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Influence of 2,4-D on the soil microflora Westlake, Donald William Speck 1955

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INFLUENCE OF 2,it-D ON THE SOIL MICROFLORA. by DONALD WILLIAM SPECK WESTLAKE A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of SOIL SCIENCE, We accept this thesis as conforming to the standard required from candidates for the degree of MA.STER OF SCIENCE. Members of the Department of SOIL SCIENCE THE UNIVERSITY OF BRITISH COLUMBIA. May, 1 9 5 5 ABSTRACT A rhizosphere effect was demonstrated for barley and corn plants at k to 6 weeks of age. No qualitative difference between the composition of the rhizosphere and indigenous flora was detected in the response of isolates to nutritional media of varying complexity. Examination of the rhizospheres of barley plants treated with 2,k-D at the rates of 0, k and 8 ounces per acre indicated that there was no differ-ence between the total counts of treated and untreated plants. However, evidence was obtained suggesting a qualitative difference between the rhizo-spheres of the 8 ounce treated plants and those of the 0 and k ounce treated plants. On the other hand, corn plants treated with 2,k-D at the rates of 1 and 2 pounds per acre showed a temporary increase in their total rhizosphere count as compared to the total rhizosphere count of untreatedjplants. The increased total counts appeared to be due to the stimulation of those organisms requir-ing amino acids and yeast-soil extract for maximum growth. The direct application of 2,k-D to soil at the rate of 100 pounds per acre appears to result in a slight decrease in the total indigenous count. Although this lowering of the total count was due to a decrease in the number of organisms i n a l l nutritional groups, some groups were affected more than others. By comparing the growth response of isolates from the indigenous and rhizosphere flora to 2,k-D at different pH levels, evidence was obtained which indicated that a physiological difference exists between these flora. A study of the rates of the decomposition of 2,k-D in different soils indicates that there is a marked variability i n the detoxication rates. An organism was isolated which was capable of decomposing 2,k-D, This isolate showed morphological and cultural characteristics which suited members of the foryHbacterium and Achromobacter genera. A C K N O M L K D G M B N f The author wishes to acknowledge Dr. D. G. Laird and Professor A. J. Renney for their suggestions and assistance in the carrying out of this study. He also wishes to thank Miss J. L. Borden and Mr. J. P. Law for their contributions to this problem. Grateful acknowledgment is also extended to the Agricultural Institute of Canada for the scholarship which was given to the author to finish this study. TABLE OF CONTENTS I INTRODUCTION 1 II LITERATURE REVIEW 2 A. The Hormone Herbicide 2,U-D 2 (a) Absorption and Translocation of 2,k-D by Plants 2 (b) Fate of 2,k-D i n Plants 3 (c) Response of Corn Plants to 2,k-D L B. Persistence of 2,k-D i n S o i l 5 (a) Leaching 5 (b) Microbial Decomposition 8 (i) Kinetics of decomposition 9 ( i i ) Mechanism of breakdown 11 ( i i i ) Isolation of an effective organism 13 (c) Physical and Chemical Combination lit. (d) Effect of Soi l Characteristics 1S> C. Effect of 2,k-D on S o i l Microorganisms 17 D. Association between Plants and Microorganisms 19 (a) Rhizosphere 19 (b) Root Excretions of Plants 20 (c) Biological Equilibrium i n Soils 22 III EXPERIMENTAL 2l± A. Characterization of the Soi l 2h B. Rhizosphere Studies 25 (a) Barley 25 (i) Experimental 25 ( i i ) Results 28 ( i i i ) Discussion 30 (b) Corn (preliminary studies) 31 (i) Experimental 31 ( i i ) Results 31 ( i i i ) Discussion 33 (c) Corn (final studies) 35 (i) Experimental 35 ( i i ) Results 35 ( i i i ) Discussion 37 C. Influence on the Flora of 2,U-D Applied Directly to the S o i l 37 (a) Experimental 37 (b) Results 38 (c) Discussion 39 D. Effect of 2,U-D on Pure Cultures of S o i l Bacteria (a) Experimental (b) Results • (c) Discussion E. Study of the Decomposition of 2,U-D i n S o i l (a) Kinetics of the Breakdown of 2,U-D i n Soils . . . . (i) Experimental ( i i ) Results ( i i i ) Discussion (b) The Nature of the S o i l Organisms Responsible for the Decomposition of 2,1|-D i n S o i l IV GENERAL DISCUSSION ¥ CONCLUSIONS . . . . VI BIBLIOGRAPHY VII APPENDIX 1 I Introduction The increased use of chemical insecticides, fungicides and herbicides i n agricultural practice has focused attention on their possible effect on the s o i l microflora and thus on s o i l f e r t i l i t y . This being almost a virgin f i e l d i n biological research, relatively few papers have appeared to date i n the literature, and those that have are largely devoted to an assessment of the influence of the chemicals on the t o t a l numbers of s o i l bacteria. Appreciating the distinction between the rhizosphere and the indigenous s o i l populations, i t was thought that perhaps specific effects of chemical treatment might be demonstrated by comparing the microflora of the rhizo-sphere of treated plants with that of untreated plants. It was f e l t that the comparison should be made not only i n respect to t o t a l numbers, but also as to the effect on the various nutritional groups of organisms as suggested by Lochhead and Chase. In view of the important part played by the s o i l microflora i n the detoxication of treated s o i l s , i t was f e l t that i t would be of interest to isolate and study the nutrition of s o i l organisms capable of doing this. Since 2,U-B i s the chemical most widely used i n weed control, i t was decided to study i t s effect upon the s o i l microflora. 2 II Literature Review A survey of the literature dealing with herbicides has disclosed that, while there are many papers on the effect of herbicides upon plants, there are few of importance relating to the effect of herbicides on the micro-flora as i t normally occurs, in soils. This may be due in part to the lack of knowledge respecting the interaction of mixed populations and in part to the lack of suitable physiological groupings whereby the microorganisms might be studied more or less in their natural habitat. To date the study has been confined to the influence of herbicides on the total numbers of organisms in the soil or on specific species under laboratory conditions. A. The Hormone Herbicide 2,U-D In l° i i2 , Zimmerman and Hitchcock 0-03) recognized the exceedingly strong hormonal and herbicidal effect of 2,lj.-D, a chlorinated derivative of the synthetic plant hormone phenoxyacetic acid. Other chlorinated deriva-tives of phenoxyacetic acid of herbicidal importance are MCPA and 2,U»5-T. The structures of these compounds are related as follows: 2,methyl-H, chloro-phenoxyacetic 2, l i ,5-tr ichlorophenoxyacetic a c i d ac id 2,U-dichlorophenoxyacetic a c i d (a) Absorption and translocation of 2,U-D by plants. One of the most unique features about 2,ii-D and other synthetic 3 hormone-like materials is their ready absorption and translocation through vascular tissues. Mitchel (62) reports that non-polar compounds of 2,h-D enter the leaf, tissue much more readily than do the polar compounds. The polarities of the commonly used compounds of 2,U-D are as follows: sodium salt) ammonium salt famine salt^ acid J esters (21). The pH of the applied solution affects the absorption and the activity of these compounds in that there is a higher degree of absorption and of herbicidal activity as the pH is lowered. The use of "adjuvants such as surfactants and cosolvents tends to increase the absorption of 2,k-D from aqueous solution (21). Grafts (22) states that i t would appear that 2,k-D and similar compounds enter plant leaves through the cuticle, not through the stomata, and that the oiL-soluble, non-polar compounds enter most rapidly and in greatest quantity. After passing through the cuticle, these compounds enter living cells, the mesophyllj and from there they are released into the phloem and thus translocated with the synthesized food materials to regions of utiliza-tion, i.e., young leaves. He further states that when these compounds are absorbed through the roots, they are translocated to the leaves where they accumulate and bring about death or injuryj and that the tissues involved in this absorption and translocation are mostly mature tissues which are characterized by a lack of response to 2,U-D. whereas in contrast, the cells that accumulate these compounds are mostly meristematic and as a result are characterized by increased water uptake, high turgor pressure, abnormal division and growth, high respiration rate, and eventually death. He suggests that hormone herbicides are sufficiently like normal metabolites to be handled by protoplasmic structures without immediate injury, (b) Fate of 2,U-D in plants. weintraub et al (98) have shown that 2,U-D (radioactive) may be fairly rapidly metabolized by plants with an attendant release of radioactive CO2. The very uniform distribution of radioactivity in young leaves of cotton plants treated by applying a droplet of radioactive 2,U-D to a single coty-ledon, suggests the possibility that such metabolically released CO2 might be synthesized into cell constituents and laid down in expanding leaves. He further points out that within a few days after the application of 2,U-D, both side chain carbon atoms labelled, that radioactivity can be found distributed amongst acids, sugars, dextrins, starches, pectins, proteins and cell wall constituents. Whatever the processes involved in the breakdown of 2,lt-D in the plant, they are apparently fairly rapid and in some cases at least, as indicated by recovery rates of treated plants, fairly complete. Undoubtedly in plants in which they account for a fair to a large proportion of the chemical appliedj they constitute an effective detoxication mechanism. They might account for the ability of some species to stand rather large doses of 2,U-D, although the herbicide is readily absorbed and translocated, (c) Response of corn plants to 2,U-D. Bashaw (33) reports that the tolerance of corn plants to 2,lt-D is not related to plant size, but rather to the rate of growth at the time of treatment. Rossman (81) found that com plants appear to be the most sensi-tive of the cereals to 2,ii-D. The data of both suggest that after corn plants have passed the six-eight leaf stage of development, their sensi-tivity to 2,U-D decreases very rapidly. When the tassel stage is reached, the plants are not susceptible to 2,U-D injury, even when concentrations of 3000 and 5000 ppm. are used. The symptoms of corn plants treated with 2,U-D at the six-eight .leaf stage of development include malformation of brace roots, defective tassel formation, lower yields, twisting and bending 5 of the leaves and stem and the proliferation and fasciation of root tissue. KLle (26) stresses the effect of environment on the action of 2,k-D upon young sweet corn plants. A summary of his results, corn plants treated with the equivalent of one pound of 2,k-D amine per acre, are as follows: (1) exposure of corn plants at the time of treatment to a temperature of 90°F. produces a markedly greater response of the plants to 2,k-D treat-ment than i f the temperature at the time of exposure was 60°F. (regardless of whether the exposure was for eight days or one hour). (2) the effect of variations in light intensity on the response of the plants to 2,U-D was slight. (3) a greater response of the plants to 2,k-D was obtained at a low relative humidity than at a high relative humidity. (k) 2,k-D had no effect upon the rate of transpiration of the plants. B. Persistence of 2,k-D in Soil It has been shown by periodic assays of soils treated with 2,k-D, both in the field and by incubation and leaching experiments in the laboratory, that there are two main factors responsible for the ultimate disappearance of 2,k-D and related compounds from soils: leaching and microbial decompo-sition. A third factor, of considerably lesser importance in soils of low organic matter content, is that of the combination of the 2,k-D with the soil organic colloids. In that the character of the soil Effects the above factors, any discussion on the persistence of 2,k-D in soil has to include the effect of the character of the soil on 2,k-D decomposition, (a) Leaching. This factor is dependent upon the intensity and the amount of the rainfall, the character of the soil, and the solubility of the herbicide used. 6 Nutman, Thorton and Quastel (78) were the f i r s t to demonstrate that most of the 2,U-D applied to a s o i l could be removed by leaching. Be Rose (23), Hanks (31), Crafts (20) and Martin (58) have confirmed this finding. Crafts (20) stressed the relationship between moisture content of s o i l and persistence of 2,U-D toxicity. He found that under the a r i d conditions found i n certain sections of California, 2yU-D treated soils retained their toxicity f o r as long as one year after treatment. While under normal moisture conditions a cultivated s o i l w i l l retain i t s toxicity for a period of time varying from a fortnight to a few months. Mitchel and Marth (66) and other workers have since confirmed these findings. Textural differences do not appear to have any direct effects on the leaching of 2,li-D from treated s o i l s . However, as textural differences influence the rate of percolation of water, they therefore influence the rate of leaching of the herbicide. S o i l type has a marked influence on the downward movement of 2,U-D i n treated s o i l s . Heavy clay so i l s and soils high i n organic matter tend to retain the compound more effectively i n the upper layers than do the l i g h t porous sandy soils or soils low i n organic matter. (20) Hernandez et a l (35) reports that 2,U-D applied at normal rates of application was leached to a depth of 5 inches i n mineral s o i l s containing 0.25 to 2.2 percent organic matterj while i n so i l s containing up to 9 percent organic matter, i t was only leached to a depth of 3 inches. Experiments by Hanks (31) with s o i l s of different pH values indicate that 2,U-D i s less readily leached from naturally alkaline soils than from those of an acid nature. Work of Martin (58) and Flieg (27) reports the opposite results. Flieg reports that an application of ll|)| ram. of i r r i g a -tion water i n seven weeks removed a l l of an 8.3 mg. application of 2,L-D from alkaline clayey and.sandy s o i l s . However, when li g h t and heavy acid 7 soils were used, kO percent and 55 percent of the applied 2,k-D was retained respectively. It i s believed by many workers that factors other than pH differences are responsible for these discrepancies. 2,k-D, either i n the acid form or as a salt or an ester, i s sufficient-l y soluble so that i t w i l l readily move downward i n the s o i l p r o f i l e , i f the opportunity arises. Minarck (60) reports that the solu b i l i t i e s of some of the commercially available salts of 2,k-D are as follows: Compound Solubility  grams/litre of B^ O 2,k-D acid 0.5 Ca (2,k-D)2 2.5 Mg (2,k-D)2 17.k • K 2,k-D approx. 30.0 Na 2,k-D n 30.0 NHk 2,k-D « 30.0 alkanolamine 2,k-D « 300.0 Zussman (10k) substantiates the s o l u b i l i t i e s of 2,k-D salts as put forth by Minarck. He further states that i f the 2,k-D acid i s applied to s o i l , calcium, magnesium, potassium, sodium, ammonium and other salts of the acid are formed. The heavy metal salts of the acid, such as iron and copper, are less soluble than the acid, but the concentration of the free heavy metal ions i n most soils i s not sufficiently high enough to t i e up more than just traces of the 2,k-D acid. Emulsifiers and solution stabilizers, which are generally present i n commercial formulations of 2,k-D, may tend to keep the herbicide i n solu-tion and, thus, would aid the downward movement of 2,k-D. The ester forms 8 of 2,lj.-D, on the other hand, are even less soluble in water than the acid and, since their hydrolysis rates are quite slow (60), they would presumably occur in the soil as oily films or globules. Their downward movement would therefore be slow. Therefore, in cultivated soils under normal conditions of moisture, leaching can reduce the concentration of 2,li-D in the surface horizons markedly, but by no means will i t account for all the 2,li-D applied. (To) Microbial Decomposition. The first indication that the breakdown of 2,lj.-D and related compounds in soil was of a biological nature was reported by Mitchell and Brown (63). They observed that the rate of detoxication of 2,li-D treated autoclaved soil was greatly retarded as compared to the rate of detoxication of 2,1+-D treated non-autoclaved soil. These results were confirmed and extended by Nutman et al (78) and De Rose and Newman (2U). The work of Newman and Thomas (72) showed that the persistence of 2,U-D in soils from which this compound had previously disappeared is of much shorter duration than the original appli-cation. Thiey postulated that the microflora responsible for the decomposi-tion of 2,li-D was highly specific as other related compounds had no effect on the persistence of 2,U-D in soil. Further evidence that the breakdown of 2,li-D and other related compounds in soils was of biological origin stems from the fact that certain field conditions exert a marked effect on the persistence of these compounds in soil. In moist soils, the toxicity is maintained for only a matter of weeks, whereas in acid soils the toxicity may be maintained for a year or more. Temperature is positively correlated with the breakdown of these compounds, as one would expect i f the process was of a biological nature. A high organic matter content also favors the rapid disipation of these compounds 9 of these compounds from s o i l . Weaver (5>6) reports that t i l l a g e of 2,k-D treated s o i l increased the rate of detoxication of the s o i l . A l l these factors suggest that the action of breakdown i s biological, and i s brought about by s o i l microorganisms which may be u t i l i z i n g the compound as a source of carbon and growth energy. (i) Kinetics of Decomposition. Audus (U,6) attempted to obtain direct evidence for the microbial decomposition of 2,1*-D and related compounds i n s o i l by u t i l i z i n g the perfusion technique of Fees and Quastel (1$). Samples of s o i l were continu-ously percolated with an aerated dilute solution of 2,U-D, MCPA and 2,U,5-T, and the changes of herbicide concentration were followed from day to day i n the perfusate by means of a biological assay. When herbicide concentration was plotted against time, a Sigmoid curve resulted. This type of curve i s taken as a strong evidence of bacterial proliferation i n response to a specific metabolite. Three periods could be recognized on the curves? a period of i n i t i a l absorption, a lag period and a period of maximum rate of decomposition of the herbicide. The period of i n i t i a l absorption occurs i n the f i r s t hour of perfusion and results i n a drop i n concentration of the herbicide of 10 percent f o r solutions of concentration of .001 percent. (Refer to section B(b) for data on the amounts of herbicides absorbed.) However, not a l l the soils under study showed t h i s period of i n i t i a l absorption as either the biologi-cal assay method used was not sensitive enough or the decrease i n concen-tration of the herbicide was masked by the effects of dilution when perfusing a moist s o i l . The lag period, i n which there i s no appreciable decrease i n the concentration of the herbicide, lasts at least for seven days. The length 10 of time of lag phases as per Audus (7) is as follows: Herbicide Time in days No. of exps. 2,U-D 13.7 i 2.8 8 MCPA 50 to 80 6 2,li,5-T * — 1 * decomposes only very slowly, 270 days plus; slight indication of enrichment. As in the case of the absorption of the herbicides by soils, there is a considerable variation in the detoxication rate, which Audus attributes to the heterogenity of the soil samples used. The maximum rates of decomposition after this lag phase is approxi-mately 3 to Ij. x 10~6 moles of 2,1+-D per gram of treated soil per day. For MCPA the rate is approximately 1/10 that for 2,U-D. This rate represents the maximum capacity of the soil to break down the herbicide under these conditions and is presumably limited by the number of 2,U-D decomposing organisms that i t can support. This limitation is thought by Audus to be due to the lack of some essential growth factor or factors in the soil itself. Audus (U,6) also noted that enriched soils tended to break down subsequent applications of 2,1|-D and MCPA at rates considerably faster than for the original application. Audus (6) convincingly corroborated this indirect evidence of bacterial action on herbicides by experiments with bacterial poisons. Enriched soils (soils capable of decomposing a herbicide at their maximum rate) capable of breaking down 2,U-D and MCPA were perfused with the usual herbicide solution containing 0.01 percent sodium ftzide. This treatment with sodium azide prevented all subsequent breakdown of the herbicide. This work by Audus is taken as constituting final proof that the 11 decomposition of 2,k-D and MCPA and possibly 2,k,5>-T i s due to the action of s o i l microorganisms. ( i i ) Mechanism of Breakdown. Studies have been made of the chemical act i v i t i e s of the adapted organisms i n s o i l by means of cross perfusion experiments. In this tech-nique, s o i l i s enriched with a herbicide and then perfused with a homologue, and the subsequent disappearance of the homologue i s followed by a biological assay procedure. It was hoped that this technique would provide means for determining whether or not one species of bacteria was responsible for the decomposition of the phenoxyacetic acid herbicides. Audus (6), using the cross perfusion technique, found that 2,k-D enrich-ed s o i l w i l l break down MCPA immediately without any occurrence of a lag phase, but w i l l not affect 2,k,5-T; and that MCPA enriched s o i l w i l l break down both 2,k-D and 2,k,S>-T equally well. However, the rates of disappear-ance of the homologues were slower than the maximum rates obtained i n so i l s directly enriched with that homologue. There i s also evidence that the activ i t y f a l l s off with continued perfusion of the foreign molecule. As the bacteria which decompose MCPA and 2,k-D show similar morphological characteristics, i t would seem l i k e l y that the same organism i s responsible for the decomposition of the two herbicides, but that different enzymer-: systems are developed i n each case i n response to the i n i t i a l perfusing molecule. Audus (7) states that these results are not inconsistent with the idea that the f i r s t step i n the breakdown of these three herbicides i s an attack upon that part of the molecule common to each of the compounds; that i s , the acetic acid side chain. The removal of this side chain i s postulated to be by hydrolysis: 12 H'OH I 2,l*,5-T Thus, i t might be expected that the corresponding phenols might accumulate, at least temporarily, during detoxication. However, no chlorophenols could be detected during the process of the breakdown of these herbicidesj yet i t i s possible that the corresponding chlorophenols are broken down as rapidly as they are formed. Brownbridge (12) found that the perfusion of s o i l with 2,h-dichloro-phenols, possible decomposition product of 2,lj.-D, produced a Sigmoid curve similar to those obtained for the i n i t i a l perfusion of the herbicides. However, the maximum rate of decomposition of the dichlorophenol was not reached u n t i l 60 days after the start of perfusion, and then the rate was only one-quarter of that of 2,U-D. When 2,k-dichlorophenol was perfused through 2,U-D enriched s o i l , there was an immediate and rapid decomposition of the phenol compound at rates equivalent to those obtained for the dis-appearance of 2,U-D from s o i l . However, the a b i l i t y of the 2,U-D enriched 13 soil to decompose dichlorophenol was lost within five to ten days after the start of the perfusion. It was thought that the concentration of phenol had become toxic to the bacteriaj whereas in 2,L-D decomposition, the concentra-tion of the dichlorophenol product at any one time is non-toxic to the detoxicating bacteria. Further evidence that the above-mentioned mechanism is possible is suggested by the work of Hansen and Buchholtz 02).. They reported that 2,U-D solutions, both en vitro and en vivo, were detoxicated by light in the presence of riboflavin. One of the end products was tenta-tively identified as 2,U-dichlorophenol, (iii) Isolation of an Effective Organism Audus (6) isolated an effective organism by spreading a drop of the perfusate from an enriched perfusion culture on an agar medium of the following composition: NH^H'gPOj^  0.1% KC1 0.02% MgSO^  0.02% Na 2,U-D 0.1% .agar to stiffen —the pH of the medium is adjusted to 7*0. The isolate was a short Gram negative rod with Gram positive bipolar granules and showed growth and fermentation reactions which placed i t in the Bacterium globiforme group. Jensen and Peterson (37), using a technique similar to Audus's isolated two different organisms capable of decomposing 2,U-Dj one of the isolates also being able to decompose MCPA (2 methyl-1; chlorophenoxyacetic acid). The isolate which was active only against 2,U-D was classified as being Flavobacterium aquatile, while the other isolate closely resembled Audus's organism in morphological, growth and fermentation characteristics. Ii* (c) Physical and Chemical Combination. The cations of the salts of 2,U-D in the soil no doubt undergo exchange reactions with the base exchange material present in soil. However, the anion part of the salt molecule, which is the active herbicide, does not appear to enter into exchange reactions or fixation reactions with the in-organic colloid fraction of the base exchange material present in the soil. There is, however, considerable evidence for this interaction of the anion part of the molecule with the organic colloid fraction of the base exchange material, thereby giving a means by which a measure of retention against leaching can be exhibited. (20,35) Audus reports (6) that, during the first hour of perfusion experiments , there is a drop in concentration of the order of 10 percent for the three herbicides, 2,h-B9 MCPA and 2,U,5-T, when perfusing solutions of .001 per-cent concentration. He was able to correlate this drop in concentration with the amount of soil perfused in that the larger the amount of soil perfused, the greater is the initial drop in concentration. He reports the following figures: * Initial Absorption of Herbicides on Soil Colloids  2,U-D 0.167 i O.Olj.8 rag/gm of soil (ave. of 8 exp'ts.) MCPA 0.028 i O.Olli » » (ave. of h exp'ts.) 2,U,5-T 0.07 " " (oneexp't.) * 50 gms. of soil perfused with 250 ml. of a 100 ppm. solution of the herbicide. This absorption occurs immediately upon the addition of the herbicide to the soil and in no way will i t account for the delayed detoxication of the herbicide. He further notes that this absorption phenomenon is not always shown by soils. Flieg (27) and Hernandez (35) have related the degree of the initial absorption of 2,U-D to the humus content of soil. Soils poor in organic matter are leached much more rapidly of 2,U-D than soils that are rich in organic matter. Lucas (56) provides further support for the absorption of 2,U-D by the organic colloids in soil. He found that activated charcoal and other finely ground similar materials readily absorbed 2,U-D. Weaver (97), using ion-exchange resins, reported a marked absorption of 2,U-D. The nature of the absorption phenomenon between 2,i|-D,and its related herbicides, and the soil organic matter is not known, nor is i t known whether or not the herbicidal activity of the absorbed 2,li-D is maintained at its full level. The work of Weaver (97) suggests that its activity is greatly reduced; whereas work by Audus (6) states that there is no reduction in herbicidal activity upon absorption. The phenomena of physical and/or chemical reaction of the 2tkrbt or its related compounds, with soil colloidal organic matter, is not ordinarily a significant factor in reducing the concentration of the herbicide in the surface horizons of soil. (d) Effect of Soil Characteristics. The speed with which 2,U-D and its related compounds are removed from a soil depends upon a wide variety of soil conditions including moisture conditions, soil temperature, organic matter content, rate of application of the herbicide and the pH of the soil. Meadows and Smith (59) working with a fine sandy loam, a peaty soil, and a combination of the same adjusted to various pH levels, report that the organic matter content was the most important of the above-mentioned factors in determining the persistence of 2,li-D in soil. Bate of application and 16 pH were of lesser importance, except i n the case of soils with high organic matter content where lowering the pH of the s o i l increased the length of time of persistence of the 2,U-D. Krone and Hamner (U7) and Kries (I46) report that heavily manured so i l s with a high organic matter content detoxicated applications of 2,1+-D much more rapidly than soils poor i n organic matter. Brown and Mitchel (63) report that the addition of cow manure at the rates of 1000, 2000 and kOOO pounds per acre greatly increased the rate of detoxication of 2,k-D, with the maximum increase occuring at 1000 pounds per acrej however, applications of 80OO pounds per acre resulted i n a marked retardation of the rate of de-toxication. No explanation was attempted for t h i s phenomenon. Soi l moisture content has a marked effect upon the inactivation of 2,k-D i n s o i l . Brown and Mitchel (63) report that a 2,lj.-D treated s o i l having a moisture content of 2.5 percent retained practically a l l i t s herbi-cidal a c t i v i t y for 18 months. On the other hand, the same s o i l at 10 percent moisture content, lost one-half i t s ac t i v i t y after one month storage; while at 30 percent moisture content a l l i t s herbicidal activity was lo s t after one month storage . (N.B. a l l the s o i l s were stored at similar tem-peratures). These workers concluded that the moisture content of s o i l can be a c r i t i c a l factor i n determining the persistence of 2,k-D and related compounds i n s o i l . Other workers have since v e r i f i e d these results. (2U,35) Soils have been stored at controlled temperatures ranging from 36° to 70°F. and the rate of inactivation of 2,U-D determined. It was found that 2,U-D treated s o i l retained a l l i t s herbicidal activity when stored at 36°P. and that the rate of inactivation increased with an increase i n temperature. (11,22*, 3 5 ) . A review of the literature with respect to the influence of pH on the rate of detoxication of 2,1|-D treated so i l s produces nothing decisive. 17 Jorgensen and Hamner (liQ) found that the pH of a soil had a negligible influence on the rate of detoxication, but that there was a slight tendency favoring the dissipation or inactivation of the toxicity at neutral or slightly alkaline pH's. Meadows and Smith ($9) report that the pH of the soil was of greater importance in determining the rate of detoxication of soils high in organic matter than in soils of low organic matter content. Kries (I46) reports that lime effects detoxication adversely, in that the rate of detoxication of limed soils was slower than that of unlimed soils. However, factors other than the presence or absence of lime could be respon-sible for the different rates of detoxication. In general, the factors that are operative in determining the level of microbial activity are operative in determining the length of persistency of 2,4-D in soil. C. Effect of 2,U-D on Soil Microorganisms. A survey of the literature reveals that 2,U-D and its related compounds, when used at herbicidal concentrations, have no effect upon the soil micro-flora as determined by either the plate count or the soil respiration tech-nique (27,28,2°,8U). These compounds have no effect upon the nitrification process (3it,H5,8U). The concentrations of these herbicides that have to be applied either in laboratory or field experiments before any deleterious effects are detectable are many times the normal rates of application. (13,27,28,29,79,8U,85). In laboratory experiments, using pure cultures of bacteria, the phenoxy-acetic acid herbicides (ester formulations excluded) are increasingly inhibitory as the pH is lowered. Therefore, i t would appear that these herbicides are inhibitory to microorganisms in the undissociated states only. The ammonium, salt of 2,i;-D was found to be more toxic than the sodium salt to pure cultures of bacteria growing on laboratory media (70). Stevenson 18 and Mitchell (67) report that 2,U-D, at 0.02 percent concentrations, ex-hibited bacteriostatic effects on many pure cultures of bacteria. Worth and McCabe (102) found that 2,1|-D, at high concentrations - 2 percent and 1 percent, inhibited aerobic organisms to a greater degree than facultative aerobic and anaerobic organismsj further that 2,1*-D at the low concentrations of 0.002 percent and 0.0002 percent stimulated the growth of aerobic and facultative aerobic organisms. However, the response of the anaerobic organisms tested varied markedly. Carlyle and Thorpe (13), and Fults and Payne (79) have showed that the herbicidal effects of very low concentrations of 2,k-D to the Leguminous plants were due to the susceptibility of the plants and not of the associated Rhizobium bacteria. They found that most members of the Rhizobium family would stand concentrations of 2,lt-D up to 0.3 percent; however, Rhizobium t r i f o l i and Rhizobium leguminosarum would tolerate concentrations of 2,k-D of 0.03 percent and 0.0U percent respectively. Concentrations of 2,U-D as low as 0.007 pounds per acre w i l l decrease the nodulation of most Leguminous plants and i n some cases, e.g., red clover, w i l l completely suppress i t . Carlyle and Thorpe (13) reported that i n bean plants where 2,U-D injury was observed, nodulation was decreased and disintegration of some of the host material was observed microscopically, accompanied by changes' i n the Gram stain reaction of the Rhizobium bacteria. To date most of the investigations conducted on the effect of herbicides on microorganisms have been concerned with their effects on the s o i l i n d i -genous population. However, Clark (15) reports that tomato plants treated with 2,li-D had higher numbers of organisms i n the rhizosphere as compared to untreated tomato plants. This increased rhizosphere population persisted for 2 to 3 weeks, when there followed a general microbial invasion and disinte-gration of the entire root system. 19 D. Association Between Plants and Microorganisms. It has been recognized for the past half century that an intimate relationship exists between the microflora of the s o i l and plant roots. Depending upon the environmental conditions this relationship may be harm-f u l or beneficial to the plant. However, while the existence of this relationship i s recognized, there i s l i t t l e information available relative to the nature and type of the organisms involved i n i t . (a) Rhizosphere. This term designates that portion of the s o i l that comes under the influence of the plant root system. It has been firmly established that this s o i l supports greater microbial activity than the s o i l more distant from the plant root. Furthermore, i t has been well-established that the effect of the growing plant varies with the kind of plant, age of plant, season, moisture conditions of the s o i l and the previous treatment of the s o i l . (i|l,l43j86,87). There i s also evidence suggesting that the rhizosphere of diseased plants may contain greater concentrations of microorganisms than healthy plants; and that varieties of plants susceptible to soil-borne diseases, even though free of disease, may show higher numbers of micro-organisms i n the rhizosphere than corresponding resistant varieties (8°). The rhizosphere effect may be shown not only by increased t o t a l counts but also by preferential stimulation of certain physiological and nutritional groups of microorganisms as judged by quantitative and qualitative bacterial methods (1U,86,87). The following phenomena may be associated with the rhizosphere effect: (1) an increase i n the t o t a l numbers of microorganisms (U3,U°,86)i (2) an increase i n the Gram-negative, non-spore~forming bacteria (h9)i 0) an increase i n the percentage of organisms that grow well on nutrient agar, of proteolytic and liquefying types, of organisms 20 . causing acid or alkaline reaction on dextrose, of motile types and of chromogenic forms (1*9); (L) an increase in the percentage of bacteria with simple nutritional requirements and of those types responding to the presence of amino acids (1*3,50,52); (5) a decrease in the percentage of organisms dependent upon the more complex nutrients provided by soil extract (1*3,50,52). Work by Lochhead's group (1*9,50,51,52,53,9k) has led to the belief that the bacterial population in the rhizosphere is possibly a selective one responding to the presence of specific nutrient materials provided by the decomposition of sloughed-off portions of root tissue and by root excretions. However, Wallace and King (95), in their study on the rhizosphere of young and mature barley and oat plants, report that bacteria capable of showing maximum growth in a simple glucose-salts medium were almost non-existent. They suggest that this difference between the general distribution of nutri-tional groups as reported by them, and that previously reported, could be due to many factors such as soil type, pH, etc. Studies pertaining to the influence of such factors on the nutritional groupings of soil bacteria s have not been reported. It is evident that the rhizosphere is a unique zone, exerting a power-ful stimulation on many soil microorganisms while suppressing others. The effects vary with the type, variety, physiological age and vigor of the plant; and the type, pH, treatment and moisture conditions of the soil as well as season. Environmental factors may exert their influence directly on the soil and thus the rhizosphere organisms, or indirectly by stimula-ting or retarding plant development, (b) Root Excretions of Plants. There is little information existing at the present time dealing with 21 the character and nature of root excretions in relation to specific plants. Indirect evidence of the excretion of broad groups of nutrients is plentiful, while proof of specific excretions is in most cases lacking. It is generally accepted that plants can excrete Inorganic ions into the soil under certain conditions (55,82,86); for example, cereals and tobacco will excrete at least potassium into the soil towards the end of the growing season (1^,101). It has been shown through the use of radioactive tracer elements that there is a continuous exchange between the nutrient ions in the soil and in the root, although the factors controlling this exchange are unknown (82). There is no doubt that the soil solution, in addition to inorganic ions, contains a variety of organic molecules. This organic fraction undoubtedly arises from the decomposition of dead plant material and from the metabolism of soil organisms and living plant roots. The presence of specific organic compounds undoubtedly plays a vital role in the intense competition that exists in soil. Many independent experiments have demonstrated that amino acids are excreted from plant roots. However, only in one instance has this excretion been definitely shown. Virtanen and Laine (92) demonstrated the excretion of the amino acidsJi-aspartic and @-alanine from the nodules of legume plants. They stated that in no instance was there any excretion of these amino acids from the roots. Lundeg&rdh and Stenlid (57) have shown that pea roots release nucleotides and flavanones. West (100) successfully showed that the roots of young flax seedlings excreted the vitamins biotin and thiamine. Sugars have been shown to be excreted by plant roots and in the case of the grass family such excretions may have very important bio-logical effects (8 ). For example, in the tropics of the Old World many grasses are semi-parasitized by a genus of plants called "Striga". The 22 striking thing is that the seeds of this semi-parasite will germinate in the presence of the living roots of a suitable grass host. It was shown that the germination of the semi-parasitic seeds is due to the presence of a pentose sugar, closely related i f not identical to d-keto xylose, in the diffusate from the host roots (8). It is to be noted that no matter how toxic an organic molecule might be, it will eventually disappear from the soil. This disappearance appears to be due to the activity of a soil flora which is resistant to its toxic action. Therefore, i t is to be expected that normally there will not be a high concentration of these organic molecules in soil. Only under abnormal conditions (e.g., water logging) will the concentrations of these molecules reach a high level. (c) Biological Equilibrium in Soils. There is increasing evidence in soil of a constant striving towards a microbiological equilibrium, for the microbial population is ever changing under the influences of temperature, moisture conditions, season, treatment of soil and the cropping system. In a soil of definite type apart from the influence of the growing plant, there exists a suprisingly uniform balance between the various morphological and nutritional groups of bacteria, even though the producti-vity may be greatly altered by manurial treatments (52). In proximity to the growing plant, however, the bacterial equilibrium in the soil is altered both quantitatively and qualitatively. In an attempt to access the bacterial equilibrium between nutritional groups, West and Lochhead (99) evolved a figure which they called the Bacterial Balance Index. This figure was obtained by assigning a negative value to the percentage occurrence of the Gram-negative, non-fluorescent bacteria capable of maximum growth in a simple glucose-salts medium, and a 23 positive value to the percentage occurrence of organisms responding to amino acids and growth factors respectively, and then adding the resulting figures. This figure, the Bacterial Balance Index, may vary with s o i l type and season (kl) and under normal conditions i s higher i n the rhizosphere i n the s o i l more distant from the plant (36,9°). I t has been found that the bacteriol-equilibrium i s altered by the presence of certain plant diseases,such as strawberry root rot and potato scab, i n that there i s a r i s e i n the Bacterial Balance Index as the severity of the diseases diminish (36,k2). It i s thought that the equilibrium between various groups of organisms i n the s o i l i s largely dependent upon the a v a i l a b i l i t y of essential nutrients. However, consideration must be given to associative and antagonistic pheno-mena i n the establishment of this equilibria. In respect to the rhizosphere with i t s increased numbers and a c t i v i t y of organisms, these phenomena could possibly be of greater importance than the a v a i l a b i l i t y of nutrients i n establishing the equilibrium. Lochhead (5>0) observed that cultural f i l t r a t e s of bacteria giving .maximum growth on a simple glucose-salts medium were stimulatory towards those organisms requiring amino acids for maximum growth. He further suggests that such effects could contribute towards the estab-lishment of biological equilibrium i n s o i l s . 2k HI. Experimental. All concentrations of 2,U-D amine (Weed-B-Gon 6k-X Weed Killer, California Spray-Chemical Corporation) used in this study are expressed on an acid-equivalent basis, and the rates of application are on an acre basis. A sandy soil of low fertility was selected for use; in order to accen-tuate any rhizosphere effect. A. Characterization of the Soil. The soil is classified as a Brown Podzolic, of glacial t i l l origin and a member of the Alderwood sandy loam series. A summary of the charac-teristics determined is as follows: pH 5.1 organic matter content 6.2$% total nitrogen 0.63$ C:N ratio 6:1 available phosphate 77.5 ppm. total exchange capacity lk. 7 meq./lOO gms. soil exchangeable H+ 9.17 " " NHj.4 2.30 " " Ca.4* 2.53 " Previous treatment of the plot from which the soil was obtained is as follows: Year Season Treatment 1952 f a l l rye stubble 1953 spring rye stubble ploughed under; application of 10-20-10 @ 300 pounds/acre; seeded to Victory oats; clean cultivated; appli-cation of NH[jN03 @ 75 pounds/acre. f a l l oats harvested, land disced and sown to rye. 195U spring rye mowed three times fa l l miscellaneous tillage, (samples were taken at this time). This soil after being potted was allowed to sit in the greenhouse at optimum moisture content for 2 weeks, in order to permit the flora to become 25 stabilized to the new environmental conditions. B. Rhizosphere Studies Accepting the premise that the rhizosphere population i s a selective one and i s characterized by the root excretions and the sloughed-off portions of the roots, any change i n the amount or composition of these materials should be reflected i n the rhizosphere. Since the application of 2,lHD to plants affects their rates of metabolism, growth, etc., i t should, there-fore, affect the rhizosphere population. (a) Barley (i) Experimental. Barley ( O l l i variety) was selected f o r study i n order to determine the effect of 2,li-D on an economic plant when used at herbicidal concentra-tions. The barley was seeded at the rate of k seeds per pot i n nine, 7-inch diameter, glazed porcelain pots containing s o i l to within 1 inch of the top. The pots were sustained i n the greenhouse at optimum moisture l e v e l through-out the experiment. When the plants were 3 weeks of age (roughly 6 inches high, 3 to h blade stage) 3 series, consisting of 3 pots per series, were sprayed with 2,U-D at rates of 0, h and 8 ounces per acre, respectively, (see appendix). Prior to treatment, the s o i l surface was covered with absorbent cotton and then with two layers of wax paper i n order to guard against the direct application of the spray to the s o i l . Two days after treatment, the s o i l was uncovered and watered. The control plants were kept several feet aray from the treated plants i n order to guard against 2,U-D contamination. One week after treatment, samples were taken for plating. For the rhizosphere population, the plants were carefully removed from the pots and the loose s o i l shaken free. The roots were then severed and placed i n a 26 dilution blank. For the indigenous population, a composite soil sample was taken from the three control pots following removal of the plant roots. This sample was well mixed and 10 grams were weighed for plating. Since this sample was taken from pots i n which plants had been growing, there i s doubt as to i t s absolute freedom from rhizosphere contamination. In order to check, to a degree at least, the reliability of this procedure, an indigenous flora was obtained from the pots treated with 2,1|-D at the rates of h ounces and 8 ounces per acre. The i n i t i a l dilution in each case was shaken for 10 minutes, and each subsequent dilution was shaken for 1; minutes, at approximately 300 oscilla-tions per minute. Three dilutions and 5 replications per dilution were plated for each sample, each plate consisting of 15 cc. of medium and 1 cc. of unknown. The composition of the plating medium was as follows: agar 12.5 grams/litre K2HP0k 0.5 grams/litre •Ksoil extract 250 cc. tap water 75>0 cc. *Preparation of the soil extract: 1 kgm. of soil + 1 l i t r e of tap water, autoclave for 30 minutes; add a l i t t l e CaSO^ , f i l t e r clear and dilute to 1 l i t r e . The plates were incubated at 28°C. for 10 days. At the end of the 10 day incubation period, a suitable dilution was chosen for each sample and counted with the aid of a Quebec Colony Counter and a tally. In order to obtain a representative population for the nutri-tional studies, a plate, representing the mean of the 5 plates counted, was selected and a l l the colonies picked into a semi-solid agar (composition as follows) and incubated at 28°C. for 5 days. 27 Semi-solid agar. agar yeast extract soil extract tap water 3.0 gram/litre 0.1 " 0.2 " 250 cc. 750 cc. Inoculations were made from these tubes into the 5 nutritional media (composition as follows) and incubated at 28°C. for 5 days. Nutritional Media. I Basal medium. glucose KpHPOi, KN03 MgSOk C a C l o NaCl FeCl 3 H20 (distilled) 1 gram/Litre 1 0.5 0.2 0.1 0.1 0.01 1000 cc. - salts added to H/jO, pH adjusted to 6.8, heated to 100°C . , cooled and filtered clear. II Amino Acid medium. basal medium casamino acids III Groxrth factor medium. basal medium cysteine thiamine biotin pyrodoxine pantothenic acid nicotinic acid riboflavin inositol Big 1 l i t re h grams/litre 1 l i t re 0.05 microgram/litre 100 " 0.1 » 200 " 100 " 100 » 200 " 0.05 gram/litre 2 microgram/litre 17 Amino acid - growth factor medium. basal medium casamino acids growth factors 1 l i t r e U gram/litre as above 28 V Soil extract - yeast extract medium. basal medium yeast extract soil extract 75>0 cc. 1 gram/litre 250 cc. A l l nutritional media dispensed in 5 cc. amounts, plugged and steri-lized at 15 pounds pressure for 15 minutes. The relative amount of growth was determined by turbidity and measured by visual examination. A value of k was given to the tube showing the maxi-mum amount of turbidity, with the remainder of the tubes for that isolate being graded 3 , 2 , 1 or 0 . Readings of 3 and k were considered to be maximum growth, while readings of 2, 1 and 0 were considered to be subminimal. In order to prevent assigning too much significance to small differences in turbidity, a difference of 2 points was considered to be significant. An isolate would then be placed in the simplest nutritional class in which i t showed a significant maximum amount of turbidity (see appendix). The data which follows refers to the classification of soil bacteria,only. Actino-myces and fungi were discarded when observed. (ii) Results. The Plants did not show any signs of injury resulting from treatment. Table 1. Oven-dry weight (gms) of soil samples used for plating. Replicate Indigenous 0 oa.2iU-D h oz.23k-D 8 oz.2,U-D Rhizosphere I 8.099 *1 5.399 6.629 5.255 II 8.222 *2 6.3U2 5.0^2 k.391 H I 8.2U6 *3 3.668 3.397 2.795 #1 indigenous sample composite of untreated pots. #2 " " " " h oz. " » *3 " « »« » 8 oz. " " 29 Table 2. Total number of organisms x 10° per gram of oven-dry s o i l . Rhizosphere Replicate Indigenous 0 oz.2,U-D: h oz.2,l4.-D :8 oz.2,U-D I 10.95 56.72 35.31; 53.61 II 10.90 27.U2 56.7U 1*5.38 III 20.79 83.37 55.91 76.97 Mean lit. 21 55.81; U9.33 58.65 Table 3» Percentage of organisms i n each nutritional group. Rhizosphere Nutritional group Indigenous 0 oz.2,11-0:1; oz.2,l;-D:8 oz.2,It-D Basal 1Q.U9 19.25 16.21 20.00 Amino acid i a . 30 37.80 36.61 50. Ik Growth factors 3.26 0.7U: l.fcL 1.73 Amino acid-growth factor 3.26 2.21 7.75 . 1.73 least extract-soil extract 3U.80 1)0.00 38.02 26.1;0 Table 1;. Numbers of organisms x 10° per gram of s o i l i n each nutritional group (mean count of a treatment x percentage/ioo °^ organisms i n each nutritional group Nutritional group Indigenous 0 oz .2 , Rhizosphere tU-B$U oz.2,1*0:0' oz.2,l;-D Basal 2.62 10.71; 7.99 11.73 Amino acid 5.87 21.10 18.06 29.UO Growth factors 0.1*6 O.Ul 0.69 1.01 Amino acid-growth factor 0.1*6 1.23 3.82 1.01 Yeast extract-soil extract k.9k 22.33 18.76 15.1*8 30 ( i i i ) Discussion. Analysis of the data i n Tables 1 and 2 shows that there i s a wide variation i n the weights of rhizosphere soils used for plating, as well as i n t o t a l rhizosphere counts. A negative correlation appears to exist between the weight of s o i l used and the t o t a l count. This relationship suggests that some indigenous s o i l , having a low t o t a l count, has been included i n the rhizosphere sample. A comparison of the t o t a l indigenous counts i n Table 2 reveals that 2 out of the 3 counts are i n close agreement while the 3rd count i s almost twice as large. I t i s possible that this may be due to the inclusion of rhizosphere s o i l , which normally has a high t o t a l count, relatively speaking. The t o t a l mean counts i n Table 2 show that the rhizosphere of k-week-old barley plants has a population 3s to U times that of the indigenous population. However, there i s no difference i n the t o t a l counts of treated barley plants, indicating that 2,k-D has had no quantitative effect upon the numbers of organisms i n the rhizosphere. The percentage of organisms as they have distributed themselves i n each nutritional group are presented i n Table 3. There would, appear to be no difference i n the percentage distribution between the indigenous and the rhizosphere floras obtained from plants subjected to the 0 and k ounce treat-ments. A comparison of the percentage distribution i n the rhizosphere of the 8 ounce treatment with that of the foregoing suggests a possible increase i n the amino acid group and a decrease i n the yeast-soil extract group of organisms. The data i n Table k i s a combination of that presented i n Tables 2 and 3, and gives the numbers of organisms per gram of s o i l i n each nutritional group. Analysis of this data shows that i n 3 out of 5 nutritional groups, there i s a kOO to J>00 percent increase i n the number of organisms i n the 31 rhizosphere as compared to the indigenous f l o r a . It also shows that there i s a proportional distribution of organisms i n the rhizospheres of the 0 and k ounce treated plants similar to that of the indigenous f l o r a . The distribution of the organisms i n the rhizosphere of the 8 ounce treated plants indicates an increase i n the amino acid group and a decrease i n the yeast-soil extract group. A similar change, i t w i l l be recalled, was ob-served with.the percent distribution data presented i n Table 3. It i s to be noted that,while the rhizospheres of 2,U-D treated plants do not d i f f e r quantitatively from untreated plants, evidence i s presented which suggests qualitative differences i n the 8 ounce treated plants, (b) Corn, (preliminary studies) (i) Experimental. Since there appears to be very l i t t l e difference between the rhizo-spheres of barley plants treated with 2,U-D at herbicidal concentrations and untreated barley plants, the response of corn plants, which are more susceptible to 2,U-D, are to be studied. The procedures used for this study are as i n part (a), except for the following: (1) no indigenous study was carried out i n this experiment; (2) only 1 plant was seeded per pot; (3) 2,U-D was applied at the rates of 0, 1 and 2 pounds per acre by means of a brush (see appendix); (U) 3 series, each consisting of 3 plants treated at 0, 1 and 2 pounds per acre, respectively, were set up with the object of sampling a series at 3 stages of growth, i.e . , 7, l U and 21 days after treat-ment, ( i i ) Results. The series that was to be sampled at 7 days after treatment became 32 contaminated and was lost. The effect of treatment was apparent in the twisted and bent conditions of the plants, following the application of 2,k-D. The plants that were treated with 2 pounds of 2,k-D indicated a greater degree of injury, and a more immediate effect than was observed with the lighter treatment. Table 5. Oven-dry weights plating. (grams) of the soil samples used in Days after Treatment 0 lb. 2,k-D 1 lb. 2,lt-D 2 lb. 2,k-D lit 1.32 21 0.72 0.78 0.3k 0.67 0.3U Table 6. Total number of organisms x 10 per gram of oven-dry soil. Days after Treatment 0 lb. 2,k-D 1 lb. 2,k-D 2 lb. 2.U-D Ht 52.3 21 2U5.8 199. k 507.9 125.5 260.8 Table 7. Percentage of organisms in each nutritional group. Days after Nutritional group Treatment 0 Ib.2,k-D 1 lb.2,k-D 2 lb.2,k-D Basal lit 0.00 1.11 0.00 21 10.00 1.20 0.00 Amino acid lit 82.00 56.67 72.30 a U3.30 25.00 It2.l0 Growth factor Ht 2.00 2.22 0.00 21 0.00 1.10 0.00 Amino acid-growth factor lit ^ 6.00 10.00 0.00 21 13.30 5.70 ' I8.2t0 Yeast extract-soil extract Ht 10.00 30.00 27.70 21 33.30 67.00 39.50 33 Table 8. Number of organisms x 10° per gram of s o i l i n each nutritional group (total count x percentage/100 of organisms i n each nutritional group). Days after Nutritional group Treatment 0 lb. 2,U-D 1 l b . 2,U-D 2 lb .2 ,U-D Basal LU 0.00 2.21 0.00 21 21+.58 1.51 0.00 Amino acid l U U2.88 112.50 367.20 21 106.UO 31-38 109.80 Growth factor l U 1.0U U.U3 0.00 21 0.00 1.38 0.00 Amino acid- ll± 3.13 19* 9k 0.00 Growth factor 21 32.69 7.15 kl.99 least extract- Hi 5-23 59.82 1U0.70 Soil extract. 21 81.85 8U.O8 103.00 ( i i i ) Discussion. Analysis of the data i n Table 5 indicates that there i s a considerable variation i n the weights of s o i l used for plating, as the weight of the heaviest sample i s 3 times that of the lightest sample. It i s suggested at the sarnie time that the low t o t a l count of the rhizosphere of the untreated plant at the LU days sampling, combined with the relatively large weight of s o i l , i s due to the possible incorporation of indigenous s o i l with'rhizo-sphere s o i l . A comparison of the t o t a l counts of the rhizospheres of treated and untreated plants i n Table 6 shows that 2,U-D has a temporary stimulatory effect on the numbers of organisms i n the rhizosphere. It would also appear that, within the limits of the experiment, the degree of stimulation i s approximately proportional to the amount of 2,U-D applied. The data presented i n Table 7, relative to the percent distribution of organisms i n the untreated rhizosphere at l U days, indicates that a very 3k large proportion of the to t a l population f a l l s into the amino acid group; whereas the corresponding figures for the rhizospheres of the treated plants are d i s t i n c t l y lower. At the same time, there i s a definite increase i n the percentage of organisms appearing i n the yeast-soil extract grouping. At 21 days after treatment, both treated and untreated rhizospheres show a marked decrease i n the percent distribution of organisms i n the amino acid group and a corresponding increase i n the yeast-soil extract. Examination of the data presented i n Table 8 shows that at the Ik days sampling the largest proportion of the t o t a l rhizosphere population of both treated and untreated plants appears i n the amino acid grouping. The data also suggests that the increased numbers of organisms found i n the rhizo-spheres of 2,k-D treated plants l k days after treatment i s due to an increase i n the number of organisms i n the amino acid and yeast-soil extract groupings. In the rhizosphere of the untreated plant at 21 days after treatment, there has been an increase i n the number of organisms i n k out of the 5 nutritional groupings. For the same sampling time i n the rhizospheres of the treated plants, there has been a decrease i n the numbers of organisms i n the amino acid groupings; whereas i n the yeast-soil extract group, there has been an increase i n numbers i n the case of the 1 l b . treated plant, and a decrease i n numbers i n the case of the 2 l b . treated plant. Because of the varied response with treatment and age, no significance i s attached to changes i n the numbers of organisms i n the amino acid-growth factor grouping of the rhizospheres of the treated plants. It i s to be noted that an increase or decrease i n the percentage of organisms i n a nutritional group does not necessarily correspond to an increase or decrease i n to t a l numbers for that group. For example, a com-parison of the percentage figures (Table 7) for the amino acid group of the rhizospheres of the untreated plants indicates a decrease i n percentage 35 between the samplings of the U±th and 21st; whereas a comparison of the numbers of organisms in this group (Table 8) shows that there has been an increase in numbers between these sampling times. This means that there has been a large enough increase in the total count (Table 6) to overshadow the effect of the decrease in the percent distribution of organisms in this grouping. (c) Corn (final), (i) Experimental. In order to substantiate or disprove the above findings, another experi-ment was conducted using corn. The procedures used for this study are as for part (a) except for the following: (1) the soil used was treated with 6-8-6 fertilizer @ 500 pounds per acre and the plants were exposed to U hours of artificial light per night; (2) three out of the 9 pots of soil were left fallow to represent the indigenous flora; the remaining 6 pots were seeded at the rate of one seed per pot; (3) 2,li-D was applied at the rates of 0 and 2 pounds per acre by means o$ a brush; (U) all sampling was done at 2 weeks after treatment; 2 dilutions per sample and 10 replicates per dilution were plated; (5) all colonies growing on a square (1 cm, x 1 cm.) were picked from each plate of the dilution counted (see appendix), (ii) Results. The stems and leaves of the treated plants differed from those of the untreated plants as in part (b). 36 Table 9. Oven-dry weights (grams) of the s o i l samples used for plating. Rhizosphere Replicate Indigenous 0 lb . 2,k-D 2 l b . 2,U-D 1 7.U6 2.81 2.33 2 7.k9 k.06 l.k2 3 7.U9 1.99 2.97 Table 10. Total number of organisms x 10 per gram of oven-dry s o i l . Rhizosphere * Replicate Indigenous 0 l b . 2,k-D 2 lb . 2»k-D 1 0.38 36.2 28.6 2 0.33 15.5 107.9 3 0.29 76.7 28.k Mean 0.33 — —• * In view of the insignificant data, further study of this mater-i a l was not proceeded with. Table 11. Percentage of organisms i n each nutritional group. Nutritional group Indigenous Basal ' 8.5 Amino Acid 22. k Growth factor 7.k Amino acid-growth factor 27.7 Yeast extract-soil extract 3k.0 37 Table 12. Number of organisms x 10* per gram of s o i l i n each nutritional group for the indigenous f l o r a ( mean table count x percentage/ioo °^ organisms i n each nutritional group). Nutritional group Indigenous Basal 0.28 Amino acid 0.75 Growth factor 0.25 Amino acid-growth factor 0.°2 Yeast extract-soil extract 1.13 ( i i i ) Discussion. Analysis of the data presented i n Tables 9 and 10 indicates a negative correlation between the weights of rhizosphere s o i l used for plating and the t o t a l count. A similar relationship was observed i n the barley studies, and a possible explanation of i t was presented i n the discussion i n part (a). The total indigenous counts as presented i n Table 10 are rel a t i v e l y uniform. The slight variation that does exist was probably due to biologi-cal variation. The percent distribution and the numbers of organisms per gram of s o i l i n each nutritional group for the indigenous population are presented i n Tables 11 and 12 respectively. C. Influence on the Flora of 2,1|-D Applied Directly to the Soi l . Since 2,ii-D i s decomposed by microorganisms, i t s addition to s o i l should induce a change i n the composition of the indigenous f l o r a . This viewpoint i s investigated i n the following experiment. (£) Experimental. Three-jpots of s o i l were treated with 2,U-D at the rates of 0 , 2. and 100 pounds per acre, by mixing the appropriate amounts of 2,U-D i n 200 cc. of d i s t i l l e d water and spreading i t evenly over the surface of the s o i l . 38 Two weeks after treatment, samples were taken f o r plating by means of sterile s o i l cores from two depths, 0 to 3.5 cm. and 3.5 to 7.0 cm. Two dilutions and 10 replications per dilution were plated for each sample. A l l the colonies were picked that appeared on opposite quarters of each plate i n the dilution series counted. The rest of the experimental tech-nique was as i n part (a). (db.) Results. Table 13. Oven-dry weights (grams) of s o i l samples used for plating. Sample 0 l b . 2,k-D 2 lb. 2,k-D 100 l b . 2,k-D 0-*3.5 cm. 7.67 7.83 7.8k 3.5-^7.0 cm. 7.59 7.69 7.98 Table l k . Total number of organisms x .30* per gram of oven-dry s o i l . Sample 0 l b . 2,k-D 2 lb. 2ak-D 100 lb. 2,k-D 0-»3.5 cm. 5.9k k.33 3.8k 3.5-»7.0 cm. 3.06 2.21 2.35 Table 15. Percentage of organisms i n each nutritional group. * 0 l b . 2,k-D 100 l b . 2,k-D ~ Nutritional group 0-3.5 cm. 3.5-7.Ocm. 0-3.5cm. 3.5~7.0 cm. Basal 8.8 k.O k .6 0.0 Amino acid 36.8 20.0 53.9 35.8 Growth factor 1.5 28.0 1.5 7.2 Amino acid-growth factor 33.8 16.0 26.2 22.2 Yeast extract-soil extract 19.1 32.0 13.8 35.8 * The nutritional c l a s s i f i c a t i o n of the isolates from the 2 lb. treated s o i l was omitted from Tables 15 and 16. 39 Table 16. Numbers of organisms x 10? per gram of s o i l i n each nutritional group (total count x percentage/loo of organisms i n each nutritional group). 0 l b . 2,It-D 100 l b . 2,1).-D """ Nutritional group 0-3.5 cm. 3 .5-7.0 cm 0-3 .£ cm. 3 .5-7.0 cm. Basal 0.52 0.15 0.1U 0.00 Amino acid 2.19 0.76 1.6U 0.8U Growth factor 0.08 1.07 0.0k 0.17 Amino acid-growth factor 2.01 0.61 0.8o 0.52 Yeast extract-soil ext. 1.13 1.37 0.1i2 0.81* (:' c i ) Mscussion. The data i n Table 13 shows that the weights of s o i l s used for plating are relatively uniform. Examination of the t o t a l counts presented i n Table Hj. indicates that there i s a negative correlation between the depth of sampling and the t o t a l count. It would appear that the application of 2,U-D to s o i l results i n a slight decrease i n the t o t a l count. It i s suggested by the data i n Table 15 that there i s a negative corre-lation between the depth of sampling and the percentage of organisms i n the basal, amino acid and the amino acid-growth factor groups; and a positive correlation between the depth of sampling and the percentage of organisms i n the growth factor and yeast-soil extract groups. I t would appear from the data f o r the 0-3.5 cm. s o i l sample that the application of 2,U-D to s o i l at the rate of 100 pounds per acre increases the percent distribution of organisms i n the amino acid group and decreases the percent distribution i n the basal, amino acid-growth factor and the yeast-soil extract groupings. The growth factor group of organisms appears to be unaffected by the addition of the 100 pounds of 2,U-D to s o i l . Analysis of the data i n Table 16 for the 0-3.5 cm. s o i l sample indicates that the large application of 2,U-D has decreased the numbers of organisms ko found i n a l l nutritional groups. The degree of depression varies with the nutritional group being considered; i . e . , the amino acid group being least affected and the basal, amino acid-growth factor and the yeast-soil extract groups showing the greatest effect. The fact that the amino acid group of organisms are the least affected of the nutritional groupings explains the increased percentage distribution of these organisms i n the treated s o i l as reported i n Table 15. The nutritional classification of the f l o r a obtained from the 3 .5-7.0 cm. sample indicates that the 2,k-D application has had no effect cn the f l o r a ; however, this data should be taken reservedly as i t i s based on a relatively small number of isolates. D. Effect of 2fk-D on Pure Cultures of Soil Bacteria. Studies were undertaken to determine the effect of pH and the concen-tration of 2,k-D upon pure cultures of s o i l bacteria growing i n a laboratory medium. Bacteria used included: (1) 50 isolates from the rhizosphere of a corn plant which had been subjected to 2,k-D treatment for 6 days; (2) 1(1 isolates representing the indigenous f l o r a of s o i l which had been treated 6 days previously with a surface application of 2,k-D. (fl) Experimental. The organisms used were selected at random from the sources outlined and were tested as to their growth response i n a f l u i d medium at k pH levels -—6.8, 6 . 0 , 5 . 5 , 5 . 0—and k concentrations of 2, k-B—0,5,25,125 PP*u A fresh stock culture of each isolate was prepared and a 2 mm. loop was used for a l l inoculations. A l l cultures were incubated at 28°C. f o r 5 days. The composition of the media used i s as follows: Ill Stock culture medium agar 1.5 grams/litre glucose 1.0 " K2HP0ii 1.0 » yeast extract 1.0 " soil extract 250 cc.. tap water 750 cc. Test Medium glucose 1.0 grams/litre K2H TOh 1.0 « KN03 0.5 " MgSO^  0.2 " CaCl2 0.1 » NaCl 0.1 " FeCl, 0.01 " H20 Tdistilled) 750 cc. soil extract 250 cc. —salts added to HJJO 4 soil extract, pH adjusted to pH 6.8, heated to 100°C. and cooled. casamino acids I4..0 grams/litre of above medium yeast extract 1.0 " " 11 growth factors as per growth factor medium —added to cooled solution, dissolvedj pH checked and adjusted to pH 6.8; filtered clear and tubed in h cc. amounts. At the end of the incubation period, the turbidity of all tubes was measured by means of a Hellige-Diller photoelectric colorimeter. A decrease in the percent transmission reading of the tubes containing 2,li-D over that of the control (0-ppm 2,l;-D) was taken as being indicative of stimulation; whereas an increase was taken as being indicative of the inhibition of the growth of the isolate by 2,li-D. In order not to assign too much importance to small variations in turbidity, the data on percent transmission readings was studied using 2 levels for stimulation, and -10# decrease in trans-mission, and 2 levels for inhibition, 45$ and 410$ increase in transmission. Thus, by recording such increases or decreases in transmission, one is able to observe trends and to discard variable results. The: following data refer to clear-cut cases of stimulation or inhibition. 1*2 (b) Results. Table 17. Effect of pH on growth of isolates. Data expressed as $ of isolates studied shewing transmission readings less than 7 % 65$ and 55$. Rhizosphere Indigenous $ Transmission >75$ > 6 » >55$ >75% >65$ PH 6.8 51*.0 18.0 6.0 71.0 58.0 56.0 6.0 61*. 0 3l*.0 6.0 85.3 82.9 78.0 5.5 81*.0 62.0 32.0 82.9 80.5 68.0 5.0 51*.o 5o.o 1*0.0 80.5 80.5 73.0 Table 18. Inhibitive effect of 2,1*-D at different pH's on the growth of the 5 0 isolates representing the rhizosphere f l o r a at 4 5 $ and 410$ transmission levels. Data expressed as $ of isolates studied showing inhibition. 2.1*-D concentrations i n ppm. 5 2 ? ^ 125 PH 5$ 10$ 5$ 10% 5$ 10$ 6.8 6 11* 1* 11* 1* 6.0 6 2 8 1* 18 12 5.5 10 1* 20 10 36 15 5.0 20 6 31* 16 1*6 20 Table 19. Stimulative effect of 2,li-D at different pH's on the growth of the 50 isolates representing the rhizosphere f l o r a at -5$ and -10$ transmission levels. Data expressed as $ of iso-lates studied showing stimulation. 2»1*-B concentrations i n ppm. 5 25 -~12T pH 5$ 10% 5$ 20% $% 10% 6.8 8 1* I* 2 6.0 30 10 26 6 16 "u 5.5 20 1U 22 8 8 6 5.0 8 1* 8 2 1* 2 U3 Table 20. Ihhibitive effect of 2,U-D at different pH«s on the growth of the Ul isolates representing the indigenous flora at 45% and +10$ transmission levels. Data expressed as % of isolates studied showing inhibition. PH 2.U-D concentrations in ppm. 5 25 125 5% 102 5% 10% 5% log 6.8 2.U 2.U 6.0 2U.U 7.S 39-0 1^5 68.5 39.0 5.5 7.3 1U.6 7.3 31.8 12.2 5.0 7.3 19.5 7.3 36.6 39.0 Table 21. Stimulative effect of 2,U-D on the growth of the Ul isolates representing the indigenous flora at -5% and 2:10% transmission levels. Data expressed as % of iso-lates studied showing stimulation. 2.U-D concentration in ppm. 5 2 ? I 2 F PH $% 10% 52 log 105? 6.8 7.3 17.1 U.9 • M B U.9 6.0 2.U — — — — — 5.5 9.8 U.9 U.9 U.9 — — 5.0 22.0 2.U U.9 —— 2.U — (:.C.: ) Discussion. The data on the effect of pH on the growth of the isolates is presented in Table 17 and Plate a. It is evident that the indigenous isolates showed £ much better growth response on the complex fluid medium used than did the rhizosphere isolates. This suggests that there was a physiological difference between the two floras. Thelbar graphs on Plate a indicate that the rhizo-sphere isolates showed their best growth at pH's 5.5 and 5 . 0 ; whereas the indigenous flora grew equally well at a l l U pH levels used. Examination of the data presented in Table 18 and Plate b relative to the inhibition of the rhizosphere isolates by 2,U-D reveals a positive PLATEa-EFFECT OF pH ON GROWTH OF ISOLATES (percent of isolates showing transmission reading less than 55 X) RHIZOSPHERE INDIGENOUS 80 U j-60 _ - . -* 40- p-. -20 - -o I n n i l 11 LU U U L 6.8 6.0 5.5 5.0 6.8 60 55 5.0 PH PLATEb- PERCENT OF ISOLATES 50 40 RHIZOSPHERE (10% level) o , 30 20 10 o 6.8 6.0 5.5 5.0 PH INHIBITED BY 2,4"D(125ppm) INDIGENOUS (lOXIevtl) 6.8 6.0 5.5 5.0 PLATEc- PERCENT OF ISOLATES STIMULATED BY 2,4"D(5ppm) RHIZOSPHERE (10% level) INDIGENOUS (10% level) °/ 20 15 10 6.8 6.0 5.5 5.0 68 6.0 5.5 5.0 P H correlation between the acidity of the medium and the tox i c i t y of 2,U-D. This correlation supports the theory that 2,1|.-D i s inhibitory to micro-organisms i n i t s undissociated state only. The ionization of 2,lj.-D i s similar to that of certain organic acids; e.vg,, benzoic acid, i n that as the pH increases the ionization of the molecule decreases. The stimulatory effect of 2,U-D upon the rhizosphere i s presented i n Table 19 and Plate c. It i s indicated that pH has an effect upon the stimu-latory action of 2,U-D, as maximum stimulation occurs between pH's 6.0 and 5>.S>. As 2,2;-D appears to be inhibitory i n the undissociated state only, i t i s probable- that i t i s stimulatory when i n the undissociated state. Therefore, the effect of pH on the stimulatory action of 2,k-D may be ex-plained by considering the concentration of the undissociated molecule present at the different pH's used and that a wide variety of material types was being studied. At pH 6.8, the concentration of undissociated 2,k-D i s low and, therefore, only a few organisms may be stimulated; whereas at pH 6.0 and the concentration has increased and thus more organisms may be stimulated. However, at pH £.0, the concentration has increased to the point where i t i s no longer stimulatory but probably inhibitory. Table 20 and Plate b present the data relative to the inhibitory effect of 2,U-D upon the growth of the indigenous isolates. Examination of this data reveals a positive correlation between the concentration of 2,U-D used and i t s toxicity, but no relationship between the acidity of the medium and the toxici t y of 2,k-D. Analysis of the data i n Table 21 and ELate c reveals no relationship between pH and the stimulatory action of 2,1|.-D on the indigenous isolates. It i s to be noted that the reaction of the rhizosphere isolates to changes i n growth conditions i s uniform, while that presented by the i n d i -genous isolates i s variable. This may be explained by considering the nature Ii5 of the f l o r a . The rhizosphere f l o r a i s thought of as being selected by the plant and, therefore, would be relatively uniform; whereas the indigenous f l o r a i s thought of as being highly diversified. Consequently, the rhizo-sphere f l o r a would present a relatively uniform reaction and the indigenous fl o r a a varied reaction to changes i n growth conditions. Independent of the pH range and the source of the isolates, the maximum inhibitive effect was demonstrated by the highest concentration of 2,U-D used; whereas the maximum stimulative effect was shown by the lowest concen-tration of 2,U-D used. E. A Study of the Decomposition of 2,1|.-D i n S o i l . Since 2,U-D i s decomposed microbiologically, studies were undertaken to determine (a) kinetics of the breakdown of 2,U-D as effected by organic matter; (b) the nature of the s o i l organisms responsible f o r the decomposi-tion of 2,li-D i n s o i l . (a) Kinetics of the breakdown of 2,U-D as effected by organic matter. The rate of detoxication of 2,U-D treated s o i l s i s important from the aspect of crop production; therefore, i t was decided to see i f there was any relationship between the organic matter content and the rate of decom-position of 2,U-D i n so i l s . (i) Experimental. Forty grams of the 0.5 to 2.0 mm. fraction of 5 Vancouver Island soils of similar texture, s o i l type and pH, but of varying organic matter contents, were perfused at room temperature with 175 ml. of a 0.01 percent solution of 2,U-D amine. The perfusion apparatus used was similar to that of Audus (3). Details of the s o i l s are presented i n Table 22. These soils had been stored i n an air-dry state for 2 years before being used i n this experiment. The loss i n volume of the perfusate during perfusion, due to evaporation and sampling, was made up by periodic additions Table 22. History and characteristics of soils used. Soil Location Classification pH QM.% History Al Co Nc Alberni Illust-ration Station Chemainus FSL Courtenay Punt ledge Illustra- FSL tion Station Nanaimo Illust-ration Station Duncan Illus-tration Station Duncan Illustra-tion Station Chemainus FSL Chemainus FSL Chemainus UFSL 5.5 11.76 Field has been operated for approx. 20 years under the following rotation: (1) hoed crop 4 16 tons manure/ acre + 6-3Q-15 @ 500 lbs/acre (2) grain 4 grass legume mixture (3) 1st year hay (k) 2nd year hay Field was in phase 2 when sampled. 5.7 9.69 19U°-oats 1950- potatoes 4 lbs/acre 1951- oats 1952- oats 6-30-15 @ 500 5.9 4.81 9 years sod - annual application of 10-20-10 @ 100 lbs/acre 1952-irrigated 4 spring applica-tion of 16-20-10 @ 200 lbs/acrej 2 summer applications of NHljNO^  at 150 lbs/acre. 5.9 3.29 1952-irrigatedj 20-1LU-72 ©100 lbs/acre; pasture. 5.9 2.59 productive fieldj irrigated in 1951; grain. of distilled water. The concentration of 2,U-D in the perfusate was deter-mined periodically by means of a biological assay (30) (refer to appendix). When these soils had decomposed their initial 2,4-D solutions, a fresh solution of the same concentration was perfused through them, and the concen-tration of 2,U-D was determined as before. Therefore, i t was possible to determine whether or not these enriched soils had the ability to decompose additional 2,U-D solutions without passing through a lag phase. 17 ( i i ) Results. The rates of the decomposition of the i n i t i a l solutions of 2,1;-D are presented on Plates 1, 2 and 3. The curves for the decomposition of the 2nd solution of 2,U-D were not presented as there was either only a very short lag phase or none at a l l . ( i i i ) Discu ssion. Analysis of the decomposition curves as presented i n Plates 1, 2 and 3 indicate that there was no relationship between the organic matter content of the soils used i n this study and the rate of decomposition of 2,U-D. The detoxication curves for the s o i l s A l , N5, Df? and D3 are similar to those reported by Audus (3,5), except f o r the marked variations i n the length of the lag phases. These curves are typi c a l of a reaction which was carried out by a rapidly proliferating bacterial population. However, the curve for the s o i l G9 would indicate that i t s f l o r a was capable of decomposing 2,U-D without undergoing any marked change. It i s to be noted that there i s a considerable variation i n time, 12 days, needed by these s o i l s to completely decompose the 2,1*-D solutions. There i s no evidence i n the description of these s o i l samples which would account f o r differing a b i l i t y to decompose 2,U-D. This data suggests a possible explanation for the observed fact that different soils require different concentrations of 2,U-D to provide adequate weed control. If the f l o r a of a s o i l were capable of decomposing 2,U-D without passing through a lengthy lag phase, i . e . , soils C§ or D5, a higher concentration of 2,U-D would have to be applied to obtain the weed control than i f the f l o r a passed through a long lag phase. The appearance of a very short lag phase when enriched soils were perfused with a second solution of 2,U-D may be explained by the fact that the results of the 2,lj.-D assay were not available u n t i l 3 days after testing. Therefore, i f the second solution of 2,U-D was added too long after the P L A T E I RATE OF 2,4-D DECOMPOSITION T I M E IN D A Y S T I M E IN D A Y S PLATE 2 i oo_ RATE OF 2,4-D DECOMPOSITION E o. o z o o o csT SOIL N5 6 8 10 12 14 16 TIME IN DAYS 18 20 l O O t r E O L O . O o o I CM SOIL D5 -L _L J I 6 8 10 12 14 16. 18 20 TIME IN DAYS P L A T E 3 RATE OF 2,4-D DECOMPOSITION T I M E IN D A Y S is i n i t i a l solution was decomposed, there would not be enough organisms present that were capable of decomposing 2,4-D to give a linear response and a short lag phase would result. The a b i l i t y of enriched soils to decompose new additions of 2,4-D very rapidly accounts for the reduced effectiveness of repeated additions of 2,4-D to s o i l . (b) The nature of the s o i l organisms responsible for the decomposition of 2,4-D i n s o i l . An organism capable of decomposing 2,4-D amine was successfully isolated by streaking 2,4-D agar plates with a loopful of the perfusate from an enriched f i e l d s o i l . This technique i s essentially that used by Audus (4) i n his isolation of 2,4-D decomposing organisms. The composition of the medium used i s as follows: * i f a s i l i c a gel plate or slopes were desired, the agar was replaced by 1$% Ludox col l o i d a l s i l i c a . After 7 days incubation at 2fj°C. two distinct types of bacterial colonies, along with a few molds, were observed growing on the streaked plates. The majority of the colonies present on the plates were shiny, cream-coloured, opaque colonies, 0.5 mm. i n diameterj the remainder of the colonies, except for the few molds, were greyish, dull-surfaced, translucent colonies. Several colonies of both types were picked onto 2,U-D agar and 2,4-D s i l i c a gel slopes and incubated at 25°C. for 1 week. Only the shiny cream-coloured colonies grew on this medium. These slopes, on microscopic and macroscopic observation, proved to be contaminated by a mold. The mold and the bacterium were separated by streaking out several of these isolates on 2,4-D agar MgS0[, 2,4-D amine #agar PH 0.1* 0.02% 0.02% 0.1% 1.5% 7.0 19 plates and picking the bacterial colonies as soon as they develop. This was possible as the mold was very much slower than the bacterium i n i n i t i a -ting growth on 2,lt-D agar. These isolates developed a salmon-pink, non-soluble pigment after 2 to 3 weeks growth. The mold isolate, upon streaking on malt agar, proved to be two separate organisms, one of which was identified as being a member of the Penicillium species, while the other mold resembled the Sporatrichium species. The relationship between these molds and 2,U-D were not studied any further. Morphological examination of the bacterial isolates showed them to be short Gram variable, motile rods of approximately 1 x 3 microns. In young cultures, Gram negative rods predominated with some of them showing Gram positive bipolar granules, a few showing a club-shaped end and the appearance of a raft formation. No coccoid forms were observed i n any of the morpho-logical studies of these isolates. The bacterial isolates showed a f a i r growth on the 2,ljr-D medium used with or without a trace of agar. However, i f 0.1 percent yeast extract, or 20:'percent s o i l extract, was added to the medium, the growth of the isolates was greatly enhanced. The organisms showed that they were capable of very good growth i f yeast extract was the only carbon source present. The optimum concentration of 2,U-D for growth of these isolates was 0.1 percent. It was noted at this time that, when the isolation medium was used as a f l u i d medium, a slight precipitate developed on autoclaving. This d i f f i c u l t y was overcome by changing the salts added to the medium, without changing the ions present. The composition of the f l u i d medium used for the remainder of the cultural studies was as follows: K2S0j1 0.1 % 0.02% 0.02% 0.1 % 7.0 50 TJI that a l l the isolates used gave a similar response to the studies so far, one of them was chosen for further s tudy. It was decided to study this isolate from the point of view of carbon sources (other than 2,k-D), nitrogen sources and general biochemical activity. The results of these studies are summarized i n Table 23. Table 23. Summary of cultural study of isolate. CARBON .. good growth (slight p e l l i c l e formation) arabinose .. .. f a i r growth SOURCE galactose .. .. " « I I it .. trace of growth ti ii it raffinose .. it it I I tt tt tt NITROGEN .. good growth SOURCE .. " " (slight reduction to NOg) BIOCHEMICAL .. no liquefaction nutrient gelatine .... no liquefaction ACTIVITIES litmus milk oxygen relationships aerobic ... positive OTHER optimum pH . 7.0 FEATURES » temperature .... 20-25°C. OF ISOLATE The organism produced very l i t t l e acid from the sugars, the pH change being from 7.0 to 6 . 5 , and no evidence at a l l of gas production. The organ-ism was capable of using nitrogen either i n the ammonium form or as nitrate nitrogen. A culture of the organism was transferred from 2,1;-D s i l i c a gel to nutrient agar and then back to 2,U-D s i l i c a gel 12 times i n a period of 50 days. Similarly, a culture of the organism was transferred from 2,U-D f l u i d medium (no yeast extract present) to nutrient broth 10 times over the same period of time. There was no loss i n the a b i l i t y of the culture to u t i l i z e 51 nor was there any Loss i n the vigor of growth on continued transfer of t h i s nature. A detoxication curve was obtained for this isolate by use of the perfusion apparatus. Two of the apparatuses used i n the earlier study were set up with glass wool replacing the s o i l sample i n the column, and perfused with 2,4-D f l u i d medium containing 0 . 0 1 percent and 0 . 1 percent 2,4-D amine respectively. Each apparatus was st e r i l i z e d i n the autoclave, cooled and then heavily inoculated with a fresh culture of the organism. The change i n the concen-tration of 2,4-D was followed by the cucumber root assay method. The organism completely broke down the 0 . 0 1 percent solution of 2,U-D within 1 2 days after the start of perfusion i n a manner similar to that of the s o i l s (refer to plate 3 ) , However, the 0 . 1 percent 2,4-D solution was only 3 0 percent decomposed at the end of 3 weeks, and no further change i n the concentration of 2,4-D was detectable. The pH of this perfusate had risen from 7 . 0 to 8 . 3 , whereas i n the former case, the f i n a l pH was 7 . 2 . 52 17 General Discussion A major problem in the study of the rhizosphere i s the limiting of the sampling to the rhizosphere itself. If indigenous soil is incorporated in the rhizosphere sample, a lower total rhizosphere count results. One has reason to believe that certain rhizosphere samples used for plating in this study were contaminated with indigenous soil, wherever this occurred a lower total rhizosphere count would follow. This difficulty might be over-come i f the soil in which the plant was growing were allowed to partially dry out prior to sampling; thus, less indigenous soil would adhere to the roots. It has been established that there i s a difference between the rhizo-sphere and the indigenous flora in that the total count for the rhizosphere is many times that for the indigenous flora. This i s in accord with the data in the literature and indicates that larger numbers of organisms find a more favorable condition for their development in close proximity to plant roots than at a distance. However, no evidence was found which indicated that either the relative proportion of the numbers of organisms or the per-cent distribution of organisms i n the nutritional groupings for the indi-genous flora differed from that found in the rhizosphere. There was no difference between the total rhizosphere counts of 2,h-D treated and untreated barley plants. However, evidence was presented which suggests a qualitative difference between the rhizospheres of plants treated at the rate of 8 ounces per acre and plants treated at the rates of 0 and U ounces per acre. It i s felt that the failure to find a greater difference between treated and untreated plants might l i e in the fact that the concen-trations of 2,U-D applied were too low. The results of the study of the effect of 2,1|-D upon the rhizosphere of corn plants parallels those of Clark (15) on the effect of 2,U-D on the 53 rhizosphere of tomato plants. In both instances, i t was possible to produce a quantitative change i n the rhizosphere by treatment of the aerial portions of plants with 2,4-D. It was evident from the comparison of the response of isolates from the rhizospheres of treated and untreated corn plants to nutri-tional media of varying complexity that the quantitative effect was due to the stimulation of certain groups of s o i l organisms. This quantitative and qualitative effect, which was induced by treatment of the plants with 2,4-D , suggests that there has been a change i n the nature of the nutrients present i n the rhizosphere which perhaps was due to a change i n the quantity and, possibly, the nature of the plant root excretions. This data emphasizes the intimate relationship between the physiological activity of a plant and the composition of the rhizosphere. It would appear that the direct application of 2,4-D to s o i l at the rate of 100 pounds per acre causes a slight decrease i n the t o t a l indigenous count. A comparison of the data on the nutritional classification of the isolates from the treated and untreated s o i l indicated that, although a l l nutritional groupings were decreased i n numbers, certain groupings were affected more than others. Since the above changes i n the indigenous f l o r a were brought about by a 100 pound per acre application of 2,4-D, i t i s suggested that herbicidal applications of 2,4-D would have l i t t l e effect. The determination of the percentage distribution of organisms i n each nutritional group does not always present a reliable estimation of a change i n the composition of the s o i l f l o r a . This i s due to the fact that, i n the determination of the percent distribution of organisms, no consideration i s given to variations i n the t o t a l count. Therefore, i n order to obtain a reliable estimation of the effect of treatment or age of plants, etc., on the composition of the flo r a , the t o t a l number of organisms i n each nutri-tional group must be determined. 5k The conflicting data i n the literature on the distribution of organisms i n each nutritional group i s f e l t to be due to the wide variety of soils and conditions used i n the various studies. As the majority of the rhizosphere population develops from the indigenous flor a , attention should be focused on the effect os s o i l characteristics on the nutritional groupings of the indigenous f l o r a . If such information were available, a more accurate evaluation of the effects of plants on the s o i l f l o r a would be, possible. The results of the study on the influence of 2,4-D at different pH levels on the growth of isolates from the rhizosphere and indigenous f l o r a indicates that there i s a difference between these two f l o r a . The isolates from the rhizosphere showed a relatively uniform growth response to 2,4-D over the pH ranges studied; whereas the indigenous isolates showed a varied growth response. This would indicate that the rhizosphere f l o r a was of a uniform nature, whereas the indigenous was of a highly diversified nature. This observations supports the idea that the rhizosphere f l o r a i s a selected one. A study of the curves on the rates of decomposition of 2,4-D i n d i f f e r -ent 'soils, suggests some interesting conclusions. For instance, the marked variation i n the length of lag phases indicates that the flo r a of some s o i l s have the a b i l i t y to break down 2,4-D at a maximum rate almost immediately on i t s application; whereas other soils require a relatively long period of time to attain their maximum rate of 2,4-D decomposition. It was impossible to account for the variation i n the length of lag phases from the data avai-lable on the history and character of the soil s used. These results are i n contrast with the accepted idea that when a l l the 2,4-D applied to a s o i l was decomposed, the f l o r a responsible for the decomposition would decline i n numbers u n t i l their normal l e v e l was reached. An organism was isolated which was capable of decomposing 2,4-D. It 55 showed morphological and cultural characteristics which suited members of either the genus Corynbacterium or the genus Achromobacter. However, the classification of this isolate to species was not followed further. The 2,U-D decomposing organism isolated i n this study differed consi-derably from those isolated by other workers. The organism isolated by Audus (it) steadily lost i t s a b i l i t y to u t i l i z e 2,U-D on repeated transfer over a 10-month period. Moreover, his isolate would not u t i l i z e 2,U-D after having been cultured on a carbon source other than 2,U-D. Similarly, the organisms isolated by Jensen and Peterson (37) lost their a b i l i t y to u t i l i z e 2,U-D on repeated transfer on a laboratory medium. However, the organism isolated i n this study did not show any decrease i n i t s a b i l i t y to u t i l i z e 2,U-D on repeated transfer over a four-month period, nor did the culturing of i t on a carbon source other than 2,U-D influence i t s a b i l i t y to u t i l i z e 2,U-D. The development of an insoluble pink pigment after 2 to 3 weeks growth on a solid medium further differentiates the organism isolated i n this study from those reported i n the literature. A study of the rate of breakdown of 2,U-D by this isolate yielded a curve comparable to that obtained for the breakdown of 2,U-D i n a normal s o i l . Thus, i t would appear that the organism isolated i s one of those responsible for the breakdown of 2,li-D i n s o i l s . ¥ Conclusions. 1. A rhizosphere effect was demonstrated for both corn and barley plants at k to 6 weeks of age. 2. There i s no quantitative difference between the rhizospheres of 2,4-D treated barley plants (U ounces and 8 ounces per acre) and untreated plants. However, there was evidence of a qualitative difference between the rhizospheres of plants treated at 8 ounces per acre and plants treated at 0 and k ounces per acre. 3. On the other hand, corn differed from barley i n that a quantitative and a qualitative difference was detected between the rhizospheres of 2,4-D treated corn plants (1 and 2 pounds per acre) and untreated corn plants. U. The treatment of s o i l with the equivalent of 100 pounds of 2,4-D per acre results i n a slight decrease i n the t o t a l indigenous count, as well as i n a change i n the qualitative composition of the indigenous f l o r a . £. No qualitative difference was detectable between the growth response of isolates from the untreated rhizospheres and from the indigenous f l o r a to nutritional media of varying complexity. 6. I t i s evident from the growth response of isolates from the rhizosphere and the indigenous f l o r a to 2,4-D at different pH levels that there i s a physiological difference between the two flo r a . 7. I t appears that soils vary i n their a b i l i t y to detoxicate 2,4-D applications. 8. An organism capable of decomposing 2,U-D was isolated which showed characteristics of both the Corynbacterium and the Achromobacter genera. °. In order to obtain a reliable estimation of the effect of treatment, 57 etc., on the composition of the f l o r a , the t o t a l number of organisms i n each nutritional group must be determined. There i s enough experimental evidence presented to indicate that a study of the effect of a chemical upon the s o i l f l o r a would provide useful information and, therefore, i t warrants further attention. 58 ¥1 Bibliography 1. Akamine, E. K., "Persistence of 2,U-D Toxicity in Hawaiian Soils/" Bot. Gaz., 112*312, 1951. 2. Anderson, G. R., and Baker, G. 0 . , "Some Effects of 2,lt-D in Repre-sentative Idaho Soils," Agron. Jour., 1*2:1*56, 1950. 3. Audus, L. J,, "The Biological Detoxication of 2»lt-D in Soil," Plant and Soil, 2, No. 1, 31, 19U9. 1*. , "Biological Detoxication of 2,1*-D in Soils - The Isola-E i o n of an Effective Organism," Nature, 166:356, 1950. 5. ' , "The Biological Detoxication of Hormone Herbicides in She Soil," Plant and Soil, 3 , 1951. 6. , "The Fate of 2,U-D ethyl sulphate in Soils," Nature, 170: 586, 1952. 7. , "The Decomposition of 2,1*-D and MCPA in the Soil, » Tour. Sci. of Food and Agriculture, 3:268, 1952. 8. , Plant Growth Substances, Leonard Hill Ltd., London, 1953. 9. Ball, W. S., "Report on 2,1*-D Experiments in the western States," Proc. 2nd Annual Weed Control Conf., 191*5. 10. Blackman, G. E., et al, "Herbicides and Selective PhytotoxLcity," Annual Review of Plant Physiology, 2:199, 1952. 11. Blouch, R., and Fults, J., "The Influence of Soil Type on the Selective Action of Chloro-I.P.C. and Sodium T.C.A.," Weeds, 2:119, 1953. 12. Brownbridge, unpub. data referred to by Audus (7). 13. Carlyle, R. E., and Thorpe, J. D., "Some Effects of Sodium and Ammo-nium 2,1*-D on Legumes and the Rhizobium Bacteria," Jour.Amer. Soc. Agron., 39:929, 19l*7. lit. Clark, F. E., "Notes on the Types of Bacteria Associated with Plant Roots," Trans. Kansas Acad, of Sci., 1*3:75, 191*0. 15. , "Soil Microorganisms and Plant Roots," Advances in Agronomy, l : 2 l*l, 19l*9. 16. Colmer, A. R., "The Action of 2,1*-D upon the Azotobacter Flora of some N Sugar Cane Soils," Appl. Microbiology, 1:181*, 1953. 17. Conn, H. J., and Darrow, N. A., "Influence of Various non-nitrogenous compounds on the Growtn or uertaxn Bacteria in Soils of Low  p7oductiffity, N. Y. Agric. Exp. Sba., Tech. Bull. 172. 1930. 59 18. Crafts, A. S., "A Theory of Herbicidal Action,»» Science, 108:85", 1&8. 19. , "Results of S o i l Treatment versus Contact Sprays i n Corn," Agr. Chemicals, 3:81, 19l;8. 20. , "Toxicity of 2,U-D i n California Soils," HJlgardia, Witt., 19U9. 21. , Symposium Report Am. Soc. Plant Phys. Meeting, Ithaca, N. T., 1952.. 22. , "Herbicides," Ann. Rev, of Plant Phys., li:253, 1953. 23. DeRose, H. R., "Persistence of some Plant Growth Regulators when Applied to the S o i l i n Herbicidal Treatments," Bot. Gag., 107:583, 19U5. 2ii. , and Newman, H. S., "A Comparison of the Persistence of Certain Plant Growth Regulators when Applied to the S o i l , " Proc. S o i l Sci. Soc. Amer., 12:222, 19U7. 25. Duda, J., and Pedzimilk, F., "The Effect of 2,lt-D and of Dinitrocresols on S o i l Microorganisms," Soils and Pert. Abst., 16:283, 1952. 26. E l l e , G. 0., "Some Environmental Factors Controlling the Response of Sweet Corn to 2,lt-D," Weeds, l : l i 3 , 1951. 27. Flieg, 0. , "The Persistence, Mobility and Decomposition of 2,U-D i n So i l with Reference to Microbial Activity,» S o i l and Fert. Abst., 15:283, 1952. 28. , and Pfaff, C , "On the Movement and Decomposition of 2,U-D i n the S o i l and i t s Influence on Microbial Transformation," Soils and Fert. Abst., 15:283, 1952. 29. Gamble, S. J. R., et a l , "Respiration Rates and Plate Counts for Determining the Effect of Herbicides on Heterotrophic S o i l Microorganisms," S o i l Sci, 7ij.:3U7, 1952. 30. Grant, V. Q., and Ready, D., "A Rapid Sensitive Method for Itetermining Low Concentrations of 2,U-D i n Aqueous Solution," Bot. Gaz., 109;39, 19U7. 31. Hanks, R. W., "Removal of 2,U-D and i t s Calcium Salts from Six Dif-ferent Soils by Leaching," Bot. Gaz., 108:186, 191+6. 32. Hansen, Jy R., and Buchholtz, K. P., "Inactivation of 2,l4.-D by Light i n the Presence of Riboflavin," Weeds, 1:237a 1952. 33. Hashaw, R. W., and Grand, A. T., '(Morphological and Anatomical Effects of 2,U-D on Young Corn Plants," Bot. Gaz., 113:65, 1951. 3U. Hernandez, T. P., and Warren, G. F., "A Comparison of 2,1*-D Pre-emergence Treatment on Onions i n Two Soils," Proc. Amer. Soc. Hort..Sci., 56:383, 1950. 60 35. Hernandez, T. P., and Warren, G. F., "Some Factors Effecting the Rate of Inactivation and Leaching of 2,U-D in Different Soils," Proc. Amer. Soc. Hart. Sci., 56:287, 15^ 0. 36. Hildebrand, A. A., and West, P. M., "Strawberry Root Rot in Relation to Microbiological Changes in Root Rot Soil by the Incorpora-tion of Certain Cover Crops," Can. Jour. Res., 012:183, l °Ul . 37. Jensen, H. L., and Peterson, H. I., "Detoxication of Hormone Herbi-cides by Soil Bacteria," Nature, 170:4°, 1952. 38. , , "Decomposition of Hormone Herbicides by Bacteria," Soils and Fert. Abst.,16:13°, 1°53. 39. Jones, H. E., "Influence of 2,U-D Acid on Nitrate Formation in a Prairie Soil," Jour. Amer. Soc. Agron., U6:522, 19U8. 1+0. Jorgensen, C. J., and Hamner, C. F., "Weed Control in Soils with 2,U-D acid and Related Compounds and their Residual Effects under Varying Environmental Conditions," Bot. Gaz., 109:325, 1°U8. UL Katznelson, H., and Chase, F. E., "Qualitative Studies on Soil Micro-organisms: VI, Influence of Season and Treatment on the Incidence of Nutritional Groups of Bacteria," Soil Sci., 58:U73, 1°UU. U2. , and Richardson, L. T., "Rhizosphere Studies and Associated Microbiological Phenomena in Relation to Straw-berry Root Rot," Can. Jour. Res., C 2l:2U°, 1S>U3 U3. , et al, "Soil Microorganisms and the Rhizosphere," Bot. Revs., 1U, No. 9, 5U3, 19U8. UU. Knowles, F., and Watkins, J. E., "Assimilation and Translocation of Plant Nutrients in Wheat During Growth," Jour. Agri. Sci., 21:612, 1931. U5. Koide, H., and Gainey, P. L., "Effects of 2,U-D and CADE, Singly and in Combinations upon Nitrate and Bacterial Content of Soils," Soil Sci., 7U-.167, 1952. U6. Kries, 0., "Persistence of 2,U-D acid in Soil in Relation to Content of Organic Matter, Water and Lime," Bot. Gaz., 108, 19U7. U7. Krone, P. R., and Hamner, C. F., "2,U-D Treatment for the Control of Weeds in Planting Gladioli," Proc. Amer. Soc. Hort., U°:370, 19U7- ~" U8. Lees, H., and Quastel, J. H., 'Biochemistry of Nitrification in Soil," Biochem. Jour., UO:803, 19U6. U°. Lochhead, A. G., "Qualitative Studies on Soil Microorganisms: IU. Influence of Plant Growth on the Character of the Bacterial Flora," Can. Jour. Res., C18;U2, 19U0. 61 50. Lochhead, A. G., and Chase, F. E., "Qualitative Studies on Soil Micro-organisms: 7. Nutritional Requirements of the Predominant Flora," Soil Sci., 55:185, 1943. 51. , and Thexton, R. H., » : VII. The Rhizosphere Effect in Relation to the Amino Acid Nutrition of Bacteria," Can. Jour. Res., C 25:20, 19U7. ~ 52. , "The Bacterial Equilibrium in Soil in Relation to Plants," Proc. Spec. Conf. in Agric, Australia, 1952. 53. , and Thexton, R. H., "Vitamin B12 as a Growth Factor for Soil Bacteria," Nature, 167:1034, 1551. 5U. , "Soil Microbiology", Annual Rev, of Microbiology, 117185, 1952. "~~ 55. Loehwing, ¥. F., "Root Interactions of Plants," Bot. Rev., 3:195, 1937. 56. Lucas, E. H., and Hamner, C. L., "Inactivation of 2,U-D acid by Absorption in Charcoal," Science, 105:340, 1947. 57. Lundegardh, H., and Stenlid, G., Ark. Bot., 31A, (10), 1, 1944. 58. Martin, J. P., "The Hormone Weed Killer, 2,4-D," Calif. Citrograph, 31 (7), 248, 261t, 191(6. 59. Meadows, M. ¥., and Smith, 0 . , 'Effect of Temperature, Organic Matter, pH, and Rates of Application on the Persistence of 2,4-D in Soil," N. E. States Weed Control Conf. Proc, 3:24, 19U9. 60. Minarck, C. E., "Pre-emergence Herbicides and Their Behavior," N. E. States Weed Control Conf. Proc, 5:29, 1951. 61. Mitchell, J. W., "2,4-D, Its Physiological Effect on Plants and Factors Affecting Its Inactivation in Soil,» N. E. States Weed Control  Conf. Proc, 2:32, 19U8. 62. , "Plant Growth Substances," lltl, Univ. of Wisconsin Press, Madison, Wisconsin. 63. , and Brown, J. B., "Inactivation of 2,4-D in Soil as Affected by Soil Moisture, Temperature, the Addition of Manure and Autoclaving," Hot. Gaz., 109:314, 19U8. 61*. , and Marth, P. C, "2,4-D as a Differential Herbicide," Bot. Gaz., 106:224, 1944. 65. , , "Germination of Seeds in Soil Containing 2,4-D," Bot7 Gaz., 107:408, 19U5. 66. , : , "Movement of 2,4-D acid Stimulus and its Relation to the Translocation of Organic Food Materi-als in Plants," Bot. Gaz., 107:393, 19U6. 62 67. Mitchell, J. W., and Stevenson, E. C., "Bacteriostatic and Bacterio-cidal Properties of 2,1*-D," Science, 101:61*2, 191*5. 68. Morita, S., and Aoki, A., "The Effect of 2,l*-D on the Microbial Action i n Soils," Soils and Pert. Abst., 15:1*00, 1952. 69. Muzir, T. J., et a l , "The Movement of 2,1*-D i n Soils," Agron. Jour., 1*3:149, 1951. 70. Newman, A. S., "The Effect of Certain Plant Growth Regulators on Soil Microorganisms and Microbial Processes," S o i l Sci. Soc. Amer. Proc, 12:217, 1°1*7. 71. , and DeRose, H. R., "Persistence of I.P.C. i n S o i l s , " S o i l Sci., 66:393, 191*8. 72. , and Thomas, T. R., "Decomposition of 2,1*-D i n S o i l and i n Liquid Media," Proc. So i l Sci. Soc. Amer., Il* : l 6 0 , 1950. 73. , , and Walker, R. J., "Disappearance of 2"7E-D and 2,i*,5-T from S o i l , " Proc. So i l Sci. Soc. Amer., 16 :21 , 1952. 71*. Noda, J., Jour. Sci. S o i l and Manure, Japan, 21:229, 1951. 75. Norman, A. G., "The Fate of Complex Organic Compounds i n S o i l , " Trans. l*th Inter. Cong. Soil Sci., 3:100, 1950. 76. Norman, A. G., and Newman, A. S., "The Persistence of Herbicides i n Soils," N. E. States Weed Control Conf. Proc, U:7, 1950. 77. . et a l , "Herbicides", Annual Rev. of Plant Phys., ITXUl, 1950. " 78. Nutman, P. S., Thorton, H. G., and Quastel, J. C., "A Comparison of Certain Plant Growth Substances with other Selective Herbi-cides," Nature, 155:1*98, 191*5. 79. Payne, M. G., and Fults, J., "Some Effects of 2,1*-D, D.D.T. and Colorado 9 on Root Nodulation i n the Common Bean," Jour. Amer. Soc. Agron., 39:52, 1°1*7. 80. Repp, G., "The Effect of 2,l*-D on T i l t h Promoting Organisms i n the S o i l , " Soils and Fert. Abst., 1 7 O l , 1951*. 81. Rossman, E. C , and Stanforth, D. W., "Effect of 2,1*-D on Inbred Lines and a Single Cross of Maiae," Plant Phys., 2l*:6o, 19l*9. 82. Russell, J. E., Soil Conditions and Plant Growth, Longmans, Green and Co., 19327 83. SLade, et a l , "Plant Growth Substances as Selective Weed K i l l e r s , " Nature, ^5:1*97, 19l*5. 81*. Smith, N. R., Dawson, V. T., and Wenzel, "The Effect of Certain Herbi-cides on S o i l Microorganisms," Soil Sci. Soc. Amer. P r o c , 10:197, 191*5. 63 85. Stapp, C , Oreter, R., "I. Effect of 2,H-D i n Soils: I I . Reaction of Soi l Bacteria to the Substance," Soils ana Fert. Abst., 16:139, 1953. 86. Starkey, R. L., "Some Influences of the Development of Higher Plants upon the Microorganisms i n the S o i l : I. Historical and Introductory," S o i l Sci., 27:319, 1929. 87. , " : II. Influence of Stage of Plant Growth upon the Abundance of Organisms," Soi l Sci., 27:355, 1929. 88. Stevenson, I. L., and Rovalt, J. W., "Qualitative Studies of S o i l Microorganisms. XI. Further Observations on the Nutritional Classification of Bacteria," Can. Jour, of Bot., 3_1:U38, 1953. 89. Timonin, M. I., "The Interaction of Higher Plants and Soil Micro-organisms: II. Study of the Microbial Population of the Rhizosphere i n Relation to the Resistance of Plants to Soil-Borne Diseases," Can. Jour. Res., C l8:)|)|lj, 19hO. 90. Thorn, C., and Humfeld, H., "Notes on the Associations of Microorganisms and Plant Roots," S o i l Sci., 3j±:29, 1932. 91. Verona, 0 . , "Effects of some Selective Weed K i l l e r s on Microorganisms with Specific Reference to those of the S o i l , " Soils and  Fert. Abst., 13:53, 1950. 92. Virtanen, A. I., and Laine, T., LIII, "Investigations on Root Nodule Bacteria of Leguminous Plants: XXII. The Excretion Products of Root Nodules," Biochem. Jour., 32:ijl2, 1939. 93. Vlitos, A. J., and King, L. J., "Biological Activation of Sodium 2,U-D Ethyl Sulphate by B. cereus boe mycoides," Contr. Boyce  Thompson Inst., 16:I;35, 1952. 9U. Wallace, R. H., and Lochhead, A. G., "Qualitative Studies of So i l Micro-organisms: VIII. Influence of Various Crop Plants on the Nutritional Groups of Soil Bacteria," S o i l Sci.,67:63, 19U9-95. , and King, H. deL., "Nutritional Groups of S o i l Bacteria on the Roots of Barley and Oats," So i l Sci. Soc. Amer. Proc., 1B:3:282, 195U-96. Warren, J. R., Graham, F., and Gale, G., "Dominance of Actinomyces the Soil Microflora after 2,U-D Treatment of Plants," Soils  Fert. Abst., 15:124, 1952. 97. Weaver, R. J., "Contratoxification of Plant Growth Regulators i n Soils and Plants," Bot. Gaz., 109:276, 19U8. 98. Weintraub, R. L., et a l , Metabolism of 2,U-D.I.C1^02 Production by Bean Plants Treated with Labelled 2,U-D," Plant Phys., 27:293, 1952. 6k 99. West, P. M., and Loehhead, A. G., "The Nutritional Requirements of Soil Bacteria - A Basis for Determining the Bacterial Equilibrium of Soils," Soil Sci., |0:lt09, l&O. 100. , 'Excretion of Biotin and Thiamine by the Roots of Higher Plants," Nature, 11 :^1050, 1939. 101. Woodford, E. K., and McCalla, A. G., "Absorption of Nutrients by Wheat," Can. Jour. Res., lit C:2lil?, 1936. 102. Worth, W. A., and Mc Cabe, A. M., "Differential Effects.of 2,4-D on Aerobic and Anaerobic and Faculatative Anaerobic Micro-organisms," Science, 108;16, 19U8. 103 • Zimmerman, P. W., and Hitchcock, A. E., "Substituted Phenoxy and Benzoic acid Growth Substances and the Relation of Structure to Physiological Activity," Contrib. Boyce Thompson Inst. 12:321, 19U2. lOlw Zussman, H. W., "Factors Involved in Sequestering 2,4-D," Agr. Chem., It (lt):17-27, 19U9. 65 VII Appendix. (1) Calculation of the amount of 2,4-D to be applied, (e.g.) U ounces 2,4-D acid equivalent per acre 1 acre - U3,560 square feet diameter of pots = 7 inches 1 U.S. gallon = 3755 cc. = 128 f l u i d ounces s 76.8 ounces of 2,4-D acid Area of pot = TTr^ = 38.46 square inches = 0.267 square feet 128 ounces of solution contain 76.8 ounces of acid 1 ounce of solution contains a ounces of acid a - 76.8 = 0.6 ounces of acid l28~" i f 1 ounce of solution contains 0.6 ounces of acid b ounces of solution contains U ounces of acid b • U s 6.66 ounces of solution 128 ounces of solution a 3755 cc. 6.66 ounces of solution = c cc. c . 6.66 x 3795 ~T2B s 197.UO cc. of solution 197.UO cc. of solution/U3560 square feet • U ounces of 2,U-D acid/acre d cc. of solution/0.267 square feet - U ounces of 2,U-D acid/acre d = 0.267 x 197.UO H3T60 s 0.00120 cc. of solution/pot equals U ounces of 2,U-D acid equivalent per acre. (2) Methods used for characterizing s o i l . pH - pH meter 4 1 pot s o i l : 2 parts water O.M. content - Wakely and Black's wet combustion method available PO^  - as per A.O.A.C. manual exchange capacity - ammonium acetate method. 6 6 Examples of nutritional c l a s s i f i c a t i o n of isolates. Isolate Number I n III IV V Classification # 1 3 2 2 U 3 AG 2 U 2 3 2 l B 3 1 3 1 u 3 A U l 2 1 u 1* AG 5 3 2 U 2 2 G 6 0 3 2 3 u IS 7 0 U 1 u 3 A assigned visual turbidity reading. Legend; I - Basal = B II a Amino acid = A III - Growth factor = G IV = Amino acid* growth factor = AG V » Yeast extract 4soil extract » YS Method of choosing squares for picking i n part III, B, (c). A square ( 6 cm. x 6 cm.) was marked off on the counting surface of v a Quebec Colony counter and divided into 3 6 squares ( 1 cm. x 1 cm.). These squares were numbered from 1 to 3 6 starting i n the upper l e f t corner and proceeding from l e f t to right. Thirty numbers were se-cured from a random numbers table, and one square, corresponding to a different number each time, was marked off with a dye on the back of each plate counted. A l l the colonies found i n these squares were picked into the semi-solid agar. Procedure for determining the concentration of 2,1;-D by the cucumber bio-assay method. The solution to be assayed i s diluted to the range of 0 . 0 0 5 to 1 . 0 ppm. 2,4-D. f i f t e e n cucumber seeds (Early Fortune variety) were placed on a f i l t e r paper moistened with 1 5 mis. of the diluted per-fusate i n a 6-inch p e t r i dish. After incubated i n the dark at 2 8 ° c 5 10 15 20 25 30 35 4 0 45 60 55 60 65 70 75 ROOT LEUGTH (mm) STANDARD CURVE FOR CUCUMBER ROOT BIO-ASSAY. 67 f o r °6 hours, the length of the primary root of the 10 most vigorously growing seeds was measured. The concentration of 2,4-D was read directly off the standard curve covering the 2,4-D concen-tration range 0.01 to 1.0 ppm. (refer to Plate 4). This technique w i l l produce accurate results over the 2,4-D concentration range 0.005 to 1.0 ppm. * * # 

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