" i v; fs '' "' ^ ^. . * t m THE UNIVERSITY OF^ILLINOIS LIBRARY 6 2> . 7 It Gt '* * VM>. I ^{5- I "8 I AGR1C ' -.4 *** . UBHUY OF THE UNIVERSITY OF ILLINOIS WON CIRCULATING CHECK FOR UNBOUND CIRCULATING COPY tit JUL 9 - 1915 UNIVERSITY OF ILLINOIS Agricultural Experiment Station BULLETIN No. 179 A BIOCHEMICAL STUDY OF NITROGEN IN CERTAIN LEGUMES BY ALBERT L. WHITING CONTENTS OF BULLETIN No. 179 PAGE INTRODUCTION 471 HISTORICAL 472 BIOLOGICAL 475 Infection 476 Inoculation as It Occurs Under Field Conditions 480 Growth of the Nodule 481 BACTERIOLOGICAL 482 Bacillus radicicola 483 Growth and Endurance of B. radicicola 484 Identity of B. radicicola 484 Enzyme Production by B. radicicola 485 Slime Production by B. radicicola 486 Isolation of B. radicicola : 486 Dissemination of B. radicicola 486 Fixation of Nitrogen Without the Legume Plant 487 Bacteroids 487 THEORIES OF ASSIMILATION, FIXATION, AND IMMUNITY 488 Theories of Assimilation by the Plant 488 Theories Regarding the Chemical Phenomena of Fixation 489 Theories of Immunity 492 PRACTICAL CONSIDERATIONS WITH REGARD TO LEGUME FIXATION 493 Mutual Symbiosis 493 Amount of Nitrogen Fixed per Acre per Year 495 Value of Legumes as Nitrogen Retainers 496 Cross Inoculation 497 Associative Growth of Legumes and Non-Legumes 498 CHEMICAL 499 EXPERIMENTAL 503 Plan of Investigations 503 PART I: STUDIES TO DETERMINE THRU WHICH ORGANS LEGUMES OBTAIN ATMOSPHERIC NITROGEN 503 General Plan of Experiments 505 Experiment I 507 Experiment II 507 Experiment III 509 Experiment IV 514 Experiment by Kossowitsch 519 General Consideration of Gas Experiments 521 Practical Application of Results 522 UNIVERSITY OF ILLINOIS LIBRARY JUL 9 - 1915 PART II: RELATIVE PERCENTAGES OF NITROGENOUS COMPOUNDS IN THE VARIOUS PARTS OF THE SOYBEAN AND COWPEA AT DEFINITE PERIODS OF GROWTH 522 Methods Employed in the Growth and Preparation of Samples. .523 Analytical Methods 525 Discussion of Some of the Methods Used. 527 Qualitative Tests 527 Series 100 (Soybeans) 528 Series 500 (Soybeans) 532 Series 700 (Soybeans) 533 Series 600 (Cowpeas) 536 Discussion of Tables 537 CONCLUSIONS . ..541 ILLUSTRATIONS PLATE PAGE I. Nodules of Robina Type on Roots of Soybeans 477 II. Nodules of Lupinus Type on Roots of Lupine Seedlings 478 III. Nodules of Robina Type on (A) Red Clover; (B) Vetch; (C) Sweet Clover 479 IV. Cowpea Seedlings in Preparation for Gas Experiments 504 V. Experiment II : Cowpeas at Harvest (37 Days) 506 VI. Experiment II: Plants Grown in Air and in GO 2 +0 508 VII. Experiment III: At Beginning and 10 Days Later 510 VIII. Experiment III: 52 Days from Beginning and at Harvest (83 Days) 511 IX. Experiment III: Roots from Plants Grown in CO 3 -f- O and in Air. .513 X. Experiment IV: At Beginning and 16 Days Later 515 XI. Experiment IV: 41 and 59 Days from Beginning 516 XII. Experiment IV : At Harvest (95 Days) 517 XIII. Experiment IV : Roots from Plants Grown in CO 2 -f- O and in N -{- CO 2 -f O 518 XIV. Typical Jar of Five Cowpeas Being Grown for Samples 523 XV. Graph Showing Soluble and Insoluble Nitrogen in Series 100 531 XVI. Graph Showing Soluble and Insoluble Nitrogen in Series 700 and 500 . 535 XVII. Graph Showing Soluble and Insoluble Nitrogen in Series 600 538 FIGURE 1. Root hair of common pea, showing infecting strand 476 2. Root cell, showing infecting strand passing thru it and the formation of lamellae 476 3. Young nodule magnified, showing affected root hair and same root hair more highly magnified 480 4. Young nodule, showing the beginning of the differentiation of its tissues. .482 5. B. radicicola, showing shape and flagella 483 6. Bacteroids, showing shape, and occurrence of vacuoles 488 A BIOCHEMICAL STUDY OF NITROGEN IN CERTAIN LEGUMES 1 BY ALBERT L. WHITING, ASSOCIATE IN SOIL BIOLOGY INTRODUCTION The investigations considered in this publication bear on the biochemical nature of the element nitrogen, especially as concerns its fixation and assimilation thru the symbiotic relationship of Bacillus radicicola and certain members of the botanical family known as Leguminosae. The sources of the element nitrogen available for agricultural purposes are numerous. Of these the atmosphere is by far the most important and most extensive. Above each acre of the earth's surface there are about 69 million pounds of atmospheric nitrogen, and science has shown that by thoroly scientific systems of management this nitrogen may be appropriated for soil improvement at a minimum expense. By growing legumes, atmospheric nitrogen may be obtained at a low cost, often at no net cost, for most agricultural leguminous crops are worth growing for feed or seed alone. In commercial fer- tilizing materials, nitrogen costs from fifteen to twenty cents per pound, an amount from two to five times greater than that expended for any of the other essential elements of plant food. It is of passing interest to note how greatly disproportionate the cost values of these elements are to the relative supplies, when the nitrogen in the air is considered. The United States spends annually, abroad, over 32 million dol- lars in the purchase of combined nitrogen for use in various opera- tions, agricultural and otherwise. 2 Of this amount 161/2 million dol- lars are expended for the purchase of sodium nitrate, which is the Submitted to the Faculty of the Graduate School of the University of Illi- nois in partial fulfilment of the requirements for the degree of doctor of phi- losophy, June, 1912. Revised to date of issuance. "Norton : Special Agent Series, Dept. of Commerce and Labor, Bur. of Manfr., No. 52, 9-11. 471 472 BULLETIN No. 179 [March, most important commercial form of inorganic nitrogen. The present world supply of this salt is estimated at 454,576,200,000 pounds. 1 How insufficient this supply is, when measured by crop require- ments, may be realized from the fact that the following nine important crops of the United States, corn, wheat, oats, barley, rye, potatoes, hay, cotton, and tobacco, in the year 1910, required for their growth 11,500,000,000 pounds of nitrogen. 2 If sodium nitrate were used for growing these crops at the rate stated above, the supply would be ex- hausted in about six years. On the other hand, the nitrogen above only one square mile, weighing 20 million tons, would be sufficient to supply what the entire world, at its present rate of consumption, would require for the next fifty years. 3 The nitrogen above four acres would furnish more than the actual yearly consumption of commercial nitrogen in the entire United States. The wonderful possibilities presented by such an extensive source of plant food, and the fact that over 100 million dollars are invested in commercial fertilizers each year in the United States, a large part of which is wasted or uselessly applied, together with the great natural losses of nitrogen that occur, tend to emphasize greatly the need of a proper utilization of this unlimited reserve supply. Further, it is well recognized that the maintenance of the nitrogen supply is the greatest of our soil problems. Nitrogen cannot be purchased on the market at a price that will permit its extensive application in growing the im- portant crops of the United States. There is only one logical and in- exhaustible source of nitrogen for the world to utilize in the produc- tion of crops. That source is the atmosphere, from which nitrogen is most economically and easily secured as a result of the symbiotic relationship between B. radicicola and leguminous plants. HISTORICAL For several centuries certain plants of the Leguminosae have been used as soil improvers. A few of the more important references to their uses are considered here. In Roman literature, among the works of Columella, 4 mention is made of the Roman farmers regarding beans as possessing the prop- erty of enriching the soil, and attention is also called to the practice of plowing under lupines. Alfalfa and vetches were observed to pro- duce similar results to those of lupines and beans. Like notations 1 Eeview of Beviews, April, 1910. 2 Yields taken from U. S. Yearbook, 1910. For calculations see Hopkins' "Soil Fertility and Permanent Agriculture" (1910), 154; also 603-604. Norton: Special Agent Series, Dept. of Commerce and Labor. Bur. of Manfr., No. 52, 9-11. 4 Marshall: Microbiology (1911), 273. 1915] A BIOCHEMICAL STUDY OF NITROGEN IN CERTAIN LEGUMES 473 may be found among the writings of Thaer and Walz. 1 Gasparin 2 constantly calls attention to the power of leguminous plants to add nitrogen to the soil. Jethro Tull 3 wrote concerning the efficiency of legumes in restoring depleted soils, mentioning especially sanfoin and alfalfa. It may be noted here that Hellriegel, who later was most promi- nent in the discovery of the relation existing between legumes and bacteria, wrote in 1863 as follows: "Clover plants may develop nor- mally and completely in mere sand to which the necessary mineral constituents of plant food have been added in assimilable forms, even when this soil contains no trace of any compound of nitrogen or of organic matter." Schultz-Lupitz 4 in 1881 reported results that were of both chemi- cal and practical significance. After growing lupines for fifteen con- secutive times on a sandy soil, without the application of nitrogenous materials, he observed that the yields did not diminish ; and when he grew cereals on the same land after the lupines, he found that the yields of the grains were two and three times the yields where no lupines had been grown. Analyses of the soils at the end of this time showed that where the lupines had been grown, the nitrogen content of the surface six inches had increased by .06 percent. Frank 5 veri- fied these results with twenty years of lupine culture on the same fields. About this time a great deal of interest centered on pot-culture experiments with legumes. Many physiologists and chemists worked on the problem of nitrogen collection by legumes. Prominent among these were the scientists Boussingault, 6 and Lawes, Gilbert, and Pugh, 7 who, owing to their great accuracy, sacrificed the possibility of becom- ing the discoverers of this important relationship. In their great care, they destroyed the vital agency (B. radicicola) necessary for the ac- complishment of this symbiotic fixation. Later, in 1886, Hellriegel and his co-worker Wilfarth 8 made the classical discovery that legumes obtain atmospheric nitrogen thru the association of microorganisms living in the nodules. In a preliminary report read before a section of scientists assembled on September 20, 1886, at Berlin, Hellriegel announced his findings ; and in a more com- 'Storer: Agriculture (1906), 2, 97. 2 Ibid. 8 Lipman: Bacteria in Eelation to Country Life (1908), 206. 4 Schultz-Lupitz: Landw. Jahrb. (1881), 10, 777. "Frank: Landw. Jahrb. (1888), 17, 501. Boussingault: Ann. Sei. Agron. (1909), 26, Ser. 3, 4, 102-130. 'Lawes, Gilbert, and Pugh: Rothamsted Experiments (1905), 6-7. 'Hellriegel and Wilfarth: Tagblatt d. Naturforscher Versamml. z. Berlin (1886), 290. 474 BULLETIN No. 179 [March, plete account rendered two years later, he made known to the world his researches. These are summarized as follows: 1 1. The legumes differ fundamentally from the grains in their nutrition with respect to nitrogen. 2. The grains (Gramineae) can satisfy their nitrogen need only by means of assimilable combinations existing in the soil, and their development is always in direct proportion to the provision of nitrogen which the soil places at their disposal. 3. Outside the nitrogen of the soil, the legumes have at their service a second source from which they can draw in most abundant manner all the nitrogen which their nutrition demands to complete that lack when the first source is in- sufficient. 4. That se_cond source is the free nitrogen the elemen- tary nitrogen of the atmosphere which is furnished to them. 5. The legumes do not possess by themselves the faculty of assimilating the free nitrogen from the air; it is ab- solutely necessary that the vital action of microorganisms of the soil come to their aid in order to attain this result. 6. In order that the nitrogen of the air can be made to serve the nutrition of the legumes, the sole presence of lower organisms in the soil is not sufficient; it is still necessary that certain among them enter into a symbiotic relationship with the plants. 7. The nodules 2 of the roots must not be considered as simple reservoirs of albuminoid substances; their relation to the assimilation of free nitrogen is that of cause to effect. Schloesing and Laurent 3 after growing legumes in a confined at- mosphere, gave out the following direct evidence of the fixation of at- mospheric nitrogen. Atmospheric nitrogen introduced into culture vessel 2681.2 ccm. Atmospheric nitrogen withdrawn 2653.1 ccm. Amount of nitrogen assimilated 28.1 ccm. (=36.5 nig.) Nitrogen in the soil and crop 73.2 mg. Nitrogen in the soil and seed 32.6 mg. Nitrogen assimilated 40.6 mg. ^ellriegel and Wilfarth: Beil. Zert. d. Verins. fur die Eubenzucker in- dustrie, Berlin, Nov., 1888; or Lafar Handbuch der technischen Mykologie (1904- 06), 3, 31. ^Nodules substituted for tubercles. "Schloesing and Laurent: Compt. Eend. Acad. Sci. (1890), 111, 750: (1892) 115, 659, 732. 1915] A BIOCHEMICAL STUDY OF NITROGEN IN CERTAIN LEGUMES 475 In addition to the scientists mentioned above, Atwater and Woods, Berthelot, Miintz, Ville, Maze, Deherain, Frank, Hartig, Nobbe, Hilt- ner, Warrington, Hopkins, and many others have done much careful work in solving the problem and applying the truths discovered. BIOLOGICAL Nodules, 1 which are the visible manifestations of infection, were observed upon the roots of legumes by Malphighi 2 as early as 1687. The investigators of those times believed that the nodules were the re- sult of pathological processes, that they were lumps, knobs, warts, and even galls. In 1853 the modern conception of the nodule as a nor- mal growth on the legume plant was established by L. C. Treviranus. 3 Various theories have been proposed as to the function of these peculiar outgrowths, some advancing the idea that they were storage reservoirs or stimuli whereby the plants obtained nitrogen from the atmosphere thru their leaves. Recently Jost 4 has called them "bac- terium galls," local hypertrophies not dissimilar to those sometimes caused by animal life. An astonishing conception has crept into the minds of the authors of certain general textbooks on bacteriology and plant physiology that nodules are abnormal growths and that their re- lationship to plants is either wholly or partially parasitic. It seems preferable, even to those familiar with the limitations of the theory, to describe this relationship as a normal condition and a true mutual symbiosis. That the formation of these nodules is due to external infection was definitely shown in 1887 by Marshall Ward, 5 who was able to inocu- late the roots of young legumes by placing them in contact with old nodules. In Germany the first attempts to grow soybeans (Glycine hispida) in the botanical gardens resulted in failure, and it was not until soil from the natural habitat of that plant was imported for inoculation that soybeans were grown successfully. 6 The history of the introduction of alfalfa culture in the states of Kansas and Illinois exemplifies in a large way this need of inoculation. From this experi- ence developed 1 lie soil-transfer method and the glue method 7 of inocu- lation, both of which are recognized today as superior to the use of so- called commercial cultures. 1 Nodules are recognized on the following non-leguminous plants: alders (Alnus glutinosa), silverberry (Eleagnus), sweet gale (Myrica Gale), sago palm, an evergreen, (Podocarpineae) , cycads (Cycacadeae) , birthwort (Aristolociaceae) . Nitrogen-fixing bacteria resembling B. radicicola have been found in the alder, silverberry, sweet gale, and five varieties of podocarpus. 2 Malphighi: Op. (1687), 2, 126, Leiden. 3 Treviranus: Bot. Ztg. (1853), 11, 393. 4 Jost: Plant Physiology (Gibson 1907), 237. "Ward: Phil. Trans. Koy. Soc. London (1887), 178, 139. "Soil inoculation experiments were instituted as early as 1887 at the Moor Culture Experiment Station, Bremen, Germany. Til. Agr. Exp. Sta. Buls. 76, 94. 476 BULLETIN No. 179 [March, Two types of nodules have been recognized by Tschirch; 1 Lupinus (lupine) represents one type and Robina (locust) the other. As may be seen by reference to Plates I and II, they differ in morphological appearance. The Lupinus type involves a swelling of the central root cylinders themselves, while in the Robina type only the epidermal and the endodermal tissues seem to enlarge. According to Tschirch, the nodules of lupines alone are of the first type, while those of all other legumes belong to the second. The figures in Plate III are sufficient to illustrate the most com- mon shapes of the Robina type. The shape varies with the different species of legumes, and to a certain extent with the individuals on the same legume plant. In the experimental work reported in this publi- cation, over twenty thousand nodules were examined closely, and it was not uncommon to find on the same plant notable variations due to external obstructions to growth. INFECTION The artificial inoculation of a plant is easily accomplished by con- tact. If the epidermis of the root is wounded and the infecting or- ganism (B. radicicola) brought into contact with the wound, nodules Fig. 1. Boot hair of com- mon pea (Pisum sati- vum'), showing infecting strand (x300) (After Prazmowski) Tig. 2. Eoot cell, showing in- fecting strand passing thru it and the formation of lamellae (x650) (After Prazmowski) Tschirch: Ber. deut. Bot. Gesell. (1887), 5, 58. 1915} A BIOCHEMICAL STUDY OF NITROGEN IN CERTAIN LEGUMES 477 PLATE I. NODULES OP EOBINA TYPE ON BOOTS OF SOYBEANS (Enlarged) 478 BULLETIN No. 179 [March, PLATE II. NODULES OF LUPINUS TYPE ON BOOTS OF LUPINE SEEDLINGS (After A. Meyer) 1915] A BIOCHEMICAL STUDY OF NITROGEN IN CERTAIN LEGUMES 479 480 BULLETIN No. 179 [March, result. Inoculation in pot cultures is attained by placing an infusion on the seed or in the medium. A similar method is successful with water cultures. INOCULATION AS IT OCCURS UNDER FIELD CONDITIONS Studies of inoculation as it occurs in the field show the following generally accepted phenomena : As the tip of the root hair of the legume pushes itself out into the soil, it chances to come into intimate contact with the organism B. radicicola. Some scientists have exploited the view that the organ- ism is attracted to the plant by chemotaxis, believing that the plant excretes a substance, probably a carbohydrate, which diffuses into the soil solution and attracts the motile organism. While it has been rather definitely shown that this organism progresses in the soil at a rapid rate, nevertheless the number of root hairs in- fected 1 is too small to lend support to a chemotactic theory. However the case may be, the organisms clus- ter at the tip of the hair and by means of an enzyme (or otherwise) rapidly dis- solve the cellulose of the cell wall, thus enabling the organism to enter the root hair. As a result, there is a decided bending of the tip, causing it to resemble a shepherd's crook. This was early observed as a sign of complete infection. It is claimed that other root hairs which form after infection are immune to the attack of other legumi- nous bacteria. 2 The organisms, by rapid division and growth, ad- vance thru the center of Prazmowski 3 found organisms in the cell Fig. 3. Young nodule magnified, showing af- fected root hair and same root hair more highly magnified (After Atkinson) the infected root hair. fierce, G. J.: Proc. Cal. Acad. Sci. II, No. 10 (1902), 295-328. Pierce found the proportion with bur clover to be 1:1000. 2 Fred: Vir. Agr. Exp. Sta. Ann. Ept. 1909-10, 123-125. "Prazmowski: Landw. Vers. Stat. (1890), 37, 160-238. 1915] A BIOCHEMICAL STUDY OP NITROGEN IN CERTAIN LEGUMES 481 sap and even in the epidermis only two days after inoculation. In this advance an infection strand (Infektion-schlauche) is formed, which consists of gelatinous material, and in the earlier stages of de- velopment this strand may be traced from the root hair into the inner tissue of the root and from cell to cell thruout the nodule. This infect- ing strand is not supposed to constitute a portion of the living tissue, nor is it a well-defined tube; but, as Fred has recently shown, it con- sists of a large number of zoogloea occurring adjacent to one another, in which separate bacteria can be distinguished. The infecting strand branches profusely, and it was this habit of growth which caused the early investigators to consider it the mycelium of a fungous growth. GROWTH OF THE NODULE The presence of B. radicicola in the tissues of the root causes a rapid cell division in the pericycle. These cells become larger and contain more protoplasm than the surrounding cells, and as growth takes place, the cortical parenchyma and epidermis are forced out- ward, thus forming a nodule. The growth of the nodule is apical. The various tissues common to the plant are present (see Fig. 4). In the central portion of the nodule is the so-called bacteroidal tissue, which is ochre, flesh, or gray in color, according to the age of the nodule, and in this portion the infecting strand (Infektion-schlauche) is distin- guished in the young nodule. It ramifies thruout the cells, causing those which it enters to lose their power of cell division but not of growth. Later, or in older nodules, the infecting strand is not visible, and the bacteroidal tissue loses its firmness. At the period when seed formation is at its height, most of the nodules are soft, and the inter- nal tissues slough off, leaving the more resistant epidermal tissue as a mere shell, which later decays. The endurance of the nodule depends upon several factors, chiefly, however, upon the kind of legume plant on which it is produced and the need of nitrogen by that plant. Pierce 1 considers the nodules as originating endogenously from the same layer of cells as the lateral roots, and as being morphologi- cally similar to them ; however, as the lateral roots rupture the epider- mis, the above statement is not entirely in accord with what actually takes place. The nodules are largest and most numerous where aeration is best in the soil. In saturated soils they occur at the surface and are often found colored green, very similar to sunburned potatoes. Nodules form in solutions, and exceptionally well in certain nutrient solutions. Several interesting instances have been brought to the attention of the Experiment Station, in which the observers believed that the nodules fierce, G. J.: Proc. Cal. Acad. Sci. II, No. 10 (1902), 295-328. 482 BULLETIN No. 179 [March, had grown above the ground. These peculiarities were undoubtedly caused by unobserved physical conditions occurring at the time of in- fection or afterward. FIG. 4. YOUNG NODULE, SHOWING THE BEGINNING OF THE DIFFERENTIATION OF ITS TISSUES (After Prazmowski) BACTERIOLOGICAL Minute bodies were first detected in nodules in 1866 by Woronin, 1 a Russian botanist. At that time bacteria were not recognized, and it was not until 1887 that they were demonstrated to be true bacteria by Wigand. 2 1 Woronin: Bot. Ztg. (1866), 24, 329. 'Wigand: Bot. Heft. Forsch, a.d.Bot. Bart. 3 Marburg (1887), 288. 1915} A BIOCHEMICAL STUDY OP NITROGEN IN CERTAIN LEGUMES 483 Immediately afterward, Beyerinck 1 isolated the organism on an artificial medium composed of a decoction of pea leaves, gelatine (7 percent), asparagine (.25 percent), and saccharose (.5 percent). He named the organism Bacillus radicicola, altho he described an organ- ism bearing a single polar flagellum. This organism became generally described as Pseudomonas radicicola, and some writers still prefer this designation. The organism has been known under a variety of terms, as Schinzia leguminosarum, Cladochitrium tuberculorum, Rhizobium radicicola, Rhizobium leguminosarum, Bacterium radicicola, Micro- coccus tuberigenus, Myxobacteriaceae, Actinomyces, and Phytomoyxa. Such inappropriate names as Rhizobacterium japonicum and Rhizo- bium sphaeroides are applied to certain special races. In commercial and general use, the organism is labeled as pea bacteria, bean bacteria, alfalfa bacteria, et cetera. Recent studies on the number of flagella possessed by this organ- ism have indicated that the organism is a bacillus, and it is there- fore desirable to adopt the original name B. radicicola, as proposed by Beyerinck. BACILLUS RADICICOLA These bacilli are rod-shaped organisms possessing numerous fla- gella (6 to 20), which are peritrichous. When full grown they vary in length from 1 to 4 or 5/*. 2 It is not uncommon to find them from .5 to .6/x wide and from 2 to 3/i long, and some have been found to measure only .18/x, wide and .9fi long. The organism is actively motile. It is strongly aerobic, and in this connection Pierce calls attention to the intracellu- lar spaces in the root, which make it un- necessary to assume, as has been done, that it must live anaerobically. It is known that this organism does not form spores, but its means of enduring in the soil has not yet been determined. The bacilli prevail in the young nodule, while the branched forms, or bacteroids (see page 487), predominate in the older structure. Fig. 5. B. radicicola, show- B. radicicola grows well on a great va- ing shape and flagella riety Qf culture ^^ perhaps begt Qn a medium of ash-maltose-agar or one of legume extract plus a sugar and dipotassium phosphate. Dextrose, saccharose, and maltose are suitable carbohydrates. The cultural characteristics of the colonies and the morphology of the organism will not be considered at this time, but it might be stated that many modifications occur on various media. Beyerinck: Bot. Ztg. (1888), 46, 725; also 741, 757, 780, 797. , or 1/25,000 of an inch. 484 BULLETIN No. 179 [March, GROWTH AND ENDURANCE OF B. RADICICOLA The optimum temperature for B. radicicola varies between 18 and 26C. The thermal death point, according to Zipfel, 1 is 60 to 62 C. Growth is perceptible between 3 and 46 C. B. radicicola is not very sensitive to the reaction of the medium, which may be either acid or alkaline. Under field conditions, the organism exists in extremely acid soils, especially the race peculiar to legumes which thrive well on very acid soils. Experiments have dem- onstrated that the bacteria can withstand any degree of acidity or of alkalinity in the soil, that the particular legume itself can endure. That the organism endures at least two years in dry soil was de- termined by Ball. 2 Harrison and Barlow 3 found that the limit of viability on ash-maltose-agar varied somewhat, but that in the ma- jority of cases it was about two years. , No doubt the organism will live much longer than this on artificial media when suitable conditions of growth are maintained. How long the clover or alfalfa organism will exist in a soil under field conditions is not yet known, but prac- tical observations indicate that it must be many years. The statement is quite generally made that B. radicicola becomes "nitrogen hungry" when cultivated thru several generations on nitro- gen-free media. This fact has not been sufficiently demonstrated to be accepted, for while Siichtung, Hiltner, and others have found that these organisms survive cultivation on nitrogen-free media for a year and at the end of that time possess the same ability to effect inocula- tion and nitrogen fixation in the legume as organisms obtained from fresh nodules, yet the bacteria had apparently made no appreciable gain in ability to effect inoculation. Garman and Didlake 4 failed to find, that nitrogen-free medium possessed any particular advantage over a legume-extract medium in causing the organism to become "nitrogen hungry." INDENTITY OP B. RADICICOLA It is believed by some that the various legumes have different species of bacteria. Evidence has been produced which indicates that nodules do not contain but a single race of infecting organisms. Gino de Kossi 5 reported the finding, in artificial cultures, of two organisms which differed in that one formed a large hyaline colony, not developing well in beef and peptone gelatine, while the other 'Zipfel: Centbl. f. Bakt. 2 Abt. (1912), 32, 97-137. Five minutes taken as the time of exposure instead of ten minutes. "Ball: Centbl. f. Bakt. 2 Abt. (1909), 23, 50. 'Harrison and Barlow: Centbl. f. Bakt. 2 Abt. (1907), 19, 429. 4 Garman and Didlake: Ky. Agr. Exp. Sta. Bui. 184, 352. "Gino de Rossi: Centbl. f. Bakt. (1907), 18, 289-314, 418-489. 1915] A BIOCHEMICAL STUDY OP NITROGEN IN CERTAIN LEGUMES 485 formed white non-transparent colonies in beef gelatine. He believed that he had found another organism associated with B. radicicola. This work has not been sufficiently substantiated to be accepted as final. Grreig-Smith 1 reported having found three races of this or- ganism in the same nodule. Hiltner and Stormer 2 classify nodule bacteria into two groups, Rhizobium radicicola and Rhizobium beyer- inckii. The former they associate with lupines, serradella, and soy- beans ; the latter with all other legumes. On the other hand, the results of many investigators 3 (especially Laurent, 4 who obtained nodules on the pea with organisms from thirty-six different legumes, and Nobbe et al., 5 who worked on the adaptability of nodule bacteria of unlike origin in different genera of Leguminosae) , seem to support the theories of the identity of nodule organisms and the presence of only one race in the nodule. On the whole, present experimental evidence is slightly in favor of the view that there is only one species of this organism thruout the entire family of legumes. 6 This conception is not easily reconciled with field observations, for under natural conditions this organism has become so modified as to make it appear that there are many species. Contamination of the nodule has undoubtedly been responsi- ble for varying conclusions in this connection. ENZYME PRODUCTION BY B. RADICICOLA Hiltner 7 reported the finding, by filtration thru porcelain, of a substance produced by B. radicicola which can dissolve the cell wall of root hairs. No proteolytic enzyme has as yet been reported. Further, Beyerinck claims that no enzyme has been found which at- tacks lime, starch, or cellulose, or which is capable of inverting sac- charose. In recent studies, Fred, 8 altho unable to detect a proteolytic enzyme, obtained slight evidence of the presence of oxidases in the slime of various legume bacteria. These results suggest the need of further studies on enzyme production by this organism. Similar to all microorganisms, it has the ability to reduce methylene blue to the colorless leuco-compound. ^reig-Smith: Jour. Soc. Chem. Indus. (1902), 26, 304-306. 2 Hiltner and Stormer: Arb. K. Gsndhtsamt, Biol. Abt. (1903), 3H. 3, 151. "Harrison and Barlow: Centbl. 2 Abt. (1907), 19, 429. Kellerman: Centbl. f. Bakt. 2 Abt. (1912), 34, 45. Buchanan: Centbl. f. Bakt. 2 Abt. (1909), 22, 371. 4 Laurent: Exp. Sta. Kee. (1890), 2, 186. "Nobbe et al.: Centbl. f. Bakt. 2 Abt. (1895), 1, 199. " " : Ibid (1900), 6, 449-457. 'Kellerman (see note 3) reported inoculation of soybean, lupine, and also of alfalfa from a culture originally isolated from alfalfa and kept on artificial media in the laboratory for six years. 'Hiltner: Centbl. f. Bakt. 2 Abt. (1900), 6, 273. "Fred: Vir. Agr. Exp. Sta. Ann. Ept. 1910-11, 123. 486 BULLETIN No. 179 [March, SLIME PRODUCTION BY B. RADICICOLA B. radicicola produces, on artificial media, a gum, or slime, which is partly soluble and party exists as a zoogloeal mass. The organisms, on suitable media, have been observed to surround themselves with definite capsules several times thicker than themselves. These cap- sules are rather distinct at first but later form a gelatinous mass. Greig-Smith 1 and Maze, 2 who studied this slime, claimed for it a nitro- genous substance. The results obtained by Buchanan, Gage, and Fred agree and are in direct refutation of the above. This is impor- tant to bear in mind in connection with the theories later to be dis- cussed. Gum is not formed from a carbohydrate containing less than five 'carbon atoms. ISOLATION OF B. RADICICOLA FROM SOIL Until recently B. radicicola had never been very successfully isolated from a normal soil except by means of a legume plant. Gage, 3 after a long, tedious process, obtained from soil an organism which was capable of producing nodules on red clover and which appeared to be identical with B. radicicola. Still more recently Greig-Smith 4 has reported the isolation of this organism from soil, but his work has not been substantiated by others. Maze early attempted the isolation of B. radicicola from sterilized and non-sterilized soils to which pure cultures had been added. He isolated the organism from the soil which had been sterilized before the addition of the culture, but he was unable to recover it from the unsterilized soil. Kellerman and Leonard 5 isolated (on the agar rec- ommended by Greig-Smith) an organism which inoculated alfalfa from soil that had been sterilized and subsequently inoculated with living organisms of B. radicicola. Lipman and Fowler 6 were able to isolate the organism peculiar to vetch (Vicia sicula) on soil-ex- tract agar and proved out the organism. They attained success in about 40 percent of the cases, judging from the condensed report recently published. DISSEMINATION OF B. RADICICOLA Those familiar with pot-culture experiments and inoculation ex- periments with legumes easily understand that legume bacteria are disseminated in many ways. In fact, sterile conditions are difficult to maintain. The layman, however, may wonder how legume bacteria 'Greig-Smith: Centbl. f. Bakt. 2 Abt. (1911), 30, 552-556. 2 Maze: Ann. Inst. Pasteur (1898), 12, 128. Gage: Centbl. f. Bakt. 2 Abt. (1910), 27, 7-48. 4 Greig-Smith: Centbl. f. Bakt. 2 Abt. (1912), 34, 227-229. B Kellerman and Leonard: Science (1913), 38, 95-98. Lipman and Fowler: Science (1915), 41, No. 1050, 256-258. 1915} A BIOCHEMICAL STUDY OF NITROGEN IN CERTAIN LEGUMES 487 not common to a certain locality creep in. A few agents concerned in the transfer of the organisms are here cited. The seeds themselves are a common means of distribution. The ruptured seed coats offer an opportunity for the various kinds of bac- teria to accompany the seeds in a most persistent manner. Sometimes wind is responsible for the dissemination of these bacteria. An inter- esting instance is cited by Ball of Texas: a wind storm blew off the roof of the culture-house in which legumes were being grown under sterile conditions, and as a consequence the various plants under ob- servation became inoculated. Water has been known to aid in inocu- lating large areas during washing and floods. The transfer of un- cleaned seed is sure to result in the conveyance of some inoculating or- ganisms in the impurities accompanying the seed. Cultivation, espe- cially harrowing, is also responsible for the spread of the organisms. The addition to soils of legume residues from either fields or stables is still another common means of dissemination. FIXATION OP NITROGEN WITHOUT THE LEGUME PLANT Maze, 1 in 1897, demonstrated that nodule bacteria have the power to assimilate atmospheric nitrogen in the absence of a legume. His researches have been verified by others, altho the amounts of nitrogen obtained in his experiments have never been equaled. Recently con- ducted experiments 2 on this question show that as an average, in liquid and in solid media, about 1.2 milligrams of nitrogen are fixed per 100 cc. of medium. A fixation in the absence of the legume plant has been found in sterile sand and in soil. How important this kind of non-symbiotic fixation is, has yet to be more fully determined. At the present time it is generally recognized as insignificant compared with symbiotic fixation in the nodules of legumes. BACTEROIDS Bacteroids are believed to be a form which appears in the devel- opment of B. radicicola. The cell activities of this form are main- tained in a way similar to that in which the cell activities of the rod- shaped form are maintained. Stefan 3 states that these bacteroids are thin-walled and capable of division when young, but that when older they become swollen and finally degenerate. Fred was able to observe the changes which occur in the organism in passing from the bacillus to the extreme vacuolized bacteroidal form. The organism at first apparently thickens at one end and then branches into the bacteroid, which is characterized by rounded outgrowths, known as vacuoles. These vacuoles appear at a definite period of growth and evidently 'Maze: Ann. Inst. Pasteur (1897), 11, 44. 2 Fred: Vir. Agr. Exp. Sta. Ann. Ept. 1909-10, 138-142. 'Stefan: Centbl. f. Bakt. 2 Abt. (1906), 16, 131-149. 488 BULLETIN No. 179 [March, are not a sign of polymorphism, but are a further development of the bacteroid. (They require special staining to be made visible.) * J~ Bacteroids occur in the nodule as we ^ as on culture media. Their morphology varies according to the constituents of the culture media. A, Some writers prefer to call these ir- & t -1& p p /] regular organisms degenerate or in- ^" Y volution forms. They were first ob- Fig G.-Bacteroids, showing d j artificia l media in 1888 by shape, and occurrence of . . J vacuoies Beyermck, 1 and have been studied by Hiltner, 2 Stutzer, 3 Buchanan, 4 Fred, 5 and others. A medium rich in carbohydrates or the glucosides of amygdalin or salicin offers very favorable conditions for bacteroid formation. Of fifteen carbohydrates tested, mannite has proved par- ticularly suited to their development. Glycerine is better than most nutrients, while the salts of organic acids have been found unsuitable. On careful observation the following factors have been found to exercise no influence upon bacteroid formation: temperature, light, osmotic pressure, decreased oxygen pressure, reaction of medium, ni- trogen-hunger, specific formative materials in the legumes, and the ac- cumulation of metabolic products. From the above observation it is evident that nutrition is a strong factor in bacteroid formation. It is claimed that each of the various legumes exhibits a different shaped bacteroid which is characteristic of that legume. In studies conducted at the Virginia Experiment Station the bacteroids pos- sessed by the Egyptian, the crimson, and the red clover were found to be very similar, while those of the vetch differed somewhat. More extended research regarding the appearance of bacteroids as connected with the beginning of nitrogen assimilation, is sorely needed. THEORIES OF ASSIMILATION, FIXATION, AND IMMUNITY THEORIES OF ASSIMILATION BY THE PLANT In brief it may be said that the two main suppositions regarding assimilation are as follows : (1) that the bacteroids are bodily absorbed by the plant fluids; and (2) that the bacteroids, by some sort of change, produce the substance containing the assimilable nitrogen which the plant utilizes. The theory that the plant absorbs these bacteroids has been chal- lenged by some on the evidence which Nobbe and Hiltner 6 produced 'Beyerinck: Bot. Ztg. (1888), 46, 725. 'Hiltner: Centbl. f. Bakt. 2 Abt. (1900), 6, 273. 'Stutzer: Ibid (1901), 7, 897. 4 Buchanan: Ibid. (1909), 23, 59-91. "Fred: Vir. Exp. Sta. Ann. Ept. 1909-10, 128-198. 'Nobbe and Hiltner: Centbl. f. Bakt. 2 Abt. (1900), 6, 449. 1915} A BIOCHEMICAL STUDY OP NITROGEN IN CERTAIN LEGUMES 489 to show that a plant had fixed 1 grain of nitrogen while its nodules weighed only .3 gram. The relationship between the amount of nitro- gen fixed and the weight of the nodules is no criterion, however, for criticism of such a theory, inasmuch as this relationship is not at all definite but varies according to the development and needs of the plant. The failure to establish the presence of a proteolytic enzyme has also been responsible for no little criticism of the first supposition. While the second supposition seems more plausible, it must be ad- mitted that it, too, is only a theory which should be studied with the hope of isolating and identifying the diffusible substance. In connec- tion with this theory Golding 1 conducted some very interesting experi- ments on the removal of the products of growth in the assimilation of nitrogen by legume bacteria. He reasoned that the plant played an important role in the removal of the products produced by bacteria in the nodule aside from the mere furnishing of suitable food. In his experiments he used a porous Chamberland filter-candle placed in a culture vessel to serve to imitate natural conditions. Aerobic condi- tions were obtained by passing purified air thru the cultures. The parts of the plants used in some of his experiments were sterilized in order to avoid the possibility of plant enzyme action. As a result of his method of experimentation, he obtained a much greater fixation of nitrogen than other experimenters had found, and the logical conclu- sion arose that the plant performs a function in the assimilation of nitrogen which is construed to be the removal of soluble products of growth. The results of Golding 's most extensive experiment are embodied in the table below: N.in grams 500.0 grams of Stems and Leaves 2.865 20.2 grams of Eoots and Nodules (quite fre:h) 094 3000.0 cc. Ammonia-free Distilled water.. .000 Total Nitrogen to start with 2.959 2870.0 ec. Filtrates and Drainings 731 566.2 grams of Wet Besidue 2.570 To'al Nitrogen after experiment 3.301 Total Gain of Nitrogen during experiment 342 THEORIES REGARDING THE CHEMICAL PHENOMENA OP FIXATION As yet, purely chemical theories of fixation are entirely hypo- thetical ; however, they deserve consideration, for even theories un- supported by facts may have a value in stimulating thought and as- sisting in the development of more rational views. Frank, 2 a prominent Frenchman, was among the first to attempt 'Golding: Jour. Agr. Sci. (1905), 1, 59-64. 'Frank: Landw. Jahrb. (1888), 17, 504-518; 19, 564. 490 BULLETIN No. 179 [March, an explanation of how the plant actually obtains nitrogen. He be- lieved that it came in thru the leaves, and even recently some isolated statements hold to this idea. His view was allied with the conception of stimulation which many held ; namely, that the organisms on the root stimulated the plant to fix nitrogen in its leaves. Stocklasa, 1 as a result of his chemical investigations, also believed that assimilation took place thru the leaves, that amides were first formed, and that .these, migrating to the nodules, reacted with glucose and produced protein, which served as the nutrient medium for the bacteria. In this connection he advanced the idea that the bacteria produced an enzyme which enabled the plant to effect this fixation. Loew and Aso 2 in 1908 suggested that ammonium nitrite was the first compound produced, the nitrous acid being readily reduced to ammonia. Little evidence has been obtained to support this theory; no evidence whatever has been found under controlled conditions. Gautier and Drouin 3 suggested that the nitrogen is oxidized to nitric and nitrous acid. Winogradsky 4 has advanced the idea that the free nitrogen in the plasma of the organism may unite with nascent hydrogen and form ammonia, which by oxidation would become assimi- lable. In connection with the two latter theories, it should be empha- sized that the presence of nitrites, nitrates, or ammonia in the nodules, roots, or tops of legumes, inoculated or uninoculated, when grown in the entire absence of combined nitrogen, has not been established. Gerlach and Vogel 5 have investigated non-symbiotic nitrogen fixation and have arrived at the conclusion that there is a direct union of free nitrogen with some organic compound inside the bacterial cell. Heinze thinks it probable that nitrogen is at once brought into com- bination with a hydrocarbon (glycogen), and suggests that a salt of carbamic acid may be formed first or that carbamic acid may be pro- duced from cyanamid. There is yet another most extraordinary theory which, owing to its somewhat recent notoriety, it seems appropriate to consider. This theory is most properly called the Jamieson theory. The rather pecu- liar views embodied in it are perhaps quite well explained in an article published in The Spokesman Review, Spokane, Washington, March 28, 1913. The Review is a bi-weekly paper devoted to agricultural inter- ests. 'Stocklasa: Landw. Jahrb. (1895), 24, 827-863. 2 Loew and Aso: Bui. Col. Agr. Tokyo Imp. Univ. (1908), 7, 567. 3 Gautier and Drouin: Bui. Soc. China. Paris 78, 84-97. 4 Winogradsky: Centbl. f. Bakt. 2 Abt. (1901), 7, 842. "Gerlach and Vogel: Centbl. f. Bakt. 2 Abt. (1902), 9, 817-821, 881-892; (1903) 10, 636-644. 'Heinze: Landw. Jahrb. (1906), 35, 907. 1915} A BIOCHEMICAL STUDY OP NITROGEN IN CERTAIN LEGUMES 4-5O gi CO Oi rH CO CM CO | .SW g x_x ' fc lg| J M* s 0? "tf ^ be a "a ^ gi^ ^^ ^ c 5 co * IH S S S gSU i a a nil? W g 33 33 333 33 3-3 "3 OO OO OOO OO fr 'll ** s^^^^ o do 1 d ^00 to ^3 11 +!++ 4- oooo^oo oo c "3 S iJ 33 CO GO CO GOC092GQ C}(l}Q}cGGGCOGQC} OOOO C^C^CjOOOOc^ oooo oia>a)OOooa) ^ a A-* [v] p-^ A"j j .1 . j frt fvj rrf p< . "i V ^ CO 0) a J ^"^ s ^ 2 ^'ojq a *> 0^0 ~ a S'loS^uii oSt~t^cococoi i I: =H 'C f^J j^ O O> Q p. I- d t-l Kl P5 rt< IO )) )> Apr. 25 May 10 May 17 May 24 May 31 38 53 60 67 74 4 leaves 6 " 8 " 10-12 leaves; an average of 5.4 pods 10-12 leaves; beans formed in pods 6 11 14 16-17 16-17 265 1 061 987 1 154 1 354 The total nitrogen determinations in the various parts of, the plants of this series at different stages of growth, together with the nitrogen fixed at each of these stages, are given in Table 15. The amount of the nitrogen fixed was determined by subtracting, from the Thompson: Jour. Am. Chem. Soc. (1915), 37, 230-235. 1915} A BIOCHEMICAL STUDY OF NITROGEN IN CERTAIN LEGUMES 529 total nitrogen found, the average nitrogen content of five soybean seeds as shown by the individual analyses of twenty seeds. Nearly all the figures in this table represent the average of six determinations. TABLE 15. TOTAL NITROGEN IN VARIOUS PARTS OP SOYBEANS AND FIXATION AT DIFFERENT PERIODS: SERIES 100 (Milligrams per jar of five plants) Harvest Lab. Nos. Nitrogen in tops Nitrogen in roots Nitrogen in nodules Nitrogen in whole plants Nitrogen in seeds Nitrogen fixed 111-112 1 114-115 87.10 13.35 28.04 128.49 57.30 1 71.19 117-118 2 121-129 204.59 22.70 47.10 274.39 57.30 217.09 3 131-139 286.91 43.44 82.95 413.30 57.30 356.00 4 141-149 356.52 40.15 60.40 457.07 57.30 399.77 5 151-159 247.82 30.82 54.56 333.20 57.30 275.90 *It would be preferable to use the analyses of uninoculated plants as a check rather than the analyses of seeds. These figures need very little explanation. The results of the first four harvests show a gradual increase in the amount of nitrogen fixed. The low results obtained at the last harvest are in accord with the results of Wilfarth and Wimmer 1 and Penny and MacDonald. 2 The separation of the nitrogen compounds into the various groups was carried out in this series as follows : The whole sample was dried for four hours at 50 C. both before and after grinding. The subsam- ples were weighed out and placed in 250-cc. beakers ; 100 cc. of water was then added and the whole heated to boiling and filtered while hot. Suction was used in filtration, and the washing was done with 50 cc. of hot water. The residue was Kjeldahlized as usual. The filtrates from some of the harvests were treated with 10 cc. of sodium hydroxid and distilled. The residual liquid in the Kjeldahl flask was trans- ferred to a 350-cc. beaker and the excess alkali neutralized with sul- furic acid, after which the regular P.T.A. method was applied. The precipitates obtained by P.T.A. were characteristic in their behavior. After the reagent had been added some one or two hours, a volumi- nous grayish white precipitate appeared, sometimes colored a yellow- ish green, and very gradually settled to the bottom in a very thin layer, leaving the supernatant liquid yellowish green in the case of 1 Wilfarth and Wimmer: Landw. Vers. Stat. (1906), 63, 1-70. 'Penny and MacDonald: Del. Agr. Exp. Sta. Bui. 86, 35. 530 BULLETIN No. 179 [March, the tops, slightly straw colored to colorless in the case of the roots, and colorless in the case of the nodules. The amount of the precipitate from the determination with the tops was much greater than that with the roots and nodules. Caution was exercised in all the determinations not to allow losses or changes due to bacterial action. A few drops of chloroform were placed on the filter and in the nitrate when the determinations were sufficiently long to be liable to bacterial action. The results of the separations are shown in Table 16. Here again, as in the case of the total nitrogen determinations, most of the figures represent the average of two determinations made upon samples from each of three jars. The total soluble nitrogen was obtained by the addition of the various determined soluble forms. The total nitrogen reported in the last column is the sum of all the separations made. These results will be discussed later with those of the other series. TABLE 16. NITROGEN SEPARATIONS: SERIES 100 (SOYBEANS) (Milligrams per jar of five plants) Har- vest Lab. Nos. Part Insol- uble nitro- gen Total solu- ble nitro- gen NaOH nitro- gen P.T.A. nitro- gen Other nitro- gen Total nitro- gen 85.91 13.90 1 111-112 114-115 Tops Eoots 61.52 8.90 24.39 5.00 4.16 .85 20.23 4.15 117-118 Nodules 15.72 11.61 3.54 8.07 27.33 2 121-123 124-126 Tops Eoots 135.15 15.49 37.99 5.67 8.11 .48 29.88 5.19 173.14 21.16 127-129 Nodules 32.83 16.03 9.66 6.37 48.86 3 131-133 134-136 Tops Roots 146.79 27.03 140.12 16.42 25.63 .93 114.49 15.49 286.91 1 43.45 137-139 Nodules 47.95 35.00 18.55 16.45 2 82.95 1 4 141-143 144-146 Tops Eoots 183.35 26.14 134.26 12.93 17.86 2.49 25.96 .85 90.44 9.59 317.61 39.07 147-149 Nodules 31.77 27.27 2.38 15.35 9.54 59.04 5 151-153 154-156 Tops Roots 151.68 21.55 95.32 14.38 12.02 1.34 29.31 1.38 5S.99 2 11.66 247.00 1 35.93 157-159 Nodules 29.23 27.21 2.00 12.13 13.08 56.44 'Taken from Table 15. 2 Obtained by difference. The accompanying graph (Plate XV) shows clearly the relation- ship in which the soluble and the insoluble nitrogen exist at the various periods of growth. In this series the first harvest was not made for thirty-eight days, and therefore the period at which fixation began is not shown. Attention has already been called to the low nitrogen fixa- 1915} A BIOCHEMICAL STUDY OP NITROGEN IN CERTAIN LEGUMES 531 SOLUBLE ^MD INSOLUBLE. IN Toi^s Ffoors /=IND NODULES OF SOYBEANS TEDIOUS OF Ser.es IOO 5o/.uBLE CH INSOLUBLE 7b/s III Tfoor-5 I I I I Z7a,. 60 Da.. t,7 U~ NODULES PLATE XV 532 BULLETIN No. 179 [March, tion found at the last harvest in this series. More results are necessary to confirm the supposition of a possible loss in the total nitrogen. The importance of the amount of soluble nitrogenous compounds at the various stages of growth has not yet been emphasized. Prelimi- nary studies have shown this soluble nitrogen to be much more rap- idly converted into ammonia and nitrates than the insoluble nitrogen. This would seem to have a direct bearing upon practical methods of handling leguminous crops in rotations when the shortest time must intervene between the turning under of the legume and the planting of the next crop. It would seem desirable to choose that period when the greatest amount of soluble nitrogen exists. SERIES 500 (SOYBEANS) Soybeans were used in Series 500. The plants were placed out of doors during pleasant days in August and September. From Table 17, showing the development at the four harvests, it will be seen that these plants made a more rapid growth than those in Series 100. TABLE 17. PLANT DEVELOPMENT: SERIES 500 (SOYBEANS) Planted Harvested Age, days Leaves and pods per plant Height, inches Nodules per 15 plants Aug. 8, 1911 > > >} Aug. 22 Aug. 30 Sept. 8 Sept. 19 14 22 30 41 3 leaves 4 " 6 " 7-8 " ; 6 one- inch pods 7 10 14 14 30 1 278 370 247 (large) Tor ten plants. The total nitrogen determinations for this series are shown in Table 18. These results agree with those shown in Table 15, altho they represent earlier stages of development. TABLE 18. TOTAL NITROGEN IN VARIOUS PARTS OF SOYBEANS AND FIXATION AT DIFFERENT PERIODS: SERIES 500 (Milligrams per jar of five plants) Har- vest Lab. Nos. Nitro- gen in tops Nitro- gen in roots Nitro- gen in nodules Nitro- gen in whole plants Nitro- gen in seeds Nitro- gen fixed 1 2 3 4 511-519 521-529 531-539 541-549 46.99 47.32 96.96 205.40 8.50 11.08 9.76 18.65 .35 11.05 17.81 26.98 55.84 69.45 124.53 251.03 57.30 1 57.30 57.30 57.30 (-1.46) 12.15 67.23 193.73 J This figure is approximate rather than exact. See note to Table 15, page 529. The separations in this series differed from those in Series 100 in that in this case a cold-water extract was made. The sub-samples 1915] A BIOCHEMICAL STUDY OF NITROGEN IN CERTAIN LEGUMES 533 were placed in shaker bottles and the same amount of water added as in the former series; the bottles were then put in a mechanical shaker for three hours. This method was considered to be more in ac- cord with natural conditions than the one used in the former series. The nodules were filtered thru a diatomaceous earth filter. The figures in Table 19 were obtained in the same manner as those in Table 16. TABLE 19. NITROGEN SEPARATIONS: SERIES 500 (SOYBEANS) (Milligrams per jar of five plants) Har- vest Lab. Nos. Part Insol- uble nitro- gen Total soluble nitro- gen NaOH nitro- gen P.T.A. nitro- gen Other nitro- gen Total nitro- gen 1 511-512 514-515 517-518 Tops Roots Nodules 19.48 4.42 25.90 4.48 3.16 .95 3.31 .33 19.43 3.20 45.38 8.90 2 521-523 524-526 527-529 Tops Roots Nodules 29.60 7.97 17.34 2.14 1.18 .00 4.96 .47 11.20 1.67 46.94 10.11 3 531-533 534-536 Tops Roots 70.04 8.56 31.64 2.29 2.67 .00 6.48 .70 22.49 1.59 101.68 10.85 537-539 Nodules 16.75 1.29 .00 .33 .96 18.04 4 541-543 544-546 Tops Roots 115.39 13.16 76.11 7.35 10.68 2.06 65.43 5.29 191.50 20.51 547-549 Nodules 22.55 2.46 .26 2.20 25.01 SERIES 700 (SOYBEANS) Soybean seeds were planted on September 6 for this series, but owing to their damping off, the jars were replanted on September 13. The seedlings that damped off were tested for ammonia, nitrites, and nitrates, with negative results. The conditions of the plants at the various harvests are shown in Table 20. TABLE 20. PLANT DEVELOPMENT: SERIES 700 (SOYBEANS) Planted Harvested Age, days Leaves per plant Height, inches Nodules per 15 plants Sept. 13, 1911 Sept. 25 12 2 leaves 5 Nodules pres- ent too small to remove Oct. 6 23 3 leaves par- tially devel- oped 8-9 254 > i j > Oct. 14 31 5 leaves 10-12 229 Oct. 25 42 7 leaves 12 288 534 BULLETIN No. 179 [March, The figures reported in Table 21 present the average of duplicates of composite samples which included the whole of the material from three jars. As will be seen, these results are concordant with those of the two series already considered. TABLE 21. TOTAL NITROGEN IN VARIOUS PARTS OP SOYBEANS AND FIXATION AT DIFFERENT PERIODS: SERIES 700 (Milligrams per jar of five plants) Har- vest Lab. Nos. Nitro- gen in tops Nitro- gen in roots Nitro- gen in nodules Nitro- gen in whole plants Nitro- gen in seeds Nitro- gen fixed 1 2 3 4 711-719 721-729 721-739 741-749 40.08 48.00 68.32 110.70 8.88 8.93 11.38 20.55 Vii 8.97 17.22 48.96 64.14 88.67 148.47 57.30 1 57.30 57.30 57.30 (-8.34) 6.84 31.37 91.17 1 See note to Table 15, page 529. The figures in Table 22 showing the nitrogen separations were ob- tained in the same manner as those reported for the nitrogen fixation in Table 21 ; that is to say, they are the averages of duplicates of com- posite samples. TABLE 22. NITROGEN SEPARATIONS: SERIES 700 (SOYBEANS) (Milligrams per jar of five plants) Har- vest Lab. Nos. Parts Insol- uble nitro- gen Solu- ble nitro- gen P.T.A. nitro- gen Other nitro- gen Total nitro- gen 1 711 714 Tops Boots 9.94 3.87 28.03 4.95 4.70 .39 23.33 4.56 37.97 8.82 717 Nodules 2 721 724 Tops Boots 23.64 5.50 21.56 2.55 11.96 .77 9.60 1.78 45.20 8.05 727 Nodules 3 731 734 Tops Boots 26.50 7.30 29.05 3.76 13.75 1.10 15.30 2.66 55.55 11.06 737 Nodules 6.30 1.79 .10 1.69 8.09 4 741 744 Tops Boots 46.45 10.67 61.97 6.31 23.75 .61 38.22 5.70 108.42 16.98 747 Nodules 14.52 2.70 .00 2.70 17.22 The close agreement of the results of Series 700 and 500 is very evident in the data presented. By reference to the accompanying graph (Plate XVI) it will be seen more easily than in the tabular form that the soluble nitrogen predominates in the early growth of the seedling. The amount decreases during this period, however, while 1915] A BIOCHEMICAL STUDY OP NITROGEN IN CERTAIN LEGUMES 535 \DOLUBLE f]ND IN TOPS T^OOT 5 frvD NODULE 5 or Db Of -L/C OOL uBL e I _ I //v5Oi UBl E To Pi . 5 /TOO' E 5 PLATE XVI 536 BULLETIN No. 179 [March, the insoluble nitrogen always increases. This is true of the roots as well as the tops. It is during the period between the twelfth and the twenty-second days that nitrogen fixation begins, according to meas- urements by the most accurate chemical methods. Detailed studies are now being made of the exact time when fixation begins. SERIES 600 (COWPEAS) Cowpeas were used for Series 600. Owing to the smaller nitro- gen content of cowpea seeds, the plants show a need of nitrogen much sooner than soybeans, and are therefore perhaps better suited to ex- perimentation of this sort. The plants grown in this series are com- parable with the soybeans in Series 500 as regards time and conditions of growth. The data in Table 23 show the development of the cow- peas when harvested. TABLE 23. PLANT DEVELOPMENT: SERIES 600 (COWPEAS) Planted Harvested Age, days Leaves per plant Height, inches Nodules per 15 plants Aug. 8, 1911 i y )> ) > Aug. 22 Aug. 30 Sept. 8 Sept. 19 Oct. 5 14 22 30 41 58 3 leaves 4 " 5 " 6-7 " 8 " 7 10 12 13-14 14 450 (very small) 892 (small) 1074 2062 1992 The results given in Table 24 show a fixation of nitrogen at the end of fourteen days from the time the seeds were placed in the sand. The increased fixation is greater with the cowpeas in this series than with the soybeans in the corresponding series (500). The other gen- eral tendencies appear to be the same as in the other series. TABLE 24. TOTAL NITROGEN IN VARIOUS PARTS OP COWPEAS AND FIXATION AT DIFFERENT PERIODS: SERIES 600 (Milligrams per jar of five plants) Harvest Lab. Nos. Nitrogen in tops Nitrogen in roots Nitrogen in nodules Nitrogen in whole plants Nitrogen in unin- oculated plants Nitrogen fixed 1 2 3 4 5 611-619 621-629 631-639 641-649 651-659 29.11 45.46 91.63 188.40 439.64 9.05 10.25 16.64 30.28 73.73 1.96 9.22 18.52 42.33 64.25 40.12 64.93 126.79 261.01 577.62 36.74 1 36.74 36.74 36.74 36.74 3.38 28.19 90.05 224.27 540.88 a This figure was used as it is a little larger than the average nitrogen con- tent for the seeds, 34.70 milligrams, which would make even a greater fixation ap- pear at the harvest. The results of the separations are shown in Table 25. The figures were obtained in the same manner as those in Series 700. 1915] A BIOCHEMICAL STUDY OF NITROGEN IN CERTAIN LEGUMES 537 TABLE 25. NITROGEN SEPARATIONS: SERIES 600 (COWPEAS) (Milligrams per jar of five plants) Harvest Lab. Nos. Part Insoluble nitrogen Total soluble nitrogen P.T.A. nitrogen Other nitrogen Total nitrogen 1 611-613 Tops 15.83 11.62 3.68 7.94 27.45 614-616 Roots 6.92 2.13 .74 1.39 9.05 617-619 Nodules 2 621-623 Tops 23.73 21.76 5.88 15.88 45.49 624-626 Eoots 9.18 1.89 .42 1.47 11.07 627-629 Nodules 7.54 2.26 .92 1.34 9.80 3 631-633 Tops 58.67 32.54 10.55 21.99 91.21 634-636 Roots 11.18 5.32 1.90 3.42 16.50 637-639 Nodules 13.69 4.83 1.22 3.61 18.52 4 641-643 Tops 97.82 93.47 32.49 60.98 191.29 644-646 Roots 21.54 7.61 2.51 5.10 29.15 647-649 Nodules 26.74 15.59 9.08 6.51 42.33 5 651-653 Tops 216.90 226.50 58.50 168.00 443.40 654-656 Roots 52.35 19.87 5.17 14.70 72.22 657-659 Nodules 32.36 31.89 20.10 11.79 64.25 The results of this series are also presented in a graph (Plate XVII). The curve of the soluble nitrogen does not show a decrease, possibly because the change upward had taken place before the end of the first fourteen days, when the first data were taken, as cowpeas show an early fixation of nitrogen and develop extremely rapidly un- der normal conditions. The same general tendencies hold thruout this series as in the others in respect to the increase of the soluble and insoluble nitrogen. When the soluble and the insoluble nitrogen ratios are considered, the results in general agree very closely in all the series regardless of time of growth and kind of legume. DISCUSSION OF TABLES The results of the total nitrogen determinations of the four series show that, as an average of eighteen harvests, 74 percent of the nitro- gen of the cowpeas and soybeans was in the tops, the remaining 26 percent being divided between the roots and the nodules. The figures show that in the first periods most of the 26 percent was in the roots, while later the nodules in some cases contained 18 of the 26 percent. In nine out of seventeen harvests, the nodules contained more nitro- gen than the roots of the same plants. The data showing the average daily fixation of nitrogen for five plants in the various series during the different growing periods are presented in Table 26. 538 BULLETIN No. 179 [March, F/HD INSOLUBLE N/TFIOOEN IN TOPS, floor 5 f)ND NnDuLEz OF Cow-?->Ea> D SOLUBLE O I To 7=5 '007-5 J PLATE XVII 191.5] A BIOCHEMICAL STUDY OF NITROGEN IN CERTAIN LEGUMES TABLE 26. AVERAGE DAILY FIXATION OF NITROGEN IN ALL SERIES 539 Series Periods in days Milligrams per jar of 5 plants 0-38 1.87 ~\r\c\ 38-53 9.72 J.UU (Soybeans) 53-60 60-67 19.84 6.16 67-74 (-17.69) 0-14 .00 500 14-22 1.51 (Soybeans) 22-30 6.88 30-41 11.50 0-12 .00 700 12-23 .62 (Soybeans) 23-31 3.06 31-42 5.43 0-14 .24 14-22 3.10 600 (Cowpeas) 22-30 30-41 7.73 14.02 41-58 17.44 The results included in Table 26 seem to indicate that the period during which the greatest total accumulation of atmospheric nitrogen takes place occurs between the fortieth and sixtieth days and coincides closely with the period just previous to seed formation. The greatest rate of increase of fixation and assimilation in these series occurred in the early periods of growth. A comparison of Series 100 with the other series indicates that the growth of the plant is closely related to the rate and the amount of nitrogen fixed. The plants of Series 100 grew much slower than the others. Those of Series 500 and 600 made the greatest growth in a given period, having had the advantage of the most favorable growing season, more especially for the cowpea, which requires a higher temperature than the soybean for optimum growth. The fixation in Series 600 for the whole period of fifty-eight days was 540.88 milligrams per five plants, or an average daily fixation of 9.32 milligrams. The greatest fixation during any one period, as is evident from Table 26, occurred with the soybeans in Series 100 be- tween the fifty-third and sixtieth days, when the daily average was 19.84 milligrams per five plants, or nearly 4 milligrams per plant per day. If this figure is calculated to an acre basis, allowing a stand of four beans per square foot, an accumulation equivalent to one and a half pounds of nitrogen per acre per day is shown. The average percentages of soluble nitrogen in the four series in terms of total nitrogen in the particular part of the plant, may be of some interest, altho it will be seen from the accompanying graphs 540 BULLETIN No. 179 [March, that the amount depends upon the stage of growth when the harvest is made. These percentages were as follows: TABLE 27. PERCENTAGES OF SOLUBLE NITROGEN IN EACH SERIES AS AN AVERAGE OF ALL HARVESTS (On the basis of total nitrogen in the given part) Series Tops Boots Nodules 100 (Soybeans) 700 (Soybeans) 500 (Soybeans) 600 (Cowpeas) 35.9 57.7 41.2 45.0 34.7 39.5 32.4 27.5 42.3 1 18.9 8.5 34.0 J The nodules in this series were not filtered thru a diatomaceous earth filter but thru an ordinary filter and are therefore not included in the average given in the conclusions. The figures for Series 100 represent soluble nitrogen obtained in a hot-water extract. Series 500 and 600 are comparable with the exception of one being soybeans and the other cowpeas. The great difference in the solubility of the nitrogen in the nodules is particu- larly noticeable. There was a gradual increase in the soluble nitrogen in the nodules of Series 600 from the first harvest to the last, the percentages on the basis of total nitrogen being 23, 26, 37, and 49. A fact not brought out in the figures showing the soluble nitrogen is that in Series 700 and 500 an extremely high soluble-nitrogen content was found in the tops and the roots at the first harvest. In Series 700 the percentage in the tops was 74, in Series 500, 57 ; while in the roots in Series 700 the percentage was 56, and in Series 500, 50. On the basis of total nitrogen, the percentage of Other nitrogen in each series, as an average of all harvests, was as shown in Table 28. Other nitrogen is the difference between the total soluble nitrogen and that precipitated by P.T.A. and NaOH. It has been shown that a part of this nitrogen consists of amino acids, but as yet the total amount is unknown. TABLE 28. PERCENTAGES OF OTHER NITROGEN IN EACH SERIES AS AN AVERAGE OF ALL HARVESTS (On the basis of total nitrogen in the given part) Series Tops Roots Nodules 100 (Soybeans) 700 (Soybeans) 500 (Soybeans) 600 (Cowpeas) 25.1 36.1 30.7 28.3 29.3 32.8 23.1 17.2 18.2 6.9 16.6 The percentages of nitrogen precipitated by P.T.A. were as shown in Table 29. The variations as regards the tops are not easily ex- plainable. Undoubtedly there is a larger error in the P.T.A. deter- minations than in the others. The nodules of Series 600 contained 1915] A BIOCHEMICAL STUDY OF NITROGEN IN CERTAIN LEGUMES 541 large amounts of nitrogen which were precipitated by this reagent, the percentages at the harvests, from the second to the last, on the basis of total nitrogen being 9, 7, 21, and 31. TABLE 29. PERCENTAGES OP P.T.A. NITROGEN IN EACH SERIES AS AN AVERAGE OF ALL HARVESTS (On the basis of total nitrogen in the given part) Series Tops Eoots Nodules 100 (Soybeans) 700 (Soybeans) 500 (Soybeans) 600 (Cowpeas) 7.00 21.10 6.75 13.40 3.0 6.6 6.0 7.3 LO 1.4 17.0 The nitrogen obtained by distillation with sodium hydroxid ap- parently is not precipitated by P.T.A., as the percentage of Other nitrogen decreases without exception when sodium hydroxid is used. However, no definite conclusions can be drawn regarding the use of sodium hydroxid. CONCLUSIONS PART I 1. The experiments reported show conclusively that the cowpea and the soybean utilize atmospheric nitrogen thru their roots and not thru their leaves. No combined nitrogen could have been assimi- lated in these gas experiments. PART II 2. The total nitrogen determinations show that about 74 per- cent of the nitrogen of cowpeas and soybeans at the time of harvest is in the tops, while the remainder is distributed between the roots and the nodules. In the earlier periods the roots contain the larger part, while later they contain much the smaller part. 3. The percentage of soluble nitrogen in soybeans and cowpeas varies with the different parts of the plant and with the period of growth. In these experiments the soluble nitrogen, as an average, constituted in the tops about 45 percent of the total nitrogen; in the roots, 34 percent ; in the nodules of the soybeans, 14 percent, and in the nodules of the cowpeas, 34 percent. 4. Phosphotungstic acid usually precipitates some form of nitro- gen. In some cases the amounts precipitated vary widely, while in others the agreement is close. In these series the nitrogen precipitated by phosphotungstic acid averaged in the tops of both soybeans and cowpeas about 12 percent of the total nitrogen; in the roots, 5.5 per- cent ; in the nodules of the soybeans 1 percent, and in the nodules of the cowpeas, 17 percent. 542 BULLETIN No. 179 [March, 5. Other forms of soluble nitrogen than those precipitated by phosphotungstic acid and sodium hydroxid occur. In these series they constituted as an average in the tops of both soybeans and cow- peas about 68 percent of the soluble nitrogen; in the roots, 77 per- cent ; in the nodules' of soybeans, 89 percent, and in the nodules of cowpeas, 53 percent. 6. Fixation takes place at a very early period in the growth of the seedling sometimes within fourteen days. It is rapid in some cases, especially with cowpeas. 7. Plants grown under the conditions of these experiments con- tain no ammonia, nitrites, or nitrates, as measured by the most ac- curate chemical methods. It is fully recognized that this work is incomplete, yet it is hoped that the study may aid in stimulating interest in some of these funda- mental problems. The lack of development of the various chemical methods used is partially responsible for some of the difficulties ex- perienced in their application. The survey presented of the chemical literature indicates a scarcity of existing knowledge regarding the more fundamental problems concerning nitrogen fixation. The biological resume points clearly to the need of more extended research along these lines. This is strikingly noticeable in the bac- teriological studies which have been undertaken with B. radicicola. Few cases are on record in which the authors actually proved out their cultures at the termination of their investigations by inocula- tion of a legume of the kind from which the organism originally came. The author takes this opportunity to express his gratitude to Professors C. G. Hopkins and J. H. Pettit for the many valuable sug- gestions they have so kindly given him. UNIVERSITY OF ILLINOIS-URBANA Q 630.7IL6B C001 BULLETIN. URBANA 166-181 1914-15 ^w "4 '?' "^ ' * >; I* *T JT'., **, ^M^ 30112019528436