THE IRONtMANGANESE RELATIONSHIP IN THE GROWTH OF OATS Thesis For the Degree of M. S. MICHIGAN STATE COLLEGE Clarence Cornelius Gray I“ 1947 THESIS This is to certify that the thesis entitled "The Iron—Manganese Relationship in the Growth of Oats." presented hg Clarence C. Gray has been accepted towards fulfillment of the requirements for ___M.'__8 °-_,_degree in $139; sci?!) C9 C.__£.W Main—1“ pi'ofesser Date Aggu§t_192 1911.7. _ M-795 ‘_“ .a—--—-‘ C 07%: ‘v- . THE IRGN ~1MKNGANESE RELATIONSHIP m m Gnowm oi? OATS 3? CIARENCE CORNELIUS GRAY III A THESIS Submitted to the School or Greduete Studies of Michigan State College of Agriculture and. Applied. Science in pertiel fulfillment of the requirements for the degree of MASTER OF 80 IENCE Depertmient of Soil Science 1947 THESIS mmm The writer wishes to express his sincere appreciation to Dr. Ray Cook for his guidance and cooperation and. to Dr. Kirk Lawton for his valuable suggestions relative to the chemical analysis. 11 111 TABLE OF CONTENTS PAGE 5.: MODUCTION............................ REVIEJOFLITERAIURE........._............... mmnmmu...n........................ RESULTS............................... Observationsduringgowth................... Fox............................ Granby.......................... Ironandmanganeeeinthesoil................. Fox. 0 O O O O O O O O O O O O O O O O O O O O O O O I O O \O\O\O (Dal-4&3” amby O C O O O C C O O O O O O O O O O O O O O O O O O O H O Ironandmanganeeeintheplants................ Fox............................10 Granby..........................10 DISCUSSION..................'...........ll SUMMARY...............................l2 CONCIUSIONS.............................l3 WE cm 0 O O O O O O O O 0 O O O O O O O O I O O O O O O 0 ll} iv LIST OF TABLES TABLE PAGE I. The Effect of Manganese and Iron Sulphatee applied to Fox sandy loam.soil on the yield of oats and the Manganese and Iron contents of the soil and the ash from the tops of the oat plants grown on the soil. . . . . . . . . . . . 15 II. The Effect of Manganese and Iron Sulphates applied'to Granby sandy loam.soil on the yield of oats and the .Manganese and Iron contents of the soil and the ash from the tops of the oat plants grown on the soil. . . . . l6 “IEE IRON - MANGANEE RELATIONSHIP IN THE (ROVER OF OATS MMON In recent years, considerable attention has been given to the role of trace elements in the growth and development of plants. Though it is generally accepted. that a definite mineral balance exists for each plant, the proportions of these trace elements to other recognized nutrient elements has been somewhat overlooked. In plants, certain metabolic disturbances, the result of a disrupted mineral balance, are evidenced by chlorotic tissue on the stems and leaves. Generally, the visual evidences of a disrupted mineral balance we specific for a puticular plant and the mineral element or elements concerned. For instance, "any-speck" or "halo blight" of oats is a charactwietic symptom of manganese deficiency. Recently, it has been shown that the intensity of manganese deficiency symptoms, on certain plants, is affected by the concentration of iron present. This suggests strongly that an inter-relationship exists between iron and manganese. Shiva (9)1 concluded that there must be a proper balance not only between the available iron and manganese in the nutrient substrate but also between the active concentrations within the plant tissues. The purpose of this investigation was to observe the effects of varying amounts of added iron and mangmese to soil on the growth and appearance of oats. 1. Figures in parenthesis refer to W CITED" p. '14. REVIEW 93 LITERATURE Twyman (12), reviewing the literature on the ironpmanganese balance in plant metabolism, reports an investigation on the effect of varying proportions of iron and manganese on oats by Balm-Herstmar in 18h8. Although Salmpflorstmar's results were significant, they were obscured by time. He found that manganese was necessary for plants fin.soils where the iron was above 1:100 and where the iron.was in contact with humus. Experimenting with the effect of manganese on the growth of Chlorella, Hopkins (2) found that manganese would not replace iron but that manganese functions physiologically in an indirect manner by its action on the state of oxidation of iron. He concluded that man- ganese controlled the ratio of ferrous to ferric iron in the culture or cell. In 1917, Johnson (3) reported.manganese as a cause of the depression of the assimilation of iron by pineapple plants. After extensive investigation into the functions of manga- nese in plants, Kelly (h) concluded that manganese acted catalytically, increasing the oxidations in the soil and accelerating the auto-oxi- dations in the plants. Upon analyzing the ash of plants on high ’manganese soils of Hawaii, he found a disturbance of the mineral balance. Increased amounts of manganese in the plant were accompan- ied by an increase in the amount of lime and a decrease in the amounts of iron and magnesium. The fact that iron.was shown to be equally available in both manganiferous and nonamanganiferous soil types of equal Hydrogen- ion concentrations led McGeorge (7) to believe that cholorsis of pineapple leaves on plants grown on manganese soils was due to a greater assimilation of lime indirectly caused by the presence of manganese in excessive amounts. He maintained that the resulting physiological disturbance was due to the greater immobility of iron caused by the excessive concentration of the lime in the leaves and ' stalk. Pugliese (8), gowing gain in water culture with Knop's solution with added amounts of manganese and iron observed that certain amounts of manganese were stimlatory when accompanied by proportion- ate amounts of iron but ‘toxic in the absence of iron. He regarded the function of manganese in the plant to be tied up with oxidation phenomena in such a manner that it acted either directly as an oxi- dizing agent or as an accelerator of oxidizing enzymes. He also pointed out that the best fertilizing results were obtained when iron and manganese were present in the proportion of 1:2.5 by molecular weight. Sideris and Irma (10) considered the toxicity of manganese in plants to be due to its chemical reaction with iron during which the iron is rendered inactive. The most recent contribution on the role of iron and manga- nese in plant metabolism is that of Somers and Shiva (11). These workers grew soybeans in solution cultures at three different iron levels, at each of which, manganese concentrations were vuied through a relatively wide range. They found that the pathological symptoms of iron toxicity were identical with those of manganese deficiency. The optimum ratio of iron to manganese in the nutrient substrate for good growth free from pathological symptoms was around 2, regardless of the total amoimts of these elements used. A similar ratio between soluble manganese and soluble iron was found in the plant tissues; fl however, no relationship existed betwosn the total amounts present. minimum. Two Michigan Soil types were selected for the experiment, an alkaline Granby sandy loam, normally manganese deficient, and an acid Fox sandy loam, normally not manganese deficient. The Ca-anby is well supplied with organic matter and has a pH of 7.8. The Fox contains only small amounts of organic matter and has a pH of 5.8. Both soils had been previously screened and were air-dry when ob- tained. Seven and nine kilograms of the Granby and Fox soils re- spectively were placed in two-gallon glazed Jars. Chemically pure mitrients were added in a circular band about 2} inches deep at the equivalent rate of ’1-00 pounds per acre of lt-16-8 fertilizer. Where iron and manganese were added, they were mixed thoroughly in the soil before the placement of the fertilizer. The soils were kept at the moisture equivalent with distilled water. i There were twenty-five treatments on each of the two soils and each treatment was replicated three times. Equal amounts of fer- tilizer were applied to all pots. The treatments , in the equivalent of pounds per acre , were 1. Check 2. 200 lbs. reason 3. 600 lbs. MnSOg, h. 1200 lbs. MnSOu 5.- 1800 lbs. MnSO), 6. 200 lbs. reach 7. 200 lbs. mason; 200 lbs. resoh 9. 10. 11. 12. 13. 1h. 15. 16. 17. 18. 19. 21. 22. 23. 2h. 25. 600 lbs. 1200 lbs. 1800 lbs. 600 lbs. 200 lbs. 600 lbs. 1200 lbs. 1800 lbs. 1200 lbs. 200 lbs. 600 lbs. 1200 lbs. 1800 lbs. 1800 lbs. 200 lbs. 600 lbs. 1200 lbs. 1800 lbs. misc;+ + 200 lbs. MnSOu +- 200 lbs. 111180), + 200 lbs. resoh MnSOh +600 lbs. MnSOh + 600 lbs. unsol, + 600 lbs. unsoh + 600 lbs. resoh Misc), +1200 lbs. MnSOh +1200 lbs. MnSOh +1200 lbs. Mason +1200 lbs. FeSOh MnSOh +1800 lbs. unsoh +1800 lbs. MnSOh +1800 lbs. MnSOh +1800 lbs. FeSOh FeSOh FeSOh 1e80,, FeSOh F0801, F9301, FeSOh FsSOh FeSOh F680), F9801, F0801, FeSOh FeSO;l Marion cats were selected because of their sensitivity to vm'ious concentrations of manganese. The cats were planted April 26, 1914?. After emergence they were thinned to an even stand of thir- teen seedlings per Jar. On May 15 all pots received MgSOh at the rate of 50 pounds per acre. On May 25 all. pots received Ca(HZPOh)2 at the rats of 100 pounds per acre and K01 at the rate of 50 pounds per acre and the pots in the Fox sandy loam received KHAN03 at the rate of 50 pounds per acre. These nutrients were added to offset the inadequate amount of fertilizer added at the beginning of the experiment. The plants were harvested on June 5 , approximately one week after heading. After hmesting, the plants were placed in paper sacks and allowed to air-dry for two weeks before weighing. Because of the lugs number of treatments, it was decided to run chemical analyses for iron and manganese on the plant tissue from only one pot in each treatment. There was little variation bstwaen replications and an effort was made to pick what was thought to be the most representative of the three. In prepmation for chemical analyses, the smples were ground in a Wiley mill, passed through a l m.m. screen, and then oven-dried at 80° C. for 2h hours. Four-grm samples were dry ashed in an elec- tric furnace and the ash dissolved in 10 ml. of 1:1 301, heated near boiling for 15 minutes, filtered, and made up to volume in a 100 ml. volumetric flask. Iron and mmgansse were determined colorimetrica‘lly on 25 ml. aliquots with the aid of a photo-electric colorimeter. Iron was determined by the O-phenanthroline method as described by Fortune and Mellon (1) and manganese by the periodate method of Willard and (heathouse (13). In order to get some idea of the availability of the iron and manganese that had been added in the treatments, it was decided to determine iron and manganese in the soils from which the plant samples had been taken. The selection of an extracting solution for the determinations presented a problem because of the uncertainty regarding what consti- tuted the "available" fractions of the total amounts of iron and manganese present. Since the mounts of iron and manganese added in the treatments were known, a preliminm‘y investigation was carried out to determine the extracting agent that would take out mounts in keeping with the known amounts present. The assumption was made that a direct relationship existed between the amoimt available and the mount added. The extracting solution finally selected was 0.1 N H01. The strength of this solution may be questioned, but it is in line with Leeper's (6) thinking that dilute acid extracts may give an esti- mate of the active manganese and also of the toxic manganese that may come into solution in an acid soil. Samples of the soil for analysis were obtained by thoroughly mixing the contents of the Jars and then screening the soil through a 2 m.m. copper wire screen. The samples were oven-dried at 110° C. Twenty-gram portions were placed in 125 ml. flasks, brought up to the moisture equivalent, and incubated for 10 days. This procedure was carried out since the availability of iron and manganese is affected by the state of the oxidation-reduction system in the soil. After incu- bation, the flasks were shaken for fifteen minutes with 80 ml. 0.1 N H01. The extracts were obtained by filtering through Buchner funnels. The extracts were evaporated to dryness and the organic matter destroyed by using Hydrogen peroxide. The residue was taken up in 0.1 N 1101, heated for fifteen minutes, filtered, and made up to volume in a 100 ml. volumetric flask. Iron and manganese were determined on aliquots in the same manner as that in the plant extracts. RESULTS Observations during growth Fox - Germination was good and the early growth was rapid. (h'owth was normal until approximately twenty days after planting, when there appeared slight traces of browning on the tips of the newer leaves of the plants on all. the treatments, including the check which received no iron or manganese. The browning increased in severity as the amount of applied MnSOh increased. The normal rate of growth continued on all pots except those treated with 1200 or 1800 pounds of MnSO;l per acre, where a definite retardation of g'owth occurred. After browning, the leaves began to die-back from the tips. At this point, the lower leaves began to turn brown. As the browning progressed, a purpling appeared. By the time the plants were harvested, the stunted plants had overcome their retardation in growth and were the same size as those in the other pots, as shown by the yield data reported in table I. @anby - Early growth was similm' to that on the Fox soil. There were no indications of any physiological disturbance until twenty-six days after planting when there appeared the chu‘acteristic manganese deficiency "grey-specks.” These symptoms appeared on sever- al of the new leaves on all of the plants that had not received Mason. As mth increased, the appearance of the "grey—specks" was more pronounced on these check plants. Surprisingly, the manga- nese deficiency symptoms and the retarded growth were less severe on the plants that had received iron. On the plants which had received 200 pounds of mach per acre, there were very slight traces of manga- nese deficiency. The plants on those pots which had received 600 pounds or more of Mason, exhibited the same symptoms that were in evi- dence on the oats gown on the Fox soil - browning and a dying-back of the tips, and a gradual purpling. Varying the rate of application of FeSOh, with the rate of application of 1411501, held constant did not affect the growth of the plants. This is shown at the 600 pound rate of MnSOu in Plate 1. At harvest, the plants on all pots had headed normally, except that the manganese deficient plants showed only a few undersized heads. In contrast to the others, the manganese defi- cient plants were darker green and much more succulent. As shown by the data in table II, yields were markedly increased by the 200 pound per acre application of MnSOu, as shown in plate 2, but were not fur- ther increased by any additional quantity. There was some indication that FeSOu caused an increase in yield where MnSOz, was not applied. Iron and Manganese in the Soil Fox - As shown by the data reported in table I , iron ranged from 37 p.p.m. in the pots which had not received iron, to 82.5 p.p.m. where 1800 pounds per acre of FeSOz, had been applied. There was con- siderable variation in the amounts of manganese obtained, even in similarly treated soil. This probably can be attributed to errors in sampling and analysis, and possibly to differences in the amounts of soluble manganese due to a shift in the manganous-manganic equilibrium. The acidity of the soils after the treatments were made varied from pH 5.91; on treatment lio. 1 to pH h.89 on treatment No. 25. @anby - Reported in table II are the results obtained from analyses of the (h‘anby soil. Iron ranged from a low of 10.8 p.p.m. in the untreated soil to a high of 28 p.p.m. where 1800 pounds per acre of Peso“ had been applied. The results of the manganese determin- ations were as erratic as those on the Fox soil. The values ranged from 3.2 p.p.m. to 99 p.p.m. The difference in the two soils as to the availability of iron and manganese can be clearly seen. The un- treated Fox soil contained as much manganese as did the Ch‘anby soil which received 200 pounds per acre of MnSOu. The depression of the alkalinity by the addition of the sulphates was from pH 7.79 to pH7.25. This, in comparison to the corresponding data for the Fox soil, indi- cated the difference in the buffer capacity of the two soils. 10 Iron and Manganese in the Plants Fox - As indicated by the data reported in table I , the total manganese in the plants increased with an increase of the element in the soil, thus indicating that the absorption of this element was de- termined largely by the concentration in the soil. The iron content of the plants showed a similar relationship; however, it did not hold in all cases. No attempt was made to establish the optimum ratio of iron to manganese for best growth since all the plants seemed to show manganese toxicity symptoms. The data show that at each of the five levels of added MnSOh, except the 1800 pound per acre application, as the addition of FeSOh was increased the total manganese in the dry tissue increased until the heaviest application of FeSOu was reached. This very high application of FeSOh reduced the manganese in the tissue. ' Apparently, from the data, no relationship exists between the iron in the plant tissue and the amount of manganese in the soil. Cs'anby - All of the plants grown on the Granby soil that exhibited manganese deficiency symptoms had, as shown by the results in table II, a total manganese content of less than 0.075 milligrams regardless of the amount of manganese added to the soil. It is inter- esting to compare the levels of manganese in plants grown on the Granby soil with those grown on the Fox soil. Even where the greatest quantity of 141180;, was applied the plants grown on the Granby contained less manganese than the control plants grown on the Fox soil. This, of course, explains why manganese deficiency did not occur on the Fox soil. Study of the data shows that there is little or no constant relationship between the ratio of iron to manganese in the tissues and the presence or absence of mmgamese deficiency symptoms. This is 11 in line with the findings of Somers and Shiva (11) that the ratios between total iron and total manganese within the tissues show no definite relation to any deficiency - toxicity condition or to normal plant growth. DISCUSSION In planning the experiment it was the intention to try to produce manganese deficiency and manganese toxicity symptoms on the oat plants by varying the concentrations and proportions of iron and manganese in the soil. Little success was attained in this connection. No preliminary investigations were carried out; consequently, the amounts of manganese and iron sulphates applied were purely reasonable approximations. Applications of iron as high as that contained in 1800 pounds per acre of FeSOh did not produce manganese deficiency on the Fox soil. Assmning that an iron-manganese balance exists for cats and discounting the interference of some unknown factor, then the amounts of iron applied were insufficient. The availability of iron and man- ganese as determined by the 0.1 N H01 extracts, and as shown in table I serves to substantiate this point of view. 0f the amounts applied to the soils only a fraction was available for utilization by the plants. A comparison of the amounts obtained shows that ”fixation” in the alka- line Ca'anby soil was much greater thm that in the acid Fox soil. This agees with the findings of other investigators that an alkaline soil reaction favors the conversion of manganese from the active mangancus form to the relatively inert, and consequently unavailable, manganic oxides. In regard to iron, the peak of its availability is considered to be reached in an acid medium. The applications of FeSOh did not seem to influence the availability of the manganese, nor did the M3801, 12 influence the availability of the iron. The gowth and appearance of the oats gown on the Fox soil was evidently the result of the presence of available manganese in excessive amounts. The chlorotic symptoms present were similar to those described by Kelly (h) on oats and barley plants gown on manga- niferous soils of Hawaii. The appearance of manganese deficiency symptoms on oats gown on the untreated Granby soil showed that the soil was lacking in ade- quate amounts of manganese. According to the theory advanced by Somers and Shive (11) these symptoms could be intensified by increasing the mount of available iron in the substrate. In this experiment, however, Just the reverse happened; as the mount of added F6801, increased the incidence of ”gay-speck" decreased. The difference in size of the gowth can be seen in the picture of cultures shown in plate 3. The results obtained are somewhat confusing in the light of the postulated theory of the iron-manganese relationship. The explana- tion may lie in the fact that soil is a complex system and experimental results obtained from natural soils may be surprisingly different from those obtained with culture solutions. SUWLARY Marion oats were gown on two different soils, one an acid Fox sandy loam and the other an alkaline Granby sandy loam. Twenty- five treatments were made on each of the two soils by increasing the mount of applied MnSOh from zero to 1800 pounds per acre at five corresponding levels of FeSOh. The oats gown on the Fox soil exhibited only manganese toxicity symptoms, apparently the mounts of FeSOh added were insufficient to produce manganese deficiency. Manganese deficiency 13 symptoms produced on the plants gown on the Granby sandy loam were corrected by additions of sufficient manganese sulfate but were not intensified by the application of FeSOh. Tests on the soils, made by extracting with 0.1 N 301 showed that greater proportions of the man- ganese and iron applied were available in‘ the Fox soil than in the (h'anby soil. The tests showed further that the untreated Fox soil contained as much manganese as did the Granby soil which had received a 200 pound per acre application of MnSOh. Chemical analyses of the plants failed to show a constant relationship between the ratio of iron to manganese in the tissue and the presence of deficiency or toxicity symptoms. CONCLUSIONS From the observations and data of this experiment it is appm'ent that: 1. Response to manganese by Marion cats is depend- ent largely on the amount of available manganese in the soil. 2. Iron in the form of FeSOh did not intensify manganese deficiency symptoms on a manganese deficient ca-anby soil. 3. Applications of F0801, up to 1800 pounds per acre did not lessen the effect of manganese toxicity on a Fox sandy loam. h. Within the limits of this experiment, Marion oats are not responsive to an iron-manganese balance in a Fox sandy loam or a Granby sandy loam. 1n LITERATURE CITED (1) Fortune, W. B., and Mellon, M. G. 1938 Determination of iron with o-phensnthroline, a spectro- photometric study., Jour. Indus. and Engin. Chem., Ana- 1313. Me 10:60-6he (2) Hopkins, E. F. ‘ 1930 The necessity and function of manganese in the gowth of Chlorella. Science 72:609-610. (3) Johnson, M. D. 1917 Manganese as a cause of the depression of the assimilation of iron by pineapple plants. Jour. Indus. and Engin. Chem. 9:147-h9. (‘4) Kelly, W. P. 1912 The function and distribution of manganese in plants and soils. Hawaii Agri. Expt. Sta. Bull. 26, 56 pp. (5) 1911+ The function of manganese in plants. Bot. Ga. 57: 213-227. (6) Leeper, G. V. 19147 The forms and reactions of manganese in the soil. Soil Sci. 63:79—9h. (7) McGeorge, W. T. 1923 The Chlorosis of pineapple plants gown on manganiferous soils. Soil Sci. 16:269-27h. (8) Pugliese, A. 1913 Biochemistry of manganese. Relation between manganese and iron in respect to vegetation. Chem. Abstr. 9:6h1-6h2. (9) Shive, J. N. 191+1 Sigificant roles of trace elements in the nutrition of plants. Plant Physiol. 16: 135-1115. (10) Sideris, C. P., and Kraus, B. H. 1931 Mineral deficiencies in plants: physiological effect of iron, titanium, manganese, boron, and fluorine on the development of Ananas Sativus and Zea Maia. Chem. Abstr. 27: 2&75. (11) Somers, I. 1., and Shive, J. W. 19h2 The iron-manganese relation in plant metabolism. Plant Physiol. 17:582-602. (12) Twyman, E. s. 19% The iron-manganese balance and its effect on the gowth and development of plants. The New phytologist h5:18-2’+. (13) Willard, H. H., md (k'eathouse, L. H. 1917 Colorimetric determination of manganese by oxidation with periodate. Jour. Amer. Chem. Soc. 39:2366-2377. the soil. The Effect of Manganese and IronSulfates applied to Fox sandy loam soil on the yield of cats and the Manganese and Iron contents of the soil and the ash from the teps of the cat plmts gown on TABLE I. PLANT ASH ANALYSIS mon" Mane/Gram “)th MMOCO-d’ MO\OOD OO\OOO\O\OCD\OOO\U\ HNWQDCD t~\o “\O bzmbzm h-Pwd) O\O\CO coma --- .mm om.m ccn.c c.ma m.cm c.mm mm.» com comm .mm cm.m smc.c c.ma m.c> c.mm he.» com coma .ea om.m smc.c m.ma c.mm c.mm cs.» com com .ma cc.m csc.c c.ma c.mm c.mm on.» com com .mH cc.: mmc.c c.ma e.m n.c Hm.» com --- .HH c>.n mmm.c c.mm c.mm c.Hm mm.m ccm coma .cH cm.m mca.c m.sH m.s> H.mm mm.» ccm coma .c cm.m mac.c c.mm m.c> c.mm an.» com com .m cm.m cec.c c.ma m.m c.mm on.» com com .s cm.» cmc.c m.ma H.m n.c m>.e com --- .m ce.ma mam.c c.mm c.mm m.mm ms.» --- coma .n cc.m mmm.c c.mm c.mm c.cm ms.e --- coma .s cc.m mnH.c c.mm c.mm c.mm cm.» --- com .m cm.c mnc.c c.mm c.mm c.Hm he.» --- com .m coJm, Hmc.c (muca {[mwmfl c.m cw.» -u- --- .H 55\ 2%: 5.5\ 9%: Emma can 4\ mph <\ mama *zcfi wmmmficfiz zomH magma: mafia scmom some: 2H 9cm mmcancc .on mmmqmcmmaa_ mHmmcaac mm< macaw mHmmcazo chm was mH< mo mm azmzaomma 7F . don on». do 855 mused" use on» no 33 on» 30.5 ass on» one flow on». no 392300 sonH one smegma: on» one case no sacs» on» so Amos secs ssqsm_anssc op sandman nopscasm_ecuu sec economesz.mo panama one .HH_mamaa Plate 1. 1? The Response of Oats to 600 pounds per acre of Mason, added to Granby sand at Five levels of FeSOu. 18 l g.’ .8 ‘ d I II on m I. I“, v. « I ' u. 'I 1' ' , ‘ ‘I a ‘% rd .‘ll‘n‘u 9', - ' f y In Plate 2. The Response of Oats to increasing mounts of MnSOh added to Granby sandy loam. No FeSOh added. Plate 3. 19 The Response of Oats. to increasing amounts of FeSOh added to Granby sandy loam. No MnSOh added. “All“ - __ _ - HMO 35H W008 III, I, L a MICHIGAN STATE UNIVERSITY LIBRARIES III II III I IIIII I | 3 1293 031161 J488