[ 13 1 ] Trans. Br. mycol. Soc. 83 (1), 131-137 (1984)
Printed in Great Britain
EFFECTS OF AUTOINHIBITION ON HYPHAL GROWTH IN COPRINUS DISSEMINATUS By GILLIAN M. BUTLER Department of Plant Biology, University of Birmingham, Birmingham B15 zTT Colonies of Coprinus disseminatus growing in agar reduced the extension rate of surface colonies which were separated from them by 'Cellophane'. Inhibition of surface colony extension was detectable when the margin was close behind that of the submerged colony and reached a maximum over parts of the submerged mycelium which were between 1 and 4 days old. The extension rate of the surface mycelium was at least partially restored by removal of agar containing submerged mycelium from beneath the growing margin of the surface colony. This autoinhibition reduced the extension rate and tip diameter of main hyphae and young primary branches. Within most treatments involving different levels of autoinhibition there was a significant linear regression between extension rate and hyphal diameter for main and primary branch hyphae separately. Between treatments there was significant heterogeneity in the slopes of these regression lines. Within treatments the slopes of the regression lines for main and branch hyphae were similar but branch hyphae extended more slowly than equally wide main hyphae. Little is known about the relationship between longitudinal and circumferential growth at the hyphal apex (Prosser, 1979; Saunders & Trinci, 1979; Koch, 1982). Indeed there is a paucity of information about the effects of environmental variables on the extension rate and diameter of hyphae. In highly differentiated colony margins on solid media the main hyphae are characteristically wider and faster-extending than their young primary branch hyphae (Steele & Trinci, 1975). Robinson & Smith (1979) have shown that the difference in width between apical compartments of main and primary branch hyphae of Geotrichum candidum is greatest at high glucose concentrations on solid media. They found no evidence of differentiation between main and branch hyphae in mycelial fragments of chemostat cultures on media with the same nutrient compositions. They suggested that differentiation on solid media could be induced by a concentration gradient of the growth-limiting nutrient and perhaps also of autoinhibitory metabolites at the colony margin. Since Butler (1961) described a difference in the relationship between extension rate and diameter for main and young primary branch hyphae of Coprinus disseminatus (Pers.: Fr.) Gray there has been no similar report. This paper explores the effects of autoinhibition on extension rate and diameter of main and primary branch hyphae in C. disseminatus.
MATERIALS AND METHODS
Dikaryon stock PI of C. disseminatus was used. In most experiments the inoculum was a 10 mm disc from the margin of a 2 % malt agar culture and the medium was 2 % Oxoid no. 2 agar in distilled water. In one set of experiments a glucose nitrate medium, D15, was used (Butler, 1981). Mycelial macerates were prepared from static 2 % malt liquid cultures. 'Cellophane' (British Cellophane P 600) was boiled for 2 min in distilled water and rinsed before autoclaving. Except where otherwise stated cultures were incubated at 22°C. In the double culture system (Butler, 1968) submerged (B) colonies were grown in 20 ml agar medium beneath 80 mm discs of 'Cellophane' and surface (T) colonies were grown on 'Cellophane' over uncolonized agar. T Surface inoculum disc
Surface (T) mycelium 'Cellophane'
Submerged inoculum disc
Distilled water agar
Submerged (B) mycelium Fig.
Diagram of the symmetric arrangement of submerged and surface colonies in a Petri dish.
Autoinhibition in Coprinus
and B colonies were combined aseptically after removal of the' Cellophane' from the B colonies. Unless otherwise stated the arrangement was symmetric, the two inoculum discs being superimposed (Fig. 1). Colony extension rates were calculated from measurements of the position of the colony margin along two diameters at right angles at 0 and 24 h after transfer. Hyphal growth was recorded by photomicrography in a controlled environment chamber at approx. 18 h after transfer. Growth of related pairs of main and branch hyphae was recorded over 5 min intervals using a x 40 objective. The first tip to be recorded was alternately that of the main and the branch in successive pairs of observations. Observations were restricted to systems in which the youngest primary branch was at least 50 p,m long. RESULTS
Activity of B mycelium When T colonies 50 mm in diameter were transferred to positions over B colonies of different sizes, T colony growth was reduced in comparison with controls transferred to water agar. The inhibition reached a maximum over z-day old mycelium (Table 1). In further experiments T colonies of different sizes were placed over standard y-day old B colonies and over control water agar (Table 2). Inhibition was detectable (t test, probability between 0'05 and 0'01) when the T margin was 3'5 mm behind the B margin. Inhibition was higher 8·6 mm behind the margin but changes in the level of inhibition further back were small. The activity of uniformly-aged B mycelium was tested by incorporating macerated mycelium in D/5 medium at the rate of 3'5 x 10· viable propagules/dish. Inhibitory activity was tested at 23'5 °C using T colonies which were previously grown over water agar for 1 day from 2 % malt agar discs 3 mm diam, Although macerated mycelium took some time to start growing there was a slight but significant inhibition over one-day old mycelium in comparison with growth over uninoculated D / 5 agar (t test, probability between 0'05 and 0'01, Table 3). Inhihition reached a maximum over 3-4-day old mycelium and subsequent changes were small. Nature of ' Cellophane' -transmissible B activity In order to determine whether the effect of B mycelium was due to removal by the submerged mycelium of some growth factor from the medium, or to the production of growth inhibitors by this mycelium, sectors of agar were removed. Wedgeshaped sectors of agar extending from the inoculum to the edge of the dish and 10 mm wide at a position beneath the margin of the T colony were removed from opposite sides of eight day old B colonies and
from uninoculated agar. Removal of uninoculated agar had no significant effect on the extension rate of four day old T colonies (Table 4). However sector removal significantly increased T colony extension rate over B colonies (r test, probability < 0'001) but only partially restored it in comparison with extension over sectors in uninoculated agar (t test, probability between 0'01 and 0'001). In a further experiment three different sorts of B colony sector were removed, complete sectors, central portions of sectors up to within 3 mm of the T colony margin and outer portions of sectors beneath and beyond the marginal 3 mm of the T colony margin (Table 5). Removal of the central parts of sectors made no significant difference to T colony extension rate when compared with the effect of intact B mycelium whereas removal of outer portions of sectors, beneath the T colony margin, appeared to be as effective as removal of the whole sector in increasing T colony extension rate. Response of T hyphae Hyphal measurements were made on T colonies approx 50 mm in diameter when transferred to different levels of autoinhibition produced by B colonies of different ages (Table 1). A small number of hyphal tips failed to grow during the period of observation. These tips, together with their partner main or branch tips, were excluded from the analysis. The samples of branch hyphae did not differ significantly in length between treatments. The mean diameters and extension rates of both main and branch hyphae were each significantly reduced by the presence ofB mycelium (analyses of variance, probabilities < 0'001 in all cases). Inhibition was at a maximum over 1- and z-day old B mycelium and was detectable within 0·6 mm of the B colony margin. The effect of presence of B mycelium on T colony margin organization was vividly demonstrated in some of the dishes containing equally aged T and B colonies. If the two colonies were not precisely superimposed the margin of the T colony was slightly ahead of the B margin at one side of the colony and slightly behind at the opposite side. In these circumstances the T mycelium had a normal smooth and dense margin where it was just ahead of the B margin in contrast with the very thin T margin containing sparse leading hyphae with branches growing at wide angles to their parent axes, where it was just behind the B margin. Taking the treatment means (Fig. 2), there was a clear association between extension rate and diameter for both main and branch hyphae. When treated as linear regressions (Table 6, 7) there was a significant regression for main and branch hyphae separately and the two regression lines did not
Colony and hyphal growth on 'Cellophane' over submerged colonies of different ages Age of submerged (B) mycelium beneath the margin of the surface (T) colonies approx. 50 nun diam (days) None
Colony extension rate (pm h- I ) Difference in colony radius
155 (11°9, 3) 6°o
115 (13°7, 3) 11°6
159 (3°° O, 3) > 19
4 179 (20°6, 3) > 19
5°22 (0°37)2 231 (22 341 (47°2) 1°48
4°8o (0"44) 204 (3°°2) 354 (47°2) 1 °74 88
4°21 (0°37) 153 (3 6°3) 291 (81°5) 1°9° 66
4°23 (0°25) 151 (35°2) 293 (111"8) 1°94 65
4°31 (0°37) 178 (47°8) 317 (112°4) 1°78 77
4°88 (0°42) 232 (25"8) 418 (71°6) 1°8o 100
131 (51°9) 4°°7 (0°46) 129 (25°8) 56
154 (106°5) 4°°7 (0"41) 141 (21°3) 69
113 (59°9) 3°57 (0°36) 87 (31°O) 57
130 (61°6) 3°61 (0°38) 9 1 (27°5) 60
135 (80°7) 3°76 (0°30) 110 (39"4) 62
144 (90°8) 4°°3 (0°37) 132 (25°5) 57
226 (13°7, 4)1
° 194 (18 0°6
No. of pairs of hyphae observed
Main hyphae Tip diam (pm) Tip extension rate (pm h- I ) Interseptal length (pm) Tip compartment generation time (h) Reduction in extension rate (% of T alone)
Primary branch hyphae Branch length (pm) Tip diam (pm) Tip extension rate (pm h- I ) Branch extension rate as % of parent extension rate
~ ttl ...:::
s.n., no. of replicate dishes.
Effect of B colonies approx, 60 mm diam on the colony extension rate of T colonies of different sizes
T colony radial extension rate (pm h~l) Mean distance of - - - - - - - - - - - - - - % of control T margin behind No B mycelium extension rate Over B mycelium (control) B margin (nun)
3"4 8·6 13°6 19'1 23°9
213 201 212 216 205 1
(20°8)1 (11°3) (7'9) (10°O) (17°1)
170 144 149 158
(26'3) (4°6) (9°6) (10'4) 128 (9'6)
S.D. for 5 replicate dishes.
72 7° 73 63 ~
Autoinhibition in Coprinus
Dis medium from macerated inoculum on colony extension rate of T
Table 3· Effect of B colonies grown on
colonies approx, 8 mm diam T colony radial extension rate measured over 24 h (urn h-') Age of B mycelium at time ofT transfer (days)
No B mycelium (control)
Over B mycelium
176 (6'9)' 178 (5"4) 167 (5'3) 15 1 (6'7) 162 (7'1) 174 (8'2) 174 (6'1)
3 4 9 17
178 17 2 107 83 85 120 110
% of control extension rate
(10'3) (5'5) (9'0) (7'8) (5,6) (5'6) (8"4)
101 97 64 55 52 69 63
S,D, for 10 replicate T colonies.
Table 4. Effect of removal of radial agar sectors on T colony extension rate in the presence and absence of B mycelium at 25°C Increase in T colony radius over 24 h (mm)
Treatment Agar intact Agar sector removed
No B mycelium
B mycelium 4 days older than T mycelium
4'9 (0'32,8)' 4'8 (0'29, 4)
2'9 (0'42, 8) 3'9 (0'25, 4)
, S.D" no, of replicate colony diameters.
Table 5. Effect of removal of different agar sectors on T colony extension rate in the presence of B mycelium at 25°C Treatment Agar intact Complete radial sector removed Central part of sector removed Outer part of sector removed (beneath T tips) i
Increase in T colony radius over 24 h (mm) 3'29 4'75 3'00 4'50
(0'36, (0'38, (0,60, (0'53,
24)' 8) 8) 8)
S.D" no. of replicate diameters.
differ significantly in slope. Since the populations of main and branch hyphae differed in mean diameter an analysis of covariance was used to calculate regression corrections for this diameter difference. There was no significant difference in the adjusted mean extension rates of equally wide main and branch hyphae. However the relationship between extension rate and diameter was not the same in different treatments. There was a significant linear regression between extension rate and diameter within nine out of twelve sets of data (Table 6), but when the regressions were compared by an analysis of variance there was significant heterogeneity (probability < 0'001). This heterogeneity persisted when the disparate data for the
treatment with z-day old B mycelium were excluded from the analysis (probability between 0'01 and 0'001). Again excluding this treatment there was still a significant heterogeneity amongst the regression lines for main and branch hyphae separately (both probabilities between 0'05 and 0'01). Table 6 shows that for both main and branch hyphae there was higher slope to the relationship between extension rate and diameter in treatments giving greater levels of autoinhibition. The slopes at these higher levels of autoinhibition were similar to that for the treatment means. In all the data where individual regressions were significant the probability that the within treatment pairs of related main and branch regressions were similar was quite high (Table 7).
Gillian M. Butler
Table 6. Slopes of regression lines between extension rate and diameter for hyphae growing under different conditions Age ofB mycelium (days)'
Extension rate of main hyphae (% control)
None 4 o 3
100 100 88 77 66 6S
* ** *** ** *** ** **
(1'56) 3'80 2'51 6,61 5'05 (2'18)
2'S9 3'45 7'48 5'19 9'20
Treatment means None (data of Butler, 1961)
** ** ** *** 0'2--0'1
, Treatments as in table 1, arranged in order of increasing inhibitory activity of B mycelium. 2 Slope of regression line. a Probabilities: * between o'oS and 0'1; ** between 0'01 and 0'001; *** less than 0'001.
Table 7. Comparisons between regression lines for main and branch hyphae within treatments
Age ofB mycelium (days)'
Comparison between Comparisonbetween adjusted mean main and branch extension rates of regression slopes main and branch (probability) hyphae (probability)'
None 4 o
O'S--o'3 0'5--0'4 0'8--0'7 0'95--0'9
Calculated difference in extension rate between equally wide main and branch hyphae (pm h-') 68 37
Treatment means None (data of Butler, 1961)
O'S--O'7 Not significant
, Treatments as in Table 1, arranged in order of increasing inhibitory activity of B mycelium, Probabilities as in Table 6.
After allowing for each joint regression branch hyphae grew more slowly than equally wide main hyphae within three out of four treatments (Table 7)· DISCUSSION
In these experiments the main nutrient supply was provided by the inoculum disc and sector removal showed that the effect of submerged mycelium on surface growth was at least partly autoinhibitory, due to products of fungal activity rather than to shortage of a growth-limiting nutrient or oxygen (Robinson & Smith, 1979). Inhibitory activity was transmitted through 'Cellophane' but either did not spread rapidly laterally or was quickly destroyed. Autoinhibition affected both extension rate and tip diameter. There was a similar relationship between mean colony extension rate and diameter of main
hyphae in a different strain of C. disseminatus when it was grown on different concentrations of the inhibitor cycloheximide (Fig. 2; Butler, 1981). Although the experiments do not provide direct information on the amounts of autoinhibitory activity at the positions of main and primary branch tips in control colonies, several features suggest that extrahyphal autoinhibition could be responsible for at least part of the diameter-associated component of the difference in extension rate between these two hyphal types. The response to inhibition was mostly a direct one by the apical 3 mm or less of the mycelial margin, the gradient of increasing inhibitory activity was steepest at the young growing edge ofthe B colony and the maximum growth reduction induced by autoinhibition was 30-40 % of the control extension rate. Previously a significant regression between ex-
Autoinhibition in Coprinus
300 0 i"
c .~ c 2 x
.<: Co >.
4 5 6 Hyphal diam (11m) Fig. 2 . Relation between mean extension rate and mean diameter of hyphae in the presence of different levels of autoinhibition at 22°C ce , main hyphae ; £, primary branch hyphae) and different cycloheximide concentrations at 25°C CO , main hyphae, data from Butler, 1981).
tension rate and hyphal diameter had been reported for the control treatment (Butler, 1961 ) . In the data presented here the regression was not quite significant (p robab ility between o · 1 ando-oyj.Ir is possible that this was due to inadequate sample size when the regression slope was low . When adjustment is made for the difference in scale of extension rate records in the earlier report, the slopes of the main and branch regression lines and their vertical separation are consistent with the trends in the results presented here (T ab les 6, 7). A curved relationship between hyphal extension rate and diameter has been described by Clutterbuck (1978) for variation amongst hyphae of wild type and some morphological mutants of Neurospora crassa and by Robinson & Smith (1980) for marginal hyphae ofcolonies of G. candidum growing at a range of glucose concentrations. In C . disseminatus each within treatment variation has been treated as a linear regression. Each treatment covers a relatively narrow range of the two parameters and the differences in slope of the within treatment linear regressions might be explained by an overall curved relationship between extension rate and diameter for main and branch hyphae separately.
However such a relationship is not a sufficient explanation of the difference between main and branch hyphae. Within treatments the regression lines for main and branch hyphae are parallel despite their difference in mean diameter and extension rate. Thus the slope of the line is a function of treatment. The treatments which show minimum slope and maximum separation of the main and branch regression lines are those in which margin extension is least inhibited by the external medium . These are conditions in which competition within hyphal branching systems is most likely to be limiting. A highly differentiated colony margin seems to be associated with a long experimentally determined peripheral growth zone (T rinci, 1971 ; Steele & Trinci, 1975). In N. crassa the peripheral growth zone includes branches (T r inci & Collinge, 1973) so that internal sharing of resources between main and branch hyphae must occur. C. disseminatus resembles N. crassa in that the experimentally determined peripheral growth zone is long, between 1400 and 1600 p,m, on both 2 % malt agar and on a glucose nitrate medium, i.e. four to five compartment lengths (M cG ro r y, pers. comm. ). In the experimental conditions used here nutrients were supplied from the inoculum disc. Primary branches arise from the subapical compartment and their relatively fast early growth and very slow increase in extension rate (Bu tler , 1968) suggest considerable dependence on the parent axis for resources. However Clutterbuck ( 1978) has shown that in N. crassa main and branch hyphae fit the same curved relationship between extension rate and diameter. He indicated that the difference between N. crassa and C . disseminatus may lie in the different sites of branching in the two fungi. In N . crassa branching occurs close to the main tips whereas in C. disseminatus primary branches develop from the subapical compartment. In the Agaricales the diameter of the narrowest passage in the dolipore septal apparatus, the holes in the parenthosome, is between 80 and 90 nm (P atton & Marchant, 1978). This would prevent intercompartmental transfer of organelles whilst permitting movement of smaller particles and molecules. Also in basidiomycetes there is pronounced polarity in the growth of excised hyphae (H arder , 1926; Yanagita, 1977). It may be that the main tip has prior use of the synthetic resources of the apical compartment whereas resources of more distant parts of the system are shared between main and branch hyphal tips. I am indebted to Dr M . J. Lawrence for helpful discussion of possible statistical approaches .
Gillian M. Butler REFERENCES
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(Received for publication 31 August 1983)