BAcrCommLOcAL RzvIws, June 1972, p. 135-145Copyright 0 1972 American Society for Microbiology

Vol. 36, No. 2Printed in U.SA.

Dmitri Josifovich Ivanovski(1864-1920)HUBERT LECHEVALIER

Institute of Microbiology, Rutgers University-The State University of New Jersey, New Brunswick, NewJersey 08903

FOREWORD.135THE FORMATIVE. YEARS.135STUDIES ON ALCOHOLIC FERMENTATION.137SOILMICROBIOLOGY. 1.38DISCOVERY OF A FILTERABLEVIRUS.139THE YEARS AT THE UTNIVERSITY OFWARSAW.141CONCLUSION.143LIST OF SELECTED PUBLICATIONS OF IVANOVSKI.144LITERATURE CTED.144

FOREWORDIvanovski is one of the discoverers of the fil-

terable nature of viruses and thus one of thefounders of virology (3), yet very little isknown about his life by persons outside of theSoviet Union. An example of this lack ofknowledge is the article published in Phyto-pathological Classics whose author was unableto find the dates of birth and death of thisimportant Russian scientist (2). All he couldtell us was that Ivanovski died during or before1924. When I drew this matter to the attentionof Dr. L. V. Kalakoutskii of the Institute ofMicrobiology of the Academy of Sciences ofthe U.S.S.R., he kindly furnished me with afew articles and a book on Ivanovski.The following sketch is based mainly on

Ovcharov's book (4), with a few commentstaken from other articles or suggested by theexamination of those papers of Ivanovski thatwe were able to secure. A list of his most im-portant publications will be found at the endof this paper. One will note that Ivanovski'sscientific interests centered around four maintopics: diseases of tobacco plants, yeast fer-mentation, soil microbiology, and photosyn-thesis. In addition, he enriched the Russianscientific literature with numerous publica-tions, both critical and encyclopedic. Ivanov-ski's name is spelled here in the simplest formthat will permit the reader to approximate theRussian pronounciation of his name. Germansuse "w" to render the "v" sound.

In looking at the list of his publications, itwill be useful to remember that a Russianscholar has to present and defend a number ofdissertations during the course of his career.

After the "candidate thesis," which is aboutthe equivalent of our Ph.D., comes the "habili-tation thesis." At this level, the candidate isconsidered capable of college-level teachingand receives the title of Docent, which mightbe compared to that of Assistant Professor.Finally, after several years' experience inteaching and research, a rather mature work isproduced, the doctoral thesis.The reader might find useful to keep in

mind that the ruble of 1890 to 1910 was worth$0.50 of the period. The author is guessing thatthe value of the dollar went down about four-fold between that period and 1972.

THE FORMATIVE YEARSDmitri Iosifovich Ivanovski (Plate I) was

bom in Russia, in 1864, as his country wasundergoing many changes under the leadershipof Alexander the Second, who was attemptinga "revolution from above" that had resulted inthe abolition of serfdom in 1861 and waschanging the whole social structure of thecountry.The exact date and location of the birth of

Dmitri Iosifovich Ivanovski seem to be indoubt. Ovcharov (4) places the happy event onthe 28th of October 1864, in the village of Niz,district of Gdov, in the region of St. Peters-burg; according to Gutina (1), Dmitri was bornon the 9th of November 1864, in Gdov itself.The difference in dates may be no more thanthe usual confusion caused by the Russians'changing from the Julian to the Gregorian cal-endar in 1918.

In any event, Dmitri's father was Iosip (Jo-135

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PLATE I. Dmitri Ivanovski as a young man. FromIzvestija Akademii Nauk SSSR, 1950 (no. 6).

seph) Antonovich Ivanovski, descendant of an

impoverished noble family from the district ofKherson.

Dmitri had two brothers, Nikolai and Alek-sei, the latter to become a famous sinologist,and two sisters, Lydia and Olga. After thedeath of the father, the family moved to St.Petersburg, where they lived modestly on a

small pension given to the widow.Dmitri attended the Larinski secondary

school and graduated with high honors in spiteof much time and energy spent tutoring otherstudents in order to enable his mother to makeends meet.

Dmitri's school years witnessed many im-portant events. In 1881, Alexander II was

murdered to be succeeded by Alexander Illwho, having seen his father's violent end, tooka reactionary course, and Russia again becamethe most despotic country in Europe to beslowly undermined by revolutionaries.

In the field of biology, Pasteur and his as-sistants were laying the foundation of a new

science, the study of the infinitely smallbeings, and were showing the infinitely largerole that they were playing in men's lives. Notonly were microbes responsible for fermenta-tions and the cause of some diseases, but itwas also demonstrated that, through the use of

attenuated strains, immunity could be con-ferred. The doctrine of spontaneous generationwas abandoned and aseptic techniques weredeveloped.

Shortly after Ivanovski entered the Depart-ment of Natural History of the Faculty ofPhysics and Mathematics of the University ofSt. Petersburg, in 1883, Robert Koch an-nounced the discovery of tubercle bacilli, andFriedrich Loffler isolated the causative agentof diphtheria. Elie Metchnikoff, an embryolo-gist attracted by the glitter of the new scienceof microbiology, proposed his famous theory ofcellular immunity. The year was 1884, and aningenious disciple of Pasteur, Charles Cham-berland, discovered the bacteriological filter,the indispensable tool for Ivanovski's work tocome.

Following the potato blight of the 1840'sin Ireland and the brilliant mycological inves-tigations of Anton de Bary, the concept thatfungi might cause plant diseases had beenlargely accepted and, in 1885, Pierre Millardetintroduced the Bordeaux mixture which was tosave so many crops. The concept that bacteriacould also cause plant diseases was not ac-cepted, and the studies of the Thomas J. Bur-rill in the U.S.A. and of J. H. Wakker in Hol-land were essentially ignored (3).

Ivanovski was a good student of meager re-sources. When he entered the University, heasked to be exempted from paying tuition, andhe applied for a scholarship since he had noproperty other than belongings which wereabsolutely necessary (4).At that time, the Department of Natural

History of the University of St. Petersburg wasstaffed with a number of famous scholars rep-resenting the best that Russian science couldoffer at the time, including D. I. Mendeleev.The professor of Botany, A. N. Beketov (1825-1902), was a dedicated teacher who wanted togive "light, and more light" to the whole Rus-sian nation.Beketov was both a phytogeographer and a

plant morphologist. He claimed that the studyof morphology should enable one to investigatethe factors controlling plant-form developmentand come to an understanding of the laws gov-erning evolution. The University of St. Peters-burg was a leader in the teaching of PlantPhysiology. This was due not only to the ef-forts of Beketov but also to those of anotherteacher of Ivanovski, A. S. Famintsin (1835-1918), well known to general bacteriologists forhis discovery of Nevskia ramosa.The influence of these outstanding scholars

had a considerable impact on young Ivanovski,

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who had already spent much time studying thegreat classics of natural sciences and philos-ophy. An entry in his student-days diary, re-lated by Ovcharov (4), revealed his maturity:"I cannot understand how one can sit with afriend for the whole evening and do nothingbut talk nonsense and enjoy it. I am bored ifconversation does not-at least to a modestdegree-nourish the mind. An evening spentin useless talk tires me."At the University of St. Petersburg, Iva-

novski served as an instructor in the laboratoryof plant physiology and anatomy while he car-ried out some undergraduate research projects.He attracted the attention of both Beketov,then the dean of the natural scientists of St.Petersburg, and Famintsin, who decided tosend him to study a tobacco disease which wasthen causing great damage.

Ivanovski was very happy to be given suchan assignment and, in 1887, he departed in thecompany of another student, V. V. Polovtsev,for Ukraine and Bessarabia. The results of thisexpedition were published in a series of papersgiving a detailed description of the pox disease(Russian = Riabukha) of tobacco as well assuggestions on how to curb it. Based on theirown observations and on interviews with farm-ers, the two students concluded that the dis-ease was most damaging in fields where to-bacco had been grown for long periods of time,frequently for more than forty years. In addi-tion, the fields planted with tobacco were oftenvery small, nothing more than a few squareyards where the peasants were growing theprecious plant "for their own pipes." Thus, themost dramatic but realistic recommendationof the two students was that the farmers intro-duce the practice of crop rotation.Returning to St. Petersburg, Ivanovski suc-

cessfully passed his preliminary examinationtowards the end of 1887 and, on the first ofMarch of the next year, he presented his Ph.D.thesis "On Two Diseases of Tobacco." TheFaculty felt that he was an outstanding candi-date and encouraged him to follow an aca-demic career. After receiving his Ph.D. degree,Ivanovski was offered a post-doctoral fellow-ship at the University that he accepted reluc-tantly, being afraid of not fulfilling the highhopes of his teachers. In 1891, he resigned toaccept a position as technician in the labora-tory of botany of the Academy of Sciences. Hethen married the daughter of a political exilewho, after having spent two years in prison,had been banished from St. Petersburg.Those were difficult times, and the leading

Russian scientists often had to resort to heroic

measures in order to carry out their investiga-tions. Mendeleev had a yearly laboratorybudget of 300 rubles, which put him in a fa-vored position, since another chemist of theUniversity of St. Petersburg, N. N. Sokolov,had no support at all and was running his lab-oratory out of his own pocket, with the assist-ance of A. N. Engelgart, the agriculturalchemist and publicist. Famintsin, tired ofasking in vain for the creation of a laboratoryof plant physiology at the Academy of Sci-ences, finally set one up in his own apartment.The lack of space, greenhouses, experimental

fields, and other facilities drove Russian bota-nists to turn to microorganisms for their physi-ological investigations. Such, indeed, had beenthe case of M. S. Voronin who brilliantly in-vestigated cabbage hernia and discovered themyxomycete-like pathogen, Plasmodiophorabrassicae, in 1878.

STUDIES ON ALCOHOLICFERMENTATION

Towards the end of the nineteenth century,few people worked on alcoholic fermentationssince all the questions one might ask seemedto have been answered by the studies of Pas-teur. However, Ivanovski felt that the exactrole of oxygen and of nitrogen-containing sub-stances still had to be elucidated. His investi-gations proceeded at a rather slow pace, be-cause he built most of his instruments himselfand purified or checked by analysis all hismedia and reagent constituents. In addition,he was satisfied by the validity of his findingsonly after having obtained the same resultsagain and again. Among the pieces of equip-ment built by Ivanovski was one which per-mitted him to carry out the continuous cultureof yeasts.

Soon Ivanovski found that his results contra-dicted some of those published by others. Du-claux was of the opinion that fermentation byyeasts starts only after they have exhaustedthe oxygen present in the medium. This pointof view was not confirmed by Ivanovski.Nageli had claimed that an increase in thegrowth rate of yeasts resulted in an increase infermentation capability. This was disputed byIvanovski who found that, at equal sugar con-centrations, increases in the quantity of thenitrogen-containing substances in the medium(peptone) resulted in an increase in growthand a decrease in fermentation. He reasonedthat, for the formation of yeast protoplasm,nitrogen-containing substances were essential.The more you give the yeast, the more it formsprotoplasm and the less it forms secondary

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metabolites such as alcohol.In general, Ivanovski felt that if the yeasts

are given ideal conditions for growth they willproduce many cells and few by-products, suchas alcohol. In addition, alcohol, if produced insmall quantities, was not toxic to the yeastand could, under some conditions, even bemetabolized by it.

Ivanovski refused to equate the anaerobicrespiration of higher forms of life with the al-coholic fermentation of yeasts because he feltthat all presumptions of this kind are moreharmful than useful, for imaginary explana-tions tend to conceal existing gaps in knowl-edge and are thereby detrimental to science.However, Ivanovski had appreciated the valueof comparative biochemistry since he statedthat obviously the same physicochemical proc-esses take place in both the cells of highly or-

ganized plants and in unicellular organisms.He also noted that, because of their ease ofhandling, microorganisms were ideal researchtools (4).

Ivanovski had selected alcoholic fermenta-tion as the subject of his habilitation disserta-tion. As he was completing his work in 1894,Nicolas the Second was crowned the Tsar ofall Russians. He continued the despotic coursetraced by his father; he was to be the last Tsar.Since 1891, in St. Petersburg, Ivanovski had amost distinguished colleague, Sergei Vino-gradski (1856-1953), who, having isolated auto-trophic nitrifying bacteria, had been appointedhead of the laboratories of general micro-biology of the Imperial Institute of Experi-mental Medicine. In 1894, Vinogradski distin-guished himself again by discovering that cer-tain anaerobic bacteria were able to fix ni-trogen.

In January 1895, Ivanovski defended hishabilitation dissertation entitled "Studies onAlcoholic Fermentation." The appointed re-viewers were Fanintsin, who was by thenAcademician, and D. P. Konovalov. Their re-port was very favorable, and Ivanovski wasappointed Privatdocent. His first task was toteach an elective course on the physiology ofthe lower plants. Within a year, due to the res-ignation of Famintsin, he was entrusted withthe compulsory course on plant physiology andanatomy.The laboratories of the University of St.

Petersburg were too poorly equipped to permitthe continuation of Ivanovski's studies on alco-holic fermentation. He solved the problem bystarting a cooperative program with the Insti-tute of Technology where, in 1896, he initiateda course on fermentation designed to train

experts for the industry. This venture wasquite successful, even attracting students fromabroad.

Ivanovski's ideas on yeast fermentation canbe summed up as follows: (i) Fermentation isnot solely controlled by the lack of oxygen, thenutrients in the medium are of considerableimportance; (ii) the fermentation products andthe intensity of the fermentation depend notonly on the cultural conditions employed butalso on the strain of yeast used.

SOIL MICROBIOLOGYIvanovski was not a soil microbiologist nor

was he an agronomist; still, his teaching inthese fields had a tremendous influence inRussia. His main contribution was in the formof lectures and discussions at the meetings ofthe Commission for Soil Sciences of the FreeEconomic Society. His expertise was such thathe was asked to join the editorial board ofPochvovedenie (Soil Science) founded in 1899,seventeen years before its American counter-part.On the occasion of a meeting of the Com-

mission on Soil Sciences in 1891, Ivanovskistated that the study of low forms of life hadchanged physiology and medicine and thatthere was no doubt that the same was tohappen in soil sciences and agriculture (4).As a plant physiologist and a microbiologist,

Ivanovski recognized that there was greatpromise in the investigations of P. A. Kos-tychev who was elucidating the role of higherand lower plant forms in the formation of thefertile black topsoil called chernozem. In hisstudies, Kostychev pointed out that soil micro-organisms were not only degraders of organicmatter but that they also synthesized newcompounds. Ivanovski supported this point ofview since he felt that, no matter how littlewas known about the role of microorganisms insoil. it was already enough to claim that micro-organisms play an obvious role in the genesisand life of the soil (4)."There is no such thing as a dead soil,"

claimed Ivanovski. "The decomposition of or-ganic matter in soil is a physiological processassociated with the life activities of innumer-able microscopic soil inhabitants" (4). Thus,Ivanovski was strongly opposed to the thenprevailing concept that the decomposition oforganic matter in soil was a strictly chemicalprocess dependent on oxygen and controlledby light, temperature, and the moisture con-tent of the soil.With his considerable flair for recognizing

competence, Ivanovski gave his backing to all

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those who, like Sergei Vinogradski, developedthe science of soil microbiology. He attackedall those who performed faulty experiments orwho tried to peddle miraculous agronomicconcoctions.At a time when many scientists doubted

that microorganisms could fix atmospheric ni-trogen, Ivanovski joined his teacher Famintsinin expressing the conviction that, in the future,good crops and rich harvests could possibly beinduced by cultivation methods assuring anabundant growth of soil microorganisms (4).

Ivanovski promoted the use of bacterial fer-tilizers and, in particular, he warmly recom-mended the application of a preparation calledNitragin which was rich in bacteria which formnodules on the roots of legume plants. He rec-ommended the use of specific bacterial fertil-izers only after thorough testing had convincedhim of the value of the product. He stoodfirmly against the premature acceptance ofany product and, after having reviewed theexperimental data collected in many countries,he discouraged the use of a preparation of thistype called Alnit.Always one step ahead, Ivanovski reminded

his contemporaries that it was easy to dip thelegume seeds in a suspension of Nitragin, buthe added that they should try to determine theconditions which would favor the developmentof the bacteria and to understand the nature ofthe processes taking place in the soil.Towards the end of his career in Warsaw,

Ivanovski returned to the study of the legumebacteria. Because he published nothing of thiswork, little is known about this part of his re-search career.

DISCOVERY OF A FILTERABLE VIRUSAlthough deeply involved in the study ol

alcoholic fermentations, Ivanovski did notforget the diseases of tobacco. At first, he paidspecial attention to the powdery mildew oftobacco. The Russian name for this disease,which is caused by an Erysiphaceae, is pepe-litsa, which means "ashening."

His observations led him to the conclusionthat the pathogen can grow only if enoughhumidity is available. He also noted that theconidial stage of the fungus (Oidium stage)attacks plants of the thistle family where itcan over-winter and serve as a source of infec-tion the following season.As a preventive measure, Ivanovski recom-

mended that farmers grow tobacco plants wellspaced, in properly drained fields. If infectionhad started, he showed that the removal ofdiseased leaves and the nicking of the top of

the plants often permitted one to curb the dis-ease through increased proliferation of healthyleaves. He advocated the destruction of all ex-cised parts by burning. Having solved thisproblem to his satisfaction, he then turned his-attention to tobacco mosaic.

In 1887, when Ivanovski was still a student,he started his work on diseases of tobacco. Hecalled the disease of tobacco that he was inves-tigating riabukha, in Russian, and Pocken-krankheit in German (literally, pox disease).Later, in 1892, Ivanovski distinguished be-tween the pox disease, stricto sensu, character-ized by the formation of brown spots on theleaves, and the mosaic disease, characteris-tically manifested by a light- and dark-greenmottling of the leaves.The name mosaic disease of tobacco had

been introduced in 1886 by Adolf Mayer, whobelieved that the disease was almost exclu-sively limited to Holland (2). Mayer rightlyfelt that names previously used in Holland,such as "bunt," "rust," and "smut," were notvery appropriate.

Referring to the mosaic disease stricto sensu,Ivanovski noted that the disease alwaysstarted in very young leaves just emergingfrom the buds and was subsequently trans-mitted to the whole plant. The main symp-toms of the disease were manifestly thechanges in color which led Mayer to coin itsname: a mosaic of dark- and light-green spotslater turning yellow.

Ivanovski repeated what were essentially theexperiments of Mayer. Diseased plants,crushed in a porcelain dish, were filteredthrough linen, and the expressed sap wasinoculated by means of capillary tubes into theleaf veins of healthy plants. This resulted inthe production of the disease within two weeksin 80% of the plants inoculated.Having convinced himself that the mosaic

disease could be passed from plant to plant,Ivanovski autopollinated diseased plants. Notone out of the one hundred planted, ripenedseeds produced diseased progeny, thus provingthat the mosaic disease is not hereditary butcontracted during the vegetative period ofplant growth.

Ivanovski was able to transmit the mosaicdisease by crushing infected leaves around theroots of healthy plants, but he was unable totransmit the disease from plant to plantsimply by growing an infected plant next to ahealthy one.

Based on these observations, Ivanovski wasin a position to stress practical control meas-ures such as the removal and the burning of all

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parts of the diseased plants, and, if all elsefailed, to-have recourse to crop rotation.

After having established that, in fields wherethe disease had been prevalent for some time,the infection is transmitted through bothleaves and roots, whereas in new fields the dis-ease was transmitted only through the leaves,Ivanovski investigated environmental condi-tions that favored the contagion. In general, hefound that the more favorable the conditionsfor the speedy growth of the vegetative part ofthe plants, the more susceptible they were tothe disease. Thus, moist soil, humid air, andbalmy temperatures were found to favor thespread of the mosaic disease.

Ivanovski had no confidence in some of theexperiments of Mayer. The latter had reporteda loss of infectivity when the sap was passedthrough a double layer of filter paper. Iva-novski went one step further and found, in1892, that, even after passage through the fil-tering candle of Chamberland, infectivity wasnot lost.

Ivanovski concluded his communication of1892 (2) with the hypothesis that the mosaicdisease was of bacterial origin and that a fil-trate passed through a Chamberland candlecontained either bacteria or a soluble toxinwhich might be capable of provoking the ap-pearance of the external manifestation of thedisease. The latter hypothesis was particularlyappealing at the time, since Roux and Yersinhad demonstrated in 1888 that diphtheria wasa disease caused by a bacterial toxin. In addi-tion, Ivanovski was unable to cultivate bac-teria from his candle filtrates.Unaware of Ivanovski's work, one of the

greatest figures of general microbiology, Mar-tinus Beijerinck working at the PolytechnicalSchool of Delft took advantage of the heatedgreenhouse of his school to continue, in 1897,some studies on tobacco mosaic disease that hehad started in 1885, in order to help AdolfMayer, when they were both at the School ofAgriculture of Wageningen.

After one year of study, in 1898, Beijerinckpublished the following general conclusions: (i)The tobacco mosaic infection is not caused bymicrobes, but by a living liquid virus. (ii) Thevirus multiplies only in growing plant organswhere cellular division takes place. (iii) Thevirus is inactivated by boiling but can be driedwithout losing its infectious properties.

In his doctoral dissertation, published in1902 in Warsaw (see List of Selected Publica-tions), Ivanovski was somewhat chagrined toremark that Beijerinck "filtered the sap of dis-eased plants through a porcelain filter and

stated that the sap, sterilized in this fashion,retained its infectivity. The author does notknow that I had already established this fact along time ago."Ivanovski repeated the diffusion-in-agar

experiments of Beijerinck and concluded thatthe agar is indeed capable of transferring theinfection. However, he was not ready to acceptthe idea that this experiment proved that thecausative agent of the disease was a fluid. Heran some experiments of diffusion in agar con-taining India ink and concluded that smallparticles are capable of motion through a gel.However, he noted that such motion was pos-sible only in old agar. He tried to use this lastobservation to determine whether the causa-tive agent of tobacco mosaic disease was par-ticulate or not.The sap of diseased plants was simultane-

ously applied to the surface of old and freshagar plates. After ten days, the upper layer ofboth agars was removed, and the lower layerwas used for infecting healthy plants. Tenplants were inoculated with fresh agar and fivewith old agar. After 11 days, all the plantstreated with fresh agar were still healthy, buttwo of the five plants inoculated with old agarwere already sick. The fresh agar-treatedplants remained healthy even after one month,and Ivanovski concluded that the cause ofinfection was particulate, since soluble sub-stances could easily penetrate through freshagar.

In another experiment, Ivanovski observedthat the sap of diseased plants, dialyzedthrough a bovine diaphragm, was still infec-tious. India ink particles could also passthrough the same membrane. It could, how-ever, be made impervious to the passage ofboth types of substances with a coating ofagar. In addition, neither substance could passthrough a layer of parchment.

Using a Chamberland candle, Ivanovski fil-tered infected sap for a period of 36 hours. Hecollected the first few drops of filtrate andthen further fractions at 12 and 36 hours.Healthy plants were inoculated with each frac-tion, but only the first cut was able to transmitthe disease. This, Ivanovski felt, supported theparticulate theory of the causative agent. Par-ticles first passed through the pores of thefilter; then, after a certain time, the pores be-came smaller and smaller as they becameclogged, and the particles could no longer pass.Summarizing the results of these experi-

ments, Ivanovski stated in the Zeitschriftpaper of 1903 (see List of Selected Publica-tions): "We see that there is not a single fact

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which supports the hypothesis on the solublecharacter of the infectious agent of mosaic dis-ease. On the contrary, the experiment with thediffusion into agar and especially the fraction-ated filtration clearly indicates that we aredealing with a contagium fixum."

Certainly, the infectious agent was particu-late, but what was its nature? At first Iva-novski was inclined to believe that it was anelusive bacterium.

In 1901 Ivanovski concluded a short note inthe Zentralblatt (see List of Selected Publica-tions) by stating: "Thus there is a specific bac-terium which causes Tobacco Mosaic diseaseand we have found that it is unnecessary toseek refuge in the completely untenable hy-pothesis of contagium vivum fluidum. A morecomplete paper will appear in 2 to 3 months."

Further experimentation was not to bringconfirmation of the discovery of the tobaccomosaic disease bacterium, and the 1903memoir in Zeitschrift (see List of SelectedPublications) ended on a more cautious note:"On the whole, the question of the artificialcultivation of the mosaic disease microbe re-mains to be solved by future investigations."

Ivanovski's microscopic observations of dis-eased cells were illustrated in Plate mI of hisdoctoral thesis which was Plate II of the 1903Zeitschrift memoir (see List of Selected Publi-cations). It is reproduced here as Plate II. Mostinteresting is the illustration of viral crystals.Some of the diseased cells were fixed with

alcohol, stained with methylene blue, washedwith alcohol and aniline, and counterstainedwith eosin. The nuclei were stained blue on apinkish background. On this rosy background,Ivanovski throught that he saw the rod-shapedbacteria of the mosaic disease stained blue(Plate II, Fig. 9 to 16). Attempts to stain thesebacteria in living cells were unsuccessful.

In 1896, Ivanovski, then only a Privatdocent,had been appointed head of a department atthe University of St. Petersburg with the un-derstanding that, within a five-year period, hewas to successfully defend a doctoral thesis.Research on tobacco mosaic was difficult andlong, and, as we have noted, Ivanovski was nota man to rush into bold conclusions. In 1901,the five years had passed and the thesis hadnot been completed. He was relieved of hisduties and replaced by V. I. Palladin. Iva-novski was a popular teacher and, uponhearing of his discharge, the students boy-cotted Palladin's lectures until they realizedthat he had nothing to do with Ivanovski'sdemotion.

Ivanovski presented his doctoral dissertation

at the University of Kiev. The two official re-viewers, S. G. Navashin and K. A. Purievich,were favorably impressed and rightly so. How-ever, Purievich went far when he wrote in hisreport that he had noted with considerable sat-isfaction that Ivanovski had refuted the hy-pothesis of Beijerinck on the existence of aninfectious agent having character of unorga-nized matter. The hypothesis of contagiumvivum fluidum, he said, was a sad chapter inthe annals of contemporary science (4).

THEYEARS AT THE UNIVERSITY OFWARSAW

In 1901, Ivanovski was appointed AssociateProfessor at the University of Warsaw. Afterhaving successfully defended his doctoralthesis in Kiev, in 1903, he concentrated mostof his research time to the study of photosyn-thesis and associated pigments. In the courseof his investigations on tobacco mosaic dis-eases, Ivanovski noted that the yellow parts ofthe leaves not only contained little chlorophyllbut also little starch. This observation mighthave been the origin of his interest in photo-synthesis, or the impetus might have comefrom his admiration for the work of one of hiscolleagues, Michael S. Tsvett.The latter was born in Italy in 1872, of a

Russian father and an Italian mother. Hestudied in Switzerland and received his Ph.D.in Botany from the University of Geneva.Tsvett, whose name means "color" in Russian,was interested in plant pigments. In 1897 hewas teaching plant anatomy and physiology atSt. Petersburg. His research on the chemistryof chloroplasts attracted the attention of I. P.Borodin, M. S. Voronin, and Ivanovski, whosaw to it that he was elected a member of theSociety of Natural Scientists of St. Petersburg.In 1901 he was appointed Assistant at theUniversity of Warsaw; six years later he be-came Professor of Botany and Agronomy. In1908 he was appointed Professor of Botany andMicrobiology at the Polytechnic Institute ofWarsaw. In 1906 he published a paper which isthe basis of chromatography, a term which hecoined (7).

In 1910, he received a Doctorate in Botanyfrom the University of Warsaw. Like millionsof others, his life was perturbed by the war andthe revolution. In 1915 he had to flee Warsawand, after some tribulations, he becameDirector of the Botanical Garden of the Uni-versity of Dorpat, in Estonia. Again, Germanoccupation forced him to flee. These trialsmust have been especially hard for him sincehe never felt that he really belonged to Russia.

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PLATE II. 1, Cross section through the green part of a diseased leaf; 2, same, through a yellow portion. 3-7,Isolated cells of the palisades parenchyma from the yellow part of a diseased leaf. Viral aggregates, calledIvanovski's crystals, can be seen in figures 3, 4, and 6. 8, Nuclei with ameboid protoplasmic structures. 9-16,Cells from the palisade parenchyma fixed with alcohol and stained with methylene blue and eosin. This is ablack and white photograph of Plate II of the 1903 Zeitschrift memoir (see List of Selected Publications). Theoriginal was in color.

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All his life he was a Swiss by heart. He died in1919, in Voronezh. Tsvett's method, which hasfound its way into all laboratories where anyinterest in biochemistry exists, was ignored formany years. Its universal acceptance probablydates back to 1931, when Kuhn and Ledererseparated carotenes and xanthophylls on col-umns of alumina and calcium carbonate (6).

In Warsaw, both Ivanovski and Tsvettstudied problems connected with the action oflight on plant pigments. Ivanovski concen-trated on the physical aspects of the problem,whereas Tsvett studied the chemistry of thepigments.

It has been observed that chlorophyll ex-tracted from plants and in laboratory solutionswas less stable to light than native chlorophyllin the plant leaves. Ivanovski found that, whenwater was added to an alcoholic solution ofchlorophyll, the chlorophyll turned from aclear solution to a colloidal one with a markedincrease in photostability. He concluded, in1909, that a colloidal solution of chlorophyllwas 15 to 30 times more stable than a molec-ular solution. He thus felt that the stability ofcolloidal chlorophyll is comparable to thatpresent in living leaves. He also observed thatthe light-absorption spectra of chlorophyll inliving leaves suggested that the chlorophyllthere was in a colloidal state. He reasoned fur-thermore, that, in the plants, chlorophyll ismuch more concentrated than in laboratorysolutions and was presumably more stable in aconcentrated form than when diluted.

It was also Ivanovski's opinion that theyellow pigments associated with chlorophyll inthe leaves played no direct role in photosyn-thesis but protected the vital chlorophyll fromthe detrimental effect of blue and violet light.He carried out light-stability experiments oncrude mixtures of leaf pigments, on pure chlo-rophyll and on chlorophyll to which the yellowpigments had been added. These experiments,made possible by the chromatographic methodof Tsvett, yielded results that clearly sup-ported his theory.

In Warsaw, Ivanovski was appointed thehead of a department and Professor of PlantPhysiology. This was at a time when Poleswere strongly oppressed. In line with his gen-eral liberal philosophy, Ivanovski did not hesi-tate to appoint Poles to his staff. He also oftenintervened in favor of Polish students whowere being refused admission to the Universitybecause their enrollment was subject to aquota.

Ivanovski tried to modernize the teaching ofBotany at the University of Warsaw by com-

bining teaching with research. His effortsmight best be termed heroic if one considersthe limitations of his budget: in the year 1907-8, for example, he had to make do with 24 ru-bles and 59 kopecks.

Ivanovski was a great teacher, popular withhis students, not only because of his liberalviews, which included fostering education ofwomen, but also because of the quality of hispresentation. One of his former students,Academician N. A. Maksimov, recalled thatIvanovski was not content merely to outlinethe established facts but, besides giving thehistorical background of any topic, tried topresent various points of view before offeringhis own opinion.

In 1915, the University of Warsaw washastily evacuated to Rostov-on-Don. In theconfusion, Ivanovski lost all his equipment, hisrich library, and many of his notebooks. Thesehardships, as well as the death of his only son,weighed heavily on him, faced as he was withthe task of organizing a laboratory under war-time conditions in unfamiliar surroundings.Still, it is under these conditions that he pre-pared his most important pedagogical contri-bution, his textbook of Plant Physiology.

Dmitri Ivanovski died in Rostov-on-Don on20 June 1920.

CONCLUSIONThe discovery of the filterable nature of the

causative agent of a disease, in 1892, by Iva-novski, was a most important contribution toknowledge. However, we find that Ivanovskihad no influence on those who made similarobservations at later dates, namely M. Beijer-inck, also working on tobacco mosaic disease,F. Loffler and P. Frosch who investigated foot-and-mouth disease, both in 1898, and W. Reedand J. Carroll studying yellow fever, in 1901(3). In addition, Ivanovski's work had less in-fluence on the development of virology thanthe studies of the above mentioned pioneers.One cannot say that Ivanovski was ignored

because he published in Russian, a languagenot often understood by scientists. He wasalways careful to publish all his importantcontributions in an "accepted" language, andhis German publications were in first-classjournals. We thus have to find elsewhere anexplanation for his minor position in the his-tory of microbiology.

It is possible that Ivanovski had little im-pact because he did not travel abroad. Vino-gradski and Metchnikoff studied in and trav-eled through many of the centers of westerncultures. They were thus better known than

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Ivanovski. Ivanovski may thus have been avictim of his poverty and lack of aggressive-ness at obtaining travel grants.

However, in Ivanovski's own publicationscan certainly be found a cause for his lack ofsuccess. He did not take a strong stand in hispaper of 1892 (2); then be kept faith in the ex-istence of an elusive bacterium, cause of to-bacco mosaic disease, which might be culturedone day. In his choice of a bacterium as thepotential causative agent of tobacco mosaicdisease, Ivanovski was showing some original-ity, since only after the studies of Erwin Smithat the tum of the century was it universallyaccepted that some bacteria were indeed etio-logical agents of some plant diseases (3).

LIST OF SELECTED PUBLICATIONS OFIVANOVSKI

A presumably complete list of Ivanovski's publica-tions was compiled by Rizhkov in 1953 (5). A largenumber of his publications were critical appraisals oftheses and books. He also contributed generously toan encyclopedia. The following is a list of some ofhis publications which seem to be the most impor-tant scientifically. Often, apparently the same paperwas published in Russian and in German. TheGerman reference is given here preferentially.Ivanovski, D., and V. V. Polovtsev. 1890. Die Pock-

enkrankheit der Tabakspflanze. (Pox disease ofthe tobacco plant.) Mem. Acad. Sci. St. Peters-bourg, Ser. 7, 37 (7):1-24.

Ivanovski, D. 1892. 0 dvukh bolezniakh tabaka.Tabachnaia pepelitsa. Mozaichnaia bolezn tabaka.(On two diseases of tobacco. Tobacco mildew.Mosaic disease of tobacco.) 19 pages. St. Peters-bourg. This paper was published both as a bookletand in Selskoe khazaistvo i lesovodstvo, St. Peters-bourg, vol. 189 (2), p. 104-121. It was probablyIvanovski's Ph.D. thesis.

Ivanovski, D. 1892. UYber die Mosaikkrankheit derTabakspflanze. (On the mosaic disease of tobaccoplant.) Bull. Acad. Imp. Sci. St. Petersbourg,Nouv. Ser. 3 35:67-70. This paper was translatedby J. Johnson (2).

Ivanovski, D. 1893. Uber die Wirkung des Sauer-stoffes auf die alkoholische Garung. (On the effectof oxygen on alcoholic fermentation.) Bull. l'Acad.Imp. Sci. St. Petersbourg, Nouv. Ser. 4 36:391-413.

Ivanovski, D. 1894. Issledovaniia nad spirtovym bro-jeniem. (Investigations on alcoholic fermentation.)76 pages. Acad. Sci. St. Petersbourg. This is Iva-novski's habilitation thesis.

Ivanovski, D. 1896. L'Influence de l'oxygene sur lafermentation alcoolique. (Influence of oxygen onalcoholic fermentation.) Monit. Sci. Paris, Ser. 410:148-149.

Ivanovski, D. 1899. Nitragin i alinit. K boprocu obudobrenii pocho bakteriiami. (Nitragin and Alnit.The question of fertilizing soil with bacteria.)Pochvovednie 1 (4):223-238.

Ivanovski, D. 1899. Uber die Mosaikkrankheit derTabakspflanze. (On the mosaic disease of tobaccoplant.) Zentralbl. Bakteriol. Parasitenk. Infek-tionskr. Abt. II 5 (8):250-254.

Ivanovski, D. 1901. Uber die Mosaikkrankheit derTabakspflanze. (On the mosaic disease of tobaccoplant.) Zentralbl. Bakteriol. Parasitenk. Infek-tionskr. 7 (4):148.

Ivanovski, D., and S. Obraztsov. 1901. Uber die Wir-kung des Sauerstoffes auf die Garung Verschie-dener Hefearten. (On the effect of oxygen in thefermentation of various types of yeasts.) Zentralbl.Bakteriol. Parasitenk. Infektionskr. 7 (9/10):305-312.

Ivanovski, D. 1902. Mozaichnaia bolezn tabaka. (Themosaic disease of tobacco.) 72 pages. Warsaw. Thisis Ivanovski's doctoral dissertation which was alsopublished the same year, in two parts, in volumes5 and 6 of Izvestiia of the University of Warsaw.

Ivanovski, D. 1902. Die Mosaik und Pockenkran-kheit der Tabakspflanze. (The mosaic and poxdisease of tobacco plants.) Z. Pflanzenkr. 12:202-203.

Ivanovski, D. 1903. Uber die Mosaikkrankheit derTabakspflanze. (On the mosaic disease of tobaccoplant.) Z. Pflanzenkr. 13:1-41.

Ivanovski, D. 1907. Uber die Ursachen der Verschie-bung der Absorbtionsbander in Blatt. (On thecause of the shift of the absorption bands in theleaf.) Beri. Deut. Bot. Ges. 25 (8):416-424.

Ivanovski, D. 1913. Kolloidales Chlorophyll und dieVerschiebung der Absorptionsbander in lebendenPflanzenblattern. (Colloidal chlorophyll and theshift of the absorption bands in living plantleaves.) Biochem. Z. 48:328-331.

Ivanovski, D. 1913. Uber das Verhalten des lebendenChlorophylls zum Lichte. (On the relationship ofliving chlorophyll to light.) Ber. Deut. Bot. Ges.31:600-612.

Ivanovski, D. 1913. Uber die Rolle der gelben Pig-mente in den Chloroplasten. (On the role of yellowpigments in chloroplasts.) Ber. Deut. Bot. Ges. 31:613-617.

Ivanovski, D. 1914. Ein Beitrag zur physiologischenTheorie des Chlorophylls. (Uber das zweite Assim-ilations-Maximum). (A contribution to the physio-logical theory of chlorophyll. On the second assim-ilation-maximum.) Ber. Deut. Bot. Ges. 32:433-447.

Ivanovski, D. 1917-1924. Fiziologiia rastenii. (Plantphysiology) 618 pages. Kharkov and Rostov-on-Don, 1917-1919. The first volume was published in1917, the second in 1919. A second edition waspublished in Moscow, by The State PublishingHouse, in 1924.

ACKNOWLEDGMENTThe writing of this paper was made possible by the lin-

guistic devotion of Jozef Balan of the Biological Institute ofthe Slovak Academy of Sciences, Bratislava, Czechoslo-vakia.

LITERATURE CITED1. Gutina, V. N. 1964. The scientist-innovator. Priroda, no.

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10, p. 15 (in Russian).2. Johnson, J. 1942. Phytopathological classics. Amer. Phy-

topathol. Soc., no. 7.3. Lechevalier, H. A., and M. Solotorovsky. 1965. Three

centuries of microbiology. McGraw-Hill Book Co.,New York.

4. Ovcharov, K. E. 1952. Dmitrui Iosifovich Ivanovskiu 1864-1920. Academy of Sciences of the U.S.S.R., Moscow(in Russian).

5. Rizhkov, V. L. 1953. D. I. Ivanovski; selections from pub-lications. Academy of Medical Sciences of theU.S.S.R., Moscow (in Russian).

6. Robinson, T. 1959. Michael Tswett. J. Chem. Ed. 36:144-147.

7. Strain, H. H., and J. Sherma. 1967. M. Tswett: adsorp-tion analysis and chromatographic methods. Applica-tion to the chemistry of the chlorophylls. J. Chem. Ed.44:238-242.

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