which is a greatly extended version of one already published in my first paper (Ingold, 1983). It is based almost entirely on my own observations of

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Fig. 6. Ustilago commelinae. A, B, Teliospores which have been on MA (for 10 d in A and for 18 d in B) and then under coverglass at zero h; drawn at indicated times in h and min; C-E, similar germinated teliospores.

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living material. The nuclear situations indicated are, however, speculative. For each box of the diagram only one species is named, but sometimes several exhibit essentially the same pattern. In what may be regarded as the basic pattern, a straight germ-tube from the teliospore becomes divided by three septa, all visible beyond the level of the spore, into a 4-celled metabasidium. Initially, a basidiospore is budded from each of its cells, then followed usually by one or more sporidia. This

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situation obtains regularly in Ustilago tragopogonis-pratensis (Pers.) Roussel (Ingold, 1985), in U. bistortarum (DC) Komicke (Ingold, 1988), in U. kuehneana and usually in U. avenae (Pers.) Rostrup and U. succisae P. Magnus (Ingold, 1983). Modifications of this basic pattern appear to have resulted from the follOWing processes: the formation of the lowermost septum of the metabasidium at or below the surface of the teliospore; the production of one septum instead of three; the fragmentation of the metabasidium into two or three pieces;

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Fig. 7. Diagram of variations in the basidium of Ustilago and its close allies. In each box a developmental pattern is illustrated. Some patterns are to be found in more than one species, but only one is indicated. The nuclear conditions shown are speculative. The box of what is regarded as the basic pattern has a double frame. Solid lines indicate how types might possibly be related. U. IOllgissima

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!--, branching of the metabasidium; and failure of its individual cells to produce basidiospores. In a single species more than one of these modifications may have occurred. Formation of a septum at or below the surface of the teliospore results in the lowermost cell of the metabasidium being contained entirely within the thick teliospore wall. This may lead to its failure to form a basidiospore, a situation sometimes seen in U. avenae (Ingold, 1983), and usually found in U. scitaminea Sydow when spores are germinated on MA (Ingold, 1988). Alternatively, a basidiospore may bud through the teliospore wall. Again this sometimes happens in U. avenae, but is of regular occurrence in U. vinosa Tul. (Ingold, 1983), in U. cynodorztis (Pass.) Henn. (Ingold, 1984), in U. hordei (Pers.) Lagerh. (Ingold, 1987) and in Sphacelotheca sorghi (Link) Clinton (Ingold, 1986). Indeed, it is one of the most frequent patterns I have encountered.

The 2-celled metabasidium with a single septum instead of three is rare. It is described above in U. commelinae and also occurs in Moesziomyces bullatus (Schr6t.) Vanky. Further, it is the regular situation in Anthracoidea spp. However, that genus with its aerial basidia seems to be unrelated to Ustilago and its close allies where the basidium is prostrate on the medium. It seems to me that Anthracoidea should not be included in Ustilaginaceae. Fragmentation of the metabasidium often occurs. The result may be a basal I-celled portion and an upper 3-celled one, as in V. violacea (Pers.) Fuckel; or two 2-celled pieces, as in U. zeae (Beckm.) Unger; or three pieces as in U. lorzgissima (Sow.) Meyen (Ingold, 1983). Branching of the metabasidium happens occasionally in U. succisae Magnus (Ingold, 1983), apparently regularly, as described above, in U. aschersoniana and often in U. bullata

Basidium development in Ustilago Berk. In the last species branching is combined with fragmentation (Ingold, 1984). Failure to form sporidia directly from the metabasidium takes various forms. In U. hypodytes (Schlecht.) Fr. there is a well-defined 4-celled metabasidium, but each cell gives rise initially to a hypha which eventually bears sporidia (Ingold, 1983). In U. tritid and U. scrobiculata conjugation occurs between compatible cells of the metabasidium and no sporidia are ever formed. From each conjugated pair, a hypha (presumably dikaryotic) grows out. A similar situation is often to be seen in teliospores of U. scitaminea germinating on W A, but in that species sporidia are freely formed on richer media (Ingold, 1988). The unusual situation in U. commeJinae, in which each cell of the 2-celled metabasidium gives rise to an unbranched hypha, has already been noted. In Ustilago and closely related genera, the recognition of what constitutes the basidium is often difficult, partly because the primary structure can rapidly be obscured by secondary development, and partly because fragmentation and branching of the metabasidium may pose problems in interpretation. In some species the analysis of the basidial structure would be difficult if the course of development of individual germinating teliospores were not closely followed. Out of 24 species I have studied only two have been encountered in which it was not pOSSible to see, in the products of teliospore germination, a structure that could be interpreted as a modified basidium. In one of these, Sporisorium puellare (Sydow) G. Deml., the germinating teliospore gave a system of branched hyphae, but no sporidia were formed at any stage (Ingold, 1988). In the (Received for publication 31 October 1988)

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other, U. crus-gallii Tracy &. Earle, although sporidia were freely formed, they did not appear to arise from a septate metabasidium (Ingold, 1987). It is a pleasure to offer my thanks to Dr J. E. M. Mordue, Dr K. Vanky and Prof. J. Webster who have given me so much help in my study of smuts. REFERENCES BREFELD, O. (1883). Untersuchungen aus dem Gesammtgebiete der Mykologie 5, Leipzig. DURAN, R. & FISCHER, G. W. (1961). The genus Tilletia. Washington: Washington State University. DURAN, R. & SAFEEULLA, K. M. (1968). Aspects of teliospore germination in some North American smut fungi. I. Mycologia 60, 231-243. INGOLD, C. T. (1983). The basidium in Ustilago. Transactions of the British Mycological Society 81, 573-584. INGOLD, C. T. (1984). Further studies on the basidium of Ustilago. Transactions of the British Mycological Society 83, 251-256. INGOLD, C. T. (1985). The basidium of Ustilago tragopogonispratensis. Transactions of the British Mycological Society 84, 731-732. INGOLD, C. T. (1986). The basidium of Sphacelotheca sorghi. Transactions of the British Mycological Society 87, 474-476. INGOLD, C. T. (1987). Germination of teliospores in certain smuts. Transactions of the British Mycological Society 88, 355-363. INGOLD, C. T. (1988). Teliospore germination in some species of Ustilaginaceae. Transactions of the British Mycological Society 91, McLAIN, D. D. (1960). The cytology and taxonomy of Ustilago commelinae. Ph.D. Thesis, University of Illinois.