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Article

Beneficial Microorganisms: Sulfur-Oxidizing Bacteria Modulate Salt and Drought Stress Responses in the Halophyte Plantago coronopus L.

by
Aleksandra Koźmińska
1,*,
Mohamad Al Hassan
2,
Wiktor Halecki
3,
Cezary Kruszyna
1 and
Ewa Hanus-Fajerska
1
1
Department of Botany, Physiology and Plant Protection, Faculty of Biotechnology and Horticulture, University of Agriculture in Krakow, Al. Mickiewicza 21, 31-120 Krakow, Poland
2
Department of Plant Sciences, Aeres University of Applied Sciences, 8251 JZ Dronten, The Netherlands
3
Institute of Technology and Life Sciences—National Research Institute, Falenty, Al. Hrabska 3, 05-090 Raszyn, Poland
*
Author to whom correspondence should be addressed.
Sustainability 2024, 16(24), 10866; https://doi.org/10.3390/su162410866
Submission received: 22 October 2024 / Revised: 28 November 2024 / Accepted: 29 November 2024 / Published: 11 December 2024
(This article belongs to the Special Issue Sustainable Agricultural Engineering: Agroecology Sustainable Development and Agriculture Environmental Impact Assessment and Measurement)
Browse Figures
Figure 1
<p>Elemental contents: sodium (<b>A</b>); potassium (<b>B</b>); chloride (<b>C</b>); K/Na ratio (<b>D</b>); sulfur (<b>E</b>); in <span class="html-italic">P. coronopus</span> subjected to drought and sodium chloride (300 and 600 mM NaCl) and sulfur-oxidizing bacteria. SOB—<span class="html-italic">Halothiobacillus halophilus</span>-inoculated substrate, non-SOB-non-inoculated substrate. Different lowercase letters indicate significant differences between plants cultivated on non-inoculated substrate by SOB within different stress treatments. Different capital letters indicate statistically significant differences between plants cultivated on substrate inoculated by SOB within different stress treatments. * Indicates statistically significant differences between inoculated and non-inoculated plants within the same stress treatment, according to Tukey’s test (α = 0.05), ±SE, n = 5.</p> "> Figure 2
<p>PCA for K/Na, S, K, Cl, and Na; KMO = 0.62; <span class="html-italic">p</span> &lt; 0.001.</p> "> Figure 3
<p>Photosynthetic pigments: chlorophyll a (chl.a), chlorophyll b (chl.b), and carotenoids (car.) in <span class="html-italic">P. coronopus</span> subjected to drought, sodium chloride (300 and 600 mM NaCl) and sulfur-oxidizing bacteria.</p> "> Figure 4
<p>PCA for GSH, GSSG, and GSH/GSSG. KMO = 0.34; <span class="html-italic">p</span> &lt; 0.001.</p> "> Figure 5
<p>(<b>A</b>). Box plot of proline (Pro), across experimental treatments. (<b>B</b>). Box plot of TPC across experimental treatments. (<b>C</b>). Box plot of MDA across experimental treatments. (<b>D</b>). Box plot of DPPH across experimental treatments.</p> "> Figure 5 Cont.
<p>(<b>A</b>). Box plot of proline (Pro), across experimental treatments. (<b>B</b>). Box plot of TPC across experimental treatments. (<b>C</b>). Box plot of MDA across experimental treatments. (<b>D</b>). Box plot of DPPH across experimental treatments.</p> ">
Versions Notes

Abstract

:
Land degradation due to salinity and prolonged drought poses significant global challenges by reducing crop yields, depleting resources, and disrupting ecosystems. Halophytes, equipped with adaptive traits for drought and soil salinity, and their associations with halotolerant microbes, offer promising solutions for restoring degraded areas sustainably. This study evaluated the effects of halophilic sulfur-oxidizing bacteria (SOB), specifically Halothiobacillus halophilus, on the physiological and biochemical responses of the halophyte Plantago coronopus L. under drought and salt stress. We analyzed the accumulation of ions (Na, Cl, K) and sulfur (S), along with growth parameters, glutathione levels, photosynthetic pigments, proline, and phenolic compounds. Drought significantly reduced water content (nearly 10-fold in plants without SOB and 4-fold in those with SOB). The leaf growth tolerance index improved by 70% in control plants and 30% in moderately salt-stressed plants (300 mM NaCl) after SOB application. SOB increased sulfur content in all treatments except at high salinity (600 mM NaCl), reduced toxic sodium and chloride ion accumulation, and enhanced potassium levels under drought and moderate salinity. Proline, total phenolic, and malondialdehyde (MDA) levels were highest in drought-stressed plants, regardless of SOB inoculation. SOB inoculation increased GSH levels in both control and 300 mM NaCl-treated plants, while GSSG levels remained constant. These findings highlight the potential of SOB as beneficial microorganisms to enhance sulfur availability and improve P. coronopus tolerance to moderate salt stress.

1. Introduction

Drought and soil salinity are among the most important abiotic stresses restricting plant growth, reducing their productivity, and significantly influencing the distribution of wild plant species in nature [1,2,3,4]. Approximately 3.6 billion of the 5.2 billion hectares of drylands utilized for farming are affected by erosion, salinization, and soil degradation. Examples of regions impacted by salinization and erosion include the Aral Sea Basin (Central Asia), the Tigris-Euphrates River Basin (Iraq, Syria, Turkey), and the Murray-Darling Basin (Australia) [5,6]. Additionally, projections suggest that by 2050, the global population will rise to 9.7 billion, which would require over a 70% increase in food production, placing even more strain on cultivable lands [7].
To achieve this goal, salt-affected, degraded, and marginal lands must be leveraged and revitalized as potential zones for food production. The restoration and rehabilitation of these saline-impacted areas for agricultural production can be achieved through a range of approaches, including the integration of halophytic plant species. Halophytes, which are specialized to flourish in saline soils, possess distinct physiological and biochemical mechanisms that enable their resilience under such extreme conditions.
Growing halophytes in salt-affected regions can improve soil quality, restore biodiversity, and generate valuable resources, such as livestock feed and renewable energy sources [8,9]. These plants possess unique structures and adaptation strategies, making them an optimal choice for marginal land exploitation in the current global context, where limited freshwater supplies, degraded soil quality, and climate change have constrained the cultivation of traditional crops [10].
However, the adaptability of halophytes to challenging soil conditions has its limits, which is why modern approaches to enhance their tolerance traits are still being explored. Among the various mechanisms to mitigate drought and/or salinity stress, managing the mineral nutrient status can be an effective defense strategy. Sulfur (S) is an essential macronutrient in plant growth and development, ranking fourth in importance after nitrogen (N), phosphorus (P), and potassium (K). Sulfur’s role remains essential for many biochemical and physiological functions in plants. It is a critical component of numerous vital compounds, including vitamins, coenzymes, phytohormones, and reduced sulfur compounds, all of which play key roles in regulating plant growth and resilience under both optimal and stress conditions [11,12]. However, sulfur (S) deficiency is becoming more common in agroecosystems globally due to factors such as agricultural practices, high biomass production, diminished sulfur emissions, and the application of non-sulfur fertilizers. This deficiency is a significant factor limiting the stress tolerance of many plants [13]. The oxidation rate of sulfur in soil, which commonly ranges from approximately 2% to 10% per year depending on environmental factors, is essential for plant bioavailability. This oxidation process is significant for plant nutrition since sulfate is an essential nutrient, particularly in protein synthesis and enzymatic functions. Sulfur is also vital in forming certain amino acids (e.g., cysteine and methionine) and secondary metabolites that help plants manage stress.
Additionally, most of the sulfur in soil (>95% of total sulfur) is bound to organic compounds, rendering it unavailable for direct uptake by plants [10]. A potential solution to this problem may involve the use of sulfur-oxidizing bacteria (SOB), which enhance the rate of sulfur oxidation in the soil, thereby accelerating the production of sulfates available to plants. Sulfur-oxidizing bacteria, such as those from the genus Bacillus and related types, can convert these non-absorbable forms into forms that plants can easily absorb. The oxidation rate of elemental sulfur (S) typically ranges from 40% to 51%, making it available for plant uptake. These characteristics of sulfur-oxidizing bacteria (SOB) highlight their potential as bioinoculants for promoting plant growth, suggesting their application as biofertilizers for sustainable crop production in agroecosystems. Conversion of reduced sulfur compounds in sulfur-oxidizing bacteria (SOB) is catalyzed by several enzymes, including quinone oxidoreductase, flavocytochrome c-sulfide dehydrogenase, dissimilatory sulfite reductase, and hetero-disulfide reductase. Environmental applications of sulfur-oxidizing bacteria (SOB) include the detoxification of hydrogen sulfide, soil bioremediation, and wastewater treatment. Recently, the use of sulfur-oxidizing bacteria as plant growth-promoting agents has been gaining attention [14,15,16,17,18,19].
Considering the above aspects, our studies were carried out to examine the implications of using sulfur-oxidizing bacteria on responses of a halophyte—Plantago coronopus L. under drought and salinity. Plantago coronopus L. is found in marine cliffs, saltmarshes, and endorheic basins at altitudes up to 800 m above sea level [20]. This annual or biennial halophyte features leaves with central veins arranged in basal rosettes measuring 2–20 cm in length. Its flowers, produced in spikes, bloom from April to October, and it has small brown seeds [21]. The plant utilizes the C3 photosynthesis pathway and contains osmolytes (sorbitol and proline) as well as antioxidants (phenols and polyamines) [22]. Moreover, Plantago coronopus is valued in biosaline agriculture for its edible leaves, which are popular in salads for their mildly salty taste, crunchy texture, and excellent nutritional value, including high levels of phenols, amino acids (phenylalanine, tyrosine), and minerals (potassium, calcium, magnesium, sodium, etc.). The above-ground parts and roots of the plant are extensively utilized in traditional medicine for their anticancer, antimicrobial, antiviral, anti-inflammatory, analgesic, astringent, expectorant, diuretic, antipyretic, and emollient properties and are also employed in the management of upper respiratory conditions [20,23]. The underlying mechanism of salinity tolerance in P. coronopus involves greater efficiency in transporting toxic ions to the leaves, the ability to utilize inorganic ions as osmotica even under low salinity conditions, and the activation of proline accumulation and K+ transport to the leaves in response to very high salt concentrations (600–800 mM) [20,22].
The aim of this research was to investigate the effect of introducing halophilic sulfur-oxidizing bacteria (SOB) Halothiobacillus halophilus DSM 6132 into the growth medium, to assess the physiological and biochemical reactions of the halophyte P. coronopus L. under drought and salt stress. We hypothesized that inoculating the soil with sulfur-oxidizing bacteria (SOB) would augment the pool of plant-available sulfur and bring about positive changes in the response of P. coronopus against applied stresses.

2. Materials and Methods

2.1. Plant Material, Experimental Design, and Experimental Factors

The experiment was conducted in a growth chamber at the University of Agriculture, Krakow, Poland. Seeds of Plantago coronopus L. (Plantaginaceae) were obtained from the La Albufera Natural Park Seed Bank, Valencia, Spain. Five surface-sterilized P. coronopus seeds were sown in 1.5 L plastic pots filled with a sterile 1:1 (v/v) mixture of sand and vermiculite. The pots were placed in plastic trays, with a total of 48 pots prepared (six pots per tray). Seeds were maintained under controlled conditions (24 ± 2 °C, 70% relative humidity, and a 16/8 h light/dark cycle) and irrigated weekly with distilled water. After germination, uniform seedlings of comparable size were transplanted into fresh pots with the same substrate (one seedling per pot). The seedlings were cultivated under the same chamber conditions used for germination. Six weeks after transplantation, stress treatments were initiated. Control plants were irrigated weekly for six weeks with 125 mL of distilled water per pot. Salt stress was applied using 300 mM and 600 mM NaCl solutions, while drought stress was induced by withholding water.
Three weeks after stress application began, two treatment variants were implemented for each condition: (1) inoculation with a sulfur-oxidizing bacterial strain (Halothiobacillus halophilus—DSM 6132) and (2) a non-inoculated control (non-SOB). For inoculation, 10 mL of bacterial inoculum (108 cfu/mL) was added to each pot, with a second inoculation applied three weeks later. Nine weeks after the start of stress treatments, plants were harvested for biochemical analysis.

Bacterial Strain

Halothiobacillus halophilus DSM 6132 was obtained from ATCC (Manassas, VA, USA), with strain ATCC®® 4987 used to inoculate the substrate. The bacteria were cultured at 30 °C on ATCC medium 1846, also known as Thiobacillus halophilus medium (Supplement S1). After incubating the culture for at least 72 h at 30 °C, the H. halophilus inoculum was prepared by suspending the cells in a 2% NaCl solution and diluting it to an optical density (OD) of 0.5 at 600 nm, corresponding to 1.5 × 108 cfu/mL. A 10 mL aliquot of bacterial inoculum (108 cfu/mL) was applied to each pot twice, in the third and sixth weeks of the experiment. The bacterial cell count was determined using the plate count method. To confirm the presence of bacteria in the substrate with cultivated plants, bacteria were isolated from the soil at the end of the experiment, following the procedure described in Supplement S2.

2.2. Analysis of Soil Electric Conductivity

Electric conductivity (EC) was measured after nine weeks of experimental treatments. Soil samples taken from pots of the same treatment were air-dried and then passed through a 2-mm sieve. A mixture of soil:water (1:5) suspension was prepared using deionized water at 20 °C and mixed for one hour at 600 u/min. Electric conductivity was determined using a Crison Conductivity Meter 522 (Crison, Barcelona, Spain) and expressed in dS m−1.

2.3. Assessing Growth Parameters

At the end of the experiment, all specimens were harvested, and the following growth parameters were assessed: stem length (SL, cm) and fresh weight (FW, g) of above-ground parts and roots separately. To obtain the dry weight (DW), a fraction of the fresh material was weighed and dried in an oven at 65 °C for 72 h. Water content percentage (WC%) was calculated using the following formula:
WC% = [(FW − DW)/FW] × 100.
The stress tolerance index (GTI) for the shoot and roots dry biomass was determined using the following formula:
GTI = (mean DW of treated plants/mean DW of non-treated plants) × 100

2.4. Assessing Elemental Content

To determine the total elemental content, dried plant tissue samples were digested using a 9 mL mixture of concentrated acids (HCl:HNO3, 1:3 v/v) via the wet digestion method in a closed system using a microwave oven (Multiwave 3000, Anton Paar, Austria). The digestion protocol was as follows: power set at 1400 u/min, temperature at 240 °C, ramp time to maximum power of 5 min, hold time at maximum power of 15 min, ventilation time of 5 min, and cooling time of 40 min. Element concentrations were measured using a Perkin-Elmer Optima 7300 DV (SpectraLab Scientific Inc., Markham, ON, Canada) inductively coupled plasma atomic emission spectrophotometer (ICP-OES). Each plant material sample was analyzed in duplicate. If the duplicate results differed by more than ±5%, an additional two analyses were conducted for the sample. Measurement quality was validated using heavy metal determinations from an internal standard and the certified reference material CRM023-050 (Trace Metals—Sandy Loam 7, Supelco, Bellefonte, PA, USA).

2.5. Assessing Metabolic Parameters

2.5.1. Photosynthetic Pigments

Chlorophyll content and total carotenoids were analyzed according to Lichtenthaler and Wellburn [24]. Fresh leaves were briefly ground in a mortar and extracted with 80% ice-cold acetone. Samples were vortexed and then centrifuged at 13,000× g for 15 min at 4 °C. The supernatants were collected and absorbance was measured at 663, 646, and 470 nm using a Genesys 10 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA).

2.5.2. Reduced and Oxidized Glutathione

The glutathione pool was measured according to Queval and Noctor [25], where 5,5-dithiobis (2-nitro-benzoic acid) (DTNB) is glutathione reductase (GR)-dependent reduced. DW tissue (20 mg) was extracted at 4 °C using 1 mL 0.2 N HCl. The samples were centrifuged at 25,255× g for 10 min at 4 °C. The obtained supernatant (0.5 mL) was neutralized with 0.5 M NaOH in the presence of 50 μL 0.2 M NaH2PO4 (pH 5.6) to reach a final pH between 5 and 6. The method allowed the measurement of the total glutathione pool (reduced plus oxidized form: GSSG + GSH) and, after pre-treatment of the extract aliquots with 2-vinylpyridine (VPD), only GSSG was measured. To measure GSSG + GSH, aliquots of 30 μL neutralized extracts were added to 300 μL 0.2 M NaH2PO4 (pH 7.5), 30 μL 10 mM EDTA, 30 μL 10 mM NADPH, 30 μL 12 mM DTNB, and 180 μL distilled water. The reaction was started by the addition of 30 μL GR (20U mL−1), and the increase in the absorbance at 412 nm was monitored for 2 min. The GSSG fraction was measured using the same routine after incubation of 200 μL neutralized extract with 3 μL VPD for 30 min at room temperature to complex GSH. Calculations were made based on standard curves plotted simultaneously for GSH and GSSG. The GSH/GSSG ratio was also calculated.

2.5.3. Proline Determination

Proline (Pro) content was measured following the method of Bates et al. [26] with slight modifications. Tissue samples (fresh leaves) were homogenized in 3% aqueous sulfosalicylic acid and then centrifuged (4 °C; 15 min; 3000× g). The resulting extracts, combined with acid-ninhydrin and glacial acetic acid (1:1:1), were incubated in a boiling water bath for 1 h. The reaction was halted in an ice bath. The mixture was extracted with toluene through vigorous mixing, and the absorbance of the toluene phase was recorded at 520 nm using a Genesys 10 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). The calculation of Pro content was based on the standard curve.

2.5.4. Phenolic Compounds

Total phenolic compounds (TPC) were measured following the method of Blainski et al. [27] through a reaction with the Folin-Ciocalteu reagent. Absorbance was recorded at 765 nm, and the results were expressed as equivalents of gallic acid (mg·eq·GA g−1 FW).

2.5.5. Lipid Peroxidation

Malondialdehyde (MDA), a final product of membrane lipid peroxidation and a reliable indicator of oxidative stress [28], was quantified following the method described by Hodges et al. [29]. After gentle agitation in a rocker shaker for 24–48 h, the methanol extracts were collected via centrifugation and combined with 0.5% thiobarbituric acid (TBA) prepared in 20% trichloroacetic acid (TCA)—or with 20% TCA without TBA for the controls—before being incubated at 95 °C for 20 min. After terminating the reaction on ice and centrifuging the samples, the absorbance of the supernatant was measured at 532 nm. Non-specific absorbance at 600 and 440 nm was subtracted, and MDA concentration was calculated using the equations provided by Hodges et al. [28], based on the extinction coefficient of the MDA-TBA complex at 532 nm (155 mM−1 cm−1), and expressed as nmol mL−1.

2.5.6. Radical Scavenging Activity with DPPH Radical

The stable free radical DPPH (2,2-diphenyl-1-picrylhydrazyl) was utilized to evaluate the radical scavenging potential of P. coronopus shoots [30]. The fresh leaf samples were extracted using 80% methanol. The changes in the absorbance of the DPPH· solution, indicating the reduction in DPPH·, were recorded at 517 nm immediately after the addition of the extract and again after 10 min, using a Hitachi (Westford, MA, USA) U-2900 spectrophotometer. The scavenging activity of the extracts was expressed as the percentage of DPPH· radical reduction per unit of fresh weight (FW).

2.5.7. Phenolic Profile

Phenolic compounds (total phenolic content—TPC, phenylpropanoids, flavonols, and anthocyanins) in P. coronopus were quantified through UV/VIS spectrophotometry [31]. Chlorogenic acid (CGA), caffeic acid (CA), and quercetin (QC) served as reference compounds for TPC, phenylpropanoids, and flavonols, respectively. The anthocyanin concentration was represented as cyanidin (CY), based on its molar absorptivity. The samples were homogenized with 1 mL of 80% methanol and then subjected to centrifugation for 15 min at 3000× g. The resulting supernatant was employed for analysis. An aliquot of the extract (0.25 mL) was combined with 0.25 mL of 0.1% HCl (in 96% ethanol) and 4.50 mL of 2% HCl (in water). After a 30-minute incubation, the absorbances were measured at 280, 320, 360, and 520 nm using a Hitachi U-2900 spectrophotometer. The phenolic content was expressed in milligrams of equivalent standard per 100 g of fresh weight (FW).

2.6. Statistical Methods

The experimental setup followed a completely randomized design (CRD). The experimental data were analyzed using Statgraphics Centurion v.16. Before performing the analysis of variance, the normality assumption was validated using the Shapiro–Wilk test, and the homogeneity of variance was assessed with the Levene test. If the ANOVA assumptions were satisfied, the significance of differences among salt treatments was determined using one-way ANOVA at a 95% confidence level, followed by post-hoc comparisons with Tukey’s HSD test. All mean values reported in the text are presented with their corresponding standard deviation (SD). If the data are normally distributed, parametric tests ANOVA can be used to detect significant differences between groups (plants exposed to different salt treatments). When investigating the stress response in plants, it is essential to ensure that the variability within each treatment group is comparable. ANOVA evaluates whether the differences in stress markers among different treatments (e.g., various salt concentrations) are statistically significant. After detecting significant differences in plant stress across treatments with ANOVA, it is important to determine which specific treatment(s) caused these differences. For example, after applying various salt concentrations, Tukey’s test can show whether a high-salt treatment led to more severe stress than a low-salt treatment. This method allows researchers to visualize the overall stress response of plants in fewer dimensions and understand which variables contribute most to plant stress. Parameters such as Pro, TPC, MDA, and DHHP were represented via box plots. To perform PCA on the dataset, the data were first standardized to have a mean of 0 and a standard deviation of 1. Then, the correlation matrix was computed, and eigen decomposition was performed to obtain eigenvalues and eigenvectors. The principal components were selected by choosing the eigenvectors corresponding to the top eigenvalues that explained most of the variance. Finally, the standardized data were projected onto these eigenvectors to form the principal components. This process reduced dimensionality while retaining the most significant features of the data. The Kaiser-Meyer-Olkin (KMO) coefficient was used to assess sampling adequacy in the PCA analysis. The calculation was performed in PQ stat version 1.8.6.

3. Results

3.1. Soil Electric Conductivity

The electric conductivity (EC) of soil samples without NaCl application (0 mM) was 0.88 dS m−1 at the end of the experiment. With the introduction of salt stress, EC increased proportionally with the sodium chloride concentration, reaching 5.18 dS m−1 in moderately salinized soil (300 mM) and 8.42 dS m−1 in strongly salinized soil (600 mM). In turn, the electrolytic conductivity of the soil after drought induction was 0.72 dS m−1. The addition of sulfur-oxidizing bacteria to the soil did not alter the EC throughout the entire experiment. With SOB, EC values are generally lower than in non-SOB treatments, particularly at 300 mM NaCl, suggesting that SOB mitigate salinity stress at moderate salt levels. However, this effect is less pronounced at 600 mM NaCl (Table 1).

3.2. Growth Parameters

Leaf water content (WC%) decreased dramatically (about 10-fold) under drought conditions compared to other treatments. Meanwhile, observed WC% was higher in stressed plants that were inoculated with SOB, in comparison to their non-inoculated counterparts. In turn, WC% in the roots decreased across all experimental variants following the application of bacteria. Inoculation with SOB resulted in a higher calculated leaf growth tolerance index (GTI) in both control plants (about 70%) and plants subjected to moderate salt stress—300 mM (about 30%). The length of the stem was 60% higher in control plants following SOB application (always in comparison to their non-inoculated counterparts) (Table 2).

3.3. Elemental Content

Sodium and chloride deposition in leaves was approximately 7–8 times higher in plants subjected to salt treatments in comparison to control and drought-treated plants. Following the SOB application, the sodium and chloride ions levels changed in plants undergoing both salt treatments, showing approximately 18% less ionic content compared to the non-SOB variants (Figure 1A,C). Potassium levels were lower in plants subjected to both drought and salinity treatments in the variant without bacteria relative to those that were inoculated (Figure 1B). It is worth noting that the K/Na ratio was significantly lower in salt-treated plants compared to the control and drought-treated plants in both the non-SOB and SOB variants. The K/Na ratio value was reduced with the increase in salt concentrations, from 0.5 to 0.05, almost a 10-fold reduction—under 600 mM NaCl treatments (Figure 1D). Drought and salinity stresses did not change the sulfur contents in leaves compared to the control in the non-SOB variant. SOB application caused an increase in S content (by ×1.5-fold on average) in all treatments except for plants treated with high salinity (600 mM NaCl) (Figure 1E).
The PCA for S, Na, K, K/Na, and Cl showed the chi-square statistic from Bartlett’s test, indicating its significance (p-value < 0.05) and the KMO measure, which suggested that the sample size is mediocre but acceptable for analysis. S and K showed a negative correlation, while the K/Na parameter exhibited a negative association with Factor 1 and a positive association with Factor 2. Cl and Na were highly correlated, particularly with Factor 1, accounting for 69.76% of the variance (Figure 2).

3.4. Metabolic Response

3.4.1. Photosynthetic Pigments

The content of photosynthetic pigments (chlorophylls and carotenoids) was higher in P. coronopus under drought and salinity conditions relative to control plants (non-SOB variant). Soil inoculation with SOB decreased this parameter in control and 300 mM NaCl-treated plants (about 18% average). The highest content of photosynthetic pigments (402 µg·g−1) was recorded in plants under moderate salt stress (300 mM NaCl) (Figure 3).

3.4.2. Glutathione

The level of reduced glutathione (GSH) was the lowest in plants exposed to the highest concentration of sodium chloride (600 mM), both with and without SOB treatment. SOB inoculation seems to have caused an increase in GSH levels in both control and 300 mM NaCl-treated plants, while GSSG concentrations remained constant. Consequently, the GSH/GSSG ratio mirrored the same trend as the overall GSH content (Table 3).
The PCA showed the chi-square statistic from Bartlett’s test, indicating its significance (p-value < 0.05) and the KMO measure highlighted the inadequacy of the sample size for analysis based on S, GSH, GSSG, and GSH/GSSG. S was negatively correlated with Factor 1. GSH/GSSG and GSH were highly negatively correlated with each other. Only GSSG showed a positive correlation with Factor 1 (explaining 64.63% of the variance) and a negative correlation with Factor 2 (Figure 4).

3.4.3. Proline, Total Phenolic Compounds, MDA, and DPPH Levels

The highest measured concentration of proline (0.4 mg·g−1) was observed in plants treated with SOB and 600 mM NaCl. A significantly lower Pro content in the leaves of P. coronopus with SOB was observed in control plants and those treated with moderate salinity (300 mM NaCl) in comparison to the non-SOB variant undergoing the same stress treatment (Figure 5A). Meanwhile, the content of total phenolic compounds (TPC) was higher in P. coronopus under applied stresses (drought and salinity). SOB soil inoculation caused an increase in TPC levels only in plants exposed to moderate salt treatment (300 mM) (Figure 5B). Plantago coronopus reacted to drought with a 16-fold higher accumulation of malondialdehyde (MDA), a product of membrane lipid peroxidation and a reliable biomarker of oxidative stress. After SOB soil inoculation, only plants treated with drought stress showed an increase in MDA levels. In the other experimental variants, SOB did not affect MDA levels (Figure 5C). Proline, total phenolics, and MDA contents were highest in plants subjected to drought, irrespective of bacterial inoculation in the soil (Figure 5A–C). The radical scavenging ability, indicated by the elevated percentage of neutralized DPPH radical (1,1-diphenyl-2-picrylhydrazyl), was significant in plants subjected to drought (over 30%) and 600 mM NaCl (over 38%). Inoculation led to a rise in scavenging activity in drought-stressed plants. Conversely, a contrasting trend was noted in plants treated with 600 mM NaCl, where the inoculation of sulfur-oxidizing bacteria resulted in reduced DPPH neutralization (approximately 40%) (Figure 5D).

3.4.4. Phenolic Profile

Total phenolics were calculated as the sum of all those measured. Our analysis of the phenolic compound profile revealed that in P. coronopus leaves, the most abundant phenolics were phenylpropanoids (regardless of treatments used), reaching 35–49% of total phenolics, followed by flavonols (25–38%) and anthocyanins (1–4%). Under the influence of high salinity (600 mM NaCl), the addition of SOB caused a lower content of total phenolics. The differences in total phenolics contents in the leaves among treated plants (all stresses and SOB treatments used) corresponded with those noted for phenylpropanoids and flavonols (Table 4).

4. Discussion

Recent studies have emphasized the potential of sulfur-oxidizing bacteria (SOB) to enhance plant resilience to stressors such as salinity and drought [17,32]. This is particularly relevant given the following: global decline in soil sulfur (S) availability due to the widespread use of nitrogen (N), phosphorus (P), and potassium (K) fertilizers that lack S; reduced use of S-containing pesticides; increased S export from soil to crops; reduced atmospheric S deposition from rainfall; and decreased sulfur dioxide (SO2) emissions from fossil fuels [33]. Global sulfur shortages harm crop yields, nutrition, plant resistance, and soil health, raising fertilizer costs and threatening food security [34]. Sulfur in soils often exists in forms that are not bioavailable to plants, leading to reduced synthesis of critical sulfur-containing compounds such as glutathione (GSH), methionine, thioredoxins, vitamins, coenzyme A, and GSH-related antioxidant enzymes. These compounds enhance the antioxidant defense system, mitigating stress effects such as salinity and drought [35,36]. Sulfur-oxidizing bacteria (SOB) convert soil sulfur into usable forms, reducing the need for synthetic fertilizers, enhancing crop quality, and supporting soil health as a sustainable, long-term solution [37].
In this study, Halothiobacillus halophilus was selected for its sulfur-oxidizing capabilities and high salinity tolerance. This genus was reclassified from Thiobacillus in 2000 due to its greater acid tolerance and phylogenetic distinctions [38,39]. SOB enhance stress resilience against salinity, drought, pests, and diseases either by producing growth-promoting substances such as IAA, gibberellins, salicylic acid, ACC-deaminase, and siderophores or indirectly by increasing sulfur-containing compound production [40,41].
The salt concentrations used in this study highlight Plantago coronopus’s remarkable halotolerance. Even under 300 mM NaCl—exceeding the 200 mM NaCl threshold for halophytes [42]—plants maintained high yield stability, particularly when inoculated with SOB, as evidenced by GTI% and WC% (Table 2). At 600 mM NaCl, close to seawater salinity, P. coronopus still exhibited signs of tolerance, demonstrating its robust stress-response mechanisms.
Our experiments confirmed increased sodium (Na+) and chloride (Cl−) ion levels in P. coronopus under salinity stress, suggesting a lack of mechanisms to restrict the transport of toxic ions to aerial parts or their retention in roots. However, under moderate salinity, SOB inoculation limited Na+ and Cl− uptake in P. coronopus. Al Hassan et al. [20] noted that Na+ and Cl− transport to aerial tissues, vacuolar accumulation, and cytoplasmic osmotic adjustment via osmolytes enable P. coronopus to develop succulence and tolerate salinity. These traits make P. coronopus a valuable candidate for biosaline agriculture [22].
An increase in Na+ content is generally accompanied by a concomitant decrease in K+ levels, as both cations compete for the same transport systems [43]. This trend was observed in P. coronopus, but varied significantly between applied treatments. In P. coronopus, a reduction in mean K+ levels was observed under drought conditions and at both applied NaCl concentrations in the non-SOB variant. However, K+ levels increased again under drought and moderate salinity when SOB inoculation was applied. Potassium (K) plays a crucial role in water management and osmotic balance, ultimately contributing to plant abiotic stress resistance such as drought or salinity [44]. Under water stress, plants have been observed to exhibit reduced K+ levels, primarily due to alterations in membrane integrity and ionic imbalance [45]. It is worth mentioning that some rhizospheric microorganisms, particularly bacteria known as potassium-solubilizing microorganisms, have the ability to release potassium in a form that plants can easily absorb [46]. Notable K-solubilizing rhizobacteria include Bacillus, Agrobacterium, Burkholderia, Enterobacter, Myroides, Pseudomonas, and Pantoea [47]. Certain bacteria, by enhancing the bioavailability of potassium, have been shown to increase its concentration in plants [48].
There are reports indicating that the steady-state levels of numerous stress-associated metabolites are already elevated in halophytes compared to glycophytes. However, growth under suboptimal conditions may trigger stress, leading to alterations in secondary metabolites such as antioxidants. Beara et al. [49] indicated that P. coronopus possesses a substantial content of minerals, flavonoids, polyphenols, and amino acids while exhibiting a strong antioxidant capability. Interestingly, the plant also contains a relatively higher chlorophyll and flavonoid content when grown in selenium-enriched media, enhancing its nutraceutical value [50,51,52,53,54]. In our investigation, the content of photosynthetic pigments increased in P. coronopus under drought and salinity conditions in comparison to the control. Soil inoculation with sulfur-oxidizing bacteria (SOB) even decreased this parameter in the control and 300 mM NaCl-treated plants. Since a relatively high pigment content was found under 300 mM NaCl stress conditions (without SOB application to the soil) compared to the control treatment, it could be determined that the content of photosynthetically active pigments is an indicator of stress in this species. Chlorophyll concentration is a key metric in stress physiology research as it reflects the plant’s photosynthetic capacity, which is directly impacted by abiotic stressors such as drought, heat, and salinity. A decrease in chlorophyll content often signals damage to the photosynthetic machinery, serving as an early indicator of plant stress [55]. Chlorophyll degradation under stress conditions is often a sign of increased reactive oxygen species (ROS), indicating oxidative damage and reducing photosynthetic efficiency. This degradation weakens the plant’s energy production. In contrast, other pigments, such as anthocyanins and carotenoids, play protective roles by scavenging ROS and enhancing antioxidant mechanisms, thus aiding in stress tolerance [56,57]. Monitoring chlorophyll helps assess plant health, stress resilience, and recovery. Studies, such as those by Murchie and Lawson [58], highlight the importance of chlorophyll in evaluating plant response to environmental stresses.
There are many reports where bacterial inoculation stimulated the antioxidant defense system by increasing the activities of enzymes (CAT, APX, GPX, PAL) or secondary metabolites such as phenolic compounds glucosinolates or flavonoids [59,60,61]. It is noteworthy that the phenolic profile of Plantago spp. exhibits a significant dependence on the specific organ and extraction solvent employed, as well as on the species under investigation [52]. For instance, a study conducted by Pereira et al. [23] demonstrated that roots contained the highest levels and diversity of phenolic compounds, followed by flowers and leaves.
It is worth emphasizing that in our studies, SOB demonstrated the capacity to enhance plant antioxidant defenses by increasing the TPC and DPPH content in P. coronopus plants subjected only to moderate salinity stress. Phenolic compounds are essential in defending plants against abiotic stressors, with flavonoids and phenylpropanoids among the most significant. These compounds combat oxidative damage by neutralizing reactive oxygen species (ROS), reducing cellular stress, and preventing macromolecule degradation. Additionally, they strengthen cell walls and act as signal molecules to trigger further stress responses, enhancing plant resilience to environmental challenges [62,63].
Increased phenolic content may also be associated with greater sulfur availability, which indirectly influences the levels of phenolic compounds and other antioxidants. For instance, sulfur is critical for the synthesis of glutathione, which in turn is pivotal for antioxidant activity and the production of protective compounds such as phenolics. On the other hand, the presence of sulfur is necessary for certain enzymatic reactions involved in phenolic biosynthesis [64,65].
Furthermore, this species serves as a significant source of amino acids (glycine, threonine arginine, leucine, lysine, phenylalanine, serine, proline, and tyrosine) and minerals (sodium, calcium, potassium, magnesium, and iron) [23], which could be a promising factor to promote their future application as an ingredient in human nutrition on a broader scale. Gil et al. [66] noted that sorbitol may contribute to the stress tolerance of Plantago crassifolia, while the salt tolerance of Plantago maritima is linked to the accumulation of proline and antioxidant enzymes in the leaves and roots, respectively [67]. Proline acts as an osmolyte by helping plants maintain cellular water balance and stabilizing proteins and membranes under stressful conditions such as drought and salinity. Its accumulation in cells under stress aids in osmotic adjustment and serves as a protective agent against reactive oxygen species [68]. This buildup also functions as an early biomarker for environmental stress in various species, including Plantago [20] and other arid-tolerant plants, signifying the onset of osmotic imbalance and helping prepare antioxidant defenses [69]. In our studies, we did not observe a significant role of proline in salinity tolerance in Plantago coronopus. The highest concentration of this osmolyte was found in Plantago coronopus plants treated with SOB and 600 mM NaCl, but it did not correlate with the increased salinity tolerance, as determined by plant growth parameters. Furthermore, SOB boosted reduced glutathione (GSH) levels without affecting oxidized glutathione (GSSG) under control and moderate salinity conditions, leading to a higher GSH/GSSG ratio and improved antioxidant defense because one of the metabolic roles of glutathione to scavenge reactive oxygen species. The GSH/GSSG ratio, which compares the reduced (GSH) and oxidized (GSSG) forms of glutathione, is a key indicator of oxidative stress in plants. A high GSH/GSSG ratio is generally considered a marker of a healthy antioxidant defense system, as it indicates a reduced oxidative state capable of scavenging reactive oxygen species (ROS) [70,71,72]. In our study, the ratio increased both with and without SOB inoculation under 300 mM NaCl and applied drought, showcasing the resilience of P. coronopus as a halophyte, and the added value SOB provide (since the ratio was slightly higher in the inoculated plants).
In our experimental setup, in principle, we did not observe a positive correlation between the sulfur pool in the plant and the level of glutathione accumulation in its organs. Glutathione levels increased in plants even without SOB application, i.e., independently of additional sulfur accumulation in plants. However, when SOB were applied to the substrate, glutathione levels increased across all experimental treatments except for the 600 mM NaCl treatment, although the increase was statistically insignificant under drought conditions. Briefly, glutathione levels in Plantago coronopus plants fluctuated under the influence of the experimental factors used (abiotic stress or the introduction of SOB to the substrate), regardless of the current level of sulfur accumulation in the plant. This may be because photoautotrophic organisms must efficiently distribute their sulfur resources between stress-response pathways and growth-promoting processes to thrive in a constantly changing environment [73]. P. coronopus plants likely allocated sulfur resources to other protein synthesis pathways rather than the biosynthesis of glutathione.
Beyond salinity and drought, SOB exhibit potential in addressing broader environmental challenges, including heavy metal contamination and temperature extremes [74].
The beneficial microorganisms such a sulfur-oxidizing bacteria are attractive candidate to increase the sustainable agricultural productivity in saline ecosystems in which halophytes occur, by modulating the uptake of ions, regulation of antioxidants activity, by the production of osmolytes and increasing sulfur availability. The novel observation that multiple parameters associated with tolerance mechanisms in halophytes are modulated by soil inoculation with sulfur-oxidizing bacteria necessitates further detailed investigation.

5. Conclusions

Our study has shown that sulfur-oxidizing bacteria of the species Halothiobacillus halophilus induce a distinct physiological response in Plantago coronopus plants exposed to drought and salinity compared to those that are not inoculated. Inoculation with SOB resulted in a GTI increase in both control plants and plants subjected to moderate salt stress. We conclude that soil inoculation with sulfur-oxidizing bacteria (SOB) is an effective intervention that enhances the total sulfur (S) content in plants and simultaneously improves the salt stress tolerance of P. coronopus. An increase in sulfur content in plants may additionally help to maintain a constant level of phenolic compounds, including phenylpropanoids, flavanols, and anthocyanins, in P. coronopus tissues. Furthermore, especially under moderate salinity conditions, soil SOB inoculation limits the uptake of sodium and chloride ions by the plant root system and also limits the loss of potassium ions. These findings provide novel insights into how halophytes respond to abiotic stress following inoculation of the growth medium with sulfur-oxidizing bacteria. The application of SOB can be an effective tool to induce salt stress tolerance.
Moving forward, it is important to expand research on sulfur-oxidizing bacteria to identify their specific role in regulating plant responses to abiotic stress.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/su162410866/s1, Supplement S1: ATCC medium: 1846 Thiobacillus halophilus medium; Supplement S2: Protocol for Bacteria isolation from the soil.

Author Contributions

Conceptualization, A.K.; methodology, A.K. and M.A.H.; software, W.H.; validation, A.K. and C.K.; formal analysis, A.K., M.A.H. and E.H.-F.; investigation, A.K. and C.K.; resources, A.K. and M.A.H.; data curation, A.K., W.H. and C.K.; writing—original draft preparation, A.K., W.H. and E.H.-F.; writing—review and editing, A.K., M.A.H. and E.H.-F.; visualization, W.H. and C.K.; supervision, A.K.; project administration, A.K.; funding acquisition, A.K. and E.H.-F. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Ministry of Science and Higher Education of the Republic of Poland—statutory funding of research activity held at the University of Agriculture in Kraków, Poland and by the Polish National Science Centre (project number 2020/04/X/NZ8/00786).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Elemental contents: sodium (A); potassium (B); chloride (C); K/Na ratio (D); sulfur (E); in P. coronopus subjected to drought and sodium chloride (300 and 600 mM NaCl) and sulfur-oxidizing bacteria. SOB—Halothiobacillus halophilus-inoculated substrate, non-SOB-non-inoculated substrate. Different lowercase letters indicate significant differences between plants cultivated on non-inoculated substrate by SOB within different stress treatments. Different capital letters indicate statistically significant differences between plants cultivated on substrate inoculated by SOB within different stress treatments. * Indicates statistically significant differences between inoculated and non-inoculated plants within the same stress treatment, according to Tukey’s test (α = 0.05), ±SE, n = 5.
Figure 1. Elemental contents: sodium (A); potassium (B); chloride (C); K/Na ratio (D); sulfur (E); in P. coronopus subjected to drought and sodium chloride (300 and 600 mM NaCl) and sulfur-oxidizing bacteria. SOB—Halothiobacillus halophilus-inoculated substrate, non-SOB-non-inoculated substrate. Different lowercase letters indicate significant differences between plants cultivated on non-inoculated substrate by SOB within different stress treatments. Different capital letters indicate statistically significant differences between plants cultivated on substrate inoculated by SOB within different stress treatments. * Indicates statistically significant differences between inoculated and non-inoculated plants within the same stress treatment, according to Tukey’s test (α = 0.05), ±SE, n = 5.
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Figure 2. PCA for K/Na, S, K, Cl, and Na; KMO = 0.62; p < 0.001.
Figure 2. PCA for K/Na, S, K, Cl, and Na; KMO = 0.62; p < 0.001.
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Figure 3. Photosynthetic pigments: chlorophyll a (chl.a), chlorophyll b (chl.b), and carotenoids (car.) in P. coronopus subjected to drought, sodium chloride (300 and 600 mM NaCl) and sulfur-oxidizing bacteria.
Figure 3. Photosynthetic pigments: chlorophyll a (chl.a), chlorophyll b (chl.b), and carotenoids (car.) in P. coronopus subjected to drought, sodium chloride (300 and 600 mM NaCl) and sulfur-oxidizing bacteria.
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Figure 4. PCA for GSH, GSSG, and GSH/GSSG. KMO = 0.34; p < 0.001.
Figure 4. PCA for GSH, GSSG, and GSH/GSSG. KMO = 0.34; p < 0.001.
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Figure 5. (A). Box plot of proline (Pro), across experimental treatments. (B). Box plot of TPC across experimental treatments. (C). Box plot of MDA across experimental treatments. (D). Box plot of DPPH across experimental treatments.
Figure 5. (A). Box plot of proline (Pro), across experimental treatments. (B). Box plot of TPC across experimental treatments. (C). Box plot of MDA across experimental treatments. (D). Box plot of DPPH across experimental treatments.
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Table 1. Soil electric conductivity after nine weeks of experimental treatments.
Table 1. Soil electric conductivity after nine weeks of experimental treatments.
TreatmentsEC (dS m−1)
non-SOB
0 mM NaCl0.88 a
300 mM NaCl5.18 b
600 mM NaCl8.42 c
drought0.72 a
SOB
0 mM NaCl0.80 A
300 mM NaCl4.92 B
600 mM NaCl8.46 C
drought0.86 A
Different lowercase letters indicate significant differences in EC on soil non-inoculated with SOB under different stress treatments. Different capital letters indicate significant differences in EC on soil inoculated with SOB under different stress treatments, according to Tukey’s test (α = 0.05). Values are presented as mean ± SE, with n = 5.
Table 2. Growth parameters in P. coronopus subjected to drought and sodium chloride (300 and 600 mM NaCl) and sulfur-oxidizing bacteria. WC%—water content (%), GTI%—growth tolerance index (%), SL—stem length (cm), SOB—Halothiobacillus halophilus-inoculated substrate, non-SOB—non-inoculated substrate.
Table 2. Growth parameters in P. coronopus subjected to drought and sodium chloride (300 and 600 mM NaCl) and sulfur-oxidizing bacteria. WC%—water content (%), GTI%—growth tolerance index (%), SL—stem length (cm), SOB—Halothiobacillus halophilus-inoculated substrate, non-SOB—non-inoculated substrate.
WC%GTI%SL (cm)
Non-SOBSOBNon-SOBSOBNon-SOBSOB
Leavescontrol95.1 c95.4 B100 a168 D*17.3 b28 C*
drought9.5 a21.8 A*100 a86.7 B*6.1 a4.8 A
NaCl 300 mM92.6 c92.3 B100 a127.4 C*21.3 c22 B
NaCl 600 mM86.8 b88.2 B100 a39.9 A*18.7 b18 B
Rootscontrol95.3 c83.1 B*100 a99.17 BWC—water content;
drought83 a45 A*100 a94.23 BGTI—growth
NaCl 300 mM95.2 c88.9 C*100 a71.88 A*tolerance index
NaCl 600 mM91.1 b89.3 C*100 a133.33 C*
Different lowercase letters indicate significant differences between plants cultivated on non-inoculated substrate by SOB within different stress treatments. Different capital letters indicate statistically significant differences between plants cultivated on substrate inoculated by SOB within different stress treatments. * Indicates statistically significant differences between inoculated and non-inoculated plants within the same stress treatment, according to Tukey’s test (α = 0.05), ±SE, n = 5.
Table 3. Glutathione (reduced-GSH and oxidized-GSSG) content and GSH/GSSG ratio in P. coronopus subjected to drought and sodium chloride (300 and 600 mM NaCl) and sulfur-oxidizing bacteria.
Table 3. Glutathione (reduced-GSH and oxidized-GSSG) content and GSH/GSSG ratio in P. coronopus subjected to drought and sodium chloride (300 and 600 mM NaCl) and sulfur-oxidizing bacteria.
GSH-Reduced Glutathione
GSSG-Oxidized Glutathione		GSH (nmol·g−1 FW)GSSG (nmol·g−1 FW)GSH/GSSG
Non-SOBSOBNon-SOBSOBNon-SOBSOB
control26 ± 2.1 b32 ± 2.1 B*1.66 ± 0.09 a1.68 ± 0.21 A15.7 ± 1.119.04 ± 1.1 B*
drought34 ± 1.5 c35 ± 4.2 B1.68 ± 0.08 a1.59 ± 0.13 A20.24 ± 0.522.01 ± 2.2 B
NaCl 300 mM38 ± 5.1 c47 ± 2.1 C*1.94 ± 0.24 ab1.88 ± 0.21 A19.59 ± 2.525 ± 1.2 C*
NaCl 600 mM19 ± 0.9 a22 ± 2.1 A2.12 ± 0.08 b1.99 ± 0.24 A8.96 ± 0.511.06 ± 1.2 A
Different lowercase letters indicate significant differences between plants cultivated on non-inoculated substrate by SOB within different stress treatments. Different capital letters indicate statistically significant differences between plants cultivated on substrate inoculated by SOB within different stress treatments. * Indicates statistically significant differences between inoculated and non-inoculated plants within the same stress treatment, according to Tukey’s test (α = 0.05), ±SE, n = 5.
Table 4. Total contents of phenols, phenylpropanoids, flavonols, and anthocyanins (mg·g−1 f.w.) in P. coronopus subjected to drought, sodium chloride (300 and 600 mM NaCl), and sulfur-oxidizing bacteria.
Table 4. Total contents of phenols, phenylpropanoids, flavonols, and anthocyanins (mg·g−1 f.w.) in P. coronopus subjected to drought, sodium chloride (300 and 600 mM NaCl), and sulfur-oxidizing bacteria.
Total PhenolicsPhenylopropanoidsFlavonolsAnthocyanins
Non-SOBSOBNon-SOBSOBNon-SOBSOBNon-SOBSOB
control15.1 ± 3.1 a15.2 ± 5.8 A7.3 ± 0.9 a7.5 ± 2.8 A6.0 ± 0.7 a5.6 ± 1.4 A1.5 ± 0.1 a1.4 ± 0.3 A
drought123.9 ± 10.2 c144.5 ± 20.1 B42.3 ± 3 c50 ± 6.4 B31.4 ± 3.2 c34.8 ± 4.3 B5.6 ± 1.1 b4.6 ± 0.4 B
NaCl 300 mM17.3 ± 0.1 a27 ± 3.5 A8.6 ± 0.3 a12.1 ± 1.9 A6.0 ± 0.1 a8.6 ± 0.8 A*0.9 ± 0.2 a1.4 ± 0.2 A
NaCl 600 mM66.1 ± 5.4 b*28.9 ± 1.4 A25 ± 2 b*11.9 ± 0.1 A17.2 ± 1.4b*8.2 ± 0.8 A2.3 ± 0.1 a*1.4 ± 0.1 A
Different lowercase letters indicate significant differences between plants cultivated on non-inoculated substrate by SOB within different stress treatments. Different capital letters indicate statistically significant differences between plants cultivated on substrate inoculated by SOB within different stress treatments. * Indicates statistically significant differences between inoculated and non-inoculated plants within the same stress treatment, according to Tukey’s test (α = 0.05), ±SE, n = 5.
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Koźmińska, A.; Hassan, M.A.; Halecki, W.; Kruszyna, C.; Hanus-Fajerska, E. Beneficial Microorganisms: Sulfur-Oxidizing Bacteria Modulate Salt and Drought Stress Responses in the Halophyte Plantago coronopus L. Sustainability 2024, 16, 10866. https://doi.org/10.3390/su162410866

AMA Style

Koźmińska A, Hassan MA, Halecki W, Kruszyna C, Hanus-Fajerska E. Beneficial Microorganisms: Sulfur-Oxidizing Bacteria Modulate Salt and Drought Stress Responses in the Halophyte Plantago coronopus L. Sustainability. 2024; 16(24):10866. https://doi.org/10.3390/su162410866

Chicago/Turabian Style

Koźmińska, Aleksandra, Mohamad Al Hassan, Wiktor Halecki, Cezary Kruszyna, and Ewa Hanus-Fajerska. 2024. "Beneficial Microorganisms: Sulfur-Oxidizing Bacteria Modulate Salt and Drought Stress Responses in the Halophyte Plantago coronopus L." Sustainability 16, no. 24: 10866. https://doi.org/10.3390/su162410866

APA Style

Koźmińska, A., Hassan, M. A., Halecki, W., Kruszyna, C., & Hanus-Fajerska, E. (2024). Beneficial Microorganisms: Sulfur-Oxidizing Bacteria Modulate Salt and Drought Stress Responses in the Halophyte Plantago coronopus L. Sustainability, 16(24), 10866. https://doi.org/10.3390/su162410866

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