Hyphal and conidial germination of ascospores in Bulgaria inquinans and of basidiospores in Ditiola peziziformis

[ 143 ] Trans. Br. mycol. Soc. 87 (1), 143-146 (1986)

Printed in Great Britain

HYPHAL AND CONIDIAL GERMINATION OF ASCOSPORES IN BULGARIA INQUINANS AND OF BASIDIOSPORES IN DITIOLA PEZIZIFORMIS 11

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

In Bulgaria inquinans, in which there are two types of ascospore in the ascus, the ascospore germinates either by a single, long germ-tube or with the production of conidia. The larger, pigmented spores exhibit lateral germination, but the smaller, hyaline spores germinate terminally. On virgin MA both types produce mainly germ-tubes, the hyaline spores giving a higher percentage germination. Under the immediate influence of a mature parent mycelium, the larger spores rarely germinate, but many of the smaller ones germinate to produce conidia. The development of blastoconidia and arthroconidia in culture is also described. In Ditiola peziziformis well-scattered basidiospores on MA germinate by hyphal germtubes, but close to a mature parent colony only conidia are formed. In Auricularia auricula-judae (Bull.) Wetts. (Ingold, 1984) and in Entomophthora pseudococci Speare (Ingold, 1986), one a basidiomycete and the other a zygomycete, spore germination on virgin 0 '2 % malt agar (MA) gives rise to long hyphal germ-tubes, whereas under the immediate influence of a mature parent mycelium both develop conidia directly. It seemed of interest to find out if the same situation occurred in an ascomycete. In search of an appropriate species, I consulted deBary's centuryold text book (deBary, 1887) and found there that the ascospores of Bulgaria inquinans Fr. are capable of forming conidia directly on germination. This determined my choice of fungus. The germination of the ascospores of th is species had been beautifully figured by Charles Tulasne (T ulasne & Tulasne, 1865). Ditiola peziziformis (Lev.) Reid, within Hemibasidiomycetes taxonomically remote from Auricularia spp. already analysed in relation to patterns of germination (Ingold, 1984), is also known to produce either germ-tubes or conidia when basidiospores germinate (Ingold, 1983). It was therefore decided to determine if here also proximity to a parental mycelium biased germination towards the conidial condition. Bulgaria inquinans B . inquinans is remarkable in producing within the ascus four large ( 11- 1 5 x 6--8pm, mean 12·6 x 7'0 pm) relatively thick-walled ascospores of a deep greenish-brown, and four smaller ones (7'5-9'5 x 4-5 pm, mean 8'1 x 4'7 pm) which are hyaline or only slightly coloured and with relatively

thin walls (F ig. 1 B, C). The two means given are based on measurements of 20 random spores freshly discharged. Both kinds of spore contain droplets of fat, larger and more numerous in the pigmented, but smaller and fewer in the hyaline ascospores. The differences between these two kinds of spore appears to be determined by two alleles of a gene. These may segregate at the first or at the second division of meiosis in the developing ascus. One of the four patterns of second division segregation is illustrated in Fig. 1 A. The larger spores tend to be bean-shaped. When viewed sideways, a germ-slit is visible along the length of the spore (F ig. 1 B). From discharged ascospores, a vigorous mycelium was soon established on MA. From a point source a colon y reached a diameter of 20-30 mm in four days at room temperature. It consisted of submerged and surface hyphae with no aerial growth. At first it was colourless, but soon, especially around the periphery, an orange colour appeared due to pigment diffused in the medium. When the colony was a few days old, conidia began to be produced in the older parts. Well back from the growing margin two kinds of hyphae were to be seen . First, there were numerous hyphae branching freely which developed thick walls with the lumen reduced to a thin line in the tapered, ultimate branches (F ig. 1 M). In these thick-walled hyphae septa were difficult to distinguish, being easily confused with bridges of cytoplasm separating long vacuoles. Secondly, there were thin-walled hyphae, with their cross-walls clearl y visible, which appeared to follow a random course on and in the

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Germination in B. inquinans and D. peziziformis

Fig. 1. Bulgariainquinans. (A) Ascus; one of the four patterns arising from segregation at the second division of meiosis; (B) larger-coloured ascospores; three show the longitudinal germ-slit; (C) smaller hyaline ascospores; (D) two larger ascospores germinating on MA after one day; (E) four smaller ascospores on MA after one day; (F) larger ascospores germinating to form blastoconidia; (G) eight smaller ascospores, after a day on an inverted MA culture of the fungus, forming blastoconidia; (H) thin-walled hyphae with conidiophores (from older parts of a mycelial colony); (I) thin-walled hyphae breaking up into arthroconidia; (J) blastoconidia; (K) blastoconidia germinating on MA after one day; (L) thin-walled unbranched hypha from the older part of a colony; (M) thick-walled hyphae from the same region. Contents shown only in A, B, C and M. All drawn from living material with the aid of a camera lucida,

C. T. Ingold agar. In these, lateral branches were rare. Fig. 1 L illustrates the middle portion, 200 p,m in length, of such a hypha which was at least 600 p,m long without any branches. It was from these hyphae that blastoconidia were produced in great numbers on tiny lateral conidiophores rarely more than spicula. The conidium developed as an apical swelling and was usually solitary, although sometimes groups of 2-5 were to be seen (Fig. 1 H). The conidia were unicellular, hyaline, mostly cylindrical and varied greatly in size (2-22 x 1-4 p,m)(Fig. 1 J). They were not aerial, being produced on the surface of the medium or submerged in the agar. In addition, arthroconidia were formed by splitting and rounding-off at the cross-walls of segments of the long, thin-walled hyphae (Fig. 1 I). Sometimes these arthroconidia bore vacated conidiophores. Arthroconidium production in the colony was on a minute scale in comparison with the production of blastoconidia. When streaked on MA, blastoconidia germinated after a day or two. For most the process involved swelling at the two ends to produce spherical vesicles giving a dumb-bell appearance (Fig. 1 K). Later a germ-tube grew out from one of these. Sometimes only a single vesicle was formed, and rarely the germ-tube developed directly from the conidium. This paper is, however, mainly concerned with the patterns of ascospore germination. After a day on MA a considerable number of discharged spores were found to have germinated. However, the percentage germination of the smaller ascospores was much higher (> 50%) than that of the larger, coloured spores « 5 %). No germination occurred on water agar. In germination a larger ascospore gaped open along the germ-slit and a germ-tube arose in a lateral position (Fig. 1 D). However, the germination of the smaller ascospores was terminal (Fig. 1 E). In most of these, swelling occurred at both ends of the spore, producing a line of three subspherical regions with slight constrictions between (Fig. 1 E). Then one of the terminal vesicles gave rise to a hypha, the other, probably because no nucleus was available, developing no further. In a spore deposit on MA after a day these triplets with their long, unbranched germ-tubes were conspicuous. However, sometimes only one vesicle was formed and occasionally a germ-tube arose directly from the ascospore without any vesicle being produced. In a spore deposit on MA the occasional ascospore of both types formed conidia on germination instead of a germ-tube. In being capable of either hyphal or conidial germination, Bulgaria inquinans resembles Auricularia spp., and

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this suggested an experiment, along the lines previously described for Auricularia (Ingold, 1984), to see if proximity to an established mycelium of the same species biased germination in favour of the conidial state. A colony on shallow (1'5 mm deep) MA having a diameter of 25 mm was used. A square (ca 10 mm side) of this colony was cut out and inverted, off centre, on MA in a fresh Petri dish. The exposed surface of this inverted colony was then subjected to a rain of ascospores for a few minutes from an overhead, inverted apothecium to give a light spore deposit. A similar deposit was produced as a control on the same dish at a considerable distance from the inverted square. The resulting situation was examined microscopically a day later. On the exposed surface of the inverted square only one of the large ascospores, out of hundreds examined, was seen to have germinated, and that by a lateral germ-tube. On the other hand, about 20 % of the small hyaline ascospores had germinated and nearly all of these (> 90 %) had produced conidia (Fig. 1 G). A few had indeed formed germ-tubes, but these were short and relatively narrow. In the control area on virgin MA over 50 % of the smaller spores had germinated, nearly all by a single long (40-100 p,m) and broad germ-tube, but an occasional spore had formed conidia. So far as the larger, pigmented ascospores were concerned over 10% had germinated, mostly by lateral germtubes, but a very few formed conidia. Thus the effect on germination of agar impregnated with the parent fungus was almost completely to inhibit germination of the large coloured spores, and strongly to bias those colourless spores that germinated towards conidium production, a situation finding a close parallel in that reported for Auricularia spp. Ditiola peziziformis Basidiospores discharged for a few minutes from a sporophore of D. pezizijormis on to MA, with a depth of 1'5 mm in the Petri dish, initiated a culture. When this was two weeks old, a square (ca 10 mm side) of agar, with a dense growth of the fungus producing abundant conidia, was cut out and inverted on the surface of MA in another Petri dish. Then from an overhead sporophore basidiospores were allowed to fall on to the exposed surface of the square to give a deposit of about five spores per mm-. A similar deposit was subsequently produced on the agar several centimetres from the inverted square. A day later examination showed that in both areas all the spores had germinated, but with a striking difference. In the square with the colony below, all the basidiospores had produced numerous spherical conidia (Fig. 2A) after the

Germination in B. inquinans and D. peziziformis

Fig. 2. Ditiola peziziformis. (A) Four basidiospores producing blastoconidia after a day on an inverted colony of the same species on MA; (B) four similar spores after a day on virgin MA; only hyphal germ-tubes are being produced. Hundreds of spores were examined in both treatments and were remarkably uniform.

manner already described (Ingold, 1983), but there was no hyphal germ-tube. On the other hand, on the virgin agar conidia were not formed, but instead broad hyphal germ-tubes were produced (Fig. 1 B). In other tests it was found that if basidiospores on virgin MA germinated under crowded conditions, a few conidia were formed in addition to the hyphal germ-tubes. DISCUSSION

It is clear that, both for the smaller ascospores of B. inquinans and for the basidiospore of D. peziziformis, germination close to a mature mycelium of the same species leads to the suppression of hyphal germ-tubes and to the encouragement of conidium development. The situation closely resembles that reported in Auricularia spp. (Ingold, 1984) and in Entomophthora pseudococci (Ingold, 1986). All may be regarded as examples of microcyclic sporulation (Smith et al., 1980) or of the telescoping of the life-cycle. The effects produced by proximity to the parent mycelium are, presumably, due to diffusible substances, and their identification is desirable. However, such work is quite beyond my own resources and capabilities.

My thanks are due to Professor J. Webster for supplying me with fresh material of the two species used in this study. REFERENCES

DEBARY, A. (1887). Comparative Morphology and Biology

of the Fungi, Mycetozoa and Bacteria (English translation). Oxford: Clarendon Press. INGOLD, C. T. (1983). Basidiospore germination and conidium development in Dacrymycetaceae. Transactions of the British Mycological Society 81, 563-571. INGOLD, C. T. (1984). Patterns of ballistospore germination in Tilletiopsis, Auricularia and Tulasnella. Transactions of the British Mycological Society 83, 583-591. INGOLD, C. T. (1986). Entomophthora pseudococci and its patterns of conidial germination. Transactions of the British Mycological Society 86, 239-245. SMITH, J. E., ANDERSON, J. G., DEANS, S. G. & BERRY, D. R. (1981). Biochemistry of micro cycle conidiation. In Biology of Conidial Fungi 2 (ed. Cole, G. T. & Kendrick, B.) pp. 329-356. New York: Academic Press. TULASNE, L. R. & TULASNE, C. (1865). Selecta Fungorum Carpologia. Paris: Imperial Press.

(Received for publication 21 November 1985)