Physiological Importance of Calcium in Legume Inoculation
THAT CALCIUM SHOULD BE an important factor in the nodulation of legumes on acid, or sour, soils is suggested by the varying results in nodulation obtained on these soils. This is indicated especially in the soils of northeastern Missouri and southern Illinois, where the many or even frequent and repeated failures of inoculation with pure cultures have been found to occur on certain predominating acid soil types; while improved inoculation results when these soils are supplied, either previous to or at the time of planting, with limestone, acid phosphate, or other calcium-bearing materials.
Data presented by Hellriegel and Wilfarth, in their original article, establishing the relation between legume root nodules and nitrogen fixation, show a stimulating effect of calcium upon nodulation and growth of serradella. Recently Alway, in comparing the effectiveness of soil transfer with that of pure cultures as inoculation for alfalfa on lime-deficient, sandy soils, found that when the land had not been limed the soil transfer method was far more effective for the first seeding. Excessive increases in the amount of culture did not make this method as effective as soil transfer. On land limed well in advance of seeding, however, the inoculations by soil transfer and by the pure culture method were of equal efficacy. This seems to indicate an inability of the organisms to establish themselves quickly in a lime-deficient soil habitat.
Fellers, in a summary of his work on the factors affecting nodulation of soy beans, states: “The bacterial infection of roots does not take place readily on acid soils even when the root infecting organisms are plentiful in the soil.” Bryan, in a study of the effect of acid soil reactions on nodulation of soy beans, found that in general the hydrogen-ion relations for the organism tend to be the same as those for the host plant. He secured a maximum nodulation at pH 6.5, and none below pH 4.9, although the critical hydrogen-ion concentration for the organisms in solution cultures was found to be pH 3.5–3.9.
Truog ventured the theory that the effect of soil reaction on the activity of the bacteria within the nodule is indirect, since the environment of the bacteria when in the nodule must be that of the plant tissue. Karraker, in examining this viewpoint, found that the root system of a single alfalfa plant, divided between a lime-deficient and a limed soil, gave differences in nodule formation corresponding to those obtained on different plants growing wholly within these different soils. He concluded that the effect of soil reaction on nodule formation must be one of localized character in the plant, a direct effect of soil pH on the bacteria in the nodules, or an antecedent effect of the soil acidity on the bacteria, while they are existing non-symbiotically in the soil.
Scanlan concluded that hydrogen-ion concentration has no direct effect upon inoculation, and analogous inoculation resulted from the use of limestone and calcium acetate where the former lessened the hydrogen-ion concentration while the latter had no effect upon it.
Falk thinks that neutralization of the acid is not the only effect of certain valuable salts, this being indicated by the fact that magnesium carbonate and phosphate improve bacterial growth more effectively than calcium carbonate. Machida demonstrated that calcium salts and not magnesium salts are effective in protecting bacteria against lethal agencies. This work was substantiated by Chambers and Rezinkoff, studying the protoplasm of Amoeba proteus.
Winslow and Falk found that concentrations of 0.4 per cent of sodium chloride alone, or of 0.2 per cent of calcium chloride alone, exerted marked lethal effects upon the growth of a typical colon Bacillus, B. communis, while, a mixture of these two, in the ratio of one Ca-ion to five Na-ions and in concentrations as high as 5 per cent of the salt, was actually beneficial in its effect upon the growth of the organism. Hotchkiss made a survey of the effects of cations upon the bacterial growth of B. coli, and found that calcium chloride in a 0.5 molar concentration limited growth completely, but stimulated growth in a 0.05 molar concentration. Scanlan found that one part of calcium chloride to 1,500 parts of solution was the optimum concentration for stimulating growth and longevity of B. radicicola.
Investigations to date give numerous observations on the effects of limed and acid soils on nodulation of legumes, with almost as many theories as to the operating causes. These conditions emphasize the need for a fuller understanding of the fundamental facts controlling the responses in nodule production by legumes and their bacteria on soils of varying degrees of soil acidity or base deficiency.
Experimentation
The work here reported bears testimony to some of the preceding viewpoints. Results analogous to those of Karraker were obtained in working with soy beans on an acid Putnam silt loam. Seedlings were grown in sterile sand for 10–14 days, when the tap roots were cut off just below the lateral roots, which had developed in good numbers and to a length of about 1 inch. These seedlings were placed over a water-tight partition in a container with one-half of the root system carefully planted into an acid soil (pH 5.14) on one side, and the other half into the same soil after it had been thoroughly mixed with calcium carbonate at the rate of 8,000 pounds per two million of soil. Liberal quantities of a suspension of inoculating bacteria were added as the soil was filled into the pans around the root system. Five seedlings with their lateral roots so divided were set into each container. In addition, ten seedlings, with their tap roots likewise cut off, were planted into the container, five on the side of the acid soil and five on the side of the limed soil. Water was maintained at the optimum by daily surface applications.
Although there was a high mortality of plants in consequence of tap root pruning and replanting, those that lived grew very satisfactorily. At the end of 5 weeks they were carefully taken up and the nodules counted. The nodule counts of the plants with divided roots and checks are summarized in table 1. The results show an increase of 208 per cent in the number of nodules formed on the portion of the roots growing in the calcium soil as compared with those formed on the portion in the untreated soil. The difference of 181.1 per cent in nodulation by the check plants, grown wholly within one kind of soil, is approximately the same ratio. The comparison of the degrees of inoculation of the divided root plants and the checks is made on this basis rather than on the actual plant units, because the average nodulation of either portion of the root of the divided root plants represents but one-half of the normal nodulation of the plants.
The reliability of these data is questionable on account of, first, the small number of plants on which the test was completed, and second, the unequal development of the two parts of the divided root systems.
The results obtained for soy beans agree well with those for alfalfa by Karraker. They indicate that, if the effect of the calcium is a physiological one through the plant, this effect is local in character, and the calcium is certainly not translocated to all the roots of the plant for equal effectiveness in improving inoculation; or, that the depressed nodulation in this acid soil is due to an effect of the soil conditions upon the bacteria before they have infected the roots of the host plant.
A test of the stimulating effect of calcium upon nodulation of soy beans on an already well inoculated soil emphasized further the possible physiological effect of this element. The soil used had a pH of 5.5, and was well stocked with the soy bean organism in consequence of well inoculated crops of soy beans on three previous seasons.
Enough of this soil was mixed with calcium carbonate at the rate of 8,000 pounds per two million to fill thirty pots, while a like number were filled with untreated soil, and each of the sixty pots was planted with five sterile, sprouted soy beans. After cautious culture for five weeks, when the plants showed a uniform healthy growth with slight superiority in color, size, and appearance in the case of those given calcium carbonate, counts of the nodules were made as given in table 2. The results show an increase of 336 per cent in the numbers of nodules as a result of liming, in spite of the fact that the soil was already well inoculated with the organisms. This agrees with Fellers’ observation, and while it demonstrates the correlation of nodulation of legumes on acid soils with the acidity, it does not establish the relation of cause and effect between them.
In order to differentiate more closely between the effect of soil reaction upon nodulation and the importance of calcium to nodulation, a study of the effect of calcium, as calcium chloride, on the viability of B. radicicola was made. For this a rather well isolated soil, mainly of residual formation from limestone, was collected from a timbered area. It was decidedly acid (pH 5.4), and sterile with reference to the soy bean organism. Sixty pots (3.5 inches in diameter) of this soil were set up, planted to soy beans, and given thorough inoculation with cultures of bacteria. One-half of these pots were given the additional treatment of a solution supplying 88 mg. of calcium chloride per pot, the calcium equivalent of 2,000 pounds of calcium carbonate per two million of soil. Eight additional pots without inoculation or calcium treatment were used as checks. The nodule counts were made after growth of five weeks.
Not a nodule had formed on a single plant in the untreated soils, and but three single nodules on as many plants in the calcium-treated soil, although a good growth of plants was obtained in all the pots. In order to test the soil for the presence of the organism, sixteen pots of each of the treated and untreated soil were immediately replanted with sprouted beans. No further inoculation or treatment was added. Again after five weeks of growth these were taken up and the nodules counted as before. The data are summarized in table 3.
The results of this trial emphasized the difficulty of establishing the soy bean organism within this soil by a single inoculation, and demonstrated that even though the organism did not exist in the untreated soil, it continued its existence in the calcium-treated soil despite no significant change in the soil reaction. This indicates, in substantiation of Scanlan’s findings, that the response by the organism must be due to effects by the calcium.
Since calcium seemed to favor nodulation, an attempt was made to determine whether or not this influence comes in consequence of its direct effect on the soil conditions, or of an indirect effect through the plant. Quartz sand was treated with hydrochloric acid for 3 hours, washed with tap water and then with distilled water until the test for chlorides was negative. The sand was dried, and heated at 3° C. for 48 hours for drying and for sterilization. Part of the sand was treated with calcium carbonate at the rate of ten thousand pounds per two million pounds of sand.
Sterilized soy beans, the progeny from a single plant, were germinated for 24 hours, planted in both the limed and unlimed sand, and then grown for 10 days. Seedlings from both the calcium-bearing and the calcium-deficient sands were washed free of adhering particles and transplanted into the respective halves of each of two flats of well prepared soils. One of these soils was a lime-deficient field soil and the other a neutral garden soil, both of which had grown well-inoculated soy beans during the three preceding seasons. Excellent growth resulted in both flats, and after 5 weeks the plants were removed and counts made of the nodules, with the results given in table 4.
On the lime-deficient soil the calcium-starved plants developed few nodules. In marked contrast to this, there were almost five times as many nodules within this soil in consequence of allowing the seedlings to grow in calcium-bearing sand and to transplant their needed calcium to the lime-deficient soil. On the neutral soil no differences in nodulation occurred as a result of either treatment to the seedlings.
In order to gain some additional information, the following chemical determinations were made: the calcium content of the bean seeds, of the 10-day old seedlings grown on the calcium-deficient sand, and of those grown on calcium-bearing sand, according to a modification of McCrudden’s method; the total dialysable base of both soils, by Bradfield’s method; and the total dialysable calcium in these soils by the same method. The summary of these analytical data is given also in table 4, with the omission of total dialysable base.
From the complete data in table 4 it will be seen that the soil on which good nodulation occurred, regardless of seedling treatment, had a pH of 7.8 and gave 24.07 mg. electro-dialysable calcium per 10 gm. of soil; while the soil which gave good inoculation only on calcium-treated seedlings had a pH of 5.5 and 11.78 mg. of electro-dialysable calcium per 10 gm. of soil.
The increased nodulation of the higher calcium-containing seedlings on the calcium-deficient soil demonstrates that the presence of calcium in the plant increases nodulation of soy beans on such a soil. On the other hand, the lack of difference in nodulation of the seedlings on the more calcium-sufficient soil indicates that either the presence of calcium in the plant does not affect nodulation on such a soil, or that if it does, the calcium-starved plants take calcium from the soil soon enough to offset the measurable effect in difference in nodulation.
The increase in calcium content of the seedlings grown on the acid-extracted sand over the calcium content of the bean seeds was due to the calcium that was carried back into the acid-extracted sand by the tap water with which the sand was washed. Although the calcium content of the seedlings grown on this extracted sand was more than expected from the analysis of the seeds, the actual amount contained was but little more than half that of the seedlings grown in the calcium-bearing sand. Improved methods on this point will probably intensify the results obtained.
After finding that the presence of calcium within the plant increased the nodulation of soy beans on an acid lime-deficient soil, a microchemical study of the seedlings was made to locate, if possible, any histological differences in the calcium-starved and calcium-fed seedlings. Parts from both stems and roots of each of these were collected at the age of 10 days, and prepared by the usual method for sectioning in paraffin. Micro-photographs of cross-sections of the stem taken near the plant crown are reproduced in figs. 1–4. These photographs are representative sections from more than forty slides. The differences shown in the figures were the same throughout the slides. The cell walls of the calcium-starved seedlings failed to retain their shape, and apparently gave way before the microtome blade, while the cells of the calcium-fed plants stood up under the treatment and gave distinctly better sections.
These differences were innate to the material, since the comparative sections of calcium-starved and calcium-fed seedlings were cut at the same time, and on the same microtome. The materials were fixed and processed as separate samples, but in duplicate of the same lot, so that variation in laboratory technique was reduced to a minimum. The usual macroscopic, or external differences in stems and roots in consequence of liberal and deficient calcium supply were noted before those of microscopic nature were studied. Miss Day emphasized the former for Pisum sativum in recent work, but she reported no significant differences in anatomical structure in these plant parts.
Figs. 1, 2.—Cross-section of stems of calcium-starved and calcium-bearing soy bean seedlings (10 days old): fig. 1, calcium-bearing, fig. 2, calcium-starved; X170.
Figs. 3, 4.—Cross-section of stems of calcium-starved and calcium-bearing soy bean seedlings (10 days old): fig. 3, calcium-bearing, fig. 4, calcium-starved; X750.
Both microchemical and staining methods for demonstrating calcium and pectate in micro-sections were employed in an attempt to discover any differences in the cell walls. Material from 10-day old seedlings is so minute in structure, however, that as yet it has been impossible to record micro-photographically and differences noted. Further work on this phase is being done, and it is expected that this observed histological difference between calcium-starved and calcium-fed soy bean seedlings can be intensified and substantiated by using seedlings more nearly deficient in calcium through growth on sand leached free of calcium with acid and distilled water, and by using seedlings of greater age to intensify their differences.
Summary
1. An experimental study was made of the effect of calcium on nodulation of soy beans on certain acid soils, with the hope of contributing to the knowledge of the role calcium plays in inoculation.
2. The divided root system of soy beans, grown in acid soil on one side and calcium-treated soil on the other, gave differences in nodule production to as great an extent as those produced when plants were grown wholly within these same soils. This indicates (1) that calcium plays some physiological role in favoring nodulation; and (2) that its effects are local or restricted in increasing the number of root infections, at least within the periods of time used in this experiment.
3. The addition of lime carbonate to an acid soil of pH 5.4, and already infected with legume organisms, gave a very marked increase in nodule production. It suggests that the effect of liming is not necessarily one of keeping alive the bacteria applied as inoculation, since in this case liming increased the number of nodules by organisms originally present in the soil. Evidently the lime carbonate exerted a physiological effect on the plants, and possibly on the organisms, to bring on the greater nodule production.
4. The addition of small amounts of calcium chloride to an acid soil (pH 5.5) increased the viability of the legume organism, B. radicicola, of soy beans, applied to the soil by pure cultures, and stimulated nodulation of the host plant.
5. Calcium taken up by the plant in its early growth influenced nodulation, since there was a difference in the nodulation of 10-day old calcium-starved and calcium-bearing seedlings when replanted to an already well inoculated lime-deficient soil of pH 5.4.
6. This functioning of calcium within or through the plant to produce increased nodulation may have a fundamental histological or physiological basis in the plant, since running parallel with the effect on nodulation by calcium given the seedlings, there is a distinct difference in the plant cell wall structure suggested by differences in ease of obtaining micro-sections of the 10-day old calcium-starved and calcium-bearing soy bean seedlings.
