THE INFLUENCE OF SOIL AND ROOT EXTRACTS ON THE ASSOCIATIVE GROWTH OF SELECTED SOIL BACTERIAL

E. C. S. C H A N , ~ H. KATZNELSON, AND J . W. ROUATT illicrobiology Research Institute, Canada Department of Agriculttrre, Ottawa, Canada

Received July 26, 1962

Abstract These studies are concerned with the growth interrelationships of mixed

cultures of five soil organisms in soil extract and root extractsof 2-, 4-, and S-week- old oats, soybeans, and wheat. Population changes of Agrobacterium radiobacter, Arthrobacter citreus, Aaotobacter cltroococct~nz, Bacillus cereus, and a Pseudomonas sp. in pure and mixed culture were followed by plating on selective media. B . cereus and A. chroococctrm grew poorly alone or in mixed culture in the extracts. In soil extract, A . citreus predominated over, or was nearly equal in nurnber to, the Gram-negative forms (Pseudomonas and Agrobacterium). In root extracts, Pseudomonas sp. always predominated over A . citreus in mixed culture. A. radiobacter was inhibited in mature root extracts (8-week-old plants) although in pure culture it recovered after a period. An antagonistic effect of Pseudo- monas sp. on A . chroococcunl plated on nitrogen-free agar medium was found to be related to the kind of agar used.

Introduction The need for studies with mixed cultures as an aid towards a better under-

standing of the ecology of soil microorganisms has been recognized for many years (12) . However, although numerous investigations with known mixtures of microorganisms have been reported in the recent literature (4 , 5 , 6, 7 , 9), relatively little work of this kind has been done with specific reference to the rhizosphere effect (1) .

Extensive studies on the microflora of soils and rhizospheres have shown (2 , 3 , 8, 1 3 , 1 4 , 15) that in the rhizosphere Gram-negative rods such as pseudo- nlonads predominate over Gram-positive rods, coccoid rods, and spore-forming types such as those in the genera Arthrobacter, Nocardia, and Bacillus. In root-free soil the latter groups predominate over the Gram-negative rods. A rationale for these ecological observations was provided in a previous study in this laboratory on the growth interactions between a Psezldomonas sp. and .4rthrobacter globiformis (1) . I t was found that the pseudomonad suppressed growth of this arthrobacter by the production of acid, fluorescent pigments, and other inhibitory substances. A shorter lag period as well as a shorter generation time also played a role in establishing the predominance of the pseudomonad. These results led us to study a wider spectruni of organisms in the model system. The present report is concerned with the growth in pure and mixed culture of five species of soil bacteria in the root extracts of different plants of various ages. The purpose of the study was to explore more defini- tively the active role of soluble root constituents in the rhizosphere effect.

'Contribution No. 511. 2National Research Council Postdoctoral Fellow, in collaboration with the Canada Depart-

ment of Agriculture, 1960-62. Present address: Department of Biology, University of New Brunswick, Fredericton, New Brunswick, Canada.

Canadian Journal of Microbiology. Volume 9 (1963)

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188 C A N A D I A S JOURNAL O F MICRORIOLOGY. VOL. 9, 1963

Materials and Methods The organisms used were from the culture collection of the h4icrobiology

Research Institute: rlgrobacteriunz radiobacter 426, Arthrobacter citreus 837, Azotobacter chroococcum 536, Baci l l z~s cereus 826, and Pseudomonas sp. Stocl; cultures of these bacteria were carried on slants of Difco Antibiotic medium 2 (Bacto-Penassay Base agar) containing 50% (v/v) soil extract, with the exception of A . chroococcum. This organism was carried on slants of azoto- bacter n~edium (see below for composition). For inocula a 96-hour slant culture of il. chroococcum and 48-hour slant cultures of the other species were used. Cells were transferred into a nitrogen-free salt solution (10) and the turbidity adjusted to a reading of 50 units, a t 420 n ~ p in a IClett-Summer- son calorimeter; 0.2 1111 of this suspension was used to inoculate each culture flask (50-1111 Erlenmeyer); 10.8 in1 of the soil or root extract per flask were used [or pure culture studies and 10.0 1111 for mixed cultures. The final voluine in each case was 11.0 1111 per flask, after the addition of inoculum. Cultures were grown a t 25' C on a rotary shaker.

Soil a n d Root Extracts Soil extract was prepared by adding 500 g of garden soil to 1 liter tap water

and heating in an autoclave for 30 minutes a t 15 lb. The slurry was then filtered through Whatman No. 5 filter paper in a Biichner funnel and the filtrate brought up to 1 liter with tap water. The extract was sterilized by passage through a millipore filter after adjustment of the pI-I to 7.0. The dry weight of this soil extract was found to be 0.53 mg/ml.

Root extracts were prepared from 2-, 4-, and 8-week-old oat, wheat, and soybean plants, varieties Garry, Acadia, and Acme, respectively. The crops were grown under greenhouse conditions with artificial illumination during the winter months; the 8-weeli-olcl plants were bearing heads or pods a t the time of harvest. After reilloval of the tops, the roots were washed in running water, air-dried (1 day) then soa1;ed in water (sufficient to cover the roots in a bealier) with occasional agitation for 2-3 hours, and the solution finally filtered through cheesecloth and No. 12 Whatnlan paper. This mild treatment resulted in the liberation of ninhydrin-positive and reducirig coi~lpounds as revealed by sinlple paper chromatography. The root extract was then centri- fuged a t 12,000Xg for 15 minutes and the pI-I adjusted to 7.0. All root extracts had an initial pI-I in the range of 6.7 to 7.2. The extracts were sterilized by passage through millipore membrane filters, and the dry weight of each extract was determined. Sterile distilled water was then added aseptically so that the final root extract used for a test had the saine dry weight as the soil extract.

Growth Determi~zat ions Growth of the pure and mixed cultures was followed by plate counts (spread

plates) on selective media. A nitrogen-free inorganic salt solution (10) was used as diluent for serial dilutions. This was necessary since both Arthrobacter cells and vegetative cells of Bacillzls are fragile in osn~otically unbalanced fluids.

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CHAN ET AL.: GROWTN OF SOIL BACTEKIA 189

Selective Media The media used to enumerate cells in a pure or mixed culture were as

follows: (a) Difco Antibiotic medium 2 (Bacto-Penassay Base agar) : on this

lnediurn il. citrez~s forlned typical bright yellow colonies in 3 to 4 days; B. cereus forlned spreading white colonies in 1 day; and Pse~~domonas sp. forlned large smooth colorless colonies in 1 to 2 days.

(b) Azotobacter medium: A . chroococcz~m fornled large brown colonies on this medium in 14 days. The colnpositioil of the medium was: I<2I-IPOa, 0.5 g; MgS0.1.7H20, 0.2 g ; NaCl, 0.2 g ; i\lInSO.I, FeS04, Nai\iIo0.1.21-120, trace; CaCO3, 5 g ; sucrose, 10 g ; agar, 15 g ; distilled water, 1000 ml. I t was sterilized by autoclaving for 15 minutes a t 15 lb.

(c) Streptomycin medium: if. rndiobacter fornled white glistening colonies on this medium; other species employed in this study were sensitive to the concentration of streptomycin used. The medium was prepared as follows: Difco Casamino Acids (vitamin-free), 5 g ; glucose, 1 g; agar, 15 g ; salt solution, 1000 1111. (The salt solution had the following composition: I<.?I-IP04, 1 g; KNOa, 0.5 g ; MgS0.1.71-120, 0.2 g ; CaCl?, 0.1 g ; NaC1, 0.1 g ; FeCla.61-120, 0.01 g. The solution of combined salts was heated to boiling, cooled to room temperature, and filtered through Whatman No. 12 filter paper. The volulne

m

? 105/ A Z ( I O N A G A R 1 :: 0 A Z ( B A C T O - A G A R )

• P P U R E M I X E D I I I I I

0 I 2 3 0 I 2 3 D A Y S D A Y S

FIG. 1. Growth interrelationships of soil bacteria in soil extract. (AG = Agrobacterium radiobacter, AR = Artltrobacter citreus, AZ = Azotobacter cizroococcum, B = Baci l lus cereus, P = Pseudomonas sp.)

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190 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 9, 1963

was brought to 1 liter with distilled water for use in the mediurn.) The pH was adjusted to 7.0. T o the medium, sterilized a t 15 Ib for 15 minutes and cooled to 55' C, was added sterile streptomycin so that the final concentra- tion of the antibiotic was 30 pg/nll.

Results Figure 1 shows the growth curves of the five test organisms in pure and

mixed cultures in soil extract. As nlay be seen, B. cerezis did not grow well and was a poor conlpetitor. A. ckoococczim apparently died rapidly in mixed culture but in later experiments in which a different kind of agar was incor- porated into the azotobacter mediulll this was found not to be the case. The true growth curve of A. c/~roococczim in mixed culture, obtained with the improved medium, is also included in Fig. 1. i l . radiobacter, A . citrezcs, and Pseudomonas sp. were unaffected in combination, growing about as well as in pure culture. The final numbers of A . radiobacter and A. citreus in both pure and mixed culture were somewhat greater than those of the Pseudomonas s P .

In preliminary studies on methods for the selective enurneration of each species of bacteria in a mixed culture, the inhibition of Azotobacter by the associated organisms was not noticed when approximately equal numbers of each species were mixed together and then plated out. Also, in the 0-day platings, there was no observed killing of 1 1 . chroococcum by the associated organisms (Figs. 1 and 3). But after 24 hours' incubation of a mixed culture in an extract (Figs. 1 and 3), Azotobacter was not recovered; even 1 : 100 dilution plates were devoid of Azotobacter colonies. Since this decrease in numbers of Azotobacter was somewhat dramatic, the question arose, was the phenomenon more apparent than real? Furthermore, it was not clear whether this was taking place in the extract or on the agar plate. These questions were resolved by the use of different kinds of agar, and various culture mix- tures grown in root extracts (from 2-week-old soybeans) for 24 hours a t 2.5' C. Table I shows counts when four different lots of agar were compared. I t is evident that A. chroococcum was 'dormant' on the Bacto agar plates and not killed in the extract. Therefore, subsequent platings of mixed cultures grown in 41 and 8-week-old plant root extracts were carried out with Oxoid Ionagar. Figure 2 illustrates the comparative recovery of A . chroococcum on plates

TABLE I Recovery of Azotobacter chroococcum from a five-culture ~nixturc irl soybean root extract

using azotobacter medium prepared with different kinds of agar

Dilutions used*

Agar Lot No. 10- lo-= 10-G

Difco Bacto agar 445232 0 0 0 0 0

Oxoid Ionagar 24 TM T M T M 32 6

Baltimore Biological 108624 0 0 0 0 0 Laboratories agar

Difco Noble agar 379633 T M T M T M 34 6

*Same dilution samples were used for all the plates; colo~iy counts are averages of duplicate plates. Plates were incubated for 14 days. TM = too many to count.

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FIG. 2. Plaic counis of llzotobncler chroococrlorl i n a 24-hour lnixccl c ~ ~ l t ~ ~ r e irl extract after 18 days' incubation a t 2.5' C. 11. Ionagar ~lsetl. B. Bacto-agar usccl.

Clian el a1.-Can. J. 3licrobiul.

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CHAN ET AL.: GROWTH OF SOIL BACTERIA 191

TABLE I1

Effect of species association and agar on counts of Azolobacler chroococcunt

Dilutions plated*

A . chroococcum plus: Agar used 10- 10- 10- 10-

A . mdiobacter Ionagar TM TM TM 69 12 Bacto-agar TM TM TM 64 7 Ionagar TM TM TM 5 1 7 Bacto-agar TM TM TM 50 5

B. cereus Ionagar TM TM TM 93 10 Bacto-agar TM TM TM 106 13

Pseudomonas sp. Ionagar TM TM TM 48 5 Bacto-agar 0 0 0 0 0

*Colony counts are averages of duplicate plates. Plates were incubated for 14 days. TM - too many to count.

from a mixed culture, using Ionagar and Bacto agar. Even though good recovery was apparently obtained with Ionagar or Noble agar, prolonged incubation was required. Where pure cultures of A . chroococc~rm would grow in 4 to 5 days, a t least 14 days were necessary for growth on a plate with a mixed culture. I t should be noted that this phenomenon occurred only with mixed cultures; A . chroococcum grew well in pure culture regardless of the kind of agar used. The data in Table I1 indicate that in the mixed culture

A Z ( B A C T O - A G A R ) A B • P P U R E M I X E D

104 I I I I I 2 3 0 I 2 3

D A Y S D A Y S

FIG. 3. Growth interrelationships of soil bacteria in 2-week-old oat root extract. (Species symbols same as in Fig. 1.)

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CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 9. 1963

B A Z

P U R E M I X E D

I 2 3 0 I 2 3 D A Y S D A Y S

FIG. 4. Growth interrelationships of soil bacteria in 4-weelc-oltl soybea~t root (Species symbols same as in Fig. 1.)

z I O 5 1 ; ; ; , , I , I 0 A R

. P P U R E M I X E D lo4

I 2 3 0 I 2 3 D A Y S D A Y S

extract-.

FIG.'^. Growth interrelatiotlships of soil bacteria in 4-week-old oat root extract. (Species symbols same as in Fig. 1.)

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CHAN ET AL.: GROWTH OF SOIL BACTERIA 193

P s e z ~ d o m o n a s sp. was responsible for the inhibition of L 1 . chroococcz~m on the azotobacter medium. (Pscudomonas sp. formed only pinpoint colonies on this substrate.) In this experiment A . c h r o o c o c c ~ ~ m was groxn with the indi- vidual species of the other genera.

Figure 3 shows the growth patterns of the five soil bacteria in root extract from 2-week-old oats; essentially similar results were obtained with the soy- bean and wheat root extracts of the same age. In all these extracts B. ccrezls was again a very poor competitor. The same phenomenon of apparent death occurred with respect to i l . clzroococcz~m; a separate experiment using Ionagar gave no evidence of death. A. radiobacter, 11. ci trez~s, and Pser~domonas sp. were not inhibited but in these extracts the maximum gro\vth reached by the pseudolnonad in mixed culture was greater than that of . l . radiobacter and A . citrez~s.

In Figs. 4 and 5 may be seen the gro\vth curves of the five species of bacteria in root extracts of 4-\\eel;-old soybeans and oats, respectively. Figure 5 (oat) is also representative of the growth patterns in 4-week-old wheat root extract. In all these extracts, B. cereus and "1. chroococcum were poor competitors. In

I " 0 I 2 3 0 I 2 3 D A Y S D A Y S

a n4-

FIG. 6. Growth interrelationships of soil bacteria in 8-week-old oat root extract. (Species symbols same as in Fig. 1.)

0 A R A Z P U R E

I

. P M I X E D

I

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CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 9. 1963

FIG. 7. Growth interrelationships of soil bacteria in 8-week-old wheat root extract. (Species symbols same as in Fig. 1.)

o A R P U RE

. A Z P U R E P M I X E D I I I I I I

I 2 3 0 I 2 3

D A Y S D A Y S

FIG. 8. Growth interrelationships of soil bacteria in 8-week-old soybear1 root (Species symbols same as in Fig. 1.)

P M I X E D

extract.

lo4 I I I I I

I 2 3 0 I 2 3

D A Y S D A Y S

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CHAN ET AL.: GROWTH OF SOIL BACTERIA 195

fact, in soybean root extract (Fig. 4) there was inhibition of B. cereus in mixed culture. Neither Pseudomonas sp. nor il. citreus was affected by the associated bacteria and the pseudomonad predominated over A . citrezls (as in the extracts of 2-week-old plant roots). Only in soybean root extract (Fig. 4) was growth of 11. radiobacter depressed in pure culture; this inhibition did not occur in mixed culture.

Figures 6, 7, and 8 illustrate bacterial growth patterns in root extracts of 8-week-old oat, wheat, and soybean plants, respectively. B. cereus grew poorly and, in soybean root extracts, was inhibited in mixed, but not in pure culture. A . chroococczlm was again not stimulated appreciably by the extracts, alone or in association with the other organisms. Although A . citreus and Psez~do7lionas sp. grew most abundantly in pure culture, in mixed culture the former was always inhibited somewhat so that tlie Pseudomonas sp. pre- dominated. The gro~vtli response of 11. radiobacter was most striking in these mature plant root extracts. In all of them, numbers of this organism, when in pure culture, were reduced in 1 day; however, after further incubation there was an increase in count. In mixed culture the reduction was not as pronounced; there was a slow decline in population (oat and soybean extracts) or stasis (wheat extract), with no recovery in numbers.

Discussion The assumption made in this study was that the root extracts, prepared

as described, were sufficiently similar to root excretions that their effect on bacterial growth may be regarded as somewhat equivalent to the 'rhizosphere effect.' The results obtained with the root extracts reflect the observed shift in the microbial equilibrium due to root excretions into soil as recorded by ecological surveys (2, 3, 8, 13, 14, 15) and do appear to lend support to the latter. In all of the root extracts, Psezldomonas sp. (representative of the Gram-negative forms in soil) predominatecl over A . citrezls (representative of the Gram-positive and coccoid rods) in mixed culture. In soil extract, the Pseudomonas sp. was somewhat lower in number than A . citrezss, while A. radiobacter, another Gram-negative bacterium, almost equalled A . citrezrs in numbers.

Another significant feature is that the age of plants played a far greater role in effecting differences in the bacterial growth patterns than the kind of plant. The growth curves of all the five species of bacteria were essentially similar in root extracts of 2-week-old plants. I-Iowever, in the extracts from the mature S-week-old plants, numbers of A . radiobacter were reduced sharply in pure culture in the first day of incubation; in mixed culture this effect was not as pronounced (Figs. 6, 7 , and S). In pure culture, counts of A. radio- bacter were distinctly lower in 4-week-old soybean extract than in the wheat and oat extracts of the same age (Figs. 4 and 5). These results suggest a gradual accun~ulation of substances toxic for A . radiobacter. In this connection it is of interest t o note that Starkey (11) reported a clecrease in numbers of B a c t e r i ~ i m radiobacter a t the late periods of plant development. The inhibition of B. cereus in 4-week-old soybean root extracts (Figs. 4 and 8) in mixed culture was also noteworthy.

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196 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 9. 1963

The bacteriostatic effect on il. ckroococcz~m by Psezldomonas sp. growing feebly on "nitrogen-free" plates, and only with certain ltinds of agar has several in~plications. I n the first place i t suggests a limitation in the technique for the isolation of ilzotobacter. Secondly, the relatively low nuillbers of i lzoto- bacter in the rhizosphere may be due t o the constant inhibition in si tu of th is species by the larger numbers of pseudoinonads (8). T h e selective inhibition of pseudomonacls could conceivably result in large increases in Azotobacter in the field. Studies on this relationship are being continued.

The consistently poor growth of i l . ckroococcz~m and B. cereus, alone or in coillpetition with other species, together with the observed predoi~~inance of P s e u d o ~ ~ o n a s over 11. citreus in root extracts, are in agreernent with the micro- bial investigations of soils and rhizospheres nlentioned earlier. T h u s it would appear tha t laboratory stuclies with mixed cultures are of value in unravelling the complex microbial interactions in the rhizosphere, for there are intriguing siinilarities between the results obtained in the laboratorv and those obtained in ecological surveys. T h e results of this investigation also suggest tha t the rhizosphere effect may be not only selectively stimulating but also selectively inhibiting.

Acknowledgments One of the authors (E.C.S.C.) acknowledges the generous support of t h e

National Research Council of Canada in a postdoctoral fellowship award. T h e authors are indebted t o Dr. I. L. Stevenson, Microbiology Research Insti tute, Ottawa, for a critical reading of the manuscript.

References 1. CHAN, E. C. S. and I<ATZNELSON, H. Growth interactions of Arthrobacler globifornzis

and Psez~do~tzo?zas sp. in relation to the rhizosphere effect. Can. J . i\iIicrobiol. 7, 759- 767 (1961).

2. CLARIC, F. E. Notes on the types of bacteria associated with plant roots. Trans. Kansas Acad. Sci. 43, 75-84 (1910).

3. LOCHHEAD, 11. G. Qualitative studies of soil microorganisms. 111. Influence of plant growth on the character of the bacterial flora. Can. J . Iies. 18, 42-53 (1940).

4. MARSHALL, I<. C. and ALEXANDER, PI. Competition between soil bacteria and Fusarium. Plant and Soil, 12, 113-153 (1960).

5. N:\I~AMURA, L. I<. and HARTMAN, P. A. Lactobacillus:yeast interrelationships. J . Bac- teriol. 81, 519-523 (1961).

6. NEVIN, T. A., HAMPP, E. G., and DUEY, B. V. Interaction between Borrelia vincelztii and an oral diphtheroid. J. Bacteriol. 80, 783-786 (1960).

7. RANSOM, J. P., FINICBLSTEIN, R. A,, CEDER, I<. E., and FORMAL, S. B. Interactions of Vibrio cholerae, Shixella f terzer i , enterococci, and lactobacilli in continuously fed cul t~~res . Proc. Soc. Exptl. Biol. Med. 107, 332-336 (1961).

8. ROUATT, J. W. and I<ATZNELSOW, H. A study of the bacteria on the root surface and in the rhizosphere soil of crop plants. J . ilppl. Bacteriol. 24, 164-171 (1961).

9. SANDERS, A. C., PELCZAR, M. J., JR., and HOEFLING, A. F. Interactions of Staphylococcus and Neisseria go?zorrhoeae in "penicillin resistant" gonorrhea. Antibiot. Chemother- an\.. 12. 10-16 (1962). - - r , r . - - -

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11. STARKEY, R. L. Some influences of the development of higher plants upon the micro- organisms in the soil: 11. Influence of the stage of plant growth upon abundance of organisms. Soil Sci. 27, 355-378 (1929).

12. TAYLOR. C. B. and LOCIIHEAD. A. G. Oualitative s t ~ ~ d i e s of soil microoreanisms. 11. A survey of the bacterial flora of soils"differing in fertility. Can. J. Res. 2, 16, 162-173 (1938). ,- - -

13. \~AGXEROVA, I!., MACURA, J., and CATSKA, V. Rhizosphere microflora of wheat. I. Composit~on and properties of bacterial flora during the first stages of wheat growth. Folia Microbial. 5, 298-310 (1960).

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CHAN E T AL.: GROWTH O F SOIL BACTERIA 197

14. VAGNEROVA, K., MACURA, J., and CATSICA, V. Rhizosphere rnicroflora of wheat. 11. Co~nposition and properties of bacterial flora during vegetation period of wheat. Folia Microbial. 5, 311-319 (1960).

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