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THE EFFECT OF ZINC ETHYLENE BISDITHIOCARBAMATE ON THE GROWTH RESPONSES OF THE TOMATO PLANT
ROBERT GEORGE EMGE B.S., University of Illinois, 1946
THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN PLANT PATHOLOGY IN THE GRADUATE COLLEGE OF THE UNIVERSITY OF ILLINOIS, 19411
UNIVERSITY OF ILLINOIS THE GRADUATE COLLEGE
I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY ENTITLED
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_ ^ . / ^ ~ In Charge of Thesis
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Recoinnicndation concurred inf Committee on Final Examination')" * Subject to successful final examination in (he case of the doctorate. t Required for doctor's degree but not for master's. 6M—12.48—40199K
THE EFFECT OF ZINC ETHYLENE BISDITHIOCARBAMATE ON THE GROWTH RESPONSES OF THE TOMATO PLANT
ROBERT GEORGE EMGE B.S., University of Illinois, 1946
THESIS SUBMl'lTED IN PARTIAL FULFILLMENT OF THE
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN P L A N T PATHOLOGY IN THE GRADUATE COLLEGE OP THE UNIVERSITY
OF ILLINOIS. 1D40
COPYRIGHTED by ROBERT GEORGE EMGE 1950
ACKNOWLEDGEMENT The author takes this opportunity to express his sincere appreciation to Professor M. B» Linn for his Interest and guidance in this investigation- It is also a pleasure to thank Professor H. W. Anderson, D. Powell, E» H. Thornberry and D. Gottlieb for their counsel during the course of graduate study and their suggestions in the preparation of this manuscript.
TABLE OF CONTENTS Page NO. I* II* III*
Zinc Deficiency Symptoms Methods and Materials
IT* V* VI. VII*
Zinc solutions and suspensions,...
Result s Discussion Summary Bibliography.
..*..6 6 6 ........,*7 .....8 13 .......14 15
...20 22 ..23
Id INTRODUCTION Very little information is available on the effect of organic zinc fungicides on the growth of plants. Heuberger (6) stated that potatoes, tomatoes, cantaloupes and cucumbers sprayed with zinc dithiocarbamate fungicides produced higher yields than when sprayed with copper fungicides even though disease control by the dithiocarbamates was inferior. Hoyman (9}, Ellis (4) and Berkely et al (I) likewise have reported increased yields of potatoes resulting from the use of organic zinc rungicides. These investigators have suggested that the zinc (6, 9 ) , carbon and possibly sulfur (6) in such fungicides may be responsible for this phenomenon, palmiter (II) reported increased yield from apple trees sprayed with Fermate and advanced the hypothesis that the nitrogen in this compound might have been partly responsible. Apparent stimulation of growth in field tomato plants sprayed 1/ with zineb (zinc ethylene bisdithiocarbamate) was noted in 1946 at Urbana, Illinois. This observation prompted further work under controlled conditions in the greenhouse. The main objective of this investigation was to determine whether tomato plants are capable of absorbing through the leaves and utilizing the zinc contained in zineb. This was done by determining the effect of this fungicide vtien sprayed on (1) plants growing in a zinc-deficient nutrient solution but not showing deficiency symptoms (2) plants
1/ Rohwer, s. A. Common names for five fungicidal chemicals* (Mimeo.) U. S. D. A. Agr. Res. Admin.: 2 pp. 1949*
also growing in a zinc-deficient nutrient solution but exhibiting severe zinc-deficiency symptoms and (3) plants growing in a nutrient solution containing zinc*
ZINC DEFICIENCY SYmPTOMS As zinc deficiency symptoms are expressed in various ways, it was thought advisable to describe the symptoms as found in several crops. Squash, mustard, cotton and tomato plants grown in zinc-deficient soils exhibit symptoms of abnormally small mottled, necrotic, leaves.
Squash and cotton particularly have extremely
mottled leaves with necrotic areas. Abnormally small leaves, either yellow or mottled, are frequently produced by tomato and mustard plants (8). In citrus however, zinc-deficiency symptoms are most apparent in the leaves which show irregular yellowing between the lateral veins.
In severe cases other ssymptoms are undersize leaves, pale
coarse fruit, multiple buds and dieback (10). The immature stage of citrus leaves destined to become mottled are often characterized by a pronounced venation, the general color of the leaf being yellowish.
As the leaf matures the green areas adjacent to the
veins spread and deepen in color with the yellow-green areas between the veins becoming more yellow, giving rise to mottled patterns (2). It was observed in the present studies that zinc-deficiency in tomatoes manifests itself in several ways.
The plants growing in
zinc-deficient solutions at first are smaller and more spindly than those growing in a complete nutrient solution (Fig. 1 ) . Later the base of the petiolules become darkened and a downward curling or epinasty of the leaves develop.
With further development, the
leaves take on a yellow-mottled appearance and develop brown
Fig* 1* Early Baltimore tomato plants growing in complete and zinc-deficient nutrient solutions and showing typical growth responses when sprayed with, organic zinc (zineb) in suspension and inorganic zinc (zinc sulfate) in solution*
necrotic or dead spots.
These symptoms are visible in 19 to 20
days after the plants were placed in the zinc-deficient culture solutions.
Although the plants growixig in the complete nutrient
solution were blooming at the end of 40 days, there was no evidence of flower buds on those growing in the zinc-deficient solution.
METHODS AND MATERIALS Plants.
The tomato (Lyoopersicon esculenturn Mill. var. Early
Baltimore) was used as the test plant in this investigation.
seedlings were started in quartz sana and watered with tap water. When they had attained a height of two to three inches, they ivere removed from the sand, the root system thoroughly washed with redistilled water and placed in the nutrient solutions. Apparatus.
Pyrex glassware and redistilled water were used
throughout the investigation.
All the glassware was thoroughly
washea in a potassium dichromate-sulfuric acid cleaning solution and rinsed with redistilled water before using.
The containers for the
culture solutions were one-liter pyrex beakers fitted with paraffindipped Masonite covers.
The outside of the beaker was wrapped with
black paper to exclude light.
Six one-inch lengths of pyrex glass
tubing (inside diameter, 7 mm.) were spaced equidistant in a circle and fitted through the covers.
The plants were held in these tubes
by wrapping the stems loosely with clean cotton.
aeration tube which extended to within a quarter of an inch of the bottom of the beaker was fitted in the center of each cover.
culture solutions were continously aerated as suggested by Durell (3) and Vlamis, et al. (15) from a compressed air line.
The air flow
was regulated so as to prevent splashing of the solution against the covers.
The air was filtered by passing it through clean cotton
plugs in the line just before it entered the solution.
light intensities are condusive to zinc-deficiency symptoms (2), supplemental light was supplied by suspending four 300 watt light
bulbs three feet above the beakers. During cloudy or rainy days the lights were left on continously and on clear days were turned on from 5:00 p.m. to 11:00 p.m. Nutrient solutions* The solution used was a modification of that of Hoagland and Arnon (7).
Potassium dibasic phosphate
(K-HPO *3H„0) was substituted for potassium acid phosphate &
(KH2P04) in order to comply with the purification procedure of Stout and Arnon (14) which is a modification of Steinberg*s (13) calcium carbonate method.
Separate sxock solutions at molar con-
centration were prepared for each salt and the amounts indicated below were used for the complete nutrient solution. Molar Solution Potassium dibasic phosphate (KgHpQ^SR^O) Potassium nitrate (KKO3) Calcium nitrate (Ca (^3)2) Magnesium sulfate (MgS04)
cc. used per liter of nutrient solution 1 5 5 2
To one liter of this basal solution was added 1 cc. of the following chemicals: Compound Boric acid (H3BO3) Manganous chloride (IvInCl2»4H20) Zinc sulfate (ZnS04'7H20) Copper sulfate (CuSO^SHoO) Molybuic acid (85$ Mo0 3 , HgMoO^-HgQ)
Grams per liter of redistilled water 2.86 1.81 0*22 0.08 0.09
Iron was added in the form of a 0.5$ ferrous sulfate solution at the rate of 1 cc. per liter of basal nutrient solution. The final concentration of the complete nutrient solution was:
N P K S Mg Fe Mn B Zn Gu
210.00 31.00 273.00 65.00 24.32 1.00 O.dO 0.50 0.05 0.02
ppm. ppm* ppm. ppm. ppm. ppm* ppm. ppm* ppm. ppm.
The reaction of the complete nutrient solution was pH 5.5. In cases where a minus-zinc nutrient solution was desired, it was necessary to omit only the zinc sulfate from the above complete nutrient solution. The water in the beakers was kept at a constant level by the addition of redistilled water. Fresh nutrient solution was added every third day. Zinc Solutions and Suspensions. Zineb and the reaction pro2/ duct from mixing nabam
(disodlum ethylene bisdithiocarbamate) witbj
zinc sulfate in water are both zinc ethylene bisdithiocarbamate which has the empirical rormula, ZnG4H6N£S4, and the following structural formula. H H S t
H-C-N-C-S^ * Zn H-C-M-C-S' i
H H S The plants were sprayed with zinc solutions and suspensions about two weeks after they were placed in the nutrient solutions. Z/ Rohwer, S. A. Common names for five fungicidal chemicals. (Mimeo.) U. S. D. A. Agr. Res. Admin.: 2 pp. 1949*
The sole exception was the plants in the recovery experiment which were sprayed only arxer zinc-deficiency symptoms were visible.
DeVilbis No* 152 atomizer was couple with a 40 cc. flask and used to spray the plants until runoff started.
Approximately 40 cc.
were required for each set of six plants. Although the zineb used in this investigation was of a technical grade (95$ zinc ethylene bisdithiocarbamate) it was washed to remove any water-soluble zinc salts.
After each series of five
washings and filtrations, the filtrates were tested for zinc by the dithizone mixed-color method (12) using a Lumetron photolometer fitted with a 650^^filter.
When no more zinc could be removed from
the zineb after thirty washings (Table 1 ) , it was dried for subsequent use* The washed zineb was compared as a spray with the unwashed compound.
Suspensions of the two were sprayed on tomato plants
growing in zinc-deficient nutrient solutions. No significant differences were found between the two treatments as far as plant growth was concerned (Table 2 ) . The reaction product of nabam plus zinc sulfate was then compared as a spray with the unwashed zineb. In order to insure complete reaction between nabam and zinc sulfate so there would not be any free zinc in the suspension, nabam was used in slight excess of the amount required for complete reaction of the two chemicals. The effect on plant growth of the unwashed zineb and that of the reaction product were not significantly different (Table 3 ) .
From the results of these two preliminary
experiments it is evident that the water soluble zinc in the 95 per cent zineb had no effect upon the growth of the tomato plant*
Table 1- Amount of soluble zinc washed with water from 95% zineb
Number of washings
Gammas of soluble zinc per gram of zineb
5 10 15 20 25 30 35 40 45 50 a/ Estimated error was 0.5
10 Do Do 10 5 5 Do Do Do Do gamraas
Table 2- Growth response of zinc-deficient tomato plants after spraying with washed and unwashed zineb
Treatment Zinc deficient solution Sprayed with washed zineb Sprayed with unwashed zineb Unsprayed Complete solution Unsprayed
Mean dry weight in grams (Roots and Shoots) 11.12 12.28 6.28 11.02
Least difference necessary for significance between means for odds of 1:19=1.87; for odds of 1:99=2*83
Table 3- Growth response of zinc deficient tomato plants after spraying with the reaction product of nabam plus zinc sulfate
Treatment Zinc deficient solution Sprayed with reaction product Sprayed with zinc sulfate Unsprayed Complete s o l u t i o n Unsprayed
Mean dry weight in grams (Roots and Shoots) 17.67 19.73 8.83 19.63
L e a s t d i f f e r e n c e n e c e s s a r y for s i g n i f i c a n c e betv/een means f o r odds of 1:19=2.35; f o r odds of 1:99=3.56
The materials used were a zineb suspension at a concentration of 377 ppm. zinc, and a zinc sulfate solution containing 136 ppm.
zinc* Experiment layout * The six experiments below included four treatments, each comprised of three replicates. The individual replicates contained six tomato plants, thus making a total of 18 plants per treatment. Two treatments consisted of plants grown in zinc-deficient nutrient solutions and sprayed with zinc solutions and suspensions. The other two treatments were unsprayed controls, with the plants of one treatment grown in a complete nutrient solution, in Experiment 6 the plants in the spray treatments v/ere grown in only complete nutrient solutions. The experiments are as follows: Experiments 1, _2 and 3. Plants receiving the spray treatments in these experiments were grown in a zinc-deficient nutrient solution.
Ten to fifteen days after they were placed in the Solution,
the plants of one treatment were sprayed with a zinc sulfate solution and those of another treatment were sprayed with a zineb suspension (Table 4). Experiments 4 and _5* The plants in these experiments were not sprayed until definite zinc-deficiency symptoms were visible. The
3/ This is equivalent to 2 lbs. per 100 gals, of commercial zineb.. which is the recommended concentration for use on tomatoes in the field. 4/ This concentration is equivalent to •§• lb. ZnS04'7H20 per gals, of water*
plants were then measured and sprayed as in Experiments 1, 2 and 3. When the plants were removed for drying and weighing, they were measured again (Table 5 ) , Experiment 6^
All the plants, except those in the unsprayed
zinc-deficient control, were grown in a complete nutrient solution from the beginning. Eighteen days after the plants were placed in the nutrient solution, those of one treatment were sprayed with a zineb suspension and those of another sprayed with a zinc sulfate solution (Table 6 ) . Plant analyses. The plants were removed from the nutrient solutions after thirty or forty days.
Weights of roots and shoots
of each replicate were taken after the plants had been dried in a forced-draft oven for twenty-four hours at 65°c. As the growing point and first expanded leaf were undifferentiated at the time of spraying and thus not receiving any spray material, they were later taken for zinc analyses (Table 4).
The procedure for the analyses
was the dithizone mixed-color method of Sandell (12), which expresses the zinc content in terms of gammas
per unit weight or
volume of the material tested. All data were treated statistically using analysis of variance (5).
5/ A gamma equals one millionth of a gram or one microgram*
RESULTS Experiments 1, 2 and 3.
In these experiments plants were
growing in zinc-deficient nutrient solutions but were not showing deficiency symptoms at the time of spraying, plants in both of the spray treatments were significantly larger (odds of 1:99) than those in the unsprayed, but there were no significant differences between the spray treatments (Table 4 ) .
The weights of the un-
sprayed plants in the complete nutrient solution were significantly greater than those of the other treatments with the exception of the zinc sulfate spray (Fig. 1). Zinc content appeared to be directly correlated with weight. The zinc-content of the plants sprayed with zinc sulfate was significantly greater than that of the plants sprayed with zineb. Experiment 4 and 5_. in these experiments, plants were growing in zinc-deficient nutrient solutions and displaying severe deficiency symptoms at time of spraying.
There were no significant
differences (odds of 1:99) between the sprayed treatments (Table 5).
The weights of the unsprayed in the zinc-deficient solution
plants were significantly less than those of the other treatments* The weights of the unsprayed plants in the complete solution were greater than those of the plants sprayed with zinc sulfate but not significantly different than those of the plants sprayed with zineb. The growth increment of the sprayed plants was significantly greater than that of the unsprayed plants. The growth increment of the unsprayed plants in the zinc-deficient nutrient solutions was the smallest.
this experiment, plants were growing in a
(complete nutrient solution at the time of spraying.
The weights of
the sprayed plants were significantly greater (odds of 1:99) than those of the unsprayed plants (Table 6).
The weights of the un-
sprayed plants in the zinc-deficient nutrient solution were significantly less than those in the rest of the treatments.
Table 4- Growth response and zinc content of zinc-deficient tomato plants after spraying with zineb
Mean dry weight in grams (Roots and Shoots)
Zinc deficient solution Sprayed v/ith zineb Sprayed with zinc sulfate Unsprayed Complete solution Unsprayed Least difference necessary for significance between means f o r odds of: 1:19 1:99
Mean zinc content in gammas per gram of dried plant material
Table 5- Growth response of tomato plants showing severe zinc deficiency symptoms at time of spraying with zineb
Mean dry weight in Mean growth incregrams (Roots and ment (inches) 20 Shoots) days after sprajring
Zinc deficient solution Sprayed with zineb Sprayed v/ith zinc sulfate Unsprayed Complete solution Unsprayed Least difference necessary for significance between means for odds of: 1:19 ^ 1:99 =
Table 6- Growth response of tomato plants maintained in a full nutrient solution and sprayed with zineb
Complete solution Sprayed with Dithane Z-78 Sprayed with zinc sulfate Unsprayed Zinc deficient solution Unsprayed
Mean dry weight in grams (Roots and Shoots) 23.03 23.55 20.32 15.50
Least difference necessary for significance between means for odds of 1:19-1.05; for odds of 1:99=1.59
DISCUSSION The evidence from this investigation indicates that tomato plants are capable of utilizing the zinc in zineb when applied as a spray on the surface of the leaves. Stimulation of plant growth under the conditions of these experiments was due largely to the zinc in this compound. Plants grov/ing in zinc-deficient nutrient solution made an apparently normal growth when sprayed with zineb, even though in some instances, zinc-deficiency symptoms were present. In most instances plants sprayed with zinc sulfate were somewhat larger than those sprayed with zineb, though the differences were not significant. This tendency is best explained on the comparative solubility in water and, hence, the avaiiibility of the zinc in the two compounds. Zinc sulfate is water soluble with the zinc largely in an ionic form when in an aqueous solution.
other hand, zineb is relatively insoluble in water. The exact degree of ionization is unknown. Decreasing amounts of zinc were removed l>y washing with water until an apparent equilibrium was reached, then the amounts of zinc removed with each successive washing were quite constant (Table 1).
Even though trace amounts
of water soluble zinc were present in zineb, it is not known if this represents an impurity in the compound or is the result of ionization of zineb.
It has not been determined if plants utilize
only the ionized zinc or if they also can absorb and utilize the non-dissociated zineb molecule. It would be difficult to determine the effect on tomato plants
of the nitrogen, carbon and sulfur in zineb. Plants grown under conditions of this investigation would not likely be deficient in any element with the exception of those which might be intentionally omitted.
In any event, it is generally recognized that plants
require considerably larger amount of nitrogen than zinc. Although no exact comparison of the two can be made, a rough approximation can be derived from HoaglandTs solution in which the ratio is about 4000 parts nitrogen to 1 part zinc. If plants in these experiments had been able to utilize any element in zineb other than zinc, it is likely that the plants sprayed with this compound should have been larger than those sprayed with zinc sulfate. This condition did not exist. Further work with plants growing in environments different from those in this investigation might present a somewhat different picture of the utilization of organic zinc fungicides by tomato plants.
In the light of this investigation that demonstrate the
utilization of zinc in organic fungicides by tomato plants, it would be logical to expect other essential micro elements in organic fungicides might be taken up and utilized by plants.
SUMMARY Tomato seedlings were grown in a complete and a zinc-deficient Hoaglands solution.
Precautions were taken to maintain an environ-
ment suitable for optimum growth and to insure in some instanoes the development of zinc-deficiency symptoms. The effect of 95$ zineb (zinc ethylene bisdithiocarbamate) on plant growth was determined by spraying one group of plants with zineb and comparing them, after a period of 20 to 30 days, with another group that had been sprayed with zinc sulfate. It was shown that a zineb spray stimulates (1) zinc-deficient plants that v/ere not showing deficiency symptoms at time of spraying; (2) zinc-deficient plants that were showing deficiency symptoms at time of spraying; and
(3) plants that were growing in a
complete nutrient solution at time of spraying. It was concluded that under the conditions of this investigation, the zinc in the zineb was responsible for the growth stimulation of the tomato plants.
ir BIBLIOGRAPHY 1.
Berkeley, G. H., R. W. Thompson and J. K* Richardson. Potato spray test in Ontario. Amer. Potato Jour* 23: 285-290. 1946.
Chapman, H. D., A. P. Vanselow and George F, Liebig jr. The production of Citrus Mottle-leaf in controlled nutrient cultures. Jour. Agr. Res. (U. S.) 55: 365-378. 1937.
3. Durell, W. D. The effect of aeration on growth of the tomato in nutrient solution. Plant Physiol. 16: 327-341. 1941. 4. Ellis, N. K. potato production on Northern Indiana muck soils. Ind. Agr. Exp. Sta» Bull. 505: 1-19. 1948. 5. Fisher, R. A. Statistical methods for research workers. Oliver and Boyd. London, 5th Ed. 199-231. 1934. 6. Heuberger, J. W. Yield response of vegetables sprayed with dithiocarbamate fungicides. Abstr. phytopath. 38: 576. 7. Hoagland, D. R. and D. I. Arnon. The water culture method for growing plants without soil. Calif. Agr. Exp. sta. Circ. 347: 1-39. 1938. 8.
, w. K. Chandler, and P. L. Hibbard* Littleleaf or rosette of fruit trees. V. Effect of zinc on the growth of plants of various types in controlled soil and water culture experiments. Amer. Soc. for Hort. sci. 35: 131-141. 1935.
9. Hoyman, William G. The effect of zinc-containing dusts and sprays on the yield of potatoes. Amer. Potato Jour. 26: 256-263. 1949. 10*
Klotz, L. J. and J. G. Johnston. Symptoms of nutrient deficiences and excesses in citrus. (Abstr.) phytopath. 31; 860. 1941.
11. Palmiter, D. H. The effects of Fermate on the yield of Mcintosh apples. (Abstr.) Phytopath. 39: 18. 1949. 12.
Sandell, E. B. Zinc. In Colorimetric Determination of Traces of Metals. 487 pp. Interscience publishers, Inc. New York, N. Y. 1944. Steinberg, R. A, Nutrient-solution purification for removal of heavy metals in deficiency investigations with Aspergillus niger. Jour. Agr. Res. (U. S.) 51: 415-425. 1935,
Stout, P. H* and D. I. Arnon* Experimental methods for the study of the role of Cu, Mn, and Zn in the nutrition of higher plants. Amer. Jour. Bot. _26: 144-149. 1939.
15. Vlamis, J. and X* R* Davis. Effects of oxygen tension on certain physiological responses of rice, barley and tomato. Plant Physiol. 19: 33-51. 1944*
VITA The author was born in San Diego, California on September 13, 1918. He attended the elementary schools of San Diego and Alhambra. California.
In 1938 he graduated from Alhambra High School and
entered Pasadena Junior College. He graduated from Pasadena Junior College in 1940 and entered the University of California at Davis. He left the University of California to be employed by the state Department of Agriculture as a Plant Quarantine Inspector* He entered the Army of the United States in 1942 and was honorably discharged in 1945. In February 1945 he entered the University of Illinois and received a B. S. degree in Entomology in September 1946 at which time he entered the Graduate College. He took up graduate work in plant Pathology and was employed as a Research Assistant in Plant Pathology, receiving the ph. D. degree in February 1950*
The author is a member of Sigma Xi, Phi Sigma and the
American phytopathological Society*