Plant Physiol. (1984) 75, 924-9280032-0889/84/75/0924/05/$0 1.00/0

Adsorption of Slow- and Fast-Growing Rhizobia to Soybean andCowpea Roots'

Received for publication March 2, 1984

STEVEN G. PUEPPKE*2Department ofPlant Pathology, University ofFlorida, Gainesville, Florida 32611

ABSTRACT

Roots of soybean (Glycine max IL.] Merr. cv Hardee) and cowpea(Vigna unguiculata IL.1 Walp. cv Pink Eye Purple Hull) were immersedin suspensions containing 10' Rhizobium cells per milliliter of a nitrogen-free solution. After 30 to 120 minutes the roots were rinsed, and thedistal 2-centimeter segments excised and homogenized. Portions of thehomogenates then were plated on a yeast-extract mannitol medium forbacterial cell counts. The adsorption capacities of four slow-growingrhizobia and a fast-growing R. meliloti strain varied considerably. Ad-sorption was independent of plant species and of the abilities of theRhizobium strains to infect and nodulate. R. Iupini96B9 had the greatestadsorption capacity, and Rhizobium sp. 3G4bl6 the least. Rhizobiumsp. 229, R. japonicum 138, and R. meliloti 102F51 were intermediate,except on cowpea, where the adsorption of strain 102F51 was similar tothat of strain 3G4bI6. The initial adsorption rates of bacteria cultured insynthetic media and in the presence of soybean roots were about thesame. Addition of soybean lectin to the bacterial inoculum failed toinfluence initial adsorption rates. Both treatments, however, reduced thenumbers of bacteria that bound after incubation with roots for 120minutes. The relationship between the logarithm of the number of strain138 cells bound per soybean root segment and the logarithm of thedensity of bacteria in the inoculum was linear over filve orders of magni-tude. Binding of strain 138 to soybean roots was greatest at roomtemperature (27°C) and substantially attenuated at both 4 and 37°C.Although R. lupini 96B9 strongly rejected a model hydrophobic plasticsurface, there were no simple correlations between bacterial binding tomodel hydrophobic and hydrophilic plastic surfaces and bacterial adsorp-tion to roots.

In most agriculturally important legumes, nitrogen-fixing rootnodules are initiated when rhizobia from the soil form infectionthreads in host root hairs. Although the mechanism of infectionthread biogenesis remains unclear, adsorption of rhizobia to hostroots seems to be of significance in the initiation of infection(10, 20). Adsorbed rhizobia are thought to induce root hairdeformation and to synthesize the enzymes that breach the cellwall of the root hair. Rhizobia have been observed microscopi-cally on the root surfaces of many legumes (for reviews, see 10,20). Although Rhizobiuim cells often bind individually, adsorbedbacterial aggregates also are found.There is evidence, mostly from combinations of legumes with

fast-growing rhizobia, that adsorption of rhizobia to roots is hostselective. Such selective adsorption of large numbers of nodulat-

'Supported by National Science Foundation Grant No. 82-00110.This is Florida Agricultural Experiment Station Journal Series No. 5423.

2 Present address: Department of Plant Pathology, University of Mis-souri, Columbia, MO 6521 1.

ing rhizobia to host root hairs is considered by Dazzo (10) to bea cardinal event in the developing symbiosis. Using light micros-copy, Dazzo and colleagues (10, 11) determined that the meannumber of infective R. trifolii cells bound per 200 Mm whiteclover (TrifoIium repens L.) root hair varied from 21 to 37,depending on the strain. For each of 18 noninfective strains,fewer than five bacteria bound per 200 um root hair. Other dataalso support the hypothesis that the adsorption of nodulating,fast-growing rhizobia to host roots is greater than that of non-nodulating strains to similar roots (15, 16, 27). The adsorptionof nonnodulating R. japonicum cells to pea (Pisum sativum L.)roots, for example, is significantly less than that of nodulating R.leguminosarum (16). Chen and Phillips (8) and Broughton et al.(7), however, found that the capacities of nodulating strains tobind to pea roots were similar.

Little is known about the adsorption of slow-growing rhizobiato roots of their legume hosts. Cells of nodulating R. japonicumstrain 110 bind to root hairs and undifferentiated epidermal cellsof wild soybean (Glycine soja Sieb. and Zucc.), but cells ofnonnodulating Rhizobium strains are reported not to bind tosimilar roots (22). Certain nonnodulating R. japonicum mutantsalso apparently fail to bind to soybean roots (23). Within I minofinoculation, cells ofnodulating R. japonicum strain 138 adsorbto roots of soybean (25). A series of capsule mutants of strain138, however, retain their abilities to bind to soybean roots andto nodulate. Between 1000 and 2300 cells of such strains bindper 1-cm root segment after incubation for I h in suspensionscontaining I0O bacteria/ml (17).The experiments reported here were designed to examine the

relationship between the adsorption of slow- and fast-growingrhizobia to soybean and cowpea roots and the abilities of theserhizobia to infect such roots. The objectives were (a) to test thehypothesis that infective, slow-growing rhizobia bind selectivelyto roots of their host legumes, (b) to determine if Rhizobiumadsorption can be modified by culture of the rhizobia with hostroots or by addition ofSBL' to the inoculum, and (c) to examinebacterial adsorption in terms of kinetics, temperature sensitivity,and possible mechanisms.

MATERIALS AND METHODS

Organisms. Seeds of soybean, Glycine max (L.) Merr. cvHardee, were from K. Hinson, USDA-ARS, University of Flor-ida. Cowpea ( Vigna unguiculata [L.] Walp. cv Pink Eye PurpleHull) seeds were purchased from Hastings Seed Co., Atlanta,GA. Five Rhizobium strains were examined. R. japonicum 138and Rhizobium sp. 3G4bl6 were from the United States De-partment of Agriculture, Beltsville, MD. Both strains infect soy-bean and cowpea roots, i.e. produce infection threads in roothairs (19). Rhizobium sp. 229, from D. Hubbell, University of

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ADSORPTION OF RHIZOBIA TO ROOTS

Florida, infects cowpea, but not soybean (19). R. lupini 96B9,from J. Burton, The Nitragin Co., and R. meliloti 102F5 1, fromC. P. Vance, University of Minnesota, infect neither soybean norcowpea. Strain 96B9 nodulates its homologous host, Lupinuspolyphillus Lindl., and strain 102F51 nodulates its homologoushost, Medicago sativa L. cv Florida 77 (S. Pueppke, unpublisheddata). The interactions of the rhizobia with soybean and cowpeaare summarized in Table I.

Adsorption of Rhizobia to Roots. Seeds were submerged in50% aqueous ethanol for 5 min and then in 2.6% aqueousNaOCl for an additional 5 min. After four rinses in deionizedH20, seeds were placed onto water agar and germinated in thedark at 25°C. After 3 d, seedlings were transferred aseptically toautoclaved plastic growth pouches (Northrup King Co., Minne-apolis, MN), each of which contained 15 ml of Jensen's nitrogen-free nutrient solution (26). The pouches were enclosed withinplastic sleeves and incubated for 1 d under fluorescent lights(approximately 500 ME/M2 s) at room temperature (27°C). Thebacterial inocula were prepared from 3-d-old liquid cultures inthe defined gluconate-mannitol medium of Bhuvaneswari et al.

(5). The cells were centrifuged at 7700g for 10 min, washed oncewith sterile, filtered Jensen's solution, and adjusted turbidimet-rically to I04 cells/ml of Jensen's solution.The adsorption assay, which was done aseptically in a laminar

flow hood, proceeded as follows: Twenty-five ml portions of thebacterial inoculum were transferred to a series of sixteen 10 cmlong x 2.5 cm diameter test tubes. Two bent paper clips, whichserved as plant supports, were hung from the lip of each tube.Seedlings then were carefully removed from the pouches andsuspended from the clips so that their roots were submerged inthe inoculum. Each treatment consisted of four tubes, eachcontaining a pair of seedlings. After 30, 60, 90, or 120 min,individual seedlings were removed from the inoculum and theirroots rinsed vigorously in a rapidly flowing 25-ml stream offiltered Jensen's solution delivered from a Brinkmann Dispen-sette. The distal 2-cm segment of the primary root of eachseedling then was excised. Two segments, one from each plantthat had been paired in an inoculum tube, were transferred to a

ground glass tissue homogenizer. One ml of filtered Jensen'ssolution was added, and the tissues were ground thoroughly.

Five 0.1 -ml aliquots of each homogenate were plated ontoyeast extract-mannitol medium (26). The plates were incubatedin the dark at 25°C, and colonies were counted after 3 to 7 d,depending on strain. Appropriately diluted bacterial inoculumalso was plated for precise determination of the initial bacterialdensity. This number was used as a constant to normalize thedata from each experiment to an initial inoculum of 104 cells/ml. Each experiment was performed three times.Three series of control experiments were done. To test for

microbial contamination of the plant roots, the bacteria were

omitted from the inoculum in some experiments. In other ex-

periments, known numbers of bacteria were homogenized in thepresence or absence of root segments. These experiments servedas controls for the possible reduction of bacterial viability due tohomogenization or to materials released from plant roots. In a

third set of controls, plants were inoculated and roots washed inthe usual way. The seedlings then were returned to fresh plasticgrowth pouches and incubated under fluorescent lights as de-scribed above. After 2 weeks, the plants were examined for thepresence of nodules.

Modified Adsorption Experiments. To determine if bacterialgrowth conditions influence the capacities of strains 138 and96B9 to bind to soybean roots, bacteria for use as inoculum were

cultured axenically in the presence of soybean roots. Seeds were

germinated for 4 d on water agar, after which individual seedlingswere transferred aseptically to 20 x 2.5 cm test tubes. Each tubecontained 15 ml of filtered, sterile Jensen's solution supple-mented with I04 bacteria/ml. An aluminum screen in each tube

held the plant shoot above the surface of the liquid. The tubeswere covered loosely with plastic film and incubated underfluorescent lights. The plants were removed after 3 d, at whichtime Rhizobium populations were about 5 x 106/ml (no contam-inating bacteria were detected). The bacteria were washed anddiluted as described above and then used immediately as inoculafor adsorption assays.The effect of SBL on adsorption of strains 138 and 96B9 to

soybean also was measured. SBL from seeds of the soybean cvDisoy was purified twice by affinity chromatography (5) anddialyzed exhaustively against filtered Jensen's solution. The lectinsolution then was sterilized by passage through a 0.2 gm filterand the protein concentration adjusted to 100 gg/ml. For theadsorption assay, this solution was mixed with the bacterialinoculum so that the final bacterial density and lectin concentra-tion were 104 cells/ml and 10 ug/ml, respectively. The assaysthen were carried out as described above.The temperature-dependence of bacterial adsorption was de-

termined as follows: Inoculum tubes containing I04 cells of strain1 38/ml were equilibrated for 20 min at 4, 27, or 37°C. Seedlingsthen were transferred aseptically into the tubes. After 1 h, theroots were rinsed at room temperature, root segments wereexcised and homogenized, and the numbers of bacteria deter-mined as above. The experiment was performed four times.The influence of inoculum density on adsorption of strain 138

to soybean was measured by preparing a series of inocula con-taining I03 to 108 cells/ml (viable cell numbers were determinedby plating). The adsorption assays were done as described above,except that the root homogenates were serially diluted in filteredJensen's solution before plating on yeast extract-mannitol me-dium. The experiment was performed three times.

Adsorption of Rhizobia to Plastic. The ability of rhizobia tobind to hydrophobic and hydrophilic plastic surfaces was esti-mated by a modification of the method of Fletcher (12). Strains102F51, 138, and 96B9 were grown for 2 d in 50-ml cultures ofliquid gluconate-mannitol medium. Strains 3G4bl6 and 229,which do not grow to high cell densities in this medium, wereincubated in media supplemented with 2 ml ofDifco SupplementB/l. Bacterial densities at the time of harvest were from 1.8 to3.1 x 108/ml. The cells were centrifuged at 7700g for 10 min,washed once with filtered Jensen's solution, and adjusted to 5 x108 cells/ml. Twenty-ml portions of cell suspensions then werecarefully poured into 9-cm hydrophobic polystyrene Petri dishes(Fisher No. 8-757-12) and into 9-cm hydrophilic polystyrenetissue culture dishes (Coming No. 25050). The dishes werecovered and left undisturbed for 2 h at room temperature.Each bacterial suspension then was carefully poured from the

dish, and 20 ml of filtered Jensen's solution was added. Eachdish was tilted gently and rotated by hand so that the liquidflowed around the dish five times. The rinse solutions then weredecanted. The bottom of each dish was heated with a hair dryeruntil the bacteria were fixed, and adsorbed bacteria were stainedwith a freshly filtered solution of crystal violet (9). The stain wasdecanted after 5 min, and the stained bacteria were washedthoroughly under a running stream of water. The dishes thenwere dried with a hair dryer. The extent of bacterial adsorptionto the dishes was estimated by spectrophotometric measurementof the A590 of the stained cells (12). Eight equidistant marks onthe rim of each dish were used for positioning, and the disheswere rotated between measurements. Each experiment was per-formed three times.

RESULTS

Strain X Host Interactions. Bacteria were not detected inhomogenates of uninoculated control roots, and the homogeni-zation process did not influence the viability of added rhizobia.The procedures for preparation of seedlings and assay of adsorp-tion thus were judged to be acceptable. Some plants that had

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Plant Physiol. Vol. 75, 1984

been inoculated and washed were returned to fresh growthpouches for 2 weeks. Nodules formed only in those combinationspreviously determined to yield infection threads and nodules( 19). This provides further evidence that the seedlings were freeof extraneous rhizobia and that the infective rhizobia whichadsorbed during the short 120-min incubation period were ca-pable of infecting and nodulating.

Although cells of each of the Rhizobium strains adsorbed tosoybean roots, adsorption varied as a function of strain (Fig. 1).The strains were divided into three groups based on adsorptionrates and the numbers of bacteria bound after 120 min. Bindingof R. lupini 96B9 was relatively rapid, so that by 120 min eachroot segment contained nearly 400.adsorbed bacteria. Strains229, 102F5 1, and 138 were intermediate in their adsorptioncapacities. The initial adsorption rate ofstrain 138 approximatedthat of strain 96B9, but the rate did not increase after 60 min.In contrast, the initial adsorption rates ofstrains 229 and 102F51were relatively slow. The rates eventually increased so that thefinal numbers of adsorbed cells of all three strains were similar.Of the strains with intermediate capacity to bind to soybean,only 138 was infective (Table I). Nodules appeared on roots of90% of the plants that were inoculated with strain 138 for 120min, washed, and returned to pouches for 2 weeks.Very few cells of strain 3G4bl6 adsorbed to soybean. By 60

min only about five cells bound per root segment, and at thetermination of the experiment, the number of adsorbed cells ofthis strain was almost an order of magnitude less than that ofnonnodulating strain 96B9. Nevertheless, 40% of the seedlingsincubated for 120 min with strain 3G4b16 were nodulated after2 weeks.The rates at which the five Rhizobium strains adsorbed to

cowpea roots were nearly linear over the duration of the experi-ments (Fig. 2), and the numbers of bacteria that adsorbed in 120min were comparable to those that adsorbed to soybean (TableI). Strain 96B9 again bound in greatest numbers, and strains 138and 229 were intermediate. The adsorption of strain 3G4bl6

1-z

4

-J0.

0

z

0

-J-JwU

FIG. 1.mean of i

experimeninocula cowas restricof the mea(5-7%), Rzobium sp

Table I. Relationship between Adsorption ofRhizobia to Roots andFormation ofInfection Threads

Soybean CowpeaStrain

Adsorptiona Infectivity' Adsorptiona Infectivityb96B9 384 ± 31 - 437 ± 24 -229 127±7 - 122± 12 +138 120±4 + 165±7 +102F51 107±22 - 47±8 -3G4bl6 56±7 + 43±5 +

'The values are the mean number (±sE) of rhizobia adsorbed per 2-cm root segment after incubation at room temperature for 2 h. The dataare from 3 experiments.

b +, Infection threads and nodules form; -, infection threads andnodules do not form.

0 30 60 90 120TIME (MIN)

FIG. 2. Adsorption of rhizobia to cowpea roots. Each point is themean of measurements made with 12 pairs of plants in 3 separateexperiments. The experiments were done at room temperature usinginocula containing 0.8 to 1.8 x 10' viable bacteria per ml, and analysiswas restricted to the distal 2-cm segment of each root. SE (as percentagesof the means) are as follows: R. lupini 96B9 (5-9%), Rhizobium sp. 229(10-12%), R. japonicum 138 (5-12%), R. meliloti 102F51 (16-27%),Rhizobium sp. 3G4bl6 (8-11 %).

./! was low and virtually identical to that of nonnodulating strainA / i q102F5 1. From 70 to 100% of the seedlings incubated for 120min with strains 138, 229, and 3G4bl6 were nodulated after 2weeks. Adsorption of the strains to cowpea roots, however, was// O |not correlated with the abilities of the strains to infect and

I, I j nodulate (Table I). Strain 229 is particularly interesting. Al-though it infects cowpea but not soybean, cowpea and soybean0 30 60 90 120 roots adsorb similar numbers ofcells of this strain.

TIME (MIN) Two procedures were used in attempts to modify the adsorp-Adsorption of rhizobia to soybean roots. Each point is the tion of infective strain 138 and noninfective strain 96B9 tomeasurements made with 12 pairs of plants in 3 separate soybean roots. In the first, adsorption assays were carried out inIts. The experiments were done at room temperature using the presence of 10 jtg of SBL/ml of inoculum, a concentrationintaining 0.7 to 3.5 x 10' viable bacteria per ml, and analysis that does not agglutinate cells of either strain (unpublishedted to the distal 2-cm segment of each root. SE (as percentages observations). In a second series of experiments, rhizobia to beins) are as follows: R. lupini 96B9 (6-9%), Rhizobium sp. 229 used in adsorption assays were cultured axenically in the presencejaponicum 138 (3-9%), R. meliloti 102F51 (20-43%), Rhi- of roots of intact soybean plants. During the initial 60 min3G4bl6 (7-13%). adsorption period, these treatments did not substantially influ-

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ADSORPTION OF RHIZOBIA TO ROOTS

ence binding (Table II). By 120 min, however, both the lectinand culture of the bacteria in the presence of roots significantlydiminished bacterial adsorption relative to controls.

Temperature- and Inoculum Density-Dependence of Adsorp-tion. The effect of temperature on the adsorption of strain 138to soybean was determined in four replicate experiments. Ad-sorption at 27°C was 160 ± 12 cells/root segment. The corre-sponding values for 4 and 37°C were 20 ± 10 and 50 ± 9 cells/root segment, respectively. Thus, compared with the control,adsorption was reduced about 90% by low temperature andabout 65% by elevated temperature.The relationship between the logarithm of the number of

bacteria per milliliter of inoculum and the logarithm of thenumber of bacteria bound per root segment was linear over fiveorders of magnitude (Fig. 3). No evidence of binding saturationwas observed. The percentage of the initial bacterial inoculumthat adsorbed per root segment increased slightly as the inoculumdensity increased. At 103 rhizobia/ml, each root segment ad-sorbed 0.4% of the applied bacteria. At 108 rhizobia/ml, thecorresponding value was 1.6%.

Adsorption of Rhizobia to Plastic. The hydrophobic and hy-drophilic properties of the Rhizobium strains were estimated byindirect measurement of bacterial adsorption to hydrophobicand hydrophilic plastic surfaces (Table III). Although the hydro-phobic and hydrophilic properties of most of the strains weresimilar, strains 96B9 and 102F51 were distinct. Strain 96B9

Table II. Effect ofRhizobium Culture Conditions and Soybean Lectinon the Adsorption ofRhizobia to Soybean Roots

Culture R. lupini 96B9 R. japonicumCulture ~~~~~~~~138Conditions

60 min 120 min 60 min 120 min

Synthetic medium 107 ± 98 384 ± 31 40 ± 4 120 ± 4Synthetic medium, SBL 86 ± 6 292 ± 18 31 ± 4 72 ± 7added to inoculum

In association with roots 122 ± 11 158 ± 12 45 ± 4 70 ± 6

'Mean number (±SE) of rhizobia adsorbed per 2-cm root segment afterincubation at room temperature for the indicated time. The inoculacontained I04 rhizobia/ml, and the data are from 3 experiments.

7

6

z 54

-J

a 4z

m0

c3

w

2

CD0

-i

0 3 4 5 6 7 8 9L O G C E L L S / M L OF INOCULUM

FIG. 3. Inoculum density-dependence of the adsorption of R. japon-icum 138 to soybean roots. The data are from 3 separate experiments inwhich analysis was restricted to the distal 2-cm segment of each root.The correlation coefficient for the linear regression is +0.953.

Table III. Adsorption ofRhizobia to Hydrophobic and HydrophilicPlastic Surfaces

Each value is the mean of 24 separate absorbance measurements ofbacteria adsorbed to three dishes. The bacteria were incubated with thedishes for 2 h prior to rinsing and staining.

Asgo (±SD) to:Strain

Hydrophobic Hydrophilic229 0.09 ± 0.002 0.04 ± 0.001138 0.08 ± 0.001 0.04 ± 0.001102F51 0.08 ± 0.001 0.16 ± 0.0043G4bl6 0.07 ± 0.002 0.05 ± 0.00296B9 0.02 ± 0.001 0.07 ± 0.003

strongly rejected the hydrophobic surfaces, although its bindingto hydrophilic surfaces was not exceptional. In contrast, theadsorption of strain 102F51 to hydrophilic surfaces was pro-nounced, but its adsorption to hydrophilic surfaces was notunusual.

DISCUSSION

The present study revealed pronounced differences in adsorp-tion of various Rhizobium strains to soybean and cowpea roots.With the possible exception of strain 102F5 1, adsorption duringa 120-min incubation period appeared to be independent of thespecies of plant that served as substrate. Adsorption also wasunrelated to the ability of the bacteria to infect and nodulate.Comparatively large numbers of noninfective R. lupini 96B9bind to roots of soybean and cowpea, and comparatively smallnumbers of infective Rhizobium sp. 3G4bl6 bind to roots ofboth plants. Strain 229 infects and nodulates cowpea but notsoybean, yet it adsorbs in similar numbers to roots of bothspecies.

All of my observations are based on short-term experimentswith a single cultivar of each host species. Thus, the conclusionsmade here may require modification in light of additional exper-iments. The results nevertheless are consistent with the data ofBroughton et al. (7) and Chen and Phillips (8) and stand in sharpcontrast to earlier reports of strong correlations between theadsorption of rhizobia to host roots and the ability ofthe rhizobiato infect and nodulate (10, 11, 15, 16, 22, 23). These correlationsare most extensive with the fast-growing rhizobia, a group oforganisms that differs substantially from the slow-growing strainsthat infect soybean and cowpea (26). Slow-growing rhizobianevertheless have been reported to bind selectively to roots ofsoybean and the closely related wild species, Glycine soja. Staceyet al. (22), for example, observed adsorbed R. japonicum cells onG. soja roots, but could not detect any bound cells of R. meliloti102F5 1 and three strains of R. lupini. The incubation period inthese experiments varied from 1 h to 4 d. Both of these Rhizo-bium species adsorb to soybean roots, and they do so rapidly andfrom a comparatively dilute inoculum (104 cells/ml in this studyversus 5 x 108 cells/ml earlier). Apart from possible differencesin the behavior of G. soja and soybean, differences in the-assaysare the most likely explanation for the discrepancy between thisand the earlier study. Stacey et al. (22) rinsed the plant roots in20 ml of plant nutrient solution and then used light and scanningelectron microscopy to examine elongated root hairs and epider-mal cells of unspecified regions of the root. The capacities ofsuch plant cells to be infected were not determined. In the presentassay, adsorbed bacteria were rinsed vigorously in a flowingstream of nitrogen-free solution. In addition, observations wererestricted to the region of the root containing cells competent tobe infected, i.e. the zone of no and emerging root hairs (4, 6,19). Although adsorption events in this zone are more likely tobe relevant to infection, the present assay does not permit iden-

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tification of the individual plant cells that serve as bindingsubstrates for rhizobia. Infective rhizobia that bind under theconditions of the adsorption assay, however, are competent tonodulate.Law et al. (17) recently reported that excised 1-cm segments

of soybean roots adsorb about 103 cells of R. japonicum 138 in1 h from an inoculum containing I05 bacterial/ml. Although therinsing procedure used in my experiments appears to be more

vigorous than that used by Law et al. (17), our results are insubstantial agreement. The present data also corrobate and ex-

tend a preliminary communication in which Bauer (1) reportedthat substantial numbers of nonnodulating rhizobia bound tosoybean roots.The rate of nodule initiation in cowpea and soybean is stim-

ulated by pretreatment of rhizobia with nutrient solutions pre-

viously used for hydroponic growth of host roots (2, 3, 14). Theeffect of these solutions on nodulation is thought to reflectchanges in the behavior of rhizobia before infection, and thus itwas of interest to determine if culture of rhizobia in similarsolutions enhanced bacterial adsorption. This clearly is not thecase in the interaction of both R. lupini 96B9 and R. japonicum138 with soybean. Growth of the rhizobia in the presence ofroots in fact reduces the numbers of bacteria that adsorb after120 min. Consequently, it seems unlikely that the effect of root-bathing solutions on nodulation is mediated by altered bacterialadsorption.

Relatively high concentrations of exogenously supplied SBLfailed to influence initial adsorption rates of strains 138 and 96B9to soybean, but they reduced bacterial adsorption after 120 min.

This nonspecific effect is difficult to explain, given that SBLbinds to cells of strain 138, but not to those of strain 96B9 (5).The lectin concentration used in the assays is theoretically satu-rating with respect to binding sites on the surfaces of 138 cells(5), and SBL-treated 138 cells would be expected to have alteredsurface properties (24). One possible explanation for the nonspe-

cific lectin effect is that the capacity of roots to adsorb rhizobiais nonspecifically reduced by exposure to SBL.There is substantial controversy about whether specific or

nonspecific mechanisms account for bacterial adsorption to plantsurfaces(10, 20). Although R. lupini 96B9 is unique in exhibitingboth a pronounced rejection of a model hydrophobic surfaceand the greatest capacity to bind to roots, there is no simplecorrelation between the abilities of the strains to bind to hydro-phobic and hydrophilic surfaces and their adsorption to roots.Over a wide range of inoculum densities, the ratio of adsorbedto unadsorbed cells ofstrain 138 is relatively constant. The failureto achieve saturation of binding sites, which also occurs in theinteraction of bacteria with leaf surfaces (13, 18), is consistentwith the postulated nonspecific nature of adsorption. In contrastto other root-bacterium interfaces (21), and to the interaction ofbacteria with plastic (12), adsorption of strain 138 to soybeanroots is markedly temperature-sensitive. The particularly sharpreduction in adsorption at4°C implies that bacterial and plantmetabolism may contribute to the process (12). Thus, the ad-sorption of rhizobia to infectible regions of soybean and cowpea

roots appears to be a complex, largely nonspecific phenomenon.

Acknowledgments-I would like to acknowledge the technical assistance pro-

vided by Ulla Benny and the valuable suggestions made by Dan Kluepfel. I thankFrank Dazzo and Gary Stacey for review of the manuscript.

LITERATURE CITED

1. BAUER WD 1982 Attachment of rhizobia to soybean roots. Plant Physiol 69:S-143

2. BHAGWAT AA, J THOMAS 1982 Legume-Rhizobium interactions: cowpea rootexudate elicits faster nodulation response by Rhizobium species. Appl Envi-ron Microbiol 43: 800-805

3. BHAGWAT AA, J THOMAS 1983 Legume-Rhizobium interactions: role of cow-pea root exudate in polysaccharide synthesis and infectivity of Rhizobiumspecies. Arch Microbiol 136: 102-105.

4. BHUVANESWARI TV, AA BHAGWAT, WD BAUER 1981 Transient susceptibilityof root cells in four common legumes to nodulation by rhizobia. PlantPhysiol 68: 1144-1149

5. BHUVANESWARI TV, SG PUEPPKE, WD BAUER 1977 Role of lectins in plant-microorganism interactions. I. Binding of soybean lectin to rhizobia. PlantPhysiol 60: 486-491

6. BHUVANESWARI TV, BG TURGEON, WD BAUER 1980 Early events in theinfection of soybean (Glycine max L. Merr.) by Rhizobium japonicum. I.Localization of infectible root cells. Plant Physiol 66: 1027-1031

7. BROUGHTON WJ, AWSM VANEGERAAT, TA LIE 1980 Dynamics of Rhizobiumcompetition for nodulation of Pisum sativum cv Afghanistan. Can J Micro-biol 26: 562-565

8. CHEN AT, DA PHILLIPS 1976 Attachment of Rhizobium to legume roots asthe basis for specific interactions. Physiol Plant 38: 83-88

9. CONN HJ 1940 Biological Stains. Biotech Publ, Geneva, NY10. DAzzo FB 1980 Adsorption of microorganisms to roots and other plant

surfaces. InG Bitton, KC Marshall, eds. Adsorption of Microorganisms toSurfaces. John Wiley & Sons, New York, pp 253-316

1 1. DAzzo FB, CA NAPOLI, DH HUBBELL 1976 Adsorption of bacteria to roots asrelated to host specificity in the Rhizobium-clover symbiosis. Appl EnvironMicrobiol 32: 166-171

12. FLETCHER M 1977 The effects of culture concentration and age, time, andtemperature on bacterial attachment to polystyrene. Can J Microbiol 23: 1-6

13. HAAS JH, J ROTEM 1976 Pseudomonas lachrymans adsorption, survival, andinfectivity following precision inoculation ofleaves. Phytopathology 66: 992-997

14. HALVERSON U, G STACEY 1984 Host recognition in the Rhizobium-soybeansymbiosis: detection of a protein factor in soybean root exudate which isinvolved in the nodulation process. Plant Physiol 74: 84-89

15. JANSEN VAN RENSBURG H, BW STRIJDOM 1982 Root surface association inrelation to nodulation of Medicago sativa. Appl Environ Microbiol 44: 93-97

16. KATO G, Y MARUYAMA, M NAKAMURA 1980 Role of bacterial polysaccharidesin the adsorption process of the Rhizobium-pea symbiosis. Agric Biol Chem44: 2843-2855

17. LAW IJ, Y YAMAMOTO, AJ MORT, WD BAUER 1982 Nodulation of soybeanby Rhizobium japonicum mutants with altered capsule synthesis. Planta 154:100-109

18. LEBEN C, RE WHITMOYER 1979 Adherence of bacteria to leaves. Can JMicrobiol 25: 896-901

19. PUEPPKE SG 1983 Rhizobium infection threads in root hairs of Glycine max(L.)Merr., Glycine soja Sieb. & Zucc., and Vigna unguiculata (L.)Walp. CanJ Microbiol 29: 69-76

20. PUEPPKE SG 1984 Adsorption of bacteria to plant surfaces. In T Kosuge, EWNester, eds, Plant Microbe Interactions. Molecular and Genetic Perspectives.Macmillan Publishing Co., New York. In press

21. SHIMSHICKEJ, RR HEBERT 1979 Binding characteristics of N2fixing bacteriato cereal roots. Appl Environ Microbiol 38: 447-453

22. STACEY G, AS PAAU, WJ BRILL 1980 Host recognition in the Rhizobium-soybean symbiosis. Plant Physiol 66: 609414

23. STACEY G, AS PAAU, KD NOEL, RJ MAIER, LE SILVER, WJ BRILL 1982Mutants of Rhizobium japonicum defective in nodulation. Arch Microbiol132: 219-224

24. TsIEN HC, EL SCHMIDT 1981 Localization and partial characterization ofsoybean lectin-binding polysaccharide of Rhizobium japonicum. J Bacteriol145: 1063-1074

25. TURGEON BG, WD BAUER 1982 Early events in the infection of soybean byRhizobium japonicum. Time course and cytology of the initial infectionprocess. Can J Bot 60: 152-161

26. VINCENT JM 1970 A Manual for the Practical Study of Root-Nodule Bacteria.Blackwell Scientific Publ, Oxford

27. ZURKOWSKI W 1980 Specific adsorption of bacteria to clover root hairs, relatedto the presence of the plasmid pWZ2 in cells ofRhizobium trifolii. Microbios27: 27-32

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