Characterization of
Rhizobium
spp. bean isolates from
South-West Spain
D.N. Rodriguez-Navarro
a,*, A.M. Buendia
b, M. Camacho
a, M.M. Lucas
c,
C. Santamaria
aaCentro de FormacioÂn e InvestigacioÂn Agraria ``Las Torres y Tomejil'', Apartado O®cial, 41200-Alcala del RõÂo, Sevilla, Spain bDepartamento de MicrobiologõÂa, Facultad de BiologõÂa, Universidad de Sevilla. Apartado 1095, 41080-Sevilla, Spain
cCentro 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 dierent 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 eectiveness 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 eective than the reference strains
Rhizobium leguminosarumbv.phaseoli TAL1121,R. etli type strain CFN42 andR. tropici type strain CIAT899. The symbiotic eectiveness 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 toR. etliby ARDRA and seem to be more competitive thanR. gallicum
or R. giardinii isolates. Additionally, a strong interaction between the bean cultivar and the native rhizobia populations was observed.72000 Elsevier Science Ltd. All rights reserved.
Keywords: Rhizobiumspp;Phaseolus vulgaris; Rhizobial diversity; SW-Spain
1. Introduction
Common bean (Phaseolus vulgaris L.) is an import-ant 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 (MartõÂnez-Romero et al., 1991), R. etli
(for-merly AmericanR. leguminosarum bv.phaseoli Ð type I strains) (Segovia et al., 1993)R. gallicumandR.
giar-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 inocu-lation (Graham, 1981; Buttery et al., 1987). This fact could be related to the promiscuity observed inP.
vul-garis (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
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* Corresponding author. Tel.: 808; fax: +34-95-5650-373.
in locations within this area where bean might be po-tentially 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 eectiveness 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 molecu-lar 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.
galli-cum 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 trap-hosts 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 eectiveness and competition experiments were main-tained 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 dis-tance of ca. 200 km. Some chemical and textural prop-erties 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 isolateRhizobiumspp. nodulating beana
Soil Location and land use pH (CaCl2) o.m. (%) CaCO3(%) Textural analysis Native rhizobia
1 Tocina, potatoes 7.7 0.8 0.2 Clay (+)
2 Cantillana, cotton 7.8 1.0 20.2 Clay±Loam ±
3 Alcolea del Rio, cereal 7.3 1.3 0.1 Clay ÿ
4 Alcolea del Rio, sun¯ower 7.6 1.0 0.2 Sandy±Clay±Loam +
5 Lora del Rio, fallow 6.6 0.7 1.0 Sandy±Loam +
6 Lora del Rio, orchard 7.6 2.5 2.7 Sandy±Loam +
7 PenÄa¯or, potatoes 7.5 0.5 12.0 Clay±Loam (+)
8 Hornachuelos, tobacco 6.4 1.3 1.1 Loam +
9 Posadas, fallow 7.6 0.8 4.1 Loam ±
10 Villarrubia, sun¯ower 7.6 1.2 1.1 Loam (+)
11 Cordoba, fallow 6.9 1.5 1.5 Sandy±Loam (+)
12 Ecija-Osuna, cotton 8.1 2.2 28.5 Clay (+)
13 Castro del Rio, potatoes 7.9 1.9 32.1 Clay ±
14 Zuheros, beans 7.9 2.0 40.2 Clay±Loam +
15 Alcolea, fallow 7.9 1.3 35.5 Sandy±Clay±Loam +
16 Villafranca, sun¯ower 8.0 1.3 34.8 Sandy±Clay +
17 San Antonio, cotton 8.0 1.8 10.2 Clay +
18 Villa del Rio, orchard 8.0 0.8 38.9 Sandy±Loam +
19 San Julian, maize 7.7 2.2 31.6 Silty±Clay ±
20 Marmolejo, cotton 8.0 1.9 35.6 Loam (+)
21 Los Villares, sun¯ower 7.0 0.6 1.7 Clay±Loam +
a
colonies were picked and routinely maintained on yeast mannitol agar (YMA) slants at 48C for further characterization.
Soil samples were further sown with cultivars Negro-jamapa and Presenta in order to increase the prob-ability of trapping dierent 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 plant-host (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 trap-plant (C, Canellini; NJ, Negrojamapa; PR, Presenta) followed by a number (nodule sampled).
2.4. Evaluation of the symbiotic properties of bean rhizobial isolates under greenhouse conditions
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 paran-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 eectiveness was esti-mated 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 bac-teria to grow in Luria Broth (LB) and peptone yeast extract (PY) minus calcium media has been assayed according to MartõÂnez-Romero et al. (1991). Rhizobial growth at dierent pH was also tested using buered (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
bacteria was resuspended in 20ml of lysis buer (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 den-sity 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% agar-ose 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.
2.7. Intrinsic antibiotic resistance (IAR)
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; strepto-mycin sulfate, 2.5 and 10; spectinostrepto-mycin, 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 polyacryl-amide gel electrophoresis (SDS-PAGE). The polysac-charide was solubilized from proteinase K-treated cells as described by KoÈplin et al. (1993). The bacterial pel-let 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 buer 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 car-ried out on a 16.5% polyacrylamide gel using a tricine buer 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
Laemmli (1970). Electrophoresis was carried out on 10±15% (w/v) gradient polyacrylamide gels. Gels were loaded with approximately 100 mg of protein (Brad-ford, 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 surface-sterilized seedlings per jar were inoculated with 109 cells per seed of a 1:1 mixture of each pair of competi-tors diering in their LPS pro®les. Also single inocula were inoculated as controls. Plants were harvested 6 weeks after planting and nodule occupancy was deter-mined by crushing surface-sterilized nodules (ca. 90 per combination) on YMA medium supplemented with either erythromycin (20mg mlÿ1) or gentamycin sulfate (5 mg mlÿ1) depending of the strain combination. In other cases, strains were identi®ed on TY plates sup-plemented 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 eective to ineective 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. How-ever, plants of cv. Canellini formed nodules in 10 soils. These 10 soils, in which both cvs. Canellini and Negrojamapa formed nodules, were tested as inocu-lants on two other cvs. Presenta and Arrocina, in order to investigate the host range of the native popu-lations. In the trial with Presenta the direct method of soil inoculation was followed as above. In the exper-iment 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 rhi-zobia gÿ1. Soil no. 14, the only soil being cropped with beans at the time of sampling, had about 4103 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. etlispecies (73%) (Table 2). Also rhizo-bia belonging to R. gallicum (10%) and R. giardinii (6%) were isolated. Surprisingly, only one isolate was ascribed to R. leguminosarum bv.phaseoli. The isolates
Table 2
Phenotypic characteristics of the Andalusian bean rhizobia isolates and reference strains used in this studya
Isolatea Rhizobiumspp.b Fixc Mel LPS LB/PYd
4C-2 R. etli + ÿ 2b +/+
4C-4 R. etli ++ ÿ mb ÿ/ÿ
4NJ-1 R. gallicum ÿ ÿ 2b +/+
4NJ-2 R.l. phaseoli + ÿ 2b ÿ/ÿ
4PR-1 R. etli + + 2b +/ÿ
4PR-2 R. etli + + 2b +/+
5C-4 R. etli ÿ ÿ 2b (I) +/+
5NJ-1 R. giardinii ÿ + 1b +/+
5NJ-2 R. gallicum + + 2b (II) ÿ/ÿ
6C-1 R. etli + + 2b (I) ÿ/ÿ
6NJ-1,6NJ-2, R. etli + ÿ 2b (II) ÿ/ÿ
6PR-1,6PR-2
8C-3,8C-4,8NJ R. etli + + 2b ÿ/+
1,8NJ-2,8PR-2
;
14C-1 R. etli ++ ÿ 2b (I) ÿ/ÿ
14C-3 R. gallicum + ÿ mb (I) ÿ/ÿ
14NJ-1 R. etli + + 2b (II) ÿ/ÿ
14NJ-2 R. etli + + 2b (I) ÿ/+
14PR-1 R. etli + + 2b (I) ÿ/ÿ
14PR-2 R. etli + + mb (II) ÿ/ÿ
15C-4 S. fredii + ÿ mb (I) ÿ/ÿ
15NJ-1 R. gallicum ++ ÿ mb (II) ÿ/ÿ
15NJ-2 R. gallicum + ÿ mb (II) ÿ/ÿ
16C-1,16C-2 R. etli + ÿ 2b (I) ÿ/ÿ
16NJ-1 R. etli + ÿ 2b (II) ÿ/ÿ
16NJ-2 R. etli + ÿ 2b (II) ÿ/+
16PR-1 R.l.viceae ÿ ÿ 2b (III) ÿ/ÿ
17C-2 R. etli + ÿ 2b (I) ÿ/ÿ
17C-3 R. etli ÿ ÿ 2b (I) +/ÿ
17NJ-1 R. giardinii ÿ ÿ mb +/+
17NJ-2 R. etli + ÿ 2b (II) ÿ/ÿ
17PR-1 R. etli ÿ ÿ 2b (II) +/ÿ
17PR-2 R. etli + ÿ 2b (II) +/ÿ
18C-1 S. fredii + ÿ mb (I) ÿ/ÿ
18C-3 S. fredii + ÿ mb (II) ÿ/ÿ
18PR-2 R. giardinii ÿ ÿ mb (III) +/+
21C-1,21C-2, R. etli + + 2b ÿ/ÿ
21NJ-1,21NJ-2, 21PR-1,21PR-2
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
aDesignation of the isolates (Section 2.3, Sampling and rhizobia strain isolation). bOn the basis of ARDRA.
cEectiveness evaluation on cv. Canellini, ++ equal to N-fertilized control, + equal or superior to TAL1121,ÿineective,not tested on Canellini. LPS, (2b) two bands, (mb) multiband. In brackets are indicated dierent types of LPS pro®les within each soil sample.
15C-4, 18C-1 and 18C-3 were considered Sinorhizo-bium fredii-like, since they had 16S rDNA patterns matchingS. fredii, although they did not nodulate soy-bean cv. Williams (data not shown). The isolate 16PR-1 has been ascribed to R. leguminosarum bv. viceae, and eectively nodulated plants of Vicia ervilia (data not shown).
R. etli strains were trapped by all the bean cultivars
used. However, other bean Rhizobium spp. (except R.
tropici) were mainly detected by using cv.
Negroja-mapa 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.
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 7.3 0 17.5 15.5
R. etli4C-2 4.1 3.0 16.5 6.0
R. etli4C-4 5.9 5.0 13.5 19.6
R. gallicum4NJ-1 1.6 3.0 4.5 20.4
R.l. phaseoli4NJ-2 4.8 3.5 9.0 18.9
R. etli4PR-1 3.3 3.5 9.0 10.3
R. etli4PR-2 4.7 2.5 9.0 12.5
R. etli5C-4 1.4 3.0 5.5 14.7
R. giardinii5NJ-1 1.5 4 4.5 9.3
R. gallicum5NJ-2 4.1 5.0 14.0 12.5
R. etli6C-1 4.6 3.5 7.5 8.1
R. etli6NJ-1 4.0 4.5 9.0 16.0
R. etli6NJ-2 2.9 3.5 4.5 14.6
R. etli6PR-1 5.4 4.5 12.0 20.4
R. etli6PR-2 3.0 3.0 6.0 13.7
R. etli8C-3 4.6 4.0 11.5 7.3
R. etli8NJ-1 4.8 3.5 9.5 16.7
R. etli8NJ-2 5.2 3.5 13.0 30.5
R. etli14C-1 6.2 3 10.5 14.1
R. gallicum14C-3 4.1 3.0 13.0 8.8
S. fredii15C-4 3.0 5.0 2.0 4.4
R. etli16C-1 4.1 4.0 8.0 3.8
R. etli16C-2 3.9 3.0 6.0 6.9
R. etli17C-2 4.6 4.0 8.0 19.9
R. etli17C-3 2.3 4.5 7.0 13.8
S. fredii18C-1 2.4 3.5 1.0 2.5
S. fredii18C-3 2.5 4.5 7.5 4.1
R. etli21C-1 4.4 3.5 11.5 7.0
R. etli21C-2 5.1 3 13.5 28.6
R. l. phaseoli 3.3 4.5 5.0 7.0
TAL1121
Untreatedd 1.3 0 1.5 8.3
LSD (0.05) 2.2 ÿ 8.7 11.8
a
Data represent mean value of two replicates (four plants). b
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
3.3. Symbiotic eectiveness under greenhouse conditions
The symbiotic performance of the bean isolate col-lection was evaluated on cv. Canellini under green-house conditions. Two experiments were carried out on dierent dates, which may explain the dierences observed in dry matter accumulation by the control treatments (non-inoculated and inoculated with the reference strain TAL1121). Nevertheless, plants inocu-lated with the reference strain TAL1121, as well as the non-fertilized (untreated) plants, yielded the same pro-portional biomass in relation to the N-fertilized plants in both trials (Tables 3 and 4). Of the isolates 80%
were eective nitrogen ®xers. Inoculation of some iso-lates, such as 4C-4, 6PR-1, 8NJ-2, 14C-1, 15NJ-1, and 21PR-2 led to plant growth that did not signi®cantly dier 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 eec-tive isolates (mentioned above). Those isolates that did not produce signi®cantly better plant growth P<
0:05than the untreated plants were classed as
ineec-tive. 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
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 H.I. (%)
N-Fertilizedd 14.1 0 13.5 0.8
R. etli8C-4 10.2 4.0 16.0 6.6
R. etli8PR-2 9.9 5.0 19.5 5.3
R. etli14NJ-1 8.0 5.0 8.5 2.7
R. etli14NJ-2 5.3 4.5 7.5 1.0
R. etli14PR-1 7.5 4.0 16.0 2.4
R. etli14PR-2 9.9 3.5 17.5 3.1
R. gallicum15NJ-1 10.9 4.5 19.0 3.9
R. gallicum15NJ-2 6.2 4.5 6.0 1.3
R. l. viceae16NJ-1 6.9 4.5 10.0 3.5
R. etli16NJ-2 10.5 4.5 17.0 4.6
R. etli16PR-1 2.6 2.5 2.0 1.0
R. giadinii17NJ-1 1.9 1.0
R. etli17NJ-2 7.0 3.5 16.5 5.7
R. etli17PR-1 2.4 1.0 1.5 0.4
R. etli17PR-2 5.0 4.5 10.0 2.0
R. giardinii18PR-2 2.0 1.0
R. etli21NJ-1 9.6 5.0 13.5 2.6
R. etli21NJ-2 9.6 4.5 14.0 2.6
R. etli21PR-1 9.9 5.0 15.0 4.2
R. etli21PR-2 11.7 5.0 21.0 7.7
R. l. phaseoliTAL1121 6.6 4.0 9.0 1.7
R. tropiciCIAT899 4.3 3.0 8.0 11.9
R. etliCFN42 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. b
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
in Tables 3 and 4. Ineective 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 toR. gallicum, except 4NJ-1, as well as isolates assigned to S. fredii-like were eective on cv. Canellini. R. leguminosarum
bv. phaseoli (4NJ-2) did not dier from the control
strain TAL1121. By contrast R. leguminosarum bv.
viceae (16PR-1) and all R. giardinii isolates were
in-eective (Table 2).
3.4. Melanin production
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.
legumino-sarum 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 theR. etli isolates (51%) were melanin pro-ducers. Isolates of this species from soils no. 16 and 17, and most of isolates from soil no. 6 did not pro-duce melanin under the assay conditions described. By
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. giardiniiand 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 rhizo-bia nodulating soybean.
3.5. Cultural characteristics
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 toR. 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
Table 5
Intrinsic antibiotic resistance pattern of bean rhizobia isolates and the corresponding reference strainsa
Antibiotics(mlÿ1) R. etli(32) CFN42 R. gallicum(5) R602 R. giardinii(3) H152
Chloramphenicol (5) 14 R 5 R 3 R
Erythromycin (10) 15 R 5 R 3 R
Gentamicin (5) 6 R 5 R 0 R
Kanamycin (5) 24 S 5 R 1 R
Neomycin (20) 3 S 5 R 2 R
Novobiocin (2) 25 R 4 R 3 R
Rifampin (5) 7 R 1 S 1 S
Streptomycin (3) 30 R 5 R 0 S
Spectinomycin (5) 19 S 0 R 0 S
Tetracycline (0,2) 7 S 4 R 3 R
aNumerical values indicate the number of resistant isolates; () total number tested; R, resistant; S, sensitive.
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 buered TY at pH 4.5, except 4PR-2, which grew 2 d after inoculation (DAI), as did CFN42 and CIAT899. Twelve R. etli isolates
andR. gallicum4NJ-1 were able to grow at pH 5.0, as
did the reference strains TAL1121, CFN42 and CIAT899, but at dierent 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 eachRhizobiumspp. 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 dierent re-sistance patterns have been recorded between the R. etli isolates. Even more, among isolates from a given location, dierent resistance patterns were recorded.
In general, R. etli isolates were resistant to strepto-mycin, novobiocin, kanamycin and spectinomycin. R.
gallicum isolates showed a totally dierent resistance
pattern than R. etli isolates, and three dierent ®nger-prints could be detected among them. As shown in Table 5 they are resistant to practically all the anti-biotics tested, except spectinomycin. Each one of the three isolates ascribed to R. giardinii species showed a
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.
dierent 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
mostR. etliisolates.
3.7. LPS pro®les
The electrophoretic mobility of LPS on polyacryl-amide 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 multi-band ladder (mb-LPS) (Fig. 2, lanes 3, 7 and 8) in ad-dition 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 beanRhizobium spp. other than R. etli (R.
gallicum14C-3, 15NJ-1 and 15NJ-2;R. giardinii
17NJ-1 and 17NJ-18PR-2) or toS. fredii-like isolates (15C-4, 18C-1 and 18C-18C-3). The isolates assigned to R. gallicum or R. giardinii, share the same LPS-pro®le that the corre-sponding reference strain (Fig. 3, lanes 4±7 and 8±10, respectively). In contrast, the LPS-pro®le ofR. etli iso-lates 4C-4 and 14PR-2 were completely dierent from that produced by the type strain CFN42 (Fig. 3, lanes 1, 2 and 3). The isolates consideredS. frediilike 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 indis-tinguishable by LPS (Fig. 2, lanes 1 and 2), however, their protein pro®les were clearly dierent (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. etliisolates 6NJ-2 and 6PR-2 seem to be the same strain (Table 2), however, their protein pro®les were dierent (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 toR. 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 mix-tures consisting of pairs of isolates from the same soil, each representing a type of LPS-pro®le, were inocu-lated on cv. Canellini plants. Thus, competition studies were performed with isolates from soils no. 4, 14 and 17 (Table 6).
Four dierent combinations were examined with lates from soil No. 4: 4C-4 (mb-LPS) with 2b-LPS iso-lates (4C-2, 4NJ-1, 4NJ-2 and 4PR-2). Table 6 shows
thatR. etli 4C-4 clearly outcompetedR. gallicum
4NJ-1 and R. etli 4PR-2, both 2b-LPS isolates occupied less than 20% of the nodules. In competition with 4NJ-2R. leguminosarumbv.phaseoli, 4C-4 was equally competitive; but in combination with the other Canel-lini isolate (4C-2) was signi®cantly P>0:95less
com-petitive. 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 bothR. etli com-petitors (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 dierent LPS-pro®le on bean cv. Canellini
Inoculum strains A/B Rhizobiumspp. LPS A/B No. nodules identi®ed % of nodule occupancy Identi®cation trait A/B
A B
4C-4/4NJ-1 etli/gallicum mb/2b 90 84 16 GenS/GenR
4C-4/4PR-2 etli/etli mb/2b 92 88 12 Melÿ/Mel+
4C-4/4NJ-2 etli/phaseoli mb/2b 63 57 43 EryS/EryR
4C-4/4C-2 etli/etli mb/2b 93 39 61 EryS/EryR
No.14,R. etli 14PR-2, was signi®cantly P>0:95less
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 ineective and easily dis-tinguishable from eective nodules. Most of the nodules formed by this mixed inoculant were eective, thus presumably formed byR. etli17NJ-2.
In summary, in all the combinations of R. etli/galli-cumor of R. etli/giardiniitested, isolates ascribed to R. etliwere more competitive nodulators onPhaseoluscv. 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 givenRhizobium 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 culti-vars 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 con-taining 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 thatR. 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 deter-mining the outcome of competition experiments, it would be necessary to con®rm the superior competitive capacity ofR. etlistrains with others bean cultivars.
Even though more isolates must be analyzed, it seems that the characterization of bean rhizobia iso-lates by ARDRA can dierentiate between Rhizobium
spp. nodulating bean as has been done by SantamarõÂa et al. (1997). Isolates of this collection have shown a high variability in N2-®xation eciency in combination
with dierent plant genotypes (RodrõÂguez-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 nodu-lation 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 (MartõÂnez-Romero 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 cul-tural 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 dierent Bradyrhizobium sp. VaÂs-quez-Arroyo et al. (1998) have dierentiated 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 com-puter programs are available to analyze pro®le simi-larities these were not found to be useful for our purpose. This technique has been used in our studies to show up dierences 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 excep-tions, shared as well the intrinsic antibiotic resistance pattern. This re¯ects good agreement between the dierent methodologies employed for strain identi®-cation, although there was no correlation between the antibiotic resistance grouping and PCR-RFLP, in fact 16 dierent 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).
LPS-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-Cer-vera 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 high-light 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 ofR. etliin 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 Span-ish 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 dierent strains ofR. 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 abil-ity. Since this is not the situation in the Andalusian region, the evaluation of the native populations in terms of symbiotic performance and competitive beha-viour might lead to the selection of superior strains, well adapted to local conditions and or bean varieties. Some isolates from this collection have provided prom-ising results under ®eld conditions with green bean varieties.
Acknowledgements
We are grateful to Dr. Jose Antonio Herrera for as-sistance with the ARDRA analysis, Dr. Noelle Amar-ger for providing some type strains and Dr. J.E. RuõÂz-SaÂinz for his helpful suggestions. Financial support was provided by the National Institute of Agricultural Research (INIA-MAPA)
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