Effect of Casuarina Crushed Nodules, Rhizospheric Soil and Leaves Compost on Salt Tolerance of Casuarina equisetifolia L. and Casuarina obesa Miq.

Soil salinization is one of the major causes of land degradation. In Senegal, this phenomenon continues to grow, making soils unsuitable for agriculture. To rehabilitate salty lands, one of the recommended strategies is amendment was added (Casuarina crushed nodules, Casuarina Rhizospheric soil or Casuarina leaves compost). Plants were subjected to saline stress. After four months of cultivation, they were harvested and morphological and physiological parameters were determined. Results showed that inoculation with Casuarina crushed nodules, Casuarina rhizospheric soil and Casuarina leaves compost improved growth, total dry biomass, total chlorophyll and proline contents of C. equisetifolia and C. obesa plants in salt stress condition. These positive effects were more important in C. obesa plants amended with Casuarina leaves compost. This study shows that Casuarina leaves compost can play an important role in the rehabilitation of saline soils by improving Casuarina trees performance in saline conditions.


Introduction
Salinity is a major abiotic stress affecting crop yield in the Sahel [1]. Salinization can be defined as a process of accumulating soluble salts in the soil at high levels that can negatively affect agricultural production or cause biodiversity degradation [2]. Lands affected by salinity are estimated worldwide at about 932.2 million hectares [3]. In Africa, salinization affects 40 million hectares of land and 15 million hectares of these lands are directly linked to anthropogenic factors [4].
In Senegal, about 1,200,000 hectares of land are reported to be affected by salinization, representing nearly 6% of the country's total area. According to Diack et al. [5], this area has increased to 1,700,000 ha, which considerably affects agricultural production potential. In arid and semi-arid ecosystems, salinization results from high water evaporation and irregular and deficient rainfall [6]. This salinization leads to land degradation and adverse effects on soil fauna and plants, resulting in decreased soil fertility and lower agricultural yields [7]. Today, with the growing need for agricultural and forestry production, land rehabilitation has become a major challenge to meet this high demand. Several methods have been used to rehabilitate saline soils, which include mechanical methods, chemical methods and biological methods.
Thus, one of the sustainable and environmentally friendly strategies for improving agricultural production and rehabilitating degraded ecosystems would be the use of salt tolerant plants associated with symbiotic microorganisms that can enhance the salt tolerance of these plants.
Plants belonging to the Casuarinaceae family are pioneer species widely used in the rehabilitation of poor soils [8] thanks to their ability to form a relationship with symbiotic microorganisms such as nitrogen fixing bacteria and arbuscular mycorrhizal fungi. These symbiotic associations improve the N and Pi nutrition  [9]. Several species belonging to this family such as C. obesa and C. equisetifolia are salt tolerant [10].  [13] and may also contain beneficial microrganisms and nutrients that improve salt tolerance of C. equisetifolia and C. obesa. These different types of amendments have several advantages: they are very accessible, easy to produce and cheap and are therefore likely to be adopted by producers.
These inocula will be an alternative to biofertilizers such as Rhizobia and AMF which are not available to farmers in Senegal and would be difficult to produce and relatively expensive. viable seeds were counted in a batch of 10 g. These seeds were not pretreated and were stored at 4˚C in the laboratory.

Cultivation Substrates
Experiments were performed using a sandy soil collected at ISRA/CNRA (Insti-

The Different Types of Amendments
The  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.

Experimentation Design
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   [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).
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 Growth Total Dry Biomass Measurement and Survival Rate
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: ( ) number of survival plants Survivalrate % *100 number total of plants = (1)

Chlorophyll and Proline Contents
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: 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].

Statistical Analysis
The data obtained was processed with the GENSTAT version 17 software (VSN International) and a Shapiro normality test was performed followed by a Levene Open Journal of Soil Science 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.

Survival Rate of C. equisetifolia and C. obesa Plants under Salt Stress Conditions
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).

Effect of Crushed Nodules, Casuarina Soil and Casuarina
Compost on the Height of C. obesa and C. equisetifolia No significant difference was noted between controls and inoculated plants in C. equisetifolia at 0 mM and 150 mM (

Effect of Crushed Nodules, Casuarina Soil and Casuarina Compost on the Total Dry Biomass of C. obesa and C. equisetifolia
The inoculation with Casuarina compost significantly increased the total dry

Effect of Crushed Nodules, Casuarina Soil and Casuarina Compost on the Total Chlorophyll Content of C. obesa and C. equisetifolia
The and 71% compared to the control plants in C. obesa at 150 and 300 mM respectively (Table 6).

Effect of Crushed Nodules, Casuarina Soil and Casuarina Compost on the Total Proline Content of C. obesa and C. equisetifolia
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 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.

Effect of Crushed Nodules, Casuarina Soil and Casuarina Compost on Survival Rate, Height and Total Dry Biomass of C. obesa and C. equisetifolia
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 dif-Open Journal of Soil Science ferent 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 Ca 2+ contents in C. equisetifolia litter could promote selective absorption of the Ca 2+ 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],

Effect of Crushed Nodules, Casuarina Soil and Casuarina Compost on Chlorophyll and Total Proline Content 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 Mg 2+ [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.

Data Availability
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.