Halotolerant PSB enhance barley tolerance to combined salinity and phosphorus deficiency via improved ion homeostasis, photosynthesis and antioxidant defense

Abstract

The use of phosphate-solubilizing bacteria (PSB) is a promising strategy to offset the harmful effect of combined salinity and low phosphorus availability and constitutes an affordable solution to enhance agricultural productivity under co-occurring abiotic stresses. In the present study, we investigate the effect of seed inoculation with different PSB isolates on the responses of barley (Hordeum vulgare) seedlings exposed to salt stress, whether individually applied or in combination with phosphorus deficiency. PSB strains used showed beneficial effect by significantly improving barley response under single and/or combined stresses. Yet, effects were strain- and organ-specific. Considering the plant growth promoting effect, GS4f isolate (Pseudomonas sp.) was the most effective strain in relationship with better water status and photosynthesis activity. Seed inoculation with PSB also reduced Na⁺ content and enhanced K⁺ content along with higher phosphorus mobilization (as P accumulation and acid phosphatase activity). This was concomitant with decreased H₂O₂ production resulting in lower MDA content in stressed roots and leaves of inoculated plants. PSB Inoculation triggered the overall plant antioxidant defense, including enzymatic (SOD, CAT and GPX) under simultaneous salinity and low phosphorus availability. Overall, our findings provide valuable information for prospective production of effective biostimulants based on halotolerant PSB and further highlight the possibility of using this promising eco-friendly approach to improve plant growth in P-deficient and salt-affected soils.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-026-09076-7.

Introduction

Barley (Hordeum vulgare) is a cereal plant species of the Triticeae tribe, widely cultivated all over the world. Barley is known as a flexible crop, with a flexible growth regime. Indeed, it is often grown in a multitude of environments [1]. Barley rates as the fourth most crucial cereal on a global scale in both area of cultivation and in quantity produced [2]. Barley is multipurpose cereal, with diverse uses, such as in the food, feed, and malting industries [3]. Partly due to the ongoing climate change, abiotic stresses are expected to significantly impact barley productivity in several countries in the world, notably the Mediterranean area [4].

Co-occurring abiotic stresses including salinity, drought, nutrient deficiency, and heavy metals strongly restrict crop production [5, 6]. About 6% of the world’s land and 20% of the irrigated areas are threatened by spreading salinity [7]. Potent harmful effects of salinity encompass osmotic stress caused by low external water potential, toxicity of sodium and/or chloride, and nutritional imbalance triggered by the interference with the uptake and transport of essential nutrients [8]. The latter is pronounced in saline and/or arid areas, where high salinity levels and poor soil physical characteristics further impact nutrient (especially P) availability [5]. Na⁺ competes with potassium (K⁺) for the uptake at roots, thus resulting in a decreased K⁺/Na⁺ ratio and disturbed K⁺ status in salt-stressed plants [6, 7]. Maintaining a high K⁺/Na⁺ ratio is directly related to salt tolerance [9]. Since salinity inhibits important metabolic processes in which K⁺ is involved, plant growth and development are consequently impacted [8, 10].

Salinity disturbs key processes such as photosynthesis, respiration, and transpiration [11]. Indirectly, elevated salt concentrations disrupt the osmotic balance, which in turn restricts water uptake in plants [12]. Morphological and biochemical changes include alterations in root and shoot length, leaf number, delayed flowering and fructification, and secondary metabolite accumulation [12, 13]. At the cellular level, Reactive Oxygen Species (ROS) are continuously generated by different abiotic stressors in plants. ROS are highly reactive to damages caused to nucleic acids (DNA/RNA), proteins, and lipids that eventually cause cell death [14, 15]. To prevent these injuries, plants tightly control ROS generation through different enzymatic and non-enzymatic antioxidants [16].

Current global changes exacerbate the impact salinity and aggravate soil calcification in semi-arid and arid areas, which limits nutrient availability for cultivated plants [17]. Phosphorus (P) is the second most important nutrient after nitrogen (N) for plant growth and development. Therefore, P deficiency is a major factor limiting crop production in a number of low-input agricultural systems worldwide, including many regions where calcareous and alkaline soils prevail [9]. It has been reported that salinity impacts P bioavailability and mobility in the plant-soil continuum, and therefore root uptake [18, 19]. Under P shortage, several vital processes are significantly altered, leading to lower plant growth and development [20]. Phosphorus deficiency in ryegrass plants leads to smaller, thinner leaves that turn blue-green due to carbohydrate accumulation and delayed protein synthesis [21]. Similarly, O₂ absorption speed is decreased, and the enzyme activity involved in respiration is altered [21]. Plants show various adaptations to take up P under P deficiency, including changes in root morphology and architecture, and secretion of protons, low-molecular-weight organic acids, and phosphatases into the rhizosphere [22]. When combined, P deficiency and salinity significantly alter the photosynthesis machinery, the mesophyll metabolism, and the ultrastructure of organelles [20, 23]. Soil P availability and salinity may affect plant P acquisition and salt tolerance in an interactive way [24]. Despite depending on stress severity and growing conditions, it is established that salinity reduces P accumulation [25] and P-uptake efficiency in several crops (Cicer arietinum) [26].

P supply to saline soil may alleviate the negative effects of salinity on plant growth and development, and hence improve salt tolerance [27]. Biological and sustainable approaches to minimize the adverse effects of salt stress and phosphorus deficiency on plant development and productivity are gaining interest, among which the inoculation with plant-growth-promoting rhizobacteria (PGPR) [28]. PGPR are a group of soil borne bacteria thriving in interaction with the plant root systems that can improve plant performance by multiple direct mechanisms such as biological nitrogen (N) fixation, phosphorus (P) solubilization, and phytohormone production (e.g. cytokinins, CKs and indole3-acetic acid, IAA) [29, 30]. Rhizospheric bacterial communities contain highly diverse populations of P-solubilizing bacteria such as Bacillus, Pseudomonas, Acinetobacter, Burkholderia, Serratia, Enterobacter, Paenibacillus, Pantoea, Methylobacterium, Azotobacter, Ochrobactrum, Rhizobium, and Acetobacter [31]. Using salt-tolerant PGPR has been documented to be effective in improving plant tolerance against salt stress [32, 33] in relationship with lowering ethylene levels, promoting elongation of root system via hormone secretion for better nutrient uptake, inducing rhizobia proliferation in soil, intensifying the number of potential rhizobium colonization sites, and pathogen suppression via antibiotic production [34, 35]. PGPR inoculation may also activate stress response in plants at morphological, physiological and biochemical levels by synthesizing antioxidants, accumulating osmolytes and producing phytohormones, ACC-deaminase and exopolysaccharides (EPS) [32].

In the context of the valorization of halo-tolerant PGPR for alleviating salt stress in crops considering sustainability goals in agro-farming practices, this study aimed to investigate the potential of inoculation with phosphate-solubilizing bacteria (PSB) isolated from extreme conditions on barley (Hordeum vulgare) grown under combined P shortage and salinity. We perform experiments in conditions that mimic natural conditions, to select an efficient bacterial strain to produce an effective biofertilizer to enhance crop production of barley with sustainable agriculture. In a recent study [36], we used the same bacterial strains to study the involvement of Phosphate solubilizing Bacteria in the adaptation of Barley cultivated under Tricalcium Phosphate (induced phosphorus deficiency). These bacteria are salt-tolerant and are characterized by the production of indole acetic acid (IAA) and P solubilization capacity, which are useful traits for improving P nutrition. We hypothesize that PSB-inoculation might mitigate the impact of P deficiency and/or salinity on plants by improving their mineral status and antioxidative response, in conjunction with the restoration of photosynthetic activity.

Materials and methods

Plant-growth-promoting rhizobacteria (PGPR) strains and their growth conditions

PGPR strains GS1c, GS3b, GS3m, GS4d and GS4f were isolated from four sites (stations), GS3b, GS3m, GS4d and GS4f were isolated from four sites (stations) located in the arid region of Gabes [36]. The phosphate solubilizing bacteria (PSB) were selected to assess their effect on barley under salinity and phosphorus deficiency. Based on 16 S rRNA gene sequencing, these bacterial isolates GS1c, GS3b, GS3m, GS4d and GS4f were identified as Bacillus sp. ADMK50 (KU851006), Bacillus subtilis V44 (MK229110), Bacillus cereus EM10 (KJ612536), Pseudomonas Brassicacearum Y21 (MH997640) and Pseudomonas sp. SA501 (MK294319) respectively [36]. More details about the identification of the used strains are provided in Table 1.

Table 1.

Closest relative of the 5 PGPR isolates based on 16 S rDNA sequence

Strain	Species	GenBank Accession N°	Identity (%)
GS1c	Bacillus sp. ADMK50	KU851006	100
GS3b	Bacillus subtilis 49-907R	KX828318	100
GS3m	Bacillus cereus SVP4	MF471326	100
GS4d	Pseudomonas Brassicacearum Y21	MH997640	100
GS4f	Pseudomonas sp. SA501	MK294319	100

They displayed potent plant growth-promoting (PGP) traits including IAA production, P solubilization, and high tolerance to salinity, pH and Temperature [36]. To reach a final concentration of 8 10⁸ CFU.mL^− 1 for each bacterial strain, a bacterial suspension was prepared in a 10 mL test tube of sterile LB medium at a concentration equivalent to the 0.5 McFarland standard, i.e., 10⁸ CFU/mL, determined using a densitometer [36].

Inoculation of barley by selected strains and plant culture

The selected bacterial isolates were inoculated into liquid culture medium for 48 h with shaking at 140 rpm. After centrifugation at 8000 rpm and 4 °C for 10 min, the obtained pellet of each isolate was washed twice with 50 mM phosphate saline buffer (PBS) and resuspended in 50 mL of sterilized distilled water at a 0.5 McF equivalent to 10⁸ CFU.mL^− 1. Control uninoculated seeds were soaked in 50 mL of sterilized distilled water.

Cultivated barley seeds (Ardaoui variety, widely cultivated in Gabes) were sterilized with 70% ethanol for 2 min, then with 1% sodium hypochlorite for 3 min, and finally rinsed five times with sterile distilled water to prevent contamination by epiphytic microorganisms. Seeds were then sown in 2 kg plastic pots (3 seeds per each) containing sterile sand (after autoclaving at 120 °C for 20 min). The support used for plant culture in this study (sand) contains a small amount of clay and was characterized as follows: electrical conductivity (EC, mS cm^− 1): 0.15; pH: 7.1; Total Organic Carbon: 0.25%; Total Nitrogen: 0.04%; Total Phosphorus: 0.07%; Total Potassium: 0.11%. Plants were grown in a greenhouse under controlled conditions (16:8 h photoperiod (day: night), 20–25 °C temperature, and 65–75% relative humidity).

A 3 × 6 factorial design (18 treatments) in randomized complete block with 5 replications per treatment was carried out to investigate the effects of the Salt stress (S), phosphorus deficiency and PGPR on barley traits (Table 2). Pots were randomly arranged within each of the five blocks according to the RCBD layout. The choice of P concentrations (5 and 180 µM) was based on previous work showing that these both concentrations were limiting and optimal for barley growth, respectively [37]. Each treatment combination was represented by five pots (biological replicates), each containing three plants, giving a total of 90 pots.

Table 2.

Experimental treatments combining three salt stress/phosphorus levels and six PGPR inoculation treatments

Salt stress / P level	PGPR treatment	Description
Control: 0 mM NaCl + 180 µM P Control: 0 mM NaCl + 180 µM P Control: 0 mM NaCl + 180 µM P Control: 0 mM NaCl + 180 µM P Control: 0 mM NaCl + 180 µM P Control: 0 mM NaCl + 180 µM P	No inoculation GS1c GS3b GS3m GS4d GS4f	Control, no PGPR Bacillus sp. ADMK50 Bacillus subtilis V44 Bacillus cereus EM10 Pseudomonas brassicacearum Y21 Pseudomonas sp. SA501
75 mM NaCl + 180 µM P (S1) 75 mM NaCl + 180 µM P (S1) 75 mM NaCl + 180 µM P (S1) 75 mM NaCl + 180 µM P (S1) 75 mM NaCl + 180 µM P (S1) 75 mM NaCl + 180 µM P (S1)	No inoculation GS1c GS3b GS3m GS4d GS4f	Salt stress only Salt + GS1c Salt + GS3b Salt + GS3m Salt + GS4d Salt + GS4f
75 mM NaCl + 5 µM P (S2) 75 mM NaCl + 5 µM P (S2) 75 mM NaCl + 5 µM P (S2) 75 mM NaCl + 5 µM P (S2) 75 mM NaCl + 5 µM P (S2) 75 mM NaCl + 5 µM P (S2)	No inoculation GS1c GS3b GS3m GS4d GS4f	Salt + P deficieny only Salt + P deficieny + GS1c Salt + P deficieny + GS3b Salt + P deficieny + GS3m Salt + P deficieny + GS4d Salt + P deficieny + GS4f

For each treatment plants were irrigated twice a week with 100 mL Hewitt nutrient solution [38]. The pH and EC (mS.cm^− 1) of the freshly prepared nutrient solution were 6.0 and 1.5 for the control and 6.1 and 7.4 for 75 mM NaCl treatment. Since sand has a high drainage rate, which contributes to its low buffering capacity. To avoid significant changes in pH levels, Good buffer as 5 mM PIPES (pH 6.1–7.5; pKa = 6.80) was added to the nutrient solution.

The difference in nutrient disorders potentially caused by the decreased solubility due to pH variation was eliminated (or buffered) by renewing the nutrient solution each 72 h and by using iron as 20 µM Fe-EDTA. EDTA (ethylenediaminetetraacetic acid), which is known to have a high stability to prevent nutrient precipitation, particularly of phosphorus [39].

Two-month-old plants were harvested and randomly separated in two groups. In the first group, leaves and roots for each plant were collected, washed, and kept at -80 °C for enzyme activity assay. The second group was intended for growth analysis and ion status of shoots and roots. Besides fresh weight (FW), dry weight (DW) was measured after 48 h of desiccation at 60 °C using a digital scale with a 0.1 mg resolution.

Tissue water content (TWC) was also calculated as:

TWC (ml g^− 1 DW) = (FW-DW) / DW

Leaf gas exchanges, chlorophyll content and ion status

Net CO₂ assimilation and stomatal conductance were recorded with a portable photosynthesis system (LCA4). Measurement conditions were as follows: 1200 µmol mm^− 2 s^− 1 photosynthetically active radiation (PAR), 378 µmol mol^− 1 ambient CO₂ concentration and 28 ± 2 °C leaf temperature. Measurements were carried out in the morning between 10:00 and 12:00 (10 replicates per treatment). Data were automatically collected every minute after the photosynthesis rate had stabilized.

Chlorophyll extraction was performed over 3 days at 4 °C in the dark to ensure complete pigment solubilization while preventing light-induced degradation. For each replicate, 100 mg of fresh leaf tissue was used and extracted with 5 mL of 80% acetone, ensuring consistent sample-to-solvent ratios for reproducibility. After centrifugation at 500 g for 5 min, extract absorbance was measured at 470, 645 and 662 nm [40].

Desiccated leaf tissues were ground to a fine powder and then broken down with concentrated 0.5% HNO₃. The resulting extract was diluted 10-fold before ion quantification. Na⁺ and K⁺ concentrations of were determined using flame emission photometry (Corning, Cambridge, UK) and expressed as mg per g dry weight (mg/g DW) of plant material.

Acid phosphatase activity and phosphorus content determination

Acid phosphatase

Leaves and roots (0.1 g) were separately ground in a mortar with 0.1 M buffer (1 g.ml^− 1 buffer), 6 mM ß-mercaptoethanol, 0.1 mM phenyl methyl sulfonyl fluoride, and 6 g insoluble polyvinylpolypyrrolidone. The homogenate was centrifuged at 30,000 g at 4 °C for 30 min. The reaction mixture contained 100 mM sodium acetate buffer (pH 5.8), 5 mM pnitrophenyl phosphate, and the enzyme in a total volume of 0.5 ml. After 30 min incubation at 30 °C, the reaction was stopped by adding 1 ml 0.5 M NaOH. Acid phosphatase activity was measured at 405 nm by monitoring the p-nitrophenol released [21].

Pi determination

Leaf samples (25 mg DW) from each plant were digested in HNO₃ at 0.5%. The inorganic phosphorous released was quantified by the molybdovanadate method at 460 nm [21]. P absorption efficiency (PAE) and P use efficiency (PUE) were also calculated [41]. PAE (µmol P mg^− 1 root DW), a useful indicator of the root capacity to absorb P from the soil solution, was calculated as the ratio between the amount of P taken up during the experiment to the mean root biomass as: PAE = Q/RDW

With Q and RDW respectively representing the quantity of P (µmol) taken up in each plant and the root dry weight (mg).

PUE was calculated as the ratio between the biomass production and P accumulation in plants (mg DW µmol^− 1 P), reflecting the dry weight increase rate per unit of P content.

H₂O₂ and MDA determination

Samples were ground with liquid nitrogen in a 0.1% TCA (w/v) trichloroacetic acid solution and then centrifuged at 10,000 g at 4 °C for 15 min. The supernatant was then incubated with 10 mM phosphate buffer (KH₂PO₄/K₂HPO₄; pH 7) containing potassium iodide (KI, 1 M). The optical density is determined at 390 nm [42]. H₂O₂ contents were quantified using a calibration curve generated from standard solutions of 0.1–1 mM H₂O₂. H₂O₂ levels in samples were expressed as µmol g⁻¹ FW, ensuring precise and standardized quantification.

The extent of lipid peroxidation was determined by measuring the MDA content formed through thiobarbituric acid reaction [42]. Leaf fresh material (200 mg) was homogenized in 2 ml of 0.1% trichloroacetic acid (TCA). Homogenates were centrifuged at 13,000 g in a refrigerated centrifuge (HERMLE Z 36 HK) for 20 min. 0.5 mL of the obtained supernatant was added to 1.5 mL 0.5% (w/v) TBA in 20% (w/v) TCA. The mixture was incubated at 95 °C for 30 min, and the reaction was then stopped in an ice bath. The mixture was centrifuged at 4° C for 10 min at 10,000 g and the absorption of the supernatant measured at 532 nm and 600 nm.

Total soluble proteins and antioxidant activities

Fresh matter was ground in a mortar with 10% (w/w) polyvinyl-pyrrolidone in 1 mL of 50 mM phosphate buffer (pH7.8) containing 0.1 mM EDTA, 0.1% (v/v) Triton X-100,1 mM phenyl methane sulfonyl fluoride (PMSF). The extract was centrifuged at 13,000 g for 20 min at 4 °C. The soluble protein content of leaves and roots was estimated according to [43]. Absorbance was measured at 595 nm to quantify protein concentration based on a bovine serum albumin (BSA) standard curve.

Catalase (EC.1.11.1.6) activity was measured by following the decline in A240 as H₂O₂ (ε = 36 M^− 1 cm⁻) was catabolized, according to [44] in a reaction mixture containing 50 µL enzyme extract in 50 mM potassium phosphate buffer (pH 7.8). The reaction was started by adding H₂O₂ and its consumption was measured for about 30 s at 240 nm. Superoxide dismutase (SOD, EC.15.1.1) activity was spectrophotometrically determined by measuring its ability to inhibit the photoreduction of nitro blue tetrazolium (NBT) at 560 nm [45]. The reaction solution contained 50 mM K-phosphate (pH 7.8), 0.1 mM EDTA, 10 mM L-methionine, 2.7 µM riboflavin and 75 µM NBT. One unit of SOD activity was defined as the amount of enzyme that inhibited 50% of NBT photoreduction at 25 °C. Guaiacol peroxidase (POX, EC 1.11.1.7) was assayed according to [46] by monitoring the enzyme activity at 470 nm during 1 min (ε = 36 M^− 1 cm^− 1). POX activity was measured as oxidation of guaiacol (9 mM) in the presence of H₂O₂ (19 mM).

Statistical analysis

Statistical analyses were performed using R software (R Core Team, 2020). A two-way analysis of variance (ANOVA) was conducted to assess the effects of Stress (C, S1, and S2), PGPR (no PGPR inoculation, GS1c-, GS3b-, GS3m-, GS4d-, GS4f-inoculation) and their interaction on each response variable using the stats package (R Core Team, 2020).

The statistical model used was:

where: is the observed value of the response variable; is the overall mean; is the fixed effect of the i^th stress level; is the fixed effect of the j^th PGPR inoculation treatment, is the interaction between salt stress and PGPR; is the random effect of block and is the random error term.

When significant effects were detected (α = 0.05), post hoc pairwise comparisons were performed with Tukey-adjusted P-values using the emmeans package. Compact letter displays for all pairwise comparisons were generated with the multcomp package [47].

All variables were standardized (mean-centered and scaled to unit variance) prior to principal component analysis (PCA) using FactoMineR [48] to explore relationships among variables and treatments. Principal components with eigenvalues higher than 1 (Kaiser’s criterion) were retained for interpretation. PCA results were visualized with factoextra. Graphical representations were produced with ggplot2.

Results

Plant growth

Significant effects of the main factors Stress and PGPR on shoot dry weight (SDW) (F_2,82 = 579.53, p < 0.001; F_5,82 = 14.48, p < 0.001, respectively) and root dry weight (RDW) (F_2,82 = 219.62, p < 0.001; F_5,82 = 26.29, p < 0.001, respectively) were found, whereas the interaction term showed no significant effects on both growth parameters (F_10,72 = 0.47, p = 0.906; F_10,72 = 1.53, p = 0.149, respectively). SDW was significantly lower in plants challenged with salinity (S1) (0.29 ± 0.02 g.plant^− 1) and combined salinity and P shortage (S2) (0.13 ± 0.01 g.plant^− 1) as compared to plants grown at 180 µmol P (C) (Fig. 1a). Concerning RDW, it was not significantly affected at S1, however under S2, it significantly decreased (0.20 ± 0.01 g.plant^− 1) as compared to the control (C) (Fig. 1b). Compared to the control uninoculated plants, PSB inoculation significantly improved SDW and RDW in plants inoculated with GS3m, GS4d and GS4f with an increase ranging from 16 to 29% (Fig. 1a-b).

Fig. 1.

Effect of PSB inoculation on Hordeum vulgare (var. Ardhaoui) growth-related traits as Shoot DW (a), Root DW (b), Shoot height (c), and Root length (d). Plants were cultivated under the following treatments: salt-free and 180 µmol P (Control, C), 75 mM NaCl and 180 µM P (S1) or 75 mM NaCl and 5 µM P (S2). Values are the means of five replicates per treatment. Bars labelled with the same letter are not significantly different at 5% according to Tukey HSD test

As for SDW, shoot height (SL) was significantly affected by the main factors Stress and PGPR (F_2,82 = 351.06, p < 0.001; F_5,82 = 25.06, p < 0.001, respectively). The highest values of SL were recorded in the control (C) while strongly decreasing in S1 (-25%) and especially S2 (-39%) (Fig. 1c). Interestingly, SL increased significantly following plant inoculation with GS4f, GS4d and GS3m bacterial strains (+ 20% to + 28% as compared to uninoculated plants) (Fig. 1c). For root length (RL), PGPR - S interaction was significant (F_10,72 = 5.69, p < 0.001). RL of uninoculated plants showed no significant changes depending on treatment (Fig. 1d). However, RL was significantly stimulated when S1 plants were inoculated by GS4f (36.2 ± 1.3 cm.plant^− 1), GS4d (33.4 ± 1.8 cm.plant^− 1) bacterial strains as compared to uninoculated ones (25.4 ± 1.5 cm.plant^− 1). This PSB-related promoting effect was more pronounced with GS4f (+ 66%), GS4d (+ 50%) and GS3m (+ 41%) and GS1c (+ 20%) strains in S2-challennged plants.

Water content

Shoot water content (SWC) was significantly affected by the interaction Stress-PGPR (F_10,72 = 5.16, p < 0.001) whereas root water content (RWC) was only impacted by the main effects of Stress and PGPR (F_2,82 = 416.21, p < 0.001; F_5,82 = 7.33, p < 0.001, respectively). PSB inoculation on SWC was significant only in S1 and S2. These effects were strain-dependent, with GS4f being the most promoting strain (+ 56% in S1 and + 37% in S2 as compared to uninoculated plants) (Fig. 2a). The highest values of RWC were recorded for plants cultivated under optimal conditions (C), before strongly decreasing upon exposure to salinity (S1) (-30%) and combined salinity and P deficiency (S2) (-53%) (Fig. 2b). Again, inoculation with PSB strains (GS4f and GS4d) resulted in a better hydration status of stressed plants. When compared to uninoculated plants, GS4f-inoculated ones showed the strongest improvement (+ 16%) in RWC.

Fig. 2.

Effect of PSB inoculation on Hordeum vulgare (var. Ardhaoui) water status and leaf pigment content. Shoot water content (a), Root water content (b), and leaf total chlorophyll content (c). Plants were cultivated under the following treatments: salt-free and 180 µmol P (Control, C), 75 mM NaCl and 180 µM P (S1) or 75 mM NaCl and 5 µM P (S2). Values are the means of five replicates per treatment. Bars labelled with the same letter are not significantly different at 5% according to Tukey HSD test

Photosynthetic parameters

Depending on the treatment, PSB inoculation had different effects on total chlorophyll content, net CO₂ assimilation (A), intercellular CO₂ concentration (Ci) stomatal conductance (gs) and transpiration (E), as revealed by the significant effect of the Stress-PGPR interaction term (F_10,72 = 6.07, p < 0.001; F_10,72 = 3.54, p < 0.001; F_10,72 = 2.44, p = 0.014; F_10,72 = 16.14, p < 0.001; F_10,72 = 2.70, p = 0.007, respectively). Salinity alone (S1) or combined with P shortage (S2) significantly impacted uninoculated plants, with up to 47% and 69%, respectively (Fig. 2c). PSB inoculation increased total chlorophyll content in plants challenged with salinity alone (S1) as compared to uninoculated plants, this mitigating effect being again more pronounced with GS4f (+ 62%), GS4d (+ 46%) and GS3m (+ 37%) strains. GS4f and GS4d strains were also the most effective under combined stresses (S2), despite to a slightly lesser extent (+ 53% and + 40% for GS4f- and GS4d-inoculated plants, respectively) as compared to S1 conditions.

Net CO₂ assimilation was maximal in the control plants, especially when inoculated with GS4f and GS4d strains (Fig. 3a). This parameter decreased significantly in uninoculated plants challenged with salinity, especially when combined with P shortage (-45% for S1 and − 70% for S2). Under S1 conditions, inoculation with GS4d and GS4f increased significantly net CO₂ assimilation by 35% and 55% respectively. This was also true under combined stresses (S2), with a stimulation reaching 40% and 60% for GS4d- and GS4f-inoculated plants when compared to uninoculated ones. Stomatal conductance sharply decreased in uninoculated plants cultivated under S1 and under S2 treatments (-48% and − 80%, respectively) as compared to their controls (Fig. 3b). However, PSB inoculation improved this parameter in S1 plants, especially with GS4f and to a lesser extent GS4d (+ 70% and + 50%, respectively). Similarly, an increase in stomatal conductance was detected in S2 plants when inoculated with GS4f (+ 80%) and GS4d (+ 50%). Generally, transpiration showed similar trends as those observed for net CO₂ assimilation and stomatal conductance (Fig. 3c). Besides, GS4f and GS4d strains were the most efficient strains in mitigating the impact of S1 stress, while the former was the most effective at reducing the effects of S2 stress on transpiration. The lowest internal CO₂ concentration was observed in plants grown under optimal P supply and salt-free conditions (control) (Fig. 3d). This parameter increased significantly in S1 (+ 30%) and especially S2 (+ 50%) plants but declined in S1 GS4f- and GS4d- and S2 GS4f-, GS4d- and GS3m-inoculated ones relative to their uninoculated counterparts (e.g. -20% with GS4f in S1 and − 15% with GS4d in S2).

Fig. 3.

Effect of PSB inoculation on leaf gas exchange parameters of Hordeum vulgare (var. Ardhaoui) as Net CO₂ assimilation (a), Stomatal conductance (b), Transpiration rate (c), and Internal CO₂ concentration (d). Plants were cultivated under the following treatments: salt-free and 180 µmol P (Control, C), 75 mM NaCl and 180 µM P (S1) or 75 mM NaCl and 5 µM P (S2). Values are the means of five replicates per treatment. Bars labelled with the same letter are not significantly different at 5% according to Tukey HSD test

Shoot Na⁺ and K⁺ content and K⁺/Na⁺ selectivity

The main effects of Stress and PGPR were significant for shoot Na⁺ content (F_2,82 = 3446.78, p < 0.001; F_5,82 = 21.89, p < 0.001), but their interaction showed no significant effect (F_10,72 = 1.67, p = 0.111). Shoot Na⁺ contents were respectively 1.23 ± 0.013 and 1.36 ± 0.039 mg.g^− 1 DW under S2 and S1 and were higher as compared to control plants (0.667 ± 0.04 mg.g^− 1 DW ) (Table 2). Besides, inoculation of barley plants with GS4f, GS4d and GS3m bacterial strains slightly restricted shoot Na⁺ accumulation relatively to uninoculated ones (-4%, -7% and − 10%, respectively).

As shown by the significant interaction term (F_10,72 = 4.59, p < 0.001), the PGPR effect for K⁺ depends on the Stress level. Shoot K⁺ content of uninoculated plants (Table 3) decreased (for S1 and S2 was respectively 28.5 ± 0.54 mg.g^− 1 DW and 25.52 ± 0.73 mg.g^− 1 DW) as compared to their control (39.05 ± 0.7 mg.g^− 1 DW), but this trend was offset following inoculation, as reflected by the significant increase of K⁺ accumulation (Table 3), especially in GS4f- (+ 25% and + 33% under S1 and S2) and GS4d-inoculated plants (+ 12% and + 15% under S1 and S2). This was also true for GS3m strain (under S1 and S2) and GS1c (under S1), despite to a lesser extent. In control conditions, only a slight increase of K⁺ content in plants inoculated with GS4f bacterial strain was observed.

Table 3.

Effect of PSB inoculation on sodium and potassium accumulation in Hordeum vulgare (var. Ardhaoui) plants cultivated under the following treatments: salt-free and 180 µmol P (Control, C), 75 mM NaCl and 180 µM P (S1) or 75 mM NaCl and 5 µM P (S2). Values are the means of five replicates per treatment. Different letters indicate significant difference at 5% according to Tukey HSD test

Treatment	Shoot Na+ content (mg g− 1 DW)	Shoot K+ content (mg g− 1 DW)	K+/Na+ ratio
Control
Uninocolated	0.667 ± 0.037 a	39.05 ± 0.7 i	57.42 ± 1.9 i
GS1c	0.665 ± 0.042 a	41.5 ± 0.65j	63.48 ± 2.35 j
GS3b	0.698 ± 0.032 a	40.4 ± 0.9 ij	62.06 ± 2.45 j
GS3m	0.657 ± 0.047 a	40.05 ± 0.82 ij	65.90 ± 2.32 k
GS4d	0.645 ± 0.025 a	41.02 ± 0.54 j	66.29 ± 3.21 k
GS4f	0.637 ± 0.029 a	41.87 ± 0.7 j	69.12 ± 1.24 l
S1
Uninoculated	1.36 ± 0.039f	28.5 ± 0.54 e	20.89 ± 0.78 d
GS1c	1.32 ± 0.022 f	31.45 ± 0.85 fg	23.79 ± 0.65 ef
GS3b	1.33 ± 0.013 f	30.02 ± 0.6 f	22.49 ± 0.42 e
GS3m	1.30 ± 0.024 ef	32.05 ± 0.75 g	24.56 ± 0.62 f
GS4d	1.24 ± 0.012 de	34.2 ± 0.66 h	27.29 ± 0.82 g
GS4f	1.22 ± 0.015 d	35.55 ± 0.57 h	29.02.24 ± 0.95 h
S2
Uninoculated	1.235 ± 0.013d	19.2 ± 0.45 a	15.7 ± 0.45 a
GS1c	1.18 ± 0.01 cd	21.02 ± 0.61 b	17.70 ± 0.68 b
GS3b	1.21 ± 0.016 cd	20.62 ± 0.73a b	16.97 ± 0.53ab
GS3m	1.19 ± 0.02 cd	22.02 ± 0.52 c	18.50 ± 0.74 c
GS4d	1.15 ± 0.017bc	23.92 ± 0.38 d	20.80 ± 0.35 d
GS4f	1.13 ± 0.008b	25.52 ± 0.73 d	22.59 ± 0.86 e
p-value
Treatment	< 0.001	< 0.001	< 0.001
Strain	< 0.001	< 0.001	< 0.001
Treatment*Strain	0.111	< 0.001	0.808

For shoot K⁺/Na⁺ ratio (Table 3), Stress and PGPR main effects were significant (F_2,82 = 3181.38, p < 0.001; F_5,82 = 26.22, p < 0.001), while their interaction effect was not significant (F_10,72 = 0.60, p = 0.808). K⁺/Na⁺ ratio was strongly reduced in plants cultivated at S1 (-60%) and S2 (-70%) following higher sodium accumulation in salt-challenged plants (Table 1). However, PSB inoculation significantly increased K⁺/Na⁺ ratio in comparison with uninoculated plants (+ 12%, + 12%, + 20% and + 26% respectively for the strains GS1c, GS3m, GS4d and GS4f).

Phosphorus nutrition

Phosphorus content

Shoot and root P contents varied significantly under Stress and PGPR interaction effect (F_10,72 = 2.21, p = 0.031; (F_10,72 = 5.78, p < 0.001, respectively). In control conditions, PSB inoculation had no significant effect on shoot and root P contents of barley plants (Table 4). In uninoculated plants grown under S1 and mostly S2 conditions, a significant decrease of shoot P content was observed (-11% and − 72% relative to control plants, respectively). This adverse effect on shoot P content was attenuated following the inoculation with GS4f and GS4d strains under both S1 (+ 10% and + 9%, respectively) and S2 conditions (+ 41% and + 38%, respectively) comparatively to uninoculated plants.

Table 4.

Effect of PSB inoculation on phosphorus nutrition-related traits in Hordeum vulgare (var. Ardhaoui) plants cultivated under the following treatments: salt-free and 180 µmol P (Control, C), 75 mM NaCl and 180 µM P (S1) or 75 mM NaCl and 5 µM P (S2). Values are the means of five replicates per treatment. Different letters indicate significant difference at 5% according to Tukey HSD test

Treatment	P Shoot content (µmol g− 1 DW)	P Root content (µmol g− 1 DW)	PAE (µmol P mg− 1 root DW)	PUE (mg DW µmol− 1)	Acid phosphatase leaves (µmol PNP mg− 1 Protein min− 1)	Acid phosphatase roots (µmol PNP mg− 1 Protein min− 1)
Control
Uninocolated	251.71 ± 4.25 hi	124 ± 4.5 g	0.38 ± 0.022 e	6.01 ± 0.21 b	2.92 ± 0.22 a	4.32 ± 0.19 a
GS1c	256.90 ± 6.1 i	129.8 ± 5.6 gh	0.41 ± 0.031 e	5.99 ± 0.12 b	3.02 ± 0.15 ab	4.49 ± 0.14 a
GS3b	256.35 ± 5.01 i	127.22 ± 6.3 g	0.39 ± 0.025 e	6.08 ± 0.25 b	2.98 ± 0.18 ab	4.22 ± 0.15 a
GS3m	262.80 ± 6.5 i	132.32 ± 5.6 h	0.40 ± 0.033 ef	6.21 ± 0.22 b	3.15 ± 0.14 b	4.71 ± 0.17 b
GS4d	255.65 ± 7.5 i	135.3 ± 4.8 h	0.42 ± 0.02 f	6.26 ± 0.14 bc	3.16 ± 0.19 b	4.74 ± 0.12 b
GS4f	262.21 ± 6.9 i	136.3 ± 5.3 h	0.43 ± 0.041 f	6.45 ± 0.28 c	3.21 ± 0.21 b	4.70 ± 0.11 b
S1
Uninoculated	224.25 ± 7.7 e	79.35 ± 3.8 d	0.22 ± 0.035 b	4.06 ± 0.16 a	4.20 ± 0.24 c	6.60 ± 0.38 c
GS1c	235.1 ± 6.4 fg	100.56 ± 4.1 e	0.26 ± 0.027 c	4.72 ± 0.34 a	4.94 ± 0.30 d	7.70 ± 0.35 d
GS3b	232.7 ± 3.9 f	100.73 ± 3.1 e	0.25 ± 0.018 c	4.38 ± 0.29 a	5.09 ± 0.29 d	7.20 ± 0.26 cd
GS3m	239.8 ± 4.1 g	112.81 ± 2.7 f	0.3 ± 0.023 cd	7.10 ± 0.19 d	6.87 ± 0.28 ef	10.26 ± 0.3 f
GS4d	243.5 ± 5.7 gh	118.91 ± 4. 5f g	0.32 ± 0.019 d	8.27 ± 0.30 e	8.55 ± 0.40 g	12.04 ± 0.35 g
GS4f	246 .± 5.4 h	124.29 ± 5.5 g	0.34 ± 0.025 d	8.95 ± 0.40 e	9.47 ± 0.38 g	12.80 ± 0.37 gh
S2
Uninoculated	69.47 ± 4.2 a	41.25 ± 2.5 a	0.18 ± 0.02 a	14.82 ± 0.26 f	6.12 ± 0.23 e	8.22 ± 0.23 d
GS1c	76.43 ± 5.8 b	42.88. ± 3.1 a	0.20 ± 0.015 a	15.21 ± 0.35 f	7.22 ± 0.31 f	9.07 ± 0.25 e
GS3b	70.3 ± 4.5 a	41.81 ± 4.1 a	0.19 ± 0.01 ab	14.90 ± 0.25 f	7.67 ± 0.25 fg	8.95 ± 0.18 e
GS3m	84.6 ± 5.3 c	57.99 ± 2.9 b	0.23 ± 0.015 bc	23.40 ± 0.38 g	8.32 ± 0.23 g	13.88 ± 0.29 h
GS4d	95.65 ± 7.1 d	60.97 ± 3.8 bc	0.27 ± 0.011 cd	32.90 ± 0.24 h	11.17 ± 0.3 h	16.63 ± 0.32 i
GS4f	98.3 ± 6.9 d	65.87 ± 4.5 c	0.31 ± 0.017 d	35.95 ± 0.48 i	12.00 ± 0.4 i	17.26 ± 0.39 i
p-value
Treatment	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001
Strain	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001
Treatment*Strain	0.301	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001

In roots (Table 4), P content decreased in uninoculated plants grown under S1 (-36%) and to higher extent S2 (-67%) conditions as compared to the control (Table 4). As for shoots, the bacterial strains improved root P content under both S1 and S2. This was especially true for GS4f (ca. +60% on average), GS4d (ca. +50% on average) and GS3m (ca. +42% on average) strains. For the other strains (GS1c and GS3b), the inoculation slightly increased this parameter in S1-challenged plants.

Acid phosphatase (AP) activity

The interaction term Stress-PGPR had significant effect on AP activity both in shoots and roots (F_10,72 = 48.86, p < 0.001; F_10,72 = 191.92, p < 0.001). In contrast to plants grown under control conditions, PGPR treatment had a significant effect on AP activity in leaves and roots of barley plants challenged with S1 and S2 (Table 4). In leaves, AP activity was stimulated in uninoculated plants grown under stress versus to the control conditions (+ 43% and + 109% respectively for S1 and S2). Regardless of the strain used, the inoculation further increased AP activity under combined stresses (S2), the highest values being recorded for GS4f, GS4d and GS3m (+ 96%, + 83% and + 36% relatively to uninoculated plants). GS3m-, GS4f-, and GS4d-inoculated plants exhibited a strong increase of AP activity under S1 with percentages ranging from + 64% to 126% (in relation to uninoculated plants).

AP activity was higher in roots than in leaves independently of the applied Stress-PGPR treatment (Table 4). S2 plants displayed the highest values as compared to S1 and control plants. Apart from GS3b, inoculation with PSB significantly increased this parameter in plants cultivated under single or combined stresses (Table 4). This stimulating effect was more pronounced for the strains GS4f under S1 and S2 (+ 94% for the first + 110% for the second), GS4d (+ 82% and + 102% respectively), and to a lesser extent GS3M (+ 55% and + 96% correspondingly).

Oxidative stress markers

ANOVA revealed significant interaction effect Stress-PGPR on H₂O₂ and MDA contents in leaves (F_10,72 = 27.33, p < 0.001; F_10,72 = 5.88, p < 0.001, respectively) and roots (F_10,72 = 8.33, p < 0.001; F_10,72 = 2.41, p = 0.019, correspondingly) of barley plants.

Hydrogen peroxide

In control plants, the inoculation with PSB showed no significant change of H₂O₂ content in shoots and roots (Table 5). Leaf H₂O₂ accumulation increased in uninoculated plants when cultivated under S1 (+ 113%) and to a lesser extent under S2 (+ 70%). PGPR inoculation resulted in a significant reduction of this parameter in plants under S1, ranging from 26 (GS3b) to 54% (GS4f). Under S2, this reduction was significant only in GS4d- (-62%), GS4f- (-60%) and GS3m-inoculated plants (-30%). In roots, H₂O₂ accumulation was observed only in S2 uninoculated plants, and it was much lower than that observed in shoots (Table 5) (+ 27%). In GS4f- and GS4d-inoculated plants, H₂O₂ content decreased for S2 plants as contrasted with their control plants.

Table 5.

Effect of PSB inoculation on oxidative stress response in Hordeum vulgare (var. Ardhaoui) plants cultivated under the following treatments: salt-free and 180 µmol P (Control, C), 75 mM NaCl and 180 µM P (S1) or 75 mM NaCl and 5 µM P (S2). Values are the means of five replicates per treatment. Different letters indicate significant difference at 5% according to Tukey HSD test

Treatment	MDA (nmol g−1FW)		H2O2 (µmol g− 1 FW)		SOD (U mg− 1 protein)		Catalase (U mg− 1 protein)		GPX (U mg− 1 protein)
	Leaves	Roots	Leaves	Roots	Leaves	Roots	Leaves	Roots	Leaves	Roots
Control
Uninocolated	3.39 ± 0.3 c	3.4 ± 0.29 de	3.6 ± 0.27c	3.1 ± 0.42 c	150.4 ± 6.9 b	143.9 ± 9.3 a	3.5 ± 0.22 a	2.63 ± 0.25 c	2.01 ± 0.11 b	4.55 ± 0.28 a
GS1c	3.1 ± 0.15 bc	3.2 ± 0.015 cd	3 ± 0.14 bc	3.16 ± 0.13 c	159.5 ± 8.5 bc	149.5 ± 8.5 a	3.8 ± 0.43 ab	2.64 ± 0.12 c	2.05 ± 0.15 b	4.65 ± 0.12 a
GS3b	3.2 ± 0.13 bc	3.3 ± 0.16 d	3.1 ± 0.2 bc	3.24 ± 0.12 c	157.4 ± 6.9 bc	148.8 ± 8 ab	3.85 ± 0.37 ab	2.53 ± 0.14 c	1.99 ± 0.2 b	4.54 ± 0.22 a
GS3m	3 ± 0.11 bc	3.1 ± 0.12 cd	2.7 ± 0.21 b	3.04 ± 0.1 c	165.8 ± 9.5 c	155.2 ± 8.4 ab	4.06 ± 0.23 b	2.7 ± 0.15 c	2.28 ± 0.12 bc	4.78 ± 0.19 ab
GS4d	2.9 ± 0.13 b	3 ± 0.11 c	2.8 ± 0.14 b	3.01 ± 0.09 c	166.9 ± 7.2 c	160.5 ± 9.2 b	4.2 ± 0.31 b	2.74 ± 0.2 c	2.06 ± 0.14 b	4.77 ± 0.13 ab
GS4f	2.8 ± 0.18 b	2.9 ± 0.14 bcd	2.5 ± 0.13 b	2.77 ± 0.06 b	170.2 ± 5.6 cd	163.7 ± 7.3 b	4.16 ± 0.2 b	2.81 ± 0.18 c	2.18 ± 0.11 bc	4.86 ± 0.21 ab
S1
Uninoculated	5.7 ± 0.53 f	4.4 ± 0.45 f	5.9 ± 0.31 h	3.63 ± 0.41 cd	134.2 ± 9.5 b	158.1 ± 6.8 ab	4.7 ± 0.24 c	2.02 ± 0.21 b	2.16 ± 0.33 bc	4.75 ± 051 ab
GS1c	4.7 ± 0.35 e	3 ± 0.3 cd	3.2 ± 0.4 cd	3.38 ± 0.2 c	147.3 ± 7.5 b	148.5 ± 7.8 b	4.04 ± 0.8 b	5.39 ± 0.64 ef	2.02 ± 0.21 b	5.4 ± 0.25 c
GS3b	4.1 ± 0.64 d	3.2 ± 0.42 cd	4.9 ± 0.41 fg	3.13 ± 0.24 c	169.9 ± 5.8 cd	161.3 ± 9.3 b	6.4 ± 0.44 e	4.18 ± 0.29 d	2.44 ± 0.3 c	4.53 ± 0.24 ab
GS3m	4.4 ± 0.29 de	3.3 ± 0.33 d	4.2 ± 0.23 e	3.41 ± 0.48 c	177.2 ± 9.6 cd	171.2 ± 8.4 bc	4.14 ± 0.21 b	4.9 ± 0.16 e	2.06 ± 0.3 bc	5.71 ± 0.27 cd
GS4d	3.2 ± 0.18 bc	3.2 ± 0.25 cd	3 ± 0.33 c	2.63 ± 0.35 ab	180.3 ± 8.6 d	159.97 ± 9 bc	5.2 ± 0.35 d	7.72 ± 0.4 g	2.76 ± 0.2 cd	4.79 ± 0.37 ab
GS4f	2.9 ± 0.26 bc	2.9 ± 0.21 c	2.9 ± 0.2 bc	2.53 ± 0.34 a	210.8 ± 9.3 e	185.4 ± 7.1 c	7.7 ± 0.29 f	10.3 ± 0.38 i	2.7 ± 0.11 cd	5.99 ± 0.26 d
S2
Uninoculated	5.02 ± 0.29 f	3.6 ± 0.19 e	5.2 ± 0.32 g	3.99 ± 0.26 d	119.3 ± 7.7 a	141.5 ± 5.2 a	4.4 ± 0.32 c	1.43 ± 0.11 a	1.02 ± 0.11 a	4.97 ± 0.35 bc
GS1c	4.1 ± 0.38 d	2.70 ± 0.6 bc	4. 5 ± 0.35 ef	3.64 ± 0.43 cd	165.8 ± 9.8 c	152.5 ± 7.8 ab	4.5 ± 0.41 c	4.49 ± 0.42 de	2.29 ± 0.16 bc	5.72 ± 0.23 cd
GS3b	4.0 ± 0.53 d	2.4 ± 0.28 b	5. 1 ± 0.3 fg	3.45 ± 0.34 c	161.5 ± 7.8 c	165.4 ± 8.6 b	6.1 ± 0.38 e	5.92 ± 0.12 f	1.9 ± 0.23 b	4.65 ± 0.22 ab
GS3m	4.3 ± 0.21 d	2.5 ± 0.3 b	3.7 ± 0.41 d	3.1 ± 0.18 c	174.5 ± 8.5 cd	166.3 ± 9.5 bc	5.2 ± 0.28 d	4.65 ± 0.15 de	2.81 ± 0.1 d	4.89 ± 0.37 b
GS4d	3.1 ± 0.22 bc	2.7 ± 0.25 bc	2.6 ± 0.12 b	2.79 ± 0.35 ab	181.2 ± 9.01 d	170.9 ± 8.2 bc	8.4 ± 0.59 f	9.33 ± 0.45 h	2.73 ± 0.24 cd	5.96 ± 0.31 cd
GS4f	1.86 ± 0.28 a	1.4 ± 0.32 a	2. 4 ± 0.18 a	2.52 ± 0.27 a	199.4 ± 8.6 de	177.9 ± 9.3 c	21.8 ± 0.75 g	11.06 ± 0.52 j	3.36 ± 0.25 e	6.08 ± 0.27 d
p-value
Treatment	< 0.001	< 0.001	< 0.001	< 0.001	0.060	0.011	< 0.001	< 0.001	< 0.001	< 0.001
Strain	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001
Treatment*Strain	< 0.001	0.019	< 0.001	< 0.001	< 0.001	0.276	< 0.001	< 0.001	< 0.001	< 0.001

Malondialdehyde (MDA) contents

MDA content (Table 5) strongly increased in leaves of uninoculated plants cultivated under S1 (+ 70%) and S2 (+ 48%) as compared to control (C). Under S1, this trend was lower in plants inoculated with GS4f (-50%), GS4d (-44%), GS3b (-31%) and GS3m (-23%) strains. This was also true for the combined treatment (S2), especially in plants inoculated with Strains GS4f (-63%) and GS4d (-38%). In roots, MDA content showed no significant changes when uninoculated plants were cultivated under S1 and S2 (Table 5). This parameter was reduced when plants were inoculated with the GS4f, GS1a (-32% and − 31%, respectively) in S1 and GS4f and GS3b (-61% and − 38%, in the corresponding order) in S2.

Antioxidant enzymes status

Superoxide dismutase (SOD)

PGPR effect on leaf SOD activity varied among Stress treatments (F_10,72 = 5.18, p < 0.001) (Table 5). In control conditions, the PGPR factor showed no significant effect on this parameter, whereas under stress conditions, PSB inoculation (except GS1c) increased significantly leaf SOD activity. The highest percentage of increase was observed for GS4f (+ 57% and + 67% in S1 and S2 plants, respectively, as compared to uninoculated ones). For roots, only the main effects of Stress and PGPR were significant (F_2,82 = 4.76, p = 0.011; F_5,82 = 10.99, p < 0.001, respectively). Root SOD activity was slightly induced in S1 and S2 (+ 5–6%) as compared to control and in GS3m-, GS4d- and GS4f-inocululated plants in relation to uninoculated ones (+ 10–19%) (Table 5).

Catalase activity (CAT)

CAT activity in roots and shoots was significantly impacted by the interaction term Stress-PGPR (F_10,72 = 116.64, p < 0.001; F_10,72 = 42.21, p < 0.001, respectively). PSB inoculation significantly affected CAT activity in the leaves and roots of barley plants challenged with S1 and S2, in contrast to plants grown under control conditions (Table 5). While GS4f and GS3b in S1 plants only slightly increased leaf CAT activity (+ 64% and + 39%, respectively), GS4f and, to a lesser extent, GS4d (+ 399% and + 93%, correspondingly) markedly increased leaf CAT activity in S2 plants as compared to uninoculated ones (Table 5).

Regardless of the bacterial strain, S1 and S2 inoculated plants showed a substantial increase in CAT activity in their roots ranging from 107% to 401% (GS3b and GS4f) and from 210% to 674% (GS3m and GS4f) as compared to uninoculated ones.

Guaïacol peroxydase (GPX)

The significant effect of the Stress-PGPR interaction term indicated that PSB inoculation had varying effects on leaf (F_10,72 = 13.27, p < 0.001) and root (F_10,72 = 5.30, p < 0.001) GPX activities depending on the stress treatment. In contrast to plants cultivated under non-stress conditions, PSB inoculation significantly affected GPX activity in the roots and leaves of barley plants challenged with S1 and S2 (Table 5).

All inoculated plants showed a considerable increase in leaf GPX activity under salinity and phosphorus deficiency (S2) as compared to uninoculated ones with GS4f as the most stimulating strain (+ 229%). When compared to uninoculated plants (Table 5), GPX activity in the roots was 26% higher in S1 GS4f- and 26% lower in S2 GS1c-inoculated ones.

Principal component analysis (PCA)

PCA was used for both single (PCA1) and combined (PCA2) stresses to further highlight potential correlations between the PSB inoculation and the determined traits (Fig. 4). Both for S1 and S2, the first principal component (PC1) accounted for a significant part of the overall variance (68.4% and 77.5%, respectively). All variables were significantly correlated with PC1 (Table S1) in both stress conditions, as evidenced by the projection of variables in the two-dimensional space generated by PC1 and PC2 (Fig. 4). In particular, MDA and H₂O₂ contents in leaves and roots, leaf Na⁺ content and Ci were negatively correlated with PC1. The second principal component (PC2) explained low proportion of the observed variance in S1 (7.9%) and S2 (6.3%) and exhibited positive correlations with root MDA content and GPX activity. For S2, PC2 was positively correlated with SL and SWC and negatively with leaf GPX activity (Fig. 4).

Fig. 4.

Principal component analysis (PCA) of the parameters analyzed in H. vulgare (var. Ardhaoui) plants under S1 and S2 treatments. Investigated parameters and treatments are projected onto the PC1 and PC2 explaining 76.30% and 83.8% of the overall variation, respectively in S1 and S2 conditions. Abbreviations: SL: shoot height, RL: root length, SDW: shoot dry weight, RDW: root dry weight, SWC: shoot water content, RWC: root water content, A: net CO₂ assimilation, gs: stomatal conductance, Ci: internal CO₂ concentration, E: transpiration, H₂O₂_L: leaf H₂O₂ content, H₂O₂_R: root H₂O₂ content, MDA_L: leaf MDA content, MDA_R: root MDA content, SOD_L: leaf SOD activity, SOD_R: root SOD activity, PC_S: shoot P content, PC_R: root P content, ChlT: total chlorophyll content, Na_L: leaf Na⁺ content, K_L: leaf K⁺ content, K/Na: K⁺/Na⁺ selectivity ratio, CAT_L: leaf CAT activity, CAT_R: root CAT activity, GPX_L: leaf GPX activity, GPX_R: root GPX activity, AP_L: leaf acid phosphatase activity, AP_R: root acid phosphatase activity

Similar patterns were observed for S1 and S2 when inoculated and PSB-inoculated plants were projected on the PCA biplots. Indeed, these plants were plotted along PC1 according to their tolerance to single/combined stress (Fig. 4). Uninoculated and plants clustered on the PC1 far opposite sides. GS4f-inoculated were the most tolerant plants and were mainly characterized by the highest values of growth (SDW, RDW, SL, RL) and photosynthetic parameters (A, gs, E), water (SWC, RWC), phosphorus (PC) and chlorophyll contents, SOD, CAT, GPX and ACP activities as well as K⁺/Na⁺ ratio. In contrast, uninoculated plants (the most sensitive plants) distinguished by the highest values of H₂O₂, MDA and Na⁺ contents (Fig. 4). GS3m-inoculated plants clustered close to the center, indicating that they were moderately single/combined stress tolerant. Midway between GS4f- and GS3m-inoculated plants, on the right side of PC1, were plotted GS4d-inoculated plants, while GS1c- and GS3b-inoculated plants were on the left side of PC1, halfway between uninoculated and GS3m-inoculated (Fig. 4). Hence, according to PC1, the relative single/combined stress tolerance level in the increasing order was as follows: Uninoculated < GS3b-inoculated/GS1c-inoculated < GS3m-inoculated < GS4d-inoculated < GS4f-inoculated plants.

Discussion

Salt stress is a major environmental challenge that limits crop productivity worldwide by causing osmotic stress, ion toxicity, and nutrient imbalances. Salinity often reduces phosphorus availability and uptake, further constraining growth, though phosphorus fertilization can mitigate some effects depending on plant species and salinity levels [36]. While much research has focused on plant responses to single abiotic stresses, how plants cope with combined stresses remains less understood. This study examines the impact of seed inoculation with different phosphate-solubilizing bacteria (PSB) on barley’s physiological traits and oxidative status under phosphorus deficiency and salt stress.

PSB seed inoculation enhances barley seedling establishment under combined salinity and P deficiency

Our results showed significant (p < 0.01) decreases in growth and tissue hydration of uninoculated barley seedlings under salinity with sufficient phosphorus (S1), with more severe effects under combined salinity and low phosphorus. Similar results were recorded in wheat plants when grown under combined P deficiency and water-deficit [49]. This decline likely results from disruptions in essential physiological processes, as salinity induces osmotic stress and ion toxicity from excessive salt accumulation, impairing cellular metabolism and overall plant function [50]. Phosphorus deficiency restricts leaf emergence and expansion, likely due to the role of vacuolar Pi reserves in maintaining cellular osmotic potential and turgor [43]. This might partly explain the observed lower chlorophyll content in barley under salinity and phosphorus deficiency, but also due to increased chlorophyllase activity [51] and pigment-protein complex instability [52]. Besides, combined stresses also inhibited photosynthesis, including CO₂ assimilation, stomatal conductance, and transpiration. Phosphorus deficiency was also documented to restrict stomatal opening and limit ATP and NADPH recycling, thereby impairing photosynthesis and growth [53, 54]. Our findings also suggest that moderate salt stress mainly diverts energy toward defense mechanisms rather than biomass production [55]. When phosphorus nutrition is adequate, growth reductions and salt toxicity symptoms are minimized but are more severe under P deficiency [56, 57]. This supports the intricate relationship between phosphorus availability, growth performance, and photosynthesis machinery.

Among the key findings of the present study, we show that inoculation with all five bacterial isolates significantly improved most growth indices, especially under stress conditions (S1 and S2) compared to uninoculated plants. Specific effects varied by PSB strain and plant organs (shoots/roots). In this regard, GS4f and GS4d strains notably enhanced growth in stressed shoots and roots, with Pseudomonas sp. (GS4f) especially under combined salinity and phosphorus deficiency. Roots, as primary sensors of soil conditions and environmental signals, are crucial to stress responses [58]. Our data corroborate previous reports of increased root growth in wheat from PSB-inoculated seeds under water deficit and low phosphorus [59]. This may be ascribed to bacterial production of organic acids, polysaccharides, and phytohormones and improved root water retention [60]. The effectiveness of PSB strains we used may also relate to their high salinity tolerance, IAA production, and phosphate-solubilizing capacity [36].

Cross-talk between PSB and ion homeostasis imparts cross tolerance to combined salinity and P deficiency

Salt stress (S1), alone or combined with P deficiency (S2), significantly (P < 0.01) increased Na⁺ and decreased K⁺ content in shoots of uninoculated seedlings, reducing K⁺/Na⁺ ratio, P content in shoots and roots, and P absorption efficiency (-53% in roots from non-treated seeds). PSB strains used enhanced K⁺ nutrition and increased the K⁺/Na⁺ ratio in stressed shoots from inoculated seeds, along with lower Na⁺ uptake and improved P nutrition. Seedlings from GS4f and GS4d strains showed higher K⁺ and P and lower Na⁺ in leaves and roots, which likely sustained growth, osmotic adjustment, and photosynthesis of barley plants under stress. It is noteworthy that the positive correlation (Fig. 4) recorded between GS4f and GS4d strains and the overall studied nutrients, revealed that these two strains were the most efficient for barley seedlings to perfectly cope with the toxic effect of salt stress. This suggests that PSB play a vital role in regulating multiple mechanisms involved in the distribution of Na⁺, K⁺, and P between shoots and roots.

Salt stress activates the Salt Overly Sensitive (SOS) pathway, key in Na⁺ exclusion and ion homeostasis [61]. On the basis of our findings, we postulate that PSB in the inoculated seed itself, triggers early events which could be associated with SOS signaling pathways. Shi et al. (2022) also suggested that seed inoculation with halotolerant bacteria induces salt tolerance in Arabidopsis thaliana through the upregulation of NHX1 and AVP1 (a vacuolar H⁺-pyrophosphatase). In the same line of ideas, the inoculation of Zea mays with two bacterial strains, Bacillus atrophaeus WZYH01 and Planococcus soli WZYH02, enhances plant responses under salt stress by significantly increasing K⁺ content and decreasing Na⁺ accumulation, thereby increasing K⁺/Na⁺ ratio and upregulating ZmNHX and ZmHKT genes [62]. Little is known about the interaction between PSB and SOS pathway in the context of seed inoculation and this remains largely unexplored. Therefore, further in-depth investigations are required to elucidate the potential cross-talk between PSB/SOS components during the inoculation phenomenon.

Seed inoculation with PSB effectively restored phosphorus availability under individual and combined stresses. Under phosphorus deficiency, barley plants from PSB-inoculated seeds showed increased phosphorus acquisition efficiency (PAE), reflecting enhanced root capacity to explore the rhizosphere and absorb phosphorus [36]. Considering the investigated PSB isolates, GS4f and GS4d were most effective in improving phosphorus availability and salt tolerance under combined salinity and P deficiency of barley plants.

Similar benefits of PSB inoculation on phosphorus accumulation were reported in wheat [63], maize [64], tomato [65] and quinoa [66].

Bacterial inoculation is known to enhance phosphorus bioavailability under individual stresses like salinity or phosphorus deficiency [67, 68]. This is the first report showing that PSB inoculation significantly improves phosphorus mobilization in barley under combined salt stress and P deficiency. PSB produce organic acids and enzymes that lower soil pH, thereby mobilizing bound phosphorus for easier plant uptake [69]. Under low phosphorus conditions, plants activate mechanisms, such as energizing plasma membrane H⁺-ATPases in roots, to maintain P metabolism and homeostasis [68]. Additionally, the phosphate transport system, regulated by transcription factors, protein kinases, and phosphatases, adjusts phosphate uptake based on availability and demand. This system involves a complex network of transcription factors, protein kinases and phosphatases, that crosstalk to regulate phosphate homeostasis in plants under stressful conditions [70]. Further experiments will be performed to assess (i) whether PSB seed inoculation may control phosphatase activity, especially when crosstalk with ROS- scavenging systems and (ii) whether barley plants derived from inoculated seeds may take advantage of these events (phosphatase/antioxidant systems) to better react against oxidative stress, subsequently generated from salinity and P-deficit stresses.

Cross-talk between PSB, ROS and acid phosphatase imparts cross tolerance to combined salinity and P deficiency

Under uninoculated conditions, salt stress alone significantly increased acid phosphatase (AP) activity in both leaves and roots, coinciding with decreased phosphorus content. Similarly, [71], reported enhanced AP activity in Lycopersicon peruvianum under 150 mM NaCl despite reduced phosphorus levels, suggesting that salt stress induces AP to mobilize phosphorus from organic sources. When salinity and phosphorus deficiency were combined, AP activity rose sharply in uninoculated plants, indicating an additive effect. Yet, physiological responses under combined stresses and salinity alone were similar, supporting [37] findings.

In the present study, AP activity substantially increased in both leaves and roots of PSB inoculated seeds and subsequently cultivated under single salinity or combined salinity and P deficiency. It is noteworthy that the highest AP was recorded in roots. Again, GS4f was to be the most efficient strain with regard increasing acid phosphatase activity under stress conditions. This effect was associated with highest P contents in both leaves and roots, reflecting the paramount role of bacteria for promoting the phosphate uptake under combined salinity and low P-stress. One could reasonably suggest that increasing AP activity is a major mechanism by which PSB convert the insoluble phosphorus into available phosphorus. PSB application was also shown to increase AP levels in wheat roots, further confirming the bacteria-induced facilitated phosphorus mobilization under nutrient-deficient conditions [67]. These observations are consistent with the reports of [72] pointing that overexpression of GmACP1 genes (encode an acid phosphatase) in Arabidopsis thaliana in roots improved phosphate absorption from the nutrient solution as well as phosphorus absorption efficiency (PAE). These genes contribute not only to phosphorus mobilization but also to broader stress response mechanisms involving reactive oxygen species (ROS) signaling pathways [73].

We show that dual phosphorus deficiency and NaCl treatment significantly increased the H₂O₂ accumulation and lipid destruction as estimated by MDA increase, in both leaves and roots arising from uninoculated plants relatively to those of control state. Interestingly, this effect was more pronounced in leaves (photosynthetic organs) than in roots. Thus, one may hypothesize that the excessive production of ROS found in leaves simultaneously exposed to salinity and P deficiency may be associated with the pronounced decrease in CO₂ availability and stomatal conductance. This response, in conjunction with the observed decreases in leaf P and K⁺ contents and the marked increase in Na⁺ accumulation, suggests that P deficiency, when coupled with salinity, further amplified the oxidative stress injuries generated by salinity alone. Similar results were found in maize seedlings grown under both salt stress and low P-availability [74]. [75] also found that both MDA and H₂O₂ contents increased in cotton seedlings when simultaneously challenged by salinity and P deficiency conditions. In the present work, salt treatment, when applied individually, generally decreased the antioxidant enzyme activities in both leaves and roots from uninoculated barley seeds. This trend was further pronounced under simultaneous salinity and low P-availability conditions as compared to control conditions. This is in agreement with the findings of [75] reporting that combined salinity and P-deficiency markedly decreased the antioxidant systems in cotton plants.

Interestingly, the oxidative stress response profile of inoculated plants contrasted strongly with uninoculated plants. All PSB treatments significantly decreased H₂O₂ and MDA levels in both leaves and roots despite stress application. This effect paralleled with amplified root and leaf CAT, SOD and GPX activities, especially under simultaneous salinity and low P-stress. It appeared that inoculation with GS4f (Pseudomonas sp) was more effective to stimulate antioxidant systems and so that restoring the oxidative damages already recorded in barley plants originating from uninoculated seeds. Under single salinity, GS4f-inoculated plants showed the lowest amounts of H₂O₂ and MDA in both roots and leaves, together with higher activities of the overall antioxidant systems. Roots from plants inoculated with GS4f and grown under salt stress showed the highest activities of SOD, CAT and GPX. Similar profile (highest root enzyme activities) was recorded when plants were exposed to combined stress conditions. This was concomitant with the lowest MDA level found in GS4f inoculated plants and co-exposed to simultaneous P deficiency and salinity. ROS are well known as essential secondary messengers that can help to trigger early defense mechanisms [76]. Further investigations are required to deepen our understanding of these crucial aspects in order to unravel the early events that may be triggered in the inoculated seed itself.

GS4f was the most potent strain regarding the stimulation of antioxidant defense in roots, the first site of stress signal perception. This beneficial effect was evidenced by the improved root growth and water uptake observed in GS4f inoculated plants grown simultaneously under P deficiency and salinity. [77] also suggested that balancing osmotic stress (as the first immediate stress caused by salinity), repairing oxidative damages and controlling hormone homeostasis in roots are the initial and main mechanisms stimulated in inoculated Triticum aestivum plants under both salinity and P deficiency. Our findings agree with [78] showing enhanced growth of stressed common bean seedlings is mediated by improving antioxidant defense of roots.

The present research strongly suggests that the beneficial effect after bacterial inoculation under interactive salinity and P deficiency may be linked to a positive ion homeostasis (enhanced phosphorus accumulation) at root level, related to absolute correction of hydration, ROS damages, as well as increased antioxidant systems. Based on these findings, further experiments should address whether the cross-talk between PSB and roots may be transferred to leaves and prevent plants from salt- and/or P shortage-induced oxidative stress. The current results showed that stress signals (both salinity and P deficiency) transported from roots to leaves lead to a spectacular increase in SOD, CAT and GPX activities. This is in line with the findings of Ahanger et al. [79] who stated that inoculating tomato plants with PSB enhanced the plant’s ability to scavenge ROS under saline and P deficiency conditions. Furthermore, PSB inoculation of salt stressed maize plants massively improved the gene expression of SOD and GPX enzymes [80]. Hence, there is an increasing evidence highlighting the active role of PSB in modulating the antioxidant defense system of plants, particularly by enhancing the activity of various antioxidant enzymes under stress conditions [81]. How PSB may control ROS homeostasis to maintain membrane stability and at the same time improve phosphorus availability needs further investigation. However, some investigations suggest that PSB can lead to a reduction in ROS levels by enhancing the plants ability to cope with salt-induced oxidative stress. This is achieved through improved nutrient uptake and enhanced enzymatic activities that scavenge excess ROS [75].

While our findings suggest a potential link between acid phosphatase (AP) activity and reactive oxygen species (ROS) signaling under combined salinity and phosphorus deficiency, this relationship remains tentative and requires further investigation. To validate this hypothesis, future studies should notably address the expression of ACP genes alongside ROS-scavenging genes such as SOD, CAT, and GPX through qPCR or transcriptomic analyses. Such integrative approaches will provide mechanistic insights into how AP and ROS pathways interact to enhance plant resilience under nutrient and salt stresses. Additionally, incorporating these investigations will strengthen the understanding of early stress signaling events in PSB-inoculated plants.

Conclusion

In summary, phosphate-solubilizing bacteria (PSB) demonstrate significant potential to alleviate the detrimental effects of combined salinity and phosphorus deficiency in barley, opening avenues for future field research (Fig. 5). This beneficial aspect was especially true for the Gs4f strain. PSB inoculation improved ion homeostasis by reducing Na⁺ accumulation while enhancing K⁺ and P uptake. This ionic balance was associated with a better water status, leading to marked growth improvement in PSB-inoculated seedlings. Furthermore, PSB modulated oxidative stress by reducing harmful ROS production and lipid peroxidation through enhanced antioxidant enzyme and likely AP activities. Despite the current laboratory findings highlight the promising role of phosphate-solubilizing bacteria (PSB) in alleviating combined salinity and phosphorus deficiency stresses, the field experiments are mandatory to confirm their effectiveness under natural conditions, where salinity is combined with other environmental constraints. Such studies will not only help to validate the mechanistic hypotheses raised in the present work, but also assess the practical applicability of PSB inoculation as biostimulants in agricultural systems.

Fig. 5.

Schematic Representation of Synergistic Interactions Mediated by PSB (GSf4) Inoculation in Barley Seeds under Combined Salinity and Phosphorus Deficiency Conditions. This diagram outlines the potential synergistic effects of PSB (GSf4) inoculation on barley seeds, focusing on its interaction with various signaling pathways, particularly under conditions of combined salinity and phosphorus (P) deficiency. The representation is divided into distinct developmental phases:

1. Seed Inoculation Phase: During this phase, barley seeds are pretreated with PSB, which may trigger early signaling events within the seed.

2. Early PSB stimulated signals: This inoculation could influence the modulation of ROS (Reactive Oxygen Species), SOS (Salt Overly Sensitive), and IAA (Indole-3-acetic acid) cross-talk.

3. Plant response under combined stress condition: At laboratory level, barley seedlings treated with PSB (GSf4) are exposed to simultaneous salt stress and phosphorus deficiency. The PSB pretreatment enhances growth establishment, water uptake, and ion homeostasis, particularly by reducing Na+ accumulation while improving K+ and P mobilization in the shoots. This response is associated with increased acid phosphatase activity, boosting Phosphatase Activity Enzyme (PAE) and Urease Activity Enzyme (UAE) parameters. As a result, photosynthesis activity improves, which is accompanied by stronger antioxidant defense mechanisms (SOD, CAT, GPX). These adaptations help minimize H2O2 and MDA levels, promoting rapid cellular damage repair.

4. Field Conditions (Future Investigations): Transitioning to field conditions, barley seedlings are expected to face more complex environmental stresses (among them salinity and P deficiency). However, with the hypothesized benefits of PSB seed treatment, these plants are expected to demonstrate enhanced resilience, particularly in their early stages of growth.

This diagram provides a comprehensive overview of how PSB inoculation influences barley’s response to combined salinity and P deficiency, from initial seed treatment under controlled conditions to potential growth in more challenging field environments.

Abbreviations

ACC

1-Aminocyclopropane-1-Carboxylate (deaminase)

AP

Acid Phosphatase

ATP

Adenosine Triphosphate

AVP1

Vacuolar H⁺-Pyrophosphatase (gene related to salt tolerance)

CAT

Catalase

CKs

Cytokinins

DNA

Deoxyribonucleic Acid

EPS

Exopolysaccharides

GPX

Glutathione Peroxidase

IAA

Indole-3-Acetic Acid

MDA

Malondialdehyde

NADPH

Nicotinamide Adenine Dinucleotide Phosphate (reduced form)

PAE

Phosphorus Acquisition Efficiency

PGPR

Plant-Growth-Promoting Rhizobacteria

PSB

Phosphate-Solubilizing Bacteria

qPCR

Quantitative Polymerase Chain Reaction

ROS

Reactive Oxygen Species

SOD

Superoxide Dismutase

SOS

Salt Overly Sensitive (pathway)

ZnNHX1

Vacuolar Na⁺/H⁺ Antiporter Gene (related to salt tolerance)

Authors’ contributions

SK and HE conceived and designed the experiments, analyzed the data and wrote the first version of manuscript; SD and CA conducted draft revision and contributed to the writing; NMA acquired funding and participated in draft revision; WT revised the manuscript and performed Statistical Analysis and Principal Component Analysis (PCA); AMH contributed to the writing and editing the manuscript; all authors have read and approved the final manuscript.

Funding

This research was supported by the Tunisian Ministry of Higher Education and Scientific Research and received financial support from the Ongoing Research Funding Program (ORF-2026-177) from King Saud University, Riyadh, Saudi Arabia.

Data availability

Data generated and/or analyzed in the framework of the present study are available from the corresponding author on request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.