Acta Agriculturae Scandinavica Section B Soil and Plant Science, 2012; 62: 179187
ORIGINAL ARTICLE
Inoculation with the native Rhizobium gallicum 8a3 improves osmotic
stress tolerance in common bean drought-sensitive cultivar
SAMEH SASSI-AYDI, SAMIR AYDI, & CHEDLY ABDELLY
Laboratoire des Plantes Extrémophiles, Centre de Biotechnologie de Borj Cedria, BP 901, 2050 Hammam Lif, Tunisia
Abstract
Symbiotic nitrogen fixation potential in common bean is considered to be low in comparison with other grain legumes.
However, it may be possible to improve the nitrogen fixation potential of common bean using efficient rhizobia. In order to
improve osmotic stress tolerance of a drought-sensitive common bean cultivar (COCOT) consumed in Tunisia, plants were
inoculated either by the reference strain Rhizobium tropici CIAT 899 or by inoculation with rhizobia isolated from native soils
Rhizobium gallicum 8a3. Fifteen days after sowing, osmotic stress was applied by means of 25 mM mannitol (low stress level)
or by 75 mM mannitol (high stress level). Fifteen days after treatment plants were harvested and different physiological and
biochemical parameters were analysed. Results showed no significant differences between the studied symbioses under
control conditions. However after exposure to osmotic stress our results showed better tolerance of COCOT to osmotic
stress when inoculated with the native R. gallicum 8a3. This can be partially explained by better water-use efficiency in both
leaves and nodules, better relative water content in nodules and better efficiency in utilization of rhizobial symbiosis as
compared with COCOT-CIAT 899 symbiosis. Hence, the present study suggested the better use of native soil isolated
strains for the inoculation of common bean in order to improve its performance and nitrogen fixation potential under
stressful conditions.
Keywords: Common bean, improvement, mannitol, nitrogen fixation, osmotic stress.
Abbreviations: ARA, Acetylene reduction activity; DAS, Days after sowing; DW, Dry weights; EURS,
Efficiency in utilization of the rhizobial symbiosis; FW, Fresh matter weight; LRWC, Relative water content of
leaves; NAR, Net assimilation rate; NDW, Nodule dry weights; NF, Nitrogen fixation; Nn, Nodule number;
NRWC, Relative water content of nodules; NWUE, Water-use efficiency in nodules; PWUE, Plant water use
efficiency; SLA, Specific leaf area; SNF, Symbiotic nitrogen fixation; SWUE, Water-use efficiency in shoots;
TSS, Total soluble sugars; TW, Turgid fresh matter weight.
Introduction
Common bean (Phaseolus vulgaris L.), a traditional
crop originating from Latin America, is the most
important food legume for human consumption
worldwide, especially in Africa, where its cultivation
as a staple food extends into marginal areas. Symbiotic nitrogen fixation (SNF) potential in common
bean is considered to be low (Pereira and Bliss 1987,
Isoi and Yoshida 1991) in comparison with other
grain legumes. However, it may be possible to
improve the SNF potential of common bean (Bliss
1993, Hardarson et al. 1993). Yield potential of
legumes depends on the rhizobia association and
plant genotype which together influence the symbiotic performance (Sadiki and Rabih 2001, Mhadhbi
et al. 2004). Therefore, inoculation with efficient
rhizobia might improve symbiotic nitrogen fixation
(SNF) and productivity of common bean.
In the Mediterranean zone, little or no rainfall
occurs during extended periods of the year. Tunisia
is mostly located in the semi-arid, arid and Saharan
climatic zones where the annual rainfall varies
from 300 to less than 100 mm (Le Houérou 1990).
Water deficits (commonly known as drought) can be
defined as the absence of adequate moisture necessary for plants to grow normally and complete their
life cycle (Zhu 2002). The lack of adequate moisture
Correspondence: Samir Aydi, Laboratoire des Plantes Extrémophiles, CBBC, BP 901, 2050 Hammam Lif, Tunisia. Fax: 00216 79 412 638; Email:
[email protected]; [email protected]
(Received 24 April 2011; revised 16 May 2011; accepted 17 May 2011)
ISSN 0906-4710 print/ISSN 1651-1913 online # 2012 Taylor & Francis
http://dx.doi.org/10.1080/09064710.2011.597425
180
S. Sassi-Aydi et al.
leading to water stress is a common occurrence in
rainfed areas, brought about by infrequent rains and
poor irrigation (Wang et al. 2005). Common bean
appears to be particularly sensitive to this stress
(Kirda et al. 1989) with considerable reduction in N2
fixation (Ladrera et al. 2007, Sassi et al. 2008b) as a
consequence of changes in nitrogenase activity and
nodule biomass (Gálvez et al. 2005).
Water deficiency and drought directly affect nodule activity and function (Davey and Simpson
1990). Regardless of the physiological mechanism
of N2 fixation inhibition by drought stress, there is
evidence that legume species have significant genetic
variation in their ability to fix N2 under drought
conditions, e.g. Pimentel et al. (1990). Several
studies have explained the effect of water stress on
plant physiology and SNF in common bean (Ramos
et al. 2003, Gálvez et al. 2005), nevertheless, few
studies have been conducted under hydroaeroponic
conditions. Hydroaeroponic environment enables
the comparison of different symbiotic associations
and the selection of the most tolerant symbiosis
under stressed conditions ( Jebara et al. 2001) which
are the main objectives of this study.
In Tunisia, Mhamdi et al. (2002) showed that
P. vulgaris is nodulated by a diversity of species
including Rhizobium gallicum, R. leguminosarum bvs.
Phaseoli and viciae, R. etli, R. giardinii, Sinorhizobium
fredii, S. meliloti and S. medicae and Mnasri et al.
(2007) showed the efficiency of the R. gallicum for
bean cultivation. The present work focused on the
enhancement of osmotic stress tolerance of a
drought-sensitive cultivar consumed in Tunisia by
inoculation with rhizobia isolated from native soils R.
gallicum 8a3 and compared with inoculation by the
reference strain R. tropici CIAT 899. For that
purpose, we analysed several physiological and biochemical traits in order (i) to look for the main traits
inducing osmotic stress tolerance amelioration, (ii) to
understand the likely mechanisms involved in such
improvement and (iii) to determine useful criteria for
genetic improvement of drought tolerance.
Methods
Plant growth and conditions for imposing osmotic stress
The biological material was bean (Phaseolus vulgaris
L.) seeds of COCOT blanc (provided by M. Trabelsi,
ESA Mateur, Tunisia). Seeds were surface sterilized
and pre-germinated in agar 0.9% then transferred in
1 dm3 glass bottles wrapped with aluminium foil to
maintain darkness in the rooting environment. The
nutrient solution contained 0.25 mM KH2PO4,
0.7 mM K2SO4, 1 mM MgSO4 ×7H2O, 1.65 mM
CaCl2, 22.5 mM Fe for macronutrients, and 6.6 mM
Mn, 4 mM Bo, 1.5 mM Cu, 1.5 mM Zn, 0.1 mM for
micronutrients. Medium pH was maintained at 7.0 by
adding 0.2 g dm-3 CaCO3. It was aerated with a flow
of 400 cm3 min 1 of filtered air via a compressor and
‘spaghetti tube’ distribution system. Plants were
grown in a temperature-controlled glasshouse with
night/day temperatures of c. 20/28 8C, relative
humidity 90/75% and a 16 h photoperiod. The
irradiance was supplied by mercury vapour lamps
(OSRAM HQI-T400W/DH).
Osmotic stress treatments
Osmotic stress was applied by means of 25 mM
mannitol (low osmotic stress level) or 75 mM
mannitol (high osmotic stress level). The water
potentials of nutrient solutions were: -0.5 MPa,
0.7 MPa and -1.2 MPa for control, 25 mM and
75 mM mannitol respectively. Mannitol is an osmotic component used generally to generate osmotic
stress when added to nutrient solution. Mannitol was
added to 15-day-old plants corresponding to the
initial period of nodules formation and the establishment of N2 fixation. Simultaneously, nitrogen source
was provided to plants as 1 mL of (Rhizobium tropici )
CIAT 899 or local (Rhizobium gallicum) 8a3 strain
that was previously isolated from the Cap Bon region
in Tunisia, characterized at the phenotypic and
molecular levels by Mhamdi et al. (1999) and kindly
provided and maintained in culture in the Laboratory of Legumes Micro-organisms Interactions
(LILM), Centre of Biotechnology Borj Cedria
(CBBC). At the beginning of flowering, 30 DAS,
plants were harvested for growth parameters determination and compared with non-stressed plants
(controls).
Dry weight and leaf area
After harvest, different plant parts were separated.
Leaves, roots and nodules were then weighed for
fresh weight determination. Leaf areas were determined with a portable Area Metre (Model LI3000A, LI COR). Dry weights (DW) of different
plant parts were determined after drying for 3 days
at 70 8C.
Relative water content
The relative water content of leaves (LRWC) and
nodules (NRWC) were measured respectively in the
second or third youngest fully expanded leaf that was
harvested in the morning and on fresh nodules
harvested at the end of the treatment period.
Inoculation with the native Rhizobium gallicum 8a3
This parameter was determined using the following
equation:
RWC ð%Þ ¼ 100 ½ðFW DWÞ= ðTW DWÞ
FW is the fresh matter weight determined within 2 h
after the harvest and TW stands for the turgid fresh
matter weight (Schonfeld et al. 1988). TW was
obtained after soaking the leaves in distilled water in
test tubes for 4 h at room temperature (c. 20 8C)
under low light condition or after 16 h at 4 8C for
nodules. Later, leaves and nodules were quickly and
carefully blotted dry with tissue paper for determining turgid weight.
The water-use efficiency
Water-use efficiency in shoots (SWUE) and nodules
(NWUE) means were calculated as the ratios of the
total dry mass produced over the total water used
(Boyer 1996).
Proline assay
Free proline was quantified spectrophotometrically
using the method of Bates et al. (1973). The
protocol is based on the formation of red coloured
formazone by proline with ninhydrin in acidic
medium, which is soluble in organic solvents like
toluene.
181
Total soluble sugars determination
Total soluble sugars were quantified using the
anthrone method. The 20 mg DW homogenate in
deionized water was incubated in a water bath at
70 8C then centrifuged at 3000 g for 10 min. 100 mL
of the supernatant was added to 4 ml of anthrone
solution (0.15 g anthrone in 100mL 80% H2SO4)
and incubated in a boiling water bath. The absorbance of the samples was determined spectrophotometrically at 620 nm using glucose as standard (Aydi
et al. 2010).
Extraction and assay of leghaemoglobin
Nodules (100 mg) were homogenized in a mortar
and pestle with 3 mL Drabkin’s solution. The
Drabkin’s solution is prepared with 52 mg KCN,
198 mg K3Fe(CN)6 and 1 mg NaHCO3 in 1 L
distilled water. The homogenate was centrifuged for
15 min at 500 g, samples of the supernatant were
adjusted to 10 mL by Drabkin’s solution, then
centrifuged at 20?000 g for 30 min. The absorbance
of supernatant was measured at 540 nm against the
Drabkin’s solution (Shiffmann and Löbel 1970).
Efficiency in utilization of the rhizobial
symbiosis (EURS)
The EURS was estimated by the slope of the
regression model of shoot biomass as a function of
nodule biomass. For a linear adjustment-curve, i.e.
y ax b, b corresponds to the shoot biomass
production without nodules (g sDW0), and a corresponds to the EURS as (g sDW g sDW0) g 1 nDW
(Aydi et al. 2004).
Nitrogenase activity and nitrogen fixed
Three plants from each treatment were used for the
assessment of nodule nitrogenase activity (EC
1.7.9.92) estimated by the acetylene reduction assay
(Hardy et al. 1968). Ten per cent of C2H2 was added
to the nodulated-root atmosphere and, after incubation, the rate of ethylene evolution was measured
using a Hewlett-Packard 4890 gas chromatograph
equipped with a Porapak-T column. This assay has
been shown to be prone to give inaccurate nitrogen
fixation (NF) measurements and, therefore, absolute
values may not be reliable (Minchin et al. 1983).
However, the results obtained herein are consistent
with plant biomass and nitrogen content parameters.
Nitrogen content was determined, according to the
Kjeldahl method, at the beginning and the end of the
osmotic treatment. Nitrogen fixed was then calculated as the N content at harvest minus the N
content of the plants at the onset of the treatment
(Sassi et al. 2008a).
Statistical analysis
Statistical analysis was carried out using the Statistica software (version 5, StatSoft, France). The
analysis of variance (ANOVA) and the lowest standard deviation (LSD) of the means were used to
determine statistical significance (p 5 5%) between
treatments. Data are presented as mean values of six
replicates (three for ARA, total soluble sugar and
proline content) and their corresponding standard
errors.
Results
Symbioses effects on growth response to osmotic stress
Table I summarized growth parameters measured
on plants growing on control nutrient solution and
those growing on the same solution with 25 mM
mannitol (low osmotic stress) or 75 mM mannitol
182
S. Sassi-Aydi et al.
Symbioses effects on nodule performance under
osmotic stress
(high osmotic stress). Under controlled conditions,
plant dry weight, leaf area, leaf number and root to
shoot ratio (R/S) did not exhibit any changes
according to the inoculated rhizobia strain. Similarly, the net assimilation rate (NAR) and the
specific leaf area (SLA) showed no significant
difference between symbioses. Such data showed
the similar effect of the studied rhizobia strain on
cv. COCOT grown under control conditions. Conversely, after 30 days of exposure to both osmotic
stress levels, a different pattern was observed (Table
I). Independently of the associated rhizobia osmotic
stress limited significantly all growth parameters of
the common bean cultivar. However, it seems that
symbiosis implicating R. gallicum 8a3 strains was
more tolerant than the R. tropici CIAT one. Plant
dry weight decreased at low (25 mM) and high
(75 mM) mannitol-induced osmotic stress in both
symbioses. However, COCOT-8a3 symbiosis exhibited lower inhibition rate mainly at high stress level
as compared with COCOT-CIAT symbiosis, reaching 42% in the former while it exceeded 60% in the
latter. This was reflected by keeping better leaf area
and leaf number under stressed conditions. Data
herein showed increased root to shoot ratio (R/S)
(Table I). This parameter showed its highest
increase under 75 mM mannitol in COCOT-8a3
symbiosis. Decreased net assimilation rate (NAR)
and specific leaf area (SLA) were also reported in
Table I, this decrease being higher in COCOTCIAT symbiosis mainly under severe stress conditions where inhibition rates reached 71% and 64%
respectively.
Nodule growth and NF parameters changes between
control and stressed conditions are given in Table II.
In general, under control conditions, no obvious
differences were observed between both studied
symbioses. Furthermore, low osmotic stress level
(25 mM mannitol) did not discriminate well between
symbioses since data did not reveal large differences.
Actually, at high stress level (75 mM mannitol),
although the inhibition rates were lower than those
of plant growth, osmotic stress induced significant
inhibition of nodule dry weights (NDW), which in
turn strongly inhibited nitrogenase activity assayed
by acetylene reduction activity (ARA). The superiority of COCOT-8a3 symbiosis is mirrored by
minor NDW decreases with only 40% paralleled
with lower ARA inhibition rates not exceeding 51%
compared with 58% and 72% respectively in
COCOT-CIAT symbiosis. In opposition, marked
inhibition was observed in the nodule number
(Nn) mainly in COCOT-8a3 symbiosis under severe
stressed conditions reaching up to 70%. In addition,
the above-mentioned symbiosis nodules kept higher
leghaemoglobin content. Nodule to root ratio (N/R)
was also determined to verify whether the inhibition
of nodule number under osmotic stress was mainly
due to lower root surface or lower aptitude of nodule
establishment by roots. The data reported in Table II
showed decreased N/R with increasing osmotic stress
level. This result revealed that osmotic stress inhibited more strongly the establishment of new nodules
generation than the root growth itself.
Table I. Effect of osmotic stress on growth parameters: PDW (plant dry weight, g. plant 1); Leaf area (cm2 plant 1); Leaf number (Leaf
plant 1); R/S (root to shoot ratio); NAR (Net assimilation rate, g DW cm 2 day 1) and SLA (Specific leaf area, cm2 g LDW 1) in a
drought-sensitive common bean ‘cv. Coco blanc’ inoculated with CIAT (reference strain) or 8a3 (native strain) and submitted to low (25
mM) and high (75 mM) mannitol-induced osmotic stress during 15 days. Values represent mean9SE (n 6). Numbers followed by a
different letter within a column are significantly different at p 5 0.05 according to LSD analysis.
Symbiosis
Coco-CIAT
Mannitol (mM)
PDW
Inhibition rate
Leaf area
Inhibition rate
Leaf number
Inhibition rate
R/S
Inhibition rate
NAR
Inhibition rate
SLA
Inhibition rate
(%)
(%)
(%)
(%)
(%)
(%)
0
2.890.2 a
622922 a
1092 a
0.290.1 c
0.1490.1 a
650925 a
25
2.190.1 b
25
506917 b
19
791 b
30
0.39 0.1 b
1.5
0.119 0.1 b
21
534921 b
18
Coco-8a3
75
0
25
75
0.990.2 d
68
31099 c
50
491 c
60
0.490.1 a
2
0.0490.1 c
71
23198 d
64
3.39 0.2 a
6559242 a
1092 a
0.290.1 c
0.1790.1 a
679922 a
2.890.2 b
15
623921 a
12
892 a
30
0.390.1 b
1.5
0.1590.1 a
12
600921 a
12
1.990.1 c
42
4879 12 b
26
691 b
50
0.590.1 a
2.5
0.0890.1 b
53
344911 c
49
183
Inoculation with the native Rhizobium gallicum 8a3
1
Table II. Effect of osmotic stress on nodule performance: NDW (Nodule dry weight, mg plant ); nodule number (nodule plant 1);
ANW (average nodule weight, mg nod 1); N/R (Nodule to root ratio); ARA (Acetylene reduction activity, mmol C2H4 h 1 plant 1); Fixed
N (mmol Plant 1); Lb (Leghaemoglobin, mg gFW 1) and EURS (efficiency of utilization of the rhizobial symbiosis) in a drought-sensitive
common bean ‘cv. Coco blanc’ inoculated with CIAT (reference strain) or 8a3 (native strain) and submitted to low (25 mM) and high (75
mM) mannitol-induced osmotic stress during 15 days. Values represent mean9SE (n 6) only for ARA determination (n 3). Numbers
followed by a different letter within a column are significantly different at p 5 0.05 according to LSD analysis.
Symbiosis
Coco-CIAT
Mannitol (mM)
NDW
Inhibition rate
Nod. number
Inhibition rate
ANW
Inhibition rate
N/R
Inhibition rate
ARA
Inhibition rate
Fixed N
Inhibition rate
Lb
Inhibition rate
EURS
Inhibition rate
(%)
(%)
(%)
(%)
(%)
(%)
(%)
(%)
Coco-8a3
0
25
75
200918 a
225924 a
0.990.01 b
0.2590.1 a
1992 a
892 a
2595a
1.490.3 a
165912 b
18
109913 b
52
1.590.05 a
70
0.2290.1 a
12
1592 a
17
591 b
29
2195 a
16
1.390.4 a
9
9899 d
51
8596 c
62
1.290.05 b
30
0.1290.1 b
52
591 c
72
292 c
71
1092 b
60
0.990.1 b
34
Symbioses effects on water relations under osmotic stress
To understand how water relations of both symbioses COCOT-CIAT and COCOT-8a3 were affected by osmotic treatment we monitored relative
water content in leaves (LRWC) and nodules
(NRWC) in both symbioses. Data from Table III
analyses indicated that the control treatments of
both symbioses showed similar values. Following
exposure to osmotic stress, both parameters decreased, this decrease being higher under the high
osmotic stress level. Nevertheless, the effect of
osmotic stress was more pronounced on NRWC as
compared with LRWC. The symbiosis COCOTCIAT exhibited the highest decreases reaching 33%
and 73% respectively at low and high osmotic stress.
In line with RWC data, PWUE as well as SWUE and
RWUE were affected by both osmotic stress levels
while controls showed similar trends in both symbioses. The one and only difference was observed in
NWUE where no significant change was detected
with both osmotic stress levels, although reduction
reached 63% as compared with controls.
Symbioses effects on osmotic adjustment under
osmotic stress
The accumulation of proline and total soluble sugars
either in leaves or in nodules of both symbioses is
shown in Table IV. Results showed no significant
differences between both symbioses under control
conditions. When compared with control leaves, the
accumulation of total soluble sugars (LTSS) ap-
0
210915 a
218919 a
1.090.03 b
0.2390.1 a
1991 a
893 a
2894 a
1.690.4 a
25
75
188911 a
10
10199 b
54
1.990.07 a
93
0.2090.1 a
13
1792 a
11
692 a
14
2594 a
11
1.590.3 a
5
131908 c
38
6695 d
70
2.090.08a
106
0.1290.1 b
48
891 b
58
491 b
43
1693 b
43
1.590.1 a
5
peared to be 3-fold and 5-fold higher in COCOTCIAT symbiosis and 2-fold and 4-fold higher in
COCOT-8a3 symbiosis respectively at low (25mM
mannitol) and high (75 mM mannitol) osmotic
stress levels. Similar trends were reported in nodules
where increased total soluble sugars content (NTSS)
exceeded 1-fold under both osmotic stress levels. On
the contrary, in both symbioses, osmotic stress had a
significant inhibitory effect on proline accumulation
either in leaves or in nodules; this effect was more
pronounced at higher osmotic stress level Likewise,
data showed no obvious differences between
both symbioses in terms of proline accumulation
(Table IV).
Discussion
Rhizobial partner involvement in growth conservation
under osmotic stress
Results presented herein revealed the negative effect
of osmotic stress on all growth parameters namely
PDW, LA, LN, NAR and SLA. This was in accordance with our previous data recently published using
four cultivars of common bean submitted to 50 mM
mannitol (Sassi et al. 2008a). Nevertheless, in the
present work, plants were inoculated with two different strains separately: (Rhizobium tropici) CIAT 899
and the local one (Rhizobium gallicum) 8a3. Under
unstressed conditions, both rhizobial partners
showed similar behaviour, indicating no significant
difference between both studied symbioses: COCOTCIATand COCOT-8a3. This result does not support
184
S. Sassi-Aydi et al.
Table III. Effect of osmotic stress on water relations: LRWC (leaf relative water content,%); NRWC (nodule relative water content,%);
WUE (water use efficiency, g DW ml 1) in a drought-sensitive common bean ‘cv. Coco blanc’ inoculated with CIAT (reference strain) or
8a3 (native strain) and submitted to low (25 mM) and high (75 mM) mannitol-induced osmotic stress during 15 days. L denotes leaves, N
denotes nodules, S denotes shoots and IR denotes inhibition rate (%). Values represent mean9SE (n 6). Numbers followed by a
different letter within a column are significantly different at p 50.05 according to LSD analysis.
Symbiosis
Coco-CIAT
Mannitol (mM)
LRWC
Inhibition
NRWC
Inhibition
PWUE
Inhibition
SWUE
Inhibition
NWUE
Inhibition
rate (%)
rate (%)
rate (%)
rate (%)
rate (%)
0
70913 a
80915 a
0.1490.01 a
0.3490.02 a
0.02290.004 a
Coco-8a3
25
67911 b
4
54910 b
33
0.0690.01c
57
0.2190.03 b
38
0.00890.002 b
64
75
3495 c
51
2297 d
73
0.0490.01 d
71
0.1790.01 c
50
0.00290.001 c
91
those of Tajini et al. (2008) that showed differences
between both symbioses under control conditions in
the field. This could be associated to various others
conditions influencing growth parameters under field
conditions mainly rhizobial competitiveness (Tajini et
al. 2008). However, under stressed conditions, our
results showed different behaviours between both
symbioses at low and mainly at high osmotic stress
levels (Table I). Actually, all growth parameters
declined in both symbioses, but it seems that reductions were lower in R. gallicumCOCOT symbiosis.
This could indicate that this symbiosis was able to
maintain higher growth potentialities even under low
water availability. This can be partially explained by
maintaining higher root to shoot ratio and lower leaf
area reduction (26%) even at high osmotic stress level
(75 mM mannitol). Indeed, it is possible that under
osmotic stress the plants spend more photosynthetic
energy on root production in search of water and/or
reducing water loss (Kafkafi 1991), which enables
common bean to avoid harmful effects of osmotic
stress (Sassi et al. 2008a).
Rhizobial partner involvement in maintaining nitrogen
fixation under osmotic stress
To estimate symbiotic effectiveness of both
symbioses, COCOT-CIAT 899 and COCOT-8a3,
nitrogen-fixing capacity and nodules features were
monitored through 30 days of exposure to low and
high levels of osmotic stress (Table II). It seems that
under control conditions, both symbioses behaved
similarly showing the typical efficiency of both
rhizobia. On the contrary, when submitted to both
osmotic stress levels, the decline in all NF related
parameters confirms the high contribution of the
rhizobial partner to the symbiotic performance
0
68914 a
81916 a
0.1790.01 a
0.2790.02 a
0.02290.005 a
25
61910 a
10
5999 b
27
0.19 0.02 b
41
0.2190.02 b
22
0.00890.001 b
64
75
3194 c
54
3595 c
57
0.0690.01 c
65
0.1590.01 c
44
0.00890.001 b
64
under stressed conditions. These results are in
agreement with reports that mention importance of
bacterial partner contribution in symbiotic effectiveness (Aouani et al. 1997, Mhadhbi et al. 2004, 2008,
Tejera et al. 2004). However, the better performance
of COCOT-8a3 symbiosis strengthens the importance of examining the interaction between the
diversity of native rhizobia with local cultivars (Tajini
et al. 2008). This suggests also the involvement of
rhizobial strain in nitrogen-fixing capacity, and
therefore selection of a suitable rhizobial partner
can increase common bean production through
improvement of symbiotic nitrogen fixation. As
well, our data suggested that the superiority of
COCOT-8a3 symbiosis was well established at a
high stress level (75 mM mannitol) and that this
superiority was not mirrored by higher nodule
number under osmotic stress since the data showed
significant reduction of this parameter under the
high level osmotic stress condition (Table II). Nevertheless, this decline was alleviated by both producing
bigger nodules (reaching 2-fold higher than respective controls) and maintaining constant the efficiency
in utilization of symbiotic rhizobia (EUSR) even
under osmotic stressed conditions. Such behaviour
has been widely reported in osmotic stress tolerant
symbioses (Saadallah et al. 2001, Aydi et al. 2004,
2008, Sassi et al. 2008a). Indeed, water stress-tolerant
N2 fixation associated with increased individual nodule
dry weight was also reported by Serraj and Sinclair
(1998) as a consequence of decreased respiration
and ureid export resulting in increased carbon
concentration in larger nodules as compared with
well-watered conditions. Thus large nodules will
favour photosynthate and water allocation, maintain
favourable nodule relative water content and provide
continued supply of water for exporting ureids in
185
Inoculation with the native Rhizobium gallicum 8a3
-1
Table IV. Effect of osmotic stress on osmotic adjustment: LTSS (Leaf total soluble sugar, mmol g DW); NTSS (Nodule total soluble
sugar, mmol g DW -1); L. Proline (Leaf proline content, mmol FW g-1); N. Proline (Nodule proline content, mmol FW g-1); in a droughtsensitive common bean ‘cv. Coco blanc’ inoculated with CIAT (reference strain) or 8a3 (native strain) and submitted to low (25 mM) and
high (75 mM) mannitol induced osmotic stress during 15 days. TRC denoted treated/control ratio. Values represent mean9SE (n 3).
Numbers followed by a different letter within a column are significantly different at p 5 0.05 according to LSD analysis.
Symbiosis
Mannitol (mM)
LTSS
TCR
NTSS
TCR
L Proline
IR (%)
N Proline
TCR
Coco-CIAT
Coco-8a3
0
25
75
0
25
75
112915 c
274917 c
0.590.01 a
1.490.1 c
324922 b
3
312922 b
1.1
0.390.01 b
40
2.890.3 b
2.0
534927 a
5
422932 a
1.5
0.190.01 c
80
5.290.5 a
3.7
152911 c
277913 c
0.590.01 a
1.790.1 c
376913 b
2
300925 b
1.1
0.390.01 b
40
2.790.1 b
1.6
554926 a
4
352921 b
1.3
0.190.01 c
80
5.990.4 a
3.5
nodule xylem (King and Purcell 2001). Actually, we
demonstrated by the presented data that even if the
studied symbioses behaved similarly under control
conditions, they did not have the same performance
under osmotic stress. This result was not in accordance with data of Mhadhbi et al. (2009) and
Pimratch et al. (2008) who reported that the superiority of a given symbiosis under stressful conditions
in terms of high biomass production and nitrogenfixing capacity was mirrored by its behaviour under
non-stressed circumstances.
Rhizobial partner involvement in keeping adequate water
status under osmotic stress
Given that the ability of plants to survive severe
water deficits depends on their ability to restrict
water loss (El Jaafari 2000), the reported work
scrutinizes the water status of cv. COCOT as
inoculated with either the reference strain CIAT
899 or the local one 8a3 and submitted to increasing
levels of osmotic stress induced by mannitol. In
accordance with growth and NF parameters, no
significant changes were observed between both
studied symbioses under control conditions (Table
III). Under stressful conditions and mainly under
higher osmotic stress level the superiority of the
symbiosis COCOT-8a3 was linked essentially to
maintaining lower NRWC reductions and constant
NWUE at high mannitol concentration in the
growing medium (75 mM). This demonstrates that
the superiority of COCOT-8a3 symbiosis in terms of
water relations is well established at nodule level
which could be the origin of the maintenance of
better NF capacity reported by this work. Indeed,
relationships between maintaining higher NRWC
and better tolerance to osmotic stress were pre-
viously reported (Sassi et al. 2008b). This could be
mainly linked to better ureid export from nodules
being easier by adequate nodule water status (Serraj
and Sinclair 1998). It should be noted also that the
maintaining of lower NRWC reduction in COCOT8a3 symbiosis was mainly attributed to the accumulation of total soluble sugars notably under a high
osmotic stress level (Table 4). Osmotic stress-induced increased soluble sugar in nodules was reported earlier (Fougère et al. 1991). It was generally
used for osmotic adjustment (OA). It was also
reported that the lowering of the osmotic potential
by osmolyte accumulation in response to stress
improves the capacity of the cells to maintain
physiological processes such as photosynthesis, enzyme activity and cell expansion (Granier et al. 2000,
Kiani et al. 2007). However, concerning COCOTCIAT 899 symbiosis the accumulation of soluble
sugar was mirrored by more than a 70% reduction in
NRWC which suggests that soluble sugar seems not
to have an important role in OA but their accumulation was consistent with the decline in sucrose
synthase activity previously reported with this symbiosis (Sassi et al. 2008b). This accumulation presents also a metabolic cost due to synthesis and
compartmentation of osmolytes (Bajji et al. 2000),
which could impede adequate nodule growth.
In conclusion, this work confirms the relationship
between osmotic stress tolerance improvement and
inoculation with native soil-isolated R. gallicum 8a3
as compared with inoculation by the reference strain
R. tropici CIAT 899. This can be partially explained
by better water-use efficiency in both leaves and
nodules, better relative water content in nodules and
better efficiency in utilization of rhizobial symbiosis.
Consequently, the present study recommends the
better use of native soil-isolated strains for the
186
S. Sassi-Aydi et al.
inoculation of common bean in order to improve its
performance and NF potential under stressful conditions. Nevertheless, further research is needed to
explain osmotic stress tolerance in common bean
symbiosis via the better understanding of the osmotic stress effect on limiting nodulation through its
effects on root-hair colonization and infection by
rhizobia.
Acknowledgements
The authors thank Dr Moez Jebara for technical
assistance in measurement of acetylene reduction
activity (ARA) and Laboratoire d’interaction Legumineuses-microorganismes in Centre de Biotechnologie de Borj Cedria (CBBC) for providing rhizobia.
This work was supported by the AQUARHIZ
project: ‘Modulation of plant-bacteria interactions to
enhance tolerance to water deficit for grain legumes in the
Mediterranean dry lands’ FP6 Project INCO-CT2004-509115, and by the Tunisian Ministry of
Higher Education and Scientific Research
(LR10CBBC02).
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