Field Crops Research 73 (2002) 121±132

Selection of bean (Phaseolus vulgaris L.) rhizobial

strains for the Brazilian Cerrados
Lilian Mostassoa,1, Fabio L. Mostassoa,1, Beatriz G. Diasa,1,
Milton A.T. Vargasb, M. Hungriaa,*
a
Embrapa Soja, Cx. Postal 231, 86001-970 Londrina, PR, Brazil
b
Embrapa Cerrados, Cx. Postal 08223, 73301-970 Planaltina, DF, Brazil
Received 26 November 2000; received in revised form 18 September 2001; accepted 24 September 2001

Abstract

The common bean crop (Phaseolus vulgaris L.) occupies 5.5 million hectares in Brazil and provides about 30% of the
population's protein needs. Yield remains low mainly due to limited availability of N and P, and to the often acid soil
conditions. Surprisingly, there have been only very limited studies in Brazil in which the isolation and evaluation of ef®cient
N2-®xing bean strains has been attempted. This paper reports the selection of bean rhizobia for the ``Cerrados'', the savannas
that represent one-fourth of Brazilian land. Two-hundred strains were selected from large pink-colored nodules collected in the
Cerrados region and biological nitrogen ®xation was evaluated under optimal (27/21 8C, day/night) and high (37/21 8C)
temperature conditions. Thirty-six strains were selected and their nitrogen-®xing capacity and competitiveness further
evaluated with black-seeded ``Negro Argel'', and colored ``Carioca'' cultivars. One-®fth of the strains showed low genetic
stability of nodulation genes, but some strains were as or more competitive than the strains currently recommended for use in
commercial bean inoculants in Brazil. The superior performance of ®ve strains was con®rmed under ®eld conditions and re-
inoculation in the second year increased bean yield. The DNA ®ngerprintings obtained by the ERIC±REP-PCR analyses
indicated a high level of genetic diversity, and among the 36 strains, six different patterns of RFLP-PCR of the 16S rRNA gene
region were detected. The 16S rDNA sequences of the most ef®cient and competitive strains were genetically similar to
Rhizobium tropici, suggesting that further studies on inoculant strains in the hot and acid soils of the Brazilian Cerrados and
Africa should emphasize this species. # 2002 Published by Elsevier Science B.V.

Keywords: Common bean; Competitiveness; Nitrogen ®xation; Rhizobium; Strain selection

1. Introduction substitution of chemical inputs by a more effective

use of natural resources. Biological nitrogen (N2)
Modern agriculture aims to increase crop yields to ®xation ®ts well in this model as it is a more envir-
satisfy the needs from a growing population, but to onmentally clean way of satisfying plant N needs.
use sustainable approaches that should include the The common bean (Phaseolus vulgaris L.) crop,
here referred to simply as bean, is very important in
* South America, especially in Brazil, where 5.5 million
Corresponding author. Tel.: ‡55-43-371-6081;
fax: ‡55-43-371-6100.
hectares are planted annually, supplying about one-
E-mail address: hungria@[Link] (M. Hungria). third of population's protein needs. Yield is low,
1
Fellowship student from EC-INCO. averaging only 710 kg ha 1 (IBGE, 1996), mainly

0378-4290/02/$ ± see front matter # 2002 Published by Elsevier Science B.V.

PII: S 0 3 7 8 - 4 2 9 0 ( 0 1 ) 0 0 1 8 6 - 1
122 L. Mostasso et al. / Field Crops Research 73 (2002) 121±132

due to poor cropping practices, and to the low avail- were provided by Dr. E. MartõÂnez, CFN, Cuernavaca,
ability of N (Hungria et al., 1997). Several restraints to Mexico. R. tropici strains PRF 35, PRF 54 and PRF 81
the improved biological N2 ®xation rates in beans have (ˆ SEMIA 4080) came from the Embrapa Soja germ-
been reported. They include poor nodulation capacity, plasm bank. R. leguminosarum bv. phaseoli USDA
competitive but inef®cient indigenous rhizobia and 2671 (ˆRCR 3644) was provided by Dr. P. van
lack of response to inoculation under ®eld conditions Berkum, Beltsville, MD, USA. R. giardinii bv. giar-
(Graham, 1981; Pereira et al., 1984; Buttery et al., dinii strain H152T and R. gallicum bv. gallicum strain
1987; Hardarson, 1993). However, an approach not R602T were provided by Dr. N. Amarger, INRA,
often considered is the isolation and assessment of Dijon, France.
ef®cient strains from local sites of bean production. A
recent selection program in Brazil has identi®ed an 2.2. Isolation of bean rhizobial strains
ef®cient and competitive Rhizobium tropici strain,
PRF 81, now commercially recommended for this A total of 200 isolates were obtained from the
crop (Hungria et al., 2000); a more continuous pro- Brazilian Cerrados Region, in the state of GoiaÂs
gram of strain selection could help to reverse the (GO), and in the Distrito Federal (DF, Federal Dis-
actual situation of low bean yield and depletion of trict). Strains were isolated from large pink-colored
soil N. nodules of ®eld-grown bean plants using standard
The ``Cerrados'', an edaphic type of savanna, cov- microbiological methods (Vincent, 1970). The purity
ers about 25% of Brazilian land, occupying 207 of the cultures was con®rmed by repeated streaking on
million hectares in the central region of Brazil. Beans yeast extract±mannitol agar (YMA) medium (Vincent,
are grown over nearly 1.2 million hectares of Cerra- 1970) with con®rmation of single type of colony
dos, with yields in the region averaging only morphology, absorption of Congo red (25 mg ml 1),
587 kg ha 1 (IBGE, 1996). Production is mainly by Gram stain reaction and growth rate. Bacteria were
small farmers and uses minimal inputs (Hungria et al., maintained in YM mixed with glycerol (1/1, v/v) and
1997). The Cerrados are quite distinct from other areas stored at 80 8C; working cultures were maintained
of Brazil, being characterized by environmentally on YMA slants at 4 8C.
inhospitable conditions, especially long periods of
water stress and high temperatures (>40 8C), as well 2.3. Nitrogen ®xation capacity under optimal and
as low pH (<5.0), aluminum toxicity and low soil P high temperature conditions
levels (Hungria and Vargas, 2000). These environ-
mental conditions can limit N2 ®xation with bean and The 200 isolates were evaluated for N2 ®xation
Rhizobium strain selection programs that emphasize capacity under greenhouse conditions. Seeds of culti-
tolerance to these speci®c constraints needed. In this var Carioca were surface-sterilized (Vincent, 1970)
paper, the selection of bean rhizobial strains for the and were inoculated with a single strain. Inoculants
Brazilian Cerrados is reported. The isolates were also were prepared in YM at a concentration of
genetically characterized in an attempt to de®ne para- 109 cells ml 1 and each seed was soaked for 20 min
meters that could assist future selection programs. in l ml of inoculum. Five seeds were planted per
sterilized Leonard jar containing sand and vermiculite
(1/2, v/v) and thinned to two plants per pot after
2. Material and methods germination. Plants were grown at 27.5/21.3 8C, aver-
age day/night temperature, received N-free nutrient
2.1. Bean rhizobia used as reference strains solution (Andrade and Hamakawa, 1994) and were
harvested 5 weeks after emergence. Controls included
Bean rhizobia reference strains in this study non-inoculated plants with or without mineral N
included: R. tropici IIA CFN 299 (ˆUSDA 9039, [30 mg of N (as KNO3)/plant/week] and plants inocu-
ˆLMG 9517), IIB CIAT 899T (ˆUMR 1899, ˆUSDA lated with strains CIAT 899 and PRF 81; the strains
9030, ˆTAL 1797, ˆHAMBI 1163, ˆSEMIA 4077, currently used in Brazilian commercial bean inocu-
ˆATCC 49672) and R. etli CFN 42T (ˆUSDA 9032) lants. Nodule number and dry weight, shoot dry
 L. Mostasso et al. / Field Crops Research 73 (2002) 121±132 123

weight and total N (N-Kjeldahl) accumulated in terracing. Five days before sowing, plots received
shoots were analyzed. 84 kg P ha 1, 60 kg K ha 1 and 40 kg ha 1 of micro-
N2 ®xation capacity under high temperature con- nutrients (containing (in %): Zn, 9.0; B, 1.8; Cu, 0.8;
ditions was also evaluated in another experiment Fe, 3.0; Mn, 2.0; Mo, 0.10). The experiment was
performed with the 200 strains grown under root carried out in a soil that had not been cultivated with
controlled temperature conditions (37 8C for 8 h of beans for 3 years, but nevertheless included signi®cant
day/21 8C, day/night) as described before (Hungria bean rhizobia. Rhizobia were estimated, in the 0±
et al., 2000). Conditions other than root temperature 20 cm depth layer, using the bean cultivar Carioca
were as described above. and the most probable number counting technique
(MPN, Vincent, 1970) in jars containing nutrient
2.4. Nitrogen ®xation capacity and competitiveness solution at pH 5.3. The experiment was repeated in
of selected strains the following year in the same plots as before to verify
the effects of re-inoculation. The pH was raised to 5.4
Thirty-six strains were selected from the ®rst two but N content was still 0.13 g cm 3. The experiments
experiments, 25 from GoiaÂs state and 11 from Federal were performed with the cultivar Carioca and ®ve
District (see Tables 1 and 2). These strains showed high-performing strains selected from those used in
best symbiotic performance under normal and high the greenhouse trials were evaluated. The experiments
temperature conditions (data not included). also included non-inoculated controls with or without
The N2-®xing capacity was again evaluated in a N fertilizers (30 kg of N ha 1 (as urea)) at sowing and
greenhouse experiment with the bean cultivars Car- 30 kg of N at 35 days after sowing, as well as a non-
ioca (meso-American colored seed) and Negro Argel nodulating bean line (NORH 54, originally from
(meso-American black seed). The experiment was CIAT, Cali, Colombia). Finally, controls inoculated
performed as described above at a mean temperature with strains CIAT 899 and PRF 81 were also included.
of 26.9/21.8 8C day/night. Non-inoculated controls, The inoculants were peat-based and prepared to a
with and without mineral N, and controls inoculated density of 109 cells g 1 of peat. Inoculants were added
with strains CIAT 899 and PRF 81 were included. to the seeds at a rate of 500 g of inoculant for 50 kg of
The experiment to evaluate competitiveness was seeds with 300 ml of a 10% (w/v) sucrose solution to
also performed under greenhouse conditions, except increase adherence. Each year, nodule number and dry
that each strain (1 ml of 109 cells ml 1 for two seeds, weight, shoot dry weight and total N (Kjeldahl) accu-
soaking for half-an-hour) was inoculated in a propor- mulation in shoots were evaluated in 12 plants/plot at
tion of 1/1 (v/v) with strain CIAT 899. The parameters early ¯owering (35±38 days after emergence). Nodule
evaluated were as described above, but, in addition, 60 occupancy was not evaluated because of cross-
nodules were randomly collected per treatment and reaction with indigenous bean rhizobial strains. Yield
analyzed for serological reaction using anti-serum of was evaluated at the ®nal harvest and values were
CIAT 899 (Somasegaran and Hoben, 1994). corrected for 13% moisture. The experiments were
All greenhouse experiments were performed using performed in a randomized block design with six
a randomized block design with three replicates, with replicates, and data were analyzed by Duncan's test
the results statistically analyzed by Tukey's test at p  0:05†.
p  0:05.
2.6. Genetic characterization of the strains
2.5. Field experiments
2.6.1. Ampli®cation with speci®c (ERIC and REP)
Two ®eld experiments were performed in a Brazi- primers
lian oxisol characterized by a low pH (4.8) and N Total DNA was extracted from each of the 36 strains
content (0.13 g dm 3). Lime was applied 3 months initially identi®ed as having promising performance
before planting, and at sowing pH was 5.3. Experi- and 50 ng used in each ampli®cation by PCR with
mental plots measured 3:0  2:0 m2 , with 0.5 m ERIC and REP primers (de Bruijn, 1992) as described
between rows, and plots separated by 2.0 m with before (Santos et al., 1999). The reactions were carried
124 L. Mostasso et al. / Field Crops Research 73 (2002) 121±132

Table 1
Nodulation and nitrogen ®xation capacity under greenhouse conditions when bean cultivar Carioca was singly inoculated with rhizobial strains
from the Brazilian Cerrados, as well as with R. tropici strains CIAT 899 and PRF 81; also nodule occupancy by each strain when co-inoculated
with CIAT 899a

Isolates Nodulation Shoot Nodule

occupancy (%)
Number (No./plant) Dry weight (g/plant) Dry weight (g/plant) Total N (mg N/plant)

Isolates from GoiaÂs state

J1 150 0.14 0.54 11.3 25
J2 123 0.13 0.38 10.2 30
J3 4 0.01 0.28 2.1 0
J4 3 0.01 0.33 1.6 0
J5 106 0.11 0.43 12.2 32
J 28 294 0.30 2.34 87.0 50
J 41 174 0.18 1.50 46.6 42
J 65 171 0.17 2.36 83.3 45
J 71 285 0.27 1.66 51.3 38
J 76 14 0.05 0.24 8.7 52
J 86 117 0.10 1.17 34.7 30
J 94 8 0.02 0.35 2.9 0
J 95 4 0.01 0.29 2.4 20
J 96 195 0.12 0.61 18.0 0
J 97 208 0.31 1.86 65.8 42
J 112 197 0.22 1.78 55.4 27
J 116 206 0.21 1.00 8.1 38
J 124 119 0.10 0.56 13.6 35
J 137 4 0.01 0.31 2.3 0
J 140 224 0.42 2.82 91.4 42
J 142 3 0.01 0.24 1.4 0
J 144 2 0.01 0.18 1.7 0
J 152 4 0.01 0.26 2.1 0
J 154 2 0.01 0.22 1.5 0
J 155 214 0.36 1.98 71.7 36
Isolates from Federal District
H 12 175 0.14 2.15 100.4 55
H 14 167 0.14 2.42 84.9 53
H 15 247 0.25 2.78 69.9 45
H 20 370 0.32 3.58 132.2 40
H 48 105 0.18 2.15 77.2 55
H 49 130 0.17 2.14 89.1 49
H 51 168 0.16 2.25 93.2 52
H 52 101 0.12 1.96 80.2 50
H 53 171 0.16 2.20 90.1 50
H 54 179 0.17 1.98 82.8 51
H 57 112 0.16 1.92 77.2 47
Strains used as comparison
CIAT 899 114 0.15 2.05 80.4 ±
PFR 81 137 0.17 2.25 92.0 55
Non-inoculated controls
Control N 0 0 0.15 1.6 ±
Control ‡ N 0 0 2.91 90.2 ±

CV (%) 51.4 45.2 15.1 16.2 41

LSD (5%, Tukey) 175 0.18 1.40 14.9 13.8
a
Plants were harvested 5 weeks after emergence and data represent the mean of three replicates.
 L. Mostasso et al. / Field Crops Research 73 (2002) 121±132 125

Table 2
Nodulation and nitrogen ®xation capacity under greenhouse conditions when bean cultivar Negro Argel was singly inoculated with rhizobial
strains from the Brazilian Cerrados, as well as with R. tropici strains CIAT 899 and PRF 81; also nodule occupancy by each strain when co-
inoculated with CIAT 899a

Isolates Nodulation Shoot Nodule

occupancy (%)
Number (No./plant) Dry weight (g/plant) Dry weight (g/plant) Total N (mg N/plant)

Isolates from GoiaÂs state

J1 23 0.04 0.35 6.5 25
J2 47 0.02 0.28 4.1 20
J3 4 0.01 0.32 1.7 0
J4 24 0.04 0.37 4.5 23
J5 4 0.01 0.29 1.4 0
J 28 200 0.15 1.68 59.4 44
J 41 185 0.20 2.26 66.9 47
J 65 165 0.12 1.45 46.5 30
J 71 214 0.08 0.76 16.3 32
J 76 4 0.01 0.32 1.9 0
J 86 170 0.16 2.03 57.6 25
J 94 141 0.10 0.72 16.0 24
J 95 196 0.21 1.32 39.2 28
J 96 282 0.21 0.80 12.2 38
J 97 180 0.13 0.97 14.4 35
J 112 170 0.08 0.70 10.7 28
J 116 5 0.01 0.34 1.9 0
J 124 162 0.08 0.60 5.3 32
J 137 35 0.07 0.59 9.2 20
J 140 204 0.14 1.23 39.6 25
J 142 170 0.08 0.47 12.2 22
J 144 4 0.01 0.36 1.8 0
J 152 56 0.03 0.59 5.5 28
J 154 4 0.01 0.31 1.4 0
J 155 206 0.14 0.95 21.3 32
Isolates from Federal District
H 12 220 0.11 2.08 72.2 51
H 14 210 0.13 1.62 60.1 48
H 15 220 0.15 1.23 48.1 50
H 20 196 0.14 1.99 77.8 45
H 48 137 0.14 1.71 65.8 45
H 49 142 0.16 1.98 74.0 53
H 51 188 0.17 2.29 85.6 40
H 52 189 0.15 1.80 71.4 48
H 53 176 0.14 2.16 80.3 51
H 54 192 0.16 2.24 82.8 50
H 57 186 0.25 2.63 100.2 52
Strains used as comparison
CIAT 899 171 0.12 1.72 62.4 ±
PFR 81 206 0.16 1.89 74.1 50
Controls
Control N 0 0 0.12 1.4 ±
Control ‡ N 0 0 2.71 80.1 ±

CV (%) 39.6 42.1 14.31 14.9 35

LSD (5%, Tukey) 130 0.12 1.24 12.8 12.8
a
Plants were harvested 5 weeks after emergence and data represent the mean of three replicates.
126 L. Mostasso et al. / Field Crops Research 73 (2002) 121±132

out in an MJ Research PT 100 thermocycler and The sequences were then aligned pairwise and com-
ampli®ed fragments were separated by horizontal pared to those of the following organisms (accession
electrophoresis on a 1.5% agarose (low EEO, type No. of the GenBank data library in parentheses):
I-A) gel 17  18 cm2 † at 100 V for 6 h. Rhizobium sp. OR 191 (X91211); R. galegae HAMBI
Cluster analyses were performed with the PCR 540T (Y12355); Rhizobium genomic species Q strain
products with the Bionumerics program (Applied BDV 5102 (Z94806); R. leguminosarum bv. phaseoli
Mathematics, Kortrijk, Belgium), using the algorithm ATCC 8002 (M55494); R. tropici IIB CIAT 899T
UPGMA (unweighted pair-group method, with arith- (U89832); R. tropici IIA LMG 9518 (X67233); R. etli
metic mean) and the coef®cient of Jaccard (J). CFN 42T (U28916); R. giardinii bv. giardinii H152T
(U86344); R. gallicum bv. gallicum R602T (U86343);
2.6.2. Restriction fragment length polymorphism and R. tropici strains PRF 81 (AF260274), PRF 35
(RFLP) analysis of PCR-ampli®ed 16S rDNA genes (AF260298) and PRF 54 (AF260275). A phylogeny
Five replicate quantities of total DNA were ampli- tree was inferred with the UPGMA algorithm using
®ed by PCR with primers Y1 and Y3, which amplify the Bionumerics program.
almost the full-length of the region corresponding to
the 16S rRNA as described before (Chen et al., 2001).
All strains produced a single PCR product with the 3. Results and discussion
expected MW. The PCR products were then digested
with ®ve restriction endonucleases: CfoI, MspI, RsaI, Of the 200 isolates obtained from effective ®eld-
NdeII and HinfI. For each enzyme, a mixture was grown bean nodules in the Brazilian Cerrados, 36 were
prepared containing: 6 ml of the PCR product; 1 ml of selected for good symbiotic performance under opti-
the speci®c buffer for each enzyme (10); 0.5 ml of mal (27.5/21.3 8C, day/night) and high temperature
the enzyme (5 U/reaction) and 2.5 ml of sterile milli-Q (37 8C for 8 h of day/21 8C, day/night) conditions
water. For NdeII, the mixture contained 1 ml of enzyme, (data not shown). Tolerance to high temperature is a
1 ml of DTT (10 mM), 6 ml of the PCR product, 1 ml of major characteristic to be considered in rhizobial
buffer and 1 ml of water. The mixtures were incubated selection program for tropical regions, as in the Bra-
overnight in the water bath at 37 8C. The fragments zilian Cerrados, since soil temperatures regularly
obtained were analyzed by horizontal electrophoresis exceed 40 8C, and often drastically decrease nodula-
in a gel 17  11 cm2 † with 3% of agarose, and carried tion and N2 ®xation rates (Hungria and Franco, 1993;
out at 100 V for 4 h. Cluster analysis was performed Hungria et al., 1993, 2000; Hungria and Vargas, 2000).
as described in the previous item. Selected strains were then evaluated for N2 ®xation
capacity and strain competitiveness, under optimal
2.6.3. 16S rDNA sequence determination temperature conditions (26.9/21.8 8C, day/night), in
The ®ve ef®cient and competitive strains used in the comparison with the two strains currently recom-
®eld experiment were submitted to direct sequencing mended for Brazilian commercial bean inoculants,
of PCR fragments obtained by the ampli®cation with CIAT 899 and PRF 81. The symbiotic performance
primers Y1 and Y3 and intermediate primers as pre- was evaluated using two-bean host plants, previously
viously described (Chen et al., 2001), and the reported as good N2-®xing hosts (Hungria and Neves,
sequences were determined in an ABI 377 (PE- 1987), but of different seed color. It is known that bean
Applied Biosystems) sequencer analyzer. The gener- seeds contain a variety of ¯avonoids related to the seed
ated rDNA sequences were con®rmed forward and color and are responsible for the rhizobial nod-gene
backward. inducing activity (Hungria et al., 1991; Hungria and
Phillips, 1993); therefore, the evaluation with two host
2.6.4. GenBank accession numbers and phylogenetic plants with different seed colors aimed to verify
analysis possible effects of distinct seed ¯avonoids. Good
The 16S rDNA sequences generated were submitted nodulation and N2 ®xation rates were veri®ed with
to the GenBank database ([Link] both cultivars (Tables 1 and 2) with no apparent effect
gov/blast) to seek signi®cant 16S rRNA alignments. of seed color on nodulation.
 L. Mostasso et al. / Field Crops Research 73 (2002) 121±132 127

When cultivar Carioca was inoculated and plants showed a very poor nodulation or lost effectiveness
were grown under greenhouse conditions, several iso- when used as inoculants under greenhouse conditions
lates ®xed as much as or more N than the two R. tropici (Tables 1 and 2).
strains of®cially recommended for use in commer- MPN counts for the ®eld experiment site were
cial bean inoculants (Table 1). Inoculation with 11 carried out using plants grown at the same pH as
different strains resulted in plant N accumulation of the ®eld soil. The area had not been cultivated with
more than 80 mg N/plant (Table 1). All strains identi- beans for 3 years, but still contained 104 bean rhizobia/
®ed as highly ef®cient were also competitive, occupy- g soil. Despite the high population of indigenous bean
ing from 42 to 55% of nodules when co-inoculated rhizobia, inoculation in the ®rst year with ®ve selected
with strain CIAT 899. Ten strains were genetically strains resulted in statistically signi®cant increases in
unstable, losing nodulation and N2-®xation capacity. nodule dry weight and yield (Table 3). In this trial, the
Similar results were obtained for the black-seeded non-nodulating bean line yielded poorly due to the low
cultivar Negro Argel (Table 2). Most of the effective N conditions, and yield in the non-inoculated plots
strains, accumulating more than 60 mg N/plant, were containing indigenous rhizobia was lower than in plots
from the Federal District, with only one strain from inoculated with selected strains. Re-inoculation in the
GoiaÂs state. Again the ef®cient strains were also following year resulted in a further increase in nodu-
competitive, occupying 40±53% of the nodules. In lation and higher yields (Table 3). In each year, the
both the experiments, plant growth and total N accu- yield of treatments inoculated with the strains from
mulated in tissues of beans inoculated with selected Cerrados was not statistically different from that of
strains did not differ statistically from plants receiving non-inoculated plots receiving mineral N. These
mineral N (Tables 1 and 2), and ®ve strains were results are similar to previous reports for Brazilian
selected for the ®eld experiments, H 12, H 20, H 53, H soils (Mendes et al., 1994; Peres et al., 1994; Hungria
54 and H 57. Although selected from effective nodules et al., 1997, 2000), in which inoculation of the bean
of ®eld-grown plants, several strains from GoiaÂs state crop can result in yield increases at low cost to the

Table 3
Effects of bean inoculation and re-inoculation on nodule number (NN) and nodule dry weight (NDW) at ¯owering (38±42 DAE) and grain
yield

Treatment Inoculation in the first year Re-inoculation in the second year

a
NN NDW Yield NN NDW Yielda
(No./plant) (mg/plant) (kg/ha) (No./plant) (mg/plant) (kg/ha)

H 12 51b 68 2344 72 82 2581

H 20 48 79 2322 65 77 2488
H 53 40 75 2054 60 89 2220
H 54 52 82 2328 64 84 2502
H 57 45 71 2100 76 93 2302
CIAT 899 40 58 2095 72 90 2070
PRF 81 51 62 2228 55 65 2350
Control Nc 35 32 1521 38 35 1612
Control ‡ Nc 8d 1d 2498 15d 3d 2600
Non-nodulatinge 0d 0d 383 0d 0d 320
CV (%) 22 17 11 25 21 9
LSD (Duncan, 5%) 13.6 19.3 325 16.7 23.1 342
a
Yield corrected to 13% of moisture.
b
All values represent the mean of six replicates and when followed by the same letter, in the same column, during the same year did not
show statistical difference (Duncan, p  0:05).
c
Non-inoculated control with or without N fertilizer (30 kg/ha of N as urea at sowing and 30 kg of N at 35 days after sowing).
d
Non-nodulating bean line NORH 54.
e
Values not considered for the statistical analyses.
128 L. Mostasso et al. / Field Crops Research 73 (2002) 121±132

farmer. It is important to emphasize that under cerrado Table 4

Patterns of RFLP-PCR detected for the bean rhizobial strains with
conditions re-inoculation was needed in soils that
®ve restriction enzymes
had quite high indigenous population levels. This app-
roach needs to be considered in other tropical regions Isolates Restriction enzymes
of South and Central America and Africa where beans CfoI MspI RsaI NdeII HinfI
are grown.
Isolates from GoiaÂs state
Isolates from this study were also genetically char-
J1 A A A A A
acterized. REP and ERIC consensus sequences have J2 A A A A A
been extensively used in ecology, genetic and taxonomic J3 F F F F F
studies, as well as for rhizobia strain identi®cation J4 D D D D D
(e.g., de Bruijn, 1992; Judd et al., 1993; Selenska- J5 F F F F F
J 28 A A A A A
Pobell et al., 1995; Laguerre et al., 1997; Hungria et al.,
J 41 D D D D D
1998, 2000; Santos et al., 1999). Ampli®cation of J 65 A A A A A
DNA of the 36 strains by PCR with the speci®c J 71 F D D D D
primers ERIC and REP resulted in several products J 76 F F F F D
per strain, except for strains H 51, H 52, J 65, J 96 with J 86 D D D D D
J 94 F D D D D
REP primer and J 1 and J 28 with ERIC primer (Fig. 1).
J 95 D D D D D
Ampli®cation of those strains using different DNA J 96 F D D D D
extractions and lysis procedures was not successful, as J 97 A A A A A
has been described before for some Bradyrhizobium J 112 A A A A A
strains (Judd et al., 1993). For the remaining strains, a J 116 F D D D D
J 124 F D D D D
high level of diversity was observed, since each strain
J 137 F F F F D
showed a unique combination of PCR products J 140 A A A A A
(Fig. 1). Clusters obtained by the analysis of PCR J 142 D D D D E
products con®rmed the observation of Laguerre et al. J 144 D D D D E
(1997) that the technique is useful for strain identi®- J 152 F F F F D
J 154 F F F F D
cation, but not for phylogenetic characterization.
J 155 A A A A A
There are ®ve described bean rhizobial species
today: R. leguminosarum bv. phaseoli (Jordan, Isolates from Federal District
H 12 A A A A A
1984), R. tropici (MartõÂnez-Romero et al., 1991), R. H 14 A A A A A
etli (Segovia et al., 1993), R. gallicum and R. giardinii H 15 A A A A A
(Amarger et al., 1997). Furthermore, other isolates H 20 A A A A A
able to nodulate bean show distinct phylogenic posi- H 48 A A A A A
tions and may well represent other species (Brom®eld H 49 A A A A A
H 51 A A A A A
and Barran, 1990; Eardly et al., 1992, 1995). The H 52 A A A A A
analysis by the RFLP-PCR of the 16S rRNA gene H 53 A A A A A
region has proved to be useful for rhizobial species H 54 A A A A A
designation, with a good agreement with the 16S H 57 A A A A A
rRNA genes (e.g., Laguerre et al., 1994, 1996, Strains used as comparison
1997), although some species are closely related, as CIAT 899TA A A A A
R. tropici and Agrobacterium cannot be distingui PFR 81 A A A A A
CFN 299 B C C C C
shed (Laguerre et al., 1997). In this study, six different
U S D AF D D D D
pro®les of RFLP-PCR of the 16S rRNA gene were 2671
obtained (Table 4). All strains from Federal District CFN 42T B E E E E
and eight from GoiaÂs state had similar pro®les to R602T F B B B B
R. tropici IIB CIAT 899. Five strains from GoiaÂs state H152T F F F F F
had similar pro®les to R. leguminosarum, four others
differed only by the restriction with CfoI and two
 L. Mostasso et al. / Field Crops Research 73 (2002) 121±132 129

Fig. 1. Dendrogram of REP±ERIC-PCR products clustered with the UPGMA method and the Jaccard coef®cient of 37 bean rhizobial strains
from the Brazilian Cerrados and of reference strains of three bean rhizobial species.
130 L. Mostasso et al. / Field Crops Research 73 (2002) 121±132

others with CfoI and HinfI (Table 4); most of those subsequent poor performance indicate that this species
strains showed a poor symbiotic performance (Tables 1 is not necessarily Fix , but can easily undergo muta-
and 2). Two strains had identical pro®les to R. giardi- tion affecting the symbiotic plasmids.
nii, while four others differed only by the pro®le The most effective strains tested under ®eld condi-
obtained with HinfI (Table 4); that species is described tions, H 12, H 20, H 53, H 54 and H 57, were also
as Fix (Amarger et al., 1997), and the strains identi- submitted to the direct sequencing of the 16S rDNA
®ed in this study were also ineffective (Tables 1 and 2). fragment. Strains H 53 and H 54 had identical
It appears therefore that we recovered three bean sequences. Strain H 12 showed a 99% similarity
rhizobial species we tested among the Cerrados iso- (1391/1403 bp) with R. tropici strain IAM 14206
lates. (accession No. D12798), while with strains H 20, H
A survey of bean rhizobia biodiversity was not the 54 and H 57 the similarity was of 98% (1386/1403,
purpose of this study, therefore the genetic analysis 1377/1396 and 1213/1227 bp, respectively) with the
aimed to relate symbiotic effectiveness of selected same strain. The ®ve strains showed mixed properties
strains with rhizobial species. Qualitative evaluations of R. tropici IIA and IIB, as has been described before
of ®eld populations were not performed, however, a for other bean rhizobia strains isolated from Brazilian
recent study with all nodules isolated from bean plants soils (Hungria et al., 2000). All of them were able to
grown in the same ®eld site indicated that despite the grown at pH 4.0 and under high temperature (40 8C),
acid conditions, the highest percentage was of R. etli but H 53, H 54 and H57 were able to grow in LB, while
(M. Hungria, I.C. Mendes, N. Amarger, M. MegõÂas, H12 and H 20 were not (data not shown).
unpublished data). This species was not detected in The phylogenetic tree built with those strains and
our study, suggesting that indigenous R. etli may be strains representative of bean rhizobial species con-
of limited ef®ciency, and were not selected. The ®rmed a higher relatedness with R. tropici species
selection of R. giardinii from effective nodules but (Fig. 2). Strain H 53 showed high relatedness with

Fig. 2. Dendrogram built with the UPGMA algorithm with the aligned 16S rRNA sequences of ®ve ef®cient and competitive bean rhizobial
strains isolated from the Brazilian Cerrados and representative strains of bean rhizobial species. Accession No. of reference strains are listed in
Section 2.
 L. Mostasso et al. / Field Crops Research 73 (2002) 121±132 131

three other Brazilian strains isolated from Parana state, Andrade, D.S., Hamakawa, P.J., 1994. Estimativa do nuÂmero de
ceÂlulas viaÂveis de rizoÂbio no solo e em inoculantes por infeccËaÄo
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R. tropici seems to be native to tropical regions of Brom®eld, E.S.P., Barran, L.R., 1990. Promiscuous nodulation of
South America (MartõÂnez-Romero et al., 1991) and in Phaseolus vulgaris, Macroptilium atropurpureum and Leucae-
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is known by a higher tolerance to high temperatures phosphorus and potassium fertilizer and to inoculation with
(MartõÂnez-Romero et al., 1991; Hungria et al., 1993; Rhizobium. Can. J. Plant Sci. 67, 425±432.
Chen, L.S., Figueredo, A., Pedrosa, F.O., Hungria, M., 2001.
Sa et al., 1993; Mercante et al., 1998; Hungria and
Genetic characterization of soybean rhizobia in Paraguay. Appl.
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and a higher genetic stability than the other bean de Bruijn, F.J., 1992. Use of repetitive (repetitive extragenic
rhizobial species, maintaining its symbiotic properties palindromic and enterobacterial repetitive intergeneric con-
under stressful conditions (SoberoÂn-Chaves et al., sensus) sequences and the polymerase chain reaction to
®ngerprint the genomes of Rhizobium meliloti isolates and
1986; MartõÂnez-Romero et al., 1991; Hungria et al.,
other soil bacteria. Appl. Environ. Microbiol. 58, 2180±
1993; Segovia et al., 1993; Hungria and Vargas, 2000). 2187.
Those are important characteristics to be considered in Eardly, B.D., Young, J.P.W., Selander, R.K., 1992. Phylogenetic
commercial inoculants. position of Rhizobium sp. strain Or 191, a symbiont of both
The most ef®cient and competitive strains from this Medicago sativa and Phaseolus vulgaris, based on partial
sequences of the 16S rRNA nifH genes. Appl. Environ.
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Microbiol. 58, 1809±1815.
R. tropici, therefore adding evidence to the results of Eardly, B.D., Wang, F.-S., Whittam, T.S., Selander, R.K., 1995.
Hungria et al. (2000), that this species seems to be the Species limits in Rhizobium populations that nodulate the
most suitable for the recommendation for tropical common bean Phaseolus vulgaris. Appl. Environ. Microbiol.
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Graham, P.H., 1981. Some problems of nodulation and symbiotic
ful conditions. Furthermore, the identi®cation of ef®-
nitrogen ®xation in Phaseolus vulgaris L.: a review. Field
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Cerrados region can also result in higher yields and Graham, P.H., Draeger, K.J., Ferrey, M.L., Conroy, M.J., Hammer,
help to reduce the soil N depletion. B.E., MartõÂnez, E., Aarons, S.R., Quinto, C., 1994. Acid pH
tolerance in strains of Rhizobium and Bradyrhizobium, and
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M. Hungria acknowledges a research fellowship Hungria, M., Franco, A.A., 1993. Effects of high temperature on
from CNPq (520396/96-0). The authors thank Ligia nodulation and nitrogen ®xation by Phaseolus vulgaris L. Plant
Soil 149, 95±102.
Maria O. Chueire, Luciano Souza, Rinaldo B. Con-
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ceicËaÄo, Rubson N.R. Sibaldelle and Jose Zucca de effects on nitrogen ®xation and transport in Phaseolus vulgaris
Moraes for their technical help. This research was L. Plant Soil 103, 111±121.
supported by EC-INCO (ERBIC18CT980321) and by Hungria, M., Phillips, D.A., 1993. Effects of a seed color mutation
PRONEX-FINEP/CNPq/MCT (41.96.0884.00). on rhizobial nod-gene-inducing ¯avonoids and nodulation in
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