Part 1

Produced by Bryan Ness, Jana Srna and the Online Distributed Proofreading Team at http://www.pgdp.net (This file was produced from images generously made available by The Internet Archive/American Libraries.)

UNIVERSITY OF ILLINOIS

Agricultural Experiment Station.

BULLETIN NO. 94.

NITROGEN BACTERIA AND LEGUMES

(WITH SPECIAL REFERENCE TO RED CLOVER, COWPEAS, SOY BEANS, ALFALFA, AND SWEET CLOVER, ON ILLINOIS SOILS).

BY CYRIL G. HOPKINS.

Work and Knowledge are a stronger team than Work and Work.

URBANA, ILLINOIS, FEBRUARY, 1904.

SUMMARY OF BULLETIN NO. 94.

1. Soil nitrogen cannot be used by plants until it is changed to the form of nitrate nitrogen by the nitrifying bacteria. Page 307

2. Atmospheric nitrogen cannot be used by any agricultural plants, excepting legumes, and even leguminous plants have no power to obtain nitrogen from the air unless they are provided with the proper nitrogen-gathering bacteria. Page 309

3. As a rule each important agricultural legume must have its own particular species of bacteria. Page 311

4. The frequent failure of red clover in normal seasons, especially on normal soils occupying the highest land, is undoubtedly due in part at least to the absence of the proper bacteria (sometimes the soil lacks lime or phosphorus). Page 313

5. On the very acid soils, where clover has never been grown successfully, applications of ground limestone should be made where legumes are to be grown. Page 315

6. Cowpeas need not be inoculated, because the cowpea bacteria are usually either present in the soil or are introduced with the seed in sufficient numbers to effect a good degree of infection if the soil is suitable and if cowpeas are seeded upon the same land for two successive years. Page 317

7. Cowpeas grown on infected soil on the University of Illinois soil experiment field contained four times as much nitrogen as the same kind of cowpeas grown on similar land which was not infected. Page 319

8. As a rule soy bean fields should be inoculated when first seeded to soy beans, otherwise they may be grown on the same land for three or four years before the soil becomes thoroughly infected. Page 320

9. Investigations, reported in this bulletin, furnish conclusive proof that infected sweet clover soil can be used for inoculating alfalfa fields and with the same results as are obtained when the infected soil is obtained from an old alfalfa field. (Sweet clover is a tall, rank-growing, sweet-scented leguminous plant widely distributed over Illinois, especially along the roadsides in the northern and central parts of the state, and in many places in bottom lands in southern Illinois.) Page 324

10. This bulletin will be sent free of charge to any one interested in Illinois agriculture upon request to E. Davenport, Director Agricultural Experiment Station, Urbana, Illinois; and, if so requested, the name of the applicant will be placed upon the permanent mailing list of the Experiment Station, so that all bulletins will be sent to him as they are issued.

Conclusions Page 327

NITROGEN BACTERIA AND LEGUMES

(WITH SPECIAL REFERENCE TO RED CLOVER, COWPEAS, SOY BEANS, ALFALFA, AND SWEET CLOVER, ON ILLINOIS SOILS).

BY CYRIL G. HOPKINS, CHIEF IN AGRONOMY AND CHEMISTRY.

Among the several different classes or groups of bacteria there are two which are of special importance to agriculture because of their relation to the element nitrogen, this being commonly considered the most valuable element of plant food.[1] These two classes of bacteria are, first, the nitrifying bacteria, and, second, the nitrogen-gathering bacteria.

THE NITRIFYING BACTERIA.

The nitrifying bacteria are those which have the power to form nitrates. In the following brief discussion of this subject we include at least three species of bacteria which by their combined or successive action have the power to transform organic nitrogen into nitrate nitrogen, which is a suitable form of nitrogen for plant food. For the exact information which we now have regarding the nitrifying bacteria we are indebted to the researches of Pasteur and Schlösing and Müntz of France, Winogradsky of Russia, Warington of England, and others.

The nitrogen in the soil is almost entirely in organic compounds; that is, the nitrogen (which is a gas in the free, or uncombined, state) is united or combined with other elements, notably with carbon, hydrogen, and oxygen, in the form of partially decayed vegetable or organic matter. (By organic matter we mean matter which has been formed by the growth of some organism, either plant or animal, as grass or flesh.) Plants cannot use the free nitrogen of the air as plant food, neither can they use the organic compounds of nitrogen which occur in the soil. There are at least three different kinds of bacteria, and also three different steps or stages involved in the process of nitrification, the nitrogen being changed from the organic compounds first into the ammonia[2] form, second, into the nitrite form, and third into the nitrate form. During the process the nitrogen is separated from the carbon and other elements composing the insoluble organic matter, and is united or combined with oxygen and some alkaline element (as calcium) to form the soluble nitrate, such as calcium nitrate, which is one of the most suitable compounds of nitrogen for plant food. Calcium is the alkaline element contained in lime or limestone. The name _calcium nitrate_ indicates just what elements this compound contains; namely, calcium, nitrogen, and oxygen. (In the names of compounds the ending _-ate_ always means oxygen.)

This is the general process of nitrification in which the nitrifying bacteria transform or transfer the nitrogen from insoluble organic compounds into soluble compounds in which it may serve as available plant food. The nitrate which is thus formed may be calcium nitrate or magnesium nitrate or potassium nitrate or even sodium nitrate, depending upon which of these alkaline elements is present in the must suitable form. If no alkaline element is present in available form then no nitrates can be made in the soil. One of the reasons for applying ground limestone to soils which are deficient in lime is to furnish the element calcium in suitable form for the formation of nitrates in the process of nitrification. Ground limestone is calcium carbonate (CaCO₃), a compound containing one atom of calcium (Ca), one atom of carbon (C) and three atoms of oxygen (O₃). This is the same form of lime which is contained naturally in limestone soils—soils which are noted for their great productiveness—and it is generally the most economical form of lime to use for correcting soil acidity and promoting nitrification.

In the process of nitrification, that is in the formation of nitrates, there is required, not only the presence of calcium, or some other alkaline element, in suitable form, but also a good supply of the element oxygen; for calcium nitrate, Ca(NO₃)₂, contains one atom of calcium (Ca), two atoms of nitrogen (N)₂, and six atoms of oxygen (O₃)₂, in each molecule as indicated in the formula, Ca(NO₃)₂. Magnesium nitrate, Mg(NO₃)₂, potassium nitrate, KNO₃ (K is from the Latin word _Kalium_, which means potassium), and all other nitrates, also, contain oxygen. The supply of oxygen for the formation of nitrates in the soil comes from the air, which consists of about twenty percent oxygen, seventy-eight percent nitrogen, and two percent of other elements and compounds, as argon, carbon dioxid, CO₂, water vapor, H₂O, etc. One of the important effects of cultivation, or tillage, is that it permits the air more freely to enter the soil, and thus promotes nitrification.

THE NITROGEN-GATHERING BACTERIA.

As stated above, the nitrogen naturally in the soil is contained almost entirely in the organic matter. Any process which tends to decompose or destroy this organic matter, such as nitrification or other forms of oxidation, will also tend to reduce the total stock of nitrogen in the soil. Because of this fact the matter of restoring nitrogen to the soil becomes of very great importance. Of course a part of the nitrogen removed in crops may be returned in the manure produced on the farm; and nitrogen may also be bought in the markets in such forms as sodium nitrate (containing 15 to 16 percent of nitrogen), ammonium sulfate (containing 20 to 21 percent of nitrogen), and dried blood (containing 12 to 15 percent nitrogen); but, when we bear in mind that such commercial nitrogen costs about 15 cents a pound, and that one bushel of corn contains about one pound of nitrogen, it will be seen at once that the purchase of nitrogen cannot be considered practicable in general farming, although in market gardening, and in some other kinds of intensive agriculture, commercial nitrogen can often he used with very marked profit.

Nitrogen is removed from the soil not only in the crops grown, but also, and frequently in larger amounts per annum, in the drainage waters, and in some other ways, as by denitrification and by the blowing and washing of the surface soil. Professor Snyder, of the Minnesota Experiment Station, has shown that during a series of years the total loss of nitrogen from some Minnesota soils in some cases amounts to several times the amount actually used in the crops produced.

Considering all of these facts, and the additional facts that there are about seventy-five million pounds of atmospheric nitrogen resting upon every acre of land, and that it is possible to obtain unlimited quantities of nitrogen from the air for use of farm crops, and at very small cost, the inevitable conclusion is that the inexhaustible supply of nitrogen in the air is the store from which we must draw to maintain a sufficient amount of this element in the soil for the most profitable crop yields.

It is often stated that leguminous plants, such as clover, have power to obtain free nitrogen from the air. This is not strictly true. Red clover, for example, has no power in itself to get nitrogen from the air. It is true, however, that the microscopic organisms[3] which commonly live in tubercles upon the roots of the clover plant do have the power to take free nitrogen from the air and cause it to unite with other elements to form compounds suitable for plant food. The clover plant then draws upon this combined nitrogen in the root tubercles, and makes use of it in its own growth, both in the tops and in the roots of the plant.

These nitrogen-gathering bacteria live in tubercles upon the roots of various leguminous plants,[4] such as red clover, white clover, alfalfa, sweet clover, cowpeas, soy beans, vetch, field-peas, garden-peas, field and garden beans, etc. These tubercles vary in size from a pinhead to a pea, varying with the different kinds of plants, being especially small upon some of the clovers, and very large upon cowpeas and soy beans. The tubercles are, of course, easily seen with the eye, but the tubercle is only the home of the bacteria, somewhat as the ball upon the willow twig is the home of the insects within. The bacteria themselves are far too small to be seen with the unaided eye, although they can be seen by means of the most powerful microscope. Several million bacteria may inhabit a single tubercle. It is not necessary to see the bacteria, because if we find the tubercles upon the roots of the plant, we know that the bacteria are present within, as otherwise the tubercle would not be formed.

Although the plant itself, as clover, for example, has no power to feed upon the free or uncombined nitrogen in the air, yet these nitrogen-gathering bacteria do have the power to absorb the free nitrogen and cause it to combine with other elements, forming nitrates or other compounds which are suitable forms of nitrogen for plant food.

It has also been demonstrated that, as a rule, there are different species of nitrogen-gathering bacteria for markedly different species of leguminous plants. Thus we have one kind of bacteria for red clover, another kind for cowpeas, another kind for soy beans, and still a different kind for alfalfa.[5]

THE RED CLOVER BACTERIA.

That clover has no power in itself to gather atmospheric nitrogen, and that the bacteria do have power to feed the clover plant with nitrogen gathered from the air is very easy to demonstrate. It is one of the regular laboratory practices of the students in soil fertility in the Agricultural College to make this demonstration. Plate 1 is an illustration of such student work. The two pots which are shown were provided with all elements of plant food, excepting the one element nitrogen. Thus far the two pots are exactly alike. Each contains no nitrogen, as indicated by the label “No N.” Each pot is planted with the same number of red clover seeds. To the right-hand pot, however, some bacteria (“Bac.”) were added, while none were added to the left-hand pot. These bacteria were obtained by taking about one pound of soil from a clover-field where abundance of tubercles were found on the clover roots, adding this soil to about one quart of pure water, shaking for a few minutes, allowing the soil to settle, then taking a small quantity of almost clear solution, and adding it to the pot which we wished to inoculate with the red clover bacteria. Aside from the addition of these microscopic bacteria to the right-hand pot, these two pots were treated exactly alike throughout the experiment. It will be plainly seen that where the bacteria were added the clover was furnished with sufficient nitrogen to make a strong and luxuriant growth, while without the bacteria the clover (in the left-hand pot) only germinated and made what little growth it could with the small amount of nitrogen contained in the seed. This result is the difference between success and failure of the clover crop.

In general the clover bacteria are well distributed over the northern and central part of Illinois, but we now have some very strong evidence that they are not well distributed in some soils of large area in southern Illinois. There is also some evidence that they were not originally present even in the soils where they are now found in great abundance; and, furthermore, it seems very probable that these bacteria may cease to live in a soil where they have once been present, provided clover is not grown on the land for several years.

It will help us to understand this matter if we bear in mind that the home of these bacteria is the tubercle upon the clover root. It is quite evident that they will continue to live upon the decaying tubercles or roots for three or four years after the clover plant has been killed. On the other hand, we have some notable evidence that the bacteria do not continue to live in a soil after five or six years’ continuous cropping with absolutely no clover growing on the land during those years. It is a simple matter for any one to determine whether the bacteria are present or not, for the tubercles which are formed if the bacteria are present are plainly seen attached to small roots. They look somewhat like miniature potatoes, varying in size from pinheads on clover to peas on soy beans or cowpeas. (See Plates 2 and 4.) It is important to remember that the bacteria live in the soil and not in the seed.

When clover is cut for seed, it is frequently left to lie upon the ground until the straw becomes half rotten and very dirty; and, consequently when it is threshed, it practically always happens that there is at least some small amount of dust and dirt taken with the seed. This dirt is almost sure to carry with it some bacteria from the soil. If these few bacteria are scattered with the clover seed when it is sowed they will inoculate at least a few plants, and if they are allowed to multiply on these plants, and especially if the same field is repeatedly seeded with clover, the soil will ultimately become thoroughly infected with the clover bacteria. Of course they may be carried from one part of the farm to another, or even from one farm to another, by various agencies, as dust or wind storms, surface drainage or flood waters, manure made from clover hay, implements used in cultivating the soil, etc., etc.

Many of the older farmers of Illinois have stated to the writer that when this country was very new it was commonly found difficult to get a “catch” of clover on new land. After a good “catch” was once gotten, then it was easier to get clover to grow on that land the next time. There was a saying among the farmers that clover would not do well until they got the “wild nature” out of the land. Their final success was undoubtedly due, not to getting anything out of the land, but rather to getting the bacteria into the land. Several Illinois farmers have reported some quite remarkable results from very light applications of the clover chaff or straw (obtained in hulling clover) in its beneficial effect on clover on land where it was otherwise difficult to get a “catch.” There is a somewhat general belief among farmers of long experience that clover straw or chaff has some special value in getting a catch of clover aside from its value as manure or for the seed which it sometimes contains.

Manager F. A. Warner of the Sibley Estate, Ford County, recently stated to the writer that they had had very great difficulty to get clover to grow when they first began growing clover on that large estate, some six or eight years ago, although, after a good crop was once secured, they rarely had any further difficulty in getting a catch of clover on the same land.

On the common gray prairie soil of the Lower Illinoisan Glaciation, in southern Illinois, the commonest type of soil in more than twenty counties, practically no red clover is grown. In the spring of 1903 we seeded red clover on that type of soil in three places; namely, on the University of Illinois soil experiment fields near Edgewood, Effingham County, near Du Bois, Washington County, and near Cutler, Perry County. On certain plots the soil acidity had been corrected with lime and an abundant supply of phosphorus (in bone meal) had been provided, potassium also having been added on some plots. These fields were carefully examined the latter part of June, and at Cutler and Du Bois the clover was found to be dead or dying, and no tubercles could be found upon the clover roots, although on the clover which had been seeded at about the same time on the University fields at Urbana the root tubercles were found in great abundance. At Edgewood a few tubercles were found and the clover appeared to be growing fairly well. Infected red clover soil was at once procured and scattered over the fields at Edgewood, Du Bois, and Cutler, but it was evidently too late to be of any marked benefit. At Cutler and at Du Bois the clover was a complete failure. (It will be tried again next year.) At Edgewood it continued to grow fairly well, and its progress next season (1904) will be watched with much interest. It should be stated that the Experiment Station has been growing clover for several years with varying degrees of failure on land adjoining the present clover field at Edgewood, and it is possible that this year’s apparent success from the start is due in part at least to the bacteria which have been incidentally introduced and multiplied year after year and scattered over the adjoining land by wind and dust storms. Before the close of the season the tubercles developed in abundance on the roots of the clover at Edgewood.

An experience reported by Professor Herbert W. Mumford, of the Animal Husbandry Department of this university, will be of interest and value in this connection. Professor Mumford commonly grows clover in his rotations on his own private farm, but he states that at one time one particular field was cropped continuously with timothy, oats, and corn for some six years or more without any clover whatever. It was then again seeded to clover, but the crop made a complete failure, although on other land where clover had been grown more recently a successful clover crop was grown from the same kind of seed seeded at about the same time. The following year this particular field was again seeded to clover. This time the “catch” was not a total failure, but it was too poor to save, and it was plowed up and the land again seeded to clover the next year, and an excellent catch of clover resulted. After this, clover was frequently grown on this field, and no special difficulty was had in getting good crops.

While the failure of clover may often be due to drouth, and in some places due to soil acidity (lack of lime), and sometimes even due to an insufficient supply of available phosphorus or of potassium, we now know with certainty that it sometimes fails because of the absence of the nitrogen-gathering bacteria, especially on land which has never grown clover, and probably also on land which has not grown it recently. We should always remember that the bacteria do not thrive in strongly acid soils. Even though they may sometimes live in such soils and perhaps produce some tubercles upon the roots of certain hardy, strong growing legumes, like cowpeas, nevertheless we are obtaining some strong evidence that in such acid soils they have but little power to gather nitrogen from the air. That ground limestone is the most economical and satisfactory material to use in correcting the acidity of soils is strongly indicated by the information we have thus far obtained. On the upland prairie soils of the Lower Illinoisan Glaciation where red clover has never been grown successfully, largely because of the acidity of the soil, it will undoubtedly be helpful and profitable not only to correct the acidity of the soil with ground limestone, but also to secure infected soil from some field of timber land or bottom land where red clover is growing, well provided with root tubercles, and inoculate the field with it. This soil should be collected to a depth of three or four inches and scattered over the prairie land at the rate of a few hundred pounds per acre at the time the clover is seeded or before.

THE COWPEA BACTERIA.

Plate 2 is made from a photograph of a cowpea root with the tubercles upon it. This illustration shows the cowpea tubercles at nearly natural size, which is about as large as the seed of ordinary garden peas.