FEMS Microbiology Ecology 48 (2004) 71–77

www.fems-microbiology.org

Attachment to plant roots and nod gene expression are not affectedby pH or calcium in the acid-tolerant alfalfa-nodulating bacteria

Rhizobium sp. LPU83

Mar�ıa Jos�e Soto, Pieter van Dillewijn, Francisco Mart�ınez-Abarca,Jos�e I. Jim�enez-Zurdo, Nicol�as Toro *

Departamento de Microbiolog�ıa del Suelo y Sistemas Simbi�oticos, Estaci�on Experimental del Zaid�ın,

Consejo Superior de Investigaciones Cient�ıficas, Profesor Albareda 1, 18008 Granada, Spain

Received 31 October 2003; received in revised form 15 December 2003; accepted 19 December 2003

First published online 20 January 2004

Abstract

Soil acidification is one of the environmental factors that more strongly hampers the establishment of an effective symbiotic

interaction between rhizobia and leguminous plants. Sinorhizobium meliloti and the acid-tolerant Rhizobium sp. strain LPU83 are

able to nodulate alfalfa plants at pH 5.6 but both exhibit a delayed nodulation and a reduction in the number of elicited nodules. We

show here that the addition of calcium (Ca) has no positive effect on the nodulation kinetics shown by LPU83 at low pH, but does

retrieve the competition capacity of S. meliloti strains in acidic media, likely by improving the ability of these bacteria to attach to

plant roots. In contrast, the attachment of the acid-tolerant strain LPU83 to alfalfa roots is not greatly affected by pH or Ca

concentration. Media acidification impairs nod gene induction in different S. meliloti strains but not in LPU83. However, the ad-

dition of Ca at low pH does not affect neither nod gene expression in alfalfa-nodulating rhizobia (S. meliloti or strain LPU83) nor the

quality of nod gene inducers exudated by alfalfa plants, in contrast to what has been reported previously. These data reveal dif-

ferential features among alfalfa-nodulating rhizobia and point out the adsorption of S. meliloti to alfalfa roots as the major limiting

step affecting its symbiotic performance in acidic conditions.

� 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Rhizobium; Nodulation; Acidity; Calcium; Attachment; nod Gene expression

1. Introduction

Progressive soil acidification is a worldwide problem

with important economical consequences for countries

which rely on alfalfa (Medicago sativa L.) production

for cattle nutrition [1,2]. The poor performance of al-

falfa plants in moderately acid soils (pH 5.5–6.5) is

mainly due to the effects of low pH on the establishment

of the symbiosis between this legume and the nitrogen-

* Corresponding author. Tel.: +34-958-181600; Fax: +34-958-

129600.

E-mail address: ntoro@eez.csic.es (N. Toro).

0168-6496/$22.00 � 2004 Federation of European Microbiological Societies

doi:10.1016/j.femsec.2003.12.010

fixing bacteria Sinorhizobium meliloti [3–6]. Low pH

causes a reduction in the rate of nodulation as well as inthe number of nodules elicited by the bacteria.

As in soybean and clover, Ca and hydrogen ions are

known to interact in the nodulation of alfalfa [7–9],

showing an increased demand for Ca as the pH de-

creases. Three stages of the Rhizobium-legume symbiosis

have been reported to be affected by Ca and pH which

possibly account for the dependence of alfalfa nodula-

tion for these factors: (i) growth of the bacteria, (ii)rhizobial attachment to roots, and (iii) the induction of

nod gene expression.

S. meliloti strains are extremely sensitive to acidic

pH, and will grow only above pH 5.5 [4,5]. In low pH

. Published by Elsevier B.V. All rights reserved.

Table 1

Bacterial strains and plasmids used in this study and their relevant

characteristics

Bacterial strains Relevant characteristics Reference or

source

S. meliloti

GR4 Wild type; Nodþ Fixþ [21]

2011 Wild type; Nodþ Fixþ J. Denari�e

LPU63 Wild type; Nodþ Fixþ [2]

Moderate acid tolerant

Rhizobium sp.

LPU83 Wild type; Nodþ Fixþ=� [2]

Acid tolerant

Plasmids

pRK2013 Helper plasmid for [22]

mobilization; Kmr

pRmM57 nodC-lacZ transcriptional [23]

fusion; Tcr

pGUS-3 PnfeD-gusA translational [24]

fusion; Kmr

pBBR1MCS-2 Broad host range cloning [25]

vector; Kmr

72 M.J. Soto et al. / FEMS Microbiology Ecology 48 (2004) 71–77

conditions, the growth rate of S. meliloti strains is in-

creased by concentrations of Ca in the millimolar range,

whereas no such increase is observed at neutral pH

[10,11]. Although it is not known how Ca assists rhi-

zobial cells to cope with high proton concentrations ithas been suggested that Ca is involved in maintaining

cell envelope stability, specifically by maintaining lipo-

polysaccharide (LPS) structure and in the expression of

outer membrane proteins [12].

S. meliloti adsorption to roots shows a strict re-

quirement for Ca and neutral pH [13,14]. It has been

suggested that neutral pH is required for stable binding,

whereas Ca could act as a bridge between negativelycharged groups on plant and bacterial surfaces, and/or

indirectly activate suitable adhesins in the bacterium.

The induction of nod gene expression has also been

reported to be affected by Ca and pH in some symbiotic

systems. Richardson et al. [15,16] demonstrated that the

nod gene induction activity of clover root exudates and

the expression of nodulation genes in Rhizobium legu-

minosarum biovar trifolii was reduced at low pH butcould be recovered in these conditions by increasing the

concentration of Ca. Similarly, Howieson et al. [17] re-

ported that an increase in Ca concentration increased the

nod gene induction activity of exudates of acid-sensitive

Medicago species (e.g.M. truncatula). Additionally, they

related the increased ability of some acid-tolerant species

of Medicago (M. polymorpha and M. murex) to achieve

nodulation in acidic soils with their ability to produceat low pH exudates with unaffected nod gene induction

capacity.

Recently, another factor has been suggested to affect

the S. meliloti-alfalfa symbiosis in some acidic soils

which has drastic effects on alfalfa yield. Acid-tolerant

but ineffective alfalfa-nodulating rhizobia have been

identified in the acidic soils of Argentina and Uruguay

[2,18]. These bacteria, which show surprising geneticuniformity [19], are related to Rhizobium sp. strain

Ori191, which was isolated in Oregon [20]. They are able

to nodulate alfalfa plants at pH 5.6 but, like S. meliloti

strains, display delayed nodulation and a reduction in

the number of nodules formed [18] at this pH. Although

poorly competitive in neutral conditions, these bacteria

efficiently compete with S. meliloti strains for alfalfa

root nodulation at low pH [18]. As these bacteria occupya higher proportion of the elicited nodules, the shoot dry

weight of the plant decreases suggesting that the pres-

ence of these types of bacteria may result in a decrease in

alfalfa yield in acidic soils.

In this work, we describe the effects of pH and Ca on

various aspects of alfalfa nodulation with S. meliloti and

the acid-tolerant Rhizobium sp. strain LPU83. Our data

suggest that attachment of S. meliloti cells to alfalfaroots is the main factor limiting S. meliloti-alfalfa sym-

biosis at low pH. Furthermore, we have found that in

Rhizobium sp. strain LPU83 neither adsorption to al-

falfa roots nor nod gene induction is significantly af-

fected by pH and Ca concentration, which raise the

possibility to use the genetic background of these bac-

teria to improve the symbiotic performance of S. meliloti

in acidic soils.

2. Materials and methods

2.1. Bacterial strains, plasmids, media and growth condi-

tions

The bacterial strains and plasmids used in this studyare listed in Table 1. Plasmid DNA was routinely iso-

lated and manipulated following standard protocols

[26]. All rhizobial strains were grown at 30 �C on tryp-

tone-yeast medium [27] or defined minimal medium

(MM) [28]. Escherichia coli was grown routinely at 37 �Cin Luria–Bertani medium (LB) [26]. Triparental bacte-

rial matings were performed using pRK2013 as a helper

plasmid [22]. pGUS-3, which contains an nfeD gusA

fusion was used in competition assays [24]. Antibiotics

were used, as required, at the following concentrations:

tetracycline, 10 lg/ml; kanamycin, 50 lg/ml for E. coli

and 200 lg/ml for Rhizobium; and streptomycin, 50 lg/ml for E. coli and 250 lg/ml for Rhizobium.

2.2. Plant assays

Alfalfa (Medicago sativa L.) seeds were sterilized,

germinated and grown on nitrogen-free medium as

described elsewhere [29], supplemented with 20 mM

Mops (3-[N-morpholino]propanesulfonic acid), for pH

7.0 and 20 mM Mes (2-[N-morpholino]ethanesulfonic

M.J. Soto et al. / FEMS Microbiology Ecology 48 (2004) 71–77 73

acid), for pH 5.6. The medium was adjusted to the

required pH with KOH prior to autoclaving. The pH

and calcium concentration of the plant mineral solu-

tion are indicated for each experiment. To test the

degree of infectivity under different conditions, 10-day-old plants (at least 20) were inoculated with a bacte-

rial suspension at a final concentration of 106 cells

ml�1. Previously, the corresponding rhizobial strain

was grown at 30 �C in TY broth with shaking to

middle exponential phase (OD600 ¼ 0.6) and then di-

luted to the required concentration with sterile water.

After inoculation, the number of nodulated plants and

the number of nodules per plant were recorded daily.To determine the competitive ability, plants and in-

oculants were prepared as described above. Sets of 12

alfalfa plants were inoculated with mixtures of S. me-

liloti 2011, GR4 or LPU63 strains�Rhizobium sp.

LPU83 at a ratio of 1:1. To distinguish between nodules

formed by the different bacteria, one of the coinoculated

strains carried pGUS3, which contains the reporter gene

encoding b-glucuronidase (GUS). To determine noduleoccupancy, roots were collected 15 days after inocula-

tion, briefly washed with water and incubated overnight

in the dark at 37 �C in 1 mM X-Gluc (5-bromo-chloro-

3-indolyl-b-DD-glucuronide) in 50 mM sodium-phosphate

buffer (pH 7.5) with 1% SDS. Nodule occupancy was

determined by counting blue and white nodules.

2.3. Root adsorption of bacteria

Root adsorption of S. meliloti and Rhizobium sp.

LPU83 was quantitatively determined as previously

described [30]. In brief, late exponential growth phase

cultures of rhizobia (OD500 ¼ 0.4//1� 109 cell ml�1) were

diluted to a concentration of 105 bacteria/ml with ni-

trogen-free Rigaud and Puppo solution [31] in which the

initial pH and the Ca concentration were modified insome experiments. Seeds of alfalfa var. Arag�on were

surface sterilized with mercuric chloride and germinated

on water agar plates [30]. Fifteen 2-day-old seedlings

were incubated in 10 ml bacterial suspension for 4 h at

room temperature, with shaking at 200 rpm. They were

then washed four times with fresh medium. The bacteria

adsorbed onto root surfaces were individually detected

as microcolonies, which developed upon culture of thewashed seedlings in embedding TY agar supplemented

with 50 lg ml�1 kanamycin plus 25 lg ml�1 cyclohexi-

mide. The number of root-bound rhizobia was deter-

mined by direct counting of the microcolonies closely

apposed to the root surface, and the percentage of in-

oculated rhizobia adsorbed to roots in the chosen ex-

perimental conditions (adhesiveness) was calculated

[30]. pH and cell numbers were determined before andafter incubation of the bacteria with the roots for each

experiment. The actual concentration of viable bacteria

was obtained by counting on TY agar plates containing

the required antibiotic. All values are given with 95%

confidence intervals.

2.4. Induction assays and measurement of b-galactosidaseactivity

For nod gene induction experiments, the plasmid

pRmM57 was used [23]. Induction was performed using

plant mineral solution supplemented with either alfalfa

root exudates or luteolin. The exudates used corre-

sponded to nitrogen-free medium in which 5 alfalfa

seedlings had been grown for 10 days under standard

conditions [29]. Plant exudates prepared in this way gaveb-galactosidase activity values 50–100-folds higher than

those previously reported [23]. When required, the pH of

exudate solutions was adjusted to pH 7.0 with KOH

immediately before use. Nod gene induction assays were

performed as previously described [17]. Briefly, rhizobial

strains containing pRmM57 were grown overnight in

MM broth to early exponential phase (optical density at

600 nm of 0.2–0.4). To induce, 200 ll of the bacterialculture was combined with 200 ll of either plant mineral

solution containing plant exudates or 2 lM luteolin in a

1.5 ml Eppendorf tube and incubated for 3 h. After this

period, b-galactosidase activity was determined by the

SDS-chloroform method as described by Miller [32].

As controls, plant nutrient solutions without inducers

were added to the rhizobial strain. Enzyme activity was

always calculated as net activity (treatments minuscontrols).

3. Results

3.1. Effect of calcium on the nodule formation efficiency

and competitive ability of Rhizobium sp. LPU83 in acidic

conditions

In this study, we tested the response of Rhizobium sp.

LPU83 nodulation on alfalfa to calcium concentration

and compared it with that of S. meliloti 2011. The ad-

dition of a higher concentration of Ca (6 mM) at pH 5.6,

significantly increased the number of nodules per plant

elicited by strain 2011 (Fig. 1(a)). In contrast, increasing

Ca concentration did not affect the nodulation on alfalfaat pH 5.6 by Rhizobium sp. LPU83 (Fig. 1(b)). Thus,

enhanced nodulation of alfalfa by increasing Ca con-

centration at low pH only occurred with S. meliloti but

not with Rhizobium sp. LPU83.

We previously reported that, at low pH, Rhizobium

sp. LPU83 competes efficiently with S. meliloti strains

for nodule formation, resulting in a decrease in alfalfa

yield [18]. As shown above, the addition of Ca within themillimolar range partially recovers the impaired ability

to nodulate alfalfa plants of a S. meliloti strain but not

of Rhizobium sp. LPU83. We investigated whether the

Fig. 1. Nodulation kinetics of S. meliloti 2011 (a) and Rhizobium sp. LPU83 (b) strains in response to various pH conditions and calcium con-

centrations, expressed as mean number of nodules formed per plant. Circles represent values obtained at pH 7.0 in the presence of either 0.7 mM

(open circle) or 6 mM (filled circle) CaSO4, respectively. Squares represent values obtained at pH 5.6 in the presence of either 0.7 mM (open square)

or 6 mM (filled square) CaSO4, respectively. Error bars represent standard errors at 95% confidence interval.

74 M.J. Soto et al. / FEMS Microbiology Ecology 48 (2004) 71–77

addition of Ca could increase S. meliloti nodulation

competitiveness under low pH against the acid-tolerant

strain, by carrying out competition assays at pH 5.6 in

the presence of 10 mM calcium sulfate. As shown in

Fig. 2, the addition of Ca at acidic pH increased the

competitive ability of the three S. meliloti strains tested

to levels close to those observed at pH 7.0. The results

were similar regardless of which strain, either S. meliloti

or the acid-tolerant Rhizobium sp. LPU83, was marked

with the pGUS3 reporter plasmid. These data provide

further evidence for the lack of effect of increasing Ca

concentration on alfalfa nodulation by LPU83 at low

pH. They may also account for an observation reported

some years ago concerning the ability of S. meliloti in-

ocula to suppress ineffective nodulation by liming acidic

soils [33].

3.2. Effect of pH and calcium on two symbiotic steps in

Rhizobium sp. LPU83

3.2.1. Adsorption to alfalfa plant roots

The adsorption of S. meliloti to roots is highly sen-

sitive to pH and higher Ca concentrations are required

at lower pH to achieve similar levels of adsorption [13].Here, we have analyzed the adsorption capability of

Rhizobium sp. LPU83 and compared it with that of S.

Fig. 2. Effect of calcium and pH on the competitive ability of rhizobial strain

by black bars. 150–200 nodules were analyzed in each co-inoculation experi

acid-tolerant Rhizobium sp. LPU83. Error bars at 95% confidence interval a

meliloti 2011 in acidic conditions (Table 2). The results

obtained with strain 2011 were consistent with previous

reports [13] as the adsorption of bacteria was almost

abolished at pH 5.6, and recovered to levels very simi-

lar to those observed at neutral pH upon increase Ca

concentration to 6 or 10 mM. Adsorption of the acid-

tolerant strain LPU83 also decreased at pH 5.6, but only

to half the value obtained in neutral conditions. Inter-estingly, and in contrast to the S. meliloti strain, the

attachment of LPU83 to alfalfa roots was not greatly

affected by Ca concentration and only a modest positive

effect was observed when it was added to the media at

6 mM. Thus, the increase in competitive ability of the

S. meliloti strain observed at low pH following the ad-

dition of Ca concentration could be explained by dif-

ferent effects of this divalent cation on adsorption inS. meliloti and Rhizobium sp. LPU83.

3.2.2. nod Gene expression

Induction of the nod genes has been shown to be af-

fected by low pH in R. leguminosarum biovar trifolii

[15,16] and in S. meliloti [17]. We decided to analyze nod

gene expression in Rhizobium sp. LPU83 in acidic con-

ditions and in response to Ca. To determine this, theexpression of a nodC-lacZ fusion was studied in re-

sponse to alfalfa exudates in cells of S. meliloti strains

s. The percentage of nodules occupied by S. meliloti strains is indicated

ment. White bars represent the percentage of nodules occupied by the

re also shown.

Table 2

Effect of pH and Ca on the adhesivenessa of S. meliloti and Rhizobium

sp. LPU83

pH/Ca2þ 0.7 mM 6 mM 10 mM

S. meliloti 2011

7.0 2.08� 0.29 2.61� 0.27 2.73� 0.35

6.5 1.06� 0.16 2.57� 0.27 2.77� 0.42

6.0 0.31� 0.01 2.05� 0.29 1.68� 0.16

5.6 0.08� 0.02 0.78� 0.13 1.80� 0.30

Rhizobium sp. LPU83

7.0 1.45� 0.17 1.04� 0.11 1.27� 0.12

6.5 1.20� 0.17 0.80� 0.10 1.07� 0.15

6.0 1.37� 0.12 1.08� 0.15 0.68� 0.09

5.6 0.72� 0.13 1.17� 0.15 0.70� 0.13aAdhesiveness values were determined as the percentage of bacteria

in the initial suspension that adhered to the roots after washing. Mean

values and standard errors at 95% confidence interval were calcu-

lated from data of 2–3 determinations in at least two independent

experiments.

M.J. Soto et al. / FEMS Microbiology Ecology 48 (2004) 71–77 75

GR4, 2011, LPU63, and in Rhizobium sp. LPU83 car-

rying pRmM57. The bacterial cells were incubated with

root exudates of alfalfa plants grown in either neutral or

acidic pH and with either normal (0.7 mM) or higher

(6 mM) Ca concentrations. The results (Table 3) show

that nodC expression in response to root exudates of

alfalfa plants grown at pH 5.6 and in the presence of low

concentrations of Ca was adversely affected in all the S.meliloti strains tested, including that isolated from acidic

soils (LPU63). The decrease in nod gene expression in S.

meliloti strains at low pH might be due to acidity

causing the quantity or quality of alfalfa exudates to be

far from optimal for these bacteria. However, when the

pH of the induction assay for alfalfa exudates obtained

at pH 5.6 was increased to neutral, the level of nod gene

expression was similar to that obtained with root exu-dates of alfalfa plants grown at pH 7.0. Thus, as it has

been observed for acid-tolerant Medicago species [17]

the potential of alfalfa root exudates to induce nod genes

was not reduced in seedlings grown at pH 5.6. In con-

trast, in Rhizobium sp. LPU83, the nodC gene was ex-

pressed to similar levels in response to alfalfa exudates

Table 3

Effect of pH and Ca on nod gene induction in rhizobia in response to alfalfa

Treatments during seedling growth b-Galactosidase a

S. melilotib

pH Ca (mM) pHc GR4

7 0.7 7 302� 59

5.6 0.7 5.6 43� 22

5.6 0.7 7 267� 99

7 6 7 192� 26

5.6 6 5.6 24� 16

5.6 6 7 122� 84a Enzyme activity (Miller units) was always calculated as net activity (treatm

interval were calculated from data of 2–3 determinations in at least two indbThe various strains harbour pRmM57.c pH during induction assay.

obtained at either pH 7.0 or 5.6 and it was independent

of the induction assay. The different effects of acidity on

nod gene expression in S. meliloti and Rhizobium sp.

LPU83 cannot be explained by differences in rhizobial

metabolism, as similar levels of lacZ gene expressionunder the control of a constitutive promoter have been

observed in the two genetic backgrounds (data not

shown).

In our system, the addition of 6 mM Ca at low pH

significantly improves alfalfa nodulation by S. meliloti

strains but not by LPU83, in which nodulation kinetics

remained unaffected (Fig. 1). We investigated whether

this was due to differences in the effects of calcium ionson nod gene induction in these two bacteria, as observed

for adsorption to alfalfa plant roots (see above). As

shown in Table 3 (row 5), the presence of higher con-

centrations of Ca during alfalfa exudate production at

pH 5.6 did not increase nod gene induction activity, ei-

ther for the S. meliloti or for the acid-tolerant strain.

Similar conclusions on effects of pH and Ca on nod

gene expression in S. meliloti GR4 and LPU83 strainswere obtained when the plant specific nod gene inducer

luteolin was used in the assays (data not shown).

Thus, the decreased nodulation observed for Rhizo-

bium sp. LPU83 at low pH cannot be explained by a

decrease in nod gene expression. Furthermore, in con-

trast to what has been reported in other symbiotic sys-

tems [15,16], nod gene expression in S. meliloti at low pH

was not improved by adding Ca. Therefore, the positiveeffect on the nodulation kinetics of S. meliloti strains at

low pH by increasing Ca concentration cannot be at-

tributed to the recovery of nod gene expression in these

bacteria.

4. Discussion

The low productivity shown by alfalfa plants in

moderately acid soils appears to be the result of two

factors: impairment in the establishment of symbiotic

root exudates

ctivitya

Rhizobium sp.b

2011 LPU63 LPU83

68� 4 120� 62 187� 71

17� 9 13� 6 148� 77

89� 5 105� 59 138� 86

32� 11 92� 43 158� 39

11� 9 10� 4 94� 60

57� 6 47� 17 77� 41

ent minus control). Mean values and standard errors at 95% confidence

ependent experiments.

76 M.J. Soto et al. / FEMS Microbiology Ecology 48 (2004) 71–77

associations with S. meliloti strains and a competitive-

ness problem related to the presence in these soils of

acid-tolerant, alfalfa-nodulating bacteria with poor ni-

trogen-fixing ability. These bacteria are able to effi-

ciently compete with S. meliloti strains for the formationof nodules at low pH, contributing to the reduction in

crop yield. Rhizobium sp. strain LPU83, recently iso-

lated from acid soils of Argentina, is one example of this

type of bacteria [2,18]. Data presented in this manu-

script show that the addition of Ca has no marked effect

on the nodulation kinetics of this bacterium and that Ca

treatment increases nodulation of alfalfa by more ef-

fective S. meliloti strains, supporting an old field ob-servation [33]. In this work, the possible basis of this

symbiotic phenotype have been analyzed. We have

found that, in contrast to S. meliloti strains, attachment

and nod gene expression in LPU83 are not severely re-

duced at low pH and they are not affected by Ca con-

centration. To the best of our knowledge, this is the first

time that an alfalfa-nodulating Rhizobium with such

characteristics has been described. Like S. meliloti,LPU83 displays an impaired nodulation phenotype on

alfalfa plants in mildly acidic conditions (pH 5.6) [2,18].

Although this result could tempt us to speculate that

alfalfa may play a role in restricting nodulation under

acidic conditions, it is also possible that the limitation of

nodulation observed for this rhizobial strain is corre-

lated to a specific bacterial problem. It was recently

shown that Rhizobium sp. LPU83 poorly infects thecentral tissue of nodules; that the symbiosomes of these

nodules are formed by several bacteroids enclosed

within a single peribacteroid membrane separated by a

medium electron-dense material, and that LPU83 bac-

teroids have morphological features typical of free-living

rhizobia [19]. Nevertheless, the genetic elements re-

sponsible for the acid tolerant traits of this bacterium

(no large reductions in adsorption and nod gene induc-tion in response to low pH) could potentially be trans-

ferred to S. meliloti strains and help them to function

more effectively under acid conditions.

It has been suggested that low pH decreases nod gene

expression in rhizobial strains by affecting the quantity

or the composition of root exudates and/or the rhizobial

response to them [34]. Our results indicate that, as for

acid-tolerant hosts, neither the quantity nor the qualityof alfalfa root exudates was severely affected by pH

during seedling growth, although we cannot rule out the

possibility that the pH of the media may have caused

some minor reversible modifications to chemical struc-

tures. On the other hand, as the growth rate of S. me-

liloti strains decreased with increasing acidity, the

decrease in nod gene expression at low pH in response to

luteolin may be an indirect effect of acidity on rhizobialmetabolism. However, the use of a reporter gene under

the control of a constitutive promoter showed that gene

expression is not negatively affected by low pH. Thus, a

more likely explanation for the decrease in nod gene

expression in S. meliloti under acidic conditions is a

limitation in the interaction between the inducers and

the activator protein, NodD, at low pH.

Growth and attachment of S. meliloti cells to alfalfaroots, as well as the expression of their nod genes are

drastically reduced at low pH, and could be responsible

for the low nodulation and competitive ability shown by

these bacteria at low pH. Here we have shown that the

addition of Ca, which has a positive effect on the nod-

ulation kinetics and competitive ability of S. meliloti,

neither alters the induction capacity of alfalfa exudates

nor nod gene expression rates. This suggests that incontrast to other symbiotic systems, the most limiting

factor in the establishment of the S. meliloti-alfalfa

symbiosis at low pH is the attachment of rhizobial cells

to plant roots. Therefore, and as little growth is required

to allow nodulation, any approach addressed to im-

prove the symbiotic performance of S. meliloti in acid

soils should be focused on solving the limitations in

attachment of the rhizobial cells to alfalfa roots atlow pH.

In summary, our results show differential symbiotic

features of the acid-tolerant Rhizobium sp. LPU83 in

acidic media and suggest that any approach addressed

to overcome the limitations of the S. meliloti-alfalfa

symbiosis in acidic soils should be focused on the im-

provement of S. meliloti adsorption to alfalfa roots in

these environmental conditions.

Acknowledgements

We are grateful to A. Lagares for kindly providing us

with the Rhizobium sp. LPU83 genomic library. This

work was funded by the European Commission Grant

TS3-CT94-0265 and Comisi�on Asesora de Investigaci�onCient�ıfica y T�ecnica of the MCyT Grant BIO2002-

02579. M.J. Soto was supported by EC (Training andMobility of Researchers) and M.E.C. fellowships.

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