J. gen. Microbiol.

(IS@), 55, 1-7

Printed in Great Britain

The Spores of Eremothecium ashbyii

By K. NORDSTRt)M*
Division of Applied Microbiology, Royal Institute of Technology,
Stockholm 70, Sweden
(Accepted for publication I 9 August I 968)
SUMMARY

Pure suspensions of spores of Eremothecium ashbyii were prepared by

filtration through glass wool; the filtrates contained several million spores/
ml. Germinating spores showed antibiotic effects. The efficiency of plating
decreased with increasing concentrations of spores but increased when the
spores were washed. The results of inactivation by ultraviolet light indicated
that the spores were haploid. Inositol deficiency was lethal to the spores.
These findings indicate that it should be possible to use E. ashbyii in genetic
work, but the lack of knowledge about the life-cycle is a serious obstacle.
INTRODUCTION

Eremothecium ashbyii produces large quantities of riboflavin and is of industrial

importance for this reason. The life-cycle and morphology of E. ashbyii has been
described by Guillermond (1935, 1936). The organism grows into quite large masses
of mycelium and after 3 to 5 days some 20 sickle-shaped spores are formed in the
sporangium which bursts and liberates the spores which are found in large numbers
in mature cultures. The significance of the spores is uncertain and no sexual reproduction has been demonstrated but, as Krneta-Jordi (1962) pointed out, this may be due
to the fact that only one isolate has been studied and that E. ashbyii may be heterothallic. Kmeta-Jordi made an extensive cytological and physiological study but she
remarked that it was impossible to get pure spore cultures. This was a serious obstacle
since it was difficult to get a standard inoculum. In the present paper it will be shown
that pure spore suspensions can easily be obtained; experiments with these spore
suspensions are reported.
METHODS

Organism. Eremothecium ashbyii our6022 was used; it was obtained from The
Department of Fermentation Technology, Faculty of Engineering, Osaka University,
Osaka, Japan.
Media. The basal defined medium (Ea) was as described by Osman & Soliman (1963).
Solid Ea-medium was made by adding 2% agar. In some experiments wort agar
(8 %, w/v, beer wort+ 2 %, w/v, agar) was used.
Dilutions and washings were made with 0.9 % (w/v) NaCl solution (saline).
Preparation of spore suspensions. Mature cultures were filtered through a sterile
I cm thick layer of glass wool. The spores passed through but mycelia and hyphae
were retained. The filtrates were in some cases used directly (or after dilution); in

* Present address: Department of Microbiology, University of UmeA, UmeA 6, Sweden.

~~

VoZ. 54, No. 3, was issued 24 January 19%

I

G. Microb. 55

K. NORDSTROM

many experiments the spore suspensions were centrifuged, the spores washed twice
with saline and then resuspended in saline (these suspensions are denoted as washed).
Total counts were made microscopically by using the Burker counting chamber
with a depth of 0.1 mm.
Viable counts of spores. Samples (0.1ml.) of a suitable dilution were spread on Ea
agar or wort agar plates which were incubated at 28". Colonies were counted after
2 to 3 days.
Eficiency of plating is defined as the ratio between the number of colonies formed
and the number of spores plated (as calculated from the total count).
Cultivation in liquid medium. Volumes (50 ml.) of the Ea medium were introduced
into 300 ml. Erlenmeyer flasks which were inoculated with spores and incubated on
a rotary shaker at 28".
Ultraviolet (u.v.)treatment. The samples that were to be u.v.-irradiated were washed
twice and resuspended in saline. Samples (10to 20 ml.) of suspension were transferred
to a 10cm. d i m . glass Petri dish and irradiated by a 10W. Hg lamp (giving about
1000ergs/mm2per min. at wavelength 2537 A at the distance used). Dilutions were
spread in the dark on Ea medium agar and incubated at 28".
Treatment with N-methyl-N'-nitro-N-nitrosoguunidine(NTG). Washed spores were
suspended in 0.2 M-acetate buffer (pH 5.0, Megnet, 1965). NTG (1-5mg./ml.) was
dissolved in the same buffer. One ml. of spore suspension (about 2 x ro6 spores/ml.)
and 2 ml. NTG solution were mixed (Megnet, 1965)and the whole incubated at 28'.
Samples were filtered through Millipore filters and the spores were washed with acetate
buffer and then resuspended and spread on Ea medium agar.
RESULTS

Preparation of pure spore suspensions

When Eremothecium ashbyii is grown in a liquid medium mycelia are formed that
are easily seen by the naked eye. After a few days hyphal tips are transformed to spore
cells which later excrete 10to 20 sickle-shaped spores each. At this stage large amounts
of riboflavin are produced, giving a yellow colour to the medium. Finally, there is a
mixture of spores, small hyphae and mycelia of various sizes (Krneta-Jordi, 1962). It
proved to be very easy to separate the spores from the other components by filtration
through a layer of glass wool about I cm. thick. The filtrate so obtained contained
virtually no other forms but spores at a concentration of several million/ml. as counted
microscopically. Kmeta-Jordi (1962)studied the germination of the spores in hanging
drops, but since she was unable to prepare pure spore suspensions this made it impossible to study the physiology of spore germination. The spore suspension prepared
by filtration as described were well suited for further studies.
Germination of spores
Pure spore suspensions were prepared by filtration through glass wool as described.
When these suspensions were diluted and plated the number of colonies formed was
much less than the total spore count. The efficiencyof plating increased with increasing
dilution, indicating the presence of some antibiotic substance in the medium where
the spores were formed (Fig. I). This conclusion was further strengthened when it
was found that the efficiency of plating increased when the spores were centrifuged

Eremothecium ashbyii spores

e.0.p. =l

100

102

106

104

No. of spores plated

10

20

Incubation time (hr)

Fig. I

Fig.

Fig. I. Eremotheciumushbyii. Viable count in spore suspensionsas a function of the concentration of spores. Dilutions were made in saline.
Fig. 2. Eremothecium ushbyii. Colonies formed on wort agar after incubation in liquid Ea
medium for the time indicated on the abscissa. Total count (microscope) showed that 350
(0)and 35 (0)spores were spread per plate.

Table

I.

Eficiency of plating of spores of Eremothecium ashbyii

The spore suspension contained 2.6 x 1oSspores/ml. by direct count microscopically.

EfEciency of plating (colony count)
Dilution
None
I/Ioo

Filtrate

Washed spores

5.0 x I O - ~
1.6~I O - ~

4-2 x I O - ~

1-0x I O - ~

and washed with saline before plating (Table I). The germination on Ea medium agar
was always rather inefficient. The spores did not germinate on wort agar but only on
the Ea medium agar; the reason for this is not known. However, wort agar could be
used as culture medium when the spores had been pregrown in liquid Ea medium
(Fig. 2).
The antagonistic effect of the growing mycelia on the germination of spores was
apparent when liquid Ea medium was inoculated with inocula of various sizes
(washed spores). After incubation for 30 hr viable (colony) counts showed that the
frequency of germination increased with decreasing size of inoculum (Fig. 3). At
greater sizes of inoculum the viable count decreased. The findings of Fig. 3 were also
verified by microscopic examination. After incubation for 30 hr, most of the spores
had not germinated and formed small mycelia when the inoculum was large (Fig. 3)
but almost all spores had germinated at the lowest concentration tested. Growth was
obtained even when the number of spores in a flask was less than 10.
1-2

K. NORDSTROM

Growth curve
The spores were formed after 3 to 4 days. The growth process can be followed by
viable count, but this method does not say anything of the increase in cell mass for
a mycelial organism. Sporulation was easily shown since the viable counts gave typical
one-step growth curves (Fig. 4). However, the efficiency of plating was low: the total
count of spores was 2-00 x ro6 per ml. in the experiment shown in Fig. 4. Since sporulation and excretion of riboflavin occurred simultaneously it was easy to know when
a culture contained fair amounts of spores.

100

100% germination

102

104

Concn. of spores (ml.-l)

Fig. 3

10

100

0.

40

Incubation time (days)

Fig. 4

Fig. 3 . Eremothecium ushbyii. Germination of spores in liquid Ea medium. The spores

were incubated for 30 hr before plating on Ea medium agar.
Fig. 4. Eremothecium mhbyii. Growth curve (viable count) for a culture starting from a small
inoculum.

Eflect of various treatments of spores

UZtraviuZet irradiation. Spores were susceptible to killing by U.V. irradiation (Fig. 9,
which was more efficient with spores which had started to germinate than with resting
spores. An apparent increase in death-rate was found already after 2 hr of pre-growth
in liquid Ea medium, the effect was fully developed after 4 hr. The germinating spores
wefe rather sensitive to U.V. irradiation; about 400 ergs/mm.2was sufficient to decrease
survival to 0.1 % as compared to about 2000
for haploid Saccharomyces
cerevisiae treated in the same apparatus (Nordstriim, 1964). Since the efficiency of
plating was dependent on the spore concentration it was meaningless to follow the
survival curve to lower values. However, the curves seemed to be linear. U.V. irradiation also had a marked effect on the germination of the spores in liquid Ea medium
(Fig. 6).
Treatment with NTG. The spores were killed by NTG; incubation in acetate buffer
with I mg. NTG/ml. for 3 hr decreased the viable spore count to about I yo of the
initial value.
Inositol-less death. 2 x 106spores/ml. were incubated in liquid Ea medium with and
without inositol (Table 2). The viable count increased about 10times in Ea medium+
inositol but decreased 50 times in Ea medium without inositol.

Eremothecium ashbyii spores

U.V. treatment (sec.)

Incubation time (hr)

Fig. 5

Fig. 6

Fig. 5 . Eremothecium ushbyii.Effect of pre-growth in liquid Ea medium on survival of u.v.irradiation.The spores were grown in liquid Ea medium for o (0),2 (a),4 (A)and 26 (0)
hr.
Fig. 6. Eremothecium ushbyii. Effect of u.v.-irradiation on germination of spores. The liquid
Ea medium was inoculated with untreated spores (0)and the same concentration of spores
u.v.-irradiated for 15sec (a) (about 250 ergs/mm*.).Viable counts were made at intervals.
The number of spores added initially was 1.79 x 106/ml.

Table 2. Efect of inositol deficiency on survival of

Eremothecium ashbyii spores
Viable counts were read at o and 5 days of incubation of spores
in the Ea medium, with and without inositol.
Viable count
7

Medium
+Inosito1
Inositol

Initial
5 . 2 10'
~

5 days

7.0~
1o6
I ' O X 108

DISCUSSION

Jinks (1952)and Griggs (1952)studied the back mutation assay method used in
microbial genetics and reported that large inocula lead to a lower efficiency of plating
than smaller inocula because of competition for limited resources in the medium.
However, Karlmark & Westergaard (1952)showed that this does not occur on media
containing excess nutrients. In such back-mutation studies the growth of a small
number of prototrophs is repressed by the presence of a large number of auxotrophs.
In the present paper competition for nutrients may be one reason for the low efficiency
of plating at large inocula but it cannot be the main reason since Saccharomyces
cerevisiae can form heavy lawns of colonies on the media used. Furthermore, the
efficiency of plating increased when the spores were washed before plating (Table I).
Thus the presence of a substance that represses spore germination seems to be reasonable. It would be a selective advantage for an organism to prevent the spores from
germination close to large masses of mycelium.

K. NORDSTROM

From the present results it can be concluded that at least some conditions needed
for a genetic study of Eremothecium ashbyii are present. It is possible to isolate single
cells (spores). However, nothing is known about the nuclear state of the spores, whether
they are haploid or diploid, etc.; Fig. 5 may suggest a haploid status since the u.v.survival curves seem to be linear. This evidence, however, is rather weak since the
result of Fig. 5 may be greatly influenced by the low efficiency of plating and by the
dilution effect (Fig. I). Krneta-Jordi (1962) showed that the spores are uninucleate.
A serious obstacle is the lack of knowledge of the life-cycle of E. ashbyii. The systematic position of this genus, and, thus, the genetic significance of the spores are
very uncertain (Lodder & Kreger-van Rij, 1952). They may or may not be formed
meiotically, etc. Guillermond (1935) has proposed that Eremothecium can be related
to either Spermophthora or Dipodascus. In the former case Eremothecium should
have lost the ability to conjugate and the spores should be vegetative spores. In the
latter case, Eremothecium should have lost its sexual differentiation. In both cases,
the organism would be difEcult to use in genetic work. However, other possibilities
may also be open. The results of U.V. irradiation showed that the spores were uninucleate and haploid (one-hit curves). Thus, it should be possible to use E. ashbyii
in mutation work. However, it cannot be a good organism for genetic work since the
efficiency of plating is low at high spore concentrations on plates, and is even lower
when the spores are not washed before plating. This makes it rather laborious to use.
One prerequisite for trying crosses with an organism is to have genetic markers,
preferably auxotrophic markers. Since inositol deficiency was lethal in the complete
medium it may be possible to select auxotrophic mutants; inositol deficiency has been
shown to be useful in other fungal systems (Megnet, 1964; Lester & Gross, 1959).
Minoura (1952) showed that inositol is a growth factor of E. ashbyii, but it has to be
shown that auxotrophs of Eremothecium really can be rescued from inositol-less death.
This work was supported by the Swedish Council for Applied Research. The skilful
technical assistance of Mrs Kerstin Ekengren-Jansson is gratefully acknowledged.
REFERENCES

GRIGGS,
G.W.(1952).Back mutation assay method in micro-organisms.Nature, Lond. 16g,98.
GUILLFUUMOND,
A.(1935).Sur un champignon nouveau, parasite des capsules du Cotonnier, d'Eremothecium ashbyii et ses relations possibles avec le Spermophthoru gossypii et les Ascomycetes.
C. r. hebd. Sdanc. Acud. Sci., Paris 200, 1556.
GUILLERMOND,
A. (1936). L'Eremothecium ashbyii, nouveau champignon parasite des capsules du
cotonnier. Revue Mycol. I, 115.
JINKS, J. L. (1952). Competitive suppression and the determination of linkage in micro-organisms.
Nature, Lo&. 170, 106.
K~LMARK,
G . & WESTERGAARD,
M. (1952).Validity of the Neurospora back-mutation test. Nature,
Lond. 169,626.
KRNETA-JORDI,M.(1962).Cytologische und physiologische Untersuchungen an Eremothecium ashbyii Guill. WirkstofTbedarf und Syntesevermogen als Funktion der Milieubeschafenheit. Arch.
Mikrobiol. a,76.
LESTER, H.E. & GROSS,
S.R. (1959). Efficient method for selection of auxotrophic mutants of
Neurospora. Science, N.Y. xzg, 572.
L~DDER,
J. & -=-VAN
RU, N.J. W.(1952). The Yeasts. A Taxonomic Study. Amsterdam: North
Holland Publishing Co.
MEONET,R. (1964). A method for the selection of auxotrophic mutants of the yeast Schizosuechromyces pombe. Experientia 20, 320.

Eremothecium ashbyii spores

MEGNET,R. (1965). Screening for auxotrophic mutants of Schizosaccharomyces pombe With 2deoxyglucose. Mutation Res. 2, 328.
MINOURA,
K.(1952).Studies on the riboflavin production by Eremothecium ashbyii. XXV. Search for
the active principle in peptone, and XXVII. Biol. Abstr. 26, 9227, 9229.
NORDSTROM,
K.(1964).Formation of esters from acids by brewers yeast. In. Formation by various
strains. J. Inst. Brewing 70, 226.
OSMAN,
H.G.& SOLIMAN,
M.H.M. (1963).Biosynthesis of riboflavin (vit BB)by Eremothecium
ashbyii. IV.The nutritional requirements of carbon and nitrogen for E. ashbyii. Arch. Mikrobiol.
46,247.