## 2 Materials and Methods
### In Vivo Assays  
#### 2.7.1 Wheat Inoculation  
Pot experiments were conducted following completely randomized block design to assess the potential of the three salt-tolerant bacteria in mitigation of salinity stress in Waha durum wheat (_Tritcium durum_). Three soils (S1, S2, and S3) with different physicochemical characteristics were used to wheat growth (Table 2). The cultures of the three selected isolates and the sterilization of the wheat seeds were applied as described above. The inoculation was carried out after immersion of the seeds for 24 h in the bacterial suspensions (BR15, OR15, RB13, BR5 + OR15, BR5 + RB13, OR15 + RB13, and BR5 + OR15 + RB13). The uninoculated seeds (control) were immersed in sterile distilled water. The treated seeds were sown at the rate of ten seeds per pot (each contains about 1 kg of soil) at a depth of 1 cm from the surface. The experiments were designed with three groups representing the type of soil (S1, S2, S3) and eight treatments indicating the type of inoculum (control, + BR15, + OR15, + RB13, + BR5-OR15, + BR5 -RB13, + OR15-RB13 and BR5-OR15-RB13) with three repetitions (72 pots in total). The experiment was conducted in a growth chamber with an average of day/night temperatures of 26 \(\pm\) 2 \({}^{\circ}\)C and 16 \(\pm\) 2 \({}^{\circ}\)C, respectively, and a photoperiod of 16 h of lighting. The soil humidity is adjusted and kept constant during the experiment by watering with water. After 45 days of growth, the plants were harvested and washed with water. The roots and leaves have been separated. The fresh, the dry (after 72 h at 65 \({}^{\circ}\)C) weights, and the length of the roots and leaves were determined.  
7.2 Photosynthetic Pigments and Biochemical Markers Photosynthetic Pigments Concentration Estimation  
Chlorophyll pigments (chlorophyll a, b, total (a + b) and carotenoids) were assessed following procedures detailed above. Absorbance of the extracts was measured at 663, 645, and 470 nm. The quantification was done as following:  
\[\text{Chl}_{\text{a}}\;(\text{mg}/\text{g})=(12.7\text{A}_{633}-2.69\text{A}_{6 45})\times(\text{V}/1000\times\text{W})\]  
\[\text{Chl}_{\text{b}}\;(\text{mg}/\text{g})=(22.9\text{A}_{645}-4.68\text{A}_{6 33})\times(\text{V}/1000\times\text{W})\]  
\[\text{Chl}_{\text{a+b}}\;(\text{mg}/\text{g})=(20.21\text{A}_{645}+8.02\text{A} _{663})\times(\text{V}/1000\times\text{W})\]  
\[\text{Carotenoids (mg}/\text{g})=((1000\text{A}470-1.9\text{Chl}_{\text{a}}-63.14 \text{Chl}_{\text{b}})/214)\times(\text{V}/1000\times\text{W})\]  
V = the final volume of the supernatant; W = fresh weight of the leaf.  
Total Soluble Sugars Content  
The plant material (0.1 g) was extracted in 3 mL of ethanol 80% incubated at room temperature for 48 h, followed by boiling at 80 \({}^{\circ}\)C until evaporation. The extract was  
\begin{table}
\begin{tabular}{l l l l} \hline \hline  & **Location Site** & **Electrical Conductivity (mS/cm)** & **pH** \\ \hline Soil 1 (S1) & Setif (tertile soil) (36.195889\({}^{\circ}\) N, 5.364722\({}^{\circ}\) E) & 0.48 \(\pm\) 0.034 & 8.075 \(\pm\) 0.055 \\ \hline Soil 2 (S2) & \
\begin{tabular}{l} Bazer Sakhra Sebkha (periphery of the sebkha) \\ (36.071722\({}^{\circ}\) N, 5.675417\({}^{\circ}\) E) \\ \end{tabular} & 3.81 \(\pm\) 0.16 & 7.735 \(\pm\) 0.064 \\ \hline Soil 3 (S3) &
\begin{tabular}{l} Bazer Sakhra Sebkha (Mechtat Zaabib: away \\ from Sebkha) (36.073579\({}^{\circ}\) N, 5.667538\({}^{\circ}\) E) \\ \end{tabular} & 2.8 \(\pm\) 0.045 & 7.885 \(\pm\) 0.015 \\ \hline \hline \end{tabular}
\end{table}
Table 2: Three soils (S1, S2 and S3) with different physico-chemical characteristics were used in this study.  
diluted by the addition of 20 mL of distilled water. After centrifugation, the total soluble sugars of supernatant (2 mL) were determined by the phenol-sulfuric method [16].