JOURNAL OF BACTERIOLOGY, Apr. 1985, p. 190-1950021-9193/85/040190-06$02.00/0Copyright © 1985, American Society for Microbiology

Vol. 162, No. 1

Strain-Specific Chemotaxis of Azospirillum spp.BARBARA REINHOLD,* THOMAS HUREK, AND ISTVAN FENDRIK

Institute of Biophysics, University of Hannover, D-3000 Hannover 21, Federal Republic of Germany

Received 29 August 1984/Accepted 21 December 1984

Chemotactic responses of three Azospirillum strains originating from different host plants were compared toexamine the possible role of chemotaxis in the adaptation of these bacteria to their respective hosts. Thechemotaxis to several sugars, amino acids, and organic acids was determined qualitatively by an agar plateassay and quantitatively by a channeled-chamber technique. High chemotactic ratios, up to 40, were obtainedwith the latter technique. The chemotactic response did not rely upon the ability of the bacteria to metabolizethe attractant. Rather, it depended on the attractant concentration and stereoconfiguration. Chemotaxis wasfound to be strain specific. Differences were particularly observed between a wheat isolate and strainsoriginating from the C4-pathway plants maize and Leptochloa fusca. In contrast to the other two strains, thewheat isolate was strongly attracted to D-fructose, L-aspartate, citrate, and oxalate. The other strains showedmaximal attraction to L-malate. The chemotactic responses to organic acids partially correlate with theexudation of these acids by the respective host plants. Additionally, a heat-labile, high-molecular-weightattractant was found in the root exudates of L. fusca, which specifically attracted the homologous Azospirilumstrain. It is proposed that strain-specific chemotaxis probably reflects an adaptation of Azospirillum spp. to theconditions provided by the host plant and contributes to the initiation of the association process.

Nitrogen-fixing bacteria of the genus Azospirillum areknown to be present in the rhizosphere of various tropicaland subtropical grasses (15). The interactions between thesebacteria and grass roots, which do not result in the formationof special visible structures on roots, as in the Rhizobium-legume symbiosis, have been described as "associative"(15). Various plants respond differently to inoculation withAzospirillum spp. Plant yield or root-associated acetylenereduction activity depends on, among other factors, thecombination of plant species or even cultivars (26, 30) withbacterial strains (7, 27).

Little information is available about the mechanisms ofinteractions and factors determining host plant specificity(24). For a better understanding of these mechanisms, moredetailed information concerning interstrain differences ofdiazotrophs is necessary (6).

Bacteria capable of chemotaxis have a competitive advan-tage if nutrient gradients exist (25). It is known that nutrientgradients in the rhizosphere are generated by plant rootexudates. Since plants can differ in the composition of theirroot exudates (28, 29) and since Azospirillum spp. showschemotaxis to oxidizable substrates (8), it can be postulatedthat chemotaxis may play a role in the adaptation of thesebacteria to their hosts. Specific chemotactic attraction tohost excretions have been demonstrated in predaceous pseu-domonads (11) and zoospores of the phytopathogenic fungusPhytophtora cinnamoni (31).The capillary assay usually applied to quantify bacterial

chemotaxis (2) is not suitable to test Azospirillum spp.because aerotaxis masks the chemotactic response (9). Toavoid this problem, a technique that uses channeled cham-bers has been recently developed. Additionally, chemotaxiscan qualitatively be demonstrated in an agar plate assay bymaking use of metabolically generated gradients which aredeveloped during bacterial growth. Thus, only attractantsserving as nutrient sources give positive chemotactic reac-tions (1).

* Corresponding author.

In the present paper chemotactic responses of threeAzospirillum strains to specific substrates were compared byusing the agar plate assay and the channeled-chamber tech-nique. Additionally, chemotaxis to high-molecular-weightroot exudates of Leptochloa fusca (commonly called Kallargrass), a highly salt-tolerant grass associated with nitrogen-fixing bacteria (21), is described. Chemotaxis was shown tobe strain specific in the genus Azospirillum.

MATERIALS AND METHODS

Bacterial strains. Azospirillum lipoferum ER15 was iso-lated from the rhizosphere of Leptocholafusca (B. Reinhold,M.S. thesis, University of Hannover, Hannover, FederalRepublic of Germany, 1984). The following bacteria wereobtained from the German Collection of Microorganisms,Gottingen, Federal Republic of Germany: Azospirillum bra-silense DSM2298 (SpT60), isolated from wheat roots; A.brasilense DSM1858 (JM6A2), isolated from maize roots; A.brasilense DSM1843 (Cd), isolated from roots of Cynodondactylon.Media and growth conditions. The synthetic malate (SM)

medium had the following composition: DL-malic acid, 5.0 g;KOH, 4.5 g; KH2PO4, 0.6 g; K2HPO4, 0.4 g; Mg S04 * 7H20,0.2 g; NaCl, 0.1 g; CaC12, 0.02 g; MnSO4 H20, 0.01 g;Na2MoO4 * 2H20, 0.002 g; Fe(III)-EDTA (0.66% [wt/vol] inwater), 10 ml; biotin, 0.1 mg; NH4Cl, 0.5 g; yeast extract(Merck, Darmstadt, Federal Republic of Germany), 0.1 g.Batch cultures were grown in 100-ml Erlenmeyer flaskscontaining 20 ml of SM medium and were inoculated with aloopful of bacteria from 24- 76-h-old SM medium agar slants,which had been incubated at 35°C. Growth conditions wereselected to avoid cell aggregation. Strains SpT60, JM6A2,and Cd were incubated at 30°C with reciprocal shaking (100rpm); strain ER15 was incubated at 37°C with rotary shaking(100 rpm).Chemotaxis medium contained 60 mM phosphate buffer

(pH 6.8) and 0.1 mM sodium EDTA. To prevent cellclumping during the washing procedure, the chemotaxis

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CHEMOTAXIS OF AZOSPIRILLUM SPP. 191

medium used for strain ER15 was modified by increasing thepH to 8.0 and adding 0.125% each of Na2SO4 and NaCl.

Culture preservation. The Azospirillum strains were main-tained on semisolid malate medium (14) at 35°C and traris-ferred every 2 weeks. Long-term preservation of the strainswas achieved by suspending exponentially growing cells infresh SM medium containing 5% dimethylsulfoxide andfreezing in liquid nitrogen.

Preparations of bacterial suspensions for chemnotaxis exper-iments. Early-log-phase cultures of the A. brasilense strains,corresponding to an optical density of 0.2 to 0.4 at 578 nm,were centrifuged at 1,500 x g for 10 min and washed threetimes with chemotaxis medium. Finally, cell density wasadjusted to 8 x 108 cells per ml as determined by opticaldensity. For testing strain ER15, stationary-phase culturescorresponding to an optical density of 3.6 to 3.9 were used.Cells were centrifuged for 6 min at 2,300 x g, washed threetimes with chenmotaxis medium, and adjusted to a density of4 x 108 cells per ml. For the chemotaxis assay in agar plates,cells of all strains were adjusted to 5 x 108 cells per ml.

Chemotaxis assay in agar plates. The chemotaxis assaywas carried out according to the method of Adler (1). Thebasic medium was semisolid SM medium (0.2% agar) with-out malic acid, KOH, or yeast extract and supplementedwith the substances to be tested at a concentration of 1 mM.In the case of organic acids, sodium salts were used. Plateswere inoculated with 20 ,ul of the bacterial suspension in thecenter and checked for the formation of a bacterial ring afterincubation at 30°C for 17 h.Chemotaxis assay in a channeled chamber. The quantitative

estimnation of chemotaxis was carried out by a modificationof the method described by Barak et al. (8). The channeledchambers were made of Teflon plates according to thedimensions given by Palleroni (23). Chambers were steri-lized by overnight exposure to UV light. Attractants weredissolved in chemotaxis medium at 1.5-fold of the givenconcentrations. In the case of organic acids sodium saltswere used as substrates. After the chambers were filled withchemotaxis medium, the attractant solution and the bacterialsuspension were simultaneously injected into the oppositewells of the sample chambers. The well to which the bacteriawere added was called the source well, and the other wasnamed the target well. Only 20 ,ul each was injected, ascompared with 30 ,l used by Barak et al. (8). In the controlchambers, the attractant solution was replaced by chemo-taxis medium. The chambers were incubated at 30°C until acountable number of bacteria reached the target well of thecontrol chambers. Strains SpT60 and Cd were incubated for4 h, strain JM6A2 was incubated for 3.5 h, and strain ER15was incubated for 1.5 h. Samples (1 RI) were collected fromthe target wells of the control and sample chambers bymicropipettes. Cell numbers were determined by directmicroscope counting in a Thoma chamber after the sampleswere diluted with 1 ,ul of 8% formaldehyde solution directlyin the counting chamber. The chemotactic ratio (Rche) wasdefined as the ratio of cell counts in the target wells of thesample and control chambers and was calculated as theaverage of 4 replicates. The chemotactic response wasconsidered positive when significantly more bacteria wereattracted in the sample than in the control.

Preparation of root exudates of Leptochloa fusca. Surfacesterilization and germination of the seeds were carried out aspreviously described (20). Five-day-old seedlings were trans-ferred into sterile tubes containing 15 ml of Fahraeus me-dium (17) supplemented with 0.1 mM NH4Cl. They wereincubated for 2 weeks in a growth chamber with an 18-h

day-6-h night cycle at 28 and 24°C, respectively, and 20 klxwith Osram HQIL lamps. Nutrient solutions were collectedaseptically at the end of the growing period. The high-mo-lecular-weight fraction (>10,000) of the substances releasedby the roots was concentrated 100-fold by ultrafiltrationthrough an Amicon PM 10 membrane and then stored at 4°C.Protein content of this fraction was 10 ,ug/ml, as determinedby the Bio-Rad protein assay (catalogue no. 500-0006; Bio-Rad, Munchen, Federal Republic of Germany).

RESULTS

Chemotaxis in agar plates. The ability of three Azospiril-lum strains to react chemotactically on oxidizable substrateswas tested qualitatively. Table 1 shows the chemotacticresponses to various organic acids, sugars, and amino acids.Several qualitative differences in the chemotactic reactionsof the strains were observed. In the case of succinate andcitrate, two bacterial rings were formed. This may possiblybe due to excretion of a metabolite during the migration ofthe first ring, with the metabolite itself acting as an attract-ant.

Chemotaxis in the channeled chambers. Responses of threeAzospirillum strains to several organic acids, sugars, andamino acids were also compared by using a quantitativeassay for chemotaxis. Results are shown in Table 2 through4.

Nearly all substrates resulting in the formation of abacterial ring in the agar plate assay revealed positivechemotactic reactions in the channeled chambers. An excep-tion was oxalate, which gave no significant reaction whentested with strain ER15 (Table 4). Attraction may occur athigher concentrations than those tested, or the degree ofattraction may be so low that it was not detected in thisassay.The chemotactic effect of a substance did not always

correspond to the ability of the bacteria to metabolize it. Forexample, trans-aconitate attracted bacteria of strain ER1Swhen tested in the channeled chambers, although no ringformation occurred in the agar plate assay; thus, the sub-stance appears not to be metabolized. L-Aspartate showedthe same effect on strain SpT60. On the other hand, sub-strates known to be utilized by Azospirillum spp., such asfructose and glucose, did not act as attractants for strains

TABLE 1. Chemotaxis by A. brasilense SpT60, A. brasilenseJM6A2, and A. lipoferum ER15 in agar plates"

Substrate A. brasilense A. lipoferoinSpT60 JM6A2 ER15

L-Malate + + +D-Malate - -Succinate + + + + +Fumarate + + +Oxalate + - +Citrate + +t-Aconitate + +D-GlucoseD-Fructose +L-Arabinose - -L-Glutamate + + +L-Aspartate - - +

a ++, Formation of two rings; +, formation of one ring; -, no ringformation.

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192 REINHOLD, HUREK, AND FENDRIK

TABLE 2. Chemotaxis by A. brasiknse SpT60 measured inchanneled chambers

Chemotactic responseSubstrate Concn Mea

Rche" ±SD Statistics'

L-Malate 1 1.1 ± 0.310 1.8 + 0.440 2.2 0.4 a

Succinate 1 1.0 + 0.210 12.9 ± 6.8 cde40 14.9 + 3.5 d

Fumarate 1 1.9 ± 1.110 6.0 ± 2.2 bc40 3.4 ± 0.7 bc

Oxalate 0.5 3.5 ± 1.9 abc1 21.3 ± 3.5 def5 3.5 0.7 b

Citrate 1 1.7 ± 1.910 4.4 ± 0.8 bc40 29.3 ± 8.7 fg

Mixture of organic acids' 1 1.2 ± 0.410 1.5 ± 0.240 1.7 ± 0.3

D-Glucose 1 1.1 ± 0.110 1.2 ± 0.340 1.i ± 0.2

D-Fructose 1 1.0 ± 0.110 13.6 ± 4.3 d40 5.0 ± 1.7 bc

L-Glutamate 1 0.7 ± 0.410 0.9 ± 0.740 2.0 ± 0.3 a

L-Aspartate 1 5.4 ± 1.8 bc10 15.4 ± 4.0 dg40 24.4 ± 3.8 ef

Control 1.0

a Values represent the mean of four replicates.b Results followed by letters are significantly different from the control at

the 5% level according to Student's t test. Values with the same letter are notsignificantly different from each other.

'Organic acids were mixed in the relative molar proportions found in theroot exudates of Leptochloa fusca: L-malate-citrate-fumarate-succinate(55:2.4:1.2:1) with L-malate at the given molarity.

JM6A2 and ER15, neither in the agar plate assay nor in thechanneled-chamber assay.Chemotaxis proved to be a stereospecific reaction in the

case of malate (Table 4). L-Malate gave significantly higherchemotactic ratios than D-malate when tested at the sameconcentrations. Although D-malate could attract the bacte-ria, no ring formatidn was observed in the agar plate assay,suggesting thAt D-malate may not be metabolized.The chemot'actic reactions depended on attractant concen-

trations. Usually the strongest chemotactic responses werefound at a concentration of 40 mM, or the Rche was at thesame level at 40 and 10 mM. For D-fructose (Table 2) andL-glutamate (Table 3) the optimum concentration for chemo-taxis was 10 mM. The attractant with the lowest optimal

TABLE 3. Chemotaxis by A. brasilense JM6A2 measured inchanneled chambers

Chemotactic responsesubstrate Concnea

Rche" ± SD Statisticsb

L-Malate 1 1.3 ± 0.9

Succinate

10 7.1 ± 3.040 15.1 ± 0.8

1 1.2 ± 1.310 3.6 ± 2.340 6.1 ± 2.0

1 1.1±0.310 3.1 ± 0.640 4.9 + 0.6

Fumarate

Oxalate

Citrate

abcd

abc

ab

1 2.1±1.410 1.6 ± 0.740 0.6 ± 0.2

1 0.9 ± 0.310 0.8 ± 0.340 1.1 ± 0.2

Mixture of organic acids'

D-Glucose

1 2.0 + 0.710 8.2 ± 1.340 19.3 ± 10.1

cc

1 1.0 ± 0.610 1.1 ± 0.440 0.9 ± d.2

D-Fructose

L-Arabinose

L-Glutamate

L-Aspartate

Control

1 0.7 ± 0.110 0.8 ± 0.240 2.2 ± 1.5

1 1.3 ± 0.510 1.1 ± 0.440 1.1 ± 0.7

1 1.0 ± 0.510 3.5 ± 1.440 0.7 ± 1.3

1 1.2±0.$10 0.8 ± 0.740 1.2 ± 0.5

1.0

ab

"Values represent the mean of four replicates.For an explanation of the letter code, see Table 2. footnote b.For composition of the mixture, see Table 2, footnote c.

concentration (1 mM) was oxalate, for strain SpT60 (Table2).The three strains showed several common chemotactic

responses. Attraction to fumarate was similar for all strains,and all strains were well attracted by succinate. L-Glutamatewas a weak attractant for the three strains, and b-glucoseproduced no significant chemotactic response.Some chemotactic reactions of the strains differed quali-

tatively and quantitatively. These differences especially oc-curred between strain SpT60 and the other two strains,which shared several chemotactic properties. Strain SpT6Owas strongly attracted by D-fructose and L-aspartate,whereas strains JM6A2 and ER15 responded weakly or notat all. Clear differences in chemotactic responses were

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CHEMOTAXIS OF AZOSPIRILLUM SPP. 193

TABLE 4. Chemotaxis by A. lipoferum ER15 measured inchanneled chambers

Chemotactic responseSubstrate Concn(MM) Mean Statistics'

Rche" ± SD

L-Malate 1 2.9 ± 0.4 a

D-Malate

Succinate

Fumarate

Oxalate

Citrate

t-Aconitate

Mixture of organic acids"

D-Glucose

D-Fructose

L-Arabinose

L-Glutamate

L-Aspartate

Control

10 14.4 ± 5.440 41.0 ± 9.1

1 1.4 + 0.610 0.8 ± 0.440 4.1 ± 1.9

1 3.5 ± 1.710 2.5 ± 1.240 14.0 ± 3.4

1 1.1±0.210 3.5 ± 0.940 4.5 ± 1.8

1 1.3 ± 0.810 0.9 ± 0.440 1.3 ± 0.5

1 1.0 ± 0.210 1.1 ± 0.840 1.2 ± 0.5

1 1.0 ± 0.510 1.7 ± 0.540 7.6 ± 2.9

1 1.6 ± 0.410 21.5 ± 1.240 29.3 ± 6.6

1 0.9 ± 0.310 1.2 ± 0.340 1.6 ± 0.4

1 1.0 ± 0.210 1.1 ± 0.740 2.0 ± 0.7

1 0.7 ± 0.210 0.9 ± 0.540 1.0 ± 0.3

1 0.8 ± 0.210 1.0 ± 0.640 1.8 ± 0.3

1 0.9 ± 0.310 0.9 ± 0.340 6.0 ± 2.0

1.0

a Values represent the mean of four replicates.For an explanation of the letter code, see Table 2, foqtnote b.For composition of the mixture, see Table 2, footnote c.

found, particularly for some organic acids (Fig. 1). Oxalateand citrate belonged to the attractants revealing the highestRches for strain SpT60, whereas the other two strains did notreact chemotactically to these substrates. In pontrast tostrain SpT60, they showed maximal attraction to L-malate.

In addition to single substrates, a mixture of organic acidswas tested, which contained acids of the same compositionand molar ratio as found in the root exudates of Leptochloafusca (20). This mixture provided results similar to thosewith L-malate.For A. brasilense Cd Rches of 14.7 ± 6.9 with 1-malate and

2.7 ± 0.5 with citrate at concentrations of 10 mM wereobtained.The high-molecular-weight fraction of the root exudates of

Kallar grass specifically attracted strain ER15. The followingchemotactic responses (mean ± standard deviation) wereobtained by using the ultrafiltrate of' the root exudates ascontrol: strain SpT60, 1.0 ± 0.7; strain JM6A2, 1.3 + 0.3;strain ER15, 5.7 ± 1.6 and 4.8 + 2.3 (values received beforeand after, respectively, testing the other two strains). Onlystrain ER15' showed a significant chemotactic response.Heating the attractant solution to 95°C for 15 min resulted inthe decrease of the Rche to 1.3 ± 0.6 for strain ER15, a valuenot significantly different from the control. Thus, the attrac-tant was heat labile.

DISCUSSION

Bacteria of the genus Azospirillum shared several proper-ties of their chemotactic reaction with Escherichia coli.Chemotaxis was found to be a stereospecific process (4). Asobserved with several sugars in E. coli, the naturally occur-ring $tereoisomer was preferred by Azospirillum spp. in thecase of malate. Due to the different experimental conditions,the optimal attractant concentrations obtained for Azospiril-lum spp. cannot be compared with those of E. coli, whichare generally lower (4). The Rches obtained for Azospirillumspp. by using the channeled-chamber technique approachvalues found in E. coli by the capillary assay, namely, up to100. Using channeled chambers, Barak et al. (8) obtainedmaximal chemotactic ratios of 14 to 15 with Azospirillumspp. For the reasons already mentioned, these bacteria gavelow values when tested by the capillary assay, not exceedinga three- to fourfold enrichment (5, 19). Therefore, theseresults are not taken into account in this discussion.The mechanism of positive chemotaxis did not rely upon

the ability of the bacteria to metabolize the substrate. Thiswas already observed in E. coli, leading to the assumptionthat the survival value of chemotaxis lies in bringing thebacteria into a nutritious environment, with the attractantspossibly signalling the presence of other undetected nutri-ents (3). Nevertheless, some substances may not have beenclassified as attractants by the assays used, because theformation of chemoreceptors may depend on culture condi-tions, as in the case of inducible sugar receptors in E. coli (4)and Salmonella spp. (16). Substantial taxis mediated bythose receptors is observed only when an inducer is presentin the growth medium.Chemotaxis was found to be strain specific in the genus

Azospirillum. Some of the differences can be correlated withthe origin of the strains. Reactions of the strains isolatedfrom the C4 plants maize and Kallar grass were similar anddiffered from those of the isolate originating from the C3plant wheat. These plants differ in the pathway of primaryphotosynthetic CO2 fixation: in C4 plants organic acidscontaining four C atoms, such as malate, are the initial stableproduct, whereas in C3 plants glycerate-3-phosphate is pro-duced. The strain isolated from wheat was well attracted bysome of the tested sugars and amino acids, in contrast to theother two strains, which were strongly attracted by organic

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194 REINHOLD, HUREK, AND FENDRIK

I Sp T6g |JM 6A21 ER 15 |

Mixture of acids

ISP T601 M6A |ER615

Oxalate

Sp T60

FIG. 1. Patterns of chemotactic response of A. brasilense SpT60, A. brasilense JM6A2, and A. lipoferum ER15 determined by thechanneled-chamber technique. A mixture of acids contained the following organic acids in the same composition and molar ratio as found inthe root exudates of Kallar grass: L-malate-citrate-fumarate-succinate (55:2.4:1.2:1) with L-malate at the given molarity.

acids only. This indicates that particularly organic acids maybe important for these strains to lead them into a favorableenvironment. But this assumption cannot be generalized toapply to strains originating from C4 plants, because A.brasilense Cd, which was isolated from the C4 grass Cynodondactylon, showed strong attraction to sugars and aminoacids as well (8).The different chemotactic responses to organic acids cor-

relate with the exudation of these acids by the host plantroots. Several C4 and C3 plants show different exudationpatterns. Malate is by far the dominating organic acidexuded by Kallar grass (20), and it is found in high amountsin maize roots (22), probably resulting in a high rate ofexudation. In contrast, oxalate could not be detected in theexudates of Kallar grass, but it is the major organic acidexuded by roots of wheat (28) and rice (10). Malate isreleased in smaller or undetectable amounts by these plants.In accordance with this, malate was significantly the strong-est attractant for the isolates originating from Kallar grassand maize, and it attracted the wheat isolate only weakly.The latter strain strongly responded to oxalate; the C4 plantisolates, however, did not. The response to citrate cannot beclearly explained, because its occurrence in wheat rootexudates varies (28) and because citrate was also detected inKallar grass exudates (20). Rice roots, however, releasecitric acid in high amounts (10). Nevertheless, the patternsof chemotactic response to organic acids found with thethree strains tested seem to be more general. A. brasilense

Cd is well attracted by malate (Rche = 12.0) and weaklyattracted by citrate (Rche = 1.9) (8). These values correspondwell with the ratios found in this paper and show thedescribed tendency of C4 plant isolates.These data show that the strain-specific chemotaxis to

low-molecular-weight substrates within the genus Azospiril-lum is more dependent on the origin of the strains than ontheir species affiliation. This strain specificity probably re-flects an adaptation of the bacteria to nutrient conditionsprovided by the host plant and may thus play a role in theestablishment ofAzospirillum spp. in the rhizosphere of theirhost, perhaps as one factor determining host specificity.

In the Rhizobium-legume symbiosis, no host-symbiontspecificity could be found in the chemotactic response tolow-molectilar-weight root exudates (18). However, a high-molecular-weight attractant identified as a glycoprotein (13)was found in the exudates of Lotus corniculatus, whichspecifically attracted Rhizobium spp. (12). The high-molec-ular-weight, heat-labile attractant described here in the rootexudates of Kallar grass specifically attracted the homolo-gous Azospirillum strain and may be analogous to theglycoprotein. Such an attractant suggests an intimate inter-action between Kallar grass and Azospirillum spp.The data obtained from the present work reveal the

possibility of chemotaxis playing a role in an initial stage ofthe establishment of association between Azospirillum spp.and grass roots. Chemotaxis may contribute to the host plantspecificity in these associations. Further elucidation can

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CHEMOTAXIS OF AZOSPIRILLUM SPP. 195

perhaps be obtained by comparative chemotaxis studieswith low-molecular-weight root exudates.

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