Popular Science Monthly/Volume 63/August 1903/Bacteria in Modern Economic Agriculture

BACTERIA IN MODERN ECONOMIC AGRICULTURE.

By ALBERT SCHNEIDER, M.D., Ph.D.,

UNIVERSITY OF CALIFORNIA.

CROPS have been cultivated for thousands of years, and from the very first the agriculturist has endeavored to get from the soil a maximum return for a minimum of labor expended. Yet scientific progress in soil fertilization and crop improvement has been exceedingly slow until very recent years. Now scientific methods are beginning to be applied not only to crop and soil improvement, but also to the allied branches horticulture, arboriculture, the dairying industries, etc. That infant science, bacteriology, in particular, gives promise of inestimable value.

Some microorganisms work in the interests of the agriculturist, while others work decidedly antagonistically to all desirable interests. Much efficient work has been done in the eradication of disease-producing organisms, and the farmer is given detailed and specific instructions how to combat organisms which are hurtful to crops, as the rusts, smuts, rotting bacteria, etc. Attempts have been made to utilize even essentially harmful organisms in working useful results, as in the extermination of chintz bugs, potato beetles, plant lice; the extermination of rats, mice and other undesirable higher animals, by means of germs which are capable of transmitting fatal diseases to the animals referred to. The department of entomology at Washington has done some very effective work of this nature in exterminating insect pests of trees and plants.

The farmer's great future problem will be to determine what beneficial organisms may be pressed into his service and what noxious organisms may be suppressed, and how such measures may be carried out most expeditiously and with the best results. There is perhaps no problem of greater interest or none which gives promise of greater beneficial results than the one pertaining to the bacteria found in the root tubercles or nodules of leguminous plants (bean family). Without entering into the history of the discovery of these organisms or the part they play in the economy of the host plant, or even dwelling upon the many points still in dispute or under discussion, I shall describe briefly some of the recent attempts at making practical agricultural use of these organisms in Europe and in this country, and outline briefly a plan of future research, pointing out the additional practical possibilities which may be anticipated with reasonable certainty, based upon results already obtained.

It has been proved experimentally that not only do the bacteria (rhizobia) of leguminous root tubercles have the power of assimilating or chemically binding the free nitrogen of the air, but also other soil bacteria and various simple algae and hyphal fungi. Undoubtedly the true ecological significance of these free nitrogen-assimilating functions of these organisms is to neutralize, balance or equalize the work of nitrifying and denitrifying (nitrogen-liberating) bacteria, which are very plentiful and widely distributed. More specifically considered, the organisms referred to chemically bind the free nitrogen of the air, forming nitrogenous compounds which may be taken up and assimilated by various plants. In the case of leguminous plants these nitrogen-assimilating bacteria (rhizobia) live within the roots (root tubercles) and supply the host directly with the enriching nitrogenous food compounds formed; in other instances the nitrogen-assimilating organisms live in the soil and the various higher plants as corn, wheat, etc., take up the compounds formed and deposited in the soil without being in actual biologic (symbiotic) association with them. These discoveries have suggested to the scientists interested in agriculture various possible improvements for increasing the yield of crops. Extensive and interesting experiments have already been made, and some noteworthy results have been obtained and, in other instances, investigations are under way which give promise of final useful results. Several processes for inoculating the soil or seeds with beneficial bacteria have been patented and, remarkable as it may seem, the slow, plodding German investigator is the first in the field with patent claims and 'practical' plans for utilizing bacteria in the interests of the farmer or the tiller of the soil.

The history of the discovery of the free-nitrogen-assimilating bacteria found in the root tubercles of leguminous plants is familiar to all botanists, but the general reader of science requires some detailed explanations and some specific statements regarding the subject in order that he may have reasonably clear ideas concerning the practical possibilities and probabilities of bacteria in modern agriculture. These necessary explanations will be given as we proceed.

The first to suggest a plan for practically utilizing root bacteria (rhizobia) and to secure letters patent for the process in Germany and in the United States were Nobbe and Hiltner, of Tharand, Germany. Since the wording of the specifications for a patent are required to be simple and intelligible to persons of ordinary technical learning, the scheme can best be presented by simply quoting the specifications. The following is the specification which forms part of letters patent No. 570,873 granted Nobbe and Hiltner in the United States, November 3, 1890:

Be it known that we, Friedrich Nobbe and Lorenz Hiltner of Tharand, pared for transport in fluid cultures. The colonies in the agar-gelatin are dis-inoculation of soil for the cultivation of leguminous plants and we do hereby declare the following to be a clear and exact description of the invention:

Since the function of the root nodules or tubercles of the Leguminosæ in the supply of nitrogen to these plants has been discovered by the fundamental researches of Hellriegel we have been working on this problem for a number of years, and have examined more especially the bacteria in said nodules or tubercles (first identified and isolated in cultures by Beyerinck) in order to determine the relationship between the bacteria and the reception of the free uncombined nitrogen of the air in the soil by the various kinds of Leguminosæ. These researches have resulted, in the first place, in the confirmation of the at that time still disputed fact that the introduction of these bacteria into the soil produces, without exception, in soil free from these bacteria, the root nodules or tubercles on the plants in question having papilionaceous flowers and enables these plants to assimilate the free nitrogen. A soil inoculated with these bacteria, even when it contains absolutely no nitrogen in an assimilable form so that the plants without any such inoculation would starve, enables the Leguminosa? to produce as rich a yield of dry material and nitrogen as they would otherwise produce if grown in a richly manured soil containing much assimilable nitrogen.

It has been established by us as an entirely new fact that the tubercle bacteria of the various Papilionacea? are of full strength (i. e., in the production of efficient nodules or tubercles) only in that species (of leguminous plant) from whose root tubercles they weie themselves obtained. With closely allied species they are of less strength and with systematically different species they are useless or inactive. Bacteria cultures from pea roots, for example, are quite useless for Robinia plants, while they promote the growth of peas in a very energetic manner, and that of the allied vetches somewhat more feebly; on the other hand, the bacteria from Robinia nodules or tubercles are quite efficient with Robinia plants, but in a lesser degree with Colutea, and are absolutely useless with peas.

At first sight it might possibly be thought that the production, transport, and distribution of such large masses of crude inoculating material as would appear to be necessary for the sufficient impregnation or treatment of large areas of land would be very difficult and costly, and therefore not practicable, while there would also be the danger that in the crude inoculating material, besides the active bacteria of the root nodules or tubercles, there would be carried from field to field, at the same time, microscopic organisms which would be detrimental to growth and would interfere more or less with the action of the inoculating material. Our process is, however, free from any such objections as those above mentioned, inasmuch as bacteria bred in quantities directly from the nodules or tubercles of the Leguminosæ in pure cultures are used as the inoculating material. Farmers are, therefore, placed in a position to make land, which was unfruitful by reason of its lack of nitrogen, fit for the cultivation of fodder and other plants belonging to the order of the Leguminosæ and to insure and increase the yield of soil. This inoculation has, moreover, an essential practical bearing in connection with the so-called 'green manuring'

Our process of inoculating land with tubercle bacteria is to be carried out as follows: The active bacteria to promote the growth of the Leguminosæ are delivered to the farmer in glass tubes or other suitable packages, which contain pure colonies thereof in agar-gelatin having suitable additions for propagating such bacteria, for instance sugar, asparagin and an aqueous extract of the green substance of the Leguminosæ. In some cases the bacteria can also be prepared for transport in fluid cultures. The colonies in the agar-gelatin are distributed in water, together with the agar-gelatin, by the user (after removing the stopper) in the proportion, for example, of the contents of one glass tube to from one to three liters of water, which is previously mixed with a suitable material, such as an aqueous extract of the green substance of Leguminosæ sugar asparagin, for propagating the bacteria. This propagating material is delivered with the bacteria tubes. Preferably the glass tube is laid in the water until the agar-gelatine is dissolved.

Immediately before sowing, the whole of the emulsion prepared as above mentioned is poured over the seeds. The amount of water added for each kind of seed is so proportioned that after the seeds have been thoroughly and uniformly moistened by a careful working over by hand, a surplus of liquid will still remain. For clover-seed, for example, for twenty kilograms of seed the admixture of three liters of water with the contents of three glasses of inoculating material (each glass containing, for instance, three cubic centimeters agar-gelatin with pure cultures) is sufficient. For more bulky seeds a somewhat larger amount of water is required. A sufficient quantity of dry sand or earth from the field to be sown is then gradually added with careful stirring, until the body of seed is in a suitable condition for sowing by hand or by means of a sowing machine.

This microbic (rhizobic) soil fertilizer for leguminous plants was given the commercial name 'nitragin,' and its efficiency was quite carefully and extensively tested and commented upon by European and American investigators. The consensus of opinion seemed to be that it was of doubtful practical utility for agricultural purposes. Some authorities maintained that it was of unquestionable value in virgin soil. In rich and otherwise favorable soil conditions it is of only slight value. It is maintained that nitragin aids very materially in developing and ripening the fruit. As becomes evident from careful consideration, the value of this microbic fertilizer depends upon whether it will cause an increased development in the number and size of root tubercles over and above those which would develop without the presence of this artificial aid. If the soil is already well supplied with rhizobia or root tubercle bacteria, as soil naturally would be if the leguminous plants under consideration had been grown in it for one or more seasons, nitragin would in all probability be of little or no value. In any case the anticipated practical results have not been realized, as I am informed by a letter from Victor Koeehl & Co. of New York City which states that 'nitragin is withdrawn from the market and is no longer manufactured.'

A second and later improvement in the method of inoculating seeds with root tubercle bacteria (rhizobia) is given by Hartleb in the following specifications forming part of letters patent No. 674,765, granted May 21, 1901, at Washington, D. C.:

Be it known that I, Richard Hartleb, a citizen of Germany, residing at the Botanisches Institute der Hochschule, Aachen, in the Empire of Germany, have invented a certain new and useful method of inoculating seeds with microorganisms, (for which I have applied for a patent in Germany, dated February 23, 1900) of which the following is a specification:

This invention relates to a method of inoculating seeds with microorganisms. For this purpose the seeds in a suitable container are covered with pure water, so that they are mechanically cleaned, and the damaged or dead seeds float to the surface of the water. The water and the impurities are then poured off and the cleaned seeds are left in the water until they begin to swell, whereupon there is a loosening of the external husk of the seed and an increase in the volume of the grain, so that the seed offers an increased surface for the microorganisms and the latter obtain easy access, owing to the loosening of the husk. The seed thus prepared is sown directly without admixture of any other substance.

The application of this method is to the inoculation of seeds with bakteroids of the microorganisms of the Leguminosæ. Very shortly after the seed has become imbedded in the soil nodule (root tubercle) formation begins. The danger of killing the organisms for the inoculation by harmful soil influences is effectively obviated, owing to the fact that these organisms in consequence of the rapid germination quickly become active. On the other hand, this danger of damage or death is always present in a seed which has been merely inoculated with the liquid and has not been allowed to swell therein, so that it is a long time in germinating.

Although not specifically stated in the above specification, it is evident that the Hartleb process is a method of applying pure rhizobia cultures to seeds of leguminous plants only. Whether the method offers any advantages over the method of Nobbe and Hiltner is questionable. In any case it would prove practically advantageous only under the conditions referred to under the discussion of nitragin. Although the method has been freely discussed and experimented upon in Germany, I am not aware that the fertilizer is on the market, certainly not in the United States.

There is on the market a third patented germ or microbic soil fertilizer of German origin known as 'alinit.' It consists essentially of a pure culture of the soil bacillus known as Bacillus Ellenbachiensis alpha or Bacillus Ellenbachiensis Caron. The germ was first brought to the attention of agriculturists by Caron, a land owner of Germany, who first isolated it and called attention to the fact that it had the power of chemically binding the free nitrogen of the air. The microbe is undoubtedly closely related to B. megatherium and perhaps also to B. anthracis. According to some authorities it is especially concerned in assimilating free nitrogen for gramineous plants (grass family, Gramineæ). If this is true it may prove of great value to grain growers.

The commercial alinit is a dry pulverulent substance of a yellowish gray color, with about 10 per cent, moisture and 2.5 per cent, nitrogen. It is evidently prepared by mixing spore-bearing pure cultures of the bacillus of Caron with a base of starch and albumen. It is used to inoculate soil either by spreading it broadcast or by sowing or otherwise planting it with the seeds. It is not a nodule or root tubercle-forming organism and does not enter into intimate symbiotic or biologic relationship with plants. Its work is simply that of binding free nitrogen, forming nitrogenous compounds which enrich the soil, thus increasing the yield of any crop benefited by such compounds. Whether alinit binds free nitrogen more actively in the presence of gramineous plants must be more accurately determined by experiment.

In 1892, through a suggestion by Professor Conway MacMillan, state botanist of Minnesota, the writer conceived the idea of modifying leguminous tubercle bacteria by special culture methods so as to induce them to develop in or upon the roots of gramineous plants, as corn, wheat, oats, rye, and barley. Investigations in this direction were begun at the Illinois Experiment Station at Champaign in 1893, under the direction of Dr. T. J. Burrill. The time granted for experimenting was much too brief for obtaining any definite results. At that time comparatively little was known of nodule bacteria (rhizobia) in artificial culture media and most of the time allotted was consumed in making cultures or attempting to make cultures of the rhizobia of different species of leguminous plants. No field experiments were attempted, but some laboratory observations were made by inoculating sprouting corn grown in vessels filled with sterile sandy soil with pure cultures of rhizobia grown upon corn extract agar media, the supposition being that the corn extract would produce the desirable changes in the organisms. After a few weeks the roots of the young inoculated corn plants were examined microscopically to determine if the presumably modified microbes showed any tendency to develop in or on the root cells. In some instances numerous microbes were found in the hair cells (trichomes) of terminal rootlets and in epidermal cells and cells in apical areas, particularly at the points of secondary root formation. While it was not experimentally proved that the microbes present were rhizobia, it is highly probable that they were, as the examination of control plants not inoculated, also grown in sterile sandy soil, showed the absence of germs in root tissues and root trichomes. It was apparent that the inoculated corn plants were thriftier and of a deeper green than the control plants or those not inoculated. Though the results are meager and far from conclusive, yet the experiments pointed toward final, more positive results. The experiment station research was now terminated, and other work kept the writer from again taking up this line of research extensively until early in 1902. At this time the investigations were begun in the bacteriological laboratory of the Northwestern University School of Pharmacy at Chicago. Pure cultures of the rhizobia of white clover (Melilotus alba) were grown upon corn extract with agar. Again considerable difficulty was encountered because of lack of information regarding the behavior of this particular variety or species of rhizobium in artificial culture media. It was not until the early part of July that definite and satisfactory conclusions were reached regarding the identity of this organism in the culture media indicated. The rhizobia were now transferred to fresh corn extract media from time to time for about six weeks; in order to effect the desired adaptive changes in the microbes. Some preliminary field experiments were carried on at a farm near Fairbury, Illinois. Plots of stubble ground were selected in which oats had been grown during the season. The ground was ploughed and harrowed repeatedly. Each plot of ground was duplicated for control purposes. On August 10 the plots were planted with white dent corn mixed with the rhizobia cultures and a small quantity of water. The seeds germinated uniformly with no appreciable differences in the various plots. Subsequent growth was carefully observed for a period of four weeks. No very marked difference was noticeable. The corn treated with rhizobia grown in neutral corn-extract agar seemed to thrive somewhat better than the rest, but the difference was not sufficiently marked to be noteworthy. The corn treated with acid agar corn-extract rhizobia showed no apparent improvement over the normal or untreated corn. The same could be said of the corn treated with crushed nodules of sweet clover. Opportunity did not present itself for making a comparative microscopic examination of the roots of the corn of the various plots. Oats was also experimented upon, but with no marked results. This in brief is the outline of the experiments of 1903 and, although no satisfactory results were obtained, the preponderance of experimental evidences again seemed to point to ultimate success. In fact so sanguine had the writer become of early marked success that he made application for letters patent for the process, but the application in the form in which it was presented was rejected on the patents by Nobbe and Hiltner and by Hartleb. The specifications filed September 29, 1903, read in part as follows:

The invention relates to the process of fertilizing the soil and increasing the yield of crops by means of specially modified pure cultures of the microbes or organisms found in the soil and in the root nodules or tubercles of plants belonging to the leguminosæ or bean family, and has for its object to render the process more effective, much cheaper and practically without the expenditure of additional labor. The living microbic fertilizer, consisting of the above mentioned modified organisms is applied by simply mixing a small quantity of the culture with the seeds just prior to planting, and introducing it into the soil simultaneously with the seed. With the germination of the seed thus treated, the modified microbes will also begin to multiply and appropriate for the use of the plant, the free nitrogen of the air.

In spite of the wonderful opportunities for the dissemination of learning, the exchange of scientific thought and plans of research and the efficient modern laboratory equipment, scientific progress is slow. To illustrate, the observations and experiments which led to the discovery of the root nodule bacteria were begun about 1863 by Hellriegel in Germany and Lawes and Gilbert in England. The nodule microbe was not discovered or recognized until 1886. At the present time we are just beginning to become scientifically familiar with the microbe and are undertaking experiments with a view to practical useful application of this microbe. In consideration of these facts it need not appear surprising that conclusive results should not, at the time, have been obtained in the line of research indicated. It is of course understood that any scientific research deserving of the name must be founded upon reasonable and sound principles. The entire experimental plan must be in harmony with the highest and best results already obtained. The following are the essential and important points for consideration and upon which the research work indicated is to be based:

1. Do rhizobia (nodule bacteria of leguminous plants) assimilate free nitrogen in artificial culture media or when not symbiotically associated with leguminous plants? Based upon the results of extensive research work, in particular by German investigators, this question is to be answered in the affirmative. A negative result would mean that it would in all probability be wholly impossible to obtain the anticipated outcome of the experiments. Since this question is, however, to be answered in the affirmative, the next question in importance is

2. Can rhizobia of leguminous plants be so modified by special culture methods as to induce them to develop in and upon the roots of other plants, as corn, wheat, rye, barley, etc.? Although, as already indicated, the experimental results thus far obtained are not conclusive, yet the indications are that they will finally prove successful. German investigators have shown that one variety of leguminous rhizobium may, by culture methods, be converted into another variety. That is, for example, the rhizobium of the bean nodules may be induced to develop nodules on the roots of the pea, and perhaps other species of closely related genera. This is, in part, denied by Nobbe and Hiltner in the specifications of patent claim, as above recorded. It is, however, generally admitted by investigators that the rhizobia of the majority of leguminous plants are morphologically very variable, and undergo very marked structural changes in different culture media and within the host root nodules at different periods of the season and of growth. Such pronounced polymorphism coincides with marked adaptive changes to new or changed environment, and it is, therefore, highly probable that these polymorphic rhizobia may be induced to change hosts, at least temporarily, which is really all that is required for the success of the experiments. That is, it is desired that i the rhizobia should live and multiply in or upon the roots of gramineous plants during one season or from the time of seed germination to i the ripening of the crop (in annuals). This question is intimately associated with the following:

3. Even should it be possible to induce the rhizobia to develop in; and upon roots of gramineous (and other non-leguminous) plants, would they still retain the power of assimilating free nitrogen? It is highly probable that this question can be answered in the affirmative, although Lafar makes the statement that the rhizobia in certain stages of existence, for instance those which exist in the infecting threads (Infectionsfäden) of nodules and predominate in young tubercles and in the apical areas of all tubercles, exist in a purely parasitic or harmful relationship with the host plant. It is, however, highly probable that this statement is not founded upon experimental proof. The assimilation of free nitrogen is an essential function of rhizobia, and it is certainly reasonable to assume that the function would continue, though perhaps in a modified degree, no matter how marked the morphological adaptive changes might be. This question can be settled very simply and easily as the experiments progress.

The following question should also be carefully considered:

4. Are there soil bacteria or other organisms, not found in leguminous root nodules, which assimilate free nitrogen and which may be especially adapted to gramineous plants? From what has already been stated, it would appear that the Bacillus Ellenbachiensis of Caron is such an organism. If it should become evident that this organism assimilates free nitrogen principally in association with gramineous plants, it would seem to give promise of great utility in the more effective cultivation of gramineous plants. This organism would be especially advantageous, because, in contradistinction to rhizobia, it forms spores. Spore-bearing cultures would be desirable, because they would keep better and longer. The microbic fertilizer could be put up in dried form and sent to farmers at great distances without danger of becoming worthless. It could be kept for months, or perhaps even a year or more, though this in itself would not be of prime advantage. Further extensive researches would be necessary to determine to what extent adaptive changes could be developed in this particular microbe.

Finally the question will arise,

5. In what morphological, biological and bio-chemical relationship would the modified organisms establish themselves with the prospective or new host plant? It is, of course, not to be anticipated that they would cause the development of root nodules or tubercles or cause any considerable morphological change in root tissues. Nor is it at all highly probable that the anticipated morphological and biological adaptive relationship would be permanent or auto-transmissible to other generations of the new host. It is probable that the relationship would be temporary only. It may be quite marked at the period of seed germination and then gradually decline more or less rapidly.

The time required to change or modify the rhizobia sufficiently to induce them to develop in or upon the roots of gramineous and other non-leguminous plants must be determined experimentally, and this is the most essential part of the experimentation. It is reasonable to assume that considerable time will be required, one year, and perhaps even a much longer period. It will also be necessary to experiment with various media in order to determine which particular culture medium will produce the desired changes most effectively and most rapidly. Present indications are that acid media are not specially indicated as was once supposed, though they induce rapid and very marked morphological changes in the rhizobia.

The agricultural conditions of Europe are quite different from those of the United States. In the plan of research suggested, the interests of the American farmer are of prime importance. Wheat and corn are the two staple farm products of the central, eastern and western states, and in the above discussion these have been primarily in mind. Later researches may be extended to other American farm products, as cotton, tobacco, potatoes, etc. Opportunity must be had to make frequent field experiments or tests along with the purely laboratory experimentation.

The successful outcome of the research will result in inestimable value to farmers. The modified microbic soil fertilizer will serve essentially as a living fertilizer; it will do away with the use of the well-known guano, manure and other chemical fertilizers, which are applied at great labor and expense. It will also do away with the need of crop rotation, which to the agriculturist is a costly process, as it necessarily reduces the cultivation of the staple farm product. It is hoped that the increase in crop yield, resulting from the use of the microbic soil fertilizer, will amount to from 5 per cent, to even 50 per cent., depending primarily upon the condition of the soil. Rich soil naturally requires no fertilizer of any kind.

The following is a brief summary of the essential points discussed in this paper with special reference to the plan of research outline:

1. Bacteria of the root nodules of leguminous plants (rhizobia) have the power of assimilating free nitrogen independent of their intimate biologic association with the host plant.

2. Rhizobia, especially the species known as R. mutabile, found in the root nodules of the majority of leguminous plants, are highly polymorphic and readily undergo marked morphological changes in different culture media and under varying environmental conditions.

3. The species or variety of rhizobium living in natural symbiotic or biologic relationship with one given species of leguminous plant may be so modified artificially as to induce it to live in a similar relationship with a different species of leguminous plant, indicating great biologic adaptability.

4. It is probable that rhizobia of various leguminous plants may be so modified by special culture methods as to induce them to develop in or upon the roots of non-leguminous plants, continuing their free nitrogen-assimilating function, thus supplying such plants with nitrogenous compounds which serve as soil fertilizer or food for the plants with which the modified rhizobia will form a temporary intimate relationship.

5. The bacillus of Caron (Bacillus Ellenhachiensis) also gives promise of great utility in future economic agriculture, especially in the cultivation of gramineous plants.