Plant Probiotic Endophytic Pseudomonas flavescens D5 Strain for Protection of Barley Plants from Salt Stress

Abstract

Soil salinity has become a global issue that is directly related to land degradation and results in many changes in climate, ecosystem services, and biodiversity. The present study focuses on the investigation of beneficial properties of a plant probiotic bacterial strain as an eco-friendly and sustainable approach to promote crop growth in saline soil. The endophytic halotolerant strain Pseudomonas flavescens D5 isolated from common chicory (Cichorium intybus L.) was able to grow on a medium containing 15% NaCl; produced indole-3-acetic acid (45.2 μg mL⁻¹) and polyhydroxyalkanoate (1.72 g L⁻¹); and had amylolytic, cellulolytic, and proteolytic activities. Polyhydroxyalkanoate had a pronounced antifungal activity against Fusarium graminearum, F. solani, F. oxysporum, and Alternaria alternata. Under salt stress conditions, inoculation with Ps. flavescens D5 increased the shoot biomass of barley plants by 8–30%, root biomass by 7–20%, chlorophyll a by 18–52%, and chlorophyll b by 7–15%. The content of proline decreased by 1.5–1.8 times. An increase in the activity of antioxidant enzymes (catalase, guaiacol peroxidase, and ascorbate peroxidase) was determined. In inoculated plants growing in saline soil, the content of Na⁺ ions was lower by up to 54.8% compared to control. This strain is promising for stimulating plant growth and protecting them from diseases and other adverse environmental factors, including salt stress.

1. Introduction

Salt stress is one of the primary environmental stresses that affects the growth, development, and productivity of crops and the quality of the crop yield. According to the FAO, more than 3% of the world’s arable soils and more than 6% of the world’s subsoil are affected by salinization [1]. Plants under salt stress have several morphological, physiological, and molecular changes that hinder their growth and development. Thus, high salt concentration affects enzyme activity, stomatal conductivity, and photosynthesis rate. Salt stress also causes oxidative stress, increasing the production of reactive oxygen species (ROS) and damaging cell membranes, proteins, lipids, and nucleic acids [2]. In addition, salinization leads to osmotic stress due to the excessive accumulation of Na⁺ and Cl⁻ ions. Therefore, it poses a severe threat to the sustainability of agriculture and food security. In this regard, it is very important to develop effective methods to reduce the adverse effects of salt stress on plant growth and development [3].

One of the strategies to solve this problem is to obtain and use new salt-resistant varieties of agricultural plants using transgenic technologies and breeding methods. However, these two approaches are insufficient as breeding is time- and labor-consuming; on the other hand, the acceptance of transgenic crops is under question due to its socio-ethical issues [4]. An innovative, effective technique that has attracted much attention in recent years is the use of microorganisms that promote plant growth [3]. These microorganisms induce plant resistance to abiotic stresses and also have several positive effects: (1) the synthesis of auxins, mainly indole-3-acetic acid (IAA), which directly promotes plant growth; (2) the effect on the activity of antioxidant enzymes (catalase, superoxide dismutase, and peroxidase), which prevents the harmful effects of ROS; (3) the effect on the content of proline, which structures the water content in plant cells and reduces the turgor pressure of plants; (4) increasing the availability of nutrients, which directly improves plant growth; (5) antagonistic activity, which protects plants weakened by stress from soil pathogens; and (6) synthesis of the 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which reduces the level of ACC and ethylene in plants, thereby reducing stress-induced plant aging [3,5,6,7].

Endophytic plant-growth-promoting microorganisms are attracting more attention because they play an essential role in the development of plants and affect their physiological and biochemical processes. The effectiveness of endophytic bacteria in increasing plant adaptation to soil salinity [8,9], as well as other abiotic stresses, such as drought, nutrient stress, extreme temperatures, and soil contamination with organic pollutants and heavy metals [10,11], has been demonstrated. Therefore, endophytes are often referred to as plant probiotic bacteria (PPB). They are widely used both as environmentally friendly biofertilizers in crop production and for solving environmental problems and protecting the environment. PPB enhance the growth of host plants, increase yields, and protect plants from abiotic and biotic stresses [12,13].

Representatives of Pseudomonas genera are a promising microbial group among plant probiotic bacteria that has a beneficial effect on the growth and development of plants under stress. In the previous studies, the ability of halotolerant strains of Pseudomona genera to reduce the effects of salt stress and increase tolerance of plants such as rice [14], soybean [15,16], camelina [17], maize [18], canola [19], hemp seed [20], pepper [21], and barley [22] was demonstrated.

Chicory (Cichorium intybus L.) is a promising plant for isolating endophytic PBB due to its high resistance to diseases and excellent acclimatization to various soil and climatic conditions, including salinization [23].

Barley (Hordeum vulgare L.) is one of the most salinity-tolerant cereal crops. Barley is the fourth most important crop after wheat, maize, and rice cultivated worldwide due to its nutritional and health benefits and as raw material for alcohol production. Barley is also an excellent model crop for studies on the mechanisms of salinity tolerance and for developing approaches to promote crop growth in saline soil [24].

Although the plant-growth-promoting properties of halotolerant bacteria are well studied, there is not enough information about endophytic bacteria that have a multifunctional positive effect on plants. The present study focuses on the investigation of beneficial properties of PPB and their ability to protect plants from salt stress. The main objectives of our study are: (1) isolation and study of probiotic properties of endophytic halotolerant bacteria; (2) investigation of the ability of the selected strain to synthesize polyhydroxyalkanoate (PHA) and characterization of its antimicrobial properties; (3) study of the response of barley treated with the selected strain under salt stress.

The present study contains new insights in the field of sustainable agriculture. The investigation of the beneficial properties of a new plant probiotic bacterial strain, as well as its application for improving plant growth and adaptation under salinity stress, is needed to expand and deepen existing knowledge. This work demonstrates for the first time the possibility of application of the halotolerant endophytic microorganism—a producer of IAA and PHA—to promote the growth of barley in saline soil.

2. Materials and Methods

2.1. Endophyte Isolation

Isolation of endophytic microorganisms from different parts (roots, stems, and leaves) of common chicory (Cichorium intybus L.) was guided by the research manuals of [25]. To sterilize the surface, individual plant pieces were previously washed with running tap water and sequentially placed in 70% ethanol solution, 2% sodium hypochlorite solution, and sterile distilled water. To isolate endophytic microorganisms, the sterilized plant fragments were plated onto nutrient agar (NA) plates. The plates were incubated at 30 °C for 7–14 days. Any present bacteria were isolated and purified.

2.2. Determination of Halotolerance of Microorganisms

To identify the halotolerance of microorganisms, the NA medium was used with the addition of NaCl at concentrations of 5%, 10%, 15%, and 25%. Microorganisms were inoculated on NA plates with different concentrations of NaCl by the streak method, and strains capable of cultivation at high salt concentrations were selected.

2.3. 16S rRNA Gene Sequencing and Phylogenetic Analysis

Bacteria were identified at the molecular level by sequencing the 16S rRNA gene. DNA was extracted and purified as described by Wilson [26]. The following PCR primers were used: 8F 5′–AGAGTTTGATCCTGGCTCAG-3′ and 806R-5′ GGACTACCAGGGTATCTAAT-3′. The sequencing reaction was performed using the BigDye^®Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Waltham, MA, USA) following the manufacturer’s instructions. Fragments were separated using an automated 3730xl DNA Analyzer (Applied Biosystems, USA). The obtained sequence was deposited to the NCBI GenBank database under accession number OP642636. The alignment of nucleotide sequences was performed using the ClustalW algorithm. The obtained sequences were compared with the related sequences of the type strains available in the GenBank database using BLAST analysis at NCBI. Phylogenetic trees were constructed using the Maximum Likelihood method in the Mega 6.0 software package [27].

2.4. IAA Production Assay

The amount of IAA produced by microorganisms was determined by the colorimetric method using the Salkovsky reagent. Strains of microorganisms were grown in a liquid nutrient medium with the addition of L-tryptophan at a concentration of 1000 µg mL⁻¹. The cultures were cultivated at 240 rpm for 2 days. After the incubation, the cultures were centrifuged at 6000× g for 20 min. The concentration of IAA was determined according to a calibration schedule constructed in the concentration range of the substance 10⁻⁸–10⁻² g L⁻¹. The concentration of IAA was expressed in μg mL⁻¹ [28].

2.5. Detection of Amylolytic Ability

The studied strain was cultivated on NA medium. The plates were incubated at 30 °C for 3 days. Lugol’s solution was used for detection of the amylolytic ability of the strain [29].

2.6. Detection of Cellulolytic Ability

The studied strain was cultured on the medium of the following composition (g L⁻¹): NaNO₃-2.0; KH₂PO₄-1.0; MgSO₄·7H₂O-0.5; KCl 0.5; FeSO₄-0.01; and agar-20.0. Sodium carboxymethyl cellulose was used as a carbon source. The plates were incubated at 30 °C for 3 days. Congo red dye was used for detection of the cellulolytic ability of the strain [29].

2.7. Detection of Proteolytic Enzymes in Microorganisms

To determine the proteolytic activity of the studied strain, a medium with the addition of casein was used. The plates were spot inoculated and incubated at 30 °C for 3 days. Proteolytic activity was determined by the appearance of halo zones around the colonies [29].

2.8. Determination of the Ability of Microorganisms to Perform Phosphate Mobilization

The fungal strains were screened for P-mobilizing ability by culturing on plates with Pikovskaya (PKV) agar medium containing (g L⁻¹): glucose, 10; yeast extract, 0.5; (NH₄)₂SO₄, 0.5; MgSO₄·7H₂O, 0.1; KCl, 0.2; NaCl, 0.2; MnSO₄·4H₂O, 0.02; FeSO₄·7H₂O, 0.02; Ca₃(PO₄)₂ 5; and agar, 15; pH 7.0. The plates were spot inoculated and incubated at 30 °C for 3 days. Phosphate-mobilizing fungi were recognized by the formation of visible halo zones around colonies [30].

2.9. PHA Production Assay

Cultures with both amylolytic and cellulolytic activities as potential PHA producers were incubated in MSM medium, at 30 °C, for 3 days [31]. The medium composition was (g L⁻¹): K₂HPO₄-1.73; KH₂PO₄-0.68; MgSO₄·7H₂O-0.1; NaCl-4.0; FeSO₄·7H₂O-0.03; NH₄NO₃-1.0; CaCl₂·2H₂O-0.02; glucose-5.0; and agar-20.0. Afterwards, Nile blue A staining plates were viewed under UV light, observing the formation of colony fluorescence [31]. Colonies that showed bright fluorescence were selected as PHA producers.

The strain was grown in a liquid MSM medium for 48 h at 150 rpm. Then, the suspension was centrifuged at 6000× g for 10 min, the supernatant was decanted, and PHA was isolated from the precipitate. Sodium hypochlorite and hot chloroform at a ratio of 1:1 were added to the precipitate, and kept at 30 °C, for 1 h. The suspension was centrifuged at 6000× g for 15 min and the upper and middle layers were removed. The sediment was precipitated with ethanol and acetone in a ratio of 1:1, dried at 35 °C, and weighed [31].

2.10. Antifungal Activity of Bacterial PHA

The antifungal activity was assayed using the plate-hole diffusion method [32]. A total of 100 μL of fungal spore suspension containing 10⁶ spore mL⁻¹ of each phytopathogenic fungi (Fusarium graminearum, F. solani, F. oxysporum, Alternaria alternata) was inoculated on an agar surface. A sterile cork borer with 10 mm diameter was used to bore holes. Then, 100 μL of 30% PHA suspension was added to each hole. Sterile distilled water was used as control. Antifungal activity was evaluated by measuring the diameter of the inhibition zone against the test phytopathogens.

2.11. Pot Experiment

Barley seeds were sterilized in a 5% chlorhexidine solution and then washed in sterile distilled water three times. The soil used in experiments had a light loam texture and the following physico-chemical characteristics: bulk density 1.24 g cm⁻³, total C 2.63%, total N 1.7 g kg⁻¹, total P 1.8 g kg⁻¹, available P 12.4 mg kg⁻¹, available K 535.5 mg kg⁻¹, CaCO₃ 7.8%, and pH (H₂O) = 8.2. Salt stress conditions were created by adding NaCl solution to the soil, reaching concentrations of 3, 4, 5, and 6 g of salt per kg of dry soil. Dry non-sterile soil (300 g) was placed in plastic pots 65 × 65 × 100 mm in size and moistened to 60% with distilled water. Surface-sterilized barley seeds were placed in pots (15 seeds per pot, 5 pots per variant) and each seed was inoculated with 1 mL of a bacterial suspension containing 10⁸ cells per ml. Control seeds were treated with sterile water. The pots were incubated in a growth chamber at 22 °C under a 16 h/8 h light/dark cycle at a 100 µmol m⁻² s⁻¹ photon flux density. After incubation for 21 days, the plants were harvested and the length and dry weight of roots and shoots were determined.

2.12. Determination of Chlorophyll Concentration

Chlorophyll concentration in plants was determined according to [19]. A total of 200 mg of the plant was triturated in 15 mL of 90% ethanol. The homogenate was centrifuged for 20 min at 6000× g. The supernatant was collected and absorbance was measured on a spectrophotometer at 650 and 667 nm. Pigment concentration was calculated using the equations:

$${\mathit{Chl}}_a=13.70\times A_{667}-5.76\times A_{550}$$

$${\mathit{Chl}}_b=25.80\times A_{650}-7.60\times A_{667}$$

2.13. Determination of Free Proline Concentration

The free proline concentration was determined using a ninhydrin reagent. Plant fresh material was homogenized in 3% aqueous solution of sulfosalicylic acid. The homogenate was centrifuged at 6500× g for 40 min. Then, 2 mL of CH₃COOH, 2 mL of ninhydrin reagent, and 2 mL of the prepared extract infusion were added. The samples were incubated for 1.5 h, at 100 °C, and cooled to room temperature. Absorbance was measured at 520 nm. A mixture of the same reagents was used as a control, without adding the extract. Proline content was calculated using a calibration curve using Sigma (Burlington, MA, USA) proline as the standard [19].

2.14. Preparation of the Extract for The determination of Antioxidant Enzymes

To isolate catalase, as well as guaiacol peroxidase and ascorbate peroxidase, an extraction medium was used, which contained: 50 mM K-phosphate buffer (pH 7.5); 1 mM EDTA, 0.3%; and 1 mM ascorbic acid. After centrifugation (20 min, 6000× g), the activity of these enzymes was determined in the resulting supernatant. All procedures for the isolation of enzymes were carried out at 4 °C [16].

2.15. Investigation of Catalase Activity

The reaction medium for determining catalase activity contained: 100 mM K-phosphate buffer (pH 7.0), 15 mM H₂O₂, and 0.1 mL of the sample [16]. The reaction was started by adding H₂O₂. The change in optical density was determined at 240 nm, for 3 min, and the enzyme activity was calculated using the extinction coefficient ε = 0.03 mM⁻¹ cm⁻¹. Enzyme activity was expressed as the amount of enzyme required to oxidize 1 mmol of H₂O₂ min⁻¹ per mg protein.

2.16. Investigation of Ascorbate Peroxidase Activity

The activity of ascorbate peroxidase was determined by the rate of ascorbate oxidation [16]. The reaction mixture consisted of a 50 mM potassium–phosphate buffer (pH 7.0) containing 0.1 mM EDTA, 0.5 mM ascorbate, and 0.1 mM H₂O₂. The reaction was started by adding 3.9 mL of buffer to 0.1 mL of enzyme extract in a quartz cuvette. The change in optical density was determined at 290 nm, for 1 min, and the enzyme activity was calculated using the extinction coefficient ε = 2.8 mM⁻¹ cm⁻¹. Enzyme activity was expressed as the amount of enzyme required to oxidize 1 mmol of ascorbate min⁻¹ per mg protein.

2.17. Investigation of Guaiacol Peroxidase Activity

The activity of guaiacol peroxidase was determined spectrophotometrically by the increasing of the absorption due to the guaiacol oxidation [16]. The reaction mixture consisted of 50 mM phosphate buffer (pH 7), 9 mM guaiacol, 10 mM H₂O₂, and 0.2 mL enzyme extract in a total reaction volume of 4 mL. The change in optical density was determined at 470 nm, for 1 min, and the enzyme activity was calculated using the extinction coefficient ε = 26.6 mM⁻¹ cm⁻¹. Enzyme activity was expressed as the amount of enzyme required to produce 1 mmol of guaiacol dehydrogenation product min⁻¹ per mg protein.

2.18. Determination of the Protein Amount

The protein content was determined by the Bradford method. A total of 1 mL of the extract was placed in a tube and 0.01 mL of Bradford reagent with 4 mL of distilled water was added. The sample was left for 30 min. Absorbance was measured on a spectrophotometer at 595 nm [33].

2.19. Element Content Assay

Plant samples were washed with running water, dried to an air-dry state, and ground to a homogeneous mass. Sample preparation was carried out by microwave digestion on a Speedwave XPERT (Berghof Products + Instruments GmbH (Eningen, Germany). Plant samples were treated with 67% HNO₃ and 35% H₂O₂; after complete digestion, the obtained samples were brought to a volume of 15 cm³ with deionized water and diluted with 1% HNO₃ in a ratio of 1:10. In the obtained solutions, the content of K, Ca, Na, Mg, Fe, Mn, Cu, Zn, Cd, and Pb was determined by inductively coupled plasma mass spectrometry on an Agilent 7700x (Agilent Technologies, Tokyo, Japan).

Statistical Analysis

The data were processed using the software Statistica version 10.0 (TIBCO Software Inc., Palo Alto, CA, USA). Student’s t-test (p < 0.05) was performed to estimate statistical differences between means. SD stands for standard deviation.

3. Results and Discussion

In most cases, sodium chloride is the primary salt toxicant, and its negative effect can be observed in the form of growth inhibition or plant death. Salinization causes a significant decrease in agricultural crop yields, especially in arid and semi-arid regions [2,3]. There are several strategies to reduce the toxic effects caused by high salinity of the environment on plant growth, among which the use of PPB is relevant [12,13,34,35].

3.1. Isolation and Plant Probiotic Properties of Halotolerant Bacteria

At the first stage, endophytic PPB with plant protective effects against salt stress were isolated. In total, 67 bacterial strains were isolated from the roots, stems, and leaves of common chicory (Cichorium intybus L.). Nine isolates showed high growth levels on media with 5% NaCl. At a salt concentration of 15%, only the strain D5 had the ability to grow. It is known that halophiles are divided into slight, which grow optimally at 3% of salt in the medium, moderate, optimal growth in the range of 3–15% of salt, and extreme, for which the optimal concentration for growth is 25% of salt [36]. According to this classification, the strain D5 was attributed to moderate halophiles.

The selected D5 strain was identified as Pseudomonas flavescens by the 16S rRNA sequence analysis.

Further, Ps. flavescens D5 was characterized for its plant-growth-promoting activities. PPB are able to provide some positive effects on plants, the main of which are: the production of metabolites with hormonal and signaling functions; increasing availability of nutrients, including phosphorus; and protective effect in conditions of biotic and abiotic stresses [12,13,37,38]. The study of the plant-growth-promoting properties of Ps. flavescens D5 revealed that the strain had amylolytic, cellulolytic, and proteolytic activities. However, the ability to solubilize phosphate was not detected.

Ps. flavescens D5 strain produced IAA in the amount 45.2 μg mL⁻¹. This concentration was higher than values of IAA for previously reported halotolerant PPB [39,40,41]. The concentration of bacterial IAA produced by PPB, which can either stimulate root development in cases where the concentration of auxins in plants is suboptimal or inhibit root development when auxin levels are already optimal, is widely discussed, as well as the probability that hormone production may change depending on the plant signals [42,43]. Thus, while searching for growth-promoting microorganisms among halotolerant bacteria, preference is given to those that produce phytohormones.

3.2. Antifungal Activity of PHA

In the present study, for the first time, in addition to the well-known properties of PPB, the ability of the halotolerant strain to produce PHA is shown. It is known that PHA as a storage compound supports the growth of microorganisms in stressful conditions, increasing their environmental stability [44]. In addition, PHA has effective antibacterial and antifungal properties [45,46]. In this study, bright blue fluorescence was observed under UV light after staining with Nile blue A, which indicates the production of PHA by this strain (Figure 1).

PHA production was confirmed by the cultivation of Ps. flavescens D5 in MSM liquid medium containing glucose as a carbon source. The results detected the accumulation of PHA at 1.72 ± 0.07 g L⁻¹, which is significantly higher than for other halotolerant strains [47,48]. At the next stage, the antagonistic activity of PHA against fungal pathogens was revealed (

Table 1. Antifungal activity of PHA against phytopathogens.

Average Size of Inhibition Halos (cm)
Fusarium graminearum	Fusarium solani	Fusarium oxysporum	Alternaria alternata
1.6 ± 0.06	2.3 ± 0.08	3.5 ± 0.1	-

).

The most significant antifungal activity was observed against F. oxysporum and F. solani (Figure 2).

In many studies, it has been shown that some PHA derivatives have bactericidal effects against Gram-negative and Gram-positive bacteria [45,46]. However, information about the antifungal activity of PHA is very limited [49]. Infections of crops caused by pathogenic fungi are among the most widespread and harmful. According to FAO estimates, 14% of global crop losses are associated with plant diseases, while fungi account for 42% of them [50]. These diseases lead not only to a decrease in yield but also to a deterioration in its quality due to the accumulation of mycotoxins dangerous to human and animal health.

Currently, the species Ps. flavescent has not been reported as a PHA producer. However, PHA production from halophilic microorganisms has added benefits due to its low-cost purification process and minimal environmental hazards.

3.3. Plant Response to Bacterial Application

At the next stage, the protective effect of the plant probiotic Ps. flavescens D5 strain on barley plants under the toxic effect of NaCl was investigated.

The investigation of inoculation with Ps. flavescens D5 showed a positive effect on shoot and root lengths of barley plants under salt stress. The shoot and root lengths were significantly higher compared with two controls—without inoculation and under non-salted conditions (

Table 2. Effect of Pseudomonas flavescens D5 inoculation on the growth parameters of barley plants in salt stressed conditions.

Treatment		Shoot Biomass, g	Root Biomass, g	Shoot Length, cm	Root Length, cm
Without NaCl	Control	5.49 ± 0.17 a	2.35 ± 0.17 a	19.7 ± 0.7 a	15.2 ± 0.5 a
	Ps. flavescens D5	6.21 ± 0.18 b	3.45 ± 0.14 b	22.9 ± 0.9 b	18.1 ± 0.7 b
NaCl 3 g kg−1	Control	4.12 ± 0.11 a	1.87 ± 0.09 a	16.2 ± 0.7 a	11.4 ± 1.7 a
	Ps. flavescens D5	4.75 ± 0.14 b	2.25 ± 0.09 b	21.5 ± 1.4 b	17.9 ± 1.1 b
NaCl 4 g kg−1	Control	3.54 ± 0.1 a	1.71 ± 0.04 a	14.5 ± 1.1 a	10.1 ± 1.3 a
	Ps. flavescens D5	3.88 ± 0.05 b	1.92 ± 0.05 b	20.2 ± 1.5 b	16.5 ± 1.1 b
NaCl 5 g kg−1	Control	3.36 ± 0.1 a	1.67 ± 0.05 a	13.2 ± 1.0 a	9.2 ± 0.9 a
	Ps. flavescens D5	3.9 ± 0.12 b	1.85 ± 0.04 b	18.8 ± 1.5 b	15.6 ± 0.9 b
NaCl 6 g kg−1	Control	2.78 ± 0.14 a	1.53 ± 0.03 a	12.5 ± 1.1 a	8.7 ± 0.7 a
	Ps. flavescens D5	3.62 ± 0.18 b	1.64 ± 0.02 b	16.5 ± 0.9 b	14.1 ± 1.3 b

Data are presented as means ± SD. Different letters in the same subcolumn indicate statistically significant differences among values according to Student’s t-test at p < 0.05.

).

With increasing salt concentration, the toxic effect increased. Inoculation with Ps. flavescens D5 strain contributed to a significant increase in the growth parameters of barley when exposed to elevated concentrations of NaCl compared with untreated plants. Shoot biomass of inoculated plants increased by 8–30% and root biomass by 7–20%. Due to the data, it can be concluded that plants under severe stress showed a noticeable delay in growth. Similar suggestions and findings were presented in previous studies [15,16], in which an improvement of the growth parameters of soybean (Glycine max L.) plants due to Pseudomonas strains inoculation in salt conditions was demonstrated.

Inoculation of barley with Ps. flavescens D5 strain enhanced the chlorophyll a and b level compared with untreated plants. The results of our research revealed a significant distinction in chlorophyll content. The highest chlorophyll a and chlorophyll b content (1.71 and 0.92 mg g⁻¹) was observed in the presence of NaCl at the concentration of 3.0 g kg⁻¹ (

Table 3. Effect of Pseudomonas flavescens D5 inoculation on chlorophyll content in barley plants in salt stressed conditions.

Treatment		Chlorophyll a, mg g−1	Chlorophyll b, mg g−1
Without NaCl	Control	2.19 ± 0.02 a	1.18 ± 0.02 a
	Ps. flavescens D5	2.37 ± 0.01 b	1.56 ± 0.02 b
NaCl 3 g kg−1	Control	1.44 ± 0.03 a	0.80 ± 0.02 a
	Ps. flavescens D5	1.71 ± 0.01 b	0.92 ± 0.03 b
NaCl 4 g kg−1	Control	1.02 ± 0.01 a	0.76 ± 0.03 a
	Ps. flavescens D5	1.55 ± 0.02 b	0.86 ± 0.01 b
NaCl 5 g kg−1	Control	0.90 ± 0.02 a	0.67 ± 0.01 a
	Ps. flavescens D5	1.35 ± 0.03 b	0.72 ± 0.03 b
NaCl 6 g kg−1	Control	0.79 ± 0.04 a	0.62 ± 0.02 a
	Ps. flavescens D5	1.15 ± 0.02 b	0.69 ± 0.01 b

Data are presented as means ± SD. Different letters in the same subcolumn indicate statistically significant differences among values according to Student’s t-test at p < 0.05.

). Similar to our results, improvements of chlorophyll content in tomato, soybean, and rice due to application of bacteria under stressed conditions have been reported by many researchers [51,52].

An increase in the proline content is one of the characteristic reactions of plants in response to various types of stress, including salt stress, which provides the first stage of plant adaptation [53,54]. In uninoculated variants under salinization conditions, the concentration of proline exceeded this value in plants grown in favorable conditions by 2.1–5.3 times and reached 164.5 ± 3 µmol g⁻¹ of fresh mass. In plants treated with Ps. flavescens D5, the proline content was 1.5–1.8 times lower than in plants without treatment and varied in the range from 40.1 to 107.1 µmol g⁻¹ depending on the concentration of NaCl (Figure 3). The results obtained indicate a reduction in the stress experienced by plants due to treatment with the PPB strain.

Enzymes such as catalase, ascorbate peroxidase, and guaiacol peroxidase are usually regarded as critical components of antioxidant plant protection. In addition, various antioxidant enzymes help to maintain a balance in the production and absorption of ROS, and it is believed that the increased activity of these enzymes contributes to protection from stress [2,3].

The present study evaluated the ability of the Ps. flavescens D5 strain to have a protective effect on barley plants under NaCl toxicity.

It was shown that catalase activity in non-inoculated plants growing at different NaCl concentrations was significantly higher compared to control plants (growing in soil without salt) (

Table 4. Effect of Pseudomonas flavescens D5 inoculation on antioxidant enzyme activity in barley plants in salt stressed conditions.

Treatment		Catalase, μmol min−1 mg of Protein−1	Ascorbate Peroxidase, μmol min−1 mg of Protein−1	Guaiacol Peroxidase, μmol min−1 mg of Protein−1
Without NaCl	Control	0.046 ± 0.001 a	7.2 ± 0.3 a	12.3 ± 0.5 a
	Ps. flavescens D5	0.049 ± 0.001 a	7.4 ± 0.2 a	12.6 ± 0.4 a
NaCl 3 g kg−1	Control	0.087 ± 0.002 a	4.6 ± 0.2 a	13.13 ± 0.6 a
	Ps. flavescens D5	0.12 ± 0.003 b	9.2 ± 0.4 b	27.1 ± 0.7 b
NaCl 4 g kg−1	Control	0.065 ± 0.001 a	5.75 ± 0.2 a	16.26 ± 0.6 a
	Ps. flavescens D5	0.23 ± 0.01 b	16.05 ± 0.7 b	34.1 ± 1.7 b
NaCl 5 g kg−1	Control	0.063 ± 0.003 a	6.3 ± 0.2 a	23.1 ± 0.8 a
	Ps. flavescens D5	0.38 ± 0.005 b	12.7 ± 0.5 b	29.2 ± 1.1 b
NaCl 6 g kg−1	Control	0.058 ± 0.002 a	8.04 ± 0.3 a	20.3 ± 0.7 a
	Ps. flavescens D5	0.37 ± 0.01 b	10.75 ± 0.4 b	25.3 ± 1.1 b

Data are presented as means ± SD. Different letters in the same subcolumn indicate statistically significant differences among values according to Student’s t-test at p < 0.05.

). The Ps. flavescens D5 strain promoted a significant increase in catalase activity (by 1.4–6.4 times) when exposed to various salt concentrations compared to untreated plants.

Data showed an increase in the activity of ascorbate peroxidase (by 1.3–2.8 times) in inoculated plants growing under salt stress conditions compared to control plants. The maximum increase in enzyme activity was observed at a NaCl concentration of 4 g kg⁻¹ (Table 4).

The increase in guaiacol peroxidase activity in non-inoculated plants correlated with the increase in NaCl concentration in the soil (Table 4). A significant increase by 1.2–2.1 times in guaiacol peroxidase activity in inoculated plants growing in saline soil compared to the control samples was revealed. It should be noted that NaCl concentration of 6 g kg⁻¹ decreased the enzyme activity in both inoculated and uninoculated plants.

In the present study, treatment with Ps. flavescens D5 strain increased the activity of catalase, ascorbate peroxidase and guaiacol peroxidase in barley plants grown in saline soil, which confirms the role of this strain in providing plant resistance to salt stress.

Similar to the data obtained, a significant increase by 5–28% in the activity of antioxidant enzymes, including catalase, ascorbate peroxidase, superoxide dismutase, and guaiacol peroxidase, was shown in corn and rice plants in the presence of the endophyte A. terreus compared to non-inoculated plants exposed to salt stress. Catalase and ascorbate peroxidase activity was increased significantly with increasing NaCl concentration [55]. The protective effect of B. subtilis 26D and B. subtilis 11VM bacteria has been detected in the plants exposed to salt stress. The analysis showed that, in plants inoculated with B. subtilis 26D and B. subtilis 11VM strains under salt stress, catalase activity increased in a range from 5 to 23%, and peroxidase activity increased by up to 48.5%, respectively [56]. Some studies have shown that overexpression of genes of various antioxidant enzymes (ascorbate peroxidase, catalase, and guaiacol peroxidase) in different plants significantly improved plant resistance to salt stress. Such a protection mechanism can be associated with the immediate decontamination of ROS or participation in regenerating low-molecular-weight antioxidants [57].

There are no data about the effect of Pseudomonas flavescens species on the antioxidant systems of plants under salt stress. Here, for the first time, the role of the endophyte strain Ps. flavescens D5 in providing plant resistance to different concentrations of NaCl due to the increase in the activity of catalase, ascorbate peroxidase, and guaiacol peroxidase is shown.

An increase in the concentration of NaCl in the soil resulted in an increase in Na content and a decrease in K ions in plants (

Table 5. Effect of Pseudomonas flavescens D5 inoculation on element content in barley plants in salt stressed conditions.

Treatment		Content of Elements, ×103 mg kg−1
		Na+	K+	Mg2+	Ca2+	Mn2+	Fe3+
Without NaCl	Control	2.9 ± 0.03 a	30.4 ± 1.9 a	1.8 ± 0.27 a	7.2 ± 0.71 a	4.1 ± 0.62 a	0.13 ± 0.02 a
	Ps. flavescens D5	3.0 ± 0.04 a	33.8 ± 1.6 a	2.0 ± 0.03 a	8.2 ± 0.12 a	4.6 ± 0.69 a	0.19 ± 0.03 a
NaCl 3 g kg−1	Control	3.8 ± 0.06 b	23.6 ± 1.8 a	2.2 ± 0.01 b	4.5 ± 0.07 a	5.24 ± 0.76 a	0.12 ± 0.03 a
	Ps. flavescens D5	1.6 ± 0.02 a	24.4 ± 1.6 a	1.7 ± 0.02 a	6.3 ± 0.09 b	5.60 ± 0.98 a	0.16 ± 0.02 a
NaCl 4 g kg−1	Control	5.4 ± 0.08 b	27.3 ± 1.0 a	3.7 ± 0.05 b	3.99 ± 0.05 a	5.4 ± 0.82 a	0.16 ± 0.02 a
	Ps. flavescens D5	2.96 ± 0.03 a	29.8 ± 1.2 a	2.6 ± 0.07 a	4.3 ± 0.03 b	4.96 ± 0.29 a	0.13 ± 0.02 a
NaCl 5 g kg−1	Control	7.6 ± 0.01 b	26.6 ± 1.7 a	3.9 ± 0.08 b	3.7 ± 0.05 a	6.6 ± 0.10 a	0.13 ± 0.02 a
	Ps. flavescens D5	5.8 ± 0.08 a	28.5 ± 1.4 a	2.1 ± 0.06 a	4.3 ± 0.04 b	5.8 ± 0.87 a	0.15 ± 0.02 a
NaCl 6 g kg−1	Control	7.8 ± 0.01 b	29.3 ± 1.3 a	3.7 ± 0.54 b	3.9 ± 0.05 a	4.6 ± 0.10 a	0.17 ± 0.02 a
	Ps. flavescens D5	7.6 ± 0.01 a	34.9 ± 1.4 b	1.7 ± 0.02 a	6.3 ± 0.53 b	5.8 ± 0.12 a	0.15 ± 0.02 a

Data are presented as means ± SD. Different letters in the same subcolumn indicate statistically significant differences among values according to Student’s t-test at p < 0.05.

). In inoculated plants growing in saline soil, the content of Na was lower by up to 54.8% compared to control plants. So in plants inoculated with Ps.flavescens D5, at a NaCl concentration of 3 g kg⁻¹ of soil, the Na content in plants was less by 42.1%; at NaCl concentrations of 4, 5, 6 g kg⁻¹ of soil, by 54.8%, 23.7%, and 2.5%, respectively, compared to untreated samples. Inoculation with Ps. flavescens D5 strain had no effect on K⁺ content in plants except the variant with a salt concentration of 6 g kg⁻¹ (Table 5). Reductions in the amount of Na input and K outflow are the most essential strategies for plants to alleviate salt stress [58,59]. Additionally, this mechanism prevents the creation of an excessive concentration of Na⁺ inside the cell, causing various physiological disorders in plants, such as a decrease in the seed germination, seedling growth, flowering, and fruiting [3]. It is known that salinization inhibits plant growth due to increased Na concentration and a low K/Na ratio in plants. Several studies have shown that inoculation with PPB contributed to the reduction in excessive Na accumulation and an increase in K concentration, as well as maintained ionic homeostasis under salt stress [16,58,59,60].

The content of Mg in barley plants increased when plants were grown in saline soils, while the content of Ca decreased (Table 5). Inoculation of plants with PPB strain had a significant effect on the content of these elements. Treatment with Ps. flavescens D5 strain contributed to the maintenance of a high Ca level and the reduction in the Mg level under salt stress conditions. Salinity did not affect the concentration of Fe and Mn in plants (Table 5). It is known that, under saline conditions, the concentration of trace elements in plants may increase, decrease, or have no effect; that is explained by the type of plant tissue, differences in plant salt tolerance, salinity levels, micronutrient concentrations in the soil, and environmental conditions [61].

4. Conclusions

It was found that the endophytic halotolerant strain Ps. flavescens D5 isolated from chicory had a complex of beneficial effects on plants, including the synthesis of IAA and enzymatic activity (amylolytic, cellulolytic, and proteolytic). For the first time, the ability of the Ps. flavescens species to synthesize PHA, which had pronounced antifungal activity, was discovered. Salinity-induced conditions significantly decreased the barley growth and contributed to the disturbance of photosynthetic pigments. The inoculation of barley with Ps. flavescens D5 improved their growth and strengthened the plant defense mechanisms. This strain is promising for stimulating plant growth and protecting them from diseases and other adverse environmental factors, including salt stress.