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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
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
e.0.p. =l
100
102
106
104
10
20
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.
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
10
100
0.
40
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.
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.
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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.
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