The sporangiophore of Pilaira species

[ 553 ] Trans. Br. mycol, Soc. 61 (3), 553-568 (1973) Printed in Great Britain

THE SPORANGIOPHORE OF PILAlRA SPECIES By H.

J. FLETCHER

School of Pharmacy, Robert Gordon's Institute of Technology, Aberdeen (With Plate 57 and 9 Text-figures) The form and behaviour of the sporangiophores of Pilaira anomala, P. caucasica and P. moreaui were examined. The stages of development resembled both Phycomyces and Pilobolus and the duration of these stages varied between the species. Rates of growth of the sporangiophores were measured. 'Stolons' were formed by abortive sporangiophores under conditions of unfavourable nutrition. The pattern of curvature of sporangiophores during positive phototropism in air and its reversal under liquid paraffin resembled Phycomyces.

Some aspects of the development, growth and tropism of the sporangiophore of Pilaira anomala (Ces.) Shrot., P. caucasica Milko and P. moreaui Ling-Young have been investigated. The stages of development of the sporangiophore of P. anomala were described by Fletcher (1969) where they were compared with those of Phycomyces (Castle, 1942) and of Pilobolus (McVickar, 1942). In the light of further studies presented here, the stages of Pilaira anomala are redefined, and estimates made of the minimum duration of each successive stage in the three species. The rates of growth of developing and mature sporangiophores have been measured. Some observations have been made on 'stolons', structures which are apparently abortive sporangiophores, which have not been reported in related genera. Finally, a detailed study of the phototropism of individual developing and mature sporangiophores has been made, similar to that for Phycomyces, extensively reviewed by Bergman et al. (1969) and to Pilobolus, reviewed by Page (1968). Previous work on these and other aspects of Pilaira anomala was surveyed by Fletcher (197 I ). MATERIALS AND METHODS

The isolates used were P. anomala (IMI 109387), and the type cultures of P. caucasica (CBS 523-68) and P. moreaui (CBS 101-26). Cultures were maintained on Sabouraud's maltose agar (SMA): maltose (Koch-Light) 40 gil; Mycological Peptone ('Oxoid') 10 gil and agar (' Oxoid' no. 3) 12 gil using 7'5 ml of medium in polystyrene 'Universal' bottles (' Sterilin' Ltd. Richmond, Surrey) for slope cultures. All media were sterilized in glassware, at I bar gauge pressure for 15 min, prior to pouring into polystyrene containers. Experimental cultures were grown on a one-fifth dilution of the SMA medium (SMA/5) in 'Sterilin' polystyrene Petri dishes containing 15 ml.

554

Transactions British Mycological Society

Plugs of mycelium on agar were cut from young colonies on SMA by means of a flamed cork borer of approximately 3 mm diam. and inoculated on to SMA medium. Most cultures were incubated in an illuminated cabinet, fitted with an 'Atlas' 3 W miniature (540 mm ) fluorescent tube. The cabinet was maintained at approximately 22 °C and fluctuations in temperature were monitored on an automatic recording thermometer (Cambridge Instrument Co.), which established that temperature variations rarely exceeded ± 1°. The light intensity within the cabinet ranged from 50 to 150 lux, according to position . In order to produce artificial light regimes (e.g. 6 h darkness and 18 h light every 24 h) the light source was fitted with a Sangamo Weston 24 h dial synchronous timeswitch. Photomicrographs were made with a Gillet and Sibert 'Photoconference' microscope which had a quartz-iodine light source, voltage regulator and light meter. This instrument allowed a wide range of magnification to be used. A dark green filter was inserted between the lamp and the condenser.

Development andgrowth of sporangiophores Plate cultures were inverted and exposed to alternating light and darkness. Each lid was wiped free from condensation and the culture, with its lid upwards, was transferred to the stage of the photomicroscope. Observations were made in a constant temperature room at 20° ± 1°C under diffuse illumination of approximately 80 lux. Selected stages were observed over measured time intervals. At the same time, other sporangiophores appeared on the negatives and their development could be followed on the enlarged prints. This particularly applied to the initiation of stage I sporangiophores. In this way the duration of up to four replicates of each stage of the three species was obtained and the elongation of sporangiophores for growth studies could also be followed.

Phototropism Plates were placed horizontally on the microscope stage and examined through their lids. Suitable early or mature sporangiophores were selected. For each experiment, the dish was orientated so that the selected sporangiophore was initially growing horizontally in a direction perpendicular to the unilateral beam of light. The subsequent positive or negative phototropic curvature of the sporangiophore occurred in an approximately horizontal plane and could be measured by observation through the microscope from above. Enlarged prints or tracings of magnifications up to x 300 were obtained. Using the terminology of Castie (1962) the curvature of the sporangiophore was analysed by measuring the slope angle along the enlarged print of the sporangiophore with a protractor at distances (Sy) on the axis of the sporangiophore, upwards from a fixed point below the growth zone. A guttation droplet that was present throughout the period of observation or a well-defined morphological feature near the sporangiophore on the surface of the substrate served as the fixed point. Up to four sporangiophores of earl y and mature stages of each species were analysed.

Sporangiophore of Pilaira. H. J. Fletcher

555

n

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I.

Key to the terminology used in the description of the stages of development of Pilaira (based on P. anomala).

RESULTS

Stages of development The new terms proposed are presented in Fig. 1. Stage II has been subdivided into IIa and lIb, to separate the transient primordial papilla (II a) which precedes the more obvious swelling of the very young sporangium (lIb). Stage III has been subdivided so that the former III can be redesignated lIla and the former IVa redesignated Illb. The terms IVa and IV b (Castle, 1942) are unacceptable in the absence of anti-clockwise rotation of the sporangium, a feature of the stage IVa of Phycomyces. The rapidly elongating phase of Pilaira is termed IV. This is terminated when dehiscence and detachment of the sporangium takes place (stage V). These stages have been described for P. anomala (Fig. 1), P. caucasica (PI. 57, fig. I) and for P. moreaui (PI. 57, fig. 2). There was no difference between the three species in the external appearance of their development from stages I to III b. There was also no difference in the next stage of development in which the sporangium borne aloft by the rapidly elongating sporangiophore dehisced from the columella during stage IV, the whole still held together by mucilage. 36

M YC 61

55 6

Transactions British Mycological Society Table

Estimated duration of intermediate stages of sporangiophore development (minutes)

I.

P. anomala

,

Stage

I lIa lIb IlIa IlIb

Mean

> 285 20 23 45 13°

>210 17 23 45 13°

>2°3 17 20 45 >72

>2°5 22 20 > 137 Total

P. caucasica

,

Stage

I lIa lIb IlIa IIIb

Mean

>5°0 38 >23 65 152

348 19 27 4° 155

381 25 45 60 120

3° 23 72 138 Total

P. moreaui Stage

I lIa lIb IlIa IIIb

226 19 22 45 117 429

4 10 24 29 59 141 663 Mean

>29° >18 35 32 113

>27° > 18 35 32 113

>253 20 32 48 113

>253 20 35 3° 113 Total

267 19 34 35 113 468

The final phase of sporangiophore development occurred when the sporangium separated from the columella; this stage V was equivalent to the forcible discharge in Pilobolus. Sporangium detachment in Pilaira occurred when the sporangiophore had elongated so as to bring the sporangium into contact with a hard surface (Ingold, 1971). There was a minor difference between P. anomala and the other two species in that in P. anomala the sporangium and spores remained together and the sporangial wall 'roofed over' the spores when it became attached to a surface. In the other two species, the mucilage appeared to be less viscous (although apparently no less efficient in holding the sporangium to the columella during dehiscence) and the spores were easily dispersed from the detached sporangium. This resulted in a droplet of spores being separated from the main mass at the time of impact.

Duration of stages In the measurement of the duration of the stages I-III b inclusive, the initial and final times of observation, of up to four sporangiophores of each stage were made. Most of the durations in Table I represent the difference between the final and initial times, but if one of these times was not available, an estimated minimum value is presented, based on observations of the previous or next stage. The values for the estimated duration of stages and their means are

Sporangiophore of Pilaira. H. J. Fletcher

557

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given in Table I. The total time taken for sporangiophores of each species to reach maturity is also given. These times are subject to the inaccuracy of the method of observation, based on photomicrographs taken at intervals, so that in the transient stages an error of the order of 10-15 min is probable. From Table I, it can be seen that stage I was the longest stage in development. It was terminated by a rapid change, when elongation ceased and a papilla appeared at the previously conical tip. Stage II was a transient stage; in stage III a the sporangium enlarged and the columella and spores were formed. Finally, the upper part of the wall darkened, the sporangium reached maximum diameter and entered the resting phase (III b). This stage was the second longest in duration. The whole developmen~ process would appear to be longer in P. caucasica than the other two species,

Rates of growth Growth curves of young sporangiophores (stages I -II a and III b) are shown in Fig. 2 for P. caucasica and P. moreaui. P. caucasica showed the most abrupt cessation of growth in the four sporangiophores studied (Fig. 2 A-D). The rapid phase of growth of stage I was terminated by the development of a sporangial primordium (stage II a). Further elongation was then temporarily arrested. In P. moreaui (Fig. 2 E, F) growth is shown during part of stage I, the measurements being made during the development of a phototropic curvature of these sporangiophores. Here there was a somewhat accelerating phase of growth during the observed period, even at maximum curvature. The growth of stage IV sporangiophores commenced in the same way as many similar structures, with an exponential phase preceding a steady 36-z

Transactions British Mycological Society ~

P. caucasica

Stolons

P. moreaui

!Omm Fig. 3. 'Stolon' production by Pilaira species on water agar, traced from photomicrographs.

linear phase of rapid elongation growth. It is uncertain whether dispersal of the sporangium occurs during the linear phase or during a subsequent decline in growth. The growth of stage IV commenced when stage III b had completed its period of cessation of both elongation and sporangial expansion. Initially, elongation was very slow in P. moreaui, a rate of less than I pm min -1 was followed by a rapid increase in rate, to a steady maximum of about 40 pm min-I. This seems the normal rate for P. moreaui and probably also for P. anomala and P. caucasica. 'When sporangiophores had elongated they became attenuated, possibly thinner-walled and were very liable to collapse.

'Stolon' production In P. anomala 'stolons' were observed on suboptimal synthetic agar media (Fletcher, 197 I) containing, for example, glucose, salts and amino acids, but lacking natural sources of growth factors such as yeast extract or peptone. On these media an early check in the radial expansion of the vegetative hyphae was usually observed, and if illuminated, structures resembling sporangiophores and sporangia were developed. Many sporangia were found to be abortive in lacking spores and the sporangiophores failed to exhibit negative geotropism and positive phototropism. These sporangiophores frequently collapsed outside the vegetative margin of the colony and new vegetative growth developed from them. PI. 57, fig. 3, shows an example of a sterile aerial' stolon' terminating in vegetative hyphae, and PI. 57, fig. 4, shows an apparently abortive sporangium which has given rise to dendroid branches possibly arising from spores. The proximal part of this' stolon' had completely collapsed on the agar and produced lateral hyphae. 'Stolon' formation was induced in P. caucasica and P. moreaui by growth on water agar, and normal vegetative mycelium was not produced (Fig. 3). The 'colony' was entirely composed of secondary mycelium developed

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F ig. 4. T racings from ph otomicr ographs of stage I sporangiopho res of Pilaira species un d ergoing phot otropi c curva ture in ai r at the time in terv als shown (mi n) . I ncid ent light horizontal fro m left ; h orizontal bar shows location of fixed m arker below th e growth zone. Sporangiophores A-C and G * ; P. anomala ; D-E: P. caucasica; F: P. moreaui. (* = under p araffin.)

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Transactions British Mycological Society

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Fig. 5. Location of curvature on stage I sporangiophores in air. Slope angle (1)), i.e. deviation from the vertical, at distance (Sy) upwards from a fixed point below the growth zone, for sporangiophores at successive time intervals. (N.B. Different symbols are used for each sporangiophore at a particular time.) Sporangiophores A~C: P. anomala; D-E: P. caucasica; F: P. moreaui.

from arched 'stolons' derived from the inoculum plug. There were also some depauperate stage IV sporangiophores. Thus the' stolon' is formed in response to poor conditions of nutrition and shows the regenerative properties of the sporangiophore.

Phototropism Tracings of the photographic record of the three sporangiophores of P. anomala in the positive phototropism of stage I sporangiophores in air are shown (Fig. 4 A-C). The slope angle (¢) in relation to height above a fixed point below the curving zone (Sy) of each sporangiophore at time intervals during the observation period is shown (Fig. 5 A-C). In this figure and in Figs. 7 and 9 the angles of deviation of sporangiophores were measured at points at regular fixed intervals from a fixed base point. The Figs. 5, 7 and 9 indicate the length of curvature, the zone between the lowest point at which no curvature was detectable and the highest point at which no curvature was detectable. These figures also indicate the development of curvature with time. In stage I there was a wide variation in results between the three sporangiophores. Curvature occurred initially just below the tip of the sporangiophore (Fig. 5 A-C). As curving continued, the curve migrated down the sporangiophore, while the upper part appeared to cease curving, although prior to this there was some upward migration of the curving

Sporangiophore of Pilaira. H. J. Fletcher

561

zone. The downward migration (Fig. 5) appeared to be abrupt as measurements were made only at widely spaced intervals along the sporangiophore. This downward migration demonstrates in Pilaira the observation of Castle (1962) on stage IV of Phycomyces. The zone of curvature lengthened during the response usually reaching a maximum value at maximum curvature. Data relating to stage I sporangiophores of P. caucasica are shown (Figs. 4 D, E; 5 D, E). Neither sporangiophore showed the downward migration of the curve. In the longer sporangiophore, the tightness of the curving zone was particularly marked, being confined to the same region throughout the period of observation. Curvature in the sporangiophore of P. moreaui is illustrated (Figs. 4 F; 5 F). The pattern of curvature was more like that of P. caucasica than P. anomala, the zone of curvature showed no sign of downward migration and upward migration was confined to the first half of the period of observation. Other aspects were similar to the other species. In the positive phototropism of stage IV sporangiophores in air, the reaction time was shorter in all three species. The rate of curvature was often faster and this was consistent with a higher rate of elongation. The phototropism in air of stage IV sporangiophores of P. anomala is shown (Figs. 6 A-D, 7 A-D). Downward migration of the curve was marked, but the curving zone was farther away from the base of the sporangium (equivalent to the tip of the sporangiophore in stage I). Sporangiophore B appeared to show some recovery of its curving zone after 7 min, which was seen in the change in the distribution of the slope angle (Fig. 6), but the possibility exists that this sporangiophore had dropped towards the agar, twisting out of the plane of measurement, producing an apparent reduction in curvature. A declination of about 20° from the horizontal plane would be sufficient to cause this effect. The results for sporangiophores of P. caucasica are shown (Figs. 6 E, F, 7 E, F). Sporangiophore E was the upper part of a long, very rapidly growing cell. It showed the location of the origin of the curve but was followed over too short a period to observe its migration. Migration of the zone of curvature in sporangiophore F, followed over a longer period, was observed. Although the average rate of growth of sporangiophore E was nine times faster than that of F and its rate of curvature was approximately five times greater, sporangiophore E had a shorter curvature zone than F. There is therefore no relationship between the measurements for growth and curvature in these sporangiophores. Results for the sporangiophores of P. moreaui are shown (Figs. 6 G-K, 7 G-K). Downward migration of the curve was seen in sporangiophores G, H,], coupled with upward migration of the upper limit of the curve of these sporangiophores. In sporangiophore K, recovery was coupled with a change in the distribution of the slope angle. Negative phototropism under liquid paraffin was observed at stages I and IV. At stage I this phenomenon has been observed in the three species of Pilaira on agar plates covered with liquid paraffin, illuminated unilaterally in an incubator. The effect was clearly marked in P. anomala (Fletcher, 1971), but only weakly developed in the other two species. A single

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Sporangiophore of Pilaira. H. J. Fletcher

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Transactions British Mycological Society A

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Sporangiophore of Pilaira. H. J. Fletcher

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Fig. g. Location of curvature on stage IV sporangiophores under paraffin. Slope angle (9), i.e. deviation from vertical, at distance (Sy) upwards from a fixed point below the growth zone, for sporangiophores at successive time intervals. (N.B. Different symbols are used for each sporangiophore at a particular time.) Sporangiophores A-B: P. anomala; C: P. caucasica; D: P. moreaui.

sporangiophore of P. anomala undergoing negatively phototropic curvature is shown in Fig. 4 (G). Results for stage IV sporangiophores of P. anomala under liquid paraffin are shown (Figs. 8 A, B, 9 A, B). In sporangiophore A it may well have been that the apparently rapid increase in growth rate between 5 and 6 min was a consequence of downward sagging of the sporangiophore over that time period, bringing a sporangiophore that was previously turned somewhat towards the observer into a plane perpendicular to the optical axis of the microscope. The method of observation employed allowed the possibility of occasional errors of this type. The rate of bending of sporangiophore B between 0-3 min was about 4'0 deg min-I. After a further interval of 12 min, the sporangiophore was recorded as turning through 90 degrees and having shown some compensatory balancing curvature. Because of the apparently abrupt increase in length at this stage, it appears probable that some sagging occurred. The results for a stage IV sporangiophore of P. caucasica are shown (Figs. 8 C, 9 C). This sporangiophore showed reversal and no obvious downward displacement of its zone of curvature, but it grew more slowly than A and B.

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Transactions British Mycological Society

Results for a stage IV sporangiophore of P. moreaui (Figs. B D, 9 D) follow a pattern similar to that of the other two species. DISCUSSION

The development of Pilaira sporangiophores resembles that of Phycomyces, in the transition from stag es I to IV. The absence of an anticlockwise stage IVa is a notable feature of Pilaira. The papilla (stage II a) formed in the course of sporangia! swelling is also seen in Phycomy ces. Pilaira more closely resembles Pilobolus in the later phases of its development (stages IV and V). The blackened upper portion of the sporangium, dehiscence of the sporangium and the mucilage surrounding the spores are similar in both genera, but the subsporangial bulb is absent from Pilaira. There is a marked specific difference in the duration of sporangiophore development in the three species, caused by a considerably longer stage I and stage III b duration in P. caucasica than in the other two species. Although the development of the sporangiophores is very rapid between stages I and III a, presumably a period of maturation is required (I II b) before the spores are fully developed. It has been observed (Fletcher, 1969) that a diurnal rhythm is shown by P. anomala with respect to sporangiophore development under natural conditions of day and night. Further work would be needed to determine whether the cycle of development, described here for sporangiophores under continuous illumination, could be modified to a natural diurnal cycle. In most of the sporangiophores studied, extension growth ceased for a time at stage III b. The continuing growth during stages II a-III a in P. moreaui contrasts with the abrupt cessation at stage II a in P. caucasica. P. caucasica had a longer period of elongation in stage I which may be correlated with the prompt termination of growth when sporangium formation was initiated. The mature sporangiophores of Pilaira exhibit remarkably high rates of growth considering their small size. While not achie ving the duration of extension growth of the sporangiophores of Phycomyces, their rapid elongation over a short period is quite outstanding. This could be of ecological advantage to the organism, since elongation of the sporangiophore is the only likely means of elevating the spores from the dung surface. The occurrence of 'stolons' might have a function under natural conditions of allowing a poorly developed colony on an exhausted fragment of substratum, unable to produce healthy sporangiophores, to give rise instead to 'stolons' which could prolong the vegetative life of the colony . The sporangiophores of Pilaira at stages I and IV resemble those of Phycomyces in that they respond rapidly to unilateral light by curving towards it until equilibrium is reached. A feature common to Pilaira anomala and Phycomyces is that the curve migrates down the sporangiophore throughout the course of the response. This occurs in both stage I and stage IV sporangiophores. Bergman et al. (1969) state that th ere is no

Sporangiophore of Pilaira. H. J. Fletcher clear explanation of the downward migration of the curve in Phycomyces and this is also true for Pilaira. Castle (1966) and other reviewers, e.g. Carlile (197 I), usually discuss the process of phototropism in terms of a light-growth reaction, although Castle (196 I) observed that phototropism does not require a transient light-growth response, since phototropism is a steady-state process. The light-growth response involves adaptation which is absent in phototropism, however, the positive light-growth response shows that one general effect of an increase in illumination of the sporangiophore of Phycomyces is an acceleration of the extension growth. Since phototropism in air is positive, the light must be having a greater effect on the distal side rather than the proximal side. This led to the concept of the lens effect, whereby the transparent sporangiophore acts as a cylindrical lens focusing the light on the distal side of the sporangiophore. It is usually considered that the region of photoperception is located in the peripheral cytoplasm, not in the wall. The lens effect can be demonstrated by immersing the sporangiophores in a liquid, usually liquid paraffin, of greater refractive index than the sporangiophore. In the case of Phycomvces, this resulted in the reversal of phototropism, due to the divergence of the rays of light, rather than the convergence through the sporangiophore such as occurs in air. Thus the sporangiophore receives maximum illumination on the proximal side under oil and curves away from the light source. This effect was confirmed by Banbury (1952) by a grazing beam of light causing a sporangiophore in air to curve almost perpendicularly away from the illuminated side. Page & Curry (1966) performed a similar experiment with stage I sporangiophores of Pilobolus. In sporangiophores of the three species of Pilaira at stage IV there is a marked reversal of phototropism under liquid paraffin. At stage I reversal of phototropism under liquid paraffin is clearly demonstrable in P. anomala, but only weakly developed in the other two species. There is no evidence, however, that the sporangiophores of P. caucasica or P. moreaui have a higher refractive index than that of liquid paraffin, as is the case in some species of Pilobolus (Page & Curry, 1966). In Pilaira, the growth under liquid paraffin may have been insufficient for a clear result. Thus the phototropism of Pilaira resembles more that of Phycomyces than Pilobolus, although little work has been done on the responses of stage I of Phycomyces. The author wishes to record his sincere gratitude to Mr G. H. Banbury of the University of Durham for his helpful advice and encouragement throughout this work. He also expresses thanks to Mr E. F. Middleton of the University of Aberdeen for the photographic printing.

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Transactions British Mycological Society REFERENCES

BANBURY, G. H. (1952). Physiological studies in the Mucorales. Part 1. The phototropism of sporangiophores of Phycomyces blakesleeanus. Journal rif Experimental Botany 3,77-94· BERGMAN, K., BURKE, P. V., CERDA-OLMEDO, E., DAVID, C. N., DELBROCK, M., FOSTER, K. W., GOODELL, E. W., HEISENBERG, M., MEISSNER, G., ZALOKAR, M., DENNISON, D. S. & SHROPSHIRE, W. (1969). Phycomyces. Bacteriological Reviews 33,99-157. CARLILE, M. ]. (197 I). The photoresponses of fungi. In Photobiology of micro-organisms (ed. P. Halldall), pp. 309-344. CASTLE, E. S. (1942). Spiral growth and the reversal of spiralling in Phycomyces, and their bearing on primary wall structure. American Journal rif Botany 29, 664-672. CASTLE, E. S. (1961). Phototropism, adaptation and the light-growth responses of Phycomyces. Journal of General Physiology 45, 39-64. CASTLE, E. S. (1962). Phototropic curvature in Phycomyces. Journal of General Physiology 45, 743-75 6. CASTLE, E. S. (1966). Light responses of Phycomyces. Science, N.r. 154, 1416-1420. FLETCHER, H.]. (1969). The development and tropisms of the sporangiophores of Pilaira anomala. Transactions of the British Mycological Society 53, 130-132. FLETCHER, H.]. (1971). Some aspects of the biology of Pilaira anomala - an extremely versatile fungus. Journal of Biological Education 5, 229-237. INGOLD, C. T. (1971). Fungal Spores - their liberation and dispersal. Oxford: Clarendon Press. MCVICKAR, D. (1942). Light controlled diurnal rhythm of asexual reproduction in Pilobolus. American Journal rif Botany 29, 372-380. PAGE, R. M. (1968). Phototropism in fungi. In Photophysiology 3 (ed. A. C. Giese), pp.65-90. PAGE, R. M. & CURRY, G. M. (1966). Studies on the phototropism of young sporangiophores of Pilobolus kleinii. Photochemistry and Photobiology 5, 31-40.

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Pilaira Fig. I. Stages of sporangiophore development of P. caucasica. Times (min) are indicated. (x 100.) Fig. 2. Stages of sporangiophore development of P. moreaui. Times (min) are indicated. (x go.) Figs. 3, 4· 'Stolon' production in P. anomala. Fig. 3. 'Stolon' terminating as vegetative hyphae. Arrow indicates aerial portion of 'stolon'. (x 110.) Fig. 4. 'Stolon' terminating as abortive sporangium. (x 110.)

(Acceptedfor publication 26 April 1973)

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