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Soil Biology & Biochemistry 32 (2000) 1601±1613
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Characterization of Rhizobium spp. bean isolates from SouthWest Spain
D.N. Rodriguez-Navarro a,*, A.M. Buendia b, M. Camacho a, M.M. Lucas c,
C. Santamaria a
a

Centro de FormacioÂn e InvestigacioÂn Agraria ``Las Torres y Tomejil'', Apartado O®cial, 41200-Alcala del RõÂo, Sevilla, Spain
b
Departamento de MicrobiologõÂa, Facultad de BiologõÂa, Universidad de Sevilla. Apartado 1095, 41080-Sevilla, Spain
c
Centro de Ciencias Medioambientales, C.S.I.C. Serrano 115 Dpdo., 28006-Madrid, Spain
Accepted 25 February 2000

Abstract
Rhizobium spp. strains able to nodulate beans (Phaseolus vulgaris L.) were isolated from Andalusian (Southern Spain) soils
with no record of recent bean cultivation (except soil 14) and no known history of bean inoculation in this area. The isolation
methodology was devised to obtain an heterogeneous rhizobia population from each soil sample, by using three di€erent bean
cultivars as trap-host. No association was found between the presence of rhizobia nodulating bean and the chemical or textural
properties of the soils. The isolates were grouped on the basis of their symbiotic e€ectiveness on bean cv. Canellini under

greenhouse conditions, intrinsic antibiotic resistance (IAR), lipopolysaccharide (LPS) and protein pro®les, melanin production,
and by ampli®ed rDNA restriction analysis (ARDRA). Most of the isolates were more e€ective than the reference strains
Rhizobium leguminosarum bv. phaseoli TAL1121, R. etli type strain CFN42 and R. tropici type strain CIAT899. The symbiotic
e€ectiveness of the isolates could not be related with other traits analyzed. Predominantly, a two bands-LPS pro®le was found
amongst the isolates. Most of them have been assigned to R. etli by ARDRA and seem to be more competitive than R. gallicum
or R. giardinii isolates. Additionally, a strong interaction between the bean cultivar and the native rhizobia populations was
observed. 7 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Rhizobium spp; Phaseolus vulgaris; Rhizobial diversity; SW-Spain

1. Introduction
Common bean (Phaseolus vulgaris L.) is an important food crop in America, Africa and Asia that can
nodulate with various fast-growing Rhizobium sp.,
such as R. leguminosarum bv. phaseoli (non-American
species capable of nodulating bean) (Jordan, 1984), R.
tropici (MartõÂ nez-Romero et al., 1991), R. etli (formerly American R. leguminosarum bv. phaseoli Ð type
I strains) (Segovia et al., 1993) R. gallicum and R. giar-

* Corresponding author. Tel.: +34-95-5650-808; fax: +34-95-5650373.
E-mail address: cifatorr@cap.junta-andalucia.es (D.N. Rodriguez-Navarro).

dinii, (Amarger et al., 1997). Bean is considered a poor
N2-®xer pulse in comparison with other grain legumes
(La Rue and Patterson, 1981; Hardarson, 1993).
Sparse nodulation or a lack of response to inoculation
in ®eld experiments has been frequently reported
worldwide, raising doubts about the bene®ts of inoculation (Graham, 1981; Buttery et al., 1987). This fact
could be related to the promiscuity observed in P. vulgaris (HernaÂndez-Lucas et al., 1995; Michiels et al.,
1998) or to other limiting nodulation factors, like the
high rate of N-fertilizer used in intensive agriculture,
which is particularly detrimental for beans (Temprano
et al., 1997).
The Guadalquivir River Valley is the area of South
Spain with the most intensive agriculture, owing to the
favourable weather conditions. We have sampled soils

0038-0717/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 8 - 0 7 1 7 ( 0 0 ) 0 0 0 7 4 - 2

1602

D.N. Rodriguez-Navarro et al. / Soil Biology & Biochemistry 32 (2000) 1601±1613

in locations within this area where bean might be potentially cropped. At the same time, there was no
available information about the characteristics of
native bean rhizobia populations that could restrict
bean inoculation practice.
Our objectives were: (1) to evaluate the size and
e€ectiveness of the native bean-nodulating population
of rhizobia in soils of the Guadalquivir River Valley
(SW-Spain) and (2) to study the diversity of these
populations by using several biochemical and molecular approaches.

2. Materials and methods
2.1. Reference strains
R. leguminosarum bv. phaseoli TAL1121 (IPAGRO,
Brazil), R. etli bv. phaseoli CFN42T, R. tropici type
IIB CIAT899T (Cuernavaca, Mexico) were used as
reference strains in greenhouse experiments. R. gallicum bv. gallicum R602T, R. giardinii bv. giardinii
H152T (N. Amarger), R. etli CFN42, R. tropici
CIAT899 and R. leguminosarum bv. viceae

ATCC10004 were used as controls for DNA restriction
analysis.
2.2. Plants and growth conditions
Four bean cultivars of P. vulgaris were used
throughout this work. White dry bean cv. Canellini,

Negrojamapa (unbred cultivar black-seeded, from
Mexico), and green bean type cv. Presenta (small
white-seeded, Asgrow S.A.) were employed as traphosts during soil rhizobia isolation. Small white-seeded
cv. Arrocina was used for most probable number
(MPN) estimations. White dry bean cv. Canellini was
mainly selected for this work because this variety is
commonly used for human consumption in this region.
For all experiments seeds were surface-sterilized with
sodium hypochlorite 10% (v/v) for 20 min and washed
with sterile water, germinated for 2 or 3 days on water
agar in the dark at 288C, and then planted. Plant sets
for rhizobial isolation, evaluation of the symbiotic
e€ectiveness and competition experiments were maintained in a greenhouse under natural light with a daily
minimum±maximum temperature of 16±288C.

2.3. Soil sampling and rhizobia strain isolation
Twenty one soil samples were collected along the
Guadalquivir River Valley (SW-Spain) covering a distance of ca. 200 km. Some chemical and textural properties from collected soils are shown in Table 1. Two
plastic pots of 200 cm3 were each ®lled with air-dried
and unamended soil. Surface-sterilized seeds of cultivar
Canellini were sown Ð one per pot Ð and placed in a
greenhouse. Plants were scored for nodulation 3 weeks
after emergence.
Two nodules were randomly excised from each plant
(when it was possible) and nodule isolates were
obtained by the procedure of Vincent (1970). Single

Table 1
Chemical and textural properties of Andalusian soils employed to isolate Rhizobium spp. nodulating beana
Soil

Location and land use

pH (CaCl2)

o.m. (%)

CaCO3(%)

Textural analysis

1
2
3
4
5
6
7
8
9
10
11
12
13
14

15
16
17
18
19
20
21

Tocina, potatoes
Cantillana, cotton
Alcolea del Rio, cereal
Alcolea del Rio, sun¯ower
Lora del Rio, fallow
Lora del Rio, orchard
PenÄa¯or, potatoes
Hornachuelos, tobacco
Posadas, fallow
Villarrubia, sun¯ower
Cordoba, fallow
Ecija-Osuna, cotton

Castro del Rio, potatoes
Zuheros, beans
Alcolea, fallow
Villafranca, sun¯ower
San Antonio, cotton
Villa del Rio, orchard
San Julian, maize
Marmolejo, cotton
Los Villares, sun¯ower

7.7
7.8
7.3
7.6
6.6
7.6
7.5
6.4
7.6
7.6

6.9
8.1
7.9
7.9
7.9
8.0
8.0
8.0
7.7
8.0
7.0

0.8
1.0
1.3
1.0
0.7
2.5
0.5
1.3

0.8
1.2
1.5
2.2
1.9
2.0
1.3
1.3
1.8
0.8
2.2
1.9
0.6

0.2
20.2
0.1
0.2
1.0
2.7

12.0
1.1
4.1
1.1
1.5
28.5
32.1
40.2
35.5
34.8
10.2
38.9
31.6
35.6
1.7

Clay
Clay±Loam
Clay
Sandy±Clay±Loam
Sandy±Loam
Sandy±Loam
Clay±Loam
Loam
Loam
Loam
Sandy±Loam
Clay
Clay
Clay±Loam
Sandy±Clay±Loam
Sandy±Clay
Clay
Sandy±Loam
Silty±Clay
Loam
Clay±Loam

a

+ Nodule formation on cvs. Canellini and Negrojamapa; (+) Nodule formation on cv. Negrojamapa.

Native rhizobia
(+)
±
ÿ
+
+
+
(+)
+
±
(+)
(+)
(+)
±
+
+
+
+
+
±
(+)
+

D.N. Rodriguez-Navarro et al. / Soil Biology & Biochemistry 32 (2000) 1601±1613

1603

colonies were picked and routinely maintained on
yeast mannitol agar (YMA) slants at 48C for further
characterization.
Soil samples were further sown with cultivars Negrojamapa and Presenta in order to increase the probability of trapping di€erent native strains, following
the same procedure as above. The numbers of native
rhizobia in soil samples were estimated by the MPN
counting technique using cultivar Arrocina as planthost (Araujo et al., 1986).
Rhizobial isolates have been assigned designations
as follows: ®rst a number indicating the soil sample,
plus a capital letter for bean cultivar used as trapplant (C, Canellini; NJ, Negrojamapa; PR, Presenta)
followed by a number (nodule sampled).

bacteria was resuspended in 20 ml of lysis bu€er (50 mM
NaOH and 0.25% SDS). Samples were heated at 958C
for 15 min. Lysed cells were centrifuged at 12,000 rpm
for 5 min and the pellet resuspended in sterile destilled
water. The suspension was adjusted to an optical density at 620 nm of 0.5 by dilution in water and was
directly used as the template for the PCR assay.
PCR ampli®cation was carried out according to
Herrera-Cervera et al. (1999). Ampli®ed DNA was
visualized by horizontal electrophoresis in 0.7% agarose gels. Aliquots of PCR products were digested with
the following restriction endonucleases (Roche): HinfI,
MspI, TaqI and NdeII. Restricted DNA fragments
were analyzed by horizontal electrophoresis in 3%
agarose gels.

2.4. Evaluation of the symbiotic properties of bean
rhizobial isolates under greenhouse conditions

2.7. Intrinsic antibiotic resistance (IAR)

Two greenhouse experiments were carried out in 2.5
l Leonard jars ®lled with a perlite±vermiculite mixture
(1/2, v/v) and watered with a N-free nutrient solution
(Rigaud and Puppo, 1975). Seedlings of cv. Canellini
were transferred aseptically to jars (three per jar) and
inoculated with 1 ml of 2 day-old yeast mannitol broth
(YMB) cultures of rhizobia to provide approximately
108 cells seedÿ1. Two non-inoculated controls were
included: non-fertilized (ÿN) and N-fertilized (+N).
Reference strains TAL1121, CIAT899 and CFN42
were included as inoculated controls. Seedlings were
thinned to uniformity to two per jar and covered with
a layer of sterilized paran-coated ®ne gravel. Jars
were arranged in a randomized block design with two
replicates per treatment. Plants were harvested 5 weeks
after inoculation and symbiotic e€ectiveness was estimated by comparing the shoot dry weights with those
of uninoculated control plants. Nodulation was scored
for abundance, size and nodule colour by the visual
rating of 1±5 as described by Redden et al. (1990).
2.5. Detection of melanin production and cultural
characteristics
Melanin production by the isolates was determined
as described by Cubo et al. (1988). The ability of bacteria to grow in Luria Broth (LB) and peptone yeast
extract (PY) minus calcium media has been assayed
according to MartõÂ nez-Romero et al. (1991). Rhizobial
growth at di€erent pH was also tested using bu€ered
(citrate±phosphate) TY medium (Beringer, 1974).
2.6. Ampli®ed ribosomal DNA restriction analysis
(ARDRA)
For sample preparation, strains were grown on agar
slopes of TY medium for 24 h at 288C; a loopful of each

Resistance to low concentrations of antibiotics was
determined by the method of Josey et al. (1979). Fresh
solutions of ®ltered sterilized (0.4 mm) antibiotics were
added to melted YMA medium to give the following
concentrations (mg mlÿ1): chloramphenicol, 5 and 15;
erythromycin, 10 and 20; gentamycin sulfate, 5 and 15;
kanamycin sulfate, 5 and 15; neomycin sulfate, 5 and
20; novobiocin, 0.5 and 1.5; rifampin, 1 and 3; streptomycin sulfate, 2.5 and 10; spectinomycin, 2.5 and 5;
and tetracycline 0.1 and 0.2. Each bacterial culture
was replicated twice per antibiotic concentration, by
dispensing 20 ml (of a 10ÿ5 dilution) per Petri plate.
Plates were incubated at 288C and scored after 3 days.
2.8. Lipopolysaccharide pro®les
Rhizobial isolates were grown in TY medium for
LPS separation on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The polysaccharide was solubilized from proteinase K-treated cells
as described by KoÈplin et al. (1993). The bacterial pellet was lysed by heating at 1008C for 5 min in 125 ml
of 1% SDS in 60 mM Tris±HCl (pH 6.8) and then
diluted to 1 ml with the same bu€er without SDS.
RNase and DNase were added and the solution was
incubated at 378C for 5 h. Proteinase K was added to
a ®nal concentration of 10 mg mlÿ1, and incubation
proceeded for a further 24 h. Electrophoresis was carried out on a 16.5% polyacrylamide gel using a tricine
bu€er system as described by Lesse et al. (1990). Gels
were ®xed and silver stained according to Kittelberger
and Hilbink (1993).
2.9. Protein pro®les
Strains were grown at 288C for 48 h on YMB.
Whole-cell protein extracts were prepared, and SDSPAGE was performed following the procedure of

1604

D.N. Rodriguez-Navarro et al. / Soil Biology & Biochemistry 32 (2000) 1601±1613

Laemmli (1970). Electrophoresis was carried out on
10±15% (w/v) gradient polyacrylamide gels. Gels were
loaded with approximately 100 mg of protein (Bradford, 1976) per lane and run at 10±15 mA for 4 h.
Bands were visualized by silver staining as described
by Switzer et al. (1979).
2.10. Nodule occupancy
A competition experiment was carried out under
greenhouse conditions described above, using the cv.
Canellini grown in 2.5 l Leonard jars. Three surfacesterilized seedlings per jar were inoculated with 109
cells per seed of a 1:1 mixture of each pair of competitors di€ering in their LPS pro®les. Also single inocula
were inoculated as controls. Plants were harvested 6
weeks after planting and nodule occupancy was determined by crushing surface-sterilized nodules (ca. 90
per combination) on YMA medium supplemented with
either erythromycin (20 mg mlÿ1) or gentamycin sulfate
(5 mg mlÿ1) depending of the strain combination. In
other cases, strains were identi®ed on TY plates supplemented with L-tyrosine (600 mg mlÿ1) and CuSO4
(40 mg mlÿ1) and melanin production was determined.
In one mixture, nodule occupancy was determined de
visu, by observing the ratio of e€ective to ine€ective
nodules.

3. Results
3.1. Rhizobial isolation from soils
Most of the studied soils (16 out of 21) had speci®c

indigenous populations of bean rhizobia (Table 1), as
plants of cv. Negrojamapa were well nodulated. However, plants of cv. Canellini formed nodules in 10 soils.
These 10 soils, in which both cvs. Canellini and
Negrojamapa formed nodules, were tested as inoculants on two other cvs. Presenta and Arrocina, in
order to investigate the host range of the native populations. In the trial with Presenta the direct method of
soil inoculation was followed as above. In the experiment with Arrocina serial dilutions of the chosen soils
were inoculated (MPN estimations). Presenta formed
nodules in eight and Arrocina in six of the 10 selected
soils.
As determined by MPN method, in most of the soils
the rhizobial population ranged between 3.6±42.4 rhizobia gÿ1. Soil no. 14, the only soil being cropped
with beans at the time of sampling, had about 4  103
rhizobia gÿ1, suggesting that the indigenous population
was stimulated by the presence of the host legume.
No relationship could be established between the
presence of native bean-nodulating rhizobia and soil
characteristics, such as pH, organic matter or CaCO3
content (Table 1).
3.2. Characterization of bean rhizobia isolates by
ARDRA
On the basis of polymorphism of 16S rRNA genes
(Fig. 1) most of the rhizobia we isolated have been
assigned to R. etli species (73%) (Table 2). Also rhizobia belonging to R. gallicum (10%) and R. giardinii
(6%) were isolated. Surprisingly, only one isolate was
ascribed to R. leguminosarum bv. phaseoli. The isolates

Fig. 1. Digestion patterns of the reference strains with the restriction enzyme Hinf I. Lanes 1, R. etli CFN42; 2, R. gallicum R602; 3, R. giardinii
H152; 4, R. leguminosarum bv. phaseoli ATCC10004. Taq I. Lanes 5, R. etli CFN42; 6, R. gallicum R602; 7, R. giardinii H152; 8, R. leguminosarum bv. phaseoli ATCC10004. Nde II. Lanes 9, R. etli CFN42; 10, R. leguminosarum bv. phaseoli ATCC10004; 11, R. giardinii H152; 12, R.
gallicum R602. Msp I. Lanes 13, R. etli CFN42; 14, R. leguminosarum bv. phaseoli ATCC10004; 15, R. giardinii H152; 16, R. gallicum R602.

1605

D.N. Rodriguez-Navarro et al. / Soil Biology & Biochemistry 32 (2000) 1601±1613
Table 2
Phenotypic characteristics of the Andalusian bean rhizobia isolates and reference strains used in this studya
Isolatea

Rhizobium spp.b

Fix

c

4C-2
4C-4
4NJ-1
4NJ-2
4PR-1
4PR-2

R. etli
R. etli
R. gallicum
R.l. phaseoli
R. etli
R. etli

+
++
ÿ
+
+
+

ÿ
ÿ
ÿ
ÿ
+
+

2b
mb
2b
2b
2b
2b

+/+
ÿ/ÿ
+/+
ÿ/ÿ
+/ÿ
+/+

5C-4
5NJ-1
5NJ-2

R. etli
R. giardinii
R. gallicum

ÿ
ÿ
+

ÿ
+
+

2b (I)
1b
2b (II)

+/+
+/+
ÿ/ÿ

6C-1
6NJ-1,6NJ-2,
6PR-1,6PR-2

R. etli
R. etli

+
+

+
ÿ

2b (I)
2b (II)

ÿ/ÿ
ÿ/ÿ

8C-3,8C-4,8NJ
1,8NJ-2,8PR-2

R. etli

+

+

2b

ÿ/+

14C-1
14C-3
14NJ-1
14NJ-2
14PR-1
14PR-2

R.
R.
R.
R.
R.
R.

etli
gallicum
etli
etli
etli
etli

++
+
+
+
+
+

ÿ
ÿ
+
+
+
+

2b (I)
mb (I)
2b (II)
2b (I)
2b (I)
mb (II)

;
ÿ/ÿ
ÿ/ÿ
ÿ/ÿ
ÿ/+
ÿ/ÿ
ÿ/ÿ

15C-4
15NJ-1
15NJ-2

S. fredii
R. gallicum
R. gallicum

+
++
+

ÿ
ÿ
ÿ

mb (I)
mb (II)
mb (II)

ÿ/ÿ
ÿ/ÿ
ÿ/ÿ

16C-1,16C-2
16NJ-1
16NJ-2
16PR-1

R. etli
R. etli
R. etli
R.l.viceae

+
+
+
ÿ

ÿ
ÿ
ÿ
ÿ

2b (I)
2b (II)
2b (II)
2b (III)

ÿ/ÿ
ÿ/ÿ
ÿ/+
ÿ/ÿ

17C-2
17C-3
17NJ-1
17NJ-2
17PR-1
17PR-2

R.
R.
R.
R.
R.
R.

etli
etli
giardinii
etli
etli
etli

+
ÿ
ÿ
+
ÿ
+

ÿ
ÿ
ÿ
ÿ
ÿ
ÿ

2b (I)
2b (I)
mb
2b (II)
2b (II)
2b (II)

ÿ/ÿ
+/ÿ
+/+
ÿ/ÿ
+/ÿ
+/ÿ

18C-1
18C-3
18PR-2

S. fredii
S. fredii
R. giardinii

+
+
ÿ

ÿ
ÿ
ÿ

mb (I)
mb (II)
mb (III)

ÿ/ÿ
ÿ/ÿ
+/+

21C-1,21C-2,
21NJ-1,21NJ-2,
21PR-1,21PR-2

R. etli

+

+

2b

ÿ/ÿ

TAL1121

R.l.phaseoli

+

+

mb

ÿ/ÿ

CIAT 899

R. tropici

+

ÿ

mb

+/+

R 602

R. gallicum



ÿ

mb

ÿ/ÿ

H 152

R. giardinii



ÿ

mb

ÿ/ÿ

CFN 42

R. etli

ÿ

+

mb

ÿ/ÿ

Mel

LB/PYd

LPS

a

Designation of the isolates (Section 2.3, Sampling and rhizobia strain isolation).
On the basis of ARDRA.
c
E€ectiveness evaluation on cv. Canellini, ++ equal to N-fertilized control, + equal or superior to TAL1121, ÿ ine€ective,
Canellini. LPS, (2b) two bands, (mb) multiband. In brackets are indicated di€erent types of LPS pro®les within each soil sample.
d
LB/PY minus calcium, + growth, ÿ no growth.
b



not tested on

1606

D.N. Rodriguez-Navarro et al. / Soil Biology & Biochemistry 32 (2000) 1601±1613

used. However, other bean Rhizobium spp. (except R.
tropici ) were mainly detected by using cv. Negrojamapa as trap-host. Seven of the 13 non-R. etli isolates
of this collection were trapped by Negrojamapa, which
indicates that this cultivar is less restrictive than the
others, as demonstrated by its greater capacity for
nodulation with native rhizobia from six more soils
than cv. Canellini.

15C-4, 18C-1 and 18C-3 were considered Sinorhizobium fredii-like, since they had 16S rDNA patterns
matching S. fredii, although they did not nodulate soybean cv. Williams (data not shown). The isolate 16PR1 has been ascribed to R. leguminosarum bv. viceae,
and e€ectively nodulated plants of Vicia ervilia (data
not shown).
R. etli strains were trapped by all the bean cultivars

Table 3
Shoot dry weight, nodulation score, pod number and harvest index of bean cv. Canellini plants inoculated with rhizobia isolates (Experiment 1)
Treatment

Shoot dry wt.a

Nodulationb

Pod number

H.I. (%)

N-fertilizedc
R. etli 4C-2
R. etli 4C-4
R. gallicum 4NJ-1
R.l. phaseoli 4NJ-2
R. etli 4PR-1
R. etli 4PR-2

7.3
4.1
5.9
1.6
4.8
3.3
4.7

0
3.0
5.0
3.0
3.5
3.5
2.5

17.5
16.5
13.5
4.5
9.0
9.0
9.0

15.5
6.0
19.6
20.4
18.9
10.3
12.5

R. etli 5C-4
R. giardinii 5NJ-1
R. gallicum 5NJ-2

1.4
1.5
4.1

3.0
4
5.0

5.5
4.5
14.0

14.7
9.3
12.5

6C-1
6NJ-1
6NJ-2
6PR-1
6PR-2

4.6
4.0
2.9
5.4
3.0

3.5
4.5
3.5
4.5
3.0

7.5
9.0
4.5
12.0
6.0

8.1
16.0
14.6
20.4
13.7

R. etli 8C-3
R. etli 8NJ-1
R. etli 8NJ-2

4.6
4.8
5.2

4.0
3.5
3.5

11.5
9.5
13.0

7.3
16.7
30.5

R. etli 14C-1
R. gallicum 14C-3

6.2
4.1

3
3.0

10.5
13.0

14.1
8.8

S. fredii 15C-4

3.0

5.0

2.0

4.4

R. etli 16C-1
R. etli 16C-2

4.1
3.9

4.0
3.0

8.0
6.0

3.8
6.9

R. etli 17C-2
R. etli 17C-3

4.6
2.3

4.0
4.5

8.0
7.0

19.9
13.8

S. fredii 18C-1
S. fredii 18C-3

2.4
2.5

3.5
4.5

1.0
7.5

2.5
4.1

R. etli 21C-1
R. etli 21C-2

4.4
5.1

3.5
3

11.5
13.5

7.0
28.6

R. l. phaseoli
TAL1121

3.3

4.5

5.0

7.0

Untreated d
LSD (0.05)

1.3
2.2

0
ÿ

1.5
8.7

8.3
11.8

R.
R.
R.
R.
R.

a

etli
etli
etli
etli
etli

Data represent mean value of two replicates (four plants).
Nodulation was estimated in basis to a scale ranging from 0 (no nodulation) to 5, taking into account the number, position, size and colour
of the nodules formed.
c
Fertilized treatment received 15 mmol of NH4NO3.
d
Uninoculated non-fertilized control.
b

D.N. Rodriguez-Navarro et al. / Soil Biology & Biochemistry 32 (2000) 1601±1613

1607

were e€ective nitrogen ®xers. Inoculation of some isolates, such as 4C-4, 6PR-1, 8NJ-2, 14C-1, 15NJ-1, and
21PR-2 led to plant growth that did not signi®cantly
di€er …P < 0:05† from the N-fertilized plants in shoot
dry weight and number of pods. Most of the isolates
ranged between the reference strain TAL1121 (45±
47.5% of the N-fertilized control) and the highly e€ective isolates (mentioned above). Those isolates that did
not produce signi®cantly better plant growth …P <
0:05† than the untreated plants were classed as ine€ective. This is the symbiotic phenotype we assign to R.
etli type strain CFN42 in our work. Results of the
symbiotic performance of all these isolates are shown

3.3. Symbiotic e€ectiveness under greenhouse conditions
The symbiotic performance of the bean isolate collection was evaluated on cv. Canellini under greenhouse conditions. Two experiments were carried out
on di€erent dates, which may explain the di€erences
observed in dry matter accumulation by the control
treatments (non-inoculated and inoculated with the
reference strain TAL1121). Nevertheless, plants inoculated with the reference strain TAL1121, as well as the
non-fertilized (untreated) plants, yielded the same proportional biomass in relation to the N-fertilized plants
in both trials (Tables 3 and 4). Of the isolates 80%

Table 4
Shoot dry weight, nodulation score, pod number and harvest index of bean cv. Canellini plants inoculated with rhizobia isolates (Experiment 2)a
Treatment

Shoot dry wt.b

Nodulationc

Pod number

N-Fertilizedd

14.1

0

13.5

0.8

R. etli 8C-4
R. etli 8PR-2

10.2
9.9

4.0
5.0

16.0
19.5

6.6
5.3

8.0
5.3
7.5
9.9

5.0
4.5
4.0
3.5

8.5
7.5
16.0
17.5

2.7
1.0
2.4
3.1

R. gallicum 15NJ-1
R. gallicum 15NJ-2

10.9
6.2

4.5
4.5

19.0
6.0

3.9
1.3

R. l. viceae 16NJ-1
R. etli 16NJ-2
R. etli 16PR-1

6.9
10.5
2.6

4.5
4.5
2.5

10.0
17.0
2.0

3.5
4.6
1.0

R.
R.
R.
R.

etli
etli
etli
etli

14NJ-1
14NJ-2
14PR-1
14PR-2

H.I. (%)

R.
R.
R.
R.

giadinii 17NJ-1
etli 17NJ-2
etli 17PR-1
etli 17PR-2

1.9
7.0
2.4
5.0

1.0
3.5
1.0
4.5


16.5
1.5
10.0


5.7
0.4
2.0

R.
R.
R.
R.
R.

giardinii 18PR-2
etli 21NJ-1
etli 21NJ-2
etli 21PR-1
etli 21PR-2

2.0
9.6
9.6
9.9
11.7

1.0
5.0
4.5
5.0
5.0


13.5
14.0
15.0
21.0


2.6
2.6
4.2
7.7

R. l. phaseoli TAL1121

6.6

4.0

9.0

1.7

R. tropici CIAT899

4.3

3.0

8.0

11.9

R. etli CFN42

2.2

2.5

3.0

0.7

Untreatede

2.0

0

1.0

0.2

LSD (0.05)

3.4

ÿ

12.3

2.9

a

Data not considered for statistical analysis.
Data represent mean value of two replicates (four plants).
c
Nodulation was estimated in basis to a scale ranging from 0 (no nodulation) to 5, taking into account the number, position, size and colour
of the nodules formed.
d
Fertilized treatment received 15 mmol of NH4NO3.
e
Uninoculated non-fertilized control.
b

1608

D.N. Rodriguez-Navarro et al. / Soil Biology & Biochemistry 32 (2000) 1601±1613

Table 5
Intrinsic antibiotic resistance pattern of bean rhizobia isolates and the corresponding reference strainsa
Antibiotics(m lÿ1)

R. etli (32)

CFN42

R. gallicum (5)

R602

R. giardinii (3)

H152

Chloramphenicol (5)
Erythromycin (10)
Gentamicin (5)
Kanamycin (5)
Neomycin (20)
Novobiocin (2)
Rifampin (5)
Streptomycin (3)
Spectinomycin (5)
Tetracycline (0,2)

14
15
6
24
3
25
7
30
19
7

R
R
R
S
S
R
R
R
S
S

5
5
5
5
5
4
1
5
0
4

R
R
R
R
R
R
S
R
R
R

3
3
0
1
2
3
1
0
0
3

R
R
R
R
R
R
S
S
S
R

a

Numerical values indicate the number of resistant isolates; () total number tested; R, resistant; S, sensitive.

in Tables 3 and 4. Ine€ective symbioses were identi®ed
irrespective of the trap-cultivar employed during the
former isolation procedure. The R. etli isolates, with
minor exceptions, all isolates belonging to R. gallicum,
except 4NJ-1, as well as isolates assigned to S. frediilike were e€ective on cv. Canellini. R. leguminosarum
bv. phaseoli (4NJ-2) did not di€er from the control
strain TAL1121. By contrast R. leguminosarum bv.
viceae (16PR-1) and all R. giardinii isolates were ine€ective (Table 2).

contrast, all the isolates from soils no. 8 and 21, and
most from soil no. 14 produced melanin (Table 2).
The isolates ascribed to R. giardinii and R. gallicum
were Melÿ, as the corresponding type strains H152
and R602, except the isolates 5NJ-1 and 5NJ-2. The
isolates classi®ed as R. leguminosarum (4NJ-2 and
16PR-1) failed to produce the pigment. None of the
three isolates considered S. fredii-like strains produced
melanin, in contrast to most of the fast-growing rhizobia nodulating soybean.

3.4. Melanin production

3.5. Cultural characteristics

All rhizobial isolates were examined for their ability
to produce melanin (Mel+), and 20 of them were
Mel+, as well as the reference strains R. leguminosarum bv. phaseoli TAL1121 and R. etli CFN42. This
ability was not restricted to isolates trapped by a given
host-cultivar: 28% of Canellini, 44% of Negrojamapa
and 54% of Presenta isolates were Mel+.
Half of the R. etli isolates (51%) were melanin producers. Isolates of this species from soils no. 16 and
17, and most of isolates from soil no. 6 did not produce melanin under the assay conditions described. By

Most of the isolates grouped with R. etli were
unable to grow in either of the media, which agrees
with the behaviour of the corresponding reference
strain CFN42. Nevertheless, we have found isolates
able to grow in either of the media like R. tropici
CIAT899 and isolates that grew in LB or in PY minus
Ca (Table 2). The three isolates ascribed to R. giardinii
had the same cultural characteristics as R. tropici
CIAT899. It means that they were able to grow in
both media, in contrast to reference strain H152. All
R. gallicum isolates, except 4NJ-1, were unable to

Fig. 2. Representative examples of lipopolysaccharide pro®les of bean rhizobia isolates. Lanes: 1, 4NJ-1; 2, 4NJ-2; 3, 4C-4; 4, 4C-2; 5, 21C-2; 6,
21C-1; 7, 18C-3; 8, 18C-1.

D.N. Rodriguez-Navarro et al. / Soil Biology & Biochemistry 32 (2000) 1601±1613

1609

Fig. 3. Multiband lipopolysaccharide pro®les. Lanes: 1, 4C-4; 2, 14PR-2; 3, CFN42; 4, 14C-3; 5, 15NJ-1; 6, 15NJ-2; 7, R602; 8, 17NJ-1; 9,
18PR-2; 10, H152; 11, 18C-3; 12, 15C-4, 13, 18C-1 14, USDA205.

grow in either of the media as was the reference strain
R602. None of the reference strains were able to grow
in PY minus calcium.
In relation to low pH tolerance, all bean rhizobia
isolates failed to grow in bu€ered TY at pH 4.5,
except 4PR-2, which grew 2 d after inoculation (DAI),
as did CFN42 and CIAT899. Twelve R. etli isolates
and R. gallicum 4NJ-1 were able to grow at pH 5.0, as
did the reference strains TAL1121, CFN42 and
CIAT899, but at di€erent rates. At 2 DAI only 4PR-1
(R. etli ), CFN42 and CIAT899 had grown. Isolates
4C-2, 4PR-2, 4C-4, 4NJ-1, 8C-4 grew at 2±3 DAI.
Most of the isolates (4NJ-2, 5C-4, 6PR-2, 8PR-2,
14NJ-1, 14NJ-2 and 16C-2) as well as strain TAL1121
grew after 8 days. At the end of the incubation most
of the isolates had raised the pH of the medium; but
interestingly, others like 4C-2, 4C-4 and 4PR-2, did
not change the initial pH.

3.6. Intrinsic antibiotic resistance patterns
Numerical results of resistance of the isolates
ascribed to each Rhizobium spp. and the corresponding
reference strain are present in Table 5. The indigenous
populations of rhizobia nodulating beans have shown
a substantial heterogeneity and at least 16 di€erent resistance patterns have been recorded between the R.
etli isolates. Even more, among isolates from a given
location, di€erent resistance patterns were recorded.
In general, R. etli isolates were resistant to streptomycin, novobiocin, kanamycin and spectinomycin. R.
gallicum isolates showed a totally di€erent resistance
pattern than R. etli isolates, and three di€erent ®ngerprints could be detected among them. As shown in
Table 5 they are resistant to practically all the antibiotics tested, except spectinomycin. Each one of the
three isolates ascribed to R. giardinii species showed a

Fig. 4. Total protein pro®les of several bean rhizobia isolates. Lanes: 1, 4NJ-1; 2, 4NJ-2; 3, 6NJ-2; 4, 6PR-2; 5, 21C-2; 6, 21C-1.

1610

D.N. Rodriguez-Navarro et al. / Soil Biology & Biochemistry 32 (2000) 1601±1613

di€erent resistance pattern, one of them shared by the
reference strain H152. In general, R. gallicum and R.
giardinii isolates were resistant to chloramphenicol,
gentamycin, neomycin and tetracycline, in contrast to
most R. etli isolates.
3.7. LPS pro®les
The electrophoretic mobility of LPS on polyacrylamide gels showed that two main LPS patterns were
clearly distinguished among the isolates: (i) the LPS I
region (the slowest-migrating area) consisting of one
or two stained bands (Fig. 2, lanes 1, 2 and 4±6), that
is the predominant pro®le among the R. etli isolates
(2b-LPS), and (ii) the LPS I region showing a multiband ladder (mb-LPS) (Fig. 2, lanes 3, 7 and 8) in addition to the fast-migrating area of LPS II. The LPS
pro®le of the isolates was not related with the bean
cultivar used during the isolation procedure.
Most of the isolates yielding a mb-LPS I have been
assigned to bean Rhizobium spp. other than R. etli (R.
gallicum 14C-3, 15NJ-1 and 15NJ-2; R. giardinii 17NJ1 and 18PR-2) or to S. fredii-like isolates (15C-4, 18C1 and 18C-3). The isolates assigned to R. gallicum or
R. giardinii, share the same LPS-pro®le that the corresponding reference strain (Fig. 3, lanes 4±7 and 8±10,
respectively). In contrast, the LPS-pro®le of R. etli isolates 4C-4 and 14PR-2 were completely di€erent from
that produced by the type strain CFN42 (Fig. 3, lanes
1, 2 and 3). The isolates considered S. fredii like had a
mb-LPS similar to that of S. fredii type strain
USDA205 (Fig. 3, lanes 11±14).
3.8. Protein pro®les
Protein pro®les, in general, help to discriminate
between isolates coming from a particular soil, e.g.
both Negrojamapa isolates from soil No. 4 were indistinguishable by LPS (Fig. 2, lanes 1 and 2), however,
their protein pro®les were clearly di€erent (Fig. 4,

lanes 1 and 2); in fact, 4NJ-1 has been ascribed to R.
gallicum and 4NJ-2 to R. leguminosarum. Similarly,
protein pro®les allow us to distinguish between isolates
belonging to the same Rhizobium spp.. By all the
characteristics studied R. etli isolates 6NJ-2 and 6PR-2
seem to be the same strain (Table 2), however, their
protein pro®les were di€erent (Fig. 4, Lanes 3 and 4).
Nevertheless, both techniques gave consistent results
in most cases, thus the six isolates of soil no. 21 once
characterized seem to be a single strain either by LPS
or protein pro®les (two examples are shown in Figs. 2
and 4, lanes 5±6), all of them were ascribed to R. etli.
3.9. Nodule occupancy
In order to investigate if isolates with the common
2b-LPS were more competitive than those having a
mb-LPS, a competition experiment between isolates
with both types of LPS was carried out. Strain mixtures consisting of pairs of isolates from the same soil,
each representing a type of LPS-pro®le, were inoculated on cv. Canellini plants. Thus, competition studies
were performed with isolates from soils no. 4, 14 and
17 (Table 6).
Four di€erent combinations were examined with isolates from soil No. 4: 4C-4 (mb-LPS) with 2b-LPS isolates (4C-2, 4NJ-1, 4NJ-2 and 4PR-2). Table 6 shows
that R. etli 4C-4 clearly outcompeted R. gallicum 4NJ1 and R. etli 4PR-2, both 2b-LPS isolates occupied
less than 20% of the nodules. In competition with
4NJ-2 R. leguminosarum bv. phaseoli, 4C-4 was equally
competitive; but in combination with the other Canellini isolate (4C-2) was signi®cantly …P > 0:95† less competitive. This result eliminates the possible distortion
of the data due to a preference of cv. Canellini for
nodulation with its homologous isolates (derived from
Canellini). Thus, R. gallicum 14C-3 (mb-LPS) was
clearly less competitive than either of both R. etli competitors (14NJ-1 and 14PR-1), forming less than 10%
of the nodules. The other mb-LPS isolate from soil

Table 6
Competition between bean rhizobia isolates with di€erent LPS-pro®le on bean cv. Canellini
Inoculum strains A/B

Rhizobium spp.

LPS A/B

No. nodules identi®ed

% of nodule occupancy
A

B

Identi®cation trait A/B

4C-4/4NJ-1
4C-4/4PR-2
4C-4/4NJ-2
4C-4/4C-2

etli/gallicum
etli/etli
etli/phaseoli
etli/etli

mb/2b
mb/2b
mb/2b
mb/2b

90
92
63
93

84
88
57
39

16
12
43
61

GenS/GenR
Melÿ/Mel+
EryS/EryR
EryS/EryR

14NJ-1/14C-3
14PR-1/14C-3
14PR-2/14NJ-1
14PR-2/14PR-1

etli/gallicum
etli/gallicum
etli/etli
etli/etli

2b/mb
2b/mb
mb/2b
mb/2b

90
94
96
91

91
96
31
40

9
4
69
60

Mel+/Melÿ
Mel+/Melÿ
EryS/EryR
GenS/GenR

D.N. Rodriguez-Navarro et al. / Soil Biology & Biochemistry 32 (2000) 1601±1613

No.14, R. etli 14PR-2, was signi®cantly …P > 0:95† less
competitive than R. etli 2b-LPS isolates 14NJ-1 and
14PR-1. In the mixture R. giardinii 17NJ-1 (mb-LPS)
and R. etli 17NJ-2 (2b-LPS) nodule occupancy was
not quanti®ed by isolation from nodules since nodules
induced by R. giardinii are ine€ective and easily distinguishable from e€ective nodules. Most of the
nodules formed by this mixed inoculant were e€ective,
thus presumably formed by R. etli 17NJ-2.
In summary, in all the combinations of R. etli/gallicum or of R. etli/giardinii tested, isolates ascribed to R.
etli were more competitive nodulators on Phaseolus cv.
Canellini than the other bean rhizobia species.

4. Discussion
The conventional method of isolating indigenous
rhizobia from soils may not re¯ect the real situation of
biodiversity of a given Rhizobium population in a soil.
Bacterial competitiveness and the plant genotype of
the trap-host probably in¯uence the ®nal results
(Michiels et al., 1998; VaÂsques-Arroyo et al., 1998).
The importance of the trap-host has been clearly
demonstrated in this study, as cv. Negrojamapa
detected native rhizobia in 16 soils in contrast to cultivars Canellini, Presenta and Arrocina that did in 10, 8
and 6 soils, respectively. In addition, our data suggest
that bean-nodulating rhizobia are widely distributed
and show a high saprophytic competence since we
have detected native populations in soils with no
recent record of bean cropping.
Competition may be particularly intense in soils containing a high density of rhizobia in contrast to those
harboring low populations. In these latter soils or by
using diluted soil samples (Muilenburg et al., 1996)
recovery of less competitive strains could be more
likely. Most of the soils of this study, except soil no.
14, had very small rhizobia populations, which may
have led to the isolation of R. gallicum, R. giardinii
and R. leguminosarum bv. phaseoli strains. Our results
of competition experiments (Table 6) show that R. etli
isolates are clearly more competitive than R. gallicum
or R. giardinii, so that the indigenous populations of
these species may have been underestimated in soil
no.14. If this were the case, the evaluation of the
nodulation competence of other bean-nodulating
species should be done with bean cultivars showing
restrictive nodulation by R. etli. Additionally, as the
plant-host genotype is of critical importance for determining the outcome of competition experiments, it
would be necessary to con®rm the superior competitive
capacity of R. etli strains with others bean cultivars.
Even though more isolates must be analyzed, it
seems that the characterization of bean rhizobia isolates by ARDRA can di€erentiate between Rhizobium

1611

spp. nodulating bean as has been done by SantamarõÂ a
et al. (1997). Isolates of this collection have shown a
high variability in N2-®xation eciency in combination
with di€erent plant genotypes (Rodrõ guez-Navarro et
al., 1999) and isolates from a given bean cultivar did
not necessarily show a better symbiotic performance
with the corresponding cultivar (Tables 3 and 4).
Our results have demonstrated as have others
(Hungria and Franco, 1993; Cubo et al., 1997) that
melanin production is not essential either for nodulation or nitrogen ®xation. Thus, it was possible to
identify Melÿ/Fix+ phenotypes, as well as Mel+/Fixÿ
strains (Table 2). The ability to grow in LB and PY
minus Ca media has been used to distinguish R. tropici
from other bean-nodulating rhizobia (MartõÂ nezRomero et al., 1991; van Berkum et al., 1994) because
it seems to be restricted to R. tropici type IIB strains.
Nevertheless, some isolates did not match with the cultural characteristics exhibited by the corresponding
type strain.
The variation in IAR enabled us to distinguish
between a range of strains present in an indigenous
population and our results demonstrate the validity
of this technique to identify strains. Boddey and
Hungria (1997) have reported the IAR as a tool
for phenotypic grouping of several isolates from
Brazil into two di€erent Bradyrhizobium sp. VaÂsquez-Arroyo et al. (1998) have di€erentiated 28
IAR patterns between R. etli isolates in Mexico.
Tolerance to antibiotics of bean-nodulating rhizobia
was found to be strain speci®c rather than species
speci®c (Amarger et al., 1997).
The electrophoresis of whole cell proteins has
allowed more discrimination than can be obtained by
using LPS pro®les distinguishing between isolates
belonging to the same Rhizobium spp.. Although computer programs are available to analyze pro®le similarities these were not found to be useful for our
purpose. This technique has been used in our studies
to show up di€erences between isolates when other
method failed to do so.
Rhizobial isolates from a soil grouped by LPS or by
protein pro®les in an individual strain, with few exceptions, shared as well the intrinsic antibiotic resistance
pattern. This re¯ects good agreement between the
di€erent methodologies employed for strain identi®cation, although there was no correlation between the
antibiotic resistance grouping and PCR-RFLP, in fact
16 di€erent resistance patterns were recorded between
R. etli isolates. These results are in agreement with the
high genomic instability reported in R. etli (Brom et
al., 1991; Herrera-Cervera et al., 1999).
The predominance of a LPS-pro®le of two bands
among the isolates of this collection, might be associated with a better saprophytic and nodulation competence, nevertheless no relationship between the LPS-

1612

D.N. Rodriguez-Navarro et al. / Soil Biology & Biochemistry 32 (2000) 1601±1613

pro®le and both the competitive ability of the strains
(Table 6) or their symbiotic phenotype (Table 2) was
observed under controlled conditions.
R. tropici was not detected among the isolates of
this collection, perhaps the better adaptation of this
species to acidic conditions might have precluded its
presence in these predominantly alkaline±neutral soils,
as has been found by Amarger et al. (1994), Anyango
et al. (1995), Hungria et al. (1997) and Herrera-Cervera et al. (1999). Interesting all of the isolates from
soil no. 4 showed a marked tolerance to low pH,
despite the neutral±alkaline pH of this soil. This highlight the lack of relationship between their ecological
origin and their in vitro behaviour. Brazilian soybean
isolates from low pH soils (pH < 4) did not show a
particular acidic tolerance during in vitro assays (M.A.
Hungria, pers. comm.).
The predominance of R. etli in these soils cannot be
explained in terms of co-evolution of Rhizobium spp.
and wild beans as in other regions of the Americas
(Aguilar et al., 1998; VaÂsquez-Arroyo et al., 1998).
More plausible seems the hypothesis of Sessitsch et al.
(1997) who suggested that R. etli strains might have
been imported to Europe as seed contaminants.
To our knowledge this is the ®rst extensive study of
native bean-nodulating rhizobial populations in Spanish soils, as the recent study (Herrera-Cervera et al.,
1999) only consider one Spanish soil sample. We have
found, like others (PinÄero et al., 1988; VaÂsquez-Arroyo
et al., 1998), that bean microsymbionts were largely
diverse; for example, in soil No. 4 members of three
bean rhizobia species were isolated and, at least, three
di€erent strains of R. etli were identi®ed.
Many reports have indicated that inoculation of
beans with Rhizobium were not successful due to the
presence of native rhizobia with high competitive ability. Since this is not the situation in the Andalusian
region, the evaluation of the native populations in
terms of symbiotic performance and competitive behaviour might lead to the selection of superior strains,
well adapted to local conditions and or bean varieties.
Some isolates from this collection have provided promising results under ®eld conditions with green bean
varieties.

Acknowledgements
We are grateful to Dr. Jose Antonio Herrera for assistance with the ARDRA analysis, Dr. Noelle Amarger for providing some type strains and Dr. J.E. Ruõ zSaÂinz for his helpful suggestions. Financial support
was provided by the National Institute of Agricultural
Research (INIA-MAPA)

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