Plant material
The study was carried out on two potato varieties Spunta (Sp), the most cultivated in Tunisia (80%; Azouz 1996) and also one of the most widely grown potatoes in the world (elornplants.com), and Claustar (Cl) variety. These plants were multiplied in vitro on MS medium (Murashige and Skoog 1962) added with Morel vitamins (Morel and Wetmore 1951) at a temperature varying from 22°C to 24°C and a photoperiod of 12h/days under a light intensity of 62 µE/m2s.
Bacterial inoculum
Plantlet roots were inoculated with Bacillus mojavensis I4 (BmI4) strain isolated from the soil of Sfax city in Tunisia and identified in the laboratory (accession number KF012872; Ghazala et al. 2016). This isolate was selected on the basis of its capacity to promote the growth of wheat plants by allowing the production of siderophores and IAA, as well as the fixation of nitrogen and the solubilization of phosphate. In addition, this strain tolerates a temperature of 55°C, 50 mg/l Cd and 10% NaCl (Ghazala et al. 2023), making it an excellent candidate for mitigating the effect of saline stress on plants. The BmI4 strain was cultivated on LB broth medium at 37°C and maintained at 4°C before use.
Plant inoculation, cultivation and application of salt stress
Plantlets of the two potato varieties, approximately three weeks-old and sufficiently rooted in MS medium, were inoculated by soaking their roots for 20 min in the BmI4 strain suspension (106 cfu/ml) previously prepared in PBS buffer (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4) for 20 min. Control plantlets which had the same age and length were treated with PBS buffer. These inoculated and non-inoculated plantlets (control) were transferred in plastic pots (12 cm in diameter) containing an autoclaved mixture of 50% sand and 50% peat (Potgrond H: Klasmann Deilmann, Geeste, Germany), and watered with tap water at a rate of 50 ml/pot twice a week. After 15 days of greenhouse acclimatization, the inoculated and non-inoculated plant groups were each divided into two groups to study the response to salt stress. Thus, the plants of each potato variety were arranged in 4 batches which underwent different treatments:
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Non-inoculated plants not subjected to salinity (control: C-)
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Inoculated plants not subjected to salinity (C+)
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Non-inoculated plants subjected to salinity (SS-)
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Inoculated plants subjected to salinity (SS+)
To apply salt stress in batches (2) and (4), the plants were irrigated with 50 ml salt solution (100 mM NaCl) twice a week the 15 days of the acclimatization until the cycle end. Whereas plants of batches (1) and (3) were kept watered with tap water. Plant culture was conducted between March 11and May 25 (2023), in a greenhouse in the presence of sunlight.
Plant growth parameters
Plant growth was evaluated for 40 days from the beginning of the treatment by measuring stem length and diameter, leaf number and area on seven plants previously selected from each batch. The tuber yield (g/plant) was also determined on these plants at the end of the cycle (60 days). At 10, 25 and 40 days of treatment, seven plants were used to measure each of leaf, stem and root fresh weights, physiological parameters and tuber weight.
Determination of chlorophyll contents
Total chlorophyll was extracted from fresh leaves (0.01g) ground in acetone (Arnon. 1949). After centrifugation at 12000 rpm for 15 min, the supernatant containing the pigments was collected and the absorbance of the samples was determined at 645 nm and 663 nm. The content of chlorophylls a (Chl a), b (Chl b) and total chlorophyll (Chl t) were determined according to the following formulas:
Chl a (µg/g FW) = [12.7* A663 − 2.69 *A645]*V/FW
Chl b (µg/g FW) = [22.9* A645 − 4.68 *A663]*V/FW
Chl t (µg∕g FW) = Chl a + Chl b
with FW: fresh weight of leaves; V: volume of sample
Detrermination of malondialdehyde (MDA) content
Lipid peroxidation was determined as thiobarbituric acid (TBA) reactive metabolites mainly MDA in leaves and roots as described by Hodges et al (1999). The fresh leaves and roots (0.15 g) were homogenized in 1.5 ml of trichloroacetic acid (TCA) 0.1%. After centrifugation at 12000 rpm for 30 min at 4°C, the supernatant was mixed with 2 ml of TBA/TCA solution (0.8% TBA and 15% TCA dissolved in 0.25 N HCl). The mixture was heated at 100°C for 15 min and then centrifuged at 12000 rpm for 10 min. The amont of MDA was measured spectrophotometrically at 532 and 600 nm, calculated using a standard curve and expressed as nmol/g FW.
Determination of H2O2 levels
The H2O2 content in leaves and roots was determined according to Lereto and Velikova (2001) method. The fresh leaves and roots (0.1 g) were ground in the presence of 2 ml TCA 0.1%. The extracts were centrifuged at 12000 rpm for 15 min. An aliquot of 0.5 ml of the supernatant was supplemented with 0.5 ml of potassium phosphate buffer (10 mM K2HPO4, 10 mM KH2PO4, pH 7) and 1 ml of KI (1 M). The absorbance was measured at 390 nm and H2O2 content was calculated using a standard curve and expressed as µmol/g FW.
Preparation of protein extract
The fresh leaves and roots (0.1 g) were ground in a mortar with 1ml Tris HCl buffer (67 mM, pH 8). After centrifugation at 12000 rpm at 4°C 30 min, the supernatant was used to assess SOD, GPX and CAT activities (Kammoun et al. 2017). Protein concentration in the extract was determined using the Bradford method (Kruger 1994).
Determination of SOD activity
The superoxide dismutase (SOD) activity was determined by monitoring the inhibition of the photo-reduction of Nitro Blue Tetrazolium (NBT) (Dhindsa et al. 1981). The reaction mix contained 50 mM phosphate buffer (pH 7), 2.64 mM NBT, 0.26 mM riboflavin and Na2EDTA-methionine. The photo-reduction of NBT was measured at 560 nm and the SOD activity was expressed as units per milligram of protein. The amount of SOD inhibiting the reaction rate by 50% was defined as one SOD unit.
Determination of CAT activity
The CAT activity was assayed by following the consumption of H2O2 at 240 nm (Aebi 1984). The reaction mix contained 100 µl of protein extract in 1880 µl of 0.1 M phosphate buffer supplemented with 100 µl H2O2 (10 mM) (Jbir-koubaa et al. 2015). One unit of catalase activity was defined as the amount that decomposes one µmol of H2O2.
Determination of GPX activity
The GPX activity was measured as described by Floh and Gunzler (1984). Indeed, 200 µl of enzyme extract were mixed with 400 µL of glutathione (GSH) solution 0.1 mM and 200 µl of phosphate buffer (67 mM, pH 7.8). The mixture was incubated at 25°C for 5 min, then 200 µL of H2O2 (1.3 mM) were added and incubation pursued for 10 min. The reaction mixture was then supplemented with 1 ml TCA (1%). After centrifugation at 3000 rpm for 10 min at 4°C, 480 µl of the supernatant was mixed with 2.2 ml of Na2HPO4 (0.32 M) and 320 µl of 5,5’-dithio-bis-(2-nitrobenzoic acid) (DTNB) (10 mM). The oxidized glutathione (GSSG) produced was determined by measuring the absorbance at 412 nm (Kammoun et al. 2017). The GPX activity was calculated as follows:
µmolreducedGSH disappeared /min/mg of protein = [(OD sample-OD control/OD control)*(0.04*5/X*10)]
with X : protein concentration (mg/ml) ; 0.04 : initial quantity of reduced GSH ; 5: to move from activity in 200 mL to activity in 1 ml; 10 : reaction time
Determination of auxin concentration
The fresh leaves and roots (0.1g) were homogenized in 1 ml of ethanol. After centrifugation at 12000 rpm for 30 min at 4°C, the supernatant was mixed with 2 ml of Salkowki’s reagent. The absorbance was measured at 530 nm. The auxin content was determined using a standard curve of IAA and expressed as µg/g FW (Ghazala et al, 2023).
Determination of proline content
Proline content was measured according to Bates et al. (1973). The fresh mass (0.1g) from leaves and roots was ground in 4 ml sulfosalicylic acid (3%) and centrifuged at 10000 rpm for 30 min. The supernatant was then mixed with 1 ml glacial acetic acid and 1 ml ninhydric acid. After incubation at 100°C for 1 h, 2 ml of toluene were added. The absorbance of upper phase was measured at 520 nm. The proline amount was calculated from a standard curve and expressed as mmol/g FW.
Determination of tuber yield, number, caliber, eyes number and skin color
Tubers of Sp plants grown under the different conditions were collected and weighted. The tuber yield (g/plant) was determined during the 40 days of monitoring and at the end of the cycle (60 days). Tuber number and caliber as well as number of eyes were determined on tubers harvested at the final step. The skin color of these tubers was also measured using a Chroma Meter CR-400/410 colorimeter (Konica Minolta). Three orthogonal coordinates define this parameter: the clarity index L* and the chromatic coordinates a* (red) and b* (yellow). They were determined by scanning the potato skin at five locations.
Determination of dry matter
The percentage of tuber dry matter of tubers was determined after drying for 72 h at 80°C. Dry matter content was determined using the following formulae: DW × 100/FW with FW : fresh weight; DW : dry weight
Chemical analysis of tubers
Lipid, total nitrogen and ash contents of potato tubers were determined according to the American Association of Cereal Chemists 2000 standard methods 46 − 30, 30 − 10 and 08 − 01 respectively (Ben Jeddou et al. 2014). The starch content was determined using the enzymatic colorimetric method described by Khabou et al. (1996). Reducing sugar content was determined by the acid 3, 5-dinitrosalicylic (DNS) method as described by Miller (1959).
Carotenoids content of tubers
To extract lipophilic compounds, 0.3 g of fresh tuber mass were ground in liquid nitrogen and then homogenized for 30 min at 4°C in 1.5 ml of acetone. After centrifugation at 10,000 rpm for 10 min at 4°C, the supernatant was collected, and the pellet was re-extracted with the same solvent. The two supernatants were pooled, and the total carotenoid concentration was determined by measuring the absorbance at 450 nm and expressed as µg/g FW (Chiab et al. 2023)
Statistical analysis
All data were presented as the means ± standard deviation (SD) of three independent biological replicates. The comparison between the different values were statistically effectuated by one-way ANOVA test using IBM SPSS Statistics (version 20). Differences were considered significant at P < 0.05.