Increased metabolic potential of Rhizobium spp.is associated with bacterial competitiveness

Jerzy Wielbo, Monika Marek-Kozaczuk, Agnieszka Kubik-Komar, andAnna Skorupska

Abstract: Of 105 rhizobial isolates obtained from nodules of commonly cultivated legumes, we selected 19 strains on thebasis of a high rate of symbiotic plant growth promotion. Individual strains within the species Rhizobium leguminosarumbv. trifolii, R. leguminosarum bv. viciae, and Rhizobium etli displayed variation not only in plasmid sizes and numbers butalso in the chromosomal 16S–23S internal transcribed spacer. The strains were tagged with gusA gene and their competi-tiveness was examined in relation to an indigenous population of rhizobia under greenhouse conditions. A group of 9strains was thus isolated that were competitive in relation to native rhizobia in pot experiments. Nineteen selected competi-tive and uncompetitive strains were examined with respect to their ability to utilize various carbon and energy sources bymeans of commercial Biolog GN2 microplate test. The ability of the selected strains to metabolize a wide range of nu-trients differed markedly and the competitive strains were able to utilize more carbon and energy sources than uncompeti-tive ones. A major difference concerned the utilization of amino and organic acids, which were metabolized by most ofthe competitive and only a few uncompetitive strains, whereas sugars and their derivatives were commonly utilized byboth groups of strains. A statistically significant correlation between the ability to metabolize a broad range of substratesand nodulation competitiveness was found, indicating that metabolic properties may be an essential trait in determiningthe competitiveness of rhizobia.

Key words: competitiveness, metabolic activity, plasmid diversity, internal transcribed spacer diversity.

Resume : Nous avons selectionne a partir de 105 isolats de rhizobiens obtenus de nodules de legumineuses cultivees cou-ramment, 19 souches stimulant la croissance vegetale symbiotique. Les souches individuelles appartenant aux especes Rhi-zobium leguminosarum bv. trifolii, R. leguminosarum bv. viciae, et Rhizobium etli presentaient des variations nonseulement dans la taille et le nombre des plasmides, mais aussi dans la taille de l’espaceur interne chromosomique transcrit16S–23S. Les souches ont ete etiquetees avec le gene gusA et leur competitivite a ete examinee par rapport a une popula-tion indigene de rhizobiums en conditions de serre. Un groupe de 9 souches qui etaient competitives par rapport aux rhizo-biums sauvages lors d’experiences en pot a ete isole. Dix-neuf souches competitives et non competitives selectionnees ontete examinees relativement a leur capacite d’utiliser differentes sources de carbone et d’energie, a l’aide de la trousse com-merciale Biolog GN2 en microplaques. La capacite des souches selectionnees a metaboliser un large eventail de nutrimentsdifferait de facon marquee et les souches competitives etaient capables d’utiliser un plus grand nombre de sources de car-bone et d’energie que les souches non competitives. Les differences majeures impliquaient l’utilisation d’acides organiqueset d’acides amines qui etaient metabolises par la plupart des souches competitives et par quelques souches non competiti-ves, alors que les sucres et leurs derives etaient utilises couramment par les deux types de souches. Une correlation statisti-quement significative entre la capacite a metaboliser un large spectre de substrats et la competitivite pour la nodulation aete trouvee, indiquant que les proprietes metaboliques peuvent etre un trait essentiel dans la determination de la competiti-vite des rhizobiums.

Mots-cles : competitivite, activite metabolique, diversite plasmidique, diversite de l’espaceur interne transcrit.

[Traduit par la Redaction]

Introduction

Legume plants possess a unique ability to establish sym-bioses with nitrogen-fixing bacteria of the family Rhizobia-ceae. These bacteria belonging to the genera Rhizobium,Bradyrhizobium, Allorhizobium, Azorhizobium, Sinorhi-

zobium, and Mesorhizobium (Young et al. 2001; Martinez-Romero 2003; Willems 2006), which are collectively re-ferred to as rhizobia, are able to form nodules on their hostplants inside of which they fix nitrogen. Bacteria–plant in-teraction resulting in nodule formation is a multi-step andcomplex process, depending not only on the symbiotic part-

Received 14 December 2006. Revision received 19 April 2007. Accepted 19 April 2007. Published on the NRC Research Press Web siteat cjm.nrc.ca on 28 August 2007.

J. Wielbo, M. Marek-Kozaczuk, and A. Skorupska.1 Department of General Microbiology, Institute of Microbiology andBiotechnology, University of M. Curie-Skłodowska, Akademicka 19, 20-033 Lublin, Poland.A. Kubik-Komar. Department of Applied Mathematics, University of Agriculture, Akademicka 13, 20-950 Lublin, Poland.

1Corresponding author (e-mail: genet@biotop.umcs.lublin.pl).

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ners but also on several environmental factors (reviewed byZhang and Smith 2002). Many plant and bacterial signalingmolecules are involved in the attachment of bacteria to roothairs, colonization of plant tissues, and establishment of ni-trogen-fixing symbiosis. Successive steps of the nodulationprocess are well characterized and have been extensively re-viewed (Perret et al. 2000; Spaink 2000). Considerably lessis known about how individual strains out compete otherstrains in the rhizosphere to establish the symbiotic interac-tion (Triplett and Sadowsky 1992; Toro 1996).

The competitive properties of rhizobia depend on bacte-rial survival in the soil and the rhizosphere, microbial rela-tions with other species present in the environment, theability to utilize nutrients, and several other factors thatcharacterize the soil (Triplett and Sadowsky 1992; VandeBroek and Vanderleyden 1995; Mercado-Blanco and Toro1996; Toro 1996). All these factors affect the effectivenessof nodulation and nitrogen fixation and for years havebecome a major drawback limiting the commercial use ofrhizobial biofertilizers (Streeter 1994; Toro 1996). To over-come this problem, numerous solutions are being examined,such as engineering of strains, enhancing bacterial-host sig-naling (Zhang and Smith 2002), constructing strains highlycompetitive for nodulation (van Dillewijn et al. 2001), orthe use of strains with an altered host range or legume gen-otype (Toro 1996).

Rhizobial populations in the soil are highly diverse at theplasmid and chromosomal level (Laguerre et al. 1993;Bottomley et al. 1994; Hartmann et al. 1998; Rigottier-Goiset al. 1998; Sterner and Parker 1999; Wang et al. 1999;Palmer and Young 2000). A suggestion that plants mayhave the potential to choose from among the functionally di-verse bacteria and in this manner enhance their competitionabilities has also been made (Simms and Taylor 2002). Evenif this were not the case (West et al. 2002), however, it iswell known that plants significantly modify their rhizo-sphere by exuding large quantities of sugars, acids, andamino acids (Gaworzewska and Carlile 1982), thus affectingthe growth of rhizosphere-colonizing bacteria (Jaeger et al.1999). Evidence also exists that rhizosphere microorganismsenhance exudation of amino acids from alfalfa roots and inthis manner can increase their access to plant carbon resour-ces (Lodwig et al. 2003; Phillips et al. 2004).

A lot of data is available concerning the effect of single, de-fined factors affecting the competitive properties of rhizobia,such as the presence of a small cryptic plasmids (Bromfield etal. 1985), the production of vitamins (Streit et al. 1996), bac-teriocins (Robleto et al. 1998; Oresnik et al. 1999), or rhizo-pins (Murphy et al. 1987). Utilization of some carbon andenergy sources, such as homoserine and rhamnose, and theireffect on rhizobial competition have been demonstrated(Hynes and O’Connel 1990; Oresnik et al. 1998).

In this paper, we describe the isolation of Rhizobium legu-minosarum bv. viciae, R. leguminosarum bv. trifolii, andRhizobium etli strains from commonly cultivated legumes,and characterization of their symbiotic activity, genetic di-versity, and competitiveness. The main objective of thisstudy was to investigate the relationship between metabolicprofiles of the rhizobial isolates and their ability to competewith indigenous bacterial populations. The capability of rhi-zobia to metabolize various carbon and energy sources was

examined by using a commercial broad-substrate rangeBiolog GN2 MicroPlateTM test. We showed that competitiverhizobial strains are capable of using more carbon and en-ergy sources, especially organic acids and amino acids, thanuncompetitive strains.

Materials and methods

Bacterial strains and plant cultivarsThe collection of 105 rhizobial isolates was obtained from

nodules of plants growing on different types of cultivatedsoils in the region of Lublin, Poland. The rhizobia wereisolated from nodules of red clover (Trifolium pratense L.‘Dajana’), pea (Pisum sativum L. ‘Ramrod’), bean (Pha-seolus vulgaris L. ‘Zlota Saxa’), field pea (Pisum arvenseL. ‘Eureka’), and tick bean (Vicia faba minor L. ‘Titus’). Intotal, 35 strains of R. leguminosarum bv. trifolii were col-lected from red clover nodules, 46 of R. leguminosarum bv.viciae strains from pea, field pea, and tick bean, and 24R. etli strains from bean nodules. After picking from roots,the nodules were surface-sterilized, crushed, and the contentwas plated on 79CA medium (Vincent 1970). Strains iso-lated from the nodules were purified by successive isolationof single colonies and clones were assayed for symbiotic ac-tivity in pot experiments.

Plant growth conditions in pot experimentsPlants were grown in plastic pots filled with 400 g (red

clover) or 600 g (pea, field pea, bean, and tick bean) ofwashed and autoclaved sand. Surface-sterilized seeds weresown in the pots and inoculated with 3 mL bacterial suspen-sion (OD550 0.1) per pot. Plants (10 seedlings of red cloverper pot, in triplicate, and in the case of other plants, 3 seed-lings per pot, in triplicate) were grown in a greenhouseunder natural light supplemented with artificial light(14 h day : 10 h night, at 24 8C : 19 8C). Plants were sup-plemented once with a sterile liquid N-free medium (100 or150 mL medium per 400 and 600 g pots, respectively) con-taining 180 mg�L–1 KH2PO4, 74 mg�L–1 Na2HPO4�2H2O,120 mg�L–1 MgSO4, 75 mg�L–1 CaCl2, 10 mg�L–1 ferric cit-rate, 1 mL�L–1 of microelement stock solution (2.86 gH3BO4, 2.03 g MnSO4�4H2O, 0.22 g ZnSO4�7H2O, 0.08 gCuSO4�5H2O, and 0.14 g Na2MoO4�2H2O suspended in 1 Lof water), pH 7.0 (Vincent 1970). Following this treatment,the plants were periodically watered with sterile water. After6 weeks, they were harvested, weighed, and root noduleswere counted. The 19 most efficient growth-promoting iso-lates (as assayed by weighing the green parts of plants)were chosen for further study (Table 1). The experiment inwhich 105 isolates were screened for their symbiotic proper-ties was not repeated.

Tagging of rhizobiaTo study nodule occupancy, rhizobial strains were tagged

by a stable plasmid vector pJBA21Tc carrying a constitu-tively expressed gusA gene (Wielbo and Skorupska 2001).The pJBA21Tc plasmid was introduced into bacteria byelectroporation. To 90 mL (about 5 � 108) of electrocompe-tent cells (Garg et al. 1999), 5 mL (about 50 ng) of plasmidDNA was added and incubated 30 min on ice. Followingthis, the bacterial cells were electroporated (at 1800 V,

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200 O, 50 mF), and then incubated for 24 h in TY (tryptone –yeast extract) liquid medium (Sambrook et al. 1989) at28 8C, and plated on 79CA medium containing 10 mg�mL–1

tetracycline. Stability of plasmid pJBA21Tc in rhizobia wasexamined as described previously (Wielbo and Skorupska2001).

Plant competition assayPlants were grown in pots filled with soil (N:P:K was

157:14:13 mg�(100 g dried soil)–1). Twenty red clover plantsper pot with 1500 g of soil and 5 pea, field pea, bean, andtick bean plants per pot with 3000 g of soil were grown. Non-sterilized seeds were soaked for 15 min in a suspension ofbacterial cells (OD550 0.1, approx. 108 cells�mL–1) and thenwere sown in the pots. The plants were grown under green-house conditions in natural light supplemented with artificiallight (14 h day : 10 h night, at 24 8C : 19 8C) and were peri-odically watered. After 6 weeks, plant roots were harvestedand stained for b-glucuronidase (GUS) activity. Whole rootsystem of clover plants (40 plants per one bacterial strain)was stained. In the case of other plants, nodules were re-moved from roots (10 plants per strain) and then stained forGUS activity in 50 mmol�L–1 sodium phosphate buffer(pH 7.2) with 50 mg�mL–1 5-bromo-4-chloro-3-indolyl b-D-glucuronide (X-Glc), 0.1 mmol�L–1 EDTA, 0.38 mmol�L–1

K3Fe(CN)6, and 0.38 mmol�L–1 K4Fe(CN)6 for about 1 h atroom temperature (Wilson et al. 1995). Competitive potentialof the tested strains was estimated by comparing the numberof blue nodules colonized by the introduced gusA-taggedstrains with white nodules colonized by native strains.

Plasmid profile and PCR fingerprintingPlasmid content of the isolates was analyzed by method

of Eckhardt (1978) and compared with standard plasmids ofR. leguminosarum bv. viciae 3841 (Young et al. 2006). Cellswere grown overnight at 28 8C in 79CA medium, 1.5 mL ofculture was centrifuged 10 min at 10 000g, and the pelletwas resuspended in 0.5 mL water. Next, 1 mL 0.3% (m/v)sarcosyl was added and cell suspension was centrifuged10 min at 10 000g. The pellet was resuspeded in 40 mL20% Ficoll in TE buffer (10 mmol�L–1 Tris–HCl, 1 mmol�L–1

EDTA, pH 8.0) and incubated for 15 min on ice. Then, 10 mLlysing solution in TE buffer containing RNase A (0.4 mg�mL–1),bromophenol blue (1 mg�mL–1), and 1.5 mg�mL–1 lysosyme(freshly prepared aqueous solution) was added. The mix-ture (35 mL) was loaded into wells of 0.7% agarose geland separated for 16 h at 7 V�cm–1.

Isolates were further characterized by PCR-RFLP of16S–23S rDNA internal transcribed spacer. For PCR ampli-fication, the following primers were used: FGPS1490 (5’-TGCGGCTGGATCACCTCCTT-3’) and FGPL132 (5’-CCGGGTTTCCCCATTCGG-3’). After primary denatura-tion for 5 min at 95 8C, 35 cycles under the following con-ditions were carried out: denaturation, 1 min at 94 8C;annealing, 1 min at 55 8C; and elongation, 2 min at 72 8C(Laguerre et al. 1996). PCR products (approx. 0.2 mg) weredigested for 1 h at 37 8C or 65 8C with 2 U of BsuRI(HaeIII) and TaqI restriction enzymes (FERMENTAS), re-spectively, in 15 mL of appropriate buffers supplemented bythe manufacturer. Restriction fragments were separated by3% agarose gel electrophoresis.

Table 1. Symbiotic properties of selected field isolates of Rhizobium.

Strain Host plantFresh mass ofshoots (g)*

Relative fresh mass ofshoots (uninoculatedcontrol plants = 1)

No. of nodules/plant*

Rhizobium leguminosarum bv. trifoliiKO17 Red clover 0.19±0.05 2.86 21±11KO18 Red clover 0.20±0.07 3.06 12±5KO24 Red clover 0.22±0.08 3.45 17±7KO25 Red clover 0.21±0.07 3.19 15±6KO27 Red clover 0.18±0.05 2.81 18±8

Rhizobium leguminosarum bv. viciaeBO03 Tick bean 6.35±1.81 1.35 25±10BO07 Tick bean 5.98±2.12 1.27 20±9BO14 Tick bean 6.21±1.86 1.32 21±9PE07 Field pea 2.17±0.42 1.15 40±16PE08 Field pea 2.19±0.61 1.16 33±17PE13 Field pea 2.17±0.33 1.15 21±7PE15 Field pea 2.24±0.59 1.18 19±9GR08 Pea 3.15±0.94 1.25 39±10GR09 Pea 3.13±1.09 1.25 31±9

Rhizobium etliFA02 Bean 3.00±0.85 1.51 32±13FA03 Bean 3.52±1.98 1.77 33±24FA21 Bean 3.02±0.70 1.52 33±8FA22 Bean 2.98±0.97 1.5 23±8FA23 Bean 3.06±1.03 1.54 19±7

*Values are mean ± standard deviation of 9 plants (tick bean, field pea, pea, and bean) and of 30red clover plants.

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Physiological testsUtilization of different carbon and energy sources by Rhi-

zobium isolates was assessed using GN2 MicroPlateTM

(Gram Negative Identification Test Panel) (Biolog, Hay-ward, California, USA) containing 95 carbon sources includ-ing sugars, amino acids, and organic acids. Bacterial cellswere grown overnight at 28 8C on 79CA agar medium, col-lected in sterile water and twice washed in water. After that,the pellet was diluted in water to an optical density at550 nm (OD550) of 0.1 (approx. 108 cells�mL–1) and 150 mLsuspension of rhizobia was added to each well in the Biologmicroplate. The cells were incubated for 24 h at 28 8C andthe results were recorded using Benchmark PlusTM micro-plate reader (Bio-Rad Laboratories, USA).

Data analysisPlasmid size estimation was done with BIO-PROFIL

BioGene Windows Application V11.01 (Vilber-Lourmat,France). For numerical analysis of PCR–RFLP results,RFLP profiles of strains were coded in the binary system.Then, similarity rates of rhizobia were calculated by SimpleMatching coefficient, and their clustering was performed bythe UPGMA method with the software NTSYS softwarepackage. Similarly, the Biolog test results were coded andcluster analysis of metabolic properties of rhizobial strainswas performed. The studied feature in statistical analyseswas the ability of strains to metabolize diverse carbon sour-ces, which were numerically represented by mean number ofsubstrates metabolized by bacteria.

The connection between competitive abilities and thenumber of the used substrates was checked by the nonpara-metric median test, based on a chi-square value, comparing2 independent samples composed of competitive and un-competitive strains. The low number of observations causedapplication of Yates’ correction for the obtained value of thetest function (Ferguson and Takane 1989). Principal compo-nent analysis (PCA) was used to investigate the ability toutilize particular substrates or their groups in modeling theprincipal components describing metabolic properties of thecompetitive and uncompetitive strains. The number of ob-tained principal components was reduced on the base ofKaiser criterion (Kaiser 1960). The varimax rotation wasthen applied to obtain a clear pattern of loadings (Morrison1990). Particular cases (strains) were plotted in the spacegenerated by the factor axes to classify them into categoriesaccording to their position in the coordinate system.Described analyses were performed with STATISTICA soft-ware.

Results

Symbiotic properties of Rhizobium isolatesIn the first step of selection, 35 field isolates of

R. leguminosarum bv. trifolii, 46 isolates ofR. leguminosarum bv. viciae, and 24 isolates of R. etli werescreened for their symbiotic efficiency on specific hostplants by estimating the mass of green parts of plants in potexperiments (data not shown). For selection of isolates forfurther studies, we chose shoot mass as the sole parameteras it is positively correlated with efficiency of nitrogen fixa-tion and is also a critical criterion in the case of various

agronomical important plants such as clover, vetch, or tickbean. Most isolates promoted plant growth, but the degreeof growth stimulation varied to a substantial extent. Thegreen mass of clover plants inoculated with various isolateswas 1.6–3.46 times higher than that of uninoculated controlplants. In the case of pea, field pea, bean, and tick beanplants, the rate of growth promotion was 0.89–1.25, 0.96–

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Fig. 1. (A) Plasmid profiles of rhizobial isolates obtained by Ec-khardt’s method. (B) Schematic representation of the plasmid pro-files. Lanes 1–5, Rhizobium leguminosarum bv. trifolii strainsKO17, KO18, KO24, KO25, KO27, respectively; lanes 6–12,R. leguminosarum bv. viciae strains GR08, GR09, PE08, PE13,BO03, BO07, and BO14; lanes 13–17, R. etli strains FA02, FA03,FA21, FA22, and FA23, respectively; lane M, plasmids ofR. leguminosarum bv. viciae 3841 used as molecular mass standard.Lowercase letters (a–f) indicate individual plasmids in strains.

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1.2, 0.92–1.58, and 0.99–1.45, respectively. Of 105 isolates,19 strains were chosen on the basis of their ability to pro-mote plant growth in the most pronounced manner (Table 1).The selected isolates seemed to be similar with respect totheir symbiotic efficiency, as assessed by comparing greenmass of inoculated plants, but other parameters such as thenumber of nodules induced on host roots were extremelyvariable within a group (a group is defined as comprisingisolates from a specific plant host) (Table 1).

Genetic diversity of the isolatesGenetic diversity among the 19 selected isolates was in-

vestigated by examining the plasmid content and PCR–RFLP polymorphism of 16S–23S rDNA internal transcribedspacer (ITS) region. The plasmid profile of each isolate wasassessed using Eckhardt gel electrophoresis (Eckhardt 1978).Plasmid profiles were obtained for 17 of the 19 isolates; theplasmid patterns could not be resolved for isolates PE07 andPE15. The analysis revealed that 17 isolates differed in plas-mid number, size, and the amount of extrachromosomalDNA (Fig. 1; Table 2). Each of the 17 isolates contained be-tween 2 and 6 distinct plasmid bands and there were 17 dif-ferent plasmid profiles. Despite substantial plasmid contentdifferences among R. leguminosarum bv. viciae GR08,GR09, PE07, PE08, PE13, PE15, BO03, BO07, and BO14strains, they infected the plants from which they were iso-lated and other ones from the same cross-inoculation group.Host specificity nodulation assays were performed underlaboratory conditions and confirmed the assignment of these

isolates to the same cross-inoculation group (data notshown).

Chromosomal diversity of 19 isolates was investigated byRFLP analysis of PCR-amplified 16S–23S rDNA ITS regionwith BsuRI (HaeIII) and TaqI restriction enzymes (Figs. 2Aand 2B). Both enzymes revealed high and comparable dis-crimination level of the selected strains (Table 2). InR. leguminosarum bv. trifolii, 3 distinct TaqI and BsuRI(HaeIII) restriction patterns were obtained (I, II, III). InR. leguminosarum bv. viciae 7 (III, IV, V, VI, VII, VIII,IX) and in R. etli 4 (IV, VII, IX, X) restriction patternswere observed. The most common restriction pattern (III)was found in both biovars of R. leguminosarum. Restrictionpatterns IV, VII, and IX were identified in bothR. leguminosarum bv. viciae and R. etli.

On the basis of RFLP pattern similarities, the selected rhi-zobia were grouped by UPGMA cluster analysis. Twogroups of bacteria were distinguished at a similarity coeffi-cient of 55% (Fig. 2C): 4 strains were grouped into onecluster, with 15 comprising the second. The similarity levelbetween strains allocated to ITS groups II, IV, and III; IXwas 91%.

Of the 2 methods used to assess genetic diversity, plasmidprofiles revealed the greatest level of variation between iso-lates. On the basis of PCR–RFLP analysis, the 19 isolatescan be grouped into 10 different genotypes (I–X). However,each isolate possessed different plasmid pattern, indicating ahigh level of plasticity within the plasmid content ofR. leguminosarum and R. etli field isolates.

Competitiveness of the selected isolatesMarker gene gusA, carried on pJBA21Tc plasmid, which

is stably maintained in rhizobial strains (Wielbo and Skorup-ska 2001), was introduced into the 19 selected isolates byelectroporation. The competitiveness of gusA-tagged isolatesin relation to native soil rhizobia was assessed in pots undergreenhouse conditions. Host plants specific for particularmicrosymbionts from the same cross-nodulation group wereused (e.g., the PE isolates were used for inoculation of fieldpea or BO isolates for inoculation of tick bean plants). Ofthe 19 selected strains, 10 appeared to be uncompetitive inrelation to native rhizobia. Those isolates were not able tooccupy nodules at all or colonized less than 1% of nodulesin a whole root system. On the other hand, competitivestrains were found in each group of isolates (i.e., inR. leguminosarum bv. trifolii strains KO17, KO18, andKO25; in R. leguminosarum bv. viciae strains BO03, BO07,and GR09; and in R. etli strains FA02, FA21, and FA23).Among strains selected as competitive, the ability of noduleoccupation varied pronouncedly; for example, BO07 straincolonized 7% nodules and KO25 strain up to 42% of nod-ules (Table 3). It was established that stability of pJBA21Tcplasmid in the competitive and uncompetitive strains wascomparable and in all cases more than 97% cells expressedGUS activity.

Utilization of carbon and energy sourcesThe capability of the selected strains to use diverse car-

bon and energy sources was examined by using commercialBiolog GN2 MicroPlateTM test. This approach revealed thatmetabolic profiles of the selected isolates varied to great

Table 2. Plasmid content and chromosomal diversity of selectedfield isolates of Rhizobium.

StrainNo. ofplasmids

TotalplasmidDNA (kb)

BsuRIrestrictionpattern

TaqIrestrictionpattern

ITStype

R. leguminosarum bv. trifoliiKO17 5 2746 A A IKO18 5 2982 B B IIKO24 4 2113 C C IIIKO25 4 2352 C C IIIKO27 3 2134 C C III

R. leguminosarum bv. viciaeBO03 3 2058 B D IVBO07 4 1869 D E VBO14 4 2316 E F VIPE07 n.o. n.o. F G VIIPE08 4 2226 G H VIIIPE13 4 2559 C C IIIPE15 n.o. n.o. C C IIIGR08 4 2867 C I IXGR09 3 2472 B D IV

R. etliFA02 4 1870 F G VIIFA03 5 1853 H J XFA21 5 2459 B D IVFA22 4 2494 C I IXFA23 6 3034 B D IV

Note: n.o., not obtained; ITS, internal transcribed spacer.

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Fig. 2. PCR–RFLP analysis of 16S–23S rDNA internal transcribed spacer (ITS) region of Rhizobium leguminosarum bv. trifolii,R. leguminosarum bv. viciae, and Rhizobium etli isolates. PCR amplification products were digested with (A) BsuRI (HaeIII) and with (B)TaqI. Lane 1, molecular size standard (Mass Ruler DNA Ladder Mix, Fermentas); lanes 2–6, R. leguminosarum bv. trifolii strains KO17,KO18, KO24, KO25, and KO27, respectively; lanes 7–15, R. leguminosarum bv. viciae strains PE15, PE13, PE07, PE08, BO07, BO14,BO03, GR08, and GR09, respectively; lanes 16–20, R. etli strains FA21, FA23, FA22, FA03, and FA02, respectively. (C) Dendrogram ofgenetic relationships among rhizobial isolates obtained from PCR–RFLP patterns of ITS region and Simple Matching coefficient. Clusteranalysis was performed using UPGMA (Unweighted Pair Group Method with Arithmetic Mean) method.

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extent, even among strains belonging to the same cross-nodulation group. Whereas mono- and oligo-sugars andpolysaccharides were commonly utilized, organic andamino acids were used relatively rarely. The level of varia-tion, with respect to the utilization of diverse substrates,was substantial in all 19 strains. The most diverse groupwas R. leguminosarum bv. viciae in which the number ofutilized substrates varied from 26 for PE08 strain to 71for GR09 strain. Among R. leguminosarum bv. trifolii andR. etli strains the range of utilized substrates varied from50 to 62 and from 47 to 65, respectively (Table 3).

After grouping the strains into the competitive and un-competitive categories, we observed that the isolates charac-terized as competitive were able to utilize from 40 to 71different carbon sources, whereas a number of substrates uti-

lized by uncompetitive bacteria was at the range from 26 to56 (Table 3). Despite the overlapping of these 2 ranges, themedians for these 2 groups were 0.621 and 0.511, respec-tively, and were statistically different at the significancelevel of P = 0.05. These medians differed from the medianfor all 19 strains, as was shown in Table 4.

On the basis of the Biolog test results, the selected rhizo-bia were grouped by UPGMA cluster analysis (Fig. 3). Ma-jority of competitive strains were located in 2 branches ofthe tree, named group 1 and group 2. In both groups, onlysingle uncompetitive strains were found. Similarity level be-tween strains clustered in group 1 and 2 was 88% and 85%,respectively.

The analysis of the metabolic profiles of competitive anduncompetitive strains did not allow us to distinguish the car-bon or energy sources that would characterize a good com-petitor strain. The principal components method applied tothe results of the Biolog test enables us to obtain 2 new fac-tors that explained 82.76% of the total variance. The firstprincipal component can be interpreted as an ability to uti-lize ‘‘easily metabolized’’ substrates such as saccharides and(or) sugars and their derivatives since the values of loadingfor mono-, di-, oligo-, and poly-saccharides as well as sugarderivatives were high and ranged from 0.847 to 0.938. Thesecond principal component can be interpreted as an abilityto utilize the substrates that were more difficult to metabo-lize than previous ones, in other words, organic acids, aminoacids, and their derivatives. The correlation coefficients be-

Table 3. Competition properties and capacity to uti-lize diverse carbon and energy sources by selectedRhizobium strains.

Biolog GN2 microplatetest

Strain

No. ofutilizedsubstrates

No. ofuniquesubstrates*

% of GUS+

nodules{

R. leguminosarum bv. trifoliiKO25 58 1 42KO18 62 3 28KO17 56 0 17KO24 57 3 <1KO27 50 0 <1

R. leguminosarum bv. viciaeBO03 63 5 15GR09 71 6 11BO07 40 1 7BO14 38 1 0PE07 54 0 0PE08 26 0 0PE13 45 0 0PE15 55 1 0GR08 36 0 0

R. etliFA23 63 3 29FA02 65 4 13FA21 59 1 9FA03 53 0 0FA22 47 2 0

*Unique substrate is utilized only by one strain in thegroup; groups are R. leguminosarum bv. trifolii,R. leguminosarum bv. viciae, and R. etli species.

{Competitive strains are defined as those whichshowed >1% nodule occupancy. Values are mean of 10plants for tick bean, field pea, pea, and bean and of 40 redclover plants. GUS+, positive for b-glucuronidase activity.

Table 4. The median test for 2 independent samples deter-mined by competitive and uncompetitive strains.

Median w2 df P-value Corrected w2 P-value

0.568 8.927 1 0.003 6.363 0.012

0.62 0.70 0.79 0.87 0.96

Coefficient of similarity

Strains

KO17*

BO14

PE08

BO07*

KO18*

FA23*

FA21*

GR09*

BO03*

PE15

KO27

PE13

KO25*

KO24

FA03

PE07

FA02*

FA22

GR08

gro

up

1g

rou

p2

Fig. 3. Dendrogram of relationships among rhizobial isolates ob-tained from the Biolog test results and Simple Matching coefficient.Cluster analysis was performed using UPGMA method. *, competi-tive strains.

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tween this factor and these substrates were at the range from0.844 to 0.880.

The values calculated in the PCA method concerning par-ticular strains were plotted in the first 2 principal compo-nents coordinate system (Fig. 4). On the basis of thisscatterplot, one can assume that, with the exception ofPE08, there were more extensive differences of the secondcoordinate values than of the first coordinate values. There-fore, the second principal component could play the role ofthe discriminatory feature for distinguishing competitive anduncompetitive strains. Most of competitive strains had posi-tive values of the second principal component, contrary touncompetitive ones (Fig. 4).

Discussion

Competition for nodulation is a very complex, poorlyunderstood phenomenon, which is dependent on many ge-netic and physiological properties of both partners (for re-view see, Triplett and Sadowsky 1992; Streeter 1994;Vande Broek and Vanderleyden 1995; Toro 1996). Little isknown about the carbon and energy sources used by micro-symbionts during the primary steps of interaction with itshost plant. It has been established that the ability to utilizesuch carbon sources as rhamnose (Hynes and O’Connel1990; Oresnik et al. 1998; Richardson et al. 2004) or galac-tosides (Bringhurst et al. 2001) affects competition proper-ties of rhizobial strains.

The main objective of our work was to assess whether arange of metabolic activities of rhizobial strains is importantfor their competitiveness in the soil. We chose the Biologmethod to examine rhizobial capabilities to metabolize car-bon and energy sources because of simplicity of this testand broad range of compounds that it allows to screen. Ofthe 105 rhizobial isolates obtained from the nodules of com-monly cultivated legumes, we selected 19 strains on the ba-sis of their ability to promote symbiotic plant growth.Individual Rhizobium strains within a species varied widelynot only with respect to plasmid sizes and numbers but also

the chromosome 16S–23S ITS region. The only commonfeature of all 19 strains was a high rate of symbiotic growthpromotion of agronomical important plants. We examinedtheir competitiveness in relation to indigenous population ofrhizobia under greenhouse conditions in an approach involv-ing tagging the strains with marker gene gusA. Of the 19gusA-marked strains belonging to R. leguminosarum bv. tri-folii, R. leguminosarum bv. viciae, and R. etli (formerlyR. leguminosarum bv. phaseoli) we isolated a group of 9strains that were competitive in relation to native rhizobiain pot experiments and were able to colonize some portionof the root systems. All the 19 selected strains, both compet-itive and uncompetitive, were assessed for their ability toutilize various carbon and energy sources. The results indi-cated a statistically significant correlation between the abil-ity to metabolize a broad range of substrates and nodulationcompetitiveness.

Nineteen strains selected from the collection of 105 iso-lates obtained from nodules of plants growing in cultivatedfields displayed high plasmid and chromosomal diversity.We observed 10 different PCR–RFLP profiles of internaltranscribed spacer and extremely variable plasmid profilesamong the selected strains. It is worth noting that all strainsbelonging to the II and IV ITS types are competitive(Fig. 2C). The range of genetic diversity among the selectedstrains could be attributed to the strains’ origin, i.e., arablesoils. There is evidence that soil cultivation results in in-crease in diversity of the indigenous population because ofbroad variation of available substrates in comparison withuncultivated soil (Kennedy and Smith 1995; Wernegreen etal. 1997; Palmer and Young 2000). The comparison of chro-mosomal and plasmid diversity of pea-nodulatingR. leguminosarum bv. viciae in arable and grassland soils re-vealed a more diverse population in the former (Palmer andYoung 2000). On the other hand, there were negative rela-tionships between rhizobial diversity and nitrogen-phosphatelevel. High levels of nutrients may decrease the diversitysimilarly to more intense fertilization (Sprent and Sprent1990). One can conclude that it is not the amount of partic-

GR08

PE07

PE08

PE13

PE15

BO14

KO24

KO27

FA03

FA22

*

** ***

**

*

GR09

KO17

KO18

KO25

FA02

FA21

FA23BO03

BO07

3

2

1

0

-1

-2

-3-4 -2 0 2 4

Fig. 4. The scatterplot of the studied strains in the principal components coordinate system. *, competitive strains; .. uncompetitive strains.

964 Can. J. Microbiol. Vol. 53, 2007

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ular carbon and (or) energy sources, but, rather their array,which endows a microsymbiont with a possibility to adaptto specific conditions in soil environment.

It seems reasonable that utilization of a wide range of car-bon and (or) energy sources creates an advantage by pro-moting bacterial growth and enhancing competitiveness, asopposed to the ability of utilization of only 1 or 2 metabolicsubstrates. Our results imply that strains selected as compet-itive were able to catabolize a large number of various com-pounds (e.g., KO18, BO03, GR09, FA02, or FA23 strains).The mean number of nutrients utilized by competitivestrains was higher than a corresponding value establishedfor uncompetitive strains. However, there were some excep-tions: competitive BO07 strain utilized markedly less nu-trients than uncompetitive strains KO24, PE07, PE15, orFA03; the noncompetitive FA22 strain utilized high numberof substrates; the competitive KO25 strain metabolized anumber of substrates comparable to noncompetitive KO24,KO27, PE13, and PE15 strains.

The ability to utilize various substrates may be linked, tosome extent, with the chromosomal type. All strains in thechromosomal type IV (BO03, GR09, FA21, and FA23) anda very similar strain KO18 of type II were identified ascompetitive. On the other hand, among uncompetitivestrains (KO24, KO25, KO27, PE13, PE15, GR08, andFA22) belonging to type III and very similar type IX, onlythe strain KO25 was competitive (Fig. 2). However, thesmall group of strains examined and their great diversitydid not allow us to find statistically significant correlationbetween chromosomal diversity and the ability to use carbonand (or) energy sources.

The differences in competitive properties between the ex-amined strains cannot be explained solely by the differencesin the ability to utilize diverse carbon and energy sources.Success or failure in nodulation competitiveness may bealso dependent on plant–microsymbiont signal exchange(Zhang and Smith 2002). Considering the very high plasmidand chromosomal diversity of the described strains, it is pos-sible that they possess different sets of nod genes and thatthey produce different spectra of signal chitolipooligosac-charides, which may be more or less suited to the plant host.

One type of compound present in legume exudates areflavonoids, which are responsible for expression of nodula-tion genes and also for early phases of legume–microbe in-teractions such as chemotaxis or growth stimulation. Theseearly functions of several classes of flavonoids may influ-ence the competitiveness of rhizobia (for review see Cooper2004). Rhizobia are capable of metabolizing flavonoids toyield a plethora of phenolic products, some of which possessnod-gene-inducing or -inhibiting properties (Cooper 2004).Flavonoids may be released as aglycones or glycoconjugateswith attached sugars, which are hydrolyzed by rhizobia,other soil microoorganisms, or plant exoenzymes (Maxwelland Phillips 1990).

Also, the ability to catabolize various specific compoundssuch as vitamins, choline, stachydrine, trigonelline, rhizo-pines, and homoserine that are exuded from nodules to pro-vide carbon and nitrogen for rhizobia may influencecompetitiveness of the microsymbionts (Jimenez-Zurdo etal. 1995, 1997; Gordon et al. 1996; Streit et al. 1996;Phillips et al. 1998; Rosenblueth et al. 1998; Fry et al.

2001; Borthakur et al. 2003). However, it is not establishedwhether these compounds, common sugars, and organicacids play an important role in supporting growth of rhizo-bia in the rhizosphere.

In our study, we found that some substrates such as sugarswere commonly utilized by rhizobia, whereas others (i.e.,amino acids or organic acids) were not. Taking into accountthe interpretation of the second principal component in PCAmethod, it may be claimed that the ability to utilize the sub-strates more difficult to metabolize is the discriminatory fea-ture for competitive and uncompetitive bacteria. Years ago,it was demonstrated that amino acids are the major compo-nents of leguminous root exudates and act as strong chemo-atractants for rhizobia (Gaworzewska and Carlile 1982);nowadays, there is evidence that amino acids are importantcompounds in plant–bacteroid exchange of carbon and en-ergy (Lodwig et al. 2003; Phillips et al. 2004). Taken to-gether, we can assume that the ability to uptake and utilizea wide range of amino acids and their derivatives, as well asorganic acids, is advantageous to rhizobia, allowing them togrow in the rhizosphere and colonize plant root hairs.Screening the isolates with the Biolog test is not a time-consuming procedure and may be an important step in se-lection of potentially competitive strains. It is not unrea-sonable, then, for the selection process of plausiblebiofertilizers to include such an examination of metabolicprofiles of rhizobial strains so that after a preliminaryscreening the strains that utilize the broadest range of car-bon and energy sources (especially amino acids and theirderivatives) might be further evaluated as inoculants withpotential competitive properties. It is tempting to hypothe-size that an adjustment of rhizobial metabolism to plantexudate contents is one of the critical elements of compet-itiveness. Detailed analyses of legume plant exudates andthe metabolic properties of the specific microsymbionts arerequired to elucidate this problem.

AcknowledgementsThis work was supported by a grant from the Ministry of

Education and Science No. PBZ-KBN-112/P06/2005.

ReferencesBorthakur, D., Soedario, M., Fox, P.M., and Webb, D.T. 2003. The

mid genes of Rhizobium sp. strain TAL1145 are required for de-gradation of mimosine into 3-hydroxy-4-pyridone and are indu-cible by mimosine. Microbiology (Reading, U.K.), 149: 537–546. doi:10.1099/mic.0.25954-0. PMID:12624215.

Bottomley, P.J., Cheng, H.H., and Strain, S.R. 1994. Genetic struc-ture and symbiotic characteristics of a Bradyrhizobium popula-tion recovered from a pasture soil. Appl. Environ. Microbiol.60: 1754–1761. PMID:16349270.

Bringhurst, R.M., Cardon, Z.G., and Gage, D.J. 2001. Galactosidesin the rhizosphere: utilization by Sinorhizobium meliloti and de-velopment of a biosensor. Proc. Natl. Acad. Sci. U.S.A. 98:4540–4545. doi:10.1073/pnas.071375898. PMID:11274355.

Bromfield, E.S.P., Lewis, D.M., and Barran, L.R. 1985. Crypticplasmid and rifampin resistance in Rhizobium meliloti influen-cing nodulation competitiveness. J. Bacteriol. 164: 410–413.PMID:2995316.

Cooper, J.E. 2004. Multiple responses of rhizobia to flavonoidsduring legume root infection. Adv. Bot. Res. 41: 1–62.

Wielbo et al. 965

# 2007 NRC Canada

Eckhardt, T. 1978. A rapid method for the identification of plasmiddeoxyribonucleic acid in bacteria. Plasmid, 1: 584–588. doi:10.1016/0147-619X(78)90016-1. PMID:107540.

Ferguson, G.A., and Takane, Y. 1989. Statistical analysis in psy-chology and education. McGraw-Hill, New York.

Fry, J., Wood, M., and Poole, P.S. 2001. Investigation of myo-ino-sitol catabolism in Rhizobium leguminosarum bv. viciae and itseffect on nodulation competitiveness. Mol. Plant–Microbe Inter-act. 14: 1016–1025. PMID:11497462.

Garg, B., Dogra, R.C., and Shama, P.K. 1999. High-efficiencytransformation of Rhizobium leguminosarum by electroporation.Appl. Environ. Microbiol. 65: 2802–2804. PMID:10347085.

Gaworzewska, E.T., and Carlile, M.J. 1982. Positive chemotaxis ofRhizobium leguminosarum and other bacteria towards root exu-dates from legumes and other plants. J. Gen. Microbiol. 128:1179–1188.

Gordon, D.M., Ryder, M.H., Heinrich, K., and Murphy, P.J. 1996.An experimental test of the rhizopine concept in Rhizobium me-liloti. Appl. Environ. Microbiol. 62: 3991–3996. PMID:16535438.

Hartmann, A., Giraud, J.J., and Catroux, G. 1998. Genotypic diver-sity of Sinorhizobium (formerly Rhizobium) meliloti strains iso-lated directly from a soil and from nodules of alfalfa (Medicagosativa) grown in the same soil. FEMS Microbiol. Ecol. 25: 107–116.

Hynes, M.F., and O’Connel, M.P. 1990. Host plant effect on com-petition among strains of Rhizobium leguminosarum. Can. J. Mi-crobiol. 36: 864–869.

Jaeger, C.H., Lindow, S.E., Miller, W., Clark, E., and Firestone,M.K. 1999. Mapping of sugar and amino acid availability insoil around roots with bacterial sensors of sucrose and trypto-phan. Appl. Environ. Microbiol. 65: 2685–2690.

Jimenez-Zurdo, J.I., van Dillevijn, P., Soto, M.J., de Felipe, M.R.,Olivares, J., and Toro, N. 1995. Characterization of a Rhizobiummeliloti proline dehydrogenase mutant altered in nodulation effi-ciency and competitiveness on alfalfa roots. Mol. Plant MicrobeInteract. 8: 492–498. PMID:8589406.

Jimenez-Zurdo, J.I., Garcia-Rodriguez, F.M., and Toro, N. 1997.The Rhizobium meliloti putA gene: its role in the establishmentof the symbiotic interaction with alfalfa. Mol. Microbiol. 23: 85–93. doi:10.1046/j.1365-2958.1997.1861555.x. PMID: 9004223.

Kaiser, H.F. 1960. The application of electronic computers to fac-tor analysis. Educ. Psychol. Meas. 20: 141–151. doi:10.1177/001316446002000116.

Kennedy, A.C., and Smith, K.L. 1995. Soil microbial diversity andthe sustainability of agricultural soils. Plant Soil, 170: 75–86.doi:10.1007/BF02183056.

Laguerre, G., Fernandez, M.P., Edel, V., Normand, P., and Amar-ger, N. 1993. Genomic heterogeneity among French Rhizobiumstrains isolated from Phaseolus vulgaris L. Int. J. Syst. Bacter-iol. 43: 761–767. PMID:8240956.

Laguerre, G., Mavingui, P., Allard, M.R., Charnay, M.P., Louvrier,P., Mazurier, S.I., Rigottier-Gois, L., and Amarger, N. 1996.Typing of rhizobia by PCR DNA fingerprinting and PCR-restriction fragment length polymorphism analysis of chromo-somal and symbiotic gene regions: application to Rhizobiumleguminosarum and its different biovars. Appl. Environ. Mi-crobiol. 62: 2029–2036. PMID:8787401.

Lodwig, E.M., Hosie, A.H., Bourdes, A., Findlay, K., Allaway, D.,Karunakaran, R., Downie, J.A., and Poole, P.S. 2003. Amino-acid cycling drives nitrogen fixation in the legume–Rhizobiumsymbiosis. Nature (London), 422: 722–726. doi:10.1038/nature01527. PMID:12700763.

Martinez-Romero, E. 2003. Diversity of Rhizobium–Phaseolus vul-

garis symbiosis: overview and perspectives. Plant Soil, 252: 11–23. doi:10.1023/A:1024199013926.

Maxwell, C.A., and Phillips, D.A. 1990. Concurrent synthesis andrelease of nod gene-inducing flavonoids from alfalfa roots. PlantPhysiol. 93: 1552–1558. PMID:16667655.

Mercado-Blanco, J., and Toro, N. 1996. Plasmids in rhizobia: therole of nonsymbiotic plasmids. Mol. Plant–Microbe Interact. 9:535–545.

Morrison D.F. 1990. Multivariate statistical methods. 3rd ed.McGraw-Hill, New York.

Murphy, P.J., Heycke, N., Banfalvi, Z., Tate, M.E., de Brujin, F.,Kondorosi, A., Tempe, J., and Schell, J. 1987. Genes for the cat-abolism and synthesis of an opine-like compound in Rhizobiummeliloti are closely linked on the Sym plasmid. Proc. Natl.Acad. Sci. U.S.A. 84: 493–497. doi:10.1073/pnas.84.2.493.PMID:16593802.

Oresnik, I.J., Twelker, S., and Hynes, M.F. 1999. Cloning andcharacterization of a Rhizobium leguminosarum gene encodinga bacteriocin with similarities to RTX toxins. Appl. Environ.Microbiol. 65: 2833–2840. PMID:10388672.

Oresnik, I.J., Pacarynuk, L.A., O‘Brien, S.H.P., Yost, C., andHynes, M.F. 1998. Plasmid-encoded cetabolic genes in Rhizo-bium leguminosarum bv. trifolii: evidence for a plant-induciblerhamnose locus involved in competition for nodulation. Mol.Plant–Microbe Interact. 11: 1175–1185.

Palmer, K.M., and Young, J.P. 2000. Higher diversity of Rhizobiumleguminosarum biovar viciae populations in arable soils than ingrass soils. Appl. Environ. Microbiol. 66: 2445–2450. doi:10.1128/AEM.66.6.2445-2450.2000. PMID:10831423.

Perret, X., Staehelin, C., and Spaink, H.P. 2000. Molecular basis ofsymbiotic promiscuity. Microbiol. Mol. Biol. Rev. 64: 180–201.PMID:10704479.

Phillips, D.A., Sande, E.S., Vriezen, J.A.C., de Bruijn, F.J., Le Ru-dulier, D., and Joseph, C.M. 1998. A new genetic locus in Si-norhizobium meliloti is involved in stachydrine utilization.Appl. Environ. Microbiol. 64: 3954–3960.

Phillips, D.A., Fox, T.C., King, M.D., Bhuvaneswari, T.V., andTeuber, L.R. 2004. Microbial products trigger amino acid exu-dation from plant roots. Plant Physiol. 136: 2887–2894. doi:10.1104/pp.104.044222. PMID:15347793.

Richardson, J.S., Hynes, M.F., and Oresnik, I.J. 2004. A genetic lo-cus necessary for rhamnose uptake and catabolism in Rhizobiumleguminosarum bv. trifolii. J. Bacteriol. 186: 8433–8442. doi:10.1128/JB.186.24.8433-8442.2004. PMID:15576793.

Rigottier-Gois, L., Turner, S.L., Young, P.W., and Amarger, N.1998. Distribution of repC plasmid-replication sequences amongplasmids and isolates of Rhizobium leguminosarum bv. viciaefrom field populations. Microbiology (Reading, U.K.), 144:771–780.

Robleto, E.A., Kmiecik, K., Oplinger, E.S., Nienhuis, J., and Tri-plett, E.W. 1998. Trifolitoxin production increases nodulationcompetitiveness of Rhizobium etli CE3 under agricultural condi-tions. Appl. Environ. Microbiol. 64: 2630–2633.

Rosenblueth, M., Hynes, M.F., and Martinez-Romero, E. 1998.Rhizobium tropici teu genes involved in specific uptake of Pha-seolus vulgaris bean-exudate compounds. Mol. Gen. Genet. 258:587–598. PMID:9671027.

Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular clon-ing: a laboratory manual. 2nd ed. Cold Spring Harbor Labora-tory, Cold Spring Harbor, NY.

Simms, E.L., and Taylor, D.L. 2002. Partner choice in nitrogen-fixation mutualisms of legume and rhizobia. Integr. Comp. Biol.42: 369–380. doi:10.1093/icb/42.2.369.

Spaink, H.P. 2000. Root nodulation and infection factors produced

966 Can. J. Microbiol. Vol. 53, 2007

# 2007 NRC Canada

by rhizobial bacteria. Annu. Rev. Microbiol. 54: 257–288.doi:10.1146/annurev.micro.54.1.257. PMID:11018130.

Sprent, J.I., and Sprent, P. 1990. Nitrogen fixing organisms: pureand applied aspects. Chapman and Hall London, UK.

Sterner, J.P., and Parker, M.A. 1999. Diversity and relationships ofBradyrhizobia from Amphicarpaea bracteata based on partialnod and ribosomal sequences. Syst. Appl. Microbiol. 22: 387–392. PMID:10553291.

Streeter, J.G. 1994. Failure of inoculant rhizobia to overcome thedominance of indigenous strains for nodule formation. Can. J.Microbiol. 40: 513–522.

Streit, W.R., Joseph, C.M., and Philips, D.A. 1996. Biotin andother water-soluble vitamins are key growth factors for alfalfaroot colonization by Rhizobium meliloti 1021. Mol. Plant–Microbe Interact. 9: 330–338. PMID:8672815.

Toro, N. 1996. Nodulation competitiveness in the Rhizobium-legume symbiosis. World J. Microbiol. Biotechnol. 12:157–162. doi:10.1007/BF00364680.

Triplett, E.W., and Sadowsky, M.J. 1992. Genetics of competitionfor nodulation of legumes. Annu. Rev. Microbiol. 46: 399–428.doi:10.1146/annurev.mi.46.100192.002151. PMID:1444262.

Vande Broek, A., and Vanderleyden, J. 1995. The role of bacterialmotility, chemotaxis, and attachment in bacteria–plant interac-tions. Mol. Plant–Microbe Interact. 8: 800–810.

van Dillewijn, P., Soto, M.J., Villadas, P.J., and Toro, N. 2001.Construction and environmental release of a Sinorhizobium me-liloti strain genetically modified to be more competitive foralfalfa nodulation. Appl. Environ. Microbiol. 67: 3860–3865.doi:10.1128/AEM.67.9.3860-3865.2001. PMID:11525978.

Vincent, J.M. 1970. A manual for the practical study of the root-nodule bacteria. IBP handbook No.15. Blackwell Scientific Pub-lishers, Oxford, England.

Wang, E.T., van Berkum, P., Sui, X.H., Beyene, D., Chen, W.X.,and Martinez-Romero, E. 1999. Diversity of rhizobia associatedwith Amorpha fruticosa isolated from Chinese soils and descrip-tion of Mesorhizobium amorphae sp. nov. Int. J. Syst. Bacteriol.49: 51–65. PMID:10028247.

Wernegreen, J.J., Harding, E.E., and Riley, M.A. 1997. Rhizobiumgone native: unexpected plasmid stability of indigenous Rhizo-bium leguminosarum. Proc. Natl. Acad. Sci. U.S.A. 94: 5483–5488. doi:10.1073/pnas.94.10.5483. PMID:9144264.

West, S.A., Kiers, E.T., Simms, E.L., and Denison, R.F. 2002.Sanctions and mutualism stability: Why do rhizobia fix nitro-gen? Proc. R. Soc. Lond. B. Biol. Sci. 269: 685–694.

Wielbo, J., and Skorupska, A. 2001. Construction of improved vec-tors and cassettes containing gusA and antibiotic resistancegenes for studies of transcriptional activity and bacterial locali-zation. J. Microbiol. Methods, 45: 197–205. doi:10.1016/S0167-7012(01)00244-5. PMID:11348677.

Willems, A. 2006. The taxonomy of rhizobia: an overview. PlantSoil, 287: 3–14. doi:10.1007/s11104-006-9058-7.

Wilson, K.J., Sessitsch, A., Corbo, J.C., Giller, K.E., Akkermans,A.D., and Jefferson, R.A. 1995. b-Glucuronidase (GUS) transpo-sons for ecological and genetic studies of rhizobia and othergram-negative bacteria. Microbiology (Reading, U.K.), 141:1691–1705. PMID:7551037.

Young, J.M., Kuykendall, L.D., Martinez-Romero, E., Kerr, A.,and Sawada, H. 2001. A revision of Rhizobium Frank 1889,with an emended description of the genus, and the inclusionof all species of Agrobacterium Conn 1942 and Allorhizobiumundicola de Lajudie et al. 1998 as new combinations: Rhizo-bium radiobacter, R. rhizogenes, R. rubi, R. undicola andR. vitis. Int. J. Syst. Evol. Microbiol. 51: 89–103.PMID:11211278.

Young, J.P., Crossman, L.C., Johnston, A.W., Thomson, N.R.,Ghazoui, Z.F., Hull, K.H., et al. 2006. The genome of Rhizo-bium leguminosarum has recognizable core and accessory com-ponents. Genome Biol. 7: R34. doi:10.1186/gb-2006-7-4-r34.PMID:16640791.

Zhang, F., and Smith, D.L. 2002. Interorganismal signalling in sub-optimum environments: the legume–rhizobia symbiosis. Adv.Agron. 76: 125–161.

Wielbo et al. 967

# 2007 NRC Canada