Mycelium, oidia and sporophore initials in Flammulina velutipes

[ 1°7 ] Trans. Br. mycol. Soc. 75 (1) 107-116 (1980)

Printed in Great Britain

MYCELIUM, OIDIA AND SPOROPHORE INITIALS IN FLAMMULINA VELUTIPES By C. T. INGOLD Department of Botany, Birkbeck College, London WC1E 7HX In Flammulina uelutipes three types of mycelial branching are recognized: below-clamp, above-clamp and between-clamp. The formation of oidia on complex aerial branch systems (mainly of the above-clamp type) is described. The development of oidia is compared with that of arthroconidia in Geotrichum and with that of the chlamydospore in Mucor. The occurrence of oidia in nature is reported. Development of sporophore primordia is described with special reference to 'aspergilloid hyphae' and to cystidia. The possible function of cystidia in translocation is considered. Flammulina velutipes (Fr.) Karst. is abundant in Britain from October to April on dead stumps and trunks of elm (Ulmus spp.). It is a fleshly agaric of outstanding interest. Many aspects of its biology have been investigated including nutrition in relation to mycelial growth and sporophore production (Plunkett, 1953; Aschan-Aberg, 1958), translocation (Littlefield, 1966), hormone regulation of stipe growth (Gruen, 1969, 1976), and bioluminescence (Foerster, Behrens & Airth, 1965). A special feature is its ability to continue producing fruit-bodies and liberate spores under the coldest of winter conditions. In tests with sporophores fully equilibrated to their surroundings, I have found that spores are discharged down to at least 2 °C below zero. This toadstool has been used extensively in genetical studies (Aschan 1952; Takemaru, 1961). It is tetrapolar with mating determined by A and B genes. In common-B matings clamp connexions occur in the restricted zone where hyphae of the two colonies intermingle, but the ability to form clamps is not transmitted to the rest of the mycelia. Another special feature is that some single-spore haploid cultures produce fruit-bodies, but these either liberate no basidiospores, or, if these are formed, they are all of the same matingtype. Extra-nuclear cytoplasmic factors may be involved in the genetical situation (Croft & Simchen, 1965). F. velutipes is further of interest in producing asexual spores, usually termed oidia, abundantly both on the monokaryon and on the dikaryon (Brodie, 1936; Aschan, 1952; Takemaru, 1954). The fungus grows and fruits readily in culture and both basidiospores and oidia germinate at laboratory temperature in water within 24 h with over 80 % forming germ-tubes. This paper is concerned with the vegetative

mycelium particularly in relation to production of oidia and of sporophore primordia. MATERIALS AND METHODS

The dikaryotic isolate studied was obtained from inner tissues of a young sporophore. Monokaryotic cultures were derived from plates seeded by holding a pileus for a few seconds above agar in a Petri dish. After incubation for a few days isolated colonies were removed for separate culture. Colonies were grown on agar containmg low concentrations of malt, usually 0'2 or 0'5 %. RESUL TS AND DISCUSSION

In a dikaryotic colony on agar the growing margin consists of mycelium, at or below the surface, the principal hyphae of which follow a roughly radial course outwards. These hyphae are, in general, distinct from the lateral branches, although it is obvious that these leaders have themselves originated as strong laterals. This continuous recruitment of new leading hyphae is an inevitable consequence of increasing colony circumference. In the hypha shown in Fig. 1 growth over a period of five and a half hours was at a fairly steady rate of 160,umfh at 17'5 "C. Details of a similar hypha are illustrated in Fig. 2A. The formation of a new clamp in the main hypha occurs 300-400,um behind the apex, and the length of individual cells in a principal hypha is 500-600 pm. If the apical cells contain a pair of nuclei, as seems clear from the cytological studies of Brodie (1936), then, at the time of the conjugate division, they are a relatively long way from the apex over whose intense activities they no doubt exercise control. Further, following division, the

0007-1536/80/2828-6190 $01.00 © 1980 The British Mycological Society

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Flammulina uelutipes. Leading hyph a on agar su rface drawnat inte rvals over a period of 5 h 25 min . Po sition of clamp s indicated by black blobs.

upper nuclear pair mu st migrate a distance of several hundred micrometre s to be in position for the formation of the next clamp. In the great majority of the principal hyphae branching is just below the clamp , a single branch being formed in thi s position (Fi g. 2B). Lateral branches usually grow much slower , and their cells are much shorter th an in the principal hyph ae, but again br anching is mainly below clamps. This type of below-clamp br anching is characteristic of the submerged and surface mycelium. However , a few hyphae, pa rt icularly at the surface of the agar, exhibit another typ e of branching that may be called ' above-clam p branching ' . This is illustrated for a pr incipal hypha in F ig. 2C and for a lateral hypha in Fig. 2D. In this type of branching there are initially no cross-walls. If at a later stage walls do appear, they are without clamps. Although above-

clamp branching is som ewhat infrequ ent amongst th e hypha e of the growing margin of th e dikaryotic colony, it app ears to be very general in the aerial mycelium producing oidial branches. Another type of br anching is mu ch less frequent . It may be termed ' between-clamp branching' (Fig. 2E) and is to be seen in th e older parts of th e colony in which it is kno wn th at the number of nucle i in th e cells may have incre ased beyond the original two. The development and cytology of the oidia of F. uelutipes were studied by Brod ie (1936). H e found that in both monokaryon and dikaryon the y were uninucleate, a fact confirmed by Takemaru (1954), and cultures from a single oidium of the dikaryon were always monokaryotic. Thus oid ial formation in the dikaryon is a process of dedikaryotization, My own observations on living material confirm the general picture presented by

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Fig. 2. Flammulina uelutipes. Hyphal branching. (A) Apical part of a leading hypha on agar surface showing three cells (i , 2 and 3) accommodated on the page by two overlapping pieces; details of celljunctions shown at higher magnification; note below-clamp branching. (B) Details of a below-clamp branch system. (C) Above-clamp branching in a principal hypha on the agar surface; the clamp (X) is just forming; later its completion was observed. (D) Above-clamp branching in a lateral developed from a principal hypha less than one mm from its apex. (E) Between-clamp branching in a hypha from a relatively old region of a culture. High-power details in (A) and (E) are to the same scale; (B-D) at a lower magnification.

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Fig. 3. Flammulina velutipes. A, Small part of the aerial system of a dikaryotic culture on 0'1 % malt agar; note rather straight horizontal hyphae sparingly branched, and also oidial branches; one horizontal hypha following a diagonal course has been converted into a line of oidia; slightly diagrammatic. B, C and D, camera lucida drawings of three oidial branch systems of a dikaryon on surface of plastic where a square of agar has previously been removed. In D development of oidia has only just started. Brodie except in relation to the overall morphology of the oidial branches. He figures these as fairly simple systems starting normally from a uninucleate outgrowth of an intercalary cell of the dikaryon. According to this model all the oidia of a chain should be of the same mating-type, and this was established experimentally by Aschan (1952). Brodie showed that the dikaryotic colony produced monokaryotic oidia of both matingtypes. His evidence suggested that these were formed in roughly equal numbers, but Aschan found that oidia of one of the two mating-types tended greatly to predominate. The basic aerial system of a colony on agar consists largely oflong, sparingly-branched hyphae running in all directions and often adhering to form hyphal strands of two or three elements (Fig. 3A). It is from this system that most of the

oidial branches arise. These are often rather complex and it is only the simpler ones that can easily be analysed under the microscope. Because the oidial system shows such a high degree of branching, and because of the tendency of branches to coil round one another and around neighbouring hyphae, it is difficult to trace the connexion of an individual branch-system with the rest of the mycelium. However, most appear to be above-clamp branches (Figs 3B-D; 4A). In the oidial branch-system there are initially no cross-walls, but later scattered, simple (unclamped) walls appear. These systems are recognizable before oidia begin to form not only because of the absence of clamps, but also because of their form, since the constituent hyphae show little tendency to taper, in contrast to those in below-clamp branching. Fig. 4A illustrates a branch-system

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Fig. 4. Flammulina oelutipes. A, Oidial hypha I system of a dikaryon befor e the formation of oidia ; only one of the three branches of the system is shown. B, Part of a similar system in a dikaryon in which oidia production has started. C, Ultimate oidial branch showing separation of cytoplasmic segment s to form oidia in a dikaryon. D , Oidia in main hypha between two clamp s. E, Small part of long horizontal aerial hypha with collapsed hyphal walls between oidia. F, H ypha on the plastic surface in a Petri dish culture of the monoka ryon in an area where the agar has been removed in advance of growth; th ere are two lateral oidial branches; outl ines of disorganizing hyphae shown with dashed lines . G, Range of oidia from a dikaryon. H , Oid ia germinating in water after 24 h at room temperature. I, Basidiospores germinating under the same conditions. Separate scales for Band F are indicated; all the rest to the same scale. Drawn from water mounts with the exception of B which was surrounded by air.

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Fig. 6. Diagram of oidial development. A,Arthroconidium development as envisagedby Kendrick (1971 ) for Geotrichum candidum, Top figure shows hypha (with a double-layered wall) before septation has occurred. Band C, Oidial development in Flammulina uelutipes, In B the separation of protoplasmic masses is slight and the final result closely resembles a row of arthroconidia. In C the intervening vacuoles are much larger and there is little resemblance to arthroconidia. In Band C the original hyphal wall is shown as a single layer, and in the final stage the disorganizing wall is indicated with a dashed line. certainly destined to produce oidia, but only one of its three sections is shown. As Brod ie observed, oidial production starts at the apex of a hypha and proceeds backwards. Fig. 4B, C shows fairly early stages in this process which would eventually involve the whole branch and might also spread to the parent, clamped, hypha. Fig. 4D shows a situation, to be seen only in the older parts of a colony, with oidia formed between two clamps in a principal horizontal hypha. These oidia are, in places, widely spaced, no doubt because in such cells the vacuole is so large that the amount of cytoplasm available for oidium formation is small. Presumably the cell has become multinucleate prior to oidial production. When an oldish culture is viewed uncovered under the low power of the microscope, long stretches of horizontal hyphae can be seen with a beaded appearance due to numerous discrete oidia with empty, and almost invisible, collapsed hyphal tubes between (Figs 3A; 4E). If in a growing culture in a plastic Petri dish a small square, of about 2 em side , is cut away at an early stage, sparse horizontal hyphae, corresponding apparently to the horizontal aerial ones, grow over the surface of the plastic and produce lateral oidial branches. Fig. 3 shows three such oidial branches and Fig. 5A, B, C and D are photographs of parts of branches all from the dikaryon. Fig. 4F is from a monokaryon, In this not only are two lateral branches converted into oidia, but

also in the principal parent hypha a few oidia are formed. The asexual apparatus is of the dry-spore type, and Brodie (1936) observed that spores could be blown from a culture. I have confirmed this using the system employed by Zoberi (1961) for the study of spore liberation in moulds, but incorporating an impactor of the kind used in a Hirst spore -trap (Hirst, 1952). It was found that the oidia were blown off by a wind of 10'2 m/sec. The liberated spores appeared on the impactor slides mostly in chains of three or more. It may be questioned whether the asexual spores of F. uelutipes are properly described as oidia, also termed arthroconidia, or thallic arthric conidia (Kendrick, 1971). Such conidia are formed characteristically by centripetal fragmentation of septate hyphae, Geotrichum candidum Lk providing the typical example. However, the asexual spores of F. uelutipes are not quite of this nature. Brodie (1936) has described the process as involving vacuolation isolating uninucleate masses of protoplasm each of which presumably acqu ires its own su rrounding wall as in chlamydospore formation in Mucor spp. If, indeed, the spores are comparable with Mucor chlamydospores, it may be doubted if any conidial term inology is appropriate, since the y are really formed internally. Fig. 6A is based on the diagram by Kendrick (1971) of arthroconidium formation in G. candidum. Fig. 6B, C represents

114

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Fig. 7. Flammulina uelutipes. Cultures on 0 '2 % malt agar; dishes inoculated on 13 April 1979, grown at room temperature and photographed on 22 May 1979. Left, inoculated in centre; right, inoculated half way between centre and circumference. In this experiment there were three replicates of both treatments and the dishes in each set were alike. my interpretation of what happens in F. uelutipes. The situation in Fig. 6B gives a chain of spores similar to arthroconidia, but, when the separating vacuoles are relatively large, the similarity is much less. Both of these conditions are to be seen in the natural system shown in Fig . 4F, the individual spores being closer together in the ultimate branches of the sporulating system, but much more widely spaced further back. Although most oidia are cylindrical to oval, 5-8 x 10-20 tun, a number are much longer (up to So zzrn) and these are often curved, no doubt because they have been formed in a section of a spiral hypha (Figs 4 G; 5D ). Again a few are more or less Y-shaped as the result of development at a point of branching. In a review of the conidial stages of Hvrnenomycetes, Hughes (in Kendrick, 1971) remarked that most are known only in pure culture. There do not seem to be previous records of the asexual stage of F. uelutipes except in culture. However, I have found it in abundance on the surface of wood of elm logs bearing sporophores in my garden. Fruit-bodies are more usually seen on the bark, not directly on the wood, but it is likely that the vegetative mycelium is in the wood below the bark and not in the bark itself. Two questions arise: first, how does the fungus get into the wood? Secondly, how do the sporophores manage to develop on the bark itself? So far as the second question is concerned, careful examination shows that normally the exit is through an old bore-hole made by a bark-beetle. Further, when, as is usual, a group of sporophores is involved, the individual stipes are compressed together in the tubular

bore-hole which passes through the bark. It seems that the minute sporophore primordia develop immediately below the bore-hole and then grow out together. So far as entry to the tree trunk is concerned, nothing is known, but bore-holes may again provide the necessary channels. Indeed, it is possible that the Scolytus beetle that disperses the Dutch elm disease fungus (Ceratocystis ulmi (Buism.) C. Moreau) may also play a part in spreading F. uelutipes. Oidia germinate readily in water within 24 h at room temperature with a germ-tube from one or both ends of the spore (Fig. 4H). In this respect they are like basidiospores which germinate just as easily and also with one or two germ-tubes (F ig. 41). In a Petri dish culture of the dikaryon grown in the light at 15-20 °C on 0'2 % malt agar and started from a central inoculum, numerous, minute brown sporophore primordia appear in 4-5 weeks, by which time the whole area of the dish is occupied by mycelium. The primordia are limited to a peripheral annular zone 1-2 ern wide. This distribution is similar to that described by Madclin (1956) for Coprinus cinereus (F r.) S. F. Gray. Indeed, on solid media in Petri dishes many fungi start to sporulate as they approach the edge of the dish. However, the production of primordia near the circumference of the Petri dish is not, or not entirely, related to a checking of growth on reaching the limits of uncolonized medium. This is demonstrated by the fact that in a dish inoculated at a point midway between the centre and the circumference, the pattern of primordia distribution is different from one resulting from central

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Fig. 8. Flammulina uelutipes, A, Knot of hyphae, representing very early stage of sporophore primordium, beset with 'aspergilloid' hyphae. B, Young primordium (compact mass of hyphae) shown in silhouette, to same scale as A, with cystidia exuding fluid. C, Silhouette of older primordium in which differentiation of stipe and cap has occurred. D, ' Aspergilloid' hyphae. E, cystidia.

inoculation. A crescent of medium, not an annulus, is occupied by primordia (Fig. 7). This suggests that it is distance from the inoculum that is significant rather than the size of the Petri dish. In a culture hundreds of primordia are produced, but only one or two develop further into mature sporophores. In the general zone of primordia production aerial hyphae of a special kind occur

which may be referred to a 'aspergilloid hyphae'. At first sight under the dissecting microscope it appears that the culture is contaminated with Aspergillus. Under high power, what seemed to be conidiophores of that mould are revealed as specialized erect hyphae. Each of these has a slightly swollen apex from which there radiate needle-shaped or feathery crystals (Figs 5 G; 8 D).

116

Oidia and sporophores of Flarnmulina

The aspergilloid hyphae seem to be limited to the dikaryon and to that part of it in which sporophore production occurs. Although a few of these hyphae may be scattered generally, they are particularly abundant in relation to incipient sporophores (Fig. SA ). The primordium first becomes recognizable, at or just above the surface of the agar, as a somewhat irregular tangle or thicket of aerial hyphae around 100 /lm across and beset with aspergilloid hyphae (Fig. SA). Later, associated with this thicket, there develops a shaft of narrow and mostly parallel hyphae of a pale brown colour bound together in a common slim e. Soon from the apex of this shaft cystidia (Fig. SE) develop, each secreting a droplet of liquid (Figs 5E, F; 8 B). As the shaft enlarges it fairly soon differentiates into a distinct stipe with an apical knob, the future pileus (F ig. 8 C). At this stage the whole primordium is densely clothed with cystidia particularly on the young cap, and the aspergilloid hyphae are no longer to be seen. The cystidia persist and increase in number during the later development of the fruit-body. They occur in large numbers on the upper surface of the mature pileus, widely scattered on the surface of the gills, and abundantly on their edges. The cystidia have a striking capacity to exude liquid and each is normally capped by a droplet. Often the droplets run together to form a macroscopic drop. In a culture on a thinly-poured agar plate left exposed in a room, drying occurs and the agar is ultimately reduced to a dry paper-thin layer. When the tiny primordia in such a culture were viewed under the microscope, it was found that, in spite of the extreme dryness of the culture generally, the cystidia were still exuding droplets. This observation suggests the possibility that the cystidia may be of functional significance in drawing water-soluble food into the developing sporophores. In the production of a fruit-body, soluble organic substances must continually be brought to it, and that such translocation does occur has been demonstrated experimentally in F. velutipes (L ittlefield, 1966). Buller (1933), working mainly with Coprinus cinereus, suggested that anastomoses, by which the mycelium is converted into a threedimensional network, facilitate translocation. However, my observations of the mycelium of F. oelutipes close to a sporophore primordium provided little evidence of anastomoses, nor were there major hyphae or hyphal strands leading directly towards it. In spite of the apparent absence or a specialized mycelial organization that

might facilit ate flow, it is clear that there must be a considerable movement through the vegetative hyphal system to the developing sporophore. Perhaps in this connexion the cystidia play a part by sucking in the aqueous fluid rich in organic substances and concentrating it by the excretion of excess water. REFERENCES ASCHAN, K. (1952). Studies on dediploidization mycelia of the basidiornycete Col/ybia uelutipes. Svensk Botanisk Tidskrift 46, 366-392. ASCHAN-ABERG, K. (1958). The production of fruit bodies in Collyb ia oelutipe s. Physiologia Plantarum 11,312- 32 8. BRODIE, H. J. (1936). The occurrence and function of oidia in the Hyrnenomycetes. American Journal of B otany 23, 310--327. BULLER, A. H. R. (1933). Re searches on Fungi. 5. London. CROFT, J. H. & SIMCHEN, G. (1965). Natural variation among monokaryons of Collybia uelutipes, The American Naturalist 99, 451-462.

FOERSTER, G. E., BEHRENS, P. Q . & AIRTH, R. L. (1965). Bioluminescence and other characteristics of Collybia uelutipes, American Journal of Botany

sz, 487-495.

GRUEN, H. E. (1969). Growth and rotation of Flammulina uelutipe s fruit bodies and the dependence of stipe elongation on the cap. M yc ologia 61, 149-166. GRUEN, H. E. (1976). Promotion of stipe elongation in Flammulina uelutipes by a diffusate from excised lameJIae supplied with nutrients. Canadian Journal of Botany 54, 13°6-13 15.

HIRST, J. M. (1952). An automatic volumetric spore trap. Annals of Applied Biology, 39, 257-265. KENDRICK, B. (E d .) (1971). Ta xonomy of Fungi lmperfecti, Toronto. LITTLEFIELD, L. J. (1966). Translocation of phosphorus-jz in sporophores of Collybia oelutipes. Phy siologia Plantarum 19, 264-27°. MADELIN, M. F. (1956). Studies on the nutrition of Coprinus lagopus Fr., especially as affecting fruiting. Annals of Botany

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PLUNKETT, B. E. (1953). Nutritional and other aspects of fruit body formation in pure cultures of Collybia oelutipes (Cu rt .) Fr. Annals of Botany 17, 193-217. TAKEMARU, T. (1954). Genetics of Collybia uelutip es, II. Dediploidization and its genetical implication . The Japanese Journal of Genetics %9, 1-7·

TAKEMARU, T. (1961). Genetical studies on fungi X. The mating system in Hymenornycetes and its genetical mechanism. Biolog ical Journal of Okayama University 7, 133- 21 1. ZOBERI, M. H. (1961). Take-off of mould spores in relation to wind speed and humidity. Annals of Botany %5, 53-64.

(R eceived for publication

2

August 1979)