UNIVERSITY OF CALIFORNIA PUBLICATIONS
IN
AGRICULTURAL SCIENCES
Vol. 4, No. 13, pp. 407-412, 1 figure in text November 20, 1923
THE TOXICITY OF COPPER SULFATE
TO THE SPORES OF
TILLETIA TRITICI (BJERK.) WINTER*
BY
FEED N. BEIGGSt
INTRODUCTION
Copper sulfate has long been recognized as an effective fungicide
for the control of Tilletia tritici (Bjerk.) Winter. The usual treat-
ment is to dip seed wheat infected by the organism in a copper sulfate
solution. That this treatment greatly diminishes the number of dis-
eased plants is certain, but just how the individual spores are affected
physiologically by the copper has never been fully determined.
REVIEW OF LITERATURE
According to Evans,* "Tessier, 1889, seems to have been the first
to use copper compounds for the prevention of smut." In a work
published in 1807, Prevost" gives a careful account of the effect of
copper on the spores of wheat smut, Tilletia tritici. Stevens^ studied
the toxicity of a large number of chemical compounds and concluded
that all the copper salts agree closely in their toxic action on fungous
spores. Duggar^ made an extensive study of spore germination as
* Thesis submitted in partial satisfaction of the requirements for the degree
of Master of Science in Agriculture, at the University of California, May, 1922.
t Assistant Pathologist, Office of Cereal Investigations, Bureau of Plant Indus-
try, United States Department of Agriculture (in cooperation with the California
Agricultural Experiment Station). The writer wishes to express his indebtedness
to the United States Department of Agriculture for the privilege of pursuing post-
graduate studies; to Professor W. W. Maekie, of the University of California,
under whom this work was done, for his kind suggestions and helpful criticisms;
to Professor J. P. Bennett for the use of his laboratory and for his many kind
suggestions.
408 University of California Publications in Agricultural Sciences [Vol. 4
affected by certain chemical, as well as physical, stimuli. He found
that fungi were stimulated very little, if any, by copper sulfate and
that they tolerate only very dilute concentrations of copper sulfate.
Clark^' ^ studied the toxicity of copper sulfate to 15 fungi which
represented fairly well the natural groups, and found that 12 forms
represented a range of lethal concentration of .0168 N to .0099 N, or
slightly less than 70 per cent variation. He also found that copper
sulfate was much more toxic when dissolved in pure water than when
dissolved in any other medium. Hawkins,^ using distilled water,
found that a .00006 N concentration of copper nitrate practically
inhibited the growth of Glomerella cingulata.
In summing up the work of previous investigators, it is readily
seen that the growth of most fungi is inhibited by rather low concen-
trations of copper compounds.
METHODS
In starting this research, the first problem was to determine the
conditions under which maximum uniform germination could be
secured.
Stakman^ writes that rather uncertain and capricious germination
was noted by Prevost, De CandoUe, Tulsane, Kiihn, Fischer von Wald-
heim, Brefeld, and others. He found that germination required from
two to four days in water at room temperature and that all nutrients
except soil infusion exerted a harmful effect on germination. Me-
Alpine" also germinated the spores in wtiter in two or three days.
Wilcox^" was not able to obtain more than 8 to 10 per cent germination
in distilled water, and that only after a period of twenty-five to thirty
days.
Culture solutions. — In preliminary experiments to determine the
best medium for germination the following solutions were used :
No. 1. Water extract from Yolo sandy loam soil.
No. 2. Same as No. 1 diluted to one-half strength.
No. 3. Same as No. 1 diluted to one-quarter strength.
No. 4. Water extract of San Joaquin sandy loam soil.
No. 5. Same as No. 4 diluted to one-half strength.
No. 6. Same as No. 4 diluted to one-quartor strength.
No. 7. Distilled water.
No. 8. Tap water.
1923] Briggs: The Toxicity of Copper Sulfate 409
Extracts of soil were made by mixing one volume of soil with two
volumes of water and autoclaving for one and one-half hours at
17 pounds pressure. ' The liquid was filtered off under pressure and
sterilized.
Solution No. 5 gave the highest per cent and most uniform
germination and was therefore used for the experiment.
Using Baker's analyzed copper sulfate, a .IN stock solution was
made up. The concentrations required for this experiment were
then made up by a series of dilutions and were prepared without the
measurement of less than 10 c.c. in any case. Standard pipettes and
volumetric flasks were used.
Temperature. — In preliminary experiments to determine the
optimum temperature for germination, tests were made at room
temperatures and controlled temperatures, 48° F., 53° F., 58° F., and
63° F. The latter temperatures were maintained by an automatically
regulated chamber placed in a 40° F. cold storage room. Based on
the results of the above experiments, it was decided to conduct one
set of experiments at a controlled temperature of 58° F. and one set
at room temperature which varied from 56° to 62° F.
Method of culture. — Two methods of culture were used: {a) the
sealed hanging drop method as described by Clark^ and Duggar^ ;
and (&) the ventilated hanging drop. The latter was prepared by
supporting the cover glass on two strips of paraffin 4 mm. square
and 25 mm. long. By heating the slides the strips were sealed to
them, and the cover glasses were made fast by pressing the edge with
a hot needle. These cultures were kept in a moist chamber, in order
to keep the drop from evaporating.
Examination of cultures.- — Cultures were examined on the fourth
day, and each day thereafter until thej' were nine or ten days old. '
Counts of ten spores were made from each of five different fields and
the average per cent of germination recorded.
410
University of California Publications in Agricultural Sciences [Vol. 4
DATA AND DISCUSSION
The data showed that there was little, if any, difference between
the germination at the laboratory temperature and that at the con-
trolled temperature. Likewise the germination in the sealed hanging-
drop cultures was not essentially different from that in the ventilated
hanging-drop cultures.
Fig. 1. Concentration of copper sulfate.
Under all conditions germination was somewhat erratic. Fre-
quently duplicate mounts showed variations of 30 to 50 per cent in
germination, but this was probably due, to some extent, to contam-
ination by moulds, which frequently occurred toward the end of the
incubation period. The technie followed in making up the mounts
undoubtedly was responsible for some of the erratic germination. A
small drop spread in a thin layer gave better germination than a large
drop. The number of spores in proportion to the amount of solution
was probably another factor. In a given drop the amount of copper
for each spore would decrease as the number of spores increased. So,
with a few spores, there might be a decided decrease in germination,
while with a large number of spores there would be little decrease.
1923] Briggs: The Toxicity of Copper Sulfate 411
Since there was so little difference in germination under the differ-
ent conditions of this experiment, the results were averaged together
and are presented in a graph (fig. 1).
It will be noted from the data represented in the graph that an
.002N solution of copper sulfate was sufficient to inhibit the growth
of the spores of Tilletia tritici under the condition of this experiment.
Cultures of .002N to .008N were kept for 20 days without any germ-
ination. A soil extract of a different concentration probably would
have given a different point of inhibition. Clark- found the lethal
concentration of copper sulfate to be .0076N when a beet decoction of
normal strength was used ; while it was .0034N, or approximately one-
half when the decoction was diluted to four volumes.
Concentrations of .0008N and .OOIN caused a decided decrease
in percentage of germination. The promycelia were very short and
distorted, in many cases never reaching a length greater than 20
to 30/x. No sporidia were found in cultures of this concentration, and
because of their weakened condition it is very doubtful if any of
these spores would be capable of infecting a wheat plant.
The concentration of .0006N copper sulfate caused many signs of
abnormal germination, but frequently a spore would germinate in
a perfectly normal manner, in so far as one could determine from
a superficial examination. In the more dilute copper solutions, ger-
mination apparently was normal with no depression of any very
great consequence in the percentage of germination, while in the more
concentrated copper solutions there was some delay in germination
and a very marked decrease until the point of inhibition was reached.
CONCLUSIONS
Under the conditions described the following conclusions may be
drawn :
1. In a culture solution consisting of a water extract of San
Joaquin sandy loam soil, a .002N concentration of copper sulfate is
sufficient to inhibit the germination of spores of Tilletia tritici.
2. In concentrations of .0008N and .OOIN there was very little
germination and that which occurred was decidedly abnormal in
character, the promycelium being very short and distorted.
3. It is doubtful if the abnormal promycelium obtained in con-
centrations of .0008N and .OOIN was capable of causing any infection.
412 University of California Publications in Agricultural Sciences [Vol. 4
4. Germination of spores in a .0006N copper sulfate solution was
abnormal except for occasional ones which seemed to develop in the
normal way.
5. In the more dilute copper sulfate solution, .00002N to .0004N,
germination apparently was normal. Occasional spores in concen-
trations of .0004N and .0006N showed some copper injury.
LITERATURE CITED
1 Clark, J. F.
1899. On the toxic effect of deleterious agents on the germination and de-
velopment of certain filamentous fungi. Botanical Gazette, vol.
28, pp. 289-327.
2 Clark, J. F.
1902. On the toxic properties of some copper compounds with special refer-
ence to Bordeaux mixture. Botanical Gazette, vol. 33, pp. 26—48.
3 DUGGAR, B. M.
1901. Physiological studies w-ith reference to the germination of certain
fungous spores. Botanical Gazette, vol. 31, pp. 38-66.
* Evans, W. H.
1896. Copper sulphate and gennination. U. S. D. A., Div. of Veg. Phys.
and Path., Bull. 10.
5 Hawkins, Lon A.
1913. The influence of calcium, magnesium and potassium nitrates upon
the toxicity of certain heavy metals towards fungus spores. Physi-
ological Eesearches, vol. 1, no. 2 (August).
6 McAlpine, D.
1910. The smuts of Australia, Dept. of Agr., Victoria, Govt. Press, Mel-
bourne, 1910, 212. pp.
"! Prevost, J. B.
1807. Memoire sur la cause immediate de la caire ou charbon des bles et de
plusieurs autres maladies de plantes et sur les preservatifs de la
caire (Montauban, 4, p. 85). Original not seen.
sStakman, E. C.
1913. Spore germination of cereal smuts. Minn. Agr. Exp. Sta. Bull. 133,
52 pp.
9 Stevens, F. L.
1898. The effect of aqueous solutions upon the gennination of fungous
spores. Botanical Gazette, vol. 26, pp. 377-406.
10 Wilcox, A. C.
1910. A method for the germination of spores of Tillctia iritici. (Manu-
script.)