FEMS MicrobiologyLetters 3 (1978) 57-60
© Copyright Federation of European MicrobiologicalSocieties
Published by Elsevier/North-HollandBiomedicalPress
57
M I C R O C Y C L E C O N I D I A T I O N IN P A E C I L O M Y C E S V A R I O T I
J.G. ANDERSON, V. ARYEE and J.E. SMITH
Department ofAppliedMicrobiology, Universityo[ Strathclyde, George Street, Glasgow, G1 1XW, Scotland
Received 9 November 1977
1. Introduction
Sporulation in micro-organisms generally occurs
following a period of vegetative growth and in
response to factors which limit the rapid proliferation
of the vegetative phase [1 ]. For some micro-organisms
conditions have been found which so suppress the
vegetative phase without preventing sporulation that
an immediate or microcycle sporulation process takes
place after spore germination. Thus in bacteria
"microcycle sporulation" has been defined as the
immediate recapitulation of sporogenesis following
spore germination [2] and in filamentous fungi as
the direct production of conidiophores and conidia
by germinating conidia [3]. These minicycles have
provided very convenient and rapid systems in which
to study developmental changes.
The induction of microcycle conidiation in several
fdamentous fungi has involved the exposure of
conidia, in a suitable sporulation medium, to a supraoptimal temperature which allows only limited
germination followed by a reduction to a temperature
which permits sporulation. This technique has been
successfully used to induce microcycle conidiation in
Aspergillus niger [3], Neurospora crassa [4] and
Penicillium urticae [5]. This paper describes the
nutritional and temperature conditions which lead to
the expression of microcycle conidiation in Paecilomyces varioti.
2. Materials and Methods
2.1. Organism
Stock cultures ofP. varioti Bainer were maintained
on potato glucose agar slopes in screw-cap bottles at
30°C. The conidial suspension was prepared and
fdtered through absorbent cotton wool to remove
hyphae and washed by centrifugation in sterile
deionised water. The conidia were counted in a
haemocytometer and in all experiments an inoculum
of 1 • lO s spores/ml medium was used.
2.2. Medium
The basal non-sporulation medium contained in 1 1
deionized water: glucose, 10 g; KH2PO4, 1.0 g;
MgSO4.7H20, 0.25 g; CuSO4.5H20,0.234 mg; FeSO4
.7H20, 6.32 mg; ZnSO4.7H20, 1.1 mg; MnC12.4H20,
3.5 rag; CaC12, 46.7 mg; (NH4)2SO4, 1.98 g. The
sporulation medium contained, in addition to the
above, glutamic acid (monosodium salt), 5 g. Where
other organic acids were used, as mentioned in the
text, they were added at a concentration of 30
#moles. When the laboratory fermenter was used the
medium contained NH4NOa, in place of (NH4)2SO4.
The pH of all media was adjusted to 4.5 with HC1.
The medium constituents were autoclaved at 121°C
for specific times depending on the volume except
for glucose which was autoclaved separately and
added aseptically.
2.3. Culture conditions
Experiments to establish the microcycleconidiating
system were carried out using a simple shaken tube
culture system. The medium was dispensed in 5 ml
aliquots in glass boiling tubes (150 X 24 mm) and
after inoculation the tubes were agitated by clamping
onto a Griffin flask shaker. Temperature control
(-+ 0.3°C) was obtained by immersing two-thirds of
the tube length into a water bath.
58
Bulk cultivation of the fungus was obtained with a
New Brunswick microferm laboratory fermenter using
a 7-1 working volume. Agitation and gas dispersion
were obtained by a standard flat-blade turbine operating at 700 rev./min in the baffled reactor vessel. A
CO2/air blender (Gallenkamp) was used to control
the gas mixture delivered to the fermenter. Silicone
antifoam was used to control foaming.
TABLE 1
Effects of medium composition and incubation temperature
on submerged spore production by Paecilornyces varioti
Variation in medium
composition at 38°C
Variation of temperature
using BM + glutamate
Medium
composition
Incubation
temperature
(0°C)
(- 106)
2.4. Morphological and medium changes
The proportion of conidia which produced germ
tubes was assessed by examination of 100 conidia per
sample. The diameters of conidia were obtained by
measurement of 50 conidia per sample using a microscope with an ocular micrometer. Numbers of conidia
produced were assessed by haemocytometer counts.
The above measurements were made on freshly
prepared samples or on samples stored in 4% formaldehyde at 4°C. The phase contrast photomicrograph was taken with a Zeiss ultraphot 11 (Carl Zeiss,
Oberkochen, West Germany) using Pan F film (Ilford).
Glucose was assayed by using a Schweizerhall
glucose oxidase kit (Unicam Instruments Ltd). Ammonia was determined as mg NHa-N/10 ml by microdiffusion [6].
3. Results
3.1. Induction o f normal conidiation
The influence of medium composition on the
morphology and differentiation ofP. varioti in submerged shaken conditions was examined. Profuse
hyphal growth occurred in the basal medium; no
sporulation occurred under these conditions. Supplementation of the basal medium with certain amino
acids and tricarboxylic acid cycle intermediates
previously shown to induce submerged sporulation in
other fungi [7,8] induced varying degrees of conidiation in P. varioti (Table 1).
3.2. Induction o f microcycle conidiation
Morphological changes during the germination of
P. varioti conidia in the glutamate-supplemented
medium were examined over a range of incubation
Spores
produced
per ml
Spores
produced
per ml
(. 106)
0
25
20
14
10
171
34
38
40
BM + citrate
29
42
BM +
86
44
178
153
111
96
46
48
59
1
Basal medium
(BM)
BM + glycine
BM + isoleucine
BM + glutamate
ct-ketoglutarate
BM + fumarate
60
32
77
95
temperatures. "Normal" germination ofP. varioti
conidia, like the germination of most fungal spores
[9], comprised an initial phase of spore enlargement
followed by a phase of germ tube protrusion and
extension. The initial enlargement phase ofP. varioti
conidia occurred at temperatures up to 50°C with
complete inhibition of enlargement evident at 52°C.
The second phase of germination, germ tube production, occurred readily at temperatures up to 42°C
but above this showed increasingly severe restriction.
Restriction of germ tube formation was accompanied
by an increased degree of conidial enlargement. The
temperature which gave the best separation of the
two phases of germination i.e. good conidial enlargement but most effective restriction of germ tube
formation was 48°C. After 24 h at 48°C only 10%
of the conidia possessed germ tubes which were very
short.
When conidia which had been held at 48°C for
24 h were incubated at the optimum sporulation temperature of 38°-40°C they displayed microcycle
conidiation. One or more outgrowths were produced
from the enlarged conidia which instead of extending
evenly formed a thickened basal portion and a thinner
apical portion from which conidia were successively
cut off (Fig. 1). Microcycle conidiation in this fungus
59
X
2.0
E
1.2
0
10
2O
3O
/,0
50
6O
7O
Time(h)
Fig. 2. Medium changes during microcycle conidiation of
~,, glucose utilisation; o
o,
ammonium utilisation; •
•, secondary conidia production.
P a e c i l o m y c e s varioti. •
Fig. 1. Mierocycle conidiation in P a e c i l o m y c e s varioti.
was therefore expressed by the direct production of
phialides from the parent conidium.
The composition of the medium and the temperature treatment were important in inducing
microcycle conidiation since sporulation did not
occur after temperature treatment of conidia in
medium lacking glutamate. A further requirement
for microcycle conidiation became apparent under
conditions of forced aeration in a laboratory fermenter. Good aeration inhibited spore production
and optimum microcycle conidiation could only be
achieved in the fermenter by using, in addition to
the sporulation medium and temperature shift treatment, a gassing mixture of 5% CO2 in air.
3.3. Medium changes during microcycle conidiation
The fermenter cultivation system was used to
examine the rates of utilisation of glucose and am.
monium from the medium relative to the time of
conidia production by the microcycle process (Fig.
2). There was no increase in spore number during
the 24-h period at 48°C. After the temperature drop
to 40°C sporulation began slowly then increased
rapidly after 40 h, and by 72 h the spore density had
reached 2 • l0 s spores/ml. The pattern of glucose
uptake was reflected in the spore density changes.
Rapid uptake of glucose occurred after 40 h but
glucose was never completely utilised in the duration of the experiment. Ammonium uptake increased
sharply after the temperature drop until 48 h when
an increase in ammonium concentration occurred
indicating some autolysis. The results demonstrated
that the onset of rapid sporulation occurred while
excess glucose and ammonium were present in the
medium.
4. Discussion
The morphological changes in P. varioti induced
by temperature and nutrient manipulation followed
60
a pattern which was similar to that which has recently
been observed with several other fdamentous fungi.
Incubation of the conidia ofP. varioti in a sporulation medium at the supra-optimal temperature of
48°C selectively allowed spore enlargement but
inhibited germ tube formation. Lowering the temperature to 38°C after this treatment induced the
production of phialides and new conidia from the
enlarged mother conidium, thus by-passing the normal
vegetative growth phase. These changes correspond
to "microcycle conidiation" which has previously
been observed in response to thermic variations in
suitable sporulation media [3-5].
The observations on the effects of temperature on
the germination of the conidia ofP. varioti and these
other species indicate that the conidial enlargement
phase of germination, the so-called spherical growth
phase [ 10], is less susceptible to high temperature
inhibition than the subsequent germ tube production
phase. It has been pointed out recently [11,12] that
this effect may be involved in the development of the
spores of certain pathogenic fungi into the enlarged
and more-or-less spherical parasitic forms which are
found in certain fungal infections.
The direct production of phialides and secondary
conidia from the enlarged conidia ofP. varioti
represent an extreme simplification of the asexual life
cycle. The microcyle processes recorded for N. crassa
[4] and P. urticae [5] involve the production of the
secondary conidia from modified apices of germ
tubes. With A. niger a true conidiophore is produced
during microcycle conidiation although this is a somewhat less complex structure than the normal form
(S.G. Deans and J.E. Smith, unpublished material).
The nature of the physiological processes which
are involved in the switch from normal germination
to microcycle conidiation are not known although an
interesting speculation has been made for N. crassa
[4]. The most general hypothesis regarding the
induction of conidiation in filamentous fungi is that
sporulation is inititated by factors, frequently exhaustion of nutrients, which limit the growth of the
fungus [13]. Analyses of nutrient levels in the present
study demonstrated that microcycle conidiation in
P. varioti was not induced by nutrient limitation. It
appears that physiological changes induced during
restricted growth at elevated temperatures predisposed
the fungus to sporulation. However, a complexity of
interacting environmental stimuli was indicated
since, in addition to the temperature check on growth,
glutamate and CO2 were required for optimal expressexpression of microcycle conidiation. Essentially
similar findings have been reported with A. niger [3].
Thus microcycle conidiation does not result simply
from growth restriction. It appears rather to be a
highly unusual response to a unique set of environmental conditions which in some way severely
restrict somatic growth and yet vigorously stimulate
those metabolic pathways which initiate and sustain
active conidiogenesis.
References
[1] Lovett, J.S. (1975) Bacteriol. Rev. 39,345-404.
[2] Vinter, V. and Slepecky, R.A. (1965) J. Bacteriol. 90,
803-807.
[3] Anderson, J.G. and Smith, J.E. (1971) J. Gen. Microbiol. 69, 185-197.
[4] Cortat, M. and Turian, G. (1974) Arch. Mikrobiol. 95,
305 -309.
[5 ] Sekiguchi,J., Gaucher, G.M. and Costerton, J.W. (1975)
Can. J. Microbiol. 21, 2048-2058.
[6] Conway, E.J. (1957) Microdiffusion Analysis and
Volumetric Error. Crosby Lockwood, Edinburgh.
[7] Turian, G. and Bianchi, D.E. (1972) Bot. Rev. 38,
119-154.
[8] Galbraith, J.C. and Smith, J.E. (1969) J. Gen. Microbiol. 59, 31-45.
[9] Smith, J.E., Gull, K., Anderson, J.G. and Deans, S.G.
(1976) In: The Fungal Spore: Form and Function,
Weber, D.J. and Hess, W.M. (eds.), pp. 301-352, Wiley
and Sons, New York.
[10] Anderson, J.G. and Smith, J.E. (1972) Can. J. Microbiol. 18,289-297.
[ 11 ] Anderson, J.G. and Smith, J.E. (1976) In: Inhibition and
Inactivation of Vegetative Microbes, Skinner, F.A. and
Hugo, W.B. (eds.), pp. 191-218, Academic Press,
London.
[12] Anderson, J.G. (1978) In: The Filamentous Fungi,
Vol. 3, Smith, J.E. and Berry, D.R. (eds.), in press.
Edward Arnold, London.
[13] Smith, J.E. and Anderson, J.G. (1973) Symp. Soc. Gen.
Microbiol. 23,295-337.