BIOLOGICAL NITROGEN FIXATION-EARLY AMERICAN STYLE'

PERRY W. WILSONDepartment of Bacteriology, University of Wisconsin, Madison, Wisconsin

ESTABLISHMENT OF THE STATE AGRICULTURAL EXPERIMENT STATIONS........................... 405ATWATER'S EXPERIMENTS ON NITROGEN FIXATION AT WESLEYAN............................... 407CONTRIBUTIONS FROM THE EXPERIMENT STATIONS .............................................. 411LITERATURE CITED........................................................... 415

ESTABLISHMENT OF THE STATE AGRICULTURALEXPERIMENT STATIONS

The year 1962 marked three noteworthy anni-versaries that have been observed with appropri-ate exercises, conferences, publications, andsimilar events at Land Grant colleges throughoutthe nation. These were: (i) the centennial oftheir authorization by the Morrill Act of 1862;(ii) the centennial of the establishment of theUnited States Department of Agriculture; and(iii) the 75th anniversary of the passage of theHatch Act, the basic legislation for the StateExperiment Stations. Since the American Societyfor Microbiology has a Division of Agriculturaland Industrial Bacteriology, it seems appropriatethat notice, even though somewhat belatedly, betaken of these far-reaching legislative events. Mycontribution, therefore, is dedicated to thosepioneers, some of whom were members of thisSociety, whose careers in the early days of theseinstitutions helped establish the patterns oftoday's research.

In 1862, agricultural experimentation was notunknown in the United States, but its typicalexpression was that of the Trustees' garden inGeorgia established in 1733 (14), rather than thatof the modern experiment station. Among themany individuals who contributed to this change,two of the leaders were S. W. Johnson, professorof chemistry at Yale (Sheffield), and his protegeW. 0. Atwater (Fig. 1).Johnson had returned to Yale in 1855 after

spending a few years in Germany where he hadbeen deeply impressed with the operation of theagricultural experiment stations, especially theone at Moeckern. His efforts to set up a similarstation in Connecticut were delayed because of

I Presented as part of a symposium on theHistory of Microbiology at the Annual Meeting ofthe American Society for Microbiology, Cleveland,Ohio, 8 May 1963, with Raymond N. Doetsch asconvener.

405

the Civil War, but by the seventies he had clearlyin mind the requirements of an "ideal" station.These were: (i) it should have a State charter andsubsidy, (ii) its sole function should be research,and (iii) it should be near to but independent of acollege (15, p. 22). In addition, Johnson proposedwhat must have been a real shocker to thosewhose idea of an experiment station was that itshould be concerned almost exclusively withvariety trials of crops and chemical analyses ofsoils and fertilizers. He stated that the stationshould be in a suburban area rather than on afarm. Johnson's reasons for this choice were thatit was important to be near facilities of library,post, telegraph, and express office, as well aspublic utilities; moreover, the experimental workcould be more effectively carried out in laboratory,greenhouse, and small garden plots than in thefield. Through the influence and subsidy ofOrange Judd, farm paper publisher and formerstudent of Johnson, a trial station was authorizedto be set up at Wesleyan University with W. 0.Atwater as its director. Apparently, Atwateryielded nothing to his former teacher in his zealfor the "ideal station"; it was written of him(15, p. 23):

The Johnsonian formula for stationorganization owed its triumph in nosmall way to the evangelical efforts ofthe young chemist, Wilbur Atwater, whoin 1873 had begun a professorial careerat Wesleyan after earning his doctoratein Johnson's laboratory in 1869 andspending 2 additional years in post-graduate work at German experimentstations. Filled with a ministerial zeal forspreading the gosepl of German agricul-tural science, Atwater volunteered hispen and his voice in the station cam-paign in Connecticut. Energetically inthe midseventies he publicized his firsthand knowledge concerning the workand current organization of the German

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Samuel W. Johnson, 1830-1909 Wilbur 0. Atwater, 1844-1907AI.ri4aM thlt vit e ofIrci dturl First tirevtor of the Office <of Ex+eri-I

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t}1"a\a - 1 t}teliltt 81 1i qisrn.1sFini}e, i1 o }~re~searchO I Hi efTots led,( to4 the es- mnent. Sttins 1-dal piInt er in ligri.-tXlII l rletI43l '7te to 1:1ithe ('onhleL 4itit eultual sztrnstttimnworkl inA} rStiat nl Ulxeri ntSlation- the nIitedSttates.lutst. in. the, Unlitediles

FIG. 1. Portraits of Johnson and Atwater. From Knoblauch, Law, and Meyer (15). This book is an

interesting and authoritative history of the state experiment stations written to commemorate the 1962 an-niversaries of the Land Grant and Hatch Acts.

stations. His definition, a product ofhis recent apprenticeship at the Germanstations, coincided with Johnson's andunderlined the central significance ofbasic research.

Note that this trial station was not yet a fullfledged State experiment station, since its supportinitially was both public ($2800 annually) andprivate (Judd gave $1000 a year and Wesleyanfurnished quarters). But 2 years later (1877) theConnecticut State Experiment Station was for-mally set up withtemporary quartersnear Sheffieldand with S. W. Johnson as its director.The next decade witnessed widespread agita-

tion in the form of meetings, memorials to Con-gress and state legislatures, and articles in thepublic and agricultural press advocating theestablishment of state stations. Progesss was

slow, primarily because of difference of opinion

among the advocates. Some states had followedConnecticut's lead in establishing independentstations; others, including eight of the states withland grant colleges, had placed them undercollege administration. Also, the question offinancing was touchy-advocates vaguely sug-gested that funds be provided by private agri-cultural organizations, by the states, and also bythe Federal government. Opponents bridled atthe latter proposal asking (15, p. 42):

Why, for example, should Congress-men, acutely aware of the historic dis-tinctions between Federal powers andStates' rights, intervene in the re-

sponsibilities of the State governmentsin order to do what the State legislatureshad not done? Why should Congress-men, politically accustomed to a Fed-eral Government of strictly limited

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functions, activate at this time, therudimentary precedents which for 20years had lain dormant? If, furthermore,American farmers desperately neededthe benefits of experimentation, whyhad not the farmers, comprising asubstantial majority of the Americancitizenry, insisted long since that sta-tions be established?

The reply was made by E. W. Hilgard, directorof the California station at Berkeley; writing toJohnson in March, 1882, he stated, "I ambeginning to feel aggressive on the subject," andin May he published an article in the AtlanticMonthly which presented cogent arguments forFederal subsidy (15, p. 41):

The Federal Government, Hilgardurged, ought no longer to leave the prob-lem of collegiate agricultural researchsolely to the jurisdiction of theindividual State legislatures. It shouldrecognize an unfortunate situation ofnationwide dimension, namely, thatthe States most in need of the improvedtechnology that only a local stationcould provide had stingy legislaturesand penniless colleges. The FederalGovernment, then, should followa corrective policy of "enlightenedintervention" and supply "substantialaid" to each land grant college for thespecific purpose of operating a station.

Finally, after much debate and politicalmaneuvering, appropriate bills were introducedin Congress-and failed to pass. Eventually,one, the Hatch Act, became law but not withoutstruggle and compromise. One amendmentprovided that the station need not be set up atan agricultural college. This was to gain supportfrom those states which had already set upindependent stations. For this writer, theunkindest cut was that Senator Spooner ofWisconsin introduced this provision on thegrounds (23, p. 129):

That the agricultural department ofthe University of Wisconsin had notbeen a success, and that it was almostimpossible to secure the attendance ofany larger number of agricultural stu-dents in classical institutions where from300 to 500 students were pursuing class-ical or scientific courses.

Another amendment provided that a state neednot participate if it did not wish. This solicitousattempt to protect states from federal subsidyproved unnecessary; in the first year, all statesand one territory approved the provisions andduly collected the appropriation.

Although the Hatch Act did not embodyall the Johnson-Atwater formulae for the"ideal" station, the really important one, thatthe stations should undertake basic research,was preserved.

ATWATER'S EXPERIMENTS ON NITROGENFIXATION AT WESLEYAN

In 1885, Atwater published results in theAmerican Chemical Journal of the first experi-ments on biological nitrogen fixation undertakenin the United States. Before looking at theseresults, a few words about the paper itself are ofinterest. First, it is lengthy (21.5 pages) andsomewhat repetitious. The essential data canbe summarized in a single table, but many othertables are included giving details of the analyses,weight of seed, and other statistics togetherwith alternative methods of calculating theresults. On reading through these details, onefinds less objectionable the modern practice ofeditors of demanding concise presentation.Second, it qualifies as a report of authenticresearch in this field as the experiments startwith an accident that casts doubt on the findings.Atwater (4) explains:

Besides the Nos. A, B and C, theseries included two others, D and E,into which, unfortunately, a quantityof rain-water fell through a leak in theroof of the greenhouse during a vio-lent shower. The water percolatedthrough the sand and was caught in thebeakers below. The accident was dis-covered by the gardener who had beencharged to watch the plants, and whopoured the contents of the beakersback upon the sand before either theassistant in charge of the experimentsor myself had learned of the matter.One of the pots received more rain-water than the other, and more fell intothe beaker below. The analysis at theend of the experiment showed in onecase a loss of nitrogen and in the othera very large gain....

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It would, of course, be unwarrantableto include these experiments with thosedescribed above (A, B and C), and, forthat matter, the possibility of such anaccident casts a suspicion upon thewhole series, so that their results canbe entitled to full credence only oncondition of their being verified byrepetition under circumstances fitted topreclude such sources of error.

Finally, the urbanity of the acknowledge-ments at the end should not be lightly dismissedin these days of sterile forms that "this researchwas supported by grant number such and suchfrom the NSF or the NIH." Space is not at apremium, so Atwater writes:

I improve this opportunity toexpress my obligations to my assistant,Mr. C. D. Woods, by whom the detailsof the experiments above describedhave been most faithfully and skillfullyperformed.

It gives me a great pleasure, also, tobear testimony to the generosity ofHon. J. W. Alsop, M.D., of this city(Middletown, Conn.), who has, bydefraying the larger part of thepecuniary cost, and by aid in otherways, made the investigations practica-able.

The results shown in Fig. 2 leave little doubtbut that Atwater obtained nitrogen fixation by

the pea plants, nor does he hedge unduly on thisaspect. He states forthrightly in his conclusion:

The outcome of these experimentsmay be concisely stated thus: 1. Theplants, peas, grown in nutritive solutionsexposed to the air, but protected fromrain and dew, contained at maturitymuch more nitrogen than was suppliedthem in nutritive solution and seed.Such were the results of a first seriesof trials, confirmed, even more strikinglyby a second series the succeeding year.For this excess of nitrogen there wasbut one possible source, namely, theatmosphere.

He observes, however, "That the plants as-similate free nitrogen is contrary to generalbelief and to the results of the best investigationsof the subject. The disagreement betweenthese results and my own may, however, bemore apparent than real. Two points in particularseem to me worthy of note."The two points were: (i) since the negative

experiments were made with little or no addedcombined nitrogen available to the plants, thismight have depressed nitrogen fixation; and(ii) the most decisive experiments were con-ducted under glass covers connected with theearth, a circumstance that probably wouldeliminate a necessary electric potential. Thislatter novel explanation was based on thefollowing consideration:

Statistics of Experimzents on the Assimilation of AtmosphericNitrogen by Peas.

Conditions of Experiments.

Larger (Conecntrated Scantily fed.

Naitogn, Dilute Sblu.. Scantily fed.

I tion. IWell fed.

Smaller rConcentrated Scantily fed.NitrogenJ Solution. I Well fed.Raitogn. Dilute Solu-3 Scantily fed.

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Total.

Mgm.207.2171.7205.7171;5130.9

93.6131.994.7

Nitrogen found atEnd of Experiment.

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FIG. 2. Atwater's table summarizing the results of the first series of experiments (1885-1886)

Appar-ent

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Berthelot has shown that free nitrogenmay be assimilated by vegetable sub-stances, dextrine, cellulose, etc., underthe influence of electricity of a potentialsimilar to that which obtains near thesurface of the earth, in the strata ofair in which our cultivated plantsgrow. The inference that the compoundsin living plants may assimilate nitro-gen in the same way is natural. Thatthis electrical tension may have beenabsent in the experiments with theplants under glass, referred to, wouldseem probable. The hypothesis of theassimilation of free nitrogen by plantsthrough the agency of electricity, andthe absence of that agency in theexperiments of Boussigault and ofLawes, Gilbert and Pugh, would, withthe effect of scanty food supply, explainthe discrepancy between their experi-ments and my own, in which the con-

ditions of growth were normal, andwould clear up the worst difficulty inthe much vexed question of the sources

of nitrogen of plants.In 1886, Atwater (5) returned to the argument

in a long paper in which he compared his own

results, including some new data dealing withliberation of nitrogen from nitrate by germinat-ing seeds (probably bacterial denitrification),with "the best investigations." The chief interestof this paper is that a new factor is offered tojoin the other two in explanation of the dis-crepancies-the possible role of bacteria isrecognized.

To recapitulate in a few words: Theexperimental testimony regarding theacquisition of atmospheric nitrogen byplants is conflicting. But the evidenceagainst it which comes from the lab-oratory and the greenhouse is basedupon experiments whose conditions were

more or less abnormal in respect tofood-supply or access of nitrogen com-

pounds or otherwise. In those whichseem most conclusive against theassimilation of free nitrogen the ar-

rangements were such as maV havehindered the action of electricity, ifnot that of nitrogen-fixing micro-organisms, two agencies toward which

late research points as possible, ifnot certain, factors in the fixation ofnitrogen. In all there is the possibility,and in some a very strong probability,that the results may have been affectedby liberation of nitrogen from seedsor plants or food supplied, a liberationwhich is sometimes, if not always, dueto ferments. This may materiallyreduce the nitrogen found at the end ofthe experiments, and with it the ap-parent gain of nitrogen from the air.

The inability of Atwater to choose between thephysical and the biological explanations probablyreflects his training and experience as a physicalscientist. The published details of his chemicalanalyses can evoke our admiration today. Butone wonders, admittedly fruitlessly, if Atwaterever discussed his experiments with his colleaguein biology, young H. W. Conn, who was begin-ning to show interest in the role of "germs" inagricultural science.On May 18, 1887, the Connecticut Assembly

passed a resolution accepting and assenting to theprovisions of the Hatch Act, and assigning one-half of the funds to the Storrs AgriculturalSchool to be used for the experimental farm andthe remaining one-half to the New HavenStation. Atwater was made the first director ofthe Storrs Station. At this time, Storrs was onlya secondary school for boys; the 2-year programincluded such subjects as general and agriculturalchemistry, natural philosophy, botany, zoology,mineralogy, surveying, theoretical agriculture,stock breeding, and English composition.Obviously, it was no "Do-the-Boys-Hall," butthe Squeers' philosophy for educating the youngwas not overlooked. The teen-agers were alsoexpected to "acquire dexterity" on the farm bylaboring for 3 hr a day in the fall semester, and5 hr each day in the spring. This work wassupervised by a farm superintendent whohad held a similar position for 27 years at theState reform school (22, p. 323). It seems asafe conclusion that, however much the boysmight have neglected their homework, the fieldwork was attended to with diligence.In the second annual report (6), Atwater and

Woods published further experiments on nitrogenfixation explaining:

The circumstances which caused thecessation of the investigation from

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1883 to 1885 were alike operative fromthe latter time until 1888, when theorganization of the chemical work ofthe Station in the chemical laboratoryof Wesleyan University, where theprevious investigations had been made,rendered their continuation possible.Meanwhile the very important in-vestigations of Hellriegel and othershad confirmed those of the first andsecond series referred to, and, whilebringing additional evidence of theacquisition of atmospheric nitrogen,had also indicated the very strong prob-ability that microorganisms are activeagents in the assimilation of nitrogen.

The results summarized in Fig. 3 are clear-cut and complete; this time the table includesinformation on the nodules, or "tubercles."The role of these is emphasized in the summary.

Peas, alfalfa, serradella and lupinecertainly, clover in all probability, cow

peas presumably, and perhaps legu-minous plants in general, are able toacquire large quantities of nitrogen fromthe air during their period of growth.That there is a connection between

root tubercles and the acquisition ofnitrogen is clearly demonstrated. Whatthis connection is, what are the relationsof micro-organism to the root tuberclesand to the acquisition of nitrogen, andin general how the nitrogen is obtained,are questions still to be solved.

If brief, the acquisition of largequantities of atmospheric nitrogen byleguminous plants which was dem-onstrated by the former experimentshere and has been since confirmed byHellriegel and others, is still furtherconfirmed by the experiments here-with reported. These experiments inlike manner confirm the observation byHellriegel of the connection between

EXPERIMENTS WITH PEAS AND ALFALFA, i888-i889.Condensed Summary of Results and Averages.

Root Tubercles.

None, - -c Few,v Fair number,

LLarge number,

, None,J Few,I Fair number,

¢ Large number,

Without Nitro-gen in Nutritive

Solutions.

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FIG. 3. Results of the second series of trials (1888-1889)-post Hellriegel and Wilfarth

Total.

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root tubercles and the acquisition ofnitrogen.

The last statement appears redundant andsomewhat weak. Although Atwater himselfprobably took an active part in the experimentsof 1888, those in 1889 must have been primarilyunder the direction of Woods who was now theacting director of the station. On October 1,1888, Atwater left for Washington to become thefirst Director of the newly formed Office ofExperiment Stations of the USDA.

CONTRIBUTIONS FROM THEEXPERIMENT STATIONS

Although Atwater must have been disap-pointed that he missed the essential observationthat would have given him priority, the manycontributions of the State Experiment Stationsin the next decade to this field should have beena source of satisfaction. In an 1893 review ofpapers published during the past 2 years,Schneider (21) stated:

I shall review foreign and Ameri-can investigations separately, as theplans and ends sought for are some-what different. European investigatorshave discovered the subject in hand andhave studied it from a thoroughlyscientific standpoint. Americans havebut very recently taken up the subjectand have treated it mainly with a viewto turning it to some practical usewhile they yet, in many cases, lacksufficient scientific data.

Since foreign investigators havemade the most important investigationsand have, so to speak, laid the founda-tion upon which future workers are tobuild, it is but just to give them ourattention first.

His first citation of "the most importantinvestigations" proved to be unfortunate-itwas one by Frank and Immendorff that al-legedly demonstrated that "all plants arecapable of utilizing more or less of the freenitrogen of the air." Their conclusion on anothercontroversial aspect, however, has stood thetest of time.

The question whether the rhizobiumhas in itself the power to assimilatefree nitrogen is not fully demonstrated.From observations upon rhizobia in

cultures, it seems that they do notassimilate more free nitrogen thanother bacteria. It is most likely thatthe presence of the rhizobium in plantsonly awakens latent assimilatingpowers.

Considering their basic nature, the citedAmerican contributions needed no apology.Schneider explained that in July, 1892, he hadsuggested that the rhizobia might be dividedinto several species. Having admitted that thiswas a bold and somewhat unwarranted thing todo (he did not say why), he went on to discussa paper that Bolley later in that year con-tributed to the program of the Association ofAmerican Agricultural Colleges and ExperimentStations titled, "Notes on root tubercules(WVurzelknollchen) of indigenous and exoticlegumes in virgin soil of the Northwest." Itcertainly impressed Schneider who wrote:

I wish to state here that the paperis remarkable, not so much for itsscientific value, as for its exact and clearcut statements. They are the statementsof a man whose preceptive facultiesare wide awake and who can describeexplicitly what he sees. It is sincerelyto be hoped that more writers willfollow his example.

Perhaps the source of his enthusiasm can befound in the following statement.

His general conclusions are thattubercle-forming organisms are freelydistributed through virgin soil, thusinfecting legumes wherever they maybe found. He also makes the suggestionthat there may be more than onespecies of Rhizobium.

He is less satisfied with the extensive studiesjust published by Atkinson observing,

His historical review is quite completeand of great importance to those whocan read only English. The originalpart of his paper is rather unsatisfactoryin many respects.

The final part of the paper is a summary of someexperiments that Schneider was making at theIllinois Experiment Station. He describes anattempt to infect maize by cultivating rhizobiaon an infusion containing extracts of corn roots.This was not successful, although he claims theorganism did invade the root hairs of the corn.

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Other trials were concerned with the physiologicalproperties of the rhizobia and the morphologyof the root nodules. Surprisingly, he stated"that it is very difficult to obtain pure culturesof any given species of rhizobia." Mentionshould also be made of two useful contributionsof this early American period: the review byAtkinson (2) of the Station at Auburn, Ala.,contained in a bulletin devoted to nematoderootgalls, and a bibliography of over 600 titlesby MacDougal (16) of Minnesota.Although the experiments discussed by

Schneider certainly could qualify for a basicresearch grant from a federal agency today, it istrue that workers at many of the stations con-cerned themselves with very practical questionssuch as the one forthrightly asked by Duggar(10): "What classes of soils in Alabama wouldbe benefitted by inoculation for clover, vetch,etc.?" Stubbs (9), in a letter to the Commissionerof Agriculture and Immigration at the BatonRouge Station in La., was more eloquent.

DEAR SIR-The leguminous plantshave been known to be restorative intheir character when used for improve-ment of soils in a systematic rotation ofcrops. Some years since it was discoveredthat the chief virtue of these plantsin abstracting and appropriatingnitrogen from the air, was due to thetubercles which occur upon theirroots. With the view of throwinglight upon this subject, and especiallyof studying these leguminous plantswhich are in common use for soilrestoration in this State, the followingexperiments were inaugurated, and havebeen successfully conducted by Prof.W. R. Dodson, Mycologist of theStation. For the purpose of diffusinginformation upon this subject amongour farmers and planters, I ask thatyou publish this report as Bulletin No.46.

Probably Bulletin 96 from the Kansas stationat Manhattan, dealing with the inoculation ofsoybeans, typifies the research (8). It has aninteresting table summarizing replies fromstate stations to an inquiry asking whethersoybeans in the region possessed nodules. Six,including the Hatch Station at Amherst, Mass.,reported that the organisms were indigenous tothe soil: two said they had obtained nodules

by inoculation: five could find no nodules; tenstated that they didn't grow soybeans (Min-nesota and Washington explaining it was toocold in their regions), and eighteen hadn'tlooked. The Kansas workers imported somesoil from the Hatch station and made someinteresting greenhouse and field experimentsfrom which they concluded:

Our experiments have not been con-ducted long enough to thoroughly testthe matter, but it is probable that afield once inoculated will always remaininoculated, and that the bacteria willslowly increase in the soil. The bacterialive for a long time in the soil after theplants are removed. We have kept dryinoculated soil in sacks two years,where it became as dry as road dust,and it had full strength in producingroot tubercles when used.

After the turn of the century, many stationspublished similar bulletins; my last choice forexamination is one at the federal level, U.S.Department of Agriculture Bureau of PlantIndustry Bulletin 71, which is certainly amixed bag (17). George T. Moore, its author,provided a valuable and scholarly historicalaccount of the use of artificial inoculation,directions for use of an inoculum put out by theU.S. Department of Agriculture, and reportsfrom satisfied customers. The names of Hell-riegel and Wilfarth are consistently misspelled,and the statement is made that, when theorganism is grown on a medium high in fixednitrogen, it loses the power to fix atmosphericnitrogen and "for this reason the mere matter ofan abundant growth is one of the least desirableconsiderations in propagating these organismsfor any practical purpose." Moore also statedthat he obtained significant fixation of nitrogenby the organism alone so that "there could belittle doubt about the power of Pseudomonasradicicola to fix nitrogen independent of anyleguminous plant." Even more interesting, heincludes a photograph showing excellent growthof inoculated alfalfa without nodules, a resultthat he ascribes to "internal infection."

Since dirt farmers were not likely to beinterested in the scientific portion of this bulletin,a companion Farmers' Bulletin 214 (18) wasissued; it was strictly business, and was illustratedwith line drawings to insure that the directionswere understood (Fig. 4). This was essential,

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//

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. .PA.

FIG. 17.-Stirring seed moistened with culture liquid to hastendrying.

FIG. 4. Illustration from Farmers' Bulletin 214

since the method advocated was a do-it-yourselfdeal in which the farmer was furnished packagedconstituents for adding to water to grow thebacteria which were supplied dried on cotton(U.S. Letter Patent 755,519). This bulletinalso carried testimonials from farmers livingat such nostalgia-invoking place names as SandyRun, S.C., Grandview, Tenn., and Simplicity,Va. Adolph Soderberg of Sister Bay, Wis.reported that "there were more pods on thevines that were treated and about 3 bushels morepeas to the acre on those that were treated,"Yas well as "about twice as many nodule forma-tions." An account of a remarkable experimentby one of the editors of Hoard's Dairyman(Fort Atkinson, Wis.) includes the followingstatement.

Our field already shows the goodeffect of inoculation. (The methodconsisted in going over an alfalfa field

that was not thriving with a sprinklingcart containing the culture liquid. .. )

Surprisingly, in view of their later interest inbiological nitrogen fixation, bacteriologists at thestation at Madison, Wis., did not contribute tothe early publications. H. L. Russell, laterDirector of the Station, published one of theearly reviews (19) on the subject, but a summaryof work by the department (1893 to 1903) listedno experimental work in this field (20). Anexamination of bachelor of science theses in theE. G. Hastings Memorial Library of the depart-ment revealed that in the period from 1895 to1915 only one dealt with nitrogen fixation,ranking this subject with "A study of thebacterial contamination of telephone trans-mitters" as the least popular areas for thesisresearch. The student, H. A. Smythe, noted:

As the Bacillus Radicicola had neverbeen grown in this laboratory, my first

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object was to secure the organism inpure culture on artificial media. ThisI found a much more difficult proposi-tion than I had anticipated.

The 22nd Annual report of the station (1905)summarized some inoculation experiments on

/1/

alfalfa and soybeans made by Russell and R. A.Moore in Agronomy. Conrad Hoffmann, thesoil bacteriologist in the department, publishedwith B. W. Hammer a research bulletin in 1910dealing with nitrogen fixation by the azotobacter,but no consistent program in this field was

i_ _~~~~~~~~~~~~~~~~~~~~~~~~~.__. ....

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FIG. 5. B. B. Fred's notes on the later experiments of Atwater and Woods. The comment in the upper righthand corner must refer to confirmation of the results of Hellriegel and Wilfarth.

414 WILSON

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BIOLOGICAL NITROGEN FIXATION

evident until young E. B. Fred joined the staffin 1913, replacing Hoffmann who had left. At theBlacksburg station in his native Virginia, Fredhad published several reports on nitrogenfixation; his first at Wisconsin appeared in 1916(12, 13). But during this time he was planningthe monograph about which Bay (7) was laterto state:

Easily the most outstanding of allworks on economic bacteriology is Hell-riegel and Wilfarth's Untersuchungenuber die Stickstoffnahrung der Gramineenund Leguminosen (1888), which openedour knowledge to the fundamentalsupply of nitrogen for the productionof protoplasm in plant organs bybacterial action. This subject hasbeen treated in a masterly manner byFred, Baldwin and McCoy, in theirmonograph, Root nodule bacteria andleguminous plants (1932), an Americanclassic.

Figure 5 reproduces one of Fred's earliest notesused in preparing this outstanding work-hiscomments on the 1888-1889 papers of Atwaterand Woods. Although off to a late start, the"school" founded, and led for many years, byFred was noted for its balanced program of basicand applied research. Such fundamental studiesas the physiology and taxonomy of the bacteria,the cytology of the nodules, and the biochemistryof the nitrogen-fixation process were made in thelaboratory and greenhouse along with the fieldtrials. Indeed, one of the first contributions wasthe production and testing of a reliable inoculumfor the farmers-during World War I as part ofthe "Food Will Win the War" campaign (1).Few of us who underwent training in this schoolescaped our tour of duty in this service toagriculture. Discontinued during World War IIwhen the department ran low on personnel andagar, it survives in the several directors oflaboratories in the firms supplying commercialinoculants. We were young, so it was all excitingand rewarding to those who participated, and Ibelieve that, perhaps, this is what Sam Johnsonand Wilbur Atwater had in mind when theydreamed of their ideal station.

LITERATURE CITED

1. ANONYMOUS. 1918. Starting a farm nitrogenfactory. The Wisconsin Farmer, March 7,1918.

2. ATKINSON, G. F. 1899. A preliminary reportupon the life history and metamorphoses ofa root-gall nematode, Heterodera radicicola(Greeff) Mull., and the injuries caused by itupon the roots of various plants. AlabamaAgr. and Mech. Coll., Agr. Expt. Sta. Bull.No. 9.

3. ATKINSON, G. F. 1893. Contribution to thebiology of the organism causing leguminoustubercles. Botan. Gaz. 18:157-166; 226-237;257-266.

4. ATWATER, W. 0. 1885. On the acquisition ofatmospheric nitrogen by plants. Am.Chem. J. 6:365-388.

5. ATWATER, W. 0. 1886. III. On the liberation ofnitrogen from its compounds and theacquisition of atmospheric nitrogen byplants. Am. Chem. J. 8:398-420.

6. ATWATER, W. 0., AND C. D. WOODS. 1889. Theacquisition of atmospheric nitrogen byplants. Conn. Agr. Expt. Sta. (Storrs),Ann. Rept. 2:11-51.

7. BAY, J. 1948. Some vital books in science:1848-1947. Science 107:485-491.

8. COTTRELL, H. M., D. H. OTIS, AND J. G.HANEY. 1900. Soil inoculation for soybeans.Kansas State Coll. Expt. Sta. Bull. 96.

9. DODSON, W. R. 1897. Leguminous root tuber-cles. Louisiana State Univ., Agr. Expt.Sta. Bull. No. 46.

10. DUGGAR, J. F. 1897. Soil inoculation forleguminous plants. Alabama Agr. Mech.Coll., Agr. Expt. Sta. Bull. No. 87.

11. FRED, E. B., I. L. BALDWIN, AND E. M. McCoY.1932. Root nodule bacteria and leguminousplants. Wisconsin Univ. Studies in ScienceNo. 5.

12. FRED, E. B., AND E. J. GRAUL. 1916. The effectof soluble nitrogenous salts on noduleformation. J. Am. Soc. Agron. 8:316-328.

13. FRED, E. B., AND E. J. GRAUL. 1916. The gainin nitrogen from growth of legumes on acidsoils. Wisconsin Univ. Agr. Expt. Sta. Res.Bull. No. 39.

14. HOLLAND, J. W. 1938. The beginning of publicagricultural experimentation in America.Agr. History 12:271-298.

15. KNOBLAUCH, H. C., E. M. LAW, AND W. P.MEYER. 1962. State agricultural experimentstations. A history of research policy andprocedure. U. S. Dept. Agr. Misc. Publ. No.904.

16. MACDOUGAL, D. T. 1894. Titles of literatureconcerning the fixation of free nitrogen byplants. Geological and Natural HistorySurvey of Minnesota. Bull. 9, Botan. Ser.II, p. 186-221.

415VOL. 27, 1963

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BACTERIOL. REV.

17. MOORE, G. T. 1905. Soil inoculation forlegumes. U.S. Dept. Agr. Bur. Plant Ind.Bull. No. 71.

18. MOORE, G. T., AND T. R. ROBINSON. 1905.Beneficial bacteria for leguminous crops.

U.S. Dept. Agr. Farmers' Bull. No. 214.19. RUSSELL, H. L. 1894. Noteworthy anatomical

and physiological researches. The fixation offree nitrogen by plants. Botan. Gaz. 19:284-293.

20. RUSSELL, H. L. 1904. Summary of experimentalwork in bacteriology for the years 1893-

1903. Abstracted from the Wisconsin Univ.Agr. Expt. Sta. Ann. Rept. 20.

21. SCHNEIDER, A. 1893. Recent investigationsconcerning rhizobia and free nitrogenassimilation. Agr. Sci. 7:549-556.

22. TRUE, A. C. 1929. A history of agriculturaleducation in the United States 1785-1925.U.S. Dept. Agr. Misc. Publ. No. 36.

23. TRUE, A. C. 1937. A history of agriculturalexperimentation and research in the UnitedStates 1607-1925. U.S. Dept. Agr. Misc.Publ. No. 251.

416 WILSON

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