Acta Agriculturae Scandinavica Section B Soil and Plant Science, 2012; 62: 179 187
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 Extre´mophiles, 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 plant genotype which together influence the symbio- tic performance (Sadiki and Rabih 2001, Mhadhbi Common bean (Phaseolus vulgaris L.), a traditional et al. 2004). Therefore, inoculation with efficient crop originating from Latin America, is the most rhizobia might improve symbiotic nitrogen fixation important food legume for human consumption (SNF) and productivity of common bean. worldwide, especially in Africa, where its cultivation In the Mediterranean zone, little or no rainfall as a staple food extends into marginal areas. Sym- occurs during extended periods of the year. Tunisia biotic nitrogen fixation (SNF) potential in common is mostly located in the semi-arid, arid and Saharan bean is considered to be low (Pereira and Bliss 1987, climatic zones where the annual rainfall varies Isoi and Yoshida 1991) in comparison with other from 300 to less than 100 mm (Le Houe´rou 1990). grain legumes. However, it may be possible to Water deficits (commonly known as drought) can be improve the SNF potential of common bean (Bliss defined as the absence of adequate moisture neces- 1993, Hardarson et al. 1993). Yield potential of sary for plants to grow normally and complete their legumes depends on the rhizobia association and life cycle (Zhu 2002). The lack of adequate moisture
Correspondence: Samir Aydi, Laboratoire des Plantes Extre´mophiles, 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 Mn, 4 mM Bo, 1.5 mM Cu, 1.5 mM Zn, 0.1 mM for rainfed areas, brought about by infrequent rains and micronutrients. Medium pH was maintained at 7.0 by -3 poor irrigation (Wang et al. 2005). Common bean adding 0.2 g dm CaCO3. It was aerated with a flow appears to be particularly sensitive to this stress of 400 cm3 min 1 of filtered air via a compressor and (Kirda et al. 1989) with considerable reduction in N2 ‘spaghetti tube’ distribution system. Plants were fixation (Ladrera et al. 2007, Sassi et al. 2008b) as a grown in a temperature-controlled glasshouse with consequence of changes in nitrogenase activity and night/day temperatures of c. 20/28 8C, relative nodule biomass (Ga´lvez et al. 2005). humidity 90/75% and a 16 h photoperiod. The Water deficiency and drought directly affect no- irradiance was supplied by mercury vapour lamps dule activity and function (Davey and Simpson (OSRAM HQI-T400W/DH). 1990). Regardless of the physiological mechanism of N2 fixation inhibition by drought stress, there is evidence that legume species have significant genetic Osmotic stress treatments variation in their ability to fix N2 under drought conditions, e.g. Pimentel et al. (1990). Several Osmotic stress was applied by means of 25 mM studies have explained the effect of water stress on mannitol (low osmotic stress level) or 75 mM plant physiology and SNF in common bean (Ramos mannitol (high osmotic stress level). The water et al. 2003, Ga´lvez et al. 2005), nevertheless, few potentials of nutrient solutions were: -0.5 MPa, studies have been conducted under hydroaeroponic 0.7 MPa and -1.2 MPa for control, 25 mM and conditions. Hydroaeroponic environment enables 75 mM mannitol respectively. Mannitol is an osmo- the comparison of different symbiotic associations tic component used generally to generate osmotic and the selection of the most tolerant symbiosis stress when added to nutrient solution. Mannitol was under stressed conditions ( Jebara et al. 2001) which added to 15-day-old plants corresponding to the are the main objectives of this study. initial period of nodules formation and the establish- In Tunisia, Mhamdi et al. (2002) showed that ment of N2 fixation. Simultaneously, nitrogen source P. vulgaris is nodulated by a diversity of species was provided to plants as 1 mL of (Rhizobium tropici ) including Rhizobium gallicum, R. leguminosarum bvs. CIAT 899 or local (Rhizobium gallicum) 8a3 strain Phaseoli and viciae, R. etli, R. giardinii, Sinorhizobium that was previously isolated from the Cap Bon region fredii, S. meliloti and S. medicae and Mnasri et al. in Tunisia, characterized at the phenotypic and (2007) showed the efficiency of the R. gallicum for molecular levels by Mhamdi et al. (1999) and kindly bean cultivation. The present work focused on the provided and maintained in culture in the Labora- enhancement of osmotic stress tolerance of a tory of Legumes Micro-organisms Interactions drought-sensitive cultivar consumed in Tunisia by (LILM), Centre of Biotechnology Borj Cedria inoculation with rhizobia isolated from native soils R. (CBBC). At the beginning of flowering, 30 DAS, gallicum 8a3 and compared with inoculation by the plants were harvested for growth parameters deter- reference strain R. tropici CIAT 899. For that mination and compared with non-stressed plants purpose, we analysed several physiological and bio- (controls). chemical traits in order (i) to look for the main traits inducing osmotic stress tolerance amelioration, (ii) to understand the likely mechanisms involved in such Dry weight and leaf area improvement and (iii) to determine useful criteria for After harvest, different plant parts were separated. genetic improvement of drought tolerance. Leaves, roots and nodules were then weighed for fresh weight determination. Leaf areas were deter- mined with a portable Area Metre (Model LI- Methods 3000A, LI COR). Dry weights (DW) of different Plant growth and conditions for imposing osmotic stress plant parts were determined after drying for 3 days at 70 8C. The biological material was bean (Phaseolus vulgaris L.) seeds of COCOT blanc (provided by M. Trabelsi, ESA Mateur, Tunisia). Seeds were surface sterilized Relative water content and pre-germinated in agar 0.9% then transferred in 1dm3 glass bottles wrapped with aluminium foil to The relative water content of leaves (LRWC) and maintain darkness in the rooting environment. The nodules (NRWC) were measured respectively in the nutrient solution contained 0.25 mM KH2PO4, second or third youngest fully expanded leaf that was 0.7 mM K2SO4, 1 mM MgSO4 ×7H2O, 1.65 mM harvested in the morning and on fresh nodules CaCl2, 22.5 mM Fe for macronutrients, and 6.6 mM harvested at the end of the treatment period. Inoculation with the native Rhizobium gallicum 8a3 181
This parameter was determined using the following Total soluble sugars determination equation: Total soluble sugars were quantified using the anthrone method. The 20 mg DW homogenate in deionized water was incubated in a water bath at RWC % 100 FW DW TW DW ð Þ¼ ½ð Þ= ð Þ 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) FW is the fresh matter weight determined within 2 h and incubated in a boiling water bath. The absor- after the harvest and TW stands for the turgid fresh bance of the samples was determined spectrophoto- matter weight (Schonfeld et al. 1988). TW was metrically at 620 nm using glucose as standard (Aydi obtained after soaking the leaves in distilled water in et al. 2010). test tubes for 4 h at room temperature (c.208C) under low light condition or after 16 h at 4 8C for Extraction and assay of leghaemoglobin nodules. Later, leaves and nodules were quickly and carefully blotted dry with tissue paper for determin- Nodules (100 mg) were homogenized in a mortar ing turgid weight. 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 The water-use efficiency distilled water. The homogenate was centrifuged for 15 min at 500 g, samples of the supernatant were Water-use efficiency in shoots (SWUE) and nodules adjusted to 10 mL by Drabkin’s solution, then (NWUE) means were calculated as the ratios of the centrifuged at 20?000 g for 30 min. The absorbance total dry mass produced over the total water used of supernatant was measured at 540 nm against the (Boyer 1996). Drabkin’s solution (Shiffmann and Lo¨bel 1970).
Proline assay Efficiency in utilization of the rhizobial symbiosis (EURS) Free proline was quantified spectrophotometrically using the method of Bates et al. (1973). The The EURS was estimated by the slope of the protocol is based on the formation of red coloured regression model of shoot biomass as a function of formazone by proline with ninhydrin in acidic nodule biomass. For a linear adjustment-curve, i.e. medium, which is soluble in organic solvents like y ax b, b corresponds to the shoot biomass toluene. production without nodules (g sDW0), and a corre- 1 sponds to the EURS as (g sDW gsDW0)g nDW (Aydi et al. 2004). Nitrogenase activity and nitrogen fixed Three plants from each treatment were used for the Statistical analysis assessment of nodule nitrogenase activity (EC Statistical analysis was carried out using the Statis- 1.7.9.92) estimated by the acetylene reduction assay tica software (version 5, StatSoft, France). The (Hardy et al. 1968). Ten per cent of C H was added 2 2 analysis of variance (ANOVA) and the lowest stan- to the nodulated-root atmosphere and, after incuba- dard deviation (LSD) of the means were used to tion, the rate of ethylene evolution was measured determine statistical significance (p 5 5%) between using a Hewlett-Packard 4890 gas chromatograph treatments. Data are presented as mean values of six equipped with a Porapak-T column. This assay has replicates (three for ARA, total soluble sugar and been shown to be prone to give inaccurate nitrogen proline content) and their corresponding standard fixation (NF) measurements and, therefore, absolute errors. values may not be reliable (Minchin et al. 1983). However, the results obtained herein are consistent with plant biomass and nitrogen content parameters. Results Nitrogen content was determined, according to the Symbioses effects on growth response to osmotic stress Kjeldahl method, at the beginning and the end of the osmotic treatment. Nitrogen fixed was then calcu- Table I summarized growth parameters measured lated as the N content at harvest minus the N on plants growing on control nutrient solution and content of the plants at the onset of the treatment those growing on the same solution with 25 mM (Sassi et al. 2008a). mannitol (low osmotic stress) or 75 mM mannitol 182 S. Sassi-Aydi et al.
(high osmotic stress). Under controlled conditions, Symbioses effects on nodule performance under plant dry weight, leaf area, leaf number and root to osmotic stress shoot ratio (R/S) did not exhibit any changes Nodule growth and NF parameters changes between according to the inoculated rhizobia strain. Simi- control and stressed conditions are given in Table II. larly, the net assimilation rate (NAR) and the In general, under control conditions, no obvious specific leaf area (SLA) showed no significant differences were observed between both studied difference between symbioses. Such data showed symbioses. Furthermore, low osmotic stress level the similar effect of the studied rhizobia strain on (25 mM mannitol) did not discriminate well between cv. COCOT grown under control conditions. Con- symbioses since data did not reveal large differences. versely, after 30 days of exposure to both osmotic Actually, at high stress level (75 mM mannitol), stress levels, a different pattern was observed (Table although the inhibition rates were lower than those I). Independently of the associated rhizobia osmotic of plant growth, osmotic stress induced significant stress limited significantly all growth parameters of inhibition of nodule dry weights (NDW), which in the common bean cultivar. However, it seems that turn strongly inhibited nitrogenase activity assayed symbiosis implicating R. gallicum 8a3 strains was by acetylene reduction activity (ARA). The super- more tolerant than the R. tropici CIAT one. Plant iority of COCOT-8a3 symbiosis is mirrored by dry weight decreased at low (25 mM) and high minor NDW decreases with only 40% paralleled (75 mM) mannitol-induced osmotic stress in both with lower ARA inhibition rates not exceeding 51% symbioses. However, COCOT-8a3 symbiosis exhib- compared with 58% and 72% respectively in ited lower inhibition rate mainly at high stress level COCOT-CIAT symbiosis. In opposition, marked as compared with COCOT-CIAT symbiosis, reach- inhibition was observed in the nodule number ing 42% in the former while it exceeded 60% in the (Nn) mainly in COCOT-8a3 symbiosis under severe latter. This was reflected by keeping better leaf area stressed conditions reaching up to 70%. In addition, and leaf number under stressed conditions. Data the above-mentioned symbiosis nodules kept higher herein showed increased root to shoot ratio (R/S) leghaemoglobin content. Nodule to root ratio (N/R) (Table I). This parameter showed its highest was also determined to verify whether the inhibition increase under 75 mM mannitol in COCOT-8a3 of nodule number under osmotic stress was mainly symbiosis. Decreased net assimilation rate (NAR) due to lower root surface or lower aptitude of nodule and specific leaf area (SLA) were also reported in establishment by roots. The data reported in Table II Table I, this decrease being higher in COCOT- showed decreased N/R with increasing osmotic stress CIAT symbiosis mainly under severe stress condi- level. This result revealed that osmotic stress inhib- tions where inhibition rates reached 71% and 64% ited more strongly the establishment of new nodules respectively. 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 gLDW 1)ina 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 Coco-8a3
Mannitol (mM) 0 25 75 0 25 75
PDW 2.890.2 a 2.190.1 b 0.990.2 d 3.39 0.2 a 2.890.2 b 1.990.1 c Inhibition rate (%) 25 68 15 42 Leaf area 622922 a 506917 b 31099 c 6559242 a 623921 a 4879 12 b Inhibition rate (%) 19 50 12 26 Leaf number 1092a 791b 491c 1092a 892a 691b Inhibition rate (%) 30 60 30 50 R/S 0.290.1 c 0.39 0.1 b 0.490.1 a 0.290.1 c 0.390.1 b 0.590.1 a Inhibition rate (%) 1.5 2 1.5 2.5 NAR 0.1490.1 a 0.119 0.1 b 0.0490.1 c 0.1790.1 a 0.1590.1 a 0.0890.1 b Inhibition rate (%) 21 71 12 53 SLA 650925 a 534921 b 23198 d 679922 a 600921 a 344911 c Inhibition rate (%) 18 64 12 49 Inoculation with the native Rhizobium gallicum 8a3 183
Table II. Effect of osmotic stress on nodule performance: NDW (Nodule dry weight, mg plant 1); nodule number (nodule plant 1); 1 1 1 ANW (average nodule weight, mg nod ); N/R (Nodule to root ratio); ARA (Acetylene reduction activity, mmol C2H4 h plant ); 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 Coco-8a3
Mannitol (mM) 0 25 75 0 25 75
NDW 200918 a 165912 b 9899 d 210915 a 188911 a 131908 c Inhibition rate (%) 18 51 10 38 Nod. number 225924 a 109913 b 8596 c 218919 a 10199b 6695d Inhibition rate (%) 52 62 54 70 ANW 0.990.01 b 1.590.05 a 1.290.05 b 1.090.03 b 1.990.07 a 2.090.08a Inhibition rate (%) 70 30 93 106 N/R 0.2590.1 a 0.2290.1 a 0.1290.1 b 0.2390.1 a 0.2090.1 a 0.1290.1 b Inhibition rate (%) 12 52 13 48 ARA 1992a 1592a 591c 1991a 1792a 891b Inhibition rate (%) 17 72 11 58 Fixed N 892a 591b 292c 893a 692a 491b Inhibition rate (%) 29 71 14 43 Lb 2595a 2195a 1092b 2894a 2594a 1693b Inhibition rate (%) 16 60 11 43 EURS 1.490.3 a 1.390.4 a 0.990.1 b 1.690.4 a 1.590.3 a 1.590.1 a Inhibition rate (%) 934 55
Symbioses effects on water relations under osmotic stress peared to be 3-fold and 5-fold higher in COCOT- CIAT symbiosis and 2-fold and 4-fold higher in To understand how water relations of both sym- COCOT-8a3 symbiosis respectively at low (25mM bioses COCOT-CIAT and COCOT-8a3 were af- mannitol) and high (75 mM mannitol) osmotic fected by osmotic treatment we monitored relative stress levels. Similar trends were reported in nodules water content in leaves (LRWC) and nodules where increased total soluble sugars content (NTSS) (NRWC) in both symbioses. Data from Table III exceeded 1-fold under both osmotic stress levels. On analyses indicated that the control treatments of the contrary, in both symbioses, osmotic stress had a both symbioses showed similar values. Following significant inhibitory effect on proline accumulation exposure to osmotic stress, both parameters de- either in leaves or in nodules; this effect was more creased, this decrease being higher under the high pronounced at higher osmotic stress level Likewise, osmotic stress level. Nevertheless, the effect of data showed no obvious differences between osmotic stress was more pronounced on NRWC as both symbioses in terms of proline accumulation compared with LRWC. The symbiosis COCOT- (Table IV). CIAT 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 Discussion RWUE were affected by both osmotic stress levels while controls showed similar trends in both sym- Rhizobial partner involvement in growth conservation bioses. The one and only difference was observed in under osmotic stress NWUE where no significant change was detected Results presented herein revealed the negative effect with both osmotic stress levels, although reduction of osmotic stress on all growth parameters namely reached 63% as compared with controls. PDW, LA, LN, NAR and SLA. This was in accor- dance with our previous data recently published using four cultivars of common bean submitted to 50 mM Symbioses effects on osmotic adjustment under mannitol (Sassi et al. 2008a). Nevertheless, in the osmotic stress present work, plants were inoculated with two differ- The accumulation of proline and total soluble sugars ent strains separately: (Rhizobium tropici) CIAT 899 either in leaves or in nodules of both symbioses is and the local one (Rhizobium gallicum) 8a3. Under shown in Table IV. Results showed no significant unstressed conditions, both rhizobial partners differences between both symbioses under control showed similar behaviour, indicating no significant conditions. When compared with control leaves, the difference between both studied symbioses: COCOT- accumulation of total soluble sugars (LTSS) ap- CIATand 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 p50.05 according to LSD analysis.
Symbiosis Coco-CIAT Coco-8a3
Mannitol (mM) 0 25 75 0 25 75
LRWC 70913 a 67911 b 3495c 68914 a 61910 a 3194c Inhibition rate (%) 451 10 54 NRWC 80915 a 54910 b 2297d 81916 a 5999b 3595c Inhibition rate (%) 33 73 27 57 PWUE 0.1490.01 a 0.0690.01c 0.0490.01 d 0.1790.01 a 0.19 0.02 b 0.0690.01 c Inhibition rate (%) 57 71 41 65 SWUE 0.3490.02 a 0.2190.03 b 0.1790.01 c 0.2790.02 a 0.2190.02 b 0.1590.01 c Inhibition rate (%) 38 50 22 44 NWUE 0.02290.004 a 0.00890.002 b 0.00290.001 c 0.02290.005 a 0.00890.001 b 0.00890.001 b Inhibition rate (%) 64 91 64 64 those of Tajini et al. (2008) that showed differences under stressed conditions. These results are in between both symbioses under control conditions in agreement with reports that mention importance of the field. This could be associated to various others bacterial partner contribution in symbiotic effective- conditions influencing growth parameters under field ness (Aouani et al. 1997, Mhadhbi et al. 2004, 2008, conditions mainly rhizobial competitiveness (Tajini et Tejera et al. 2004). However, the better performance al. 2008). However, under stressed conditions, our of COCOT-8a3 symbiosis strengthens the impor- results showed different behaviours between both tance of examining the interaction between the symbioses at low and mainly at high osmotic stress diversity of native rhizobia with local cultivars (Tajini levels (Table I). Actually, all growth parameters et al. 2008). This suggests also the involvement of declined in both symbioses, but it seems that reduc- rhizobial strain in nitrogen-fixing capacity, and tions were lower in R. gallicum COCOT symbiosis. therefore selection of a suitable rhizobial partner This could indicate that this symbiosis was able to can increase common bean production through maintain higher growth potentialities even under low improvement of symbiotic nitrogen fixation. As water availability. This can be partially explained by well, our data suggested that the superiority of maintaining higher root to shoot ratio and lower leaf COCOT-8a3 symbiosis was well established at a area reduction (26%) even at high osmotic stress level high stress level (75 mM mannitol) and that this (75 mM mannitol). Indeed, it is possible that under superiority was not mirrored by higher nodule osmotic stress the plants spend more photosynthetic number under osmotic stress since the data showed energy on root production in search of water and/or significant reduction of this parameter under the reducing water loss (Kafkafi 1991), which enables high level osmotic stress condition (Table II). Never- common bean to avoid harmful effects of osmotic theless, this decline was alleviated by both producing stress (Sassi et al. 2008a). bigger nodules (reaching 2-fold higher than respec- tive controls) and maintaining constant the efficiency in utilization of symbiotic rhizobia (EUSR) even Rhizobial partner involvement in maintaining nitrogen under osmotic stressed conditions. Such behaviour fixation under osmotic stress has been widely reported in osmotic stress tolerant To estimate symbiotic effectiveness of both symbioses (Saadallah et al. 2001, Aydi et al. 2004, symbioses, COCOT-CIAT 899 and COCOT-8a3, 2008, Sassi et al. 2008a). Indeed, water stress-tolerant nitrogen-fixing capacity and nodules features were N2 fixation associated with increased individual nodule monitored through 30 days of exposure to low and dry weight was also reported by Serraj and Sinclair high levels of osmotic stress (Table II). It seems that (1998) as a consequence of decreased respiration under control conditions, both symbioses behaved and ureid export resulting in increased carbon similarly showing the typical efficiency of both concentration in larger nodules as compared with rhizobia. On the contrary, when submitted to both well-watered conditions. Thus large nodules will osmotic stress levels, the decline in all NF related favour photosynthate and water allocation, maintain parameters confirms the high contribution of the favourable nodule relative water content and provide rhizobial partner to the symbiotic performance continued supply of water for exporting ureids in Inoculation with the native Rhizobium gallicum 8a3 185
Table IV. Effect of osmotic stress on osmotic adjustment: LTSS (Leaf total soluble sugar, mmol g-1 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 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. 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 Coco-CIAT Coco-8a3
Mannitol (mM) 0 25 75 0 25 75
LTSS 112915 c 324922 b 534927 a 152911 c 376913 b 554926 a TCR 35 24 NTSS 274917 c 312922 b 422932 a 277913 c 300925 b 352921 b TCR 1.1 1.5 1.1 1.3 L Proline 0.590.01 a 0.390.01 b 0.190.01 c 0.590.01 a 0.390.01 b 0.190.01 c IR (%) 40 80 40 80 N Proline 1.490.1 c 2.890.3 b 5.290.5 a 1.790.1 c 2.790.1 b 5.990.4 a TCR 2.0 3.7 1.6 3.5
nodule xylem (King and Purcell 2001). Actually, we viously reported (Sassi et al. 2008b). This could be demonstrated by the presented data that even if the mainly linked to better ureid export from nodules studied symbioses behaved similarly under control being easier by adequate nodule water status (Serraj conditions, they did not have the same performance and Sinclair 1998). It should be noted also that the under osmotic stress. This result was not in accor- maintaining of lower NRWC reduction in COCOT- dance with data of Mhadhbi et al. (2009) and 8a3 symbiosis was mainly attributed to the accumu- Pimratch et al. (2008) who reported that the super- lation of total soluble sugars notably under a high iority of a given symbiosis under stressful conditions osmotic stress level (Table 4). Osmotic stress-in- in terms of high biomass production and nitrogen- duced increased soluble sugar in nodules was re- fixing capacity was mirrored by its behaviour under ported earlier (Fouge`re et al. 1991). It was generally non-stressed circumstances. used for osmotic adjustment (OA). It was also reported that the lowering of the osmotic potential by osmolyte accumulation in response to stress Rhizobial partner involvement in keeping adequate water improves the capacity of the cells to maintain status under osmotic stress physiological processes such as photosynthesis, en- Given that the ability of plants to survive severe zyme activity and cell expansion (Granier et al. 2000, water deficits depends on their ability to restrict Kiani et al. 2007). However, concerning COCOT- water loss (El Jaafari 2000), the reported work CIAT 899 symbiosis the accumulation of soluble scrutinizes the water status of cv. COCOT as sugar was mirrored by more than a 70% reduction in inoculated with either the reference strain CIAT NRWC which suggests that soluble sugar seems not 899 or the local one 8a3 and submitted to increasing to have an important role in OA but their accumula- levels of osmotic stress induced by mannitol. In tion was consistent with the decline in sucrose accordance with growth and NF parameters, no synthase activity previously reported with this sym- significant changes were observed between both biosis (Sassi et al. 2008b). This accumulation pre- studied symbioses under control conditions (Table sents also a metabolic cost due to synthesis and III). Under stressful conditions and mainly under compartmentation of osmolytes (Bajji et al. 2000), higher osmotic stress level the superiority of the which could impede adequate nodule growth. symbiosis COCOT-8a3 was linked essentially to In conclusion, this work confirms the relationship maintaining lower NRWC reductions and constant between osmotic stress tolerance improvement and NWUE at high mannitol concentration in the inoculation with native soil-isolated R. gallicum 8a3 growing medium (75 mM). This demonstrates that as compared with inoculation by the reference strain the superiority of COCOT-8a3 symbiosis in terms of R. tropici CIAT 899. This can be partially explained water relations is well established at nodule level by better water-use efficiency in both leaves and which could be the origin of the maintenance of nodules, better relative water content in nodules and better NF capacity reported by this work. Indeed, better efficiency in utilization of rhizobial symbiosis. relationships between maintaining higher NRWC Consequently, the present study recommends the and better tolerance to osmotic stress were pre- better use of native soil-isolated strains for the 186 S. Sassi-Aydi et al. inoculation of common bean in order to improve its Fouge`re, F., Le Rudulier, D., & Streeter, J. G. (1991). Effect of performance and NF potential under stressful con- salt stress on amino acid, organic acid and carbohydrate composition of roots bacteroids and cytosol of alfalfa ditions. Nevertheless, further research is needed to (Medicago sativa L.). Plant Physiology, 96, 1228 1236. explain osmotic stress tolerance in common bean Ga´lvez, L., Gonza´lez, E. M., & Arrese-Igor, C. (2005). 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