SOA'IE LETHAL EFFECTS OF ULTRA-VIOLET RADIATION ON FUNGUS SPORES - PNAS
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VOL. 27, 1941 BOTANY: DIMOND AND D UGGAR 459
SOA'IE LETHAL EFFECTS OF ULTRA- VIOLET RADIATION ON
FUNGUS SPORES
By ALBERT DIMOND AND B. M. DUGGAR
DEPARTMENT OF BOTANY, UNIVERSITY OF WISCONSIN
Communicated August 22, 1941
Smith' has thoroughly reviewed the literature up to 1935 on the effects
of radiation on the fungi. Discussion in relation to the work here reported
has been limited to recent papers dealing with yeast and with the filamen-
tous fungi.
Several studies have been made with the object of determining the shape
of the survival curve in an attempt to disclose the mechanism by which
radiation effects the killing of single cells. Smith2 used the full spectrum of
the quartz mercury vapor lamp and irradiated spores of Fusarium. She
studied not only the shape of the survival curve at a constant tempera-
ture, but also the variation in the shape of this curve with increasing tem-
perature, so that temperature became a contributing lethal factor. She
obtained sigmoid survival curves at lower temperatures and logarithmic
curves when the temperature was increased. Oster3 also studied the ef-
fect of temperature and calculated the coefficients for killing of yeast by
monochromatic ultra-violet radiation.
Oster constructed a curve relating wave-length to the efficiency of killing
of yeast. Hollaender and Emmons4 worked with Trichophyton mentag-
rophytes and have recently established a similar curve for this organism.
Both of these investigations indicated that a maximum killing efficiency
is attained at wave-length 2650 A. These results have recently been con-
firmed for Ustilago zeae.5
Various investigators have published data indicating that the survival
curve of yeast and the fungi is sigmoid in form.3
The relation between the age of the fungus and the amount of energy
required to kill cells was studied for yeast by Oster3 and by Schreiber.7
Whereas Oster observed that increased amounts of energy were required
to kill older cells, Schreiber noted little effect of age.
In our studies the method of irradiation employed was essentially that
used by Duggar and Hollaender.8 The source of radiation was an 85-
watt type H-4 mercury vapor lamp made by the General Electric Company.
The outer glass casing of the lamp was removed. Monochromatic radia-
tion was obtained by means of a Bausch and Lomb quartz monochromator,
kindly loaned for this work by the Department of Physics of the Univer-
sity of Wisconsin. Throughout this work, monochromatic radiation of
wave-length 2650 A was employed. A thermopile in conjunction with a
Leeds and Northrup high sensitivity galvanometer was uised for determin-
Downloaded by guest on May 28, 2021460 460BOTANY: DIMOND AND D UGGAR PROC. N. A. S.
ing the amount of radiation absorbed by the spore suspension in the ex-
posure cell. The amount of energy absorbed was determined by the stand-
ard photochemical procedure. Irradiation was carried out at a temperature
of 250C.
Three species of fungi were used in this work. A culture of Rhizopus
suinus Niels., originally obtained from Utrecht and maintained in culture
here for several years, was used because of its rapid growth, repeatable
behavior in culture and prolific spore production, the spores being only
moderately pigmented. The minus strain of Mucor dispersus Hagem, ob-
tained through the kindness of Prof. A. F. Blakeslee, was used because its
spores were free of any pigmentation. Both R. suinus and M. dispersus
produce multinucleate spores. Aspergillus melleus Yukawa, obtained from
the collection of Aspergilli maintained by the Department of Agricultural
Bacteriology of this university, was also used for the reason that the spores
were pigmented and available in large quantities. Thom and Church9
state that, according to Dangeard, the spores of many of the Aspergilli
are uninucleate. We have assumed the production of uninucleate spores
by this species of Aspergillus.
Stock cultures of Rhizopus and of Aspergillus were grown on a liquid
medium and were incubated at 28°C. Since the mycelium of the Mucor
tended to grow submerged in liquid media, it was grown on potato dextrose
agar in Erlenmeyer flasks. Except for this procedure, the method of
treatment was identical with that used for the other two fungi.
Spore suspensions for irradiation were prepared in the following manner:
The liquid from a flask stock culture was poured off and the mycelium was
rinsed with a few cc. of physiological salt solution8 diluted to half strength.
After two rinsings under sterile conditions, fresh salt solution was added.
The flask was stoppered with a sterile cork and shaken thoroughly. The
resulting suspension was then filtered through sterile absorbent cotton to
remove mycelial fragments and clumped spores. The density of spores
in the suspension to be irradiated was determined by means of a hemocy-
tometer. The suspensions to be irradiated were of such concentration that
complete absorption of the incident beam of radiation did not occur. This
procedure was adopted in order that the spores in the beam of radiation
would be "saturated" with lethally effective quanta. Fungous spores of
these species are far more resistant to ultra-violet radiation than are vegeta-
tive bacterial and yeast cells, and it is therefore necessary to use suspen-
sions of low spore concentration in order to effect high killing in a reasonable
time. Such a procedure also minimizes effects other than those of radiation.
In making intensity measurements, the difference between the deflection
of the galvanometer when the theromopile was placed behind the cell filled
with spore suspension and that obtained behind the cell filled with salt
solution was used as a measure of the light absorbed by the fungous spores
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after making suitable corrections for reflection of light by the exposure cell.
Since a stirring mechanism in the beam of incident radiation would ab-
sorb and scatter considerable quantities of radiation, no mechanical stirrer
was used to agitate the spore suspension in the exposure cell. Frequent
agitation of the spore suspension was necessary to prevent sedimentation
of spores and was effected by means of a pipette during the course of ex-
posure.
After a suitable time interval, l/1o-cc. samples of spore suspension were
removed from the exposure cell. These were diluted with a potato dex-
trose solution consisting of the broth of 200 g. of potatoes, 20 g. dextrose
and 1 g. agar in 1000 cc. of distilled water. This quantity of agar was just
sufficient to cause gelation of the medium so that sedimentation of the
spores in the hanging drop was prevented. Ten volumes of this nutrient
were added to one volume of spore suspension. This medium was used
directly in making hanging drop cells, according to the standard procedure.
Preliminary experiments, carried out in order to ascertain the amount of
time required for the process of germination to become complete, showed
that a period of 15 to 20 hours was adequate, the time being dependent upon
the dosage of radiation given the spore suspension. This was true under the
experimental conditions employed for each of the three species studied.
Hanging drop cells were incubated at 28°C. for 15 to 20 hours and counts
were then made of the number of germinating spores as well as of the total
number of spores. A minimum of 200 spores was counted for each hanging
drop culture and such cultures made in duplicate, or in greater replication,
were prepared for each type of exposure. The average value of the per-
centage germination was determined in each case.
Control suspensions of spores were made up and were treated identically
with exposed suspensions in every way, except that they were not exposed
to radiation. Exposed suspensions were irradiated for as long as 3 hours,
but no effect of standing in physiological salt solution for this time on
unirradiated spores was observed either for the time required for germina-
tion or on the germinability.
The dosage of radiation given the spore suspension was calculated when
the time of exposure, intensity of the beam of radiation, proportion of the
beam absorbed by the suspension and the number of spores responsible for
this absorption were known.
In order to determine the shape of the lethal curve, experiments were
carried out in triplicate with Rhizopus suinus. Cultures which had been
incubated for 4 days after inoculation of the nutrient were used as a source
of spores for irradiation. Germination counts were made of spores in the
hanging drops. The criterion of germination used required that the germ
tube produced by a spore must equal the diameter of the spore in order
that the spore be considered germinated. The dosage of radiation in ergs
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absorbed by each spore was compared with the survival of spores in the
suspension, and curves showing the relation of these two factors were
plotted.
The curve of figure 1 shows the results of 3 experiments carried out
under identical conditions. The smoothed curve is of sigmoid shape.
L.I
F-
z E0
LJi
U 00E
CC
LLi
a.
z
0
F-
0)
z 0\ 4
cx: ** * 4E 0
Lli
U
o
FIGURE 1
Composite curve of data of three experiments on the
survival of spores from 4-day cultures of Rhisopus
suinus.
A similar series of experiments was carried out using spores of the minus
strain of Mucor dispersus. Spores for this series of experiments were simi-
larly obtained from 4-day stock cultures. Results of 3 parallel experiments
are presented by means of figure 2. As with Rhizopus suinus, the curve is
sigmoid in shape.
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Spores of Aspergillus melleus were likewise obtained from 4-day cultures.
Suspensions to be irradiated were considerably more dense than the ones
used for the mucoraceous fungi for the reason that the spores were much
smaller and that absorption was less. Since these spores germinated in
about the same time as those of the other species used, they were treated
throughout in an identical manner. The results of a series of 3 experiments
are presented graphically in figure 3. The survival curve for spores of
Aspergillus melleus is of logarithmic form, and is quite different from that
for spores of Rhizopus or Mucor. The significance of this behavior will be
discussed later.
1
U.
I-
z
LUI
Lii
a.
z
0
I-
Z 50 0
0
25 0.1 0
ERGS PER SPORE
FIGURE 2
Composite curve of data of three experiments on the
survival of spores from 4-day cultures of the minus
strain of Mucor dispersus.
Another point of interest, shown by a comparison of figures 1, 2 and 3,
is the difference in resistance of spores of the 3 species of fungi. Following
a customary procedure, we shall compare the amounts of energy required
for a 50% killing of the spores exposed to radiation, and for Rhizopus
suinus this quantity is 0.09 erg per spore. The minus strain of Mucor dis-
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persus, however, required a larger dosage of radiation, viz., 0.12 erg per
spore, to cause the same amount of killing. Aspergillus melleus, on the
other hand, is much the most susceptible of the three species employed,
since 50% killing of the spores is effected by the absorption of 0.0064 erg
per spore.
U
I-
z
lJ
U
0-
50
z
0
I-
z
cx:
Li
U
o
_ ._.& .-
ERGS PER SPORE
FIGURE 3
Composite curve of data of three experiments on the
survival of spores from 4-day cultures of Aspergillus
melleus.
The differences in resistance observed led the authors to make a quanti-
tative comparison of the properties of the spores of these 3 species. Mea-
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surements of the dimensions of large populations of spores grown in the
standard manner were made by means of an ocular micrometer. Spores of
Rhizopus were found to be of the size reported by Nielsen10 in his original
description of this fungus. They are of ellipsoidal shape and vary in size
over a considerable range, from 5.5 to 9.0 IA by 3.5 to 7.0 ,u. Those of As-
pergillus were found to be of the size given by Thom and Church' and range
from 2.5 to 4.0 ,u in diameter, being spheroidal in shape. Spores of Mucor
were by far the largest of the three, but showed a greater variation in size
than is reported by Saccardo.1' Spores were observed ranging from 9.0 to
15.4 ,u in diameter and were spheroidal in form.
Since mucoraceous spores generally contain more than one nucleus, an
attempt was made to determine the average number of nuclei in spores of
Mucor dispersus produced under the conditions used for these experiments.
Spores were stained by means of a modification of Feulgen's nuclear reac-
tion and counts were made of the number of nuclei per spore in a popula-
tion of spores. Counts of 9 to 20 nuclei per spore were obtained, and the
average number of nuclei per spore was 13. Data comparing the proper-
ties of spores of the 3 species on the assumption that the spores of Asper-
gillus are uninucleate are given in table 1.
TABLE 1
A COMPARISON OF THREE SPECIES OF FUNGI WITH RESPECT TO RESISTANCE TO RADIA-
TION, SIZE OF SPORES AND NUMBER OF NUCLEI PER SPORE
BROS PBR SPORE FOR MEAN VOLUME OP NUCLEI
50% KILLING SPORE PER SPORE
SPECI (R&ATIO) (/$S) (ATIO)
Aspergillus melleus 0.0064 1.0 8 1.0 1 (?)
Rhizopus suinus 0.088 13.7 28 3.4
Mucor dispersus 0.12 17.5 113 14.1 13
The ratios recorded in the table were obtained by setting the values for spores of
Aspergiilus melkeus equal to 1 and by raising the corresponding values for spores of the
other fungi in proportion.
From this table it is seen that the differences in resistance between
species is not correlated directly with the volume of the spore or with the
number of nuclei. Very possibly differing degrees of resistance between
spores of various species are correlated not only with the size of spores and
the number of nuclei contained, but also with the kind and amount of pig-
mentation in the spore wall.
Differences in the types of killing curves obtained for Aspergillus and
for the mucoraceous species used in this study merit some consideration.
The killing curves recorded in the literature have been variously inter-
preted as indicative of a multiple quantum hit-to-kill relation and as in-
dicative of variation in the susceptibility to radiation on the part of a popu-
lation of spores. Other workers have postulated an ideal shape of killing
Downloaded by guest on May 28, 2021466 BOTANY: DIMOND AND DUGGAR PROC. N. A. S.
curve as characterizing a unimolecular reaction. This would involve a
logarithmic order of deathr such as was obtained for Aspergillus melleus
(Fig. 3). Departures from this type of curve have been explained as being
attributable to various factors, such as the amount of pigmentation of
spores, the temperature at which irradiation was carried out, age of the
spores and so on. Factors such as the size of the spores and the number
of nuclei which they contain may also influence the shape of the survival
curve.
If it is assumed that the nucleus is the part of the cell which must be hit
by quanta in order that death may result, any factors which reduce the
number of quanta hitting the nucleus will also influence the effectiveness
of an injurious wave-length. The volume of protoplasm about the nucleus
might act in this way by absorption of quanta, and through its action on
the nucleus might alter the shape of the survival curve.
In the case of multinucleate spores the question arises whether or not
more than one nucleus need survive irradiation in order that the spore may
germinate. Since a population of mucoraceous spores shows a wide varia-
bility with respect to the number of nuclei per spore, there seems no a
priori reason for believing that more than one nucleus need survive irra-
diation in order to insure normal germination of the spore, even though
growth in the early stages may be somewhat retared as a result of the
treatment. If this is the case, it may well be that ability of the spore to
survive treatment, although all but one of the contained nuclei are inac-
tivated, may alter the shape of the survival curve. When the spore contains
a large number of nuclei, the centrally located nuclei are more protected,
and the probability is much less that a quantum entering the spore will
hit one of them than is the probability that the quantum will hit one of the
outer nuclei. The effect of inactivation of one or more of the nuclei in a
spore on the subsequent behavior of the spore is unknown.
The work of Smith,2 referred to in the introduction, may provide an ex-
planation for the difference in the shape of the survival curves obtained for
Aspergillus in the present study as contrasted with the shape of survival
curves obtained in other studies on uninucleate fungous spores. She,
working with Fusarium, and Hollaender and Emmons,4 with Trichophyton,
obtained sigmoid survival curves at irradiation temperatures below 20°C.
If spores of both these fungi are uninucleate, results obtained with spores
of Aspergillus in the present study might be expected to be similar to those
obtained by these authors. At irradiation temperatures lying between
200 and 50°C., Smith obtained a gradual transition from sigmoid to
logarithmic shape of the survival curve. In the present investigation,
irradiation was carried out at a temperature of 25°C. This was
somewhat higher than the temperature at which irradiation was carried
out by Smith and by Hollaender and Emmons, and the possibility exists
Downloaded by guest on May 28, 2021VOL. 27, 1941 BOTANY: DIMOND AND DUGGAR 467
that the higher temperature at which irradiation was carried out was re-
sponsible for the logarithmic shape of the survival curves for spores of As-
pergillus. Zahl, Koller and Haskins"2 have recently given results of irradia-
tion of spores of Aspergillus niger showing survival curves of a transition
type between sigmoid and logarithmic shape. The temperatures employed
are not stated.
The relation between resistance to radiation and the age of spores was
studied, using cultures of Rhizopus suinus. The data of a previous experi-
ment were used to represent the behavior of a 4-day culture. Spores from
cultures which had been incubated 12, 14, 21 and 34 days after inoculation
were irradiated and data on percentage germination resulting from this
treatment were accumulated in the manner described. The germination
obtained in control hanging drops of older cultures was less than in the
younger cultures, owing to loss of viability of spores. Consequently, the
germination percentages of control hanging drops were in all cases regarded
as 100, and the values for irradiated suspensions of spores from these cul-
tures were raised in proportion.
The results of these experiments are presented in table 2. The table
shows how resistance to radiation increases with age of the culture from
which the exposed spores are taken. These results confirm those of Oster,3
and are in disagreement with the results of Schreiber,7 both of whom used
yeast as the test organism. The resistance does not change markedly dur-
ing the first few days of growth; the difference in the table for cultures
4, 12 and 14 days old probably are not in themselves significant. The
trend is definite, however, and extends throughout the series.
TABLE 2
kHE RELATION BETWEEN RESISTANCE TO RADIATION AND AGE OF SPORES OF
Rhizopus suinus
AGE OF CULTURE ERG PER SPORB
(DAYS) FOR 50% KIrLLING
4 0.075
11 0.08
14 0.09
21 0.11
34 0.21
Increased resistance to radiation with age may be caused by a number of
factors. The spores may continue to increase somewhat in size after
being formed in the sporangium. Any considerable increase in size not
accompanied by nuclear division would result in increased resistance of
the spore to radiation. The mass color of spores of Rhizopus suinus deep-
ens with age, and an increased amount of pigment would also increase re-
sistance to radiation. Possibly the drying of spores with age alters the
protoplasm in such manner that greater protoplasmic absorption of radia-
tion occurs with resultant decrease in nuclear absorption.
Downloaded by guest on May 28, 2021468 CHEMISTRY: ZECHMEISTER, ET AL. PROC. N. A. S.
Summary.-Spores of the fungi Rhizopus suinus, Mucor dispersus and
Aspergillus melleus were tested with respect to their resistance to ultra-
violet of wave-length 2650 A. Sigmoid survival curves were obtained for
the fungi Rhizopus suinus and the minus strain of Mucor dispersus when
spores from 4-day cultures were irradiated. Survival curves describing a
logarithmic order of death were exhibited by spores from 4-day cultures of
Aspergillus melkeus.
The three species of fungi showed marked differences in susceptibility to
radiation (2650 A). A combination of the differences in pigmentation, size
of the spores and the number of nuclei may account for the differences in
susceptibility observed.
A trend of increasing resistance to radiation with increased age was ob-
served for spores of Rhizopus suinus.
'
Smith, E. C., in Biol. Effects of Radiation, 2, 889-918 (1936).
2 Smith, E. C., Bull. Torrey Bot. Club, 62, 45-58, 151-164 (1935).
3 Oster, R., Jour. Gen. Physiol., 18, 71-88, 243-250, 251-254 (1934).
4Hollaender, A., and Emmons, C. W., Jour. Cell. and Comp. Physiol., 13, 391-402
(1939).
6 Landen, E. W., Ibid., 14, 217-226 (1939).
8 Luyet, B. J., Radiology, 18, 1019-1022 (1932).
7Schreiber, H., Strahlentherapie, 49, 541-595 (1934).
8 Duggar, B. M., and Hollaender, A., Jour. Bact., 27, 219-239, 241-256 (1934).
9 Thom, C., and Church, M. B., The Aspergilli (1926).
10 Nielsen, N., Virchow's Arch. f. path. Anat. u. Physiol., 273, 859-863 (1929).
11 Saccardo, P. A., Sylloge Fungorum, 21, 821 (1912).
12 Zahl, P. A., Koller, L. R., and Haskins, C. P., Jour. Gen. Physiol., 22, 689-698
(1939).
PROLYCOPENE, A NATURALLY OCCURRING STEREOISOMER
OF L YCOPENE
By L. ZECHMEISTER, A. L. LEROSEN, F. W. WENT AND LINUS PAULING
THE GATES AND CRELLIN LABORATORIES OF CHEMISTRY* AND THE KERCKHOFF
LABORATORIES OF BIOLOGY OF THE CALIFORNIA INSTITUTE OF TECHNOLOGY, PASADENA
Communicated September 3, 1941
In this paper we record the observation that there occurs in the variety
of tomato called "tangerine tomato" a carotenoid, prolycopene, which is an
isomer of lycopene; the isomeric relationship is similar to that between
lycopene and neolycopene,I and in our opinion prolycopene is to be classed
as a naturally occurring neolycopene, being the first observed natural
neo form of a C40-carotenoid.
The color of ripe tomato fruits is determined primarily by two sets
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