Effect of Sodium Chloride on Growth, Nutrient Accumulation, and Nitrogen Fixation of Common Bean Plants in Symbiosis with Isogenic Strains

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1 Journal of Plant Nutrition, 28: , 2005 Copyright Taylor & Francis Inc. ISSN: print / online DOI: / Effect of Sodium Chloride on Growth, Nutrient Accumulation, and Nitrogen Fixation of Common Bean Plants in Symbiosis with Isogenic Strains N. A. Tejera, 1 R. Campos, 1 J. Sanjuan, 2 and C. Lluch 1 1 Departamento de Fisiología Vegetal, Facultad de Ciencias, Universidad de Granada, Spain 2 Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, CSIC, Granada, Spain ABSTRACT The effect of sodium chloride (NaCl)-salinity on growth responses and tissues organic solutes and mineral content was investigated in common bean plants inoculated with salttolerant Rhizobium tropici wild-type strain CIAT899 and four mutant derivatives having decreased salt-tolerance (DST). Under non-saline conditions two mutants formed partially effective (HB10, HB12) and another two almost ineffective (HB8, HB13) nodules. A great variation of NaCl tolerance in the different symbiosis tested was observed at harvest, 32 day after planting. Common bean plant responded to salinity by decreasing the content of dry plant biomass, nodule number and the nitrogen fixation, and increasing the root to shoot ratio. The salt dose of 25 mm produced an increase of total soluble sugar and free amino acids content. This result suggest that these metabolites might be related with a nodule osmotic adjustment response under saline conditions, however cannot be excluded that the increase of amino acids content could be a consequence of protein degradation. In the other hand, sodium, calcium and phosphorus contents in shoot increased under the saline treatments. Potassium (K) and calcium (Ca) contents, unlike phosphorus (P) content, in shoot were not related with the symbiotic efficiency of mutant, however the decrease of P content suggest that these symbioses have limited their P absorption process independently of the saline treatment. NaCl tolerance associated with a retention of sodium and maintenance of potassium selectivity seem to be a strategy used for the salt stressed common bean plants in symbiosis assayed here. Keywords: common bean root nodules, Phaseolus vulgaris, salt stress Received 30 July 2003; accepted 24 September Address correspondence to N. A. Tejera, Departamento de Fisiología Vegetal, Facultad de Ciencias, Universidad de Granada, Campus de Fuentenueva s/n, Granada, Spain. natejera@ugr.es 1907

2 1908 N. A. Tejera et al. INTRODUCTION Soil salinity is one of the major drawbacks for agriculture in arid an semi-arid regions of the world (Epstein, 1985), because salinity decreases the rate of photosynthesis and plant growth to various degrees. Legumes are classified as salt sensitive crop species (Lauchli, 1984) and their production is particularly affected by salt stress since these plants depend on symbiotic N 2 fixation for their nitrogen requirement (Elsheikh and Wood, 1995). The limitation in productivity is associated with a lower growth of the host plant, poor symbiotic development of root-nodule bacteria (Georgiev and Atkins, 1993) and consequently with a reduction in the nitrogen-fixation capacity (Delgado et al., 1993). In addition to these physiological responses, saline stress accelerates greening of the nodules and lowers the leghemoglobin content (Delgado et al., 1993), which is considered an index of nodule senescence. Despite decades of research into the effects of salinity on crop plants, the causes of sodium toxicity remain controversial. Plants are stressed in two ways under high salt environmental conditions: by the increase in osmotic potential of the rooting medium as a result of high solute content, and by the toxic effect of high concentration of ions (Demir and Kocacaliskan, 2002). The specific symptoms of sodium toxicity include high tissue sodium concentration and low K:Na ratios, inhibition of root elongation, and shoot calcium deficiency (Maas and Grieve, 1987). Plant species adjust to high salt concentrations by lowering tissue osmotic potential with the accumulation of inorganic as well as organic solutes (Samaras et al., 1994). The accumulation of some organic solutes has been considered as an adaptation of plants against osmotic stress (Gilbert et al., 1998). Amino-acids such as proline, asparagine and γ -aminobutyric acid, can play an important role in the osmotic adjustment of the plant under saline conditions (Gilbert et al., 1998). Other organic compounds that accumulate in response to stress, such as soluble sugars, apparently play a role in the development of salt tolerance (Hu and Schmidhalter, 1998). The strategies used in the last few years to reduce the effect of salt stress on legume production has been conducted by the selection of host genotypes that are tolerant to high salt conditions (Cordovilla et al., 1995). Thus, an increase of tolerance to salinity of rhizobial bacteria might constitute another approach to improve plant productivity under symbiosis. In free-living conditions some strains of Rhizobium meliloti (Breedveld et al., 1991) and Rhizobium tropici (Graham, 1992) can tolerate up to 500 mm of NaCl. In the present study, we used the wild-type strain CIAT899 (salt tolerant) and its decreased salt-tolerance (DST) mutant derivatives, which were selected for their reduced growth in media supplemented with variable amounts of NaCl (Nogales et al., 2002). However, legumes are generally more sensitive to salinity than their rhizobial counterparts and, consequently, the symbiosis is more sensitive to salt stress than free-living rhizobia (Zahran, 1999).

3 Effect of Sodium Chloride on Common Bean Plants 1909 Rhizobium tropici CIAT899 is a rhizobial strain that forms effective symbiosis with Phaseolus vulgaris and Leucaena tress (Martínez-Romero et al., 1991). This strain has been shown to be tolerant to several abiotic stresses, including high temperatures (Riccillo et al., 2000), low ph (Graham et al., 1994), and salinity (Khadri et al., 2001). Many reports have shown that hightolerant strains are symbiotically more efficient than salt-sensitive ones under saline conditions (Chien et al., 1992). However, few reports have investigated isogenic or nearly isogenic strains with regard to salt tolerance and symbiotic effectiveness. The objective of this study was to evaluate the effect of inoculation with wild-type and DST mutant strains on growth, nutrient accumulation, and nitrogen fixation of common bean plants cultivated under controlled saline conditions. The contribution of both organic and inorganic solutes to osmotic adjustment is also discussed. MATERIALS AND METHODS Plant Growth and Inoculation Common bean ( P. vulgaris cv. Contender) was purchased from Semillas Bolívar S.A. (Granada, Spain). Surface-sterilized seeds were germinated, planted in Leonard jars (Leonard, 1943) with vermiculite and nutrient solution (Rigaud and Puppo, 1975) (1.5 mm KH 2 PO 4 ; 0.81 mm MgSO 4 7H 2 O; 2.7 mm KCl; 0.70 mm CaSO 4 2H 2 O; 72.2 µm Fe-EDTA; 16.5 µm Na 2 MoO 4 2H 2 O; 10.7 µm MnSO 4 2H 2 O; 8.0 µm CuSO 4 5H 2 O; 10.4 µm ZnSO 4 7H 2 O; 0.29 µm H 3 BO 3 ; 0.59 mm CoCl 2 4H 2 O) and inoculated with one ml (about 10 9 cells) of a log-phase culture of either R. tropici CIAT899 or one of the four DST mutant derivative. The R. tropici CIAT899 was described as salt tolerant and its DST mutants as having different levels of salt tolerance (Nogales et al., 2002). Plants were cultivated in controlled environmental chambers with a 16 8hlight-dark cycle, C day-night temperature, relative humidity 55 75% and photosynthetic photon flux density ( nm) of 450 µmol m 2 s 1, supplied by combined fluorescent and incandescent lamps. Experimental Treatments and Harvest Salt treatments consisted of plants grown in N-free nutrient solution with NaCl concentrations of 0, 25, or 50 mm added to the nutrient solution at the beginning of the culture. Nutrient solution was renewed each 4 5 days and ph was maintained at a constant value of 7±1 through the experiment. Eight plants of each salt treatment were grown in symbiosis with CIAT899, HB8, HB10, HB12, and HB13 strains. Non-inoculated plants were used to compare results.

4 1910 N. A. Tejera et al. Plants were harvested 32 days after transplanting, coinciding with the onset of flowering. Plants were removed from the jars, the roots thoroughly rinsed with water, blotted dry on filter paper, and nodules picked and kept on ice. Shoot, root and nodule dry weight were recorded after 24 h at 70 C. The experimental layout was a randomized complete-block design for wild-type strain and DST mutants. Nitrogen Fixation Assay The acetylene reduction activity (ARA) was assayed using a Perkin-Elmer 8600 gas chromatograph equipped with a flame ionization detector and with a Poropak-R column (Ligero et al., 1996). Nodulated roots were incubated in six replicates, at 25 Cin25mLrubber-capped vials containing C 2 H 2 (10% of volume) in air. After 10 min and 20 min, 200 µlofgas samples, appropriately diluted, were subjected to C 2 H 4 analysis. The enzyme activity was referred to a standard curve of ethylene ( nmol) and the results expressed as µmol C 2 H 4 plant 1 h 1. Although the use of such a closed system for measuring acetylene reduction does create problems related to an acetylene-induced decline in nitrogenase activity (Minchin et al., 1983), it is still useful for comparative purposes, especially when the assay time is short. Organic and Mineral Solute Analysis Nodule extracts were prepared using 12 ml of extracting buffer for each1gof nodule fresh weight. Proline and free amino-acids were measured with ninhydrin reagent by the colorimetric methods of Irigoyen et al. (1992) and Yemm and Cocking (1955), respectively. In these cases, standards curves with L-proline and L-glutamine were prepared for the calculation of proline (µg proline g 1 NFW) and amino-acid (mg asparagine g 1 NFW) concentrations, respectively. Assay for total soluble sugars (TSS) was followed by the colorimetric method of Irigoyen et al. (1992) with anthrone reagent, and the calculation of concentration (mg glucose g 1 NFW) was performed according to a standard curve of glucose µgml 1. For nitrogen (N), phosphorus (P), sodium (Na), potassium (K), and calcium (Ca) analysis, dried shoot samples were ground to pass a 20-mesh sieve and digested with a mixture of H 2 SO 4 -H 2 O 2 using microwave energy, modified technique of Lachica et al. (1973). Total nitrogen was analyzed by the Kjeldahl method (Bouat and Crouzet, 1965). The phosphorus content was determined colorimetrically with an auto-analyzer (Capitan and Martínez, 1954) on digested samples. Sodium and potassium were measured by flame-emission photometry using an EEL spectrometer according to CIETA (1969). Calcium was measured on acid-digested samples by atomic absorption

5 Effect of Sodium Chloride on Common Bean Plants 1911 spectrophotometry in a Perkin Elmer Analyst 800 (Perkin Elmer Inc., Wellesley, MA) spectrophotometer equipped with a PE6017 lamp, and measured at nm. Statistical Design and Analysis The experimental layout was a randomized complete block design. The growth values and parameters related to nitrogen fixation (nodule number and acetylene reduction activity per plant) were means of six replicates per treatment. Three replicates were performed for the assays of soluble sugar, amino-acid and proline content and for nutrient determinations. All results were subjected to a two-way analysis of variance with a least significant difference (LSD) test between means. Also, the correlation coefficients among the parameters studied were calculated and are presented in Figures 1 3. Figure 1. Correlation between shoot dry weight (SDW) and acetylene reduction activity per plant (ARAP) or nodule number (NN) in plants of common bean inoculated with the R. tropici CIAT899 strain and DST mutants.

6 1912 N. A. Tejera et al. Figure 2. Correlation between nitrogen percent in shoot and shoot dry weight (SDW) or nodule number (NN) in plants of common bean inoculated with the R. tropici CIAT899 strain and DST mutants. RESULTS The efficiency of each DST mutant in symbiosis with Phaseolus vulgaris plants was investigated. Plant-growth parameters, shoot dry weight (SDW), root dry weight (RDW) and root to shoot ratio (RSR) are shown in Table 1. Plants inoculated with the wild-type strain CIAT899 had two-fold higher SDW than did non-inoculated plants. With HB10 and HB12 DST mutants, SDW was 35 45% reduced with respect to the wild-type symbiosis, whereas plants inoculated with HB8 and HB13 DST mutants reached the same growth as non-inoculated plants. The adverse effect of salinity on SDW was significant in all treatments. However, with 25 mm NaCl, the shoot biomass of plants inoculated with DST mutants was higher than non-inoculated ones, except for HB8 mutant. In addition, RDW in all symbiosis tested reached the same values than did non-inoculated plants,

7 Effect of Sodium Chloride on Common Bean Plants 1913 Figure 3. Correlation between proline content and potassium or calcium concentration in shoots of common bean plants inoculated with the R. tropici CIAT899 strain and DST mutants. being 30% to 45% lower than values obtained in the symbiosis with wildtype strain. The RSR increased with the salt dosage in all symbioses tested. In the CIAT899, HB10, and HB12 symbioses, increases in this ratio were not statistically significant at all salt levels with respect to control (0 mm NaCl). In these symbiosis the negative effect of saline treatment in the SDW and RDW were lower compared with the other mutants. On the contrary, RSR of plants in symbiosis with strains HB8 and HB13 significantly increased (44% and 47%, respectively), as well as did non-inoculated plants (74%). Nitrogen fixation parameters (acetylene reduction activity per plant and nodule number) and the content of organic solutes (proline, total soluble sugars and amino acids) are shown in Table 2 as percentage with respect to control without salt. The negative effect of salt stress on plant nitrogen fixing capacity was clearly observed in all strains used. Under salt concentration of 50 mm nitrogenase activity of wild-type strain, HB8 and HB13 symbioses, was inhibited

8 1914 N. A. Tejera et al. Table 1 Dry weight of shoot [SDW] and root [RDW] expressed as mg plant 1 ; and root:shoot ratio [RSR] in plants of common bean inoculated with the R. tropici CIAT899 strain and DST mutants treated with three NaCl concentrations. NI (non inoculated plants) Strain NaCl SDW RDW RSR NI CIAT HB HB HB HB LSD (P 0.05) in a range of 85% to 95%. However in mutants forming partially effective symbiosis (HB10, HB12) this inhibition was lowered to 35%. Regarding to nodule number it was found that salt doses provoked a higher decrease in HB8 and HB13 as compared with other symbiosis tested. Despite of all CIAT899-derived mutants established symbiosis with reduced nitrogen-fixing capacity (data not shown), all of them were able to form nodules, which looked normal in size and shape as compared with wild-type nodules. The relative percentage of proline content decreased with salt application in all symbiosis tested (Table 2). However with a moderate salt level (25 mm), this solute kept stable in nodules formed by wild-type strain and ineffective symbiosis HB8 and HB13. This effect was not observed in the partially effective symbiosis (HB10, HB12) where a decrease was shown. The highest inhibition in proline content (30% to 40% with respect to control 0 NaCl) was shown with 50 mm of NaCl in nodules of HB10, HB12, and HB13, whereas in nodules of CIAT899 and HB8 the content of this metabolite was 15% lower. The content of total soluble sugar (TSS) responded positively to the saline treatment. The increase in this parameter was observed in nodules of mutants

9 Effect of Sodium Chloride on Common Bean Plants 1915 Table 2 Acetylene reduction activity per plant (ARAP), nodule number (NN) and content of proline, total soluble sugar (TSS) and amino acid (AA) in nodules of common bean plants inoculated with the R. tropici CIAT899 strain and DST mutants. Plants were treated with three NaCl concentrations and the results are expressed as percentage respect to the control without salt treatment Strain NaCl ARAP NN Proline TSS AA CIAT HB HB HB HB HB10, HB12, and HB13 with 25 mm of NaCl, however in the mutant HB8 this solute remained stable with this salt dose. Higher NaCl concentration diminished TSS in a similar way in HB8, HB13 and wild-type strains. In addition, salt application to the nutrient solution did not alter the soluble amino-acid content in nodules formed by CIAT899, HB8 or HB10 (even in the highest NaCl dose of 50 mm), whereas in the rest of symbioses tested this metabolite increased with both saline treatments. The sodium percentage in shoot increased with salt in all symbioses tested (Table 3). Plants inoculated with the CIAT899 strain had two- to five-fold higher sodium concentration with salt treatments of 25 and 50 mm respectively as compared with non-inoculated plants. In plants inoculated with the strain HB12 a similar result was observed. Higher differences were registered when results were expressed in mg plant 1. Sodium content increased to eight-fold in CIAT899, whereas this value rose two- to four-fold (data not shown) in mutants. On the other hand, K content in the shoot increased with the salt application in the nutrient solution in all strain tested. With 50 mm of NaCl the mutants HB8 and HB13 reached the maximum increase in the K percent, 125% and 130%, respectively. Ca percentage remained similar in all symbiosis tested without salt application, however this mineral element increased with the saline treatment.

10 1916 N. A. Tejera et al. Table 3 Percentage of sodium (Na), potassium (K), calcium (Ca), nitrogen (N), and phosphorous (P) in shoots of common bean plants inoculated with the R. tropici CIAT899 strain and DST mutants, and treated with three NaCl concentrations. NI (non inoculated plants) Strain NaCl Na K Ca N P NI CIAT HB HB HB HB LSD (P 0.05) Higher percentages were detected in plant inoculated with the mutant HB13 (NaCl 50 mm) and in non-inoculated plant at both salt doses assayed. The N percentage in inoculated plants with wild-type strain CIAT899 was four-fold higher than non-inoculated control plants (Table 3), but this value increased to eight-fold when results were expressed in mg plant 1 (data not shown). The major reductions in the nitrogen concentration in the shoot were shown by plants inoculated with all CIAT899-derived mutants, particularly in the case of mutants HB8 and HB13. The N concentration in the shoot of inoculated plants tended to decrease with salt. With 25 mm NaCl the highest concentrations of this element were observed in the CIAT899, HB10 and HB12 symbioses, while in the presence of the highest salt dosage the percentage of nitrogen differed in the mutants tested. Plants inoculated with the wild type strain CIAT899 had 87% more phosphorus content than did those inoculated with the mutant HB8, HB12, and HB13, and 30% more than HB10 symbiosis and non-inoculated plants. In general, the percentage of P in shoot increased with the salt application in all strains, except in plants inoculated with HB10 strain where values remained constant.

11 Effect of Sodium Chloride on Common Bean Plants 1917 DISCUSSION Common bean plant formed by all decreased salt-tolerance (DST) mutants of R. tropici showed lower dry plant biomass (SDW and RDW) and nitrogenase activity (data not shown) than plant inoculated with wild-type strain. According to results reported by Nogales et al. (2002), these mutants are classified as Fix d, however our results showed that symbioses formed by strains HB10 and HB12 were partially effective, whereas those induced by mutants HB8 and HB13 were virtually Fix. The little nitrogen fixation efficiency of these later nodules was corroborated by the significant reduction in the dry weight of shoots and roots (Table 1) or nitrogen content (Table 3) respect to the other symbiosis assayed. In this sense, total plant N is a suitable indicator to estimate nitrogen fixation (Bliss, 1993). In several important grain legumes, including Phaseolus vulgaris, have been reported by Delgado et al. (1994) an important reduction of plant growth and dry-matter accumulation under saline conditions. The salt effect on shoot dry weight was more pronounced than on root dry weight, thereby increasing the root/shoot ratio (Table 1). Increases in RSR were even more significant in the least efficient symbioses (formed by strains HB8 and HB13) and in noninoculated plants. The higher sensitivity of shoot than root to salinity found here is in accordance with previous reports of Cordovilla et al. (1994) and Soussi et al. (1998). Results of this study showed a narrow relationship between shoot dry biomass and salt dose applied. In addition, SDW showed a positive and significant correlation with nodule number and acetylene reduction activity per plant (Figure 1), and consequently, with the nitrogen percentage in this organ (Figure 2). On the other hand, the nitrogen percentage in shoot showed a high and positive correlation with the nodule number (Figure 2) and hence the nitrogen fixation depended on a great manner on the ability of each mutant to establish the symbiosis. The increase of total soluble sugars and free amino acid content with the saline treatment could be related to the mechanism of tolerance of these plants to salt stress (Gilbert et al., 1998; Hu and Schmidhalter, 1998). The selective accumulation of intracellular amino acids and other organic and inorganic solutes is a widespread physiological response to osmotic stress (Hu and Schmidhalter, 1998), given that they participate in osmotic adjustment (Gilbert et al., 1998). However, taking into account that total free amino acids content increased in nodules of both partially effective and ineffective symbiosis, and that the protein content decreased with salt application in all symbioses tested (data not shown), we suspect that the increase in amino acid content may probably be a result of protein degradation in these conditions. Therefore, this result could be an effect of stress, as has been reported for soybean nodules (Gordon et al., 1997), and for chickpea nodules (Soussi et al., 1999). Despite the increase in the total soluble sugars in nodules of all mutants with 25 mm of NaCl, the greater values obtained in partially effective nodules than

12 1918 N. A. Tejera et al. ineffective nodules support the role of these substrates in osmotic adjustment in saline conditions. In addition, the clear accumulation of carbohydrates in these nodules under salinity suggests that the decline in nitrogenase activity per plant was not caused directly by a lack of photosynthates (Hunt and Layzell, 1993). Contrary to the experimental result that often regarded proline as an compatible osmolite associated with the salt-resistance mechanisms (Samaras et al., 1994), and can also help to lower the osmotic potential of the cells (Gilbert et al., 1998), this study found a decrease in this amino acid content under saline conditions. This organic solute, as the same as rest of free amino acids, more than to be a compatible osmolite could be a consequence of protein degradation caused by salt stress. Proline content in nodules showed a negative and significant correlation with the content of potassium (0.81 ) and calcium (0.77 )inthe shoot (Figure 3). The finding of a sodium progressive accumulation in shoots of all symbiosis assayed, with the NaCl dose in the nutritive solution, also has been reported for several legume species grown under saline conditions (Cordovilla et al., 1995). Because plants inoculated with more effective strains (CIAT899, HB10, and HB12) registered the highest increase in biomass production, coinciding with an increase in shoot sodium content, it is considered that the higher tolerance experimented by inoculated plants with these strains is related with a minor water potential and their ability to exclude sodium from the shoot. On the other hand, Cordovilla et al. (1995) found that the most sensitive legumes to NaCl were P. sativum and V. faba, which accumulated Ca in shoot. In addition, Gadallah (1999) reported a enhanced content of Ca 2+ with the increase of salinity in Vicia faba plants, which is in accordance with our results. On the contrary Maas and Grieve (1987) detected a shoot calcium deficiency induced by sodium in salt stressed corn plants. As a conclusion, these results indicate that a) the content of potassium and calcium in shoot increased under saline treatments, however it did not depend on the efficiency of nitrogen fixation; b) the decrease of P content in plants inoculated with DST mutants suggest that these symbioses have limited their P absorption process independently of the saline treatment; c) the nitrogen content in shoot is useful indicator of N 2 fixation, even in symbiosis with mutants differing in nitrogen fixation efficiency. In addition, proline content in nodule can not be considered an indicator of saline stress as has been found in other plant tissues and species, while mean total soluble sugar indeed contributed to osmotic adjustment under saline conditions. ACKNOWLEDGMENTS Financial support was obtained through the Andalusian Research Program (AGR-139) and the Spanish Ministry of Education and Culture grant BOS C02-02.

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