Calcium and Hydrogen-Ion Concentration in the Growth and Inoculation of Soybeans
THE COMMON FAILURE OF legumes on sour soils has led us to believe that the soil acidity, or degree of reaction, is responsible. Little attention has been given to the deficiency of calcium as the possible cause, and further consideration needs to be given the question whether legume failure is caused by the harmful effects of the excessive degree of acidity, or by the failure of the plants to obtain sufficient calcium. In mineral, humid soils, the increased deficiency of calcium usually parallels the excessive degree of acidity. Also the use of calcium carbonate on sour soils functions both to supply calcium and to reduce the degree of acidity. Thus, the two possible causes have not been differentiated, and it is readily possible that casual significance has been ascribed to the wrong one of two contemporaneously variable factors. It seems highly essential that we separate these to learn whether legume failure can be ascribed wholly to the excessive hydrogen-ion concentration of the soil or to the deficiency of calcium and its proper functioning in the legume growth.
The particular function of calcium in the plant can not be so readily determined because of the difficulty of controlling accurately the calcium in the soil, and the fact that its ionic form in a water culture may function differently than the adsorbed soil form. Recent developments in the technic of manipulating soil colloids suggested a means of controlling calcium conditions to greater refinement than possible in simple quantitative chemical methods, and a study of the importance of calcium in growth and nodule production of legumes was undertaken with the hope of understanding better the significance of the element calcium in these crops.
Previous work has pointed to calcium as important in soybean inoculation and suggested that it does not exercise this importance so much through its direct influences on the bacteria. These remained viable in an acid soil even though they failed to produce nodules. Inoculated sour soils which failed to produce nodules gave a nodulation increase of 300% when given calcium but no additional bacteria. That the lime functions through the plant more than through its effects on the bacteria is indicated by a significant increase of nodules on the plant grown for 10 days on a calcium-bearing sand and then transplanted to an acid, but well inoculated soil. This same preliminary treatment of the plants for 10 days in calcium-bearing medium also gives greater growth and greater nitrogen fixation in the plants’ early history. These facts led to a more careful study of legume growth and inoculation by the use of electrodialyzed colloidal clay as a means of supplying calcium under conditions controlled for (a) the reaction and (b) the amount of calcium supplied.
Methods
The electrodialyzed clay titrated with different amounts of calcium hydroxide served to give varied hydrogen-ion concentration (pH), and an inversely varied amount of calcium. The colloid of this type contains mainly adsorbed calcium and hydrogen ions and introduces no other significant disturbing factors. The selection of different degrees of calcium saturation made possible constant but different pH values, while the use of different quantities of clay per seed gave different but constant amounts of calcium. Clays so selected were mixed with a leached silica sand and served as a medium for the growth of soybeans previously germinated in plant-food-free sand. The treated clay permitted an approximate range in pH from 3.5 to 7.0. The concentrations of clay in the sand ranged from almost insignificant amounts to quantities never large enough to disturb the good physical condition of the sand. In the early work this never exceeded 2%.
No other plant foods were added, since inoculation may take place under normal conditions of the soybean as early as 14 days, and the cotyledon probably carries a reserve of the elements necessary for this short period of growth. Ionic calcium in the form of solutions of acetate and chloride titrated with their acids was used for comparison with that adsorbed on the clay colloid. The plants were grown in different trials, first, by varying the amount of calcium and inversely the degree of acidity, second, by varying only the acidity at constant amounts of calcium, and third, by varying only the calcium at constant acidity.
Fig. 1.—Growth of soybean plants on varying amounts of calcium and inversely varying acidity, maximum calcium 0.0215 M.E. per plant.
Experimental Results
In the first trials, which used the clay titrated to different degrees of saturation, the amounts of calcium were so low that difficulty occurred in obtaining growth of the plants. Their very earliest growth seemed normal but was followed by a diseased condition resembling damping-off. The number of plants with apparently normal growth varied with the treatment, becoming more numerous with increased calcium and lessened acidity. This effect was far more pronounced for the calcium as acetate than for the calcium in the colloidal clay, suggesting the more ready availability of calcium in the acetate or free form than in the adsorbed form. The growth, or weights, ran parallel with the percentage of healthy plants, as shown in Fig. 1. A trial along this same plan, supplying large amounts of calcium, produced good growth with most of the plants normal as shown in Fig. 2. The introduction of the proper bacteria failed to produce nodules. Very few of the plants grew to significant size and the study was directed to work out more fully the relation of growth and nodulation to calcium and the acidity.
Clays were prepared at different degrees of saturation, and therefore different degrees of acidity within a pH range from 3.84 to 6.94. The calcium was maintained constant as amounts per seed by using more clay of the higher acidity and less of the lower acidity. Two levels of calcium were taken, viz., low calcium at 0.014 M.E. per plant and high calcium, at 0.35 M.E. per plant. The results of this trial are given in Figs. 3 and 4, and point clearly to the fact that with the low supply of calcium the growth, measured in terms of both normal plants and weights, was erratic, irrespective of pH, while with the higher supply of calcium, the growth was good regardless of the degree of acidity.
Fig. 2.—Growth of soybean plants on varying amounts of calcium and inversely varying acidity, maximum calcium 0.20 M.E. per plant.
Fig. 3.—Percentage of normal of soybean plants as grown on low and high amounts of calcium per seed through a pH range from 3.84 to 6.94.
Fig. 4.—Weights of soybean plants grown on low and high amounts of calcium per seed through a pH range from 3.84 to 6.94.
Since the previous trial points to the importance of the calcium more than to the hydrogen-ion concentration, it was necessary to determine the significance of the amount of calcium in the plant growth. Another trial was undertaken in which this was varied in one series through a range from 0 to 0.042 M.E. per seed at a pH of 6.94 (neutral) followed with a duplication at pH 6.92 through calcium from 0–0.35 M.E. per plant grown as individual plants, and another series through a variation from 0.0086 to 0.0308 M.E. at a pH of 4.4 (acid). The results in this trial as given in Fig. 5 and in Tables 1 and 2 show that the growth is associated with the amount of calcium (within the ranges used) and that growth is quite independent of the pH. The acetate serves better to supply the calcium than does the clay colloid at these low amounts of calcium. This points rather definitely to the significance of the calcium in producing the growth and emphasizes the fact that if this element is present in either the free or adsorbed form in sufficient amount, growth seems normal irrespective of the degree of acidity.
Fig. 5.—Growth of soybean plants on varying amounts of calcium per seed at pH 6.94.
Inoculation was applied in all these trials, but no significant nodule production occurred except that there was a suggestion that the nodulation will occur as larger amounts of calcium (approaching 0.30 M.E.) are available to the plant. Growth was terminated after relatively short intervals, though long enough to permit nodulation under normal conditions. Since growth was obtained with difficulty, it is readily possible that even this growth was an abnormal condition for nodule production.
Since growth in these trials was closely correlated with the presence of calcium and its concentration and not with the hydrogen-ion concentration, it points out clearly that calcium is far more effective in producing growth than excessive hydrogen-ion concentration is in prohibiting it.
In the preceding trials attention was directed only to calcium and hydrogen. There arises also the question whether other nutrient cations might not function likewise and play a role correspondingly as important as that of calcium. Methods of test, similar to those used previously, were employed to try the importance of potassium and magnesium as chlorides in comparison with calcium in this and the acetate forms, all at initial pH 7.00. The results are given in Figs. 6 and 7. Increasing amounts of each salt improved the growth. With potassium chloride this ranged from 6 to 26% normal, with magnesium chloride from 4 to 88% and for calcium chloride and calcium acetate marked improvement occurred with every increment, while normal growth was obtained as soon as a certain minimum of calcium (approximately 0.023 M.E. calcium per seed) was reached.
Nodulation was observed in these trials testing calcium, magnesium, and potassium only in the higher concentrations of calcium as acetate (at 0.017 and 0.043 M.E. calcium and above). Consequently, the sands as used were given calcium carbonate, reseeded, but not reinoculated. Growth was normal and nodulation developed, according to the data in Table 3. This points clearly to the greater significance of calcium in larger amounts to bring about nodulation that had previously failed.
Gedroiz used oats, mustard, and buckwheat plants in soils dialyzed free of their calcium but saturated with other bases and reports that, “Plants are thus able to utilize the elements magnesium and potassium in a soil from which the exchangeable bases of these elements were removed practically completely. When the other nutritive elements were provided, high yields were obtained. Totally different results are obtained concerning the role of calcium in the plant nutrition; if practically all the exchangeable calcium is removed from the soil and is then replaced by a base, the presence of which does not prevent the development of the plant in one way or another, then…the plant dies entirely when no calcium salts are added to the soil.” This points to a similar significance of calcium for non-legumes, such as oats, mustard, and buckwheat, as suggested for soybeans in the tests reported here. Gedroiz places the importance of calcium not only over magnesium and potassium, but also over the other bases sodium, aluminum, iron, hydrogen, manganese, lithium, and ammonium. This can not be with the same force in connection with strontium, which he believes “may to a certain extent take the place of calcium for plant growth.” Nevertheless, of a total list of 16 bases used to saturate the soil fully, he believes that “the plant…points to a very special position of exchangeable calcium among all the bases tested.”
Fig. 6.—Percentage of normal of soybean plants as grown on salts of potassium magnesium, and calcium supplying varying amounts of each, initial pH 7.00.
Fig. 7.—Weight of soybean plants grown on salts of potassium, magnesium, and calcium supplying varying amounts of each, initial pH 7.00.
As for the base hydrogen, Gedroiz believes this ion injurious, contrary to the results suggested in the studies here presented. In failing to obtain crop growth on a soil fully saturated with hydrogen-ions and given no calcium carbonate, he says, “Two causes prevented their growth: the absence in such a soil of available calcium, and the acid reaction.” He points to the injurious effects by the acid reaction, since his introduction of calcium sulfate in place of calcium carbonate into a hydrogen-saturated soil fails to produce a crop. Yet, in his explanation, it is suggested that the sulfuric acid formed from the introduction of calcium sulfate into a hydrogen-saturated soil may be the cause of plant growth failure, which is a detrimental factor quite different from the adsorbed hydrogen-ion of the soil, and does not necessarily prove the hydrogen-ion in acid soil as the injurious factor.
These results thus far emphasize the significance, first, of the element calcium itself; second, of the quantity of calcium present; and third, of its association with different cations (acetate, chlorine, clay). The latter point suggested a trial of calcium in different forms to include (a) the acetate, providing calcium in the free, diffusible, ionic form; (b) calcium permutit, with calcium in the adsorbed form, becoming free through exchange with other cations; and (c) anorthite, carrying calcium in the insoluble crystal form of this alumino-silicate mineral. The individual plants were grown at pH 7 with a supply of calcium varying through a range from 0.0125 to 2.50 M.E. per plant. The calcium acetate produced normal plants, the permutit produced such only at higher concentrations, while the anorthite failed at all concentrations. This points out clearly that the “availability” of the calcium or degree of being free is a significant factor as well as the amount of this element per plant.
Though inoculation was applied regularly in all the preceding work, irregularity of growth was the outstanding result and nodules failed to develop in most cases, except when increasing amounts of calcium were applied. This led to other attempts to produce nodulation by still larger amounts of calcium per seed applied by means of clay. The plants were grown in the individual containers and given variable amounts of calcium at the constant pH 7.0 (neutral) and associated with different cations. Two different trials are represented. The results are given in Table 4. In these trials nodulation was obtained in few cases of the lower calcium concentration, but increased with increasing amounts of calcium. These confirm, in part, the previous trials in which nodulation failed in the lower calcium concentration but developed in the calcium acetate above 0.043 M.E., and in the calcium clay above 0.05 M.E. per plant (Table 1). At this concentration and all those above it, nodulation occurred, suggesting this as a possible lower limit for nodulation under the conditions of the experiment.
Again the amount of the calcium, its form, and the associated cation emphasize themselves. The ionic, free forms produce nodulation at a concentration lower than that of the adsorbed form. It is particularly interesting to note that the calcium carbonate was as effective, if not superior, to all other forms. At the much larger amounts of calcium used in these trials growth was always normal and nodulation very satisfactory. Further trials of these larger calcium amounts at other hydrogen-ion concentrations are needed.
Summary
The data as a whole suggest that, with the minimum amounts of calcium used, the plants are readily attacked by disease and that only poor growth without nodulation occurs. With increased amounts of calcium, growth improves and seems to be normal, but only as still greater amounts of calcium are available to the plant will nodulation occur. Though only two trials of larger amounts of calcium at one hydrogen-ion concentration were used to bring about nodulation, they suggest a minimum necessary calcium for nodulation with improved nodule production as this is increased. This suggests that the calcium supply must first meet the requirements for growth and then an additional amount of this element is needed to permit the nodulation. Further, the data point out clearly that the significance of calcium for the soybean plants rests on its function as an element in the plant’s activities rather than on that of reducing the hydrogen-ion concentration of the soil or growth medium.
