Popular Science Monthly/Volume 38/February 1891/Progress in Agricultural Science
By Dr. MANLY MILES.
THE progress recently made in tracing the interdependent relations of living organisms is clearing up some of the obscure problems in the nutrition of plants that have a direct bearing on the processes of evolution and the applications of science in agriculture.
Since the discovery of the composition of the atmosphere, the problem of the sources of the nitrogen of vegetation has given rise to a wider range of experimental investigation and discussion than any other in vegetable physiology. The evidence appeared to be conclusive as to its source in certain families, including the cereals, while the larger supplies of nitrogen obtained by leguminous plants were not fully accounted for.
The experiments of Boussingault, in France, and the elaborate investigations at Rothamsted, in England, seemed to show that atmospheric nitrogen is not appropriated, to any extent, by the leaves of plants, and that the soil is the main or sole source of the nitrogen of vegetation.
Wheat and barley were the leading cereals under experiment, as field crops, at Rothamsted; and it was found that, while they contained less nitrogen in their composition than leguminous crops, they were specifically benefited by nitrogenous manures. On the other hand, leguminous crops, which obtained larger supplies of nitrogen from the soil, were not benefited by nitrogenous manures, and they grew luxuriantly on soils that did not furnish the cereals with their comparatively limited supplies of nitrogen.
These apparently paradoxical results are now explained, in part at least, by investigations made within the past five years by Hellriegel and Willfarth, Ward, Prazmouski, and others, which have been fully verified by experiments at Rothamsted which are still in progress. Former experiments showed that leguminous plants obtained nitrogen from some source, or under conditions that were not available for the nutrition of the cereals, and it was evidently not obtained from the atmosphere.
It was suggested that the tubercles observed on the roots of leguminous plants had a direct relation to the appropriation of nitrogen; but most observers looked upon them as abnormal and of no physiological significance.
The latest investigations, however, show, beyond the shadow of a doubt, that these "tubercles" or "nodules" are the results of infection by microbes, and that "the relation between the roots and the bacterial organisms is a true symbiotic one, each developing more vigorously at the expense of the other," and that free nitrogen is appropriated by the microbes.
In 1883 Hellriegel began experiments with leguminous plants in pots of washed quartz sand, to which no nitrogen was added. Marked differences were observed in the growth of the plants under these conditions, but tubercles were found on the roots of the plants that made the best growth, while they were ab sent in other cases. He was then led to attempt the production of the root-tubercles by seeding or inoculating sterilized sand with a water-extract of a soil in which leguminous plants were growing. To some of the pots, in which peas and vetches were planted, from twenty-five c. c. to fifty c. c. of a water-extract of a fertile soil were added. When this soil-extract was not sterilized, there was a luxuriant growth of the plants in the pots to which it was applied, with abundant formation of root-nodules; but when the soil-extract was sterilized, this result was not obtained.
This soil-extract, however, was without effect on lupines and some other plants; but when the lupine pots were inoculated with an extract of a soil in which lupines were growing, the plants made a luxuriant growth, and root-tubercles were abundantly developed. In all cases the nitrogen supply of the plants was coincident with the development of root-tubercles, that were produced by inoculation with the extract of a fertile soil.
In 1888 a preliminary series of experiments, on the same lines, were begun at Rothamsted by Sir John B. Lawes and Prof. J. H. Gilbert; and in 1889 they were continued, on a more extended scale, with modified conditions suggested by the results of the preceding year. Their first experiments were made with peas, blue lupines, and yellow lupines, in pots seven inches high and about six inches in diameter. For our present purpose we need only call attention to the experiments in 1888 with peas.
Pots 1, 2, and 3 were filled with a washed yellow sand, to which was added 0*5 per cent of the ash of pea plants to furnish the required mineral constituents. Pot 4 was filled with a rich garden soil. Distilled water was used for watering the plants, and no other application was made to pot 1. Care was taken to determine the nitrogen of the soils, and of the seeds planted, which we need not describe in detail.
An extract of a rich garden soil was prepared by shaking in a stoppered bottle one part of soil with five parts of distilled water, and, after the coarser particles had subsided, twenty-five c. c. of the liquid was applied to each of pots 2 and 3. A chemical analysis of this soil-extract showed that the amount of plant food contained in it was so small that it could be safely neglected as an element of plant growth, and that its effect must be attributed solely to the soil microbes it contained.
There was a considerable development of roots in the upper part of pot 1, and a number of root-tubercles were formed, owing to the fact, as proved by subsequent experiments, that the sand was not sterilized before planting the peas. The roots in pots 2 and 3, inoculated with soil-extract, were more abundant than in pot 1, and the root-tubercles were decidedly more numerous and frequently in clusters. The above-ground growth was more luxuriant in pots 2 and 3 than in pot 1, and "in the total vegetable matter there was in pot 2 more than twice, and in pot 3 nearly twice as much, nitrogen as in pot 1 without soil-extract."
A comparison of the total nitrogen in the soil and plants at the close of the experiment with the original nitrogen in the soil and seeds showed that "in pot 1, with the impure and not sterilized sand, but without soil-extract, there was more than three times as much nitrogen in the products as in the soil and seed; in pot 2, with soil-extract, there was about five times as much; and in pot 3, also with soil-extract, there was more than four times as much." There was very little difference in the amount of nitrogen in the soils at the beginning and the close of the experiments, and, neglecting this, it appears that "the nitrogen in the substance grown was, in pot 1, nine and one half-fold; in pot 2, nearly eighteenfold; and in pot 3, nearly fifteen-fold that supplied in the seed."
In 1889 similar experiments were made with peas, red clover, vetches, blue lupines, yellow lupines, and lucern. For the lupines and lucern glazed earthenware pots, six inches in diameter and fifteen inches deep, were provided, and for the other plants the same pots were used as in 1888.
"The sand used was a rather coarse white quartz sand, from which the coarser and the finer portions were removed by sifting, and more of the finer by washing and decantation, first in well, and afterward in distilled water.
"In each case the sand was mixed with 0·1 per cent of the plant-ash, and 0·1 per cent of calcium carbonate." The prepared sand was sterilized by keeping it for several days at a temperature of nearly 100° C. in a water-bath.
"There were four pots of each description of plant." Of the peas, clover, vetches, and lucern there was one pot of each of the prepared quartz sand without inoculation with soil-extract; two pots of the prepared quartz sand inoculated with the microbes of a garden-soil extract; and one pot of garden soil itself. Of the blue and the yellow lupines there was one pot of each of the prepared, but not inoculated, quartz sand; two pots of the prepared quartz sand inoculated with an extract of a soil from a field where lupines were growing; and one pot of the lupine soil itself, to which was added 0*01 per cent of lupine plant-ash.
"The soil-extracts were in all cases added on July 9th, before the sowing of the seed; twenty-five c. c. in the case of the peas, the vetches, and clover, and fifty c. c. in that of the lupines and lucern. The seeds, carefully selected and weighed, as in 1888, were sown on July 10th—that is, about four weeks earlier than in the previous year, but still not as early as was desirable."
Ten seeds of clover, three of the lupines, and two each of the peas, vetches, and lucern, were put in each pot. "No analytical results of the experiments of 1889 are as yet available," and we can only notice the relative growth of the plants under the different conditions. The pots of clover and lucern were left for a second year's growth, and their roots could not, therefore, be examined. A photograph of the four pots of peas was made October 22d (a copy of which is given in Fig. 1), and the plants were taken up for examination October 23d and 24th.
The relative growth and development of the plants in the different pots are clearly shown in the photograph. "Unlike the result obtained in pot 1 in 1888, with the impure and non-sterilized sand, the plants in the purer and sterilized quartz sand (pot 1, Fig. 1) show extremely limited growth." The plants in pots 2 and 3, inoculated with a soil-extract containing microbes, began to show enhanced growth, when compared with the plants in pot 1, before the end of July. Finally, the plants in pot 1 were eight inches and a quarter, and eight inches and a half high; in pot 2, fourteen, and fifty inches and a half; in pot 3, fifty-two inches and a half, and fifty inches and a half; while in the garden soil, in pot
Fig. 3.—Yellow Lupines.
4, they made a somewhat less extended growth than those in pots 2 and 3 in a sterile sand inoculated with soil-microbes. It should be remarked, however, that "the plants in pot 4 were more vigorous, and, while they flowered and seeded, neither of those in pots 2 or 3 did so."
A photograph of the vetches was taken October 25th (a copy of which is given in Fig. 2), and they were harvested for examination the following day. The plants in pot 9, which was not inoculated with soil microbes, were eleven inches and a quarter, and ten inches and a half high; those in pot 10, in a sterile but inoculated quartz sand, were fifty-two inches and a half, and sixty-seven inches high; those in the duplicate pot 11 were sixtyone inches and a half, and fifty-one inches high; while those in pot 12, in a garden soil, were only fifty-three, and thirty-six inches high. As in the case with the peas, the plants in pot 12 flowered and seeded, while those in pots 10 and 11 did not.
Most of the blue lupines, as in 1888, failed to grow. After some reseeding two plants of yellow lupines were grown in each pot. Their relative development, November 29th, is shown in the photograph copied in Fig. 3.
The plants in pot 17, in the sterilized sand not inoculated with soil-microbes, were one inch and a half, and two inches high, "scarcely showing over the rim of the pot"; those in pot 18, in the inoculated quartz sand, measured twenty-four, and eighteen inches, "both spreading much beyond the width of the pot"; in pot 19, also in inoculated quartz sand, one plant was more than two feet and the other but little more than eight inches high; while in pot 20, in a soil from a field where lupines were growing, one plant was but sixteen inches and the other only eighteen inches high, and both less branching than those in pots 18 and 19.
"Unlike the peas and vetches, the yellow lupines, with soilextract seeding (pots 18 and 19, Fig. 3), flowered and podded freely. One plant in pot 18 had nine small pods, and one in pot 19 four large and three small ones. There were also in pot 20, with lupine soil, on one plant five pods and on the other six. Thus, in the quartz sand with lupine soil-extract seeding, the plants not only produced a great deal more vegetable matter than those in the lupine sand itself, but they as freely flowered and seeded." This was probably owing to the less porosity of the lupine soil when watered in the pot.
The root development and root-tubercles in the different pots may be briefly described as follows: In pots 1 of the peas, 9 of the vetches, and 17 of the lupines, no root-tubercles could he found, and the roots were decidedly less developed than in the inoculated pots 2, 3, 10, 11, 18, and 19.
In pot 4 of the peas in the garden soil the roots were abundant, but the root-tubercles were not as numerous as in pots 2 and 3' In pot 12 of the vetches, also with garden soil, the root-tubercles were less numerous, and the roots were not as well developed as in pots 10 and 11. In pot 20 of the yellow lupines, in a soil from a field where lupines were growing, the root-tubercles were not as numerous, and there was less root development than in pots 18 and 19.
In their "preliminary notice" of the results of these experiments, Sir J. B. Lawes and Prof. J. H. Gilbert say: "It will be admitted that the results so far brought forward are abundantly confirmatory of those obtained by Hellriegel; and that the fact of the fixation of free nitrogen in the growth of Leguminosæ under the influence of microbe seeding of the soil and of the resulting nodule formation on the roots may be considered as fully established."
The results obtained by the inoculation of the prepared quartz sand with the microbes of a fertile soil, or of one in which lupines were growing, as shown in the increased growth of the plants in pots 2, 3, 10, 11, 18, and 19, when compared with those in pots 1, 9, and 17, which were not inoculated, are striking; but a comparison of the plants in the inoculated pots with those in pots 4 and 12 in a garden soil, and pot 20 in a "lupine soil," furnish still more significant indications of the futility of purely chemical considerations in discussing the nutritive processes of plants and their relations to the soil. The peas and vetches in a rich garden soil flowered and seeded, but the plants were not as large, and the root-tubercles were not as numerous, as in the sterile quartz sand inoculated with microbes from a fertile soil; and the lupines made a better growth in the inoculated quartz sand than in soil from a lupine field.
The biological factors concerned in the elaboration of plant food seem to be quite as important as the chemical elements provided in the soil itself; and a revision of the accepted theories of plant growth, and the relations of soils to their processes of nutrition, is evidently needed from this standpoint.
It should be remarked, however, that the root-tubercles produced by microbes are not confined to the Leguminosæ, as they have, in fact, been observed in several natural orders of plants. Moreover, there are indications that several varieties or species of symbiont microbes are concerned in the production of tubercles on the roots of leguminous plants, and it is probable that each species has its own favored form.
Hellriegel failed to grow lupines in a nitrogen-free soil inoculated with a fertile soil-extract; but, when the inoculation was made with an extract of a sandy soil in which lupines were growing, a luxuriant growth was obtained.
In the Rothamsted experiments on land where red clover had been grown repeatedly, and its yield of nitrogen was reduced to but 22 pounds per acre, vetches, on an average for three years, obtained 120 pounds of nitrogen per acre; lucern yielded as high as 340 pounds, and made an average for six years of 150 pounds of nitrogen per acre; and Bokhara clover yielded crops of 130 and 145 pounds of nitrogen per acre. On land where beans had been grown almost continuously for thirty-two years, and had practically failed "to grow, their yield of nitrogen per acre having been reduced to about 16 pounds on the unmanured plot, and less than 27 pounds on the plot with mineral manure but without nitrogen, very large crops of red clover were grown containing about 300 pounds of nitrogen per acre.
If attention is directed exclusively to the root-tubercles of plants and the roots to which they are attached, it is difficult to understand the manner in which the free nitrogen of the air permeating the soil is made available by the microbes for the nutrition of the more highly organized hosts with which they are associated; but the problem is simplified when we take into consideration the interdependent relations of living organisms arising from their habits, and different requirements in their processes of nutrition.
The influence of cats on the growing of clover seed, as pointed out by Darwin, furnishes a good illustration of dependent relations in the struggle for existence. Cats prey on field-mice that destroy the nests of humble-bees, and the bees are known to be important factors in the fertilization of the clover plant. Quite as marked relations of dependence have been observed among microbes, but the sequence of organisms may be brought about by a different process.
In the ordinary processes of putrefaction we find an orderly succession of living organisms engaged in the work of disintegration in which relations of dependence are clearly manifest. The microbes that initiate the putrefactive process appropriate the materials required for their own growth and multiplication, and the residual mass soon becomes better fitted for the nutrition of other species which succeed them. These are, for similar reasons, succeeded by other forms that are better adapted to the changed conditions, and a series of organisms, of diverse habits, is required to reduce the organic compounds to their elements. Each species performs a specific rôle, "the earlier ones preparing the pabulum, or altering the surrounding medium, so as to render it highly favorable to a succeeding form" while their own activities are checked by the changed conditions.
The term symbiosis, as now used, is limited to the immediate and direct relations of certain species that are mutually beneficial in their processes of nutrition and growth; but this interdependence of vital activities and interests, in many cases at least, seems to extend to more remote relations through a series of organisms, each of which may have an influence on the well-being of the others. An increased growth of clover in a nitrogen-free soil has been obtained by seeding it with an extract from a root-crop soil; and this, in connection with the facts already presented, is certainly suggestive in explaining the advantages arising from crop rotations.
The micro-organisms that are found in great variety in soils must have an important influence on the processes of metabolism that are constantly taking place in the soil itself; and the results of their activities, which are not limited to processes of putrefaction and nitrification, can not be measured solely by the amount of nutritive materials appropriated. In my own experiments with soil-microbes they have proved their ability to take their required supplies of lime and potash from solid fragments of gypsum and feldspar, and even from the glass tubes in which cultures were made, which were deeply etched by their action.
The roots of plants undoubtedly aid in determining conditions of the soil that favor the vital activities of certain microbes, and interfere with the well-being of others of different habits; and the plants, in their turn, are presumably benefited by the activities of the microbes best adapted to the prescribed conditions. In the struggle for existence the dominance of these favored forms can not, however, be indefinitely maintained. The roots of one species of plant and their associated microbes, in appropriating their required supplies of nutritive materials, induce a metabolism of the soil that, sooner or later, renders it better fitted for other species of plants and other microbe associates; and these, in their turn, prepare the way for species of still different requirements in their processes of nutrition.
Soil metabolism, and the involved liberation or elaboration of plant food, will thus be promoted by a succession of plants of different habits of growth, each with its associated microbes; and the elements of fertility stored in, or permeating the soil, must, under such conditions, be more completely utilized.
It is practically misleading and inaccurate to say that leguminous plants appropriate the free nitrogen of the atmosphere. The evidence clearly shows that the soil-microbes which find favorable conditions for the exercise of their vital activities in the vicinity of, or in contact with, the roots of leguminous plants, are able to make use of the free nitrogen that permeates the soil, and that it is thus made available as combined nitrogen in the nutrition of the higher chlorophyl-bearing leguminous plants. The latest investigations are, therefore, strictly in accordance with the earlier experiments by Boussingault, and at Rothamsted, in showing that the soil is the source of the nitrogen of plants, and we must look to soil conditions as essential factors in determining the vital activities of the microbes that bring free nitrogen into the combined form that is available for the nutrition of the higher plants.
It must be admitted that red clover appropriates nitrogen that has been prepared for it from the free nitrogen of the soil through the agency of its symbiont microbes, but it is well known that it will not grow for many years in succession on the same land, and other crops must be introduced to put the soil in suitable condition for growing it again. The cereals with their different requirements, through their reactions upon the soil, which are undoubtedly aided by their associated microbes, and even the roots and companion microbes of other leguminous species, may have a direct influence in determining conditions of the soil that favor the nutritive processes of the clover roots and their specific symbiont microbes.
The interdependent biological relations of different farm-crops, and of the soil-microbes that find favorable nutritive conditions in the vicinity of their roots, appear to be quite as important factors in farm economy as the chemical composition of soils and crops, and the conditions of the soil that influence these relations are of great practical interest.
In the light of our present knowledge, it must be obvious that the applications of science to agriculture, so far as crop-growing is concerned, will be best promoted by investigations relating to the life history of these microbes, and their immediate and remote relations to the roots of plants of different species, and to processes of metabolism in the soil under different conditions.
The suggestion made by Dr. M. T. Masters, in his Plant Life on the Farm, that in the future the farmer may be able to apply the ferment-producing germs to his soil, to promote the growth of his crops, with greater advantage than he now derives from the application of chemical manures, seems to be fully warranted by the results of recent experiments; and it may be that the breeding of beneficial microbes may come to be of as great practical interest to the farmer as the breeding of yeast now is in the manufacture of beer.
We must not, however, be misled by the plausible inferences that may be made from the evidence presented in regard to this recently discovered source of nitrogen supply to leguminous plants under special conditions. It is not safe to assume that the nitrogen removed from the soil by crops and by drainage, or otherwise, is fully restored by corresponding amounts derived from free nitrogen through the agency of microbes, or that this is the sole or even the main source of the nitrogen of leguminous crops on average soils.
The Rothamsted experiments show that the previous accumulations of combined nitrogen in the soil must be the source of a large proportion of the nitrogen of leguminous crops, and that the frequent repetition of such crops does not prevent an appreciable diminution of the nitrogen of the surface soil.
The evidence we now have seems to indicate that, under ordinary conditions of farm practice, the microbes concerned in working up the accumulated stores of combined nitrogen in the soil are quite as significant factors in the nutrition of leguminous plants as their symbiont microbes that appropriate free nitrogen; and the conditions of soils and plants that determine the exercise of these diverse biological activities, in one direction or the other, present a promising field for future investigation. With every advance in knowledge there is increasing evidence that the transformations of matter and energy taking place in the normal processes of living organisms are so exceedingly complex that they can not be expressed or defined in simple formulæ relating to a single department of science, and this fact must be recognized if any real progress is made in solving the problems presented in the applications of science to agriculture.