4% ah =a > Copy 1 WINE VY Guo ty a LEARN Poet, ae ey The Growth of Field Corn as Affected by Iron and Aluminum Salts BY - CHARLES HOMER ARNDT , i] Tay A THESIS © PRESENTED TO THE FacubLTy OF THE GRADUATE SCHOOL IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR Tur DrecReE or Doctor or PHILOSOPHY In BoTANY PRESS OF ©: THE NEW ERA PRINTING COMPANY LANCASTER, PA. 1922 by. Iron, and. Aluminu A - S CPhivaduelion - wines ‘ f Eee ee i at | " Tibtorical Review ; a bes : ) The Present Investigation (Methods and Materials). * sonition: Calta Sees Sand Cultures Sank o i A eat epetnettn Data ei 8 mn dees! ESCO : a oe Bffect of the Composition of the Nutrient Solution upon . . CMe Se cate i tion «Ep Nous Nee end ATS & Ao he a: Solution. Cultures. BORE | ai eit * Sand Cultures. . oe ay Desnene” SV Ts eres nn naa a es carers , i Shinlaie care Le : ei ion ce Ae Nea iShe te : » oy Bibliography . % bd ee Oo ca Tn toot ee pias : as i {Reprinted from the AMERICAN JOURNAL OF Borany, 9: 47-71, February, 1922.) \"\ \ \ 5 STEN aie THE GROWTH OF FIELD, CORN -AS AFFECTED: BY IRON AND ALUMINUM SALTS CHARLES HOMER ARNDT (Received for publication May 17, 1921) INTRODUCTION The investigations of Hoffer and Carr (’20) on corn diseases have shown that a brown or brownish-purple discoloration of the lower portion of the nodal tissue is frequently associated with evidences of malnutrition and of root rot. This discolored area they have designated zone “B.’’ Chem- ical analyses showed that a high iron or aluminum content was associated with such a discoloration. The injection of iron salts produced a similar brownish discoloration, increased the catalase and oxidase activities, and reduced the H-ion concentration. Ferrous salts produced these effects more readily than did ferric salts. The injection of aluminum salts pro- duced no discoloration, but had an effect similar to that of iron salts upon the physiological activities. Stalk- and root-rot organisms were usually associated with the accumulation of iron and aluminum in zone “‘B.”’ Bordnar (’15) earlier reported a similar correlation between a high aluminum content of the sugar beet and its infection by bacterial organisms. His analyses showed that a high aluminum content preceded the infection, which indicates that an increased aluminum content is in some manner related to the decreased resistance to infection. The present investigation was undertaken to determine whether toxic concentrations of iron and aluminum salts would produce a similar patholog- ical condition in corn. With this object in view, a study was made of the effect of the composition of the nutrient solution upon the toxicity of sulphuric, nitric, and hydrochloric acids and of their corresponding salts with iron and aluminum. HISTORICAL REVIEW Some investigators of the toxicity of aluminum salts believed that the toxicity is due largely to the acid liberated in the hydrolysis of the salts. Abbott, Conner, and Smalley (’13) investigated the effect of alumi- num nitrate and nitric acid on field corn. Their results indicated that nitric acid is as toxic as the same normality of aluminum nitrate. They [The Journal for January (g: I-46) was issued February 21, 1922.| 47 48 AMERICAN JOURNAL OF BOTANY [Vol. 9 concluded that the toxicity is due to the hydrolysis of the aluminum salts with the accompanying liberation of nitric acid. Miyake (’16) has com- pared the effect on rice seedlings of the same normality of aluminum chloride and hydrochloric acid. The toxicity of these two solutions was not greatly different. The H-ion concentration produced by the acid was three times as great. This indicated that the toxicity must be due to something other than the H-ion concentration. A similar conclusion was reached by Hart- well and Pember (’18) in their study of the effect of aluminum sulphate on barley and rye. The sulphuric acid produced a H-ion concentration four times as great as the same normality of aluminum sulphate. The two substances were, however, alike toxic to barley. The acid depressed the growth of rye similarly to that of barley, but the salt had little effect on the rate of growth of rye. This indicated that plants may vary greatly in their tolerance of aluminum salts. Duggar (11) states that the various soluble inorganic salts of the same metal are of about equal toxicity. Rothert ('06) has shown that this may not be strictly true for aluminum salts. Aluminum chloride was found to be much more toxic to corn than the sulphate. The toxicity of the salts was also dependent upon the method . of application. They were most toxic when used alone in distilled water; less toxic in Knop’s solution; and least toxic in soil cultures. The toxicity of iron salts, particularly of the ferrous salts, when present in excess, is well known. Hartwell and Pember (’08) determined the effect of ferrous sulphate upon the growth of rye and barley. Katayama (06) found that a concentration of ferrous sulphate of less than 0.01 percent stimulated the growth of barley. Higher concentrations were toxic. Clover, according to Rupprecht (’15), is seriously injured under certain conditions by ferrous salts in a concentration above 4 p.p.m. Iron hydrate when added to sand cultures is toxic to spinach. Analyses by Czadek (04) have shown that iron in this form is easily absorbed by spinach. Iron salts, as aluminum salts, are readily hydrolyzed. Thus, they produce an increased acidity of the sand or solution cultures. Boiret and Paturel (’92) suggested that the toxicity of ferrous sulphate is due to the acid radicle. Ferric salts are even more easily hydrolyzed and precipitated in nutrient and soil solutions. The generally recognized greater toxicity of the ferrous salts, as compared to the ferric salts, indicates that at least in the case of the ferrous salts the toxicity is possibly due to something other than the acidity. This may be due in part, as has been suggested by Awatsu (’14), to an abnormal stimulation of the physiological activities. The ferrous salts produce the greater effect on these activities. Maquenné and Demousy (’20) found that the addition of small amounts of copper sulphate to solutions containing a toxic concentration of ferrous sulphate reduced the toxicity. The addition of the copper to like solutions of ferric salts did not reduce the toxicity. They believe that the unlike effect is due to the catalytic action of the copper which hastens the oxidation of SRE ee oy APE | Feb.,. 1922] ARNDT — THE GROWTH OF FIELD CORN 49 the ferrous salt to the less toxic ferric salt. The addition of calcium salts and phosphates to toxic solutions of both salts also lessened the injurious effect by aiding the formation of insoluble iron salts. It has already been noted that most soluble iron and aluminum salts are readily hydrolyzed with an accompanying liberation of the acid radicle. Daikuhara (’14), Abbott, Conner, and Smalley (’13), Hartwell and Pember (18), and Mirasol (’20) state that all soils which indicate injury to plants by aluminum are acid. This acidity is usually reported as the lime require- ment of the soil. Recent work by Joffe (’20) indicates that this lime re- quirement is closely related to the H-ion concentration. Duggar (’20) has shown that the H-ion concentration may be a limiting factor in plant growth and that the effect of any particular concentration upon plant growth varies with the plant used. Good yields were secured with corn with H-ion concentrations varying from a pH of 3.2 to one of 7.1. The optimum H-ion concentration depended upon the nutrient solution and possibly to some extent upon the environmental conditions. Hoagland (’17) reported that a H-ion concentration of 0.3 X 107% is toxic to barley seedlings. These facts emphasize the necessity of determining the depression caused by a H-ion concentration in the nutrient solution, which lacks the toxic salt, equal to that which is produced by the hydrolysis of the iron and aluminum salts, if we wish to determine the toxicity of the salt itself. A comparison of the depression caused by the salt with that of an equal acidity produced by an acid whose anion is of little or no importance as a depressing factor, should give some indication of the toxicity of the salt itself. Because of the importance of a suitable source of iron in nutrient solu- tions, it was necessary to ascertain the proper source of iron and the amount necessary to secure the optimum growth of corn. Mazé’s (’19) experi- ments with corn lead him to recommend ferric sulphate rather than ferrous sulphate as a source of iron. Shive (15), Tottingham (14), and others, in the study of the salt requirements of plants, have used ferric phosphate as a source of iron. Duggar (’20) has noted that corn grown in Shive’s solution becomes chlorotic. The efficiency of ferric and ferrous phosphate has been compared by Corson and Bakke (’17). They secured the best yield with the ferric salt. Shive and Jones (’21) have found lately that the use of ferrous sulphate as a source of iron in wheat cultures gives superior growth to that secured in cultures in which ferric phosphate is used. Pre- liminary experiments showed that ferric phosphate is not a suitable source of iron for corn in solutions of Type I as recommended by the Committee of the National Research Council on the Salt Requirements of Representa- tive Agricultural Plants. Consequently, experiments were made to de- termine the form of iron necessary to assure a sufficient supply to prevent a lack of it from limiting the growth of the plants. 50 AMERICAN JOURNAL OF BOTANY [Vol. 9 THE PRESENT INVESTIGATION Solution cultures were used in the main part of this experimental work. These results were checked with sand cultures to determine to what extent the solution cultures might indicate the toxicity of these salts in the soil. In all cases the weight of the plants grown in the unmodified solution was taken as the control. The effect of the acid or salt is indicated as the per- centage of the weight of the plants grown in solutions containing them, as compared with the control culture. The weight in the control was taken as 100 percent. For the tops the green weight was used as a criterion of the relative growth, as this probably gives the best indication of the condition of the plants. Comparisons of the green and dry weights gave similar relative weights. As a criterion of the root development, the dry weight was used. The roots were placed in weighed test tubes and dried at 100° C. for several days. The H-ion concentration for the solutions was determined by means of the Lubs and Clark (Clark, ’20) series of indicators, using the buffer mixtures recommended by them. ‘These were carefully prepared from re- crystallized salts. Standards were kept in Pyrex test tubes of Io cc. capacity, the solutions to be tested were placed in similar test tubes, and the same concentration of the indicator was added to them. The H-ion concentration of the sand cultures at the time of the renewal of the solutions was determined by adding distilled water to bring the sand to 60 percent of its water-holding capacity; 50 cc. of the solution was drawn off by suction through a hose previously rinsed with distilled water; the new solution was then added, and the remaining 450 cc. was withdrawn. The pH value is reported for the 50 cc. and for the total 500 cc. drawn off. All solutions were clear. Consequently, turbidity did not interfere with the determina- tions. The pH values given to show the effect of the growth of the plants upon the reaction of the solution were determined just previously to the time of harvesting the plants. This was usually at the end of a warm, sunny period favorable for growth, as these conditions seemed to have an important effect on the results. The seed corn used in these experiments was Reid’s Yellow Dent, a variety of yellow dent (or, according to Sturtevant (’94), Zea mays var. indentata), which was furnished by Dr. G. N. Hoffer. It gave practically 100 percent germination. Very few grains showed any infection by parasitic organisms. The salts used were Baker ‘‘analyzed’’ chemicals. The ferric phosphate was prepared as recommended by the Committee of the National Research Council on Salt Requirements of Representative Agri- cultural Plants. All stock solutions of the salts and acids were made up as N/10 solutions. ‘These were made up fresh at least twice a week, except the ferrous sulphate which was made up immediately before it was used. The distilled water was prepared with a Barnstead still and was stored in glass containers. Feb., 1922] ARNDT — THE GROWTH OF FIELD CORN 51 In the solution cultures, pint Mason jars and colorless cylindrical museum jars of 900 cc. capacity were used. ‘These were treated with clean- ing fluid for several hours before each experiment. The jars were com- pletely covered with opaque black paper. A loose shell of stiff light-brown cardboard was placed over this. Flower pots were used for the sand cultures. These were thoroughly impregnated with heated paraffin and then well coated on the inside with a thin layer of the same. The sand was procured from the Whitall Tatum Company. It is the brand known as ‘‘Juniata.’”’ It was prepared as recommended by the Committee on the Salt Requirements of Representative Agricultural Plants. It had a water- holding capacity of 32 percent. It was kept at 60 percent of its water- holding capacity throughout the experiment by the method recommended by the above-named Committee. The relative transpiration of the plants grown in the various solutions was determined from the amount of water added daily to replace the water which had been lost. The relative transpiration here reported is based on the loss for a certain period immediately preceding the time when the plants were harvested. During this period the evaporating power of the atmosphere was determined by standardized spherical white and black atmometer cups. To keep the conditions of light and temperature as nearly uniform as possible for all cultures, as no rotating tables were avail- able, the plants were shifted daily in a systematic manner so that each plant occupied the same position for about the same period during the experiment. ; Each solution culture contained four plants. Five plants were grown in each pot in the sand cultures. In most cases the cultures were run in duplicate series, so that the relative growth is based on the total weight of eight or ten plants. The weights reported in the tables are based on the mean weight of one plant. Germination The seed used was carefully selected for uniformity of size and shape. The seed was soaked for 15 minutes in tap water, drained, and allowed to stand at room temperature for two hours. It was next treated for 15 minutes with a 5 percent calcium hypochlorite solution (5 g. per 100 cc.). This was removed by washing the seed several times with boiled tap water. It was finally soaked for 12 hours in a shallow covered dish in just sufficient boiled water to cover the seed. After soaking, the seeds were distributed on filter paper in germinating dishes and covered with a layer of filter paper. The dish was placed in the greenhouse, covered with glass, and flooded once a day with water. When the radicles were one centimeter long, the seeds were removed to a paraffined germination net made of coarse netting. This was stretched over an inverted twenty-liter bell jar, and filled with a solution of one tenth the concentration of the solution R.S; 52 AMERICAN JOURNAL OF BOTANY [Vol. 9 of the Type 1 solutions recommended by the Committee on the Salt Re- quirements of Representative Agricultural Plants. In addition, 2 g. of calcium carbonate was added to each jar. Tap water was used instead of distilled water. Consequently, the exact composition of this solution is not known, but as it gave excellent results, it was probably as good as any solution which can be used until we have some definite information con- cerning the effect of various solutions on the germination of corn. The seedlings were left on the net until the shoots were about 6 cm. long. This required from 6 to 9 days. Ten times as many seeds were soaked as were finally used. This number was reduced by one half at the time the seedlings were transferred to the net. At the time the seedlings were transferred to the solutions a second selection of the best seedlings was made. For the solution cultures these were wrapped loosely with cotton above the seed and placed in one-half-inch holes in paraffined corks. When they were to be grown in sand, the seed was placed just below the surface. Solution Cultures (a) Solution ‘‘H.’ The solution which Hartwell and Pember (’18) ' found satisfactory for studying the effect of aluminum sulphate on barley and rye, was found to be well adapted for the growth of corn. It was modified slightly to secure a better growth of corn. Its composition, as used, is as follows: CaleWdOWnoaseaeeneeoos 0.00005 WM INOS @frcneiny see ees reece 0.0008 M @a(NO oes er et ae as OOnS Mo Sona (SOs) aku es oan ee OOOOOR ND INEUIN@ 3) ects tha cl7-cere ee OO lee VEE INS Oper ero eres wane .oooolr ~M 1G ied ake ae da oy 0 8 0008 M ZS Ogencey pte ete aie .000005 M Total 0.004168 M To this was added 7 mg. of FePO, per liter. For convenience, this solution will be referred to as solution “H.’’ Preliminary experiments indicated that Hartwell’s solution gave the best results when aluminum, zinc, and manganese were added. The work of Mazé (’15) on the salt requirements of corn suggested this modification. The phosphate con- centration of the solution used is about twice that recommended by Hart- well and Pember. There was no sign of precipitation when aluminum salts were added and the solution was allowed to stand a week. Again, all solutions were changed every other day, or at one half the interval used by Hartwell and Pember. The ferrous-sulphate solutions were changed every day to prevent, as far as convenient, the change from the ferrous to the ferric condition. It was impossible to prevent precipitation when the ferric salts were used. Consequently, the results may not truly represent the relative toxicity cf the ferrous and the ferric salts. (b) Solution ‘A.’ A number of preliminary experiments demonstrated that with solutions of Type I as recommended by the Committee on the Feb., 1922] ARNDT — THE GROWTH OF FIELD CORN ao Salt Requirements of Representative Agricultural Plants, a solution of one half the molecular concentration of R2S3 was well within the range necessary for the optimum growth of corn. A solution was used whose partial volume-molecular proportions were as follows: KH»PO,, 0.0024 M; Ca- (NO3)o, 0.0036 M; MgSQOu,, 0.0035 M. This solution probably had an osmotic pressure close to 0.5 atmosphere. In this solution it was impossible to prevent precipitation when the various salts were added. Consequently, the results of the experiments in which this solution was used are not strictly comparable with those in which solution ““H’”’ was used. They are comparable when considered in relation to the effect of the composition of the nutrient solution upon the tolerance of acidity by corn. Because of this precipitation, a renewal of the solutions at small intervals would have had little effect on the amount of the salt in the solution. To economize time, the ferrous sulphate solutions of o.oo1 N and higher were changed twice a week; the others were changed once a week. Sand Cultures Solution “‘H’’ was used in all sand cultures. The method of McCall (16) as modified by Johnson (’20) was used for changing the solution. The cultures containing ferrous sulphate were renewed every day; the others, every third day. The sand did not contain sufficient iron for the optimum growth of corn. Ferric phosphate was added to the solution as in the solution cultures. The ferric phosphate was added to the ferrous- sulphate cultures every third day, as most of the phosphate probably was held in the sand and was not withdrawn when the solutions were renewed. After 2000 g. of sand was placed in each pot, it was thoroughly washed a second time by drawing 3 liters of distilled water through it. The sand used for the acid cultures was treated for 48 hours with strong sulphuric acid and then thoroughly washed until the water withdrawn was neutral. These sand cultures were not covered with a wax seal. This was thought desirable in order to simulate soil conditions as far as possible. A wax seal would have reduced the aération and induced conditions in the soil which would undoubtedly have increased the toxicity of the ferrous salts. In order to secure some idea of the relative amount of water lost through evaporation and transpiration, a pot without plants was placed in the series and treated as the others. The amount lost through transpiration was calculated by deducting the loss from this pot from the total loss of the other cultures. The error introduced by this method of determining the transpiration should be small, as the loss through evaporation was relatively small in comparison to the total water loss. The writer wishes to express to Dr. J. W. Harshberger his sincere appre- ciation of the interest and assistance which made possible the experiments, and to acknowledge his indebtedness to Dr. G. N. Hoffer of the Office of 54 AMERICAN JOURNAL OF BOTANY [Vol. 9 Cereal Investigations of the United States Department of Agriculture for the suggestion of the problem and for much valuable information. Dr. Alice M. Russell has added greatly to the interest of the investigation by her mycological study of the discolored nodes. To Dr. R. H. True and to other members of the Botanical Department of the University of Pennsyl- vania, the writer is greatly indebted for generous help and criticisms. TaBLe 1. The Effect of the Composition of the Nutrient Solution upon the Availability of Iron in Ferric Phosphate Total Weight Relative Weight | Mg. FePO: Solution per are at : = | | aie Liter Tops Roots Tops | Roots lbTe an sercereracs 7 82.6 gm. | 3.84 gm. 100% 100% SAN to Bree mse te Ba5 ad ee Cena meseam es | 42% 58% bb ” ‘ic ‘ | a4 at Pet eee 14 | 43-9 © | De * 53 70 | 59% iNgars arabe ete | 35 59.84 | 3.68 | 72% | 97% EXPERIMENTAL DATA _I. The Effect of the Composition of the Nutrient Solution Upon the Avail- ability of Iron in Ferric Phosphate Four cultures were set up in duplicate as shown in table 1. The plants were grown in the 800-cc. jars during the period from February 7 to March 5, 1921. The ‘‘H”’ solutions were changed twice a week; the others every ve ral Tops —— ae, (2091S — Initial pH------ Go PH aft. growlh ——— = TF AGT 9 35 0.00005 0.0001 0.9002 9.0005 9.90/ 9.00005 9.0005 2.901 0002 Ss SS eee EEE ane mgm. perl, Fe POg Fe SOa Fe (NO;), Fic. 1 (left). Relative growth of roots and tops with FePO, in solution “A” (3.5, LA, 35)sandeaH G7): : Fic. 2 (right). Relative growth in solution “A” with FeSO, and Fe(NOs;)3. Also H-ion concentration before and after growth. Feb., 1922] ARNDT —_ THE GROWTH OF FIELD CORN 55 week. All the plants in solution ‘‘A’’ were unquestionably chlorotic, which fact indicated that they did not secure sufficient iron. The plants grown in solution ‘“‘H”’ were of a normal color. Other experiments showed that an increase of iron in this solution did not increase the yield. An increase in the amount of ferric phosphate in solution “A” did increase the yield. The color of the plants, however, indicated that the iron was not available in sufficient quantities. Plate IV and also figure 1 show clearly the difference of growth in the various concentrations. II. The Effect of Iron Salts in Solution ‘‘A”’ Two preliminary experiments, in which ferrous sulphate was used as a source of iron, were made in the fall of 1920. This was a poor period for growth because of the great amount of cloudy weather. The results are of interest in comparison with those of the experiment performed under more favorable conditions during March, 1921. In this experiment, the plants were grown for 25 days in 80o0-cc. jars. The first two experiments will be referred to in table 2 as series 1 and 2 respectively; the latter as series 3. The relative transpiration is reported for the last week of growth for series 3. During this period the average daily loss from the white and the black atmometer was I1.4 cc. and 13.6 cc. respectively. TABLE 2. The Effect of Iron Salts upon the Relative Yields of Tops and Roots and upon Transpiration in Solution ‘‘H"’—Also the Initial pH and the pH After Growth | pH : Yield of | Trans- |—_ as ON: : | Yield of Tops Roots | piration | | After Growth Salt | Nor- ; a a petoalittys ||) Ser-alee. | 2 3 3 3 fee ee 2 3 O00. oe | L35% eae 5 FeSQ4.... 0.00005 | 74% 88% Sno | es | 6.7 Pere LOOOK iy 75 le 92 77 See We oh) 6.6 Geer ner 0002 66% 88 100 Saar a 526% | afr KO005 |e || TOO% © |! 100 100 100 (TOORy-ee ule5e5n) eae (2.6 gm.) (4.21 gm.)|(24.7 gm.)|(0.48 gm.) (790cc.)| | s .OOI 68 66 54 67 ee 5 5.28) 5.2 ‘i .002 62 he Ae} | Foocmea amb elas I Zia cs 004 47 | Wissen ee Peete OOS 21 | [Sees Fe(NOs3)3.| .00005 le er 38 ps8 Wie, | 6.8 “c -| .0O005 68 728 | 70 | 3.9 | 6.7 ‘ .OO1 71 74 Vez | 325: 6.6 $ .002 |? 50 63 be53 212) 4.8 All plants grown in the solutions containing ferric nitrate were chlorotic. This fact, and the low yields associated with it, indicated that ferric nitrate was much inferior to ferrous sulphate as a source of iron in solution “A.” In all cases, the best yield was secured with 0.0005 N ferrous sulphate. The above-noted results for series 3 are shown graphically in figure 2. A comparison of figures I and 2 shows that the lack of iron does not usually 56 AMERICAN JOURNAL OF BOTANY [Vol. 9 depress root development as much as it does that of the tops. It is in- teresting to note the poor root development in 0.0005 WN ferric nitrate. There is little difference in the relative growth with either salt in 0.oo1 NV and 0.002 N. It is singular that ferric nitrate should begin to depress growth before it supplies sufficient iron for normal growth. 10 ono 100 Ae vier 9 pte Acs Initial pH ------ 90} Nl oe ee ak 80 A Tene cml : Ain Roots 7 ———— Sale Gen H $0, HNO HCl 2,50 HNO HC] AlzG04), 1, (50,), AI0.), Al (03); Tap rol SS : | | | eee ea ee ee 0.0005N 0.001N. 0.0004 0,0008N 9.00/6N 90.0008M. Fic. 3. Relative growth and the H-ion concentration upon the addition of acids and aluminum salts to solution ‘‘A.”’ The H-ion concentration is given for the initial solution and at the end of the first and last weeks of growth. TABLE 3. The oe Ss Acids and Aluminum Salts in Solution “A” pH Sol. “A” Nor- Be |) Rel: After Growth plus | mality Transpir ul ation Ini- | | Tops | Roots | tial Ist wk. | 4th wk. Ol00n ye eens | 100% 100% 100% 4.8 6 | 6.4 (10.1 gm.) | (0.24 gm.) | (560 cc.) Fs © a eaes 210. OL0005 95 | 105 | 89 3.6 5.4 6 Bea hhc OOo! Tih 73 75 3:3 Av 6 HNO 0005 87 77 89 3.4 4.8 6.2 Re ae oo! 45 38 53 3.2 3.6 5.2 FCI ae tee 0005 90 100 34 Bale Bee 6.2 vi oo! 46 | 38 44 3.2 3.6 4.8 Als(SOx) 0004 98 93 | 89 3.9 6 6.2 r .0008 87 81 | 83 aM 5.2 6.2 r .OO16 65 | 69 82 3.4 5 5.2 Al(NOs); 0008 OI | 89 85 3.9 5.2 6.4 AICI; 0008 93 | 98 88 3.8 5.6 6.2 Feb., 1922] ARNDT — THE GROWTH OF FIELD CORN 57 III. The Effect of Aluminum Salts and Acids Upon Growth in Solution “‘A”’ The object of this series of experiments was to determine the relative toxicity of hydrochloric, nitric, and sulphuric acids and their corresponding aluminum salts. The solution was modified by the addition of the acids and the salts as shown in table 3. As previously stated, the aluminum salts were precipitated in this solution, and the concentration added does not represent the amount remaining in solution. The results, consequently, do not represent the real relation between the toxicity of the acid and that of the salts. The plants were grown for 30 days during the month of April in a shaded greenhouse. Pint Mason jars containing 400 cc. of the culture solu- tions were used. The results are shown graphically in figure 3. The weather was very favorable during the first portion of the period. There was practically no sunshine during the last week. This fact, undoubtedly, accounts for the small change in the pH value for the last week of growth. TABLE 4. The Effect of Acids and Iron and Aluminum Salts upon Growth in Solution “H” pH Yield of Tops | Af Sol. ““H” Nor- Sys 3, Vield of | Transpir- Gronth plus mality Roots ation Ini- is ' Ser. 1 2 2 2 tial | wD 4 Controle san. 100% | 100% 100% 100% | 4.9 | 4.9 | 6.2 (17.5 gm.) | (20.2 gm.) | (0.58 gm.)| (745 cc.) | | ToS Ose a nie 2) O,0002 97 | Baap sl vibes | i ne alee eee 0004 71 | 78 71 74 BES PAns! hore eS ee ey LOOOG 88 | 67 65 69 AAI Beisel DIN Oisesteec se's .0002 go 3:7 | 4.6) Reet eran ih yA OOOA S| 82 76 69 | 68 Brau EO u .0006 68 62 55 51 B22) hARSe | 5.0 HCl .0002 97 Bory aes | ee eh as ee ael(i 0004: 82 88 71 84 ALE Bere || x2 eee | 0006 69 63 53 49 Bw) | BeAN eed: FeSO,. 0002 72 | 71 66 | 60 4.6 | 4.8 | 4.4 ab at iee Seen 0004 62 Ip hats 54. 50 4.6 | 4.6 | 3.8 Aisin 0006 41 * | 4.2 | 4.3 | Bes (SOs) sae ees .0002 100 Avs Mave mem ich Mie: .0004 72 7 66 52 207s WACSuletey, ope Wiabeets, Wigs .0006 75 7 61 67 Baa, aay, || ala Fe(NOs) 3... 3.4, .0002 07 4 4.3 site Oe me .0004 69 62 65 54 Balt Aco AGO. ehh Save ysl tee .0006 52 64 Or 59 Wese Shee Sinle Aa ie Gla ise yc4| .0002 95 | 4 4.2 Pn arden ee eee .0004 86 63 61 56 eee Sone Wie hee a wh ls Der te Oe ae .0006 67 51 51 54 [PSG hl 355 laste AlX(SOg se - .OOOI gI | 88 88 84 | 4.2 | 4.8 | 5.8 teat cane .0002 84 en eae 73 66 Quik | Ange |aane prt cote .0004 | 73 62 59 52 (Sem eon eae2 ve aa Pee Sete .0006 64 57 67 | 53 2.9 13nd ALCNO3) 3. 225-8 .0002 63 66 67 | 63 Ae20 4.7 AO ee aR RUIN CY .0004 53 57 50 | 2 ALD. ie eat any eee ay i it .0006 50 lee ae 48 lesen S| Ale eA 2 AICI eee .0002 hp yoke 74 66 | 4.2 4 ERY .0004 Wiss | 9a 62 2 ie les) ¥ .0006 [pas es 60 49 | 3.9 | 3-5 38 AMERICAN JOURNAL OF BOTANY [Vol. 9 The pH value at the end of the first week is also given. The water loss by transpiration is given for the entire growth period. The average daily loss from the white and black atmometers was 5.83 cc. and 6.49 cc., re- spectively. IV. The Effect of Iron and Aluminum Salts Upon Growth in Solution “H”’ (a) Solution Cultures. As solution ‘‘H”’ has been previously described, only a brief account of the experiment and the environmental conditions will be given here. The pint jars and 450 cc. of the culture solutions were used inall cases. Series 1 was run for 37 days during January and February. Conditions for growth at the beginning of the experiment were not very ff T “alla on 90 | | ss Tops Initial p}----- so \V/ [— pH after growth——— 1 Z\\ 7 ApS / “~~ + \ BO aa © .A-S H-W H-CFtS FrrS F-V E-€ A-S A-N A-C_H-S = = COUTHSF-N OE-G) (AES aN A 0.0002 i Sig |, Nas NOROD DEAS at Fic. 4. Relative weight of tops and H-ion concentration before and after growth in solution ‘‘H’’ as they were affected by the addition of acids and salts in 0.0002 Nand 0.0006 N concentration. In this and the following figures, the following notation is used: C, control; H, F, and A denote the cations H, Fe, and Al. The anions are indicated thus: S = SOs; N = NO;; C = Cl. Ferrous and ferric sulphate are distinguished by plus signs; F++ S = ferrous sulphate, F+++ S = ferric sulphate. favorable. Conditions were very favorable for growth during the 37-day period in which series 2 was grown. The plants grew rapidly and seemed to be in excellent condition until several days before the end of the experi- ment, when the leaves of some of the plants were unable to open because of the formation of a mucilage-like substance. This was most noticeable in the solutions containing aluminum. This pathological condition was associated with a number of bright sunny days during which it was im- possible to keep the greenhouse temperature below 32° C. It never ex- ceeded 35° C. The relative transpiration is given for series 2 and is based upon the transpiration for the last week of growth. During this period the average daily loss from the white and the black atmometers was I1.4 cc. and 13.6 cc., respectively. Feb., 1922] ARNDT —- THE GROWTH OF FIELD CORN 59 (b) Sand Cultures. The plants in the sand cultures were grown for 40 days in April and May. The modifications of solution ‘‘H’”’ and the pH Tops 90 Roeors —— Transpir in —-— 50 Initial pt ----- pH 2ft. growth —— mee wee oe KO ee af 1 i 2k eae] € ESM -W UAC A RES SECS EAN F- CO) ASS iV ie Fic. 5. Relative growth of tops and roots, transpiration, and the H-ion concentration before and after growth in solution ‘‘H”’ upon the addition of 0.0004 N acid and salts. For notation see figure 4. results are given in table 5. The transpiration is reported for the last 16 days of growth. The total average loss by evaporation from the pots with- out plants, which was deducted from the total loss of the others to determine the amount lost by transpiration, was 405 cc. During the period of growth the average daily loss from the white and black atmometers was 7.83 cc. and 8.5 cc. respectively. The small difference between these values is due to the fact that the plants were grown in a well shaded greenhouse and to the large proportion of cloudy weather during the first part of the ex- periment. On sunny days the temperature ranged from 30° C. to 35° C. This was probably too high for the optimum growth of corn. The pH values are given for the first 50 cc. and for the total 500 cc. drawn off at the end of the second week, and also at the time of the last renewal for which the pH value of the last 50 cc. is also given. The change in the pH values of the initial solutions by plant growth was much greater in the sand cultures than in the solution cultures, as is well shown in figure 6. The depression of growth of the tops is also on an average less, and particularly so in case 60 AMERICAN JOURNAL OF BOTANY [Vol. 9 TABLE 5. The Effect of Acids and Iron and Aluminum Salts upon Growth im Sand Cultures | | | | ne | | ; | End 2d wk-| End last wk. Sol: “H” | Nor- Weight Root | Transpi- | Plus mality of Tops | Score ration | Ini- | Woe tial | 50 | 500 | fst | 500 | Last | (See |) es lee cc.| cc. |50 cc. Controls 5 100% 21 -| 10095, |e45Q 6:2) "6:21 6.45 |5Gn a06 | (12.8 gm.) (1410 cc.) | | els SO ene ete ee cen? 0.001 gI 16 100 3 5-4 | 3-8 | 5.6 | 3.8 | 3.6 A ae eee .002 59 VBteeese byl toi PaSy Bebe) Sele eh. als) LUN @airper re fies deoe| .OO1 101 (a ate 98 3 5 3.8 186 Ne SES FG arte avets 2 .OO1 89 Ii sieht 86 3 Seb ee) sol) So) | SS) FeSO, .0002 102 eae 99 A.O-165.8..| 5:08 )|'4-8)|420.) nano ‘ .0004. 88 2 79 HA Om 05.00) 5. On e4 Ae eqn Ae MV, ice it nce ihe .0006 49 Oo 60 4.2 |(3480)| 23:6 |"3. aula cal leaie Fes(SO,)3.:.. .0004 79 10 75 8 Fale 5:8] 520, | See oe2ae5 Fe(NOs)3... .0004 80 8 69 Bf 65:02 | 25e8) lease |e 4.8 |r Gl eee eae alee! 0004 66 6 61 Be hG.) (9528: 125.60) Saw eee AS SOA) asecm fee e .0002 69 4 70 Aste NOP |KO 62.6. "538 oe Meteo he: .0004 66 2 69 3:9) | 5-8 | 5-8 15.8 | 5-2 14.8 CoN, Shae Aa ae .0006 57 58a) satan | acto | 34e a INLGNO sean ces oe .0002 73 6 TAG A280 KON 10.2" 10. gm aes A ae rb ee .0004 54 5 64 Par 2e Or Wo 5.4 | 4.8 | 4.8 eg .0006 54 58 Aon Ad Seeal aN er ee ere ta .0002 80 Orta 7 | A258. 110 6 5.8 | 5.4 5 ee RE 0004 | 63 I 67 Vast 6.2 | 6:27 h'5.4° | 4uleas CER Ca RE 0006 56 48 ela ar oliea of the peculiar behavior of ferrous sulphate at 0.0002 N and 0.0004 N. This difference is probably accounted for by the better aération of the sand cultures. It is still more unusual to have the lower concentration give a slightly higher yield than the control, and 0.0004 N a higher yield than the same concentration of the ferric salts. This is probably to be explained by the fact that the ferrous solutions were renewed every day; the others every 3 days. The addition of sand to the culture solution changed greatly the toxicity of the acids. The average depression in the sand cultures for 0.001 N acid was 6 percent, and in the solution cultures for 0.0004 WN it Was 21 percent. The aluminum salts produced approximately the same depression in both types of cultures. The corresponding average depressions in the sand and solution cultures for 0.0002 N are 26 percent and 28 percent, and for 0.0004 N, 39 percent and 40 percent, respectively. The extensive researches of Shive (’20) have indicated that this similarity in effect in sand and solution cultures is to be expected when the solutions are changed at frequent intervals, and that such factors as chemical action and pre- cipitation must not be considered. The inhibiting effect upon root development of the acids and of the iron and aluminum salts was much less in the sand than in the solution cultures. A 0.0006 N concentration of ferrous sulphate in the sand cultures in- ' These results are taken from a series run previous to the time the other cultures were grown. Feb., 1922] ARNDT — THE GROWTH OF FIELD CORN 61 hibited their development relatively less than 0.0002 N in the solution cultures, 0.001 N H2SQ, less than 0.0006 N; and 0.0004 N of the aluminum salts much less than 0.0002 NV. In the solution cultures the dry weight was used as a criterion of relative root growth. The results, for reasons to be Ser aT: | A 700 aT f At So/ Cult. We. Tops | fa + Ar 4 al oh » « pH after grth ——— Nel =I [ Sand Cult We. Tops —ma em t; | Ro | " « PH aft. grth 70 \-- \ 7 —~ 60} $ eas ae $0 =a eS : t i —!! 5 ae XN Sei orig “holes sL---- scree a se , Neetea kl | 4 sod toot fond SS \ a avila rade Ase 3 i" === I 3 CHEZ SAAC) ESE SV KG AS AEN ACME Sie cS ata) Van iG A-S —$ $A A 7? eS eet 90002 NV. O00004N 0.0006/¥ 0.001N 0.002. Fic. 6. Relative growth in sand and solution cultures and the effect of plant growth upon the H-ion concentration. For notation see figure 4. discussed later, were not satisfactory. In this experiment the roots were washed free of sand and then compared by the method devised by Free (15). A comparison can readily be made by reference to the numbers reported. The highest number indicates the best root development. DISCUSSION It is plainly evident from the experiments described in the preceding pages that the effect of any particular salt upon plant growth depends largely upon the composition of the solution in which the plants are grown. One of the best examples is the difference in the availability of the iron in ferric phosphate in solutions ‘‘A’’ and “H.” Something in solution “A” prevents the plant from absorbing the iron. This is not due to any in- herent property of the ferric phosphate. At the time series 2 of experiment 4 was run, plants were grown in a modified solution ‘‘H.’’ The calcium phosphate was replaced by an equivalent molecular weight of ferric phos- phate. The growth of the tops in these cultures relative to the control was 95 percent; of the roots, 68 percent, and the transpiration was 96 per- cent. The plants were as well developed as those of the controls. The lower nodes of these plants did not show any discolored nodes, such as were found when the plants were grown in the solution with this concentration 62 AMERICAN JOURNAL OF BOTANY [Vol. 9 of the other iron salts. This behavior of ferric phosphate was very interest- ing when it formed as a precipitate when ferric nitrate was added directly to solution ‘‘A.’”” A 0.001 N concentration did not produce plants of a normal green color. A 0.002 N concentration was decidedly toxic. It is singular that growth should be depressed by the addition of an iron salt, before sufficient iron is available for the growth of the plant. When the efficiency of ferric nitrate as a source of iron is compared with that of ferrous sulphate, the result is striking. At 0.0005 N, the yield in the latter is 68 percent of that in the former; while at o.oo1 JN, the ferrous salt be- comes very toxic and the ferric salt gives the better yield. This greater physiological activity of the ferrous salt may be correlated with its greater influence on catalytic activities. Other factors, however, may be con- cerned. The ferrous salt is less readily precipitated than the ferric. There may also be a difference in the solubility of the two phosphates which may be influenced by the concentration of phosphorus and calcium in the solu- tion. Any attempt at an explanation of this difference can be only specu- lative until more data are available. The concentration of ferrous sulphate necessary to give the maximum yield with corn in solution ‘‘A”’ is probably close to 0.0005 N. This is “approximately 14 mg. per liter. The iron requirements of corn are evi- dently very different from those of wheat and probably also from those of rice as determined by Shive and Jones (’21) and Gile and Carrero (’20). The findings of Corson and Bakke (’17) apparently are not applicable to corn. It is somewhat difficult to interpret Mazé’s recommendation of a ferric rather than a ferrous salt. He used a concentration of 50 mg. of ferrous sulphate per liter, an amount which would have been extremely toxic in solution ‘‘H.’”’ Mazé (13) noted that everything depends on the relative proportion of all elements. This is well illustrated by the behavior of ferrous sulphate in solutions ‘‘H’’ and “‘A.”’ A concentration of it in solution ‘‘H,”’ two fifths of that required for optimum growth in solution “A,” reduced the yield 25 percent to 30 percent. A doubling of this con- centration in solution ‘‘H’’ reduced the yield almost 50 percent. The three ferric salts are about alike toxic, and their effect upon growth in solution ‘““H,’’ when compared to ferrous sulphate, is practically of the same order as the relative efficiency of ferric nitrate and ferrous sulphate in promoting growth in solution ‘‘A.’’ Ata o.0002 N concentration they are practically without effect upon the yield; and at 0.0004 N, they gave about a 15 percent better yield than the ferrous salt. The toxicity of aluminum salts at the higher concentrations does not seem to be a function of the concentration. The average depressions are: 0.0001 N 10 percent, 0.0002 N 28 percent, 0.0004 N 40 percent, 0.0006 N 42 percent in the solution cultures, and 0.0002 N 26 percent, 0.0004 N 39 percent in the sand cultures. There is an increase in toxicity of aluminum salts, as measured by yield of tops, with increasing concentration; but it is Feb., 1922] ARNDT — THE GROWTH OF FIELD CORN 63 not at all proportional to the increase in concentration. The effect on the development of the roots is more nearly related to the concentration. This difference in root development is much greater than is actually indicated by the small differences in the relative root yields. The development of the small secondary roots is inhibited at the higher concentrations; but coinciding with the stunting of the secondary roots there is a thickening of the main roots and an increased formation of prop roots, which increases the weight of the root system. Because of this fact, the dry weight is a poor criterion of the root development and of the number of feeding roots. In the solution cultures, the roots were barely able to penetrate a solution of 0.0006 N, no secondary roots formed, and the root tips were frequently swollen and recurved. Nevertheless, the roots were able to absorb sufficient material to produce a stunted growth. The poor development of the roots in the higher concentration probably accounts to some extent for the tendency of chlorosis to be associated with aluminum injury. It is likely that the capacity of aluminum salts to antagonize the action of iron salts, as has been noted by Stoklasa (’18), may be the most important factor. The effect of aluminum salts on root development described above is similar to that noted by Rothert (’06) in his study of the effect of aluminum chloride and sulphate upon the growth of Zea mays. He states that the shoots suffer the least and the secondary roots the most.