Retraction-septa in various fungi

Notes and brief articles

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Fig. 3. Tremella mesenterica. (A) discharged basidiospores. (B) four discharged basidiospores 3-6 h after deposition on 0'2 % malt agar; in the lowermost example five conidia have been liberated and a further three are seen developing from the conidiogenous cells. (C) yeast-like budding of conidia following a period of rest; shown mounted in indian ink to reveal the sheaths of mucilage. (D) germinating basidiospores scraped from the hymenium of a sporophore; in two cases a retraction-septum has formed and the old basidiospore has collapsed.

Scrapings from the hymenium of a hydrated sporophore reveal numerous stranded basidiospores, many of which have germinated. However, none of these has germinated in the manner described above for spores on agar. Instead, from each basidiospore a germ-tube is formed which narrows to give a fine sterigma at the end of which a ballistospore is produced (Fig. 3 D). This type of germination on the hymenium agrees exactly with what happens in Auricularia spp. and in E. glandulosa. On the basis of basidiospore form, pattern of germination, and shape and development of the conidia, there is nothing to suggest that Exidia glandulosa and Tremella mesenrerica should be placed close together in any taxonomic arrangement. If there is validity in the concept of the holomorph (Kendrick, 1979), E. glandulosa must be

considered closer to Auricularia spp. than to T. mesenterica. My thanks are due to Professor J. Webster for providing material of both the fungi considered in this note, and to Dr D. Reid for confirming the identification of the Tremella on Ulex.

REFERENCES

BANDONI, R. J. & BISALPUTRA, A. A. (1971). Budding and fine structure of Tremella mesenterica haplonts. Canadian Journal of Botany 49, 27-30. INGOLD, C. T. (1981). Basidiospore germination and conidium development in Auricularia. Transactions of the British Mycological Society 77, 161-166. KENDRICK, W. B. ed. (1979). The Whole Fungus. Vols. 1 and 2. National Museum of Canada, Ottawa.

RETRACTION-SEPTA IN VARIOUS FUNGI BY C. T. INGOLD I I

Buckner's Close, Benson, Oxford OX9 6LR

Hughes (1971), in discussing chlamydospore formation, quoted from Lyman (1907): 'in Mucor racemosus. . . the condensing protoplasm draws Trans. Br. Mycol. Soc. 19 (2), (1982).

away from the ends of the cell and concentrates in the middle region where the side walls bulge to receive it. Here a resistant wall (endospore) forms

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374

Notes and brief articles

about the encysted cell within and adnate to the clear that there were two types of chlamydospore hyphal walls at each end. Continued contraction of differing not only in form but also in development. theprotoplasm may causethe abandonment oftheseend The first, mycelial chlamydospores, are formed walls and the formation of new walls further in . . . ' singly on short, prostrate, lateral branches of the (my italics). Hughes refers to the use by Dr J. H. vegetative mycelium. At 20°C they are produced Ginns of the term 'retraction-septa ' for abandoned within 2 mm of the margin of the growing colony walls of this nature. Recently (Ingold, 1981) I have which has a daily extension of 8 mm. These are, drawn attention to the formation of similar septa indeed, the first spores to be formed, sporangioduring the germination ofbasidiospores in Auricul- phores not arising from the mycelium until it is two aria spp. This paper gives more details of this days old. Each chlamydospore is first recognizable processinA.auricula-judae(Bull.exStAm.)Wetts. as an ovoid swelling a short distance from the tip and describes the formation of these septa in some of a lateral branch (Fig. 3 F, G). Its protoplasm is other fungi. seen to be somewhat denser than in the rest of the WhenbasidiosporesofAuriculariaauricula-judae hypha. It is then delimited on either side by are allowed to fall from a sporophore to give a light cross-walls. Although fairly soon the contents of deposit on 0'2 % malt agar in a Petri dish, the lateral hypha, apart from the chlamydospore, germination occurs during the following days. The become clear, there is no evidence of migration of situation can be observed microscopically in an protoplasm into the chlamydospore. At maturity its inverted unopened dish using a 16 mm objective. wall becomes highly refractive, although not much In spores germinating with a single germ-tube, thickened. The chlamydospore is oval (12-15/lm retraction-septa are formed (Fig. 1 A). By the time diam) with narrow ends 3-4/lm across. At a later stage chlamydospores of the second branching starts (Fig. 1 B, C) the living, branched system is seen to be subtended by a dead type (sporangiophoric) are formed in the mature unbranched part. This dead part is just visible in aerial sporangiophores. Very occasionally one is the undisturbed state as a pale track lacking definite seen within the columella itself. In the production outline and interrupted at short intervals by of a chlamydospore the protoplasm, in a section of refractive bars each corresponding to a retraction- the sporangiophore, appears to aggregate as a septum. In one case the dead region was seen to be separate protoplast with two convex surfaces. Each 750/lm long, with 35 retraction-septa (Fig. 1 C). develops a retraction-septum. If the protoplast is When a square of agar bearing germinating relatively small and condensed, the two septa define spores is cut out and covered with a coverglass, the the young chlamydospore. If, however, the protodead parts of the germ-tube, but not the original plast is longer and not so dense, retreat may occur spore, become completely invisible (Fig. 1 D). It from one or both of these walls before the seems that the walls of the vacated hyphal segments chlamydospore is formed. The end walls of the undergo a process of dissolution, no doubt due to sporangiophoric chlamydospore are normally enzyme action. retraction-septa, unlike the limiting walls of the Germinating basidiospores of Polyporus squamo- mycelial chlamydospore. However, both end walls sus Huds. ex Fr. behave like those of Auricularia are not necessarily of this nature. Normal crossspp. (Fig. 2). This was described by Buller (1906) walls are not infrequent in the sporangiophore. If and I can confirm his observations. Mostly a spore an isolated protoplasmic mass is blocked on one gives rise to a single germ-tube, apical or basal, side by a normal wall, this may contribute one end but sometimes one grows from each end of the of the chlamydospore, the other being a retractionelongated spore. The first retraction-septum to be septum. The sporangiophoric chlamydospores are formed is usually close to the point of emergence barrel-shaped to spherical (Fig. 3 B-E). They are of the germ-tube, but occasionally it is within the strikingly different from those found in the young spore. All septa are concave when viewed from the vegetative mycelium. apex of the germ-tube. Where germination is by Retraction-septa are also formed in the promytwo polar hyphae, no retraction-septa are formed. celium (metabasidium) of Tilletia caries (DC.) Tul. This is exactly what happens in A. auricula-judae Chlamydospores were allowed to germinate on (Ingold, 1981). The breakdown of the walls of the tap-water agar in a Petri dish. A square of medium dead hyphal segments, a feature of A. auricula- was then cut out, placed on a slide and, with the judae, does not happen in P. squamosus. Once the minimum of added water, covered with a coverslip. germ-tube starts to branch to give a mycelium, Under these conditions growth of the promycelium, ordinary cross-walls (primary septa) are formed in prior to basidiospore formation, continues apparthe hyphae. ently in a normal manner for several hours and is Mucor racemosus Fres. was cultured on 0'2 % easy to follow microscopically. An example is malt agar to study chlamydospore formation. It was shown in Fig. 4 illustrating the production of a Trans. Br. Mycol. Soc. 79 (2), (1982).

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375

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Fig. 1. Auricularia auricula-judae. (A) Germinating basidiospore with two retraction-septa. (B, C) Undisturbed germinated basidiospores in which branching of the germ-tube has started; the original spores (each with one retraction-septum) can be seen, also a number of retraction-septa appearing as dots. (D) Similar germinated basidiospore (a) mounted in water ; no retraction-septa now visible between the living hypha (b) and the dead basidiospore ; the hypha I tip (c), the base of the living hypha (d) and the spore (e), with two retraction-septa, are shown at higher magnification .

Trans . Br , My col. Soc. 79 (2) , (198:7.).

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Notes and brief articles

Fig. 2. Polyporus squamous. Germinating basidiospores four days after sowing on 0'2 % malt agar and kept at room temperature. Except for the spore at top right, all germ-tubes show retraction-septa. In one a retraction-septum is in the middle of the spore.

number of retraction-septa. A vacuole appears above a septum and enlarges rapidly to develop a clear zone below the granular protoplasm. Thus at time 85 min (Fig. 4) the withdrawing protoplasm presents a concave surface. At this stage there is certainly a layer of protoplasm lining the wall, but this is too thin to be recognizable under the microscope. At 95 min the protoplasm has flattened. At 113 min it has become convex and is probably delimited by wall material. However, it is impossible to be sure just when a wall is formed. This sequence of events has been observed repeatedly. After 425 min the promycelium has seven septa, but it would be incorrect to say that it is composed of eight cells. It is, in fact, basically unicellular, the living region being subtended by a number of empty hyphaI segments successively cut off by retraction-septa. The germination of the chlamydospore of T. caries was carefully studied by Bullerand Vanterpool (Buller, 1933) and my observations are in general agreement with theirs. Retraction-septa also occur in some members of the Entomophthorales. The growth of the mycelium of Basidiobolus ranarum Eidam has been described in beautiful detail by Robinow (1963) who noted that two types of septa are formed. He described how the protoplast moves forward in the hyphaI tube and' leaves in its wake a long chain of empty Trans. Br. Mycol. Soc. 79 (2), (1982).

cell chambers' for periodically' a transverse septum is laid down across the cell along the lower edge of the contracted protoplast, separating the inhabited from the empty portion of the cell wall tube'. Each leading hypha in a growing colony has a single, large, oval nucleus situated 50-100 pm from the tip and visible, unstained, even under low power. Robinow described how a transverse septum grows inwards from the cell wall between daughter nuclei towards the end of mitosis. A branch develops below this septum and into it the lower of the two nuclei passes, while above the cell wall protoplasmic withdrawal occurs and a retraction-septum is formed, followed by further retreat and a repetition of the process. This hyphal situation was observed at the growing margin of a colony on o· 2 'Yo malt agar. The wall formed during nuclear division is a z-ply structure and the two halves tend to round off, resulting in partial disarticulation. The net effect of all these processes is that the growing mycelium comes to consist of a number of separate uninucleate portions. In Entomophthora coronata (Cost.) Srin. & Thirum. the situation is rather similar, but each growing hypha has many small nuclei which cannot be distinguished without staining. Fig. 5 illustrates the growth of a leading hypha observed in an inverted, unopened Petri dish containing a thin

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Notes and brief articles

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layer of 0' 2 % malt agar. At the start of observations the hypha has one normal septum, just above the uppermost of two laterals, and a single retractionseptum. After 70 min another normal septum has formed 360 Ilm from the apex. Thereafter, during the next 105 min, three retraction-septa are formed above the normal one. Thus the mycelium becomes broken up to form a number of isolated living Trans. Br. Mycol. Soc. 79 (2), (1982).

regions separated by short lengths of empty hyphal segments. It is not quite clear how retraction-septa are formed. There is no doubt that the first stage involves the development of a large vacuole. One possibility is that a wall is formed within the protoplasm just above the vacuole cutting off a hypha I segment which, though apparently empty,

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Notes and brief articles

Fig. 4. Tilletia caries. Chlamydospore on agar germinating to produce a promycelium. Drawn at intervals over a 425 min period.

Trans. Br. Mycol. Soc. 79 (2), (1982).

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Notes and brief articles has its wall still lined by an extremely thin layer of cytoplasm. This segment, however, soon dies. Then the positive pressure in the living hypha on the other side causes the new cross-wall to bulge while it is still soft. The second possibil ity is that the large vacuole finally bursts, somewhat as a contractile vacuole does. The protoplasm then rounds off, and on its surface a wall is organized, just as a wall is regenerated on a naked protoplast. In this model the retraction-septum is curved from the start. In practice it is difficult to decide which of the two mechanisms is operating, largely because it is impossible to determine precisely when a wall becomes established. Buller and Vanterpool (Buller, 1933), from their observations on Tilletia, supported the first view. My own observations on both Tilletia and Entomophthora suggest that the second possibility cannot be excluded. It is to be noted that the formation of a retraction-septum in these fungi is rapid, occupying only a few minutes. It is quite possible that in some cases the first mechanism, and in others the second, is involved.

It is a pleasure to express my thanks to Professor John Webster for supplying me with cultures and for helpful advice in preparing this paper. REFERENCES

BULLER, A. H . R. (1906). The biology of Polyporus squamosus, a timber-destroying fungus. Journal of Economic Biology 1 , 101-138 . BULLER,A. H. R. (1933). Researcheson Fungi, V. London: Longmans, Green. HUGHES, S. J. (1971). Phycomycetes, basidiomycetes and ascomycetes as fungi imperfecti. In Taxonomy of Fungi Imperfe cti (ed. B. Kendrick), pp. 7-36. Toronto : University of Toronto Press. INGOLD, C. T . (1981). Basidiospore germination and conidium development in Auricularia. Transactions of the British Mycological Society 77, 161-166. LYMAN, G . R. (1907). Culture studies on polymorphism of Hyrnenomycetes. Proceedings of Boston Society of Natural History 33, 125-209. ROBINOW, C. P. (1963). Observations on cell growth, mitosis, and division in the fungus Basidiobolus ranarum. The Journal of Cell Biology 17, 123-152.

Fig . 5. Entomophthora coronata. Leading hypha from the margin of a young colony on 0'2 % malt agar, drawn from an unopened, inverted Petri dish at 22°C at the indicated number of minutes from the start. A normal septum has formed (360 lim from the apex) after 70 min just above an incipient lateral. After 175 min three retraction-septa have formed above the normal septum. Tran s. Br . Mycol. Soc. 79 (2), (1982). Printed in Grear Brirain