'EHE EFFECT OF 2, 4-DICH'LGRPHENOXYACETIC ACID ON THE UFTAKE AND DISTRlBifiTlON OF CALCEUM AND PHOSPHGRUS EN QARLEY Thai: for flu Dogma :2? Ph. D. MECL‘ilfiAN STATE UNEV§RS§TY Lawrence 6‘3. Smifi'h 195:9 -. .Nwfi ....... MM This I. 16 certify, that the I thesis entitled THE EFFECT or 2,h-DICHLORPHENOXYACETIC ACID ON THE UPTAKE AND DISTRIBUTION or CALCIUM AND Paosmoaus IN HARLEY presented by LAWRENCE B. SMITH has been accepted towards fulfillment of the requirements for Eh. Do degree in Em Crops [ .4/41 , :f’Z 4,414/2k, Major professor / /., ~ Date / filé 1‘3 f/ L I B R AR Michigan State University 0-169 PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE E H _ i MSU Is An Affirmative Action/Equal Opportunity Inuitutlon 4t ”I .. .‘ _ . “a .' . 7? EFFECT OF 2,h-DICHLORQPHENOXYACETTC ACID ON SEEDLING DEVELOPMENTe AND UPTAKE AND DISTRIBUTION OF CALCIUM AND PHOSPHOHUS IN HARLEY By K "r LAWRENCE H.“ SMITH A THESIS Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Farm Crops Year 1959 f. 3 \ ' . U \ i 3-; (I W (W 'x 1‘ \-~.; (“‘- 2" LAWRENCE H. SMITH A study was made on the effect of 2,hrD applied as a pre-emsrgence spray and at the three- and five-leaf stages of barley upon the uptake and distribution of calcium and phosphorus. It was found that the 2,hrD applied as pre-emergence spray signifi- cantly reduced the dry weight, and ash, calcium and phosphorus content. Radiograms indicated a failure of the plants treated with 2,er to trans- locate calcium!"5 and phOSphorus32 to the above ground portion of the plant. However, there was no discernible effect of 2,bsD upon the distribution of these two elements within the plant. Histological sections of root tissue revealed extensive prolifera- tion of cells and lateral root formation initiating from the pericycle in all plants treated with 2,bPD. hS and calcium Microradiograms disclosed the fact that both phosphorus32 'were concentrated in the proliferated lateral root areas of those plants treated with 2,h-D. Two,er applied to barley at the three-leaf stage also effected a significant reduction in dry weight and ash, calcium and phosphorus content of the treated plant, as compared to the check. However the dry weight, ash, phosphorus and calcium content of the plants treated with 2,LPD, expressed as percentage of the check plants, was greater than that found to occur in the plants treated with a pre-emergence spray. A failure to translocate phosphorus32 and calciumh5 from the roots to the above ground portions was also observed in those plants treated at the three-leaf stage. Barley treated with 2,bPD at the five-leaf stage showed no significant effects of the 2,hrD upon the dry weight, ash, calcium and phosphorus content or distribution of calcium)"5 and phosphorus}2 within the treated plants. It was found that the greatest effect of 2,hrD upon the calcium and phosphorus content in barley occurred in the pre-emergence application 'with the least effect at the five-leaf stage with the three-leaf application being intermediate in response. It is suggested that 2,er directly affects the uptake and transloca- tion mechanism in barley. ACKNOWLEDGMENT The author wishes to express his sincere appreciation to Dre. C. M. Harrison and R. S. Bandurski for their advice and counéel during the course of this investigation. He is especially indebted to Dr. Bandurski for the procurement of radioisotopes and the use of his laboratory facilities and equipment. Grateful acknowledgment is also due to Dr. E. J. Hanna and staff for their assistance and counsel in the chemical assays required for this study. INTRODUCTION The advent of chemical weed control has greatly essed.man's endless struggle to eliminate competition by undesirable plants‘with those spe- cies which he immediately wishes to cultivate. Of the many chemicals available to the producer, none is used more often or mere carelessly than 2,hpdichlcrophenoxyacetic acid (2,b9D). Its selectivity in destroying broad-leaved weeds in preference to many narrowbleaved species renders it especially valuable in the production of small grains. However, little is known of its mode of action or inr fluence upon the metabolism of the small grain plant. Recent experiments studying the effect of 2,hrD upon nutrient up- take of broad-leaved plants hare indicated that there is a decided in- fluence upon nutrient uptake and specifically upon certain ions in preference to others. It is the purpose of this study to determine the influence of 2,beD on the uptake and distribution of calcium and phosphorus in barley treated at various stages of plant growth. REVIEW OF LITERATURE Fang and Buttsal), utilising carboxyl (Cm) labeled 2,Lr.-D, studied the absorption and translccation of this compound in corn and wheat. Using bean plants for comparison, it was found that 2,Lr-D was absorbed by the monocotyledons, but at a slower rate than that exhibited by the bean plants. The translocation of Cm to the apical regions of corn and wheat plants was very slow. There appeared to be a block in young plants at the intercalary meristem of the monooot stems and leaves. Fmiberg and Clsrk(5) found that when soybean plants were treated with 2 ,Lr-D the absorbing capacity of the roots was apparently changed, as indicated by the failure of treated plants to increase in total nitro- gen. Stahler and Whitehead( 12) reported that when sugar beet leaves were erroneously sprayed with a solution of 2,14-D, the nitrate content in- creased. In studies on the effect of 2,14-D on phosphorus content in cranberry bean plants by Banner, Rebstock and Sillw) , the total amount of phos- phorus in the leaves of the treated plants was lower than that of the non- treated plants. In stem tissues, however, the results were strikingly different. The total phosphorus content of the stem tissue from plants treated with 2,1.r-D was higher than that of the non-treated plants. The phos- phorus content in the roots of the treated plants was not appreciably different from that found in the non-treated plants. Fang and Butts(3), with bean plants, found that in all cases, the 32 phosphorus activity of leaves from plants treated'with.2,hrD'was much less when compared to leaves of control plants, while the activities in either the stem or root from those two groups of plants showed no sig- nificant differences. The distribution of phosphorus32 in plants treated 'with growth regulator differed from that found in control plants. (7) Loustalot, Moria, Garcia and Pagan have evidence that 2,er pro- meted phosphate accumulation in the roots, leaves and stems of white beans after forty-eight hours. However, one week after treatment, the phosphorus content in roots had increased significantly and this coin- sided with a sharp decline of phosphorus in the leaves, indicating that it may have been translocated from leaves to roots. Excised roots of four day old wheat seedlings showed a marked inhi- bition of nitrate absorption by 2,hPD‘within three hours after treatment as described by Nance(1o). Further experiments with potassium chloride indicated that 2,hrD inhibition of ion uptake was not a specific nitrate effect. (15) ‘Weldon, Hemner and Bass , working with tobacco seedlings, reported that the taps of treated plants contained a lesser percentage of potassi- um, sodium and phosphorus and a greater percentage of boron and iron than did untreated plants. Roots of treated plants accumulated.more calcium and less copper than roots of untreated plants. There was little or no difference in the accumulation of calcium, copper, magnesium and zinc in the tops of the plants, nor in the accumulation of boron, potassium, manganese, magnesium and phosphorus in the roots of treated and untreated plants. Plant roots contained greater percentages of boron, capper, iron, magnesium,:manganese, sodium and potassium than taps, whether treated or untreated. Calcium accumulated to a greater amount in roots than in the teps of treated plants, although no differences were noted in un- treated plants. Potassium accumulated to a greater extent in tcps than in roots of untreated plants, while no differences were noted in treated plants. The content of phosphorus in the taps of the treated plants was much less than that of untreated plants. MATERIALS AND PROCEDURE In order to evaluate the effect of 2,)4—D on the uptake and distri- bution of calcium and phosphorus in the plant, the following techniques were employed: (1) Chemical analysis for calcium and phosphorus of above and below ground parts of the plants to determine the relative amounts and proportion of calcium and phosphorus within these two segments of the plant. (2) Histological sections to determine the effect, if any, of 2,14-D on various plant cells and tissues which might in some way affect uptake and/or distribution of calcium and phosphorus within the plant. (3) Radioactive isotopes, eg. calcium)"5 and phosphorus32, to determine the effect of 2,14-D upon the distribution of these elements within the plant and to evaluate the uptake of these ions over a short period of time. (h) Microradiograms, utilizing nuclear track plates, to trace the distribution of calcium)"5 and phosphorus? at the cellular level. The experiment consisted of five rates of application of 2,14-D; sero, one-quarter, one-half, one and two pounds of the sodium salt of 2,14-D per acre at three stages of growth: pre-emergence, three-leaf stage, and five-leaf stage, with four replications. In order to avoid the normal fluctuation of temperature, length of day, light intensity, humidity and etc., prevailing under general green- house conditions--all of which directly and indirectly affect the metaboliam of plants and thus their mineral nutrition--an attempt at con- trolled or at least of reproducible conditions was made. A.double unit seed germinator was selected as the basic unit for a controlled chamber, and a bank of twelve daylight florescent tubes and six incandescent bulbs provided the necessary light. These lights pro- vided an average of'nine hundred foot candles of light, measured four inches from the false floor of the unit. Day length was varied with a time clock and after preliminary investigations, a sixteen hour day was decided upon. To gain some control of temperature and humidity, cold tap water was continually circulated through the water jacket enclosing the germinator. This water averaged 63 degrees Farenheit throughout the dur- ation of the experiment. The walls of the germinator were colder than the rest of the area and a fan was added to recirculate the air and thus maintain a satisfactory uniformltemperature throughout the germinator, 'whioh varied from 78 degrees Farenheit plus or minus 2 degrees with the lights on to 68 degrees Farenheit plus or minus 2 degrees with the lights off. .A false floor consisting of one-quarter inch mesh hardware cloth over a‘wooden frame raised the plants one inch above the germinator floor, thereby allowing for pot drainage and circulation of air. The chamber is depicted in Figure l. Figure 1: Growth Chamber Seed of Kindred barley, a melting type spring barley, was obtained from foundation seedrstock. Seeds were selected on the basis of uniform- ity, apparent freedom from disease, blemishes and mechanical injury. Plastic containers with a capacity of one and one-half pints were used as ''pots" and #'7‘Wausan* silica send as the growth medium. The rate of 2,er used with each culture was inscribed on the containers and these were used for similar treatments in repeated experiments. The pots were cleaned after each experiment by a thorough detergent and securing powder brushing, followed by a nitric acid rinse and three distilled *American graded Sand Company, Chicago, Illinois water rinses. Initially the presence of 2,)4-D remaining in the pots was checked by the application of the final distilled water rinse to tomato seedlings; however, since there was no herbicide present, as measured by the tomato seedling response, it was felt that the cleaning treatment was effective in removing the 2,h-D and the seedling tests were discontinued. Holes were cut through the bottom of the plastic pots with a cork borer and glass wool was placed over the Opening to allow for drainage. Six seeds were planted at a depth of one-half inch and on germina- tion were thinned to four plants per culture. The planting was staggered so that all three stages of growth were treated with 2,)4-D at the same time. The plants were watered at two day intervals with a modified Hoagland's nutrient solution applied through a sprinkler head and thor- oughly flushed with distilled water twice a week. Prior to the application of the 2,14-D, rubber steppers were placed in the drainage holes of the pots to make a sealed container. The plants were then ranked according to vigor and general condition within each growth stage. The 2,LL-D was applied as an aqueous spray to the tops at forty pounds per square inch, utilizing the pressurized tank and gun in Figure 2e Figure 2: Spray Application Equipment Imsdiately after treatment the plants were watered with nutrient solution and again thereafter at two day intervals. Fourteen days after treatment the plants , in the three-leaf and five- leaf treatments, were harvested and the tops and roots separated. The plants from the pro-emergence treatments, however, were retained intact. The entire experiment was repeated four times in order to secure adequate material for chemical analysis. Chemical analysis for calcium and phos- (1) phorus were run, utilising standard A.0.A.C. methods , as modified by the Department of Agricultural Chemistry, Michigan State University. - 10 - In the tracer experiments, the plants were handled in the usual man- ner with twenty ,ucuries cf the tracer being added directly to the culture at the end of the fourteen day treatment period, separate plantings being required for both calcium)"5 and phosPhcrusaz. Plants utilised for radio- grams were harvested in the normal manner, pressed, dried and placed in contact with nc-screen xrray film for a period of seven days. The film was then developed. Sections of plant tissue for micrcradicgrams were taken at harvesttime and immediately frozen by placing in containers which had been previously stored upon dry ice. The sections were then frozen dry and embedded directly into paraffin in the apparatus pictured in Figure 3. Figure 5: Freeze-Drying Equipment - 11 - The tissue was sectioned, placed on slides, the paraffin removed in xylene and the preparation coated with celloidan. The slide was then taped to a one by three nuclear track plate for seven days, after which the nuclear track plate was developed. The remaining plants were then ground, ashed, and dissolved in con- centrated hydrochloric acid and made up to a valume of twenty-five milli- lliters. No millilliter aliquots were then counted to determine the radioactivity present. All counts obtained were extrapolated back to zero time to avoid decay corrections. EXPERIhENTAL RESULTS Experiment I - Pro-emergence Treatment with 2,1i-D External appearance. The general condition of the check plants were excellent and, in general, two new leaves had been produced during the fourteen day treatment period. However, in the plants treated with 2,h-D, a slight chlorcsis was evident as well as definite stunting of growth. As the rate of application of 2,h-D was increased, the severity of stunt- ing increased. At the two pound rate, the plants had made no new growth during the treatment period; whereas at the one-quarter and one-half pound levels, one new leaf had been produced. At the one pound level a new leaf was in the process of being produced, but was still in the rolled stage. The roots of the check plants were normal in development, whereas the roots fran plants treated with 2,h-D were short and thick with num- erous protuberances along the root. This reaction to 2,14-D increased in severity with increasing amounts of 2,14-D until at the two pound level the roots were, on the average, from one-half to one inch long with num- erous protuberances. From histological sections, it was determined that these protuberances were lateral roots which had been stimulated by 2,h-D. D31 weight. The stunting effect of 2,14-D upon barley plants when applied as a pro-emergence application was very evident from its influ- ence upon the dry weight of the plants as indicated in Table I. -13- TABLE I INFLUENCE OF 2.1.4) UPON DRY WEIGHT (IN MILLIGRAMS) 0F HARLEY Pounds of 2,114) Applied per Acre ROPBO‘tion 0e 00 0.25 0 I000 2e 00 I 1156-11 750-5 730-7 615.11 5111.14 II 11.03.? 670.11 668.2 637.9 631.11 III 1325.11 70h.6 6116.0 6118. 3 612.6 Iv 127L1. 3 736.8 671.0 701.6 636.2 new 1290.0 710.5 67900 6me8 @5014- R.E. (5% . 8.57 P = 5 c.v. : 7.2% (1% = 12.111 The data indicates that upon the application of 2,1.1-D, the dry weight decreased significantly from the control. Within the 2,).1-D treat- ments, the two pound application significantly lowered the dry weight when compared to the one-quarter pound rate. Although a trend of decreasing dry weight with increasing rate of application of 2,14-D was evident, there was no significant difference between the one-half, one and two pound rates of 2,11-D and its effect upon dry weight. 5111. The data in Table II indicates that the ash content, as well as dry weight, decreased upon the application of 2,14-D. TABLE II was EFFECT OF 2,)4-0 UPON ASH CONTENT OF 31mm a 11 ti p.00 2.00 °" °‘ “as; g was. 7° as- ms- 7° 115- 7° I 137-7 16-2 78.3 10-7 514-0 7J4 58-9 9-6 50.6 9.11 II 238.2 17.0 68.9 10.2 1.8.0 7.2 63.3 9.9 1.9.0 7.8 III 205.8 15.5 59.2 8.11 58.6 9.1 77.8 12.0 146.11 7.6 IV 198e6 15e6 103.14 meo 66e6 9e9 70e2 10.0 1.18.9 7e? Mean 207.6 16.1 77.5 10.9 56.8 8.1.1 67.6 10.1. 1.18.7 8.1 ReEe (5% 3 214-035 P = 5 CeVe I 5e36% (1% = ital-L9 The ash content, calculated as percentage of dry weight and.milli- grams of total ash, decreased upon application of the 2,hrD when compared to the check except at the one pound rate where a non-significant increase in both percentage and total milligrams was evident. .As in the dry weight, the decreases in ash content of plants caused by the 2,er treatments ‘were significantly different from that of the control. The ash content of plants treated with the one-quarter pound rate was significantly high- er than the two pound rate, with a non-significant trend of decreasing ash content with increasing rate of 2,hrD over the'whole experiment. Calcium. The data presented in Table III indicates the influence of 2,er upon the calcium.content of barley when applied as a pro-emergence treatment. TABLE III THE EFFECT OF 2,174) UPON 0110qu CONTENT OF BARLEY f FEM: of 2,1131) Applied per Acre . 0.00 0.25 0.50 300 2.00 Replication