S65! ct ‘ ae ira INFLUENCE OF NITRATES ON NITROGEN-ASSIMILAT- ING BACTERIA! By tvs ., Eieus.? Research Bacteriologist, Idaho Agricultural Experiment Station INTRODUCTION RELATION OF NITRATES TO VARIOUS FORMS OF PLANT LIFE The importance of nitrogen to plant life can not be overestimated. It is one of several elements essential to plant growth, one, moreover, which is apt to be deficient in arable soils. These facts are well brought out by the almost innumerable investigations which have been made concerning the source of nitrogen for plants. The influence of nitrate nitrogen on various plants has been the con- trolling idea in many of these experiments. Very little attention has been placed on the effect of nitrates on the lower plants, especially the bacteria. Because of the relation that exists between higher plants and bacteria it seems advisable to consider the effect of nitrates on the soil bacteria. Indeed, progress in the knowledge of nitrogenous fertilizers depends on a study of the effect of the fertilizer on the soil organisms as well as on the higher plants. The action of fertilizers on the different groups of soil organisms, the relation of these organisms to higher plants, and the separation of the important from the unimportant groups are some of the factors involved in the problem of soil fertility. REVIEW OF LITERATURE The relation of nitrates to the germination of seeds has been studied by De Chalmot (rr)*, who found that corn germinated in solutions con- taining nitrate was more robust than corn germinated under similiar conditions without nitrate. He also noted that if too concentrated solutions of nitrate were used germination was retarded rather than hastened. The presence of nitrate also increased the amount of al- buminous material in the seed. The direct influence of nitrate nitrogen on the growing plant is too well known to justify any lengthy discussion here. Jost (26, p. 134) gives the results of experiments made by Boussingault, who grew the sunflower (Helianthus argophyllus) in sand with and without nitrate. 1Major portion ot a paper submitted in partial fulfillment of the requirements for the degree of doctor of philosophy in bacteriology in the Graduate School of the University of Wisconsin, December, 1916. 2 The writer wishes to acknowledge his appreciation of the suggestions and criticisms obtained through- out the progress of this work from Prof. EK. B. Fred and E. G. Hastings, of the University of Wisconsin. 3 Reference is made by number (italic) to ‘Literature cited,’’ pp. 227-230. Journal of Agricultural Research, Vol, XII, No. 4 Washington, D.C. Jan. 28, r918 Ir Key No. Wis.—1e (183) 184 Journal of Agricultural Research Vol. XII, No. 4 During the three months’ growth of the plants 1.40 gm. of potassium nitrate were added. At the end of the period the dry weight of the plant supplied with nitrate was nearly 60 times greater than that of the plant where no nitrate was added. The relation between the growth of nonleguminous plants and the amount of nitrate nitrogen supplied is shown in a very striking manner in the following table taken from Hell- riegel and Wilfarth (21, p. 53-54): Nitrogen as Ca (NOsz)2 added to pots, ATM ois satcale cieisiew eitiarelusvele sti wierleaionrenis None 0. 056 O. 112 o. 168 ©. 224 0. 336 : . 5+ 9024 Dry weight of oats (grain and Lannea 0- 3605 aes Io. 9814 \ ee { pial \ Sneaace Be ae yatta iatercmttete enc paatere gm.. 5. 2867 But little work has been done on the direct influence of nitrates on the development of the Eumycetes. Some investigations have been made as to the ability of certain fungi to assimilate nitrate nitrogen directly. Ritter (42) studied many species and found that some forms would assimilate nitrate directly, while others reduced it to nitrite and am- monia. He found some forms which failed to grow on media containing nitrate. Kossowicz (28) found that various fungi utilized nitrates and that nitrite and ammonia were produced. Miinter (36) studied the influence of inorganic salts on the growth of various Actinomycetes. He found that potassium and sodium nitrates in quantities equivalent to 5 per cent permitted good growth of the organisms ‘but retarded spore formation. Calcium, barium, and stron- tium nitrates in small quantities affected some species but not others. Small quantities of these nitrates did not affect growth to any extent, but larger quantities were detrimental to growth and spore formation. Silver nitrate in all amounts studied almost entirely prohibited growth. Nitrates appear to exert some influence on the yeasts. Drabble and Scott (73) studied the effect of sodium nitrate on these organisms. They found that the greatest reproduction took place in solutions containing 0.2 gram-molecule of the nitrate. Increasing amounts of the salt led to a decrease in reproductive activity until with 0.7 gram-molecule present no reproduction took place. From their results it is evident that small quantities of nitrate stimulated reproduction, whereas larger amounts proved detrimental. Kayser (27) studied the effect of man- ganese nitrate on yeasts. He found that the amount which produced the maximum increase in the alcoholic fermentation of sugar varied with the strain of yeast employed. He likewise found that manganese nitrate produced greater increase than did the same quantity of potassium ni- trate. Fernbach and Lanzenberg (14) concluded that nitrates hindered the rapidity of cell multiplication of yeasts but greatly accelerated the action of the zymase. More alcohol was formed in the presence than in the absence of nitrate. According to Kossowicz (28), nitrates are not a suitable source of nitrogen for yeasts. n OK Jan. 28, 1918 Nutrogen-Assimilating Bacteria 185 The direct influence of nitrates on bacteria has been studied to a limited extent. The influence of various nitrates on soil bacteria has been studied by Greaves (19). He added sodium, potassium, calcium, mag- nesium, manganous and ferric nitrates to soil in varying quantities. The amount added to the soil was such that in each case equivalent quantities of the anion (NO,) in the various forms were added. ‘The effect of these salts on the bacteria was determined by using ammonifi- cation as an index of the bacterial activity. He found that sodium- potassium, manganous and ferric nitrates in small amounts, approxi- mately 0.97 to 5.5 mgm. of nitrate in 100 gm. of soil, slightly stimulated ammonification. Greater concentrations of these salts proved toxic as evidenced by a decrease in the amount of ammonia formed. Sodium nitrate was much more beneficial to ammonification than potassium nitrate. From his results as a whole Greaves concludes that it is the electronegative ion which stimulates bacterial activity. Calcium and magnesium nitrates proved toxic in all concentrations studied. However, a majority of the investigations have been directed toward a determination of the effect of the bacteria on the nitrates. But little work appears to have been done on the direct action of nitrates on bacteria. Pfeffer (38, p. 351) cites some experiments showing the repellant action of potassium nitrate toward certain bacteria. Spir- wlum undula was repelled by a solution of potassium nitrate having an osmotic concentration equivalent to 0.5 to 1.0 percent. With Spirillum volutans a much higher concentration was necessary to bring about the same reaction. It was found that different organisms required different quantities of the same nitrate to repel them. | It can be readily seen that by far the greatest amount of work on the relation of nitrates to plant growth has been done in the realm of the higher plants. Obviously further investigations should be made in respect to the effect of nitrates on the lower forms of plant life, especially the bacteria. In this paper an attempt is made to set forth the results secured in a study of the influence which nitrates exert on certain groups of soil bacteria, including not only their reproduction but also some of their physiological properties. EXPERIMENTAL WORK OUTLINE OF PROBLEM The results of much careful experimentation show that nitrate nitro- gen is most readily assimilated by higher plants. As a rule it seems to stimulate the plant to increased activity. In some cases this is un- doubtedly due to increased nutrition, while in others it is a result of nuclear stimulation with a consequent cell multiplication. No sharp line can be drawn between these two effects. Probably one overlaps the other, and the increased growth of the organism can be attributed to a combination of the two actions. 186 Journal of Agricultural Research Vol. XII, No. 4 From a practical standpoint the relation of nitrates to the nitrogen- assimilating organisms of the soil is of importance. Hence, it was arranged to study the effect of nitrates on soil bacteria, especially those forms concerned with the fixation of atmospheric nitrogen. ‘The work naturally falls into two rather distinct lines of investigation. First, the influence of nitrates on Azotobacter was determined. Here studies were made of the effect of nitrates on the growth of the organism in soil and also the effect of these salts on the nitrogen-fixing property of these bacteria. ‘The action of Azotobacter on nitrates in solution, the relation of nitrates to pigment production and to the formation of volutin bodies were studied. Second, the influence of nitrates on the growth of Bacillus yadicicola in soil was studied. The action of B. radicicola on nitrates in solution and the possible nitrogen-assimilating properties of the legume in the presence of nitrates were investigated. Also the influence of nitrates on gum production was determined. The latter part of the investigations included a study of the relation of nitrates to nodule formation on alfalfa. METHODS USED IN EXPERIMENTS Nitrates were determined by the reduction method with Devarda’s alloy and also by the phenolsulphonic acid (colorimetric) method. The total nitrogen content of all samples was determined by the modified Kjeldahl method with sulphuric acid, salycilic acid, sodium thiosulphate, and copper sulphate. Where nitrate nitrogen was present, 50 c. c. of concentrated sulphuric-salycilic acid (25 c. c. of concentrated acid plus 25 c. c. of distilled water) were added to the cultures slowly and with constant stirring. This acid was allowed to react for a few days, after which the usual procedure was carried out. Digestion was continued for five to six hours subsequent to the clarification of the liquid. The amount of ammonia was determined by distillation with steam in the presence of magnesium oxid. Nitrites (qualitative test) were tested for with Trommsdorf’s reagent. In all distillations N/r4 acid and alkali were used. In the preparation of agar cultures of alfalfa seedlings the seeds were treated with a 0.25 per cent solution of mercuric chlorid and rinsed in sterile distilled water. Three bacteria-free seeds were transferred to the surface of soft mannit agar (0.7 per cent agar) in each tube. The nitrates were added in solution to all cultures. Gram-molecular quantities of potassium, sodium, calcium, and ammoniun nitrates (Merck’s) were weighed into sterile distilled water. These solutions were prepared in such a manner that 5 c. c. contained 450 mgm. of nitrate. In all nitrate solutions the nitrate radical, or anion, was present in the same quantities, while the cation, or metal, was present in varying quantities, depending upon the particular salt. Jan, 28, rox8 Nitrogen-Assimilating Bacteria 187 Fe eS as spent ta it SN ear SA ee Plate counts of all soil cultures were made by weighing 20 gm. (dry weight) of the soil into a 200-c. c. water blank. From this suspension all subsequent dilutions were made. Mannit agar * was used for the plate counts in the cultures of Azotobacter and B. radicicola. Duplicate plates were made for each dilution poured. SOIL USED Only one type of soil was employed, Miami silt loam obtained from the Experiment Station farm. No chemical analyses of the soil were made other than an estimation of its organic matter content, which was approximately 2.75 per cent. The soil was neutral in reaction and its nitrate content was approximately 1.5 mgm. of nitrogen as nitrate in 100 gm. of the dry soil. ISOLATION OF AZOTOBACTER AND BACILLUS RADICICOLA AZzOYOBACTER.—(1) Strain A was isolated from a silt loam soil. This strain grew well on mannit agar, but produced no pigment after three weeks’ growth. (2) Strain B was isolated from a sandy loam soil. This strain grew equally well on mannit agar and produced a brownish black pigment within one week’s growth. Both strains assimilated practically the same amount of atmospheric nitrogen under laboratory conditions. BACILLUS RADICICOLA.—A stock laboratory culture of B. radicicola was replated twice before taking the final culture. The nodule produc- ing power of the organism was determined by inoculating bacteria-free alfalfa seedlings (in soft agar). After sufficient incubation nodules were produced in abundance. INFLUENCE OF NITRATES ON AZOTOBACTER INFLUENCE OF NITRATES ON THE GROWTH AND REPRODUCTION OF AZOTOBACTER IN STERILIZED SOIL What effect do nitrates have on pure cultures of Azotobacter in ster- ‘lized soil? Do these salts cause a decrease in the numbers of the organ- isms? Do they cause an increase in numbers? Or do they exert no particular influence one way or the other? It is difficult to believe that the latter could be true, inasmuch as nitrates have such a profound effect on higher forms of plant life. Such readily soluble and assimilable substances as nitrates could hardly remain without affecting either an sncrease or a decrease in the number of organisms existing in their presence. With the idea of determining what effect nitrates might have on Azotobacter when grown in sterilized soil, the following experiments were planned. In this work both strains of the Azotobacter (described on 1 FreD, E. B. A LABORATORY MANUAL OF SOIL, BACTERIOLOGY. P. 108. Philadelphia and London, 1916. 188 journal of Agricultural Research Vol. XII, No. 4 p. 187) were employed and conditions governing the preparation and incubation of the cultures were similar in the case of each strain. The only variation was the periods used in incubating the cultures. Counts were made after one and two weeks’ incubation with strain A and after one, two, and three weeks with strain B. TABLE I.—Influence of potassium nitrate on the growth of Azotobacter (strain A) in sterilized soil ede Wee Number of organisms in 1 gm. of dry soil. (nitrate Culture No. in 100 J Faso Peas After 1 week. Relative. After 2 weeks. Relative. Mgm. , 3 Per cent. Per cent. ites ° 15, 600 25, 000 315, 000 Joie ° I5, 600 935, 000 } Mie { 360, 000 \ hate eee 10 15, 600 I, 500, 000 \ { I, 175, 000 Ani 10 iis} Wola) hide solos Ab Ga coooe raha | eee ee 348 Bete 25 15, 600 4, 200, 000 { 12, 350, 000 Ge. 25 15, 600 5, 000, 000 523 IO, 750, 000 3, 418 > pee 50 15, 600 20, 400, 000 { 27, 750, 000 Sut 50° 15, 600 18, 900, C00 1 CE NS) an meen na 8, 210 Oi isGacan ids. 100 15, 600 II, 000, 000 { Q, 000, 000 TOC eee eciocit 100 15, 600 I1, 820, 000 ENz95 9; 150, 000 2, 085 Tis seeks 150 THOS NWA Nis, darn \ a { 25, 000 ae 5 OA ERIE Ore 150 15, 600 I, 575, 000 79 55, 000 eu eae te 200 15, 600 225, 000 M { fe) TAM are oe daskatuley: 200 15, 600 250, 000 7 ° c Te tea at 300 15, 600 ° 3} ° Ome caciee 300 15, 600 ° ° 2 a TaBLE II.—Influence of sodium nitrate on the growth of Azotobacter (strain A) in stertlized soil Treatment Number of organisms in x gm. of dry soil. (nitrate Culture No. in 100 h ae a4 as After 1 week. Relative. After 2 weeks. Relative. a eR EN i a Mogm. : Per cent. 1 Per cent. ites ° 13, 800 310, 000 425,000 |} Bree ° 13, 800 225, 000 \ seat { 490, 000 }f hk Bits Io 13, 800 575, 000 875, 000 Ae 10 13, 800 430, 000 SB NA ea Ui aOR (ole 25 13, 800 2, 850, 000 2, 250, 000 6... 25 13, 800 5, 800, 000 HN AU ae ae a8 [oe 50 13, 800 15, 200, 000 15, 500, 000 (5 DE 50 13, 800 12, 750, 000 5 217 13, 300, 000 3) 150 Oo commie 100 13, 800 I7, 750, 000 9, 850, 000 TOn cise eine ots 100 13, 800 16, 200, 000 6; 335 15, 750, 000 oe i oqo ogjus See 150 13, 800 550, 000 Bae 690, 000 Bayh ate. sont hla I50 13, 800 400, 000 77 375, 000 ary ti Aetna ae 200 13, 800 ° y fo) 14 200 13, 800 fo) * fo) . I Ga BOOTS os 300 13, 800 ° ° TOR aces ae ee 300 13, 800 ° ° ° i Jan. 28, 1918 Nitrogen-Assimilating Bacteria 189 TaBLE III.—Influence of calciwm nitrate on the growth of Azotobacter (strain A) in sterilized soil Treatment Number of organisms in x gm. of dry soil. (nitrate Culture No. in 100 : Pose ee After x week. Relative. After 2 weeks. Relative. Mgm. Per cent. Per cent. yo. ° IO, 000 260, 000 310, 000 owe: fo) I0, 000 330, 000 ric 260, 000 1 Far 000 lefe) 3 Bie) IO, 5; 800, o 1, 966 975, 000 362 As Io TONOOON)|in 6 sistelers eve ster I, O90, 000 Beier 25 I0, 000 IO, 700, 000 Q, 200, 000 Ones 25 IO, 000 9, 600, 000 31 440 8, 600, c00 3,442 Gib 50 10, 000 13, 250, 000 13, 200, 000 822. 50 IO, 000 IT, 600, 000 | 4; 213 12, 600, 000 4, 526 OROIOOOT ole 10, 0 6, 600 (ole! 9 100 , COO , 500, 000 2, 144 8, 750, 000 2,938 TO actsevsiaetercts 100 IO, OCO 6, 050, 000 8, 000, 000 Te CD ORC 150 10, 000 3, 500, 000 2, 000, 000 6 I, 254 793 HONe Sa Cena Boose 150 10, 000 3, 900, 000 2, 350, 000 Mais toneiierel ate | 200 10, 000 ° Ps ° Ps TA creates sic ets 200 10, 000 O | ° Te EER DOODLE 300 10, 000 ° | ° ° ° UGibaadmecmune 300 10, 000 ° ° One hundred and fifty gm. of soil (dry weight) were weighed into 500- c. c. Erlenmeyer flasks and the nitrates added in solution, as indicated in the following tables. At the same time 1 per cent of mannit was added in solution and the moisture content was raised to approximately 18 per cent. The flasks were allowed to remain at room temperature for one day, when the contents were thoroughly mixed. The flasks and contents were then sterilized at 15 pounds’ pressure for three hours. Upon cooling they were inoculated with 5 c. c. of a suspension of the organisms in sterile distilled water. The cultures were incubated at 28° C. and counts made at the intervals already indicated. Mannit agar was used in pouring the plates. Each number in the following tables represents an average of duplicate plates. Tables I, II, and III show the results of the work with strain A and Tables V, VI, and VII the results with strain B. It will be seen at a glance that all three nitrates exerted an enormous influence on the growth of the Azotobacter. The smallest concentration _ did not appear to exert much influence either in increasing or decreasing the number of Azotobacter. There was a slight gain, but it was not so marked as that brought about by higher concentrations of nitrates. When 25, 50, and 100 mgm. of nitrate were present in 100 gm. of soil, very large increases were obtained in practically all instances. In one instance sodium nitrate caused the greatest relative gain, but the most consistent increase was produced by calcium nitrate. Beginning with 150 mgm. the number of Azotobacter began to decrease. ‘This decrease was especially noticeable in the cultures containing potassium and sodium nitrates. At the end of the first week, Azotobacter organisms 190 Journal of Agricultural Research Vol. XII, No. 4 were still found in the potassium-nitrate cultures where 200 mgm. were present. However, at the end of the second week the organisms were dead. The same concentration of sodium and calcium nitrates proved even more toxic. No evidences were secured, indicating that these organisms can resist concentrations in excess of 300 mgm. of nitrate per 100 gi. of soil. The question may be raised in regard to the influence of sterilization on the nitrate present in the soil, Does the prolonged heating in the presence of soil organic matter reduce the nitrate? In order to study this point, a few cultures were prepared similar to those already described. They were subjected to sterilization under pressure of 15 pounds for two, three, and five hours. Nitrate determinations at the end of these periods failed to show any reduction. In the presence of 1 per cent of mannit the nitrate content remained unchanged during sterilization. From these results it is evident that small amounts of nitrate up to 150 mgm. of nitrate in 100 gm. of soil greatly increased the reproduction of Azotobacter. In regard to the toxicity of higher concentrations, sodium nitrate appeared to exert the greatest influence in this direction, followed by calcium and potassium nitrates in the order named. ‘The results of the experiment are recorded in Table IV. TABLE IV.—Influence of ammonium nitrate on the growth of Azotobacter (strain A) in sterilized soil Treatment Number of organisms in 1 gm. of dry soil. (nitrate Culture No. in 100 j Ary soit): ace After 1 week. Relative. After 2 weeks. Relative. | Mgm. Per cent. Per cent. Ts ° 18, 500 I, 400, 000 fo 975) 000 ans Bios fo) 18, 500 I, 050, 000 I, 100, 000 Bh hs 25 18, 500 5, 600, 000 |’ 3 5, OCO, 000 - ah 25 18, 500 4, 900, O00 |} gagil 3, 900, 000 43 ec 100 18, 500 2, 900, 000 3, 950, 000 |) (OIE bofe) 18, 500 2, 600, 000 223 4, 100, 000 f 388 hele 200 18, 500 I, 100, 000 8 875, 000 86 Sis: 200 18, 500 950, 000 4 QI5, 0co | That the nitrate radical and not the combined metal was the causal agent in the increase in the number of Azotobacter was indicated from the results of the next test. Here ammonium nitrate was used. It will be seen from the data of this experiment that ammonitm nitrate caused an increase in the number of Azotobacter when present in small amounts. However, the increase in the presence of ammonium nitrate was less marked than when equal quantities of the other nitrates were used. Since the experiments with ammonium nitrate were not made at the same time as the preceding experiments (discussed on pp. 189-190), it is possible that conditions varied sufficiently to account for the less pro- nounced results: When 200 mgm. of nitrate were present in 100 gm. of Nitrogen-Assimilating Bacteria IQI Jan. 28, 1918 soil the number of Azotobacter showed a decrease. Apparently ammo- nium nitrate is more toxic than potassium, sodium, and calcium nitrate. However, the main point at issue seems fairly well established—namely, that the increase in the number of Azotobacter is caused by the nitrate radical and not by the combined metal. TABLE V.—Influence of potassium nitrate on the growth of Azotobacter (strain B) in sterilized soil Treat- Number of organisms in 1 gm. of dry soil. ment Culture Garrats No. : : ao fone peace After 1 week. |Relative.} After 2 weeks. |Relative.| After 3 weeks. eae soil). Mogm. Per cent. Per cent. Per ct. Lee © | 12, 600 235,000 || 144 II2, 500 = { 116, 000 ae 28 OETA OOO ELS weliicareuel ie | ILO, 500 II7, 000 ae 10 | 12,600 | 3,750,000 | { 2, 100, 00O { 875, 000 Ars Io 12, 600 | 3, 300, 000 ts 3T9 2, 250, 000 19959 I, 260, 000 926 Roe 250 \et2;/600) 1) )5,1750,,000 { I, 575, 000 I, 700, 000 \ Gy 25 | 72,600 | 5,700, 000 2, 436 I, 950, 000 1, 581 I, 325, 000 rede wink 50 | 12,600 | 3, 100, cco 3, 250, 000 3, 525, 000 Sa 50 | 12,600 | 3,200, 000 \r, 340 { 4, 900, 000 \;, 655 2, 960, 000 to, 783 eve 100 | 12,600 | 3, 200, 000 4, 000, 000 2, 500, 000 \ ZO. . 100 | 12, 600 3, 000, 000 \r, 320 { 3, 500, C0O i, 363 { 2, 9OO, 0CO 2,317 Tee I50 | 12,600 | 2, 100, 000 2, 000, 000 I, 500, 000 12): I50 12, 600 I, QOO, 000 \ 851 { 2, 100, 000 \1, 838 { 2, 000, 000 \,, a8 Ties 200 | 12, 600 875, 000 { 800, coo { 650, 000 \ TA). 200 | 12, 600 880, 000 \ 373 750, 000 G95 700, 000 580 meh 300 | 12, 600 ro) \ ot { ro) \ e { ) \ é TOn 300 | 12, 600 ° ° fo) TABLE VI.—Influence of sodium nitrate on the growth of Azotobacter (strain B) in sterilized soil Treat- Number of organisms in r gm. of dry soil. ment Culture Gat N ‘ aig. ae Pee After x week. |Relative.| After 2 weeks. |Relative.| After 3 weeks. ate soil). Mom. 3 Per cent. Per cent. Per ci. Bist © | 15, 600 158, 000 110, 500 { 112, 500 Ate ° 1s, 600 149, 000 \ ray { 126, 000 \ ae 115, 000 \ aii Zh. 10 | 15,600 | 1, 250, 000 \f I, 750, 000 { 5) 000, 000 \ Arie | 10 15, 600 990, 000 (Fi I, 350, 000 T5379 6, 600, 000 51°97 Bi 25 | 15, 600 | 1, 765, 000 6, 600, coo { 9, 150, 000 olan 25 rs, 600 1, 825, 000 I, 165 |L 5, 300, 000 p02 7, 150, 000 7; 161 “len 50 | 15, 600 | 1, 875, 000 { 2, 025, 000 \ (is) 2a oee \ 4 Sie 50 1s, 600 | 2, 250, 000 T, 338 | 3, 040, 000 aay 14, 600, 000 13, 423 Ges 100 | 15, 600 | 2, 200, 000 \ 7 { 2, 775, 000 5, 800, 000 \ Io... 100 | 15, 600 | 1,950, 000 T, 35° 3, 200, 000 24525 5) 250, 000 4, 860 Vine I50 | 15, 600 165, 000 { 530, 000 \ 3, I00, 000 \ Te 150 | 15, 600 170, 000 fo 785, 000 559 2, 750, 000 24573 ER 200 | 15, 600 fc) \ i ° \ { ° \ TA. 200 | 15, 600 ° ” ° ¥ ° = rer 300 | 15, 600 ° { ° } { ° \ 10.. 300 | 15, 600 ° \ \ ° ° ° e | 192 Journal of Agricultural Research Vol. XII, No. 4 TABLE VII.—Influence of calcium nitrate on the growth of Azotobacter (strain B) in sterilized soil a, Number of organisms in 1 gm. of dry soil. Treat ment Culture (nitrate No. in i roo gin.| At begin- : Rela- aS pa After 1 week. |Relative.| After 2 weeks. |Relative.| After 3 weeks. ray soil). Mom. Per cent. 2 Per cent. Per ct. B Top © | 22,000 905, 000 100 |J 11475, 000 too | 11 130, 000 vila PD oe © | 22, 000 860, c00 I, 460, 000 I, 157, 500 ae 2,000 | 23, 200, 000 2 ° 050, co 3 TO | 22,00 ae ? \., 423 { é; 000, 000 \,, 18r ae 2 , COO is, 002 Abs: IO | 22,000 | 19, 600, 000 36, 000, C00 34, 600, 000 as 2 2, 000, 00 2 : 5 25 | 22,000 | 17, 200, 000 2, 084 2s , COO 3) 255 9, 750, 000 2, 273 Big ¢ 25 | 22,000 | 19, 600, 000 43, 500, 000 22, 250, 000 i (ole) I, 800, 000 22, 500, 00 ©, 400, 000 }) 7 50 | 22,000 | ITI, ? I, 461 7 500, 900 zi, 448 30, 400, 009 2, 633 (le 50 | 22,000 | 14, 000, 000 20, 000, 000 29, 850, 000 \ uy lofe) 00, 000 2, 000, 00 21, 750, 00 9 I0o | 22,0 7) 509; I, 053 I2, 000, 000 818 »75 ° I, 780 iifO) 4 HOON | 22) COO!) 1, OOO NOOO! |Ifin? sie niet enche eel 18, 950, 000 Tiers I50 | 22,000 | 2,550, 000 300, 000 800, 000 I 5 ? 2 559, 342 a oe") 402 4; ’ 420 UP), I50 | 22,000 | 3,500,000 EOOLOOOM NINN yy Hal iseleverneter ote « uigloe 200 | 22, 000 107, 500 rr | 21752 000 es I30, 000 Ee TAG 200 | 22, 000 87, 500 3, 225, 000 3 120, 000 Ege. 300 | 22, coo ° 2 ° ~ { ° n TORN 300 | 22, 000 ° | ° fo) A glance at the figures of Tables V, VI, and VII shows that the small- est concentration of nitrate used produced a much more marked relative increase in numbers with strain B than it did with strain A. On the other hand, the greater resistance of this strain to the higher nitrate concentrations is clearly evident. In the potassium- and calcium- nitrate cultures the organisms were present in an active state where the nitrate was added in amounts equivalent to 200 mgm. of nitrate in 100 gm. of soil. However, this same concentration of sodium nitrate prevented the development of the Azotobacter. ‘The first five concen- trations of all three nitrates caused a very large increase in the number of Azotobacter when compared with control cultures where no nitrate was added. In one instance an enormous increase was noted after three weeks’ incubation in the presence of 50 mgm. of nitrate as sodium nitrate. This increase far excelled that noted with other concentrations of the same salt. The writer can offer no conjecture as to this occurrence. Similar results were obtained by the writer in 1914 (23) with a strain of Azotobacter isolated-from a silt loam soil at the Pennsylvania Experi- ment Station. It was found that soil and liquid cultures containing small amounts of potassium, sodium, and calcium nitrates caused an increase in the number of Azotobacter in pure culture compared with control cultures containing no nitrate. An increasing concentration of the nitrates continued favorable to the growth of the organism up to a certain limit, but higher concentrations retarded its growth. Finally a nitrate concentration was attained at which Azotobacter growth altogether ceased. Jan. 28, 1918 Nitrogen-Assimilating Bacteria 193 The results of the study of nitrates and their influence on Azotobacter in sterilized soil show very clearly that small amounts of nitrate cause a great increase in the number of Azotobacter cells. Higher concentra- tions are not so favorable to the growth of the organisms, and the highest concentrations studied prevented the development of the Azotobacter in sterilized soil. From a study of the results of these experiments, it seems that the increase in number of Azotobacter in the presence of small amounts of nitrate is a direct result of nuclear stimulation. Later studies to be cited (pp. 205-208) show that nitrates exerted considerable influence on the internal structure of the Azotobacter cell. It appears reasonable to expect that the nitrate affected the nuclear structure in such a manner that an increase in cell multiplication resulted. It seems probable that the action of nitrate as a simple nutrient would be shown by a slower increase in cell multiplication. INFLUENCE OF NITRATES ON THE FIXATION OF NITROGEN BY AZOTOBACTER It has been shown in the preceding paragraphs that the presence of small quantities of nitrate in sterilized soil bring about a large increase in the number of Azotobacter. This increase was noted in the case of both strains of Azotobacter. It would be of interest to know whether the increase in bacterial numbers was accompanied by a corresponding increase in the amount of nitrogen assimilated. The results secured by a few investigators indicate that in the presence of combined nitrogen as nitrates the nonsymbiotic nitrogen-fixing organisms will not fix atmospheric nitrogen. Stoklasa (44, p. 492-503) studied the influence of Azotobacter on sodium nitrate in aerobic and anaerobic liquid cultures. He found only a small gain in organic nitro- gen and from these results he concluded that in the presence of nitrates Azotobacter could not assimilate atmospheric nitrogen. It has been shown by Hanzawa (20) that in a liquid culture containing 12 mgm. of nitrate (from potassium nitrate) in 100 c. c. of medium, a mixed culture of Azotobacter fixed 5.25 mgm. of nitrogen. Under the same conditions with 60 mgm. of nitrate present in 100 c. c. of medium he found but 5.35 mgm. of nitrogen fixed. He concluded that nitrates remained, as far as small quantities were concerned, almost without influence on the amount of atmospheric nitrogen fixed by Azotobacter. Some studies have been carried on with respect to the influence of ni- trates on the nonsymbiotic anaerobic nitrogen-assimilating organism, Clostridium spp. Bredemann (9) showed that ammonium nitrate in solution caused a decrease in the amount of nitrogen fixed by species of Clostridium. Pringsheim (go) grew cultures of C. americanum in solutions containing potassium nitrate. He found that in the presence of available energy the organism fixed some nitrogen when nitrate was 194 Journal of Agricultural Research Vol. KIL, No. 4 present but to a less extent than did control cultures containing no nitrate. From these results it appears that nitrates do not stimulate the nitro- gen-assimilation of the nonsymbiotic nitrogen-fixing bacteria. Inasmuch as nitrates in small amounts caused such an increase in the number of Azotobacter in sterilized soil, it was thought advisable to determine just what influence these salts exert on nitrogen fixation by Azotobacter. Accordingly, experiments were carried out with Azotobacter on agar films, in soil cultures and in solution. AGAR-FILM CULTURES.—In this work both strains of the Azotobacter were used. One hundred c. c. of mannit agar were placed in liter Erlenmeyer flasks and nitrates of potassium, sodium and calcium added in varying quantities. The flasks and contents were sterilized at 10 pounds’ pressure for 25 minutes, cooled, and inoculated with 10 c. c. of a suspension of the organism in sterile distilled water.. The flasks were incubated at 28° C. for three weeks. The weight of both inoculated and uninoculated flasks was maintained throughout the experiment by the addition of sterile distilled water. At the end of the incubation period total nitrogen analyses were made. Because of the high nitrate content dilute sulphuric-salycilic acid was added slowly and carefully to prevent loss of nitrogen by the evolution of gaseous oxids of nitrogen. ‘The acid was allowed to react for a few days before continuing the total nitrogen determination. The results of the experi- ments are presented in Tables VIII and IX. TaBLE VIII.—Influence of nitrates on the fixation of nitrogen by Azotobacter (strain A) on agar films Nitrogen contained in 100 c. c. of medium. Cul- A ture | Treatment (nitrate in rooc. c. of medium). Inoculated. Uninoculated. N ans No. ed. Found. | Average. | Found. | Average. Mam. Mom. Mo». Mam. Mom. Fl Oeste caaronrecie isla gee Lar mie ae dia ache a Re 13. 00 4.9 BHO P eiavaress oadaars caches Mian s eee 12.70 12. 80 4.9 4. O5 8. 75 GolRC Hono uo ‘ a dentate Stal eee yep a aie ters atone 12. 60 At 4 | 50mgm.o otassium nitrate.| 18. 50 . 0 (| ee cea Os eroete 1 uy Wee eS eS ace ae 18. re \ 18. 45 { b nis \ fot TI. 35 6 | r1oomgm.of NO; potassium nitrate] 27. 6 6.8 Rie At TMOG hes: SOE iarpor eee. It aia oe a3 8 | 50 mgm. of NO, sodium nitrate...| 18.6 : Aan ane eh seaaar eu Sit een od) Aid aaeee Io | 100 glee of NO; sodium nitrate.| 27. 00 15. 00 Ls Pas ae cL Ovstotader test acta laa a eetestenty araaeeas 27. 65 27-35 15. 20 \ Toe iis 12 | somgm.of NO, calcium nitrate..} 13. 8. 00 Se Pal ha aR OM ae ayo ae PANTS eae ee iS \ 13-75 { 8. 50 \ es 5: 5° 14 | roomgm.of NO, calcitim nitrate.| 18. 80 8 14. 50 IGG eats OWE oferta ccltieyer steele autres ane ederode 1g. 15 12.99 14. 30 \ ag 455 Jan. 28, 1918 Niirogen-Assimilating Bacteria 195 TABLE IX.—Influence of nitrates on the fixation of nitrogen by Azotobacter (strain B) on agar films | Nitrogen contained in 100 c. c. of medium. | Cul- ture | Treatment (nitrate in 10oc. c. of medium). Inoculated. Uninoculated. a ei No. ed. Found. | Average. | Found. | Average. Mom. Mom. Mom. Mam. Mom. HW Ol bu tcc SORU TC OUC Cb Cao ana One rk 15. 50 | 6. 50 2) Wi@binaso bon BORED oouD CON CH oan anes 15.70 |¢ 15.60 6. 30 6. 40 Q. 20 Bil Cscoe? Socapcocuocedcs apo hcouoR 15. 60 6. 40 4 | 75 mgm. of NO; as potassium ni- WZUG osonebasnonebcoododoMese 25. 20 13.00 Rl ee OME arenes cid ecoms aes ieisie ciate Sister & a 40 \ a0 3° { a 70 \ 13. 85 aS 6 | 150 mgm. of NO, as potassium ni- EMTS is ears cityrare tapsuevaceyesfasiey evelevejeke-ene 36. 40 24. 00 y ; [ol Pec GOs Sehsc ae ecrehsle bea os es sisal 36. 90 \ 3&- 05 { 23.20 |f 73 Go £395 8 mgm. of NO, assodium nitrate| 25. 60 12. 80 9 oe ae Sebo cinhy ipl eit rca re 70 \ 25,05 { I3. 20 15. o0 12. 65 mgm. of NO, as sodium nitrate .6 6. ee al hell. coon (tO Ge aol Sd 12 | 75mgm.of NO,ascalcium nitrate} 20. 10 | 12. 00 13 # Fi AC (0G) lh eit eee ico Hise ney 1g. 60 IEE i t23 70 12. 35 7- 5° 14 | 150mgm.of NO,ascalcium nitrate] 32. 80 | 24. 50 | HG cusicuse: Odinnn icine So helen Gabe Diol tae 33. 30 33-95 { 25. 20 as ete A glance at the results (Tables VIII and IX) shows that an increase in nitrogen fixation occurred where potassium and sodium nitrates were present, whereas a marked decrease in the total nitrogen content was observed where calcium nitrate was used. Whether the calcium itself is detrimental to an increase in organic nitrogen or whether it is the com- bination of calcium with nitrate can not be stated. It is significant, however, that this decrease in fixation of nitrogen was noted throughout all the experiments where calcium nitrate was employed. It is very evident that calcium nitrate exerts some detrimental effect on the nitro- gen assimilating properties of the organism. There seems to be but a slight difference in the nitrogen-fixing ability of the two strains studied. In the absence of nitrates the amount fixed varies but little. Also in the presence of potassium and sodium nitrates the relative increase in amount of nitrogen fixed remains about the same. Calcium nitrate offers an exception where it is employed. The detrimental effect seems to be more marked in the case of strain A than with strain B. Strain A under normal conditions fixed slightly less nitro- gen than strain B, so it may be possible that this strain is weaker. The formation of pigment by the Azotobacter in the presence of the nitrates is of interest. Strain A normally produced no pigment by the end of three weeks’ incubation. But when grown on the agar films in the presence of nitrate a most marked pigment production appeared. This. pigment was especially noticeable in the presence of the calcium 196 Journal of Agricultural Research Vol. XII, No. 4 salt. Since strain B normally produces a good pigment, the influence of nitrate on this strain was not very marked. The relation of nitrates to pigment formation will be taken up later (pp. 203-205). From the results of the experiments with agar films containing various amounts of nitrate, it seems apparent that potassium and sodium nitrates in amounts of 50 and 100 mgm. of nitrate in 100 c. c. of medium cause a small increase in the amount of nitrogen fixed. However, this increase in fixation is not at all parallel with the increase in number of Azoto- bacter caused by nitrates in sterilized soil. It may be concluded that an increase in the number of Azotobacter in sterilized soil as a result of nitrate stimulation does not mean a corre- sponding increase in nitrogen fixation on agar films. Soll CULTURES.—The conditions obtaining in these experiments were strictly comparable with those heretofore cited dealing with the influence of nitrates on Azotobacter in sterilized soil (pp. 187-193). The fixation of nitrogen was studied in pure culture in sterilized soil and in unsterilized soil. One hundred and fifty gm. of soil (dry weight) were weighed into 1-liter Erlenmeyer flasks, nitrates were added in vary- ing amounts from 10 to 200 mgm., and 1 per cent of mannit was also added. ‘Triplicate flasks were prepared for each amount of nitrate studied. The moisture content was raised to approximately 18 per cent and the flasks allowed to remain at room temperature for one day. The con- tents were then thoroughly mixed and a fine crumb structure produced. The flasks for the experiments with pure cultures in sterilized soil were immediately sterilized at 15 pounds’ pressure for three hours. After cooling, two of each set were inoculated with 5 c. c. of a suspension of Azotobacter (strain A) in sterile distilled water. The remaining flask of each set was not inoculated, but was incubated at 28° C. with the inocu- lated flasks. The moisture lost by evaporation was replaced from time to time by the addition of sterile distilled water. At the end of the incu- bation period the soil was removed and spread out in thin layers and allowed to dry. It was then thoroughly ground in a porcelain-ball mill for one hour. At the end of this time all of the soil passed through a 100-mesh sieve. Soil cultures used in the study of the effect of nitrates on nitrogen fixation in unsterilized soil were prepared in a similar manner, except that the flasks were not sterilized. Previous to incubation a small inoculum of Azotobacter (strain A) was added to insure the presence of the nitrogen-fixing organism in the soil cultures. The proper moisture content was maintained in the same manner as in the case of the pure cultures in sterilized soil and the incubation period was the same for both. The results are given in Tables X, XI, XII, and XIII. Jan. 28, 1918 Nitrogen-Assimilating Bacteria 197 TABLE X.—I nfluence of sodium nitrate on the fixation of nitrogen by Azotobacter in stertlized soil 1. Total nitrogen in 100 gm. of dry soil. : ea LUM Cf eS ee itrogen Culture No. San at Inoculated. Uninoculated. Leelee ee Found. Average. Found. Average. ae Mgm. Mom. Mogm. Mgm. Mom. Mogm. Tete eRe ce Seis rs at ° 135.0 132.0 TO Nr Seis oaks done ° 134.0 133-7 131. 5 TaTag 207 DAD eps Reaches acy clas ° 132.0 I3I. Zena Mace Oo aie ee ° TEZVOry abe ell [ hes itare sts cel laarsrome ee Dig Noma se Nave tees esate ° 137.0 TBO’ clare yorsre aceite is | ae eee B35 BE eta eaenisienols OR Pscrncinicecra UMN MAMINe ater cteta chai tty ce otatharacse Biers orate he inj sha) as 10 137.0 134. 0 Bue sealers ee are's Io 136.0 136. 6 133.5 D337 2.9 Fie troeysteeisvep create cron 3 Be) 137.0 134.0 EGR ee. eee a be) 136. 5 | Ubngia Wis « de x) eset teren e | Ae reas a he EI ies ae) rea7a5 TBINO' iors, vic orevatols) cblehe Suctoeens 343 RRS AD ae OEP ROE RCTS Be) LA 7HOV Need 6m Actes ha cesar d | tins ae aati Sea Sleeve ciaieiee an enshale 50 149. 0 140. 0 eras rana te canter aut ie 50 149. © 149. 0 137.0 138. 5 10. 5 Basta e aieityater sis nase sateen 50 149. 0 138. 5 Os rrsete Sects masts sien 50 LZ Lean a alot Arai | (ried nis ping pictotmod ss Ohne abate sdon 50 T49. 5 149. 2 pes sae eheherahs | 10. 7 Oi aaa Gen oon 50 TAG N Sell ey manta UiWestetevetere) a erates [done haat: 7 I50 163. 0 152.0 (SiGe Ee oO as Ua oor 150 162.0 162. 3 150. 0 I5I. 5 to. 8 7 | PSEA Ae ents oe 150 162.0 152.5 Beane cee Sesto dameceers 150 TOD Sele Mee seul ekerets.oyeneiapcve’sl| eyeretey nieta's oes ene Der ee 150 163.0 MICE N ice toi oy cakes | steaks II.0 8 150 LOZ SOMME meow Nal Mate talencs oot eae scala ee TABLE XI.—Influence of sodium nitrate on the fixation of nitrogen by Azotobacter in unstertlized soil treat Total nitrogen in 100 gm. of dry soil. Nitrogen Culture No. trate ce Inoculated. Uninoculated. bagi? too gM. Of | AMA A |] . dry soil).|_ Found. Average. Found. Average. ony Bell Mogm. Mom. Mgm. Mom. Mom Mom LR Se sreheta evel seve ae at aah ° 132.0 130. 0 TIE Sige teh shes et amatoes satohets et & ° 135.0 134.0 133-5 131.8 2.2 Re essa oh. sith se s8y ata sok ° 135.0 132.0 Bese fore are fe aietsisien tere ° TAZA wees. Whe li[rats rarewetset ote. terete ate ote 2) I ORY aE ce RET fo) 134. 0 DS Sa Sinton viie!siek\cieie sere Tag 2 SEE SARI ree Ore ° DAA OMAR Wis. alii Mnasstohbuctrotas wl aiarore tater ete Bi eens sts Pyare ad BOs 10 137 5 134.0 cL oe er Se eel RE Ee be) 138. 8 ESS 133.0 133-3 4.5 Sees ISi ce sPobenG ae honey toni Bie) 138. 8 133.0 FT coir ts SCR eRe Eat eR Io 134505 kr Auphecstats [ui eeteis ech are 7S APP RCS ott AMER Cee be) 137. 5 TSI IN o.6,.0: hehe) «|| tears lal are ehens 4.4 Ale «Salou SROs a ahs 018) se) FQSAOs il) Maree OA Meei seis eaters) all reset si econwes ote By ais avers aint ceeharerate say aurkeG) & 50 150.0 140. 0 I SACRO ero ORATOR 50 I5I.0 150. 3 140. 5 140. 8 9-5 Gcvonst chsdore se oto RSMAS: 5° 150.0 142.0 One ahaten ap awrentesnohe aut obals 50 FAGLON ALN Vy Mil Revotoctetsts «(ins stsroaha sina OO iaia sistant eee attenaeeaat cial 50 149. 5 TAQ a eta tatede axl score aye rege ae i 8.9 UP RAS cichtn Decor nn 50 TROL SE MARI eos Nerecerttiste crnlistn xix ste sys a Tic kiat Bo (Bika Me Pay oer eS 150 169. 0 148. 0 CALE SO SC IO TEE 150 167.0 168. 0 154.0 151.8 16. 2 Fala bite) Disraloice nate eee a 150 168. 0 2533/5 SAR arnrata abo: SNe) Saran obs 150 DOSS bame arena we iis cecie te lla’ cra rots tances Do igeasl clave era sie Neth oye eee 150 168. 0 168. 0 Sub San ene on 16. 2 Gere aloha erie al gaa centers 150 TOS St Hiei wee |Neaane ate spo Ss ien ais nin clan a 198 Journal of Agricultural Research Vol. XII, No. 4 TaBLeE XII.—Influence of calcium nitrate on the fixation of nitrogen by Azotobacter in sterilized sotl pees ‘Total nitrogen in 100 gm. of dry soil. Nitrogen Culture No. trate = Inoculated. Uninoculated. Leese Age din, ff) 2: ee | Se . dry soil). Found. Average. Found. Average. dey sott Mgm. Mam. Mgm. Mom. Mam Mom TRAGER Aor Mesa cutieaaion ° 133.0 131.0 TRA BoeeRen eo Aas cries ° 133. 6 133513 131.0 T3t43 2.0 Tasos ae eerayes et Siete wigs ° 13353 132.0 Bie Nessie lobia ss csusnend eh oasiols fo) ESS COMM Wenn Ni Geeta esate Mel cea yeeraieyalotare Dee IAA Sreceperanecnesurle fo) 134. 2 TZN Ml nay aver ehoralencremstere cases 2512 Pie hee Vea ees ° ES SUAG Ney MAb alibetetetel ac) eeltctsyskotals necens SA NEE AOS a Sees ro 137.0 135.0 Belcojeedad cans doduagdo ro 137.0 136. 8 134. 0 134. 7 2a Fe ae a Gos ERA Sere le 5 Ae) 136. 5 135.0 BT aatte i senct es ay alia) Stel bast se) TS OOS aA dealt ITH (fou dorsiereden speller edobcheis) siete UIE eae le PE Sa eae sie) 137.0 137.0 Mele le| aleve ola aichate (aye) 4c 2u98 7 a hd 5 ORS DIP RRL EEA SE ike) TO7esS A Ae wm Ueno teva ve liatoisteter cpeierer Hae AH Oh ABO Ba Soly ayaa 50 148. 0 T40. 5 or acvcie CI aL Ap more Aen ate 50 148. 5 148. 5 | I4I.0 140. 7 acs Each citer eysgl slaielorsl ache 5° 149. 0 140. 5 Oa osbocia nlae ela Oblate 50 TASH S| Ml mn nge wp ltarcreteiere tobe aliae sumusvencterate ORGS ohare sein eyacic 50 149. O 148. 5 Siete sisges Alice erm auacanelas | 7.8 OTB R NAA se et ate atch 50 TAGAONI | M Memy Re a allllieeaterets) scroll ai ccmteravatene rc Pays ences oho teie, ea hecho ts 200 173.0 163.0 OF farses teh ys Gerevevar ofc wie’ Shs 200 173.0 73a7 164. 0 163. 8 9-9 Wiawhatees abaya cha jannre Medals 200 174.0 164. 5 Gehl clei iabas| ch chaneds) oe nol oye 200 rig pet tol VEE fe eela gol allsiois ca conla 4 Qe nant aar shave stats t tlevats 200 173.0 | ube Is WH ences cra are nk a onebal ate 9.7 CHE Bar eme picks Ercteois Maer 200 rer Wo | WAS oo analivaaigho gd bc TaBLe XIIL—Influence of calcium nitrate on the fixation of nitrogen by Azotobacter in unstertlized soil Treat- | Total nitrogen in 100 gm. of dry soil. ? ment (ni- Nitrogen Culture No. trate in Inoculated. Uninoculated. Bae yoo seem OF | AA) over dry soil).| Found. Average. Found. Average. EAs Mam. Mam. Mam Mam. Mgm Mam Tne aebetrclewel eters vaNesetatsiots fe) 134.5 134.0 Tapeh schesrateinsiel ssletcistoets ° 136.0 eT Uy TASH 22.2 Dy is Dtsch scat Oech ae NM ale fo) 130. 5 3220 Aowelsdo SOO BO LOOUC Dap fo) Ta SORIA Ns wert all teusksuetckodstetalekaheaonsVelaney ate BS rat eeien etal ater er sb cnetscereve’ ° 135.5 1B ell MAcioce omellod CoualU cide Bie at Zeer cece cece ree scees ° Dee TS Sne al) eet ete Oey Bil USio ouotniord.o-6l loci to's DIGion. Bde alc Cae taNalons Slat svat eheuste Io 138. 5 T2365 Bier ioalys cnt asece si cise Io 138.0 138. 5 133.0 133. 2 5-3 a as TOON Me Age) 139. 0 133-0 Ab OW ob ropa BAUR bUUOOH SE ro Tigkobrelaillih eave Il ciernie6 d.0 olleamion Uolmion Al seiatedeielits. 303} anst Aisi eve atoiats ie) ea 138. 0 Ss todh Balloo ioe 4.8 ads a8an 50K bbUGounUGse se) wes A Whe bo Neos eed do die ave bigot Ce Aaiee EE a vee erie maay tied 50 rit Bi | 140. 5 | Scoso0 cd ooh ono aoou. 50° 152.0 151.5 I4I.0 I4I.0 10. 5 Lagasse nos SUR coca: 50 151.0 I4I. 5 Geer recto ste(s onereneiciec totes 50 UFO NP LN Neo ooo doe ollolao auto mc Gy Bodco bb oonlgconoooe 50 I5I. 5 | ENON Op |\weteyalistene fe Saligeioay bins a4 9. 8 Gadel ae oe dic cos p ac see 50 TieAKo i) ay Be Take aS & 685 calla pgiola oat Fo Halos bogs HoeaEU uC 200 177.0 164. 0 Fpibe Saco OS Banoo COBO 200 178. 0 177.0 165.0 164. 3 L217 Tid oe aoudlwa Gas Sob oNCIoO 200 176.0 164. 0 lo alway Sadlod wa Maae bak 200 MORES WAC TW Soba tals aialfo ols oyaism clos ELGG NAG Beat hep ic bioaeee 200 177.0 | 177.2 SK) 5 olais Solita oig'o'e alsiglc | 12.9 Bape eaten Sis atelier 200 TIS OVP NC Oey eee eee pea a Jan. 28, 1918 Nutrogen-Assimilating Bacteria 199 It will be seen at a glance that a greater relative increase in nitrogen fixation in the presence of nitrates occurred in the soil cultures than on the agar films. But in the latter instance the amount of nitrogen as- similated in the absence of mistakes is far in excess of that assimilated in the soil cultures under similar conditions. The amount of nitrogen fixed in the soil cultures is surprisingly low, but as relative increases or decreases are desired this does not materially influence the results. The influence of sodium nitrate on the fixation of nitrogen by pure cultures of Azotobacter in sterilized and unsterilized soil is brought out very clearly in the figures of Tables X and XI. In both cases, where no nitrate was added, an equal fixation of nitrogen occurred. Where 1o mgm. of nitrate were added to 100 gm. of soil, slightly more nitrogen was assimilated in the unsterilized soil than in sterilized. The reverse seemed to be true when 50 mgm. of nitrate were added. But in the presence of 150 mgm. of nitrate, the fixation by the pure culture in sterilized soil did not increase materially in comparison with that which occurred in the 50 mgm. of nitrate concentration. Evidently the maximum fixation under these conditions had been reached. The gain in the unsterilized soil at the highest concentration of nitrate studied almost doubled the amount fixed in the pure culture. It appears evident that the presence of sodium nitrate causes a greater fixation of nitrogen in unsterilized soil than it does under similar conditions in sterilized soil inoculated with Azotobacter. In the case of calcium nitrate, somewhat comparable results were ob- tained. The fixation where no nitrate was added was equivalent to that obtained in the controls for the sodium nitrate. Where nitrate was added in amounts equal to 10 mgm. of nitrate in 100 gm. of soil, an increased fixation was obtained in the unsterilized soil, but practically no increase occured in the pure culture in sterilized soil. Fifty mgm. of nitrate in 100 gm. of soil produced an increase in fixation. In the highest concentration of calcium nitrate the difference in nitrogen fixed between the pure culture in sterilized soil and unsterilized soil was not so great as in the case where sodium nitrate was used. In the sterilized soil where the two nitrates were present in equal amounts it can be seen that more fixation took place in the presence of sodium nitrate. The difference is not marked, but it exists neverthe- less. It will be remembered that calcium nitrate had a detrimental effect on nitrogen fixation by Azotobacter on agar films. However, in soil cultures this same nitrate stimulated Azotobacter to an increased assimilation of nitrogen. This difference is not suprising as it has been shown repeatedly that bacterial activities in soil and in artificial cultures are not always comparable. From the results of the experiments performed with reference to the influence of nitrates in soil on the fixation of nitrogen therein, it appears 200 Journal of Agricultural Research Vol. XII, No. 4 evident that in pure cultures both sodium and calcium nitrates in the amounts studied produced an increase in the amount of nitrogen fixed. The sodium salt stimulated this process to a slightly greater extent than did the calcium salt. In unsterilized soil nitrates exerted the same action but to a more marked extent. The amount of nitrogen fixed under these conditions was generally in excess of that fixed under similar conditions in sterilized soil inoculated with a pure culture of Azotobacter. Such large relative increases in total nitrogen in the soil in the presence of nitrates would not normally take place under field conditions for here no accumulations of nitrate occur in quantities sufficiently large enough to influence this process. Summing up all the experiments performed in relation to the influence of nitrates on the fixation of atmospheric nitrogen by Azotobacter, it appears that the increase in total nitrogen in the presence of these salts is by no means comparable to the increase in the number of organisms in sterilized soil under the same conditions. An increase in the number of Azotobacter does not mean a parallel increase in the amount of nitrogen fixed. INFLUENCE OF AZOTOBACTER ON NITRATES IN SOLUTION Attention has been thus far directed toward the influence exerted by nitrates on the growth and nitrogen-assimilating power of Azotobacter. The following points are now to be considered: Do the nitrogen-fixing bacteria reduce nitrates to nitrites and ammonia? Is there an increase or decreasé in the amount of organic nitrogen as a result of the presence of nitrate in the medium? Beijerinck and Van Delden (5) found that Azotobacter chroococcum reduced nitrate directly to ammonia. Stoklasa (44, p. 492-503) studied the changes in a nutrient solution containing 0.2 per cent of sodium nitrate inoculated with Azotobacter. He found under an- zerobic conditions that the nitrate was largely reduced to nitrite and ammonia and that a very small amount of organic nitrogen was formed. Under erobic conditions there was more nitrite formed than under anerobic conditions and very little ammonia or oganic nitrogen. He concluded, therefore, that Azotobacter did not fix atmospheric nitrogen in the presence of nitrates. The following experiments were performed in an endeavor to answer the questions raised in the initial paragraph of this section. To Erlen- meyer flasks of 500-c. c. capacity, containing 100-c. c. portions of mannit solution, sodium and ammonium nitrates were added in amounts equiva- lent to 150 mgm. of nitrate in 100 c. c. of the solution. Nine flasks were prepared for each nitrate and the same number for the controls containing no nitrate. The flasks and contents were sterilized at 10 pounds pres- sure for 30 minutes. After cooling, six of each set were inoculated, three Jan. 28, 1918 Nutrogen-Assimilating Bacteria 201 with strain A and three with strain B, and the remaining three were left uninoculated to serve as controls. The flasks were incubated at 28° C. for 21 days. The total weight was maintained throughout the- experi- ment by the addition of sterile distilled water from time to time. At the end of three weeks the contents of each set of triplicate flasks were poured together and 50-c. c. samples drawn for analysis. Nitrate ammonia and total nitrogen were determined as given under “‘Methods.”’ ‘The results are shown in Tables XIV, XV, and XVI. TABLE XIV.—I lea i of Azotobacter on nitrates in solution, giving the quantity of nitrate lost Nitrate in roo c. c. of medium. Strain A. train B. Cul Treatment (ni- S B ah mie se SSS SSS ture! «ce of me A No. dium) Inoculated. | Uninoculated. Inoculated. | Uninoculated. tela Nitrate Aver- Aver-| ost. Aver- Aver-| lost Found.) “) Be: Found.) “) oy Found.) “) fs Found.) *) ee Mogm. | Mgm.| Mam. | Mgm.| Mgm. | Mam. | Mgm.| Mam. | Mgm.| Mom. ae id chavelevareseatecaieta bis Sige S08 rie \ 0.00 BEES See alae ee Bas ie ro-18 | x50gm.of NOs as sodium ni- trates... 3): 80.9 150.4 ie fees 6 \ ie 50-4 \ Pr to-18 |..... do.. ‘iNO -| 80.6 V0. 75 ee 3 150.8 |9—70+05 105.2 |f7054 |\rsr.3 150.8 BO RAS 19-27 | 150Imgm. 3 asammonium nitrates... I00. 3 149-6 \ As as I \ fae: 6 \ cl TQ—27) |e clee donne 102.1 \ror.2 ee ° 149.8 |?—48- 60 130-7 130-9 150-0 149-8 |¢—18-90 @ Strong NO: reaction. bMedium NO: reaction. ¢ Slight NO» reaction. TABLE XV.—Influence of Azotobacter on nitrates in solution, giving the quantity of ammonia produced Nitrogen as ammonia in roo c. c. of medium. Strain A. Strain B. eat Treatment (ni- Hak i trate oy 100 Sa Chee d. | Uninoculated. Inoculated. | Uninoculated. No. dium), Inoculate ninocula : : —$$<$_$_—_—_|——_—— monia |_| monia Aver- Aver- | PTO. Aver- Aver- | _,PTO- Found. age Found. ae duced. | Found. age Found age. duced Mgm. | Mgm.| Mam. | Mgm.| Mgm. | Mgm. | Mgm.| Mam. | Mgm.| Mgm = 2 Liataaret sais. satehis-s ees oss aie Aes ozs me ae _— ea ats ro-18 | r50mgm. of NOs as sodium ni- UrAcera sects nie < 2.00 —.10 2-20 —.I0 \ to-18 |..... do sao NOs 1-80 |f 7°9° 20 \ °5 1.85 { 2.40 |f 2°3° 20 05 2-25 19-27 | rs0 mgm. 3 asammonium ss TItratey, nd. 13-90 ihe \ ; r is . eae \ : ‘ 1927 |....- dOmaedcerrs a 13-95 |f73°97 |\ 13.90 |f73" 9 °7 1 13.80 |f%5 80 1) 13.90 |f73°9° a 202 Journal of Agricultural Research Vol. XU, No. 4 TABLE XVI.—Influence of Azotobacter on nitrates in solution, giving the quantity of nitrogen fixed Total nitrogen in roo c. c. of medium. ee ee Ce Strain A. Strain B. reatment (ni- Cul- trate) (in 100! 5 eisai eee ULE |e. cs af, ane : : No. dium) Inoculated. | Uninoculated. Inoculated. | Uninoculated. NESE SD ney ey ans aoe ee : Aver- Aver- ae Aver- Aver-| fixed. Found. Gee! Found. are Found. ape. Found. wa Mam. | Mgm.| Mom. | Mgm.| Mom. | Mom. | Mgom.| Mom. | Mgm.| Mam. Be Sarees tia agsea alte 2) Mica poly ses ile eA SilNunescoul S225: eee. ollhi 2:08) ausaeae to-18 | rs0mgm.of NOs as sodium ni- sorts | doe | F288 fen ss TES [pease | as if 288 ffer-oe | 498 [fea x0 | 5-80, 19-27 | rs0mgm.of NO3 asammonium Nitrate... 47-00 \ 43-20 \ 48-10 i {33-20 \ 19-27 |..... Oe piiameme'« 46-90 46.95 42-90 43-05 3-90 48-20 ane 42-90 43-05 770 Table XIV showing the effect on the total nitrate content will be discussed first. Strain A differed widely from strain B in its ability to reduce nitrates. It will be noted that strain A reduces nitrate more readily than strain B in the presence of both sodium and ammonium nitrate. In order to determine the nature of the reduction of the nitrates, qualitative and quantitative tests were made. The reduction of nitrates by Azotobacter takes place with the formation of nitrites aS shown in Table XIV. Strain A effected a Strong reduction of nitrate to nitrite with both sodium and ammonium nitrate. Strain B also reduced nitrate to nitrite, but to a lesser degree than did strain A. An inspection of the data in Table XV indicates that the reduction of nitrates ceased with the formation of nitrite, since no appreciable amounts of ammonia were produced by either strain of Azotobacter. In regard to the fixation of atmospheric nitrogen by these strains of Azotobacter, it was found that nitrogen was assimilated both in the presence and absence of nitrate. In the presence of nitrate there was a large increase in the total organic nitrogen. Sodium nitrate stimulated both strains, although strain B fixed the larger amount. Similar results were obtained when the fixation of nitrogen on agar films was studied. In the presence of ammonium nitrate the amount of nitrogen fixed was considerably decreased, but the amount fixed was in excess of the control cultures containing no nitrate. It seems evident that sodium and ammonium nitrate in the amounts studied did not prevent the fixation of atmospheric nitrogen. In fact, the presence of these salts seemed to stimulate the process. 3 Under aerobic conditions both strains of Azotobacter studied caused a reduction in the total amount of nitrate present in the solution. This reduction may be accounted for in two ways: (1) The reduction of nitrate to nitrite and (2) the assimilation of nitrate by the organisms. Practically no ammonia was formed under the conditions of these experi- ments. These results agree with those of Stoklasa. However, in con- Jan. 28, 1918 Nitrogen-Assimilating Bacteria 203 i un Ra II aid Da NRA ME Sg Pe ee EE RE trast to the work of Stoklasa, both strains of Azotobacter assimilated more atmospheric nitrogen in the presence of nitrates in solution than in the absence of these salts. INFLUENCE OF NITRATES ON THE PRODUCTION OF PIGMENT BY AZOTOBACTER It has already been noted in the experiments dealing with the effect of nitrates on the fixation of atmospheric nitrogen on agar films that nitrates favor pigment production. This was true in the case of both strains of the Azotobacter. Moreover, it has been observed by other investigators that Azotobacter when grown in the presence of nitrate will produce a darker pigment than when grown in its absence. Beijerinck (4, p. 575) states that Azotobacter in pure culture will form a dark-brown pigment in the presence of glucose and a small amount of nitrate. Sackett (43) found that nitrate caused an increase in pigment production by Azotobacter. In media without the nitrate the pigment formation was materially decreased and in some cases practically eliminated. He also noted that the amount of nitrate present has a direct influence on the intensity of the pigment formation. He found that when sodium nitrate was added to a suitable medium to give a content of 0.0, 0.01, 0.03, 0.05, 0.08, 0.1, 0.3, and 0.5 per cent, with glucose used as the source of energy, the organisms produced pigment. Streak inoculations were made, and after 14 days’ incubation he found that the maximum of color was obtained at 0.05 to 0.08 per cent and that greater concentrations did not increase the intensity of the brown-black pigment. From his results it is evident that sodium nitrate caused an increase in pigment formation by azotobacter. In order to determine the possible effect of potassium, sodium, and cal- cium nitrate on pigment formation with strains A and B, the following experiment was performed. Under normal conditions on mannit agar free from nitrate strain A produced little or no pigment even after three weeks’ growth. At the end of this time dirty-yellow streaks occurred throughout the growth, but no brown pigment was produced. However, with strain B at the end of two or three weeks a decided brown to brown-black pigment was produced in the absence of nitrate. Agar slope cultures containing increasing amounts of potassium, so- dium, and calcium nitrate, as indicated in Table XVII, were prepared. These were inoculated with both strains of Azotobacter and incubated at 28° C. for 10 days. Daily observations were made for first evidences of pigment formation. In some of the cultures of strain A growing on media containing calcium nitrate this pigmentation was observed as early as 48 hours subsequent to inoculation. The following day pig- mentation developed in strain B. The cultures on the potassium and sodium-nitrate media began to show evidence of pigmentation in four to six days. ‘The final results, obtained after 10 days’ incubation, are found in Table XVII. Vol. XII, No. 4 Journal of Agricultural Research 204 ‘OGh ae Sp ee ee optics | cc umosg ye fo poetq-umorg foo Opa ce:| > eeu op?***: oof 8 “yovjq-umorg [oc **smMorq yreq [oo Ope cee eee Ops ioe as See Ope eae ope: 00z L “umorq yseq fo oprtfeccctsssess opts :|cer ss trmosq ye foo umosq umipayy [oo opt ae 9 ‘od ear cetinn cn) Chor curs cc op PerececiG| KO.Choror dros) caacecap op Ghee tedes|\apmiic mur elalleNataynrens op ane, altetie|| qgielelysiane ef telme ince op sie) aperie||\eualeteire UMOIq MN Ipeyl OOE ¢ ‘od BMoveelaletunsia: ia seia op @) ojfeis. e||\eMe (e\ | ¢ (agama (0/tauie) = 4a op ee eeelensae UMOIG mInIpeyl cece eee uMmoiq SVT Powhou phonon Cho op SeceOson oS 54 “umorq umIpeyy fo oprtfcce teeessopretesfesrss es mMmorg UBT | ope fice umosq 7Srq | Sz ¢ “MOG Beg, |) -- Ops ar -Hee an 3 see acops es * "|r: uMoIq 181 AIDA “+ [MOIq WySt AIDA -*TMOIg 131] AIDA or Zz “umorq wnTpayy | °° umorq wmrpey [°° *° umes ummpayy [ct cauony foe amo fos auoyy | © ; = “ud TT ‘ayesyIM TIMED ‘ayeIIM UANIpog ‘aye1]10 MANIsse}0Og ‘ayeqIT MINED ‘ayeIqIa mENnIpog ‘ayeqIa mIMIsse}Og (mmtpeur jo > ‘2 | ‘ON "@ BLES : “y bres cor ur | ain} “HOIJEUTIO; JUSUITId UI asvaIOUT ATIL[OW, SS as a a rt a ee «ee a ee ee SSS ee sajongopozy ur quambrd fo uoynmsof ay} Uo sayoaziu fo aouanyuJ—JTIAX Hav Jan, 28, 1918 Nutrogen-Assimilating Bacteria 205 A general idea may be gained from Table XVII concerning the relative increase in pigment formation in the presence of the nitrates. A study of the table gives a fair idea of the relative differences in pigment pro- duction. Very interesting results were obtained with strain A. It will be seen from Table XVII that no pigment was produced in the control culture after 10 days, while in the presence of nitrates pigmentation was noted. The intensity of the pigment varied with the increase of nitrate up to 150 mgm. Beyond 150 mgm. there was no increase. Potassium and sodium nitrate did not exert such a decided influence on pigment production as calcium nitrate. The latter salt produced an intense dark-brown to brownish-black pigment. In the case of strain B the influence of nitrate was not so pronounced since this strain normally produced considerable pigment in the absence of nitrates. Potassium and sodium nitrate caused a slight increase in pigment formation. Here, again, the calcium salt brought about most pronounced increase. However, the relative increase in pigment forma- tion in strain B was not so pronounced as in strain A. Where the nitrate was present, a much more spreading growth was obtained. A heavy bacterial growth accumulated at the base of the slope except in the two cultures in which the highest concentrations were used. In the latter instances the accumulation was less than those in cultures growing on media containing no nitrate. Although the original inocula- tion could not be made absolutely uniform, so far as number of organ- isms was concerned; nevertheless it was evident that on those slopes containing 10, 25, 50, and 100 mgm. of nitrate in roo c. c. of the medium a much more abundant growth was obtained than on those slopes free from nitrate. Here, again, it is seen, in a rough, comparative way, that the smaller amounts of nitrates caused an increase in the number of Azotobacter. The results of this work on pigment production are quite in accord with those of Sackett. Potassium, sodium, and especially calcium, nitrates in varying amounts increase pigment formation by Azotobacter with an increase in nitrate concentration. ‘This effect is especially marked in strain A, which under normal conditions does not produce any pigment. INFLUENCE OF NITRATES ON THE FORMATION OF VOLUTIN BODIES IN AZOTOBACTER The presence of volutin bodies, or metachromatic granules, in Azoto- bacter has been shown by Bonazzi (7). These substances, according to Meyer (34, p. 238), are reserve food materials other than fat droplets, glycogen, and similar substances reacting with iodin stain which occur in the cytoplasm of the cells of various bacteria. With Millon’s reagent they give no reaction. He believes that these bodies are composed of nucleic-acid compounds, but are not nuclear proteids. 206 Journal of Agricultural Research Vol. XII, No. 4 In connection with the foregoing investigations concerning the influ- ence of nitrates on pigment formation by Azotobacter, it was thought that some results of cytological interest might be obtained in regard to the effect of varying amounts of nitrates on the volutin bodies. Slope cultures of mannit agar were prepared containing the different nitrates as indicated in Table XVIII. These slopes were inoculated with both strains of Azotobacter and incubated at 28° C. for 10 days. At the end of this time each culture was stained and examined micro- scopically. The following method was used for demonstrating the presence of the volutin bodies. The organisms to be examined were air dried on a glass slide and then fixed in the flame of a Bunsen burner. The preparation was then flooded with a 1 to 10 aqueous solution of methylene blue (Merck’s) prepared by adding ro c. c. of a saturated aqueous solution of methylene blue to 90 c. c. of distilled water. The stain was washed off after five minutes with a 1 per cent solution of sulphuric acid and immediately rinsed in distilled water. The prepa- ration was dried and examined with the oil-immersion objective. The volutin bodies appeared within the cytoplasm as very dark blue dots, the outline of the cell wall was a lighter blue, while the cell net work was stained a very light blue. Guignard’s stain! was also used to demonstrate the presence of the volutin bodies. Fresh smears on a giass slide were fixed over 10 per cent osmic acid for three minutes. The preparation was then air-dried and fixed te the slide by rapidly passing the latter a few times through a Bunsen burner. The preparation was covered with the stain which was allowed to react for five minutes. The stain was then washed off with distilled water, dried, and examined with the oil-immersion objec- tive. The outline of the cell as well as the net work within-was stained light purple. The granules within the cytoplasm were a reddish purple. The results are given in Table XVIII. TaBLeE XVIII.—Influence of nitrates on the formation of volutin bodies in Azotobacter in IO days ‘Treat- Strain A. train B. ment Culture |(itrate No 1n I00 (s c.c. of | Potassium Sodium Calcium Potassium Sodium Calcium me- nitrate. nitrate. nitrate. nitrate. nitrate. nitrate. dium) Mom. Dae es o | Present.@....| Doubtful....| Doubtful....| Present@....| Doubtful....| Present. Beikeseeios ide) Baar do.¢@,.... Present @,...} Present 2 a, Do. @ Dataneaa Baoan dog ae. dois eas does. oslo doi@cn|. 20 GO. Do. @ PBS Satine eh| esaon Goi alicace do.>.....] Doubtful rf H Do.2 BeltaMacies ¢ TOO! |e aisle GO. Gye ssloe he dolore Ee Present @,...]..... Goo ees doo: Do.@ Oevcniwnte ao) IR A AE Cola Hel ere CGO ee ealaneien Goseeeleteee Cok es ee doe: Do.d Pow RAEI Oe AGO}. Gora each Ra Co Rt ae ete Bad a (ole W ye Cmwel ie ste oko Oat 8 aia) ta ie ovo. Do.2 Scere 300 ||..04). ole pOs Geel lagen ro (oat ata Pa Ce Foy A ea (ota eee donner Do.> @ Representing an approximate average of two volutin bodies per cell. bd Representing an approximate average of four volutin bodies per cell. Guignard’sstain. Fifty c. c. of 2 per cent fuchsin in 1 per cent acetic acid; 4oc. c. of 0.2 per cent methyl green in x per cent acetic acid; 1 c. c. of glacial-acetic acid. Distilled water was used in making the 1 per cent acetic-acid solution. Jan. 28, 1918 Nitrogen-Assimilating Bacteria 207 It will be seen that all three nitrates exerted considerable influence on the formation of volutin bodies. Not only was the number of bodies increased, but also the size. The relative increase in size of the granules was much more marked than was the numerical increase. In Azoto- bacter cells grown on mannit agar containing no nitrate the number of volutin bodies in each cell averaged about two; in the presence of nitrate four to five volutin granules were found. The greatest increase in num- ber, as well as size, occurred where the nitrate concentration was highest. With both strains sodium nitrate apparently caused the greatest increase. This was true in the lower as well as in the higher concentrations. The volutin bodies in strain B seemed to respond to the presence of nitrates more noticeably than did those of strain A, especially in the presence of potassium nitrate. It is evident that nitrates of potassium, sodium, and calcium cause an increase in the number and size of volutin bodies in Azotobacter cells. Do these salts tend to hasten the appearance of these bodies, or do they at first retard their development? The following experiment was carried out in an endeavor to determine this point. Only sodium nitrate was used, since this particular salt proved most beneficial to the forma- tion of volutin bodies. Agar slopes were prepared containing the different amounts of nitrate as indicated in Table XIX. The cultures were incu- bated at 28° C. and examined daily for the presence of volutin bodies, The methylene blue—1 per cent sulphuric acid—method of staining was employed. The results of the experiment are given in Table XIX. TABLE XIX.—Influence of sodium nitrate on the rate of formation of volutin bodies in Azotobacter Nitrate in roo c. c. of medium. Time Strain A. Strain B. SREEEAEM Glas DAlEl Waka Gi fi Gama oMgm. | 25 Mgm! | 10o Mgm.| 300 Mgm.| oMgm. | 25 Mem. | too Mgm. | tion Day Eases Soe Absent...| Absent...} Doubtful.| Doubtful.| Absent...) Absent...) Doubtful.) Dbtful. 2h ecloe LeSPOAe Present @,| Present @.| Present@,|...do.0...|...do..... Present @.|...do.>...| Do.b. Be theres sae v0rs ...do.¢.,..|...do.¢,...]...do.¢....|...do.5...| Present@.)...do.b...)...do.b...| Dob. greta es aie .-.do.4....|...do.@,...]...do.0...|...do.0...)...do.2.... (do; 8's ace Do.b. @ Representing an approximate average of two volutin bodies per cell. b Representing an approximate average of four volutin bodies per cell. A study of Table XIX shows that it is rather doubtful whether the nitrate present tended to hasten the appearance of the volutin bodies. No convincing evidence has been presented for or against this statement. No granules were seen, in the first day’s growth of strain A, although the next day they were present in all four cultures. In strain B more convincing proof is furnished that the sodium nitrate hastened the appearance of these reserve food substances. The volutin bodies were not present in the control and lowest nitrate concentration cultures the first day, but they were very noticeable in the culture containing the highest concentration of nitrate and doubtful in the remaining one. On the second day volutin bodies were present in all cultures grown on 208 Journal of Agricultural Research Vol. XII, No. 4 nitrate media, while the control culture was still free from them. The third day showed the presence of volutin bodies in all four cultures. Strain B offers the better proof that sodium nitrate tends to hasten the appearance of volutin bodies in the cells of Azotobacter. Further experiments were not made in an endeavor to determine what influence nitrates might have on the cytology of the Azotobacter cell. The brief studies reported here were made in connection with the pigment forma- tion experiments, but do not bear any particular relation to them. The increase in number and size of volutin bodies may bear some relation to the increased amount of nitrogen fixed or assimilated by Azotobacter in the presence of nitrates. INFLUENCE OF NITRATES ON BACILLUS RADICICOLA INFLUENCE OF NITRATES ON THE GROWTH AND REPRODUCTION OF BACILLUS RADICICOLA IN STERILIZED SOIL One hundred and fifty gm. (dry weight) of the soil were weighed into 500-c. c. Erlenmeyer flasks and the nitrates added as indicated in Tables XX-XXII. Duplicate cultures for each amount of nitrate were prepared. One per cent of mannit (in 5 c. c. of distilled water) was also added. The flasks were kept at room temperature for one day and the contents then thoroughly mixed. The flasks were steri- lized at 15 pounds’ pressure for three hours. Upon cooling they were inoculated with 5 c. c. of a suspension of Bacillus radicicola in sterile distilled water. The number of bacteria in the inoculum was deter- mined. The moisture content was then approximately 18 to 20 per cent. The flasks were incubated at 28° to 30° C. and mannit-agar plates poured at the end of one and two weeks. The results of these experiments are given in Tables XX, XXI, and XXII, in which each figure represents an average of duplicate plates. TABLE XX.—Influence of potassium nitrate on Bacillus radicicola in sterilized soil Treat- | Number of organisms in r gm. of dry soil. ment (ni- Culture No. trate in 7 aoe pees After 1 week. Relative. After 2 weeks. | Relative Mgm 2 Per cent | Per cent ML at sie cars ro) 10, 670 680, 000 { 8, O15, 000 \ Phe ni srern tc atc fo) 10, 670 825, 000 a 7, 000, 000 oe Bry eiarayayie oars ere 10 10, 670 2, 195, 000 " 14, 600, 000 ie Bes a TeF ee): OF 10 10, 670 3, 410, 000 37 II, 050, 000 7 (Heer Ap 2 10, 670 3) 900, C00 { 15, 400, 000 Gee Sieneteraisiecs 25 10, 670 3; 885, 000 St7 14, 800, oo |f 797 Fore Sint Pecan 50 10, 670 I, 555, 00 Ba { II, 500, 000 \ a ees pS 50 10, 670 T, 585, 000 i 14, 400, 000 73 Cay al SSN 100 10, 670 445, 000 a { 2, 680, 000 p DOW fete. MRS 100 10, 670 320, 000 5 3, 290, 000 4 4 MR ee Oe RAST 150 10, 670 375) 000 6 { 560, 000 8 IPO OOG ASOD 150 10, 670 330, 000 4 790, 000 a TSP eye cis ciate 200 10, 670 135, 000 fs go, 000 as A 83 emer eG 200 10, 670 170, 00O a ; Tee eer. eee 300 10, 670 45, 000 { 25, 000 1 OG ara ic 300 10, 670 50, 000 6.3 30, 0CO "ae a Contamination. Jan. 28, 1918 - Nitrogen-Assimilating Bacteria 209 TABLE XXI.—Influence of sodium nitrate on Bacillus radicicola in sterilized soil Culture No. seers eres eee eereeoers ee eevee ecose Treat-_ ment (ni- trate in At begin- ning. T5, 500 15, 509 152,500 15,500 15,500 15, 500 T5, 500 15, 500 T5, 500 T5, 500 15, 500 I5, 500 15, 500 T5, 500 15, 500 T5, 500 Number of organisms in 1 gm. of dry soil. After 1 week. I, 500, 000 I, 250, 000 2, 560, o00 3, 000, C00 6, 150, 000 52 375, 000 4, 850, 000 51 570, 000 2, 000, 000 I, 850, 000 I, 060, 000 835, 000 760, 000 725, 000 250, 000 365, 000 Sener oe eee ere ee eee on” Relative. Per cent. 100 201 418 378 140 69 54 22 a Contamination. —_——s Seo ees eee oes 5 = After 2 weeks. 6, 750, 000 51 959, 900 10, 000, 000 12, 500, 000 14, 650, 000 15, 700, 00O a 8, 500, 000 I, 520, 000 I, 650, 000 850, cco 940, 000 500, 000 620, 000 150, 000 210, 000 Relative. Per cent. 100 177 240 134 25 14 8.8 2.8 ——e—aew— Oe eer eee ee eee ee eee” TaBLE XXII.—Influence of calcium nitrate on Bacillus radicicola in sterilized soil Treat- Number of organisms in 1 gm. of dry soil. ment (ni- Culture No. trate in , | | | seoem ct At be aa After 1 week. Relative. After 2 weeks. | Relative. Mgm. Per cent. | Per cent SER EPCRRSTCEBOORC ° IO, 00O 960, 000 Baa | 4,675,000 | 104 Bis ve. arwisiebehenre fo) IO, 000 850, 000 4, 590, 000 Be ona dean ese Io IO, 000 3, 650, 000 419 6, 000, C00 | coh 7 hae ae aa Io I0, 000 3; 940, 000 5) 450, 000 tao dp ano 25 IO, 000 5, 500, 000 A 10, 650,000 || _, 6 6 74 eS 274 PEt ree 25 10, 000 , 700, 000 14, 700, 000 (aA 50 10, 000 4, 000, 000 : { 9, 350,000 || J. a GPA eRe Hise 5° 10, 000 3; 500, 000 ne 8, 670, 000 | 95 Qe aide ase aes 100 10, 000 I, 200, 000 ay { I, 500, 000 | TOV aye a cosiatsieste 100 10, 000 2, 050, 000 I, 750, 000 | 35 ERA a Ne ny deve 150 10, 000 865, 000 aan { 765,000), BRIN OE HREOC Hae I50 10, 00 I, 050, 000 800, 000 7 DF i esciaieaa'e 200 10, 000 375, 000 | { 350, 000 ‘ TA ore y aie ever asi 200 IO, COO 260, 000 35 300, 000 | 7: Tse cy tea Balas 300 10, 000 35; 000 | an { 25, 000 | as TOW Wevelresste vieheds 300 IO, 000 47, 000 5 40, 000 ‘ An inspection of all three tables reveals two marked differences from the results obtained in similar work with Azotobacter. First, it will be noted that nitrates do not appear to exert such a marked stimulating effect with B. radicicola as with Azotobacter. The numerical increase due to the presence of the nitrate is clearly shown in the percentage columns. Second, it will be noted that B. radicicola does not seem to be so sensitive to higher concentrations of nitrates as does Azotobacter. In all instances at concentrations equivalent to 300 mgm. of nitrate in 210 Journal of Agricultural Research Vol. XII, No. 4 roo gm. of soil the legume organisms were still alive, although present in numbers far below those of the control cultures. In all Azotobacter cultures no organisms survived this concentration. No one nitrate produced an excessive stimulation in comparison with the others. The calcium salt present as 150 mgm. of nitrate in 100 gm. of soil at the end of the first week gave the greatest stimulation for con- centrations of that amount. However, at the end of the second week this concentration had caused a marked decrease in the number of or- ganisms. In the case of all three nitrates the concentration representing 25 mgm. of nitrate in 100 gm. of soil produced the greatest stimulation. This resulting stimulation also held true throughout the second week. The decrease in number below those of the control cultures, due to in- creasing concentrations of nitrate, began first in the presence of potassium nitrate at 100 mgm. of nitrate per 100 gm. of soil, then with sodium nitrate at 150 mgm., and lastly with calcium nitrate at 200 mgm. But the number of organisms present in the soil cultures containing sodium nitrate in amounts equivalent to’ 100 mgm. and calcium nitrate at 100 mgm. at the end of the second week was below those of the control cultures. | It therefore appears from these results that small amounts of potassium, sodium, and calcium nitrate stimulate the reproductive activity of B. vadicicola. Concentrations of nitrates greater than those amounts which produced maximum stimulation cause a decrease in the number of or- ganisms. The highest concentration of nitrate studied did not entirely prevent the growth of the bacteria, but it reduced the number of organ- isms far below those contained in control cultures where no nitrates were added. Ammonium nitrate was also employed. The soil cultures were pre- pared as already described and inoculated with B. radicicola. The cul- tures were incubated at 28° to 30° C. and counts were made at the end of one and two weeks’ time. The results of the study with ammonium nitrate are given in Table XXIII. TABLE XXIII.—Influence of ammonium nitrate on Bacillus radicicola in sterilized soil Treat- Number of organisms in 1 gm. of dry soil. ment (ni- Culture No. trate a Too gm. 0 At begin- : : cy ene parte After 1 week. Relative. After 2 weeks. | Relative. Mgm Per cent. Per cent. Tie ro) 10, 000 850, 000 saad { I, 365, 000 ee a: ° TO, 000 765, 000 I, 400, 000 Bes 25 10, 000 2, 500, 000 4 { 5, 060, 000 8 Abs oe 25 IO, 000 3, 050, 000 343 4, 320, 000 33 Sea 100 IO, 000 I, 350, 000 148 I, 030, 000 ‘ Gre. I00 10, 000 I, 050, 000 950, 000 7 Cienae 200 IO, 000 700, 000 84 635, 000 4 haa: 200 IO, 000 655, 000 605, 000 5 Jan, 28, 1918 Nitrogen-Assimilating Bacteria 211 From the results as a whole it appears that it is the nitrate radical and not the combined salt which causes the increase in the number of B. radicicola when small amounts of nitrates are present. A stimulation occurred, resulting in an increase in number which is quite comparable to that obtained with potassium, sodium, and calcium nitrates. The highest concentration of ammonium nitrate used did not appear to have such an inhibiting effect as did the corresponding concentrations of the three other salts. . : Throughout the work with B. radicicola in sterilized soil compara- tively low numbers of these organisms were found. Whether or not this depression was due to toxic substances formed as a result of steriliza- tion can not be stated. If this decrease in numbers as a result of the presence of toxic substances is true, it is very evident that the detrimental effect had not become materially lessened at the end of the incubation period. However, in any event the validity of the outcome is not im- paired, since comparative and not absolute data are of importance and since in all probability the same conditions obtained throughout the cultures. It seems certain from the results of these studies on the effect of potassium, sodium, calcium, and ammonium nitrates on the growth of B. yadicicola in sterilized soil that small amounts of nitrate stimulate the growth of the organisms. It is also shown that B. radicicola is much more resistant than Azotobacter to higher concentrations of potassium, sodium, calcium, and ammonium nitrates. INFLUENCE OF BACILLUS RADICICOLA ON NITRATES IN SOLUTION The series of soil culture experiments just discussed served to give an idea concerning the effect of nitrates on the legume organism. It was found that in small amounts nitrates stimulated the bacteria to increased reproduction. But no study was made as to theeffect of Bacillus radicicola on the nitrate. Does the organism break up the nitrate, reducing it to nitrite or ammonia? Does it cause any loss in nitrate when grown in a solution containing that salt? Beijerinck (2, p. 762) as a result of physiological experiments with B. radicicola, states that the organism does not reduce nitrate. Prucha (41) also states that B. radicicola does not reduce nitrates. However, Zipfel (49) found that B. radzcicola will reduce nitrates to nitrites but not to ammonia. , The following experiments, somewhat similar to those already cited in relation to Azotobacter, were carried out in an endeavor to answer these questions. To twenty 500-c. c. Erlenmeyer flasks containing 200 c. c. of mannit solution, potassium, sodium, calcium, and ammonium nitrates were added as indicated in Tables XXIV, XXV, and XXVI. Quadruplicate flasks were prepared for each concentration of nitrate and for the control cultures without nitrate. The flasks and contents were sterilized at 212 Journal of Agricultural Research Vol. XII, No. 4 10 pounds’ pressure for 30 minutes. After cooling, two of each set of four flasks were inoculated with 5 c. c. of a suspension of B. radicicola in sterile distilled water. The remaining two flasks of each set (uninocu- lated) served as controls. The flasks were incubated at 28° C. for 21 days. The total weight of the flasks was maintained throughout the incuba- tion period by the addition of sterile distilled water from time to time. At the expiration of the period of incubation the nitrate, ammonia, and total nitrogen contents were determined as given under “Methods used in experiments.” The contents of the duplicate inoculated flasks were poured together and 50 c. c. samples drawn for analysis. The same procedure was followed in the case of the uninoculated flasks. The results are given in Tables XXIV, XXV, and XXVI. TABLE XXIV.—I pfluence of Bacillus radicicola on nitrates in solution giving the quantity of nitrate lost Nitrate in roo c. c. of medium. Cul- C ture| ‘Treatment (nitrate in 100 c. c. of medium). Uninoculated. Inoculated. asa oO. . Found. | Average. | Found. | Average. Mam. Mam. Mam. Mom. Mgm. niall ilo) a toy eM Mort Rene Gieees 6 CIA I EIA tole oO. 00 oO. CO Os ee (cVO pe aise Mit men stdin TAs Lie aes . 00 eee . 00 Ons Onee omgm. of NO, as potassium nitrate .| 151. I1I7.0 ial = ..do : Sette ter fesse : SAIC Holo Ha aoWad 6 aa Z \esr. . ee fe) bony, eas Iso mgm..of NO, as sodium nitrate. .| 148.8 114. 3 : Ec os eee St Ae nM casale apie sheet 8 \rg8. 8 oe : bina, = i eee Ico mem.of NO, as calcium nitrate.) 154.8 6.6 Bel telat ae SHOR Ur i Rea 6 hiss. 2 { 16.8 \ 727 aR os 9 | r50mgm.of NO,asammonium nitrate] 151. 4 142. 6 EG) ices or GOs Vos. ah wu ce Mee ee ee ere TETNOMuM ens finde: O° fot ise cea TABLE XXV.—I nfluence of Bacillus radicicola on nitrates in solution giving the quantity of nitrogen as ammonia formed Nitrogen as ammonia in roo c. c. of medium. fede ees EP SN thor Cul- P : : genas ue Treatment (nitrate in 100 c. c. of medium). Uninoculated. Inoculated. ammo- oO. nia formed. Found. | Average. | Found. | Average. Te a ed Sa i Li INONE est brscnseetn ee \ 73: 92 { Bs } Se eae Jan. 28, 1918 Nutrogen-Assimilating Bacteria S15 TABLE XXVI.—Influence of Bacillus radicicola on nitrates in solution giving the quantity of nitrogen fixed Total nitrogen in roo c. c. of medium. Cul- f : Nitro- fare Treatment (nitrate in 100 c. c. of medium). Uninoculated. Inoculated. gen oO. ed. Found. | Average. | Found. | Average. —§——— | | | None... aa ecenene 2. 40 3 Beats wae) gusgeantenent IN Soi} 24s S30 ]} 3-40] 095 150 mgm. of NO, potassium nitrate..| 18. 00 8. : ae Shisha 3yae's ; eth Se ee ee 17.90 \ 41.99 sa \ ee sg 5 | 150 mgm. of NO; sodium nitrate....| 16. 80 6 1g. 30 : Gales OS Bode Belts DOD TD BDEO De tpare noe 17.00 |f 779° 1) 10. 20 |f 7% 25 | 2-35 r15o'mgm. of NO, calcium nitrate...| 1x 6 Oder oa Bee: a ET 14 80 |t 339° rk go |t x4 65 | +75 9 | 150 mgm. of NO;ammonium nitrate.| 40. 50 { 4I. 30 \ TOM erereras Oa oa ncadlonoaK Ac OOD OMe Ono Sen aD 4I. 20 40. 85 41.70 at 65 The data in Table XXIV show that a rather large reduction in the total nitrate content took place. This reduction varied rather markedly among the four different nitrates studied. The greatest reduction oc- curred where calcium nitrate was used. Potassium and sodium were next in order; the loss was almost the same for both salts. Ammonium nitrate was last with but a very small comparative reduction in total nitrate. The question arises as to whether the nitrate is reduced to nitrite, ammonia, or elemental nitrogen or whether the reduction in amount is due to a natural assimilation of the nitrate by the organisms. ‘The first possibility was precluded when qualitative tests for nitrites were made and none found. Table XXV reveals the fact that no ammonia was produced. Table XXVI shows no loss in total nitrogen. Therefore it seems obvious that reduction in total amount of nitrate present is brought about by the assimilation of those compounds by the organisms. An inspection of Table XXVI, which gives the results of the total nitrogen determinations, shows that a slight fixation of atmospheric nitrogen took place. This fixation is entirely possible, as will be shown later when the influence of nitrates on the fixation of nitrogen is taken up. In the presence of potassium, sodium, and ammonium nitrates the amount of nitrogen assimilated is somewhat decreased. But in the case of sodium nitrate a large increase in the amount of total nitrogen seems to have taken place. This is interesting in the light of results to be presented later. . From the results of the work on the effect of B. radicicola on nitrates it may be concluded that the organisms do not reduce the nitrates to nitrite or ammonia or elemental nitrogen under aerobic conditions. 214 Journal of Agricultural Research Vol. XII, No. 4 INFLUENCE OF NITRATES ON THE FIXATION OF ATMOSPHERIC NITROGEN BY BACILLUS RADICICOLA : The ability of B. radicicola to fix atmospheric nitrogen in the absence of the host plant has been studied by numerous investigators. From the results of their work it seems fairly probable that the legume organ- ism can fix nitrogen to a slight extent when growing in a nonsymbiotic state. Beijerinck (3) was one of the earliest to make a study of the possible fixation of atmospheric nitrogen by B. radicicola under these conditions. He found that a small quantity, 0.91 to 1.82 mgm. of nitro- gen was fixed per 100 c. c. of the medium. Prasmowski (39, p. 55) and Berthelot (6) concluded as a result of their experiments that when the organism was grown outside the host plant the gain in nitrogen was small. The greatest gain in nitrogen was found by Mazé (32) who re- ported an increase of 23.4 mgm. of nitrogen per 100c. c. of the medium in 16 days. Lewis and Nicholson (30) found by incubating the cultures for a considerable length of time that a large increase in fixation occurred. Bottomley (8) found that a pure culture of B. radicicola fixed approxi- mately 1 mgm. of nitrogen in 15 days. Fred (17) made a study of the possible fixation of nitrogen by the legume organism and found that it fixed approximately 1.2 mgm. of nitrogen in 100 c. c. of the medium. He found that on agar films a greater fixation occurred than when the organisms were grown in a liquid medium. A few investigators, however, found that no increase in nitrogen occurred when B. radicicola was grown outside the host plant. Frank (16) states that in a nitrogen-free medium the legume organisms did not fix enough nitrogen to be accurately measured. Immendorf (25) also found no increase in nitrogen when pure cultures of B. radicicola were grown in soil containing a nitrogen-free solution. It will be seen that the majority of investigators, especially the more recent ones, found that a slight amount of atmospheric nitrogen was fixed or assimilated by B. radicicola when grown outside the host plant and on a medium suitable for its development. i It has already been shown that nitrates cause an increase in the num- ber of B. vadicicola when grown in pure culture in sterilized soil. Does such an increase in the number of organisms necessarily mean an 1n- creased fixation of nitrogen? ‘Three experiments using agar films were carried out in order to determine this point. Erlenmeyer flasks of 1-liter capacity containing 100 c. c. of mannit agar were used. Before the medium solidified, the nitrates were added in the proportions indi- cated in Table XXVII. Six flasks for each different quantity of nitrate were prepared, except in one case, as shown in Experiment II. The flasks were plugged with nonabsorbent cotton and sterilized at 10 pounds’ pressure for 30 minutes. After cooling, three of each set were inoculated with 5 c. c. of a suspension of B. radicicola in sterile distilled water. The organisms had been growing on mannit agar at 28° C. for six days. The flasks in Experiments I and IiI (Table XXVII) were incubated at Jan. 28, 1918 Nitrogen-Assimilating Bacteria 215 Geri ois SN aD gg se ee 28° C. for three weeks and those in Experiment II for two weeks. The moisture lost by evaporation in both inoculated and uninoculated flasks was replaced from time to time by the addition of sterile distilled water. At the expiration of the incubation period the total nitrogen was deter- mined as given under ‘‘Methods used in experiments.” The results of the experiments are given in Table X XVII. An inspection of the data reveals the fact that B. yadicicola in pure culture fixed a small amount of nitrogen when growing in a nonsymbiotic ‘state with no nitrate present. In the presence of nitrates there was an increased fixation. Although the increase in total nitrogen is small, because of the number of determinations made, it may be considered as positive. The potassium and sodium salts seemed to be more effective than the calcium nitrate, with one exception (Table XXVII, Experiment I). It will be remembered that the latter salt appeared to depress nitro- gen fixation by Azotobacter and the two former somewhat to favor it (p. 194-195). TABLE XXVII.—Influence of nitrates on the fixation of nitrogen by Bacillus radicicola, giving the increase in nitrogen EXPERIMENT I Total nitrogen in roo c. c. of medium. fie Sei ee eee aoa Treatment (nitrate in 100 c. c. of medium.) Uninoculated. | Inoculated. Sky Found. |Average.| Found. | Average. Mam. Mogm. | Mom. ere Mgm. Tt {fal foytlS 5 AY Ghee hte ba oe oO ak OOo 4. 5 tia PMNS sete GLO Rahs BI id Be Oeics CUO Uns 5 4.4|¢ 4.45 |} 4-6 |¢ 4.60 0. 15 Brivsetes GOR Fae eis beeheraei Serene 4.4 1 Laer Bas 4 | 75 mgm. of NO, as sodium nitrate ...| © 8.7 II. 9 Ne ee finan aeelsecda ey =n ety Vas = ies Fs 8.7 | 8.70 |) 11.8 |pIt. 75 3. 05 (Olle aie AG (ce Geren eeocible dlc ees be eco meigioianig 8.6 11. 6 7 | 150 mgm. of NO; as sodium nitrate..) 12.5 14.9 | Sell stare CL ae ares ate beatles iene cue nye: sis 12.7 |}12.60 |) 14.6 |¢14. 70 2.10 ra ea arte 7 bs lgotee(ho Alas Wiebe iran eee hee ear ene cin | AaRa RoS \\ 14.7 10 | 75 mgm. of NO, as calcium nitrate .. 8.8 1233 eB lois (si OIE OMI ee ea SEO SC ARN oie 8.9 |} 8.90 |) 12.8 |/ 12. 40 3. BO il WP (OE SHA TO Sine ei OMe Cee bia Hiar ste g. 0 12. I 13 | 150 mgm. of NO,as calcium nitrate ..| 13.3 |{ 14.5 BAL sevsoene Free SP 2 ci ete Oe ets eee cea 13. I [13.20 |} 14.0 |f14. IO 0. 90 i behead (6 (0), Saisie Lda Weis ao. aig cloroscaneac mio 13. 2 13. 8 TRS a ea | an ene a LS eR a EXPERIMENT II Ni | 10 A POE eRe ac oie clnj- nee esis gs + = . go : ; j hs ee NHR IE Seah ay Mee chain, o804S, sv ecailee 2 a \4 9° { rf 05 \s. O73 its 3 | 75 mgm. of NO, as sodium nitrate 8. 70 { Q. 20 \ r eT Hinteen 2 Sue ets ge aha = 8. 50 & mo g. 80 D.59 | eas § | 350 mgm. of NO, s sodium alirate...) 13-30 Uhr. 25 WIE 2 lbs. 35 | %-20 z | 25mgm of NO, a5 calcium nitric | 1115 hon nag 078 [hon 65 | 0.535 19 | 350 mgm. af NO, as calcium nitrate.) 14,7 hey 70 HE to [ft 5 | % 55° a Lost by breakage during sterilization. 216 Journal of Agricultural Research Vol. XII, No. 4 TABLE XXVII.—Influence of nitrates on the fixation of nitrogen by Bacillus radicicola, giving the increase in nitrogen—Continued EXPERIMENT III Total nitrogen in roo c. c. of medium. Nitrogen Culture : : 5 . : No. Treatment (nitrate in 100 c. c. of medium): Uninoculated. Inoculated. ieee Found. | Average.| Found. | Average. Mam. Mam. Mom. Mom. Mom. El) SINGLE ot fe sre fend cl cletayeis jn tele ee olovel eiehe et 5. 10 5. 50 CLS lh ate oes ope mister crehmens sncrerlsicr store tale 5. 10 |? 5.07 5. 40 5. 50 0. 43 Bal nee an GO atte ete ais etait beers: +s] 5-00 5-45 4|75 mgm. of NO; as potassium ni- | . (Sega Hosea sua hiol oD BEBO BOE Gc 9. 35 To. 85 Ci ies CREE ee Tend MOLTEN ‘| 9. 50 |? 9-37 |} 10.90 |¢ 10.90 I. 53 Ou |eye cere (3 OC RT OER AIO CREO 9. 25 10. 95 7 | 150 mgm. of NO; as potassium ni- ELAS res Reta eyeie aoe csmuttatevealle snore selave te 14. 50 15. 65 toed Lede ro LO Aistci BENG Le Coed oan ciienen cater 14. 20 |>14. 28 |) 15.30 |p 15,45 Hi, ait7) Gules GO Aires se manic Menle es ies ane 14. 15 15. 40 10 | 7s mgm. of NO; as sodium nitrate.| 8.50 A} CE S5 sD ieee: CLO sian eileonals Racy ice rsiciestanepksvere cays 8. 30 |? 8. 38 g. 90 9. 83 I. 45 LON ethos Oram ergata tes tare rnustec spay stceans 8. 35 9. 70 13 | 150mgm. of NO;as sodium nitrate.| 12. 35 12.95 DAG Se crete (i (oS a een TOES emer mea ericRa echt I2. 40 | p12. 33 |} 13-10 |? 13. 03 0. 70 Bisel oe OO Ag nical he apnee sae elels sei 12. 20 13. 05 16 | 75 mgm. of NO; as calcium nitrate.| 8.95 9. 85 1 ee Losey Nae eeN ea ite hee NRF 9. 10 |? 9. OT 9. 90 9. 93 0. 92 TSH ere CLO cy tetie Goes ecode she he cis ear ce trcuato ret g. 00 IO. 05 19 | 150mgm. of NO,as calcium nitrate.| 13. 90 14. 40 SON Pet Oe ataints Siete iet ts SAREE aa ca tite 13. 80 |? 13. 80], 14.50 |? 14. 42 0. 62 DE Al aksrave LOU eteegstcinie a arevarsishetebauelvaevonals 13. 70 T4. 35 It has been shown that, when nitrates are added in varying quantities to sterilized soil, the number of B. radicicola are increased. Provided the the organism can fix a small amount of nitrogen in the absence of nitrate nitrogen, is it not possible that this increase in nitrogen fixation may be due merely to the increase in the number of cells? It seems that this is true according to the results in Table XXVII. It appears probable that the increase in nitrogen fixed in the presence of nitrates is very likely because of an increase in the number of bacterial cells and not to any physiological change brought about in the organism itself. There was a marked increase in bacterial growth on the media con- taining the nitrate compared with the same media free from nitrate. The growth on the latter medium exhibited a normal, whitish watery appearance, characteristic of this organism. On the cultures containing nitrates a much more profuse growth occurred. In,many instances a pinkish tint was observed. This pigment production was especially marked in the case of the culture containing the sodium salt. After the first experiment had been completed, it was thought that possibly this pigmentation was due to an impurity in the culture. Therefore the two remaining experiments were made, using a subculture from the original. Jan. 28, 1918 Nitrogen-Assimilating Bacterta 217 This culture was plated three times, each plating being made from a well- isolated colony. ‘The final subculture was taken from a similar well- isolated colony. However, pigment formation in the presence of nitrate persisted in the two final experiments, showing clearly that some reaction took place between the nitrate and the organism grown on the medium. It is of interest to note that the pigment formation in the presence of nitrate was observed in later work where the influence of nitrates on nodule formation was investigated. Prucha (gr) found that on agar slopes of medium containing 0.5 per cent of potassium or calcium nitrate, the growth of B. radicicola became opaque and that an iridescent tint was produced. Although the results of these experiments may vary somewhat among themselves, taken as a whole it appears evident that B. radicicola may fix a small amount of atmospheric nitrogen when grown without the host plant and on a suitable medium. The addition of various amounts of nitrates as indicated increased somewhat the amount of nitrogen assimilated by B. radicicola. INFLUENCE OF NITRATES ON THE PRODUCTION OF GUM BY BACILLUS RADICICOLA , Since nitrates, especially in smaller amounts, cause an increase in the number of B. radicicola in pure culture, it was thought advisable to determine what influence these salts have on the production of gum. In culture media favorable to the growth of B. radicicola these bacteria will produce a gelatinous substance which is readily precipitated with 95 per cent alcohol or acetone. Upon the addition of either of these precipi- tants a fairly heavy, water-white, frothy gelatinous mass is formed which soon rises to the surface of the liquid. Upon standing, this mass contracts somewhat, and portions of it may fall to the bottom of the liquid from which it has been precipitated. Chemical analyses, according to Buchanan (zo), have shown that this gum is a carbohydrate. Upon hydrolysis with 2 per cent sulphuric acid and 15 pounds’ pressure for one hour, Fehling’s solution is reduced, showing the presence of a sugar. ‘The gum does not give proteid reac- tions with the Millon, biuret, or xanthoproteic tests. Hence, the gum is not protein in character; nor does it contain nitrogen in the combined form. Clearly it is a nonnitrogenous body. In the experiment undertaken to determine whether nitrates influence the formation of gum only relative differences are noted. No attempt was made to obtain quantitative results. Erlenmeyer flasks of 1-liter capacity containing 200 c. c. of mannit solution were used. ‘The cultures contained various quantities of nitrate as indicated in Table XXVIII. Triplicate flasks for each amount of nitrate were prepared. In this table these three flasks are represented as “a,” “b,” and ‘‘c.” After sterilization at 15 pounds’ pressure for 25 minutes the flasks were cooled and inoculated with 5 c. c. of a suspension 218 Journal of Agricultural Research Vol. XU, No. 4 A sae Vi Sc 8 A AERA ESAS ee of B. radicicola in sterile distilled water. The cultures were then incubated at room temperature (approximately 25°C.) for eight weeks. At the expiration of the incubation period the contents of the flasks were poured into hydrometer cylinders of equal depth and diameter. One hundred and fifty c. c. of acetone were added to precipitate the gum. After careful shaking, the cylinders were covered with inverted petri dishes to prevent evaporation. At the end of 24 hours the amount ot gum precipitated was observed. The relative amounts are recorded in Table XXVIII. TABLE XXVIII.—Influence of nitrates on the production of gum by Bacillus radicicola Relative production of gum—precipitated by Cul- acetone. ture Treatment (nitrate in 100 c. c. of medium). a Flask a. Flask b. Flask c. Tae iy NIV es be b atalasiare af exgihiaie cial eye large... ; Barge scan Large. 2 | 7s mgm. of NO, as potassium nitrate .| Very large.| Very large.| Very large. 3 | 450 mgm. of NO, as potassium nitrate.| Large...... Warceserer Large. 4 | 75 mgm.of NO,as sodium nitrate ....| Very large.|...do....... Very large. 5 | 450 mgm. of NO; as sodium nitrate...| Large......|... doses... Large. 6 | 75 mgm.of NO, as calcium nitrate...|... AOWrsietersfatorete (0 Lo pa Do. 7 | 450 mgm. of NO, as calcium nitrate. .|... ds es Co nsider- | Co n sider- able. able. From the results it is certain that the nitrates, especially in the smaller of the two concentrates, caused a very considerable increase in the amount of gum produced by B. radicicola. ‘The nitrates of potassium and sodium caused a production of more gum than did the calcium salt. It will be remembered that in the experiments where the influence of nitrates on the fixation of atmospheric nitrogen by B. radicicola was studied, less nitrogen was fixed in the presence of calcium nitrate than in the pres- ence of the other two salts. Here again the greater stimulative action of potassium and sodium nitrates is emphasized. Buchanan in his investigations on the formation of gum by B. radici- cola has found that varying amounts of potassium nitrate in a 2 per cent saccharose solution or in a 2 per cent saccharose-clover-extract solution caused a slight increase in growth and in gum production. It seems probable that the increased gum production in the nitrate cultures is caused not only by an increase in bacterial cells but also perhaps by an increased stimulation in the formation of gum by the cells themselves. The relative increase in the amount of gum produced in the presence of nitrates seems to be greater than the actual increase in number of organisms brought about by the stimulating effect of the nitrate. In the latter instance this stimulating effect has been deter- mined in soil cultures only and so a fair basis of comparison can not be Jan. 28, 1918 Nitrogen-Asstmilating Bacteria 219 found. Had the influence of nitrates on the growth and reproduction of B. radicucola been determined in liquid culture, as well as in soil cultures, then a comparison could have been made. Furthermore, the divergencies in the time element, eight weeks’ incubation in the liquid cultures and three weeks in the soil cultures, are such as to render futile any attempt at correlation. It may be that the large formation of gum was due to the prolonged incubation. A shorter period of three weeks undoubtedly would show a relatively smaller amount of gum produced as a result of the presence of the nitrate. However, from the results of the experiment it is certain that potas- sium, sodium, and-calcium nitrate influence the formation of gum by B. radicicola. ‘The three nitrates studied caused a large increase in the amount of gum obtained by precipitation with acetone. Calcium nitrate caused the least stimulation, but the difference was not large. INFLUENCE OF NITRATES ON NODULE FORMATION The xesults of numerous investigations have shown that nitrates retard and oftentimes entirely prevent the formation of nodules on leguminous plants when grown in soil or liquid cultures. Vines (45), working with the horse bean, found that the use of large amounts of nitrate in the form of potassium nitrate retarded nodule formation. He concluded that a decrease in the amount of nitrates meant an increase in the number of nodules. Woods (48) found that leguminous plants assimilated more nitrogen when they were grown in the absence of potassium and calcium nitrate than*when thus supplied. His results seem to indicate that nodule development was retarded somewhat by these salts. Similar results were obtained by Frank (16). Nobbe and Richter (37) in 1902 grew soybeans in a rich garden soil and found upon inoculation that a gain of 74.7 per cent of nitrogen cecurred. However, upon the addition of nitrates this gain was considerably reduced, the extent of the reduction corresponding to the amount of nitrate added. About this same time, Wohltmann and Bergené (47) using many different types of soils, found that nodules were not formed on the roots of peas when ammonium nitrate was added. Creydt (r2) found that sodium nitrate retarded nodule formation on yellow lupines when these legumes were grown in soil. Fred and Graul (z8) found that very small amounts of nitrates did not appreciably decrease nodule formation, but that larger amounts proved detrimental and finally prohibited entirely the development of nodules. The presence of nitrates in culture solutions has also been found to reduce and oftentimes to inhibit the formation of nodules on leguminous plants. Marchal (3z) concluded that alkaline nitrates in concentrations of 1 to 10,000 in liquid cultures prevented the formation of nodules on peas. Flamand (z5) grew vetch and beans in a nutrient solution and 220 Journal of Agricultural Research Vol. XII, No. 4 found that nitrates in the following amounts prevented nodule forma- tions: potassium nitrate, 1 to 10,000, sodium nitrate 1 to 2,000, ammo- nium nitrate 1 to 2,000, and calcium nitrate 1 to 2,000 and 1 to 10.000. Hiltner’s (24) experiments showed that 5 mgm. of nitrogen as potassium nitrate per liter prevented nodule formation on peas. In contrast to these experiments Bassler (z) claimed that results obtained from his work indicated that no effect was noticed by adding nitrates to lupines growing in quartz sand. The question naturally arises whether this condition is due to the weakening of the organism brought about by growth in a nitrated environment and to a consequent impairment or entire loss of its infect- ing power, or whether it is caused by some interreaction between the salt and the plant root, tending to increase the latter’s resistance to the attack of this particular organism. INFLUENCE OF NITRATES ON THE INFECTING POWER OF BACILLUS RADICICOLA Some investigations have been carried out to determine what effect nitrates have on the legume organisms themselves. Wilson (46) showed that although nitrates inhibit the formation of nodules, the organisms capable of producing nodules did not lose their vitality or nodule-pro- ducing power when grown in the presence of nitrates. The results of Prucha (47) are in accord with those of Wilson. He found that B. radt1- cicola does not seem to lose its infecting power when grown on media containing nitrate. During the course of his work he found that potas- sium and sodium nitrates inhibited the formation of nodules. Further evidence that the organisms appear to retain their vitality in the pres- ence of nitrates is produced by the results of Mazé (33, p. 15-17), who showed that legume bacteria were able to fix a slight amount of nitrogen when grown in a soil extract solution containing 1 per cent sodium nitrate. Herke (22) states that potassium. nitrate favors the growth of nodule bacteria. However, other investigators state that nitrates have a harmful effect on B. radicicola. Laurent (29, p. 134) found that legume organ- isms failed to grow in a pea or lupine decoction containing nitrate in the form of potassium and sodium salts in amounts equivalent to 1 to 500 and 1 to 1,000. Moore (35) in his experiments demonstrated that nitrates at 1 to 10,000 were sufficient to prevent nodule formation. He states that B. radicicola loses its power of infection when grown in a medium containing nitrates. From the results cited it can be seen that there is some disagreement as to the influence exerted by nitrates on B. radicicola. In some cases the organism seems to retain its vitality in the presence of nitrates, while in others it appears to have become weakened. It must be ad- Jan. 28, 1918 Nitrogen-Assimilating Bacteria 221 mitted, however, that the evidence seems to favor the former contention— namely, that nitrates do not cause the bacteria to fe their nodule- producing power. In order to determine whether or not nitrates weaken these organisms, the following experiments were made: Slopes of mannit agar (in test tubes) containing various amounts of sodium and calcium nitratesas indi- cated in Table XXIX were inoculated with B. radicicola. ‘These cul- tures were incubated at 28°C. for one week, when transfers were made to fresh nitrate media and incubated at 28° C. for another week. At the expiration of this time, three 4-day-old seedlings of alfalfa were inoculated with three drops of a suspension of the organism in 5 ¢. c. of sterile distilled water. The same slope cultures were incubated at 28° C. and used for all subsequent inoculations in this experiment. The inoculated seedlings were placed in the greenhouse under cheese- cloth covering. The temperature here during the daytime averaged approximately 30° C. The seedlings were examined for the first appear- ance of nodules and in no case did they appear before 18 to 20 days. A total count of nodules on all plants was made at the end of 45 days. Three subsequent inoculations were made under the same conditions. In this way organisms in contact with nitrate for varying lengths of time could be used. The results of the inoculation experiments are given in Table XXIX. TaBLE XX1IX.—Influence of nitrates on the infecting power of Bacillus radicicola Number of nodules after 45 days. ee Treatment (nitrate in 100 c. c. of medium). l No. Inoculated | Inoculated | Inoculated | Inoculated June 3. June ts. July xr. July 17. Bias | eINOT eseeeee ys Perce peter Pere etireta evar eireccs 5 4 Tat 3 2 | 15 mgm. of NO; as sodium nitrate. . . 5 4 5 | 5 3 | 37 mgm. of NO, as sodium nitrate... 7 6 I5 5 4 | 75 mgm. of NO, as sodium nitrate... 4 5 3 II 5 | 150 mgm. of NO, as sodium nitrate. . 5 8 4 8 6 | 225 mgm. of NO, as sodium nitrate. . 6 5 5 7 7 | 450 mgm. of NO, as sodium nitrate . . 2 4 6 3 SOM G ec cu cuiaeieeiae te odie Laie wis + o-2 2 7 8 7 8 9 | 15 mgm. of NO, as calcium nitrate. . 4 4 9 4 ro | 37 mgm. of NO; ascalcium nitrate. .. 9 4 8 6 Ir | 75 mgm. of NO, as calcium nitrate .. 5 5 II 8 12 | 150 mgm. of NO,‘ascalcium nitrate. . 7 5 9 9 13 | 225 mgm. of NO, as calcium nitrate. 5 7 4 3 14 | 450 mgm. of NO, as calcium nitrate. 6 8 6 3 Poy e Spskirclo\e v2) 72s Ne ° ° ro) ° TO) erates RAGS eh.) 3) sii ,c ein df sn) 6)'nc) 00,0 ie ° ° ° re) From the results given in Table XXIX it is very evident that under the conditions of the experiment the legume organisms did not lose their power of producing nodules when grown on a medium containing 222 Journal of Agricultural Research Vol. XII, No. 4 varying amounts of sodium and calcium nitrates. The numbers of nodules produced on the alfalfa plants by organisms grown on media containing nitrate do not vary widely from those on the plants inocu- lated with organisms grown on media containing no nitrate. Not only did the organisms fail to lose their nodule-producing power, but from all appearances their infecting power did not seem to be materially weakened. It therefore seems apparent that an explanation for the failure of nodules to develop on leguminous plants in the presence of nitrates is not found in the theory that the organisms producing these nodules are weakened when grown in the presence of nitrates. INFLUENCE OF NITRATES ON ALFALFA ROOTS AND NODULE FORMATION The next step taken would naturally be in the direction of a study of the influence of the nitrates on the plant roots themselves in order to determine whether or not they thus are made more resistant to the attack of these organisms. A review of the literature shows that almost nothing has been done touching this phase of the question. Wilson (46), studying the effect of certain salts on nodule production, states that possibly the salt has some effect on the root, making it resistant to the attack of the organism. Mazé (33, p. 15-17), who also concluded that nitrates did not cause B. radicicola to lose its infecting power, says that nodules do not develop on roots of legumes when nitrates are present because the carbohydrate in the roots is changed into protein material by the absorption of the nitrate. Alfalfa seedlings (Medicago sativa) growing in soft agar containing potassium, sodium, and calcium nitrates, as indicated in Table XXX, were used in this study. Quadruplicate tubes were prepared for each amount of nitrate. The higher concentrations of the nitrate were not used, since it was found that germination and subsequent growth were considerably impaired in the presence of such large amounts. The tubes with the mannit agar and nitrate were sterilized at 15 pounds’ pressure for 30 minutes. These were cooled and sterilized alfalfa seeds planted as given under ‘‘Methods used in experiments.” The tubes were then placed in the greenhouse under cheesecloth covering and the seeds allowed to germinate. Germination took place in all instances, although it was retarded somewhat by the presence of the nitrate. At the end of five days the first tube of each set was inoculated with three drops of a suspension of B. radicicola in sterile distilled water. Subsequent inocu- lations were made as indicated in Table XXX. These were made at different intervals in order to allow the roots of the seedlings to remain for a longer time in contact with the media. It was hoped that in this way an idea might be obtained as to the time when the root first became resistant. The results are given in Table XXX. Jan. 28, 1918 Nitrogen-Assimilating Bacterva 2 i) ww TaBLE XXX.—Influence of nitrates on alfalfa roots and nodule formation ral a ee eee see ee ee SEL SNE REEL i ‘Total number of nodules in each tube of | seedlings inoculated after— pers Treatment (nitrate in roo c. c. of medium). 5 days’ 1o days’ | 18days’ | 22 days’ | growth. growth. growth. | growth. | INO Teer ea eerie tevec chat ee 9 er tery b ro mgm. of NO as potassium nitrate .....| 25 mgm. of NO, as potassium nitrate .....| 50 mgm. of NO, as potassium nitrate ..... roo mgm. of NO, as potassium nitrate .... 150 mgm. of NO; as potassium nitrate .... ro mgm. of NO, as sodium nitrate ........ 25 mgm. of NO; as sodium nitrate........ | 50 mgm. of NO, as sodium nitrate ........ roo mgm. of NO; as sodium nitrate ....... } (2) 150 mgm. of NO, as sodium nitrate ....... Neo (ey) ro mgm. of NO, as calcium nitrate ........| 13 | 25 mgm. of NO, as calcium nitrate........ 14 | so mgm. of NO, as calcium nitrate........ rs | roo mgm. of NO, as calcium nitrate ....... (0) 16 | 150 mgm. of NO, as calcium nitrate ....... (5) ° | Oo ° 5 lomm ro) (2) 12} (b) eo0o00000 OW HHH NHOW ON OUSW NH OOWODODDOHOAODODOIOHN oo} (oN) eo0o0OOH OOO OW | | | | Qa 0 010: .G)0r0) 0) Oc) Gr0 @ Fungus contamination. b Plant died after few days’ growth. It will be seen that in a few instances where a high concentration of nitrates occurred the development of the seedlings subsequent to ger- mination ceased. ‘This condition may have been due to too high a con- centration of soluble salts or to inferior seed. However, losses were not sufficiently serious materially to affect the outcome of the experiment. In all cases the seedlings grown in agar without nitrate produced nodules when inoculated with B. radicicola. A few nodules appeared on seedlings in cultures containing the lowest concentration of all three nitrates. The number of nodules in these cases was less than in the control cultures. No nodules whatever developed in any concentration above 10 mgm. of nitrate in 100 ¢c. c. of medium. Under normal condi- tions in test-tube cultures the nodules make their appearance at about 18 to 20 days after inoculation. The incubation of all cultures was extended 4o days after inoculation in order to make certain that no further nodule development would take place. The nonproduction of nodules was not due to the failure of the inoc- ulum. In all cases an excellent inoculum growth was obtained, espe- cially in the case where nitrate was present in the medium. Indeed, it was so luxuriant that in many cases the organism grew in considerable quantity far down into the root zone. In many cases where nitrates were present the same pink coloration was produced that was discussed under another caption, on page 216. As has been already stated, seedlings of varying ages were inoculated for the reason that it was thought that a more or less prolonged contact of the roots with the nitrate in the medium might serve as an index to 224 Journal of Agricultural Research Vol. XII, No. 4 the time in the growth of the seedling when permanent resistance to attack of the organisms was established. The results obtained do not seem to indicate that seedling roots 18 to 20 days’ old are any more resistant to the attack of the organisms than are those that are younger. Evidently if any reaction takes place between the nitrate and the plant root it occurs during the very early stages in the development of the plant. These results seem to point to the conclusions that the nonformation of nodules in the presence of nitrates is due not to a weakening of the vitality of the organism, but to some reaction between the plant root and nitrate. One naturally queries whether the plant root cells are made more resistant to the bacteria seeking to gain entrance there or whether the naturally occurring carbohydrate food supply to be used by the organisms after gaining entrance is diminished by its conversion into protein material by the absorption of nitrate? Further studies were not made in an endeavor to solve this question. INFLUENCE OF NITRATES IN SOIL ON ALFALFA NODULES AND ON THE REFORMATION OF NODULES Additional studies were made with nitrates in relation to their influ- ence on nodules already formed and on the redevelopment of nodules once removed from alfalfa plants. The experiments were carried out in an endeavor to determine whether nitrates prevented an increase in the number of nodules on plants possessing nodules and whether they prevented the reformation of nodules when removed. Experiments revealed clearly that removed nodules were replaced by new ones pro- vided the plant was carefully replaced in the soil (soil with normal low nitrate content) and the proper amount of moisture maintained. In these experiments 1-gallon earthenware jars were used. These were filled to within an inch of the top with 1,800 gm. of soil of a low ‘ nitrate content. Different amounts of the nitrates to be studied were added in the quantities indicated in Table XXXI. Concentrations of 100 and 300 mgm. of nitrate in 100 gm. of soil were also used, but the transplanted alfalfa seedlings were unable to withstand such excessive concentration, with the result that all died within a week or ten days after transplanting. Quadruplicate pots were prepared for each con- centration of nitrate. The nitrates in solution were mixed with the proper amount of distilled water which, when added to the pots, brought the moisture content to approximately 20 per cent. The pots were then allowed to remain undisturbed for one day at room temperature to allow the water containing the nitrate to become well diffused through- out the soil mass. Into two pots of each set were transplanted young alfalfa plants from which the nodules had been removed. The two remaining pots contained transplanted alfalfa plants with the nodules left on and their location noted. The plants used in this experiment a Jan. 28, 1918 Nitrogen-Assimilating Bacteria 225 were removed from an alfalfa plot, the soil of which was a sandy loam. Previous to transplanting the roots of the young plants were carefully washed in running water and immediately transplanted. The pots were kept well watered, and after two or three days they were removed to the greenhouse. Here they were watered when necessary. Trans- plantations were made on the 27th of June and the experiment termi- nated on the 3d of August. The plants were removed from the pots, the roots carefully washed and examined for the presence of nodules. The results are presented in Table XX XI. TABLE XXXI.—Influence of nitrates in soil on alfalfa nodules and on the reformation of nodules Number of nodules— Pot No. Nitrate in roo gm. of dry soil. area of 7 begin fei ning NST a hath tas PUTO Tyas sie Sen oye ia oh S= Wh cans vane Sia fea era Removed....... ° 3 GO Way stray si) (nretotets CO eryane ores serfs seca spaucs ous) sit, a olist yah iat onaysiffoietereta Oe Se sets ° 4 DAIS Nites eae ats ORR Ere aR hemes are Dhaka eotgereets Not removed 4 8 1 A Rane eee Da Sateraior cach Gar helaxe Bate olexere Magiaye ob ahes lan goeer PY Sasha 3 7 Biles ans: 25 mgm. of NO, as potassium nitrate Removed.... re) ° Bi2irorscteretel|lors ones DEN GRR TOMS ataape wah lal valatebel da ea (IME et daprisaah o| (2) 12 ea re Oe tenia eferevsinidatgels dete oom aiopsks sates Not removed 4 3 SPA wera tell diate LO MaSP Ree TE Eee rte eric dar erthetenerele adie ements GO emoiee 8 5 87: OME nee 50 mgm. of NO, as potassium nitrate. .... Removed ... ro) ro) Li Pee on I atte ie Meh nt cath, Bh Rea eae he yl leiepinede Loe 5 a fe) ° Byres ye ecienerora Ea carat eerste erate tice aaah Se Not removed 4 2 Coa ra aoe LOE te hae ea et GRP TEEN. #, cows caren GO Axe TI I Dir: 25 mgm. of NO, as sodium nitrate....... Removed oO | ° Dae elec albisesters Oe tates oe ales) else ite MEMOS lak orks lena co oleae Ames ) ° DS a aha UG Raa east, Hck tay fete aava oa P eta Not removed 4 2 1D AVR Ae ree at MAGN reason te Na ayy stata thc) wp cake faye 29 lays tos arelfiory stone AG 5 te brats 5s 5 I eh Leggs a 50 mgm. of NO, as sodium nitrated ...... Removed.... fe) ° TRACY: | aaarae (3 USNR Bee SOLO NS SS RL ee a Fee dalek ak o} (2) Waa ne vedas tay Cele rhinitis, Shc hda Ph inccas)'S piacied btiaté = Not removed 2 I LD ae eg) Le Ya Ree ra (er ro aes | 6 3 1 a ee 25 mgm. of NO, as calcium nitrate ...... Removed.. .. fe) ° [SE PRESSES FEN (eee Oat ee deity te cin ciate mje 2:5) 4 she lst snore dO). theouns ro) ° 1 eS ereael eae Meare ee ema Felker aU NS o's fale, oo sia Not removed 2 2 EAS eres lacie CLO RIN ities atcha wid bcin eee eal GOR oT 4 | 3 Sete Be sseys 50 mgm. of NO, as calcium nitrate ...... Removed.... o | ° GlOBe create leet scete (toe ee Pas SUA Ua ae arene) Rem dOWs santas fe) fr) GaP Ei aliases AS aia ite tose whew ewients dele eek Not removed 4 3 Cees a ONES TOA oy Soren oat a ee EE, ee eae (eee Oss nde se. 2 I @ Plants died. It will be seen in the control pots, where no nitrate was present (ex- cept the small amount normally present in the soil at the beginning of the experiment), that if the nodules were removed, new ones formed. The location of the nodules before their removal was noted, and the new ones were found to occupy the same place. However, when nitrates were added to the soil no new nodules were formed. This statement holds true for both concentrations of all three salts in all experiments. 226 Journal of Agricultural Research Vol. XII, No. 4 Se Hebe ote Syl gsr EA Oh NA 2 Es AC IAM RIS aS i Some interesting results were obtained where the nodules were not removed. In the control pots an increase in nodule formation took place. It can not be stated definitely whether the new nodules appeared as a result of inoculation from the soil or whether the organisms had already gained entrance to the roots before the plants were removed from the field soil previous to transplanting. Nevertheless, it is shown that the number of nodules increased as compared with the number present at the time of transplanting. But where nitrates were added a reduction in number occurred rather regularly throughout all the pots. In two instances the number remained constant, in 10 it was reduced, and in none was it increased. The calcium salt appeared to effect the least reduction in number of nodules. Conclusions concerning the comparative in- fluence of the three salts in this regard can not be drawn because of the small number of determinations made. It is sufficient to note that nitrates present in amounts equal to 25 and 50 mgm. of nitrate in 100 gm. of soil did not permit an increase in number of nodules, but rather caused a decrease. The conclusions drawn from the experiments relative to the influence of nitrates on nodule formation are: (a) the presence of nitrates is detrimental to the formation of nodules by alfalfa; (b) the nonformation of nodules is not due to a weakening of B. radicicola when grown in the presence of nitrates; (c) some reaction takes place between the nitrates and the plant root, thus preventing nodule formation; (d) nitrates in the soil prevent.the re-formation of nodules once removed and also cause a decrease in the number of those already present. SUMMARY (1) Small quantities of potassium, sodium, and calcium nitrates caused a great increase in the number of Azotobacter in sterilized soil., Ammonium nitrate in the same quantities caused a less marked in- crease. Higher concentrations were not so favorable to the growth of the organisms. (2) Potassium and sodium nitrates in the concentrations studied caused an increase in the amount of nitrogen assimilated by Azoto- bacter on agar films. Calcium nitrate in the same amounts brought about a decrease in the amount of nitrogen fixed to a point even below that representing the amount assimilated in the absence of nitrates. In soil cultures nitrates of sodium and calcium caused an increase in total ni- trogen, which was more marked in the unsterilized cultures than in those cultures sterilized and inoculated with a pure culture of Azotobacter. However, the increase in total nitrogen is not commensurate with the increase in the number of Azotobacter noted under the same conditions. (3) Under aerobic conditions Azotobacter in liquid cultures reduced nitrate to nitrite, but not to ammonia. More atmospheric nitrogen was assinwilated in the presence of nitrate than in the absence of this salt. . ; Jan. 28, 1918 Nutrogen-Assimilating Bacteria 227 (4) Pigmentation occurred when potassium and sodium nitrates, and especially calcium nitrate, were used with Azotobacter, the colora- tion increasing with the concentration of the salt. This effect was more marked in Azotobacter strains which produce little or no pigment in the absence of nitrates. (5) All three nitrates studied caused an increase in the number and size of volutin bodies in Azotobacter cells. From all appearances these salts also tended to hasten the development of these bodies. (6) The number of Bacillus radicicola in sterilized soil was increased by the addition of small quantities of potassium, sodium, ammonium, and calcium nitrates. This increase was not so marked as in the Azoto- bacter cultures. B. radicicola appeared to be much more resistant to higher concentrations of nitrates than Azotobacter. (7) B. radicicola under aerobic conditions did not reduce nitrates in solution to nitrite, ammonia, or elemental nitrogen. The presence of nitrates did not materially influence the small amount of atmos- pheric nitrogen fixed under these conditions. (8) When grown on agar films, B. radicicola fixed a small amount of nitrogen, varying from 0.15 to 0.43 mgm. of nitrogen in 100 c. c. of the medium. The addition of various amounts of potassium, sodium, and calcium nitrates increased to a slight extent the amount of nitrogen assimilated. (9) In liquid cultures all three nitrates caused a large increase in the amount of gum obtained by precipitation with acetone. (10) The presence of large amounts of potassium, sodium, and cal- cium nitrates proved detrimental to the formation of nodules on alfalfa. B. radicicola did not appear to lose its infecting power when grown on media containing varying amounts of sodium and calcium nitrates. Alfalfa seedlings grown in the presence of large amounts of nitrate did not produce nodules when inoculated witha viable culture of B. radicicola. _ Nitrates in soil cultures prevented the re-formation of nodules once removed and also caused a decrease in the number of nodules already present. LITERATURE CITED (1) BASSLER, P. 1895. SANDKULTURVERSUCHE UBER DIE STICKSTOFFASSIMILATION DER GELBEN LUPINE IM STERILISIERTEN UND GEIMPFTEN BODEN BEI DARGEBOT WECHSELNDER MENGEN VON SALPETERSAUREN SALZEN. In Jahresber. Agr. Chem., n. F., Bd. 18 (Jahrg. 38), p. 131. (2) BEIJERINCK, M. W. 1888. DIE BACTERIEN DER PAPILIONACEEN-KNOLLCHEN. Jn Bot. Ztg., Jahrg. 46, No. 48, p. 758-771. (Continued article.) (3) 1891. OVER OPHOOPING VAN ATMOSPHERISCHE STICKSTOF IN CULTUREN VAN BACILLUS RADICICOLA. Jn Verslag. en Meded. Kx Akad. Wetensch. [Amsterdam], r. 3, deel 8, p. 460-475. 228 Journal of Agricultural Research Vol. XII, No. 4 (4) BEIJERINCK, M. W. I90I. UEBER OLIGONITROPHILE MIKROBEN. In Centbl. Bakt. [etc.], Abt. 2, Bd. 7, No. 16, p. 561-582, 1 pl. and DELDEN, A. van. 1902. UEBER DIE ASSIMILATION DES FREIEN STICKSTOFFS DURCH BAKTERIEN. In Centbl. Bakt. [etc.], Abt. 2, Bd. 9, no. 1/2, p. 3-43. (6) BERTHELOT, M. P. E. 1893. RECHERCHES NOUVELLES SUR LES MICRORGANISMES FIXATEURS DE L’ AzOoTE. In Compt. Rend. Acad. Sci. [Paris], t. 116, no. 17, p. 842-849. (7) Bonazz1, Augusto. 1915. CYTOLOGICAL STUDIES OF AZOTOBACTER CHROOCOCCUM. In Jour. Agr. Research, v. 4, no. 3, p. 225-239. Literature cited, p. 238-2309. (8) BoTToMLEY, W. B. 1909. SOME EFFECTS OF NITROGEN-FIXING BACTERIA ON THE GROWTH OF NON- LEGUMINOUS PLANTS. In Proc. Roy. Soc. London, s. B, v. 81, no. 548, p. 287-289. (9) BREDEMANN, G. 1909. BACILLUS AMYLOBACTER A. M. ET BREDEMANN IN MORPHOLOGISCHER, PHYSIOLOGISCHER UND SYSTEMATISCHER BEZIEHUNG. Jn Centbl. Bakt [etc.], Abt. 2, Bd. 23, no. 14/20, p. 385-568, 13 fig., 2 pl. Literatur- verzeichnis, p. 559-560. (10) BucHanan, R. E. 1909. THE GUM PRODUCED BY BACILUS RADICICOLA. Jn Centbl. Bakt. [etc.], Abt. 2, Bd. 22, No. 11/13, p. 371-396. Citations, p. 395-396. (11) CHatmot, G. de 1894. THE INFLUENCE OF NITRATES ON GERMINATING SEEDS. In Agr. Science, v. 8, no. 10/11, p. 463-465. (12) CrEYDT, Bodo. 1915. UNTERSUCHUNGEN UBER DIE KALKEMPFINDLICHKEIT DER LUPINE UND IHRE BEKAMPFUNG. Jn Jour. Landw., Bd. 63, Heft 2, p. 125-191, 6 pl. (1g) DRABBLE, Eric, and Scott, Daisy G. 1907. ON THE EFFECT OF ACIDS, ALKALIS, AND NEUTRAL SALTS ON THE FER- MENTATIVE ACTIVITY AND ON THE RATE OF MULTIPLICATION OF YEAST CELLS. Jn Biochem. Jour., v. 2, no. 7/8, p. 340-349, 1 fig. Literature P- 349- (14) Fernpacnu, A., and LANZENBERG, A. I9I0. DE L’ACTION DES NITRATES DANS LA FERMENTATION ALCOOLIQUE. In Compt. Rend. Acad. Sci. [Paris], t. 151, no. 17, p. 726-729. (15) FLAMAND, Henri. 1905. UBER DEN EINFLUSS DER ERNAHRUNG AUF DIE ENTWICKLUNG DER KNOLLCHEN DER LEGUMINOSEN. In Centbl. Agr. Chem., Jahrg. 34, Heft 11, p. 738-740. (16) Frank, B. 1892. DIE ASSIMILATION FREIEN STICKSTOFFS BEI DEN PFLANZEN IN IHRER ABHANGIGKEIT VON SPECIES, VON ERNAHRUNGSVERHALTNISSEN UND VON BODENARTEN. Jn Landw. Jahrb., Bd. 21, E I-44. (17) FRED, E. B. 1913. A PHYSIOLOGICAL STUDY OF THE LEGUME BACTERIA. In Va. Agr. Exp. Sta., Ann. Rpt., 1911/12, p. 145-173, fig. 34. Literature, p. 172-173. and GrauvL, E. J. 1916. THE EFFECT OF SOLUBLE NITROGENOUS SALTS ON NODULE FORMATION. In Jour. Amer. Soc. Agron., v. 8, no. 5, p. 316-328. Literature cited, Pege7—sec- (5) ~ (18) Jan. 28, 1918 Nitrogen-Assimilating Bacteria 229 ms (19) GREAVES, J. E. 1916. THE INFLUENCE OF SALTS ON THE BACTERIAL ACTIVITIES OF THE SOIL. In Soil Science, v. 2, no. 5, p. 443-480, 4 fig. (20) Hanzawa, J. 1914. EINIGE BEOBACHTUNGEN UBER STICKSTOFF-BINDUNG DURCH AZOTOBAC- TER IN STICKSTOFFARMEN UND IN STICKSTOFFREICHEN SUBSTRATEN. In Centbl. Bakt. [etc.], Abt. 2, Bd. 41, No. 18/23, p. 573-576. (21) HELLRIEGEL, H., and Wi-Fartu, H. 1888. UNTERSUCHUNGEN UBER DIE STICKSTOFFNAHRUNG DER GRAMINEEN UND LEGUMINOSEN. In Ztschr. Ver. Riibenzuckerindus., Beilageheft, Nov., 234 p., 6 pl. (22) HERKE, 8S. 1913. CONTRIBUTIONS ON NITROGEN FIXATION AND NUTRITION OF BACILLUS RADICICOLA AND ON BACTERIAL TESTS OF NITRAGIN AND AZOTOGEN. (Abstract.) In Exp. Sta. Rec., v. 29, no. 8, p. 733. (Original article in Kisérlet. Kézlem., v. 16, no. 3, p. 311-322, 1913. Not seen.) (23) Hus, T. L. THE INFLUENCE OF NITRATES ON THE GROWTH OF AZOTOBACTER. Un- published. Offered for publication in the Ann. Rept. of the Pa. Agr. Exp. Sta. (24) HILTNER, L. 1900. UEBER DIE URSACHEN, WELCHE DIE GROSSE, ZAHL, STELLUNG UND WIRKUNG DER WURZELKNOLLCHEN DER LEGUMINOSEN BEDINGEN. In Arb. K. Gsndhtsamt., Biol. Abt., Bd. 1, Heft 2, p. 177-222, pl. 3. (25) ImmMENDoR®F, H. 1892. BEITRAGE ZUR LOSUNG DER “‘STICKSTOFFRAGE.”” In Landw. Jahrb. Bd. 21, p. 281-339. (26) Jost, Ludwig. 1907. LECTURES ON PLANT PHYSIOLOGY. Translated by R. J. H. Gibson. 464 p., 172 fig. Oxford. Bibliography at the end of each lecture. (27) Kayser, E. 1910. INFLUENCE DES NITRATES SUR LES FERMENTS ALCOOLIQUES. Jn Compt. Rend. Acad. Sci. [Paris], t. 151, no. 19, p. 816-817. (28) Kossowicz, ALEXANDER. 1914. UBER DAS VERHALTEN VON HEFEN UND SCHIMMELPILZEN ZU NITRATEN. In Biochem. Ztschr., Bd. 67, Heft 4/5, p. 400-419. (29) LAURENT, Emilé. 1891. RECHERCHES SUR LES NODOSITES RADICALES DES LHGUMINEUSES. In Ann. Inst. Pasteur, année 5, p. 105-139, 3 fig. (30) Lewis, L. L., and Nicnorson, J. F. 1905. SOIL, INOCULATION. TUBERCLE-FORMING BACTERIA OF LEGUMES. Okla. Agr. Exp. Sta. Bul. 68, 30 p., 8 fig. (31) MarcHaL, Emilé I90I. INFLUENCE DES SELS MINERAUX NUTRITIFS SUR LA PRODUCTION DES NODOSITES CHEZ LE pois. Jn Compt. Rend. Acad. Sci. [Paris], t. 133, MO. 24, Pp. 1032-1033. (32) Mazé&, P. 1897. FIXATION DE L’AZOTE LIBRE PAR LE BACILLE DES NODOSITES DES LEGUMINEUSES. Jn Ann. Inst. Pasteur, année 11, no. I, p. 44-54. (33) 1898. LES MICROBES DES NODOSITES DES LEGUMINEUSES. In Ann. Inst. Pasteur, ann. 12, no. I, p. 1-25, t fig. (34) Meyer, Arthur. 1912. DIE ZELLE DER BAKTERIEN. 285 p., 34 fig., 1 col. pl. Jena. Literatur, p. 267-282. 230 Journal of Agricultural Research Vol. XII, No. 4 (35) Moor:, G. T. 1905. SOIL INOCULATION FOR LEGUMES; WITH REPORTS UPON THE SUCCESSFUL USE OF ARTIFICIAL CULTURES BY PRACTICAL FARMERS. U. S. Dept. Agr. Bur. Plant Indus. Bul. 71, 72 p., 10 pl. (36) MGnrER, F. 1916. UBER DEN EINFLUSS ANORGANISCHER SALZE AUF DAS WACHSTUM DER ACTINOMYCETEN. DI. Ju Centbl. Bakt. [etc.], Abt. 2, Bd. 44, No. 24/25, p. 673-695, 9 fig. (37) Noss, F., and Ricurer, L. 1902. UBER DEN EINFLUSS DES NITRATSTICKSTOFFS UND DER HUMUSSUBS- STANZEN AUF DEN IMPFUNGSERFOLG BEI LEGUMINOSEN. In Landw. Vers. Stat., Bd. 56, Heft 5/6, p. 441-448. (38) PFEFFER, W. 1906. THE PHYSIOLOGY OF PLANTS. ed. 2. Translated by A. J. Ewart. v. 3. Oxford. , (39) PrazMowsk1, Adam. 1891. DIE WURZELKNOLLCHEN DER ERBSE: II. TEIL. DIE BIOLOGISCHE BEDEU- TUNG DER WURZELKNOLLCHEN. In Landw. Vers. Stat., Bd. 38, p. 5-56. (40) PRINGSHEIM, Hans. 1914. ZUR STICKSTOFFASSIMILATION IN GEGENWART VON SALPETER. In Centbl. Bakt. [etc.], Abt. 2, Bd. 40, no. 1/8, p. 21-23. (41) Prucwa, M. J. 1915. PHYSIOLOGICAL STUDIES OF BACILLUS RADICICOLA OF CANADA FIELD PEA. N. Y. Cornell Agr. Exp. Sta. Mem. 5, 83 p. Bibliography, p. 79-83. (42) Rirrer, G. I909. AMMONIAK UND NITRATE ALS STICKSTOFFQUELLE FUR SCHIMMELPILZE. In Ber. Deut. Bot. Gesell., Bd. 27, Heft 10, p. 582-588. (43) SACKETT,W. G. I9I5. THE PIGMENT OF AZOTOBACTER CHROOCOCCUM. Jn Proc. 35th Ann. Meeting Soc. Prom. Agri. Sci., 1914, p. 80-88, 2 col. pl. (44) STOKLASA, Julius. 1908. BEITRAG ZUR KENNINIS DER CHEMISCHEN VORGANGE BEI DER ASSIMI- LATION DES ELEMENTAREN STICKSTOFFS DURCH AZOTOBACTER UND RADIOBACTER. In Centbl. Bakt. [etc.], Abt. 2, Bd. 21, no. 15/16, p. 484-511. (45) VINES, S. H. 1888. ON THE RELATION BETWEEN THE FORMATION OF TUBERCLES ON THE ROOTS OF LEGUMINOSAE AND THE PRESENCE OF NITROGEN IN THE som,. Jn Ann. Bot., v. 2, no. 7, p. 386-389. (46) Wison, J. K. I9I5. PHYSIOLOGICAL STUDIES OF BACILLUS RADICICOLA OF SOY BEAN. (Ab- stract.) Jn Science, n.s., v. 41, no. 1048, p. 180. (47) WOHLTMANN, Ferdinand, and BERGENE, 1902. DIE KNOLLCHEN-BAKTERIEN IN IHRER ABHANGIGKEIT VON BODEN UND DUNGUNG. In Jour. Landw., Bd. 50, Heft 4, p. 377-395. (48) Woops, C. D. 1892. THE ACQUISITION OF ATMOSPHERIC NITROGEN BY GROWING PLANTS. In Conn. Storrs Agr. Exp. Sta., 4th Ann. Rpt., 1891, p. 17-28. (49) ZieFEL, Hugo. IQII. BEITRAGE ZUR MORPHOLOGIE UND BIOLOGIE DER KNOLLCHENBAKTERIEN DER LEGUMINOSEN. Jn Centbl. Bakt. [etc.], Abt. 2, Bd. 32, No. 3/5, P- 97-137. Literature, p. 136-137. shat. ae (Ate , ae eB, wn "LIBRARY OF CONGRESS WMI O000e7S5b6514)