Introduction

One-third of the world’s arable land resources are affected by salinity (Qadir et al. 2000). Salt tolerance in plants depends mainly on the capability of roots for (a) restricted or controlled uptake of Na+ and Cl−, and (b) continued uptake of essential elements, particularly K+ and NO3 − (Jeschke and Wolf 1988). Consequently, the preferential uptake of K+ over Na+ has generally been considered as an important trait contributing to salt tolerance in various halophytes and non-halophytes (Greenway and Munns 1980; Jeschke 1984). Considering the potential of bacterial exopolysaccharides (EPSs) to bind cations including Na+ (Geddie and Sutherland 1993), it may be envisaged that increasing the population density of EPS-producing bacteria in the root zone would decrease the content of Na+ available for plant uptake, and thus help alleviating salt stress in plants growing in saline environments. Although, the beneficial role of EPS-producing bacteria in removing toxic heavy metals from wastewaters is relatively well established, and attributed to the cation-binding capacities of bacterial EPSs (Kuhn and Pfister 1989; Loaëc et al. 1998; Suh et al. 1999), the possible role of these bacteria in restricting Na+ uptake and thus alleviating salt stress in plants growing in saline environments has not yet been established. Using five strains of EPS-producing bacteria belonging to Aeromonas and Bacillus, the present study was carried out to elucidate the role of EPS-producing bacteria in alleviating salt stress in wheat seedlings grown in a moderately saline soil.

Materials and methods

The moderately saline soil used in this study was obtained from the salt-affected area at Pacca Anna, 30 km southwest of Faisalabad. Some physicochemical characteristics of the air-dried and sieved (<2 mm) soil were: sand, 56.3%; silt, 29.2%; clay, 12.0%; maximum water-holding capacity, 20%; organic C, 2.9 g kg−1; total N, 0.34 g kg−1; mineral N, 18.6 mg kg−1; pH (saturation paste), 8.2; electrical conductivity (saturation extract), 8.0 d S m−1; sodium absorption ratio, 11.7; and water-soluble Na+, K+, Ca2+, Mg2+, Cl−, and SO4 2− were 45.7, 3.4, 11.1, 4.2, 11.4, and 22.7 mmol kg−1, respectively. The soil was not sterilized before use. We compared five EPS-producing bacterial strains viz. A. hydrophila/caviae (strain MAS765), Bacillus insolitus (strain MAS17), and Bacillus sp. (strains MAS617, MAS620 and MAS820). The bacteria were isolated from the rhizosphere soil (RS) of wheat grown in a salt-affected soil and identified as described earlier (Ashraf et al. 1998). Briefly, the isolates were classified into Gram-negative and Gram-positive by aminopeptidase test, and identified by the phenotypic system of bacterial identification (bioMérieux, France). The Gram-negative A. hydrophila/caviae MAS-765 was identified by API20NE strips, and Gram-positive Bacillus spp. by API50CHB strips. Polysaccharide production was observed by spot plate method on RCV-sucrose medium (modified Weaver’s medium; Amellal et al. 1998) containing 40 g l−1 sucrose and 15 g l−1 NaCl.

Tryptic-soy-broth, supplemented with 15 g l−1 NaCl was used for culturing the EPS bacteria, and the population density after a 24-h incubation at 30°C was 109 colony-forming units (CFU) ml−1. The soils as well as the wheat seedlings were inoculated. For inoculation of the soil, bacterial cultures were grown for 12 h, cells harvested by centrifugation (16,000g, 10 min), washed twice with sterile distilled water (SDW), and resuspended in biological saline (0.85% KCl). Soils in sterile polyethylene bags were sprayed with the respective bacterial suspension (final bacterial count, 108 CFU g−1 soil) and after a thorough mixing, 250-g portions distributed in plastic pots. Soil sprayed with SDW served as control. Seeds of wheat (Triticum aestivum L. CV Inqalab-91) were surface sterilized in 2% Ca (OCl)2 (2 h), rinsed in SDW, soaked in 11% H2O2 (20 min), and washed thoroughly with SDW. The seeds were germinated in sterile petri plates, and after germination the sterility of seedlings checked by overnight incubation in liquid broth. Five-day-old sterile seedlings were soaked for 2 h in an actively growing bacterial culture (109 CFU ml−1) and transplanted into the respective inoculated soil (four plants pot−1). For a control, seedlings soaked in SDW were transplanted into the uninoculated soil. Plants were grown in a growth chamber with day/night temperatures of 15/21°C and an 8-h light period (450 μmoles m−2 s−1). The soil moisture was maintained at 60% WHC throughout the experiment.

After a 25-day growth, plants with intact roots were recovered from the pots and shaken on a vibrating-arm shaker (1 min) to remove the loosely adhering soil from roots. The soil more tightly adhering to the roots (RS) was recovered by washing with SDW. Shoots, roots, and the RSs of four plants from each replicate pot were pooled before analyses. Potassium, Na+, and Ca2+ concentrations in plant tissues (acid digests) and in RSs (1:1 soil/water extracts) were determined by flame-photometry (Philippine Council for Agriculture and Resource Research 1980). Water-insoluble saccharides and Na+ in the RS were determined from the air-dried residual soil after filtering the RS-water suspension. Saccharides (as glucose equivalents) were measured by phenol-sulphuric acid method (Šafařík and Šantrůčková 1992). The population densities of EPS-producing bacteria in the RS and on the rhizoplane were determined by MPN method as described by Gouzou et al. (1993). Results are reported as means of six replicate pots. The significance between treatments was determined by ANOVA using a PC package CoStat (CoHart Software, Berkeley, United States).

Results

Inoculating EPS-producing bacteria substantially increased the dry matter yield of roots (149–522% increase), and shoots (85–281% increase; P <0.01; Table 1). A much more pronounced effect of inoculation was on the mass of RS that showed a 176–790% increase over control (P <0.01; Table 1). Comparing different strains, inoculation with MAS617 showed the least effects on different yield parameters, whereas MAS820 was the most efficient. Except with MAS617, the inoculation also increased the population density of EPS-producing bacteria on the rhizoplane (P <0.01; Table 1). In RS, however, inoculation with MAS617 caused the highest population of EPS-producing bacteria, followed by MAS17 (P <0.01), whereas other strains had no effect (results not shown). The population density of EPS-producing bacteria on the rhizoplane, but not that in the RS, was significantly correlated with the mass of RS (r =0.920, P <0.01), and the dry matter yields of roots (r =0.975, P <0.01) and shoots (r =0.807, P <0.02). Moreover, the population density of EPS bacteria on the rhizoplane, and the dry weights of RS, roots and shoots were significantly correlated with the content of water-insoluble saccharides in the RS (P <0.01).

Table 1 Effect of inoculating wheat seedlings with exopolysaccharide- (EPS-) producing bacteria on different yield parameters
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The EPS bacteria varied greatly in affecting the concentration of major cations, both in the RS and in the plant tissues (Table 2). Inoculation with MAS617 decreased the K+ level in the RS, whereas other strains caused 183–270% higher K+ levels in the RS (P <0.01). The K+ concentration in roots was not affected by inoculation, whereas in shoots, it was either not affected by inoculation (with MAS617), or it was higher (with other strains, 26–64% increase, P <0.01). Inoculation consistently decreased the Na+ concentration of the RS (soluble Na+, 27–36% decrease), and plant tissues (in roots, 21–61% decrease; in shoots, 33–60% decrease; P <0.01; Table 2). Inoculation also decreased the Ca2+ concentration of the RS (soluble Ca2+, 48–82% decrease, P <0.01) and shoots (13–26% decrease, P <0.05). The effect of inoculation on the root Ca2+ level, however, varied with strains (Table 2). The level was either not affected (MAS617 and MAS620 treatments), was higher (45% increase with MAS17) or lower (48–61% decrease due to MAS765 and MAS820) than the control (P <0.01).

Table 2 The concentrations of K+, Na+, and Ca2+ in the rhizosphere soil (RS), and in wheat roots and shoots as affected by inoculation with EPS-producing bacteria
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With the exception of the MAS620 treatment, roots of inoculated compared to control plants maintained a higher K+/Na+ ratio and K+-Na+ selectivity (the K+/Na+ ratio of the plant tissues divided by K+/Na+ ratio of the soil used), the most pronounced effect being with MAS820 (Table 3). However, in shoots, all the strains caused a higher K+/Na+ ratio and K+-Na+ selectivity, again with MAS820 showing the maximum effect (P <0.01). Moreover, probably due to the preferential transport of K+ over Na+ from roots to shoots, the latter always maintained a much higher K+/Na+ ratio and K+-Na+ selectivity than the roots. However, such an effect was relatively less pronounced in the control and in MAS617 treatment (10–11 times increase), and most pronounced with MAS17 and MAS620 (20–22 times increase). For the root tissue, the strains varied in affecting the Ca2+/Na+ ratio and Ca2+-Na+ selectivity (the Ca2+/Na+ ratio of the plant tissues divided by Ca2+/Na+ ratio of the soil used), but all the strains invariably increased the Ca2+/Na+ ratio and Ca2+-Na+ selectivity of the shoot tissue (P <0.01).

Table 3 Effect of inoculating EPS-producing bacteria on K+/Na+ and Ca2+/Na+ ratios and selectivities of wheat roots and shoots
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The experiment was repeated with two strains (the most efficient, MAS820, and the least effective, MAS617) showing similar effects, i.e. the inoculation stimulated wheat seedling growth and reduced the Na+ uptake (results not shown). Also, in a parallel study using the same soil we compared six other strains of EPS-producing bacteria belonging to Bacillus amyloliquefaciens, B. insolitus, Microbacterium sp. and Pseudomonas syringae. These strains produced a similar stimulatory effect on the growth of wheat seedlings, which was attributable mainly to the restricted Na+ uptake by roots and its transport to shoots (unpublished data).

Discussion

There are diverse reports on the effect of salinity on EPS-producing bacteria. Salinity negatively affects the attachment of Azospirillum brasilience to maize and wheat roots by alterations in the EPS (Jofré et al. 1998). Due to increased cellular and extracellular saccharide contents under salt stress, several cyanobacteria are known to thrive under salt stress (Padhi et al. 1997). In the present study too, the EPS might have played a role in the salt tolerance of the bacterial strains used.

The increased soil adhesion to roots resulting in higher mass of RS is attributable to EPSs (Watt et al. 1993; Amellal et al. 1998; Bezzate et al. 2000; Vanhaverbeke et al. 2003). All the bacterial strains tested, except MAS617, also increased the RS mass/root mass ratio (P <0.05; Table 1), which was significantly correlated with the content of water-insoluble saccharides in the RS (r =0.831, P <0.02), indicating the role of EPSs in soil aggregation around roots. As evidenced from the data on the RS mass, the strains differed in their ability to form soil aggregates, most probably due to variable physicochemical characteristics of their EPSs (Chenu and Guerif 1991; Czarnes et al. 2000). We may not rule out the indirect role of inoculation in improving the soil adhesion to roots, as enhanced root exudation in response to inoculation is known to increase the population of EPS-producing bacteria around roots of various plant species (Haynes and Francis 1993).

The most obvious physiological effect of EPS bacteria was the restricted Na+ uptake by wheat roots, which was more discernable when the Na+ concentration of RS and plant tissues was expressed as mM, i.e. on the basis of moisture content (Table 4). Due to the lower moisture level in the RS of inoculated plants (most probably due to higher water consumption by their comparatively higher plant biomass; Table 1), the concentration of plant-available Na+ in the RS solution was actually much higher in all inoculated treatments as compared to the control. On the other hand, in contrast to the control, Na+ concentration in the root and shoot tissues of inoculated plants was consistently lower than that of RS, which confirms the restricted uptake of Na+ due to inoculation.

Table 4 Sodium concentration (mM) in the rhizosphere soil (RS) and in wheat roots and shoots
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The similar levels of water-insoluble Na+ in the RS of uninoculated and inoculated plants indicate that binding of Na+ with the EPSs in the RS was not responsible for the observed lower Na+ uptake by roots of inoculated plants. Moreover, the lower Ca2+ level in the RSs and shoots of the inoculated compared to uninoculated plants, and the variable effect of different strains on the root Ca2+ level indicate that the restricted Na+ uptake was not attributable to the ameliorative effects of Ca2+ under salinity (Epstein 1998). The lower Na+ uptake by roots of inoculated than uninoculated plants most probably resulted from a decreased passive (apoplasmic) flow of Na+ into the stele due to a higher proportion of the root zones being covered with soil sheaths in inoculated treatments. The hydraulic conductivity of the root zones lacking soil sheaths is reportedly 100 times greater than those covered with soil sheathes (Wenzel et al. 1989). The significance of the apoplasmic pathway for the uptake of Na+ into the stele in the basal root zone, as well as the role of transpiration pull in the uptake and translocation of Na+ are well established (Marschner and Schafarczyk 1967; Yeo et al. 1987; Marschner 1999). As supported by the data on the RS mass/root mass ratio (Table 1), the roots of inoculated compared to control plants possessed a much higher proportion of the sheathed zones, thus lowering the passive influx of Na+ into roots. However, the role of EPS bacteria in restricting the apoplasmic pathway of Na+ uptake needs further studies with different salt-sensitive plant species.

Most of the strains increased the K+/Na+ ratio and K+/Na+ selectivity in roots, whereas all increased the K+/Na+ ratio and K+-Na+ selectivity of the shoot tissue. The effect was attributable mainly to the restricted Na+ uptake by roots and to the preferential translocation of K+ over Na+ from roots to shoots. However, the lack of such an effect in roots under MAS620 treatment, which also showed substantial yield increase, suggests that other factor(s) were also involved in the observed plant growth stimulation.

All the strains decreased the content of soluble Ca2+ in the RS, which may be attributed to its binding with the bacterial EPSs (Geddie and Sutherland 1993). In saline environments, the competitive effect of Na+ may cause Ca2+ deficiency in plants (Marschner 1999), and some strains indeed decreased the root Ca2+ level, as well as the Ca2+/Na+ ratio and Ca2+-Na+ selectivity. In shoots, though the inoculation invariably decreased the Ca2+ level, it improved the Ca2+/Na+ ratio and Ca2+-Na+ selectivity, probably due to the preferential transport of Ca2+ over Na+ from root to shoots. Nevertheless, the observed negative effects of inoculation on the tissue Ca2+ level were probably not large enough to cause Ca2+ deficiency or to affect the dry matter yields.

EPS-producing bacteria can play both beneficial and detrimental roles in nature (Bryers 1993; Sutherland 2001). Inoculating EPS-producing bacteria has recently been reported to alleviate drought stress in plants (Amellal et al. 1998; Alami et al. 2000; Bezzate et al. 2000; Vanhaverbeke et al. 2003). Inoculating wheat seeds with Azospirillum brasilense Cd was found to alleviate noxious effects on germinating seeds caused by compost application by possibly transforming the composition of humic acids in the compost (Bacilio et al. 2003). Also, the salt-tolerant EPS-producing Azospirillum spp. is known to promote the growth of the oilseed halophyte Salicornia bigelovi (Bashan et al. 2000). The results of the present study indicate yet another beneficial role of EPS-producing bacteria that is the remarkable potential of EPS-producing bacteria to restrict Na+ influx into roots, thus enabling the otherwise salt-sensitive plants to survive and flourish under salt stress.