Physiological observations on Sphaerobolus

Physiological observations on Sphaerobolus

[ 4 11 ] Trans. Br. mycol. Soc. 57 (3),411-416 (1971) Printed in Great Britain PHYSIOLOGICAL OBSERVATIONS ON SPHAEROBOLUS By JUDITH M. ALLOWAY AND ...

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[ 4 11 ] Trans. Br. mycol. Soc. 57 (3),411-416 (1971) Printed in Great Britain

PHYSIOLOGICAL OBSERVATIONS ON SPHAEROBOLUS By JUDITH M. ALLOWAY

AND

C. T. INGOLD

Birkbeck College, London (With 4 Text-figures) Absorption, in the visible region of the spectrum, by a methanol extract of Sphaerobolus is described. There is no significant absorption above 580 urn; below this there are peaks at 420, 452 and 480 nm, The course of pigment development in cultures following exposure to light is examined, the concentration rising to a peak and falling sharply as discharge commences. It later increases again as the next crop of sporophores develops. Localization of glycogen and of the orange carotinoid pigment is illustrated. In the palisade layer of the inner peridium pigment is limited to the upper part that is devoid of glycogen. Growth of isolates readily referable to S. stellatus is slower at the optimum and the temperature optimum is lower than in three isolates of a larger species, one from Nigeria, one from South Africa, and one from W. Pakistan.

PIGMENTATION

Sphaerobolus exhibits sensitivity to light in a rather complex manner. For sporophore initiation and early development light is essential, and it has been shown (Alasoadura, 1963) that the blue end is the effective region of the visible spectrum. However, during the second of the 2 weeks of the full developmental period, at 18-20 cC, light ceases to be essential but remains strongly stimulatory (except for the very last day) and during this period it is light of longer wavelength, in the yellow to red spectral regions, that is especially effective. Again light usually determines the direction in which the glebal mass is discharged from the sporophore. Early in development the base-apex axis of the nearly spherical sporophore becomes oriented parallel with the incident light, and further, in the very last stages of development light appears to determine the position of the centre around which stellate opening finally occurs prior to discharge. Either or both of these responses may determine discharge towards the light (Nawaz, 1967). It is light at the blue end of the visible spectrum that governs the direction of glebal mass discharge. Even in the very last stages, orange light is without effect (Ingold, 1969). It is to be noted, however, that whereas in isolates from Britain, Nigeria and the United States, readily referable to S. stellatus Tode ex Pers., this phototropic response is very striking, in two large African isolates of Sphaerobolus with black glebal masses (Ingold, 197 I) this reaction is feeble or even non-existent. Clearly, pigments are of interest in Sphaerobolus as possible photoreceptors in the light-sensitive processes. In connexion with sensitivity to blue light 26

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a yellow-orange photoreceptor might be envisaged, but with sensitivity to orange or red light a blue receptor seems indicated. A conspicuous feature of fruiting cultures is the orange colour of the ripening sporophores. Friederichsen & Engel (1957) have published an absorption spectrum of a petrol-ether extract of S. stellatus below 600 nm. This shows strong absorption in the region 420-510 nm with peaks at approximately 440, 460 and 490 nm. This type of absorption curve is highly characteristic of coloured carotinoids. In methanol extracts of fruiting cultures we confirm their findings, the peaks being at 420, 452 and 480 nm (Fig. I). It is to be 1·8 1·6 1-4 1-2

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Sphaerobolus, Absorption spectrum of methanol extract ofsporophores using SP8oo.

noted that the exact position of these peaks is influenced by the solvent used. In this work no absorption was found in the visible region above 580 nm, even when highly concentrated extracts were made from cultures at a stage when the sporophores were particularly sensitive to light from a sodium lamp (585 nm). It was considered of interest to follow the course of pigment production in cultures exposed to light. In darkness no carotinoid pigment appears to be formed and, indeed, its production in the light seems to be restricted to the sporophores. Further, in illuminated cultures the agar itself assumes a distinct yellow colour, probably due to a water-soluble pigment, but it has not yet proved possible to concentrate this sufficiently to obtain an absorption spectrum. In this work the following procedure was used. Petri dish cultures on oatmeal agar were inoculated each with a single, centrally-placed glebal mass and incubated at 18-20° in the dark. On the seventeenth day half the dishes were transferred to continuous light (500- 1000 Ix from' daylight'

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fluorescent tubes), the rest remaining in the dark. Samples (each of ten dishes) were taken (one from light and one from darkness) at intervals of 2 or 3 days, any glebal masses discharged being also recorded. For a particular pigment determination each of the ten dishes had cut from it a square of 2'5 em side with the original inoculum at the intersection of the diagonals. These squares were extracted with 10 ml methanol and during the process they were ground up with sand to facilitate extraction. The concentration of pigment was read at 452 nm from absorption spectra. 1·8 1·5 1·2 0·9 0·6 0·3 0

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Fig. 2. Sphaerobolus. Three separate experiments showing amount of pigment (in arbitrary units) plotted against time (e-e), using previously dark-grown cultures exposed to continuous light. Daily rate of glebal mass discharge (0-0) also indicated.

The results of three experiments of this kind are illustrated in Fig. 2. It will be seen that following exposure of dark-grown cultures to light, orange pigment began to be formed, increased and reached a maximum at the onset of discharge. The decline in pigmentation of the culture as discharge commenced can be attributed to the breakdown ofthe exhausted sporophores which apparently also involves the destruction ofthe associated carotinoid. Although the discharged glebal masses are themselves coloured, the colour is not due to pigments extractable with methanol. At no stage was any absorption recorded in extracts from the dishes kept in darkness. It has already been noted (Alasoadura, 1963; Ingold & Peach, 1970) that under constant illumination at 18-20° successive crops of'sporophores, spaced 10-12 days apart, ripen and discharge their glebal masses. It is not 26-2

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surprising, therefore, that after the first peak ofpigmentation, and following the decline associated with discharge from the first crop of sporophores, the pigment present in the cultures tended to increase again although in a somewhat irregular manner. The distribution of orange colour in the mature, but still unopened fruit-bodies was studied in thin sections, cut vertically, using a freezing A

Fig. 3. Sphaerobolus. Vertical section of mature, unopened sporophore (based on freezing microtome sections offresh material). Left halfshows cellular details; right only outlines the tissue and indicates distribution of orange pigment (small dots) and glycogen (large dots). Irregular central cavity shown is where some glebal contents hav e fallen out during sectioning. A, E, C, layers of outer peridium forming outer peridial cup; D, E, F, layers of inner peridium (F breaks down during opening) ; G, glebal wall; H, spores and gemmae of gleba.

microtome. At the same time other sections, mounted in iodine dissolved in potassium iodide, indicated the distribution of glycogen which is stained chestnut-brown in that reagent. The carotinoid pigment is located in the upper parts of the mature but

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unopened sporophore (Fig. 3). It occurs particularly in the upper region of the middle layer of the inner peridium, a region where the palisade of elongated cells (arranged radially in the unopened fruit-body) gives place to pseudoparenchyma. However, the orange colour extends also to the outer layers of the peridium. When the sporophore opens, the pigmented regions of the inner peridium are freely exposed, since the carotene is largely in the teeth which are attached to those of the outer peridium. 3·5

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Fig. 4. Sphaerobolus. Temperature and extension growth. Three isolates: A, from South Africa (IMI 1551°3); B, from Ibadan in Nigeria (IMI 155104); and C, from W. Pakistan (IMI 158763), all with large sporophores producing black glebal masses. Also three isolates: D, from Britain (IMIISSIOI); E, from He in Nigeria (IMII55I02); and F, from California (IMIISSIOS), all with much smaller sporophores producing brown glebal masses.

This may be of significance if the carotene is the effective photoreceptor and if Nawaz (1967) is correct in thinking that even after a sporophore opens it can respond directionally to light.

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The storage of glycogen on a considerable scale appears to be confined to the palisade region of the middle layer of the inner peridium. As noted by Walker & Andersen (1925), and easily confirmed, the inner peridium ceases to give the glycogen reaction by the time actual discharge occurs. The glycogen is apparently converted to sugar (Engel & Schneider, 1963) thus building up the osmotic pressure and increasing the tissue tension between the layer of palisade cells straining to expand and the outer layer of fine and mainly tangential hyphae. TEMPERATURE AND GROWTH IN CERTAIN ISOLATES

Two types of Sphaerobolus isolate have been studied in this laboratory and almost certainly two distinct species are involved (Ingold, 197 I). Three isolates of one type have relatively large sporophores. From these black glebal masses are discharged each with a distinct cap and each containing spherical 'cystidia' (Buller, 1933). Of these three, one is from South Africa, another from Nigeria (Ibadan) and the third from West Pakistan. All other isolates, including several from Britain, one from California and one from He in Nigeria, have much smaller sporophores that are strongly orange in colour. Further, the brown glebal mass has no cap and contains no 'cystidia'. It was considered of interest to study some of the growth characteristics of these isolates. When the isolates were grown at various temperatures on oatmeal agar, it was found (Fig. 4) that the three with the bigger fruit-bodies had a higher optimum for extension growth (around 30°) whilst all the isolates of the smaller form, even those from warmer regions (Nigeria and California), had a lower optimum (around 25°). Thus the differences in optima would seem to be species-related and not essentially connected with the prevailing temperatures in the countries from which the isolates originated. At the optimum the growth rate of the isolates of the larger form was considerably greater than that of the isolates of the smaller one. REFERENCES

ALASOADURA, S. O. (1963). Fruiting in Sphaerobolus with special reference to light. Annals ofBotany 27, 125-145. BULLER, A. H. R. (1933)' Researches onfungi, 5. London. ENGEL, H. & SCHNEIDER,]. C. (1963). Die Umwandlung von Glykogen in Zucker in den Fruchtkorpern von Sphaerobolus stellatus (Tode) Pers. vor ihrem Abschusz. Berichte derDeutschen botanischen Gesellschaft 75, 397-400. FRIEDERICHSEN, I. & ENGEL, H. (1957). Beitrage zur Kenntnis des Abschussrhythmus und Farbstoffs von Sphaerobolus stellatus (Tode) Pers. Planta 49, 578-587. INGOLD, C. T. (1969). Effect of blue and yellow light during the later developmental stages of Sphaerobolus. American Journal ofBotany 56,759-766. INGOLD, C. T. (197 I). The glebal mass of Sphaerobolus. Transactions oftheBritish Mycological Society 56, 105-113. INGOLD, C. T. & PEACH,]. (1970). Further observations on fruiting in Sphaerobolus in relation to light. Transactions of the British Mycological Society 54, 2 I 1-220. NAWAZ, M. (1967). Phototropism in Sphaerobolus. Biologia 13, 5-14. WALKER, L. B. & ANDERSEN, E. N. (1925). Relation of glycogen to spore-ejection. Mycologia 17, 154-159.

(Acceptedfor publication 8 March 1971)