MATERIALS AND METHODS

The study was conducted in Faculty of Science, University of Douala, Cameroon, between August, 2007 to May, 2008. Three bambara groundnut (Vigna subterranea L. Verdc.) landraces were used in this experiment: White Seed Coat (WSC), Black Seed Coat (BSC) and light Red Seed Coat (RSC). Seeds were obtained from Agronomic Institute for Research and Development (IRAD, Dschang). Seeds were sterilized in 3% sodium hypochlorite for 20 min and then washed twice with distilled water. They were germinated in Petri-dishes on wet No. 1 Watman filter paper. Germination was carried out during 1, 2, 3, 4 and 5 days, seedlings developing their first couple of leaves were separated in four groups of 15 individuals each. For each group, five seedlings were randomly selected and sowed together in the same pot. Pots were kept in laboratory (temperature: 26±3°C, light: 5000 lux for 12 h alternating periods and relative humidity of 51-70%)and supplied every three days with nutrient solution containing 0.4 mM of KNO₃, 0.2 mM of KH₂PO₄, 1.0 mM of Ca₂NO₃ and 0.4 mM of MgSO₄ (Wacquant, 1974). Pots filled with clean sand (1000 g) were irrigated with NaCl solutions of different concentrations of 0, 50, 100 and 200 mM. The plants were irrigated with the de-ionized water. Roots, stems and leaves were harvested six weeks after sowing and weighed. The fresh material, plants were washed and 2.0 g of samples were taken for total chlorophyll determination. The remaining plants were oven dried at 70°C and digested with a mixture of HNO₃ and HCl0₄ acid (4:1) in order to assess the water content and Na⁺ and K⁺ concentrations (Turan et al., 2007b).

Sodium and potassium concentrations were determined by flame photometer (Jenway), as described by Taffouo et al. (2008).

Total chlorophyll of plants was extracted in 80% (v/v) aqueous acetone and absorption was measured in Thermospertronic Heλios β model spectrophotometer at 645 and 663 nm (Arnon, 1949).

Parameters assessed under field conditions were dry weight, number of pods per plant, number of seeds per pods, 1000 grains weight, grains and pods yield. The trial was conducted in the experimental field of the University of Douala (4°01 N, 9°44 E, altitude: 13 m above sea level, with total annual rainfall of 3597 mm and average temperature of 27°C). The soil of experimental site has a sandy soil. Its chemical and physical soil properties are shown in Table 1. The experimental design was a complete randomized block design with two treatments and three replicates. Plots were 8 x 8 m surfaced. The experimental plots were supplied with 50 mM NaCl and the control with simple distilled water (0 mM NaCl). The trials were carried out in pure culture without fertilization. Plants were watered once a week at a rate of 20 L m^-2 (Taffouo et al., 2009).

Data are presented in term of mean (±standard deviation). Multiple comparisons of several means was set up using analysis of variance (ANOVA) and the post-hoc pairwise analysis was set up using the Student-Newman-Keuls procedure when the normality and equal variance conditions passed. Multiple comparisons of data noted in experimental groups versus those recorded in the single control group were set up using the Dunnett's procedure (Sigma Stat software 2.03).

Table 1:	Physical and chemical properties of the experimental sites (0-20 cm of depth)

RESULTS

Ionic distribution of bambara groundnut landraces organs
Sodium, potassium concentrations and K/Na ratio: NaCl affected ion concentrations of roots, aerial parts (stems and leaves) of three Bambara groundnut landraces (Table 2). K⁺ uptake and transport to the aerial part of the Bambara groundnut were significantly (p<0.001) reduced with the increased NaCl concentration. In this experiment, salt stress, induced by increasing NaCl in the soil solution, resulted in an increase in Na⁺ concentrations. K/Na ratio was significantly decreased as NaCl concentration increased in aerial parts (Table 2).

Water content of bambara groundnut landraces organs: Water content was significantly reduced (p<0.001) in roots, stems and leaves of BSC and RSC landraces on contrary to increased amount of NaCl applied (Fig. 1a-d). The highest reduction at both landraces was observed at 200 mM NaCl treatment. Water content of WSC landrace organs grown under saline conditions was not significantly different from that of control plants (Fig. 1).

Growth and yield of Bambara groundnut landraces: Sodium chloride has a depressive effect on growth and yield component in BSC and RSC landraces (Table 3). For these species, it decreased significantly (p<0.001) dry weight of whole plant, the number of pods per plant, the number of seeds per pods, the 1000 grains weight, the chlorophyll content, the pods yield and grains yield (Table 3). Nevertheless, WSC landrace was observed to have relatively higher tolerance average of all parameters than others (Table 2). For this plant species, the depressive effect of salt was less marked on dry weight and yield component (p>0.001) except total leaf chlorophyll content.

Table 2:	Distribution of Na, K and their ratio K/Na in roots, aerial parts (stems and leaves) of three Bambara groundnut seedlings as affected by sodium chloride rates
Based on Dunnett’s test, values headed by differ significantly (*p<0.05 ; **p<0.01; ***p<0.001). Values are Mean±SE

Table 3:	Comparisons of growth parameter (Plant dry weight (g plant-2)) and Yield components (number of pods per plant, number of seeds per pods, 1000 grains weight (kg), total chlorophyll concentrations (mg g MS-1), grains yield (t ha-1) and pods yield (t ha-1) of three bambara groundnut under salt stress. n =10 for each landrace
Based on Dunnett’s test, values headed by differ significantly (*p<0.05 ; **:p<0.01; ***p<0.001), ns = p>0.05. Values are Mean±SE

Fig. 1:	Salt effects on the water content of organs of three Bambara groundnut seedlings. (a): Control medium (0 mM NaCl); (b): 50 mM NaCl; (c): 100 mM NaCl and (d): 200 mM NaCl; n = 5 for each landrace; White Seed Coat (WSC), Black Seed Coat (BSC) and Light Red Seed Coat (RSC). ns = p>0.05 ; *:p<0.05; *p<0.01; ***p<0.001

DISCUSSION

NaCl affected ion concentrations of roots, aerial parts (stems and leaves) of three bambara groundnut landraces (Table 2). The data have shown that K⁺ uptake and transport to the aerial part of bambara groundnut landraces were significantly (p<0.001) reduced with increased NaCl concentration. Similar results were reported by Carvajal et al. (2000), Taffouo et al. (2004, 2008, 2009), Al-Khateeb (2006) and Turan et al. (2007b). This implies a competition between Na⁺ and K⁺ absorption in bambara groundnut plant, resulting in a Na/K antagonism (Al-Karaki, 1997; Mezni et al., 2002). The reduction of K⁺ uptake caused by Na⁺ is likely to be the result of the competitive intracellular influx of both ions (Cerda et al., 1995). It is well established that many K⁺ transport systems have significant affinity for Na⁺ (Schachtman and Liu, 1999). Wolf and Jeschke (1987) reported that in shoot of salt stressed barley there was recirculation of K⁺ through phloem from shoot to root. Part of this recirculated K⁺ was transferred from phloem to xylem within the root and returned to the shoot through xylem. In this experiment, salt stress, induced by increasing NaCl in the soil solution, resulted in an increase in Na concentrations. There is an overwhelming amount of evidence to indicate that NaCl induced salinity increases Na uptake by plants (Taffouo et al., 2004, 2006, 2008; Al-Khateeb, 2006; Turan et al., 2007a, b). K/Na ratio was significantly decreased as NaCl concentration increased in aerial parts (Table 2). Similar results were reported in Medicago sativa (Al-Khateeb, 2006).

Water content values were decreased significantly (p<0.001) in roots, stems and leaves of BSC and RSC landraces on contrary to increased amount of NaCl applied (Fig. 1). The highest reduction at both landraces was observed at 200 mM NaCl treatment. Similar results for decreased water content values of plants organs were reported by Taffouo et al. (2004) in two salt-sensitive species (Phaseolus vulgaris and Glycine max). Water content values of WSC landrace organs grown under saline conditions were not significantly different from the control plants (Fig. 1). Similar results were found in organs of Phaseolus adenanthus and Avicennia germinans, two natural halophytes (Taffouo et al., 2004, 2007) and Lagenaria siceraria, salt-tolerant plant (Taffouo et al., 2008). It is attributed to the significant storage of monovalent cations in leaves, facilitated by the increase in Na⁺ concentrations from roots to leaves.

The present study showed that sodium chloride has a depressive effect on growth and yield component in BSC and RSC landraces (Table 3). For these species, it decreased significantly (p<0.001) dry weight of whole plant, the number of pods per plant, the number of seeds per pods, the 1000 grains weight, the chlorophyll content, the pods yield and grains yield (Table 3). These results showed that BSC and RSC landraces, in common with certain other plant leguminous (e.g., beans), is highly sensitive to salt with severe effects even at 50 mM NaCl (Levitt, 1980). For these species, salinity may reduce the growth and crop yield by upsetting water and nutritional balance of plant (Alam et al., 2004; Khan et al., 2007; Zadeh and Naeini, 2007) and loss of photosynthetic capacity (Alam et al., 2004). In the presence of salt, the available of nutrients to plant is restricted and the plant has to spend more energy to sustain itself. According to Munns (2002) this unfavourable effect of salt could be due to the deficiency in calcium, nitrate, phosphate and sulphate. Similar results were found in two salt-sensitive species of tropical cucurbit, Cucurbita lanatus and Cucurbita moshata (Taffouo et al., 2008) and two salt-sensitive leguminous plants, Phaseolus vulgaris and Glycine max (Taffouo et al., 2004). Nevertheless, WSC landrace was observed to have relatively higher tolerance on average of all parameters than others (Table 3). For this plant species, the depressive effect of salt was less marked on dry weight and yield component (p>0.001) except total leaf chlorophyll content. Similar observations for dry weight were reported in two halophytes, Ceriops roxburghiana (Rajest Arumugam and Venkatesalu, 1998) and Phaseolus adenanthus (Taffouo et al., 2004) and in Lagenaria siceraria, salt-tolerant plant species (Taffouo et al., 2008). There are a great number of plant species which are regarded as salt tolerant, the most competitive being those that are able to become established, grow to maturity and survive until they are able to reproduce (Turan et al., 2007a). Salinity decreased the total leaf chlorophyll concentration of all Bambara groundnut landraces. This effect of salt was attributed to a salt-induced weakening of protein-pigment-lipid complex (Strogonove et al., 1970) or increased chlorophyllase enzyme activity (Stivesev et al., 1973). Similar results were reported for total leaf chlorophyll concentration of three tropical cucurbite (Taffouo et al., 2008) and wheat and lentil plants (Turan et al., 2007a, b).

The present findings showed that two landraces of bambara groundnut, BSC and RSC are less sensitive to salt stress at vegetative and harvesting phases. For these landraces, reduced growth and yield may be the result of disturbed water consumption and K⁺ uptake within saline medium. An analysis combining growth and yield components parameters measured, suggested that WSC cultivar was relatively more tolerant to salinity than others. WSC cultivars could be grown in the coastal and semi-arid saline soils. This cultivar could be studied further as a source of genes for salt tolerance that could be exploited in breeding programs. The addition of NaCl resulted in a decrease of the K/Na ratio, water content partitioning of BSC and RSC landraces, Na⁺ was increased by treatment with NaCl, while K⁺ and total chlorophyll concentrations were decreased in all bambara groundnut landraces.

ACKNOWLEDGMENTS

This research was supported by the International Foundation of Science (IFS), Stockholm, Sweden and Swedish International Development Cooporation Agency Department for Natural Resources and the environment (SIDA NATUR), Stockholm, Sweden through a grant No. C/3817-1 to Dr Victor Désiré TAFFOUO.