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UNIVERSITY OF ILLINOIS
Agricultural Experiment Station

BULLETIN No. 179

A BIOCHEMICAL STUDY OF NITROGEN IN
CERTAIN LEGUMES

By ALBERT L. WHITING

URBANA, ILLINOIS, MARCH, 1915

CONTENTS OF BULLETIN No. 179

PAGE
EN TRODUCREON 2. 5 tiieccvecorarnicrererscersyss yew ssi toes oS oie oe) tele G biote ages esate. alae 471
EME CLC AM yori cise co sarel oe sieteroneisisleisiwlarsuals racer ole teaieta ale aeieioate aieue tion srwelese 472
ES TOT OGG AT arte, feats cee asaye eta eee aie tateiel anede ard cssar anchors angi sid alan bun okestaccretehova stays Hiete 475
HE CEL ON tere occ faus cocci cresobener oes aie osua ote csalsasrageer tata aT ere Cie orotate tote sta sr eraie 476
Inoculation as It Occurs Under Field Conditions.....................4. 480
GOW th «OL tH 7 IN OCUIO 5 :c;ccratesn sc siatens fevsie erotic Hel cncresaher ot ycicn aeatenst casts tome sarejanete 481
BACT WRIOGOGICAUH gos cscvescrachaicieos aisraieieteaisloteas(orcts or aici cicereiasa ehepesaratereie ease 482
Bacillus PAdicieola se scsicscpsvtisis aks Siepst ane ios cosets Mees aieased e eine Oe Maneiareiaieiee See 483
Growth: and; Endurancesof B. tadicicolay ...c2.2).cnsis ace cide oer nas steers wisuere 484
EGOUDULGY§ OL bose TAG CLCOLA ic cise ror ct a fal orale oi stay oh ol cxs say od eet one MopoR ee oeie pees io oneal oe pone 484
ENZVINO- ETOCUCLLONT DY. 5s -TACICLCOlA rs crore acai foyeqous ues als elores toys ysies ci jaws 485
Slime:Production.by Bb: Tadicicolans seas cies prise cette reir ereie a een eioneele 486
Isolation of B. radicicola..... RS issllhy ganenis sfevedeva Neue. Oievere ismeuee eres raiers Tee ee acetate te 486
Dissemination: Of: Bi TAAICICOL A... 5 s0.ete 5 iiss wsle ce shed &. oveesiatoh suave tevin ay eueders eieiais ace 486
Fixation of Nitrogen Without the Legume Plant...................... 487
SACEONOLOS ters ciersioys erotes otonsle stars Searetate wie cto tere nts cots tareratintan spayarel sree Se foratalavanctarsie ts 487
THEORIES OF ASSIMILATION, FIXATION, AND IMMUNITY........ 488
Theories of: Assimilation: by. the’ Plant ss.5c25 3)2)2 2s ssisrs 34 se -gisac epee cleo 488
Theories Regarding the Chemical Phenomena of Fixation.............. 489
PHEOLICS OL -LINMUNI GY! oor: sia accinis cate sec eter erate occceta oiaiaie “slonef oholegsna? ciel sneve.s ener et= 492
PRACTICAL CONSIDERATIONS WITH REGARD TO LEGUME
TENTEXGATISTIOUNG oie cost. Saris eu'es octets vate aitas oes aa o5/ctieca; cee anelevaliel® aber ora tahay acsioter netetsrars 493
Mattia (Sym Diss ey j cise ces tesco css ohecste lea siacs oie te vero carte a etetaieee ie atele eras etoile eeeetens 493
Amount of Nitrogen Fixed per Acre per Year.................ccceees 495
Value of Legumes as Nitrogen Retainers. ...............cccceeccecees 496
Cross! INOCULATION. (<:s12:015 ote aieierei se t)6 aie wheres DCR ig Meek wae 497
Associative Growth of Legumes and Non-Legumes..................... 498
RTS MEL CAUSA prcecirav stot scck at yaa etevesto Suz ie ator tan cleuct el wistera tata iets laierskeng stabs Biacere otaenies esters 499
ee LEN EAE rs oO cre a ata aia ceiy 8 Seat aha ae lata tn aigia se eratae alee neikes eae covlaly aay 503
PlanGoLolnvesti ations sfsiiva 7 foysioie ts (ere ie stare ete eis “elocelslaveie releis e/ee aera ateteeenersts 503
Part I: SrupIES TO DETERMINE THRU WHICH ORGANS LEGUMES OBTAIN
‘ATMOSPHERIC NITROGEN 9. cic cicislsisicls ores Gristarcisiafe sie alar sveveicitraruw isis cise iets 503
General ‘Plan: of “Experiments «ffir tied cite wee oils MS chess eae 505
EXP OVUM Ont vhosvececens jerevote w claters cies siete ape rare ale Oretor etal wiatavaeiatecia atte ts 507
PSXPOELM OME HE licsrersyeiei cs ovetsyepcin menparstetnia whe ceiesaie ate ate feceyelele wists ads lores iocels 507
Dab giv sire aaa lB ESS Aaa erro ioe sree Si ben hearye seis Tore Co poe CP eee 509
ER DGTaOWNe: V6 0555584 5 as ops 45h oe 4S ER EN RAS Saws aS 514
Experiment by OnsOwitaenl....vaiccoiss vicki sd ae beens slesien ses 519
General Consideration of Gas Experiments.................... 521

Practical Application Of Tosulte. .i0o.i <. bs cs cnet cto sdacvces 522

UNIVERSITY OF ILLINOIS LIBRARY

JUL 9 - 1915 Be

Part II: RELATIVE PERCENTAGES OF NITROGENOUS COMPOUNDS IN THE
VARIOUS PARTS OF THE SOYBEAN AND COWPEA AT DEFINITE PERIODS

OP AMROWTE oes ei cn cs ncce sho 0 ue pp mMMEOR eS oss 6 cate 5 ¢e- sue 522
Methods Employed in the Growth and Preparation of Samples. .523
Analytical Methods: «os... ..scc « seeeeemetelaleare ois kale sia-aseveio eles 525
Discussion of Some of the Methods Used................0-6- 527
Qualitative Testa 2 scsi. occu coke eee ee ta eaten sane min eee 527
Series 100 (Soybeans)... 6. cs sces dav c wae cee wale ume perma 528
Series 500 (Soybeans)... ... oo. ess cele a Seles a) q alate eae 532
Series: 700: (Soybeans) sii. v..ce sdccrerecal titlohe rete leceheeoeeeremaiateie seers 533
Series 600. (Cowpeas) ....5 6.20 ter cee bee pn ale ener atanions 536
Discussion Of “Tables s.c:s sia 5scccssce alate gree ase nclets) a ace eae eae 537

CONCLUSIONS 54:4 aiew one e waareea wees ceo eae wea ear * 541

ILLUSTRATIONS

PLATE PAGE
I.—Nodules of Robina Type on Roots of Soybeans................-4.- 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. cts setsraie sie sip she Ee ee ee Ce eR eee 479
IV.—Cowpea Seedlings in Preparation for Gas Experiments............. 504
V.—Experiment II: Cowpeas at Harvest (37 Days)................6- 506
VI.—Experiment II: Plants Grown in Air and in CO,+0............. 508
VII.—Experiment III: At Beginning and 10 Days Later................ 510
VIII.—Experiment III: 52 Days from Beginning and at Harvest (83
DPV) Biase rciccets tevale leeciatace tinier ttateretaleae Goer oteoeteer a eimetele itoeiare eis totetee 511
IX.—Experiment III: Roots from Plants Grown in CO, + 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)............cc cece eee ewes 517
XITI.—Experiment IV: Roots from Plants Grown in CO,+O and in
Be OO el Oar te alanine ane PTC IOC OR ee ET IT 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
Ge INOOUAG 5 52a isis ss ids sin bbe ODE OT AT VME aN oes 6 Whe RO RENE 476

3.—Young nodule magnified, showing affected root hair and same root
hair more. Dighly magnified . 53). ea gasn< os.os de gobs ales's.a d's ote ee es 480

4.—Young nodule, showing the beginning of the differentiation of its tissues. .482
5.—B. radicicola, showing shape and flagella.................ceeeceeseees 483

6.—Bacteroids, showing shape, and oceurrence of vacuoles...............++: 488

A BIOCHEMICAL STUDY OF NITROGEN
| IN CERTAIN LEGUMES!

By ALBERT L. WHITING, AssociaTE 1n Sort BioLtocy

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 sourees 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.? Of this amount 1614 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.

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.? 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. 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,* mention is
made of the Roman farmers regarding beans as possessing the prop-
erty of enriching the soil, and atténtion 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

*Review of Reviews, April, 1910.

“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.

*Marshall: Microbiology (1911), 273.

1916] A BIocHEMICAL Srupy oF NITROGEN IN CERTAIN LEGUMES 473

may be found among the writings of Thaer and Walz. Gasparin?
constantly calls attention to the power of leguminous plants to add
nitrogen to the soil. Jethro Tull? 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* in 1881 reported results that were of both chemi-
eal 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® 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,® and Lawes, Gilbert, and Pugh,’
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® made the
classical discovery that legumes obtain atmospheric nitrogen thru the
association of microdrganisms 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.

*Tbid.

‘Lipman: Bacteria in Relation to Country Life (1908), 206.
“Schultz-Lupitz: Landw. Jahrb. (1881), 10, 777.

‘Frank: Landw. Jahrb. (1888), 17, 501.

*Boussingault: Ann. Sci. 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. 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 second 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 microdrganisms 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? 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? after growing legumes in a confined at-
mosphere, gave out the following direct evidence of the fixation of at-
mospherie nitrogen.

Atmospheric nitrogen introduced

into culture. vessel, i...0 6600555 oiler are os 2681.2 cem.
Atmospheric nitrogen withdrawn.............. 2653.1 ecm
Amount of nitrogen assimilated.............. 28.1 cem.
(=36.5 mg.)

Nitrogen in the soil and crop...............-. 73.2 mg.
Nitrogen in the soil and seed................ 32.6 mg.
Nitrogen aasimilated: < scs'ssus ss Giwedien ko b6 ee 40.6 mg.

*Hellriegel and Wilfarth: Beil. Zert. d. Verins. fiir die Rubenzucker in-
oan a Berlin, Nov., 1888; or Lafar Handbuch der technischen Mykologie (1904-
06), 3, 31.

*Nodules substituted for tubercles.

*Schloesing and Laurent: Compt. Rend. Acad. Sci. (1890), 111, 750; (1892)
115, 659, 732.

1915] A BrocHEMIcAL Stupy oF NITROGEN IN CERTAIN LEGUMES 475

In addition to the scientists mentioned above, Atwater and Woods,
Berthelot, Miintz, Ville, Mazé, Dehérain, 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,! which are the visible manifestations of infection, were
observed upon the roots of legumes by Malphighi? 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 lL. C. Treviranus.®

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* 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 deseribe 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 the soil-transfer method and the glue method’ of inocu-
lation, both of which are recogmzed today as superior to the use of so-
called commercial cultures.

*Nodules are recognized on the following non-leguminous plants: alders
(Alnus glutinosa), silverberry (Eleagnus), sweet gale (Myrica Gale), sago palm,
an evergreen, (Podocarpineae), eycads (Cycacadeae), birthwort (Aristolociaceae).
Nitrogen-fixing bacteria resembling B. radicicola have been found in the alder,
silverberry, sweet gale, and five varieties of podocarpus.

*Malphighi: Op. (1687), 2, 126, Leiden.

*Treviranus: Bot. Ztg. (1853), 11, 393.

‘Jost: Plant Physiology (Gibson 1907), 237.

*Ward: Phil. Trans. Roy. 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 ;! 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, mene 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—Root hair of com- Fig. 2.—Root cell, showing in-
mon pea (Pisum sati- fecting strand passing thru it
vum), showing infecting and the formation of lamellae
strand (x300) (After (x650) (After Prazmowski)
Prazmowski)

*Tschirch: Ber. deut. Bot, Gesell. (1887), 5, 58.

1915] A BiocHEMICAL Stupy oF NITROGEN IN CERTAIN LEGUMES 477

PLATE I.—NopULES OF ROBINA TYPE ON Roots or SOYBEANS
(Enlarged)

‘

478 BuuLeTin No. 179 . [March,

Puate II.—NopuLes or LuPINus Type oN Roots or LUPINE SEEDLINGS
(After A. Meyer)

A BIOCHEMICAL Stupy oF NITROGEN IN CERTAIN LEGUMES 479

1915]

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480 BuLueTIn 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 FreLp ConpDITIONS

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! 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.”

Fig. 3.—Young nodule magnified, showing af- The organisms, by rapid
fected root hair and same root hair more Broint d wth d-:
highly magnified (After Atkinson) Ivision and growth, ad-

vance thru the center of

the infected root hair. Prazmowski® found organisms in the cell

*Pierce, G. J.: Proc. Cal. Acad. Sci. II, No. 10 (1902), 295-328. Pierce found
the proportion with bur clover to be 1: 1000.

*Fred: Vir. Agr. Exp. Sta. Ann. Rpt. 1909-10, 123-125.
*Prazmowski: Landw. Vers. Stat. (1890), 37, 160-238.

1915] A BIocHEMICAL Stupy oF 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 pericyecle. 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! 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

*Pierce, G. J.: Proc. Cal. Acad. Sci. II, No. 10 (1902), 295-328,

482 : BuLuEeTIN 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.

EOD
ae

ae
eS
re <x

S
e

“

Ee

esse

o

Xyferm
Fie. 4.-—Young NopuLE, SHOWING THE BEGINNING OF THE DIFFERENTIATION
oF ITs TissuES (After Prazmowski)

BACTERIOLOGICAL

Minute bodies were first detected in nodules in 1866 by Woronin,}!
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.?

*Woronin: Bot. Ztg. (1866), 24, 329.
*7Wigand: Bot. Heft. Forsch, a.d.Bot. Bart. 3 Marburg (1887), 288. °

1916] A BiocHEMICcAL Srupy or NITROGEN IN CERTAIN LEGUMES 483

Immediately afterward, Beyerinck! 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 Phytomoyza.
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 5y.2_ It is not uncommon to find them from .5 to
.6% wide and from 2 to 3y long, and some have been found to measure
_ only .184 wide and .9n 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

LZ (see page 487), predominate in the older
structure.
Fig. 5.—B. radicicola, show- B. radicicola grows well on a great va-

ing shape: soe Sears riety of culture media, perhaps best on

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.
74—=micron, 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 26°C. The thermal death point, according to Zipfel,! 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® 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* 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 OF 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 Rossi® 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.

‘Garman and Didlake: Ky. Agr. Exp. Sta. Bul, 184, 352.

*Gino de Rossi: Centbl. f. Bakt. (1907), 18, 289-314, 418-489.

1915] A Biocnemica, Srupy or NirrogeN 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. Greig-Smith! reported having found three races of this or-
ganism in the same nodule. Hiltner and Stormer? classify nodule
bacteria into two groups, Rhizobium radicicola and Rhizobium beyer-
inckti. 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* (especially
Laurent, 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’ reported the finding, by filtration thru porcelain, of a
substance produced by B. radicicola which ean dissolve the cell wail
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,® 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 microérganisms, it has the ability to reduce methylene blue to the
colorless leuco-compound.

1Greig-Smith: Jour. Soc. Chem. Indus. (1902), 26, 304-306.

*Hiltner and Stérmer: Arb. K. Gsndhtsamt, Biol. Abt. (1903), 3H. 8, 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.

- ‘Laurent: Exp. Sta. Rec. (1890), 2, 186.
"Nobbe et al.: Centbl. f. Bakt. 2 Abt. (1895), 1, 199.
77-97-99 = Thid (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. Rpt. 1910-11, 123.

486 BuuuetTiIn 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! and Mazé,? 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 Sor

Until recently B. radicicola had never been very successfully
isolated from a normal soil except by means of a legume plant. Gage,®
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*
has reported the isolation of this organism from soil, but his work
has not been substantiated by others.

Mazé 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® 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® 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.
*Mazé: Ann. Inst. Pasteur (1898), 12, 128.

*Gage: Centbl. f. Bakt. 2 Abt. (1910), 27, 7-48.
*Greig-Smith: Centbl. f. Bakt. 2 Abt. (1912), 34, 227-229.
“Kellerman and Leonard: Science (1913), 38, 95-98.
“Lipman and Fowler: Science (1915), 41, No. 1050, 256-258.

1915] A BI0OcHEMICAL Stupy 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 OF NITROGEN WITHOUT THE LEGUME PLANT

Mazé,! 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? on this question show that as an average, in liquid
and in solid media, about 1.2 milligrams of. nitrogen are fixed per
100 ee. 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-symbiotie 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* 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

*Mazé: Ann. Inst. Pasteur (1897), 11, 44.
*Fred: Vir. Agr. Exp. Sta. Ann. Rpt. 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.)

, Bacteroids occur in the nodule as

V4 well as on culture media. Their

morphology varies according to the

co @ onl LA constituents of the culture media.

ee 9 & a writers same to eall these ir-

; regular organisms degenerate or in-

Vv be ? 2 sets } volution forms. They were first ob-

Fig. 6.—Bacteroids, showing — served in artificial media in 1888 by
shape, and occurrence of A ’

vaeucien Beyerinck,! and have been studied by

Hiltner,? Stutzer,? Buchanan,‘ Fred,®

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,
osmotie 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® produced

*Beyerinck: Bot. Ztg. (1888), 46, 725.

*Hiltner: Centbl. f. Bakt. 2 Abt. (1900), 6, 273.

*Stutzer: Ibid (1901), 7, 897.

“Buchanan: Ibid. (1909), 23, 59-91.

*Fred: Vir. Exp. Sta, Ann. Rpt. 1909-10, 128-198.

‘Nobbe and Hiltner: Centbl. f. Bakt. 2 Abt. (1900), 6, 449.

1915] A BIOCHEMICAL Stupy or NITROGEN IN CERTAIN LEGUMES 489

to show that-a plant had fixed 1 gram of nitrogen while its nodules
weighed only .38 gram. The relationship between the amount of nitro-
gen fixed and the weight of the nodules is no eriterion, however, for
eriticism 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! 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 rdle 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. Aerobie 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

000,0 -grams:of Stems. and: eaves 2:2: i sates 6 oid os aan as oaks ee cae eee 2.865
20.2 grams of Roots and Nodules (quite freth)................. 00.00% 094 -

3000:0. cc, Ammonia—free Distilled water... 5... 0.05 tein ct ees ces wine eletene .000

Total Nitrogen to start: With: 6:5. 0.02 Fs 25s Mey eka en ca ope 2.959

een0.0 cc. _Kiltrates. and Draimngs. .<.. 59:25. Scie + so ares A were sin ale oe 731

566.2 prams. Of Wet. Residue sis:s ice oid Sir 's%0. 02s. 2e ered ne Sib, ease ccd ais cobays Neca 2.570

Total Nitrogen’ after experiments oo... dette wissen oo piss os oO 3.301

Total Gain of Nitrogen during experiment............. ‘age ae Be sabe 342

THEORIES REGARDING THE CHEMICAL PHENOMENA OF 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,? 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,! 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? 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? suggested that the nitrogen is oxidized to
nitric and nitrous acid. Winogradsky* 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® 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 eyanamid.

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.

*Loew and Aso: Bul. Col. Agr. Tokyo Imp. Univ. (1908), 7, 567.
*Gautier and Drouin: Bul. Soc. Chim. Paris 78, 84-97.
*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 BiocHEemMicaLt Srupy or NITROGEN IN CERTAIN LEGUMES 491

‘¢Do PLANTS DirEcTLY ABSORB FREE NITROGEN FROM THE AIR?
’ ¢¢ScorcH SCIENTIST IS AT ODDS WITH THE COMMON BELIEF

‘¢The doctrine that plants directly absorb free nitrogen from the air con-
flicts with the earlier beliefs. It is held that plants, with the exception of
legumes, cannot utilize the nitrogen of the air, the explanation in the case of
legumes being that by the aid of nitrogen in organisms on their roots these
plants utilize atmospheric nitrogen.

‘‘Thomas Jamieson, Director of the Agricultural Research Association of
Scotland, takes issue with this belief. Mr. Jamieson has made a life study of
the problems of plant nutrition. An abstract of his views is given in the New
Zealand Journal of Agriculture. Mr. Jamieson proceeds to show:

‘“1, That the legume-tubercular theory is untenable.

‘¢2. That the nitrogen of the air is directly used.

‘¢3. That the application of this knowledge is valuable to the agricul-
turalist HHEKEKEK EE

‘‘Mr. Jamieson disagrees with the theory that the tubercle formation on
leguminous plants is a normal growth containing a net structure of plant food
thru the union of fungus and a legume. He says:

‘¢ <T regard the tubercles as abnormal growths. I hold that no ‘‘symbiotic’’
action takes place; that the fungus is not a fixer of the nitrogen; that the
legume plant is itself the fixer, and that it sends its manufactured albuminous
products to heal up the wound or to counteract the drain of the parasitic fungus;
and that the tubercle has nothing to do with the fixation of the nitrogen of

the air KREKKKKKKKKKKKKEK)

‘*As to the explanation of tukercles of leguminous plants Mr. Jamieson

says:
‘¢ «The plant keing attacked by the fungus, a wound is made, the fluid of
the plant courses to repair it, and not only is the leguminous fluid of the plant
rich in nitrogen, but its most nitrogenous fluid, albumen, is just a plastic material
like the white of an egg, especially suited to heal the wound and to form a sac
round the invader.

‘¢ «There is nothing exceptioral in the bearing of tukercles by the legume.
The nodules, or tubercles, are well displayed. The legume is a plant specially
sought by fungus demanding nitrogen. It is provided with a means of supplying
the element, hence it is specially attacked.’ ;

‘‘Further investigations lead Mr. Jamieson to the conclusion that nature pro-
vides special means for all plants to absorb nitrogen. Even the hardest leaves
are soft in the earlier stage. The cultivated members of the legume family have
broad, soft leaves studded with apertures, supposed to serve for exhalation. It
is accepted that the green cells or the chlorophyl contained by these cells de-
compose the carbonic-acid gas. Cannot a similar action extend to nitrogen?

HKKHKKKKKKKKKKKKE

‘‘The effect on the soil of producing certain crops, as cereals and grasses,
is to reduce the available plant foods. Of these, in their simple forms, the most
important supplied by fertilizers are nitrogen, phosphorus, and potash, and of
these the farmer can avoid the expense of the purchase of nitrogenous manures
by the adoption of a rotation to include those plants that are rich in nitrogen.
This is not new. Legumes have been availed of from the earliest recorded time
as preparatory to cereals. What is new is that there is a wider field of plants
for selection and the farmer knows why these plants enrich the soil for the ni-
trogen-demanding cereals. The plants, among others mentioned by Mr. Jamieson,
are rape, mustard, and turnips.’’

' The above theory has received more contradiction and less sup-
port than the others reviewed.

*Henry: Ann. Sci. Agron. (1909), 26, 102-130.
Vageler: Centbl. f. Bakt. 2 Abt. (1909), 22, 452.
Kovessi: Compt. Rend. Acad. Sci. (1909), 149, 56.
Kny: Ber. deut. Bot. Gesell. (1909), 27, 532.

Mameli and Pollacci: -Ann. Sci. Agron. (1914), 31, 141.

492 BuuuetTiIn No. 179 [ March,

Three possible chemical processes in fixation have been consid-
ered by scientists:
1. Reduction
2. Oxidation
3. Direct union into an organic compound

The first two possible processes have received no chemical verification,
while the third is supported only by data which are of an eliminative
character. More data of a similar nature will be found in Part II
of the experimental section of this bulletin.

It is interesting to note that opinion seems to be scenpthenlae
in support of the theory of the direct union of nitrogen gas into
organic combination, in spite of the fact that such a combination is
unknown in chemistry to take place at ordinary temperatures. It is
possible, however, to unite nitrogen into organic combination at at-
mospheriec pressure, altho a high temperature is required.

THEORIES OF IMMUNITY

Reasoning from animal life, it seems logical for one to believe
that the relative strength of a legume plant or of B. radicicola may
vary under certain conditions so that the plant will resist the entrance
of the organism. Inoculation experiments have produced data show-
ing that B. radicicola causes a certain resistance on the part of the
plant, making it necessary in some cases to employ organisms of
greater efficiency in order to produce inoculation.

Hiltner! has given the six following conditions as instances in
which immunity demonstrates itself.

1. The organisms cannot get into the plant.
2. The organisms gain admission into the plant but do not pro-

duce nodules because the plant, by its greater resistance, absorbs the
bacteria.

3. The organisms enter the plant and produce nodules, but no
fixation of nitrogen occurs.

4. The organisms enter, produce nodules, and nitrogen is fixed
and assimilated by the plant.

5. The organisms are so efficient in comparison with the plant
that the latter is injured.

6. The organisms are parasitic and the plant is actually killed.

In the pursuance of the investigations reported in this bulletin
no indication of the existence of any of these conditions, except No.
4, was observed, and in no instance under normal conditions did in-
oculation fail to produce nodules and cause a fixation and an assimi-
lation of nitrogen.

*Lafar: Handbuch der technischen Mykologie (1904-6), 3, 45.

1915] A BIocHEMICAL Strupy or NITROGEN IN CERTAIN LEGUMES 493

Various other similar theories of resistance have been proposed, all
of which are permeated with the idea of natural or acquired immunity.
Prominent among these might be mentioned Siichtung’s theory of
equilibrium. Siichtung assumed that the bacteria produced a toxin
and the plant an antitoxin, and that the degree of equilibrium deter-
mined the extent of nodule formation, the plant becoming immunized _
by an antibody and not by a substance produced by the bacteria;
further, that the nitrogen supply in the plant was regulated by the
production of this antibody. It is conceivable that in some of the cases
observed the apparent immunity may have been due to a weakness on
the part of the bacteria rather than to resistance by the plant. Siich-
tung’s equilibrium theory considers varying conditions of virulence on
the part of the bacteria and varying degrees of resisting ability by the
plants. His theory was advanced as the result of carefully conducted
experiments which showed that there were variations in virulence
between organisms of the same kind when grown upon artificial media
and when obtained from fresh nodules.

The inoculation of legumes in solution is inhibited by potassium
nitrate, tho a convincing explanation of this inhibition has not yet
been offered. Some experimenters believe that the immunity of a
plant is strengthened by its nitrogen nutrition; others hold that
bacteria find another source of nitrogen nutrition in the nitrate and
hence do not seek the plant. It has been recently shown, however,
that the organism will produce nodules after the concentration of the
nitrate has been reduced by the plant, which would tend to show that
the immunity of a plant is not strengthened and that the organism
is not permanently injured by a solution of potassium nitrate suit-
able for the plant.

Inoculation under field conditions is no doubt inhibited by physi-
eal and antagonistic biological factors, which have been considered
only briefly by most investigators.

The subject of immunity in plants has been given little attention
thus far, but the increasing number of bacterial diseases in the plant
kingdom will undoubtedly lead to research in this direction. It is well
known that bacterial diseases of plants are the most difficult to control ;
the need of investigation in this unexplored field of immunity as an
aid in their control is imperative.

PRACTICAL CONSIDERATIONS WITH REGARD TO
LEGUME FIXATION

Mutua SyMBIOsIsS

Mutual symbiosis may be defined as the contiguous association of
two or more morphologically distinct organisms not of the same kind,

*Siichtung: Centbl. f. Bakt. 2 Abt. (1904), 11, 377.

494 BULLETIN No. 179 _ [March,

resulting in an acquisition of assimilated food substances. It implies
that the organisms concerned have the power of independent exist-
ence, but that both are benefited by the close association.

The relationship existing between B. radicicola and legumes is
one of mutual symbiosis. The facts which bear out this belief are too
convincing to need explanation. However, some prefer to call the re-
lationship a truly parasitic condition, while others consider it to be
parasitic in the beginning and later a true mutual symbiosis. This
latter conception would seem to be plausible, yet no exact data have
been produced to show that a parasitic condition exists at any stage.!

The result of this mutual symbiosis is wonderfully characteristic
of nature as well as astounding when one considers the corresponding
chemical process, in which the energy expended is so apparent and the
temperature required so high.2 The energy values in the symbiotic
fixation of nitrogen by B. radicicola and legumes have never been de-
termined. When B. radicicola and Azotobacter are grown under simi-
lar conditions, apart from their respective hosts,? less organic carbon
per unit of nitrogen fixed is oxidized by B. radicicola than by Azoto-
bacter. Present knowledge indicates that a very great amount of
energy is necessary for the fixation of atmospheric nitrogen by Azoto-
bacter. |

Table 1 presents the amounts of some of the ecmmon materials
that must undergo rather complete oxidation in order to furnish suf-
ficient energy for the addition of fifteen pounds of atmospheric nitro-
gen to the surface soil of an acre by Azotobacter. The figures show
that in the case of dextrose 6624 times as much organic matter is re-

TABLE 1.—AMOUNTS OF MATERIALS NECESSARY FOR THE FIXATION OF FIFTEEN
POUNDS OF ATMOSPHERIC NITROGEN PER ACRE By AZOTOBACTER

Kind of material Pounds required
Dextrose: (SUlar ))< 5 cancel oeicices «is sie’ oiolevete tsi srecss ste 1 000
IBTOSN-. CLOVER: COPS <2 01 o/<'s terse oe 3iec Ure stetayare otoists ote ont 1 212
Pree? IUDINO. LONE 25's 0.5) 4,0 Sinaia eae Monee 2 000
Wik OS Gi SUES Wee cas ocsic's:o xtzo sols cates nese icit ei aici 4 300
CCOTNT STOVER veces e tie he Ste eS en alwieia he elaoesiene 5 500
ORB OA VOR niente Ree Pein artes gine tote emt arate ete views 11 500

*This figure represents the minimum amount of dextrose consumed per unit
of nitrogen fixed; in other words, 1 gram of dextrose (yielding 3,750 calories)
is necessary for the fixation of 15 milligrams of atmospheric nitrogen by Azoto-
bacter.

*Experiments are now in progress at this station with the view of obtaining
data on this question.
_ “The most recent figures show that 1 kilowatt hour yields 70 grams of nitrogen
in the form of cyanamid; in other words, 1.35 horse-power yields 70 grams, or
8.74 horse-power per hour yields 1 pound of nitrogen.
; “Algae are understood as the host for Azotobacter. The word host as used
in this publication is not intended to convey the idea of parasitism.

1915] A BIocHEMICAL Srupy OF NITROGEN IN CERTAIN LEGUMES 495

quired as there is nitrogen fixed, and in the ease of oak leaves, 766
times as much. In the oxidation of such large amounts of organic
carbon it is easily seen that the volume of organic matter in the soil is
greatly reduced.

It has been definitely shown that Azotobacter lives in a symbiotic
relationship with algae. It is also well known that our normal soils
possess an abundant algal flora. In view of these two facts it may
possibly be found that nitrogen is accumulated by Azotobacter with-
out the above reduction in the volume of the organic matter of the soil.

Whatever the future may disclose, the only fact that now re-
mains to be pointed out is that in the symbiosis between legumes and
B. radicicola, instead of there being a decrease in the organic matter
of a soil, a material increase is bound to result. While fixation pro-
eresses, organic matter is being manufactured by the plant, which is
later returned to the soil. This process is, then, a constructive one, as
compared with the destructive non-symbiotic fixation.

AMouNT OF NITROGEN FIXED PER ACRE PER YEAR

The amount of nitrogen added to a soil depends in part upon the
relative supply of that element in the soluble and decomposable forms,
organic as well as inorganic. The poorer the soil the greater the
amount of nitrogen that will be fixed, tho in a rich soil in which the
nitrogen is not in an available form, large amounts may be fixed.
For instance, altho a peat soil contains in one acre some thirty
thousand pounds of nitrogen in the surface million pounds (0 to 634
inches), yet because of a lack of proper organisms in the soil to
decompose the organic matter, which resists natural decay and there-
fore does not readily furnish nitrogen to plants, a legume crop may
add large amounts of that element.

Where nitrates are present in large amounts, they are taken up .
by legumes; but where they are present in only small amounts, as is
the case during dry seasons even on the common prairie corn-belt land,
atmospheric nitrogen is fixed by legumes. The fixation varies with
the seasonal conditions, a hot, moist season being best suited to the
summer legumes. Among other factors the kind of legume and the
duration of its growing period affect the amount of nitrogen added.
The annual legumes must necessarily fix nitrogen much faster than
the biennial or perennial legumes. The yield does not necessarily in-
dicate the amount of fixation, as some legumes which yield much less
hay and seed than others may have a greater total nitrogen content.

The most reliable data which now exist indicate that two-thirds
of the nitrogen in legumes grown on soils of normal productive power
is obtained from the air.1 These figures, contributed by the Illinois

*Hopkins: Ill. Agr. Exp. Sta. Buls. 76 and 94.

496 . BULLETIN No. 179 [ March,

Experiment Station, were obtained by analyzing inoculated and unin-
oculated legumes from like areas of normal soils, and as a result of pot
experiments. Computed by these data, a 3-ton erop of cowpea hay
adds 86 pounds of nitrogen per acre, a 25-bushel crop of soybeans
with 214 tons of straw adds 106 pounds, a 4-ton clover crop adds 106
pounds, and a 4-ton alfalfa crop adds 132 pounds.

A nitrogen gain of 200 pounds per acre has been reported by the
New Jersey Experiment Station* with crimson clover. At the Rhode
Island Experiment Station,’ as a result of a pot-culture experiment, it
was found that nitrogen had been added at the rate of 400 pounds per
acre per year. This experiment extended over five years, and legumes
were grown both in the summer and in the winter. The tops of the
summer legumes (cowpeas and soybeans) were removed from the soil,
while the winter legume (vetch) was turned back into the soil. It
should be noted, however, that an acre'of this soil to a depth of 6%
inches contained only a little over three thousand pounds of nitrogen.
- Moreover, in this experiment optimum conditions were established,
and no losses were possible from drainage,—which factors would tend
to make these results much higher than would be obtained under field
ecnditions.

In considering soil enrichment by clover, ten years’ results of a
field experiment at the Experimental Farms, Ottawa, Canada,* are
important. In this experiment a light, sandy loam with a sandy sub-
soil was planted to clover continuously, being reseeded every two
years. The clover was cut and left to decay on the land. In ten
years the nitrogen content of this soil was doubled. The yearly gain
of nitrogen was fifty pounds per acre. It was found that from two
to three times that amount was added, but that all but fifty pounds
was dissipated by bacterial activities and in other natural ways.
Analyses of the clover crop also brought out the fact previously men-
tioned that the amount of nitrogen fixed is influenced in part by the
season.

VALUE OF LEGUMES AS NITROGEN RETAINERS

Legumes have a very great value aside from their réle in the
nitrogen-fixing process. It is well known that they require more-
nitrogen for their growth than other ordinary farm crops, and that
they therefore contain more of it per ton. It seems very appropriate,
therefore, to select legumes for such purposes as holding soil from

*N. J. Agr. Exp. Sta. Rpt. 1894, 158.
*R. I. Agr. Exp. Sta. Bul. 152.
*Experimental Farms, Ottawa, Rpt. 1912, 145.

1915] A BIOCHEMICAL Srupy oF NITROGEN IN CERTAIN LEGUMES 497

washing and preventing sands from shifting, for not only do they
serve these purposes well, but at the same time they conserve rela-
tively more nitrates from loss than non-legumes. Of course a con-
dition might occur in which a legume would draw all its nitrogen
from the soil, but even in such a case a legume would be preferable
to a non-legume, as by its use relatively more nitrogen would be kept
from leaching and so saved for future crops.

Cross-INOCULATION

There are relatively few cases of cross-inoculation that have been
definitely determined as occurring under natural conditions. The
most important example is the cross-inoculation that takes place be-
tween the sweet clovers and alfalfa. Bur clover (Medicago lupu-
lina) is another source of inoculation for alfalfa. The wild vetches
serve for inoculation of the cultivated vetches. It would seem that
many such cases may exist in which the wild specie of a legume con-
tains the organism for the inoculation of the cultivated legume of the
same specie or even of an entirely different specie sr genera. Inves-
tigations along this line have not been carefully undertaken as yet.

It has been possible under laboratory conditions to ecross-inocu-
late in many different ways. The data furnished by Laurent, re-
ferred to in an earlier part of this publication (page 485), together
with that furnished by Moore, Kellerman and Leonard, and others,
is of interest. Laurent produced nodules on the pea with the organ-
isms from thirty-six different species of legumes. Moore! produecd
nodules on many legumes with the pea organism, among which were
erimson clover (Trifolium incarnatum), white clover (Trifolium re-
pens), red clover (Trifolium pratense), berseem (Trifolium Alexan-
drinum), alsike (Trifolium hybridium), sweet clover (Melilotus alba),
cowpea (Vigna catjang), alfalfa (Medicago sativa), broad bean (Vicia
faba), common bean (Phaseolus vulgaris), fenugreek (Trifolium foe-—
num graecum), hairy vetch (Vicia villosa), scarlet vetch (Vicia ful-
geus), and yellow vetch (Vicia lutea). The results published by
Kellerman and Leonard represent the extreme in ecross-inoculation at
the present time. It will be recalled that they have reported the
inoculation of the soybean, the lupine, and alfalfa with an organism
originally obtained from alfalfa nodules,? altho it has been quite
generally believed that the soybean was representative of a special
class as regards its inoculation, and the same can be said regarding
the lupine. Legumes may be grouped as follows according as their

*Moore: U.S. Dept. Agr. Bur. Plant Indus, Bul. 71.
*See page 485, note 6.

498 BULLETIN No. 179 [ March,

bacteria are interchangeable for the purposes of inoculation:

Group 1 Alfalfa, sweet clovers, bur clover,
black medick

All true clovers

Cowpea, partridge pea

Soybean

Bean

Peas (garden and field), vetches (cultivated
and wild), sweet peas, lentils

Lupines

Sanfoin

Locust

9?
9?
9?
9?)
9)

9?)
39
”

oon BD Ol Go dO

Nobbe, Hiltner, and Schmid! obtained inoculation and nitrogen
fixation with the locust and vetch cross-inoculated with each other and
with pea bacteria, as shown below:

Inoculated with Locust Vetch Pea-bacteria
Nitrogen Locust (Robina) 232.1 13.5 21.2
assimilated Vetch (Vicia) " 12.9 264.0 22.6.

Some prefer to divide the legume bacteria into two classes accord-
ing to the beneficial or detrimental effect produced by lime upon the
legume. In this classification, alfalfa would represent one type and
serradella the other.

The question of cross-inoculation is far from settled. It is easily
seen that a great many interesting problems, aside from the purely
scientific studies of the laboratory, are presented for the soil biologist
in the pursuance of this field of research.

ASSOCIATIVE GROWTH OF LEGUMES AND NoN-LEGUMES

The associative growth of legumes and non-legumes has been
given renewed notoriety in recent publications. Practical observa-
tions of long standing have indicated that a non-legume benefits by the
presence of a legume during the second year of its growth—as might
reasonably be anticipated. The proof of a benefit by association dur-
ing the first season is not sufficiently established for a generalization,
for errors in sampling, in methods of experimentation, and other un-
favorable conditions have crept in and overshadowed the full value of
the data reported.

The problem of associative growth involves many details that
must be further studied. The stimulation caused by the struggle for
existence in association may increase the height of the crops or the
amount of the organic matter produced, yet not necessarily the nitro-
gen content. In the work under observation at this station, it appears
that the nitrogen which is returned to the soil as the nodule sloughs
off could hardly be utilized by an ordinary annual non-legume crop.
It is yet to be determined whether either the legume itself or its

*Nobbe, Hiltner, and Schmid: Landw. Vers. Stat. (1894), 45, 12.

1915] A BIocHEMICAL Srupy oF NITROGEN IN CERTAIN LEGUMES 499

nodules exude nitrogenous compounds during their active period of
growth.

CHEMICAL

The chemical composition of legumes from the standpoint of their
nitrogenous constituents has been investigated to some extent, but the
studies closely related to this point are relatively few. The follow-
ing data are very general in character and relate to studies concern-
ing the total nitrogen content of the different parts of legumes at dif-
ferent periods of growth. Studies upon some of the various nitroge-
nous compounds are also included.

In 1895 Stocklasa,! working with lupines (Lupinus luteus
and Lupinus augustifolius), found that the nodules were richest in
the element nitrogen at the time of blooming, while the roots appeared
to be richest in that element at the fruiting period. His results are
given in Table 2. The figures for the nodules indicate that the nitro-
gen is either taken up by the plant for seed production or diffused
into the soil.

TABLE 2.—ToTaL NITROGEN IN LUPINUS LUTEUS: RESULTS OBTAINED
By STOCKLASA

(Percentage on dry basis)

Period Roots _ Nodules
Blooming esiectacis ss se9s05 - 1,64 i:
WPUIGIN 5 eats hicee soe ties 1.84 2.61
Maturity. osc.a felsic wesieuere nine 1.42 1.73

Stocklasa also determined protein, amides, and asparagine in lu-
pine nodules. The protein was obtained by the Stutzer method, the
amides by the Kjeldahl method, and the asparagine by calculation
from the ammonia obtained by distillation with magnesium oxid.
Table 3 shows his results.

TABLE 3.—NITROGEN COMPOUNDS IN LUPINE NODULES: RESULTS OBTAINED
BY STOCKLASA

(Percentage on dry basis)

Period Protein Amides | Asparagine
PORSOMUDE . i550 ss ses 3.99 35 34
MIATHGEEY Hciostsisi sides 1.54 15 Trace

The presence of asparagine in the nodule is important, as it is
thought to be intimately related with the formation of protein.

In 1901 Wassilieff? studied the nitrogen compounds in white
lupine (Lupinus alba) seeds and seedlings. He found that the seeds
contained 7.68 percent of total nitrogen’; and that of this, 6.89 percent
was in the form of protein and .53 percent was precipitated by phos-

1Stocklasa: Landw. Jahrb. (1895), 24, 827-863.
*Wassilieff: Landw. Vers. Stat. (1901), 55, 45-77.

500 BuLteTIN No. 179 [March,

photungstie acid, leaving a difference of .26 percent, asparagine. The
occurrence of asparagine in large amounts in the seedlings is shown
by the data given in Table 4.

TABLE 4,—NITROGEN COMPOUNDS IN FOoURTEEN-DAyY-OLD GREEN SEEDLINGS OF
WuitEe LUPINES: RESULTS OBTAINED BY WASSILIEFF

(Expressed in percentage on dry basis)

nds a ; : Total
Parts nitrogen Asparagine | Protein nitrogen
FO 5 tS Pe 58 1.45 4.11 6.57
COERMIDORMR ss Soi s's tce cass .63 3.83 2.44 7.83
SSUGTIN oy ck ahd Sie cede Sg 42 4,57 1.56 6.77
MROGUES Weicccta ait sc'e\s se a eisie-s's7rs 46 2.20 1.87 5.40

1p.T.A.: This abbreviation for phosphotungstic acid will be used thruout
this publication.

Wassilieff also demonstrated the presence of leucine and tyrosine
in the cotyledons of one-week-old seedlings of white lupines. These
and other amino acids would be expected to be present when the pro-
tein of the seed is breaking down for the nutrition of the seedling.

Knisely! analyzed the leaves, pods, stems, roots, and nodules of
lupine plants for total nitrogen at three distinct periods of develop-
ment. His results show better than the others presented where the
nitrogen accumulates as the plant matures.

TABLE 5.—ToTaL NITROGEN IN LUPINES: RESULTS OBTAINED BY KNISELY
(Expressed in percentage on dry basis)

Period | Leaves Pods Stems | Roots | Nodules
MOM SHOOM ac tyes oka ae3 4.02 _ 38.07 1.15 92 5.17
Pods: well. formed... .%6.05.6. 3.70 3.38 88 .83 4.29
POGS. Very IATIO 2 062s. cis, 8.6'2 53018 3.41 3.68 90 66 3.70

Schulze and Barbieri? examined lupine and soybeans seeds and
seedlings for nitrogen and obtained the results shown in Table 6.

TABLE 6.—NITROGEN IN LUPINE AND SOYBEAN SEEDS AND SEEDLINGS: RESULTS
OBTAINED BY SCHULZE AND BARBIERI

(Expressed in percentage on dry basis)

: Total : tof ot Filtrates
rs nitrogen Protein nitrogen | from P.T.A.

Lupine seeds.............. 8.63 8.17 24 22
IGT IMMNN Sac ONG ic Sy ee ens 6.73 6.32 13 .28
Lupine dark seedlings

11 to 12 days old........ 10.64 3.40 1.60 5.64
Lupine dark seedlings

12 Gaye: O86 5 ose 10.51 2.33 2.17 6.01
Soybean seedlings

OR Ce ee er ee 7.42 3.86 56 - 3.00

*Knisely: Ore. Agr. Exp. Sta. Rpt. 1909, 30-31.
*Schulze and Barbieri: Landw. Vers. Stat. (1881), 26, 241.

1915] A BiocHEMIcAL Stupy or NirrogEN IN CERTAIN LEGUMES 501

They also found a large amount of asparagine in both the lupine
and the soybean seedlings.

Schulze! has made a careful study of the compounds in plants,
and has formulated the hypothesis that the same decomposition
products arise from protein in the plant as outside it, but that in
the plant the compounds are further altered, thereby affecting in
varying degree the individual products of the hydrolytic decomposi-
tion. A comparison of the analyses of pea seedlings one week old
and those three weeks old showed the following differences:

Leucine Tyrosine Arginire Asparagine
= 1 WOOKs i. FoeirseX abundant little present absent
3 WEOKS 5 05:6 s.d:dise much less absent almost absent very abundant

Arginine and amido acids were shown to be present in the lupine
cotyledons, but asparagine was absent, altho the latter substance was
found in the stem of the seedling. It has been suggested that the oc-
currence of asparagine is associated with the disappearance of amido
acids and not of protein. Phenyl alanine, tyrosine, and tryptophane
have been reported in the white lupine (Lupinus alba), tyrosine and
tryptophane in vetch (Vicia sativa), and tryptophane in the garden
pea (Pisum sativum) .?

Smith and Robinson? found 4.19 percent of nitrogen in soybean
nodules and 3.90 percent in cowpea nodules. They observed that inocu-
lation inereased the protein content of soybean plants without in-
creasing the yield of beans. This has been noted by other experi-
menters. ;

Hopkins‘ has reported the analyses of cowpea plants for total
nitrogen with and without inoculation. The nodules, roots, and tops
were analyzed separately, as will be seen.by reference to Table 7.

TABLE 7.—NITROGEN FIXATION BY COWPEAS: RESULTS OBTAINED BY HOPKINS
(Expressed in egs.)

Nitrogen

Treatment Tops Roots | Nodules fixed
Ten plants with bacteria............... 146 9 11 125
Ten plants without bacteria............ 38 3 ae Sr
Ten plants with bacteria............... 171 10 18 140
Ten plants without bacteria............ 55 4 ‘a eee
Ten. plants with bacteria............... 143 8 17 124
Ten plants without bacteria........... 40 4 es sek

~-WSehulze: Zeits. f. Physiol. Chem. (1895), 24, 18; 30, 241.
' *8chulze et al: Zeits. f. Physiol. Chem. (1887), 11, 43; (1906), 48, 387, 396;

(1910), 65, 431.
Gorup Besamez: Ber. deut. Chem. Gesell. (1887), 10, 781.

‘Smith and Robinson: Mich. Agr. Exp. Sta. Bul. 224, 125-132.
‘Hopkins: Ill. Agr. Exp. Sta. Bul. 94, 319.

502 Butietin No. 179 { March,

The inoculated plants contained a much greater percentage of
nitrogen than the uninoculated, the average content of the inoculated
being 4.24 percent in the tops, 1.48 percent in the roots, and 5.92 per-
cent in the nodules, while the average content of the uninoculated was
2.48 percent in the tops and .88 percent in the roots.

The ash and the ash constituents of the nodules and the roots of
lupines have been determined by Stocklasa,! as presented in Table 8.
The total ash of the nodules was found to be 6.32 percent, while that
of the roots was found to be 4.55 percent.

TABLE 8.—ASH CONSTITUENTS IN LUPINE NODULES AND Roots:
RESULTS OBTAINED BY STOCKLASA

(Expressed in percentage)

Constituents Nodules Roots
Sie her ee 1.59. « 1.90
Rar sran etatecote sets ene 4.90 6.38
Pie ia cette wiikey 6.51 4.28
Keke nee Goran Mio al 12.05
IN tenet gs ee ers 16.94 19.94
MG Waysrotemiaues 7.41 7.05
Gas Sate ee eed Bo ds 7.64 12.04
NG Sy iy nteecrs sete es .83 NES

The analyses of red-clover nodules show a potassium content of
2.63 percent in the dry matter.2 The nodules, therefore, are rela-
tively rich in mineral elements as well as nitrogen compounds; and
Stocklasa’s results (see Table 8) show that the chief differences
between the roots and the nodules in the composition of the ash
constituents are in phosphorus, potassium, calcium, and sodium.
The nodules are richer in the first two elements and the roots in the
latter two. The differences in nitrogen content of the various parts of
the plant have already been brought out somewhat, but they will be
dealt with more fully in the results presented under the experimental
portion of this bulletin. The presence of the bacteria would in itself
be sufficient to account for these differences.

In brief, the chemical data which have been considered, altho
small in amount, show the relative richness in nitrogen of the nodule
as compared with other parts of the plant. They point to the acecumu-
lation of nitrogen in the seeds, at the expense of the other parts, as
the plant matures. That the nitrogen exists in the form of protein,
asparagine, and other soluble forms, is also clear. The presence of
various aliphatic and carbocyclic amino acids has been mentioned.

‘Stocklasa: Landw. Jahrb. (1895), 24, 827-863.
“Analyzed by Aumer, Ill. Agr. Exp. Sta. (unpublished data).

1915] A BiocHEMIcAL Stupy oF NITROGEN IN CERTAIN LEGUMES 503

EXPERIMENTAL
PLAN OF INVESTIGATIONS

The experimental studies herein reported are for convenience di-
vided into two parts. Part I consists of studies made in order to de-
termine thru which organs legumes obtain their nitrogen from the air.
Part II is concerned with an attempt to determine more definitely the
mechanism of the reactions occurring in the fixation and assimilation of
atmospheric nitrogen by B. radicicola and legumes, a process concern-
ing which science is greatly in the dark. This phase of the problem has
attracted the attention of plant physiologists, physiological chemists,
and other scientists outside the field of agricultural research. No les-
ser chemist than Emil Abderhalden! has written concerning it as fol-
lows: ‘‘It would be very interesting to know the compounds into
which these organisms convert the nitrogen. At present we have no
knowledge of this. We assume that the final substance produced is
protein, which is then in part assimilated by the plants with the help
of fermentation.’’ Any light which may be thrown on this question
will be of great value toward its final solution.

PART I

STUDIES TO DETERMINE THRU WHICH ORGAN LEGUMES OBTAIN
ATMOSPHERIC NITROGEN

For a long time it was believed that the nitrogen fixed by legume
bacteria and assimilated by the plant was obtained thru the leaves, and
even now many hold to this belief. Frank and Otto? in 1890 obtained
- analytical results which seemed to them to be proof of this theory.
They believed that the bacteria were only incidentally connected with
the process, acting perhaps as stimuli.

The first experiment resulting in data of a contradictory nature
was made by Kossowitsch? in 1891, but the results of this investigation
were not generally accepted. Nobbe and Hiltner* in 1899 added fur-
ther evidence to the existing knowledge, but their conclusions, drawn
from physiological differences, have not been substantiated by chemi-
cal data, which seem more reliable than those of a physiological nature.

*Abderhalden: Physiological Chemistry, Trans. by Hall, 198.

‘*Frank and Otto: Ber. deut. Bot. Gesell. (1890), 8, 331.

*Kossowitsch: Bot. Ztg. (1892), 50, 697-702, 7138-723, 729-738, 745-755,
771-774,

*‘Nobbe and Hiltner: Landw. Vers. Stat. (1899), 52, 455-465.

SLNENIMIAXY SV UO NOWVAvdHYd NI SONTIGGUG VIEMOO—'AT GLVIG

1915] A BIocHEMICAL Srupy oF NITROGEN IN CERTAIN LEGUMES 505

Experiments on this question were conducted by the author in
1911-1912. The general plan was the same thruout each experiment;
the various modifications are considered under the individual -experi-
ments.

GENERAL PLAN OF EXPERIMENTS

The plants used were the soybean and the cowpea. Uniform seeds
were carefully selected and inoculated with an infusion placed directly
in contact with them. They were then planted in heakers containing
nitrogen-free white sand. Mineral plant food was added in solution.
When the seedlings had developed two leaves and possessed small nod-
ules, they were carefully washed from the sand and transferred to
the apparatus.

The apparatus! was arranged as follows: Woulfe bottles, placed
inside battery jars painted black in order to obviate the influence of
light, were connected with drier bottles, which in turn were connected
with a gasometer. An outlet tube from each bottle was provided,
the external end of which was immersed in water. In the first ex-
periment two Woulfe bottles were used and in the others six. Sterile
nitrogen-free sand containing calcium carbonate was placed in the
Woulfe bottles and the young seedlings carefully transplanted, one
to each. The plants were then sealed gas-tight by means of rubber
tissue placed double thick about the stem. Rubber cement was also
used to make all joints tight.

Plant food, with the exception of nitrogen and calcium, was added
in solution. This solution was sterilized, boiled, and cooled just pre-
vious to its being used in order to prevent the addition of absorbed
gases. The plant-food solutions and sterile, distilled, nitrogen-free
water were added from the outer end of the outlet tube, with the gas
flowing in order to avoid the possible admittance of air. The moisture
content of the sand was maintained at about 12 percent. When the ©
apparatus had been made tight, the gas was started and allowed to
flow gently for eight to ten hours per day; at night it was entirely
shut off. By this method the plant roots were kept constantly in the
same atmosphere.

The gas mixture used in the first three experiments consisted of
96 to 98 percent oxygen and 2 to 4 percent carbon dioxid. For the
purpose of comparison, air was passed thru part of the bottles in these
experiments. The gas mixture was made in the laboratory, great care
being exercised to eliminate nitrogen, air, and other impurities. The
oxygen was made from potassium chlorate and manganese dioxid, and
the carbon dioxid was generated from marble and hydrochloric acid.
The air, when used as a source of nitrogen, was passed thru sulfuric
acid before entering the gasometer and after leaving it. In order to

*Plate V- shows the apparatus in use.

506 BULLETIN No. 179 [ March,

TT died
; Slee =
eo 77\"\ ia

Ree

| B: ~

PLATE V.—EXPERIMENT II: COWPEAS AT THE TIME OF Harvest (37 Days)

UNINOCULATED

1915] A BiocHEemicaL Srupy or NirroGEN IN CERTAIN LEGUMES 507

dispel any possible doubt as to the oxygen mixture being too strong,
a fourth experiment was conducted in which the effect of a mixture
made of 90 percent oxygen, 7 percent nitrogen, and 3 percent carbon
dioxid was compared with that of a.mixture made of 97 percent
oxygen and 8 percent carbon dioxid.

EXPERIMENT I

In Experiment I, soybeans were used. Three plants twenty-one
days old were placed in position on September 1, 1911, one in each of
two Woulfe bottles and a check plant left uninclosed. Thruout the
experiment these plants were kept out of doors during the day. The
experiment was continued for twenty-eight days. At the end of that
time the plants were analyzed for total nitrogen by the official Gun-
ning! method. The average of individual analyses of twenty soybean
seeds was taken as the criterion from which to calculate the amount
of nitrogen fixed by the plants. The results are presented in Table 9.

TABLE 9.—FIXATION* OF NITROGEN By SOYBEANS: EXPERIMENT I

(Results expressed in milligrams)

Nitrogen in Nitrogen 3
— Treatment plant at end, in check | x Pan
; 28 days seeds
1 CO, +O 10.43 11.4 (-.97)
2 co, +0 10.65 11.4 (-.75)
3 Air 17.61 11.4 7.07

*The word fixation is used in this publication in its broader sense and should
be understood as meaning the fixation of atmospheric nitrogen by bacteria and
the assimilation of the nitrogenous compounds formed by the plant.

The error in Plants 1 and 2 is partially accounted for by a slight
injury to these plants by grasshoppers and red ants. There is, how-
ever, a small experimental error which is difficult to eliminate, as will
be observed in the other experiments.

EXPERIMENT IT

The experience gained in Experiment I led to the selection of
cowpeas for the later investigations, since they are less subject to
injury by red ants than are soybeans. Experiment II was started on
November 23, 1911, and continued until December 29, thirty-seven
days. Six two-liter Woulfe bottles were planted with seedlings
twenty-four days old. Air was passed thru three of the bottles and the
gas mixture thru the other three. The average of individual analyses of

*In preliminary tests the Gunning and Kjeldahl methods modified to include
nitrates gave no higher results than the official Gunning or Kjeldahl methods.

508 BuLLeTIN No. 179 [ March,

PuaTe VI.—ExXPERIMENT II: PLANTS ABOVE GROWN IN AIR; THOSE BELOW
Grown In CO,+ 0

1915] A BIocHEMICAL Stupy or NITROGEN IN CERTAIN LEGUMES 509
fifteen cowpea seedlings seventeen days old was used as a basis from
which to calculate the nitrogen fixed by the plants during the experi-
ment. Seedlings of this age were taken for analysis in order that the
results of this experiment might be comparable with those of the
others, altho the seedlings of the experiment when transplanted were
somewhat older.

TABLE 10.—FIXATION OF NITROGEN By COWPEAS: EXPERIMENT II

(Results expressed in milligrams)

Nitrogen in

Nitrogen in

hve Treatment plant at end, | check seedlings sy
i 37 days at beginning
1 co, + O 9.21 7.90 1.31
2 CO, + 0 13.03 © 7.90 5.13
3 co, + 0 9.43 7.90 1.53
4 Air 24.84 7.90 16.94
5 Air 23.61 7.90 15.71

Note.—Plant 6 was lost in distilling thru the breaking of the flask, caused
by sand adhering to the roots.

The fixation shown by Plant 2 is attributed to a leak discovered
around the stem. of this plant some few weeks after it had been put in
place; trouble was had thruout the experiment in keeping it gas-tight.
The evident fixation in the case of Plants 1 and 3 is within experi-
mental error; yet since these plants were twenty-four days old when
placed in the apparatus, while the check seedlings analyzed were only
seventeen days old, it is reasonable to assume, from the results obtained
in the next experiment, that a part at least of the assimilation of this
nitrogen had taken place before the seedlings were transferred.

On the plants receiving air the nodules became well developed.
The accompanying photograph (Plate VI), taken at the termination
of the experiment, shows the comparative development of the roots
and the tops grown in the gas mixture and those grown in the air.
The most interesting part of this experiment was the very evident
translocation exhibited by the plants growing in the mixture of carbon
dioxid and oxygen, as shown by their color. The same phenomenon
was observed in the later experiments and is discussed under the
general consideration of the gas experiments.

EXPERIMENT III

. | Experiment III was conducted with cowpeas in a manner similar
to that of the preceding experiments. It was started on March 5, 1912,
with six seedlings seventeen days old and discontinued after eighty-
three days, May 27, 1912. Air was passed thru three of the bottles
and the gas mixture thru the other three. In order to obtain the best
possible check on the results, fifteen additional seedlings of the same

510 BULLETIN No. 179 [ March,

CARBON DIOXIDE |
- & ORYGEN

CARBOW DIOXIDE |
&-O), YGEN

PLATE VII.—EXPERIMENT III: LOWER FiguURE SHOWING EXPERIMENT AT BEGIN-
NING; UPPER FIGURE SHOWING EXPERIMENT 10 Days LATER

1915 | A BiocHEMICAL Stupy or NITROGEN IN CERTAIN LEGUMES 511

: 1S a ~

PLATE VIII.—EXPERIMENT III: UPPER FIGURE SHOWING EXPERIMENT 52 DAYS
FROM BEGINNING; LOWER FIGURE SHOWING EXPERIMENT AT HARVEST
(83 Days)

512 BULLETIN No. 179 [ March,
lot as those transplanted to the Woulfe bottles, grown from seeds 185
milligrams in weight, were analyzed individually at the beginning of
the experiment. The results showed the presence of an average of 7.90
milligrams of nitrogen, while the average nitrogen content of twenty
seeds of the same weight analyzed individually equaled 6.94 milli-
grams, making an average fixation of .96 milligram of nitrogen by
these seedlings in the first seventeen days.

TABLE 11.—FIxATION OF NITROGEN BY COWPEAS: EXPERIMENT IIT

(Results expressed in milligrams)

Nitrogen in

Nitrogen in

Sona Treatment plant at end, check seedlings sais,
; 83 days at beginning
i CO, + 0 9.48 "7,90 1.58
2 C0260 7.49 + 7.90 (-.41)
3 co, + O 8.49 7.90 59
A Air Roots 74.27
Tops 112.59
186.86 7.90 177.96
5 Air Roots 71.99
Tops 166.03
238.02 — 7.90 230.12
6 Air Roots 66.71
Tops. 120.51 ;
187.22 7.90 179.32

The figures in Table 11 show to what extent fixation took place.
Plants 1 and 3 may have contained more than 7.90 milligrams of nitro-
gen as seedlings, altho it cannot be proved that they did, owing to the
impossibility of analyzing and growing the same seedling. There was
always another possible source of error in the dissolved nitrogen gas
in the water used for pressure in the gasometers.

It is well to observe that in all these experiments the gases were
passed thru sulfuric acid, which eliminated the possibility of ammonia
playing any part in the fixation. This is claimed by many to occur;
yet the first experiment ever made for the purpose of showing that
legumes obtain nitrogen from the air was so conducted that combined
nitrogen was eliminated.

The plants in the carbon dioxid and oxygen mixture were from 3
to 4 inches in height and possessed two leaves at the end of the experi-
ment, while those growing in the air measured from 8 to 9 inches in
height and possessed nine leaves.

1915] A BIocHEMICAL Stupy oF NITROGEN IN CERTAIN LEGUMES

PuatTeE [X.—ExXpERIMENT III: ON THE LEFT, Roots FRoM PLANT GROWN IN
CO,+0; ON THE RicHT, Roors rroM PLANT GROWN IN AIR

514 BuLLETIN No. 179 [ March,

In order to test the viability of B. radicicola after it had grown
on the plant under extreme oxygen conditions, organisms were re-
moved from the nodules of Plants 1, 2, and 3, and an infusion made in
sterile water. Portions of this infusion were applied to cowpea seeds
that had been sterilized and planted in sterile sand. Sterile conditions
were maintained thruout this test. Profuse nodule formation resulted,
demonstrating that no harmful results had been produced upon the
organism by its long exposure to an atmosphere with a high content of
oxygen.

EXPERIMENT IV

Having made certain in Experiment III that no detrimental ef-
fects had been produced upon B. radicicola by long exposure to an
atmosphere high in oxygen, Experiment IV was instituted in order to
determine if there could have been any possibility of injury to the
plants in the previous experiments from the use of gaseous mixtures
high in oxygen. ;

The plan involved a comparison of the effect of a mixture of 97
pereent oxygen and 3 percent carbon dioxid, and that of a mixture of
90 pereent oxygen, 7 percent nitrogen, and 3 percent carbon dioxid.
The nitrogen used was obtained from the air; otherwise this experi-
ment was similar to Experiment III. Each gas mixture was passed
thru three of the Woulfe bottles. The experiment was begun on Sep-

TABLE 12.—FIXATION OF NITROGEN By COWPEAS: EXPERIMENT IV
(Results expressed in milligrams)

Nitrogen in

Plant Nitrogen in : Nitrogen
Treatment check seedlings ai
No. plant at beginning fixed
First Harvest (26 Days)
2 co, + O 10.00 7.90 | 2.10
4 N+C0,+ 0 14.94 7.90 7.04
Second Harvest (28 Days)
3 | CO,--O 8.06 7.90 16
6 | N+4+CO0O,+0 33.51 7.90 25.61
Third Harvest (95 Days)
1 CO, + 0 13.97 7.90 6.07
5 N+CO,+0 | Leaves 129.26
Stems 45.17
Tops 174.43
Roots 32.93
Nodules 112.02
319.38 7.90 311.48

1915] A BrocHEemicaL Srupy or NITROGEN IN CERTAIN LEGUMES 515

90 YoOxy en, 7%, Nitrogen, : 97% Oxygen,
i ' 1

. 8% Car, on Dioxide. j ay Car ton Dioxide.

PLATE X.—EXPERIMENT IV: UPPER FIGURE SHOWING EXPERIMENT AT BEGINNING;
LOWER FiguRE SHOWING EXPERIMENT 16 Days LATER

516 BULLETIN No. 179 [ March,

PLATE XI.—EXPERIMENT IV: LOWER FicurE SHOWING PLANTS 41 Days FROM
BEGINNING; UPPER FIGURE SHOWING PLANTS 59 Days FROM BEGINNING

1915] A BiocHEMICAL Stupy or NITROGEN IN CERTAIN LEGUMES 517

AYA A \.

. =0, 4
90 YyOr: feo, 7, Nitrogen, 1 977% Orygen,

3% Carbon Dioxide. — x
*%

: a%,Ca bon Dioxide. e

PuLatTE XII.—ExXpPERIMENT IV: At THE TIME OF HarvEsT (95 Days)

[ March,

BULLETIN No. 179

518

ON THE RicHT, Roots FROM PLANT GRowN IN N-CO,+0

PLATE XIII.—EXPERIMENT IV: ON THE LEFT, Roots. FROM PLANT GROWN IN
CO,-L0;

1915) A BiocHEemican Srupy or NirrogEN IN CERTAIN LEGUMES 519

tember 7, 1912, with six cowpea seedlings eleven days old, and con-
tinued for ninety-five days. The plants were harvested two at each
of three periods.

The results given in Table 12 need no explanation, tho it might
be well to call attention to the individual differences in the plants in
the amounts of nitrogen fixed. During the ninety-five days of the ex-
periment, Plant 5 fixed fifty-one times as much nitrogen as Plant 1.
The following comparison between the growth of these two plants is
of interest.

Plant 1 attained a total height of 5 inches, possessed one leaf, and
on the roots were counted 60 nodules. Plant 5 reached a height of 61
inches; its leaves measured as follows:

2 measured 5 inches along midrib, 4 inches at base
9? 4% rae 9?) 9? 3-314 9) 9? 2?
4

EF +? 9? 3 a7: 22 2)

3

1

3 9? 3% 9? 9? 9? 2% Be 3 * he d 22
2 3

9) 2? 9? 9? 2 9? a, 2?)

3 9? 23%, 9? 9?) 9? 1% 2?) PA J 7)
1 3

9? ed 9? 9? 9 2?) 9?) 2?

9?)

*s

Several pods were formed, as may be seen by reference to Plate
XII, one of which measured 41% inches in length and was partially
filled with seeds. The roots were so large that the Woulfe bottle had
to be broken in order to obtain them. The plant possessed 32 large
nodules, 46 medium to large, 66 medium, and 144 small; 288 in all.

EXPERIMENT BY KOSSOWITSCH

Reference has been made to a laboratory experiment conducted
by Kossowitsch in the summer of 1891 (see page 503). As his work
has been accepted by some and ignored by others, it is of particular
interest.

Peas were started in a mixture of four-fifths sand and one-fifth
soil in which peas had been grown the year before. When the plants
had developed good nodules, they were transferred to jars containing
nitrogen-free sand. In some eases the roots were enclosed and in others
the tops, a similar means being used in each case to make the joints
air-tight. Over the tops of the jars bell-jars were placed. These
were connected thru drier bottles with a gasometer. Because of mois-
ture collecting in the bell-jars, absorbents were used to keep the
atmosphere normal. The gas mixtures used were hydrogen and
oxygen in some cases; hydrogen, oxygen, and carbon dioxid in some;
and air in others. Combined nitrogen was also used to check up
the possible abnormal condition due to the necessary manner of
experimentation. Great accuracy was displayed in arranging the ap-
paratus and in analyzing the gases.

[ March,

179

BULLETIN No.

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HOSLIMOSSOM Ad LNANINAAXY JO SLINsSAY—'El AAV],

1915] A BIOCHEMICAL StupDy oF NITROGEN IN CERTAIN LEGUMES 521

A study of the results of Kossowitsch’s experiment, given in Table
18, discloses very little data bearing strictly on the question, thru
what organs plants obtain atmospheric nitrogen. Plants 1 and 2
tend to show that nitrogen cannot be obtained thru the leaves. Plant
2 shows a fixation, but it may be within experimental error. Plants 3,
4, 6, and 10 may be eliminated, as assimilation should have taken place
in these cases. Plant 5, the leaves of which were in hydrogen, oxygen,
and carbon dioxid, fixed nitrogen, but Kossowitsch states that this re-
sult is not reliable, owing to the leaves not having been sufficiently iso-
lated. Plants 7, 8, and 11 show normal results, and, like Plant 9,
indicate, aside from the experimental error, that the nitrogen must be —
brought into contact with the roots in order to effect fixation. Plant
12, the leaves of which were enclosed in hydrogen, oxygen, and carbon
dioxid, also showed a fixation. Thus it will be seen that the data .
bearing on the question were obtained from only seven plants.

Somewhat related to this problem is the work of Nobbe and Hilt-
ner,! who grew lupines in solutions. They thought that it was neces-
sary to raise the nodules above the solution in order to obtain a
fixation, and this seemed to indicate to them that legume plants obtain
atmospheric nitrogen thru their roots rather than thru their tops.

GENERAL CONSIDERATION OF GAS EXPERIMENTS

The plants in the mixture of carbon dioxid and oxygen usually
developed two and sometimes three leaves before they seemed to be
checked in their growth. Soon an interesting translocation set in.
Each plant removed the nitrogen from the lower leaves and developed
a new leaf of a normal green color. The green of the old leaves disap-
peared from the margins first; soon the whole leaves became yellow,
and shortly dropped from the plant. This process repeated itself un-
til there was not nitrogen enough left in a translocatable form to give
green color to another leaf, when a pale green or even a yellow leaf
appeared. The duration of this process was remarkably long in some
of the plants. The appearance of the plants was in every case of
especial note, even to a layman. .

It should be emphasized that no combined nitrogen was present
or could have been assimilated in these experiments. The slight amount
of nitrogen reported as fixed by the plants in carbon dioxid may have
been either actual fixation or experimental error. From close observa-
tion and study made in order to reduce this error, it would seem that
its only possible source, disregarding Plant 2 in Experiment II,
already mentioned, would be the dissolved air in the water used for
pressure in the gasometers. This was unavoidable. It may also account
for the error in Kossowitsch’s experiment. Plant 1 in Experiment IV

*See page 503, note 4.

522 BuLLEeTIN No. 179 [ March,

would seem to indicate that the above view is correct, as it showed a
content of 6 milligrams of nitrogen after having remained under ex-
periment for ninety-five days. In this length of time there were a
great many changes of the gasometers and the use of a large amount
of water, which would tend to increase the atmospheric nitrogen and
thus perhaps contaminate the other gas mixtures. The nodules on all
the plants were normal. On Plant 5, Experiment IV, they were very
large, and the spongy sutures were highly developed as if to present
as large an absorbing surface as possible.

Finally, it should be noted that had the plants growing in the
mixture of carbon dioxid and oxygen possessed any ability to take in
nitrogen thru their leaves, they should have made as good a develop-
ment as those growing in the air, and those in the oxygen, nitrogen,
and carbon dioxid mixture.

PrRAcTICAL APPLICATION OF RESULTS

The practical application of the results obtained in these experi-
ments would appear to rest in the proper aeration of the soil in order
that greater amounts of nitrogen may be fixed. As the plants obtain
their nitrogen thru their roots, it is essential that the soil contain
plenty of air at all times.

PART II

RELATIVE PERCENTAGES OF NITROGENOUS COMPOUNDS IN THE
VARIOUS PARTS OF THE SOYBEAN AND COWPEA AT
DEFINITE PERIODS OF GROWTH

It has seemed advisable to determine, if possible, in what forms
atmospheric nitrogen exists in the various parts of the legume, for it
is believed that this knowledge would throw much light upon the whole
process of fixation. To do this, various compounds in the nodules,
roots, and tops were determined at definite periods in the growth of
the soybean (Glycine hispida, Maxim) and the cowpea (Vigna un-
guiculata, Walp). As it would be quite impossible to determine all
these compounds separately, they were grouped and determined as
follows,—total, insoluble, and soluble nitrogen. In the soluble-nitro-
gen group was included the nitrogen precipitated by P.T.A. and
Other nitrogen. The constructive and destructive metabolisms in the
plant doubtless give rise to other nitrogen compounds than pure pro-
tein; so it is quite possible that in addition, proteoses, peptones, pep-
tides, acid amides, amino acids (mono and di), guandine residues, pig-
ment nitrogen, alkaloids, ammonia, nitrites, nitrates, and other nitro-
gen compounds may have been present.

Because of the great importance of controlling conditions under
which experimental plants are grown, all the factors in this work were,
controlled except the influence of sunlight.

1915] A BiocHEMicaL Stupy oF NITROGEN IN CERTAIN LEGUMES 523

MetHops EMPLOYED IN THE GROWTH AND PREPARATION OF SAMPLES

Plants Used.—The soybean and the cowpea were selected for this
work because of their adaptability to the conditions under which it
was necessary to carry on the investigations. The rapidity and habit
of growth, as well as the nodule formation of these plants, makes them
especially desirable for experiments of this sort. The cowpea is an im-
portant crop, especially in southern Illinois, while the soybean is being
grown more and more each year in central and northern Illinois. The
seed used in all the experiments conducted was produced in 1910 on
some of the experimental fields of this station. The soybeans were of
the Medium Green variety, the cowpeas, the Whippoorwill.

Methods Used in Growing the Plants.—4.7 kilos of pure nitrogen-
free sand, in which 10 grams of chemically pure calcium carbonate had
been thoroly mixed, were placed in each of a number of 6-inch cylin-

Cow Pea

PLate XIV.—TypicaAL Jar oF Five CowPEas BEING GROWN FOR SAMPLES

524 BuLueTiIn No. 179 [March,

drical glass battery jars. These jars were painted black in order to
keep the light from the roots and check the growth of algae. A mois-
ture content of 12 percent was maintained thruout the growth of the
plants. This was done by weighing each jar at least every week, some-
times every four days, and adding sufficient nitrogen-free, distilled
water! to restore the original weight of the jar. During the interim
each jar was given the same amount of water.

Mineral plant food in solution was applied once a week. Each
jar was given the following solutions:

10 ec. each of—
25 grams CaH, (PO,), per 2500 ce. water

20 grams MgSO, per 2500 ce. water

50 grams K,SO, per 2500 ec. water
1 ce. of—

-l1 gram FeCl, per 250 ec. water

These amounts were diluted with water and added at the same
time that the plants were made up to weight. The jars in several
series were kept out of doors during the pleasant days, but all plants
were kept in the greenhouse during the night.

Planting and Inoculation.—Five seeds of average size were se-
lected and planted in each jar. In the earlier work, in order to insure
a proper germination of the five seeds and avoid the possibility of some
decaying and leaving organic matter in the sand, moistened filter
papers were placed over the seeds until germination was assured, when
the seeds were covered with one-half inch of sand. Later this method
was found unnecessary owing to the excellent germination of the seeds;
and by starting more jars than needed in the series, the required num-
ber containing five plants was insured. By always covering the seeds
with the same amount of sand, more uniform plants were secured.

Inoculation was attained by the following methcd: Plants were
grown in a soil in which they would form nodules. These nodules
were then removed from the plant, thoroly washed, and dipped in
alcohol. After burning off the alcohol by passing the nodules thru a
flame, they were crushed in sterile distilled water. The inoculum was
then diluted to about a liter and 5 ee. applied to each seed just be-
fore it was covered with sand. When this method of infection was
carried out, no failures were experienced and an abundance of nodules
was always obtained.

Uninoculated material was obtained by planting a few jars sim-
ilarly to the inoculated, except that the seeds were dipped in
alcohol and the alcohol then burned off, and that a sterile spatula was
used in planting. The sand in these jars was not sterilized, but the
jars were placed in vessels of water in order to prevent possible infec-

*Whenever water is mentioned in this publication nitrogen-free distilled
water is always meant, unless otherwise stated.

1915] A BriocHEmMicaL Stupy oF NITROGEN IN CERTAIN LEGUMES 525

tion by ants and red spiders. This method gave very good results;
in no ease did inoculation occur.

Harvesting Samples.—As soon as the seed coats were free from
the young seedlings, they were removed from the jars, labeled accord-
ing to the jars from which they came, and later were analyzed with
the roots from that jar. In like manner the cotyledons and all the
leaves which had dropped were analyzed with the tops from the cor-
responding jar.

The periods at which the plants were harvested were regulated ac-
cording to the development of the leaves. Harvests were made from
jars as nearly uniform as possible, in most cases selected in triplicate.
The jars were taken to the laboratory, where the tops of the plants
were cut one inch above the surface of the sand and placed in a suit-
able receptacle to air-dry. A stream of water was then carefully di-
rected on the jars in order to wash the sand from the roots. After all
the visible nodules had been carefully removed with forceps, counted,
and placed away to air-dry, the roots were also placed away to air-dry.

Laboratory numbers were given as follows: odd hundreds to soy-
bean series, even hundreds to cowpeas; the tens to the number of the
harvest ; and the units 1, 2, 3, to tops; 4, 5, 6, to roots; and 7, 8, 9, to
nodules. Thus, 129 refers to soybean nodules of the second harvest.
The roots of the same are numbered 126, and the tops, 123.

Preparation of the Sample.——After complete air-drying, all the
samples were ground so that they would pass thru a 100-mesh sieve.
No difficultities were experienced in the grinding except with that
part of the stem which extends a little above and below the surface of
the sand. At this place where the plant needs the greatest strength to
sustain its upright position, the fiber is very tough. The difficulty in
grinding this part of the stem was overcome by adding some grains of
pure sand before grinding the tops and roots. All possible care was
exercised in grinding, yet some slight loss was inevitable. This was
especially true with the soybean leaves, as they are pubescent and small
hairs will sometimes float in the air. However, it is doubtful whether
the error due to this loss was relatively as great as that due to chemi-
cal manipulation. As the results given in the tables all refer to milli-
grams of nitrogen per jar, the presence of sand in some samples does
not affect them. After the samples had been ground they were placed
in air-tight jars until needed for analysis, when the total weight of
each was taken.

ANALYTICAL METHODS

Total Nitrogen—When determining total nitrogen, duplicate
subsamples for each determination on each sample were weighed out
at the same time in order to avoid error from possible moisture
changes. For the tops and roots .5 gram was taken and for the

526 BULLETIN No. 179 [ March,

nodules .1 or .2 gram. The total nitrogen was determined by the Jodl-
bauer method.! Glass beads were used to prevent bumping in digest-
ing. The hydrochloric acid and the ammonium hydroxid used in
titrating were one-tenth normal. Lacmoid was used thruout as the
indicator. The roots were not so easily oxidized as the other parts,
but the use of permanganate to complete the oxidization was avoided,
except in the case of a very few determinations.

Insoluble Nitrogen.—In determining insoluble nitrogen, a .2-gram
sample was weighed out and placed in a 400-cc. shaker bottle, 150 ce.
of water was added, and the bottle was then placed in a mechanical
shaker for three hours. In some cases a smaller sample was used, es-
pecially with the nodules, and sometimes with the roots; in these cases
a smaller amount of water was used. The contents of the bottle were
then filtered onto an S. & S. filter paper and platinum cone, suction
being used when necessary. The residue and filter paper were trans-
ferred to a Kjeldahl flask and the insoluble nitrogen determined by
the Kjeldahl method.

Soluble Nitrogen.—The total soluble nitrogen was obtained by ad-
ding P.T.A.? and Other nitrogen. In the few cases where distillation
was made with sodium hydroxid, the nitrogen obtained is also in-
cluded under the soluble nitrogen.

Nitrogen by Distillation with NaOH.—In several of the harvests
of the first two series, the filtrate from the insoluble nitrogen was dis-
tilled with sodium hydroxid. The nitrogen obtained by this treatment
is reported as nitrogen by sodium hydroxid. This nitrogen may rep-
resent various compounds, as will be explained later.

P.T.A. Nitrogen.—In order to determine P.T.A. saieaderl. the
filtrate from the insoluble residue was made up to 200 ec. with water.
Five grams of concentrated sulfuric acid per 100 ee. of solution were
added and then 10 ee. of a solution containing 20 grams of P.T.A.
and 5 grams of sulfurie acid per 100 ec. of water. In the beginning
30 ec. of P.T.A. solution was used; later this amount was reduced to
10 ee. and in some eases to only 5 ec. with the roots. This precipita-
tion was made in the cold and the solution allowed to stand for forty-
eight hours in order to obtain a complete precipitation of arginine.
At the end of that time the precipitates were filtered off thru S. & S.
filter papers and washed with a solution containing 2.5 grams of
P.T.A. and 5 grams of sulfuric acid per 100 ec. Later, washing was
carried out by using part of the mother liquor. The precipitates on
the filters when thoroly washed and dried were transferred to Kjel-
dahl flasks, and the nitrogen determined by the Kjeldahl method.

*The Jodlbauer method was used at first, as it was thought nitrates might
be present, but later it was discontinued except for determining total nitrogen.

*See note to Table 4, page 500.

1915] A BiocHEMIcAL Srupy or NITROGEN IN CERTAIN LEGUMES 527

Other Nitrogen.—In determining Other nitrogen, the filtrate
from the P.T.A. precipitate, after having been evaporated to about
30 ec. and then made up to 50 ec., was divided into two parts in the
first series, and a Kjeldahl determination made on one half and an
amino-nitrogen determination on the other. This proved unsatisfac-
tory because of the small amount of nitrogen present in the filtrate for
an amino-nitrogen determination. Owing to the excess of P.T.A. in
this filtrate, serious bumping occurred during digestion. Altho glass
funnels were placed in the necks of the flasks to prevent loss, some de-
terminations were lost. Digestion was continued for four to seven
hours with this filtrate.

A few modifications of the above methods are eonsidered in con-
nection with the series in which they occurred. In all the analytical
work the reagents were carefully and constantly checked up for nitro-
gen, altho the methods were applied under the same conditions at all
times.

DISCUSSION OF SOME OF THE METHODS UseEp

The insoluble nitrogen represents certain proteins and probably
other insoluble nitrogenous compounds. The nitrogen obtained by dis-
tillation of the filtrate from the above might represent nitrogen from
acid amides or volatile organic bases or basic amino nitrogen (arginine
and cystine). The presence of volatile organic bases was eliminated
by qualitative tests. Thus it would seem that the nitrogen found by
the distillation represents an amide or basic nitrogen. The presence
of asparagine in soybean seedlings has already been pointed out, but
it cannot be assumed that the nitrogen thus determined was aspara-
gine, as arginine and cystine may have been present.

P.T.A., as already stated, precipitates various nitrogenous com-
pounds. The reagent, however, does not completely precipitate alka-
loids, peptones, proteoses, peptides, and diamino acids (basic nitro-
gen) ; further, Van Slyke! has shown that the diamino acids are-ap-
preciably soluble in this reagent. Osborne and Harris? have demon-
strated that P.T.A., while subject to various criticisms, nevertheless,
when used under constant conditions, gives very good comparative
results.

The filtrate from the P.T.A. precipitate contains all soluble nitro-
gen not precipitated by P.T.A. This nitrogen may be made up of
amino acids, pigment nitrogenous compounds, and possibly other ni-
trogenous compounds.

QUALITATIVE TESTS

A large number of plants and parts thereof, grown under the
same conditions as those harvested for quantitative determinations

*Van Slyke: Jour. Biol. Chem. (1911-12), 10, 15-56.
*Osborne and Harris: Jour. Am. Chem. Soc, (1903), 25, 323-333.

528 Buuuetin No. 179 [ March,

were tested for ammonia, nitrites, and nitrates. Nessler’s reagent was
used to test for ammonia and diphenylamine sulfurie acid for nitrites
and nitrates. All these tests were negative. Ten grams of tops were
treated with 800 cc. of water in a liter flask. The solution was boiled
but no ammonia was obtained. Upon the treatment of the filtrate
from this solution with sodium hydroxid, a large amount of ammonia
was found but no volatile organic bases, as the distillate gave the typi-
eal test for ammonium chlorid when absorbed in hydrochloric acid
and left no carbonaceous residue. Zine sulfate gave no precipitate in
this filtrate. The filtrates from the P.T.A. precipitates were tested in
the Van Slyke apparatus with results which indicate that some of the
nitrogen in the filtrate was in the form of primary amines.

Since the completion of these experiments, a study of the total
amino nitrogen in the seedlings of the Alaska pea, as obtained by the
Van Slyke apparatus, has been reported by Thompson.! His results
indicate that as high as 43.3 percent of the total nitrogen may be in
the form of primary amines. The percentage of amino nitrogen found
in the seed was only .088, increasing in the seven-day seedlings to
28.27.

Series 100 (SoyBEANsS)

The conditions under which the soybeans used in Series 100 were
grown have already been explained (see page 523). Their develop-
ment at each of the five harvests is shown in Table 14.

TABLE 14.—DEVELOPMENT OF SOYBEANS: SERIES 100

F Nodules
Age, Leaves and pods Height,
Planted Harvested days per plant inches Pants
Mar. 18, 1911 | Apr. 25 38 4 leaves 6 265
ental <TRES & May 10 53 Ge? 11 1 061
Srl pants May 17 60 ae 14 987
en eee May 24 67 10-12 leaves; an
average of 5.4 pods | 16-17 | 1 154
apes cea! a May 31 74 10-12 leaves; beans
formed in pods 16-17 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 Srupy or 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.—ToTat NITROGEN IN VARIOUS Parts oF SOYBEANS AND FIXATION
AT DIFFERENT PERIODS: SERIES 100

(Milligrams per jar of five plants)

; ; : Nitrogen | ,,. :
: Lab. | Nitrogen | Nitrogen | Nitrogen |. ~~ Nitrogen | Nitrogen
Harvest | Nos. | in tops |in roots |in nodules|" Whole |’; ) sceas| fixed
plants
111-112
i 114-115 87.10 13.35 28.04 128.49 | 57.30% 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! and Penny and MacDonald.?

The separation of the nitrogen compounds into the various groups
was ecarried 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-ce. beakers; 100 ce. 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 ce.
of hot water. The residue was Kjeldahlized as usual. The filtrates
from some of the harvests were treated with 10 ec. of sodium hydroxid
and distilled. The residual liquid in the Kjeldahl flask was trans-
ferred to a 350-ce. beaker and the excess alkali neutralized with sul-
furie 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

*Wilfarth and Wimmer: Landw. Vers. Stat. (1906), 63, 1-70.
Penny and MacDonald: Del. Agr. Exp. Sta. Bul. 86, 35.

530 BuLuetTIn 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 filtrate 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)

Insol- | sotua- | NaO A.| oth
solu- aOH | P.T.A. ther Total
sax) Jah, Part | Uble ble nitro- | nitro- | nitro- nitro-
vest | Nos. nitro- F
Hn nitro- gen gen gen gen
8 gen
1 | 111-112 | Tops 61.52 24.39 re 4.16 20.23 85.91
114-115 | Roots 8.90 5.00 pleads 85 4.15 13.90
117-118 | Nodules 15.72 11.61 oose 3.54 8.07 27.33
2 | 121-123 | Tops 135.15 37.99 av ae 8.11 29.88 173.14
124-126 | Roots 15.49 5.67. via eft 48 5.19 21.16
127-129 | Nodules 32.83 16.03 seae 9.66 6.37 48.86
3 | 131-133 | Tops 146.79 | 140.12 se-. | 25.63 | 114.49 | 286.91*
134-136 | Roots 27.03 16.42 are 93 15.49 43.45
137-139 | Nodules 47.95 35.00 seve | 18.55 16.45? 82.95%
4 | 141-143 | Tops 183.35 | 134.26 | 17.86 | 25.96 90.44 | 317.61
144-146 | Roots 26.14 12.93 2.49 | 85 9.59 39.07

147-149 | Nodules 31.77 27.27 2.38 | 15.35 9.54 59.04

5 | 151-153 | Tops 151.68 95.32 12.02 | 29.31 53.99? | 247.007
154-156 | Roots 21.55 14.38 1.34 1.38 11.66 35.93
157-159 | Nodules 29.23 27.21 2.00 | 12.13 13.08 56.44

*Taken from Table 15.
*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 StTuDyY oF NITROGEN IN CERTAIN LEGUMES 531

So.uBLE 4p InsocuBLE Nit ROGEN
iN Tors Floors AND NonULES OF SOYBEANS
AT
DiereRrenr Permions of LlevELOPNENT

Series /OO
LEGEND

So.usce CJ InsocuBle

aula

38 Ja. 53 Te. 60 De. 67 De. 74 Das
Noputes

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, |Leaves and pods Height, Nodules per
days per plant inches 15 plants
Aug. 8, 1911 |Aug. 22 14 3 leaves 7 301
of edt oe Aug. 30 22 ae 10 278
Ore al Sept. 8 30 oa 14 370
mee Li ed Sept. 19 41 7-8 ’’ ; 6 one- 14 247 (large)
inch pods

*For 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)

sae. Lab. Nitro- Nitro- | Nitro- plea Nitro- | Nitro-
dak on gen in genin | genin whole | genin gen
: tops roots | nodules seeds fixed

plants
1 511-519 46.99 8.50 35 55.84 57.307 (-1.46)

2 521-529 47.32 11.08 11.05 69.45 57.30 12.15

3 531-539 96.96 9.76 17.81 124.53 57.30 67.23

+ 541-549 205.40 18.65 26.98 251.03 57.30 193.73

‘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 BrocHEMicaL Stupy or 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.
19.—NITROGEN SEPARATIONS:

TABLE Series 500 (SoyBEANs)

(Milligrams per jar of five plants)

Insol-

Total

NaOH | P.T.A. | Other Total

Har- Lab. Part uble soluble nitro- | nitro- | nitro- nitro-
vest Nos. nitro- nitro- on wah

gen | gen | S™ | gem | gen | gen

1 511-512 | Tops 19.48 25.90 3.16 8.31 | 19.43 45.38

514-515 | Roots 4.42 4.48 95 sae 3.20 8.90

517-518 | Nodules eae Saere at ue See HA

2 521-523 | Tops 29.60 17.34 1.18 4.96 | 11.20 46.94

524-526 | Roots 7.97 2.14 .00 47 1.67 10.11

527-529 | Nodules fees Sh eae ae Stays eravaie Sree

a 531-533 | Tops 70.04 31.64 2.67 6.48 | 22.49 101.68

534-536 | Roots 8.56 2.29 .00 .70 1.59 10.85

537-539 | Nodules 16.75 1.29 .00 .00 .96 18.04

4 541-543 | Tops 115.39 76.11 10.68 | 65.43 191.50

544-546 | Roots 13.16 7.05 2.06 5.29 20.51

547-549 | Nodules 22.55 2.46 .26 2.20 25.01

Serres 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.
various harvests are shown in Table 20.

The conditions of the plants at the

TABLE 20.—PLANT DEVELOPMENT: SERIES 700 (SOYBEANS)

] Age Leaves Height Nodules per
Planted Harvested days per plant inches 15 plants
Sept. 13, 1911 | Sept. 25 12 2 leaves 5 Nodules pres-
ent too small
to remove
Pe at ee Oct. 6 23 3 leaves par- 8-9 254
tially devel-
oped
a ie Oct. 14 31 5 leaves 10-12 229
29), PGR Eh Oe 2 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 OF SOYBEANS AND
AT DIFFERENT PERIODS:

SERIES 700

(Milligrams per jar of five plants)

FIXATION

Ena Maen Nitro- | Nitro- | Nitro- penta Nitro- | Nitro-
eat Nua gen in genin | genin | Viole gen in gen
tops roots | nodules plants seeds fixed

1 | 7ii-719 | 40.08 | 888 | .... | 48.96 | 57.30 | (-8.34)
2 | 721-729 | 48.00 | 893 | 7.21 | 64.14 | 57.30 6.84
3 | 721-739 | 6832 | 1138 | 897 | 88.67 | 57.30 | 31.37
4 | 741-749 | 110.70 | 20.55 | 17.22 | 148.47 | 5730 | 91.17

1See 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)

Insol- Solu-
Har- Lab. Parts uble ble pein bind brit
vest Nos. nitro- nitro- ae ey. rained
gen gen 8 gen se
af (fail Tops 9.94 28.03 4.70 23.33 37.97
714 Roots 3.87 4.95 39 4.56 8.82
ralys Nodules ees dia Sete hte Siots see's
2 721 Tops 23.64 21.56 11.96 9.60 45.20
724 Roots 5.50 2.55 ard § 1.78 8.05
127 Nodules arate 2 Ea a; exe see savers
3 731 Tops 26.50 29.05 13.75 15.30 55.55
734 Roots 7.30 3.76 1.10 2.66 11.06
737 Nodules 6.30 1.79 .10 1.69 8.09
4 741 Tops 46.45 61.97 28.75 38.22 108.42
744 Roots 10.67 6.31 61 5.70 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 BrocHEmicaL Stupy or NITROGEN IN CERTAIN LEGUMES

So.us.e Ano Insocus.e Nit ROGEN

IN Tors. Floors AND Nonuces OF OovBEANS
AT
LD) rr eRpent Pe FIODS OF Te vELOPME NT

SocuB.e LC] Inso.usie

SE HES 700

ee fe

Tors
——_ cm cme -
Ffoo7s
— | i
72 fla 23 Tlo- 31De. 9? Ta.
Nor ek S

Se RIES 500

Tors

c—_ ee —E |
Floor 5

14 Da. 22 Ta 30 Ta 41 Da.
Noaures

PLATE XVI

535

536 ButieTin 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 (CowPras)

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)

Age, | Leaves Height, Nodules per

metal Harvested days | per plant | inches 15 plants
Aug. 8, 1911 Aug. 22 14 3 leaves a. 450 (very small)
Oe ran ee Aug. 30 22 a ee 10 892 (small)
Feet ge? Sept. 8 30 aa 12 1074
i i el Sept. 19 41 6-7: 27 13-14 | 2062
ee ee Oct. 5 58 ene hg 14 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.—TotTaL NITROGEN IN VARIOUS PARTS OF COWPEAS AND FIXATION
AT DIFFERENT PERIODS: SERIES 600

(Milligrams per jar of five plants)

Nitrogen
Sarva Lab. Nitrogen | Nitrogen | Nitrogen | Nitrogen} in unin- | Nitrogen
Nos. in tops | in roots jin nodules|in whole | oculated |_ fixed
plants | plants
i 611-619 29.11 9.05 1.96 40.12 36.744 3.38
2 621-629 45.46 10.25 9.22 64.93 36.74 28.19
3 631-639 91.63 16.64 18.52 126.79 36.74 90.05
4 641-649 188.40 30.28 42.33 261.01 36.74: 224.27
5 651-659 439.64 73.73 64.25 577.62 36.74 540.88

*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 BrocHEMICAL Stupy oF NITROGEN IN CERTAIN LEGUMES 537
TABLE 25,—NITROGEN SEPARATIONS: SERIES 600 (COWPEAS)
(Milligrams per jar of five plants)

Waveked Lab. Part | Insoluble eer: P.T.A. | Other Total

Nos. nitrogen | J+. ogen nitrogen | nitrogen | 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 SAPS Saat ‘i oi pisate

2 621-623 | Tops 23.73 | 21.76 5.88 | 15.88 | 45.49
624-626 | Roots 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
imsoluble 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 eases 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.

BULLETIN No. 179

So.uBLe AND LNs0.uBLeE Nit ROGEN

in ToPs Floors ano Nonures oF CowPe 45
AT

Whe rerenr Feriops of DEVELOPMENT fron

Series 600
LEGEND
SOLUBLE cl INSOLUBLE Ped

= oe

Froors

-_at I

14 Da. 30 Ta 41 De. § Ta
erie ES
PLATE XVII

[ March,

1915]

A BIOCHEMICAL StupDyY OF NITROGEN IN CERTAIN LEGUMES

TABLE 26.—AVERAGE DAILY FIXATION OF NITROGEN IN ALL SERIES

539

. Periods Milligrams per
Rerits in days jar of 5 plants

0-38 1.87
38-53 9.72

100
53-60 19.84
Leeyueans) 60-67 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
22-30 7.73
(Wawpeas) 30-41 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 Roots Nodules
100 (Soybeans) 35.9 34.7 42.3*
700 (Soybeans) 57.7 39.5 18.9
500 (Soybeans) 41.2 32.4 8.5
600 (Cowpeas) 45.0 27.5 34.0

*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) 25.1 29.3 ae
700 (Soybeans) 36.1 32.8 18.2
500 (Soybeans) 30.7 23.1 6.9
600 (Cowpeas) 28.3 17.2 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 OF 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 Roots | Nodules -
100 (Soybeans) 7.00 3.0 oiea
700 (Soybeans) 21.10 6.6 0
500 (Soybeans) 6.75 6.0 1.4
600 (Cowpeas) 13.40 7.3 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 eases the amounts precipitated vary widely, while in
others the agreement is close. In these series the nitrogen precipitated
by phosphotungstie 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 searcity of existing knowledge regarding the
more fundamental problems concerning nitrogen fixation.

The biological resumé 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.

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BULLETIN. URBANA
166-181 1914-15