Seeds of Casuarina equisetifolia (L. Johnson) harvested at Notto Gouye Diama (14˚59'29.89"N, 17˚00'35. 97"O), batch SN/2008/0018/D in the north of the Thiès region were provided by PRONASEF (National Forest Seed Program). Casuarina obesa (Miq.) seeds (lot 15777) were provided by CSIRO (Australian Tree Seed Center). They were harvested in the locality of Mullewa, Australia, at a latitude of 28˚15' North, a longitude of 115˚38' East and an altitude of 250 m. About 2800 viable seeds were counted in a batch of 10 g. These seeds were not pretreated and were stored at 4˚C in the laboratory.

Experiments were performed using a sandy soil collected at ISRA/CNRA (Institute Senegalese of Agriculture Research/National Center for Agronomical Research, Bambey, Senegal) (14˚71 North - 16˚48 West) at the horizon (0 - 30 cm). This soil was sieved through a 2 mm mesh and homogenized to eliminate plant debris. The physicochemical characteristics of the soil sample were determined at laboratory of soil, plant and water, ISRA/CNRA, Bambey, Senegal (Table 1).

The soil was autoclaved at 120˚C for 2 hours and stored at −30˚C to eliminate microorganisms.

The fresh nodules and the Casuarina rhizospheric soil (CRS) (0 - 20 cm horizon) used in this experiment were collected from C. equisetifolia adult plants at 12˚8' and 16˚41' north latitude and 11˚21' and 17˚32' west longitude in the Niayes area where a Casuarina plantation was established in 1920. Casuarina leaves compost was delivered by the Horticultural Development Centre (ISRA/CDH); its physical and chemical characteristics were determined at the laboratory of soil, plant and water, ISRA/CNRA, Bambey, Senegal (Table 2). The fresh nodules were washed several times with tap water, then 3 times with sterile distilled water. 20 g of nodules were crushed in a mortar. The crushed nodules thus obtained were suspended in 500 ml of sterile distilled water before inoculation. Soil collected under the C. equisetifolia trees was sieved to 2 mm and the fine soil was mixed with autoclaved Bambey sand respectively 1/4 and 3/4.

C. obesa and C. equisetifolia seeds were germinated in 23.5 cm × 9.5 cm polyethylene bags filled with sterile soil. Soils were previously sterilized by autoclaving at 120˚C for 20 mn to prevent colonization by native AMF/ectomycorrhizal fungi and/or Frankia, and thus to analyze the effects of the amendments. Two months old seedlings were transferred into pots (25 × 12 × 50 cm) filled with sterilized soil and kept in a greenhouse at ≈30˚C at the ISRA/CNRA, Bambey.

Different treatments were carried out: plants inoculated with Casuarina leaves compost (hereafter called Casuarina compost), Casuarina rhizospheric soil (hereafter called Casuarina soil) or Casuarina crushed nodules (hereafter called crushed nodules). The quantity of C. equisetifolia leaves compost or Casuarina rhizospheric soil added in the polyethylene bags filled with sterile soil was 1/4 of the substrate. For crushed nodules, a suspension of 5 ml (0.4 g of nodules/mL) was added directly to the substrate during transfer near the root system to promote contact of symbiotic microorganisms and the plant. Control plants were transplanted directly to the sterile substrate.

The plants were watered with distilled water for 1 month, to allow the establishment of symbiosis and allow time for the plants to adapt to post-transplant stress. Then, plants were subjected to salt stress by applying different treatments of sodium chloride (NaCl) (0 mM, 150 mM and 300 mM) until the end of the

experiment. Salt stress was gradually applied to avoid osmotic shock. For the first application, a concentration of 25 mM corresponding to 1.46 g/L was used. This concentration corresponds to a threshold beyond which most non-salt-resistant plant species cannot develop [14]. This concentration was increased gradually as described by Djighaly et al. [10] until reaching the desired concentration (150 mM or 300 mM). These concentrations constituted the daily watering solution for the plants and the salt concentration was regularly checked for saturation with an Extech portable salinity refractometer Z741839-1EA (Sigma).

The design included three factors: plant species (C. equisetifolia and C. obesa), amendment (crushed nodules, C. equisetifolia rhizospheric soil, C. equisetifolia leaves compost and controls), and salinity (0, 150, 300 mM NaCl). Twenty four (24) treatments (2 × 4 × 3) were carried out with nine (9) replications per treatment.

After four months of growing in greenhouse, the plants were harvested and the following parameters were evaluated: survival rate, height growth, aerial and root biomass, chlorophyll and proline contents.

Shoot length was measured using a graduated ruler. After four months of growing, C. equisetifolia and C. obesa plants were harvested and the shoot and root systems were separated, washed in deionized water and dried at 70˚C for 72 h. The dried biomass of each sample was evaluated separately using an electronic precision scale. The survival rate was determined using the formula:

Survivalrate ( % ) = number   of   survival   plants number   total   of   plants * 100 (1)

The chlorophyll content (a), (b), and (a + b) levels in C. equisetifolia and C. obesa plants were determined with 100 mg of fresh leaves as described in Djighaly et al. [10].

Total chlorophyll contents (Chlorophyll a and b) were calculated from Arnon [15] according to the following formula:

C H t   ( mg ⋅ g − 1 ) = [ 8.02 × O D   663   nm + 20.2 × O D   645   nm ] × V / M (2)

where V is the volume of the total extract, M the mass of the fresh material and OD the optical density (nm).

To determine the concentration of proline in the plants, 100 mg of fresh shoots were ground and proline concentrations were quantified by spectrophotometry (520 nm) according to Monneveux and Nemmar [16].

The data obtained was processed with the GENSTAT version 17 software (VSN International) and a Shapiro normality test was performed followed by a Levene test to check the equality of the variances. Finally, all data following the normality was analyzed using a two-way ANOVA test and a Newman and Keuls test to assess the effects of NaCl concentration and their interaction on survival rate, shoot height, total dry biomass, chlorophyll content and proline content with a significance threshold set at 0.05.

After four months of growing in greenhouse, the survival rate of C. equisetifolia and C. obesa plants was evaluated. In the absence of NaCl, a survival rate of 100% was observed in C. equisetifolia and C. obesa (Table 3).

Lowercase letters (a - c) indicate significant differences between the control and treated plants at P < 0.05.

At 150 mM of NaCl, a survival rate of 100% was observed in C. equisetifolia plants inoculated with Casuarina compost, followed by the plants inoculated with crushed nodules (88.9%) and plants inoculated with Casuarina soil (88.9%).

A survival rate of 100% was also observed at 150 mM of NaCl in control plants. At 300 mM, plants inoculated with Casuarina compost and Casuarina soil had the same survival rates of 22.3% in C. equisetifolia. A lower survival rate 11.2% was recorded in plants inoculated with crushed nodules. At 300 mM, no C. equisetifolia plants survived after four months of cultivation.

Higher survival rates were noted in C. obesa plants compared to C. equisetifolia in saline soil for all NaCl concentrations. At 300 mM, the highest survival rate was observed in C. obesa plants inoculated with Casuarina compost (100%), followed by plants inoculated with crushed nodules (66.7%) and Casuarina soil (55.6%).

No significant difference was noted between controls and inoculated plants in C. equisetifolia at 0 mM and 150 mM (Table 4). At 300 mM, mortality of all control plants was observed while the inoculated plants survived. A significant decrease in height growth was noted at 300 mM compared to control plants in C. equisetifolia. In C. obesa, inoculation with Casuarina compost improves the height at 300 mM compared to controls plants. A comparison between the two species showed that height of C. obesa plants was significantly bigger than C. equisetifolia at all concentrations. In plants inoculated with Casuarina compost, 83% of difference was noted in height between C. obesa and C. equisetifolia at 300 mM NaCl.

The inoculation with Casuarina compost significantly increased the total dry

Each value represents the height mean of plants (n = 9) used for each treatment. Lowercase letters (a - d) indicate significant differences between the control and treated plants at P < 0.05.

plant biomass by 51% and 58% respectively at 0 and 150 mM of NaCl in C. equisetifolia (Table 5). At 300 mM, the control C. equisetifolia plants did not survive. The plants inoculated with Casuarina compost showed a significant increase in biomass of 51% 58% and 29.8% respectively at 0, 150 and 300 Mm NaCl for C. obesa. At 300 mM of NaCl, a difference of 94% and 92% was noted between C. obesa and C. equisetifolia biomasses respectively in plants inoculated with Casuarina compost and those inoculated with Casuarina soil.

The results show higher total chlorophyll contents in C. equisetifolia plants inoculated with Casuarina compost compared to control plants at 0 and 150 mM of NaCl. The inoculation with Casuarina compost increased the chlorophyll content by 23% and 28% compared to the control C. equisetifolia plants respectively at 0 and 150 mM in. However, at 300 mM, control plants did not survive in C. equisetifolia (Table 6).

The inoculation with Casuarina compost improved the total chlorophyll content of plants in the presence of salt in C. obesa at 150 and 300 mM. The inoculation with Casuarina compost increased the chlorophyll content by and 12.65% and 71% compared to the control plants in C. obesa at 150 and 300 mM respectively (Table 6).

In the absence of salt stress and in the presence of 150 mM of NaCl, inoculation with Casuarina compost significantly increased the proline content of the plants compared to control plants in C. equisetifolia (Table 7). An increase of 89% and 52% was noted in C. equisetifolia plants inoculated with Casuarina compost at 0 and 150 mM respectively compared to control plants. In C. obesa, inoculation

Each value represents the total dry biomass mean of plants (n = 9) used for each treatment. Lowercase letters (a - f) indicate significant differences between the control and treated plants at P < 0.05.

Each value represents the chlorophyll content of plants (n = 9) used for each treatment. Lowercase letters (a - g) indicate significant differences between the control and treated plants at P < 0.05.

Each value represents the proline content mean of plants (n = 9) used for each treatment. Lowercase letters (a - g) indicate significant differences between the control and treated plants at P < 0.05.

with Casuarina compost increased proline content by 32%, 20% and 82% respectively to 0, 150 and 300 mM of NaCl compared to the control plants (Table 7). A comparison between both species shows higher proline content in C. obesa compared to C. equisetifolia at 0, 150 and 300 mM NaCl.

The survival rate after four months of greenhouse cultivation is higher in C. obesa compared to C. equisetifolia plants. This higher survival rate under salt stress conditions in C. obesa is probably due to a better salt tolerance of this species. Better tolerance of C. obesa compared to C. equisetifolia, C.cunninghiamiana and C. cristata has already been described by Van der Moezel et al. [17]. This difference was related to a better regulation of the Na/K ratio. Among the different types of amendments used in this experimentation, a better survival rate was noted in C. obesa plants inoculated with Casuarina compost at 300 mM. The result is possibly linked to the fact that Casuarina compost contains organic matter (OM) and also many nutrients such as N, C, Ca etc. for the plant [18]. The results of Lal [19] showed that the amount of OM is a determining factor in agricultural productivity. The characterization of the Casuarina leaves compost used revealed the presence of essential nutrients such as nitrogen, phosphorus, potassium and organic carbon. These essential nutrients in compost probably help to rebuild the physicochemical properties of the soil and restore microbial activity [20] [21]. In saline stress conditions, the OM contained in the compost promotes the flocculation of clays [22] [23], which increases air circulation in the soil and is essential for plant growth but also for the functioning of soil microorganisms.

Similar results were found by Sall et al. [24] and Soumaré et al. [25] who showed that organic matter had a significant impact on the structure and activity of the microbial community. The decomposition of C. equisetifolia litter under semi-arid conditions results in the release of many nutrients in the following order: Ca > N > K > Mg > Na > P > Fe > Zn > Cu > Cr [26]. These high Ca²⁺ contents in C. equisetifolia litter could promote selective absorption of the Ca²⁺ ion during exchange and reduce Na⁺ absorption [27] under saline stress conditions. This phenomenon, which is a saline stress resistance mechanism, limits the toxicity of Na+ cations and could explain the higher survival rate, height and biomass in C. obesa and C. equisetifolia plants inoculated with Casuarina leaves compost under salt stress conditions. The better growth of plants inoculated with Casuarina leaves compost could also be explained by nitrogen nutrition due to the effect of assimilable nitrogen. Our analysis of the chemical characteristics of the compost showed a high level of nitrogen (0.973%). Thus, the work of Diallo et al. [28] showed a higher nitrogen mineralization in soils previously amended with Casuarina litter. Compared to other species such as Faidherbia albida (Del.) Chev., Azadirachta indica A. Juss, Andropogon gayanus Kunth, Eragrostis tremula Hochst. ex Steud, these authors found higher nitrogen levels in soils amended with C. equisetifolia. Phosphorus that can be released by amending with C. equisetifolia plays a very important role in degraded soils. In combination with nitrogen, they improve plant performance. These results confirm those of Brito et al. [29] which showed that use of Acacia waste compost as an alternative component for horticultural substrates.

The mortality observed in control plants of C. equisetifolia at 300 mM could be related to the threshold tolerance level of this species. Djighaly et al. [29] showed that increasing NaCl concentration decrease C. equisetifolia growth but inoculation with AMF but inoculation with AMF improves their performance in saline condition.

Results also showed that high NaCl concentrations reduced height growth of C. equisetifolia and C. obesa plants. Same results were obtained by Ly et al. [30], which provide information on the negative effect of salinity on the height growth of Jatropha curcas L. seedlings. However, it is important to remember that decreasing height growth would constitute an adaptation strategy to saline stress [31]. It allows the plant to reduce energy and resource costs and thus avoid stress. However, inoculation with Casuarina leaves compost improved the stress tolerance of plants of C. obesa and C. equisetifolia.

This study also revealed that the two species studied do not react in the same way for the synthesis of chlorophyll under saline stress conditions. In C. equisetifolia, the decrease in chlorophyll synthesis was observed under different salt concentrations. In contrast, in C. obesa, the highest chlorophyll levels were observed at 150 and 300 mM. This could be explained by the better salt tolerance that was reflected in the growth and survival rate under saline stress conditions for this species. However, the decrease in chlorophyll levels may be due to the inhibition of certain enzymes involved in the synthesis of photosynthetic pigments [32] due to the accumulation of Na+ ions.

However, inoculation with Casuarina leaves compost improved the chlorophyll levels of both species even at high concentrations of NaCl (150 to 300 mM). This positive effect of Casuarina leaves compost on chlorophyll synthesis in the presence of salt could be related to the fact that Casuarina leaves compost contains Mg²⁺ [26] which play an important function in photosynthesis [33] [34] [35].

As for proline, the levels vary with NaCl concentrations, the type of amendment and also according to the species. The amino acid proline is one of the most accumulated osmolytes in plants, in response to salinity and drought [36] [37]. Its accumulation allows plants to regulate intracellular osmotic pressure in order to avoid water losses that can cause cells desiccation [38] [39]. In the presence of salt, the highest proline levels were found in plants inoculated with Casuarina leaves compost in both C. equisetifolia and C. obesa. However, at high NaCl concentration (300 mM), the highest proline contents were found in C. obesa plants inoculated with Casuarina leaves compost. This greater accumulation in proline confirms the salt tolerance of this species and its performance in saline soil. This proline maintains the homeostasis of the cells for the plants, allowing them to continue growing in a saline environment, compared to control plants.