Raising Beet Tolerance to Salinity through Bioaugmentation with Halotolerant Endophytes

: Increasing land salinization in recent decades has led to a decrease in crop productivity worldwide. We hypothesized that bioaugmentation of beetroot ( Beta vulgaris ) with halotolerant endophytic bacterial strains isolated from the obligatory halophytic plant Salicornia europaea L. may mitigate salt stress in new host plants. Therefore, we investigated the e ﬀ ects of inoculation with Pseudomonas stutzeri ISE12 or Kushneria marisﬂavi CSE9 on B. vulgaris growth in substrates enriched with various NaCl concentrations (0, 50, 150, 300 mM). The results of this study indicated that bioaugmentation with either bacteria resulted in improved growth parameters and increased chlorophyll content, as well as decreased proline and hydrogen peroxide concentrations, in B. vulgaris organs. However, K. marisﬂavi CSE9 was more e ﬃ cient in achieving salt stress mitigation than P. stutzeri ISE12. In conclusion, the range of salinity tolerance seems to be a key parameter in the selection of strains for beet inoculation. The selected halotolerant endophytes ( P. stutzeri ISE12 and K. marisﬂavi CSE9) isolated from the roots of obligatory halophytic S. europaea may be employed for plant growth promotion, especially in saline areas, and have potential applications in sustainable agriculture.


Introduction
The beginning of the 21st century has been marked by increasing global scarcity, environmental pollution and salinization of arable soil and water resources [1]. It is estimated that 800 million ha of land in the world (nearly 7% of all land) has been degraded by salinity, including 32 million ha (40%) of all agricultural land [2][3][4]. Soil is recognized as saline when its electrical conductivity in solution reaches 4 dS m −1 (equivalent to approximately 40 mM NaCl) [5]. Soil salinity can be a consequence of natural (e.g., geological and hydrological changes) or anthropogenic processes (such as deforestation and irrigation using saline water) [6,7]. Salinity negatively affects the physiology and biochemistry of plants and results in reduced crop productivity [8,9]. The response of plants to salt stress depends on the species, variety and phase of plant growth [6]. Only a small group of plants, the halophytes, can tolerate salinity exceeding 200 mM NaCl, while most known terrestrial plants (and the majority of arable crops) are glycophytes that have the ability to grow only in a slightly saline environment. Possible means of increasing salinity tolerance of crops are the domestication of halophytes or the
Bacterial inoculum was prepared from three-day-old cultures incubated on R2A medium (Difco TM ) supplemented with 2% NaCl at 25 • C. Bacterial cells were suspended in 2% NaCl solution and diluted to OD = 0.5 (OD-optical density, measured at 600 nm, equivalent of 1.5 × 10 8 cells/mL). Plants were inoculated twice: first inoculation-surface sterilized seeds were incubated (45 min) in bacterial suspension with shaking, second inoculation-two weeks after sowing 1 mL of bacterial suspension was added in the proximity of the plant root system. Control variant was prepared by treatment of seeds in sterile 2% NaCl solution.

Plant Material
Seeds of beetroot (B. vulgaris), Red Ball variety (Torseed ® , Toruń, Poland), were surface-sterilized with a mixture of 70% ethanol and 30% hydrogen peroxide (1:1, 4 min) and washed six times with sterile distilled water. The liquid obtained after the last wash was used to evaluate the efficiency of the sterilization process by plating the rinse on agar plates containing R2A (Difco) and Martin (BTL) medium.

Pot Experiment
B. vulgaris plants were cultivated in three variants of inoculation: control (noninoculated), inoculated with P. stutzeri ISE12, and inoculated with K. marisflavi CSE9 and four variants of salinity (0, 50, 150, and 300 NaCl mM) in sterile mixture of sand and vermiculite (1:3). Plants were cultivated in plastic trays (30 × 46 cm) containing 54 pots (each pot-0.077 l; 5.5 cm height, 3 × 3 cm base width, 4.5 × 4.5 cm top width). One tray with 54 plants per each variant was prepared in this experiment. Plants were maintained under controlled environmental conditions (temperature: 24 • C ± 2 • C and light/dark: 16/8) and irrigated with Hoagland medium (once per week) and distilled water (according to plant requirements) [32]. After eight weeks, plants were collected and analyzed according to

Plant Growth Parameters
The number of germinated seeds was determined daily (always at the same time) until the end of the 10th day after sowing. The germination percentage (GP) was calculated in accordance with the International Seed Testing Association (ISTA) method: GP = number of normally germinated seeds/total number of seeds sown × 100 The germination index (GI) was calculated using the following formula: where Gt is the number of seeds germinated on day t, and Tt is the number of days.
After eight weeks of cultivation, the chlorophyll content index (CCI) was measured in the third leaf of each plant (Chlorophyll Content Meter CCM-200, Opti-Sciences, Tyngsboro, MA, USA), as described by Richardson et al. [33]. The obtained CCI values were based on absorbance measurements at 660 and 940 nm on a surface of 0.71 cm 2 .
To evaluate growth parameters, plants were carefully removed from the pots, roots were washed to remove the rest of the growth substrate, and the following parameters were measured: fresh and dry (72 h, 85 °C, 24 h) weight, root and stem length and number of leaves.

Plant Growth Parameters
The number of germinated seeds was determined daily (always at the same time) until the end of the 10th day after sowing. The germination percentage (GP) was calculated in accordance with the International Seed Testing Association (ISTA) method: GP = number of normally germinated seeds/total number of seeds sown × 100 The germination index (GI) was calculated using the following formula: where G t is the number of seeds germinated on day t, and T t is the number of days.
After eight weeks of cultivation, the chlorophyll content index (CCI) was measured in the third leaf of each plant (Chlorophyll Content Meter CCM-200, Opti-Sciences, Tyngsboro, MA, USA), as described by Richardson et al. [33]. The obtained CCI values were based on absorbance measurements at 660 and 940 nm on a surface of 0.71 cm 2 . To evaluate growth parameters, plants were carefully removed from the pots, roots were washed to remove the rest of the growth substrate, and the following parameters were measured: fresh and dry (72 h, 85 • C, 24 h) weight, root and stem length and number of leaves.

Determination of Proline and Hydrogen Peroxide Levels
In the roots, stems and leaves, the levels of proline and hydrogen peroxide were determined immediately after collecting the plants. The analyses were performed in three biological and three technical replicates (six in total).
The proline level in B. vulgaris organs was assessed according to the methodology proposed by Abraham et al. [34] with modifications. Leaf, stem and root tissues were homogenized in a mortar using liquid nitrogen and extracted with 3% (w/v) aqueous sulphosalicylic acid (5 µL/mg of fresh plant material). The obtained homogenate was transferred to Falcon tubes (15 mL) and centrifuged at 4 • C, 12,000× g for 5 min. Then, 100 µL of supernatant was added to the previously prepared mixtures containing 100 µL 3% sulfosalicylic acid, 200 µL glacial acetic acid and 200 µL acidic ninhydrin reagent. Acidic ninhydrin reagent was prepared as described by Bates et al. [35]. The mixture was mixed and maintained at 96 • C for 30 min. The reaction was terminated by placing the samples on ice for 3 min. The chromophore was extracted with 1 mL of toluene and quantified spectrophotometrically at 520 nm. The proline content was evaluated using the standard curve for proline in the concentration range of 0-40 µg/mL. Proline concentrations were presented in µg of proline per gram of fresh weight.
Hydrogen peroxide content in plant organs was examined according to Anderson [36] with modifications described by Ngo and Lenhoff [37]. The analysis was performed directly from 0.5 g of fresh plant material (roots, stems, and leaves), homogenized in liquid nitrogen and extracted with 0.1% trichloroacetic acid (5 mL) at 4 • C. The obtained homogenate was transferred to Falcon tubes (15 mL) and then centrifuged at 4 • C, 10,000× g for 10 min. An aliquot of the supernatant was introduced into new Falcon tubes (50 mL), neutralized with 0.4 M KOH to pH 7.5 and centrifuged (1000× g, 1 min). Neutralized supernatant (200 µL) was placed in microtiter plate and 50 µL 19.8 mM DMAB in 0.5 M phosphate buffer (pH 6.5), 46 µL 0.456 mM MBTH and 0.4 µL peroxidase (1 kU/mL) were added. After a 20 min incubation at 25 • C, the H 2 O 2 content was measured using a Magellan ® spectrophotometer at 590 nm. A standard curve was prepared from serial dilutions (range of 0-0.039 µM H 2 O 2 ), and the final H 2 O 2 concentration was calculated and expressed in nmoles g −1 of fresh weight (FW) of plant material (roots, stems, leaves).

Statistical Analysis
The obtained results are presented as the mean with standard error values for growth (4-50 replicates) and biochemical (three random replicates) parameters. To determine differences among treatments, parametric ANOVA with Tukey post hoc comparisons (p < 0.05) was performed. The statistical analyses were performed using the Statistica 10.0 software package, StatSoft 2006. Evaluation of the effect of inoculation (P. stutzeri ISE12 and K. marisflavi CSE9) on the growth and biochemical parameters of B. vulgaris was performed by multivariate statistical analysis-a principal component analysis (PCA) (Canoco 5.0) [38].

Salinity Tolerance of Bacterial Strains
Analysis of the tolerance to salinity revealed significantly higher growth of P. stutzeri ISE12 in comparison to K. marisflavi CSE9 in all studied variants of our experiment (Table S1). The highest growth of P. stutzeri ISE12 was observed in salinity corresponding to 150-600 mM of NaCl. Significant growth inhibition of P. stutzeri ISE12 was observed in media containing 800 mM NaCl. Optimal conditions for K. marisflavi CSE9 growth were observed in the range of 150-800 mM NaCl, while the lack of NaCl in the medium significantly inhibited bacterial growth (Table S1). K. marisflavi CSE9 was characterized by Agronomy 2020, 10, 1571 6 of 17 higher salt stress tolerance than P. stutzeri ISE12 based on an analysis comparing bacterial growth in NaCl supplemented media with the growth of bacteria in a control media (without NaCl) expressed as percentage growth inhibition or stimulation ( Figure 2). Agronomy 2020, 10, x FOR PEER REVIEW 6 of 16 Figure 2. Growth of P. stutzeri ISE12 and K. marisflavi CSE9 in media supplemented with different NaCl concentrations (50-800 mM) was expressed as a percentage increase/decrease compared to bacterial growth in media without salt.

Inoculation Effect on Germination and Growth Parameters
The positive influence of the two selected PGPEs (P. stutzeri ISE12 and K. marisflavi CSE9) on the growth of B. vulgaris manifested in improvement of seeds germination rate, growth and biochemical parameters (Figures 3 and 4, Tables S2 and S4) in the absence and presence of salt. Alleviation of the negative effects of salt stress on the plant was observed in all investigated growth parameters, including germination percentage and index, chlorophyll content, number of leaves, length of roots, stem, fresh and dry weight, in comparison to their noninoculated variants (control) ( Figure 3, Table  S3). The K. marisflavi CSE9 strain was more efficient in promoting overall plant growth and development than P. stutzeri ISE12 (Figure 3). K. marisflavi CSE9 inoculation significantly increased the seed germination rate and germination index at all salt concentrations compared to the noninoculated controls and P. stutzeri ISE12 inoculations. Plants inoculated with K. marisflavi CSE9 showed significantly enhanced growth parameters at all the tested salinity levels (with the exception of chlorophyll level at 150 mM NaCl and stem length at 0, 50 and 300 mM NaCl) compared to the noninoculated variants ( Figure 3). The obtained values were several times higher for the roots length (4.3-11.2), fresh (5.3-7.6) and dry weight (5.5-11.5) of K. marisflavi CSE9-inoculated plants. (Figure 3).
Inoculation with the P. stutzeri ISE12 strain significantly stimulated B. vulgaris root length and fresh weight in all variants of salinity, while the increase in the value of other parameters was related only to selected NaCl concentrations. In general, the positive effects of P. stutzeri ISE12 treatment were observed (with the exception of stem length) on the growth parameters of plants growing in the

Inoculation Effect on Germination and Growth Parameters
The positive influence of the two selected PGPEs (P. stutzeri ISE12 and K. marisflavi CSE9) on the growth of B. vulgaris manifested in improvement of seeds germination rate, growth and biochemical parameters (Figures 3 and 4, Tables S2 and S4) in the absence and presence of salt. Alleviation of the negative effects of salt stress on the plant was observed in all investigated growth parameters, including germination percentage and index, chlorophyll content, number of leaves, length of roots, stem, fresh and dry weight, in comparison to their noninoculated variants (control) (Figure 3, Table S3). The K. marisflavi CSE9 strain was more efficient in promoting overall plant growth and development than P. stutzeri ISE12 (Figure 3). K. marisflavi CSE9 inoculation significantly increased the seed germination rate and germination index at all salt concentrations compared to the noninoculated controls and P. stutzeri ISE12 inoculations. Plants inoculated with K. marisflavi CSE9 showed significantly enhanced growth parameters at all the tested salinity levels (with the exception of chlorophyll level at 150 mM NaCl and stem length at 0, 50 and 300 mM NaCl) compared to the noninoculated variants ( Figure 3). The obtained values were several times higher for the roots length (4.3-11.2), fresh (5.3-7.6) and dry weight (5.5-11.5) of K. marisflavi CSE9-inoculated plants. (Figure 3).
with P. stutzeri ISE12 and K. marisflavi CSE9) (Figure 3). In contrast, an increase in the chlorophyll content in B. vulgaris leaves (plants inoculated with P. stutzeri ISE-12 and control variant) and the number of leaves (in both inoculated variants) was correlated with increasing NaCl concentration in the growth substrate ( Figure 3, Table S3). However, no effect of salt stress was observed with respect to dry weight and root length (with the exception of the variant inoculated with K. marisflavi CSE9). The influence of salinity on fresh weight was dependent on the variants of the experiment (noninoculated-decrease in value related to the increase in the salinity level; variant inoculated with P. stutzeri ISE12-no effect; variant inoculated with K. marisflavi CSE9-the highest weight in 150 mM NaCl; Figure 3, Table S3).

Figure 3.
Growth parameters (germination percentage and index, number of leaves, chlorophyll content, length of roots and stems, fresh and dry weight) of B. vulgaris inoculated with P. stutzeri ISE-12 and K. marisflavi CSE-9 cultivated in different NaCl concentrations (0, 50, 150 and 300 mM NaCl). Significant differences (based on p < 0.05, df, MS and p value, one-way ANOVA with Tukey post hoc comparisons) between variants of experiment (noninoculated control; inoculated with P. stutzeri ISE-12 and K. marisflavi CSE9) in each NaCl concentration are denoted by different marks * and **, while . Growth parameters (germination percentage and index, number of leaves, chlorophyll content, length of roots and stems, fresh and dry weight) of B. vulgaris inoculated with P. stutzeri ISE-12 and K. marisflavi CSE-9 cultivated in different NaCl concentrations (0, 50, 150 and 300 mM NaCl). Significant differences (based on p < 0.05, df, MS and p value, one-way ANOVA with Tukey post hoc comparisons) between variants of experiment (noninoculated control; inoculated with P. stutzeri ISE-12 and K. marisflavi CSE9) in each NaCl concentration are denoted by different marks * and **, while the effect of salinity for each variant is marked with different letters (including gray font for control variant, red font in the case of P. stutzeri ISE-12 treated variant and blue font for K. marisflavi CSE-9 inoculated plants). Mean values ± standard error are presented (n = 4-55, depending on the tested parameters). vulgaris plants: root and leaves > stems) (Figure 4).
B. vulgaris plants exposed to saline conditions exhibited an increase in proline (except for the variant inoculated with P. stutzeri ISE12-roots, and noninoculated variants-leaves) and hydrogen peroxide (with the exception of noninoculated variant and inoculated with K. marisflavi CSE9-stems) concentrations in the investigated organs associated with increasing substrate salinity (Figure 4, Table S5).  Inoculation with the P. stutzeri ISE12 strain significantly stimulated B. vulgaris root length and fresh weight in all variants of salinity, while the increase in the value of other parameters was related only to selected NaCl concentrations. In general, the positive effects of P. stutzeri ISE12 treatment were observed (with the exception of stem length) on the growth parameters of plants growing in the highest NaCl concentration (300 mM) (Figure 3).
Substrate salinity led to a decrease in seed germination percentage and index, as well as the length of B. vulgaris stems for all the tested variants (control plants-noninoculated, inoculated plants with P. stutzeri ISE12 and K. marisflavi CSE9) (Figure 3). In contrast, an increase in the chlorophyll content in B. vulgaris leaves (plants inoculated with P. stutzeri ISE-12 and control variant) and the number of leaves (in both inoculated variants) was correlated with increasing NaCl concentration in the growth substrate ( Figure 3, Table S3). However, no effect of salt stress was observed with respect to dry weight and root length (with the exception of the variant inoculated with K. marisflavi CSE9). The influence of salinity on fresh weight was dependent on the variants of the experiment (noninoculated-decrease in value related to the increase in the salinity level; variant inoculated with P. stutzeri ISE12-no effect; variant inoculated with K. marisflavi CSE9-the highest weight in 150 mM NaCl; Figure 3, Table S3).

Inoculation Effect on Biochemical Parameters
The influence of PGPE inoculation was tested for biochemical parameters, such as proline and hydrogen peroxide levels. The results showed that PGPE inoculation in B. vulgaris reduced the levels of proline (in roots and stems) and hydrogen peroxide (in roots and leaves) compared to the control variants ( Figure 4, Table S4). However, the proline content in B. vulgaris leaves differed among the experimental variants. In the case of K. marisflavi CSE-9 inoculated plants, the lowest proline concentrations were observed in those growing in the substrate without NaCl (0 mM) and that with 50 mM NaCl. In contrast, in the noninoculated plants, the lowest proline levels were detected at higher NaCl concentrations (150 and 300 mM) (Figure 4, Table S5). The hydrogen peroxide content in stems of B. vulgaris inoculated with P. stutzeri ISE12 in saline substrates (50300-mM NaCl) was higher than in other variants (control, inoculation with K. marisflavi CSE-9) (Figure 4, Table S4). In general, the levels of proline and hydrogen peroxide in plant organs decreased in the following order: stems > roots > leaves (with the exception of the content of hydrogen peroxide in noninoculated B. vulgaris plants: root and leaves > stems) (Figure 4).
B. vulgaris plants exposed to saline conditions exhibited an increase in proline (except for the variant inoculated with P. stutzeri ISE12-roots, and noninoculated variants-leaves) and hydrogen peroxide (with the exception of noninoculated variant and inoculated with K. marisflavi CSE9-stems) concentrations in the investigated organs associated with increasing substrate salinity ( Figure 4, Table S5).

Comparison of the Strain Effects
The principal component analysis (PCA) combined the results of the investigated growth and biochemical parameters obtained for all three variants of the experiment (noninoculated variants, inoculated variants with P. stutzeri ISE12 and K. marisflavi CSE9) revealed visible differences between the control variant (noninoculated) and the other two ( Figure 5). The PC1 ordination axis (56.72% variation explained) separated the inoculated and noninoculated variants of B. vulgaris. The PC2 axis (24.01%) separated the salinity variant of each treatment. There was a positive correlation between the length of the stem, the germination percentage and index and the inoculated variants cultivated in 0 and 50 mM NaCl (Ps-0, Ps-50, Km-0, Km-50). The dry and fresh weight, root length, number of leaves and chlorophyll level in leaves are associated with the K. marisflavi CSE9 variants at high salinity (150 and 300 mM NaCl) ( Figure 5). Both inoculated variants at high salinity (Km-300, Ps-150, Ps-300) demonstrated a positive correlation with the level of hydrogen peroxide in stems and the level of proline in leaves ( Figure 5). Control variants of B. vulgaris grown at lower salinity (0 and 50 mM) were negatively correlated with the level of chlorophyll and hydrogen peroxide in stems, root length and number of leaves, while variants cultivated at 150 and 300 mM NaCl (C-150 and C-300) were positively associated with the proline levels in all investigated plant organs and hydrogen peroxide in roots and leaves ( Figure 5). The PC1 axis clearly demonstrates the effect of inoculation on the B. vulgaris response. The control variant characterized by the lowest growth and the highest proline and hydrogen peroxide accumulation is opposite to the K. marisflavi CSE9 variants in the ordination diagram. P. stutzeri ISE12 variants occupy the intermediate position ( Figure 5). length and number of leaves, while variants cultivated at 150 and 300 mM NaCl (C-150 and C-300) were positively associated with the proline levels in all investigated plant organs and hydrogen peroxide in roots and leaves ( Figure 5). The PC1 axis clearly demonstrates the effect of inoculation on the B. vulgaris response. The control variant characterized by the lowest growth and the highest proline and hydrogen peroxide accumulation is opposite to the K. marisflavi CSE9 variants in the ordination diagram. P. stutzeri ISE12 variants occupy the intermediate position ( Figure 5).

Effect of Salinity on the Growth and Biochemical Parameters of B. vulgaris
The detrimental impact of salinity on plants manifests in osmotic, ionic and oxidative stress which, in turn, cause several changes in metabolic and physiological processes [39]. A negative influence of salinity on several growth and biochemical parameters of B. vulgaris was observed in our study. We did not observe adverse effects of salt stress in the case of chlorophyll content, number of leaves, length of roots, or dry weight, which was surprising due to the widely known unfavorable effect of salinity on plant growth [40][41][42][43][44]. However, B. vulgaris exhibits high innate tolerance to salinity, which probably results from genetic similarity to its halophytic ancestor, the sea beet [45,46]. The most detrimental impact of salinity was observed in the case of germination percentage and index. Germination is a crucial point in seedling formation, determining the health and vigor of a plant [47]. A decrease in germination was observed in the case of different B. vulgaris genotypes, which is in line with our results [13,48]. In addition to the inhibition of B. vulgaris seed germination, salinity disrupted the growth of stems. The highest concentration of NaCl (300 mM) significantly decreased the length of stems. Shoot growth reduction is one of the known mechanisms of plant response to salt stress and is probably connected with hormonal signals generated by the roots [49]. It might be expected that the roots that are directly exposed to salt will be more vulnerable, but they are characterized by a surprisingly robust and relatively good ionic status [49]. The higher sensitivity of stems to salinity compared to roots was confirmed in several reports [28,[49][50][51].
Good tolerance of B. vulgaris to salt stress was confirmed by our results. The increase in proline level associated with an increase in substrate salinity was observed primarily in the case of stems and roots regardless of the variant. According to Wu et al. salinity caused a more significant increase in the level of proline in the shoots than in the roots of three sugar beet cultivars [52]. The accumulation of compatible solutes, including proline, is a very important defense mechanism against salinity stress and is responsible for intracellular osmotic equilibrium maintaining [52,53]. An increase in proline levels in plants growing under salt stress was observed in different plant species, including canola (Brassica napus), dill (Anethum graveolens), mulberry (Morus alba cv Khonpai), pistachio (Pistacia vera L.) and Jerusalem artichoke (Helianthus tuberosus) [28,39,[54][55][56][57]. The accumulation of proline in plants growing under salt stress is associated with the low activity of antioxidant enzymes [58], which is accompanied by an increase in the level of ROS [21,59]. The increase in hydrogen peroxide concentration in response to salt stress was observed with other plant species, e.g., mung bean (Vigna radiata), rice (Oryza sativa), and mulberry (Morus sp.) [54,60,61]. The results of our study indicated a positive correlation between the level of hydrogen peroxide in roots, stems and leaves of B. vulgaris and NaCl concentration. The rise of hydrogen peroxide is usually associated with impairment of cell homeostasis, but H 2 O 2 also acts as a central player in stress signal transduction pathways responsible for the activation of multiple acclimatory responses [59,62]. The studied red beet was able to maintain a balance between H 2 O 2 production and scavenging in a less saline environment (50 mM). A significant effect of salinity could be observed only at higher NaCl concentrations (150 and 300 mM). These observations demonstrate the high tolerance of B. vulgaris to salt stress.

Effect of K. marisflavi CSE9 and P. stutzeri ISE12 Bioaugmentation on the Growth and Biochemical Parameters of B. vulgaris
Inoculation of plants with PGPE is a notably cost-effective and ecologically sensitive technique that is helpful in salt stress alleviation and the improvement of crop yield [63,64]. Bioaugmentation of B. vulgaris with P. stutzeri ISE12 and K. marisflavi CSE9, which was performed in this study, enhanced all studied growth parameters regardless of NaCl concentration. Furthermore, bioaugmentation with selected PGPEs caused significant enhancement of plant root length and fresh weight in the presence of NaCl compared to noninoculated plants. It was shown that salinity causes a reduction in endogenous levels of phytohormones, including auxins, gibberellins, jasmonic acid and salicylic acid, some of which play crucial roles in the growth of roots [65]. A strong correlation between a decline in phytohormones level and reduction in plant root length was noted by Egamberdieva [66,67]. There are also reports on the beneficial influence of exogenous phytohormones application on root system enlargement in plants growing under salt stress [47,68]. IAA-synthesizing bacteria are preferred in promoting plant growth in saline conditions. For example, bacterial IAA plays a major role in salt stress tolerance and stimulates resistance against fungal pathogens of cotton [65]. Briefly, the application of IAA to plants undergoing salt stress stimulates their growth, and cell enlargement improves protein content, promotes stem and hypocotyl elongation, increases height and dry weight, and promotes the development of the root system and seedling fresh and dry weight [47,69,70]. As the tested bacterial strains belong to genera able to produce IAA, we suggest, that they may contribute to salt stress alleviation in B. vulgaris by IAA level modification.
The inoculation of B. vulgaris with P. stutzeri ISE12 and K. marisflavi CSE9 affected proline levels in roots and stems and, to a lesser degree, in leaves, which was probably observed because leaves have the greatest distance from the stressful factor. Decreased in proline content in PGPB-inoculated and salt-stressed plants, indicate that the synthesis of osmotically active substances was not enhanced despite the presence of the salt. This finding has been reported by many scientists and was connected with the alleviation of salt stress [26,71]. We propose that the PGPE strains used by us activate a mechanism responsible for salt stress alleviation that is different from the stimulation of proline synthesis.
Proline synthesis is the reaction to the osmotic component of the salt stress. Since the increase in proline content is prevented by bacterial inoculation, it may be suggested that bacteria mitigate the osmotic stress. Consequently, the osmotic adjustment is not required since the synthesis of osmoprotective substances (including proline) is restricted, the energetic cost of acclimation to salinity is reduced. Saved resources may be redirected support processes of growth and development. Resolving the underlying mechanism will require further experiments. Osmotic stress is triggered by the salt present in the soil and it results in decrease in water uptake by roots. Therefore, it is plausible that the bacteria-dependent, osmotic stress alleviation is due to some mechanism(s) facilitating water uptake from the salinized soil. Studying water relation in plants will be required to address this issue. The increase in proline content and decrease in H 2 O 2 in chickpea plants bioaugmentated with Mesorhizobium ciceri and Bacillus subtilis were related to the adaptation of their saline stress mechanisms [72]. It is widely known that salt stress stimulates plant ROS production and that PGPE can reduce their synthesis and indirectly protect membrane lipids from peroxidation [72]. The positive effects of bioaugmentation with different PGPB species, e.g., Bacillus subtilis and Pseudomonas frederiksbergensis OS261, on H 2 O 2 decreased under salt stress conditions, which was observed in different plants, such as chickpea and caliph media [3,73]. In our study, B. vulgaris inoculated with P. stutzeri ISE12 or K. marisflavi CSE9 were characterized by lower levels of hydrogen peroxide in roots and leaves under both growth conditions (nonsaline and saline: 50, 150, and 300 mM NaCl). Interestingly, our results are consistent regarding the beneficial effects of the bioaugmentation of plants with selected endophytes, while contradicting results were obtained for the levels of H 2 O 2 in stems. We assume that the higher concentration of H 2 O 2 in the stems of inoculated plants compared to the control variant may be associated with a more advanced lignification process, but further studies are warranted to investigate this possibility [74].
Selected endophytes strongly stimulated B. vulgaris growth, leading to better overall conditions, but the effect was strain-specific. The positive effect of P. stutzeri ISE12 had already been demonstrated in the case of rape and beet growing under high-salinity conditions [13,28]. In general, there are many scientific reports indicating the beneficial influence of Pseudomonas species on plant growth under salt stress conditions [73,[75][76][77][78]; however, there are no studies considering the effect of Kushneria sp. As revealed in our earlier studies, P. stutzeri ISE12 possesses more plant growth-promoting properties (e.g., N 2 fixation, synthesis of siderophores and IAA, and cellulolytic activity) than K. marisflavi CSE9, for which only selected activities were noted (e.g., synthesis of IAA and siderophores) and at a considerably lower level. However, a stronger beneficial influence on the growth and biochemical parameters was observed for plants inoculated with K. marisflavi CSE9 than P. stutzeri ISE12. This may be explained by the fact that K. marisflavi occurs inside halophytes, where it probably plays an important role in plant adaptation to the saline environment [31,79,80]. This halotolerant strain likely evolved a wider range of adaptation mechanisms for survival in salt-affected environments and heavily synthesizes compounds that are important in promoting plant growth under abiotic stress conditions. It is possible that K. marisflavi CSE9 has other metabolic traits that we did not study. For example, inorganic phosphorus solubilization capacity was observed in the case of halotolerant Kushneria sp. YCWA18 isolated from the sediment of the Daqiao saltern (China) [81]. It is also possible that K. marisflavi CSE9 forms a stronger symbiotic interaction with B. vulgaris. In summary, we showed that red beet bioaugmentation with halotolerant endophytic bacteria K. marisflavi or P. stutzeri alleviates salt stress and improves biochemical and growth parameters.

Conclusions
Salinity negatively influences B. vulgaris growth and development, which is confirmed through our results on plant growth parameters and biochemical analysis. Nevertheless, this plant species has a high tolerance to salt stress, as can be observed in the various investigated parameters (e.g., stem length, germination percentage and index, and proline content), and the strongest effects of salinity on the plant were seen at the highest NaCl concentration (300 mM) in the substrate.
Bioaugmentation of B. vulgaris with both selected PGPE strains mitigated the adverse effects of salinity, leading to increased plant growth and a decrease in proline and hydrogen peroxide concentrations in plant organs. The present study demonstrated that K. marisflavi CSE9, characterized by higher salinity tolerance, is more effective in reducing salt stress in B. vulgaris than is the less salt-tolerant endophyte P. stutzeri ISE12. This study is the first to consider the role of K. marisflavi in plant growth promotion and development, as well as in alleviating salt stress in plants. These results indicate that this strain represents a very valuable resource for future research attempting to identify alternative methods for sustainable crop production.