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Genotypic variability for tolerance to salinity of
N_2-xing common bean (Phaseolus vulgaris)
Kaouthar Saadallah, Jean-Jacques Drevon, Mokhtar Hajji, Chedly Abdelly
To cite this version:
Kaouthar Saadallah, Jean-Jacques Drevon, Mokhtar Hajji, Chedly Abdelly. Genotypic variability for
tolerance to salinity of N_2-xing common bean (Phaseolus vulgaris). Agronomie, EDP Sciences,
2001, 21 (6-7), pp.675-682. �10.1051/agro:2001160�. �hal-00886140�
Original article
Genotypic variability for tolerance to salinity
of N
2
-fixing common bean (Phaseolus vulgaris)
Kaouthar SAADALLAH
a
, Jean-Jacques DREVON
b
, Mokhtar HAJJI
c
, Chedly ABDELLY
a
*
a
INRST, Laboratoire d’Adaptation et d’Amélioration des Plantes, Nutrition Minérale, BP 95, Hammam-Lif 2050, Tunisia
b
INRA, Laboratoire Symbiotes des Racines et Sciences du Sol, 1 place Viala, 34060 Montpellier Cedex 1, France
c
Département de Biologie, Faculté des Sciences de Tunis, Campus Universitaire, 1060 Tunis, Tunisia
(Received 16 November 2000; revised 4 May 2001; accepted 5 June 2001)
Abstract – The common bean (Phaseolus vulgaris L.) is often subject to various environmental constraints in the soil. One of these
constraints is salinity which is a major limitation for grain legumes yield, especially when the plant growth depends upon N
2
fixation.
In order to confirm the variability of the response to moderate salinity, and identify the morphological and physiological criteria asso-
ciated with tolerance, 9 common bean lines (BAT477, Coco blanc, DOR585, ABA16, NAG310, Flamingo, BRB17, Candide and
Striker) were inoculated with
Rhizobium tropici CIAT899, and grown in a glasshouse with or without 25 mM NaCl on sterilized
sand. A genotypic variation in tolerance to salt was found: ABA16 and BRB17 were the most sensitive lines whereas BAT477 and
Flamingo were the most tolerant. The estimated parameters of growth and symbiotic nitrogen fixation showed that (i) some lines
which exhibited vigorous growth under the control treatment were greatly affected by salt treatment, (ii) the relative tolerance of
some lines to salt seems to depend on the ability to maintain an adequate leaf area and to develop an abundant and efficient nodular
system.
Phaseolus vulgaris / salinity / N
2
fixation / genotypic variability
Résumé – Variabilité génotypique de la tolérance au sel chez le haricot (
Phaseolus vulgaris), en condition de fixation symbio-
tique de N
2
. Le haricot (Phaseolus vulgaris L.) est souvent soumis dans le sol à diverses contraintes environnementales. L’une des
ces contraintes est la salinité qui représente une limitation majeure du rendement des légumineuses à graines, particulièrement quand
la croissance des plantes dépend de la fixation symbiotique de l’azote. Pour confirmer la variabilité de la réponse à une salinité modé-
rée et identifier les critères morphologiques et physiologiques associés à la tolérance, 9 lignées de haricot (BAT477, Coco blanc,
DOR585, ABA16, NAG310, Flamingo, BRB17, Candide et Striker) sont inoculées avec une souche efficiente
Rhizobium tropici
CIAT899, et cultivées sur sable stérile sous serre vitrée en absence ou en présence de 25 mM NaCl. Une variation génotypique dans
la tolérance au sel a été observée : ABA16 et BRB17 sont les lignées les plus sensibles alors que BAT477 et Flamingo sont les plus
tolérantes. Les paramètres estimés relatifs à la croissance et à la fixation symbiotique de l’azote montrent que (i) certaines lignées
exprimant une croissance vigoureuse en milieu témoin sont sévèrement affectées par le traitement salin, (ii) la relative tolérance au
sel d’autres lignées repose particulièrement sur leur aptitude à maintenir une importante surface foliaire et à développer un système
nodulaire abondant et efficace.
Phaseolus vulgaris / salinité / fixation symbiotique de N
2
/ variabilité génotypique
Agronomie 21 (2001) 675–682 675
© INRA, EDP Sciences, 2001
Communicated by Gérard Guyot (Avignon, France)
* Correspondence and reprints
chedly.abdelly@inrst.rnrt.tn
K. Saadallah et al.
676
1. INTRODUCTION
The limitation of symbiotic nitrogen fixation (SNF)
by environmental constraints, especially salinity,
restricts the development of a sustainable agriculture,
and the extension of this legume cultivation, particularly
in Mediterranean areas. Salinity affects the initiation,
development and function of nodules, as well as the pho-
tosynthetic capacity of leaves, though SNF was found to
be more affected by salt than plant growth [6, 10, 25,
40]. Generally, nodular activity is less affected by salt
than nodulation [2, 13, 30]. Thus, the infection process
seems to be the most sensitive to salt [33, 41].
Nevertheless, a large genetic variability in salt tolerance
was found among legume species and lines [6, 18].
The enhancement of legume productivity in salty
zones requires the development of salt-tolerant symbio-
sis. This approach implies the genetic improvement of
the two partners of symbiosis. However, it is generally
admitted that the
Rhizobium is relatively more tolerant to
salinity than their macrosymbiont [30]. The latter seems
to constitute the most determinant factor for the symbio-
sis efficiency [2, 19].
Exploration of the variability in salt response would
permit us not only to identify some tolerant species and
lines, but also to determine useful criteria for genetic
improvement of salt tolerance. Several works carried out
on interspecific variability showed that the tree legumes,
such as
Prosopis and Acacia spp., are highly tolerant to
salinity [20]. Grain legumes have generally been consid-
ered either sensitive or moderately tolerant to salinity
[17]. Common bean, chickpea, and pea were the most
sensitive legumes [3, 32], whereas soybean was the most
tolerant one [3, 6].
Studies carried out on intraspecific variability in salt
responses are few especially within bean species. Among
19 lines of common bean cultivated for 13 days on a
nutrient solution supplemented with 0, 40 and 80 mM
NaCl, variability was observed for height, number and
dry matter of leaves, stems and roots [38]. Variability in
sensitivity to salt stress was also found among beans
grown in Tunisia, a local line showing a sensitivity index
(SI) of –21% versus –46% for Gabriella [31]. However,
in both studies, the performance of lines under salt stress
which was established in early stages of development
was not maintained in later stages. The aim of the pre-
sent work was to explore the genotypic variability in salt
tolerance in 9 common bean lines and to investigate the
effects of NaCl on the behavior of common bean lines:
growth of the whole plant, leaf area, nodulation and
nitrogen accumulation in different tissues.
2. MATERIALS AND METHODS
2.1. Culture conditions
The common bean lines used in this study were Coco
blanc that is extensively cultivated in the Mediterranean
basin, Candide (Klauss, France), Striker (supplied by C.
Lluch, University of Granada, Spain), ABA16, BAT477,
DOR585, NAG310, Flamingo and BRB17 (supplied by
M. Trabelsi, ESA Mateur, Tunisia, from a collection ini-
tially supplied by B. Voyssest from CIAT, Colombia).
Experiments were carried out in a glasshouse in 1 L pots
filled with sterilized sand. Bean seeds were surface-ster-
ilized in 3% (w/v) calcium hypochlorite for 15 min,
moistened with sterilized water, and inoculated with
1 ml of liquid inoculant containing approximately
10
9
bacteria of Rhizobium tropici CIAT899. The
seedlings were irrigated with the following N-free nutri-
ent solution: KH
2
PO
4
(0.36 mM), CaCl
2
(1.65 mM),
MgSO
4
(1 mM), K
2
SO
4
(0.7 mM), H
3
BO
3
(4 µM),
MnSO
4
(4 µM), ZnSO
4
(1 µM), CuSO
4
(1 µM), CoCl
2
(0.12 µM), NaMoO
4
(0.12 µM), FeEDTA (40 µM). In
addition, plants received 2 mM urea as starter-N supply
during the first 2 weeks, i.e. before nodule emergence.
When the first trifoliate leaf appeared, about 21 days
after sowing (DAS), plants were distributed into two
plots: the first one was irrigated with the above nutrient
solution (control) and the second was watered with the
same solution supplemented with 25 mM NaCl. There
were ten replicates for each line and each treatment.
Plants were irrigated every two days with 100 ml per pot
leading to a light out-flow. The day/night temperatures
and relative humidities were 25/20 ± 5 °C, 65/85 ± 5%,
respectively.
2.2. Measured parameters
Two harvests were made: (i) at the beginning of treat-
ment (21 days after sowing, DAS) and (ii) at the flower-
ing stage, 45 DAS. Plants were separated into leaves,
stems, roots and nodules. Leaf area was measured using
an Area Meter (LI-COR model LI-3000A).
After desiccation at 65 °C during 72 h, the dry weight
of organs as well as the total number of nodules were
determined. The tissue content in total nitrogen was
determined by the Kjeldahl procedure. The symbiotic
nitrogen fixation (SNF) was estimated as the difference
between N quantities (mmol
⋅plant
–1
) at 45 and 21 DAS.
Effect of salinity on N
2
-fixing common bean
677
2.3. Parameters of result analysis
The mean relative growth rate, i.e. the rate of increase
in total dry weight per unit of plant dry weight, was cal-
culated according to the following expression [15]:
RGR = (lnW
2
– lnW
1
) / (t
2
– t
1
), RGR in mg⋅mg
–1
⋅d
–1
);
with W, total plant weight (mg), and t, the time (days);
the subscripts 1 and 2, initial and final harvest.
The net assimilation rate, i.e. the rate of dry matter
production per unit of leaf area, calculated according to
the following expression:
NAR = (W
2
– W
1
) / (t
2
– t
1
) × [ (LA
2
– LA
1
) /
(Ln LA
2
– Ln LA
1
)] in mg DW⋅cm
–2
⋅d
–1
;
with LA, the leaf area (cm
2
); subscripts 1 and 2 denote
respectively initial and final harvest.
The sensitivity index, i.e. the difference between dry
matter production of plants raised in solution containing
25 mM NaCl and the control one expressed in percent of
this latter, was calculated according the following
expression [31]:
SI
NaCl
= [100 × (W
NaCl
– W
control
)] / W
control
.
2.4. Statistical analysis
All values are means of 10 replicates per treatment.
After a two way ANOVA analysis, means were com-
pared at the 0.05 probability level using a Tukey HSD
test.
3. RESULTS
3.1. Growth and leaf area
The bean lines expressed different growth potentials
on control treatment (Fig. 1): Candide, Flamingo,
ABA16 and Coco blanc were significantly more produc-
tive compared to BRB17. Except for BAT477, salt sig-
nificantly decreased (p < 0.05) the growth of all other
lines. In order to rank the lines along a scale of sensitivi-
ty to salt, we calculated the sensitivity index (SI). This
parameter was more negative when the line was sensitive
to NaCl. According to SI values, the lines ABA16 and
BRB17 were sensitive to salt treatment whereas BAT477
and Flamingo were relatively tolerant.
The accumulation of dry matter during a treatment
depends upon the initial size of plants, the treatment
duration and the rate of growth during treatment.
Relative growth rate (RGR) eliminates differences in
biomass production related to treatment duration and/or
initial plant size (at the beginning of salt treatment). For
such reasons, RGR gives a relative basis for comparison
of the effect of salt on plant growth among species and
genotypes [15]. Salt significantly reduced RGR
(
p < 0.05) in all lines, except for Flamingo and BAT477
(Tab. I). The sensitivity index established on the basis of
relative growth rate (not shown) was perceptibly identi-
cal to the one determined on the basis of the final dry
matter. Therefore, the variability of the response to salt
depended more on the growth activity during the salt
treatment than on the initial vigor of plants.
The RGR is a function of the net assimilation rate
(NAR) and the leaf area [4]. In the present study, salt
significantly reduced (
p < 0.05) leaf area for most lines
of common bean (Tab. I). Flamingo and BAT477 were
not affected, whereas ABA16 was the most sensitive to
salt stress. The calculated sensitivity index on the basis
of the leaf area (not shown) was related to those estab-
lished previously. In order to test whether the limitation
of the growth resulted essentially from a reduction of
photosynthetic area, we calculated the net assimilation
ratio (NAR). The data in Table I show that except for
Coco blanc, NAR values were significantly affected
(p < 0.05) by salt. However, genotypic differences were
less than with previous ratios. Our results suggest that
the limitation of plant growth (estimated by dry matter
weight at the end of treatment or RGR) was essentially
attributed to the decrease in photosynthetic area. In
Figure 1. Effect of salt on whole plant growth (g DW⋅plant
–1
)
for 9 lines of common bean. Numbers in histograms corre-
spond to sensitivity index (SI) to salt. Values are means of
10 replicates
± standard deviation. Means with the same letter
are not significantly different (
p < 0.05).
K. Saadallah et al.
678
addition, salt lowered photosynthetic performance for
the remaining leaf area.
3.2. Nodular development and nitrogen status
Salt significantly decreased (p < 0.05) the nodule dry
matter of all bean lines (Fig. 2), though ABA16, BRB17
and Striker were more affected than BAT477, DOR585
and Flamingo. BRB17 maintained the lowest nodular
growth with or without salt. For the majority of lines, the
reduction of nodule dry matter was significantly larger
than that of the nodule number (Fig. 3). Thus, salt inhib-
ited not only the nodulation, but also the nodule growth.
Salt did not significantly modify the N content in
shoots and roots whatever the bean lines (Tab. II). By
contrast, it generally significantly decreased (p < 0.05)
nitrogen accumulation in nodules, except for Flamingo
and ABA16. The reduction of plant growth by salinity,
that was associated with the constant N content, particu-
larly in the shoots that represent the major part of the
plant, suggested that the dry matter production may be
determined by the lines’ capacity to fix N
2
.
SNF was significantly (
p < 0.05) decreased by salini-
ty, except for Flamingo (Fig. 4). BAT477 was also less
affected, confirming the higher salt tolerance of these
two lines compared to other genotypes, particularly
ABA16 and BRB17. The salt-induced decreases in SNF
were higher than those in growth for all lines, suggesting
that the SNF was more sensitive to salt than the host-
plant growth.
Figure 5 shows that Candide, Coco blanc, NAG310,
BRB17, Striker and ABA16 were more affected by salt
for nodule growth and for SNF. The data for these lines
were very close to the regression line, indicating that the
inhibition of SNF by salt constraint was particularly
linked to the decrease in the nodular growth. By contrast,
Table I. Changes in the mean relative growth rate (RGR in mg⋅mg
–1
⋅d
–1
), the net assimilation rate (NAR in mg⋅cm
–2
⋅d
–1
) and leaf
area (LA in cm
2
⋅plant
–1
) with medium salinity. Values are means of 10 replicates ± standard deviation in parenthesis. For each para-
meter, values not followed by the same letter differed significantly at the 0.05 probability level.
Lines Treatments RGR NAR LA
Coco blanc Control 0.08 (0.01) a-c 0.63 (0.08) de 332 (66) a
25 mM NaCl 0.05 (0.01) d-f 0.56 (0.08) e-g 191 (52) c-f
Candide Control 0.07 (0.01) b-d 0.74 (0.07) bc 281 (47) b
25 mM NaCl 0.05 (0.01) ef 0.58 (0.08) ef 179 (39) d-f
ABA16 Control 0.07 (0.02) b-d 0.65 (0.08) c-e 309 (21) ab
25 mM NaCl 0.02 (0.01) g 0.41 (0.09) h 63 (8) h
Striker Control 0.08 (0.01) ab 0.74 (0.11) bc 211 (56) c-e
25 mM NaCl 0.05 (0.02) d-f 0.48 (0.1) f-h 116 (28) g
BAT477 Control 0.07 (0.01) b-d 0.63 (0.07) de 235 (52) c
25 mM NaCl 0.05 (0.02) d-f 0.46 (0.07) gh 204 (35) c-e
DOR585 Control 0.08 (0.02) a-c 0.78 (0.08) ab 237 (45) c
25 mM NaCl 0.05 (0.01) d-f 0.47 (0.07) f-h 165 (44) ef
NAG310 Control 0.09 (0.01) a 0.82 (0.13) ab 193 (16) c-f
25 mM NaCl 0.05 (0.02) ef 0.47 (0.10) f-h 81 (18) h
Flamingo Control 0.07 (0.01) b-d 0.71 (0.05) b-d 241 (27) c
25 mM NaCl 0.05 (0.02) d-f 0.56 (0.09) e-g 230 (43) cd
BRB17 Control 0.08 (0.01) ab 0.87 (0.09) a 146 (37) fg
25 mM NaCl 0.04 (0.01) f 0.49 (0.06) f-h 73 (14) h
Figure 2. Effect of salt on the nodule growth (g DW⋅plant
–1
)
for 9 lines of common bean. Other details as in Figure 1.
![Figure 4. Changes in symbiotic nitrogen fixation SNF (mmol⋅plant–1) with medium salinity. Numbers in histograms correspond to sensitivity index established on the basis of the SNF. SINaCl = [100 × (SNFNaCl – SNFcontrol)]/SNFcontrol; SNF, symbiotic nitrogen fixation estimated as difference between N quantities (mmol⋅plant–1) at 45 and 21 DAS. Other details as in Figure 1.](/figures/figure-4-changes-in-symbiotic-nitrogen-fixation-snf-mmol-2607s6tq.webp)
