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Quality changes in forage crops following applications of 2,4-D Vaartnou, Herman 1953

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QUALITY CHANGES IN FORAGE CROPS FOLLOWING APPLICATIONS OF 2,4-D by HERMAN VAARTNOU A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN AGRICULTURE In the Department of Agronomy We accept th i s thesis as conforming to the standard required from candidates for the degree of MASTER OF SCIENCE IN AGRICULTURE. Members of the Department of Agronomy THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1953. ABSTRACT Q u a l i t y Changes i n Forage Crops F o l l o w i n g A p p l i c a t i o n s o f 2,4-D by Herman Vaartnou A T h e s i s Submitted i n P a r t i a l F u l f i l m e n t o f the Requirements f o r the Degree o f Master of Science i n A g r i c u l t u r e In the Department of Agronomy The U n i v e r s i t y o f . . B r i t i s h Columbia A p r i l 1953. Very few i n v e s t i g a t i o n s have been made which r e p o r t d i r e c t l y on the e f f e c t o f the p l a n t growth r e g u l a t o r 2,4-D on a great group o f p l a n t s , p r i n c i p a l l y g r a s s e s and. legumes, the f o r a g e s used by l i v e s t o c k . Under c e r t a i n c o n d i t i o n s such growth r e g u l a t i o n , substances may e f f e c t c o n s i d e r a b l e changes i n the chemical composition o f h i g h e r , p l a n t s . The ; f a c t that forage value may be d e l i b e r a t e l y a l t e r e d by a p p l y i n g s u b - h e r b I c i d a l c o n c e n t r a t i o n s o f growth r e g u l a t o r s seems not to have been c a r e f u l l y s t u d i e d . An i n v e s t i g a t i o n appeared warranted and i s the s u b j e c t of t h i s r e s e a r c h . Under f i e l d c o n d i t i o n s s e v e r a l s u b - h e r b i c i d a l 2. l e v e l s of 2,4-D were applied at several stages i n the development of red and white clover, perennial ryegrass, Kentucky bluegrass, and timothy growing on Ladner clay and Alderwood loam. Appreciable increases In dry matter and nitrogen y i e l d obtained in certain treatments. Other s i g n i f i c a n t effects were noted i n flowering and seed prod-uction. Confirmation of the f i e l d r e s u l t s was obtained i n greenhouse studies on red clover and O l l i barley. A search f o r the source of the increased nitrogen did not reach a definable end but work with the influence of treated plants on s o i l Rhizobia and Azotobacter suggest that 2,4-D applied to forages may i n some instances modify the ecology of the s o i l f l o r a . ACKNOWLEDGEMENTS The writer wishes to acknowledge h i s appreciation of the assistance so generously provided by members of the " Department of Agronomy, i n p a r t i c u l a r , Dr. V. C. Brink, under whose d i r e c t i o n t h i s research was undertaken, and Dr. D. G. L a i r d , whose interest i n the bacteriological"aspects was • a stimulus. To my wife I must accord, not only patience and interest, but help i n the management of the plots i n the f i e l d . I am g r a t e f u l , too, to my stenographers, Miss Velma Smith and Miss Brownie Carnell, who have given most generously of t h e i r time and energy, and who have helped me over many of the d i f f i c u l t i e s i n my English. The assistance of Mr. Tom W i l l i s , Superintendent of the Dominion Range Experiment Station, Kamloops, B. C , i s recorded f o r sending me several hundred pounds of a special s o i l from the B. C. I n t e r i o r . TABLE OP CONTENTS Page I. THE INTRODUCTION. 1 I I . THE REVIEW OP LITERATURE 2 1. The Background f o r Modern Studies on Growth , Regulation in Plants 3 2. D e f i n i t i o n and Structural Requirements of Growth Regulators 7 3. Forms of Auxin In the Plant 9 4. Auxin Physiology •••• • • 9 a) The Fundamental Roles........ 9 b) Auxin Translocation .10 c) Auxins i n Ga l l and Nodule Formation. ...... 11 d) Auxins and Enzymes..;.. 12 e) Auxins and Vitamins 12 f) Auxins i n Mineral N u t r i t i o n 13 g) Auxins i n Nitrogen Metabolism...... 13 h) Auxins i n Carbohydrate Metabolism 16 i) Auxin and S o i l Micro-organisms 17 j) Auxins i n A g r i c u l t u r a l Practice. 19 I I I . THE REPORT ON CERTAIN FIELD, GREENHOUSE AND LABORATORY STUDIES 21 1. F i e l d T r i a l s , 1950-52 21 a) Location and Materials. 21 TABLE OF CONTENTS Page b) Methods... 22 c) R e s u l t s 25 Red C l o v e r . .. 23 White Clover,....,. , 25 Kentucky B l u e g r a s s . . 27 P e r e n n i a l Ryegrass 27 Timothy. 28 Seed P r o d u c t i o n o f Red and White C l o v e r . . 38 Seed P r o d u c t i o n o f the. Grasses........... 38 IV. GREENHOUSE TRIALS 45 Objects 45 M a t e r i a l . . 45 Methods.............................. • 45. a) . Red C l o v e r '•• 48 b) . O l l i B a r l e y 53 V. DISCUSSION , 64 VI , A SUMMARY'STATEMENT OF OBSERVATION AND CONCLUSIONS 67 LITERATURE' CITED. .. .. .. 70 TABLES: I... THE EFFECTS' OF. 2,4-D CONCENTRATION ON RED CLOVER 29 I I . THE EFFECT OF TIME OF 2,4-D.APPLICATION ON RED CLOVER ... 29 TABLE. OP- CONTENTS . Page TABLES: I I I . THE EFFECTS OF 2,4-D CONCENTRATION ON WHITE CLOVER .............. .( .... 30 IV. THE EFFECT OF TIME OF 2,4-D APPLICATION ON WHITE. CLOVER ., 30 V. THE EFFECTS OF 2,4-D CONCENTRATION ON KENTUCKY BLUEGRASS. .......................... 31 VI. THE EFFECT OF TIME OF 2,4-D APPLICATION ON KENTUCKY BLUEGRASS. 31 VII. THE EFFECTS OF 2,4-D CONCENTRATION ON. PERENNIAL RYEGRASS •••••• • •• 32 VIII. THE EFFECT OF TIME OF 2,4-D APPLICATION ON. PERENNIAL RYEGRASS......., ... 32 IX. THE EFFECTS OF 2,4-D CONCENTRATION ON TIMOTHY. ............ ..... 33 X. THE EFFECT OF TIME OF 2,4-D APPLICATION 0N; TIMOTHY 33 XI.. SEED PRODUCTION AND QUALITY IN RED CLOVER TREATED WITH Na 2,4-D IN DIFFERENT CONCENTRATIONS • • • • • • •. 40 XII. SEED. PRODUCTION AND QUALITY IN RED CLOVER. TREATED AT DIFFERENT STAGES OF DEVELOP-MENT WITH .1 LB. Na 2,4-D p.a.. 40 TABLE OF CONTENTS Page TABLES: XIII. SEED. PRODUCTION AND QUALITY IN. WHITE GLOVER TREATED WITH Na 2,4-D IN DIFFERENT CONCENTRATIONS ... 41 XIV., SEED PRODUCTION AND QUALITY IN • WHITE CLOVER TREATED AT DIFFERENT STAGES OF DEVELOPMENT WITH .1 LB. Na 2,4-D p.a .41 .. XV." SEED PRODUCTION. AND QUALITY IN KENTUCKY BLUEGRASS TREATED WITH Na 2,4-D IN DIFFERENT CONCENTRATIONS 42 XVI. SEED PRODUCTION AND QUALITY IN KENTUCKY BLUEGRASS TREATED AT DIFFERENT STAGES OF DEVELOPMENT WITH 1.0 LB. Na 2,4-D p.a. .... 42 XVII. SEED PRODUCTION AND QUALITY IN PERENNIAL RYEGRASS TREATED WITH Na 2,4-D IN DIFFERENT CONCENTRATIONS 43 XVIII. SEED PRODUCTION AND QUALITY IN PERENNIAL RYEGRASS TREATED AT DIFFERENT STAGES OF DEVELOPMENT WITH 1.0 l b . Na 2,4-D p.a. ... 43 XIX, SEED PRODUCTION AND QUALITY IN TIMOTHY TREATED WITH Na 2,4-D IN DIFFERENT CONCENTRATION '. 44 XX. SEED PRODUCTION AND QUALITY IN TIMOTHY TREATED AT DIFFERENT STAGES OF DEVELOP-MENT WITH 1.0 LB. Na 2,4-D p.a 44 TABLE OF CONTENTS Page TABLES: XXI. XXIA, XXII, XXIIA. XXIII. XXIV. NODULATION AND CRUDE PROTEIN PRODUCTION IN RED CLOVER GROWN IN THE GREENHOUSE, ON THREE SOIL TYPES TREATED WITH TWO CONCENTRATIONS OF Na 2,4-D AND. HARVESTED AT FOUR DIFFERENT STAGES. OF DEVELOPMENT.. 49 PRODUCTION OF RED CLOVER GROWN IN THE GREENHOUSE, ON THREE SOIL TYPES, TREATED WITH TWO CONCENTRATIONS OF Na 2,4-D AND HARVESTED AT FOUR DIFFERENT STAGES OF DEVELOPMENT........ 50 YIELD•OF DRY MATTER IN OLLI BARLEY GROWN IN THE GREENHOUSE.IN FOUR SOIL TYPES, WITH AND WITHOUT AZOTOBACTER VINELANDII AND A. CHROCOCCUM, WITH AND WITHOUT 2,4-D 56 YIELD OF CRUDE PROTEIN IN OLLI BARLEY GROWN IN THE GREENHOUSE IN FOUR SOIL TYPES, WITH AND•WITHOUT AZOTOBACTER VINELANDII AND. A. CHROCOCCUM, WITH, AND WITHOUT 2,4-D.. 57 CRUDE PROTEIN IN OLLI BARLEY, GROWN IN THE GREENHOUSE IN FOUR SOIL TYPES, WITH AND WITHOUT AZOTOBACTER VINELANDII AND A. CHROCOCCUM, WITH AND WITHOUT 2,4-D. 58 ESTIMATED AZOTOBACTER PER GRAM OF DRY SOIL FOLLOWING 2,4-D TREATMENTS OF PLANTS GROWN ON THE SOIL 59 TABLE OP CONTENTS FIGURES: Page 1. DRY MATTER YIELDS OF RED CLOVER GROWN ON LADNER CLAY AND TREATED ON MAY 12 WITH SEVERAL CONCENTRATIONS OF 2,4-D . 34 2. CRUDE PROTEIN YIELD PER ACRE IN RED CLOVER GROWN ON LADNER CLAY AND TREATED MAY 12 WITH SEVERAL CONCENTRATIONS OF 2,4-D 35 3. DRY MATTER YIELDS OF TIMOTHY GRASS ON LADNER CLAY AND TREATED WITH SEVERAL CONCENTRATIONS .OF 2,4-D 36 4. CRUDE PROTEIN YIELD PER ACRE IN TIMOTHY GROWN ON LADNER CLAY AND TREATED ON MAY 12 WITH SEVERAL CONCENTRATIONS OF 2,4-D 37 5. RED CLOVER GROWN IN THE GREENHOUSE IN POTS OF ALDERWOOD GRAVELLY LOAM AND TREATED AT THE THIRD TRUE LEAF STAGE WITH 2,4-D 51 6. RED CLOVER GROWN IN THE GREENHOUSE IN POTS OF LADNER CLAY AND TREATED AT THE FOURTH TRUE LEAF STAGE WITH 2,4-D 52 7. OLLI BARLEY GROWN ON ALDERWOOD GRAVELLY LOAM IN THE GREENHOUSE 60 8. OLLI BARLEY GROWN IN THE GREENHOUSE ON MUCK SOIL 61 9. OLLI BARLEY GROWN ON LADNER SOIL IN THE GREENHOUSE 62 10. A GRAPHICAL SUMMARY OF THE RESPONSES, IN TERMS OF DRY MATTER YIELD, OF GREENHOUSE GROWN OLLI BARLEY TO FOUR DIFFERENT SOILS, TO SOIL INOCULATION WITH AZOTOBACTER AND TO DIFFERENT CONCENTRATIONS OF 2,4-D 63 QUALITY CHANGES IN FORAGE CROPS FOLLOWING APPLICATIONS OF 2,4-D. . I THE INTRODUCTION Since 1935, when Kogl, Haagen-Smitji and Erxleben announced the Isolation of auxin a (auxentriolic acid) , many substances have been found to possess growth regulating properties, thousands of papers have been published about them,and dozens of uses with many species of plants have been described f o r them. However, very few papers report d i r e c t -l y on t h e i r e f f ects on a great group of plants, p r i n c i p a l l y grasses and legumes, the forages used by li v e s t o c k . Under s p e c i f i e d conditions growth regulating sub-stances may effect considerable changes i n the chemical composition of the plant. The fact that plants treated with such materials may have an altered n u t r i t i o n a l value has been given scant attention. The fact too that forage value might be altered a f t e r applying low concentrations of growth regulators seems not to have been entertained. An i n v e s t i -gation of the p o s s i b i l i t y appeared warranted and Is the subject of t h i s research. A f i r s t step to the solution of the problem of modifying the n u t r i t i o n a l value of forage using growth regulators was to define the nature and magnitude of the chem-i c a l changes and the circumstances under which they could be effected. The growth regulator used i n our studies was 2,4-D (2,4-dichlorophenoxyacetic acid) because i t i s 2. Inexpensive and widely used. Changes i n nitrogen metabolism and the associated changes i n carbohydrates were the subject of the most intensive studies. The special attention directed towards nitrogen metabolism arose as a r e s u l t of the fact that, on occasion at least, the crude protein or Kjeldahl Nitrogen percentage of a forage could often be increased without loss of dry matter y i e l d follow-ing l i g h t 2,4-D a p p l i c a t i o n . Since protein percentage i s a major factor i n determining forage q u a l i t y , special attention i n t h i s d i r e c t i o n seemed warranted. Attempts to f i n d reasons f o r the crude protein increases have l e d to studies with s o i l microorganisms known to be important i n nitrogen f i x a t i o n , notably the Rhizobia and Azotobacter. Forage whose composition had been modified by 2,4-D was not i n t h i s series of studies, subjected to that which should ultimately follow, feeding t r i a l s «with grazing animals. I I . THE REVIEW OF LITERATURE There are several excellent reviews on the subject of plant growth regulators. (103), (109), (81), (36). The following review i s therefore, i n a sense, a summary account, i n which special reference i s made to studies which appear to bear rather d i r e c t l y on t h i s research. 1. The background f o r modern studies on growth regulation i n plants. Julius Sachs, nearly a century ago, from a series of bold experiments, decided that there are special sub-stances which regulate the growth of d i f f e r e n t plant organs. His "lead", however, was not pursued very e f f e c t i v e l y and growth and movement i n plants, usually considered as d i s t i n c t l y d i f f e r e n t phenomena, were viewed as a r e s u l t of external s t i m u l i . Some support for Sachs* views were given by Ch. Darwin i n h i s t r e a t i s e (1881), e n t i t l e d , "Power of Movement i n Plants", wherein he noted that a l a b i l e substance i n certain plants moves from lower parts to upper parts causing movement of leaves and bending of shoots. Prior to the work of F i t t i n g (1907) l i t t l e penetrating investigation was undertaken, although the works of P f e f f e r (1890), Haberlandt (1905) and Oltmanns (1897) maintained interest In growth phenomena. F i t t i n g shed 4. new l i g h t on the conduction of the "phototropic stimulus" i n the coleoptiles of graminaceous plants. He found that the stimulus could be transmitted l a t e r a l l y as well as -lengthwise and that i t was not Impeded by two i n c i s i o n s on opposite sides of a c o l e o p t i l e up to 3/4 of the width. He found, too, that i n a plant exposed to a sublethal o temperature of 40 C an active p r i n c i p l e was i n h i b i t e d or Inactivated. Modest success attended F i t t i n g ' s e f f o r t s to chemically characterize a growth regulating substance i n orchid flowers. From the laboratory of F.A.F.C. Went, i n the second decade of t h i s century, came a series of investigations ascertaining threshold value and presentation time required f o r stimulation by l i g h t i n e t i o l a t e d coleoptiles of Avena 3 a t i v a . T h e techniques developed became fa v o r i t e s for experimentation r e l a t i v e to the auxin hypothesis. A t r u l y decisive step came i n 1926 when F.W.. Went isol a t e d i n agar cubes a substance, termed then, auxin. The problem, says Weevers, soon became one f o r the a n a l y t i c a l chemist. After a lengthy and d i f f i c u l t chemical research Kogl and h i s associates were able to prepare the auxin i n c r y s t a l l i n e form, but i n amounts so small that i t could not be f u l l y characterized - although over 100,000 maize coleoptiles were used In the i s o l a t i o n . Then, from a convenient source, human urine, Kogl, Haagen-Smith and Erxleben i s o l a t e d an active substance, l a t e r termed auxin-a, 5. i n s u f f i c i e n t quantities to f u l l y characterize i t . Auxin-a was shown to be a c i d i c , to have a molecular formula of C 1 8 H 3 2 0 5 a n d a Probable st r u c t u r a l formula of CH- H 0 CH, l 5 | 3 CH 3 - CHg - CH - GH CH - GH - CHg - CH 3 I H> H H HC C - G - GH - C - C - COGH 0 2 0 0 H H H Other auxins were obtained l a t e r . Prom maize o i l Kogl and h i s associates i s o l a t e d the less active auxin-b i n 1934, a substance with a molecular formula of C 18 H30^4 and a probable s t r u c t u r a l formula of H 2 GH_ p CH_ f 3 / ° \ I 3 CH 3 - CHg - CH - CH' CH - C - CHg - CH^ H H 0 CH - — • C - C - C - C - CH - COOH O H 2 H In 1934, too, Kogl Isolated hetero-auxin or Indole acetic acid. In p a r a l l e l work by Neilson, who i s o l a t e d i t , and by Thimann, who characterized i t , the same substance was isola t e d from cultures of the fungus, Rhizopus. suinus. It proved to have the formulae: C^QHQO^N and H G / \ HO c C I I I HC C CH • \/\:/ G N H H I s o l a t i o n of auxin-a and auxin-b has not been repeated, for i t soon became apparent that hetero-auxin, i n nature, was the most common growth substance. There i s no5 doubt to-day that i t i s of general importance,as a natural plant growth substance. Not long a f t e r the i s o l a t i o n of indole acetic acid i n 1934, several groups of workers were able to show that a number of chemicals possessed growth regulating a c t i v i t y ; some of them are synthetic and have not been demonstrated to occur n a t u r a l l y i n plant t i s s u e s . Bonner places these active substances, which to-day form a long l i s t , i n four categories: a) indole derivatives other than indole acetic acid b) naphthalene derivatives c) phenoxyacetic acid derivatives d) substituted benzoic acids At present emphasis i s being placed on the synthesis of homologs and analogs of the n a t u r a l l y occurring auxins, and on the role of auxins i n i n t e r -mediary metabolism. _ CH - COOH 2 7. 2. D e f i n i t i o n and structural requirements of growth regulators. As has been noted, growth substance a c t i v i t y i s shown by many quite d i f f e r e n t substances, some occurring i n nature and some synthetic. D e f i n i t i o n , however, of growth substance a c t i v i t y i s not simple. Bonner (17), probably correctly, implies that a l l growth regulators are active i n the pea curvature t e s t , i . e . are e f f e c t i v e i n causing c e l l elongation. He points out, of course, that any given active compound reacts d i f f e r e n t l y to the other "growth, tests", such as the tomato p e t i o l e bending test, the e t i o l a t e d Avena col e o p t i l e curvature t e s t , the Avena section t e s t , the root i n i t i a t i o n t e s t , etc. The differences may sometimes be quite spectacular; f o r example, naphthalene acetic acid i s only 2.5% as active as indoleacetic acid i n the oat curvature t e s t , but i t i s more e f f e c t i v e than indoleacetic i n the s p l i t pea curvature t e s t . In part, the differences i n response may be explained by differences i n " t r a n s p o r t a b i l i t y " of the d i f f e r e n t growth substances, by differences i n d i s s o c i a t i o n constants and so f o r t h . To possess primary a c t i v i t y Koepfli,,Thimann and Went (47) i n 1958 postulated that the following minimum structural features must be possessed by a growth substance: (&) a ring system as a nucleus, (b) a double bond i n the r i n g 8 system, (c) a side chain, (d) a carboxyl group on the side chain, or a group r e a d i l y converted to a carboxyl group, at least one atom removed from the r i n g , and (e) a p a r t i c u l a r space relationship between the ring and the carboxyl group. Subsequently Thimann (103) has suggested that the carboxyl group requirement should be broadened and stated merely as an acid group which i s not too highly dissociated.. Veldstra (101), too, would extend the str u c t u r a l requirements f o r a c t i v i t y . He notes that the basal ring sys-tem must have a high surface a c t i v i t y and that the unsaturation of the ring i s also necessary f o r a c t i v i t y . This unsaturation cannot be replaced by unsaturation on the side chain. Van Overbeek' (94) finds, that i n general Increasing the length, of the side chain decreases a c t i v i t y . Insofar as pH may modify the structure of auxins, i n a sense, i t too, i s a "s t r u c t u r a l " requirement f o r penetration and a c t i v i t y of the auxins (Blondeau (15) '). Varied though the chemical nature and a c t i v i t y of the many "growth substances" may be, i t appears that these compounds, variously, but synonymously, termed "growth substance", "growth regulator" and "auxin" have i n common, the physiological c h a r a c t e r i s t i c of promoting,elongation of c e l l s and the structural c h a r a c t e r i s t i c of having a s p e c i f i c type of molecule,. 9 . Auxins, i t i s now clear, are but a- class of phytohormone. Other kinds of organic compounds which regulate plant p h y s i o l o g i c a l processes are being i s o l a t e d and characterized v i z . the reproduction carotenoid hormones, c i s and trans dimethyl crocetin, the wound hormone, traumatic acid, and others. 3 . Forms of auxin i n the plant. Auxins and related compounds occur i n the plant i n a variety of forms: i n a free molecular form, and i n association with other molecules forming a complex (17). The group of free molecular forms contains acids such as indoleacetic a c i d , auxin-a, etc. and neutral forms such as indoleaceticaldehyde, auxin-a lactone, etc. The bound forms y i e l d free auxin only aft e r autolysis of the plant ti s s u e s . Van Overbeek ( 3 6 ) states that free auxin has been isol a t e d a f t e r enzymatic digestion of proteins isol a t e d from seeds and from spinach leaves. He notes, how-ever, that some bound forms may not contain auxin i n a more or le s s free acid form, but may require a series of oxidations to make the conversion. Further to t h i s he adds that the status of bound auxin i s very much i n doubt, but that a high l e v e l of free auxin i s frequently associated with a high l e v e l of auxin a c t i v i t y . 4. Auxin physiology, a) The fundamental r o l e s . From the point of view of applied physiology the 10. auxins play a multitude of roles and each growth regulating substance may play the roles with d i f f e r e n t i n t e n s i t i e s . It seems now that auxins exert t h e i r multiple e f f e c t s as a r e s u l t of c o n t r o l l i n g a few fundamental phy s i o l o g i c a l processes. J. Bonner (18) and associates i n 1946 Isolated an auxin protein with the properties of a phosphatase, an enzyme clos e l y associated with the release of energy from phosphate bonds. Prom t h i s and l a t e r work i t appears that auxins regulate the energy "flowing" to a number of synthetic processes. In van Overbeek's words "auxin would be the foot on the t h r o t t l e . " These researches confirm and extend those of Thimann and associates (89), who, i n 1939 were able to demonstrate that auxin treated oat coleoptiles showed Increased r e s p i r a t i o n rates and that there was a linkage with the Krebs cycle. Elongation remains as the most d i r e c t l y record-able general role of the auxins. They appear to make c e l l wall more p l a s t i c , which, i n turn, r e s u l t s i n increased d i f f u s i o n pressure d e f i c i t inside the c e l l . The further r e s u l t of t h i s Is f o r the c e l l to take up water and elongate. What, then, i s usually referred to as the "growth e f f e c t " of auxins i s r e a l l y c e l l elongation, b) Auxin translocation. Auxins are transported "rapidly" inside the plant i n at l e a s t three d i f f e r e n t ways: i 1 1 . l ) through the phloem, 2 ) through parenchimatous tissue devoid of vascular bundles and 3 ) through the xylem trans-p i r a t i o n stream. Weaver and de Rose ( 9 9 ) found they could pass upward through dead tissue but not downward. Oddly enough too, they found that stomata were not portals of entry f o r l e a f sprays of the hormone. A number of investigations have shown that auxins are present constant-l y i n a l l l i v i n g plant t i s s u e s , but are most abundant i n the meristems. c) Auxins i n g a l l and nodule formation Of some physiological interest i s the role of auxins i n g a l l and nodule formation. Observations relevant to t h i s phase are of special interest to our researches reported upon l a t e r . Crown g a l l s caused by Phytomonas tumafaciens have a high auxin content. Auxins, however, do not produce the g a l l s d i r e c t l y : i t has been concluded ( 3 9 ) that the production of g a l l s involves two phases, a) the bacteria change normal host c e l l s to tumour c e l l s and b) the auxin produced by the host induces the neoplastic c e l l s to actual tumour formation. Auxin, too, plays an important role i n nodule formation on legumes. Here again, the Rhlzobium. although quite capable of producing auxin i n v i t r o seems to change the c e l l s of the host i n such a way that they are "conditioned" to grow. Coincidentally with the increased auxin production i n the developing nodule Is a very exact chromosome doubling i n 12. bacteroid c e l l s (Wipf and Cooper (100) ). It may be the effect ~te the chromosomes i s a dir e c t one and mutagenic. •>;,• (see Unrau (100) )'. d) Auxins and enzymes. In plants, as has been noted, auxins appear to function by regulating enzymatic processes e s p e c i a l l y those involved i n energy releasing processes. The researches of Thimann et a l (89) have demonstrated the probable r e l a t i o n of auxins to the energy y i e l d i n g Krebs cycle, and Bonner et a l (18) have further supported the argument by associat-ing auxin d i r e c t l y with phosphatase a c t i v i t y . Puzzling relationships of auxins to enzymes, however, remain. Euster (34) found for example that auxin retarded the action of isolated diastase, but enhanced the a c t i v i t y of the diastase adsorbed on charcoal. Berger and Avery (14) found that naphthaleneacetic acid i n h i b i t e d glutamic acid and i s o c i t r i c dehydrogenases, but that indoleacetic acid Inhibited only the f i r s t named enzyme. e) Auxins and vitamins. In animals the relationships of hormones to vitamins and to enzymes have been the subject of intensive study. In plants, however, the relationships have not been accorded the same attention. Luecke, Hamner and S e l l (53) however, have found that the auxin hormones, when applied to leaves of the bean, generally reduced the thiamine, r i b o f l a v i n , n i c o t i n i c acid and pro-vitamin A content, but 13 that they increased the pantothenic acid content. 2,4-D, fo r example, reduced the carotenoids by as much as 1/3. Mitchell et a l (61) found that auxin treated f r u i t a f t e r a period of storage contained 45% more ascorbic acid than the untreated f r u i t s i n storage. Hey and Hope (44) were able to relate the h e r b i c i d a l effectiveness of auxins to vitamin K le v e l s i n plants - the plants with the high vitamin K contents succumbed e a s i l y to h e r b i c i d a l auxin l e v e l s . f) Auxins i n mineral n u t r i t i o n . Auxins may s i g n i f i c a n t l y influence the mineral n u t r i t i o n of plants. Brunstetter (23) found that leaves treated with 3-indoleacetic acid showed increases i n K, Mg, Mn and B. Some increases were also recorded f o r P, A l , Pe, and Cu. Went (102) found some evidence that auxin, added to the nutrient solution, p a r t i a l l y overcame zinc and boron d e f i c i e n c i e s . Rhodes, Templeman and Thurston (75) found that increasing concentrations of methoxone reduced i potassium uptake, but did not appreciably a l t e r the uptake of Na, Mn, Ca, and Pe. They admitted to a number of j possible interpretations of t h e i r data, admission'which seems to be c h a r a c t e r i s t i c of most of the studies i n th i s ' f i e l d . g) Auxins i n nitrogen metabolism. The relationships of auxins to nitrogen a s s i m i l -ation are being a c t i v e l y sought for and delimited. As 14 yet the fundamental body of thought seems to be l a r g e l y i n the making and some incoherence i n the l i t e r a t u r e accordingly may be expected. How Intimately auxins relate to nitrogen metabolism and assimilation i s problematical. It Is, of course, obvious that they influence It i n d i r e c t l y through t h e i r well-known influence on carbohydrate metabolism. It i s a frequent observation, following f o l i a r applications of auxins to growing higher plants, to see the fol i a g e green i n t e n s i f y i n g . These and other observations can often be associated with metabolic changes involving protein synthesis. In more detailed observations, M i t c h e l l (65) found beans treated with auxin had higher nitrogen l e v e l s than untreated beans. Smith et a l (84), Borthwick, Hamner and Parker (19) and Wort (106) found nitrogen to decrease i n bean leaves, but to increase i n other plant parts following auxin applications. Rasmussen (74) found 2,4-D applications to be associated with increases i n t o t a l soluble nitrogen i n dandelion leaves. Davis, (86), using bean seedlings treated'with indoleacetic acid, found nitrogen moving from plant upper parts to lower. Gordon (58), studying rooting responses of Hibiscus cuttings to auxins, found soluble nitrogen highest at the base of the treated cuttings, but not on the controls. S e l l (79) 15. and others found protein and amino acids accumulating i n the stems of auxin treated bean plants and noted declines i n aspartic acid but increases i n l y s i n e , v a l i n e , methionine and phenylalanine. Borthwick and associates (19) under somewhat d i f f e r e n t conditions found marked increases In protein i n meristematic areas subjected to auxin influences.. Recording the changes i n nitrogenous constituents following auxin treatments seems, not yet, to have taken physiologists very f a r i n r e l a t i n g these phytohormones to nitrogen a s s i m i l a t i o n . Although good experimental evidence i s yet forthcoming, Chibnall (29) has suggested a very probable hypothesis that the protein l e v e l s of leaves are determined by phytohormones. Whether or not auxins are the hormones involved cannot yet be stated. A somewhat similar viewpoint i s to be found i n the suggestion by All-Zade (4) that hormones, possibly auxins, control the nitrogen metabolism of legume root nodules. I f nitrogen metabolism i s i n fact, intimately r e l a t -ed to the action of auxins, and, since auxins are so extensively used i n a g r i c u l t u r e , one might expect to f i n d records of qu a l i t y changes i n crops. Casual references are, i n f a c t , quite numerous, but few detailed studies are available. Deepening of the green colour of lawns common-l y follows h e r b i c i d a l applications. To mention a few other observations: Aberg, Hags end and Vaartnou (1.) 16 found 2,4-D sprays increased the nitrogen content of wheat, f l a x and peas. Erickson, Seeley and Klages (33), using 2,4-D i n wheat, obtained increases i n the protein of the grain of 4.6$ (from 10.9$. in the control to 15.5$ i n the treated plants) and voiced the view that such responses should be taken into consideration i n the control of. weeds i n wheat. On the Great Plains, 2,4-D i s used extensively to control annual weeds in grain crops and q u a l i t y in the wheat crop i s , In some measure, associated with i t s protein content. It i s i n t e r e s t i n g to notethat Aitken, Meredith and Olson (2) found s i g n i f i c a n t increases i n the crude protein of wheat, barley and oats which had been sprayed f o r weed control. They suggested that there i s a period during the growing period when applications of 2,4-D may cause changes i n the q u a l i t y of bread wheat and malting barley, h) Auxins i n carbohydrate metabolism. One of the commonly recorded changes in chemical composition of higher plants following 2,4-D application i s a rapid increase i n the percentage sugars i n leaves. M i t c h e l l and h i s associates i n a series of papers (62), (63), (66) have, fo r example, recorded that under cert a i n treatments with indoleacetic acid that in leaves placed i n darkness, the percentage sugar Increased to twice the percentage i n the controls within four hours. The source of sugars appear to be the starchy reserves which are depleted as the sugars increase. When, however, 17. the starch reserves are depleted, often i n the f i e l d a f t e r two weeks, decreases i n the sugars ensue. It i s f a i r l y generally believed that h e r b i c i d a l l e v e l s of auxins height-en r e s p i r a t i o n a c t i v i t y to the point where the carbo-hydrate reserves are depleted and death through starv-ation i s a r e s u l t . S e l l et a l (79) found that as t o t a l sugars decreased t o t a l protein increased and suggested that protein synthesis was "fostered" at the expense of carbohydrates i n the plant. In the experiments by S e l l et al, crude f i b e r , too, decreased i n kidney beans treated with 2,4-D. Most workers, however, found that crude f i b e r changes very l i t t l e following auxin treatments (Aberg, Hagsand and Vaartnou ( l ) ). i) Auxin and s o i l micro-organisms Patterns of auxin a c t i v i t y i n the s o i l and i n s o i l micro-organisms are not yet well developed. Many s o i l ' bacteria are capable of producing auxin i n v i t r o . B a c i l l u s radlobacter. f o r example, which occurs in the s o i l and i n legume roots, i s a "free" producer of auxin. Rhizobium too, which i s d i r e c t l y or i n d i r e c t l y responsible for legume root nodulation produces le s s (Wilson (104) ). I t probably follows from i n v i t r o r e s u l t s such as the above, that many s o i l organisms produce auxin i n the s o i l . The addition of auxins to the s o i l , a r t i f i c i a l l y , often effects considerable change i n the ecology of the s o i l microflora. According to Worth and McCabe (107) 18. aerobic bacteria are usually "sensitive to" added 2,4-D on the s o i l , but that anaerobic organisms are much less s e n s i t i v e . M a r c e l l i (56), Smith et a l (85) report, what appears to be a f a i r l y common observation, that i f 2,4-D Is added a r t i f i c i a l l y to s o i l - that the populations of micro-organisms reduce very r a p i d l y i n size at f i r s t , but that l a t e r the populations become larger than before 2,4-D treatment. The recovery of the m i c r o f l o r a l populations from s o i l s which were not leached^ according to Newman and Norman (68) i s due to the f a i r l y rapid decomposition of 2,4-D by micro-organisms. Amounts of auxins required to considerably modify the plant ecology of the s o i l are low: Payne and Pults (72), f o r example, found that .075 l b s . of 2,4-D per acre of s o i l could prevent nodulation of common beans. Carlyle and Thorpe (27) also note that .5. p.p.m. 2,4-D i n the s o i l w i l l seriously interfere with nodulation and growth of beans, peas, red clover, and a l f a l f a . Very small amounts of 2,4-D added to the s o i l or in cultures i n v i t r o may "benefit" some organisms. Thus B a l l (11) finds that the addition of very small amounts of indoleacetic acid i n a glucose-tryptophane medium more than doubled the number of viable Escherichia  c o l i c e l l s i n 24 hours. Anker (8) found that endogenous r e s p i r a t i o n of "starving" glycogen containing yeast c e l l s was stimulated by heteroauxin. Stimulation of reproduction was noted i n Ch l o r e l l a vulgaris and Coccomyxa simplex by 19. Brannon and Bartseh (21) when growth substances were added to cultures i n v i t r o . No Investigations were found which report upon the effects on s o i l microflora following applications of auxins to the f o l i a g e of higher plants, j) Auxins i n a g r i c u l t u r a l p ractice Only a b r i e f reference can be made to the many uses to which auxins have been placed. Went and Thimann i n 1934, soon a f t e r indole acetic a c i d was i s o l a t e d and c r y s t a l l i z e d , discovered that t h i s hormone promoted root formation In i s o l a t e d stem sections of pea seedlings. This discovery has been followed by an Immense amount of study with the result, that to-day, several growth regulating substances are widely used i n the vegetative propagation of many kinds of plants, herbacious and woody. In somewhat similar fashion a e r i a l buds may be i n i t i a t e d (102), (45) by applying auxin paste to cut stem surfaces. On the other hand, bud development may be suppressed by t h e i r use v i z . naphthalene acetic a c i d i s used to prevent bud development and i n i t i a t i o n i n potatoes i n storage. The role of auxins i n flower formation i s f a r from clear. That they are i n some way involved i s un-doubted. Auxin-induced flower formation nas only been established with certainty i n the pineapple (95). In most plants there appear to be other agents which are rather d i r e c t l y associated with the auxins i n flower formation (51). 20 Par from Inducing flowering i n some plants, auxin applications may delay maturity and the onset of flower-tog. (87), (99), (71) Flowers of solanaceous plants l a s t f o r an abnormally long time a f t e r being treated with growth regulating substances (40), (102) and i n some species parthenoearpic development of f r u i t s may ensue. To a limit e d extent t h i s fact has been commercially exploited with the development of parthenoearpic f r u i t . Abscission of many organs, f r u i t s , flowers, petals, and leaves, i s sometimes associated with a low cfe 6& c£ auxin l e v e l . Abscission of thi s type may be oorrobtod by an auxin spray. U t i l i z i n g t h i s information orchardists have economically controlled pre-harvest drop of tree f r u i t . The effectiveness of certain hormone herbicides i s too well known to more than mention i t fo r the sake of completeness of the review. The persistence of 2,4-dichloro-phenoxy acetic acid (2,4-D) and 4 chloro-2-methyl phenoxy acetate (Methoxone) Is probably a prime factor i n t h e i r effectiveness as herbicides f o r they accumulate i n meristematic zones and eff e c t an exaggerated r e s p i r a t i o n , which, since i t i s long continuing, i s associated with starvation and eventual l e t h a l i t y . 21 I I I . THE REPORT ON CERTAIN FIELD, GREENHOUSE AND LABORATORY STUDIES  1. F i e l d T r i a l s , 1950-52 T r i a l s were maintained over three growing seasons (1950-52) with a view to assessing, under f i e l d conditions, the effects of 2,4-D, at various times and concentrations, on the chemical composition of several forage grasses and legumes commonly grown i n the Lower Fraser Valley. a) Location and Materials: The t r i a l on the Alderwood gravelly loam ( g l a c i a l t i l l and outwash) of the U.B.C. farm In Point Grey was duplicated on Ladner s i l t y clay (alluvium) close to the Fraser River North Arm at the foot of Blenheim St., Vancouver, B. C. The U.B.C. s o i l was low i n f e r t i l i t y and subject to severe summer drought. The s i l t y clay was productive and moist but possessed a pH l e v e l somewhat below that considered desirable. The forage species used i n the t r i a l s Included two legumes, Red Glover (T r i f o l i u m pratense) and White Glover (T. repens) and three grasses, timothy (Phleum pratense). perennial ryegrass (Lolium perenne) and Kentucky Bluegrass (Poa p r a t e n s i s ) . A l l seed used was "commercial". Pr i o r to seeding, the land at both locations received 800 l b s . of a g r i c u l t u r a l lime per acre. 22 The sodium s a l t of 2,4-dichlorophenoxyacetic acid was the growth regulator used, largely.because i t i s r e a d i l y available and commonly used. b) Methods: Ultimate plots f o r a l l treatments were 9' x 18', of which an area, 6' x 15' was harvested f o r y i e l d . Each treatment was r e p l i c a t e d f i v e times but only four r e p l i c a t e s were used for y i e l d ; one was used f o r studying seed production and v i a b i l i t y . A l l seeding was in 10" rows using a one-spout "Planet Junior" "push seeder". Only the Kentucky Bluegrass seeding on the Ladner clay f a i l e d to produce a good stand i n the spring of 1950; i t was reseeded i n August 1950. The plots were kept free of weeds by "push hoe" and "hand p u l l i n g " so that stands were, i n a l l cases, "pure" stands. The 1951 growing season started slowly, following snows i n late March, but i n May growth was very rapid and the f i r s t spraying was accomplished on May 12th. A l l spray applications were made with a low pressure knapsack sprayer and aquaeous solutions of the sodium 2,4-D were always used. In one series of experiments, 2,4-D i n d i f f e r e n t concentrations was employed. The range of concentrations on clovers was from .01 to 1 l b . and on grasses from .1 l b . to 8 l b s . parent acid per acre. In a second series of experiments, 2,4-D was applied at d i f f e r e n t times.. Spraying in t h i s series was undertaken i n May and i n June at 7-day i n t e r v a l s . 23. The plots on the Ladner clay were harvested on June 23rd and on the Alderwood gr a v e l l y loam on July 1st. Fresh weights were taken and 1-kilogram samples were taken for determining dry matter, crude protein, and invert sugar. Records were taken from time to time on flowering behavior and seed s e t t i n g . The chemical analyses were accomplished i n the winter of 1951-52. O f f i c i a l A.O.A.C. methods were used. Analyses were In duplicate 1 or t r i p l i c a t e . In the summer of 1952 observations were made on a l l p lots (including those of the "bienn i a l " red cl o v e r * ) . E f f e c t s of 2,4-D which might "carryover" into .the second year a f t e r treatment were sought f o r . In -addition, a new series of treatments was superimposed on the 1951 U.B.C. treatments, the object of which was, primar i l y , to determine the effects of 2,4-D on flowering i n t e n s i t y and maturation. c) Results: Red clover - v i d . Table I - the effects of 2,4-D concentration on red clover and Table II - the effects of time of 2,4-D application on red clover. An examination of the data In Tables I and II w i l l show that y i e l d and protein l e v e l s of red clover, a biennial tap-rooted legume, may be materially modified by 2,4-D appl i ations. s-Many of the red clover plants persisted f o r a t h i r d year on the p l o t s . 24. Marked v a r i a t i o n i n f e r t i l i t y occurred over short distances i n the Alderwood s o i l s . Nonetheless, significantincreases i n mean y i e l d over the control were recorded with a l l concentrations of 2,4-D (applied May 12th when the plants were yet vegetative) except with the highest concentration. With the control yielding, on the average • 3805 l b s . per acre, the highest y i e l d i n g concentration, giving 5435 l b s . per acre, was .05 l b s . 2,4-D per acre. Ladner s o i l was more homogeneous and while again s i g n i f i c a n t increases i n mean y i e l d over control were recorded f o r low concentrations of 2,4-D the Increases were not so striking.. Where the average y i e l d of controls was 6400 l b s . per acre .1 l b s . 2,4-D per acre increased the y i e l d to 7200 l b s . per acre. Higher concentrations of 2,4-D ( v i z . .5 and 1.0 l b . per acre) markedly depressed the red clover y i e l d s . Crude protein percentage, as the tabled data w i l l show, increased s i g n i f i c a n t l y . There was no i n d i c a t i o n from the harvest taken i n a more or les s optimum hay cutting time, weeks af t e r the actual 2,4-D was applied, that t h i s increase was at the expense of the sugars i n the plant. Acre y i e l d s of protein again were s i g n i f i c a n t l y increased under a l l applications of 2,4-D but with the higher concentrations the Increases are small. Optimum concentration f o r protein y i e l d per acre appears to be 25. around .1 l b . per acre f o r red clover. Again, the res-ponse on Alderwood s o i l was more marked than on Ladner s o i l . 2,4-D was applied on red clover on f i v e occasions (representing f i v e stages of development from early l e a f to mid-flowering) at the rate of .1 l b . per acre. Very d i f f e r e n t responses were noted on the two s o i l s . On the red clover grown on Alderwood s o i l , applications made early (May 12) were seemingly e f f e c t i v e i n elevating dry matter y i e l d s and protein per acre y i e l d s as were a l l applications made a l a t e r date. However, p l o t v a r i a b i l i t y was so great that increases could not be regarded as s i g n i f i c a n t . The early application on the Ladner clay grown red clover resulted i n a s i g n i f i c a n t increase i n y i e l d and protein per acre. Later applications were depressive. It would appear that the time of application and the stage of growth of the red clover plants determines to a very considerable extent the nature of the response to 2,4-D. It i s to be noted that i n Tables I and I I , and i n some of the others to follow, that both Kjeldahl nitrogen and crude protein are reported. The reason f o r t h i s i s that the form of the nitrogen following 2,4-D applications i s not known; It may be that non-proteiri nitrogen only i s increased. White clover - v i d . Table III - the e f f e c t s of 2,4-D concentration on white clover and Table IV - the e f f e c t s 2 6 , of time of 2,4-D application on white clover. The effects of 2,4-D on white clover, a creeping rooted perennial legume, were not so marked as those on red clover, a tap-rooted b i e n n i a l . Possibly because the shallowness of the root system of white clover and the dryness of the season, the plot y i e l d s on Alderwood s o i l were v i s i b l y about as variable as one could hope to f i n d , and yet v i s i b l y sharp qu a l i t a t i v e differences could be accorded treatment, undoubtedly the t r i a l s should be repeated under more favourable conditions. Nonetheless i t would appear that on Ladner clay grown white clover, intermediate ^concentrations of 2,4-D increased both y i e l d and protein per .acre. On Alderwood grown white clover, the reaction Is not clear - but increases i n y i e l d and protein were obtained at the higher concent-rations. Consistently, too, the r e l a t i v e l y low concentration of 2,4-D. applied to Alderwood grown white clover, .1 l b . per acre, proved to be depressive of y i e l d and protein per acre i n the "time of application series". Protein percentage Increases are recorded on the white clover grown on the Ladner clay. 2,4-D applied early (May 12) increases y i e l d of dry matter, protein per acre and protein percentage. Late applications WOEC depressive. 27. Kentucky Bluegrass - v i d . Table V - the eff e c t s h of 2,4-D concentration on Kentucky bluegrass and Table VI -i the e f f e c t s of time of 2,4-D application on Kentucky Bluegrass. 6 When spraying was started, the Kentucky Bluegrass was well developed, with panicles well developed and, on the Alderwood p l o t s , past flowering. The stage of develop-ment was probably too advanced to influence chemical composition very much. The processes of maturity had not advanced so f a r i n the plants growing on the Ladner s o i l . Nonetheless, a s i g n i f i c a n t increase i n y i e l d was noted on Alderwood grown Bluegrass when 4.0 l b s . per acre was applied. On Ladner clay-grown Bluegrass, however, increases i n y i e l d s , protein per acre and protein percentage Stvena-2 of were obtained at the higher rates of ap p l i c a t i o n . Early applications appeared to be most e f f e c t i v e i n increasing y i e l d and protein on both s o i l s . Perennial R y e g r a s s - v i d . Table VII - the ef f e c t s of 2,4-D concentration on perennial ryegrass and Table VIII -the effects of time of 2,4-D application on perennial ryegrass. Maturation processes were well advanced i n the perennial ryegrass before the plot treatments were madej es p e c i a l l y was t h i s the case i n the grass on the upland s o i l . Nonetheless the growth regulator caused some recordable changes i n y i e l d of dry matter, on protein per acre, and percentage protein. A l l concentrations, f o r example, applied May 12th, increased y i e l d s and crude protein of perennial 28 ryegrass grown on Ladner clay but only the high coneen- . tr a t i o n s increased them on Alderwood s o i l . In general, the early applications tended to increase y i e l d and protein and the la t e applications to depress them. The most s i g n i f i c a n t response was obtained with the e a r l i e s t a pplication on the grass grown on Ladner clay. Timothy - v i d . Table IX - the ef f e c t s of 2,4-D concentration on timothy and Table X - the ef f e c t s of time of 2,4-D application on timothy. Timothy matured more slowly than Kentucky Blue-grass and perennial ryegrass. It was, therefore, noted with interest that the "low applications" of 2,4-D were quite e f f e c t i v e i n modifying the y i e l d and chemical composition of t h i s species. Both i n Alderwood and Ladner s o i l s a l l concentrations of 2,4-D applied early (May 12) increased dry matter y i e l d s and, as well, on the Ladner s o i l s , crude protein per acre and protein percentage. Early applications of the auxin tended to increase dry matter y i e l d , crude protein percentage and t o t a l protein per acre ; but l a t e r application on grass grown on both S o i l types were depress-ive. TABLE It THE EFFECTS OF 2,4-D Na 2,4-D ! Dry Matter Y i e l d s ! Crude P r o t e i n as % Dry Matter C o n c e n t r a t i o n s l b s . % of D e v i a t i o n i n j % % D e v i a t i o n i n l b s * p.a. p.a. c o n t r o l l b s . from c o n t r o l ! dry of % from c o n t r o l + — j matter c o n t r o l •> mm a)On Alderwood S o i l j 1 0 3805 100.0 0 0 10.55 100.0 0 0 • 01 4730 124.3 925^ 9.66 91.6 .89* •05 5435 142.8 1630* 11.59 109.8 1.04* .1 ! 4525 118.9 720 11.18 105.9 • 2 j 4815 126.5 1010 14 . 2 3 134.8 3.68* • 5 i 4735 124.4 930 13.31 126*1 2.76* 1,0 i 3785 99.4 20 12.76 120.9 2.21* b) On Ladner * > > 1 > S o i l j 0 j 6400 100.0 0 0 i 13.58 100.0 0 0 •01 J 6390 99.8 10 14.27 105.0 .69* •05 j 6975 108.9 575 14.92 109.8 1.34* .1 7200 112.5 800* 13.96 102.7 •38* .2 j 6555 102.4 155 16.02 117.9 2.44* • 5 5985 93.5 415^ 15.91 117.1 2.33* 1.0 5040 78.7 1360* | 15.79 116.1 2.21* TABLE II» THE EFFECT OF TIME OF Time of Dry Matter Y i e l d s Crude P r o t e i n as % Dry Matter A p p l i c a t i o n (.1 lbs.Na 2,4-D; l b s . p.a. % of c o n t r o l D e v i a t i o n i n l b s * from c o n t r o l % dry % of D e v i a t i o n i n % from c o n t r o l P.a.) •» m matter c o n t r o l a) On Alderwood S o i l ! 0 May 1 2 , 1951 It tt M 26 • June 2, w n o it 3 8 0 5 4 5 2 5 4 0 4 5 4400 3 9 3 0 3 3 6 0 100.0 118.9 106.3 115.6 103.2 88.3 0 720 2 4 0 595 1 2 5 0 4 4 5 10.55 11.18 11 . 7 3 11.00 1 2 . 2 5 11 . 9 2 1 0 0 . 0 105.9 111.1 104.2 116.1 112.9 0 . 6 3 * 1.18* . 4 5 * 1 . 7 0 * 1 . 3 7 * 0 b) On Ladner S o i l 0 May 1 2 , 1951 » 19 " « 26 n June 2 B n 9 n 6 4 0 0 7200 6 2 4 0 6145 5410 5592 100.0 112.5 97.5 96.0 84.5 87.3 0 800* 0 1 6 0 2 5 5 ^ 9 9 0 * 808* 13 . 5 8 1 3 . 9 6 13.44 11 . 7 8 i 11.41 i 1 3 . 6 2 100.0 102.7 98.9 86.7 84.0 100.2 0 • 3 8 * 0 .14* 1.80* 2 . 1 7 * •04 CONCENTRATION ON RED CLOVER 29. I n v e r t S Acre Y i e l d s of Crude P r o t e i n K j e l d a h l K j e l d a h l Sugar l b s . % D e v i a t i o n i n l b s . N N Percent i n p.a. of from co n t r o l l b s . p . a . % i n Dry Dry Matter c o n t r o l Matter 2.0 4 0 1 100.0 mm 64.1 1.68 1.9 457 113.9 73.1 1.54 1.9 630 157.1 229* 100.8 1 . 8 5 2.1 506 126.1 80.9 1.78 2.0 685 170.8 284* 109.6 2 . 2 7 1.8 630 157.1 229* 100.8 2.12 1.9 483 120.4 82 77.2 2.04 2.1 . 869 .100.0 139.0 . 2.17 2.1 912 104.9 kK 145.9 2.28 2.0 3040 119.6 171* 166.4 2.38 1.8 3005 115.6 1 3 6 * 160.8 2 . 2 3 2.1 30 5 0 120.8 181* 168.0 2.56 1.9 9 5 2 109.5 83* 152.3 2.54 1.9 8 5 2 98.0 17 f 136.3 2 . 5 2 2,4-D APPLICATION ON RED CLOVER In v e r t Acre Y i e l d s of Crude P r o t e i n KjeldahlS K j e l d a h l Sugar l b s . ' % D e v i a t i o n i n l b s . N „ N * Percent i n p.a. of from c o n t r o l l b s . p . a . % i n Dry Dry Matter c o n t r o l - Matter 2.0 4 0 1 100.0 64.1 1.68 2.1 506 126.1 1 0 5 80.9 1.78 1.8 474 118.2 73 75.8 1.87 2.0 484 120.6 83 77.4 1 . 7 6 2.0 481 119.9 80 76.9 1 . 9 6 1.9 400 99.7 1 64.0 1.90 2.1 869 100.0 mm 139.0 2.17 1.8 3005 115.6 1 3 6 * 160.8 2 . 2 3 1.8 839 96.5 3 0 134,2 2.15 1.9 724 83.3 145* 115.8 1.88 2.1 617 71.0 2 5 2 * 98.7 1.82 1.8 7 6 2 87.6 107* 121.9 2.18 M,S.Dy a) 1. - 1591 l b s . p . a . 2. - .14 % 3. - 212 l b s . p . a . b) 1. - 608 l b s . p . a . 2. - .09 % 3. - 61 l b s . p . a . M tS.D t a) 1 . - 1 0 2 7 l b s . p . a . 2 . - .14 % 3. - 143 l b s . p . a . b) 1. - 601 l b s . p . a . 2 . - .09 % 3. - 55 l b s . p . a . TABLE I I I : THE EFFECTS OF 2, 4^:1lT;CoNCENTRATlON ON WHITE CLOVER Na 2,4-D Dry Matter Y i e l d s Crude Protein as % Dry Matter Concentrations l b s . % of Deviation i n % % Deviation i n lb s • p.a. p.a. control l b s . from control dry of % from control T - matter control r •* a) On Alderwood S o i l 0 3940 100.0 0 0 14.09 100.0 0 0 .01 3140 79 .6 300* 13.94 98.9 **15* .05 3235 82.1 705* 13.61 96.5 .48* .1 3265 82.8 675* 12.67 89.9 1.42* .2 4185 106.2 245 13.01 92.3 1.08* .5 4000 101.5 60 15.01 106.5 .92* 1.0 3045 77.2 895* 16.79 119.1 2.70* b) Cn Ladner S o i l * 0 3930 100.0 0 0 16.80 100.0 0 0 .01 4055 103.1 14.49 86.4 2.31* .05 4335 110.3 405* 14.99 89.3 1.81* .1 4295 109.2 365* 17.89 106.6 1.09* .2 4105 104.4 175 18.25 108.8 1.45* ' .5 - - =3645 92 .7 285* 18.17 108.3 1.37* 1.0 3255 82.8 675* 14.62 87.1 2.18* TABLE IV: THE EFFECT OF TIME OF 2y-4«rD—APPL-IGA-T-ION- ON WHITE CLOVER. Time of Appli c a t i o n (lbs.Na 2.4-r-D p.a.; a) On Alderwood S o i l May 12, 1951 19, « 26, » June 2, " tt 9 j n b) Cn Ladner S o i l May 12, 1951 " 19, " M 26, M June 2, 11 „ Q j tt l b s . p.a. 3940 3265 3360 2735 3170 2425 3930 4295 3870 3775 3535 3245 Dry Matter Y i e l d s % of control 100.0 82.8 85.2 69.4 80.4 61.5 100.0 109.2 98.4 96.0 89.9 82.5 Deviation i n l b s , from control 0 675* 580* 1205* 770* 1515* 0 365* 60 155* 395* 685* Crude Protein as % Dry Matter % dry matter 14.09 12.67 15.12 15.44 14.30 12.61 16.77 17.89 16.91 16.72 16.81 16.37 % of jDeviation i n c o n t r o l \ % from control 100.0 89.9 107.3 109.5 101.4 89.49 100.0 106.6 100.7 99.7 100.2 97 .6 1 .03* 1.35* .21* 0 1.09* .14* .04 0 1.42* 1.48s Invert SuVar Percent i n Dry Matter .05 .40" Acre Y i e l d s of Cruce Protein l b s . p.a. % \ of | c ont r o l 1.7 1.7 1.6 1.9 1.8 1.7 1.8 1.7 1.9 1.8 1.8 1.7 Deviaticn i n l b s . from coritrol  555 414 508 422 453 306 660 768 654 631 594 531 100.0 74.5 91.5 76.0 81.6 55.1 100.0 116.3 99.0 95.6 90.0 80.4 141* 47* 133* 102* 249* 108"1 6 29* 66* 129* Kjeldahl N lbs.p.a. 88,8 66.2 81.2 67.5 72.4 48.9 105.6 122.8 104.6 100.9 95.0 84.9 Kjeldahl N % i n Dry Matter 2.25 2.02 2.41 2.47 2.28 2.01 2.68 2.86 2.70 2.67 2.68 2.61 Invert [ Acre Y i e l d s of Cruje Protein Kjeldahl Kjeldahl Sugar l b s . % Deviatib i n l b s . N N Percent i n 1% 1# M 1 _ P.a. of from c o t r o l lbs.p.a. % i n Dry Dry Matter •control , 4- - , Matter , 1.7 555 100.0 88.8 2.25 1.8 437 78.7 118* 69.9 2.23 a) 1.9 440 79.2 115* 70.4 2.17 1.7 414 74.5 171* 66.2 2.02 2.0 544 98.0 11 87.0 2.08 1.8 600 108.1 45 96.0 2.40 1.7 511 92 .0 44 81.7 2.68 1.8 660 . 100.0 105.6 2.68 b) 1.7 587 j 88.9 73* 93.9 2.31 1.9 650 * 98.4 i 10 104.0 2.39 1.7 768 f 116.3 108* 1 • • 122.8 2.86 1.8 749 j 113.4 89* 119.8 2.92 J 2.0 662 1 100*3 I • -2 • — I- . -- - - — 105.9 2.90- t r 476 1 72.1 | I 184* 76.1 2.33 1 M.S.D. 1. 567 lbs.p.a. 2. .09 % 3. 50 lbs.p.a. 2. J2 % 3. 30 lbs.p.a. a) b) M.S.D. 1. 399 lbs.p.a. 2. .09 % 3. 36 lbs.p.a. 1. 129 lbs.p.a. 2. ,12 % 3. 16 lbs.p.a. T A B L E 7* THE E F F E C T S OF 2,4w® Na 2,4-D D r y Mai t t e r Y i e l d s i C r u d e P r o t e i n a s % D r y M a t t e r C o n c e n t r a t i o n s l b s . .% o f D e v i a t i o n i n i i D e v i a t i o n i n l b s * p . a . p » a « c o n t r o l l b s . f r o m c o n t r o l I d r y e f % f r o m c o n t r o l 1 m a t t e r . c o n t r o l . • a ) Cn A l d e r w o o d j S o i l 0 1729 100.0 0 0 2.70 100.0 0 0 .1 2058 119.0 329 1.70 62.9 1.00* .5 2093 121.0 3*4 1.68 62.2 1.02* 1.0 2198 1 2 7 . 1 469 1.67 62.2 1.03* 2.0 2149 124.2 4 2 0 1.71 62.9 .99* 4.0 2624 151.7 895* 2.33 86.2 .37* 8.0 2212 127.9 483 2.84 105.1 .14* b ) On L a d n e r S o i l 0 3052 100*0 0 0 5.44 100.0 0 0 »1 2744 89.9 308 \ 5.40 ' 99.2 > ' . 0 4 • 5 2905 95.1 147 ! 5.34 98.1 1.06* •10 1.0 3283 107.5 231 j 6.50 119.4 2.0 3465 ! 113.5 413 6.82 125*3 1.38* 4.0 1 3332 ! 109*1 280 1 5.72 105.1 .28* 8.0 [ 3101 [ 101.6 49 ? 5.53 101.6 .09 T A B L E V I : THE E F F E C T OF T I M E OF T i m e o f A p p l i c a t i o n (1.0 l b s . fe2,A-Dp.a.) a ) Oi A l d e r w o o d S o i l M a y 12, 1951 it 1 9 it « 26 w J u n e 2 " H 9 tt b ) On L a d n e r S o i l May 12, 1951 » 19 « • 2 6 » J u n e 2 • n C; w l b s © p . a . 1729 2198 1561 1659 1701 1659 3052 3283 2709 3157 2898 3024 D r y M a t t e r Y i e l d s T ~ o f c o n t r o l 100.0 127.1 90.2 95.9 98.3 95.9 100.0 107.5 88.7 103.4 94.9 99.0 D e v i a t i o n i n l b s * ) f r o m c o n t r o l 0 469 0 231 105 168 70 28 70 0 343 154 28 \ d r y m a t t e r C r u d e P r o t e i n a s % D r y M a t t e r % o f c o n t r o l D e v i a t i o n i n % f r o m c o n t r o l 2.70 1.67 1.79 1.86 1.75 1.93 5.44 6.50 5.29 5.44 5.13 5.17 100.0 62.2 66.2 68.8 64.8 71.4 100.0 119.4 97.2 100.0 94.3 95.0 0 U 0 6 * 0 1 . 0 3 * •91* .84* .77* .15 o . . 3 1 .27 3 1 . CONCENTRATION ON KENTUCKY B L U E G R A S S I n v e r t S u g a r t r c e n t i M a t t e r A c r e Y i e l d s o f C r u d e P r o t e i n 1.4 46 100.0 76.0 1.5 35 1.4 35 76.0 1.4 38 82.6 1.3 37 80.4 1.5 61 132.6 1.5 63 136.9 D e v i a t i o n i n l b s . f r o m c o n t r o l  1.5 166 100.0 1.5 148 89.1 1.4 155 93.3 1.6 213 128«3 1.5 236 142.1 1.4 190 114.4 1.4 171 103.0 K j e l d a h l N l b s . p . a . 2 6 . 5 2 3 . 6 2 4 . 8 3 4 . 0 3 7 . 7 3 0 . 4 2 7 . 3 K j e l d a h l N % i n D r y M a t t e r i>4 »D A P P L I C A T I O N ON KENTUCKY B L U E G R A S S I n v e r t S u g a r P e r c e n t i n D r y M a t t e r 1.4 1.4 1.6 1.3 1.5 1.5 1.5 1.6 1.4 1.5 1.4 1.3 A c r e Y i e l d s o f C r u d e P r o t e i n ! "% I D e v i a t i o n i n l b s l b s . p . a . 46 38 30 31 30 32 166 213 143 172 149 156 o f ifrom c o n t r o l ! -f c o n t r o l 100.0 82.6 65.2 67.3 65.2 69.5 100.0 128.3 86.1 103.6 89.7 93.9 47 6 8 16 15 16 14 23 17 10 K j e l d a h l N l b s . p . a . 7.3 6 . 0 4.8 4.9 4.8 4.9 2 6 . 5 3 4 . 0 2 2 . 8 2 7 . 5 2 3 . 8 2 4 . 9 K j e l d a h l N % i n D r y M a t t e r .43 • 26 .28 .29 • 28 .30 .87 1.04 .84 .87 .82 .82 M . S . D . a ) 1. - 489 l b s . p . a . 2. — .12 % 3 . - 58 l b s . p . a . b ) 1. 2. 3. 669 l b s . p . a . .25 % 167 l b s . p . a . M . S . D , A ) 1. - 222 l b s . P . A . 2. - a12 % 3. 26 l b s . p . a . b ) 1. 2. 3. 850 l b s . p . a . • 25 % 212 l b s . p . a . TABLE V I I : THE EFFECT OF 2,4-P Na 2 ,4-D Con c e n t r a t i o n s lbs.p.&« a) On Alderwood S o i l 0 .1 .5 1.0 2.0 4.0 8,0 b) On Ladner S o i l 0 .1 • 5 1.0 2.0 4.0 8.0 l b s . p.a. 10206 9636 8760 9462 10950 10698 9822 10542 10854 12174 11100 11412 10776 10710 Dry Matter Y i e l d s % of c o n t r o l D e v i a t i o n i n l b s * from c o n t r o l 100.0 94.4 85.8 92.7 107.2 104.7 96.2 100.0 102.9 115.4 105.2 108.2 102.2 101.5 744 792 0 312 1632* 558^ 870* 234 168 0 570 1440* 744 384 Crude P r o t e i n as % Dry Matter dry matter 4.14 4.34 3.97 3.95 6.65 4.90 5.15 5.82 5.27 6.32 6.46 6.59 6.61 5.88 of c o n t r o l 100.0 104.8 95.8 95.4 160.6 118.3 124.3 100.0 90.5 108.5 110.9 113.2 113.5 101.0 D e v i a t i o n i n % from c o n t r o l 0 .20* 2.51* .76* 1.01* • 50* .64* .77* •79* .06 .17* .19* 0 • 55 TABLE V I I I : THE EFFECT OF TIME OF Time of A p p l i c a t i o n (1.0 l b s . Na 2 ,4-D p.a.) a) Cn Alderwood S o i l May 12, 1951 • 19 n • 26 w June 2 n w 9 n b) On Ladner S o i l 0 May 12, tt 1 9 • 26 June 2 " 9 1951 n n n it Pry Matter Y i e l d s % of c o n t r o l Crude P r o t e i n as % Dry Matter D e v i a t i o n i n l b s , from c o n t r o l 10206 9462 10224 9012 9744 10140 10542 11100 10746 10752 10152 9768 100.0 92.7 100.1 88.3 95.4 99.0 100.0 105.2 101.9 101.9 96.3 92.6 0 18 0 558* 204 210 dry matter 0 744 1194* 462 66 3.90 774* 4.14 3.95 4.10 4.63 4.17 3.93 5.82 6.46 5.81 5.93 5.91 6.37 % of c o n t r o l 100.0 95.4 99.0 111.8 100.7 94.9 100.0 110.9 100.0) 101.8 101.5 109.4 D e v i a t i o n i n from c o n t r o l .49" . 0 3 0 .64* .11* •09* .55* 0 .19* • 04 .21* .01 CONCENTRATION ON PERENNIAL RYEGRASS In v e r t Sugar Percent i n Dry Matter 2.4 2.6 2*5 2.3 2.3 2.4 2.3 Acre Y i e l d s of Crude P r o t e i n l b s . p.a. 4 2 2 418 3 4 8 3 7 4 728 524 509 2.6 613 100.0 2.6 572 93.3 2.8 769 125.4 2.5 717 116.9 2.7 752 122.6 2.7 712 116.1 2.5 (630 102.7 of c o n t r o l D e v i a t i o n i n l b s . from c o n t r o l  1= 100.0 99.0 82.4 88.6 172.5 124.1 120,6 306* 102* 87* 156* 104* 139* 99* 17 K j e l d a h l N l b s . p . a * 6 74 48 41 67.5 .66 66.8 .69 55.6 .63 59.8 .63 116.4 1.06 83.8 .78 81.4 .82 98.0 .93 91.5 .84 123.0 1.01 114.7 1.03 120.3 1.05 113.9 1.05 100.8 •94 K j e l d a h l N % i n Dry Matter 2,4-D APPLICATION ON PERENNIAL RYEGRASS In v e r t I Acre Y i e l d s of Crude P r o t e i n Sugar l i b s . ; Percent i n l p . a . of Dry Matter; fc o n t r o l 2*4 2.3 2.4 2.2 2.3 2,1 2.6 2 05 2.7 2.4 2.4 2.6 422 374 419 417 406 398 613 717 624 638 600 612 j100.0 I 88.6 99 98 96 94 D e v i a t i o n i n l b s . from c o n t r o l  K j e l d a h l N l b s . p . a . K j e l d a h l N % i n Dry Matter 48 3 5 16 24 100.0 116.9 101.7 104.0 97.8 99.8 104* 11 25 13 1 67.5 59.8 67.0 66.7 64.9 63.6 98.0 114.7 99.9 102.0 96.0 97.9 .66 .63 .65 .74 .67 .62 .93 1.03 .93 .95 •94 1.01 H tS.D y a) 1. - 957 l b s . p . a . 2. - .09 % 3. - 85 l b s . p.a. b) 1. - 628 l b s . p.a* 2. - .07 % 3. - 43 l b s . p.a. M.S.D. a) 1. - 988 l b s . p.a. 2. - .09 % 3 . - 89 l b s . p.a. b) 1. - 458 l b s . p a . 2. - .07 % 3 . - 3 1 l b s . p.a. TABLE IX: THE EFFECT OF 2,4-D Na 2,4-D Concentrations l b s . p . a . l b s » p.a. a) On Alderwood S o i l 0 • 1 .5 1.0 2,0 4.0 8*0 b) On Ladner S o i l 0 •1 .5 1.0 2.a 4.0 8.0 4140 4 4 7 6 4 2 6 0 4 6 2 0 4518 4 3 2 0 4 5 9 6 4308 4740 14992 5 5034 14836 114644 ! 4062 Dry Matter Y i e l d s % of c o n t r o l 100.0 108.1 102.8 111.5 109.1 104.3 111.0 100.0 110.0 115.8 116.8 112.2 107.3 94.2 D e v i a t i o n i n l b s , from c o n t r o l 0 336 120 480 378 180 456 0 432^ 684* 726* 528* 336 Crude P r o t e i n as % Dry Matter % dry matter 246 4 . 59 4.34 4 . 2 6 4 . 6 7 4.14 4.28 4 . 2 9 4.82 4 . 8 7 7 . 7 2 7 . 7 8 7 . 2 3 7 . 7 2 7 . 6 3 % of c o n t r o l 100.0 94.5 92.8 101.7 90.1 93.2 93.4 1 0 0 . 0 1 0 1 . 0 1 6 0 . 1 1 6 1 . 4 1 5 0 . 0 1 6 0 . 1 1 5 8 . 2 D e v i a t i o n i n % from c o n t r o l .08 0 2.90* 2.96* 2.41* 2.90* 2.81* 0 . 2 5 * .33* . 4 5 * .31* .30* CONCENTRATION ON TIMOTHY In v e r t Sugar Percent i n Dry Matter TABLE X: THE EFFECT OF TIME OF Time of A p p l i c a t i o n (1.0 l b s . Na 2,4-D p.a.) a) On Alderwood S o i l May 12, 1951 " 1 9 w • 26 « June 2 n n 9 n b) On Ladner S o i l May 12, 1951 • 19 " " 26 « June 2 B tt C; tt l b s«» ! % of p. a . I c o n t r o l Dry Matter Y i e l d s 4140 4620 4134 4404 4056 3528 4 3 0 8 5 0 3 4 4608 4 6 6 2 4 2 3 6 3966 100.0 111.5 99.8 106.3 97.9 85.2 100.0 116.8 106.9 108.2 98.3 92.0 D e v i a t i o n i n l b s , from c o n t r o l 0 480 264 Crude P r o t e i n as % Dry Matter dry matter % of c o n t r o l 0 726* 300 354 84 612* 72 3 4 2 4 . 5 9 4 . 6 7 4 . 2 4 4 . 2 9 4 . 2 4 3.90 4.82 7.78 6.83 6.98 5.73 4.72 s Loo.o 101*7 92.0 93.4 I 92.0 ] 84.9 iioo.o J161.4 0.41.7 144.8 0.18.8 97.9 D e v i a t i o n i n % from c o n t r o l 0 .08 0 2.96* 2.01* | 2.16* .91* .35* .30* •35* .69* .10 2.1 2.1 2.3 2.0 2.0 2.2 2.1 Acre Y i e l d s of Crude P r o t e i n l b s . p.a. of c o n t r o l 2.3 207 100.0 2.4 231 111.5 2.1 385 185.9 2.3 391 188,8 169.0 2.3 350 2.1 358 172.9 2.1 310 149.7 D e v i a t i o n i n l b s . from c o n t r o l , 4 25 24 178 184 143 151 103 3 5 K j e l d a h l N l b s . p. a. 3 0 . 4 31 .0 28.9 3 4 . 4 29.9 29.6 3 1 . 5 33.1 36.9 61.6 62.5 56.0 57.2 49.6 K j e l d a h l N % i n Dry Matter .73 .69 .68 .74 .66 .68 .68 .77 .77 1 . 2 3 1 . 2 4 1 . 1 5 1 . 2 3 1 . 2 2 2,4~D APPLICATION ON TIMOTHY of Crude Proteinfj K j e l d a h l e v i a t i o n i n l b s . B N In v e r t Sugar Percent i n Dry Matter Acre Y i e l d s l b s . p.a. 2,1 190 100.0 2.0 215 113.1 1.9 175 92.1 2.0 189 99.4 1.8 172 90.5 1.9 137 72.1 2.3 207 100.0 2.3 391 188.8 2.2 315 152,1 2.0 325 157.0 1.9 243 117.3 2.2 187 90.3 of c o n t r o l D ati from c o n t r o l 25 184 108 118 36 i l b s . p . a . 15 1 18 53 30.4 34.4 28.0 30.2 2 7 . 5 21.9 K j e l d a h l N % i n Dry Matter .73 .74 . 6 8 . 6 8 . 6 8 . 6 2 20 33.1 .77 62.5 1.24 50.4 1.09 52.0 1.11 38.8 .91 29.9 [ .75 M.S.D. a) 1. -2. -3. -b) 1. 2. 3. 548 l b s . p . a . .07 % 37 l b s . p . a . 510 l b s . p . a . .07 % 3 5 l b s . p . a . M.S.D. &) 1. - 608 l b s . p . a . 2. - .07 % 3. - 64 l b s . p . a . b) 1. - 533 l b s . p . a , 2. - .07 % 3. - 37 l b s . p . a . •v.. Figure 1. - Dry matter y i e l d s of red clover grown on Ladner clay and treated on May 12 with several concentrations of 2,4-D. Ill O < e UJ o. CO CD I-O or 0 . UJ o ae o 1100 1090 1000 5 950 9 0 0 890 800 .01 .05 .2 W0 2,4-D LBS. PER ACRE. -<L: Figure 2. - Crude protein y i e l d per acre i n red clover grown on Ladner clay and treated May 12 with several concentrations of 2,4-D. Figure 3» - Dry matter y i e l d s of timothy grass on Ladner clay and treated on May 12 with several concentrations of Figure 4. - Crude protein y i e l d per acre i n timothy grown on Ladner clay and treated on May 12 with several concentrations of 2,4-D« 38, Seed production of Red and White Clovers - v i d . Table XI - seed production and q u a l i t y i n red clover treat-ed with 2,4-D i n d i f f e r e n t concentrations and Table XII-seed production and q u a l i t y i n red clover treated with .1 l b . 2,4-D at d i f f e r e n t stages of development. Table XIII - seed production and q u a l i t y i n white clover treated with 2,4-D i n d i f f e r e n t concentrations. Table XIV - seed production and q u a l i t y i n white clover treated with .1 l b . 2,4-D at d i f f e r e n t stages of development. The e f f e c t s of 2,4-D on the seed produced i n the plots of red and white clover, of e a r l i e r mention, are very simi l a r . Seed size and weight were not m a t e r i a l l y influenced by the 2,4-D applications, but heavy concentrations and l a t e applications reduced flowering and hence seed production. While seed size and weight, as has been noted, were not Influenced, s i g n i f i c a n t changes i n hard seed percentage were recorded: i n red clover the hard seed percentage was consistently increased but i n white clover i t was sometimes decreased. Again, however, with l a t e applications the e f f e c t s were not pronounced. Seed production of the grasses - v i d . Tables XV, XVI, XVII, XVIII, XIX and XX - the e f f e c t s of con-centration and time application of 2,4-D on seed from the Kentucky Bluegrass, perennial ryegrass and timothy p l o t s . 39. In general, the seed q u a l i t y of the grasses was not materially influenced by the applications of 2,4-D. There are, probably, a series of subtle interactions here which our data do not reveal - pr i m a r i l y for the reason that sea\l y i e l d per plot was not taken. I t Is probable that, where seed set i s light^ then seeds which are pro-duced are plump and viable and where seed set i s high single, seed weights and v i a b i l i t y may not be much dif f e r e n t from those where seed set i s low. In timothy, seed production e f f e c t s were not noticeable, although timothy y i e l d and protein modification was most notice-able. Again, early, l i g h t and heavy applications increas-ed seed set; l a t e r applications did not, even when "heavy". 40. TABLE XI SEED PRODUCTION AND QUALITY IN RED CLOVER TREATED WITH Na 2,4«D IN DIFFERENT CONCENTRATIONS Na 2,4-D Concentration lbs.p.a* On Alderwood Soil iGermination Sprouted % Hard Seed % 1000 Seeds Weight gms, Seeds from 50 Heads gms. Estim. Flower Inten-sity 1-10 On Ladner Soil Germination Sprouted % Hard Seed % 1000 Seeds Weight gms, T 0 .01 .05 • 1 • 2 .5 1.0 72 54 46 36 35 48 50 25 39 44 55 55 46 46 1.560 1.380 1.085 1.400 1.425 1.430 1.325 100.0 88.4 69.5 89.7 91.3 91.6 84.9 4.372 3.200 3.162 3.891 4.201 3.488 2.806 10 10 10 9 10 8 6 73 61 53 35 38 43 52 23 33 40 58 51 50 42 1.490 1.330 1.140 1.420 1.330 1.455 1.560 100.0 89.2 1S6.5 95.3 89.2 97.6 104.6 TABLE XII SEED PRODUCTION AND QUALITY IN RED CLOVER TREATED AT DIFFERENT STAGES OF DEVELOPMENT WITH .1 LB. Na 2,4-D p.a. Time of Application On Alderwood Soil Germination Sprouted H a n S e e i 1000 Seeds Weight gms Seeds from 50 Heads gms Estim. Flower Inten sity 1-10 On Ladne r Soil Germination Sprouted Hard Seed % 1000 Seeds Weight gms. May 12, 1951 » 19 " » 26 " June 2 " it Q n 72 3 6 52 58 54 62 25 55 39 37 39 34 1.560 1.400 1.335 1.590 1.685 1.945 100. C 89.7 85. 101.9 108. C 124.. 4 . 3 7 2 3.891 2.259 2.579 2.716 2.899 10 9 9 8 6 5 73 35 47 75 58 57 23 58 43 23 3 6 38 1.490 1.420 1.295 1.590 1.630 1.915 100.0 95.3 86.9 106.7 109.3 128.5 TABLE XIII 4 1 . SEED PEODUCTION AND QUALITY IN WHITE CLOVER TREATED WITH Na 2,4-D IN DIFFERENT CONCENTRATIONS Na 2,4-D Concentration lbs.p.a. On Alderwood Soil Germination Sprouted!Hard % |Seed % 1 0 0 0 Seeds Weight gms, % Seeds' from 50 Heads Estim. Flower Inten-sity 1-10. On Ladner Soil Germination Sprouted % Hard Seed % 1000 Seeds Weight gms. T o .01 .05 .1 •2 .5 1.0 25 3 6 4 6 3 6 3 2 29 30 70 56 45 54 62 64 63 .665 .720 .670 .625 .660 .640 .610 100.0 108.2 100.7 93.9 99.2 96.2 91.7 1.206 .736 .801 .846 1.139 .970 .634 10 10 10 10 10 9 7 32 33 42 37 39 17 14 64 58 46 54 57 77 80 .685 .670 . 6 4 0 .565 .620 .665 .620 100.0 97.8 93.4 82.4 90,5 97.0 90.5 TABLE XIV SEED PRODUCTION AND QUALITY IN WHITE CLOVER TREATED AT DIFFERENT STAGES OF DEVELOPMENT WITH ,1 LB. Na 2,4*»D P.A. Time of Application On Alderwood Soil Germination Sprouted! Hard % Seed 1 0 0 0 Seeds Weight gms. T Seeds from 50 Heads gms • Estim. Flower Inten-sity 1 - 1 0 On Ladner Soil Germination Sprouted Hard Seed % 1 0 0 0 Seeds Weight gms . T o May 12, 19 26 June 2 « 9 N It 1951 II n it it 25 36 38 39 42 33 70 54 58 54 51 60 .665 .625 .705 .690 .685 .660 100.0 93.9 106.0 103.7 103.0 99.2 1.206J .846 .950 1.203 .903 1.260 10 1 0 1 0 10 7 6 3 2 3 7 3 2 44 3 7 33 64 54 62 49 55 61 .685 .565 .650 .645 .700 .680 100.0 82.4 94.8 98.5 102.1 99.2 42. TABLE XV SEED PRODUCTION AND QUALITY IN KENTUCKY BLUEGRASS TREATED WITH Na 2,4-D IN DIFFERENT CONCENTRATION ' On Alderwood S o i l On Ladner S o i l Na 2,4rD Ger- 1000 Seeds Seeds Ger- 1000 Seeds Concentration min- Weight from min- Weight lbs.p.a. ation gms. 50 ation gms . i % Panicle % gms. 0 87.5 .365 100.0 1.609 85 .415 100.0 .1 82 .450 123.2 2.218 82 .410 98.7 i . 5 85.5 .385 105.4 2.570 83 .410 98.7 1.0 84.5 .435 119.1 1.763 86.5 .430 103.6 2.0 82 .425 116.4 1.814 81.5 .405 97.5 4.0 84 .450 123.2 1.801 85.5 .490 118.0 8.0 82.5 [•440 120.5 2.345 85 .420 101.2 TABLE XVI SEED PRODUCTION AND QUALITY IN KENTUCKY BLUEGRASS TREATED AT DIFFERENT STAGES OF DEVELOPMENT \ WITH 1.0 LBS. Na 2,4*D P.A. i On- Alderwood S o i l I — . On Ladner S o i l Na 2,4-D Ger- 1 0 0 0 Seeds Seeds Ger- 1 0 0 0 Seeds Concentration\ min- Weight from min* Weight lbs.p.a. ation gms. % 50 ation gms.~ % % Panicle % 1 gms . ;o 8 7 . 5 • 365 1 0 0 . 0 1.609 85 .415 1 0 0 . 0 May 1 2 , 1 9 5 1 8 4 . 5 . 4 3 5 119 .1 1 . 7 6 3 8 6 . 5 . 4 3 0 1 0 3 . 6 tt 1 9 n 83 . 4 2 0 1 1 5 . 0 1 . 9 2 0 84 .410 9 8 . 7 « 26 " 8 7 * 5 .340 9 3 . 1 2 . 0 0 6 8 5 . 5 • 3 8 5 9 2 . 7 June 2 w 82 .5 . 3 7 5 1 0 2 . 7 1.521 83 . 3 8 0 91 . 5 It 0 It 80 . 3 8 5 1 0 5 . 4 1 . 6 8 4 81 • 405 9 7 . 5 i i 43. TABLE XVII SEED PRODUCTION AND QUALITY IN PERENNIAL RYEGRASS TREATED WITH Na 2,4*-D IN DIFFERENT CONCENTRATIONS Na 2,4-D Concentration, lbs.p.a. On Alderwood S o i l On Li idner S o i l Ger-min-ation 1000 . Weial Seeds i t s Seeds from (panicle; gms. Ger-ming , at ion % 1000 I Weia Seeds i t gms. . i .gms. % 0 ol .5 I.O 2.0 4*0 8.0 92 87.5 88.5 88.5 100 90 92.5 2.070 2.150 1.960 1.930 2.110 2.210 2.200 i 100.0 103.8 94.6 93.2 101.9 106.7 106.2 • 3.664 6,431 5.584 5.853 4.742 3.923 4.199 89.5 91 95.5 97.5 89.5 93 96,5 2.160 2.120 1.820 2.220 2.000 2.280 2.280 100.0 98.1 84.2 102.7 92.5 105.5 105.5 TABLE XVIII SEED PRODUCTION AND QUALITY IN PERENNIAL RYEGRASS TREATED AT DIFFERENT STAGES OF DEVELOPMENT WITH 1.0 LBS. Na 2,4»D P.A. Time of Application On Alderwood S o i l On L adner S o i l Ger-min-ation % 1000 Wei Seeds eht Seeds from W.50 /panicle Ger-ming ation % 1000 Seeds Weight gms. % gms. 0 May 12,1951 " 19 w N 2 6 " June 2 n II Q tt 92 88.5 89.5 89.5 100 92 2.070 1.930 2.070 1.800 1.890 1.940 100.0 93,2 100.0 86.9 91.3 95.1 3.664 5.853 3.288 3.980 2 . 4 3 2 2 . 9 2 1 89.5 97.5 89 100 93 90 2.160 2.220 2.210 1.810 1,930 1.920 100.0 102.7 102.3 83,7 89.3 88.8 44. TABLE XIX SEED PRODUCTION AND QUALITY IN TIMOTHY TREATED WITH Na 2,4-D IN DIFFERENT CONCENTRATION On Alderwood S o i l On Ladner S o i l I Na 2,4-D Ger- 1000 Seeds Seeds Ger- 1000 Seeds § Concentration min- . Weight from min- Weight 1 lbs.p.a. ation gms. t 50 ation gms. i % Heads % gms. 0 100.0 .515 100.0 6.191 100.0 .505 100.0 .1 98.5 .475 92.2 5.564 98 .465 92.0 .5 97.5 . 4 4 0 85.4 7.289 99 . 4 5 0 89.2 1.00 98 .415 80.5 8.198 99 .415 82.1 2.0 98.5 .415 80.5 8.476 98.5 .420 83.1 4.0 98.5 .445 86*4 7.596 99.5 .465 92.0 8.0 99.5 .460 89*3 8.800 99.5 .445 ' 88.1 TABLE XX SEED PRODUCTION AND QUALITY IN TIMOTHY TREATED AT DIFFERENT STAGES OF DEVELOPMENT WITH 1.0 LBS. Na 2,4-D p.a. - On Alderwood S o i l On Ladner S o i l 1 Time of Ger- 1000 Seeds Seeds Ger- 1000 Seeds 1 Application min- Weight from min- Weifl ht 1 ation gms. % 50 ation gms • i ( % Heads I gms. 1 1 0 100.0 .515 100.0 6.191 100.0 .505 100.0 1 May 12,1951 98 .415 80.5 8.198 99 .415 82.1 § n 19 " 98.5 .425 82.5 6.882 98 .405 80.1 I w 26 " 100 .440 85.4 7.241 98 .395 78.2 1 June 2 M 98.5 .380 73.7 5.175 98 .390 77.2 1 II 9 " 98.5 .365 70.8 5.578 99 .380 75.2 § 45 IV. GREENHOUSE TRIALS Objects: The purpose of t h i s series of experi-ments, conducted i n the greenhouse, were twofeid. F i r s t of a l l , I t was thought that an experiment should be set out, under the more uniform conditions under glass, whieh would serve as a check on the f i e l d t r i a l s . Also, since nitrogen uptake appeared to be an important factor i n the f i e l d t r i a l s , i t seemed that a study should be made of the effects of 2,4-D sprayed plants, on the f r e e - l i v i n g , nitrogen-fixing, s o i l bacteria such as Azotobacter v i n e l a n d i i and A. chrococcum and symbiotic nitrogen-fixing bacteria such as the Rhlzobia of the legumes. Materials: One legume, red clover, and one cereal grass, barley var. O l l i , were chosen as the test plants. Cultures of Azotobacter v i n e l a n d i i and A. ffaroeoccum were obtained from the U.B.C. S o i l Bacteriology laboratory stocks. Alderwood s o i l came from the U.B.C. area, the muck s o i l from a half-bog i n West Point Grey, the Ladner clay from the Fraser River alluvium at the foot of Blenheim Street i n Vancouver and the I n t e r i o r pedocal from the Dominion Range Station lands at Kamloops, B. C. Methods: The Alderwood s o i l , pH 5.6, the muck s o i l , pH 4.2, and the Ladner s o i l , pH 4.8, some weeks before seeding were given an application of superphosphate, 50 mgs. per l b . of s o i l . The pH of a l l s o i l seeded to legumes was adjusted to Ca pH 6.0 by adding 175 mgs. 46. a g r i c u l t u r a l lime per l b . to Alderwood s o i l , 2.0 gms. per l b . to muck s o i l , and 700 mgs. per l b . to Ladner clay. Pri o r to seeding, legume seeds and s o i l s were inoculated with the Rhizobium-containing "Nitragin". Somewhat more a g r i c u l t u r a l lime was added to the s o i l s seeded to O l l i barley; v i z : Alderwood, 200 mgs. per l b . , muck, 2.6 gms. per l b . , and Ladner clay, 800 mgs. per l b . The lime was added on June 26th and on the date of planting, July 17th, the pH readings on the s o i l were as follows: Alderwood, 6.5, Ladner clay, 6.3, muck, 6.3 and In t e r i o r pedofal, 6.3. Pri o r to planting the barley, one-half of the seeds were soaked f o r 24 hours i n an Azotobacter v i n e l a n d i i and A. chrococcum suspension made from the surface growth of cultures i n Ashby's mannitol agar. Also 5 c c . (20 m i l l i o n c e l l s per c c . ) of the Azotobacter suspension were added to one-half of the pots of s o i l i n which O l l i barley was seeded. In the red clover experiment^ each treatment was accorded three pots, and 25 seeds were planted i n each pot, but a f t e r the seedlings were well established they were thinned to 5 plants per pot. 2,4-D ranges were as follows: 0.0 l b . , .01 l b . , and .1 .b. per acre and applications were made by aqueous*on July 25, 1952 when the plants were s t i l l s t r i c t l y vegetative - 4 true leaves on the plants grown on Ladner and muck s o i l s and 3 true leaves on the plants grown on Alderwood s o i l . Cuttings were made at 4 47 di f f e r e n t times: the f i r s t was accomplished on August 7th, while the plants were s t i l l vegetative, the second on September 6th, just before the flowering of the plants on Alderwood s o i l and early flower f o r the plants on the Ladner and muck s o i l s . Plants whose f i r s t cut was taken August 6th were again cut on October 6th when Alderwood-grown plants were vegetative, and Ladner and muck s o i l grown plants were pHre-bud. The l a s t cutting was made on plants which were i n milk seed stage on Alderwood, and on plants which were mature on Ladner and muck s o i l s . In the greenhouse, clover sprayed with only .1 l b . per acre of 2,4-D showed marked narrowing of leaves while i n the f i e l d t r i a l s the morphological response was only noted aft e r much heavier applications. Roots of a l l harvested plants were marked c a r e f u l l y and t h e i r nodules studied; they were c l a s s i f i e d as to white, green and pink and t h e i r lengths i n m.m. recorded. In the barley experiment the 2,4-D was applied with an atomizer on July 23, 1952 when the barley on a l l s o i l s was i n the 3rd l e a f stage. The concentrations of 2,4-D used were 0, .1 and 1.0 l b s . per acre. Half of the barley was harvested at pre-boot stage, 15 days afte r spraying, and the other h a l f at maturity, 57 days a f t e r spraying. Pour pots were assigned to each treatment and 20 seeds were planted per pot. 10 seedlings only, however, were permitted to develop. One-half of the pots i n each 48. treatment were taken f o r the early cutting treatment, the other h a l f were allowed to develop d i r e c t l y to maturity, a) Red Clover Vid. Table XXI and Table XXIA. - Nodulation, crude protein percentage, dry matter, and t o t a l nitrogen, In red clover grown i n the greenhouse, i n 3 s o i l s , treated with two concentrations of 2,4-D, and harvested at 4 di f f e r e n t stages of development. Pew s i g n i f i c a n t percentage protein changes were recorded. Depression of protein percentage occurred i n plants from Alderwood and Ladner s o i l s , but increases occurred i n plants grown on the muck s o i l s . However, the percentage composition table scarcely portrays the true picture, f o r the maturation process was altered by the 2,4-D, as well as the nitrogen uptake and t o t a l dry-matter y i e l d . Accordingly, a somewhat better idea of the interactions isobtained by an examination of the photos, which show the marked differences i n blooming time of the red clover under treatment, and of the Table XXIA which shows the dry matter elaboration and t o t a l nitrogen uptake. When these are appraised i t i s noted that again, the 2,4-D early applications frequently r e s u l t i n greater nitrogen uptake and greater y i e l d s . No differences i n nodulation were observed; the size, number and kind of nodules were much the same under a l l treatments. The 2,4-D concentrations of t h i s experiment was not high enough, apparently, to i n t e r f e r e with nodule formation. TABLE XXI MODULATION AND CRUDE PROTEIN; PRODUCTION IN RED CLOVER GROWN IN THE GREENHOUSE, ON THREE SOIL TYPES TREATED WITH TWO CONCEN AND HARVESTED AT FOUR DIF 49. TRATIONS OF Na FEBENT STAGES OF DEVELOPMENT Alderwood Soil Unclassified Muck Soil Na 2,4* Concen-tration Crude Protein % of Dry lbs.p.a.iMatter % of Control Deviation from Control Number per Plant Woflules Length Range in m.m, Cr.ude Protein % of Dry Matter Nodules Crude Protein % of Control Deviation from Control Number per Plant Length Range in m.m. % of Dry Matter % of Control Deviation from Control Nodules Numberj Length per j Range Plant Bin m.m. Harvested 15 Days After Spraying 0 • 01 .1 23*80 24.93 23.94 100.0 104*7 100.5 0 L I S * .14 0 3.1 4.2 2.4 i « l | §-2§ 1*1 1 123.86 23.43 23.0-6 100.0 98.1 96.6 0 0 .43 •80: 78.9 100.7 52.8 J 4 i 4 -4 124,22 22.33 [23.85 100.0 92.1 98.4 0 0 1.89* • 37 16 , 6 11.9 6,9 : 4 b-2i • **2 Harvested 45 Days After Spraying 0 .01 .1 19.64 18*83 20*85 100.0 95.8 106.1 0 0 .81 43.2 36»3 34.2 H 1 g — D 1 i-5 J 16.21 17.50 18.10 100,0 107.9 111,6 0 L . 2 9 * L,89* 0 69.7 56.7 6 i . l 1-6 1*6 1-6 16.14 14.93 14*49 100.0 92 , 5 89.7 0 0 1,21* 1.65* 38.1 29.3 29,8 1 - 7 I«7 iHarve sted 75 Day After £ 3praying 0 .01 • 1 19.82 17.48 17.34 100.0 88.1 87.4 0 0 2.34* 2.48* 42.0 23.0 34.3 |*4 1 - 5 16.27 20.05 18,29 100.0 123.2 112.4 0 3.78* 2.02* 0 60,6 309.2 '83.0 15.81 15.70 16.22 100.0 99.3 102 . 5 0 .41 0 .11 63.9 63.0 75.0 |-4 0 .01 .1 18.75 17.69 19.56 100.0 94.3 104.3 0 .81 0 1,06* 36.2 35.4 35.1 i * 5 21,55 22,55 '21,64 Harvested 70 Days (Second Cut) After Spraying 100,0 104.6 100.4 0 LOO .09 65.2 84.0 9 2 . 1 i-5 18.56 100.0 0 0 60.2 i-4 f«5 19.28 103.8 .72 62.8 i-5 i-5 19.22 103.5 .66 67.1 1-5 M.S.D. (5% point) 1.0397% * TABLE XXIA 50. Production of Red Clover Grown i n the Greenhouse, on Three S o i l Types, Treated with Two Concentrations of Na 2,4-D> and Harvested at Four Different Stages ofDevelopment. 2,4-D Alderwood S o i l Unclassi So iiedMuck •11 Ladner S o i l p.a. : Dry Mattel 5 plants gms. r Crude Pro-tei n per 5 plants gms. Dry Matter per 5 plants gms. • Crude Pro-tein per 5 plants gms. ^  Dry Matter per 5 plants gms. Crude Pro-tein per 5 plants gms. Harvested 15 Days After Spraying 0 .01 .1 i .794 .678 ; .666 .188 .169 .159 2.719 3.014 2.208 .647 .706 .509 2.257 2.316 1.380 .546 .517 .329 Harvested 45 Days After Spraying 0 .01 .1 2.583 3.475 1.501 .507 .65U .312 -8.115 9.219 9.102 1.315 1.613 1.647 8.610 8.415 8 .420 1.389 1.256 1.220 Harvested 75 Days After Spraying 0 .01 •1 2.736 3.731 2.770 .51*2 .652 .480 8.234 9.475 9.925 1.339 1.899 1.815 11.500 12.506 12.811 1.818 1.963 2.077 Harvested 70 Days (Second Cut) After Spraying 0 .01 •1 2.454 2.339 2.356 .460 .413 •460 2 .245 2.301 2.710 .483 .518 .586 3.405 4.082 3.462 .631 .787 .665 M. S. D. ($% point) Dry Matter - .3109 gms. Crude Protein .3232 gms. 51. Figure 5. - Red Clover grown i n the greenhouse in pots of Alderwood gravelly loam and treated at the t h i r d true l e a f stage as follows: 1. control, no 2,4-D, 2 . .01 l b . 2,4-D per acre 3 . .1 l b . 2,4-D per acre. Photo taken four weeks after treatment. 52 Figure 6. - Red clover grown i n the greenhouse in r>ots of Ladner clay and treated at fourth true l e a f stage as follows: 1. control, no 2,4-D, 2 . .01 l b . 2,4-D per acre, 5 . .1 l b . 2,4-D per acre. Photo taken four weeks afte r treatment. 53. b) O l l i Barley Vid. Table XXII: Y i e l d of dry matter In O l l i barley, grown i n the greenhouse, i n four s o i l s , with and without Azotobacter v i n e l a n d i i and A. chrococcum, with and without 2,4-D. Vid. Table XXIIA - y i e l d of crude protein i n O l l i barley, grown i n the greenhouse, i n four s o i l s , with and without Azotobacter v i n e l a n d i i and A. chrococcum, with and without 2,4-D. Vid. Table XXIII - as for Table XXIIA preceding but the expression i s i n terms of percentage of protein. To the eye, q u a l i t a t i v e l y appraising,this green-house experiment, the r e s u l t s were s t r i k i n g . Much of the error v a r i a t i o n of the f i e l d t r i a l s was avoided and the responses showed a welcome uniformity. The quantitative appraisal, as recorded i n the tables mentioned above, i s also s t r i k i n g . Not only are the variables of 2,4-D l e v e l s and of Azotobacterinoculation interesting per se, but also t h e i r interactions with the dif f e r e n t s o i l s . Azotobacter inoculation of the Alderwood s o i l increased the y i e l d of the barley grown on the s o i l promptly afte r inoculation (pre boot stage). On a l l s o i l s the i n f l u -ence of the inoculation was generally observed, i n the increased y i e l d of seed and of haulm, and i n larger, t a l l e r plants. 54. 2,4-D e f f e c t s , per se, were most pronounced on the Alderwood s o i l . Apparently, as f a r as Influence on y i e l d i s concerned, 2,4-D was somewhat more e f f e c t i v e on the plants of coastal s o i l s than was inoculation of s o i l with Azotobacter. Azotobacter and 2,4-D i n combination did not give an additive response, or i f so, not an appreciable one. Curiously, i n an Int e r i o r s o i l , well populated with Azotobacter, 2,4-D depressed y i e l d . Expressed as percentage composition protein, the effects of 2,4-D and Azotobacter are not' s t r i k i n g . Some percentage increases are to be noted, e s p e c i a l l y on the muck s o i l and on the Ladner clay. However, f a r more revealing, are the ef f e c t s expressed i n terms of the nitrogen taken up. Quite generally with both treatment series alone and i n combination, increased nitrogen uptake by the barley i s noted. It i s observed a d d i t i o n a l l y that i n the Int e r i o r alluvium, barley treated with 1.0 pounds 2,4-D per acre took up l e s s nitrogen than the control. Azotobacter populations - v i d . Table XXIV: estimated Azotobacter per gram of dry s o i l following 2,4-D treatments of plants grown i n the s o i l . In an attempt to record the changes on the s o i l f l o r a following 2,4-D application to plants, the populations of the f r e e - l i v i n g , nitrogen-fixing, Azotobacter were followed. The«record i s somewhat inconclusive f o r a l l but the Alderwood s o i l . Here the populations increased rapi d l y following 2,4-D 55. application u n t i l at 15 days a f t e r treatment, there were ca. 7x as many organisms i n the s o i l supporting 2,4-D treated plants, as i n the control s o i l . 72 days a f t e r spraying, the population differences i n the s o i l s were n e g l i g i b l e . Possibly because of the infrequent sampling, but possibly f o r other inherent reasons," appreciable differences i n Azotobacter populations were not recorded f o r the other s o i l s 56. TABLE XXII YIELD OF DRY MATTER IN OLLI BARLEY, GROWN IN THE GREENHOUSE IN FOUR SOIL TYPES, WITH AND WITHOUT AZOTOBACTER VINELANDII AND A. CHROCOCCUM, WITH AND WITHOUT 2,4-D Treatment Azotobacter 2,4-D l b S e P . a . Pre Boot Stage 15 Days After Spraying gms e Mature 57 Days After Spraying Seed gms. Halm Only gms. Length of Halm 57 Days After Spraying cm. ALDERWOOD GRAVELLY LOAM (COASTAL) Without 0 2,111 1.967 2 , 3 2 0 4,298* 35.0 t  .1 2.334 6.118* 47.7* it 1,0 2 a456* 7.051* 4.486*: 51.0* With 0 4.289* 5.590* 3.780* 45.0* n .1 4,875* 6.130* 4.294* 48.7* it 1.0 4.779* 5.401* 4.402* » 51.5* UNCLASSIFIED MUCK (COASTAL) Without it it With II ti 0 .1 1 . 0 0 o l 1 . 0 4 * 5 8 2 4 . 7 8 4 4 * 3 6 9 4 . 9 9 1 * 5 . 7 0 9 * 4 . 5 2 5 I* 630 5.518* 6.139* 4.634* 5.737* 6.707* &• 580 34.7 3.883* 53.0* 3.841* 57.0* 3.726* 51.5* 3.946* 55.7* 3.781* 53 .0* LADNER CLAY (COASTAL) Without 0 4.090 6.369 4.100 52.2 ti • 1 5.104* 7.637* 5.056* 57.7 ti 1.0 5.854* 8.091* 5.636* 63.7* With 0 4.251 6 . 5 2 1 * 4.178 54.5 it .1 4.247 6.711* 4.222 48.7 n 1.0 5.951* 7.513* 4 . 9 2 2 * 54.7 SILTY PEDOCAL (ALLUVIUM FROM THE "DRY INTERIOR") Az, occur- 0 6.784 6.896 5.762 66.7 ring natur- 1.0 5.988* 4.687* 4.677* 59.5* a l l y M.S.D. % Dry Matter (5% point) a) Pre Boot Stage .3141 gms, b) Seed .2364 " c) Halm only ,2627 11 d) Length (Halm) 5.9165 cm. TABLE' xxnr 57< Yield of Crude Protein in OIL! Barley Grown in the Greenhouse in Four Soil Types, with and without Azotobacter Vinelandii and A. Chrococcum, with and without 27H-D , Treatment Preboot Stage l 5 days after spraying, gms. Mature 57 days after spraying Azotobacter 2,4-D lbs: p.a. Seed gms. Halm only gms. Alderwood Gravelly Loam (Coastal) J Without n n With tt n 0 .1 1.0 0 .1 1.0 .302 «33° .299 . 6 1 3 * . 6 4 9 * . 6 7 9 * .148 . 4 5 8 * . 5 3 0 * . 5 2 3 * . 5 2 2 * . 4 6 3 * .064 . 1 2 1 * . 1 3 4 * •106 .115* . 1 2 2 * Unclassified Muck (Coastal) Without N It With n N 0 .1 1.0 0 .1 . . 1.0 .397 .453 .411 .469 . 5 3 4 * .434 .170 .064 . 5 1 8 * .097 . 5 2 3 * .091 . 4 7 3 * .086 j . 5 3 5 * .085 . 7 5 1 * .094 j Ladner Clay (Coastal) j Without n tt With n it 0 .1 1.0 0 .1 1.0 .339 .416 .567* .448 .434 .671* .575 .719* . 6 7 6 * •628* . 6 3 9 * •691* .101 •122 .137 .104 .101 ! - .155* Silty Pedocal (Alluvium from the "Dry Interior") kz• Occurring 0 Naturally J 1.0 .765 .674 .628 ,202 i . 4 6 2 * .203 1 3 1 M.S.D. ($% point) A.) Crude Protein Preboot Stage .112 gms. B.) " • Seed ,043 •» G.) » « Halm Only .038 • TABLE XXIII CHUDE PROTEIN IN OLLI BARLEY, GROWN IN THE GREENHOUSE IN FOUR SOIL TYPES, WITH AND WITHOUT AZOTOBACTER VINELANDII AND A. CHROCOCCUM WITH AND WITHOUT 2,4-D ~ CRUDE PROTEIN Treatmi ants Let . 15 Dai if vPre Boot; rs ^ af ter Spraying Seed 57 Days_.after Spraying ] 57 Da: i&lm. Only Fs^after Spraying Azotobacter 2,4-D lbs.p.a. % oi dry matter oi control deviation from control dry .matter 7» 61 control deviation from control $61 dry matter Jb of control DeVi atIon from . control ALDER1 tfOOD GRAVELLY LOAM (COASTAL) Without n 11 With it tt 0 .1 1.0 0 .1 ' i.o -14.34 14.55 12.17 14.31 13.33 14.21 100.0 101.4 84.8 99.7 92.9 f 99.0 + 0 j 7.56 4- .21 7.50 -2.17* 7.53 - .03 9.36 -1.01* 8.52 • - .13 I 8.58 i 100.0 J <• 0 99.2 1 - .06 99.6 - .03 123.8 4-1.80* 112.6 4- .96* 113.4 1 t-1.02* 2.77 2.83 3.00 2.83 2.70 | 2.78 100.0 102.1 108.3 102.1 97.4 \ ioo r3 i 0 + .06 * .23* * .06 | - .07 t • «• .01 • • II UNC] LASSIFIED MUCK (COASTAL) 1 Without j 0 8.67 100.0 * 0 i10.43 100.0 4- 0 2.50 100.0 o I tt j .1 9.48 109.3 + .81* 1 9.40 90.1 «*1.03* 2.52 100.8 ** .02 |, II j 1.0 9.41 108.5 , * .74* ! 8.53 81.7 -1,90* 2.38 95.2 - .12 | With 1 0 9.41 108.5 * .74* I10.22 97.9 - .21* 2.33 93.2 .17* P It j .1 9.36 107.9 1 «• .69* I 9.34 89.5 -1.09* 2.17 86.8 .33* I tt J 1.0 9.60 110.7 i 4. .93* 111.21 107.4 * .78* 2.51 100,4 4» • 01 LADNER CLAY (COASTAL) 1 Without 1 0 8.31 100.0 ! * 0 j 9.04 1 100.0 * 0 2.48 100.0 ± 0 I « i ] .1 8.17 98.3 ! - 1 9.42 104.2 * .38* 2.42 97.5 .06 I II 1.0 9.69 116.6 *1.38* 1 8.36 92.4 - .68* 2.44 98,3 m .04 j With 0 10.56 127.0 J •2.25* I 9.64 106.6 * .60* 2.51 101.2 .03 " i .1 10.22 122.9 i *1.91* 1 9.53 ! 105.4 2.40 96.7 ,08 I i 1.0 S11.28 135.7 1 4-2.95* 1 9.21 | 101.8 4. .17® 3.15 127.0 *• .67* SILTY PEDOCAL (ALLUVIUM FROM THE "DRY INTERIOR") Az. occurring 0 11.29 i 100.0 ! * 0 1 9.11 100.0 /s * 0 3.51 100.0 ± 0 naturally 1.0 11.27 I 99.8 I — .02 I 9.87 1 108,3 + .76* j 4.36 124.7 *> .85* M.S.D. % Dry Matter (5% point) a) Crude protein leaf .3593 b) " " seed .1823 c) n " halm .1483 59 TABLE XXIV ESTIMATED AZOTOBACTER PER GRAM OF DRY SOIL FOLLOWING 2-4-D TREATMENTS OF PLANTS GROWN ON THE SOIL TREATMENT Na-2-4-D lbs* p.a. Number of Azotobacter chroococcum f Azotobacter vinelandii per Gram Soil in Millions 15 Days After Spraying 72 Days After Spraying Dilutions 1:100.000 Dilutions 1:100,000 Dilutions 1:1,000,000 Number| Difference Number 1 Difference! Number 1 Difference Alderwood Gravelly Loam (Coastal) 0 • 1 1.0 3.3 21.7 12.9 • 0 «• 18.3* * 9.5* 2.7 18.3 14.0 * 0 «• 15.6* • 1 1»3* 2.7 2.3 3.3 • 0 • .4 * .6 Unclassified Muck (Coastal) 0 .1 1.0 4.2 3.5 1*1. •• 0 * .67 » 3.07* 3.7 3*7 -1.7 * 0 * 0 * 2.0® 1.3 2.0 1.3 • 0 • .7 1 o Ladner Clay (Coastal) 0 .1 1.0 8.8 7.6 9.7 t 0 - 1.2 * .9 9.3 9.0 9.0 1 o * .3 • .3 2.3 4.3 2*3 i o *> 2.0* t o S i l t y Pedocal (Alluvium from the "Dry Interior") 0 1.0 .8 1.97 * 0 7 1.14 1*0 1.7 * 0 * .7 4.3 3.3 * 0 - 1.0 M.S.D. (Number of Azotobacter in millions per gram of soil) (a) 15 days; d i l . 1:100,000 1.6 (b) 15 days; d i l . l:l;000,000 2.3 (c) 72 daysj d i l . ltlOODOO 2.0 6 0 , F i g u r e 7. - O l l i b a r l e y grown on Alderwood g r a v e l l y loam i n the greenhouse. Treatments as f o l l o w s : 1. C o n t r o l , no 2,4-D, no s o i l i n o c u l a t i o n w i t h A z o t o b a c t e r . 2. Azoto-b a c t e r i n o c u l a t i o n o f s o i l p r i o r to p l a n t i n g . 3. 2,4-D a p p l i e d at .1 l b . per acre a t t h i r d l e a f stage, and s o i l i n o c u l a t e d w i t h A z o t o b a c t e r p r i o r to p l a n t i n g . Photo taken 5 weeks a f t e r 2,4-D treatment. Note immaturity and s m a l l e r s i z e o f the c o n t r o l . Note the u n i f o r m i t y o f 2 and l a c k o f i t i n 3. Figure 8. - O l l i barley grown i n the greenhouse in muck s o i l . Treatments as follows: 1. Control, no 2,4-D and no s o i l i noculation. 2 . S o i l inoculated p r i o r to seeding with Azoto-bacter and, 3» s o i l inoculated p r i o r to seeding with Azotobacter and plants treated at t h i r d l e a f with .1 l b . 2,4-D per acre. Photo taken 5 weeks a f t e r 2,4-D treatment. Note immaturity and smaller size of the control plants. 62. Figure 9 . - O l l i barley grown on Ladner s o i l i n the greenhouse. Treatments as follows: 1. Control, no s o i l inoculation and no 2,4-D. 2. Azotobacter inoculation p r i o r to seeding and, 3 . Azotobacter inoculation p r i o r to seeding and 2,4-D at ,1 l b . per acre at t h i r d l e a f stage. Photo taken 5 weeks a f t e r 2,4-D treatment. The control i n this case matures at about the same time as the treated plants but i s the lowest i n y i e l d . Plants of 3 are heaviest y i e l d e r s of dry matter and crude protein. UNCLASSIFIED MUCK SOIL W I T H A Z O T O B A C T E R . I W I T H O U T A Z O T O B A C T E R . I W I T H A Z O T O B A C T E R . I W I T H O U T A Z O T O B A C T E R . I 0 I 2 3 4 S OMS. | j \ YIELD. /' Figure 10. - A graphical summary of the responses, i n terms of dry c n matter y i e l d , greenhouse grown O l l i barley, to four d i f f e r e n t s o i l s , to s o i l inoculation with Azotobacter and to d i f f e r e n t concentrations of 2,4-D. 64. V. DISCUSSION Many authors have pointed out that 2,4-D and other auxins are organic regulators of physiological processes and are not i n themselves, l i k e carbohydrates, energy y i e l d i n g . Therefore, i t becomes of some interest to determine the manner i n which the 2,4-D i n certa i n of our experiments increased dry matter y i e l d s and nitrogen uptake. Several possible answers to the question are offered. In the f i r s t place, It may be asked i f i n our experiments the energy p o t e n t i a l of the plant was increased through the application of 2,4-D. Does auxin concentration l i m i t the metabolic a c t i v i t y of the forage plant during the grand period of i t s growth? Does 2,4-D added a r t i f i c i a l l y , at an early stage in development, meet t h i s deficiency which, when wet, permits greater synthetic a c t i v i t y by the plant? Our experiments neither support nor detract from such a possible explanation but c e r t a i n l y 2,4-D acting i n t h i s way could increase the energy p o t e n t i a l of a plant. Other p o s s i b i l i t i e s would permit auxin to act i n such a manner that t o t a l plant energy i s increased. Virtanen and others i n extensive, well-known experiments, have estab-l i s h e d to t h e i r s a t i s f a c t i o n , but not to that of many others, that a number of organic substances are excreted from the roots of plants in general, and i n p a r t i c u l a r , from the 6 5 nodules of legumes. The excretion, they say, i s to be noted at times when the plant i s growing vigorously. I f such plant excretions are a f a c t , then i t seems possible that they might profoundly modify the f l o r a and fu-ana of the rhizosphere. 2,4-D i t s e l f might be excreted following i t s applica-t i o n to a e r i a l portions and t h i s might produce a heightened nitrogen f j ^ a t i o n i n the s o i l and an increase i n nitrogen uptake. Two items came to mind which might favour t h i s view, v i z . (a) that 2,4-D added to s o i l s i n non-herbicidal concentrations produces y i e l d and nitrogen responses similar to those obtained when 2,4-D i s applied to the a e r i a l parts of forages, and (b) that Rhizobia and Azotobacter, nitrogen-f i x i n g bacteria, are known to' produce auxin i n larger quantities than many, bacteria. Sugars^might be the excreta,, for 2,4-D, i t , i s well known, increases the sugar leve l s i n plants at the expense of reserve carbohydrates., Sugars, too, might modify the ecology of the. s o i l organisms. In. any event, the p o s s i b i l i t y exists that the,increases in" dry matter y i e l d and nitrogen uptake by plants treated with ..sub-' h e r b i c i d a l l e v e l s of 2,4-D result*? from a modification of the ecology of the s o i l microflora. In an analagous way, a n t i b i o t i c s are known to modify the ecology of i n t e s t i n a l die f l o r a and f a a n a o f young animals and thereby increase t h e i r rate of gain.. Another possible answer to our:, question l i e s i n the kind of substances which are found in the plant. Auxins could profoundly, modify the synthetic processes i n the plant 6 6 without materially a l t e r i n g the energy pote n t i a l of the plant. It may be that structural compounds of the plant might be elaborated at the expense of reserve carbohydrates, i . e . ce l l u l o s e , l i g n i n , protein,, etc., might increase at the expense of starches, fructosans, sugars, etc.. Such a re-dir e c t i o n of the synthetic function of the plant could possibly explain increased dry matter y i e l d s . Forage produced of such r e d i r e c t i o n would, of course, be of less value to the grazing animal. Calorimetric studies should quickly determine whether or not synthetic" r e d i r e c t i o n i s an answer. That redirection.occurs i s well brought out by van Overbeck when he related carbon-nitrogen r a t i o s to auxin l e v e l s and to f r u i t f u l n e s s . But whether i t Explains dry matter and y i e l d increases or not i s , quite possibly, another aspect. The topic seems to be worthy of pursuit. The vistas of usefulness which control of maturation processes., nitrogen uptake, and dry matter production appear to open up are.intriguing. 6 7 . VI. A SUMMARY STATEMENT OF OBSERVATIONS AND CONCLUSIONS 1. 2,4-D applied i n sub-herbicidal concentrations to the forage plants of these experiments when"young and a c t i v e l y growing resulted i n an eventual increase i n dry matter y i e l d , and nitrogen uptake. "Too l i t t l e " 2,4-D gave no response, "too much". 2,4-D was depressive.. 2. The i n i t i a l e f f e c t of even the "low" concentrations was to depress y i e l d s but increased synthetic a c t i v i t y followed. 3. As the plants matured, lar g e r amounts of 2,4-D were required to produce eventual increases i n synthetic a c t i v i t y and the point was-reached a t - l a t e r stages of growth where such 2,4-D additions were simply depressive. 4. 2,4-D, i t i s apparent, in our experiment, impinged on the maturation processes of the forages but not i n very predictable ways. Applied when the plants were young i t seemed to hasten maturity; when applied slater, to delay i t . However', these effects appoarod to be related In some subtle way to the s o i l , the phot©period and probably the C/N ra t i o of the plant. 5. To obtain " b e n e f i c i a l " responses related above, 5 to 10 times as much 2,4-D was required f o r grasses as f o r clovers. 6. Percentage composition was. not a r e l i a b l e guide to the effects of'2,4-D on the forage. --Total y i e l d s of dry matter and protein are important factors i n effect appraisal. 68. 7. Kjeldahl nitrogen, expressed as crude protein, did not. adequately indicate the effect of 2,4-D on the nitrogen metabolism of the plant. Non-protein nitrogen would be worthy of appraisal, but was not determined for our experi-ment s. 8. In general, i t appeared that plants growing i n the r e l a t i v e l y i n f e r t i l e Alderwood s o i l showed more marked responses when young to 2,4-D than those i n the more f e r t i l e Ladner clay or muck s o i l . Maturation processes apparently proceeded more r a p i d l y with season on the upland Alderwood s o i l so that plants were responsive f o r a shorter period to the sub-herbicidal concentrations of 2,4-D. 9. Not only did responses to 2,4-D d i f f e r with d i f f e r e n t s o i l s but also with d i f f e r e n t forage species. This may have been due to d i f f e r e n t rates of maturation of the species. 10. E f f e c t s of 2,4-D on see.d,..prpduction were variable. High concentrations unquestionably .reduced seed y i e l d but not necessarily seed si z e . The responses were confounded somewhat with time of flowering. In general, i t may be said that some responses are marked and worthy of further s p e c i f i c study. An interesting aside was the e f f e c t of 2,4-D on the percentage of hard seeds of red and white clover and the uniformity of development. 11. 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