Two contrasting Frankia strains were used in this study. CcI3 strain, whose isolation was reported by Zhang et al. (1984) is sensitive to salt stress while CeD, isolated by Diem et al. (1982) is salt tolerant in our cultivation conditions. Both Frankia isolates were grown in liquid BAP medium which contained (at final concentration) 1.4 mM CaCl₂·2H₂O, 0.2 mM MgSO₄·7H₂O, 0.195 mM FeNaEDTA, 5.6 mM KH₂PO₄, 3.2 mM K₂HPO₄, trace elements (H₃BO₄, M_nCl₂·4H₂O, ZnSO₄·7H₂O, CuSO₄·5H₂O, Na₂MO O₄·2H₂O, and CoSO₄·7H₂O) and vitamins (thiamine-HCL, pyridoxine-HCL, folic acid, Ca panthotenate, nicotinic acid, biotin, and riboflavin) at a final pH of 6.7 (Murry et al., 1984). Sodium propionate (5 mM) and NH₄Cl (5 mM) were used as carbon and nitrogen sources, respectively. For rapid hyphal growth, this nutrient BAP medium was modified and supplemented with phosphatidyl choline (3.33 g/L) and MES-Tris buffer (0.5 M, pH 6.8; Schwencke, 1991). Cultures were maintained at 28 ± 1°C, in darkness under stirring conditions.

C. glauca seeds (seed lot 15,934, ref. 086-5929) were collected at the Myall Lakes National Park in Australia and provided by the Australian Tree Seed Centre (ATSC, CSIRO). C. equisetifolia seeds (seedlot SN/2011/0014/D) were collected in Louga area in Senegal and provided by the National Tree Seed Program (PRONASEF).

For experiments with non-trangenic plants, C. glauca and C. equisetifolia seeds were germinated under semi axenic conditions in a plastic tray (53.5 × 27.5 cm) containing a sterile mixture of compost (ref EN 12580) and sandy soil (v/v; 120°C, 60 min). They were watered daily with a quarter-strength Hoagland liquid medium (Hoagland and Arnon, 1950) to promote germination and initial growth of the seedlings.

Genetic transformation of C. glauca was performed using an Agrobacterium tumefaciens strain containing a ProCg12:GFP construct (Svistoonoff et al., 2003). Six independent C. glauca transgenic lines were generated as described previously (Smouni et al., 2002). For each transgenic line, GFP expression was analyzed. All plants showed the expression pattern described in Svistoonoff et al. (2003). The ProCg12:GFP line showing the highest expression levels of GFP was clonally propagated as described (Svistoonoff et al., 2010). Similarly for ProCgNIN:GFP, we used the transgenic line previously described (Clavijo et al., 2015) which showed the highest GFP expression.

One month after seed germination, C. glauca seedlings were uprooted from the soil, gently washed 5 times with distilled water. Seedlings were individually transferred in hydroponic conditions, into Gibson glass tubes filled with a 50 mL liquid BD medium supplemented with KNO₃ (5 mM) as nitrogen source, at pH 6.7 (Broughton and Dilworth, 1971). They were incubated in a growth chamber at 28 ± 1°C with 16 h day/8 h night photoperiod and a 74 μmol m⁻² s⁻¹ light intensity. The BD medium was renewed every 2 weeks to avoid nutrient depletion and pH drift. After 1 month, salt stress was applied gradually through the weekly increment of one concentration of NaCl at 0, 50, 100, 200, 300, 400, and 500 mM. When 500 mM NaCl was reached, the plants were placed in nitrogen free-BD medium before being inoculated separately either with CcI3 or CeD Frankia strains.

C. glauca nodulation was performed as described previously (Ngom et al., 2015). Before inoculation, homogenized cells of CcI3 and CeD were suspended in sterile water with a final absorbance of 0.2, measured at λ = 595 nm for each strain. To establish actinorhizal symbiosis, inoculum of each strain was first brought into contact with the root system for 2 h. Plants were replaced back into Gibson tubes replenished with a 45 mL of nitrogen free-BD medium +5 mL of each bacterial suspension. Nodulation rate or the percentage of nodulated plants (total number of nodulated plants/total number of inoculated plants × 100) and the mean nodule number (average number of nodules per plant) were followed for about 2 months after inoculation. All experiments were repeated twice and 22 plants were used for each salt treatment per experiment.

C. glauca seedlings were placed in hydroponic culture. Salt stress was applied gradually and plants were inoculated separately with either Frankia strain CcI3 or Frankia strain CeD, as described above. Two days after inoculation, root hair deformation was evaluated through micrographs of small lateral roots acquired with a Micro Publisher 3.3 RTV digital camera (QImaging) and a BX50F microscope (Olympus). For each treatment, five plants were used and three lateral roots were analyzed per plant. A total of 180 lateral roots and 12,217 root hairs were observed. Root hair deformation intensity was evaluated as described in Clavijo et al. (2015). For each micrograph, root hairs were observed and the following scoring was used: 0, no deformation; 1, straight root hair with tip swelling; 2, only one change in growth direction; 3, more than one change in growth direction but no bifurcation; 4, one or more bifurcations. At least two independent experiments were performed.

Transgenic lines expressing ProCgNIN:GFP or ProCg12:GFP fusions were propagated and grown hydroponically in BD medium as described previously (Svistoonoff et al., 2010). Two NaCl concentrations (0 and 50 mM) were applied for 7 days. Plants were inoculated either with Frankia strain CcI3 or CeD, as described above. For each transgenic line, four plants per treatment were used. Activation of ProCgNIN:GFP was monitored 24, 48, and 72 h after inoculation. Activation of ProCg12:GFP was observed 3, 7, and 14 days after inoculation and nodule sections were examined for GFP fluorescence. GFP expression was observed using an AZ100 epifluorescence microscope (Nikon) and a GFP filter.

C. glauca seedlings were cultivated in hydroponic conditions and were nodulated with Frankia strains CcI3 or CeD, as described above. A batch of 22 uninoculated plants was used as controls. After inoculation, nodule formation was monitored weekly. Twenty-five days after inoculation, all of the plants were nodulated and treatment with NaCl was initiated. Salt stress (0, 50, 100, 200, 300, 400, and 500 mM NaCl) was applied gradually, as described above, to avoid osmotic shock. Morphological and physiological parameters of growth such as length of aerial parts, shoot and root dry weight, chlorophyll, and proline contents were evaluated as described below. Independent experiments were performed twice with 22 plants each treatment per experiment.

One month after seed germination, C. equisetifolia seedlings were transplanted into plastic bags containing sterile sandy soil (120°C, 1 h). The experiments were conducted in a nethouse (Bel-Air experimental station, 14°44′N–17°30′W, Dakar, Senegal). Seedlings were watered daily and inoculation was applied 1 month after transplantation. Suspension of crushed nodule was used as inoculum. Nodules (20 g) were collected from C. glauca plants grown in hydroponic conditions and inoculated separately with Frankia strains CcI3 or CeD. Nodules were surface-sterilized with 5% sodium hypochlorite for 20 min then rinsed 3 times in sterile distilled water as described by Ng (1987). Grounded nodules were resuspended in 500 mL sterile distilled water. A 5 mL suspension was added into each bag according to the Frankia strain except for uninoculated plants. A batch of 8 plants was used for each treatment. As for C. glauca, the establishment of the symbiosis was monitored before gradually applying salt stress (0, 50, 100, 200, 300, 400, and 500 mM NaCl), as described above. Morphological and physiological parameters of growth such as length of aerial parts, shoot and root dry weight, chlorophyll, and proline contents were evaluated as described below.

Length of aerial parts were measured every 2 weeks. Four months after inoculation, plants were harvested. Shoot and root systems were collected, washed in deionized water, surface-wiped with blotting paper, and dried at 70°C for 72 h. The dried biomasses of each samples (C. glauca n = 22, C. equisetifolia n = 8 per sample) were weighed separately.

Chlorophyll content was determined using Arnon's method (1949). Fresh leaves (100 mg) were crushed in 10 mL of acetone at 80%. Samples were incubated overnight at 4°C and centrifuged at 6000 g for 10 min. The absorbance of chlorophyll (a) and (b) was measured using a UV-1800 spectrophotometer (UVisco) at λ = 663 and 645 nm, respectively. Total chlorophyll content (C. glauca n = 5, C. equisetifolia n = 4 per sample) was calculated according to Arnon (1949).

Fresh leaves (100 mg) were crushed in 2 mL of methanol at 40%, and the samples were immerged in a water bath at 85°C for 1 h. After cooling, 1 mL of leaf extract was mixed with 1 mL of ninhydrin at 2.5% and 1 mL of the reaction mixture (48 mL distilled water, 32 mL acetic acid, and 120 mL orthophosphoric acid). A second incubation was done in a water bath at 100°C for 30 min. Samples were cooled on ice, then a 5 mL toluene was added to the mixture. The upper phase was collected after vortexing and dehydrated with anhydrous sodium sulfate. Absorbance of leave samples was measured using a spectrophotometer at λ = 520 nm, as described by Monneveux and Nemmar (1986). Proline contents (C. glauca n = 5, C. equisetifolia n = 4 per sample) were calculated and determined through a calibration straight graph constructed from a standard range of proline concentrations (Monneveux and Nemmar, 1986).

Nitrogen fixation was measured using the acetylene reduction assay described by Hardy et al. (1973). C. glauca plants were placed in tightly closed 150 mL jars. In each jar, 10% of the air (15 mL) was removed and replaced with acetylene. Plants were incubated at 28°C for 3 h. From each jar, 1 mL was withdrawn and assayed for ethylene using a gas chromatograph (Agilent 6850, GC System). Nodules were removed from plant roots and dried at 70°C for 72 h. Nitrogenase activity was calculated per nodule dry weight and expressed as nmoles ethylene/nodule (g).

Statistical analyses were performed on dry weight, chlorophyll and proline data. Statistical tests were performed using the XLSTAT 7.2 software. The Student-Newman–Keuls test at p < 0.05 was used to evaluate the differences between inoculated and uninoculated plants and between NaCl treatments.

First we analyzed the growth of 8 Frankia strains isolated from several Casuarina species under saline conditions. All of the strains showed a reduced growth in response to salt treatment (data not shown). Two Frankia strains (CcI3 and CeD) were selected on the basis of their different sensitivity to salt and osmotic stresses. As shown in Figure 1, the growth of both Frankia isolates was reduced by increasing the NaCl and PEG concentrations in the medium. At 100 and 200 mM NaCl or PEG, the growth of CeD was significantly less impacted than the growth of CcI3 (Figures 1A,B). At high concentrations of NaCl and PEG (300, 400, and 500 mM), no or reduced growth was observed for both strains, with a more pronounced effect in presence of PEG 4000 in the medium. Altogether, our data indicated that CeD is more tolerant to salt and osmotic stresses than CcI3.

The impact of different NaCl concentrations on the nodulation of C. glauca plants by Frankia strains CcI3 and CeD was studied (Table 1). In both plants inoculated with Frankia CcI3 and CeD, the control plants (0 mM NaCl) had higher mean nodule number and rate of nodulation than NaCl-treated plants. The number of nodules formed increased over time at different rates with more nodules on seedlings inoculated with Frankia strain CeD for 63 days. At 50 mM NaCl, the mean nodule number declined by 66.6 and 60.3% in plants inoculated with CcI3 and CeD, respectively, compared to control plants. Nodule formation did not occur in seedlings inoculated with strain CcI3 at NaCl concentrations above 100 mM, whereas some plants inoculated with strain CeD were still forming nodules at 200 mM NaCl after 49 days of inoculation. Mean nodule number and nodulation rate of seedlings were reduced by increasing the salinity level.

Because salinity inhibited nodulation, the effects of salt stress during the early stages of the actinorhizal symbiosis establishment was investigated. Root hair deformation responses in C. glauca plants treated with several concentrations of NaCl and inoculated with Frankia strains CcI3 and CeD was first analyzed. Regardless of the presence of Frankia, salt treatment reduced the number and size of root hairs in both uninoculated and inoculated C. glauca plants (Figure 2A). In C. glauca plants inoculated with Frankia strains CcI3 and CeD, extensive root hair deformation was detected 2 days after inoculation, in small lateral roots of no salt-treated plants. Increased salinity reduced the amount of deformation in seedlings inoculated by both strains with a more pronounced effect for plants inoculated with CcI3 (Figure 2B). Deformation was particularly low at 200 mM NaCl, which also showed previously the smallest number of nodules in plants inoculated with CeD and no nodule development in plants inoculated with CcI3. No or few deformation were observed on uninoculated plants treated with various levels of NaCl.

To further investigate the effects of salinity during the early stages of the establishment of symbiosis, we studied the impact of salt stress on the expression of two early symbiotic marker genes: CgNIN (Clavijo et al., 2015) and Cg12 (Svistoonoff et al., 2003) using transgenic plants of C. glauca expressing ProCgNIN:GFP and ProCg12:GFP. CgNIN gene is a pre-infection marker which is early expressed in root hairs competent for Frankia infection (Clavijo et al., 2015) and Cg12, an infection marker associated with root hairs and cortical cells infection by Frankia (Svistoonoff et al., 2003).

Observations revealed that ProCgNIN:GFP was activated in both control and 50 mM NaCl treated plants, from 24 to 72 h after inoculation with Frankia strains CcI3 and CeD (Table 2A), suggesting that CgNIN expression was not repressed by salt treatment (50 mM NaCl). Expression of ProCg12:GFP was observed 14 days after inoculation in both control and 50 mM NaCl treated plants. A lower number of fluorescent spots per plant were detected in NaCl treated plants (Table 2B), pointing to a possible inhibition of infection and ProCg12 expression by salinity. We were not able to detect any differences regarding the pattern or the intensity of ProCg12 activation in prenodules or nodules when comparing control and NaCl-treated plants, as shown in Figure 3.

In addition to C. glauca, C. equisetifolia was studied because it is the most introduced Casuarina species worldwide for land reclamation and reforestation programs including Senegal (LADA, 2003; National Research Council, 1984). Furthermore, C. equisetifolia is also highly tolerant to salt stress (El-Lakany and Luard, 1983). The effects of prior inoculation with Frankia strains CcI3 and CeD on the salt tolerance of C. glauca and C. equisetifolia was studied to see if these nitrogen-fixing bacteria could be used to increase the salt tolerance of Casuarina species. Both Frankia strains CcI3 and CeD improved the growth of C. glauca at all concentrations of NaCl tested compared to the control plants (Figure 4A). In control plants, growth decreased with increased NaCl concentrations. There was no growth above 50 mM NaCl, 12 weeks after inoculation. Positive effect of inoculation with Frankia CcI3 and CeD on plant height started to be observed 4 weeks after inoculation. Plant height increased progressively at all NaCl concentrations in inoculated plants compared to the control, but growth gradually decreased with increasing NaCl concentrations. For instance, an increase by 65.5 and 44.5% was observed in 500 mM NaCl treated plants inoculated with Frankia strains CcI3 and CeD, respectively compared to controls. In contrast, in C. equisetifolia plants only Frankia strain CeD increased plant growth at all NaCl concentrations as compared to the control (Figure 4B).

Inoculation with Frankia strains CcI3 and CeD improved C. glauca shoot and total biomass significantly in all NaCl treatments compared to control (Table 3A). Compared to control, Frankia strains CcI3 and CeD significantly increased root biomass in all NaCl treatments except for 0, 50, and 100 mM salt-treated plants inoculated with CeD. In both control and inoculated plants, root, shoot, and total biomass decreased with increasing salt concentration, but the change was not significant in plants inoculated with strain CeD and in root dry biomass of control plants. On the other hand, only CeD increased significantly shoot and total dry biomass of C. equisetifolia plants for some NaCl treatments (0–200 mM) compared to the control and the plants inoculated with CcI3 (Table 3B). As observed for C. glauca plants, root, shoot, and total dry biomass decreased with increasing salt concentration in both control and inoculated plants. The change was significant between NaCl treatments in general and between low (0 and 50 mM NaCl) and high salinity (300 and 500 mM NaCl) in particular.

Chlorophyll and proline contents were determined in order to appreciate physiological state of non-nodulated and nodulated plants in saline conditions. The chlorophyll content (a, b, and total) was significantly increased in C. glauca plants inoculated with Frankia strains CcI3 and CeD, as compared to control (Figures 5A–C). However, there was no significant difference between NaCl treatments in C. glauca plants. In C. equisetifolia plants, the chlorophyll a was significantly increased by strain CeD only in no salt-treated plants (Figures 5D–F). No significant difference was observed between NaCl treatments. However, Frankia strain CeD increased the total chlorophyll content in 0, 50, 100, and 200 mM NaCl treated plants, compared to control and plants inoculated with CcI3.

As what was observed in chlorophyll content, there were significant changes in proline content between control and inoculated plants (Figure 6). Frankia strains CcI3 and CeD increased the proline content of C. glauca at all concentrations of salt tested (Figure 6A). Proline content increased with increasing salt concentrations in the control and inoculated plants, but the change was not significant in C. glauca plants inoculated with strain CeD. Similarly, in C. equisetifolia plants, proline content increased with increasing salinity (Figure 6B). There were significant differences between NaCl treatments in both control and plants inoculated with strains CcI3 and CeD. Only Frankia strain CeD increased proline contents significantly in 0, 50, and 300 mM NaCl treated plants.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.