Journal of General Microbiolog y ( 1 9 7 6 ) 185-192
~~~
Printed in Great Britain
Nuclei, Septation, Branching and Growth of Geotrichum candidurn
By CAROLYN FIDDY AND A. P. J . TRINCI
Microbiology Department, Queen Elizabeth College, Campden Hill,
London W8 7AH
(Received 13 May 1976)
SUMMARY
A study was made of growth, septation and branching in Geotrichum candidum,
a mould which forms physiologically complete septa. A correlation was observed
between septation and branch initiation ; branches were almost invariably formed
just behind septa. Primary branches and their parent intercalary compartments
initially increased in length at an exponential rate before eventually attaining a
constant rate of extension. The whole branching system (which eventually contained seven tips) produced by an intercalary compartment increased in length
exponentially until it attained a total length of at least 1-5mm.
The total length and the number of nuclei of undifferentiated mycelia increased
exponentially at the same specific growth rate. The results suggest that nuclei
divide just before or just after arthrospore formation.
INTRODUCTION
Trinci (1971, 1973) showed that the extension rate (KT)of a hypha was a function of the
length of its peripheral growth zone (w)and the mould's specific growth rate (a).Thus,
Kr = wcc
(1)
The maximum length of the peripheral growth zone of a hypha of Geotrichum candidurn,
unlike that of most species, can easily be measured since its septa lack central pores (Bracker, 1967) and therefore, only apicd compartments can contribute to tip extension.
Primary branches of G. candidurn initially double in length at a very fast rate (Trinci,
1970). It follows from equation (I) that the initial extension rate of a branch will be a funo
tion of the length of its parent intercalary compartment and of the mould's specific growth
rate. A primary branch and its parent intercalary Compartment would be expected initially
to increase in length at an exponential rate. Thus,
+
lnL, = lnLo a(tl-to)
(2)
in which Lo is the combined length of the branch and its parent intercalary compartment
at time t o and & is the combined length of the branch and compartment at time tl. Equation
( 2 ) would also describe the growth of the whole branching system produced by an intercalary compartment.
The present study was made to establish the validity of equation (2) for describing the
growth of primary branches and branching systems, and to determine the relationship
between septation and branch initiation.
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186
C. FIDDY AND A.
P. J. TRINCI
METHODS
Organism and media. Geotrichum candidum (Queen Elizabeth College, strain FI) was
grown on DM medium (Trinci, 1971)~
omitting the vitamins. Solid medium was prepared
by adding agar (15g 1-l).
Septation, branching and branch growth. Drops of dilute spore suspensions were dispersed
with a sterile glass spreader over the surface of media which had previously been overlaid
with sterile cellophane (PT 300, British Cellophane). The dispersed spores were overlaid
with a second sheet of sterile cellophane so that the mycelia grew as a sandwich between two
layers of cellophane. The septa of mycelia grown in this manner could be seen more clearly
than those formed by myeelia grown on a single layer of cellophane or without cellophane.
A 35 mm Shackman Mark I time-lapse camera (Shackman and Sons, Chesham, Buckinghamshire) was used to record growth of the hyphae, and measurements were made from
enlarged photographs.
Apical and intercalary compartment lengths were measured using a travelling micrometer
eyepiece.
Measurement of the ratio of hyphul length to nuclear number (HJ. Undifferentiated mycelia were grown in Iiquid media as described by Trinci (1972). Microscope slides were
coated with 10% (wlv) crystalliied egg albumen (preserved with a crystal of phenol) and
allowed to dry. Samples of G. candidurn were then spread on these slides and air dried. The
samples were cold treated, fixed with acetic acid/alcohol, hydrolysed with HCl and stained
with Gurr’s Giemsa as described by Fiddy & Trinci (1976).
RESULTS
Branch initiation
The lengths of apical compartments of undifferentiated (Steele & Trinci, 1975)mycelia
and leading hyphae of colonies were 233 & 37 and 372 & 46 pm respectively. Septa were
formed at the rear of apical compartments, forming new intercalary compartments (Fig. I) ;
synchronous or near synchronous septation (King & Alexander, I 969 ; Clutterbuck, 1970;
Fiddy & Trinci, 1976)was very rarely observed (Fig. 4).
Each intercalary compartment initially produced a single branch (Fig. I); the lag between septation and branch initiation was remarkably constant, being 24 & 6 min for intercalary compartments (mean length, 64& 10pm) of undifferentiated mycelia and 28 & 9 min
for intercalary compartments (mean length, I 23 & 28 pm) of leading hyphae at the margin of
colonies. Branches were almost invariably initiated just behind septa (Fig. I ; Table I),
suggesting that compartments were highly polarized. This polarity was slightly less marked
in compartments of undifferentiated mycelia than in compartments of leading hyphae at the
margin of colonies (Table I).
Intercalary compartments usually produced a second branch in the manner illustrated in
Fig. 2, i.e. a second branch was initiated behind a new (intercalary) septum formed within
the original compartment. However, an intercalary compartment sometimes produced a
second branch without prior formation of a septum.
Branch growth
Figure 3a shows a comparison of the observed growth of a primary branch and its
parent intercalary compartment (which was about 75 pm long) with their growth as predicted by equation (2) ;mycelia grown between cellophanesheetshada specificgrowth rate of
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i
Growth of Geotrichum candidurn
Time (min)
Time (min)
Fig. I
Fig. 2
Fig. I. Septation and branch initiation during growth of a hypha of an undifferentiated mycelium
of G.candidurn. Tracings made from photographs.
Fig. 2. Formation of first and second branches from an intercalary compartment.
Table I . Position of branchesformed by intercalary compartments of
leading hyphae and hyphae of undiferentiated mycelia
The number of branches formed in each compartment region is expressed as a percentage of the
total number of branches.
No. of branches (%) in each region
Region of intercalary compartment*
...
Intercalary compartments of hyphae of undifferentiated mycelia
Intercalary compartments of leading hyphae of colonies
A
f
\
I
2
3
4
5
71
92
I3
I2
2
2
I
0
5
3
* Each intercalary compartment was divided, visually, along its length into five equal regions,
nearest the hyphal tip.
I
being
0.39 h-l. Equation ( 2 ) adequately described growth of the primary branch until it attained a
length of about 325 pm (Fig. 3a). Deceleration of primary branch growth from its initial
exponential rate was correlated with septation, i.e. with a reduction in the length of the
.
I
branch continued to increase until it attained a length of about 700 pm and had an apical
compartment (peripheral growth zone) which was about 325 to 41opm long (Fig. 3).
Subsequently the branch extended at a linear rate.
The total length of the branching system (eventually consisting of seven hyphae) produced by the intercalary compartment increased exponentially at the predicted rate (equation 2 ) until it was at least 1.5 mm long (Fig. 3a).
Figure 4 shows the variation in the length of successive apical compartments formed by a
leading hypha of G. candidurn which was extending at a constant rate. The mean interval
between the formation of successive septa was usually significantly less than the doubling
time of the mycelium (Fig. 4) ; in Aspergi12u.s nidulans the mean interval between successive
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I
e
I
8
w
0
I
$
0
0
I
m
Length of apical compartment of
primary branch, B1 (pm)
vl
0
-z+E
+K
Extension rate of primary branch, B1 (pm h-1) Length of primary branch, B1 or of entire branch system, B1 to B7 @m)
Growth of Geotrichum candidurn
2mt
100
1
2
Time (h)
Fig. 4
1
3
4
Fig. 5
Fig. 4.Length of successive apical compartments formed by a leading hypha. The vertical arrows
indicate the times at which septa were formed in the apical compartments.
Fig. distribution of nuclei in a germling. The nuclei were stained with Giemsa. Camera lucida
drawing.
50
-
a
200
300
400
500
Total hyphal length of undifferentiated mycelium (,urn)
Fig. 6. Variation in the number of nuclei with the total hyphal length of Undifferentiated mycelia.
The correlation coefficient (0.99)is highly significant (I"c 0'001).
septation cycles in a hypha was approximately the same as doubling time of the mycelium
(Fiddy & Trinci, 1976). In G. candidurn, unlike A . nidulans (Fiddy & Trinci, 1976), septation
rarely if ever divided the apical compartment of leading hyphae into regions of approximately equal length (Fig. 4).
Ha values of undiflerentiatedmycelia and arthrospores
Nuclei were distributed more or less uniformly throughout the cytoplasm of undifferentiated mycelia (Fig. 5). The direct relationship which was observed between the number
of nuclei and the total mycelial length (Fig. 6) indicates that both parameters increase
exponentially at the same specific growth rate.
The number of nuclei and the hyphal length per nucleus (HB)values of apical and intercalary compartments of undifferentiated mycelia are shown in Table 2. The mean distribution of nuclei in intercalary compartments which had not formed branches is shown in Fig.
7.Nuclei were evenly distributed throughout the length of the compartment.
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C. F I D D Y A N D A. P. J. TRINCI
I90
o\a
0
o - = t o o
Fig. 7. Diagramatic representation of the mean distribution of nuclei within intercalary compartments. Distances are given in pm.
Table 2 . Numbers of nuclei and H, values of apical and intercalary
compartments of undirerentiated mycelia of G. candidurn
Apical compartment
Intercalary compartment
Mean length @m)
Wf35
825 10
Mean no. of nuclei
19+ 5
7f2
8.0+ 1.2
12.3+2
Mean hyphal length per nucleus, iY,(pm)
Mean hyphal length per nucleus of entire myoelia,
HnoLm)
9'3*
* Calculated for mycelia which varied in length from about 20 to over 600 pm.
Table 3. Number of nuclei, length and H, values of arthrospores of
G. candidum formed in submerged culture and on solid media
No. of nuclei per spore
A
r
Frequency of spores (% of sample)
On solid media*
In submerged culturet
Mean spore length @m)
On solid media
In submerged culture
Spore length per nucleus, H, (pm)
On solid media
In submerged culture
2
3
4
5
6
7
3
44
61
29
9
I7
I
5
I
I
9-2
9.1
11.6
15.4
15.6
4'6
4'5
3'9
3'7
3'9
3'1
29
7'3
7.0
7'3
* I I 3 and
7
I
7'0
11-2
70 spores in sample.
Arthrosporeformation
Geotrichum candidurn forms arthrospores by fragmentation of vegetative hyphae (Trinci
& Collinge, 1g74a; Cole, 1975). Arthrospores formed on solid media contained I to 7
nuclei whilst those produced in submerged culture contained I to 4 nuclei (Table 3). With
the exception of uninucleate arthrospores, the H, values of the spores (Table 3) were approximately half those of their parent intercalary compartments (Table 2).
DISCUSSION
We have established that the initial extension rate of a primary branch of G. candidum is
a function of the length of its intercalary compartment and of the mould's specific growth
rate. Increase in the extension rate of a primary branch is correlated with an increase in the
length of the peripheral growth zone (Fig. 36). Eventually a hypha attains a constant rate
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Growth of Geotrichum candidurn
191
of extension (Fig. 3b) although its apical compartment (the putative peripheral growth zone
of the hypha) continues to vary in length (Fig. 4). However, the cytoplasm in the distal
regions of long apical compartments is usually highly vacuolated and hence may not make
an appreciable contribution to tip extension.
The vesicles observed at the tips of fungal hyphae are thought to contain wall precursors
and/or the enzymes required for the insertion of these precursors into the tip wall (BartnickiGarcia, 1973). The variety of branching patterns produced by compartments of hyphae of
Neurospora crassa recovering from an osmotic shock suggests that a branch can potentially
be formed from any part of the hyphal wall, includingthe septum (Trinci & Collinge, 1g74b)
and that sites of branch initiation are determined by cytoplasmic events (possibly vesicle
accumulation). The imposition of barriers to apical vesicle transport in a hypha may result
in the accumulation of vesicles behind the barrier (Trinci & Collinge, 19743) or in their
fusion with the hypha behind the barrier. In G. candidum, branch initiation is clearly correlated with the formation of complete septa (Fig. I) which form effective barriers to protoplasmic streaming. There may be a lag between septation and branch initiation because a
critical concentration of vesicles may have to accumulate before branch initiation. Figure 2
and Table I suggest that the polarity of vesicle transport in intercalary compartments is
maintained after septation, i.e. vesicles accumulate immediately behind septa. In Aspergillus nidulans (Fiddy & Trinci, 1976) branches may commonly be initiated from all regions
of intercalary compartment walls because the septa are incomplete, i.e. initially, at least,
they do not form complete barriers to vesicle transport (Trinci & Collinge, 1973).
No evidence was obtained which indicated that nuclei divided synchronously in G.
candidum although it should be remembered that most arthrospores had more than one
nucleus.
The difference between the Hnvalues for apical and intercalary compartments (Table 2)
may be correlated with the observation that the cytoplasm of intercalary compartments was
more vacuolated than that of apical compartments. Except for the uninucleate arthrospores,
the H, values for spores were approximately half those observed for the intercalary compartments (Table 2) from which they were formed. This observation suggests that the nuclei
divide during arthrospore formation. Uninucleate arthrospores, however, had an Hn ratio
(Table 3) which was almost identical to the value observed for intercalary compartments
(Table 2).
We thank the Science Research Council for financial support.
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