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UBC Theses and Dissertations

The influence of Wildiers' bios on nodule bacteria and legumes West, Philip Manthorne 1937

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T H E I N F L U E N C E O F W I L D I E R S * B I O S OS N O D U L E B A C T E R I A A N D L E G U M E S by • P. M. WEST S U B M I T T E D I K P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R O F S C I E N C E I N A G R I C U L T U R E 1937 TABLE OF COMTENTS I n t r o d u c t i o n Page H i s t o r y and procedures. 1 - 9 P a r t 1 - The I n f l u e n c e of B i o s on Rhlzobium t r i f o l i i 1 G i a n t Colony S t u d i e s 10 - 14 I n f l u e n c e of a l f a l f a e x t r a c t 10 - 12 I n f l u e n c e of Crude B i o s 2 1 2 - 1 3 I n f l u e n c e of B i o s 2(a) and 2(h) 13 - 14 2 P l a t e Counts - The R e l a t i o n of G i a n t Colony S i z e to C e l l M u l t i p l i c a t i o n 14 - 16 3 Importance of P r e v i o u s H i s t o r y of the Organism, i n Dete r m i n i n g i t s A c t i v i t y 16 - 17 4 N i t r o g e n F i x a t i o n S t u d i e s 17 - 25 5 Sugar Fer m e n t a t i o n S t u d i e s 25 - 30 6 Ammonia P r o d u c t i o n S t u d i e s 30 - 35 7 B i o s as an Ac c e s s o r y F a c t o r f o r R h i z o b i a 36 - 38 P a r t 11 - The I n f l u e n c e of B i o s on Legume S e e d l i n g s P u b l i c a t i o n a t t a c h e d 39 P a r t i l l - The I n f l u e n c e o f B i o s on the Symbiotic A s s o c i a t i o n between Rhlzobium t r i f o l i i and Red C l o v e r . P r e l i m i n a r y experiment 40 - 42 References 43 - 44 1 THE INFLUENCE OF WILDIER3' BIOS'OK NODULE BACTERIA AND LEGUMES INTRODUCTION. An unknown organic substance, required by Saocharomyees oerevisiae in minute quantities, but nevertheless indispensable for growth, was demonstrated by Wildiers in 1901. To this accessory growth factor for yeast, Wildiers gave the name "Bios." The properties Wildiers assigned to Bios are: 1 Soluble in water. 2 Insoluble in absolute alcohol and ethert soluble in 80$ alcohol. 3 Organic in nature. 4 Resistant to boiling for hour with 5$ sulfuric acid, but is destroyed through boiling with 20$ acid. 5 Not altered by boiling hour with 10$ sodium hydroxide. 6 Not precipitated by lead acetate. Neither is i t precipitated by phosphomolybdio or phosphot ungstic acids; nor by silver nitrate in acid, basic or neutral solution* 7 Dialyzable. 8 Occurs in meat extract, commercial peptone and beer wort. 9 Yeast does not synthesize any more bios during growth; i t i s therefore not a substance the yeast c e l l can manufacture. 10 Bios i s not urea, asparagine, aniline, a nuolein base, adenine, guanine, nucleic acid, or oreatin. 2 WiIdlers showed that, If a minimal inoculation of yeast were made into a medium lacking Bios, growth would be very gradual, one c e l l await-ing the disintegration of another before being able to divide. Theoret-, ioally, a single c e l l would be unable to grow at a l l . If, however, a sterile extract of yeast cells was added to the medium, growth and re-production took place normally, no matter how small the inoculum used might be. Since i t is now recognized that Bios is generally distributed throughout a l l plant and animal tissues* Wildiers, therefore, did not necessarily have to use yeast extract. Anand and Ide (1902) and Devloo (1908) confirmed Wildiers 5 findings in. sa far as the presence of a v i t a l factor was concerned. Narayanan and Drumnond (1930) went a step farther and outlined a detailed method for isolation of a Bios conoentrate. As other-prbfeedures.will be describ-ed, that of the above workBrs is designated for future reference as Procedure 1. Procedure 1* Extract 600 gm. al f a l f a meal with 1600 ml. 95% ethyl alcohol under a reflux condenser at 67° - 70°C for 4 hours. Filter, wash with 55% alcohol ( by volume) and re-extract with 900 ml. 50% alcohol. Free combined fil t r a t e s from aloohoj. by d i s t i l l a t i o n in vacuo, and make the alcohol - free - extract up to 800 ml. Add 190 gm. baryta i n 15.0 ml. d i s t i l l e d water., to the ab0V6 extract and hydrolyze i n auto clave 3 hours at 15 lbs. pressure. F i l t e r and free of baryta with sulfuric a c i d . Saturate f i l t r a t e with 20% neutral lead acetate. Filter, free from lead with hydrogen sulfide, neutralize with 2N soda and concentrate i n vacuo to 450 ml* ,3 Add an excess of phosphotungstio acid crystals and allow to stand 12 hours, ihis treatment precipitates the active principle. Free pre-oipitate from phosphotungstic acid with baryta and reduoe volume in vacuo. •Add 10 ml. concentrated silver nitrate solution, saturate with baryta, and add more silver nitrate until precipitation is complete. Free filtrate from barium and silver with sulfuric acid and hydrogen sulfide respect-ively. By measuring yeast growth, Narayanan and Drummond found this filtrate to be active. A more recent development, is the procedure advanced by W. L* Miller (1933). This method (Procedure 2), differs in the following respects from that of Narayanan and Drummond. 1 Bios is not precipitated by any of the reagents used. 2 Bios is separated into two distinct entitles, each oapable of producing stimulation of yeast cells alone, but together producing an effect greater than additive. Procedure 2t Extract 500 gm. alfalfa meal with 2 11. distilled water in flowing steam for 2 hours and at 15 lb. pressure for 1 hour. Reduce the hydrolysate in vacuo to approximately 175 mil and add 350 ml. alcohol (95$ by volume). Filter and wash with 66% alcohol. Reduce combined filtrate and washings to dryiness in vacuo. Dissolve, neutralise with soda and dilute to 180 ml. Hydro lyze by adding 36 gm. baryta and boiling on water bath. 3 hours. To the hot hydro ly sate add 20 ml* 96% alcohol and thoroughly shake the mixture. Filter and reextract the residue with •water* Combine ;:-";-T.. tkfc-.-A washings, free from barium with sulfuric acid and neutralize. This solution is Crude Bios 1. 4 Free the filtrate of the baryta hydrolysate from barium with sulfuric aoid. Evaporate the solution repeatedly to dryness in vacuo to remove volatile acids. Dissolve the residue, neutralize and make up to 25 ml. ;This solution is Crude Bios 2. To the Crude Bios 2 solution add 1 ml. of 2 H sulfuric, acid and 1 gm. Darco charcoal. Shake 15 minutes, filter, and wash with 15 ml. distilled water. ShalsB filtrate again for 15 minutes with 0.5 gm. charcoal. Combine all filtrates and washings, free from sulfuric acid with baryta, evaporate to dryness in vacuo and mate residue ®p to 80 ml. This solution is Crude Bios 2 (a). Shake the charcoal from the two above operations for 30 minutes with 40 ml* freshly prepared Sparling's reagent (5 ml. cone* ammonium hydroxide, 35 ml. distilled water, and acetone to 200 ml.). Filter, wash charcoal with. 10 ml*. Sparling9 s reagent and shake charcoal once again with 25 ml. of the reagent for 30 minutes, filtering and washing as before* Combine all extracts and washings and evaporate repeatedly to dryness in vacuo to remove acetone and ammonia* Dissolve the residue in distilled water, neutralize with uA soda and make up to 25 ml. This solution is Crude Bios 2 (b). Following three years additional work, Miller (1936) published a revised procedure for the fractionation of Wildier's Bios. This new procedure avoids the use of costly ethyl alcohol, and in addition enables the separation of another active factor which he calls Bios 5. The complete procedure is not fully outlined below, but only that portion pertaining to fee preparation of the crude fractions as used throughout certain sections of this paper. For the complete procedure, covering the further purification of Bios 2(a) the reader Is referred to the original reference* For the treatment of the tannic acid precipitate containing Bios 5 see E.2*a£r:! li (1936). Procedure 3» To the contents of 6 tins of tomato juice (about 6.5 lbs* each), add 135 gm. tannin dissolved in 600 ml. hot water; shake the juice from each tin separately with one sixth of the tannin* Let stand for 24 hours, then separate the tannin precipitate from the tannin f i l t r a t e in a centrifuge. The precipitate contains Bios 5 (from which tannin i s removed by lead aoetate treatment)* To the f i l t r a t e add 640 gm. crystalline lead acetate dissolved in 800 ml. hot water. Shake 1% hours, centrifuge, and discard the precipitate. Bemove most of the lead by gradually adding 55 ml. cone* sulfuric acid dissolved in 240 ml. water; after shaking for \ hour, the precipitate i s easily removed by f i l t e r i n g through paper, i f some well crystallized lead sulfate has been added. Precipitate the remainder of the lead by adding 30 ml. of fresh, saturated barium sulfide solution, shaking j? hour and fi l t e r i n g through paper. Remove hydrogen sulfide from the f i l t r a t e by d i s t i l l i n g off about 3 11. of water in vacuo. Add 2 ml. cone, sulfuric acid per l i t e r of solution; shake f i r s t with 100 gm., then with 75 gm. Nuchar or Norite ohareoal. The charcoal adsorbs Bios 2(b), the f i l t r a t e contains Bios 2)a) and inositol* Wash the charcoal with hot water in a soxhlet (12 runs) then shake hour with Sparling's Acetone - Ammonia reagent* F i l t e r through paper wife, suction, and immediately evaporate to dryness in wacuo. Dissolve the residue in a l i t t l e water and evaporate again. For the f i r s t portion of oharcoal use 6 1250 ml. of reagents This extraction removes 50 - 60$ of Bios 2(h) present in the tannin filtrate, a second extraction of the same charcoal with 940 ml. of the reagent yields 10 - 15$ more* Extraction of the second portion of charcoal with 940 ml. of reagent yields another 10 - 15$. The charcoal filtrate is the Crude Bios 2(a) solution; the charcoal eiuate is Crude Bios 2 (b). The following modified procedure for the isolation of the Bios 2(a) and Bios 2(h) fractions of Miller which carries the purification further and includes the removal of inosite from the Bios 2(a) fraction, was suggested and success fully used by Eagles and Wood (unpublished data 1936). Procedure 4: To 3500 ml. agueous extract of alfalfa laeal, add 210 gm. finely powdered lead aoetate. Shake, allow to stand 24 hours, then filter. Treat the filtrate with 1000 gm. basic lead solution (180 gm. lead acetate, 110 gm. litharge). Allow to stand for •§ hour and filter* Then, add 150 gnu baryta dissolved in 350 ml. water* Remove lead and baryta with 5$ sulfuric acid. Then add 20 ml. 20$ sulfuric acid, 10 ml. 10$ phosphotung-stic acid per 100 ml. of solution. After filtering , remove excess phos-phortungstic acid and sulfuric acid with baryta. Reduce the volume of the solution to 500 ml. by distillation in vacuo* Add 20 ml. 2N sulfuric acid, shake with 50 gm; then with 25 gm. Darco charcoal* Remove Crude Bios 2(b) from the charcoal by treatment with Sparling's reagent, first eluting with 400 ml. and the second with 200 ml* of the reagent* Remove acetone and ammonia by repeated distillation in vacuo to dryness* Mke up to 100 ml* wilk distilled water, and neutralize with N/4 soda* Free the Bios 2(a) as contained in the charcoal filtrate, from sulfuric acid with baryta. Distill in Vacuo as with Bios 2(b), make up to 100 ml. and neutralize. 7 Kogl (1936) l ias .published a method of i s o l a t i n g a c r y s t a l l i n e substance having Bios a c t i v i t y as tes ted on yeasts , which he c a l l e d " B i o t i n . " The essent ia l d i f ference between Kogl ' s technigue and that of M i l l e r i s that the former carr i e s out a l l p r e c i p i t a t i o n s i n very concentrated s o l u t i o n , whereas the l a t t e r works w i th large volumes. By us ing concentrated phosphotungstic a c i d in concentrated s o l u t i o n Kogl has p r e c i p i t a t e d the act ive material completely. It i s probably due to the d i f f erent cone en-t r a t i o n s involved that there Is no p r e c i p i t a t i o n of a c t i v i t y by t h i s reagent i n Pro oe dure 4. Kogl , moreover* does not seem concerned wi th the mult iple nature of B ios , s ince a f t e r removing jiiospho tungstic a c i d from h i s f i l t r a t e and charcoal ing , the B i o t i n i s aocording to Kogl , completely adsorbed on ohareoal and no a c t i v i t y remains in so lu t ion . Procedure 5: Cook 1000 fresh hens eggs f o r 15 minutes and separate out the y o l k s . Divide yolks i n four port ions and extract each twice with 5 l i water by b o i l i n g f o r 1 hour. F i l t e r the mixture while hot , and a f t e r coo l ing , a l low to stand w i t h h a l f i t s volume of acetone. A f t e r several hours, the f locoulent p r e c i p i t a t e i s f i l t e r e d o f f and the c l e a r l i g h t greenish yel low f i l t r a t e evaporated In vacuo to dryness. The residua i s d isso lved In 1 11. water and allowed to stand over night with 4 11. absolute a l c o h o l . F i l t e r the prec ip i ta te o f f and evaporate f i l t r a t e to dryness In vacuo. Dissolve the residue i n 3 l i . water and treat w i t h an excess o f concentrated lead acetate s o l u t i o n . A f t e r oentr i fug ing , remove lead from the f i l t r a t e wi th hydrogen s u l f i d e , f i l t e r and evaporate to dryness in vaoao. 8 Dissolve the residue in 150 ml. 5$ sulfuric acid, and with continued shaking add 200 ml. 50$ phosphotungstic acid in 5$ sulfurio acid. Wash precipitate with about 40 ml* water acidulated with hydrochloric acid* .Remove phosphotungstic acid from the precipitate by grinding in a mortar with baryta solution, f i l t e r , and bring the action filtrate to dryness. Dissolve the active residue from the phosphotungstic precipitate In 500 ml* water and shake for one hour with 50 gai* charcoal. Filter and wash with water. Shake charcoal for two hours wife 1 lie 50$ alcohol* After washing with alcohol, eluate charcoal three times with 1 l i . acetone-ammonia reagent (60$ acetone - 2*5$ ammonia) by shaking ijg hours each time. Evaporate to dryness in vacuo and remove acetone-ammonia by repeated dis-tillation. This is the material containing Biotin* For the remainder of the procedure concerning the isolation of Biotin, see reference. The work reported upon in this paper involved only that portion of the procedure given above* * * * A study of the role of growth factors in relation to the physiology of micro-organisms is now being recognized as a very important field of bacteriological research. Although the geneial occurrence of Wildiers9 Bios in a l l living cells had been known for some tins, its physiological influence had not been studied until the last few years, except in the case of yeasts. Orla Jensen's (1936) monograph on the vitamin require-ments of the lactic acid bacteria is undoubtedly the fi r s t most detailed contribution to appear on the subject* He showed that a i l lactic acid •Stoaptococgl, Strentobaoterla and Thermobacteria require the factor, vitomin Bg or Bios for their most intensive metabolism* If vitamin Bs 9 is a part of the Bios complex, as he supposes It to be. It would appear to correspond to Bios 2(b) of Miller* He also suggests that milk contains another heat stable, alkali fast factor which is not absorbed on charcoal and which Is required by the lactic acid bacteria he studied. This factor would appear to correspond to Bios 2(a) of Miller. Eagles and co-workers (1936), also studying the lactic acid organisma, were the first to show that Bios is of vital importance in the metabolism df certain strains of Betaoocous cremoris. They followed Procedure.1 for the preparation of Bios concentrates, but were not entirely satisfied with the results. Procedure 2 was then tried out with better success, and adopted throughout their work for the preparation of a l l Bios fractions. They were able to confirm, by the use of this procedure, the contention of Miller regarding the multiple nature of Bios. In more recent, unpublished work. Procedure 4 was developed and used extensively for the preparation of the Bios 2(a) and 2(b) fractions of Miller. It was with this background, that the writer undertook an investig-ation of the role of the growth factor or factors known as Bios, in the vital processes of the root nodule bacteria and the leguminous plants which they infect. 10 PART l : THE INFLUENCE OF BIOS ON RHIZOBIUM  TRIFOLII Bight stock strains of Rhizobium Trifolil, were selected for study. Considerable difficulty was encountered at the outset in selecting a suitable method for measuring the effects of the aotive fractions on the physiology of the organism* iince the distinguishing feature of this organism, under natural conditions, is its ability to utilize atmospheric nitrogen, one might expect to determine the influence of Bios by measuring its effect on nitrogen fixation* This was not possible, however, as these organisms are not capable of fixing nitrogen when growing on laboratory artificial media apart from the host (see pages 26 - 28). As a means of determining stimulation in the preliminary investigations, the giant colony technique was employed. Extreme care must be taken with the amount of inoculum used, and considerable experience with this technique is necessary before uniform colonies result* This method permits a measure of cell multiplication rather than a measure of the products of cell metabolism* , N (l) SIANT COLONY STUDIES  Influence of Alfalfa Extract. Before proceeding to a study of the Bios concentrates, the activity of the original alfalfa hydrolysate was determined. In this, as in al l similar tests, an unenriched medium was used as a basis for comparison. Yeast water, which Is usually used as an enrichment for the Hhizobia was omitted because of the growth factors contained thereto. The following constitutes the basal medium used throughout this paper. Mannitol — • — . iO#0 gm. K HPO^  — ~ 0.5 gm. Mg So^  = - • • — — — ~ — 0.2 gm. Sa CI — « - — * o.l gm. Tap water — - 1000 ml. An enriched medium is one carrying a definite percent of the Bios concen-trate. All fractions as well as the basal medium are adjusted to pH 7.0 Using alfalfa extract in 10$. concentration, 6 enriched and 4 control plates were poured for giant colonies of each of eight strains of Hhizoblum Trif o l i i . Bach plate contained 20 ml. of medium. Inoculations were made from the respective organisms cultured in basal medium for two previous o transfers. Plates were incubated at 28 C, and measurements made at 4, 6, 8 and 10 days. Uniform circular colonies were obtained in most instances. The figures in the following table represent the average area of giant colonies in square millimeters, resulting from the above treatment. TABLE 1 EFFECT OF ALFALFA EXTRACT ilOJ) ON STRAINS OF HHIZOBIUM TRIFOLII STRAIN 4 days 6 days 8 days 10 days Final Increase EXTRACT 26.7 57.6 116.2 142.7 *C1 22B CONTROL 6.2 9.0 11.1 12.o' 11.9 times EXTRACT 23.5 112.2 170.5 221.6 f Bel £02 CONTROL 0.0 2.1 4.3 6.0 35.2 " EXTRACT 33.2 97.1 116.5 185.8 R ol 205 CONTROL 14.0 24.5 24.7 25.3 7.4 " EXTRACT 31.0 74.3 138.2 220.8 B d 227 CONTROL 8.1 9.5 11.8 12.3 17.9 " EXTRACT 9.1 80.8 154.5 237.8 R ol 224 CONTROL 2.5 14.2 17.5 21.0 11.3 " EXTRACT 18.4 52.8 99.6 173.2 R cl 230 CONTROL 0.0 6.2 9.2 13.7 12.6 »» EXTRACT 20.8 76.3 138.4 213.2 R cl 231 OOKCBOL 4*2 4.7 9.7 10.3 20.7 » EXTRACT 21.4 76.6 129.6 185*0 R ©1 2&G3 CONTROL 3.0 8.5 10.0 11.7 15.8 » •*Kxceptlonally good nitrogen fixing strain. see &BAPH 1. t Distinctly poor nitrogen fixing strain. 12 From these results it is apparent that a l l strains of Bhizoblum T r l f o l l l tested, are stimulated by alfalfa extract. The amount of stimul-ation induced in the various strains bears no relation to the amount of growth on unenriched medium. It is interesting to note also that the response of the poor nitrogen fixing strain i s three times greater than that of the good nitrogen fixing strain. M i ether or not this ia of any significance, has not yet been determined. ffllLUBTOB OF CRUDE BIOS 2 Procedure 2 was employed in the preparation of the Bios concentrates used in this test. Prior to separation of the active f i l t r a t e into two distinct entities by charcoal adsorption (see Procedure), the f i l t r a t e is known as Crude Bios 2. It was thought advisable to test this crude fraction to determine whether or no t a l l the factors present i n the original extract of a l f a l f a , were s t i l l present. 0.5$ Bios 2 was used as enrichment, and for purposes of comparison 10$ yeast water enrichment was also tested. Two typical strains of Rhizobium T r i f o l i i were selected for this exper-iment, viz. strains CI 22B and R c l 227. Same technique used as in previous experiment. TABLE 2 - EFFECT OF MILLER'S CRUDE BIOS 2 ON, RHIZOBIUM TRIF0L33 SmmB RjglJ27 AND OL 22B, TREATMENT 2 4 6 8 10 . 1Z I4days Bios 2, .5$ 17.6 56.5 110.8 170.2 235.3 317.1 395.7 Yeast 10$ 14.7 39.2 60.0 90.3 117.7 145.6 161.1 Control 5.1 6.8 . 13*0 16.0 19.2 Bios, 2 5$ 14.2 18.7 20.8 •21*3 22.0 22.6 23.3 Yeast, 10$ 30.3 51.3 62.6 73.2 89.0 103.7 '109.9 Control 4.0 6.7 9.8 12.2 13.0 see 14.6 graph 2. 23.1 13 It is Interesting to note that the good nitrogen fixing strain used in this particular instance does not respond to Grade Bios 2, while a poorer strain so used Is stimulated* Later work has shown that this effect may have been oaused by too high a concentration of the factor (see Page 50), resulting in toxic material masking the stimulating effect. Since, however, the size of the colonies are the same as the controle, it is possible that this particular strain requires growth factors originally present in the plant extract and removed by treatment with heavy metals. This variation in groth factor requirements within the species may be a profitable clue to follow in studying characters of the strains in relation to nitrogen fixation. In both cases, yeast extract gives a considerably lower stimulation than the plant extract due possibly to lack of a factor in the former which the organism, by its association with plants, has come to require for its best growth. It is to be noted, that in the case of strain R cl 227, greater stimulation is brought about by the additions of 5 ml. Crude Bios 2 per liter, than where 100 ml. of plant extract was used in the previous experiment, indicating a concentration of the active principle of approximately twenty times at this point in the procedure. The organism ohosen for further study was strain R ol 22?' because of its ready response to Bios enrichment. IOTLPEHOB OF BIOS 2 fa) AFP BIOS 2(b). Having selected the organism for Intensive study, the finally concen-trated Bios 2 (a) and Bios 2(b) fractions (Procedure 2) were tested for activity by the same giant colony technique as previously described Bios 1 (Eastman's ash-free inositol was made up in the concentration of .05 gm. to 50 ml water, and this solution added to the medium at the rate of 20 ml. per liter. The Bios 2 (a) and Bios 2 (b) fractions were added at 0.5$ 14 The Bios 2(a) and Bios 2(b) fractions are in a l l probability chemically distinct due to the selective nature of charcoal adsorption. Since both were shown by this experiment (see Graph 3) to stimulate Rhizobium g r l f o l l i Miller's conception of the multiple nature of Bios i« confirmed. However, Miller's observation that Bios 2(a) and 2(b) when acting in combination, have a stimulating power on yeasts greater than what would be expected by mare addition does not apply in the case of the nodule organism studied. m PLATE COUNTS - THE RELATION OF GIANT COLONY DATA TO RATE OF CELL MULTIPLICATION The stimulation of yeasts is measured by the weight of the centrifuged yeast crop and, since no gum is produced, that value is a direot expression of the number of cells present. Since the Hhizobla produce considerable gum, i t was thought possible that the stimulation noted in giant colony studies might have been due to gum. production rather than c e l l multipli-cation. It was therefore considered desirable to determine whether or not a direct correlation existed between size of colony and c e l l division. The organisms were grown in 10 ml. fluid cultures using 0.5$ Crude Bios 2 (Procedure 2) as enrichment, and the basal medium as oontrol. To each tube, 2 drops of a rich suspension of R 01 227 (grown for two transfers on slopes of basal medium with no enrichment) were added and o the cultures incubated at 28 . Plates were poured in dilutions of 1:1,000,000 and 1:10,000,000 at 24 hours, 2 days, 3 days and 4 days on yeast water mannitol agar (basal medium to which 100 ml. yeast extract added per l i t e r ) . 1 5 T A B L E 3 - E F F E C T O F B I O S 2 O N O E L L M J L T I P L I O A T I O N I N  C U L T U R E S O F R H I Z O B I U M T R I F O L I I A G E C O U N T I N M I L L I O N S P E R M L . C O N T R O L B I O S 1 D A Y 1 ® 3 9 2 D & Y S 2 7 8 1 3 tt 2 9 2 1 8 4 tt 2 9 2 1 5 see Graph 4 . It Is to he noted from the above results that there is very close agreement between the figures obtained by this method and' those obtained by giant colony teohnigue. At four days, the ratio of cell numbers in controls to c e l l numbers in Bios cultures in the above instance is 2 9 1 2 1 5 , The corresponding ratio between the size of the same organism at the same age i s 6 . 8 : 5 6 . 5 . Considering the difference in approach, there is apparently l i t t l e doubt that the giant colony measurments give a reason-ably accurate indication of actual cell multiplication for this organism. At 3 days the aotual number of living cells present "'cached a maximum and at 4 days the counts were the same or showed a slight decline in both Bios and control tubes. Naturally, such a curve i s not presented by plotting giant colony data since such data includes the non-living cells as well as the living. Although the aotual number of living cells causes a consistent increase in the size of the giant colony until lack of nutrients or accumulations of toxic produots of c e l l decomposition become limiting factors. 16 Although the medium on viSiich both the Bios 2 enriched and control cultures were plated was the same, the colonies from the enriched culture were in most cases about four times greater in size than the colonies from control plates. Sinoe it does not seem likely that stimulating factors could be carried through in sufficient quantity after dilution to account for the great differences in colony size, i t appears that the previous history of the organism should be considered in expressing results* In previous work, care was taken to grow the organism in an unenriched medium for at least two transfers, to avoid the effects of active substanoes carried over in the inoculum as would, be the case i f the normal yeast water enriched medium were used. To clarify this point, an experiment was begun to determine the influence of the previous history of the organism on its response to stimulating factors. (3) IMPORTANCE OP PREVIOUS HISTORY OF THE ORGANISM  IN DETERMINING ITS ACTIVITY Strain R 01227 was inoculated from a stock yeast water mannitol slant into two 50 ml. fluid cultures, one containing the basal medium and the other containing the same medium enriched with 10% yeast extract. After three days, transfers were made from the two cultures into new flasks of the respective media. After three days growth in the second series* the cultures were used as sources of inoculum for giant oolonies on control plates and 10$ yeast extract enriched plates. Measurements of giant colonies were made on alternate days for two weeks. These results (see Graph 5} demonstrate the importance of considering the previous treatment of the organism when studying stimulating factors. I O 34 G R A P H P A P K f t . S M I T H . D A V I D S O N ft W R I G H T . L T D 17 Also, an explanation ie afforded for the differences existing between the size of colonies obtained when making plate counts on Bios enriched and control fluid cultures. In general, it appears that when the organ-• ism is in a highly active state because of growth in a medium containing Bios, it is more responsive to further stimulation than if it had not been previously activated. There is also better growth, even in an unenriched medium following previous stimulation, than where the organism has not contacted Bios for several transfers. One might expect that the organism when grown in a yeast water medium would come to depend on the enrichment and grow more poorly on an unenriched medium, than an tan-activated organism which had opportunity to adopt itself to lack of the growth stimulating factors. That the reverse is actually the case is clearly brought out by this experiment. (4) HTBOQEN FIXATION STOPIES The Bhizobia, when growing in the absence of the host plant, have never been satisfactorily shorn to fix atmospheric nitrogen. Allison (1929) carefully investigated this question and concluded that his ex-periments "in no oase gave any evidence that Bhizobia can fix notrogen when grown apart from the host." He tested many different strains of Hhizobiura species, utilized some sixteen carbohydrates as energy sources, and enriched his media with various plant extracts. His conclusions were based on an analysis of over nine hundred cultures. Some workers had previously reported the oocurrenoe of fixation of nitrogen by Bhizobia on ordinary laboratory media, but M. Lohnis (1930) 18. definitely demonstrated the error of such experiments. She found that uninoculated control cultures lost nitrogen in some volatile form during incubation, Whereas tfeose containing growth, retained this nitrogen. Tims, when controls were analyzed at the end of the tests, It was found that they contained less nitrogen than the inoculated flasks J and the difference was interpreted as a gain in notrogen due to the nitrogen fixing a b i l i t y of the nodule organism. That this was only "apparent fixation" became obvious when such experiments were repeated and controls analyzed at the outset of the test. When this was done, there was found to be no differ-ence in notrogen content between inoculated and uninoculated flasks. Wilson, Hopkins and Fred (1932) before considering the matter settled , attempted by many various methods to provide the necessary conditions for fixation to occur. The works of Olara and of Golding which had com® to be considered almost classical experiments demonstrating fisation were care-ful l y repeated and the findings of the original workers not confirmed. Along with the Rhizobia in the Nodule, various contaminating organisms nay possibly be found, and i t was thought that more natural conditions and perhaps the essential conditions might be provided by mixed cultures. Rhizobia were grown together with Bacillus radlobaoter. Pseudoraonas flnoresoens. Clostridium Pastorianum and various protozoa, but no nitrogen gains were observed. The possibility that the presence of respiring plant tissue with i t s accompanying enzymes might, by a complimentary action enable the Rhizobia to be active in u t i l i z i n g atmospheric nitrogen, was investigated but with no positive results. Nodules were taken from roots and grown in f l u i d cultures under aerated conditions, and even those nodules, once severed from the plant, although intact, could not f i x 19 nitrogen. Wilson, Hopkins and Fred therefore concluded that the solution to the problem would have to be found in another angle of attack, since in no instance did they receive positive results. The writer made rather extensive investigations to determine whether or not Bios concentrates would induce Rhlzobium T r i f o l i i to f i x nitrogen when grown apart from the host plant. In the preliminary experiment, 5% alfalfa extract was used as enrichment for the basal medium. Twenty 5 0 ml. fl u i d cultures in 1 11 ErllnmeyerB were inoculated with R 01 227, The cultures were incubated at room temperature and shaken by hand at frequent intervals to provide aeration. Analyses for nitrogen were made at 4,10 and 14 days by the Salicylic modification of the Kjeldahl technique. TABLE 4 - NITROGEN CONTENT OF CULTURES OF RHIZOBIUM TRIFOLII ) ST RAIN R Cl 227) CULTURE AGE NITROGEN CONTENT (MGM) Inoculated, basal medium 4 days 0 . 5 1! ti n 1 0 tt 0 . 8 tl «? tt 1 4 it 0 . 5 tt alfalf 'a extract 4 it 9 . 5 IS it n 1 0 tt 9 . 5 tt . it tt 1 4 tt 6 . 6 Unlnoculated ., basal medium 1 0 tt 0 . 8 s o It was concluded from the above results that with the technique employed, no nitrogen was fixed by R01 227. There was definite stimul-ation of growth in the presence of alfalfa extract during the first four , days, but at ten days an examination of the culture showed the viability to be very low* A similar experiment to the above was conducted with the actual Bios fractions 2(a) and 2(b) (Procedure 2). These fractions were added at the rate of 0.5 per cent, and In addition, inosite was used in some cultures to the extent of 0,04 mgm* per ml. Aeration was accomplished by tying the flasks on a tilted, continually revolving plane as an improvement over the hand shaking method used previously. Cultures of botfo strain ROl 227 and strain Cl 22B were grown in this experiment. Results are expressed as milligrams of nitrogen per 25 ml. of culture. TABLE 5, - NITROGEN CONTENT Og CULTURES. OF RHIZOBIUM TRIFOLII Inoculated: Bios 1(inosite) Bios 2(a) + 2(b) Bios l+-2(a) + 2(b) Yeast extract (10$) Control STRAIN ROl 227 STRAIN, Cl 22B ,10 "da. 4 da..'" 10 da. 0.00 0.10 0.05 • 0.107 0.00 0.00 0.00 0.00 2.5 2.6 2.7 2.4 2.4 2.8 2.7 2.7 2.3 2.3 2.8 3.2 2.3 2.4 2.6 3.5 3.0 2.8 2.9 3.2 2.0 2.9 2.9 2.8 0.10 0.10 0 0 . 0 0 0.10 0.10 0.10 0.05 0.05 21 igABLE 5 (QON?£l Uninoculated s 4 da. 0.2 Bios £; los . 0.0 .1 -;.o 5 2«7 Bios 2(a) 2(b) 2.5 2.3 Bios 1 2(a) 2(b) 2.4 0.7 Yeast extract 3.7 0.1 Control 0.1 From these results, i t was ooncluded that there is no definite indication that Bios stimulates Rhizobium t r i f o l i i strains to f i x nitrogen apart from the host plant when grown in f l u i d culture for ten days. Since growth was slow at ten days the experiment n s discontinued. It was considered possible that lack of aeration nay have been the factor limiting growth and inhibiting nitrogen fixation in the previous experiments with f l u i d cultures. Plate cultures were therefore sub-stituted, and a medium with 1.5$ agar added, but otherwise similar to that used previously was employed. Bios 2(a) and 2(b) - 0.5$ - and yeast extract, 10$ were used as enrichments. 50 ml. portions of the medium were poured into eight inch petri plates, and after the agar hardened, 2 ml. of inoculum (strain R01 227) was smeared as evenly as 'o possible over the whole surface. Plates were incubated at 28 C for twelve days, then the contents transferred to Kjeidahl flasks and nitrogen determinations, made as before by the modified salicylic method to include nitrates. A l l plates analyzed at twelve days. 22 TABLE 6 - MITHOSM _QOmmT OF OOMmBTMjw m^PJLIUjLTaiPOLn (BQ1 227 1 — .—PJfflEHRJL. MEM. K. TKT PL/WR! Bios plate, inoculated 4.3 »» i i tt 4.5 !L_ .. uninoculate^ 2.5 Yeast plate, inoculated 3.7 « H f |) 3.6 »_. " . unlnnaalated 3.0 Apparently atmospheric nitrogen was fixed In this experiment, "beyond experimental error, especially in the case of the concentrated Bios enrichment. In view of the work of M. Lohnis of Wisconsin, however, another experiment was begun in which the controls were analyzed at the outset as well as after incubation to see i f the "fixation" resulted from nitrogen losses in the controls or whether gains were actually made. This experiment was conducted in essentially the same manner as the a b o v e • Share were extra controls however, so that analyses for nitrogen could be made at the time of inoculation as well as after the incubation period which was 6 days. The Davison - Parsons modification of the Kjeldahl procedure ( 1919 ) was used in tt*Is instance. Although this method is undesirable from the standpoint of its complexity, i t is thought by many investigators that a more accurate evaluation of nitrogen is afforded by its use where materials containing a high percentage of water are analyzed. zz TABLE 7 - NXTBOGEN CONTENT OF armrnmM nv HHjZOBIUM TRIFOLII fftfll 3?.7|. • WLgUftq M_E_IN MYS ISM, N. IN PLATE ,.Bios plates, uninoculated 0 4*2 4.4 6 2.4 2.2 —2 inoculated s 3.9 4.«0 Yeast plates, uninoculated 0 5.8 6.5 6 6.1 5.4 — — H i fapculated 6 4.2 5.Q Control plates, uninoculated 0 0.0 0.0 6 0.8 0.8 —- — " « tooguiasafl 6 0.6 1.0 It w i l l be noticed that this experiment gives a perfect check on the previous one when the additional information, supplied by analyses of controls at the outset is considered, it is found that both inoculated and uninoculated plates lost nitrogen during incubation; since the latter lost nitrogen more rapidly than the former an "apparent fixation" occurs, confirming Lohnis' contention. In the case of the unenriohed controls, a slight gain in nitrogen after six days took place, probably due to adjustment of equilibrium between the agar and the combined nitrogenous gases of the laboratory atmosphere. Having obtained a very active Crude Bios 2 preparation by Procedure 4, this fraction «s tried in plaoe of Miller's 2(a) and 2(b) (Procedure 2) in a final attempt to obtain nitrogen fixation on synthetic media. Thirty, eight inch fluid plates were poured using the Crude Bios 2 fraction in 5.$ 24 concentration. Agar was left out of the madiura to permit growth throughout the plate. Twenty plates were inoculated with strain B01 227 from a yeast extract enriched culture, instead of from the usual unen-riched culture. The remaining plates were reserved as controls. Analyses were made at 0, 7, 10 and 14 days. TABLE 6 - NITROGEN CONTENT OF OULTUBES OF J^OBIUM TRIFOLII (ROl 227^ MSM* N. U N I N O C . P L A T E S I N O C U L A T E D P L A T E S 10.2 0 days 10.2 10.2 10.3 10J2_ 7 days 9.5 10.0 9.4 9.9 9.9 10.0 9.2 9.3 9.8 — — _ : 9.9 10 days 8.7 9.4 9.1 .9.1 9.1 9.6 9.9 14 days 9.1 8.7 8.5 9.0 9.5 The organism produced such quantities of gum in this experiment, that after one week incubation the plates could be Inverted, the viscous mass having formed a gel. Even with such growth as this, no nitrogen was 25 fixed* It was finally concluded that, with the technique employed in these experiments, that no fixation occurs either with or without the addition of Bios to the medium* Any "fixation" that was observed was only "apparent fixation*" the loss of nitrogen from the controls giving rise to this effect* The form in which this nitorgen was lost was not deter-mined* (5) SUGAR FERMENTATION STUDIES In order to determine further, the influence of Bios on the physiology of Rhizobium t r l f o l l l 227., and In an attempt to find a new method of measuring stimulation, sugar fermentation reactions were studied. Thirteen different carbon sources were used, each in three media different in respect to growth factors. The sugars were: glycerine, S^lose, glucose, sucrose, lactose, maltose, trehalose, raffinose, dextrin, mannitol, adonitol, sa l i c i n and starch. The basal nitrogen free medium, with other sugars substituted for mannite, was compared to a similar medium containing 2$ Crude Bios 2(Procedure 4) and to another enriched with yeast extract• The yeast extract contained 1.72 mgra. introgen per ml* and Crude Bios 2,1.06 mgm. nitrogen per ml* Yeast extract was added in such a concentration, as to ensure the same nitrogen content in the two enriched media. Three l i t e r s of the basal medium (without sugars) were prepared and divided into 3, 1 l i t e r portions. Bios 2 (2$) and yeast extract (nitrogen equivalent) were added to these respective flasks while the third was retained as control. These were autoolaved at 15 lbs. for 15 minutes, then filtered, and the reaction adjusted to pH 6.8. Three, 0.4 gm. 26 quantities of each sugar were added to 40 ml. of each of the three media which enabled the test to be conducted in triplicate. 0.5$ of an alcohol solution of brom thymol blue was added and the medium tubed in 10 ml. quantities. After autoolaving at 15 lbs. for 15 minutes the tubes containing glucose, lactose, and maltose changed to pH 6.4 and xylose to below pH 6.0. fhes9 tubes were discarded. The balance retained their original reaction. Each tube was inoculated with one drop of suspension of ROl 227 from an unenriched mannitol agar slant. o After four days incubation at 28 C, the following changes were noted: Controls: No change - starch, adonitol, dextrin, raffinose. S l i ^ i t acid production - Sucrose, glycerine Considerable acid " - Salicin, mannitol, trehalose. Yeast - no change - starch. Very slight acid production - trehalose, adonitol, mannitol. Slight " i i « Sucrose, salicin, dextrin. Considerable " " - raffinose. Slight alkali " " - glycerine. Bios - no change - glycerine, raffinose, destrin, adonitol, starch. Slight acid production - sucrose. Considerable acid " - mannitol, trehalose, salicin. From the above observations, i t would appear that yeast extract provides factors other than Bios 2 which affect the organism. Whereas the degree of fermentation of the various sugars in the control conforms with the Bios enriched tubes, the yeast tubes present an almost reverse situation. Only starch and sucrose retain their relative positions 27 throughout. I/annitol and trehalose from which the greatest amount of aoid i s produced i n the Bios and control tubes, are very s l i g h t l y acted upon in yeast tubes. Conversely, raffinose, which does not appear to be attached i n the Bios and control tubes, i s the one showing fee most highest degree of fermentation i n the presence of yeast extract fermented when yeast extract i s present. A f t e r seven days incubation, the tubes were t i t r a t e d with 0.0025 N sodium hydroxide. This was prepared immediately p r i o r to t i t r a t i o n by d i l u t i n g 10 ml. of standard 0.25 N soda to 1 l i t e r with boiled d i s t i l l e d water.. A l l tubes, including controls, were t i t r a t e d to pH 7.2. The difference i n t i t r a t i o n values between controls and inoculated tubes, was taken as a measure of a c i d produced. Positive values as presented i n table 9 represent ml. 0* 01 N acid, negative values ml. 0.01 N A l k a l i produced per l i t e r . TABLE 9 - INFLUENCE OP YEAST EXTRACT AND BIOS 2 ON  SUGAR FERMENTATION BY RHIZOBIUM TRIFOLII 227 SUGAR CONTROL Control Tnnn RamiH YEAST BIOS 2 GLYCERINE 2.25 3.5 + 1,25 ooncroj. 3.0 Inoe.Result 2.0 - 1.0 uont. 3.0 Inoe.Result 2.0 -1.0 SUCROSE 2.0 7.5 + 5.5 4.0 5.2541.25 2.5 5.0 +2.5 TREHALOSE 0 5.75 + 5.75 0 4.0 •4-4.0 1.0 3.25 +2.25 RAFFINOSE 2.0 2.0 0 0 7.25 +7.25 2.0 1.75 -0.25 DE20EXN 5.25 5.75 + 0.5 0 1.75 + 1.75 2.75 0 -2.75 MANNITOL . - 2.5 3.75 + 6.25 2 .75 5.25 +-3.0 0 4.5 +4.5 ADONITOL 0.4 0.0 - 0.4 2.5 4.5 +-2.0 3.5 1.5 -2.0 SALIC IN 0 6.25 \ 6.25 3.75 6.25 + 2.50 2.75 8.5 +5.75 28 In the visual observations made at four days, it n s not possible to consider the buffer capacity of the various mediae For; this reason, the results tabulated above present a more exact account of what is taking plaoe. In general, the two agree closely, but many additional details are emphasized by the titration values* Raffinose is the outstandingly interesting carbon source* and may prove to be a key sugar in respect to determining the nature of various activating fractions* In accordance with this data at least, the growth factors have no effect whatsoever in altering the organism's ability to produce acid from raffinose, whereas additional faotors, possibly of vitamin nature, which are oontained in yeast extract enable i t to ferment raffinose strongly. Salicin and mannitol, particularly the former, present the reverse situation. The presence of yeast extract greatly inhibits acid production from these sugars whereas in both Bios enriched and control tubes, considerable acid production in Bios enriched tubes than in the controls. There was, however, considerable alkaline fermentation of glycerine, adonitol, and dextrin in the presence of Bios, the production of alkaline products from the last two energy sources being distinctive of Bios enriched tubes alone. The possible "Key" sugars suggested for future studies of this nature are therefore: raffinose, adonitol, mannitol, dextrin and salicin. By means of these sugar reactions, information regarding the type of activating factors present in a stimulating fraction might be obtained. If factors other than Bios are concerned, there appears to be a very different effect than where Bios alone is Involved* It is interesting also, to contemplate what information might be gained by a study correl-29 ating sugar fermentation of different strains in the presence of various activators, to nitrogen fixing ability, phage sensitivity, etc. of these strains. At the time titrations were made, the tubes were examined for amount of growth by observing the degree of cloudiness, in order to assess; .the degree of the stimulation of strain R 01 227 in media containing sugars other than mannitol. The Bios tubes which definitely showed great in-crease in growth over the controls were those containing raffinose, dextrin, mannitol, and adonitol. Slight stimulation was apparent in the case of sucrose and trehalose. The others showed no differences. Actual number of cells present is apparently not correlated with sugar ferment-ation. In other words the nature of the substrate and the presence of emzyms activators is more Important than amount of enzyme produced. Attempts to demonstrate the appearance of reducing sugars in cultures of the more complex sugars at the age of four weeks were without success. Likewise, negative results were obtained in an at tamp t to detect loss of reducing sugars in cultures containing glucose after one month. 30 (6) AMMONIA PRODUCTION STUDIES Due to the small amounts of aoid produced by the Rhizobia, and the complex nature of the results obtained using various energy sources and enrichments, the need was s t i l l felt for some simple method that would involve a measure of metabolic activity. Plate counts and giant colonies while giving desired results, were objectionable from the standpoint of time and equipment involved. Since Rhizobia contain urease, it was thought possible that deter-minations of ammonia production from urea in the presence and absence of Bios might afford a ready means of measuring activation as well as re-vealing further physiological effects of Bios on the organism* The preliminary experiment relative to this study was rather cumber-some, due to the use of large cultures and the method of estimating ammonia production. Ten 1 '- l i t e r flasks were used, each containing 100 ml. of the basal medium, five of which were enriched with 5$ of Bios 2 (Prooedure 4). After autoclaving, urea solution, sterilized separately and neutralized with sterile hydrochloric aoid, was added aseptically so that each flask contained 0.2$ urea. Each was inooulated with 1 ml. of a 4 day old unenriched culture of RC1 227. The flasks were incubated at 28° C. After two days, the Bios flasks contained heavy growth while the controls showed no apparent cloudiness. At four days, both were equally cloudy (see Graph 6)• Ammonia determinat ions were made at the outset, and at 2, 4, 6 and 8 days by placing the ufaole culture in a Kjeldahl flask, adding 2 gm. magnesium carbonate to liberate ammonia, and d i s t i l l i n g into N/7 Ijydro-31 ohlorio acid. Paraffin ana pumioe were added to the d i s t i l l i n g flasks to prevent excessive foaming and humping. Results are expressed as milligrams of nitrogen per 100 ml. of cultures TABLE 10 - AMM3NIA PRODUCT ION BY RHIZOBIUM TRIFOLII 227. MGM. AMMONIA NITROGEN PER 100 Mfeg. Ag-Ej CONTROL BIQS„.__ 0 days 1.2 1.2 2 •» 3.2 0.0 4 « 5.0 3.2 6 « 27.6 12.0 § '1 , 11«0 . See Graph 6 A study of the table indicates that, in general, there is a gradual accumulation of ammonia nitrogen in the control flaska while a similar though markedly smaller accumulation occurs in the presence of Bios. This would appear to be due to the utilization of ammonia nitrogen by the organ-isms in the Bios cultures. The above procedure for determining ammonia production was modified considerably in the following experiment and permitted a detailed study of the various Bios fractions in a large number of different concentrations. The Basal medium, containing in addition to the usual constituents, 2 gm. of urea per l i t e r , was adjusted to pH 7.0 with brom thymol blue. S$ of a saturated solution of Cresol red was added directly to the medium. A l l enrichments used, were carefully adjusted to pH7.0 and the medium sterilized by passage through a Berkefeld f i l t e r . By this means, the medium in 10 ml. quantities was added to test tubes. The same f i l t e r was I p 50 \ ' n >Diot I c c)\ /I-)h)o proli-ne Oil 32 used for a l l dilutions of one enrichment, the lowest concentration being f i l t e r e d f i r s t , and the f i r s t two tubes of each subsequent dilution dis-carded to ensure retaining only the dilution required® Inoculations were made with 2 drops of a rich suspension of the organism from an agar slant. o After 46 hours incubation at 28 0, the pH of the tubes was recorded. In this time, the controls had not changed, whereas the cultures containing activators in optumum concentration ranged from pH 7.8 to 8.8 Fourteen days incubation were required for the controls to reach pH 8.0 In again testing the activity of various cultures in accordance with the Slant Colony procedure, i t was observed that 101 227 on enriched basal media demonstrated immunity to stimulating factors. In the light of this observation i t was considered possible that the state of activity or the previous history of the inoculum might explain this peculiar situation. Therefore, transfers of this inactive culture direct from giant colonies, in addition to normally functioning strains were subjected to the urease test. These data are presented in table 11. TABLE 11 - CHANGE IN REACT ION OF CULTURES OF RHIZOBIUM TRIFOLII DUE TO UREASE ACTIVITY.. l A i a s 2(b) ^Bios 2(bi •DH .01 7.0 .6 7.7 .05 7.0 .8 7.8 .1 7.0 1.0 8.0 .2 7.0 1.5 7.7 .3 7.0 2.0 7 . 4 . 4 7.0 3.0 7.0 CONTROL pH 7.0 35 A l l the cultures of RC1 227 produced identically the same results insofar as the effect of Bios fractions on urease activity was revealed by pH changes. Moreover, the optimum concentration for Bios 2(b) relative to increase i n pH correlates with the optimum for giant colony growth. Shis method therefore, provides a quick, convenient means of testing the activating power of various Bios fractions. It is d i f f i c u l t to explain how, in this test, stimulation of the organism causes a rise in pH, whereas in the previous experiment the opposite was observed. At the same time growth in one case is correlated positively with ammonia production, in fee other case negatively. This has not yet been satisfactorily ex-plained. Growth i s greatest in these tubes, producing most ammonia. A complete l i s t of data with respect to a number of active fractions tested by this method i s given below. Figures represent pH values. TABLE 32 - INFLUENCE OF BIOS ON AMMONIA PRODUCTION .QE HH. TRIFOLII 227 FROM URFA SUBSTANCE TESTED .ni .05 .1 .2 CONCENTRATION f .4 .6 .8 1.0 1.5 2.0 Control Bios 2A(Proe.4alfalfa) 7.4 77.8 8.0 7.7 7.7 7.0 7.0 7.0 7.0 Bios 2 ( » ) 7.0 7.0 7.0 7.7 7.8 8.0 7.7 7 . 4 7.0 Bios2A(Proe«3tomat0) 7.6 7 . 8 7.5 7.0 7.0 7.0 7*0 7.0 7.0 Bios2B '» 7.0 7.2 7.9 7.6 7.6 7.3 7.0 7.0 7.0 7.0 7.0 Bios 5 " . 7.7 7.8 7.7 7«0 7.0 7.0 7.0 7.0 7.0 Active f i l t r a t e (.Pro c»5—Esrer) 8.2 7.8 7.6 7.4 7 .4 7 . 4 7.6 7.6 7 .4 7 .4 * These concentrations represent amounts of actual preparations used . and a l l are not st r i c t l y comparable. For example, 1 ml. Bios 2(a) (Proc.3) represents 11ml. tomato juice,. 1 ml. 2(b) equals 88 ml. and 1 ml. 5 equals 30 ml. of original material. These results are brought to a common basis in accompanying graphs (7-1S inclusive). 34 She question that immediately a r i s e s i s : Does Bios stimulate enzyme production or does i t stimulate the a c t i v i t y of the enzyme. Shis question had arisen previously i n connection with the production of l a c t i c acid "by the Streptocoooi and Beracocci i n the Department of Dairying, hut the complexity o f the enzyme reactions involved did not permit of a ready solution. In t h i s instance, however, the s i m p l i c i t y of the reaction, and the f a c t that urease can be obtained in r e l a t i v e l y pure state, offered the p o s s i b i l i t y of supplying a d e f i n i t e reply to this interesting question concerning the a c t i o n of Bios. Urease tablets (B. D. H.) f o r estimating urea in blood were obtained f o r t h i s study. She medium was made up exactly as before, and 5 ml. amounts were pipetted into tubes. Since the reaction is quite rapid, s t e r i l i z a t i o n of the medium i s of course, unnecessary. A f t e r adding Bios enrichments as required, 1 ml. of a one to ten d i l u t i o n of a urease tablet (one tablet dissolved i n 10 ml. water) was added to each tube. After two hours incub-o at ion a t 28 0 pH readings were made. Figures i n the following table represent pH values obtained upon te s t i n g a number of Bios f r a c t i o n s i n t h i s manner. 35 IABLE 13 - INFLUENCE OF BIOS ON THE ACTIVITY OP um&tm. • .•'••-.* CONCENT RATION % SUgSTANCE TrjSTET) 0.1 . 4 .6 .8 1. 2 ' 4 ' 10 Omii^t Bio82 (a) (Proo4Alf.7.4 7.5 7.8 7.9 8.0 8.1 8.3 8.6 8.6 7.3 Bios2(b) " ) 7.3 7.5 7.6 7.6 7.7 7.7 7.6 7.3 7.3 7.3 Bios2{a)ProcSTomJ 7.3 7.4 7.6 7.7 7.8 7.8 8.0 8.0 7.8 7.3 Bios2(b) » J 7.9 7.9 8.2 8.2 8.2 8.1 7.7 7.5 7.4 7.4 Bios 5 '» ) 7.3 7.3 7.5 7.6 7.6 7.9 8.0 7.3 7.3 7.3 Bios2(a)(Proc3Eggs7.8 8.4 8.4 8.4 8.4 8.4 8.4 8.2 7.9 7.4 Bios2(b) " 7.4 7.4 7.5 7.7 7.8 7.8 8.4 8.4 8.4 7.4 t Filtrate(Proc5 M 8.0 8.6 8.6 8.6 8.6 8.6 8.6 8.6 8.4 7.3 Precipitate »• 7.4 7.4 7.4 7.4 7.4 7.4 7.6 7.8 8.0 7.4 Yeast extract 7.4 7.5 7.8 8.0 8.4 8.4 8.4 8.4 8*4 7.4 *See footnote Table 12. t 0.01$ - 7.4 0.05$ 'ft 7.7 In order to compare the influence of Bios on the production of ammonia by the Bhizobia and by the pure enzyme, a l l concentrations were brought to a comparable basis, and the increases in pH beyond the controls expressed on a percent basis. For the coordinated results see Graphs 7 to 18 inolusive. The results would appear to indicate that a l l Bios fractions induce increased product ion of urease by cultures of Rhlzobium t r i f o l i i 227. Otherwise the curve representing the ammonia production of the organism would exactly follow the pure enzyme curve, at least to a certain point. In addition, the situation is rendered more complex by the fact that Bios stimulates the enzyme i t s e l f . Therefore, the curve showing ammonia M M W N O i 4* * « S * I V « I W r f V W S V"g O N O * " ! J M « 1 « M W N O S a l A V O ' M J L I W * ' U S r f V d M d V U S * ( O H -fB444+fffhH-ff A N .•3 <3 1-5 >o e g p ) L J i •v. *--p I " S i •3 "3 <3 Cs 4 — - - BSC 3 0 3 c 36 produced by the organism represents the net r e s u l t of various degrees of a c t i v a t i o n of varying amounts of enzyme. The Bhizobia are activated by a lower concentration of Bios than the enzyme and they have a decidedly narrower optimal range. The concentration of Bios required f o r optimum stimulation of the enzyme, has i n a l l cases but one, proven inhibitory for the organism. Bios 2(a) has a greater a c t i v a t i n g power than Bios 2(b) when prepar-ations are compared on an equal volume basis. This relationship might not hold true i f Bios 2(a) and 2(b) could be compared on an equal weight basis, as there might be a small amount of Bios 2(b) and a r e l a t i v e l y large amount of Bios 2(a) present in equal volumes of the concentrates. Eggs appear to be much higher i n substances of the Bios group than either tomato jui c e or A l f a l f a extract. (7) BIOS AS AN ACCESSORY FAQ TOR FOB BHIZOBIA. i n order to e s t a b l i s h a basis for discussion, an accessory food factor i s defined as a substance required by an organism in minute amounts, i n the absence of which, metabolism is impaired to such an extent that death r e s u l t s . Vitamins are therefore accessory food factors for man; Bios i s an accessory faotor for certain yeasts. Thome and Walter (1936) have presented data to show that Bhizobia require no accessory food factors. They state, however, that t h e i r inoculum consisted of about f i v e m i l l i o n organisms from a yeast extract enriched medium. It does not seem l i k e l y that they appreciated the im-portance of small amounts of growth factors which could be transferred 37 in such an inoculum and which would probably prove sufficient for growth. Their work was checked, giving consideration to the amount and source of inoculum. The following medium, advocated by Thome and Walker was used :-0. P» Sucrose — — — — — lo gm. K HPO — — ; — — - — 20 gm. 2 4 MgS04 7 HgO — — — — — 0 . 1 gm. Ca 01 ——-—• .—0.1 gm. 2 (ML. L s o ' — —-2.0 gm. Fe Gig — — • -0.01 gm. Distilled water — — 1000 ml. Rhizobium t r i f o l i l 227 was transferred four times on an agar slope of the above unenriched medium. After four days incubation, 5 ml. of sterile water were added to the most recent slope, and the scanty growth scraped from the surface of the medium and brought into suspension. This suspension was then diluted out in sterile water blanks to 1:100,000 and 1:1,000,000. Several tubes of Thome and Walker's fluid medium were inoculated with 1 ml. from one or other of the two dilutions, and at the same time, 1 ml. quantities of each of the dilutions were plated on yeast water mannltol agar to determine the number of organisms in the inoculum. Counts showed that 240 and 25 organisms were used as inocula from the 1:100,000 and 1:1,000*000 dilutions reopectively. After five days incubation growth was apparent In a l l tubes, and after seven days, considerable cloudiness had developed. Apparently RC1 227 is able to grow and multiply in a medium consisting of minerals and carbohydrate only. 38 To confirm this point, 15 plates of Thorne and Walker's medium and 15 plates of the same medium enriched with 1% Bios 2(h) (Procedure 4 -Alfalfa) were poured. Starting with a much thinner suspension of the organisms, dilutions were made as before* Then five drops of the 1:1,000, 000 culture were smeared* over the surface of each plate. After four days incubation, the counts were as follows :-Bios plates: 3, 4, 7, 5, 6, 5, 4, 6, 7, 4, 6, 5, 6, 5, 4, Average 5.06 Unenriched plates: 2, 2, 1, 1, 3, 2, 1, 1, 2, 2, 0, 1, 1, 1, 1, Average 1.40 Further incubation failed to bring up more colonies on any of the plates. The colonies on the Bios plates, as would be expected, were much larger than those growing in the absence of Bios. Results obtained by attacking the problem from this direction show that there are about seventy-five per oent of the organisms which f a i l to grow in the absence of Bios, whereas the remaining twenty-five per cent produce oolonies. The same experiment was repeated using Thome and Walker's medium which had been charcoaled to remove any possible traces of Bios 2(b) that might be in the sugar. Similar results were obtained as before. It Is rather surprising to find that within a single strain, indiv-idual cells vary in their Bios requirements to such an extent. It is well known that the progeny of a single Rhlzobium c e l l produoe organisms which vary in oolony characteristics, germ production etc., but more constancy was anticupated with respect to the fundamental requirements. Whether or not a culture might be maintained indefinitely on a Bios-free medium is a point worthy of consideration, but has not as yet been determined. SUMMARY (PART 1) A l l strains of Hhisobium t r i f o l i i tested, were stimulated by alfalfa extract as measured by giant colony growth. No relationship was apparent between the degree of stimulation induced in the various strains and the amount of growth on unenriched medium. Not a l l strains responded to enrichments of Crude Bios 2. A good nitrogen fixing strain used in this particular instance, did not respond, whereas a poorer strain so used, was stimulated. The latter strain (RC1 227) was chosen for more intensive study of the influence of Bios fractions. Both Bios 2(aJ and Bios 2(b) fractions activated Rhizobium t r i f o l i i  227. thus lending confirmation to Miller's conoeiption of the multiple nature of Bios. Giant colony measurements gave a reasonably accurate indication of actual c e l l multiplication of Rhizobium t r i f o l i i 227 as indicated by comparison with plate counts. When the organism was in a highly active state because of growth in a Bios enriched medium, i t was more responsive to further stimulation than i f i t had not been previously activated. With the technique employed, no fixation of atmospheric nitrogen occurred either with or without the addition of Bios to the medium. Any "fixation" that was observed, was only "apparent fixation" due to loss of nitrogen from the controls, giving rise to this effect. The sugar fermenting ability of the organism was altered in the presence of yeast or Bios enrichments. The carbon sources which appeared SUMMARY CON'S. most interesting as possible "key" sugars in determining the nature of various active factors were: raffinose, adonitol, mannitol, dextrin, and salicin. Large cultures of ROl £27 consisting of basal medium containing 2 peroent urea, showed a gradual accululation of ammonia nitrogen in control flasks, while a similar, though markedly smaller accumulation occurred in the presence of Bios. lest tube cultures revealed that rapid increases in alkalinity took place in cultures enriched with optimal amounts of Bios, while scarcely any activity occurred in controls. She. optimum concentration of Bios relative to increase in the latter case, in pH correlated with the optimum for giant colony growth. It was demonstrated that Bios activates the pure enzyme, urease. Results showed that Bios apparently affects ammonia production by ROl 227 both by stimulating the organism to produce more enzyme and by stimulating the enzyme produced. Bios as an accessory food factor is discussed on the basis of preliminary experimental data obtained. 39 P A H T 1 1 * ^ - i m S a S J j Q F BIOS ON I^QOMB,.aEgBLJSflS PUBLISHED WORK ATTACHED INFLUENCE OF BIOS ON NODULE BACTERIA AND LEGUMES Ai 111E I N F L U E N C E OF BIOS OlvT t L E G U M E S E E D L I N G S i B Y D . G . L A I R D A N D P . , M . W E S T -h. i \ I C A N A D A . r' ' i \ i ' I 1 > • . , , l< •< v ' ' , < \ 1 1 [ v - i V - \ j U i J l ' 1 , v , , , * - I f ' 1 f l I 1 ^ ' * x ~ 1 ' t s ' , f , - - ' I ,', ^Reprinted from the , _ Asi? V L - i : , CANADIAN'JOURNAL1"OF.! RESEARCH- '--W^-^hJd • • V ' v i f C, 75 : 1-6. 1937 . Reprinted from Canadian Journal of Research, Sec. C, Vol. 15, January, 1937 THE INFLUENCE OF BIOS ON NODULE BACTERIA AND LEGUMES1 A. T H E I N F L U E N C E O F BIOS O N L E G U M E S E E D L I N G S B Y D . G . L A I R D 2 A N D P . M . W E S T 3 Abstract The hypocotyls of red clover seedlings, when sprouted on a seed bed enriched with crude Bios 2, grew upwards in a vertical position, while the cotyledons rested on the surface, supporting the inverted plant and possibly absorbing nutrients for its growth. From the tip of the upturned primary root, secondary roots developed about ten days after seeding. These new roots grew down-wards, and after approximately one week, they penetrated the substratum in a normal fashion. The concentration of crude Bios 2 necessary to cause maximum hypocotyl bending, was observed to be approximately four times that required to produce optimum stimulation of the nodule bacteria. When plants were allowed to start in an unenriched growing medium super-imposed on a layer of agar enriched with crude Bios 2, no upward bending of the roots occurred as they reached the Bios layer. Bios 2(b) alone appears to be the factor responsible for the hypocotyl bending phenomenon. Bios 1, Bios 2(a), pantothenic acid, various amino acids, and miscellaneous compounds were tested and did not appear active in this respect. Though causing a different form of root aversion, hetero-auxin, like Bios 2(b), actually prevents the hypocotyls from entering an enriched medium. While the two substances bring about a certain similarity of physiological effect, they cannot be considered identical, and chemically, they are absolutely distinct. A drop of Bios 2(b) placed on the sensitive parenchymous lining of a bean pod caused rapid cell multiplication, resulting in the production of a wart-like pro-tuberance. Similar, though somewhat less distinct results followed the pricking of such tissue with a pin. These results would appear to strengthen the hypothesis advanced by Went in which Bios was assigned the properties of a "wound hormone". A study devoted to the influence of the Bios complex on nodule bacteria, alone and in association with red clover (unpublished data), suggested to the writers that Bios might exert a specific influence on the character of the host. While the Bios of Wildiers is commonly known to occur in the tissues of higher plants (4, 5, 11), its effects on the growth processes of such plants are as yet unknown. Kogl (6), has suggested that Bios may be physiologically related to auxins (a) and (b), and hetero-auxin, which appear to function both as organizators and as phyto-hormones of cell expansion. Went (12), on the other hand, has presented the hypothesis that Bios might be considered 1 Original manuscript received October 30, 1936. Contribution from the Department of Agronomy, The University of British Columbia, Vancouver, Canada. 2 Associate Professor,Department of Agronomy (Soils), The University of British Columbia, Vancouver, Canada. 3 Carnegie Scholar, Department of Agronomy (Soils), The University of British Columbia, Vancouver, Canada. 2 CANADIAN JOURNAL OF RESEARCH. VOL. 15. SEC. C. a wound hormone. A consideration of these facts and suggestions, and a desire for increased knowledge regarding the important interrelationships between bacteria and legumes have been the inducements for a study, as reported below, of the influence of Bios on the growth of red clover seedlings. The Bios fractions used in preliminary work by Eagles el al. (3), were prepared after the manner of Miller and his associates (9). As the work progressed, however, the original procedure for the isolation of Bios con-centrates was altered in order to obtain fractions with fewer impurities. These efforts resulted in the development, by Eagles and Woo4 (unpublished data), of an improved procedure. The Bios 2(a) and 2(b) concentrates pre-pared according to the new method were found to exercise the same biological effects as the respective fractions prepared after the manner of Miller when tested on bacteria, but possessed a distinctly greater activating capacity. Bios obtained by the revised procedure of fractionation was used in the plant studies subsequently reported upon. The following method was employed to determine the influence of Bios 2 preparations on the growth of red clover: Using washed sand cultures, the seeding was carried out under aseptic conditions; the required nutrients were supplied in the. form of modified Crone's solution (2), containing, except in the case of the controls, 2% of an active solution of Bios 2, i.e., a solution containing both Bios 2(a) and Bios 2(b). The rate of germination was normal, but the seedling roots growing on a Bios medium absolutely refused to penetrate the sand, with the result that they were unable to support the plant. After the surface of the sand had dried slightly, the plants wilted and died. This experiment was conducted in 4-inch porcelain pots, each treatment replicated four times, and about 50 seedlings grown in each pot. The test was repeated to guard against any possible error. As sand did not appear to constitute a satisfactory growing medium, it was replaced by a modified Crone's nutrient solution to which was added 0.75% Bacto agar. The Bios enrichment used was the same as in the previous experiment. Red clover seeds were then placed on the surface of the medium which had been autoclaved in quart sealers covered by a Petri plate. The effect of the Bios became evident after three to four days. In every case the hypocotyls ascended vertically or nearly so, while the cotyledons remained in contact with the agar surface (see Fig. 1). This phenomenon is referred to throughout the remainder of the paper as "root bending", since it is the root tissue of the hypocotyl that undergoes subsequent modification and not the stem tissue. The upturned hypocotyls differed further from the normal in being slightly swollen and in possessing no root hairs. The upward growth continued for approximately ten days, and after that, one or more secondary roots were produced which grew downwards and penetrated the substratum in an apparently normal fashion. In most cases, the first true leaf and the subsequent trifoliate leaf had developed when the secondary roots entered the nutrient medium (see Fig. 2). Until this time, the absorption of water and any possible nutrients required must have taken place through the coty-LAIRD AND WEST: INFLUENCE OF BIOS 3 F I G . 2 . Red clover seedlings 21 days old, on Bios 2 enriched medium. Note secondary roots growing down in a normal fashion through the medium which caused hypocotyls to bend upwards. 4 CANADIAN JOURNAL OF RESEARCH. VOL. 15, SEC. C. ledons, or the stem immediately beneath them, or through both. Thousands of seedlings have been used in this study. The experiment has been repeated numerous times and always with identically the same result. In fact, in work now in progress, the hypocotyl behavior as described is being used in determining the character of certain fractions as isolated from plant and animal materials. It became evident, during the investigation, that red clover seedlings vary in their response to Bios enrichment, and this observation led to a detailed study of the effect of different Bios concentrations. The concentration of any preparation required for maximum "root bending" was observed to be roughly four times that required to produce an optimum stimulation of the nodule organism. Fractions of unknown activity were, therefore, first tested on bacteria to obtain an indication regarding the amount required to produce "root bending". By varying the concentration of Bios in the medium from 0.25 to 8% the maximum "root bending" effect was observed to occur with a 2% enrichment. Beyond 2% there was evidence of slight toxicity, while below 2% only the more "sensitive" plants responded, the roots of the others merely extending along the surface of the substratum. Since the activity of different Bios solutions may, and does, vary to some extent, the above figures refer only to the preparation used in these experiments. While a positive correlation was observed to exist between the Bios concentrations required for optimum bacterial growth and "root bending", the possibility is not to be overlooked that more than one factor might be concerned. Likewise, it seemed desirable to determine the effect of Bios on the growth of plant roots after the plants had become established in the absence of Bios. For this purpose, a layer of Crone's agar medium containing Bios 2 enrich-ment in optimum concentration was placed in the bottom of a quart jar, and another layer, without Bios addition, was superimposed on this. Seeding was carried out as previously described. The seedlings grew normally, and when, after one week, the roots reached the layer containing Bios, they neither turned up nor did they show any other observable effect from the Bios con-centration. It would seem probable, under certain conditions at least, that the factor which causes such marked changes in the hypocotyl as observed in the primary stages of growth, does not exercise any influence on the root tissue once the early seedling stage has passed. Although, when tested separately on bacteria, no distinct difference had been observed in the action of Bios 2(a) and Bios 2(b), it was considered possible that one or the other of these factors might be specifically responsible for the "root bending" phenomenon. Experimental results showed that an agar seed bed enriched with Bios 2(b) alone brought about bending of the hypocotyls, while the addition of Bios 2(a) alone exercised only a very slight influence, if any, in this respect. Crude Bios 2(a) and 2(b) preparations obtained after the manner of Miller el al. (9), were likewise tested and gave similar results, though crude Bios 2(b) was not as active in producing "root bending" as the more refined Bios 2(b). This finding lends support to the LAIRD AND WEST: INFLUENCE OF BIOS 5 conception of Mil ler that Bios 2 is of a multiple nature, and it further indicates that the new fractionation method results in a reasonably complete separation of two distinct entities as represented by Bios 2(a) and 2(b) respectively. Recently Mil ler (10), made the suggestion that the active constituents of Bios 2(a) might be /3-alanine and /-leucine. These amino acids were included in the tests along with aspartic and glutamic acids, arginine, cystine, tyrosine, tryptophane, and histidine; none of these compounds caused bending of the hypocotyls. Negative results were also obtained with Bios 1 (inosite). Similarly, the pantothenic acid of Williams which has been reported to stimulate the growth of alfalfa (8), and Riccia (13), did not possess the effect of Bios 2(b) as shown on red clover plants. Various compounds, carnosine, indole, scatole, guanine, ergothioneine, and glutathione were also found to be inactive. In order to determine whether or not the "root bending" phenomenon might be due to the toxicity of the heavy metals used, and possibly carried through in small quantities into the Bios preparation, these metals (barium, mercury, and lead) were added to the medium in minute amounts. General evidence of toxicity was observed, but otherwise the metals caused no change in the normal root development. In the light of the work of Kogl (6, 7), and Went (12) regarding the effect of auxins on plant growth in general, it was thought desirable to enquire whether or not a corresponding "root bending" phenomenon might be caused by these substances. A priori it was realized that the treatment of auxins with heat, in the presence of dilute acid and alkali respectively, results in their destruction, while Bios 2(b) is quite stable under similar conditions. Besides, auxins are soluble in ether, and Bios 2(b) is not. Thus on the basis of chemical properties it did not seem probable that any of the known auxins could be responsible for the "root bending" of the type referred to above. However, the response of red clover seedlings to hetero-auxin (/3-indolyl acetic acid) was studied in a manner similar to that already described for Bios, except that while the latter was used in a 2% concentration, the hetero-auxin, after being tested at six different concentrations ranging from 0.0001 to 0.01 mg. per c c , was used at a concentration of 0.0005 mg. per cc. Germination of the seeds revealed, that while hetero-auxin, like Bios 2(b), does not permit the hypocotyls to enter the substratum, there are, at the same time, apparent differences in the action of the two agents on red clover seedlings. As previ-ously reported by other workers (12), hetero-auxin causes the development of thick, heavily swollen hypocotyls not observed in the case of Bios 2(b). The latter, again, compels the hypocotyls to extend upwards from the cotyledons, which remain resting on the seed bed, while hetero-auxin, although not permitting the hypocotyls to enter the seed bed, causes a slight horizontal twisting of the hypocotyls and allows the cotyledons to occupy their normal upright position. Finally, Bios 2(b) inhibits the formation of root hairs on the primary roots, while hetero-auxin has no such inhibitory influence. Hetero-auxin and Bios 2(b) may, therefore, be distinguished on the basis of either their chemical properties or their physiological effects on the develop-ment of red clover seedlings. 6 C A N A D I A N J O U R N A L OF R E S E A R C H . V O L . 15, SEC. C. After having demonstrated the dissimilarities between the auxins and Bios 2(b), the attention of the writers was called to the recent work of Bonner (1) who, in seeking an ideal medium for plant tissue cultures, found that additions of plant extract were essential to the successful growth of his cultures. His statement regarding the properties of the active compound in the extracts employed indicates a close relationship to Bios 2 fraction, and his reference to the adsorption of the active substances on charcoal would suggest that the compound with which he was concerned might be identical with Bios 2(b). B y applying a drop of his extract to the parenchymous lining of the cup-like depressions inside a bean pod, Bonner reports the production of a "wart-like protuberance". A similar effect was observed following the pricking of such cells with a pin. The auxins, vitamins B i and B 2 and pantothenic acid were shown by him to be inactive in this respect. On the assumption that Bios 2 (b) might be the causative factor, the writers conducted a similar test using this activator, with hetero-auxin, water, and pin-pricks as controls. Bios 2(b) produced protuberances which appeared to be similar in all respects to those described by Bonner, while hetero-auxin and water were ineffective. Histological preparations showed that an in-tensive cell multiplication occurred from the addition of Bios 2(b) where Phaseolus vulgaris was tested, while in the case of Phaseolus coccineus, cell elongation was chiefly responsible for the swelling. Pin-prick controls gave evidence of a small amount of cell multiplication. These results would appear to strengthen the hypothesis advanced by Went in which Bios was assigned the properties of a "wound hormone". Acknowledgments The authors wish to express their thanks to Professor P . A . Boving for much helpful criticism in the preparation of the manuscript; to Dr . R . J . Williams of Oregon State College for /3-alanine and pantothenic acid, and to Dr . B . A . Eagles for many of the remaining compounds used in this study. References 1. B O N N E R , J . Proc. Nat. Acad. Sci. 22 : 426-430. 1936. 2. B R Y A N , O. C . Soil Sci. 13 : 271-285. 1922. 3. E A G L E S , B. A. , W O O D , A . J . and B O W E N , J . F . Can. J . Research, B, 14 : 151-154. 1936. 4. E A S T C O T T , E . V . Proc. and Trans. Roy. Soc. of Canada, Section III, 3rd Series, 17 : 157-158. 1924. 5. F U L M E R , E . I., D U E C K E R , W . W . and N E L S O N , V . E . J . Am. Chem. Soc. 46 : 723-726. 1924. 6. K O G L , F . Ber. 68 : 16-28. 1935. 7. K O G L , F . , H A A G E N - S M I T , A . J . and E R X L E B E N , H . Z. physiol. Chem. 214 : 241-270. 1933. 8. M C B U R N E Y , C. H . , B O L L E N , W . B. and W I L L I A M S , R. J . Proc. Nat. Acad. Sci. 21 : 301-304. 1935. 9. M I L L E R , W . L . , E A S T C O T T , E . V . and M A C O N A C H I E , J . E . J . Am. Chem. Soc. 55 : 1502-1517. 1933. 10. M I L L E R , W . L . Proc. and Trans. Roy. Soc. of Canada, Section III, 3rd Series, 30 : 99-103. 1936. 11. S A D L E R , W . , E A G L E S , B . A. , B O W E N , J . F . and W O O D , A. J . Can. J . Research, B, 14 : 139-150. 1936. 12. W E N T , F . W . Botan. Rev. 1 : 149-182. 1935. 13. W I L L I A M S , R. J . and R O H R M A N , E . Plant Physiology, 10 : 559-563. 1935. 40 PART I H - mamm 3E OF BIOS ON IHEj SYMBIOTIC ASSOCIATION BETWEEK RHIZOBIUM TRIFOLII AND RED CLOVER Naturally i t was f e l t that major attention should be directed to Parts 1 and 11 before any extensive investigation of combined effects were undertaken. Thus the work presented under t h i s heading i s only to be considered as a preliminary for extended studies in t h i s d i r e c t i o n . A f t e r observing that the concentration of Bios causing "root bending" was four times that required for optimum stimulation of the nodule or-ganism,, i t became possible to grow plants with normal root development i n the presence of Bios, and at the same time provide optimum conditions f o r the Bhizobia. A study was therefore undertaken to determine the effect of Bios, i f any, on the nodulation of Red Clover plants when added i n the above concentration. Eighteen pots f i l l e d with washed s i f t e d sand, were auto-claved f o r s i x hours at f i f t e e n pounds pressure and then placed i n a s t e r i l e plant chamber. Ten s t e r i l e red clover seeds were planted in each pot. The treatment was as follows J 3 pots uninoculated, watered with N-free Cron's Solution. 3 " " " " Bios enriched " 3 " " " " Crone's Solution to which nitrogen was added as sodium n i t r a t e (nitrogen euqivalent to that i n Bios). 9 " treated as above, but each inoculated with 1 ml. of Bhizobium  t r i f o l i i s t r a i n ROl 227 from an a c t i v e l y growing culture. Pots were watered with the solutions indicated ( s t e r i l i z e d ) at the outset, and every other week thereafter® Optimum moisture content was 41 maintained through the use of sterile water whenever necessary. At the outset Crude Bios 2 (Procedure 2) was used in lf> concentration, hut with subsequent applications, 4$ Crude Bios 2 was added. .Observations, ? At the end of the first week a l l plants began normally with the exception of an occasional "seedling" which had difficulty in producing normal root development. In two weeks a l l plants had developed their first true leaves. Those to which nitrate of soda had been added showed the best growth. Ho significant differences were apparent between unenriched and Bios enriched cultures. Pour Weeks After four weeks a l l inoculated plants showed distinct gains over uninoculated plants. In both groups, Bios enriched cultures gave better growth than controls, but those to which nitrate of soda had been added were better than either of the other two. The uninoculated Bios enriched plants were approximately the same in development as the inoc-ulated unenriched. At the end of eight weeks the following observations were made: (a) Uninoculated - unenriched cultures produced plants averaging is/4 to 2 inches in height, with decidedly yellow foliage. Bios enriched plants averaged 3 3/4 to 4 inches in height? had good color. - Sodium nitrate treated plants were about 5 inches in height? had good color. (b) Inoculated - detailed measurements were made of height of tops, depth of roots, and number of culms with no significant differences being observed in the whole group. Modulation was carefully studied, the number, 4S, size and shape of nodules on the tap root, on the first two inches of •branching roots, on the second two inches and below four inches, being recorded. Striking differences were noted in the type of nodulation of the three groups of plants. Tjipical specimens from each group are shown in the accompanying photographs (see Plates 1-3 inclusive). She nodules in the case of the unenriched plants were small, from one to two millimeters in diameter, spherical, and scattered uniformly on the fibrous roots of the plants. She nodules of those enriched with Bios were large, cylindrical and in many cases four to five and even six millimeters in length. They were chiefly concentrated in the upper two inches of the root system, especially on the main tap root. Since it is in this position that most efficient fixation is believed to occur, these results may indicate the importance of growth factors in determining nodular size and distribution. That these results are not due to the nitrogen supplied in the Bios fraction is shown by the nodulation of those plants fertilized with an equivalent amount of nitrogen as nitrate of soda. They show nodules largely situated in the upper portion of the root system, but much smaller in size and distributed on the root hairs, rather than concentrated on the tap root. AG KM0 Y/LED GEMEN TS The author wishes to express h i s thanks to Dr. D.G. L a i r d and P r o f e s s o r P.A. Boving f o r much h e l p f u l c r i t i c i s m i n the p r e p a r a t i o n of the manuscript; to the U n i v e r s i t y of Wis c o n s i n f o r the stock c u l t u r e s p r o v i d e d ; to Dr. H.J. W i l l i a m s of Oregon S t a t e C o l l e g e f o r B- a l a n i n e end pantothen-i c a c i d , and to Dr. B.A. Eagles f o r many of the remaining compounds used i n t h i s study. 43 H E F E R E H O E S b Allison, F« E. Jour Agr. Res. 39, 893 - 924. 1924. Bonner, J . Proc. Hat. Aead. Sci 22 , 426 - 430. 1936. Bryan* 0. C. Svil Sci. 2£, 271 * 285. 1922. Davisson, B. S., and Parsons, J . T. J . Ind. Eng. Chem. 11, 306-311. 1919. Eagles, B. A., Wood, A. J., and Bowen, J . P. Can. J . Research B. 2&, 151 - 154. 1936. Eastcott, E. V. Proc. and Trans. Hoy. Soc. of Canada, Section 111, 3rd. Series, 17, 157 - 158. 1924. Elder, M. L. Trans. Roy. Soc. of Canada, Section 111, 3rd, Series, 3fi,e 89 - 97. 1936. Fulmar, E. I., Lueoter, W. W., and Nelson, V. E. J . Am. Chem. Soc. 4£,, 723 - 726. 1924. Kbgl, F. Ber. 68.* 16 - 28. 1935. Kogl, F., Jaagen - Smit, Av J . P and Erxleben, H. Z. physiol. Chem. 214. 241 - 270. 1933. Kbgl, F. and Tonnis, Benno. Z. physiol. dhem. 241. 43 - 73. 1936. Lohnis, M. P. Soil Sci. 2J>, 37 - 57. 1930. MoBurney, C. H.» Bollen, W. B., and Williams, R. J. Proc. Nat. Acad. Sci. 21, 301 - 304. 1935. Miller, W. L«, Eastcott, E. V., and Maconachie, J. E. J . Am. Chem. Soc. 55, 1502 - 1517. 1933. Miller, W. L. Proc and Trans. Roy.-Soc. of Canada, Section 111, 3rd, Series, 30,, 99 - 103. 1936. Narayanan, B. T. Bioohem J . £&, 6-18. 1930. Sadler* W., Eagles, Bi A.i Bowen, J . F«, and Wood, A. J . Can® J. Research," B, 1&, 139 - 150. 1936. 44 Thome. D. W., and Walter, H. H. Soil Sol. 42, 231 - 240. 1936. Went, F. W. Botan. Rev. 1, 149 - 182. 1935. WiIdlers, E. La Cellule, 18, 313 - 331. 1935. Williams, R. J«, and Bohrman, E. Plant Physiology 10,, 559-563. 1935. Wilson, P. W., Hopkins, E. W., and Fred, E. B. Arohiv. fur MLkrohiol. S» 322-340. 1932. 

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