SB 608 .P25 P7 Copy 1 *—=HYSIOLOGICAL STUDIES OF BACILLUS RADICICOLA OF CANADA FIELD PEA A THESIS PRESENTED TO THE FACULTY OF THE GRADUATE SCHOOL OF CORNELL UNIVERSITY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY BY ae No MARTIN J.-PRUCHA JUNE, 1913 (Reprinted from Memoir No. 5, March, 1915, of Cornell University Agricultural Experi- ment Station as Contribution No. 15 of the Laboratory of Plant Physiology): PHYSIOLOGICAL STUDIES OF BACILLUS RADICICOLA OF CANADA FIELD PEA A THESIS PRESENTED TO THE FACULTY OF THE GRADUATE SCHOOL OF CORNELL UNIVERSITY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY BY MARTIN J. PRUCHA JUNE, 1913 (Reprinted from Memoir No. 5, March, 1915, of Cornell University Agricultural Experi- ment Station as Contribution No. 15 of the Laboratory of Plant Physiology) ty e Oo a CORNELL UNIVERSITY AGRICULTURAL EXPERIMENT STATION De. EXPERIMENTING STAFF BEVERLY T. GALLOWAY, B.Agr.Sc., LL.D., Director. ALBERT R. MANN, B:8.A., Secretary. JOHN H. COMSTOCK, B.S., Entomology. HENRY H. WING, M.S. in Agr., Animal Husbandry. T. LYTTLETON LYON, Ph.D., Soil Technology. JOHN L. STONE, B.Agr., Farm Practice. JAMES E. RICE, B.S.A., Poultry Husbandry. GEORGE W. CAVANAUGH, B.S., Agricultural Chemistry. HERBERT H. WHETZEL, M.A., Plant Pathology. ELMER O. FIPPIN, B.S.A., Soil Technology. G. F. WARREN, Ph.D., Farm Management. WILLIAM A. STOCKING, Jr., M.8.A., Dairy Industry. CHARLES 8. WILSON, A.B., M.S.A., Pomology WILFORD M. WILSON, M.D., Meteorology. RALPH S. HOSMER, M.F., Forestry. JAMES G. NEEDHAM, Ph.D.; Entomology and Limnology. ROLLINS A. EMERSON, Ph.D., Plant Breeding. HARRY H. LOVE, Ph.D., Plant Breeding. ARTHUR W. GILBERT, Ph.D., Plant Breeding. DONALD REDDICK, Ph.D., Plant Pathology. EDWARD G. MONTGOMERY, M.A., Farm Crops. WILLIAM A. RILEY, Ph.D., Entomology. . MERRITT W. HARPER, M.S., Animal Husbandry. J. A. BIZZELL, Ph.D., Soil Technology. GLENN W. HERRICK, B.S.A., Economic Entomology. HOWARD W. RILEY, M.E., Farm Mechanics. CYRUS R. CROSBY, A.B., Entomology. HAROLD E. ROSS, M.S.A., Dairy Industry. KARL McK. WIEGAND, Ph.D., Botany. EDWARD A. WHITE, B.S., Floriculture. WILLIAM H. CHANDLER, M.S. in Agr., Pomology. ELMER 8S. SAVAGE, M.S.A., Ph.D., Animal Husbandry. LEWIS KNUDSON, Ph.D., Plant Physiology. KENNETH C. LIVERMORE, B.S. in Agr., Farm Management. ALVIN C. BEAL, Ph.D., Floriculture. MORTIER F. BARRUS, Ph.D., Plant Pathology. CLYDE H. MYERS, M.S8., Ph.D., Plant Breeding. GEORGE W. TAILBY, Jr., B.S.A., Superintendent of Live Stock. EDWARD 8S. GUTHRIE, M.S. in Agr., Ph.D., Dairy Industry. JAMES C. BRADLEY, Ph.D., Entomology. PAUL WORK, B.S., A.B., Vegetable Gardening. JOHN BENTLEY, Jr., B.S., M.F., Forestry. EARL W. BENJAMIN, Ph.D., Poultry Husbandry. EMMONS W. LELAND, B.S.A., Soil Technology. CHARLES T. GREGORY, Ph.D., Plant Pathology. WALTER W. FISK, M.S.A., Dairy Industry. ARTHUR L. THOMPSON, M.S. in Agr., Farm Management ROBERT MATHESON, Ph.D., Entomology. HORACE M. PICKERILL, B.S., Dairy Industry. MORTIMER D. LEONARD, BS., Entomology. FRANK E. RICE, Ph.D., Agricultural Chemistry. V. B. STEWART, Ph.D., Plant Pathology. BRISTOW ADAMS, B.A., Editor. LELA G. GROSS, Assistant Editor. The regular bulletins of the Station are sent free on request to residents of New York State. 3 CONTENTS ‘ PAGE LEAT DERE AIO, Ge Rae a a gg la at I ee Pi 9 Deane omtae INnVestIgation... 6... 44.2.) 62. ae vane ye sre a wa ee vec ds 9 iicinedcotsin vestigation: <2... os. 5.05 el oes dad nee e.es ae le 10 CPE TEST TR PSN rhs Re rR A ea CRN eR ca Og OR, ae scree 10 VAST Ee EN 3 tio HCA Mie: See RA op OR CdSe 2 y Wie 10 SEMA tiOnyOl Utensils sin 6 ek ee eel ee Pk oe cate oe 10 ie tanlizevt OM ObsSeCUt heats heat ase mn re oe ab uct tata Racy ote ys 10 Sieciization of media. and soil... s..04<4 0.05.04. ocak es 1 Method of srowing the plants; >2.. ....%. <<. i. os de 12 ) aVEYORULEN ACCT OVSIER eka caien Fe sO ca ER Re TR OY nce trl ee 13 Hyamination of plants:for nodules 7... 70. 2s... 2b. co we 13 Special method for growing plants under sterile conditions... 18 Part I. Isolation and identification of the organism............... 14 ieolation of bacillus radicicola......... 2... 0i.escteiw cata. 14 iidentiiication of the organism... 25. 065..0. 00. 6.00 ete bee 15 Morphology ‘of the organism. 26.0 .0.26. 6 6a ok op a ok 16 Cultural‘and biochemical features........%.......2...-5. 18 Part II. Influence of certain factors on nodule production.......... 19 Experiment 1. Influence of aération in light and in darkness.... = 21 Experiment 2. Influence of some nutrient solutions in light and BET UKM Se es 6h 5. ahead Gs Le ae Opel iad te Ss 22 Experiment 3. Influence of potassium nitrate in light and in USIP SITIES oo aa Se So ae OE, rR or a ee 24° Experiment 4. Influence of potassium nitrate and calcium nitrate TEL PSNGSBICIBESRASTG) NU GUG) 0 Aa wap Se an eee oe oe a ee er 25 Experiment 5. Influence of various concentrations of Pfeffer’s solution, both with and without nitrates..<...............:. 26 Experiment 6. Influence of Pfeffer’s solution in which the essen- iraleclementstwene: AUSeMt oc. \ faa. 2 by dee dos ese aoa 26 Experiment 7. Influence of moisture......... a eee ae 27 Experiment 8. Influence of certain substances in varying CPTI TEIES I ok ak mee es, oat ene Se inate Ri Experiment 9. Influence of certain additional substances. ..... 29 General discussion of results of experiments.................. 32 5 6 CONTENTS PAGE Part III. Influence of various media on the infecting power and the vitality of Bacillus radicicola.: 242.2. 0%. se eee 32 Experiment 10. Influence of clay, loam, sand, and carborundum 39 Experiment 11. Influence of hydrochloric acid, sodium hy- droxide, and cane sugar, in varying concentrations ......... 41 Experiment 12. Influence of some other media............... 47 Experiment 13. Influence of media 300, 310, 335, and 400..... 68 UMIAIIATY sc iss. s e.g se ve aha HE a we ee eS Be 73 Generalidiscussion . f.ij.6c..) 6s ole eee nee ene 75 Aeknowledgments: 2. 6.0.0 o0. oe Ca on ee eee 78: Bibliography. <2 2.0.5 6. os Shas eae eo ee 79 PHYSIOLOGICAL STUDIES OF BACILLUS RADICICOLA OF CANADA FIELD PEA PHYSIOLOGICAL STUDIES OF BACILLUS RADICICOLA OF CANADA FIELD PEA! Martin J. PRucHa INTRODUCTION Ever since the discovery that the formation of nodules on the roots of legumes is associated with a definite microorganism, there have appeared on the market pure cultures of this organism for the artificial inoculation of leguminous plants. Nobbe and Hiltner (1897)? first caused to be placed on the market a pure culture called nitragin,’ in which the medium employed was gelatin. The efficiency of the culture was low, and Nobbe and Hiltner explained this by ascribing to the culture a loss of infecting power due to the medium used. Moore (1905) stated, as a result of his work, that the virulence of the organism was impaired when it was prop- agated on nitrogenous media, and consequently he used a nitrogen-free medium. The same opinion has been held by various other investigators, and is observed by all firms and institutions that distribute pure cultures of the legume organism. Despite the fact that media low in nitrogen have been employed, the results with pure cultures have not always been satisfactory, and Kellerman (1912) has stated that the pure cultures have not been as efficient as field soil. It would seem, then, that the conditions of isolation and cultivation of the legume organism must influence the organism, or that possibly the soil conditions would exert some influence on the efficiency of the organism to effect inoculation or would have an influence on the plant to resist infection. SCOPE OF THE INVESTIGATION With these ideas in mind, the investigation herewith reported was undertaken. It is concerned with the following: (1) Isolation and iden- tification of the organism causing nodule development on the roots of Canada field pea; (2) A study of the influence of various factors on nodule 1 Laboratory of Plant Physiology, Contribution No. 15. 2 Dates in parenthesis refer to bibliography, page 79. 3 This is not the nitragin now on the market. 9 10 Martin J. PRUCHA development in Canada field pea when the plant is grown in water or soil cultures; (3) A study of the influence of various environmental con- ditions on the infecting power of the organism. METHOD OF INVESTIGATION Organism Bacillus radicicola from Canada field pea was used for all the experiments except number 11, for which the organism from alfalfa was used. The organism was isolated as described on page 14. The stock culture was kept in the laboratory on agar slopes of medium 335, and was exposed to diffused light during the entire investigation. It was transferred at irregular intervals of time varying from one to three months. Media The various kinds of media used are referred to in the text by their laboratory numbers. In table 1 are given the laboratory numbers and the composition of the principal media employed. In addition to these a number of other media were used, and these are described subsequently. All the media were prepared according to the recommendations on the Descriptive Chart of the Society of American Bacteriologists. Sterilization of utensils Strict precautions were observed in sterilizing all the utensils. Glassware was sterilized for two hours at from 150° to 170° C. Sealpels and similar instruments, which resist high temperature, were heated in the flame. For work requiring air free from dust, a room was used into which a stream of steam was allowed to flow until the air was saturated; after condensation of the steam, the air in the room was conspicuously free from any dust. Sterilization of seed The experiments in Part III required seed free from B. radicicola. It was necessary, therefore, to sterilize the seed. In experiments 10 and 11, 95-per-cent alcohol was used, the seed being treated for thirty seconds. In experiments 12 and 13 a solution of bleaching powder was used. The author is indebted for this method to Professor J. K. Wilson, of Cornell University. The solution is prepared by mixing 8 grams of bleaching Stupies oF Bacituuus RADICICOLA OF CANADA FIELD PEA TABLE 1. Lasoratory Numpers, Composition, AND REACTION OF MEDIA 11 Laboratory number Medium 101 Medium 300 Medium 310 Medium 331 Medium 334 Medium 335....... Medium 337 Medium 400 Crone’s solution... . Pfeffer’s solution... . Composition 0.2 gram MgSO, 1 gram KH.PO, OLOMorampbeC hare ayertes shee ae 1000%cc: distilled water:..2.:........-..:... iL FOTAMIS TAWA ve toes Mens es oe he ee 3 grams Liebig’s beef extract 10 grams Witte’s peptone 1000 cc. distilled water Reaction Not changed | +1, Fuller’s scale ) Same as 300, plus 2 per cent dextrose Neutral POP ETAMNS LAC AT Ae Ney. oak, erent gn pitta ne 10 grams cane sugar eramyiCHse Oy Sige ee er he Bete ee es 0.2 gram MgSO, 0.2 gram MgSO; 0. 0. 0 1LO0O;ccy tape waterxess se ets see ase Same as 334, plus 20 grams cane sugar | | Not changed Not changed Not changed Same as 335, plus 10 grams Witte’s peptone.. | Not changed Oper AISA rans. «e leOM eee tea. De 1 ee 3 grams Liebig’s beef extract 20 grams Witte’s peptone 1000 ce. distilled water 0.25 gram CaSO, (gypsum) 2000) celdistilled water\-).. 52.0... 55.0262- | +1, Fuller’s scale t Not changed Averamsi Ca (INO; sternc oc sth oreetoee ee [i erarne KON Osea ane, cians ioe ta: 1 gram MgSO, OPororamuKi@ li tga en eee Meg eek Ox0GyerambieC lye oni eon an ee ne GOO0*cekdistilledwaters. 42 rsa oe | Not changed 12 Martin J. PRUCHA powder with 140 cubic centimeters of water; after standing for a few hours, the clear liquid is decanted and is then ready for use. Seed was kept for three hours in this solution. According to Professor Wilson’s results, seed treated in this way is completely sterilized. This method of seed sterilization will later be published in detail. That the methods of seed sterilization employed were effective was proved by a large number of experiments, and particularly by experiments with soy beans. The organism producing nodules in soy bean is not present in the soil of this region, and in all the experiments made not a single nodule developed in the cultures in which sterilized seed was used unless the plants were subsequently inoculated. Unsterilized seed oc- casionally produced plants with nodules. Sterilization of media and soil All the media used for pure cultures, if in test tubes of small volume, were sterilized in an autoclave for fifteen minutes at 120° C. In experiments 10, 11, 12, and 13 it was necessary to sterilize the soil in which the plants were grown. Three-inch flowerpots and glass tum- blers, each containing about 300 grams of soil, were used for this purpose. These were sterilized for three hours at 120° C., in a large canner’s retort. This retort was found very useful, since several hundred of the flowerpots could be sterilized at one time. Method of growing the plants In experiments 1, 2, 3, 4, 5, and 6, the plants were grown in water cultures; in experiments 7, 8, 9, 10, 11, 12, and 13 they were grown in soil. For the water cultures glass vessels were employed. The vessels were filled with the nutrient solution and the opening was covered with par- affin paper. The seed was then germinated in a moist chamber, and when the radicle was about three centimeters long it was inserted into the solution through a small hole in the paraffin paper, allowing the coty- ledons to rest on top of the paper. For the soil cultures flowerpots and glass tumblers were used. These were filled with sandy soil, were covered with paper, and, for experiments 10, 11, 12, and 13, were sterilized. The seed was planted directly in the soil, the paper covers being kept on until the seedlings began to push them off. StrupiEs oF Bacituus RADICICOLA OF CANADA FIELD PEA 13 Distilled water was used for watering the plants in experiments 1, 2, 3, 4, 5, 6, 7, 8, and 9. In experiments 10, 11, 12, and 13, boiled tap water was used. The plants in all the experiments were grown in the greenhouse. When special precautions were necessary to guard against contamination during the growing period, the plants were kept in an especially constructed culture room. For this purpose a part of the greenhouse was set off by a partition. Cracks in the walls and around the panes of glass were filled with plaster of paris. For ventilation, two panes of glass were replaced by a special frame fitted with a layer of cotton held between two pieces of cheesecloth. Inoculations Inoculations were made at the time of planting the seeds or within a day or two following. The culture of the organism to be used for in- oculation was introduced into sterile water, and the infusion was then added to the soil or the water culture in which the plants were grown. In some experiments quantitative inoculation was made, in which a spe- cific amount of the infusion was added. Examination of plants for nodules Nodules usually appeared in about two weeks. The plants were exam- ined at the end of three weeks. The roots were washed and the nodules were counted, and a note was made of the size and the place of attachment of the nodules. Since the soil in the vicinity of Ithaca is well inoculated with the Canada field pea organism, plants kept longer than three weeks were subject to some contamination. Special method for growing plants under sterile conditions For certain experiments it was necessary to maintain absolutely sterile conditions throughout the period of experiment. The method employed was as follows: A large glass cylinder, 65 centimeters high and 10 centimeters in diameter, was used as a growth chamber. In the bottom of the cylinder a few pieces of broken flowerpot were placed and were just covered with water, and on the top of these was set a four-inch pot, filled with a sandy soil. The cylinder was plugged with cotton, through which was passed a glass tube 7 millimeters in diameter and 65 centi- meters long, the lower end resting on the surface of the soil in the pot 14 Martin J. PRUCHA and the upper end protruding above the cotton plug. The tube was plugged at its upper end with cotton. The whole was sterilized in the autoclave for five hours at 15 pounds pressure. The seeds were sterilized by the bleaching-powder method. The steri- lized seed were dropped into the pots through the glass tube, and by manipulation of the tube they were buried in the soil. The soil of the pots was inoculated by introducing through the tube a few cubic centi- meters of water containing the nodule-forming organisms. PART I. ISOLATION AND IDENTIFICATION OF THE ORGANISM ISOLATION OF BACILLUS RADICICOLA On October 10, 1910, a field pea plant 30 centimeters high, with a great abundance of nodules, was procured. The whole plant was washed thoroughly in running water. One of the nodules, of firm consistency, was selected and cut off in such a way as to leave about 3 centimeters of the root on each side of the nodule; cut off in this way the nodule is more easily manipulated. The nodule was then disinfected in a solution of one part of formalin to forty parts of water, for five minutes. Four petri dishes were prepared, each containing a few drops of sterile water. The nodule, after being disinfected, was washed in sterile water, placed on a filter paper, and cut open, and with a pointed scalpel a part of the central tissue was removed and placed in petri dish 1. This nodule tissue was crushed and mixed with the water. Three loopfuls of this infusion was transferred from plate 1 to plate 2, three loopfuls from plate 2 to plate 3, and three loopfuls from plate 3 to plate 4. Ten cubic centimeters of medium 331 were then poured into each petri dish, and after sufficient agitation to effect equal distribution of the organisms the plates were allowed to incubate at 20° C. In three days a few colonies charac- teristic of the nodule organism became visible on plate 1; on the other plates plenty of colonies were present, but were visible only by the micro- scope. In ten days the small colonies became large enough to be con- veniently transferred. As far as could be ascertained from the general appearance of the colo- nies, all the plates contained only one organism. ‘The large colonies that developed on plate 1 appeared to be giant colonies, having started from small pieces of the nodule tissue in which a large number of the organisms were held. In order to obtain a pure culture of this organism, one of the SrupiEs oF Bacituus RADICICOLA OF CANADA FIELD PEA 15 small colonies was introduced into 5 cubic centimeters of sterile water in a test tube, and from this infusion a number of petri dishes of different dilutions were again prepared. From one of the colonies that developed on these petri dishes a transfer was made on agar slope with medium 335. This culture was used in all the experiments except experiment 11. The stock culture was kept on a shelf in the laboratory, and consequently was exposed for nearly three years to diffused light and to the ordinary variations of temperature and other atmospheric changes. The trans- ferring of the stock culture was made at irregular intervals of time varying from one to three months, and the cultures were kept in test tubes on agar slopes of medium 335. IDENTIFICATION OF THE ORGANISM In order to be certain that the organism isolated was the causal organism of the nodules on Canada field peas, the following procedure was followed: (1) Canada field peas were grown under sterile conditions and were inoc- ulated with this organism; (2) from one of the nodules that developed under sterile conditions a culture, No. 2, was isolated by the same method as described above; (3) the original culture and the culture No. 2 were again tested as to their ability to cause the development of nodules on - Canada field peas under sterile conditions; (4) the two cultures were compared in the laboratory with respect to their morphological characters and physiological activities. Ten Canada field peas were grown under sterile conditions according to the method described on page 13, one plant in each of ten glass cylinders. Five of the plants were inoculated with the above organism, and five were left without inoculation, as controls. At the end of six: weeks the plants were examined. They had made a fair growth, having reached a height of 60 centimeters. They were spindling, however, the leaves were small, and the root system was very poorly developed. All the moculated plants had nodules on their roots, while the controls were free from any nodules. Each plant and the soil in which it was grown were examined for contamination. One of the controls was found to be contaminated with a mold, and one of the inoculated plants was contaminated with a yellow organism. Four of the controls were sterile, and four of the in- oculated plants were found to be sterile with respect to organisms other than B. radicicola. 16 Martin J. PRUCHA A pure culture of the organism was isolated from a nodule found on one of the plants grown under sterile conditions. This organism was again tested as to its ability to produce nodules on Canada field peas. Fifteen Canada field pea plants were grown under sterile conditions again, in a similar manner to that described above. Five were inoculated with this organism, five were inoculated with the original organism, and five were left as controls. Again all the plants inoculated. with both the cultures developed nodules on their roots, while the controls had none. The organism that was isolated on October 10, 1910, therefore, was the causal organism of nodules on Canada field pea plants, and as far as could be determined by laboratory methods it was also a pure culture. The two cultures of the organism—the one isolated originally, and the other isolated from a nodule of a plant grown under sterile conditions —were studied in the laboratory with respect to their morphology and their physiological activities. An exhaustive study of this phase was not undertaken, the study being carried only far enough to show whether the two cultures were the same organism. The study consisted in propa- gating the two cultures on various media and in comparing and describing their cultural and biochemical features. The descriptions were recorded on the Descriptive Chart adopted for such use by the Society of American Bacteriologists (1907). 3 Morphology of the erganism Bacillus radicicola of Canada field pea produces no spores when prop- agated on the artificial media in the laboratory. In a young culture on artificial media, the organism is in the form of small rods about one micron long. In this form it is able to multiply by fission, like other bacteria. Under certain conditions —for example, with the addition of certain nutrients, such as sugar, to the media — some of these small rods develop into large cells, which are generally called bacteroids. Some of the bac- teroids assume the characteristic X and Y forms, the same as are found in the nodules. The development of the bacteroids seems to be largely a matter of nutrition, and the bacteroids are not a degenerate form, but a normal form, of the organism. In a culture twenty-four hours old on an agar slope the organism is very motile. As the agar-slope culture gets older, fewer and fewer of the organisms show motility, until in a culture about two weeks old no motility STUDIES OF BacrtLus RapDicicoLA OF CANADA FIELD PEA ily is detected. Motility seems to be influenced by environmental conditions. This point has not been studied extensively, beyond the observation that on nitrogenous media the motility is lost sooner than on nitrogen-free media. Since the generic name of the organism depends on the presence and the place of attachment of flagella, and since there has been so much un- certainty on this point, an effort was made to demonstrate the number and the arrangement of the flagella. Beyerinck (1890) was the first who claimed to have isolated the organism in a pure culture. He described it as having one polar flagellum, and named it Bacillus radicicola. In 1905, Moore, agreeing with Beyerinck as to the number of flagella and wishing to conform to Migula’s classification, changed the generic name from Bacillus to Pseudomonas. Edwards and Barlow (1909) found only one long, whiplike flagellum, thus agreeing with Beyerineck and Moore. De’ Rossi (1907) was the first investigator who found the organism of Vicia faba to have about eight flagella with a peritrichic arrangement. Zipfel (1912) agreed with de’ Rossi, stating that the organism has numerous flagella. In 1912 Kellerman, using a special method, also succeeded in staining the organism of several leguminous plants. He likewise found the organism to have several flagella peritrichically arranged. In this investigation the following method was used for staining the organism of Canada field pea for flagella: An agar slope culture twenty hours old, on medium 335 at 24° C., was very carefully transferred into 3 cubic centimeters of sterile distilled water in a test tube. This was allowed to stand for about four hours at a con- stant temperature, without being shaken or disturbed in any way. A drop of this infusion was placed on a cover glass and allowed to dry at room temperature, and when dry it was stained. It was essential that the cover glasses should be clean. To this end they were treated with a cleaning solution of potassium bichromate and sulfuric acid, washed in water, placed in alcohol, and finally dried with a piece of cheesecloth which had been treated with ether in order to get rid of any fats. The cover glasses were then placed in a petri dish and baked in the oven for three hours at 200° C. If cover glasses are treated in this way a large amount of difficulty in the staining of flagella is avoided. Pitfield’s mordant as modified by Muir, and carbol fuchsin, were used for staining. The mordant has the following composition: 18 Martin J. PRUCHA Cubic centimeters Tannic acid, 10 per cent aqueous solution............. 10 Corrosive sublimate, saturated aqueous solution....... 5 Alum, saturated aqueous solution.) - 27> =. 20. trees ser 5 Oarbol fuchsin >. 20) Ss ee ee ee 5 The mordant was applied for six minutes and the preparation was then very thoroughly washed with water. Carbol fuchsin was then applied for nine minutes. It was found that the organism from Canada field pea has peritrichic arrangement of flagella. The largest number of flagella observed was six, arising from any part of the organism, and the indications were that the organism may have ap even larger number. According to Migula’s classification the organism is Bacillus. Cultural and biochemical features The surface colonies in a petri dish on agar medium 335 are colorless, watery, and very viscous. When ten days old, at 20° C., they are about 3 millimeters in diameter, although occasionally colonies 10 millimeters in diameter may develop. The colonies under the surface are invariably of spindle shape. Under the 16-millimeter objective the microscopic structure appears granular. On agar slope with peptone and beef extract, the growth is watery, scanty, and colorless at first; after long standing it becomes brownish. In standard gelatin stab the growth becomes brownish. On agar slope with medium 335 to which 0.5 per cent of potassium or calcium nitrate had been added, the growth becomes opaque and iridescent. In all the standard media free from sugar the growth is scanty. In the presence of dextrose, saccharose, maltose, or glycerin, much more abundant growth takes place. Lactose and galactose increase the growth only slightly, while the addition of levulose tends to inhibit it. Two per cent of levulose added to media entirely inhibits the growth. Whether this is due to this sugar or to some impurity in it was not determined. The organism does not produce any indol, hydrogen sulfite, or ammonia. It does not reduce nitrates and does not liquefy a 12-per-cent gelatin stab at 20° C., but when grown in milk it partly digests the casein without curdling the milk. It does not produce any gas from the sugars in fer- SrupDIES OF BACILLUS RADICICOLA OF CANADA FIELD PEA 19 mentation tubes. From dextrose, maltose, and saccharose it produces a slight amount of some acid. Neutral litmus milk becomes alkaline after the organism has grown in it for about two weeks. The organism of Canada field pea does not have any strikingly charac- teristic colony features by which it can be distinguished from other microorganisms. The group number of the organism, according to the chart of the Society of American Bacteriologists, was found to be By 222.2322033. PART II. INFLUENCE OF CERTAIN FACTORS ON NODULE PRODUCTION The literature on the general subject of legume inoculation is very extensive; yet knowledge concerning the factors that influence nodule production is suprisingly meager. For the most part the investigations on this point. are incidental and fragmertary. It has been shown by various investigators, however, that the nature of the medium in which the plant is grown has an influence on the production of nodules. Rautenberg and Kiihn (1864) grew Vicia faba in various nutrient solutions, and incidentally observed that in the nitrogen-free solution the beans developed an abundance of nodules, while in the solutions con- taining nitrogen no nodules were produced. Hugo de Vries (1877) made a similar observation. He grew red clover in nutrient solutions, and the plants developed a large number of nodules in the nitrate-free solution but only a few or no nodules in the solution containing nitrates. Vines (1888-1889) tested the influence of potassium nitrate on nodule development on Vicia faba grown in nutrient solutions and also in the soil. His experiments showed that potassium nitrate tends to inhibit nodule development, both in soil and water cultures. Frank (1889) grew lupines and peas in humus soil and in humus-free soil. He found that the plants grown in the humus-free soil developed an abundance of nodules, and those grown in the soil rich in humus had no nodules. He offered the following explanation for this: ‘‘ The lupines, and also the peas, obtain the same benefit from the nodule fungus as they do from the humus. Where humus is present, the plants prefer to obtain the nourishment from the humus and no nodules are developed; where humus is wanting, however, the nodule fungus infects the plants.’”* 4 Translation from the original German, 20 Martin J. PRUCHA Hiltner (1900) showed that the addition of potassium nitrate to the nutrient solution in which legumes are grown has an injurious influence on nodule development, and he thinks this is due to the fact that the formation of bacteroids in the small nodules is hastened by the presence of the nitrate. He considers the bacteroids as degenerate and inactive forms of the nodule-forming organism. A somewhat more extensive investigation of this subject was under- taken by Nobbe and Richter (1902). They attempted to determine the influence of potassium nitrate and of humous substances on the fixation of nitrogen by soy beans. They grew the plants in flowerpots in a rich soil and in a poor soil. For rich soil they used garden soil. The poor soil was prepared by mixing 4000 grams of sand and 2500 grams of garden soil. Potassium nitrate was added to the poor soil in the proportions of 500 and 1000 milligrams to 6500 grams of the soil. When the plants were harvested the total amount of dry substance and of nitrogenous matter was determined in each plant. The results of this experiment show that the function of the nodules for nitrogen fixation is injured to a high degree by the presence of potassium nitrate. The influence of humous substances is similar, but not so great. The observation made by Frank, by Nobbe and Richter, and by others —namely, that soil rich in humous substances has a deleterious effect on nodule development — was confirmed also by Moore (1905). He grew soy beans in rich nitrogenous soil, i poor clay soil, and in poor sandy soil. Very few nodules developed on tne soy beans grown in rich soil, while in the poor clay soil and in the poor sandy soil the plants developed an abundance of nodules. The development of nodules may also be affected by other agencies. Gain (1893) attempted to determine the influence of moisture on nodule development on Pisum sativum, two varieties of Lupinus albus, and Faba vulgaris. He grew the’ plants in a field located in a region where rain was very scarce during the early part of the summer. One-half of the plat planted with each legume was watered artificially, and the other half was exposed to drought. Examination of the plants showed that the plants watered artificially had five and one-half times as many nodules as those not watered. Marchal (1901) determined the influence of fifteen different nutritive mineral salts on nodule development on peas grown in Sachs’ nutrient - StupiEs oF Bacittus RapIcIcoLA OF CANADA FIELD PEA 21 solution. He concluded from his experiments that nodule development is inhibited by the addition of the following nutrient salts in the given concentrations: Alkaline nitrates, concentration 1 to 10,000 Ammonium salts, concentration 1 to 2,000 Potassium salts, concentration 1 to 308 200 Sodium salts, concentration 1 to 260 20 The influence of phosphates was variable, and calcium and magnesium salts stimulated nodule development. Marchal was of the opinion that the variation of the osmotic pressure, due to the presence of the different salts, may be the cause of this phenomenon. Moore (1905) states that the addition of 1 per cent of sodium and potassium salts often entirely inhibits the formation of nodules, and smaller quantities considerably reduce their formation. The addition of calcium and magnesium salts, on the other hand, greatly favors nodule formation. Moore states further that this is not true with all the legumes, since the addition of calclum and magnesium carbonates is injurious to the formation of nodules on lupines and other plants adapted to acid soil. . In the following experiments the influence of various factors on nodule production has been investigated. The factors studied are light and darkness, aération, moisture, various concentrations of nutrient solutions, and a considerable number of chemical substances. EXPERIMENT 1 INFLUENCE OF AERATION IN LIGHT AND IN DARKNESS In this experiment Canada field peas were grown in an aqueous soil extract. The extract was prepared by taking one part of soil and four parts of water, by weight. The mixture was allowed to stand for two hours, and the liquid was then decanted and filtered. Twelve Erlen- meyer flasks of 300 cubic centimeters capacity were filled, and one pea was planted in each flask. Six of the flasks were covered with black paper and the other six were exposed to diffused light. Three flasks n each of the two series were aérated by passing a current of air through the liquid during the entire experiment. All the plants were inoculated with 22 Martin J. PRUCHA a pure culture of Bacillus radicicola. After twenty-four days the plants were examined for nodules. Results All the plants developed plenty of nodules. The inoculation with the pure culture had no apparent effect on the number of nodules. The soil extract was not sterilized and apparently had plenty of the organisms. The plants whose roots were kept in darkness had a greater abundance of nodules than those whose roots were exposed to light. The aération as supplied in this experiment had no stimulative effect on either the number or the size of the nodules. EXPERIMENT 2 INFLUENCE OF SOME NUTRIENT SOLUTIONS IN LIGHT AND IN DARKNESS Ninety Erlenmeyer flasks of 300 cubic centimeters capacity were divided into five series, with eighteen flasks in each series. These flasks were filled with the following solutions, respectively: series 1, with medium 101; series 2, with Crone’s solution; series 3, with Pfeffer’s solution; series 4, with tap water; series 5, with soil extract (the same as was used n experiment 1). Six flasks from each series, three covered with black paper and three not covered, were inoculated with a pure culture of Bacillus radicicola. A second group of six flasks from each series were prepared in the same manner, but each flask was inoculated with 5 cubic centimeters of soil extract. A third group of six flasks from each series were prepared in the same manner but were not inoculated. One plant was grown in each flask. The water of transpiration was replaced each week. At the end of four weeks the plants were examined for nodules. The results are given in table 2. Results Not all the plants that were inoculated developed nodules. A few nodules developed on the plants grown in medium 101, in Crone’s solution, and in Pfeffer’s solution. In the soil extract all the plants developed nodules, although the plants grew better in Crone’s solution and in Pfeffer’s solution. In the tap water no nodules developed. More nodules developed on the roots kept in darkness than on those exposed to the light. Stupies oF BacitLtuus RapDIcICOLA OF CANADA FIELD PEA 2a TABLE2. INFLUENCE oF Some Nutrient So.tutions on NopuLE DEVELOPMENT Culture solution* Medium 101...... Number of ; nodules Crone’s solution... Pfeffer’s solution. . Soil extract....... Treatment Exposure = Plant 1 | Plant 2 | Plant 3 (| Light None None None Inoculated with B. radicicola «.|-—————— — Dark None None None Light None None None Inoculated with soil extract. — ~ Dark None None Few Light None None None Not inoculated............ ——— — oo Dark None None None Light None None None Inoculated with B. radicicola ——_—— Dark Few Few Few Light None None None _} Inoculated with soil extract. ——|— | Dark Few Few Few | = _ | Light None None None || Not inoculated............ SO | Dark None None None Light None None None Inoculated with B. radicicola < |— ——|—— Dark None None Few | Light None Few Few Tnoculated with soil extract. —— | Dark None Few Few Light None None None Not inoculated............ a a Dark None None None Light None None None Inoculated with B. radicicola « |——-———|— —|———— — Dark None None None Light None None None Inoculated with soil extract. ._ |—————— a Dark None None None Light None None None Not inoculated............ — = Dark None None None i Light | Present | Present | Present Inoculated with B. radicicola ¢_ || |_| || Dark | Present | Present | Present Light | Present | Present | Present Not inoculated............ —— | Kg |}—— Dark | Present | Present | Present * See page 11, 24 Martin J. PRUcHA EXPERIMENT 3 INFLUENCE OF POTASSIUM NITRATE IN LIGHT AND IN DARKNESS In this experiment twelve glass cylinders, each of 5 liters capacity and 50 centimeters in height, were used. Six of these were filled with Crone’s full nutrient solution, and the other six were filled with the same solution except that potassium chloride was substituted in place of potassium nitrate. Five plants were grown in each cylinder. The experiment was arranged in the following manner: Series 1. Three of the cylinders filled with Crone’s full nutrient solution were covered with black paper. Two of these were inoculated, and one was not inoculated. Series 2. The other three cylinders filled with Crone’s full nutrient solution were treated as was series 1, but were not covered with black paper. Series 3. Three of the cylinders filled with Crone’s solution in which potassium nitrate was replaced by potassium chloride, were covered with black paper. Two of these were inoculated, and one was not inoculated. Series 4. The remaining three cylinders, with the same solution as was used for series 3, were treated as was series 3 but were not covered with black paper. Results When the plants were three weeks old, those grown in the solution with nitrate looked green and healthy, while those grown in the solution without nitrate were turning yellow and the lower leaves were dropping off. No difference in appearance was observed between the inoculated and the uninoculated plants. The uninoculated plants had no nodules; those grown in the presence of nitrate and inoculated had one or two nodules each; those grown in nitrate-free solution had about fifteen nodules each. Six weeks after planting, the plants grown in nitrate solution had thick, green leaves and thick roots, and no more nodules had developed on the inoculated plants. The plants grown in nitrate-free solution were yellowish except for the upper leaves, which were green; the roots were longer and more abundant than on the plants grown in nitrate solution. Nodules were abundant, continually developing on the new roots. The uninoculated plants in nitrate-free solution had no nodules and were practically dead. a | STuDIES OF BaciLLusS RADICICOLA OF CANADA FIELD PEA 25 The plants grown in nitrate-free solution with their roots exposed to light were slightly shorter than, and did not have quite as many nodules as, those grown in the same solution but with their roots kept in darkness. In the presence of the nitrate the development of certain green alge interfered somewhat with root growth. EXPERIMENT 4 INFLUENCE OF POTASSIUM NITRATE AND CALCIUM NITRATE IN PFEFFER’S SOLUTION The procedure in this experiment was the same as in experiment 3. Only one plant was grown in each cylinder. Calcium nitrate was replaced by calcium chloride, and potassium nitrate was replaced by potassium chloride. The plants were kept until they began to blossom. Results In the solution with the nitrates, two or three nodules developed on each plant within twelve days after inoculation. No more nodules developed after that. In the cylinders not covered with black paper, alge developed in abundance, and, surrounding the roots, dwarfed the plants. In the cylinders covered with black paper, also, some algz developed in time, but they were far less abundant. In the nitrate-free solution there was an abundant development. of nodules. The nodules were more numerous on the plants grown in the cylinders covered with black paper than on those the roots of which were exposed to light. The root system of the plants grown in the nitrate- free solution was more developed than that of the plants grown in the nitrate solution. An interesting point observed in this experiment and in experiment 3 was that the nodules developed, both in the nitrate solution and in the nitrate-free solution, immediately after moculation. In the nitrate solu- tion, however, no further development of nodules took place, while in the nitrate-free solution there was a continual development of new nodules on the new rootlets as time went on. This-would seem to indicate either that the nodule-forming organisms were made inactive by the nature of the solution, or that the solution in some way affected the resisting power of the plants. 26 Martin J. PRUCHA EXPERIMENT 5 INFLUENCE OF VARIOUS CONCENTRATIONS OF PFEFFER’S SOLUTION, BOTH WITH AND WITHOUT NITRATES Wide-mouth bottles of 500 cubic centimeters capacity were used in this experiment. They were all covered with black paper. Two series were prepared, one with Pfeffer’s full nutrient solution and the other with Pfeffer’s solution in which the nitrates were replaced by the chlorides - of the same metals. The following concentrations in each series were employed, taking the concentration of the full nutrient as 1: 4, 3, 4, 3, 1, 2, 3, 4, and 8. Five plants were grown in each bottle, and all were inoculated. The duration of the experiment was three weeks. Results In the full nutrient solution a few nodules developed in the ;, concen- tration. In the other concentrations no nodules appeared within the three weeks. In the nitrate-free solution nodules developed best in concentration 1. In the $ and } concentrations a few nodules appeared. EXPERIMENT 6 INFLUENCE OF PFEFFER’S SOLUTION IN WHICH THE ESSENTIAL ELEMENTS WERE ABSENT In this experiment the same vessels were used as in experiment 5. The solutions were prepared as follows: { Ca(NO3)2 was replaced by CaCh | KNO; was replaced by KCl K NOs was replaced by NaNO; For solution minus potassium + KH,PO, was replaced by NaH2PO, KCl was replaced by NaCl For solution minus phosphorus, KH2:PO, was replaced by NetePO, KE For solution minus sulfur, MgSO, was replaced by MgCl. | For solution minus magnesium, MgSO, was replaced by Mge@l. Ne, SO For solution minus iron, FeCl; was replaced by NaCl For solution minus nitrogen One plant was grown in each bottle. The plants were examined three weeks after planting. STupIES oF BaciLLusS RaADICICOLA OF CANADA FIELD PEA pare Results Nodules developed only in the nitrate-free solution. The plants in most of the solutions in this experiment and in experiment 5 did not grow well and normally. It is possible that slightly different results might have been obtained had the length of time of the experiment been extended. It was observed, however, in these and in the other experiments, that the pumber and the size of the nodules on a plant are influenced by the rate and the amount of growth of the plant. In other words, any disturbing factor in the normal functions of a plant tends to hinder the development of nodules. EXPERIMENT 7 INFLUENCE OF MOISTURE In experiments 7, 8, and 9, the plants were grown in glass tumblers. In each tumbler was placed 300 grams of air-dry sandy soil containing less than 0.5 per cent of moisture. The following percentages of moisture were used: 5, 10, 15, 20, 25, 30, 40, 50, and 60, the percentage being based on the air-dry soil. Three tumblers were used for each percentage of moisture, and five plants were grown in each tumbler. The plants were kept in the greenhouse. They were watered every other day, the neces- sary amount of water to be added being determined by weighing. The soil is naturally well inoculated with the Canada field pea organism, but in addition to this each tumbler was inoculated with a pure culture of the organisin. The duration of the experiment was four weeks. Results The best growth took place in 15, 20, and 25 per cent of moisture. In 5 and 10 per cent of moisture the plants grew very slowly, while in 50 and 60 per cent the roots rotted. Nodules were present on all the plants. The number of the nodules on each plant increased with the percentage of moisture up to 40 per cent. These results agree with those of Gain (1893), who found that a larger number of nodules develop when the plants are abundantly watered. EXPERIMENT 8 INFLUENCE OF CERTAIN SUBSTANCES IN VARYING QUANTITIES The same soil and the same kind of vessels were used in this experiment as in experiment 7. Three hundred grams of the air-dry soil was in- 28 Martin J. PRUCHA troduced into each glass tumbler; the substance to be tested was dissolved in the proper quantity of water, and this was added to each tumbler. Five plants were grown in each tumbler, and all the cultures were made in triplicate and were inoculated. The plants were allowed to grow for four weeks. The kind and the amount of the substance added, together with the results, are given in table 3: TABLE 3. INFLUENCE ON NoDULE DEVELOPMENT OF CERTAIN SUBSTANCES IN VARYING QUANTITIES Quantity added to Substance used 300 grams | Condition of plants Nodule development of soil (grams) 0.25 | Good growth........ No nodules KNO i 0.50 | Poor growth........ No nodules SNS EER a aD ee 100" Nolgrowtht. ee No nodules 2.007) sNolsrowthie, sce No nodules 0.25 | Good growth........ Few nodules 0:50) Poor growths: 5... 2. No nodules Ca(NOs)2........- 26.2. ) 1.00 | No growth.....:.... No nodules 2000 Noterowthees sees No nodules 0.25 | Good growth........ Nedules abundant Meso 0.50 | Good growth........ Nodules abundant CO a ASAE Reeser 1.00 | Good growth........ Nodules abundant 2.00 | Good growth........ Nodules abundant 0.25 | Good growth........ Nodules abundant KH:PO { 0.50 | Good growth........ Nodules abundant PA ie Soa ae Rane 1.00 | Good growth........ Nodules abundant 2.00 | Good growth........ Nodules abundant 0.25 | Good growth........ Nodules very abundant CaCO, 0.50 | Good growth........ Nodules very abundant isk See PTAs ) 1.00 | Good growth........| Nodules very abundant 2.00 | Good growth........ Nodules very abundant 0.25) | No erowthteseseee No nodules NH.Cl ] O50) SNorterowthe. meee No nodules Sa Dare aaa ee cama ooo 1.00 | No growth..........| No nodules 2.005 No! growths...5ass5- No nodules 0:25. Nojgrowth, sae ee No nodules FeCh..... 02505) Nojerowths sess No nodules oF 7 Oy area ees Ee No growth..........| No nodules 00" | No’ growth*.... No nodules SrupiEs oF BacriLLusS RADICICOLA OF CANADA FIELD PBA TABLE 3 (concluded) 29 Quantity added to ; Substance used 300 grams | Condition of plants Nodule development of soil (grams) | OF25) | Hairierowth....- 2. - Few nodules a es 0.50 | Poor growth........ No nodules Witte's peptone.......... 1008 |SRoorierowthee.o.5 4. No nodules 200: |) Poor growth. .:... .. No nodules 0.25 | Good growth........| Nodules present 0.50 | Good growth........ Nodules present 1.00 | Good growth........ Nodules present CTS GUT eeeO Oa 2.00 | Good growth........| Nodules present 4.00 |-Poor growth........ Nodules present 8.00 | Poor growth........ No nodules 16.00 | Very poor growth... .| No nodules (COORNUEO) [Ea ree en a | Nothing | Good growth........ Nodules present added Results The addition of MgSO,, KH.PO,, and CaCO; in the concentrations used in the experiment had a beneficial effect on the development of nod- ules. Cane sugar at low concentrations had apparently no effect. At the concentrations of 4, 8, and 16 grams of sugar in 300 grams of soil, cane sugar was injurious, probably due to fermentation products and to stimulation of the development of microorganisms injurious to the plants and also to the development of nodules. The addition of NH,Cl and FeCl; completely inhibited the growth of the plants. In the case of KNO; and Ca(NO3z)2 the concentration of + gram in 300 grams of soil had a beneficial effect on the growth of the plant, but an injurious effect on the development of nodules. A few nodules developed in the presence of Ca(NOs3)2, but none in the presence of KNO;. The higher concentrations of Ca(NOs3)2 and KNO; inhibited nodule development and also caused injury to the plants. EXPERIMENT 9 INFLUENCE OF CERTAIN ADDITIONAL SUBSTANCES The method used in this experiment was the same as in experiment 8. Several additional chemicals were tested. In examining the plants, the 30 MartTIn J. PRUCHA number of nodules was counted and both the total number of nodules and the average per plant for each concentration of the substance were calculated. The results follow in table 4: TABLE 4. INFLUENCE OF CERTAIN ADDITIONAL SUBSTANCES ON NODULE DEVELOPMENT Quantity added to a ‘ i Number} Condition Chemical ae cham of plants} of plants (grams) | | 0/95. |) 1.10) | iSman) 4 0.50 | Sai omallenes Ca(NOs):...... 1.00 | 0 Plants killed ( 2.00 | 0 | Plants killed 0.25 | Tel Goods ; 3 0.50 | Sl Goods. se Tannic acid... . 1.00 1 || Goodie en. 2.00 [3 |Goodseee ne 0.25 6: | Goode. >- 0.50 Vell GOOGa reac KH:POu....... 1.00 Ag Senne e ee 2.00 As\ssmeallenonss ( 0.25 145 |e Goodernas= 0.50 1 Goodec- hacen 1.00 199) G@ood. 2.00 13 | Good 0.25 Ce-|) fSroaesl ls ee KCl 0.50 0 | Plants killed Sa cera ° 1.00 0 | Plants killed 2.00 0 | Plants killed 0.25 14 Goode ere 0.50 Take || SSaaslll SS KNOs........ 1.00 3 | Plants killed 2.00 0 | Plants killed ie 12e\eGooda. se 50 el eomalleases NH.Cl........ 1.00 0 | Plants killed 2.00 0 | Plants killed 0.25 1S Good #ersne ee 0.50 ll |kGoode ass. Witte’s peptone 1.00 9 | Small... 2.00 1’ Very small. . small OO Oe) Dy ech co Number of large Total number of nodules | nodules eee eee Pe Oech cl nodules per plant COR coe —7— —se — > = 00D © naone Oo Orr o> alwrorn!worol vornn 0 leyrapim) ape @ (6) eis (playa! © @ o36 ee) eee eee Strupres OF BacitLtLus RapicicoLaA oF CANADA FIELD PEA 31 TABLE 4 (concluded) ny ee ave Number | Number Total Sis 3 umber ondition of of | number Chemical used er ed of plants} of plants small large | — of nodules (grams) nodules | nodules | nodules ae 0.25 12 Goode s.a: 35 50 | 85 all KOH 0.50 LOM Goods: ae: 10 45 | 55 Deo pages so 1.00 Sh oma eee ae 11 25 36 12.0 2.00 On| FRlantsikilled!ae ats. Uk oa. eee neo eee | eee ere 0.25 ie eGoode seme 4 0 4 0.4 0.50 14> Goods: 1 0 1 Ost Fe(NOj)s...... 1.00 Gulecuiatlas 4) 0 0 0 0 2.00 ste rapa lh fay eae cll es ans ENA =| Be pete oe | ge er eee 0.25 So |*Goodi.. ... 6 45 51 6.4 0.50 14 | Good 90 94 184 ial Ca(H2PO,):.... 1.00 iP e@eodes Le 54 Goren Tie 9.5 2.00 5) 4) isyaorllls 5 oo. 8 11 19 3.8 0.25 Il | MG@erel 5 Sec 22 65 87 19 0.50 | 10 | Good...... 15 60 75 a5 CaSO......... iOGS ere 14. | AGaod. tae 57 50 107 74 2.00 10 | Good...... 21 45 66 6.6 0.25 Ob *Blants killed ese. ol). cee lle ceaeces lee 0.50 Os sPlants=killed |r gee es ns | ea oe ell meer e FeCls.......... 1.00 @; lePlanitasbeilled| ehoce Piel cence es ee ee ee 2.00 OulBlantsckalledi aes See eens. oo renee 1.00 12>) Good)...... 40 82 122 10.2 Starch. ...<... 2.00 13) Goode se. 59 AO 99 7.6 4.00 SuleGood. .aaee 18 42 60 eo Glitaneee 1S Good jens sue cee eee 113 6.3 Controls. ..... 4A in cca TAB Good? {sa al ake 2 owl tose 107 36 { Results The results of experiment 9 are similar to those of experiment 8. The following chemicals added to the soil at the concentrations used in the experiment were injurious to the plants and tended to inhibit the de- velopment of nodules: Ca(NOs3)o, KCl, KNO;, NH,Cl, Witte’s peptone, Fe(NO3)3, and FeCl;. On the other hand, tannic acid, KH:PO,, MgSOu,, KOH, Ca(H2PO,.)o, CaSO, and starch exerted a beneficial influence on nodule development and appeared to have no injurious effect on the plants. a2 Martin J. PrRucHA GENERAL DISCUSSION OF RESULTS OF EXPERIMENTS The nine experiments reported in Part II; on the factors affecting the development of nodules on Canada field peas, are not extensive enough to allow any broad and general deductions, but the results point to several conclusions. Nodules develop readily on Canada field peas in nutrient solutions, provided the proper nutrient salts are added. Varying the concentration appears to have a marked influence on nodule development. Aération has no appreciable effect. Nodules developed on the long roots at as great a depth as 30 centimeters below the surface of the nutrient solu- tion. If air is essential for the development of nodules, enough of it was dissolved in the nutrient solution under the conditions in the experiments. 7 The presence of nitrates in the nutrient solution or in the soil tends to | inhibit the development of nodules; the reason for this is not known. If the plants are grown in the presence of nitrates for about a week and | then inoculated, a few nodules will develop, but no further development of nodules takes place; in water cultures without nitrates and inoculated, a continuous nodule development takes place as long as the roots grow. ; The chemical composition of the various soils used for agricultural | purposes differs. What influence this has on the various groups of the | nodule-forming organism and on nodule development has never been extensively investigated. The results of the foregoing experiments em- phasize its importance. The limited distribution of the different groups of the nodule-forming organism in some soils, the failures in inoculations, and the difficulty in growing certain legumes, may be explained in certain — cases as being due to the composition of the soil. It is known that the addition of lime to certain soils has a beneficial effect on nodule develop- ment and on the growth of some legumes. It is highly probable that the addition of other substances to the soil may be beneficial to other legumes. PART III. INFLUENCE OF VARIOUS MEDIA ON THE INFECTING POWER — | AND THE VITALITY OF BACILLUS RADICICOLA : As indicated previously, and again further developed in the discussion | of this subject, the view has been maintained that the infecting power, — or “ virulence,” of the nodule-forming organism may be impaired by cultivating it on certain media. In order to determine whether or not — | | Stupies oF Bacititus Rapicrcota oF CANADA FIELD PEA 33 ‘such is the case, and also to determine the media most favorable for msintaining the vitality of the organism, the following experiments were made. The term virulence has been used by previous investigators to mean the ability of the organism to penetrate the root and produce nodules. Since the term virulence in this connection, as also suggested by Edwards (in Marshall’s Microbiologie), does not correctly apply to the legume bacteria, the term infecting power will be used throughout this paper. Beyerinck (1890) was the first to isolate a pure culture of an organism from a nodule. Prazmowski (1890), Frank (1889), and Nobbe and others (1891), stimulated by Beyerinck’s success, were also able to obtain pure cultures from the nodules of various legumes, and to produce nedules by inoculating the plants with the pure cultures. The experiments on the inoculation of legumes by pure cultures at once raised a question as to the classification of the nodule-forming organism, which question is in- timately connected with the subject of the infecting power of the organism and the resistance of the plants. Do all the different organisms from the various species of legumes belong to several species, or do they belong only to one species but to several races or varieties? Can the organism from one species of lerumes cause nodules on a different species of legumes? Is the relation between the legume and the organism a case of symbiosis or a case of parasitism? Does the organism have the biological or physio- logical character called virulence as understood by pathologists, and can this be altered or destroyed without injuring or destroying the other physiological activities? Do the host plants have a resistance in a patho- logical sense, and can this resistance be altered by the environmental factors without altering the morphology, the structure of the tissues, and the physiological activities of the plants? Is the resistance against the entrance of the organism into the root tissues different from the resistance against the development of the organism inside the root tissues? These and similar questions formed the foundation of the numerous investigations that were undertaken subsequently to the isolation of the pure culture of the nodule-forming organism, and nodule production by pure cultures. Even before the isolation of the nodule-forming organism by Beyerinck, it was observed by Hellriegel (1886) that when peas, vetch,- beans, clover, serradella, and lupines were inoculated with an infusion from the same 34 MartTIn J. PRUCHA - soil, all the plants developed nodules except the serradella and the lupine. From this Hellriegel inferred that important differences must exist between the nodule bacteria of the different legumes. Beyerinck (1888) was of the opinion that the nodule-forming organisms of the legumes belonged to one species, but that there were several groups and in each group a number of varieties. From the results of his sub- sequent investigation (1890) he was forced to change his former opinion. He considered the organism of Ornithopus and that of Vicia to be two_ distinct species. Frank’s investigations (1899) led him to believe that there was only one species among the nodule-forming organisms. Salfeld (1888) grew peas and horse beans in ‘‘ Hochmoorboden,” and inoculated one part of them with sandy soil in which peas were grown and the other part with sandy soil in which lupines were grown. Both | the peas and the horse beans inoculated with the pea soil developed nodules, while those inoculated with the lupine soil were free from nodules. Laurent (1901) could produce nodules on dwarf peas by inoculating — them with material from nodules of thirty different leguminous species, © but he claimed that the number, size, and appearance of the nodules | was influenced by the inoculating material of the different sources. | Kirchner (1896) grew about one hundred different species of legumes | in the Hohenheimer botanical garden. He observed that all the different — species of legumes developed nodules in the garden soil except the soy beans, although these had been grown in the garden for ten years. The soy beans did not produce nodules until they were inoculated WHE soil on which Japanese soy beans had been grown. The investigations of Mazé (1898) led him to divide the nodule-forming organisms into two groups — those adapted to a neutral or an alkaline soil, and those adapted to an acid soil; the former infecting the plants that favor neutral and alkaline soil, and the latter infecting the plants that favor acid soil. Nobbe, Schmid, Hiltner, and Hotter (1891) undertook a very extensive series of investigations on the general subject of nitrogen assimilation by leguminous plants. Much of the present information on this subject is due to these men, especially to Hiltner and Nobbe. They showed that the only way to study the relations between the nodule-forming organisms of the different legumes and the different species and varieties of legumes | Stupies oF Bacituus RapDIciIcoLA OF CANADA FIELD PEA 35 was to use pure cultures of the organisms for inoculation purposes, and not ihe soil infusions as was done by a number of previous investigators. _ In a subsequent paper, Nobbe, Hiltner and Schmid (1895) arrived at the following impcrtant conclusions on the relation of nodule-forming organisms to the different species of legumes: “The infecting power of the nodule bacteria of the various groups and species of legumes cannot be differentiated absolutely, but only in degree. The pure cultures from nodules of different species of legumes do not represent different species, but only different forms. We have not the least doubt that all the nodule bacteria of the different legumes we have studied, even those of Mimosz, are one species, all belonging te Bacillus radicicola of Beyermek. These bacteria, however, are influenced by the plants in whose roots they live to such a degree that their descendants are able to infect readily only that species of legumes to which the former host plant belonged, at the same time losing partly or completely the power to infect other species of legumes. When the legume is grown in a suitable soil, nodules will develop on the roots only wher. either those nodule bacteria are present which have lived previously on that legume species, or when the neutral nodule bacteria are present. The latter will be found in the soil where legumes have never been grown or where they have not been grown for a long time. If one legume is preponderantly grown:;in a soil, most of the neutral bacteria become influenced by this legume, and when a different legume is planted which is not closely related to the former no nodules will be formed, or only very few and faulty ones, and these will appear so late that they will be of very little value to the plants.” ® By means of extensive experiments (Nobbe and Hiltner, 1896) it has been demonstrated that effective moculation is obtained only when the plants are: inoculated with bacteria from the nodules of the same species of legumes. Moore (1905) conducted extensive cross-inoculation experiments, and maintains that “it is possible to cause the formation of nodules upon practically all legumes, no matter what was the source of the original organisms, provided they were cultivated for some time upon a synthetic nitrogen-free medium.” He states further: “It is undoubtedly true that the long adaptation of the bacteria to the special conditions obtaining 5 Translation from the original German. -_ 36 Martin J. PRUCHA in a particular species of legume enables such organisms to produce more abundant nodules in a shorter length of time than bacteria isolated from some other legume and grown upon nitrogen-free media. While this is of considerable practical importance, and will probably always make it neces- sary to distribute the specific organism for the specific crop, it does not in any way indicate that the bacteria found in the nodules of beans, peas, clovers, ete., are separate species. The most that can be maintained is that there is a slight physiological difference due to the long association with a plant of a peculiar reaction which enables the bacteria more easily © to penetrate the host upon which they have been accustomed to grow. 7 These slight racial characteristics can readily be broken down by culti- vation in the laboratory, and it is entirely possible to secure a universal organism capable of producing a limited number of nodules upon all the | legumes which now possess these growths.” Hopkins (1904) found that the organism from sweet clover readil inoculates alfalfa. Nobbe and Hiltner (1900) undertook to train the nodule-forming | organism of peas and that of beans so that the former may cause nodules on beans and the latter on peas. They succeeded in doimg this, and _ drew the following conclusions: 1. The nodule-forming organism from peas can be trained to produce © nodules on beans, and that from beans to produce nodules on peas. 2. Although some nodules are produced in both cases, the organisms do not assimilate any nitrogen ‘at first. . 3. If the pea organism that’ caused nodules on beans is isolated and beans are inoculated with it a’second time, the organism then infects the 7 beans more readily than at the first inoculation and its power to assimilate nitrogen is increased. The organism of beans behaves in the same manner | when made to infect peas. / Kellerman (1912) reports that Mr. Leonard has succeeded in securing abundant inoculation on soy beans, lupines, and alfalfa from an organism of a culture originally isolated from the alfalfa nodule and kept on an — artificial medium in the laboratory for about six years. Kellerman, therefore, is of the opinion that the nodule-forming organisms of all the Leguminose should be considered as a single species. The evidence from the investigations mentioned above points to two conclusions: (1) that, with some exceptions, the nodule-forming organism Srupigs oF BAcILLUS RADICICOLA OF CANADA FIELD PEA 37 fram one legume does not cause nodules on another legume; (2) that ‘the organism from one legume may be trained to cause nodules on any other legume. The evidence for the latter conclusion, however, is ‘not final. About 1895 a German company placed nitragin on the market — a pure culture of the nodule-forming organism for inoculation purposes. The cultures were propagated on gelatin and their preparation was based on the results of the investigations of Nobbe and Hiltner. These cultures were extensively tested both in Germany and in other countries, and, as judged by the reports of these tests, the cultures proved only partially ‘successful. As a result of these adverse reports on nitragin, Nobbe and Hiltner (1899) undertook to ascertain the cause of the low efficiency of their cultures. They bad already shown that the nodule-forming organism can be trained to infect other legume species than that of its host plant, when they trained the organism from peas to produce nodules 'on beans and that from beans to cause nodules on peas. They went a step further and demonstrated that the infecting power of the organisms |can be altered in degree. They stated that the propagation of the ‘organism on artificial media increases or diminishes the vitality, and 'that in general nitrogenous media are injurious to the vitality of the organism. Moore (1905) also reports that as a result of numerous trials it has been found that, although the bacteria increase most rapidly on a medium rich in nitrogen, the resulting growth is usually of very much reduced vitality, and when put into the soil these organisms have lost the ability to break up into the minute forms necessary in order to penetrate the root hairs. In a further study of this subject, Hiltner (1900) was led to believe that this variableness in the infecting power of the nodule-forming organism _is the determining factor of the number and size of the nodules on every plant when grown under otherwise favorable conditions. He took some older plants that already had nodules on their roots, and placed them in a nutrient solution without any nitrogen. Repeated inoculation with its own organism did not produce any nodules on the new rootlets. When fall came, and the leaves began to turn yellow and drop, and the organisms in the nodules became weaker than those in the solution, nodules began to develop on the rootlets. When Hiltner took older 38 Martin J. PRUcHA plants that had no nodules on their roots and placed them in a similar | solution to that used with the other plants, an immediate development ~ of nodules took place on the new rootlets. From this and other experi- © ments, Hiltner concluded that “the active nodules produce immunity | in the plant against the bacteria that possess the same or a lower degree of infecting power than those already living in the nodules of that plant. | Only bacteria of higher infecting power are able to enter the root | tissue. Suchting (1904), believing that Hiltner’s theory of the infecting power of the organism and its relation to nodule development was not sound, | undertook a series of interesting experiments on this subject, as well © as an elaborate discussion of Hiltner’s theory and of his own theory. | In his experiments Stichting attempted to ascertain three points: (1) Have the organisms that produce the first nodules on the plant less infecting | power than those that produce nodules on the same plant subsequently? (2) Does the symbiosis with the plant influence the infecting power of the organism’? (3) Does the artificial medium influence the infectirg power of the organism? 196 From his experiments Siichting drew the following conclusions: 1. The infecting power of the bacteria is not proportional to the age of the nodule. 2. The passage of the bacteria through the host plant does increase their infecting power. Their infecting power does not vary at the different — stages of the plant’s vegetative period, and the feeding of the plant by potassium nitrate is injurious to the bacteria in the nodules. 3. When propagated on artificial media the lupine bacteria lose their infecting power on some media and may exist in a so-called pseudo form. On neutral media the bacteria retain their infecting power better than on acid media. The horse-bean bacteria do not behave in the same manner, | but keep their infecting power for a long time on suitable media. Lewis and Nicholson (1905), on the other hand, found by their experi-— ments that “It seems that the presence or absence of nitrogen in the — culture media is not the determining factor in maintaining the activity — of the germ. Cultivation in the presence of the amount of nitrogen | usually present in bouillon with from two to five per cent of cane sugar 6 Translation from the original German, StuDIES OF BaciLLus RADICICOLA OF CANADA FIELD PEA 39 or glucose, preferably the former, has given best results in a1 of the work connected with the experiment.”’ In the following pages data are presented on experiments conducted through several years in an attempt to alter the ‘“ virulence ’’— that is, the infecting power — of the organism. In experiments 10, 12, and 13, Bacillus radicicola of Canada field pea was used; in experiment 11 that of alfalfa was used. The organisms were propagated and kept on various media. Their infecting power was tested and measured by the nodule development in plants grown in a sterilized sandy soil. EXPERIMENT 10 INFLUENCE OF CLAY, LOAM, SAND, AND CARBORUNDUM In this experiment the organism was grown on clay, sandy loam, sandy soil, fine quartz sand, coarse quartz sand, and carborundum. One hundred grams of each substance, air-dried, was introduced into Erlenmeyer flasks of 300 cubic centimeters capacity. After sterilization the media were heavily seeded with B. radicicola. This was accomplished by introducing into each flask the growth of B. radicicola from one agar slope, along with the necessary quantity of water. The amount of moisture added to each medium was about five per cent less than its capacity for holding water. Two series of flasks were prepared. In series 1 the media, as soon as seeded with the organism, were spread on sterile paper and allowed to dry at room.temperature. The time required for their complete drying was about six hours. In series 2 the media were left in the flasks, plugged with cotton, and allowed to stand in the laboratory. Drying of the media in this series was very gradual. The infecting power of the organism in these cultures was tested by inoculating plants. For this purpose Canada field peas were grown in sterilized soil in flowerpots, and were inoculated with the respective cultures at the time of seeding. Inoculation was accomplished by scattering one gram of the inoculating material over the soil in the flowerpots. The first test was made when the cultures were ten days old and the second test when the cultures were forty- six days old. When the plants were three weeks old they were “examined for presence of nodules. The results are presented in tables 5 and 6: 40 Martin J. PRUCHA TABLE 5. Resvuuts or First INocutation Test. THE Cuttures WERE Ten Days OLD Narber Deas Total Number Plants inoculated with of plants meee ajiees plants Je nodules | per plant (O) are ee ae aeRO ne ETA irae cP PE NIOO Oc 17 4 31 1.8 SSI SETAG ha lor Tah Abra Stet lanth Araceae ¢ 23 13 81 3.5 TiAl MAME SOW ss ait Canta enc eats eters he 14 il 45 3.2 Seu Hine Quartz, SANG: cv Yas ne tnaee 15 3 9 0.6 al @oarseiquarty Sal. 0... scree ee ae 19 0 0 0 Carborundumivne.s2c8- cae ee iby) 2 25 23 Clays eso havaray tet elaacs hosters Se ee 10 6 51 5.1 Sandy loamenssac aise sue cece mere 15 10 90 6.0 Fah EL Ye BOL i goo sean ysraenete natn tat aa eae 18 10 67 347 ai Ane quanta Sand)... weitere te 13 7 92 tol ‘= |) @oagse quartz sand.....2o.2s0. 8. ks 18 12 94 5.2 ou M@ArHOLunGym seco eke Seine eee 14 4 23 1.6 (Acarislope Cultureeniaioeee er eee 19 15 90 4.7 Controlssey eo Pee oe ee 13 3 15 1.2 TABLE 6. ReEsutts oF Seconp InocunatTion Test. THe Cuttures WERE Forty-six Days OLp Number Niet of fies ee Plants inoculated with of plants ais 7 4 1 plants with cj ee agialen nodules per plant (OEM be mira SOs BE caOeP io ne o aerate ores 5 5 53 10.6 be POANGY TOAM oa slo. cas seco h Stabsaee & ee 12 11 79 6.6 mah MOAI VASO ced =1 Py tele tee eee 12 10 151 12.6 “et iiMine. Quartz Bande. ./c's..jo fea ace oeeks 10 10 58 5.8 ry oaree: quartz Sand gehen, se 4 4 41 10.3 @arborundumstpe se. a eee eee 11 7 118 10.3 Bi ee Ne ee 3| 3 20 6.7 Eat madly: loan 10 penta Seta eter 4 3 45 ES 2 Samy Sollee aes meena Mee one teat ree 14 14 188 13.4 “Hii mBIM ey qUanog Sala de mite see ereen reer h ip 8 7 190 |. 23.8 ca, | oarse quartz Sandin ee oP sels ee 7 6 78 HL i @arborun dum sens cere: eee ee 10 4 32 S02. COnirOle te sue nee ad bee ete ae 36 13 120 3.3 StupiEes oF Baci~tus RapDicicoLtA OF CANADA FIELD PEA 41 Results In both tests there was a certain amount of infection due to other sources than the inoculating materials. In the first test three of the thirteen plants used as controls developed nodules, while in the second test thirteen of the thirty-six control plants developed nodules. It was noticed, however, that, as a rule, if contamination took place subsequently to the inoculation and the plants were examined within four weeks after planting, the nodules due to the contamination were small and developed on the lateral roots near the surface of the soil, whereas nodules resulting from inoculation always appeared first on the taproot and were larger. Nevertheless, the results as shown in the two tables do not allow any clear-cut deductions. The plants of the first test (table 5) were examined three weeks after planting; the plants of the second test (table 6) were kept for four weeks, which probably accounts for the larger number of nodules on those plants. It appears certain that B. radicicola remained alive and retained its infecting power in practically all the substances for forty-six days. Car- borundum gave the poorest results. The plants inoculated with this substance developed only small nodules, mostly on the side roots — a fact pointing to subsequent infection. As regards the two series, much better inoculation was obtained from series 2 in both tests. In the first test 99 plants were inoculated with the cultures of series 1. These plants produced 191 nodules, which is an average of 1.9 nodules per plant. The 88 plants inoculated with the cultures from series 2 produced 417 nodules, which is an average of 4 7 nodules per plant. From similar calculations for the second test, it is found that the average number of nodules per plant was 9.2 in series 1, and 12 in series 2. In both tests the plants inoculated with the cultures of series 2 produced more nodules than those inoculated with the cultures of series 1. The drying of the substances in series 1 either reduced the infecting power of B. radicicola, or reduced the number of the organisms, or had both results. EXPERIMENT 11 INFLUENCE OF HYDROCHLORIC ACID, SODIUM HYDROXIDE, AND CANE SUGAR, IN VARYING CONCENTRATIONS In this experiment B. radicicola of alfalfa was isolated and identified according to the procedure described in Part I of this paper. The 42 Martin J. PRUCHA organism was propagated on media 334, 335, and 337 (page 11), to which were added various amounts of hydrochloric acid (HCl) and sodium hydroxide (NaOH). Cane sugar was added in various amounts to medium 334. Ten cubie centimeters of the media were introduced into each test tube and sterilized. While the agar was still melted the various additions were made to the tubes, and, after thorough mixing, the tubes were sloped. All cultures were made in duplicate. At the end of three weeks stain preparations were made from each slope for morphological study, carbol fuchsin being used for staining. The media employed and the results of the morphological study are given in table 7: TABLE 7. MorpHoLoGicaL VARIATION OF B. RADICICOLA ON THE DIFFERENT MEDIA Medium Morphological appearance Multiplication Medium 334, 10 ce. = Ee ETE rae Small ishorticellso sue 1 eee ee Fair Pee Lce N/A Hel aa Short rods; ‘slightly, stameds: = sacl seme Fair =e): SICCHIN, EL Olicgs sass Shortirodssshehtly staineduas a5 ene Poor Sy ole Orcem N/T ELC ae Short rods; shghbtly, stained] 4...) eee None ee aL Gy olos IN AR Ns (Oy Sse cis Short-rods\ slightly; stained = .cn.a-e arene None Sara recs Ni LaClis= =a Short rods: shiehtly stained ae cree renee None +. 3.0cc. N/1 HCl...... Short rods, slightly stained....:............ None +0 Ice. N/l NaOH. + .| ‘Short-rods,awell*stamed-.4.. 2.15-- eee Good 44 0.5.ec, N/i NaOH. ....) Short.rods; well stained’) 557 gee ee Good =— 1 0ec) N/t NaOH... 5 Shortirods* wellistaeden mess ee eee Poor => ce N/T NaOH... <|(Shortirods) well staimed= =). eee Poor + 2.0cc. N/1 NaQH....) Short rods, well stained, few small bacteroids..| Very poor + 3.0cc. N/1 NaOH....| Short rods, well stained, few small bacteroids. .| Doubtful + 0.1 per cent cane sugar| Cells vary in size, bacteroids present......... Good + 0.5 per cent cane sugar} Cells vary in size, bacteroids present......... Good + 1.0 per cent cane sugar} Cells vary in size, bacteroids present......... Good + 1.5 per cent cane sugar| Cells vary in size, bacteroids present......... Good + 2.0 per cent cane sugar} Cells vary in size, bacteroids present......... Good + 3.0 per cent cane sugar| Cells vary in size, bacteroids present......... Good + 4.0 per cent cane sugar| Cells vary in size, bacteroids more abundant. .| Good + 5.0 per cent cane sugar) Cells vary in size, bacteroids more abundant..) Good + 6.0 per cent cane sugar) Cells vary in size, bacteroids more abundant..| Fair + 8.0 per cent cane sugar) Cells vary in size, bacteroids very abundant. .| Fair + 10.0 per cent cane sugar.| Cells vary in size, bacteroids very abundant. .| Poor STupiEs oF Baciuuus RaApDIcICcOLA OF CANADA FIELD PEA 43 TABLE 7 (concluded) Medium Morphological appearance | Multiplication = | | Medium 335, 10 cc. Tee SRR SE RERDES fairs acl Variation in size and shape, few bacteroids....) Good = 0.1 ce: N/1 HCl....... Variation in size and shape, few bacteroids..... Good aOuonces N/T ELC f. . Wietyal CwaLOGsinmia te coe acpi e A Sala Gt oe | Poor leOrees N/le Clo. . i... INOforzanisms: visiblemanneeencaeaken. wales. Doubtful silences Nil EiClin. +5... INOzOLzanisms visiblew watson ee ee ae None eae ONGCCHIN iy ELC... 20 INGIOrZanismsnvisiblecepee a ee eae ee None Sore (eCCa IN Me@l.) Novorgantems visible. 1-2. < + wasp. me 5 | None + 0.1 ec. N/1 NaOH......)| Variation in size and shape, few bacteroids....| Very good + 0.5 ec. N/1 NaOH.....| Variation in size and shape, few bacteroids....| Very good +1.0cc. N/1 NaOH...... Greater variation, more bacteroids than in 0.5) Good +1.5ece. N/1 NaOH......) Greater variation, more bacteroids than in 0.5, Good + 2.0 cc. N/1 NaOH......| Greater variation, more bacteroids than in 0.5) Poor + 3.0cec. N/1 NaOH......| Cells stained deeper than others, bacteroids jS)RESCCLAN ee cot cattery Acre Bigs Gielen, emer orate | Doubtful Medium 337, 10 ce. SE (Uy Sea IGe eee Pronounced variation in shape and size of cells.; Good + 0.1 ce. N/T HIGH. shea. Small cells, irregular shape and size.......... Good +0.5cc. N/1 HCl....... Very slender cells, irregular shape and size....| Poor tT alOrec. Ni /ASTICK. . 2 8. extremely snrallicelisheee esse serie oer Very poor otmleosces N/A, HEI. . aN. ixtremelys smallicellst eos ecm. oe en eee Doubtful aoa Ovee. N/ IHC). . .. 2. bxtremely7smallicellsena eee 1. 2) ei ree None eta) ce: NL IC: 22. 22. INororganismspvisibless es. 2c emaeeee ee | None +0.lce. N/1 NaOH..... Shorusirreswlanicells, es see eee: Abundant + 0.5 cc. N/1 NaOH...... Shorteireesul arecellsieee ee Seer ae | Abundant +1.0cc. N/1 NaOH......| Shape and size extremely varied, many bac-) POLOIDS oe. Poste eC Sh RSE ORE ES Abundant +1.5cc. N/1 NaOH...... Large cells, varying in shape and size, many a At ene oe rhae Reo ek, i oe ena eet Good + 2.0 cc. N/1 NaOH...... Large cells, varying in shape and size, many DAGULETOIUS.8 atin oc Ae er acres See pose Good + 3.0cc. N/1 NaOH....:.| Very large cells, varying in shape and size, IM ATVs DACECEOICS rn: Selmer rn are acer va Poor In order to test the infecting power of the different cultures, three tlowerpots of alfalfa plants grown in sterile soil were imoculated with the organism from each test tube. plants were examined for nodule development. Four weeks after inoculation the The results of the in- oculations are présented in table 8. Fifty-eight plants in flowerpots These plants were grown in sterile soil and were not inoculated. The control plants were distributed among the other were used as controls. 44 Martin J. PRucHA plants in order to see to what extent an infection from neighboring flower-_ pots may take place. TABLE 8. _ Inrectinc Power or Various CuLtures. B. RADICICOLA WAS PROPAGATED FOR THREE WEEKS ON THE DIFFERENT MEDIA Number Total Number of number of Medium plants of nodules inoculated | nodules | per plant Medium 334, 10 ce. te TR CE bade gsi Una ats. LOR ee ae i Oe ME 42 265 6.3 a OL Meer NT CHCIS, 2352.5 3 ee ee eee See 50 320 6.4 — =toy Oe nC CERIN LEN Gly 5h ec 2 SRS er ere eRe 43 188 4.3 EL OicesiNy/ UoCHs. 23h sue. Ste crete eae cere ere 32 0 Oa =f iL 85). GO> IN TAI @ Sis neck concep eens ee Soe ee 38 6 0.2 ol SePAL ON clr aN) ial s ©) Leama RE ryt EN eRe Bay Vm: 53 22 044 =m st OKeGepNI ELC letras, steerer minrs seer e aMenN ns Buea 35 7 0.2 = ae Ue en “het aE ee ar Mt ROA Oren A ey Ar ie 37 184 5.09 etme Neck aNy A. Nia © Elen x mace 2 areata are 43 275 6.4 et ON ONCOMING (1 NAO Eo ra te) cease Oni pee (een 48 234 (4.9 — =e alvOcewN/ 1 NaOH. Sea Oak Sek a ee aes 47 304 6.5 Ste sorcer NL INGOHE Se ees oe eee een em 34 168 4.9 Stee ence INU Na Oie ® 50s ee es Eee 20 94 3.5 a OM OLECING EIN AO cts crs sate etal meee a Rene ah 75 «2S =~ a0sleperscenticane SUtaATs.. ee Fe eee eee eee 46 182 4.0 — = SOO uperscenticane Sugar! a eee aes 35 124 i505) =) wl#Oiperscenticanessugars:.% 50.0 dun: ome ee 32 117 3a ==) le Ssperscent Cane SUPAr. 4.0 ee ee eee 48 198 4.1 nl) PEMD SKE o Go ooadhoonatcasscacdscae 38 140 3.7 —- SouOhper (cent; Cane SURAT. a. 02 pata eee 29 125 4.3 -1-)s440) percent cane suparves. «seemless tiene ee 35 138 39°49 =1- O80) Denycentecane Suc ares |e era e en res en eee ne 33 132 4.0 S80) Percent. Cane SUPAL= He Hiei a nici ease 33 110 3.3 ={=l OROMperacentacanessugarsac. oe. aeeeaae eenoe nee 30 85 2.8 Medium 335, 10 ce. SPS IO Sorte Roan ioe occa toa th ctivntor de antler a 29 132 4.6 — =fe a oCe: INL EOL 4s) Ske ir, ees near ae 34 183 5.4 st 0)5 CoN AGE Cl sitions ike: Woe eee se ee 50 6 0.1 sole Occe. NiCr om eae Bees Shree sea 68 0 0s =jMe eco INT ECT: ce. oc Le = ee te eee 50 22 0.4 — sfx OiCes N/M ELC) neve a. og ae eons ie 56 5 0.1 Stores OLCOY SIN / Lee Ole nae, | peed Pee acer tee On 58 4 Osi =O miee IN le Nia O Hie 05.02 ed 2 ee a 33 186 5.64 re MOPAR CALING / Ls IN OIE 35 ree steht ds era aa 32 131 4.154 Stel OC esa NG 1) INI ODS 531: sorter ste cect ate et 30 212 (ee! rb oreee NYU NSOR Ee 0edy SE ee 36 284 7.99 Sere Ole IN ALSNGOL 4 shoes eel eiin.cunntaciee ie ee ne 30 180 6.09 =f AD CG. IN /JLSINGOEN 5 oot vir cho ney ee ee 21 109 5.2 9 aes es SFR? STUDIES OF BaciLLUS RapicicoLA OF CANADA FIELD PEA 45 TABLE 8 (concluded) Number Total Number of number of Medium plants of nodules inoculated | nodules | per plant Medium 337, 10 ce. als (UL Se sinaast tie ae BA a ee ae ae eee 23 54 Die =U) aL COMING ELNINO Der rce ops cg 3 octs un co are tise, Souk besttavetnvee 32 50 1.6 STM ORCC HON OIMEN Cline weet RBS tice oar seattle, slats ey esos 37 110 3.0 = mln RCOMOING AMEN [Ette) ye eeeetde ot ee Pe EO 36 dl Deal SEI! 2 ByiCereR INIA" 8 | ©) Ea eae PPA ST bec OR Oo iencate ewer 36 23 0.6 aks BP. (EO MINT 1c 13k ©) [ROR itean en eaten seen eee aan et rw 33 94 2.8 ROM ONCCRONG A Clastge sack setts myneteen eee ie iS 32 132 41 SEU CCHRUING cH IN LOL tet a5 0! atch teveng cee w lov love acoteraee oe destinuade se 26 80 3.1 = OORCCE ON PMN AOS, oe on, Mikes. face athe wes oe avons 27 120 4.4 ee ORCCHING MINA OTHE ens ol. pam see ee eed 39 174 4.5 ALA TT 15} GCOS INI/AILSIN ENO} 8 Fear as ae are are nan ee Rn ene 31 114 Sei) Seo ONCOMING MIPUN A OUN. cesa s/c .as @ute sobs s send ccuein Udi te ae 32 154 4.8 Fron ORCC IN ANA OEDs au ceeae. | ent aedt 2s SE | oe 33 87 2.6 “Charnes or aac eae gh Sa a OS | 735 *6§24 0.8 * Nodules were present in only thirteen out of fifty-eight pots Results The extent of multiplication and the description of the morphological characters of the organisms in the various media are summarized in table7. No visible increase in the number of organisms was found when 1 cubic centimeter or more of the normal solution of HCl was added to 10 cubic centimeters of each medium. The few organisms found on these slopes were probably those that were introduced when the slopes were inoculated. In medium 337 slight multiplication took place on slopes of 10 cubic centimeters of the medium plus 1 cubic centimeter of the normal solution of HCl. The best multiplication occurred in media 335 and 337 when 0.1 to 1 cubic centimeter of the normal solution of NaOH was added to 10 cubic centimeters of the medium. This was particularly noticeable in medium 337. The addition of 3 cubic centimeters of the normal solu- tion of NaOH to 10 cubic centimeters of the medium practically inhibited multiplication. The bacteroids developed more readily when NaOH was added. 46 Martin J. PRUCHA The addition of sugar to medium 334, up to 10 per cent, caused more rapid multiplication and also the development of bacteroids. In medium 334 multiplication of the organisms was very slow and only a few bacteroids developed in the three weeks. In medium 337 multipli- cation was abundant and bacteroids developed. The results of inoculation are given in table 8. There were two hundred and eight flowerpots, which were crowded together because of lack of space. Fifty-eight flowerpots contained control plants, which were scattered among the inoculated pots. The plants in forty-five of the control pots developed no nodules; thirteen of the control pots were contaminated, and these were located mostly among the flowerpots inoculated with the organisms grown on medium 337. This contamination occurred in spite of the precautions taken to prevent the organisms from being carried from one flowerpot to another when the plants were watered. In these experiments and in others not reported in this paper, it was found that when a large number of flowerpots were used at one time it was difficult to prevent infection from other sources than that of the inoculating material. This was particularly true in the case of those legumes that produce an abundance of nodules in the soil of this region, and when the plants were allowed to grow for longer periods than three weeks. The results in this experiment are marred by a certain amount of con- tamination. The data in table 8 are so arranged as to show the total number of nodules on all the plants inoculated with the same material, and also to show the average number of nodules to each of these plants. Basing the conclusions on the mere number of nodules overemphasizes: the importance of the contamination. When the plants were examined, and the size, location, and evenness of distribution of the nodules among the plants in the same flowerpot were noted, in addition to their total number, much more reliable evidence was obtained. In view of this, the following conclusions seemed to be warranted: When B. radicicola of alfalfa is propagated on media 334, 335, and 337 and kept for three weeks, multiplication of the organism takes place and the infecting power is not lost. Many | Many 2.0 per cent dextrose, hi SUES Oe saree aes Many | Many Few Oper centlevuloseker: jntaate ut. gustan Site hier aoe None None None AiO yoysiecersy sure ENG oe ey Caeteeiidicts Qa Toa od oIIOl mr OloIe d\os0.0Lc Many | Many | Many Qa uper cent) eHlactose fees ae oe sc clas Sasa arise wae Many | Many | Many LOsOmmer centicane SUgaR + 6. jovi. tablets cote eee eres Many | Many Few PO EOnpericent Cane SUGAR ai ek nn nese ewan hee een Few Few Few SOnOtper icent «cane sugars... << ¢in .ing: facac ere ane Few Few Many ANBOperveent/ cane. sugar: f. jsxy «sch ek eee ee Ee aera Few Few Few WO per cenh, SACI: fo) ciicpa cele ete peel eee RE ence Many | Many | None 0.5 per cent amygdalin../... 2.20. 6/ 20.020. 2 eee Many Few Few OrdipercentimesOrclan shook: nae lta ones ee melee eer eta None | None Few (e2oper cenh palorOglucinin. tape esta lat ett erates [a ew, Few Few 0.5 per cent potassium oxalate...........0...... 6.0. eee. | None None Non2 0.5 per cent potassium citrate... 1.2.0.0... 0... sees eee. None None None Oplepen cent pocassiumenitrate a. 46 on ase aati eerie eer | Many | Many | Many O52 per cent potassium: nitrate: 2.2 se Gee oer eee | Many | Many Few . O86 percent potassium nitratey 6) go/s teria eee ene Few Few Few | Omlepertcent cal ctuminitraten sees eevee er iteneciee Many | Many Few { O22 ipertcenticalchum nitrate sss ts eer warreracre oeian a eet Many | Many Few OvGipericenticalenmmitratesia. mer anaes nors eee) terete Many | Many | Many iO; percent Wittels peptone a. cesta: sae eres ee ee Many | Many | Many | 2. OpervcentWatte’s"peptone ect... 2 ocr esriets bhatt Few Few None 5 O:per cent, Witte’s| peptone 2... Geo isis eee et None None None | O}2 percent; Merck's peptone s=.2.-6 te ce Many | Many | Many | 1.0)per’ cent Merckis;peptone.-4)4 56.05 2s ete ener Many | Many | Many 3.0 per cent Merck’s peptone staal e SNA eitoath Siesta eet ase PO g A O|_g 2.4 cn pe So & 5 oO)" 2, 2H Gs seo sella ~ mA Seer 2/92 jor Be @! ret £5 ks BE | ys | as | ao] oa | a | een eee ene ee £8 ss Sa |8/)8|8|8 18 | $8 |s3eleae 4 a 4 BR RI | A|A |< < 6, Medium 334 + 20 per cent 10 8 ] Si) 2S 22) VASA cane sugar PD) 5) rol Se BO 26) (on2| oe 12). $4) 0 NOt Ah e Olga Si 6 Medium 334 + 40 per cent PS LT KBE OSes 33|° 82 ; . cane sugar A TS lees S12 | ae Sel |G 54 7 13 so) aoe 15) 8) sal. Ble 8G a3l0 Oo Medium 334+ 2 per cent 16, O| 14 15) 10 9 ASS "OR6 dextrose AGQE soeli() | fel |e eerie 43) OR A terse 18 4 8 4 Ginreae 22 5.5 Medium 334+ 2 per cent) HE Os SINE ESI ee g =2.2 lactose PAD 2845 SSL 90) AL -S} 9)< LS heel 21) 0) 6) 5h 0) 0. Li eae Medium 334+ 2 per cent| 22; 5| si 7/....1....| 201. 6.7 galactose 23) LO ANG Ae A> 10.2 eon 2A 8) -2, 7910s eae 56) 11.2 Medium 334-+ 2 per cent 28; 3/ 2) 16) 14 14 49-98 glycerin 29). 15s 85) 20/7 210 60} 15.0} 12.1 BUS rar alesse Eamets ae {tha ani Weck | Medium 334 + 0.5 per cent 31} 0| O--o| Of Ol Q) >. 0 potassium oxalate 32a | ae heed Al Se 9 22| ORG | dale Ol 20) = Oley 0) aaa 0} 0 Medium 334 + 0.2 per cent, So) Sole oe te Sree 127: phloroglucin 30}. OF «8OF 6}. CO 0} 0 2.2 36, £) Spee bieads 26), 5.2 Medium 334 + 0.5 per cent) Ale tO lee tO Bie eae 23) ond resorcin 38] 0) i) 9 40 eel eee 11) 2 Det 2a 39}. <0) 270) \cOe a0 aaa 0) 0 Medium 334+1 per cent} 40 0| 2) 4| O| 3 Oras salicin Aa AO | ee Ole Oieear el ealie Zi Oo 42} 0} 2}. 0} 4) 6]. 1.5 Medium 334 + 0.5 per cent 43| 0]. 8) 14) 229/06 40; 8.0 amygdalin 44, 10) 15) 24) 15) 8 72| 14.4) 12.0 Jo 451 14) 20) 00). es |) al iad dae Laboratory number of culture —_ 19) StuDIES oF Bacttuus RapicicoLtaA oF CANADA FIELD PEA Medium in which B. radi- cicola was propagated Medium 334 + 0.1 per asparagin Medium 334 + 0.1 per potassium nitrate Medium 334 + 0.2 per potassium nitrate Medium 334 + 0.6 per potassium nitrate Medium 334 + 0.1 per calcium nitrate Medium 334 + 0.2 per calcium nitrate Medium 334 + 0.6 per calcium nitrate Medium 334 + 0.5 per potassium citrate Medium 334 + 0.2 per Merck’s peptone Medium 334 + 1 per Merck’s peptone Medium 334+ 1 per Witte’s peptone cent cent cent cent cent cent cent cent cent cent 55 TABLE 12 (continued) One| eth & wd lessee 5 rae 3 jas |RaS 2 o O's, ise ors lous 2 g 2g tee ne gq 3 aS s $5 |e |SSo A sao 5B me 2 ed $ A oS = eared Po sV jan,0/e05 oe ag eats SOs ce [pos lt | 15. |e Ss. l'on o'eo as ~ ~ +» ~ ~ fd So 5) co 8 Sa/ai/a|)s| a) s| 58 |$sa\sss 4 Pla |/e/al|a}a ja < 46| 16 10! 6] 18|. 10|- 60| 12.0 wn 10 95). 8-10] 2. |= 83.8.2) 8:8 48} 6 oO! 12] 2 10} 30/ 6.0 ADE 793) AQ) ae] 015| ee 47,70. 4 Saas 5h 6) Olen0|-.- 23h 4-6)" 659 Bil iged5|) 20 OF S|) 33h) 16.6 521 2) 2| 6 9] 10- 29| 5.8 Ga)eel PS) «Sled Ns 30.6.0) 5:8 Ai OL go] 1k 8} tol, ~ 21) 4.2 55| “10! 16) 10) 18] . 21" . 56) 11.2 56| 13| 16] 2) 30)....| 61] 15.2| 10.6 B10. 0) Ble ESI AD. Sila 6.2 5S maliot 2, 528 65 83h, Bb 7h FO: Ogi Biel 5.2 66, O| O| 6 10) 5] 21) 4.2 Geordie wal are ee Cioltors 63 Os OOF --0! 0 0 o| 0.7 Ol. Ole Ol Ole =0).. 0 0 Ohies Tl Al 27. Bl Pa a 98 9 Beal HAO: Oh Spe sles he 1D a8 CON 4 aed 72° Ole Ol: Bla. Fl WO 28) G44 73\ Olea Blt ag| seta. Silo .16s2 TAN DOs. hoe o © Je) 4 Cn sep fe -oid yuvjd Jed sain a = ac} o o ts AN au ° o 0 -pou jo Joquinu asvioaAy jodiemog [NOW N [one [oox pre re, TaN ny | One eee yore ul que|d Jod soyn mNANe ono ee Ds oONo ooo oD S2 CO ONS oto (<> j[—=}[—=) arvae) Om - -pou jo Jaquinu osei0Ay COD |/SODSOl/AND [MON [SOO l/ADSO (RCO lHMOl|OOn |] OMD o jyodsaMoy yore ut ae Ons SH | ro eae | aS sajnpou jo Jequinu |ej07, ; ia =i nl a i od ll SK ee — I Sooo SonlooSn Sma smKelco ° oD iN . re cl N ¢ yueld : : HOS | OHO |] BOO (MES [COD [ANDI DANO [OOO lLOOOIANAM! Om uR Ome re boo oe oe | ret F PUB | guejd yore SMD, OOO ]/SOMn [Ono /OCOSOlTNOMIAROC!]ORO]OOO [OND OO uo saynpou € que[g ripe | o 19quin = ers ee ail | 3 4 N AMM | OOO |HOO 1/OOO|/SCOO /SOSCSO / NOC TOSCO l/OOn | OCOD LOM re re mN re gaurd | | SNS [ONO [ONO ISON [SOOO [OMNMILNOO [HOO lOOOl/NOFT | OO ri ee hem | re Se) T Jueld Ont |NOtH | mOn | DOS! HAM | HHS | RDS SaN|/ea2|/eS52/20e70 sjodiamo MOO | DOD | DDD | DDH | ADD 1 AAD | Aah oo 1|S00/S00/] On B Soe Bh oe oo | Soe HE ee I oo | Soe I oe Bh | See jo sequinu A10ye10qGeR'T : Ss, ees 2 ; ee) =| =| =| mn 3) o ® S | Co Co Co fo} 5 Ld & oF oF Py 3 S| 8 S 2 3 mae i= | 2 ue 2 19> Ep © Gi | paye3edoid sem 070919 are se == “o a s Bs po. g YoryMurwunipayy | SE | se | x ec Cale S Zi = tA oo ar) z > a a B 5 C} = = a = op) | 2 @) 3) op) '@) oO S fa =e eee) = Pica oF ~ 1D Wo) = rove) fan) aanyyno A | oO on or) | oD se) | oF on | on oD jo Joequinu £10}B10q8T | 37| StrupiEs oF Bacrittus RApDICcCICOLA OF CANADA FIELD PEA TABLE 12 (concluded) hi “J 5 sy a Sa E si 5 2 A xe Sa Bis a8 £5 gs as 33 Crs nai ge ae od 4 = 40, Wheat middlings 41) Compost | 42| Partly decomposed cow feces 43) Fresh cow feces 44) Muck | 45 Sandy soil 46, Controls 47 Controls © S84 hea Hn SS o PES 2, FE 23% = Os [=| * Sad = “ ge asf - N oD = sé/al/aals Sa ia|a| se] s | 4 Ay Ay AY false | 112} 6 18] 20) 4! 113| 20; 12} 5} 6 1148 Ole ez |" 23 115) 16 3| D0 116) “12|- ~ 4) 15|...°8 117} 10| 14] 12) 16 118) 5 6 7 5 119) 6 3 14 8 120) 8} 6 3 4 i} of oF 7 oO DI ee ees Fl et 0 ile ed 123} 20/ 10] 12) 12 124, 101 8] 10) 12 125) 265 Gl. Sl 9 126), 3), 12) i883 197|\ 22a rG) 0) 128] 10). ©O 8) . 4 129 Hebe) xian le 4 130} 0). 0.39 Giexs0) 131] 0 0 Oo O 132] 0} 3 0 0 133} oO] 0. arc 134) U0 GO. | 20k 20), 135)'> Ol Oy 10h uO aoe Total number of nodules | in each flowerpot Average number of nod- ules per plant in each flowerpot Average number of nod-| plant pro-) duced by the culture ules per 9.8 The average numbers of nodules per plant for all the plants inoculated with each of the given cultures appear in the last column in table 12. In table 13 these numbers are rearranged in numerical order according to the average number of nodules per plant produced by each culture: 58 Martin J. PRucHA TABLE 13. Average NuMBER oF NopULES PER PLANT IN SECOND TEST, ARRANGED IN NUMERICAL ORDER Medium Medium 334 + 1 Medium'334:--: 5) per centigelatim=.. 5.0572 2h. . 5. ee eee Medium 334 + 0.2 per cent calcium nitrate...................... Mediumiss4-|-2 percent se lycenni eam ee eeint ee ee ere Medium 334 + 0.5 per cent amygdalin......................--+: Medium 334 + 0.6 per cent potassium nitrate.................... Medium 334 -- 10 per cent cane sugar.....-....<2..-.5----++--s+6 | Cornstarc hls 38sec scchiksce eee ee DU OE ee per cent Witte’s peptone........5..5.52..4-2624. Pp Wiheat middling); 3: suet eee eee 1M Ki) ee Re area Meee Lees KOU naa hts Avi bige ina bia o.oo 6 Canada field peasrootsiics «8 Lhe. sh am he en rtacaaes ead d area aoe ee | Medium 334 + 0.1 per cent potassium nitrate.................... Medium 334 + 0.1 per cent calcium nitrate...................... Medium 334 + 1 per cent Merck’s peptone...................... Medium 334 + 0.2 per cent potassium nitrate.................... Me a a Z a St tee 36 & oie) ~ 53° as ake & § a & as Dus Ole: 3 5 Sq | a 0 H Ye) Re) ~ a Sit ep 3 33 Seige lale le le \el se] ss |es) s4 a oF cape | sto lest, Pest icaets |e tera et | 6. |e Pe | Be 4 | a 4 Bla |e RR |e |e | & <—e| 1/ Medium 334 +2 per, il 10) GIA “Bt. Pai 31) 6.2 cent cane sugar.... 2 i) 0 0 4' 10 2.0 4.5 3| 8 14 0 (0} eee Ae a 22 5.5 — |—— —— | 3) Medium 334 + 2 per AN SO Tale 6154" ONee o 30] 7.5 cent cane sugar 5 6 1 0 (De ec allo ace 7 Ue) 4.8 Be 8) = Cie SOT SB) Sh: fo alee 25, 5.0 4, Medium 334 + 10 per a) 0 0| Oo (0,1 Vek ectame ee aed Petar ee [Oi cent cane sugar. ... SOS OR Ole OER 5 Se 823i 7 Sel + 9| 0 0 0. 0 Oye hate etl nee ey are cllls. casters 5 Medium 334 + 10 per FO EROPS. ABI slim Bee hina 15/327 cent cane sugar 11 0 0. Oo; 60 OWS 0 0 1.4 12 0 0 3 0, 3 Ole ee 6 1.0 6 Medium 334 + 20 per DS) S0|) Ole Ol “Ole 40} 0} 0 cent cane sugar 14 0 0 Ol Gise 77 13 2.6 3.0 06 SoBe) So Sli elt 2 | 26] 8.7 7, Medium 334 + 40 per 16| 0). ‘Ol Ok> O]° OL) Oo. Oo 6} 0 cent cane sugar 17 0 0 0 0. P 0 0 0 18 0 0 CP esdcsH None een | melita] (eo a 0 0 8| Medium 334 +2 per 19 8 4 4 0}. Salle 16 .0 cent dextrose 20 0 6 6 8 hs 20 5.0 4.4 QU A. Siemeciatscieiltneladtemeerd le ae 4 .0 9| Medium 334 +2 per 22 a 4; 12 6 8 Olenc 35 5.8 cent lactose 23 8 6 0 0 4 0 3) 21 3.0 §.3 2a 8) 40) E162 10/2 eerie: Aa Bb 10| Medium 334 +2 per| 25 12) 10, 6] 16)....1........) 44] 11.0 cent galactose 26 5 Al 15 6 0], 5 8 43 6.1 5.9 Zi oOF SObeOlers aly Tas a ld|ae273 11| Medium 334 +2 per| 28| 10] 1{ Oj] 10) 14: 7|.. 42) 7.0 cent mannite 29) 12 5 2 8) Dieters 29 5.8 7.6 30} 16 6217 a 1 ee aa Re) [Ee 36) 12.0 12) Medium 334 +2 per Sie oleeS eels vinmo eos 13 58] 8.3 cent glycerin 32 Sit 4s Vole 15 16)" 10 8 SOE t2s3|eelOeD 33 8 9 7 11 ils) (ms IC: 4 eee (G¥ilee shhey 13) Medium 334 + 0.5 per aa @ OO) Oia ssalyoe | Ol, 20 cent potassium oxa- 35 0 0 0) Oe) 0}. 0 0 0.3 late = Sal 50) = Ol— Olas Gee ele oo Aleons Bh 1s 14) Medium 334 + 0.2 per 37 0 0 0 0, 0 0}. 0 cent phloroglucin oS 0 0 Sita 16h: sacle es | 18 3.6 1.6 avcuetsl|fetevctatltenenceey|ice cle Sve cko eal Sle iostalio ceupians | Po eirsoucis 15| Medium 334 + 0.5 per £0\ OK FO) Ole Oe eaee ot eet: | 0} 0 cent resorcin 41 0 0 0 0 OVA ales 0 0 0 42 0; 0 0 0 ORs Salton 0 0 16] Medium 334 +1 per 43/= Sol? 0) Ok Ol Om Ol. 0) 60 cent salicin 44 OE er eee eal od oe alle rR () en) 0 45 0 0 OU sae icrsvlioeers 0 0 intro- of organisms duced into each flowerpot Number 324,000 324,000 324,000 259,200 259 , 200 259 , 200 90 ,000 90 , 000 90 ,000 52,000 52,000 52,000 2,000,000 20,000 20,000 ecoolicooco!looo!ooo 64 Martin J. PRucHA TABLE 15 (continued) ere E 2 72 Ey 3 & g 3 2 | 8. | Ba | ee 3 | 3 3 ge Slee eae B oe | = 3 ue) 35 asl n Pa 3 | : cS sa = of | od BE be isa) & 25 a = ae 2G ts abe, g PaaS) ‘S Weis) 6a Re I 29 g 3s reve a SI Pre = aS 3} istite 3 fa Hey ey eect [ISS O68 a a A 3° | 28 | ae | a BG) B Ag B é Sees = ° e) “= Q oad 2 g 5 Bi aes Prieae |cset (6 esl aa lie oe a Se | Se sy . 38 ss iei/e/8/8/8/8)|8) 88) G8) 6e] Ba ah oe ai er See ecm teliee cea Ten ae il ice =. ae 4 a 4 Be a |e la] a | | ee < <4 Zz 17) Medium 334 +0.5 per AGI EEO hrs O00 en 10 ee [pee 0| oO 0 cent amygdalin 47 0 0 OF O} VOle 0 0 0 0 23)" 0| 's 30/0 Olaaael acon etee io 0 0 0 a Peete 18) Medium 334 + 0.1 per 49 Ol) LO Sade s Ul Oe 63/7 10e5 10,800,000 cent asparagin 50 6) Ss ee e160 Gl Reale 49 9.8} 10.0 108,000 51 8 OV LGE si2\5 rs AT rom 58 9.7 108,000 19} Medium 334 + 0.1 per D2) W2ale eka 6 CO heel leis lo Siac 48} 12.0 7,200,000 cent potassium 53 0 8) 10 Ol, 26% SeRAR 44 8.8 9.5 72,000 nitrate 54; 20) 12) 10 4 4 er 50 8.3 72 ,COO 20) Medium 334 + 0.2 per 55 3 5 SielOleeao ollee 29 4.8 224,000 cent potassium 56 5 5 0 0 ease 10 Pha 15) 3.9 224 ,000 nitrate 57|... 012+. ae ae SECERE aA pes 9 [a bl | gra 224,000 21| Medium 334 +0.6 per) 58; 0! 10/ 12 4| 14! O.... 40| 6.7 14,400,000 cent potassium 59] 14: Bl cD sees ercllhcgecces |e 29 9.7 6.7 144 ,000 nitrate 60 6; 10 0, ZliFatoe eee lla 18 4.5 144 ,000 22) Medium 334+ 0.1 per 61 12) 12 Sipe, Co) teas | (et sree 40. 8.0 48, 600 ,000 cent calcium nitrate 62 2 1 7 9 5 Siena 27 4.5 Gaia 486 ,000 63] 8 4; 16 0 SP Taree 47 7.8 486 ,000 23| Medium 334 + 0.2 per 64 2 1 Slen Fay, Vela Gee 53, 8.8 10,800,000 cent calcium nitrate 65| 20 8 5 6} 16} 18 6 C9 lds 8.6 , 000 66, 5 3 6} 14 3 8 1 40, Se 108 ,000 24 Medium 334 +0.6 per| 67; 6| 10/ 98] 11l....|....|.... 356 8.7 13,000,000 cent calcium nitrate 68 3 7 8 Tia 7 8 53 7.6 9.2 130 , 000 69" 13) 6! '8) 0) 1G | a2 ee eee tomes 130,000 25\ Medium 334 +0.5per| 70 12| 12] 16] 4! 6j....|....] 50) 10.0 300 , 000 cent potassium TW TABI Sli E20 peas ion |e 5OV) 12 95| Geom 3,000 citrate 72 1 0 Sie UWers a3 earns 28) 5.6 , 000 26 Medium 334 + 0.2 per 73 1 0 Uh 125) 10 OW As 24 4.0 800 , 000 cent Merck’s pep- 74 3 Sie 2h sD vere eee eee 32, 8.0 5.9 , 000 tone Tones 0 Ole te BGO eee 38, 56} 8,000 27, Medium 334 +1 per 76 2 ANS LO) Lae eas eee 30| Tao 32,400,000 cent Merck’s pep- Etim 10 Weed 2 ens eS mee Alera eee 59) Deak Ste) 9.5 324,000 tone hsv ae), NS) OR eee ieee 25' 88.3 324,000 28! Medium 334 -+1 perl 79] 3] 7] 3! Ol S/o 2 18 Sse 21,600,000 cent Witte’s peptone 80 4). 32) 10 2) V4) 15) 220 87, 12.4 9.4 216,000 81) 12 Sia Gieer eh | Pen | iris ay 36) 2-0 - 216,000 29 Medium 334 +2 per 82). 12 4 4| 10}.. Seve ne Ae 30 hes) 64, 800,000 cent Witte’s peptone 83 4 0; 12 to epg (Eigen [eco le 24 6.0 7.9 648 , 000 84, 10) 14 Gl eaalllares le eal eet BEI o salt (0) 648 , 000 30) Medium 334 +5 per 85 0 0 0 2 0 Olen 2 0.3 (6) cent Witte’s peptone 86 OH 0} 0 0 0 Olen 0 0 O22 0 87 0} 0 0 0 0 0 0 0 0 0 31| Medium 334 +5 per 88 3 2 2} 10 ieee ee 30 5.0 6,480,009 cent gelatin 89)/) 5) S| aie Glee viene Be 36] 6.0] 6.6 64,800 | 90): - 9! 18) a2). cae able eles 46 9.2 64,800 STUDIES OF BAcILLUS RADICICOLA OF CANADA FIELD PEA TABLE 15 (concluded) 65 ro) 8 f 7 mn a & a) ' 5 S$ 5 = A a, 25 & 3 is é 3 eis |ss| #3 ° = = ag 3 | Be =s° Pp. Fo S oo 3 Lo} Lee} cS bro s 5 3 4 Ol | 3. + 0 | oO] & a | WE we 3 > 2 33 See lcotrarladoleaalad \acgled lod ee | oe lobe | og | 32) Soy bean roots, ground 91 0; 0 Olee MOl ace lle Oi 0 0) S2MSO EEO al es Ole cles 0 0 ) 0 | CA a) Ol Or We ea) Sol o| 0 0 SS. ——.|§ | ———— |} ———_ ] ——_ | ——_ | ———. | _ 33 Soy bean hay, ground 94 0 o| oO 0) Ole 0 0 0 95 COV Ca}! on (OVI a) eee ce 0 0 ) 0 | GIS OF Gl Ol) OlesJ0l) Olaee | 0) 0 ee eae SE EL ESS S$) SS SS ) | |] | 34, Canada field pea roots, 97 0 0) 0 0 0 0 0 0, 0) 0 ground SS ie ORO) 10 Se Reegircetalee. 3 o| oO 0 0 99} 0] Oo] Of Oj. fe | fee Or 6 0 35| Canada field pea hay, 100 0 0 Ol erence cesta ces | 0) 0 0 | ground 107} “| 0} 0 | alee 0 0 | 1 SO Or Gi Oy Or Oke: 0 0 marl | | ae! Pee Feat pa cee eae eee: ee ee ae SGieSawdusti.. ssc: 4 SOG a ee hs | see ote be {aera 15) 15.0 350,000 10D Sg Ol es5lh eas 8 See 26 5.2 6.6 3,500 | NOS babe sl 52: asze| sock eaealeees anes 5 0 500 | ESS eet | eh eae |S el aes eee | es a7) (Cormmmeal......./..) 106] 6] 10| af 16)....|....|....| 40| 10.0 9,700,000 107 E 1 2) Sir ee 14 37 6.2 7.5 pene | TOS helo ler 8 ee Sie ll seen 35 0 ,000 et aed eee | pa i eee eae 38] Cornstarch.......... 109) 12 ts PUA ee ee et eal Ve Se lies bende Sol) 0 9,700 ,000 TO SPS Gloria eeeals nec sal fh Shay | ae )ea 97,000 ALT eS a7 | 1 | erase ere ae, 20 eaeoue7 | 97 ,000 39| Wheat bran......... 112 fea USN LAO | ia reces erat oh eteneee tas 29 9.7 60,000 , 000 Dea ely 2) OPPS lta eats 54 33 8.2 9.8 po, 000 1A EA ENOO| ST One tiles ke sete es 6| 12.0 ,00 — eo eee ee i | —_—_— SSS 40| Wheat middlings..... Vis SON Ol eo lbatO|beesie =| 42| 10.5) | 42,000,000 LG f US| ele |e Ll esselgeliae salen) 43| 14.3] 12.0) 420,00 a : 117] 12| 10) 13). se ae Se eet | AAT 420,000 Atl Compost. isc. 6... Ais) sO cota le eB, Ol a Ntesbot an 34] 8.5 12,000,000 | PPO [Se Bis hr Slike Petersen Glee 16 5.3 73 jes ieh HO one Osten 0) ee elle ieee 23 iy 20, 42; Partly decomposed| 121, 5| 4] 5)....]....].-..|.... 14, 4.7 324,000,000 cow feces 122) SGI 121-17 eal Gla oe oe 64 10.7 Sas eee 125 )SG| eeOlerTS|-e12| Zl ne. i ,240, 45| Fresh cow feces...... ALO ee 121 Seas aealloeepewe| ae ile fae 44) 11.0 81,000 , 000 A | Ofc Gh) a SN ee | Bill] yeh erat! eee 1G Ree soon Sa es oes | cea ete A cae He eA Fg AO ‘ — | ——— | ——_ | —_ |__| —— ee 127 eel Hh Ol Ge Ole aleee ee | 4} 1.0 24,000,000 128/56 28/2812] 4 HO aOR sale lpae | 20 5 0 3.5 a ae LZQ| ok AN SRW 77 ens | ene val a [eee 1 4. 3 = ee el Pe ee pera eieaes eS 45| Sandy soil........... 130\Goi| 5-40 merle Oe al ohoatee 44| 11.0 2,000,000 131 I e714 1G | en | eae | ella AD\pn2)2) 21263 20,000 EE a Sy aA as aril oe all are 42] 14.0 20,000 66 Martin J. PRUCHA In this test a known number of organisms was introduced into each flowerpot at the time of inoculation, in order to trace the relation between the number of nodules developed and the number of organisms used for~ inoculation. It was hoped that in this way some means might be found | of measuring not only the loss, but also the degree, of infecting power. | From the data in table 15 it is seen that absolutely no relation exists” between the number of organisms introduced into the flowerpots and the | number of nodules that developed, so that the only measure of infecting | power is the presence or the absence of nodules. This, however, does not | measure the degree of infecting power, but only its presence or absence. The average number of nodules per plant produced by each culture, as | shown in table 15, are rearranged in numerical order in table 16. It is found on comparing tables 13 and 16 that the results of the third test of infecting power showa general agreement with the results of the second test. — If it is considered that the presence of three or more nodules per plant indicates that the culture was efficient, the following cultures are seen to — have lost their efficiency: > Soy bean hay Soy bean roots -Canada field pea hay Canada field pea roots Medium 334 + 10 per cent cane sugar Medium 334 + 40 per cent cane sugar : Medium 334 + 0.2 per cent phloroglucin Medium 334 + 1 per cent salicin Medium 334 + 0.5 per cent amygdalin Medium 334 + 0.5 per cent resorcin a Medium 334 + 0.5 per cent potassium oxalate om Medium 334 + 5 per cent Witte’s peptone The cultures that had any living organisms at the time of inoculation — produced nodules practically in all cases in the three tests. The cultures in which no living organisms were found produced no nodules, or only a few | unevenly distributed. It must be remembered that a certain amount of — contamination may occur, and that the method of determining the presence of living organisms by plate cultures is not absolutely accurate. The cul- tures in this test were ten weeks older than those in the second test. Th results show that the nodule-bacteria cultures can be kept for at lea twenty weeks, and the bacteria will still be efficient in producing nodules | UE SEB spieit oe gat hee) ae eg TEEPE ELS Sa, 2 eo ge gn Medium 334 + 0.1 per cent calcium) nitrate....:4.............: Medium 334 + 0.6 per cent potassium nitrate.................. Medium 334 + 5 per cent gelatin............................| ENTS fe Sac 4 2 Aaa OR ec Se Medium 334 + 2 per cent galactose........................... Medium 334 + 0.2 per cent Merck’s PEDUON Gs Sys 5 seus ands see Medium 334 ++ 2 per cent lactose............................. Medium 334 + 2 per cent cane sugar......................... Medium 334 + Z per cent cane SUB AIS cgtcasretovcen cy soe eh sents amtonea, Medium 334 +- 2 per cent dextrose........................... Medium 334 + 0.2 per cent potassium nitrate................ BES ct eS ee Medium,334 + 10 per cent cané SUS AT sey Penn ee ee heen Medium 334 + 20 per cent cane BUCA TS cet spre erate eee | Medium 334 + 0.2 per cent phlovophucin 20... dohowie on oe Medium 334 + 10 per cent cane SUG AP ey sri seria, tee Rae a, 2b Medium 334 + 0.5 per cent potassium oxalate..............._ Medium 334 + 5 per cent Witte’s WEPLONE | Hoot » | tageo | x - UI so[npou jo Jequinu [eo], 3 oe: a9 : CO rl a) I —] l=) = les =| a r= = a > ma > yas Jo Jaquinyy | | sselintaiaan Suachaals j Len! eINyNo yo raquinu A10}VIOGeT | bai = om Se ° 8 =) 8 8 g | =) (=) =} = =} =) =} 3 =) : S S (=) ' S Meg care es eee ies has |e A a lhe " S io) S So S ro] So Ne: Le e420 8 o°8 ees Wee aceite : 9ing[no aq} Ul punoy =} o =} So =) 3 S cs) =) ° ; SUISIUBZIO SUIAT JO Jequinyy oO o aS o a oO o a So SS a a fon} Pan] om cal ww Y N for) sH ica) LY wD om a a 19 Gr} ro) ro o oO 4 ; H nN om ; 4 a) i a a a a Yo} 1D oO a = = ro) ainjjno ay} Aq paonpoad yueyd o is © 2 =| > = ss S Oo = : ise eas ra) Saale — N oD + rete alfa as ra) a 4 ainjjno jo Jaquinu A10}v10qUT a a = mee | oN N | nN a | nN a | N ro) a ros) STUDIES oF BaciLLus RADICICOLA OF CANADA FIELD PEA 71 Results The nature and the composition of the four media used in experiment 13 are given in table 1, page 11. No nitrogen in any form was added to medium 335. The other three media received 0.3 per cent of Liebig’s beef extract, and, in addition to this, media 300 and 310 received 1 per cent and medium 400 received 2 per cent of Witte’s peptone. Medium 335 contained 2 per cent of cane sugar, and medium 310 contained 2 per cent of dextrose. As seen from table 17, the age of the individual cultures varied between fifteen days and one hundred and fifteen days; but the organisms in all the cultures, except 27, 28, and 29, had been under the influence of their respective media for one hundred and fifteen days. Cultures 27, 28, and 29 were fifteen days old and had been under the influence of their respec- tive media for only fifteen days, having been grown previously on medium 335. The influence of these four media on the organism is shown in table 17. In order to bring out the relations more clearly, a part of. these data are rearranged in table 18: TABLE 18. INFLUENCE oF AGE OF CULTURES ON THE GIVEN MEDIA eatok Average Number of Laboratory number of culture Culture | culture number hving medium (da of nodules organisms ys) : per plant in culture Db cs ’ dct CRIES TERRORS as ee 335 15 4.5 576 , 000, 000 TO) oi ee ee ee nea 335 49 | 6.2 216,000,000 SO Pee corey Sogasicr clerk fook Ralees ons 335 61 9.8 396 , 000, 000 De 83 oro AES eae eae 335 81 6.9 186,000,000 ia 5 poG@ee et + SUCRE Oo ees 335 105 9.5 360 , 000 , 000 Ee eae SE poe oe ake lain ons 335 110 (3 350 , 000, 000 1s Bo AGEL CRRA RGR ARES RL a eae a ee 335 115 9.8 300 , 000 , 000 abe did Sagan Pe ono ORC ae arc are eRe 300 15 8.5 | 4,320,000 ,000 LO), £8 obah etree Ae ip pie ee ane a eRe 300 49 11.2 16,000,000 19) 3.6. GuRehe LE ee Oc ees 300 81 0 0 “Sig "a ee RS Aes Ean 300 105 0 0 OEE ES erica eT oes eee eS 300 110 0 0 Peo bic ait otha Bb Sarno Eee Pee oy GESTS 300 115 0 0 2Do oe Sénetloaew GST GREE eae 400 15 5.8 | 2,592,000,000 2H OMe eae ech RCI Ea en 400 49 6.8 252,000 , 000 171 5 soe had oOo oO a ee eee 400 61 Chall 10,000 , 000 Ree ANe eT ACTeMa eases alee She wae 400 81 8.5 1,000,000 72 Martin J. PRUCHA of TABLE 18 (concluded) ; * -| Average Number of Culture Age of number living Laboratory number of culture ees Ee of nodules | storeaaeean! aye per plant in culture Hileseeek Ole..; Meta ee Ryne BAIR hee 400 98 | 0.5 90, 000 Orie Mn Wael ee | ice AO Aes Nae yA 400 105 | 0 0 (Til neue erete Rie MRAM ONE yeh alae ec RN | 400 110 | 0.6 0 Seta: 4 AB ere SIBLE ah, Ascari cles Laie 400 115 | 0 ED repentance iano ie CRmege ee aes Sch eaves 3 Sear 310 ilsy 6.2 | 3,240,000,000 — De ii Stet Ai, Ate 8 aa Rae 5 205 22 | 310 49 | 7.8 8,000 , 000 LOS carne ate ode RISC EAT acne mee ee 310 61 0 0 UG Boss Siete tna cue pata tie retin wert) CURES rl 310 81 10.7 540 , 000 , 000 Ope 5 Wi ahs HE thas om ee 310 105 10.7 144,000,000 CO fal.