TRIAZINE HERBICIDES AND THE MINERAL NUTRITION 0F CONIFERS Thesis for the Degree of Ph. D. MECHTGAN STATE UNiVERSITY BOBBY JOE CONNER 1969 MlChlfg; ...y— This is to certify that the thesis entitled TRIAZINE HERBICIDES AND THE MINERAL NUTRITION OF CONIFERS presented by Bobby Joe Conner has been accepted towards fulfillment of the requirements for & degree in m Major professor Date February 7, 1969 LIB]? {R l‘ : Statc Lllix Slty ~~P (2 t}. me nee 11nd 43. ABSTRACT TRIAZINE HERBICIDES AND THE MINERAL NUTRITION OF CONIFERS BY Bobby Joe Conner In controlled environment experiments, low level soil applications of 2—chloro-4,6 bis (ethylamino)-s-tria- zine (simazine) and 2—chloro-4—ethylamino-6-isopropylamino- s-triazine (atrazine), and their interaction with ammonium and nitrate sources of N, influenced the growth and foliar N nutrition of newly germinated slash (Pinus elliottii Engelm.)and loblolly pine (giggg E3393 L.) seedlings. Herbicidal rates of pre-emergence applied simazine and atrazine did not significantly influence the foliar mineral nutrition of Scotch pine (Eiggg sylvestris L.), white spruce (Eigga glauca (Moench) Voss), or balsam fir (£2325 balsamea (L.) MillJ nursery transplants, except for the foliar concentration of Mg. Simazine (0.05 - 0.8 ppm) applied without supple- mental N increased the foliar N concentration (% N in needles) of 10- and lZ-week-old slash pine seedlings grown under controlled environment. The maximum increase was 43.8 percent at the highest rate of simazine. Simazine 0f th the gre ”as herb Bobby Joe Conner applied at 0.8 ppm with both ammonium and nitrate sources of N (84 ppm N) increased the foliar N concentration of seedlings more than the N sources applied alone. Green and dry foliage weights were increased by simazine treat- ments of 0.4 ppm, but decreased by 0.8 ppm. Soil applied atrazine (0.4 ppm) increased the fo- liar N concentration of ll—week-old slash and loblolly pine seedlings. However, atrazine (0.1 and 0.4 ppm) treat- ments depressed both the growth and foliar N accumulation of loblolly pine seedlings. In the greenhouse, soil applications of 0.5, 1.0, and 2.0 ppm simazine increased the foliar N concentration of 16-week-old slash pine seedlings. The maximum increase was 91 percent at the highest simazine level. This increase was greater than where 84 ppm N of either ammonium or ni- trate sources of N was supplemented in the nutrient solu- tion. tThe foliar N concentration of seedlings treated with simazine (0.5 - 2.0 ppm) and both levels (28 and 84 ppm N) of either ammonium or nitrate N was greater than where these N sources were applied alone. Simazine applied at 0.5 and 1.0 ppm did not affect the foliar N accumulation of seedlings. Decreases in both green and dry seedling weights were found when simazine was applied at concentrations of 0.5 to 2.0 ppm. When a herbicidal rate of simazine (2.0 ppm) was applied to slash Pine seedlings, the significant increase in foliar N Bobby Joe Conner concentration was primarily a function of growth reduction. Simazine treatment depressed root weight more than foliage weight. Simazine treatment at 2.0 ppm increased foliar nitrates. No differences were found in foliar nitrates between ammonium and nitrate treated seedlings, either alone or with simazine. This implies that possibly the ammonium ion was as effectively utilized with simazine as was the nitrate source of N. In the field, the lower rate of soil applied atra- zine (2.25 kg/ha) increased the foilar Mg concentration of Scotch pine, white spruce, and balsam fir over the con- trol. Simazine applied at herbicidal rates (4.50 and 9.00 kg/ha) did not significantly alter the foliar concentration of any element for either species the first growing season. Neither did simazine treatment affect the foliar N concen- tration of seedlings the second year. A greater herbicidal injury and mortality occurred in white spruce than the other species. Since the phyto— toxic effect of triazine treatment on this species appeared syreater when the herbicides were applied alone, it is pos- sible that supplemental N to the triazine plots might have cnounteracted herbicide phytotoxicity. Ammonium nitrate applied to simazine plots signifi- <3antly decreased the foliar concentrations of P, Ca, Cu, Zn, and B for various species. Supplemental N applied with Bobby Joe Conner atrazine significantly decreased foliar P and B, and in- creased foliage Mg and Mn for some species. Ammonium nitrate applied with simazine and atrazine the first year did not affect the foliar concentrations of N, K, Na, Ca, Fe, or Al for either species. Neither did N applied to simazine plots affect foliar Mg or Mn, or N applied with atrazine affect foliar Cu or Zn of any seedlings. TRIAZINE HERBICIDES AND THE MINERAL NUTRITION OF CONIFERS BY Bobby Joe Conner A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Forestry 1969 ACKNOWLEDGMENTS The author is indebted to the Chairman of his Guidance Committee, Dr. D. P. White, for his sustained encouragement and guidance throughout the course of this study. He is also grateful to the other members of the Guidance Committee--Drs. H. D. Foth, J. W. Hanover, and S. K. Ries--for their valuable assistance and suggestions during the course of this work. The author extends his appreciation to Dr. C. E. Cress for his guidance and assistance with the statistical aspects of the project. Acknowledgment is made to Geigy Agricultural Chem- icals, Ardsley, New York, for their financial support and continued interest in this study. ii .J L ‘ y. 'x 1;“ *9 VITA Bobby Joe Conner Candidate for the Degree of Doctor of Philosophy Final Examination: February 7, 1969 Guidance Committee: H. D. Foth, J. W. Hanover, S. K. Ries, and D. P. White (Major Professor) I)issertation: Triazine Herbicides and the Mineral Nutrition of Conifers Outline of Studies: Major subjects: Forest Soils Minor subjects: Herbicides, Plant Nutrition Biographical Items: Born January 28, 194% Kings Mountain, North Carolina Home town: Wilmington, North Carolina Undergraduate Studies: Wilmington Junior College, 1958-1960 A.A. Associate in Arts, 1960 North Carolina State University, 1960-1963 B.S. Forestry, 1963 B.S. Soil Science, 1963 Graduate Studies: North Carolina State University, 1964-1966 M.S. Soil Science, 1967 Michigan State University, 1966-1969 Ph.D. Forestry, 1969 Experience: Professional Baseball Pitcher, Washington Senators (Middlesboro, Kentucky), 1962: U. S. Coast Guard Re- Serve, June-November, 1963; Assistant Area Forester, Container Corporation of America (Starke, Florida), January—September, 1964; Graduate Research Assistant, North Carolina State University (Raleigh, North Caro- lina), 1964-1966; Graduate Research Assistant, Michi- gan State University (East Lansing, Michigan), 1966 to date. Member: Xi Sigma Pi (Secretary-Fiscal Agent, 1967-1968) Zklpha Zeta Society of American Foresters Soil Science Society of America iii TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . . . . . . . . Vi LIST OF FIGURES . . . . . . . . . . . . . . . . . . Viii LIST OF APPENDICES . . . . . . . . . . . . . . . . . x CHAPTER I . INTRODUCTION . . . . . . . . . . . . . . . . 1 II. LITERATURE REVIEW . . . . . . . . . . . . . 4 Background . . . . . . . . . . . . . . Triazine Herbicides--Plant Growth and Mineral Nutrition . . . . . . . . . . . 5 Triazine Absorption, Translocation, and Metabolism . . . . . . . . . . . . . 7 Triazine Herbicides--Mechanism of Action . . . . . . . . . . . . . . . . . 10 III. METHODS OF INVESTIGATION . . . . . . . . . . 15 Growth Chamber . . . . . . . . . . . . . . 15 Experiment 1- I O O O O C O O O O C O O O 17 Experiment 2 . . . . . . . . . . . . . . 22 Experiment 3 . . . . . . . . . . . . . . 23 Greenhouse o I I I I I I I I I I I I I I I 27 Field I I I I I I I I o I I I I I I I I I 31 IV. RESULTS AND DISCUSSION . . . . . . . . . . . 38 Growth Chamber . . . . . . . . . . . . . . 38 Experiment 1 O O D O O D C O C C I O C C 38 Experiment 2 . . . . . . . . . . . . . . 44 Experiment 3 . . . . . . . . . . . . . . 53 iv CHAPTER Greenhouse . . . . . . . . . . . . . . . Foliar Nitrogen Content . . . . . . . Foliar Nitrates . . . . . . . . . . . Simazine and Growth Reduction . . . . Field I I I I I I I I I I I I I I I I I Foliar Mineral Nutrition . . . . . . . Interaction of Nitrogen and Herbicide Treatments with Mineral Nutrition . Mulching and Mineral Nutrition . . . . V. SUMMARY AND CONCLUSIONS . . . . . . . . . First Growth Chamber Study-~Slash Pine with Simazine . . . . . . . . . . . . Second Growth Chamber Study--Slash Pine with Simazine . . . . Third Growth Chamber Study--Slash and Loblolly Pine with Atrazine . . . . . Greenhouse Study--Slash Pine with Simazine . . . . . . . . . . . . . . . Foliar Nitrogen Content . . . . . . . Simazine and Growth Reduction . . . . 'Field Study—-Simazine and Atrazine at Herbicidal Rates . . . . . . . . . . . Foliar Mineral Nutrition . . . . . . . -Si1vicu1ture Implications . . . . . . . LITERATURE CITED I I C I O I I O . I I C I l I O 0 APPENDIX I I I I I I I I I I I I I I I I I I I I I Page 55 55 64 7O 7O 78 83 84 84 85 86 87 87 88 89 89 90 93 98 TABLE 1. 10. 1].. LIST OF TABLES Nitrogen and simazine treatments Experiment 1 . . . . . . . . . Preparation of nutrient solution ments used in Experiment 1 . Basic nutrient solution . . . . Nitrogen and simazine treatments Experiment 2 . . . . . . Preparation of nutrient solution ments used in Experiment 2 . . Nitrogen and atrazine treatments Experiment 3 . . . . . . . . . Preparation of nutrient solution ments used in Experiment 3 . . Nitrogen and simazine treatments Greenhouse Experiment . . . . Preparation of nutrient solution used in treat- used in treat- used in treat- used in treat- ments used in Greenhouse Experiment . Fertilizer and weed control treatments used in Field Experiment . . . Significance of experimental factors on the foliar nitrogen content and foliar percent dry weight of slash pine seed— lings grown in the growth chamber for 10 weeks (Experiment 1) Significance of experimental factors on the green and dry weights of slash pine seedlings grown in the growth chamber for 10 weeks (Experiment 1) . vi Page 18 19 20 23 24 26 26 29 3O 34 39 43 II TABLE 13. 14. 15. l6. 17. 18. 19. 20. 21. Significance of experimental factors on the foliar nitrogen content and foliar percent dry weight of slash pine seed- lings grown in the growth chamber for 12 weeks (Experiment 2) . . . . . . . . Significance of experimental factors on the green and dry weights of slash pine seedlings grown in the growth chamber for 12 weeks (Experiment 2) . . . . . . Significance of experimental factors on the foliar nitrogen content and foliar percent dry weight of slash and lob- lolly pine seedlings grown in the growth chamber for 11 weeks (Experiment 3) . . Significance of experimental factors on the green and dry weights of slash and loblolly pine seedlings grown in the growth chamber for 11 weeks (Experiment 3) . . . . . . . . . . . . . . . . . . . Significance of experimental factors on the foliar nitrogen content, foliar per- cent dry weight, height, and stem diam- eter of slash pine seedlings grown in the greenhouse for 16 weeks . . . . . . Significance of experimental factors on the green and dry weights of slash pine seedlings grown in the greenhouse for 16 weeks . . . . . . . . . . . . . . . . Changes in the foliar mineral nutrition of field planted SCOTCH PINE from am- monium nitrate and weed control treatments I I I I I I I 'I I I I I I I I ‘ Changes in the foliar mineral nutrition of field planted WHITE SPRUCE from am— monium nitrate and weed control treatments . . . . . . . . .,. . . . . . Changes in the foliar mineral nutrition of field planted BALSAM FIR from am- monium nitrate and weed control treatments . . . . . . . . . . . . . . . vii Page 45 50 53 56 57 65 71 72 73 FIGURE 1. 2. LIST OF FIGURES General view of 9-week-old slash pine seedlings in the growth chamber . . . . . General view of Field Experiment at the Tree Research Center . . . . . . . . . . Relationship between simazine application rate and the top dry weight and N con- tent of 12-week—old slash pine seedlings grown in the grOwth chamber without sup- plemental N (Experiment 2) . . . . . . . Increase in foliar N over the control for slash pine seedlings treated with com- binations of simazine and nitrate or ammonium sources (ppm N) and grown in a growth chamber (Experiment 2) . . . . . . Relationship between simazine application rate and the N content of lG-week-old slash pine seedlings grown in the green- house without supplemental N . . . . . . Changes in the foliar N concentration (%) and N uptake (mg N/top) from the con- trol for slash pine seedlings grown in the greenhouse and treated with combina- tions of simazine and nitrate or ammonium sources (ppm N) . . . . . . . . . . . . . Relationship between simazine application rate and the decrease in green and dry weights from the control for slash pine seedlings grown in the greenhouse with- out supplemental N . . . . . . . . . . . Effect of simazine application rate on the top and root growth of 16-week-old slash pine seedlings grown in the green- house without supplemental N . . . . . . viii Page 16 32 46 48 58 60 61 66 FIGURE Page 9. Effect of simazine and nitrate and am— monium treatments on the top growth of 16-week-old slash pine seedlings grown in the greenhouse . . . . . . . . . 68 10. Effect of simazine and nitrate and am- monium treatments on the top and root growth of 16-week-old slash pine seedlings grown in the greenhouse . . . . 69 ix APPENDIX A. LIST OF APPENDICES Effect of simazine and nitrogen treat- ments on the foliar nitrogen content and growth of slash pine seedlings grown in the growth chamber (Experi- ment 1, Tables I-III). . . . . . . . . Effect of simazine and nitrogen treat- ments on the foliar nitrogen content and growth of slash pine seedlings grown in the growth chamber (Experi- ment 2, Tables IV—VI) . . . . . . . . Effect of atrazine and nitrogen treat- ments on the foliar nitrogen content and growth of slash and loblolly pine seedlings grown in the growth chamber (Experiment 3, Tables VII-IX) . . . . Effect of simazine and nitrogen treat- ments on the foliar nitrogen content and growth of slash pine seedlings grown in the greenhouse (Tables X-XIII) Effect of nitrogen and weed control treatments on the foliar mineral nutrition of field planted Scotch pine, white spruce, and balsam fir (Tables XIV-XIX; Analysis of vari- ance--Tables XX-XXI) . . . . . . . . . Page 98 101 104 107 111 CHAPTER I INTRODUCTION Triazine herbicides have certain characteristics such as selectivity, persistence, low hazard and economy that have promoted their wide acceptance as herbicides in many forestry operations. Early forestry research in chemical weed control in this country dates back to at least the 1920's when Toumey and Korstian (1942) were experimenting with sul- furic acid and copper and zinc sulfates. During the period preceding the second World War the scarcity of labor and successful use of volatile oils for weed control in vege— table crops resulted in the introduction of these materials for use in forestry (Eliason, 1954). In recent years, researchers have found 2-chloro-4,6 bis (ethylamino)-s-triazine (simazine) and 2-chloro-4-ethyl— amino-6-isopropylamino-s-triazine (atrazine) very effective in eliminating a broad spectrum of grasses and broad—leaved weeds in forestry operations. These herbicides are classi- fied as mono-chloro triazines and are the most important triazines from the standpoint of commercial use. Simazine and atrazine are widely used for pre- and post-emergence weed control in forestry operations. The selectivity and long residual action of simazine are desirable traits which lend themselves well to tree plantation establishment and rnaintenance. However, many species do not show complete tolerance to this chemical, particularly at a young age, and its use is not recommended on 1-0 seedlings (Winget. g£_gl., 1963). Although the triazines have only very lim- ited application in tree nursery seedbeds, they have been used for weed control in nursery transplant areas. The major uptake of triazines is by root absorp— tion and therefore good soil moisture is essential to insure their effectiveness. Simazine has a water solubility of 5 ppm and atrazine 70 ppm (Gysin, 1962). The leaves of plants are unable to absorb measurable amounts of simazine while atrazine shows a considerable effect through the leaves. Affected plants first show chlorosis of the foliage, followed by death of the affected tissue, and with seedlings, death of the plant. Vigorous tree growth following the use of these compounds in tree plantations has been generally attributed to a reduction in competition (White, 1960). There is now evidence that other physiological processes may be involved in growth stimulation by these compounds (Ries gt_al., 1967). In the early 1960's, White (1960) observed improved growth and foliar color following triazine treatment in coniferous plantations. Research by Ries et a1. (1963) with fruit trees and agricultural crops seems to substantiate a "fertilizer effect" following the control of weeds with triazine compounds. The effect manifests itself in im- proved plant vigor, foliage color, and N nutrition over and above that observed with similar weed control by me- chanical means or other types of herbicides. Other workers have reported a "bonus effect" of growth stimulation from triazine treatment (DeVries, 1963; Ries and Gast, 1965; Tweedy and Ries, 1967). This study attempted to define the growth stimulat- ing effect of triazine treatments on coniferous tree species grown under growth chamber, greenhouse, and field conditions. Under controlled environment, the growth and nitro- gen nutrition effects from low level applications of sima— zine and atrazine and their interactions with nitrogen were measured on slash (Pinus elliottii Engelm.) and loblolly pine (Pinus Eagdg L.) seedling. In the field, herbicidal levels of triazines were applied both with and without supplemental nitrogen in attempt to detect their influence on coniferous plant nutri- tion. Three species, Scotch pine (Pinus sylvestris L.), white spruce (Picea glauca (Moench) V055), and balsam fir (Abiesbalsamea(In) Mill), representing three different genera, were used. CHAPTER II LITERATURE REVIEW Background Triazine compounds were first synthesized in Swit- zerland in 1952. No group of herbicides has been more extensively studied or more widely used. The structure of triazine herbicides is based on cyanuric chloride, the starting material from which these compounds are made (Gysin, 1962). Simazine and atrazine are classified as mono-chloro-s-triazine derivatives and are synthesized by replacing two of the three chlorine atoms of cyanuric chloride with two aliphatic amino-groups. The only structural difference between these two compounds is that atrazine has one ethylamino and one isopropylamino- substituent, where simazine has two ethylamino-substituents. However, this small change in structure causes a drastic difference in the physical and biological properties of simazine and atrazine (Gysin, 1962). The high biological effectiveness of these substituted symmetrical triazines against a wide spectrum of plants has aroused much interest in their use. While they have been found useful as soil 4 sterilents, they are particularly valuable as selective herbicides on a number of crops. Triazine_Herbicides--Plant Growth and MineraI'Nutrition Triazine herbicides have the unusual property of influencing the growth and N content of some plants when applied at sub-toxic levels (Ries et al., 1967). Ries gt El. (1963) reported that both peach (Prunus sp.) and apple (M3135 sp.) trees treated with simazine had a higher leaf N and more growth than trees where weeds were controlled by other means. Bartley (1957) found that corn (Egg gays L.) treated with rates of simazine up to 17.9 kg/ha was greener and taller than where no simazine was used. Tweedy and Ries (1967) found simazine applications increased the dry weight and N content of corn grown at sub—optimal tem- perature in low levels of nitrate. They found nitrate reductase activity in corn growing on sub-optimal levels of nitrate to increase with simazine concentration. Gram- 1ich and Davis (1967) reported from field studies that corn and Johnsongrass (Sorghum halepense) treated pre—emergence with atrazine were smaller and contained a higher percentage N than untreated plants. Plants treated with high rates of atrazine (17.9 kg/ha) had less N uptake (mg N/plant) than the untreated controls. Freney (1965) reported applications of l to 5 ppm simazine to the soil of greenhouse pots, in— creased the dry matter yields and N uptake in corn only when additional N was applied to the soil. He found sima- zine applied at 0.06 ppm in solution culture increased the yield of corn tops by 36 percent, the uptake of N by 37 percent, P by 25 percent, Mg by 24 percent, and K by 41 percent. Simazine treatment had no effect on the yield of roots. DeVries (1963) found that simazine applications generally reduced the dry weight of corn and Monterey pine (Pinus radiata D. Don), but increased the N content of the shoot. Kozlowski and Kuntz (1963) reported that pre- emergence soil spray of simazine and atrazine at 1.1 kg/ha to red (Pinus resinosa Ait.) and white pine (Pinus strobus L.) seedlings in greenhouse flats cause more injury and re- duction in dry weight than a delayed herbicide application. Conner and White (1968) found low level soil applications of simazine (0.8 ppm) increased the foliar N content of slash pine seedlings grown in the growth chamber. The dry vveight of seedling tops was not changed by simazine appli- cations but a reduction in dry root weight was observed. Ries gt_§1. (1967) reported that rye plants (Secale (zereale L.) receiving a 0.10 uM (0.02 ppm) application of simazine contained 45 percent more water-extractable protein Iper plant than controls. The fresh weight per plant was not cfluanged, but there was a progressive decrease in percent dry vnaight with increasing simazine concentration. Simazine tJreatments increased the respiration rate more than 10 per— ceant without affecting the respiratory quotient. Increased fl respiration may account for the decrease in dry weight. The increased rate of respiration combined with the lower rate of carbohydrate accumulation suggests a greater energy requirement in simazine treated plants. They found simazine applications increased protein accumulation in plants grown with nitrate, but not in plants grown with ammonium as a N source. This response implies that the action of simazine involves some step in the reduction of nitrate to ammonia, or perhaps earlier in the uptake of nitrate but no step in the further conversion of ammonia. The fact that the effect of simazine decreased as the ni— trate level approached the optimum suggests that simazine treatment enhances nitrate utilization by the plant (Ries et al., 1967). Gramlich and Davis (1967) found in nutrient culture studies with corn that 4 and 8 ppm of atrazine added to the nutrient solution increased the percentage foliar nitrates without an accompanying decrease in free ammonia as might be expected if atrazine inhibited the enzymatic conversion of nitrate to ammonia. They also concluded that the increased percentage total N of atrazine treated plants was primarily due to increased nitrate. (triazine Absorption, Translocation, and Metabolism Various workers have shown by the use of radioactive- Ilabelled triazines that the chloro-triazines are absorbed through the roots and migrate to the leaves through the xylem in the transpiration stream (Davis gt_al., 1959; Sheets, 1961). Davis 3E_31. (1959) have shown 14C-labelled simazine applied in nutrient culture moved from the roots to leaves of cucumber (Cucumis sp.) plants in less than 30 minutes. Simazine moved rapidly into the roots but almost no absorption occurred through intact leaves, however, simazine did enter when the cuticle was broken. Ragab 22 3;. (1961) found from labelled l4co2 studies with corn and cucumber seedlings that more radioactivity was present in roots than leaves and stems, and more activity from cucum- ber extracts than from corn. Sheets (1961) observed that 14C-ring labelled simazine was absorbed by both oat (Aygna sativa L.) and cotton (Gossypium hirsutum L.) seedling roots and the 14C was distributed throughout seedling oats within three hours. Absorption and translocation of sima- zine upward through the treated seedlings was greater at 37°C than at 26°C, and with temperature constant at 37°C, absorption and translocation were greater in plants which were grown in a 66 percent relative humidity. The 14C in leaves of oats was approximately three times that in the leaves of cotton when both species were treated alike. They found the amount of 14C in leaves to be dependent on transpiration and the simazine molecule was apparently changed more rapidly in roots than in leaves. Montgomery and Freed (1961) demonstrated using 2.25 and 9.0 kg/ha of l4 . . C-labelled SimaZlne and atrazine that the total concen- tration of 14C in corn plants decreased after about 30 days. They confirmed these findings with ion exchange and paper chromatography studies by showing that only trace amounts, if any, of these materials remained unchanged in the plant. In later work, Montgomery and Freed (1964) concluded that although there was a good correlation be- tween resistance and extent of metabolism, even the highly susceptible plants have a limited capacity for degrading these chemicals. Negi gt_§l. (1964) studying the metabolism of 14C-labelled and unlabelled atrazine in soybeans (Glycine m3§_Merr.), beans (Phaseolus sp.), and oats (susceptible), peanuts (Arachis hypogaea L.) and cotton (intermediate), and Johnsongrass, grain sorghum (Sorghum vulgare), and corn (resistant), found atrazine residues in all plants 11 days after a pre-emergence application of 1.12 kg/ha. Unaltered atrazine found in the plants was roughly correlated with plant susceptibility. All plants converted some atrazine to hydroxy-atrazine and the amount of this material formed was somewhat correlated with resistance as the three resis- tant species converted at least twice as much atrazine to hydroxy-atrazine as did the susceptible soybeans and oats. Although triazines have been found to be absorbed by plants rather rapidly, only a small portion of the herbicide applied is actually taken up by the plant. For example, if maize seedlings were grown in nutrient solution containing 2 ppm 10 Simazine, between 0.25 and 0.75 ppm simazine would prob- ably be recovered in the leaves (Gysin, 1962). Triazine Herbicides--Mechanism of Action Once an absorbed triazine molecule reaches the leaves, it enters the living cell and causes a drastic change in theplantsnetabolism in the presence of light. Ashton gt_al. (1960) found with kidney beans (Phaseolus vulgaris L.) that treatments of simazine and related tria- zines drastically inhibited CO fixation in the light. The 2 degree of inhibition increased with higher herbicide concen- tration and longer exposure time. This is undoubtedly an important factor in the phytotoxic characteristics of the triazine compounds. Zweig and Ashton (1962) showed that 0.1 ppm of atrazine applied to kidney beans did not influence CO2 fixation, while 1 ppm caused 90 percent inhibition after 2 days. From chromatography work, these authors showed that high concentrations (10 ppm) of applied atrazine greatly changed the synthesis of various organic acids in kidney bean leaves. While glycine practically disappeared from the leaves a great increase of aspartic acid formation occurred. These investigators are of the opinion that atrazine affects products of the tricarboxylic acid cycle and interpret the disappearance of sucrose and glyceric acid and the forma- tion of aspartic and malic acid as a result of phosphoenol- pyruvic carboxylase activity. In addition, Roth (1958) has 11 Shown that plants with high peroxidase activity are rela- tively resistant to the effects of simazine and other chloro—triazines. He extracted from maize what he called a polyphenol fraction which was able to break dowm simazine in yitrg and showed that simazine detoxification was of non- enzymatic nature. He also reported a high catalase activity in these simazine treated plants. Later, Eastin §£_31. (1964) reported a significant increase in the catalase activity of a resistant strand of corn treated with atrazine, where the catalase activity of a susceptible strand decreased with atrazine treatment. The phytotoxic effects of triazines are due, at least in part, to an interference in the photosynthetic process. Moreland gt_al. (1959) have shown that the con- version of sugar to starches can continue in the presence of triazines but that formation of the sugar is blocked. They demonstrated that by feeding carbohydrates to an en— tire living barley plant (Hordeum vulgare L.) through severed leaf tips the phytotoxicity of simazine could be reduced. Gast (1958) also showed that the accumulation of starch is inhibited by simazine treatment to coleus (Coleus blumei) plants. He also demonstrated that starch- free coleus chloroplasts kept in the dark in a saccharose solution were able to form starch in the presence of sima- zine, which proves that triazines inhibit sugar formation. The reduction of the photochemical activity by triazine treatment can be measured using isolated chloroplasts in 12 the presence of redox—dyes (with the so—called Hill reac- tion). Exer (1958) showed that the triazines inhibit the Hill reaction in the same order of magnitude as the urea herbicides of the CMU type, findings which were confirmed later by Moreland §E_al. (1959). Moreland g£_al. (1959) found simazine treatment reduced the photochemical activity (Hill reaction) of isolated barley chloroplasts by 50 per- cent at 4.6 x 10_6M. Within the group of the active chloro- triazines, atrazine inhibits photolysis at a lower concen- tration than simazine. There is, however, not always a correlation between ability to interfere with the Hill re- action and herbicidal activity. Although the interference with photosynthesis may not be the only mode of action, it is thought to be a principal one. Ashton et al.(l963), studying the structural changes in kidney beans induced by applications of atrazine, found that chloroplasts of both developing and mature primary leaves were ultimately dis- integrated in plants treated with atrazine in the light. Comprehensive reviews of the literature on the nature of the triazines in relation to their phytotoxicity and effect upon biological plant processes has been provided by Gram- lich and Davis (1967), Gysin (1962), Hamilton (1964), and Montgomery and Freed (1964). Some crops, notably corn, show a high level of tolerance to assimilated simazine and atrazine through detoxication of the parent molecule (Montgomery and Freed, 13 1961). The rate of plant metabolism must be rapid enough to prevent accumulation of the lethal concentration of the triazine in the plant, and the pathway of metabolism must alter the parent molecule to a less or non-phytotoxic form. Montgomery and Freed (1960) found that atrazine undergoes extensive conversion to a new compound in the plant. On the basis of the chromatographic behavior of this compound, it was suggested that the product was the hydroxy analog of atrazine, the chlorine atom having been replaced with a hydroxyl group. These observations were confirmed by Cas- telfranco g£_31. (1961), who characterized the constituent responsible for the conversion. The properties of the active constituent were subsequently isolated and identified by Roth and Knulsi (1961), and independently by Hamilton and Moreland (1962). It was found to be the cyclic hydroxamate, 2,4-dihydroxy-3-keto-7 methoxy-l,4-benzoxazine. Hamilton and Moreland (1962) showed that the triazine tolerant corn could convert simazine to 2-hydroxy-4,6-bis(ethylamino)- s-triazine (hydroxy-simazine) which is non-toxic. Later, Ries and Gast (1965) reported hydroxy—simazine treatment did not affect the N content in corn plants. Funderburk and Davis (1963) found corn to metabolize both ring and side- chained labelled simazine. Paper chromatography of the extracts of treated plants indicated that hydroxy-simazine and an unidentified 14C product are formed with either type of labelled herbicide. They concluded that all portions of 14 the triazine ring are subject to complete oxidation by corn, cotton, and soybean plants and appreciable amounts of radio- active 14CO2 is given off showing that these plants can completely metabolize a portion of the absorbed triazine. Hamilton (1964) reported the tolerance of several species of Gramineae to atrazine was not related to the ability of their excised roots to metabolize l4C-simazine. He found the content of benzoxazinone derivatives to be directly related to the ability of excised roots to form hydroxy- simazine. Shimabukuro (1967) found atrazine in corn is rapidly detoxified by hydroxylation, whereas susceptible species such as soybeans and intermediately susceptible species such as peas (Pisgm sativum L.), dealkylate atra- zine to produce still a somewhat toxic product. Even though simazine may be dealkylated by susceptible species, there is still sufficient activity to affect the protein content (Ries §E_§1., 1967). Although the mechanism whereby triazine herbicides may affect the increase in foliar N and protein content has not been fully established, recent research has pro- vided some of the physical and biochemical changes that occur when various plants are treated with very low concen- trations of triazines (Ries §E_al., 1967; Ries and Gast, 1965; Tweedy and Ries, 1967). CHAPTER III METHODS OF INVESTIGATION Growth Chamber The influence of varying levels of triazine on slash and loblolly pine seedlings was measured in three controlled environment chamber (Sherer CEL 512-37) studies. Seedlings were grown in 236 m1 plastic containers contain- ing 700 g of equal parts Spinks loamy sand (Al horizon) and quartz sand (Figure l). The original mineral soil was obtained from Baker Woodlot, Michigan State University, air-dried and screened through a 2 mm mesh prior to mixing. A sample of this mixture was analyzed for NO3-N by the Brucine method. P was determined by the Bray P1 technique, and K, Ca, and Mg, by the flame photometer. pH was read with a pH meter using a 1:1 soil-water ratio and organic matter was determined by loss on ignition at 500°C. Soil analyses by the Michigan State University Soil Testing Laboratory showed the following soil characteristics: Available Nutrients (ppm) Organic pH Matter % NO3-N P K Ca Mg 5.6 1.3 15 20 4 323 29 15 i ii? i i ’1)?1|’1.€«|. ll ill} (1)11 16 1111.1” 111 1111111 ((((( 111 1 _ 1:1-11111111111111. 111111111111 1111 m 5. A‘ ‘1 1&1 “a. r , 1““( UK ‘(1 ’1’) .-..~, ' '. r- {'l.‘ _ \ . . D 1 K 1 “11 1111111111 "1.1.1.1 .1111111 11111 111.11 /'/‘.\. .L. 1111‘ )I . -..C. 1111 ‘ 1 1 111111. + General view of 9—week-old slash pine seedlings in the growth chamber. Figure l. 17 The environmental conditions maintained throughout these experiments were a 16 hour light period at 28°C, an 8 hour dark period at 22.5°C, and a relative humidity of approximately 70 percent. The light intensity was approxi- mately 3148 foot candles at the plant foliage level (76.2 cm). Slash (Dodge G-4) and loblolly pine (Meriwether G—6) seeds were obtained from the Forest Science Laboratory, Southern Forest Experiment Station, Athens, Georgia. These seeds were dry stratified for 2 months in the refrigerator at 2°C prior to germinating in the growth chamber (approxi- mately 7 days) in a glass dish containing a 7.6 cm layer of quartz sand. The seedlings were transplanted 3 days fol— lowing emergence. Experiment l.—-Newly transplanted slash pine seed- lings were grown for 4 weeks (May 23 to June 20, 1967) in the growth chamber prior to treatment (Tables 1 and 2). The pots were treated with several low levels of simazine (0, 0.05, and 0.1 ppm) and N was supplied at both 14 and 42 ppm N. The herbicide was included in a slightly modi- fied nutrient solution (Table 3) prepared according to solution No. l of Hoagland and Arnon (1938). To obtain a solution of simazine that would mix with the nutrient solu- tion, 25 mg of pure simazine1 (99.2 percent) was dissolved in 50 ml of chloroform. Ten milliliters of this solution . 1Obtained from Geigy Agricultural Chemicals, Ardsley, New York. 18 Aamm m.wv m>nmmlz + Az 8mm mvv cmmouuflc evanesfid + Aemm oa.ov mnflnmsflm Hm AZ 8mm mvv cmmouuw: ESHQOEEd +.Aemm oa.ov mGwNmem om Aemm v.av m>nmmlz + AZ Ema way cmmonuflc EsHGOEE¢ + “Ema oH.ov mnwumawm ma AZ Ema way cmmouuflc Eswcofifid + Afimm.oa.ov maflnmfiflm ma Az Ema mvv cmmoupflc mumuuwz + Aemm oa.ov maflumeflm 5H AZ San «av cwmouuwc mpmuuflz + Aamm oa.ov mcflNmEHm ma Aemm oa.ov maaumsflm ma Aamm ~.vv m>ummlz + AZ 8mm N¢V cwmouuflc Edwcoead + Aemm mo.ov wqflNmEHm wa AZ 8mm va cmmonuwz EdHcoEE¢ + Aamm mo.ov mcwumawm ma Aemm v.av m>ummuz + AZ Ema way cmmouuac EsflcoEE< + Aamm mo.ov waflumfiwm NH Az Ema way :mmouqu EddeEEd + Aemm mo.ov mcflumfiflm Ha AZ San mwv cmmonufic mumuuflz + Aemm mo.ov wawNmEHm 0H AZ Ema way cmmonuflc muMHUflz + Asmm mo.ov mcflnmaflm m Aamm mo.ov mcflnmaflm m Aemm ~.vv m>ummnz + Az Emu va cmmonuwa anacoEE¢ 5 AZ 8mm mvv zmmouuwc fizflaofia¢ m Afimm v.av m>ummlz + AZ Ema «av :mmouufic 65H20854 m Az 8mm way cmmouuflc Edacofisd v AZ 8mm va ammouuwc mumuuwz m AZ 8mm way cmmouuwc mumnuwz m Az ummoxw muzwfluusc Hamv Houuqou H unmfiummua .oz .H pawfiflummxm ca 0mm: munmfiumwnp mchmem can cmmouufiZIu.H magma Dltlllli‘lltuli‘ii ‘ Table 2.--Preparation of nutrient solution treatments used in Experiment 1. Nitrogen Source Simazine N-Serve .5§p(NH4)ZSO (5 ppm) (50 ppm) .;____-__-__--____--;.-éml/l;_—_-_; ________________ 1 _ - - 2 1 - - - 3 3 - - - 4 - l - - 5 - 1 - 28 6 - 3 - - 7 - 3 - 84 8 — - 10 - 9 l - ~10 - 10 3 - 10 - ll - l 10 - 12 - 1 10 28 13 - 3 10 - l4 - 3 10 84 15 - - 20 - 16 1 — 20 - l7 3 - 20 - 18 - l 20 - 19 - l 20 28 20 3 20 - 21 3 20 84 5.0; KOH, able 3). 2.0: CaCl 0.2; Fe-EDTA, All treatments contained the following nutrients (1M solution, ml/liter: 1.0; K SO °2H O, 5.0; 3 Manr WW—v “.7 ,2 a . .. n r 20 Table 3.--Basic nutrient solution.1 Reagent_ ‘Concentration_ Stock _ Nitrate Ammonium 3| g/l ---—é-ml/1+ ------- KNO3 1 101.0 2.0 - (NH4)2SO4 1 132.0 - 1.0 MgSO4'7H20 1 246.0 2.0 2.0 CaClZ-ZHZO 1 147.0 5.0 5.0 KH2P04 1 136.0 1.0 1.0 K2504 1 87.0 5.0 5.0 KOH 1 100.0 0.2 0.2 Fe-EDTA 1 0.25 5.0 5.0 Minor Elements2 1.0 1.0 H3BO3 2.86 MnC12°4H20 1.81 ZnSO4°7H20 0.22 CuSO4-5H20 0.08 H2Mo4-H20 0.02 1The amount of KNO3 and (NH )2SO varied with the N level desired. éhe sent 28 ppm N. stock solution used amounts above repre- 2Minor element stock solution was prepared by adding the listed amount of each element and making the total volume to 1 liter with distilled water.~ 21 was mixed with 1 liter of distilled water in a 2 liter round bottom evaporating flask. The chloroform was evaporated from the mixture in 30 minutes using a rotary film evaporator with a water bath temperature of 50°C. The remaining solution was made up to a volume of 1 liter with distilled water to obtain a 5 ppm stock solution. N was supplied with KNO3 as the nitrate source, and (NH4)ZSO4 as the ammonium source. The pH of all nutrient solution treatments was adjusted with KOH to pH 6.3. Since ammonium was compared to nitrate as a N source, 2—chloro-6-(trichlor- omethyl)pyridine1 (N-Serve) was used to prevent oxidation of ammonium by Nitrosomonas sp. (Goring, 1962). Since N- Serve is relatively insoluble in water, 50 mg of pure N- Serve (99 percent) was dissolved in 50 ml of benzene. This solution was mixed with 1 liter of distilled water in a 2 liter round bottom evaporating flask. The benzene was evaporated from the mixture in 30 minutes using a rotary film evaporator with a water bath temperature of 50°C. The remaining solution was made up to a volume of 1 liter with distilled water to obtain a 50 ppm stock solution. The N- Serve was included in the ammonium treatments at a rate of 10 percent of the respective N level. The treatments were pipetted onto the soil surface at a rate of 100 ml per week (2-50 m1 applications) for 6 weeks (June 20 to August 1, 1967). 1Obtained from Dow Chemical Company, Midland, Michigan 22 Approximately 550 ml per container of treating solu- tion was applied during this period. The pots were periodically watered with distilled water to adjust soil moisture to approximately 15 percent by weight as deter- mined by weighing the pots. The experiment was arranged in a randomized com- plete block design with 4 replications. After 10 weeks, green and dry weight measurements were made of seedling fo- liage,stems, and roots (2 seedlings per pot). The plants were dried at 70°C for 48 hours in a mechanical convection oven, ground in a Wiley mill, and total N was determined on the foliage by the micro-Kjeldahl method. The data was subjected to analysis of variance and treatment means were compared using planned orthogonal contrasts and Tukey's w-procedure. Experiment 2.—-The experimental methods and mate- rials used in this study were the same as that described previously with the following exceptions: Newly trans- planted slash pine seedlings were grown in the growth chamber for 3 weeks (August 4 to August 25, 1967) prior to treatment (Tables 4 and 5). Higher levels of both simazine and N were used and the plants were treated for 9 weeks at a rate of 100 ml per week (2-50 m1 applications). Approximately 900 ml per container of treating solution was applied during this period. Simazine (0, 0.2, 0.4, AEmm v.mv m>umm|z + AZ San «my cmmonufla Edflcofiad + “Bag m.ov mcwnmeflm om AEmm m.mv m>nmm|z + AZ Ema mmv cmmouuflc ESHcOEEd + Aamm m.ov mcwumEHm ma Az 8mm emv comonuflc mumuuflz + “Ema m.ov mcflumEHm ma AZ San mmv cmmouwflc wumuuflz + Asmm m.ov wcwumEHm ha . Aamm m.ov mcflumfiwm ma Aamm «.mv 0>Hmmlz + Az Ema vmv cmmonuwc Esflcoeam + Aamm v.ov mcfiumEHm ma AEmm m.mv.m>nmmlz + AZ Ema mmv cwmouuwc Edficoafid + Afimm v.0v mcwnmEHm ea Az Ema «my cmmouuflc mumuuflz + Afimm v.ov mcflumfiwm ma Az Ema mmv comouufla opmuuflz + Afimm v.ov mswnmem NH lama v.ov maaumsflm Ha AEQQ w.mv m>nomtz + AZ Ema wmv somonuwc Edflcofiem + AEmm m.ov msflumeflm 0H AEQQ m.~v w>ummlz + AZ Ema mmv cmmonuflc financead + AEQQ ~.ov ocflnmem m AZ San «my cmmouuflc mumnuflz + AEQQ m.ov mcflnmaflm m AZ San may cmmouufic mumuuflz + fiend ~.ov mswumEHm n lama ~.ov mannmsflm m Aamm «.mv w>ummlz + AZ Ema vmv cmmonuwc EdadOEEd m AEQQ m.mv m>ummlz + Az Ema mmv cmmouuflc financesd v AZ San vmv cmmouuflc mumnuflz m AZ San mmv ammonuflc ohmnuflz m AZ umwoxw mandamus: HHMV Houucoo H usmEummHB .oz II .N psmfiflnmmxm cw pom: mucmEummHu msfiumfiflm can cmmouuHZII.¢ magma 24 Table 5.--Preparation of nutrient solution treatments used in Experiment 2. Nitrogen Source 1 Simazine N-Serve No. 1M_KNO3 .51:1(NH4)2SO4 (5 ppm) (50 ppm) -g-_-___-_-___-_ _____ ”Ml/1‘ _____________________ 1 _ _ _ - 2 2 - — - 3 6 - - - 4 - 2 - 56 5 - 6 - 168 6 - — 4o _ 7 2 - 40 - 8 6 - 40 - 9 - 2 40 56 10 — 6 40 168 ll - - 80 - 12 2 - 80 - l3 6 - 80 - 14 - 2 80 56 15 - 6 80 168 16 - - 160 - l7 2 - 160 - 18 6 - 160 - l9 - 160 56 20 — 6 160 168 1All treatments contained the following nutrients (1M solution, ml/liter): MgSO4-7H20, 2.0; CaC12°2H20, 5.0; KH PO , 1.0; K 80 , 5.0; KOH, 0.2; Fe-EDTA, 5.0; Minor elgmefits, 1.0 lSeg Table 3). 25 and 0.8 ppm) was included in a slightly modified mutrient solution prepared according to solution No. l of Hoagland and Arnon (1938) as described in Experiment 1. Nitrate (KNO3) and ammonium ((NH4)ZSO4) sources were supplemented in the nutrient solution at both 28 and 84 ppm N. N-Serve was used in the ammonium treatments as described previously. The experimental design, physical growth measurements, plant chemical analyses, and tests for significance among treatment means were the same as described previously. Experiment 3.--This experiment was similar to Ex- periment 2 except a second species, loblolly pine, was iné cluded, and atrazine was substituted for simazine as the herbicide. Slash and loblolly pine seedlings were trans- planted into each pot on August 4, and 8 respectively. The seedlings were grown in the growth chamber for 3 weeks until treatments were begun on August 25, 1967 (Tables 6 and 7). Treatments were applied for 8 weeks at a rate of 150 ml per container per week (3-50 m1 applications) with a total volume of 1150 ml per container being applied during the treatment period. Atrazine (0, 0.1, and 0.4 ppm) was in- cluded in a slightly modified solution prepared according to solution No. 1 of Hoagland and Arnon (1938) as described previously. Nitrate (KNO3) and ammonium ((NH4)ZSO4) sources were supplemented in the nutrient solution treatments at 0 and 84 ppm N. N-Serve was used in the ammonium treat- ments as described previously and the physical growth 26 Table 6.——Nitrogen and atrazine treatments used in Experiment 3. No. Treatment 1 Control (all nutrients except N) 2 Nitrate nitrogen (84 ppm N) 3 Ammonium nitrogen (84 ppm N) + N-Serve (8.4 ppm) 4 Atrazine (0.1 ppm) 5 Atrazine (0.1 ppm) + Nitrate nitrogen (84 ppm N) 6 Atrazine (0.1 ppm) + Ammonium nitrogen (84 ppm N) + N-Serve (8.4 ppm N) 7 Atrazine (0.4 ppm) 8 Atrazine (0.4 ppm) + Nitrate nitrogen (84 ppm N) 9 Atrazine (0.4 ppm) + Ammonium nitrogen (84 ppm N) + N-Serve (8.4 ppm) Table 7.-—Preparation of nutrient solution treatments used in Experiment 3. Nitrogen Source 1 Atrazine N-Serve No. 1M KNo3 .sg (NH4)ZSO4 (5 ppm) (50 ppm) ml/l 1 _ _ _ _ 2 6 - - - 3 - 6 _ 168 4 - - 20 — 5 6 - 20 - 6 — 6 20 168 7 - _ 80 _ 8 6 — 80 - 9 - 6 80 168 1All treatments contained the following nutrients (1M solution, ml/1iter: MgSO4'7H20, 2.0; CaC12-2H20, 5.0; KH_PO4, 1.0; K 504, 5.0; KOH, 0.2; Fe-EDTA, 5.0; Minor elements, 1.0 lSee Table 3). 27 measurements, plant chemical analyses, and tests for sig- nificance among treatment means were the same as described for Experiment 1. The experiment was arranged in a split— plot design with 4 replications. Greenhouse Newly germinated slash pine seedlings were grown for 16 weeks (April 1 to July 22, 1968) in 3.78 liter plastic greenhouse containers containing 3200 g of equal parts Spinks loamy sand (Al horizon) and quartz sand. A sample of this soil mixture analyzed by the Michigan State University Soil Testing Laboratory using the techniques described previously (except for organic matter, which was determined by a Leco Carbon Analyzer using high induction combustion with thermal conductivity quantitation) showed the following characteristics: Available Nutrients (ppm) Organic pH Matter % No3-N P K Ca Mg 5.5 2.4 10 l 24 322 24 The same slash pine seed source was used as des- cribed for the growth chamber studies. Drainage was pro- vided in each container by 3 holes (1.27 cm diameter) equally spaced around the outer bottom circumferences of the container. The seedlings were transplanted in 28 ‘greenhouse pots 3 days after emergence and grown for 3 weeks prior to treatment (Tables 8 and 9). Treatments were applied to the soil surface for 13 weeks. A 5 ppm stock solution of simazine in water was prepared as des- cribed in Experiment 1. Weekly applications of simazine at rates of 0, 0.5, l, and 2 ppm were applied independent of the nutrient solution treatments in a single applica- tion of 200 ml per container. Approximately 2600 ml per container of simazine solution was applied during this 13 week period. The soil moisture level in each pot was periodically adjusted with distilled water to approximately 15 percent by weight as determined by weighing the pots. Nitrate (KNO3) and ammonium «NH4)ZSO4) sources at both 28 and 84 ppm N were supplemented in a slightly modified nutrient sOlution prepared according to solution No. l of Hoagland and Arnon (1938) as described in Experiment 1. These treat— ments were applied on alternate weeks in a single applica- tion at a rate of 200 ml per container. Approximately 1300 ml per container of nutrient solution treatments were applied during the 13 week period. A stock solution of N-Serve in water was prepared as described previously. This material was included in all nutrient solution treat- ments at a rate of 10 percent of the 84 ppm N level. Sev- eral pairs of fluorescent lights were arranged parallel in the center of the plant bed 63.5 cm from the plant foliage level. These lights were automatically controlled and 29 Table 8. Nitrogen and simazine treatments used in Green- house Experiment. 1 No. Treatment 1 Control (all nutrients except N) 2 Nitrate nitrogen (28 ppm N) 3 Nitrate nitrogen (84 ppm N) 4 Ammonium nitrogen (28 ppm N) 5 Ammonium nitrogen (84 ppm N) 6 Simazine (0.5 ppm) 7 Simazine (0.5 ppm) + Nitrate nitrogen (28 ppm N) 8 Simazine (0.5 ppm) + Nitrate nitrogen (84 ppm N) 9 Simazine (0.5 ppm) + Ammonium nitrogen (28 ppm N) 10 Simazine (0.5 ppm) + Ammonium nitrogen (84 ppm N) 11 Simazine (1.0 ppm) 12 Simazine (1.0 ppm) + Nitrate nitrogen (28 ppm N) 13 Simazine (1.0 ppm) + Nitrate nitrogen (84 ppm N) 14 Simazine (1.0 ppm) + Ammonium nitrogen (28 ppm N) 15 Simazine (1.0 ppm) + Ammonium nitrogen (84 ppm N) 16 Simazine (2.0 ppm) 17 Simazine (2.0 ppm) + Nitrate nitrogen (28 ppm N) 18 Simazine (2.0 ppm) + Nitrate nitrogen (84 ppm N) 19 Simazine (2.0 ppm) + Ammonium nitrogen (28 ppm N) 20 Simazine (2.0 ppm) + Ammonium nitrogen (84 ppm N) l 10% of the 84 ppm N level. All treatments contained N-Serve at a rate of I. 30 Table 9.--Preparation of nutrient solution treatments used in Greenhouse Experiment. Nitrogen’Source 1 Simazine No. 1MDKNO3 .SI‘_’I__(NH4)ZSO4 (5 ppm) -_-;-;--__-_----__;;m1/1g--__--____-__a__a___ l - — - 2 2 - - 3 6 - - 4 - 2 - 5 - 6 - 6 - - 100 7 2 - 100 8 6 - 100 9 - 2 100 10 - 6 100 ll - - 200 12 2 - 200 13 6 - 200 14 - 2 200 15 - 6 200 16 - - 400 17 2 - 400 18 6 - 400 19 - 2 400 20 - 6 400 1 (1M solution, ml/liter): All treatments contained the following nutrients: MgSO4'7H20, 2.0; CaC12'2H20, 5.0; KH3P04, 1.0; K2SO4, 5.0; KOH, 0.2; Fe-EDTA, 5.0; Minor ele- ments, 1.0 (See Table 3). All treatments contained N-Serve at a rate of 10% of the 84 ppm N level (168 ml/l of 50 ppm stock solution). I‘IIIIII I I. .I.II.\.I((I|IIA 31 Synchronized with the daily photoperiod to provide artifi- cial light for a 16 hour period per day. The experiment was arranged in a randomized complete block design with 4 replications. After 16 weeks, seedling height (measured from soil surface), diameter (measured 2 cm above root col- 1ar), and green and dry weights of seedling foliage, stems and roots were recorded (3 seedlings per container). The plants were dried at 70°C for 48 hours in a mechanical con- vection oven, ground in a Wiley mill and total N was deter- mined on the foliage by the micro-Kjeldahl method. Foliar nitrates were determined by the method of Lowe and Hamilton (1967). The data was subjected to analysis of variance and means were compared by Tukey's w-procedure. Field Field plots were located at the Michigan State Uni- versity Tree Research Center (Figure 2). The soils (Kala- mazoo and Spink series) were well-drained with sandy loam plow layers. Soil samples taken from the control plots within each replication were analyzed by the Michigan State University Soil Testing Laboratory using the same techniques as described for the growth chamber. The general fertility level of these soils is described below: Available Nutrients (kg/ha) Organic Lime Requirement pH Matter % (metric tons/ha)_NO 3-N P K Ca Mg 5.1 2.1 6-3 13-2 24.2 83.2 548.1 84.1 General view of Field Experiment at the Tree Research Figure 2. Sub—plots are 1.67 sq m; mulch is from fresh hardwood chips. Center. ‘ .33 The experimental area was covered by a sown timothy- bromegrass sod and various weed species common to the area. No fertilizer or herbicide treatments had been applied to the area during the past five years. On April 5, 1967, the site was prepared for experimental planting by plowing, discing, and packing lightly. The experiment was arranged in a split-plot design with 5 replications. Individual plots within each repli- cation contained l.67 sq m of area (0.91 m x 1.83 m), and each plot was separated from the adjacent plot by a 0.61 m buffer strip. The replications were separated by a 1.07 m strip or walkway. One plant each of Scotch pine, white spruce, and balsam fir was handplanted in each plot on April 22, 1967. The spruce and fir were 2-3 nursery stock from a northern Wisconsin seed source and the Scotch pine was a 2-0 nursery stock from a southern France source. The fertilizer and weed control treatments includ- ing simazine, atrazine and wood chip mulch were applied on April 29, 1967 (Table 10). Ammonium nitrate (33.5%N) was the only fertilizer used as a variable in the study and was broadcast by hand at rates of 0, 112, and 336 ng/ha. Additional applications of ammonium nitrate were applied on July 7, 1967, and April 10, 1968, at the same rate. Superphosphate (8.8%P) and sulfate of potash (44.8%K) were broadcast by hand over all plots (except controls) at 34 .so m.s mo spams 8 pm muoam may lacs Goddamn 925 £0.25: mfino @003 .ucmflomumsufl 0?.308 0» .»Hm>fiuommmmu 6cm mn\mmx mm mo moumu um “Mwm.v¢v nmwuom mo ovumazm mumnmmonmquSm pm>flmomu AN pom H mucmeummua umwoxmv muoam Ham N .ms\xmx mwa 6cm Ammm.mc Hm>o maauom momma mmumu mUwOfinumm .mpmum nonmawuumm mMS AZwm.mmv mumuuwc SSHGOEada Amaxzmx mmmc mumnuaa ssflcossa + Ams\mx om.¢v sow mcaumuua ma Ann\zmx «Hav,mumnuac ssaaossa + Ama\mx om.ev sow mcaumuua as . Amn\mx om.vc sow mcaumuua GH Amn\zmx 8mm. mumuuaa suacossa + Amn\mx mm.mv sow mcaumuua ma Amn\zmx «Hat mumuuac sancossa + Amsxmx m~.~c sow manumuua «a Amn\mx m~.~V sew manumuua ma Amnxzmx ammo oumuuac sancossa + Ams\mx oo.mv sow mcaumsam NH Amn\zmx mafia mumuunc asagossa + Ama\mx oo.mv sow mnaumsnm as Amnxmx oo.ac sow mannmsam OH Amn\zox mmmv mumuuac sancosam + “ma\mx om.¢c sow wannmsnm m Amn\zmx mane mumuuac sswqossa + Ama\mx om.wv sow mcaumsnm m Amn\mx om.vv sow mannmsam a noasa mflno @003 + Amn\zmx mmmv mumuuac Edam08§¢ m Amg\zmx wmmv mumuuflc ESHGOEEd m scans mflno @003 + Amn\zox NHHV mumuufic ESHGOEE£ v Ams\zmx made mumuuac ssflcossa m sodas mane poo: m Houucoo H NusmEummHB .oz H.usmefluomxm pamflm ca poms musmaumwuu Houucoo pmw3 can nonmafluumm .oa magma 35 7rates of 28 kgP/ha and 149 kgK/ha, respectively. Atrazine 80W and simazine 80W were applied pre- emergence both alone and in combination with the three N levels. Atrazine was applied at rates of 0, 2.25, and 4.50 kg/ha, and simazine at 0, 4.50, and 9.00 kg/ha active ingredient. Additional applications of simazine and atra- zine were applied on April 15, 1968, at the same rate. All materials were sprayed uniformly over the plots and seedlings using a calibrated hand sprayer. A fresh hard— wood chip mulch was used as a non-phytotoxic weed control treatment. This material was applied uniformly over the plots at a depth of 7.6 cm. Although triazine treatments provided excellent weed control, plots not receiving these herbicides required sanitation weed control to provide more uniform growing conditions. Sanitation herbicide and insecticide treatments were applied to the experimental area on the following dates: May 23, 1967 - Applied chlordane (40%) @ 3.4 kg/ha to soil surface. June 6, 1967 - Sprayed Amitrol-T (21.1%) @ 18.7 liters/ha around border and in walkways. July 6 and September 1, 1967, and June 3, 1968 - Sprayed paraquat (42%) @ 4.7 liters/ha around border and in plots and walkways. June 8, 1968 — Foliar spray of Lindane @ 4.7 liters/ha. 36 August 27, 1968 - Sprayed Amitrol-T (21.1%) @ 18.7 liters/ha around border and in plots and walkways. Total precipitation at the field plot site for the 1967 growing season was normal as compared to the period of record. However, early spring precipitation was par- ticularly heavy, followed by a dry period in May and an- other above normal period in June. Total precipitation for the 1968 growing season was above normal as compared to the period of record. Heavy precipitation was recorded in May and June, followed by a normal period in July. Above normal precipitation was again measured in the late growing season during August and September. A comparison between the experimental seasons (1967 and 1968) and the period of record is shown below: Growing Season Precipitation (cm) April May June July August Sept. Total Period of Record (1921-1950) 7.2 9.5 8.6 5.8 6.8 7.8 45.7 1967 Growing Season 904 209 15.7 2.0 804 7.9 46.3 1968 Growing 37 On September 19, 1967, and October 4, 1968, needles of the current years lateral growth were taken from each tree for nutrient analysis. The samples were dried in a mechanical convection oven at 70°C for 48 hours and ground in a Wiley mill. Total N was determined by the micro- Kjeldahl method. Potassium was determined by the flame photometer and all other elements (P, Na, Ca, Mg, Mn, Fe, Cu, B, Zn, and Al) by a direct reading spectrograph. The. data was subjected to analysis of variance and means were compared by planned orthogonal contrasts. CHAPTER IV RESULTS AND DISCUSSION An unusual characteristic of triazine herbicides is that they have been found to increase the growth and N content of some plants when applied at sub-toxic levels (Ries gt_31., 1967). Although similar effects have been postulated previously in the N nutrition of conifers, this research presents experimental evidence that triazine treat- ment significantly affects the growth and mineral nutrition of conifers. Experimental results from growth chamber, greenhouse, and field experiments are discussed. Growth Chamber Experiment 1.--Neither the foliar N concentration (% N in needles) nor the foliar N accumulation (mg N/top) of lO-week-old slash pine seedlings raised on a soil-quartz sand mixture in a growth chamber were significantly altered by soil applications of non-phytotoxic levels (0.05 or 0.10 ppm) of simazine (Table 11 and Appendix A). However, a trend of increasing foliar N concentration and N accumula- tion was observed with increasing simazine applied. The maximum increase in foliar N concentration was 11.1 percent 38 39 Table ll.--Significance of experimental factors on the foliar nitrogen content and foliar percent dry weight of slash pine seedlings grown in thel growth chamber for 10 weeks (Experiment 1). Source df % N mg N/top % Dry Wt. Rep 3 Simazine (S)2 2 NS NS NS Nitrogen (N) 6 ** ** * Control vs N 1 ** ** ** NO3(14 vs 42 ppm N) 1 NS NS NS NH4(14 vs 42 ppm N) 1 NS NS NS NO vs NH 1 NS NS NS N-Serve 1 NS 'NS NS NH x N-Serve 1 NS NS NS S X fi 12 *1! ** *- Error 60 CV (8) 5.6 14.1 2.9 1See Appendix A for data. 2Orthogonal contrasts. * Factor is significant at .05 level. ** Factor is significant at .01 level. NS Factor is not significant. 4O V1.35 to 1.50%N) with 0.10 ppm simazine. This increase in foliar N concentration was approximately one-third that ob- tained when either nitrate or ammonium sources were supple— mented in the nutrient solution at a rate of 42 ppm N. The maximum increase in foliar N accumulation from simazine application (0.10 ppm) was one-half that obtained where 42 ppm N as either nitrate or ammonium forms were supplied in the nutrient solution. The increased N accumulation with increasing simazine applied appears to be a result of an increase in both foliar N concentration and dry weight (Appendix A). Thus, it appears that simazine concentra- tions up to 0.10 ppm under these experimental conditions provides a stimulating effect upon both the growth and foliar N content of slash pine seedlings. Similarly, Tweedy and Ries (1967) found low levels of simazine applied to the root zone of corn plants grown under both sub-optimal temperature and low nitrate levels increased the N content and dry weight of plants 20 to 25 percent. When simazine was applied in combination with the lower level of nitrate (14 ppm N) in the nutrient solution, the foliar N concentration of seedlings was greater than where an equivalent rate of N was applied alone (Appendix A). The foliar N concentration of seedlings treated with the ammonium source (14 and 42 ppm N) was greater than the nitrate, particularly where the lower level of N was applied with 0.10 ppm simazine. Simazine (0.10 ppm) applied with 41 ‘both levels of ammonium N (14 and 42 ppm N) enhanced the foliar N concentration of seedlings more than did nitrate N and simazine at similar levels. Both levels of ammonium N applied with 0.10 ppm simazine significantly increased the foliar N concentration over the control. The maximum increase in foliar N concentration was 45.9 percent where 0.10 ppm simazine and 42 ppm N as ammonium N were applied. No differences in foliar N concentration were found between the ammonium and nitrate sources (at either 14 or 42 ppm N) applied with 0.05 ppm simazine. Nitrate N at 42 ppm and ammonium N at both 14 and 42 ppm N applied with 0.05 ppm simazine significantly increased the foliar N concentration over the control.. N-Serve supplemented in the ammonium treatments had no significant effect upon the foliar N concentration of slash pine seedlings (Table 11). This implies that possibly ammonium N is as effectively utilized with sima- zine as is the nitrate source, or that N-Serve did not ef— fectively control nitrification. It is also possible that the seedlings were fulfilling their N requirements from an endogenous nitrate supply contained in the unsterilized soil mixture. Sabey (1968) reported that a fall applica- tion of N-Serve to soil buried in plastic bags under field conditions and allowed to incubate over winter, effectively delayed nitrification for about one month in the spring. Thus, it is possible that under our experimental conditions 42 ‘where the N-Serve was applied with the nutrient solution, there was an insufficient incubation period for nitrifica- tion suppression. Simazine applied with both 14 and 42 ppm N as either nitrate or ammonium N in the nutrient solution did not significantly alter the foliar N accumulation of seed- lings over where nitrate or ammonium forms were supplemented alone. The maximum increasein foliar N accumulation in simazine treated pots was 65.3 percent where 0.05 ppm simazine and 42 ppm N as nitrate N were applied (Appendix A). However, when 42 ppm N as ammonium N (with N-Serve) was used alone, a foliar N accumulation of 76.4 percent was observed. Since both the 14 and 42 ppm N levels of the ammonium source alone significantly increased N accumu- lation over the control, and no differences were observed between these levels of N and where the same N levels were applied with simazine, the stimulating effect of simazine applied with N in this experiment on foliar N accumulation is probably very slight. The effect of simazine on seedling growth is shown in Table 12 and Appendix A. Although increasing soil ap- plied simazine (0.05 and 0.10 ppm) did not significantly affect either green or dry seedling weights, the 0.10 ppm simazine application increased both the green and dry foliage weights of seedlings 22.0 and 19.4 percent, re- spectively, over the control. The total green and dry 43 .uGMUAMHcmHm you we Houomm mz .mumo now m xflpcwmmm mom H 3% 53 8.3 3mm 83 53 93 8% 23 5.3 3: >0 om Hounm m2 m2 m2 m2 m2 m2 m2 m2 m2 m2 NH 2 x m mz m2 m2 m2 m2 m2 m2 mz m2 m2 8 nomoufiz m2 m2 m2 m2 m2 mz m2 m2 m2 m2 m wcnumfim m mmm uoom Hmuos uoom Emum womaaom Doom Hmuoa uoom 88pm mmmwaom up womsom \meHHom \0mmwaom 13 £383 who 3 ”2363 53o H.AH ucmfiwuwmxmv mxmmB ca MOM Hwnfimno auzoum on» Ga csoum mmsflapwwm mafia gmmam mo musmflmB Map cam cmoum mnu so muouumw amusweflummxw mo oOGMOflmwcmflmnn.NH magma 44 seedling weights were increased 22.6 and 14.0 percent, re- spectively, when 0.10 ppm simazine was applied. These in- creases in both foliage and total seedling weights were greater than when 42 ppm nitrate N was supplemented in the nutrient solution. Freney (1965) also reported that 0.06 ppm simazine in solution culture increased the yield of corn tops 36 percent and N uptake by 37 percent without affecting root growth. Experiment 2.--In this experiment, higher non- phytotoxic levels of simazine were used than in the pre- vious experiment. The foliar N concentration (% N in needles) of slash pine seedlings grown on a soil—quartz sand mixture in the growth chamber was increased by sima- zine treatments of 0.2, 0.4, and 0.8 ppm (Table 13, Figure 3). The maximum increase was 43.8 percent (1.30 vs 1.87%N) at the highest rate (0.8 ppm) of simazine. This increase was almost equivalent to adding 84 ppm N of either ammonium or nitrate N to the nutrient solution (Appendix B). N accumulation (mg N/top) was increased 26.2 per- cent over the control when 0.4 ppm simazine was used in the nutrient solution. Increased foliar N as a result of simazine treatment at sub-toxic levels has also been shown for other crOps (Ries gt_al., 1967; Tweedy and Ries, 1967). The decrease in N accumulation between the 0.4 and 0.8 ppm simazine treatments, although an increase over the control, is probably a result of a reduction in foliar dry weight 45 Table 13.--Significance of experimental factors on the foliar nitrogen content and foliar percent dry weight of slash pine seedlings grown in thel growth chamber for 12 weeks (Experiment 2). Source df % N mg N/top % Dry Wt. Rep 2 3 Simazine (S) 3 ** * ** Control vs S l ** * ** 0.2 vs 0.4 and 0.8 ppm 1 ** * NS 0.4 vs 0.8 ppm 1 ** NS * Nitrogen (N) 4 ** NS ** Control vs N 1 ** NS N03 (28 vs 84 ppm N) l ** NS NH4 (28 vs 84 ppm N) l ** * NO VS NH4 1 NS ** S x 8 12 * NS NS Error 57 CV (%) 5.2 18.7 3.5 1See Appendix B for data. 2Orthogonal * Factor is ** Factor is NS Factor is contrasts. significant at .05 level. significant at .01 level. not significant. Ir 46 WEIGIT ‘0 " '0“ 8 30. M1) .338 E E E I E“ m" .22 g 8 ‘10- "(Hi/RP) “1 0 1 J l 0 0 .2 .4 .8 SIMZIIEW‘D Figure 3. Relationship between simazine appli- cation rate and the top dry weight and N content of 12-week-old slash pine seedlings grown in the growth chamber without supplemental N (Experiment 2). 47 at the 0.8 ppm simazine level (Figure 3). A decrease in seedling dry weight from simazine treatment has also been reported by other workers (DeVries, 1963; Kozlowski and Kuntz, 1963). Ammonium and nitrate N at a concentration of 84 ppm N, when applied both alone and with simazine in the nutrient solution, increased the percent foliar N over an equivalent 28 ppm N treatment, except where 0.4 ppm simazine and 84 ppm N as ammonium N were applied (Figure 4). Simazine treatment appears to enhance N accumulation in seedling foliage. Both nitrate and ammonium additions at 84 ppm N increased the foliar N concentration by as much as 70.8 and 65.4 percent, respectively, when 0.8 ppm simazine was added to the soil (Figure 4). It was established early by Addoms (1937) that loblolly pine seedlings grown in sand culture for 29 months were capable of utilizing both nitrate and ammonium forms of N at concentrations of 136 and 195 ppm, respectively. The trend of N accumulation in Figure 4 suggests that the 84 ppm N treatment used in this experiment lies below the optimal level for this species. However, accord- ing to Fowells and Krauss (1959) this level of N should be sufficient to maintain adequate growth. They found the maximum growth of one-year-old loblolly and Virginia pine seedlings grown in sand culture occurred between 25 and 100 ppm N. Tweedy and Ries (1967) found the addition of c. . 48 .AN usmfifluomxmv Hwnfimso nuBoum 6 Ga GBOHm can AZ Emmy mQUMDOm ESHGOEEM Ho mpmnuas paw mcflnmaflm mo mGOHum: IHQEOU nuw3 pwumwuu mwsflapmwm mafia amMHm Mom Houucoo on» Hm>o z HMHHOM Ga wmmmuocH .v mnsmfim a)nmm3amlmmml IR l is 252; A 2.5 ”25% ad :J _ . . l . . . . . . x/ / 1 ca 2 .329 2 .3 S r is a w , l -am 1 ml, 1 [gemA . m_ IR 1 _ L8 7 1.3327. 8... Ena¢w Ennwm .2 A “I: A 49 simaZine to the root-zone area of corn plants grown under both sub-optimal temperatures and low nitrate levels in- creased the N content and dry weight of the plants by 20 to 25 percent. This increase is thought to be associated with an effect on nitrate reductase. They did not find ' any increase from simazine treatment when an ammonium source was added. In contrast to the findings of Tweedy and Ries (1967), simazine treatment in this experiment increased the foliar N content of slash pine seedlings grown on am— monium as well as nitrate N. However, it is possible that either the N-Serve did not effectively control nitri- fication or the seedlings utilized an endogenous supply of nitrate contained in the unsterilized soil mixture as dis- cussed in Experiment 1. Ries gt_gl. (1967) also found the effect of simazine on protein accumulation in rye plants decreased as the nitrate level approached the optimum. A similar N accumulation response was found for seedlings in this study where simazine was applied with nitrate N. A greater foliar N accumulation was found in slash pine seed- lings treated with the lower (28 vs 84 ppm N) nitrate level, applied either alone or with increasing simazine in the nutrient solution (Figure 4). For example, 0.8 ppm sima- zine and 28 ppm N as nitrate applied in combination in- creased foliar N accumulation 46.9 percent (Table 14). 4.uc80HMHcmHm uoc mH Houomm mz .Hw>mH Ho. um unmoHMHsmHm mH uouomm 44 .Hm>mH mo. um unmoHMHcmHm mH Houowm 4 .mummuucoo Hmcomonuuo m .mumc How m prcmmmd mmmH H.mH o.om m.mm m.Hm m.mH o.mH o.mH N.w~ «.mm m.mH Hwy >0 , hm Hounm mz mz m2 m2 m2 m2 m2 m2 m2 m2 NH Z x m m2 m2 m2 44 m2 m2 H vmz m> moz m2 m2 m2 m2 m2 m2 AZ Ema m w H m> mmv mz Hz and m 44 44 44 m2 44 «4 H m> mmv oz m2 m2 m2 m2 m2 m2 H z m> HOHHGOU mz 44 4 m2 44 4 4 m2 m2 44 4 «123 cmmouqu 44 44 H saaam.o m> H.o E m.o can m2 m2 H e.o m> ~.o m2 4 H m m> Houuaoo ¥¥ m2 m2 m2 m2 ¥¥ m2 m2 m2 m2 m NAmV QGHNMEHm m mam uoom Hmuoa uoom Ewum mmmHHom uoom Hmuoa uoom Emum meHHom up moudom \mmmHHom \mmMHHom Amy uanmz who _ . Amy uanmz ammuw .Am unmEHHmmxmv mxwm3 NH mom Hmnamno nusoum may QH c3oum mmcHHpmmm mch nmmHm mo musmHmz who can somum mnu so muouomm HmucwEHummxo mo mocmoHMHcmHmll.wH mHQma 51 At the high rate of N application, without any herbicide added, there is the expected development of highly succulent tissue with a relatively high concentra— tion of N in the tissue, but a low actual N accumulation (Figure 4). In these same high N treatments the addition of increasing amount of herbicide results in a large in— crease in N accumulation with a small but linear increase in N concentration. In contrast to the low nitrate treat- ments where simazine increased N assimilation, the herbicide seemed to depress N assimilation in pots treated with a low level of ammonium N. However, the N accumulation of slash pine seedlings was greatest when the highest levels of both ammonium N (84 ppm N) and simazine (0.8 ppm) were applied in combination. This increase in foliar N with increasing herbicide application occurs without any signif- icant change in seedling mass (Table 14 and Appendix B). Increasing soil applied simazine (0.2 to 0.8 ppm) did not significantly alter either green or dry seedling weights. However, simazine applied at 0.4 ppm increased both green and dry foliage weights 9.6 and 8.1 percent, respectively, over the control. Although the total dry seedling weight was not increased when 0.4 ppm simazine was applied, total green seedling weight was increased 23.0 percent. These increases in both green and dry foliage and total seedling weights were greater than when 84 ppm N of either nitrate or ammonium N was supplemented in the 52 nutrient solution. When the concentration of soil applied simazine was increased to 0.8 ppm, both green and dry foliage, stem, root, and total seedling weights were de- creased. The depressing effect of simazine treatment on seedling root weight is shown by the increasing foliage/ root ratio with increasing simazine concentration. Experiment 3.—-This experiment was similar to the previous study except a second species, loblolly pine, was included and atrazine was substituted for simazine as the herbicide. Non-phytotoxic levels of soil applied atrazine (0.4 ppm) increased the foliar N concentration of ll-week- old slash and loblolly pine seedlings grown in a soil- quartz sand mixture in the growth chamber (Table 15). How- ever, the maximum increase was 3.2 and 13.0 percent for slash and loblolly pine seedlings, respectively (Appendix C). This increase was considerably less than where either nitrate or ammonium (84 ppm N) sources were used alone. Atrazine at 0.1 and 0.4 ppm applied in combination with both nitrate and ammonium (84 ppm N) sources, signif— icantly increased the foliar N concentration of both slash and loblolly pine seedlings over the control (Table 15 and Appendix C). The maximum increase was 53.4 percent for loblolly pine seedlings when 0.4 ppm atrazine and 84 ppm N as nitrate N were applied. Since both nitrate and ammo— nium forms alone significantly increased the foliar N con- centration of both species, the effect of atrazine on the 53 Table 15.--Significance of experimental factors on the foliar nitrogen content and foliar percent dry weight of slash and loblolly pine seedlings grown in the gr (Experiment 3). gwth chamber for 11 weeks Source df % N mg N/top % Dry Wt. Rep 2 3 Atrazine (A) 2 ** * NS Control vs A 1 ** NS 0.1 vs 0.4 ppm 1 ** ** Nitrogen (N) 2 ** ** ** Control vs N 1 ** ** NS N03 vs NH4 (84 ppm N) 1 * ** ** A x N 4 NS NS NS Error (a) 24 Species (S) 1 NS ** NS A x S 2 NS NS * N x S 2 NS * NS A x N x S 4 NS NS NS Error (b) 27 cv (%) Error (a) 3.9 23.7 4.7 Error (b) 5.3 24.6 3.3 1See Appendix C for data. 2Orthogonal contrasts. * Factor is ** Factor is NS Factor is significant at .05 level. significant at .01 level. not significant. . 54 enhancement of foliar N in these seedlings is very slight. However, it appears that a combination of atrazine and supplemental N to the soil generally increased the foliar N concentration more than when N was applied alone. The foliar N accumulation in slash pine seedlings was increased by 0.4 ppm atrazine applied both alone or with supplemental N in the nutrient solution. For example, when 0.4 ppm atrazine was applied with ammonium N (84 ppm N), the foliar N accumulation of slash pine seedlings was increased 80.6 percent over the control (Appendix C). Ex- cept for the previous example, the increase in foliar N accumulation in slash pine from nitrate or ammonium sources applied with atrazine did not exceed that where N was ap- plied alone. In loblolly pine, all N and herbicide treat- ments decreased foliar N accumulation, except where 0.4 ppm atrazine was applied with either nitrate or ammonium forms (84 ppm N). This decrease in foliar N accumulation in loblolly pine seedlings was probably due to the depressive effect of both atrazine and N on the foliage dry weight of seedlings (Appendix C). No differences in the foliage dry weight of slash pine seedlings were found when these same atrazine and N treatments were used. Gramlich and Davis (1967) found similar results from field experiments in which atrazine treated corn plants were smaller and con- tained a higher N concentration, but less N accumulation, than untreated plants. 55 The effect of atrazine on the growth of both slash and loblolly pine seedlings can be observed in Table 16. Although no significant differences were found in either green or dry foliage, root, or total seedling weights when atrazine was applied to either species, the green and dry foliage and total weights of slash pine seedlings were in- creased when 0.4 ppm atrazine was applied to the soil (Appendix C). In contrast to slash pine, both the green and dry foliage, root, and total weights of loblolly pine seedlings were decreased with increasing concentrations of atrazine applied. Greenhouse Foliar Nitrogen Content.—-The foliar N concentra— tion (% N in needles) of 16-week-old slash pine seedlings raised in the greenhouse was increased by soil applications of 0.5, 1, and 2 ppm simazine (Table 17 and Figure 5). The maximum increase was 91 percent (1.26 vs 2.41 % N) at the highest simazine level. This increase in foliar N concen- tration was greater than where 84 ppm N of either nitrate or ammonium N was used in the nutrient solution (Appendix D). In fact, 1 ppm simazine applied to the soil was equivalent to supplying 84 ppm N of either nitrate or ammonium N in the nutrient solution. This stimulating effect of simazine applications on the N concentration in other crops has also been shown by other investigators (DeVries, 1963; Ries gt_al., 1963). .ucmoHMHcmHm no: mH Honomm mz .Hm>mH Ho. um ucmoHMHcmHm mH uouomm 44 .HmbmH mo. um UGMOHMHsmHm mH Houomm 4 .mummuucoo HmsomocuHON .mumo mom 0 prcmmm4 mom H m.mH o.¢~ m.mm.m.m~ m.m~ v.5H m.hm H.mm m.¢m v.m~ Any Houum m.mH N.wm H.Nm N.hN m.m~ m.mH m.hN m.wm m.Hm w.m~ Adv Houhm Hwy >0 pm Ant uouum m2 mz « wz m2 m2 m2 m2 m2 m2 v m x Z x 4 m2 wz m2 4 4 m2 m2 m2 m2 m2 N m x 2 m2 m2 m2 m2 m2 m2 m2 m2 m2 m2 N m N d m2 4* as as «s «s as «s 8* at H Amv mmflowmm am Amy Houum % m2 m2 m2 m2 m2 4 m2 m2 m2 m2 4 z x 4 m2 Hz and 44V *4 as «4 s «4 #4 H vmz m> mOZ m2 m2 m2 m2 8 m2 4. H Z m> HOHHGOU 4 4 4 4 4 44 m2 4 m2 mz m mxzv cmmounHz m2 m2 H and 4.o m» H.o * a H d m> HOHHGOU m2 m2 m2 4 m2 m2 m2 m2 4 m2 m «lav mcHumuua m mmm uoom Hmuoe uoom Emum ommHHom uoom Hmuoa,uoom Emum mmMHHom up wousom \omMHHom \meHHom: . 4. . _ , Amy uanm3 who . Amy pamHmz dwmuu H.Hm yams (Hummxmv mxmms HH you Honsmno guzoum may cH csoum mmcHHpmom och MHHoHnOH 0cm . smMHm mo muzmHmz who can somhm on» so muouOMM HmucmSHHmmxm mo mOGMOHMHcmHmII.mH mHnma .HCMOHMHsmHm Hos mH H0pomm mz .Hw>wH Ho. um ucmonHcmHm mH Houomm 44 .Hw>mH mo. um ucmonHcmHm mH Houomm 4 .muwp new 0 xHUstm< mom H m.m m.o n.m o.mH m.vH H.v Hwy >0 7 5 mm Houum m2 m2 m2 m2 m2 m2 NH Z x m 4 4 m2 m2 44 44 v cmmOHqu 44 44 44 44 44 44 m OCHNMEHm m dmm nwquMHo Emum ucmHom uanmz who 4 ZLmoz mou\z ma 2 w up condom H.mxmms mH How wmsoacmwum wnu sH szoum mmaHHUmmw osHm ammHm mo kuoEMHp Ewum tam .panma rucmHm3 who psmouwm umHHOM 4usmpsoo cmmouuHs HMHHow msu so muouomm HmucmEHHmmxm mo cosmoHMHsmHmul.bH mHnma g) m N w v m E . E 4. § ’55 20 E m 0 -m -m -E 58 - I” I I I )- ” ’I ’I I I ’I h N (I) 'z I I - I I I I I ' I p l D I I h I ’ I .l I _’ . I N (rs/m) ’ , I ................ a” I , --------------- pun...- ...’.’. a; i. ’ fl, ‘.\. "' n- ‘_ m3 (us/9) . . '4 l l l 0 .5 1 2 $WVHE(HM) 5. Relationship between simazine application rate and the N content of 16—week—old slash pine seedlings grown in the green— house without supplemental N. 59 An increase in foliar N concentration was also found with increasing levels of simazine applied with both 28 and 84 ppm N as either nitrate or ammonium sources (Figure 6). The maximum increase was 118 percent where 2 ppm simazine was applied in combination with 84 ppm N as ammonium N. Simazine applied without supplemental N did not significantly increase foliar N accumulation (mg N/top) over the control (Figure 5). However, a trend of increas- ing foliar N accumulation was observed in plants treated with both 0.5 and 1 ppm simazine. An additive effect on foliar N accumulation was observed where 0.5 ppm simazine and 84 ppm N as ammonium were applied in combination. This treatment increased foliar N accumulation 80 percent and was greater than supplying 84 ppm N of either nitrate, or ammonium N in the nutrient solution (Figure 6). When simazine was applied at a standard herbicidal rate (2 ppm) both alone and in combination with either nitrate or am- monium N, there was a significant depressing effect upon N accumulation. This decrease in foliar N accumulation is probably due to the reduction in foliar dry weight where 2 ppm simazine was used (Figure 7). Thus, the 2 ppm simazine concentration applied to slash pine seedlings at this stage of development was probably phytotoxic. Simi- lar effects were observed on red pine seedlings by-Winget et a1. OBGBL From the depressive effect of simazine on 6O 2.5 mania .Hz Emmy moOHSOm EDHGOEEM no oumuuHc 6cm wcHNmEHm mo mGOHumcHnEoo nqu powwow» paw mmsoncwwum on» CH szoum mocHprmm qum nmmHm How Houusoo may Eoum AQOU\Z 08v mxmums 2 was Hwy COHumnuswocoo z HMHHOM mnu :H momsmso N: Z me I 383 95525:: 4:48 SRBRQRBS (Z) mum mu mnwm 'é" ’f a 8 ESE 2 53m wusmHm O gasssesassas (DINNER/DWI 7 ‘ .11! .‘1" I‘ll 41", .z HmucmEmHmmsm usonqu mmson Icmmum may :H csonm mmcHHpmmm mch smMHm mom Houucoo may Scum muanmB HuanHV who can HummHv :mwum CH mmmmnomp on» can mums cOHumoHHmmm msHumEHm cmm3umn mHnmcoHuMHmm .m musmHm Etfiflfifm (D 11mm [Elifllfifllflmlskfl EEVEHEB N H m. . H o EYIMKDHdeMflEflflBEHEBB 62 the growth and N accumulation of seedlings in this study, it is apparent that the increased foliar N concentration from increasing simazine applied was primarily a function of decreased dry weight. Gramlich and Davis (1967) found corn treated pre-emergence with high rates of atrazine (17.9 kg/ha) also had less N uptake than the untreated . controls. i Accumulation of Foliar Nitrates.--Simazine applied j at 2 ppm without supplemental N increased the foliar ni— EJ trates (ug NO3/g dry weight) of slash pine seedlings (Fig- ure 5). However, a maximum increase of 25.4 percent was obtained where 2 ppm simazine and 84 ppm N as ammonium were applied in combination. No differences were found in foliar nitrates of seedlings supplied with nitrate and ammonium sources, either with or without simazine. Ac- cording to the technique of Low and Hamilton (1967) for the determination of nitrates in plant extracts, no nitrates should have been found in the ammonium treated seedlings if only the ammonium ion was utilized. The fact that fo— liar nitrates were found in ammonium treated seedlings indicates that either: (1) the N-Serve did not effective- ly control nitrification, (2) the seedlings were utilizing an endogenous supply of nitrate contained in the unsteri- lized soil mixture, or (3) the ammonium source was as ef- fectively utilized with simazine as was the nitrate source. If the latter is true, then the effect of simazine on 63 foliar N accumulation is not necessarily a function of in- creased nitrate reductase activity accelerating nitrate uptake by the plant as suggested by Ries gt_§l. (1967). Tweedy and Ries (1967) found in experiments with corn, that responses to simazine occurred in plants grown with nitrate, but not ammonium as the N source. They found nitrate reductase activity to increase almost ten-fold in plants exposed to simazine for 7 days. Also, Gramlich and Davis (1967) found in nutrient culture studies with corn that atrazine treatment at 4 and 8 ppm increased the percentage foliar nitrates without an accompanying decrease in free ammonia as might be expected if atrazine inhibited the enzymatic conversion of nitrate to ammonia. They con— cluded that the increased percentage total N of atrazine treated plants was primarily due to increased nitrate. However, it has been shown that conifers receive most of their N in the form of ammonium as little nitrification occurs under acid forest conditions (Addoms, 1937). Bengt- son1 found that slash pine seedlings grown in the green- house for 270 days had the highest N uptake and dry matter production when fertilized with ammonium sulfate at 600 mg N/pot. Also, Tiedjens (1934) found that the ammonium ion ‘was assimilated most satisfactorily in tomato and apple plants from nutrient solutions having a pH of 5.0 to 6.5 1G. W. Bengtson, TVA, personal communication, 1968. . 64 Since the acid forest soil mixture used in our studies had a pH of 5.6 and the nutrient solution treatments had a pH of 6.3, conditions appear favorable for ammonium ion ab- sorption by the seedlings. Simazine and Growth Reduction.--The effect of sima- zine on the growth of lG-week-old slash pine seedlings is shown in Table 18 and Figure 7. In contrast to the growth chamber studies where low levels of simazine (0.05 - 0.8 ppm) did not affect green or dry weights of slash pine seedlings, decreases in both green and dry foliage, stem, root, and total seedling weights were found where slightly higher concentrations of simazine (1 to 2 ppm) were applied in the greenhouse. In fact, 1 ppm simazine significantly lowered both the green and dry foliage, stem, root, and total seedling weights (Figure 7). This decrease in seed- ling growth may be a result of increased respiration. Ries gt_§1.(l967) have shown that increasing applications of simazine progressively decreased the percent dry weight of rye plants. Simazine also increased the respiration rate of the plants more than 10 percent without affecting the respiratory quotient, which they thought accounted for the decrease in dry weight. A 10.7 percent increase in seedling height was found where 0.5 ppm simazine was applied (Figure 8). Plants treated with 0.5 ppm simazine were taller than where 84 ppm .uGMOHMHsmHm no: mH Houomm mz .Hm>mH Ho. um uGMOHMHcmHm mH Houomm 44 .Hm>mH mo. um unmoHMHcmHm mH Houomm 4 .mumt How a xHUsmmmd mmm H m.mH m.HH. ¢.hH h.mH m.HH m.wH w.mH m.mm m.mH m.mH . Amy >0 hm Houum "w m2 m2 .mz m2 m2 m2 m2 m2 m2 m2 NH 2 x m m2 44 m2 4 44 m2 44 m2 4 44 v GOmOHuHZ 44 44 44 44 44 44 44 44 44 44 m mcHNmEHm m mum uoom Hmuoa uoom Ewum mmMHHom uoom Hmuoe. boom Emum mmMHHom up mousom \4444H04 \444HH04 Amy uanmz mun Amy uzmHm3 smmuu H.Mmms mH Hom.mmsonsmmnm may cH c3oum mmcHHpmmm mch gmMHm mo musmHm3 who pcm,cmmnm mnu so muouomm HmucoEHHmmxm mo mocMOHMHcmHmII.mH mHQMB Figure 8. Effect of simazine application rate on the top (upper) and root (lower) growth of l6—week—old slash pine seedlings grown in the greenhouse without supplemental N (Treatments: A=Control, B=Simazine @ 0.5 ppm, C=Simazine @ 1 ppm, D= Simazine @ 2 ppm). ‘JJ‘. )(n' \t -.\ Effect of simazine and nitrate (upper) and ammonium (lower) treatments on the top growth of 16—week-old slash pine seedlings grown in the greenhouse (Treat— ments: Upper, A=Control, B=NO3-N @ 28 ppm N, C=NO3—N @ 84 ppm N, D=Simazine @ 0.5 ppm; Lower, A=Control, B=NH4-N @ 28 ppm N, C=NH4—N @ 84 ppm N, D=Simazine @ 0.5 ppm). 4 69 .Hsmd N a mcHNmsHmna .2 add 44 a 2-4mzuo .z and 44 a Zumoz um .Honpcouud .usmHm “Ema m.o w ocHnmEHmuo .z Ema «m w zlwmzuo .z Ema 4m @ zlmoznm Honpsoonfl .ummq "mpcmfiummufiv mmsogsmmum map CH c30uq mmsHHpmmm mch QmMHm pHolxmmznoH wo £u3oum poou ps8 mow mun so musmfiummnu EsHsoEEm pcm oumuuHc can msHNmEHm mo pomwmm .OH wndem 70 It is possible that the mycorrhizal habit in conifers may alter uptake of simazine by degradation within the fungal mantle or by altering the absorption pattern. Freeman 33 El; (1964) suggest the presence of mycorrhizae may enhance the resistance of white pine seedlings to simazine. They 14C-labeled simazine in noninoculated found the uptake of plants was more than double that of inoculated ones. Since the slash pine seedlings in these studies had well- developed mycorrhizae, the influence of these fungi upon simazine absorption and degradation is a possibility. Field Foliar Mineral Nutrition.--Simazine applied pre- emergence at herbicidal rates without supplemental N did not significantly alter the foliar mineral nutrition of field planted Scotch pine, white spruce, or balsam fir nursery transplants (Tables 19, 20 and 21; Appendix E). The lower rate of soil applied atrazine (2.25 kg/ha) increased the foliar Mg concentration of all species over the control. Earlier, DeVries (1963) reported significant increases in the Mg uptake of corn plants grown on limed soils treated with simazine. Freney (1965) also found simazine applied at 0.06 ppm in solution culture increased the Mg uptake of corn by 24 percent, N by 37 percent, P by 25 percent, and K by 41 percent. However, in this study simazine did not significantly affect either N, P, or K t , ‘8‘ .4 .. . . 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I '4. . .. _ Fpklis:4tkil .ommmn was usmfiumouu may SOH£3 com: msHm> may no mOHH an :oHu IomuHo mHmsHm 8 GH oomcmno umnu manoeummuu unmoHMHsmHmlcoc How ch0 UmcHEumumU mucous mmcmzo oz 0 .csmuu mchmmuomo HIV .Ho>mH mo. um mmmmuomp UGMOHMHsmHm I .psmuu msHmmmHocH A+v .Ho>mH.mo. um mmmmnocH unmoHMHsmHm + .msHm> cOHumuucmocoo z psoomm umooxm .mmMHHOM dowmmm mcHonm nmmH .mGOHm mmUHOHnHmz m>HuommmmH on» ou nommmmu nqu who mOvaz + movHOHoumn map .GOHumuunmocoo mUHOHnHmn mchmmHosH mo HHSmoH m was Hz usonuHsv mcHumuum pom msHN ImEHm .Honucoo on» on uncommon €53 mum mHm>mH mOvaz can 301:: “How mmmcmnu N .Am xHosmmmd mmmv mummnucoo Hmcomonuuo EOHMH o o luv 0 o + o o o . 1.5 + o Amaxzmx ammo m024mz + mcHumuua H+c o Hut 0 14V 0 o o o n o o o Ama\zm4 NHHV m024mz + maHNmuu4 o o Hue o o o u o o o o I o Ama\mx om.4 can mm.mv mcHNmuua o . ch . o o H45 o luv 4 o 4 o 18a\zmx 8mmv M02442 4 mcHumeHm HIV HIV HIV luv 0 o 14V H+v o o o o o Hmn\zmx NHHV m024mz + mcHNmsHm 1.5 o 1.3 H+V luv o o Hue 1+5 0 o o o Amaxmx oo.m 6am om.4c mcHumsHm o . 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Hut luv H+V luv H+V 14c “no I o o o Hma\zm4 NHHV mozsmz o o Hub 0 u o u n H+v + o o + HUHsz Houucoo H4 as m so as as as mo 82 m s Hmm.vz z ucmsummue NGOHumuucmocoo HmHHom .mucmeumouu Houucoo @003 pamwumnuHc ESHGOEEM Eonm muammm maHms pmusm*m pHmHm «0 coHannsc HmumcHE HMHHom may cH momcmsonn.om mHnms .memn mm3 usmfiumwuu mnu SOan coma mnHm> may no wOHH an GOHH Iooqu mHuch m :H oomcmno umnu mucmsumwuu HGMOHMHcmHmIcoc How ch0 pmcHEHmpmp mucosa .mosmno oz o .psmuu mchmmuomo HIV .Hm>mH mo. um mmmonomp unmoHMHsmHm I .pcmnu mchmmHosH A+V .Hm>mH mo. um mmmmuosH usMOHMHsmHm + .mnHm> QOHumuusmocoo z ocoomm ammoxm .mmMHHom common msH3onm hmmH .mcon mopHoHnHmn m>HuommmmH map on nommmmu nuH3 mum mOvaz + mmoHOHnHma may .QOHumuucmosoo mUHoHQHmn msHmmmHosH mo uHSmou m mum Hz usozuH3V.wcHumupm can mcHumEHm .Houucoo on» on pommmmu cuHs mum mHm>mH mOZsz pom noHoE “Mom momcmso N .Am xHUammmd momv mummnucoo Hmcomonuuo EoumH Mu A+V HIV I HIV 1+5 + o o HIV I o o o “maxzmx mmmc mozvmz.+ mcHumuua o o HIV HIV 0 o + H+V HIV 0 o o o Hmnxzmx mHHv m024mz + mcHNmuua o o o o b o. I o .o o A+V o o Hmn\mx om.4 can mm.mv mcHumnua l+c o AIV H+V A+v 14c o o H+V o AIV o o Hma\zmx ammo m02312 + mcHnmsHm o o I 1+3 0 l+v o x+v H+V o o o o Ama\zmx NHHV m024mz + mcHumsHm o 3 o A: o H: o o o o o o 0 85385 65... 8.3 65883 I I I I 14v 4 A+V H+V AIV I 14V 0 + Hmaxzmx mmmc moZsz I I I o o o o 0 HIV I 1+3 o o Hmn\zmx NHHV oz mz o o AIV o I o I I H+V + o o + aoHsz Houpcoo Hm an m 50 mm :2 oz mu 82 m M Amo.vz z . , ucmsuomua mcoHumnpamoaou “MHHom H H.mucmEummHu Honusoo @883 can mumuuHs EchOEEm Eoum mHm Equsm pmucmHm UHon mo cOHuHHusc HmumcHE HMHHom map :H momcmsUII.Hm MHQMB 74 assimilation. Neither did atrazine applied pre-emergence at herbicidal rates to all species, have any significant effect upon the foliar content of any of the elements de- termined during the 1967 growing season other than Mg. Approximately three months following herbicide treatment in the first growing season, a severe chlorosis was observed in white spruce seedlings. This foliar dis- coloration, followed by needle cast and eventually death, was associated more with simazine (particularly at the higher rate) than atrazine and was more severe when the herbicides were applied without supplemental N. The de- pression of N assimilation coupled with injury at the higher rates of simazine and atrazine suggests that white spruce may be more susceptible to these herbicides than the other two species. This phenomenon was also observed following a second application of the herbicides in the spring of 1968. In the second growing season (1968), in which only foliar N was determined on the seedlings, again simazine treatment did not significantly change the foliar N con— centration of either species (Tables 19. 20 and 21). How- (ever, in contrast to the previous year, a delayed increase in the foliar N of Scotch pine and white spruce was found from ammonium nitrate (336 ng/ha) applied to the simazine Plots. Freney (1965) reported similar results when appli- cations of 1 to 5 ppm simazine to the soil of greenhouse 75 pots, increased the N uptake of corn only when additional ‘N was supplied to the soil. In our study the higher rate of N (336 ng/ha) applied with simazine also significantly increased the foliar N concentration of Scotch pine and white spruce seedlings over where the lower rate of N (112 ng/ha) was applied similarly, or where simazine was applied alone. No differences were found in the foliar N of these species between the low level of ammonium nitrate (112 ng/ha) applied with simazine and where simazine was applied without supplemental N. Ammonium nitrate applied to sima- zine treated balsam fir did not significantly alter its foliar N concentration during the second growing season. Ammonium nitrate fertilizer (336 ng/ha) applied alone (without herbicide) to Scotch pine, White spruce, and balsam fir seedlings in the early spring of 1967, sig- nificantly increased their foliar N concentration over where no N was applied (Tables 19, 20 and 21). No differ- ences were found in the foliar N of Scotch pine and balsam fir between the two levels of supplemental N (112 vs 336 ng/ha). However, the higher rate of ammonium nitrate (336 ng/ha) significantly increased the foliar N concen- ‘tration of white spruce over where the lower level of N (112 ng/ha) was applied. In the second year, ammonium nitrate treatment significantly increased the foliar N Concentration of only Scotch pine and white spruce seed- lings over the control. The 336 ng/ha ammonium nitrate VT“ "arr—a: 4...-..“ n1 .. I 4“; .“ application 5 species over I to balsam fir centration as Alth alter the fc first growix the second I centration and high In N° Signs-1 Pine Or be le‘Jels. applied t ioliar N by Soil Reid, 1 tatiOn 91:0in and t‘ atraq Zine “V194 Ere 315 Si} 76 application significantly increased the foliar N of these species over where 112 ng/ha.was applied. Supplemental N to balsam.fir seedlings did not affect their foliage N con- centration as was found in the previous year. Although atrazine treatment did not significantly alter the foliar N concentration of seedlings during the first growing season, the low rate of herbicide (2.25 kg/ha) the second year significantly increased the foliar N con- centration of white spruce seedlings over both the control and high herbicide (4.50 kg/ha) application (Table 20). No significant differences in foliar N of either Scotch pine or balsam fir were found between the two atrazine levels. However, the high rate of atrazine (4.50 kg/ha) applied to Scotch pine seedlings significantly decreased foliar N over the control (Table 19). It is possible that degradation of the triazines by soil organisms (Burnside gt_al., 1961; BurSChGII 1961; Reid, 1960) coupled with leaching by above normal precipi- tation at the test site during the early spring of both growing seasons, might reduce triazines to sub-toxic levels and thus contribute to the increased N assimilation in atrazine treated white spruce. The concentration of atra- Zine and simazine under these environmental conditions Inight have approached that used in the growth chamber or greenhouse where significant increases in the foliar N of Slash pine were obtained with low level applications of Simazine. '_ i ; Hg! n-‘A‘ni’ka ‘ -. M-_.|..— , 4 W“ \I -— —.-_._- 77 It is unusual that atrazine should significantly increase the foliar N concentration of white spruce seed— lings during the second growing season as this species ap— peared more susceptible to herbicidal injury throughout both growing seasons. It is possible that differential ‘uptake and/or distribution of both atrazine and simazine occurred among the three species. According to Freeman et al. (1964) using 14C-labelled simazine, the 14C was fairly evenly distributed throughout red pine seedlings after 40 days, but in white pine a much higher percentage was retained in the non-photosynthetic organs (roots and stems). The needles of red pine contained approximately three times more radioactive material than white pine. Although the uptake and distribution of the herbicides were not measured in our study, it is possible that the injury and mortality in white spruce from simazine and atrazine treatment was a result of greater absorption and/or accumulation of the chemicals in the photosynthetic areas of these seedlings. Since triazines are thought by some workers to kill by inhibiting the Hill reaction dur- ing photosynthesis (Moreland gt_gl., 1959), if differential herbicide distribution did occur among these species, the fact that white spruce is less tolerant to these herbicides is not unreasonable. The increased herbicidal injury and mortality that occurred in plots on the lower lepe also suggests that 78 lateral soil movement of the herbicides probably occurred. Heavy precipitation following treatment application in the early spring of both growing seasons (See Chapter III) very likely contributed to herbicide movement and concentration on the lower slope. Interaction of Nitrogen and Herbicide Treatments with Mineral Nutrition.--Broadcast applications of ammonium nitrate fertilizer (336 ng/ha) to simazine and atrazine treated plots during the first growing season, significantly reduced the foliar P concentration of Scotch pine and white sprce seedlings over where the herbicides were applied alone (Tables 19 and 20; Appendix E). The depressive ef- fect of N on foliar P has also been observed by other workers (Dumbroff and Michel, 1967). The high N level (336 ng/ha) supplemented with atrazine also significantly decreased the foliar P of balsam fir, where N applied to simazine treated balsam fir did not affect its foliar P content (Table 21). The effect of ammonium nitrate applied with either simazine or atrazine on reducing the foliar P concentration of the affected species was greatest at the highest level of N (336 ng/ha). Although no differences were found in the foliar P of either species between the two levels of ammonium nitrate applied with atrazine, the high rate of N (336 ng/ha) applied to the simazine plots significantly reduced foliar P in both Scotch pine and white spruce seedlings over where the lower rate of N 79 (112 ng/ha) was applied similarly (Tables 19 and 20). No differences were found in the foliar P of these seedlings between the lower rate of N (112 ng/ha) applied with simazine and where simazine was used alone. However, the 112 ng/ha application of ammonium nitrate applied with atrazine significantly decreased the foliar P of white spruce seedlings over where the herbicide was applied alone. Similar to where N was applied with simazine and atrazine, ammonium nitrate applied alone during the first year sig- nificantly lowered foliar P for all species over where no N was applied. This decrease in foliar P was greatest at the highest level of N (336 ng/ha). Also, the high rate of supplemental N (336 ng/ha) significantly reduced foliar P in balsam fir over the lower rate (112 ng/ha). The foliar Ca concentration of white spruce seed- lings was significantly decreased by 336 ng/ha of ammonium nitrate applied with simazine over where 112 ng/ha was applied similarly (Table 20). N supplemented to the sima- zine plots did not affect the foliar Ca of either Scotch pine or balsam fir seedlings (Tables 19 and 21). Also, ammonium nitrate applied both alone and with atrazine did not significantly alter the foliar Ca concentration of the seedlings. The lower rate of ammonium nitrate (112 ng/ha) applied with atrazine significantly increased the foliar Mg concentration of both white spruce and balsam fir over 80 where the higher N rate (336 ng/ha) was applied with atra- zine (Tables 20 and 21). However, ammonium nitrate applied both alone and with simazine did not significantly alter the foliar Mg concentration of any species. The high rate of ammonium nitrate (336 ng/ha) applied with atrazine significantly increased the foliar F“ Mn concentration of white spruce and balsam fir seedlings over where the herbicide was applied alone (Tables 20 and 21). The 336 ng/ha ammonium nitrate treatment (without herbicide) significantly increased the foliar Mn concen- tration of balsam fir. A trend of increasing foliar Mn was observed in both Scotch pine and white sprce seedlings where this same rate of supplemental N (336 ng/ha) was applied. Also, the 336 ng/ha treatment significantly in- creased foliar Mn in these seedlings over the 112 ng/ha application. Ammonium nitrate applied with simazine did not significantly affect the foliar Mn concentration of either species. However, a trend of increasing foliar Mn was observed in balsam fir with increasing N applied with simazine (Table 21). Ammonium nitrate (336 ng/ha) applied with sima- zine treated white spruce significantly lowered its foliar Cu concentration over where the herbicide was applied alone (Table 20). This same treatment combination significantly reduced the foliar Zn concentration of both Scotch pine and white spruce seedlings. These decrease in foliar Cu and Zn . 81 were greatest at the higher level of N (336 ng/ha). Sim— ilarly, ammonium nitrate applied alone (without herbicide) significantly reduced the foliar Zn of white Spruce and balsam fir, and foliar Cu of balsam fir over the control. The high level of supplemental N (336 ng/ha) was more ef- fective in decreasing the foliar Cu of these seedlings. The foliar Cu and Zn concentrations of seedlings treated with ammonium nitrate and atrazine were not significantly affected. Ammonium nitrate applied to simazine treated plots of Scotch pine and balsam fir in the first growing season significantly decreased their foliar B concentration over where simazine was applied without supplemental N (Tables 19 and 21). The decrease in the foliar B of Scotch pine was greatest at the higher rate of N (336 ng/ha), where the lower level of N (112 ng/ha) applied with the lower simazine level (4.50 kg/ha) was more effective in reducing the foliar B of balsam fir. The higher rate of ammonium nitrate (336 ng/ha) applied with atrazine significantly lowered the foliar B concentration of balsam fir over where the herbicide was applied alone. N additions to the atrazine plots did not significantly alter the foliar B of either Scotch pine or white spruce, although a decreasing trend in foliar B was observed with increasing N applied (Tables 19 and 20). Supplemental N alone also significantly reduced the foliar B concentration of Scotch pine and balsam fir seedlings. 82 Broadcast applications of ammonium nitrate to sima- zine and atrazine treated seedlings during the first grow- ing season did not significantly alter the foliar concen— trations of N, K, Na, Ca, Fe, or Al for either species (Tables 19, 20 and 21). Similarly, as N applied to sima- zine plots did not significantly affect foliar Mg or Mn, N applied with atrazine did not affect the foliar Cu or Zn concentration of either species. Supplemental N alone (without herbicide) broadcast over the field plots did not significantly alter the foliar assimilation of K, Na, Ca, Mg, or Fe for either species in the first growing season. However, N additions alone to balsam fir seedlings signif— icantly lowered their foliar Al concentration over the control (Table 21). This decrease in foliar Al was great- est at the lower level of N (112 ng/ha). It is apparent that combination herbicide and N treatments applied to the field plots significantly changed the concentration of many of the foliar elements. However, it should be noted that these changes in foliar mineral nutrition were primarily a response to added N and not herbicide. Although, no explanation is offered to describe the interaction effects of herbicide and N treatments on the changes in the foliar mineral concentrations in these species, these observations may prove useful to future researchers. 83 Mulching and Mineral Nutrition.--Mulching used as a non-phytotoxic weed control treatment, significantly in- creased foliar N and P and decreased foliar Ca, Mg, and Fe for all species during the first growing season (Tables 19, 20, and 21; Appendix E). Mulching had no significant effect upon the foliar concentrations of K, Na, Mn, Cu, B, Zn, or Al for either species. The effect of mulching on N concentration was not apparent in the second growing season. Concentrations of other foliar elements were not measured after the first year. The lack of a greater gain in N assimilation in conifers from triazine compounds used under these field conditions may be a result of the high herbicide rates along with surface dispersion of herbicides by abnormal precipitation. It is possible that as triazine levels are further reduced by leaching and degradation with time, there may be a delayed nutritional gain in subsequent growing seasons. P ’f “"113." L. Lgfim‘ ”0‘? I £“h‘.!i_“.. .! '4 1.11! I CHAPTER V SUMMARY AND CONCLUSIONS The objectives of this study were to determine: (1) the effect of low level soil applications of simazine and atrazine, and their interaction with ammonium and ni- trate sources of N, on the growth and foliar N nutrition of slash and loblolly pine seedlings grown under controlled environment; (2) the effect of pre-emergence herbicidal applications of simazine and atrazine, and their interac- tion with ammonium nitrate fertilizer, on the foliar min- eral nutrition of Scotch pine, white spruce, and balsam fir nursery transplants. First Growth Chamber Study--Slash Pine wItH’Simazine Non-phytotoxic applications of simazine (0.05 and 0.10 ppm) did not significantly alter either the foliar N concentration (% N in needles) or foliar N accumulation (mg N/top) of 10-week-old slash pine seedlings raised on a soil-quartz sand mixture. However, simazine at 0.10 ppm appeared to enhance the growth and foliar N content of slash pine seedlings. Simazine (0.10 ppm) applied with the 84 85 ammmnium source of N (14 and 42 ppm N) tended to increase the foliar N concentration of seedlings more than did ni- trate N and simazine at similar levels. Simazine applied 'with.both l4 and 42 ppm N of either nitrate or ammonium N did not significantly increase the foliar N accumulation of seedlings over where the N sources were applied alone. Simazine treatments (0.05 and 0.10 ppm) did not signifi- cantly affect either total green or dry seedling weights. Second Growth Chamber Study--Slash Pine witHWSimazine In this study slightly higher levels of simazine were used.» The foliar N concentration of lZ-week-old slash pine seedlings was increased by simazine applications of 0.2, 0.4, and 0.8 ppm. The maximum increase was 43.8 percent at the highest simazine level. This increase was almost equivalent to adding 84 ppm N of either nitrate or ammonium N in the nutrient solution. N accumulation was increased 26.2 percent when 0.4 ppm simazine was applied to the soil. Simazine appears to enhance N accumulation in seedling foliage. Increased foliar N content from sub- toxic applications of simazine has also been shown for other crops (DeVries, 1963; Ries 32431., 1967). A decrease in foliar N accumulation between the 0.4 and 0.8 ppm sima- zine treatments (although an increase over the control) was a result of decreased foliage dry weight at the 0.8 ppm simazine level. Simazine applied at 0.8 ppm with both 86 nitrate and ammonium additions (84 ppm N) increased the foliar N concentration of seedlings 70.8 and 65.4 percent, respectively. The effect of simazine on foliar N accumula- tion decreased as the nitrate level approached the optimum. Conversely, at the higher levels of both ammonium N (84 ppm N) and simazine (0.8 ppm), N accumulation was greatest. This increase in foliar N with increasing herbicide appli- cation occurs without any significant change in seedling mass. Increasing concentrations of soil applied simazine (0.2 - 0.8 ppm) did not significantly alter either green or dry seedling weights. However, the higher simazine rate (0.8 ppm) decreased both the green and dry weights of all plant parts. Third Growth Chamber Study-—Slash and Loblollprine‘with Atrazine In the final growth chamber study a second species, loblolly pine, was included and atrazine was substituted for simazine as the herbicide. Low level soil applications of atrazine (0.4 ppm) increased the foliar N concentration of ll—week-old slash and loblolly pine seedlings. How- ever, this increase was considerably less than where either nitrate or ammonium (84 ppm N) sources of N were applied alone. The foliar N accumulation in slash pine seedlings was increased by 0.4 ppm atrazine applied both alone and with supplemental N in the nutrient solution. However, atrazine and N treatments depressed the foliar N accumulation 87 in loblolly pine seedling, except when 0.4 ppm atrazine was applied with either nitrate or ammonium sources of N (84 ppm N). Atrazine (0.4 ppm) treatment decreased the green and dry weights of loblolly pine seedlings. Greenhouse Study--Slash Pine with SimaZine Foliar Nitrogen Content.--The foliar N concentra- tion of 16-week-old slash pine seedlings was increased by applications of 0.5, l, and 2 ppm simazine. The maximum increase was 91 percent at the highest simazine level. This increase in foliar N concentration was greater than when 84 ppm N of either nitrate or ammonium N was applied in the nutrient solution. The foliar N concentration of seedlings treated with simazine (0.5 - 2 ppm) and both levels (28 and 84 ppm N) of either nitrate or ammonium N was greater than when these N sources were applied alone. Simazine treatment at 0.5 and 1 ppm did not af— fect the foliar N accumulation of seedlings. However, 2 ppm simazine applied both alone and with nitrate or ammonium sources of N significantly depressed N accumulation. Thus, simazine applied at this concentration to seedlings at this stage of development was probably phytotoxic. It is apparent that the significant increases in foliar N concen- tration from simazine treatment in this study were primarily a function of decreased seedling biomass. 88 Simazine treatment at 2 ppm increased foliar ni- trates. No differences were found in foliar nitrates be- tween nitrate and ammonium treated seedlings, either alone or with simazine. This implied that possibly the ammonium ion was as effectively utilized with simazine as was the nitrate source of N. If this is true, the effect of sima- zine on foliar N accumulation is not necessarily a function of increased nitrate reductase activity accelerating nitrate uptake by plants as suggested by Ries gt_§l. (1967). How- ever, it is possible that N-Serve added to inhibit nitrifi- cation of ammonium N was not effective and part of the growth and increased N from ammonium N and simazine was actually a nitrate response. Simazine and Growth Reduction.--In contrast to the growth chamber where low levels of simazine (0.05w-0.8 ppm) did not affect the green cn: dry weights of slash pine seedlings, decreases in both green and dry seedlings weights were found where slightly higher concentrations of simazine (1 - 2 ppm) were applied in the greenhouse. Simazine treatment depressed root weight more than foliage weight. For example, simazine applications at 0.5 ppm significantly reduced both green and dry root weights, but did not effect either green or dry foliage weights. Since N additions to simazine treated plants failed to significantly improve growth, simazine as these concentrations is probably inter- fering in some way with physiological growth processes. 89 Field Stud --Simazine and Atrazine 1 aE HerbicifiaI Rates Foliar Mineral Nutrition.—-The lower rate of soil \ applied atrazine (2.25 kg/ha) increased the foliar Mg con- centration of field planted Scotch pine, white spruce, and balsam fir over the control. Atrazine treatment did not affect the foliar concentration of any other elements the first season other than Mg. However, the 2.25 kg/ha atra- zine application the second year significantly increased the foliar N concentration of white spruce over the control and 4.50 kg/ha treatment. Simazine applied at herbicidal rates (4.50 and 9.00 kg/ha) did not significantly alter the foliar concentration of any element for either species I the first growing season. Neither did simazine treatment affect the foliar N concentration of seedlings the second year. A greater herbicidal injury and mortality occurred in white spruce than the other species. Since the phyto- toxic effect of triazine treatment on this species appeared greater when the herbicides were applied alone, it is pos- sible that supplemental N to the triazine plots might have counteracted herbicide phytotoxicity. It is also possible that differential uptake and/or distribution of the herbi- cides occurred among the three species as the white spruce was most significantly affected. Ammonium nitrate fertilizer applied to simazine Plots significantly decreased the foliar concentrations 90 0E P, Ca, Cu, Zn, and B for various species. Supplemental N applied with atrazine significantly decreased foliar P and B, and increased foliar Mg and Mn for some species. Ammonium nitrate applied with simazine and atrazine the first year did not affect the foliar concentrations of N, K, Na, Ca, Fe, or A1 for either species. Neither did N applied to simazine plots affect foliar Mg or Mn, or N applied with atrazine affect foliar Cu or Zn of any seedlings. A fresh hardwood chip mulch, used as a non-phytotoxic weed control treatment, significantly increased the foliar concentrations of N and P, decreased foliar Ca, Mg, and Fe, and had no effect upon foliar K, Na, Cu, B, Zn, or Al for either species. Silvicultural Implications The majority of sites on which coniferous trees are planted are inherently low in fertility or degraded by past use. Nitrogen is one of the most important ele- ments in plant nutrition and a commonly deficient element in trees growing on poor soils. From growth chamber re— sults where non-phytotoxic levels of simazine (0.1 - 0.4 ppm) enhanced the foliar N accumulation of slash pine seedlings without significantly altering seedling biomass, it appears that a nutritional gain from low level applica- tions of simazine might be an efficient way of supplying ,) ‘1”.-. 4.441.411 IA, A all.“ . . I 11:31le x 4... 91 available N to trees growing on low fertility soils. Also, low level applications of triazines to coniferous seed beds may provide some weed control in addition to a nutri- tional gain. However, the differential tolerance among various species to triazine compounds must be considered. At present, additional research is needed to further sub- stantiate a "nutritional bonus" in addition to weed control from triazine compounds applied at sub-toxic levels. Low level applications of triazines and their interaction with both soil and applied nutrients should be tested in the field using a variety of species under different soil and climatic conditions. When a herbicidal rate of simazine (2 ppm) was ap- plied to seedlings in the greenhouse, the significant in— crease in foliar N concentration was primarily a function of growth reduction. Although foliar N concentration is not always correlated with growth, triazine treatment to coniferous trees at herbicidal rates may significantly in- fluence: (l) resistance to disease, (2) resistance to insect attack, (3) seed production, and (4) frost resistance. In addition, the use of herbicidal rates of simazine and atrazine for weed control in young tree plantations may influence a delayed nutritional gain in subsequent growing seasons after triazine compounds are degraded to lower levels in the soil. . ll IIIIIII 92 Although low level applications of triazines in this study were tested only under controlled environment, this research will provide some useful guidelines for other investigators. ligl( .. I LITERATURE CITED Addoms, R. M. 1937. Nutritional studies on loblolly pine. Plant Physiol. 12:199-205. F1 Ashton, F. M., E. M. Gifford, Jr., and T. Bisalputra. 3 1963. Structural changes in Phaseolus vulgaris induced by atrazine I. Histological changes. Bot. Gaz. 124(5):329—335. Ashton, F. M., G. Zweig, and G. W. Maspg. 1960. The ef- “ fect of certain triazines on C 02 fixation in red kidney beans. Weeds 8:448-451. Bartley, C. E. 1957. Simazine and related triazines as herbicides. Agr. Chem. 12:34-36, 113-115. Burnside, O. C., E. L. Schmidt, and R. Behrens. 1961. Dissipation of simazine from the soil. Weeds 9:477-484. Burschel, P. 1961. Studies on the behavior of simazine in soil. Weed Research 1:131-141. Castelfranco, P., C. H. Foy, and D. B. Duetsch. 1961. Non-enzymetic detoxification of 2-chloro-4, 6-bis (ethylamino)-s-triazine (simazine) by extracts of £93 may . Weeds 9:580—591. Conner, B. J. and D. P. White. 1968. Triazine herbicides and the nitrogen nutrition of conifers. Quart. Bull., Mich. Agric. Exp. Sta. 50(4):497-503. Davis, D. E., H. H. Funderburk, Jr., and N. G. Sansipg. 1959. The absorption and translocation of C - labeled simazine by corn, cotton, and cucumber. Weeds 7:300—309. DeVries, M. L. 1963. The effect of simazine on Monterey pine and corn as influenced by lime, bases and aluminum sulfate. Weeds 11:220-222. 93 94 Dumbroff, E. B., and B. E. Michel. 1967. The expression of interionic relationships in Pinus elliottii. Plant Physiol. 42:1465-1471. Eastin, E. F., R. D. Palmer, and C. O. Grogan. 1964. Ef— fect of atrazine on catalase and peroxidase in resistant and susceptible lines of corn. Weeds 12:64-65. Eliason, E. J. 1954. The use of oil spray to control weeds in coniferous seedbeds. Proc. 8th Annual Meeting N. E. Weed Control Conf. Exer, B. 1958. Der Einfluss von Simazin auf den Pflanzen- stroffwechsel. Experientia 14:136—137. tion of loblolly pine and Virginia pine with special reference to nitrogen and phosphorus. Forest Sci. 5(1):95~112. I" Fowells, H. A., R. W. Krauss. 1959. The inorganic nutri- g. Freeman, F. W., D. P. White, and M. J. Bukovac. 1964. Uptake and differential distribution of C14-1abeled simazine in red and white pine seedlings. Forest Sci. 10(3):330—334. Freney, J. R. 1965. Increased growth and uptake of nu- trients by corn plants treated with low levels of simazine. Aust. J. Agri. Res. 16:257-263. Funderburk, H. H., r., and D. E. Davis. 1963. The metab- olism of Cl chain-and—ring-labeled simazine by corn and the effect of atrazine on plant respiratory systems. Weeds 11:101-104. Gast, A. 1958. Beitrage zur Kenntnis der phytotoxischen Wirkung von Triazinen. Experientia 14:134-136. Goring, C. A. 1962. Control of nitrification by 2-chloro-6- (trichloro—methyl) pyridine. Soil Sci. 93:211-218. Gramlich, J. V. and D. E. Davis. 1967. Effect of atra- zine on nitrogen metabolism of resistant species. Weeds 15:157-160. Guillemat, J., M. Charpentier, P. Tardieux, and J. Pochon. 1960. Interactions entre une chloro-amino—triazine herbicide et la micro-flore fongique et bacterienne du sol. Annales dex Epiphyties. 261—295. 95 Gysin, H. 1962. Trizaine herbicides; their chemistry, biological properties and mode of action. Chem- ‘istry and Industry, pp. 1393-1400. 1Hami1ton, R. H. 1964. Tolerance of several grass species to 2-chloro-s-triazine herbicides in relation to degradation and content of benzoxazinone deriva- tives. J. Agri. Food Chem. 12(1):14-17. Hamilton, R. H., and D. E. Moreland. 1962. Simazine degradation by corn seedlings. Sci. 135:373-374. Hoagland, D. R., and D. I. Arnon. 1938. The water cul- ture method for growing plants without soil. Calif. Agr. Exp. Sta. Circ. 347. Kozlowski, T. T., and J. E. Kuntz. 1963. Effect of sima- zine, atrazine, prOpazine, and eptam on growth and development of pine seedlings. Soil Sci. 95:164- 174. Lowe, R. H.. and J. L. Hamilton. 1967. Rapid method for ' . determination cf nitrate in plant and soil ex- tracts. J. Agr. Food Chem. 15:359-361. Montgomery, M. L., and V. H. Freed. 1964. Metabolism of triazine herbicides by plants. J. Agr. Food Chem. 12(1):11-14. Montgomery, M. L., and V. H. Freed. 1961. The uptake, translocation and metabolism of simazine and atrazine by corn plants. Weeds 9:231-237. Montgomery, M. L., and V. H.-Freed. 1960. The metabolism of atrazine by expressed juice of corn. Res. Prog. Rept. Western Weed Control Conf. p. 71(abstr.) Moreland, D. E., W. A. Gentner, J. L. Hilton, and K. L. Hill. 1959. Studies on the mechanism of herbi- cidal action of 2-chloro-4, 6-bis (ethylamino)-s- triazine. Plant Physiol.. 34(4):432-435. Negi, N. S., H. H. Funderburk, Jr., and D. E. Davis. 1964. Metabolism of atrazine by susceptible and resis- tant plants. Weeds 12:53-57. Ragab, M. T. H., and J. P. McCollum. 1961. Degradation of C14-labeled simazine by plants and soil micro- organisms. Weeds 9:72-84. 96 Reid, J. J. 1960. Bacterial decomposition of herbicides. Proc. North Eastern Weed Cont. Conf. 14:19-30. Ries, S. K., H. Chmiel, D. R. Dilley, and P. Filner. 1967. The increase in nitrate reductase activity and protein content of plants treated with simazine. Proc. Nat. Acad. Sci. U. S. 58:526-532. Ries, S. K., and A. Gast. 1965. The effect of simazine on nitrogenous components of corn. Weeds 13:272- 274. ~ Ries, S. K., R. P. Larsen, and A. L. Kenworthy. 1963. The apparent influence of simazine on nitrogen nutrition of peach and apple trees. Weeds 11:270- 273. Roth, W., and E. Knulsi. 1961. Beitrag zur Kenntnis der Resisteanhanomene einzelner pflanzen gegenuber dem phytotoxischen Wirkstoff Simazin. Experientia 17:312-313. Roth, W. 1958. Recherches sur l'action selective de sub- stances herbicides du groupe des triazines (A dissertation) Universite de Strasbourg. pp. l-92. Sabey, B. R. 1968. The influence of nitrification sup- pressants on the rate of ammonium oxidation in midwestern USA field soils. Soil Sci. 32:675-679. Sheets, T. J. 1961. Uptake and distribution of simazine by oats and cotton seedlings. Weeds 9:1-13. Shimabukuro, R. H. 1967. Atrazine metabolism and herbi- cidal selectivity. Plant Physiol. 42:1269—1276. Tiedjens, V. A. 1934. Factors affecting assimilation of ammonium and nitrate nitrogen, particularly in tomato and apple. Plant Physiol. 9:31-57. Toumey, J. W., and C. F. Korstian.. 1942. Seeding and planting in the practice of forestry. Third Edi- tion, John Wiley and Sons, Inc., New York, N.Y. Tweedy, J. A., and S. K. Ries. 1967. Effect of simazine on nitrate reductase activity in corn. Plant Physiol. 42:280-282. 97 ‘White, D. P. 1960. Effect of fertilization and weed con— trol on the establishment, survival and early growth of spruce plantations. Proc. VII Congr. Int. Soil Sci. 3:355-362. ‘Winget, C. H., T. T. Kozlowski, and J. E. Kuntz. 1963. Effect of herbicides on red pine nursery stock. Weeds 11:87-90. Zweig, G., and F. M. Ashton. 1962. The effect of 2-chloro- 4—ethy1amino-6-isoprppylamino-s-triazine (atrazine) on distribution of C compounds following C1402 fixation in excised kidney bean leaves. J. Exptl. Botany 13:5—11. APPENDIX 1.18 'V— V ‘5‘“.303‘8. -1 a 4. .43 APPENDIX A Appendix Table I.-—Effect of simazine and nitrogen treatments on the foliar nitrogen concentration and accumulation of slash pine seedlings grown in the growth chamber for 10 weeks; Experi- ment 1 (means of 4 replications). TreatmentIi Foliar Nitrogen2 _ I 3 % N Increase No. Simazine NO3 NH: Conc. Accum. over Control ppm --ppm N-— % mg/top Conc. Accum. 1 -- -- -- 1.35 4.84 -— —— 2 -- l4 —- 1.63 7.27 20.7 50.0 3 -- 42 —- 1.85* 7.31 37.0 51.0 4 -- -- 14 1.88* 8.06** 39.3 66.5 5 -- —— 14 1.77 7.47 31.1 54.5 6 -- -- 42 l.95** 7.30 44.4 50.8 7 -- —- 42 1.87* 8.54** 38.5 76.4 8 0.05 -~ -- 1.43 5.39 5.9 11.4 9 0.05 14 —- 1.78 7.18 31.8 48.4 10 0.05 42 -- 1.87* 7.99** 38.5 65.3 11 0.05 -— 14 1.68 6.93 24.4 43.0 12 0.05 —- 14 1.85* 7.44 37.0 53.7 13 0.05 -- 42 l.93** 6.90 43.0 42.6 14 0.05 —— 42 1.80 6.84 33.3 41.3 15 0.10 —- —— 1.50 6.36 11.1 31.4 16 0.10 14 —- 1.78 6.77 31.8 39.9 17 0.10 42 -- 1.80 7.10 33.3 46.7 18 0.10 -- 14 l.93** 7.96** 43.0 64.5 19 0.10 —- 14 1.90* 7.05 40.7 45.7 20 0.10 ' -- 42 1.97** 7.02 ' 45.9 45.0 21 0.10 -- 42 l.92** 7.49* 42.2 55.0 Tukey's w (.05) 2.65 (.01) 3.04 1Treatments 5, 7, 12, 14, 19, and 21 contained N— Serve at a rate of 10% of the respective N level. 2Determined‘on a dry weight basis. See Table 11 for statistical significance. 3Significance determined from transformed data (arc- sin /§), thus no Tukey's w shown. * Significantly greater than control at 0.05 level. ** Significantly greater than control at 0.01 level. 98 E. “MI-Utah“ IIHIIHIRIMII' .wosmoHMHcmHm HMOHumHumum Mom NH mems mom N .Hw>wH z 0>Huommmmu on» no wOH mo mums 8 us w>uwmIz wasHmusoo HN pom .mH .vH4NH .h 4m mpcmsumoHBH mH.m om.H Nv.o mH.o NN.H Nv II oH.o HN mv.m Hh.H mm.o mH.o MN.H Nv II CH.o 0N mm.m Nh.H mm.o NH.o vN.H «H II 0H.o mH mm.m Hm.H Nv.o MH.o hm.H 4H II OH.o mH Hm.m mm.H o¢.o mH.o Hv.H II Nv OH.o hH oH.v an.H mm.o «H.o om.H II 4H 0H.o mH mN.m mo.N bv.o mH.o 4v.H II II OH.o mH mH.m vh.H mm.o MH.o NN.H Nv II mo.o 4H om.m om.H N¢.o MH.o VN.H Nv II mo.o mH wm.m mm.H ow.o vH.o ov.H vH II mo.o NH wN.m mo.N om.o mH.o Nv.H 4H II mo.o HH W em.m No.N ow.o NH.o me.H II Nv mo.o OH ow.m NN.H Hw.o 4H.o Nm.H II vH mo.o m mm.N Hm.H mm.o HH.o 0H.H II II mo.o m mm.m wH.N mv.o mH.o mm.H Nv II II n Nm.m om.H ow.o HH.o mN.H Ne II II o hm.m em.H ov.o mH.o mm.H «H II II m OH.m mo.N om.o mH.o mw.H 4H II II 4 Nm.m om.H Hv.o mH.o om.H II Nv II m mv.m mm.H Nw.o mH.o N¢.H II 4H II N mH.m mm.H mm.o NH.o mH.H II II II H m IIz EmmII Ema uOOM\meHHom Hmuoa uoom Ewum mmMHHom wmz , moz ocHNmEHm .oz NusmHmS smwuo Husmaummua .AmCOHumoHHmmH v mo mammfiv H usmEHmexm umxmw3 OH How nmnfimno zpsonm may sH szoum mmcHprww msHm AmMHm mo usmHos comma as» so mucosumouu somouuHc pom mcHumsHm mo pooMMMII.HH mHnt prsmmms II... IIII.I. III. .III .mosmoHMHcmHm HmoHumHumum mom NH can HH menma mom N .Hm>mH z m>HuommmmH may mo 40H «0 mums m an m>nmmIz pmchusoo HN can .mH 40H .NH .5 .m mucmeummHBH 0N.N H0.0 mH.0 m0.0 mm.0 0.0N Nv II 0H.0 HN mm.N mm.0 vH.0 v0.0 hm.0 h.mN N4 II 0H.0 0N mm.N mm.0 mH.0 00.0 0m.0 0.0N 0H II 0H.0 0H HN.m H0.0 0H.0 m0.0 Nv.0 0.0m «H II 0H.0 0H 00.N mm.0 mH.0 m0.0 mm.0 0.0m II N0 oH.o NH NN.m 0m.0 MH.0 v0.0 mm.0 v.mN II vH 0H.0 .mH hm.N m0.0 hH.0 00.0 mv.0 0.0m II II 0H.0 mH 00.N mm.0 mH.0 00.0 mm.0 0.0m Nv II m0.0 4H mn.N mm.0 mH.0 00.0 0m.0 5.0N N0 II m0.0 mH m0.N No.0 hH.0 m0.0 Hv.0 «.mN 4H II m0.0 NH n4 mm.N m0.0 0H.0 m0.0 H¢.0 5.0N «H II m0.0 HH nu H0.m no.0 mH.0 m0.0 00.0 N.mN II N0 m0.0 0H 1. mn.N H0.0 0H.0 m0.0 Hv.0 n.0m II 0H m0.0 m ev.N wm.0 0H.0 40.0 mm.0 H.0m II II m0.0 m 00.N No.0 mH.0 40.0 00.0 0.0N N4 II II n hm.N 0m.0 mH.0 m0.0 hm.0 m.mN Nv II II 0 mm.N No.0 mH.0 m0.0 Nv.0 0.0m «H II II m m0.m m0.0 mH.0 m0.0 mv.0 0.0N vH II II 4 N0.m mm.0 4H.0 m0.0 04.0 N.Hm II N0 II m 0n.N no.0 eH.0 m0.0 mv.0 0.Nm II 4H II N hH.N hm.0 hH.0 00.0 0m.0 H.0m II II II H IIIIIIIIIIIII mIIIIIIIIIIII w IIz EmmII Ema uoomxmmmHHom Hmuoa boom swam omMHHom wmz moz mcHNmsHm .oz Nuanmz who Hucmsummus .HmsoHumoHHmmu 4 mo mommav H ucmsHummxm «mxoms 0H Mom Honfimao nusonm 0:» 2H csoum mmcHHpmmm mch cmMHm mo uzmHm3 auc may no mucmEumoHu cmmouuHc pom mcHnmfiHm mo uommmmII.HHH mHoma prsmmm< APPENDIX B Appendix Table IV.--Effect of simazine and nitrogen treat- ments on the foliar nitrogen concentra- tion and accumulation of slash pine seedlings grown in the growth chamber for 12 weeks; Experiment 2 (means of 4 replications). Treatment1 Foliar Nitrogen2 _ 4_ -——————§ % N Increase NCM Simazine NO3 NH4 Conc. Accum. Over Control ppm --ppm N-- % mg/tOp Conc. Accum. 1 -- -- -- 1.30 4.93 -- -- 2 -- 28 -- 1.58 5.79 20.8 17.4 3 -- 84 -- 1.97** 5.05 51.5 2.6 4 -- -- 28 1.55 7.30 19.2 48.1 5 -- -- 84 l.90** 5.37 46.2 8.9 6 0.2- -- -- 1.63 5.55 24.6 12.6 7 0.2 28 —- 1.50 6.16 15.4 24.9 8 0.2 84 -- 2.00** 5.94 53.8 20.5 9 0.2 -- 28 1.70 5.77 30.8 16.8 10 0.2 -- 84 2.02** 6.16 55.4 24.9 11 0.4 -— -- 1.55 6.22 19.2 26.2 12 0.4 28 -- 1.53 6.17 16.9 25.2 13 0.4 84 -- 2.07** 5.30 59.2 7.5 14 0.4 -- 28 2.13** 6.52 62.3 32.2 15 0.4 -- 84 1.95** 7.27 50.0 47.5- 16 0.8 -— —- 1.87** 5.86 43.8 18.9 17 0.8 28 -- 2.07** 7.24 58.5 46.9 18 0.8 84 -- 2.22** 7.18 70.8 45.6 19 0.8 -- 28 1.92** 5.79 47.7 17.4 20 0.8 -- 84 2.15** 8.22* 65.4 66.7 Tukey's w (.05) 3.07 (.01) 3.52 1Treatments with NH4—N contained N-Serve at a rate of 10% of the respective N level. 2Determined on a dry weight basis. statistical significance. See Table 13 for 3Significance determined from transformed data (arc- sin /§), thus no Tukey's w shown. * Significantly greater than control at 0.05 level. ** Significantly greater than control at 0.01 level. 101 .mosmoHMHamHm HMOHumeMUm Mom 0H mHama mmmN .Hm>mH z m>Huommm Imu mau mo 40H mo mums m um m>nmmIz pmsHmuaoo ZI0mz auHs muamaummns H 00.N 00.H 00.0 MH.0 NN.H 00 II 0.0 0N 00.N mm.H hm.0 HH.0 00.H 0N II 0.0 0H 00.m 0m.H Hm.0 HH.0 mH.H II 00 0.0 0H 00.0 NN.H 0m.0 MH.0 0m.H II 0N 0.0 5H m0.m 00.H 0m.0 HH.0 00.H II II 0.0 0H mm.N N0.H 00.0 NH.0 HN.H 00 II 0.0 mH 0m.N mm.H 00.0 HH.0 00.H 0N II 0.0 0H wm.m Nm.H 0N.0 HH.0 Nm.0 II 00 0.0 MH H0.N 00.H 00.0 NH.0 Hm.H II 0N 0.0 NH 2 00.N hm.H 00.0 MH.0 mN.H II II 0.0 HH m 00.N Hm.H mm.0 HH.0 H0.H 00 II N.0 0H HH.m m0.H mm.0 HH.0 0H.H 0N II N.0 m HH.m 0m.H 0m.0 HH.0 m0.H II 00 N.0 m Hm.N mm.H 00.0 0H.0 mm.H II 0N N.0 h hm.m mh.H hm.0 MH.0 0N.H II II N.0 0 mm.N Nm.H 0m.0 00.0 00.0 00 II II m NH.m mm.H m0.0 mH.0 0m.H 0N II II 0 00.m hm.H 0m.0 HH.0 N0.0 II 00 II m m0.N NN.H 00.0 MH.0 0N.H II 0N II N 0m.N Nm.H 0m.0 MH.0 0H.H II II II H m IIz Emmll Ema uOOM\mmmHHom Hmuoe uoom Emum mmmHHom Nmz moz maHNmEHm .oz Nuavaz ammuw Husmfiummua .AmsOHumoHHmmu 0 mo mammEv N usmEHHmmxm “mamms NH MOM HmaEmao auzoum mau sH asoum mmaHHpmmm maHm amem no uamHmS ammum may no mucmaummum ammonuHa 0am maHNmEHm mo uommmmII.> mHama prammma .mUGMOHMHamHm HMUHumHumam How 0H 0am mH mmHama mmmN .Hm>mH z m>Huommm Imu may no 40H «0 mums m as m>ummIz pmaHmuaoo zI0mz auHs muamaummueH mm.N mm.o mH.o m0.0 mm.0 m.mN 00 II 0.0 0N mm.N 00.0 NH.0 00.0 om.0 m.mN 0N II 0.0 0H mH.m 00.0 HH.0 no.0 Nm.0 o.NN II 00 0.0 0H 0N.N Nm.0 NH.0 00.0 0m.0 0.0N II 0N 0.0 0H HH.m m0.0 oH.o no.0 Hmwo 0.0N II II 0.0 0H 0N.N mm.0 hH.o m0.0 mm.0 0.0m 00 II 0.0 mH mm.H Hm.0 oH.o 00.0 Hm.0 m.oN mN II 0.0 0H 00.N 00.0 00.0 00.0 0N.0 m.mN II 00 0.0 NH 0m.N 00.0 oH.o 00.0 00.0 0.0m II 0N 0.0 NH 0m.N 00.0 oH.o 00.0 00.0 >.Hm II II 0.0 HH 1. HN.N 00.0 0H.0 00.0 Hm.0 0.0m 00 II N.0 0H m HEN 8.0 NHS 00.0 0m.o ~.mm mm II N.0 m 00.N 00.0 NH.0 00.0 om.0 0.0N II 00 N.0 m m0.N H0.0 oH.o no.0 00.0 m.mN II 0N N.0 h N0.m Hm.0 NH.0 00.0 mm.o 0.0N II II N.0 0 0m.N 00.0 HH.0 no.0 0N.0 0.Nm 00 II II m 00.N H0.0 mH.0 m0.0 00.0 0.0m 0N II II 0 0m.N 00.0 HH.0 00.0 mN.0 m.>N II 00 II m 00.N hm.0 oH.o m0.0 hm.o 0.0N II mN II N No.N 00.0 oH.o 00.0 hm.0 m.Nm II II II H I IIIII I IIIII mIIIIIIII IIIII m IIzgammII Ema- uoom\mmMHHom Hmuoe uoom Emum mmMHHom wmz moz maHnmEHm .oz NuamHmB who I. . . . . . Huamaummua .AmGOHummHHmmH,0-uo mammev N pamaHummxm.xmamm3 NH How umaEmao apsoum mau aH ozonm mmaHHpmmm maHm amMHm mo uamHms map map so muamsummuu ammouuHs 0am maHNmEHm mo uommmmII.H> mHama prammmd IlII'lII‘I‘ .1(Ill‘|.I II APPENDIX C Appendix Table VII.--Effect of atrazine and nitrogen treat- me ti lo nts on the foliar nitrogen concentra- on and accumulation of slash and blolly pine seedlings grown in the growth chamber for 11 weeks; Experiment 3 (means of 4 replications). l . . 2 Treatment Foliar Nitrogen _ + 3 % N Deviation No. Atrazine NO3 NH4 Conc. Accum. from Control ppm -—ppm N-- % mg/top Conc. Accum. Slash Pine 1 -- -- -- 1.58 3.91 —- —— 3 -- —- 84 2.07** 4.97 32.5 27.1 4 0.1 -- -- 1.50 3.72 -4.4 -4.9 5 0.1 84 —— 2.35** 3.98 49.0 1.8 6 0.1 -- 84 2.00** 4.58 27.4 17.1 7 0.4 -— -- 1.63 4.42 3.2 13.0 8 0.4 84 -— 2.32** 4.21 46.5 7.9 9 0.4 -1 84 2.12** 7.07** 35.0 80.6 Loblolly Pine 1 -— —— -- 1.63 3.15 —- -— 3 -- -- 84 2.10** 2.62 30.4 —16.8 4 0.1 _-' —- 1.63 2.39 0.6 -2401 5 0.1 84 —- 2.05** 2.37 26.7 -24.8 6 0.1 -- 84 2.20** 2.88 36.6 -9.2 7 0.4 -- -- 1.82 2.42 13.0 -23.2 8 0.4 84 -- 2.47** 3.63 53.4 14.9 9 0.4 -— 84 2.20** 3.21 36.6 2.2 Tukey's w (.01)4 2.57 l 10% of th 2 Treatments with NH -N contained N-Serve at a rate of e N level. 4 Determined on a dry weight basis. 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IIz Small ,Emm uoomxmeHHom Hmuoe room swam mmmHHom Nmz moz mcHNmuua .oz NuamHmB nun Hwameammna .HmaOHuMOHHQmH 0 mo mammev m usmEHnmmxm «mammz HH MOM uma IEmao ausonm map aH ozonm mmaHHpmmm maHm nHHoHaOH 0am ammHm mo pamHms and map so musmEummHu smonuHa 0am maHanum mo pomMMMII.xH mHama xHUammma APPENDIX D Appendix Table X.—-Effect of simazine and nitrogen treatments on the foliar nitrogen concentration, ac- cumulation, and nitrates of slash pine seedlings grown in the greenhouse for 16 weeks (means of 4 replications). Treatment1 Foliar Nitrogen2 _ _ + 3 % N Deviation No. Sima21ne NO3 NH4 Conc. Accum. Nitrates from Control ppm --ppm N— % mg/top ug/g Conc. Accum. 1 —- -- -- 1.26 13.15 13.86 -— -- 2 -- 28 -- l.73** 19.09 13.70 37.3 45.2 3 -- 84 -- l.7l** 16.17 12.29 35.7 23.0 4 —- -- 28 1.43 15.43 13.33 13.5 17.3 5 -- -- 84 1.85** 21.49** 11.08 46.8 ‘63.4 6 0.5 -- -- 1.61 13.67 12.65 27.8 4.0 7 0.5 28 -- l.89** 17.68 11.55 50.0 34.4 8 0.5 84 -— 1.86** 18.37 11.97 47.6 39.7 9 0.5 -— 28 l.78** 17.30 12.86 41.3 31.6 10 0.5 -- 84 2.21** 23.66** 11.60 75.4 79.9 11 1.0 -— —- 1.99** 14.04 13.54 57.9 6.8 12 1.0 28 -— 2.13** 13.85 13.97 69.0 5.4 13 1.0 84 -- 2.23** 14.07 11.60 77.0 7.0 14 1.0 -- 28 2.08** 15.31 11.92 65.1 16.4 15 1.0 -- 84 2.26** 18.76 14.33 79.4 42.7 16 2.0 -- -- 2.41** 9.44 17.22 91.3 -28.2 17 2.0 28 -— 2.38** 11.12 12.60 88.9 -15.4 18 2.0 84 -- 2.63** 9.86 16.01 108.7 -25.0 19 2.0 —— 28 2.58** 10.57 16.64 104.8 -l9.6 20 2.0 -- 84 2.74** 11.64 17.38 117.5 -ll.5 Tukey's w (.05) 6.01 5.32 (.01) 6.90 6.11 1 All treatments contained N-Serve at a rate of 10% of the 84 ppm N level. 2Determined on a dry weight basis. statistical significance. 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Treatment1 . . - + . 2 . 3 No. SimaZine NO3 NH4 Height Diameter ppm ---ppm N-—— cm 1 -- —- -- 18.24 0.29 2 -- 28 -- 18.88 0.31 3 -— 84 -— 16.58 0.30 4 -- -- 28 18.81 0.30 5 -— 84 18.77 0.31 6 0.5 -- -- 20.20 0.26 7 0.5 28 -- 18.90 0.27 8 0.5 84 -- 18.41 0.28 9 0.5 —- 28 18.70 0.28 10 0.5 -- 84 19.38 0.29 11 1.0 —- -- 18.55 0.23** 12 1.0 28 -- 18.95 0.23** 13 1.0 84 -- 17.81 0.23** 14 1.0 -- 28 19.19 0.25 15 1.0 -- 84 19.55 0.26 16 2.0 -- -— 15.63 0.18** 17 2.0 28 —- 16.66 0.20** 18 2.0 84 -- 15.91 0.17** 19 2.0 -- 28 16.59 0.18** 20 2.0 -— 84 16.26 0.18** Tukey's w (.05) 3.11 0.046 .01) 3.56 0.052 1 of the 84 ppm N level. 2 . Measured from 3011 surface. 3Measured 2 cm above root collar. 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