Inoculation of Legumes as Related to Soil Acidity

The significance of soil acidity as a factor influencing legume growth and inoculation has long been recognized. Reaction, or degree of acidity, as it influences the behavior of the legume bacteria both within the plant and outside of it on special media has been widely studied. Ranges in reaction tolerated by the plants^8,9,14 and similarly for their particular variety of bacteria have been specified.^8,12 Inconsistencies in these reaction ranges^8,9,10,15 with differing other factors in the experimental conditions have pointed to the need for further study of the influence of soil acidity as a factor affecting legume inoculation.

Phases of the Problem

Effective inoculation, or the entrance of the legume bacteria into the host plant and their function in nodule production and nitrogen fixation there, requires, first, the presence of the specific viable bacteria, and second, the healthy growing plant which is susceptible to the entrance of bacteria and able to bring about development of nodules with nitrogen fixation within them. If soil acidity exercises detrimental effects, it may do so by its effects on the bacteria, on the plant, or on both. It may either destroy the bacteria or lessen their ability to effect entrance. It may bring about a non-susceptibility of the plant, or it may do both.

Soil acidity may be considered as representing roughly (a) an environmental condition in which the excessive hydrogen-ion concentration is intolerable and (b) an irregularity in nutrition of either or both the bacteria and the plant, possibly through shortage of basic elements replaced by the hydrogen, or through injury to, or disturbance of, the plant’s mechanisms for absorption and utilization of the necessary nutrient elements. Since soil acidity represents a loss of basic elements while the content of hydrogen increases, the question naturally arises whether the injurious effect of acidity to inoculation may not be due to an irregularity in the plant’s supply of bases as well as to the presence of the excessive hydrogen-ion concentration. The following work was undertaken in an attempt to separate and measure, in part, the effects of these two phases of soil acidity on legume inoculation.

Previous Work

As the supply of bases in the soil naturally decreases with the development of soil acidity, calcium is the one base more readily removed, hence it presents itself as a possibly deficient element.^15,16 The importance of calcium over many other bases in connection with inoculation has been pointed out.^2,6 Its influences on this process without apparent change of, or influence upon, the soil’s reaction^4,5,6 have served to direct thought to its importance as a nutrient element in the inoculation process as related to soil acidity. That this importance manifests itself not so much through the bacteria as through the plant³ is indicated by the observation that ineffective inoculation or complete inoculation failure on acid soils have resulted when bacteria were plentifully present as revealed by their successful inoculation of the following crop through the simple addition of different forms of calcium salts to the soil. Moderately good inoculation on acid soil has been improved decidedly by calcium addition only.³ Also, the growth of the soybean plants for but 10 days in a liberal supply of calcium increased their growth, nodule production, and nitrogen fixation over those grown similarly on a deficient calcium supply, when both kinds of plants were transplanted to an inoculated acid soil.¹ It has also been shown in studies with a variable supply of calcium under constant degrees of acidity² that larger amounts of calcium were needed to bring about inoculation of soybeans than were necessary for apparently normal growth.

These observations of the significance of calcium in connection with inoculation prompted the following attempt to study the effects of different levels of calcium through a wide range of different degrees of acidity on the inoculation of soybeans.

Methods Used

The soybean plant was selected in consequence of previous studies of it under tests with a controlled supply of calcium and controlled reaction⁶ which revealed the behavior of this plant over a range in its calcium supply and at different degrees of reaction. Limits in these respects for this plant under familiar experimental conditions were thus already established. The control of the pH, and of the supply of calcium, was possible through recourse to colloidal clay as previously used.^2,6

The electrodialyzed clay,⁷ with an initial pH of 3.35, was titrated with calcium hydroxide to produce Ca-H clays of the degrees of acidity desired, according to the titration curve in Fig. 1. These clays of different degrees of acidity were then taken in such amounts as needed to supply the desired amount of calcium per plant and mixed into constant amounts of quartz sand for growing 50 plants. Three different amounts, or levels, of calcium, viz., 0.05, 0.10, and 0.20 M.E. (milliequivalents), were provided through a pH range from 4.0 to 6.5 by intervals of 0.5 pH. These amounts of calcium represented low, medium, and high levels as indicated by previous trials.2 These standardized sand-clay media were each planted with 50 soybeans which had been sterilized, soaked in distilled water, and germinated between filter papers until the radicals were about 1 cm long. The moisture content was maintained at optimum by controlled weights of distilled water added daily. The growth period was 4 weeks.

Fig. 1.–Titration curve of hydrogen clay and calcium hydroxide.

 

Experimental Results

Growth Differences, First Series

Differences in growth were noticeable early and persisted through the period of observation. Brown spots along the lighter green edges were a symptom that seemed to increase with lesser calcium and greater degree of acidity. The height and weight of plants were related to both the degree of acidity and the amount of calcium, as shown in Fig. 2. None of the plants died, but with the highest degree of acidity at all three levels of calcium they failed to grow much beyond the production of the first pair of small leaves just above the cotyledon. Growth improved with decreasing acidity, so that at nearly neutral reaction and even with the lowest amount of calcium the growth was very good. This points out that the sensitivity of the plants to soil reaction is greater as the calcium supply is less, in accordance with the observation Mitscherlich13 reports for acidity and the nutrient supply.

Fig. 2.–Soybean growth according to varying degrees of acidity at different calcium levels.

 

The influence of the varying amounts of calcium on the growth was decidedly significant. This was without influence at pH 4.0 and 4.5 which gave very poor growth, but of decided influence at all pH figures tested above these. The effects of the degree of reaction were displaced or offset by increasing amounts of calcium.

This is clearly evident from Table I. The 0.20 M.E. calcium at pH 5.5 produced the equivalent in height of and were superior in weight to that with 0.10 M.E. calcium at pH 6.5. An increase of 10 times in acidity was offset in its effects on growth by merely doubling the amount of calcium. Many other comparisons within this table point to similar differences, suggesting calcium from 2 ½ to 5 times as significant in growth as is the degree of acidity within the ranges tested.

Table I–Nodulation and growth of soybeans (first crop) as influenced by the calcium and by the pH of calcium-clay soils.

Plant characters		Calcium per plant, M.E. (milliequivalents per plant)	pH at outset (first crop)
			4.0	4.5	5.0	5.5	6.0	6.5
Nodules, 50 plants		0.05	0	0	0	0	7	14
		0.10	0	0	0	8	28	40
		0.20	0	0	0	60	69	127
Height, cm		0.05	11	26	28	31	36	36
		0.10	9.5	27	34	42	44	45
		0.20	8	25	40	45	48	52
Weight of 50 plants in grams	Tops	0.05	4.8	6.3	6.8	7.0	7.9	7.6
		0.10	4.2	6.3	7.3	8.9	9.5	8.7
		0.20	4.6	6.0	8.7	9.2	9.4	9.9
	Roots	0.05	1.5	2.5	2.0	2.0	4.0	3.6
		0.10	1.7	2.2	2.1	4.3	4.3	4.2
		0.20	1.0	1.7	2.5

 

Differences in Nodule Production, First Series

The production of nodules points out clearly that, as in the case of growth, both the degree of reaction and the amount of calcium are significant. Though all plants were similarly treated with bacteria at the outset, no nodules were produced by the soybean plants at pH 5 or greater acidities, suggesting a possible critical limit in acidity beyond which no nodule production may be expected. Above this pH figure nodule numbers increased with lessening acidity at constant calcium levels. Far more significant however, was the increase in nodule numbers at constant pH levels with the increasing amount of calcium. The variation in calcium may even cause a decided shift in the suggested initial acidity limit. According to Table I this limit falls between pH 6.0 and 5.5 when 0.05 M.E. of calcium are present, but falls between pH 5.5 and 5.0 when 0.10 or 0.20 M.E. of calcium are present. Irregularities in past attempts to specify or to duplicate pH limits for inoculation may have been due to variations in the calcium supply.

With reference to the number of nodules produced, the effect of calcium is far more outstanding than that of acidity. An examination of the table reveals that merely doubling the amount of calcium at a constant pH figure approximately doubled the nodule numbers, while at a constant calcium level a corresponding improvement occurred with a 0.5 pH increase, or a reduction in degree of acidity by one-third. This suggests that, in general, calcium is about 1.5 times as influential on nodule numbers as is the hydrogen-ion concentration.

With 0.1 M.E. of calcium per plant present, the nodule numbers per unit calcium increased 100% in going from pH 5.5 to 6.0 and about 70% in going from pH 6.0 to 6.5. With 0.2 M.E. of calcium per plant present, these corresponding pH changes represented roughly 3% and 25% increases, respectively, or a lesser variation due to the pH change as the amount of calcium was increased. These variations occurred in consequence of changing the acidity by one-third at constant amounts of calcium. By using constant pH figures while the calcium was varied, doubling the latter at pH 5.5 meant an increase of 600% in nodule numbers per unit calcium; doubling it at pH 6.0 meant 300% increase; and at pH 6.5, this same rise in calcium amount increased by 200% the nodules per unit of calcium. This points out clearly the greater influence of the calcium in contrast to that of the hydrogen-ion concentration on the nodulation of the soybeans within these higher pH figures and conditions of the experiment.

Soil Reaction Changes in Consequence of Crop Growth

After the crop was removed, electrometric determinations of the pH of the soil were made. Some of the original clays as made up, titrated at the outset and stored without mixing into the sand, were also tested for their pH. They showed no significant changes from their original record. Mixing these into the sand caused no change in degree of reaction. Hence changes of pH in the sand-clay mixtures growing the plants could not be innate to the clays nor due to the mixing of them with the sand. Their changes in this respect must be ascribed to the activities of the growing plants.

It is interesting to note the changes in the pH of these different soils as given in Fig. 3. The more acid soils, those below pH 5.5 became less acid, while those less acid, above this figure, changed to a more acid condition. These changes were greater as the original pH figures were further above or below 5.5. The changes were greater above 5.5 with the extreme one in the most nearly neutral soil. By using the calcium analyses of the seeds and clay at the outset and of the final plants as a means of calculating the calcium left in the clay, and thus its corresponding pH (Fig. 1) at the close of the growth of this series, the pH figure for the clay by determination was higher or the clay was less acid than by the calculations based on the assumption of calcium removal and its substitution by hydrogen. This was true for all of the 18 trials except for the one at pH 6.5 with 0.20 M.E. of calcium. The difference between the actual and calculated figures was relatively constant, ranging from 0.2 to 0.8 pH, or an average of almost 0.5 pH. As for the cause of these differences, theoretical discussion must be omitted here and further work is necessary to establish such.

Fig. 3.–Changes in pH of calcium clay soils in consequence of soybean growth (first series). 

 

Growth Differences, Second Series

Following the growth of the first series and the complete removal of the crop, another crop was planted without any change of the soil or procedure. Soon after the plants were started many of them showed irregularities in growth and symptoms of calcium shortage which might be mistaken for “damping-off,” as previously described.⁵ The number of plants so affected was, in general, inversely related to the amount of calcium in the substrate at any one constant pH figure and to the changes in this figure. Many plants were thus eliminated due to calcium shortage, showing the influence of the calcium depletion by the first crop upon the second. After 3 weeks, the crop growth was widely different in appearance and in weights according to the calcium supply and pH of the soil as given in Fig. 4.

Growth was very poor on all the pans, but it was decidedly better on those same pans where the best growth had occurred previously. Again the growth differences were related more pronouncedly to the calcium than to the pH. Due to the changes in the reaction brought about by the previous crop, 4 pans in each series, or 12 pans in all were of nearly equal acidity, pH 5.4. Even though their reaction was nearly uniform, yet at the low calcium level two of these four gave better growth; at the medium calcium level, three of the four grew better; and at the higher calcium level all four pans had the better growth. These differences are shown by the figures for height in Table 2. Such a disparity in growth at almost constant pH certainly suggests little control over the growth by the pH and points forcibly to the importance of the calcium in bringing about these growth differences at this rather acid figure of pH 5.4.

Fig. 4.–Soybean growth of second crop according to varying degrees of acidity at different calcium levels.

 

Table 2–Nodulation and growth of soybeans (second crop) as influenced by the calcium and by the pH of calcium-clay soils. 

Plant characters	Calcium per plant, M.E.	pH at outset (second crop)
		4.6	4.8	5.4	5.4	5.45	5.45
Nodules, per pan	0.05†	0	0	0	0	4	0
	0.10	0	0	0	0	4	6
	0.20	0	0	0	0	0	3
Dead plants	0.05	14	16	6	9	3	16
	0.10	20	8	1	1	2	1
	0.20	35	14	1	1	0	0
Average height, cm	0.05	6.0	9.0	7.5	7.5	18.5	17.0
	0.10	7.5	10.0	7.5	20.5	21.0	20.0
	0.20	5.0	10.5	20.5	22.0	21.0	22.0
Weight per plant, grams	0.05	0.0767	0.0880	0.0740	0.0820	0.0946	0.1058
	0.10	0.0792	0.0772	0.0752	0.0754	0.0896	0.0996
	0.20	0.0596	0.0819	0.0993	0.1055	0.1074	0.1233

† The calcium supply was reduced by the first crop to a range of 0.047-0.03 in the 0.05 series, to 0.095-0.068 in the 0.10 series, and to 0.195-0.151 in the 0.20 series.

 

Nodulation of the Second Series

Although many nodules had been produced by the previous crop and although the 18 pans were reinoculated at the second planting, nodules developed in only 4 of them. Nodules were formed on less than a dozen plants. The failure of nodulation on plants of medium or poor growth in the first series suggests that nodulation could scarcely be expected in this second series when the growth was so much poorer. The growth period of only 3 weeks may have been too short for nodules to develop. The growth of the second crop was so much inferior to that of the first and nodulated crop at the same age, however, that the low calcium supply suggests itself as the responsible factor rather than the age of the plants.

Reaction Changes in Consequence of Second Crop

Since the growth of the first crop brought about significant changes in the acidity of the soil, determinations of the pH of the soil were made at the close of the second crop. These determinations, together with those at the close of the first series and at the outset, are brought together in Fig. 5.

It is interesting to note that the second crop brought additional changes in reaction in the same direction as was true for the first crop. Further, these changes in reaction tended to make the degree of reaction more nearly the same for all the pans. This leveling effect on the degree of acidity after the second crop was greatest with the largest calcium supply and the greater pH. The three series of six pans each whose initial pH figures ranged from 4.00 to 6.50 were reduced by the two crops to the narrow pH range of 4.46 to 5.00. This suggests a figure of pH 5.00, or slightly below it, as the probable acidity limit of the soil under test below which only a poor activity of the young crop of soybeans will be possible.

Fig. 5–Changes in the pH of calcium clay soils in consequence of growth of two crops of soybeans.

 

Summary

As a result of these studies on nodule production by soybeans as correlated with the degree of acidity and the available calcium supply in the soil, it is evident that the degree of soil acidity is responsible as an environmental factor for nodulation failure on excessively sour soils. In the experiments reported herewith, the acidity at which this failure occurred was at pH 5.0 and lower values. With pH figures larger than this, or soils less acid, the nodulation failure was brought about not so much by the degree of acidity as by the deficiency of the available calcium in the soil. These data point to a decided effect of the element calcium on nodule production in soils with a pH of 5.5 and higher and to an increasing nutritional influence of this element as the soils are less acid. These experiments separate for the first time the effect on nodulation of hydrogen-ion concentration from that of available calcium and further serve to direct attention to the supply of available calcium of the soil as one of the essential conditions for growth and thorough inoculation of soybeans, or possibly other legume crops. They indicate a significance of calcium in symbiotic nitrogen fixation as has been shown for it in the non-symbiotic process.¹¹ They emphasize need for consideration of fertilizing with calcium on the less sour soils as well as changing the reaction in those of higher degree of acidity, if soybeans and possibly other legume crops are to grow well and to be thoroughly inoculated.

 

 References Cited:

1. Albrecht, W. A.: “Nitrogen fixation as influenced by calcium.” Proc., 2d Internat. Cong. Soil Sci., 3: 29-39, 1930.
2. —— “Calcium and hydrogen-ion concentration in the growth and inoculation of soybeans.” Jour. Amer. Soc. Agron., 24: 793-806, 1932.
3. —— and Davis, F. L.: “Physiological importance of calcium in legume inoculation.” Bot. Gaz., 88: 310-321, 1929.
4. ——, —— “Relation of calcium to the nodulation of soybeans on acid and neutral soils.” Soil Science, 28: 261-274, 1929.
5. —— and Potwar, E. M.: “Fractional neutralization of soil acidity for the establishment of clover.” Jour. Amer. Soc. Agron., 22: 649-657, 1930.
6. —— and Jenny, Hans: “Available soil calcium in relation to the ‘damping off’ of soybean seedlings.” Bot. Gaz., 92: 263-278, 1931.
7. Bradfield, R.: “An inexpensive cell for the purification of colloids by electrodialysis.” Ind. and Eng. Chem., 20: 79, 1928.
8. Bryan, O. C.: “Effect of different reactions on the growth and nodule formation of soybeans.” Soil Science, 13: 271-302, 1922.
9. —— “Effect of reaction on growth, nodule formation and calcium content of alfalfa, alsike clover and red clover.” Soil Science, 15: 23-35, 1923.
10. —— “Effects of acid soils on nodule forming bacteria.” Soil Science, 15: 37-40, 1923.
11. Burk, Dean: “The energy and chemical mechanism of nitrogen fixation by azotobacter.” Proc. 2d Internat. Cong. Soil Sci., 3: 67-71, 1930.
12. Fred, E. B., and Davenport, Audrey. Influence of reaction on nitrogen assimilating bacteria. Jour. Agr. Res., 14: 317-336. 1918.
13. Mitscherlich, E. A.: “Discussion by K. Aso on the different behavior of barley and rice toward the reaction of soils.” Proc. 2d Internat. Cong. Soil Sci., 4: 18, 1930.
14. Salter, R. M., and McIlvaine, T. C.: “Effect of reaction of solution on germination of seeds and on growth of seedlings.” Jour. Agr. Res., 19: 73-96, 1920.
15. Scanlan, R. W.: “Calcium as a factor in soybean inoculation.” Soil Science, 25: 313-324, 1928.
16. Shed, O. M.: “Deficiency of plant food calcium on soils.” Ky. Agr. Exp. Sta., 32nd Ann. Rpt., 29-31, 1919.