Exploring the Interactive Mechanisms of Halophilic Bacterium SPSB2 and Mannitol in Mitigating Sodium Chloride and Arsenic Stress in Tomato Plants

Exploring the Interactive Mechanisms of Halophilic Bacterium SPSB2 and Mannitol in Mitigating Sodium Chloride and Arsenic Stress in Tomato Plants | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Exploring the Interactive Mechanisms of Halophilic Bacterium SPSB2 and Mannitol in Mitigating Sodium Chloride and Arsenic Stress in Tomato Plants Lubna #, Muhammad Aizaz, Shima Ahmed Ali Alrumaidhi, Rawan Ahmed Mohammed Alhinai, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4798297/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Agricultural productivity is adversely affected by soil salinization and contamination with heavy metals, emphasizing the necessity for environmentally friendly technologies. This study investigates the impact of sodium chloride (NaCl) and arsenic (As) stress on tomato seedlings and explores the stress-alleviating effects of mannitol and a halophilic bacterium, Nitratrieducator aquimarinus SPSB2. Our results revealed that bacteria strainSPSB2 establishes a symbiotic relationship with tomato plants, which modulates the secondary metabolites and antioxidant system in tomato plants exposed to both NaCl and As stress. Under the NaCl and As stress tomato seedling growth was significantly reduced, although this reduction was mitigated by bacteria strain SPSB2 and mannitol treatment. When exposed to NaCl stress, the bacterial strain enhances shoot and root length by 84.8% and 152.5%, respectively. Similarly, under the As stress conditions, bacteria strain SPSB2 inoculation increased the shoot and root weights by 63.1% and 45.5%, respectively. Bacteria strain SPSB2 inoculation also significantly enhanced the chlorophyll a, b, and carotenoid contents by 76.3%, 78%, and 50%, respectively, compared to their non-inoculated counterparts under As stress conditions. Furthermore, during NaCl and As stress conditions, treatments with SPSB2 and mannitol increase the levels of enzymatic components (catalase, polyphenol oxidases) and non-enzymatic components (flavonol protein, sugar, starch), indicating a stress-alleviating effect of bacteria strain SPSB2 and mannitol. In the current study, the bacteria strain SPSB2 was more effective than mannitol in improving tomato plants' salinity and heavy metal tolerance regarding growth and physiological attributes. The symbiotic relationship between SPSB2 and tomato plants positively impacted various parameters, including plant growth, chlorophyll content, and antioxidant system activity. Moreover, the study suggests that SPSB2 is more effective than mannitol in improving tomato plants' salinity and heavy metal tolerance. These findings contribute to the understanding of environmentally friendly strategies for managing soil salinization and heavy metal contamination in agriculture, and the potential use of SPSB2 in microbial-assisted phytoremediation of polluted saline soils. halotolerant bacteria tomato salt and heavy metal stress mannitol Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Threats to agricultural sustainability in the 21st century arise from the expanding human population and decreasing agricultural land. Agriculture's problems are worsened by the worldwide water shortage, environmental contamination, and the increasing soil and water salinity (Shahbaz and Ashraf 2013 ). Salinity stress negatively impacts seed germination, plant vigor, and agricultural productivity (Chakma et al. 2019 ). There are two ways in which soil salts might prevent plants from flourishing. First, salt in soil solutions reduces plants' ability to absorb water, hindering their growth and development. Second, suppose too much salt enters the plant via the transpiration stream. In that case, it may cause damage to the cells and the transpiring leaves, leading to a further slowing of development. This effect is described as the ion-excess or salt-specific impact of salinity (Greenway and Munns 1980 ; Patil et al. 2016 ). In addition to salt stress, heavy metals like As, Ni, Cd, Fe, Mn, Cu, Co, Zn, and Hg have been accumulating in soils as a result of the disposal of industrial waste. However, many metals are essential micronutrients needed in trace amounts for numerous biological processes. Still, an excess of these metals adversely affects senescence, metabolism, physiology, and plant growth and development (Ghori et al. 2019 ). Tomatoes are the most popular and economically significant fruits and vegetables worldwide. The demand for tomatoes has increased significantly worldwide in recent years due to their wide range of uses in raw, cooked, and processed foods (Chaudhary et al. 2019 ). Being a temperate crop, tomatoes are grown in various climate zones, making their production more difficult, and abiotic stressors, including drought, salt, and heat, significantly negatively impact agricultural productivity and output (Rao et al. 2016 ). Many unconventional tomato-producing sites have embraced greenhouse-based agriculture to guarantee a consistent year-round supply. Therefore, improving tomato cultivars' stress tolerance is more financially viable and sustainable. The principal problems limiting tomato production include extreme water and temperature regimes, nutritional imbalances, various abiotic stresses, elemental toxicity, and high salt in the soil substrate (Conti et al. 2023 ). Abiotic stresses become more complex in agricultural settings due to the frequent coexistence of many types of stresses. To feed the world's population, we need to breed crops that can withstand and recover from a wide range of abiotic stresses while still producing high yields (Patil et al. 2016 ). Population growth is outpacing the increase in demand for agricultural products and food security (Pawlak and Kołodziejczak 2020 ). Farming methods have changed in recent decades to consider this and respond to this global problem. Applying chemical fertilizers and agrochemicals can improve food production by boosting crop yield, enhancing quality, and prolonging shelf life. However, these actions are costly in terms of ecosystem destruction, global warming, soil erosion, and biodiversity loss (Fasusi et al. 2021 ; Mitter et al. 2021 ). For decades, soil health has steadily declined, primarily due to the careless use of agrochemicals (El-Ghamry et al. 2018 ; Guha et al. 2020 ). Microbes, with their eco-friendly, cost-effective, and protective attributes against various biotic and abiotic pressures, may offer a viable alternative approach to overcoming constraints on agricultural output (Zaidi et al. 2014 ). Halotolerant plant growth-promoting rhizobacteria (HT-PGPR) are a varied group of saltwater soil microorganisms that help plants survive in salt habitats and boost their development and other soil-related properties (Hidri et al. 2022 ). The role of HT-PGPR in mitigating the adverse effects of salt stress on crops has become prominent in recent years. Through the production of exopolysaccharides, siderophores, volatile organic compounds (VOCs), suitable osmolytes, and phytohormones, HT-PGPR boosts the productivity of the saline-agroecosystem (Bhat et al. 2020 ) directly by preventing the impacts of the phytopathogens, or indirectly by controlling the expression of stress-related genes (Prasad et al. 2019 ). Rhizobium , Arthrobacter, Flavobacterium, Alcaligenes, Pseudomonas , and Azospirillum are only a few halotolerant bacteria reported to increase crop salinity tolerance (Saghafi et al. 2019 ). When assessing halobacteria for their potential to promote plant development and shield certain crops, toxicity to heavy metals has also been considered. In fact, (Masmoudi et al. 2019 ) found that B. velenzensis and B. subtilis were effective in boosting the development of tomato plants ( S. lycopersicum ) subjected to salt stress and heavy metal contamination (Co, Ni, Cu, and Cr). Endophytic and rhizospheric bacteria may decrease metal toxicity in plants via their metal resistance mechanism and boost plant development under metal stress; hence, phytoremediation with bacterial strains has recently been highly recommended for cleaning up metal-polluted soils(Asaf et al. 2023 ). Because they are affordable, eco-friendly, and capable of protecting plants from biotic pressures, microorganisms offer a viable alternative approach to overcoming the constraints on the agricultural output caused by abiotic stress and for improved plant health and protection (Zaidi et al. 2014 ). Moreover, the most effective method for removing hazardous metals. This approach is favored due to its natural, eco-friendly nature, cost-effectiveness, and widespread acceptance as a technology for metal removal. Utilizing bacteria for the bioremediation of heavy metal-contaminated soil to encourage plant growth is one such method (Adesemoye et al. 2009 ; Chamkhi et al. 2021 ; Shaffique et al. 2022 ) . In the current study, we proposed that the SPSB2 halophilic bacterial strain has the potential to serve as an effective mitigator of salt and heavy metal stress in tomato plants. The purpose of the present studyis that bacteria strains may positively influence plant growth, modulate the antioxidative system, and stimulate the production of phytohormones. This contribution could advance the development of an eco-friendly phytoremediation strategy for efficiently addressing salt and heavy metal stress while promoting the growth of tomato plants. We investigated the in vitro and in vivo plant growth-promoting abilities of the Nitratrieducator aquimarinus SPSB2 strain in a greenhouse experiment, subjecting tomato plants to both control and high NaCl and As stress conditions. Materials and Methods Isolation of halophilic bacteria Halophilic bacteria were isolated from Muscat, Oman, as described in recent studies. Sediment weighing 10 gm was mixed into 90 mL of 5% or 15% NaCl (w/v) sterile brine and incubated at 37°C for an hour while being shaken at 200 revolutions per minute. To isolate bacteria, aliquots of the resultant slurry (0.1 mL) were distributed onto Petri plates and serially diluted with sterilized NaCl brine (5% or 15%, w/v). Colony counts were triple checked by plating three copies of each medium on separate plates. NaCl (5 or 15% w/v) and nystatin (50 mg/L) were added to all agar plates to prevent the development of non-bacterial fungi. One to six days were spent in a 37°C incubator with the Petri plates. Size and color were used to select colonies for further purification on inorganic salt-starch agar or TSA with 5% or 15% (w/v) NaCl. Long-term preservation of the purified colonies was accomplished by suspending them in glycerol (20% v/v) and stored at -20°C. Screening bacterial strains for IAA production The examination of bacterial strains for their IAA synthesis using the colorimetric method (Asif et al. 2023 ). The bacterial strains were cultured in nutrient broth at 30°C of bacterial suspension. After seven days, the samples were filtered, and the IAA concentrations were determined by adding 1 mL of Salkowski reagent to 2 mL of each culture filtrate, followed by 30 minutes of incubation in the dark. The optical density was determined at 530 nm using a UV spectrophotometer. IAA production was calculated using a standard curve based on indole acetic acid (Gordon and Weber 1951 ). Phosphate solubilizations activity The quantitative assessment of phosphate (P) solubilizations was performed using the vanado-molybdat phosphoric method (Murphy and Riley 1962 ). we autoclaved a 50 mL volume of NBRIP broth medium in a 250 mL flask and pH-adjusted to 7.0 and after that, a new 200 µL inoculum was added, and the flask was shaken for seven days at 28°C and 120 rpm. An autoclaved and uninoculated NBRIP broth medium was used as a control. After the incubation period ended, 2 mL of the culture was centrifuged at 10,000 rpm for 15 minutes, and the molybdenum blue colorimetric technique was used to determine the amount of soluble Phosphate present. A UV-VIS spectrophotometer was used to determine the optical density at 430 nm. Triplicates of all conditions were carried out throughout the experiment. Siderophore production The Chrome-Azurol S (CAS) medium and the Universal Chemical Assay were used to evaluate the siderophore synthesis of all of the bacterial strains (Senthilkumar et al. 2021 ). Four independent tests were performed to ensure reliable findings. In a nutshell, bacterial strains were cultured into CAS plates for four days at 28°C. An orange ring around the colonies indicated were production of siderophore. Bacterial identification and phylogenetic analysis The isolated bacterial culture was hydrolyzed using enzymes to isolate their genomic DNA as prescribed by (Saito and Miura 1963 ). To amplify the whole 1.4–1.5 kb 16S rRNA gene, universal bacterial primers 27 F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1541R (5′-AAG GAG GTG ATC CAG CCG CA-3′) were used in a PCR reaction. To amplify the 16S rRNA region, a DNA engine gradient thermal cycler was used (BIO-RAD, USA). The 50 µL PCR reaction mixture comprised 4 µL of 2.5 U/µLTaq DNA polymerase (Tiangen, Beijing), 5 µL of 10× buffer (Tiangen), 1 µL of 20 mM dNTPs (Tiangen), 37 µL of SDW, 1 µL of 50 µM per primer, and 1 µL of the template was prepared. Initial denaturation was performed at 94°C for 4 minutes, followed by 30 amplification cycles at 94°C for 1 minute, 56°C for 1 minute, and 72°C for 2 minutes, and finally elongation was performed at 72°C for 10 minutes. The amplified region was sequenced by Macrojen company, South Korea. The GenBank databases ( http://www.ncbi.nlm.nih.gov/genbank/ ) were searched for matching data to compare the sequences. The MEGA-11 software (Tamura et al. 2013 ) with Clustal-W alignment and the neighbor-joining (NJ) technique was used to construct the phylogenetic tree of the SPSB2 bacterial strain with other downloaded matching bacterial sequences. Each node in the phylogenetic trees was statistically supported using 1,000 bootstrap replications. Seed sterilization and germination The experiments were conducted within the confines of the Biotechnology and OMICs laboratory's greenhouse at the University of Nizwa Oman. High yielding of tomato seeds cultivar ( S. lycopersicum cv.Yegwang ) was acquired from Kyungpook National University, South Korea and assessed for viability before use. HgCl 2 solution was used for surface seed sterilization for one minute, followed by a double wash with 70% ethanol and two additional washes in ddH 2 O. The seeds were transferred to autoclaved filter papers and placed in Petri dishes, and 3 mL of distilled water was added. After five days, germinated seeds were carefully transferred to 10 × 9-cm plastic pots filled with horticulture substrate containing coco peat moss (10–15%), coco peat (45–50%), perlite (35–40), NH+ (ca.0.09 mg/g), KO (c.0.1 mg/g), zeolite (6–8%) with NO3 (ca. 0.205 mg/g), and PO (ca.0.35 mg/g) autoclaved soil. The pots were placed in a growth chamber and maintained environmental conditions with day and nighttime temperatures range and relative humidity between 60% and 70%. Experimental design In this experiment, we used different stress conditions on tomato plants: (a) control group, treated with dH 2 0, (b) Inoculated plants with SPSB2 bacterial strain, (c) Plant treated with 100 mM mannitol, (d) Plant treated with 400 mM NaCl, (e) Plant treated with 0.5 mM As, (f) Plant treated with NaCl + SPSB2, (g) Plant treated with NaCl + mannitol, (h) Plant treated with NaCl + bacteria + mannitol, (i) As treated plants, (j) Plant treated with As + SPSB2, (k) Plant treated with As + mannitol, (l) Plant treated with As + SPSB2 + mannitol. As a result, eleven distinct treatments were administered, each with five replicates, requiring a total of 35 pots for implementation. Determination of plant biomasses and relative water contents (RWC) Plants were harvested after 60 days and various growth parameters such as root and shoot length, and fresh weight of root and shoot were measured. The collected samples were quickly placed into liquid nitrogen and preserved at -80°C for future analysis. RWC of the leaves was assessed using a previously established method (Silveira et al. 2003 ). Fresh leaves from the plants were immersed in distilled water for 4 hours to note their turgid weight. Subsequently, the leaf samples were subjected to a 24-hour drying process in an oven maintained at a temperature of 80°C to acquire their dry mass. The following formula was used to calculate the RWC. RWC (%) =(FW − DW)/(TW − DW) × 100 Determination of stomatal density and size With slight modifications, we used the nail polish and stick tape method as described by (Silveira et al. 2003 ), to determine the stomatal density. Three mature fresh tomato leaves from each experimental plant were carefully selected and removed from the stem. A thin layer of nail polish was spread on the lower leaf surface. When the nail polish was dried, a strip of clear stick tape was placed over the leaf area, and the stick tape was immediately pressed down to contact it. Carefully removed, the tape was fixed to the microscopic slide and then the stomatal density and structure were examined under a compound microscope. Determination of chlorophyll contents Fresh tomato plant leaves were ground in liquid nitrogen, and a 200 mg sample was mixed with 1 mL of 80% acetone in a 1.5 mL test tube. After a brief vertexing, the tube was centrifuged at 1,000 rpm for 5 minutes. Subsequently, 150 µL of the supernatant was transferred into a 96-well microplate. Data was recorded using a spectrometer for chlorophyll a, b, and carotenoids at 663 nm, 645 nm, and 470 nm, respectively. Photosynthetic pigment concentrations were calculated using the extension coefficients and equation given in Barnes’s method (Barnes et al. 1992 ) with slight modifications. Determination of protein and catalase contents The method described by (Bradford, M. M., 1976) was used for protein content estimation. Fresh leaves (200mg) were crushed into fine powder in liquid nitrogen and added with 1mL of extracted buffer containing (50 mM Tris HCl (pH 7.0), 10%, glycerol, 3mM, MgCl 2 , 1mM EDTA, and 1% pvp) and mixed gently. The homogenate samples were centrifuged at 4,000 rpm for 10 minutes at 2°C. Transfer 240 µL supernatant in a new tube and mixed with an equal volume of phosphate buffer of 0.1 mM (pH 7.0) and 120 µL of 0.2 M H 2 0 2 , and the optical density (OD) was measured at 240 nm using spectrophotometry. Determination of sugar, starch, and flavonols Sugar and starch contents were analyzed using the ethanol method. A 0.5 g grained tomato plant sample was taken, added with 1mL 80% ethanol, and kept at room temperature for 24 hours. The ethanolic solution was centrifuged at 3,000 rpm for 10 minutes. After centrifuging the solution, sugar and starch content were measured at 490 nm and 630 nm by spectrophotometer. The method described by the previous research (Levon and Klymenko 2021 )was used to extract and quantify total flavonol contents. Fresh leaf samples were meticulously ground using a mortar and pestle with liquid nitrogen. Subsequently, 0.5 g of the resulting powder was added with 1 mL of 80% methanol, and this mixture was left at room temperature for 24 hours. Following this incubation period, the mixture underwent centrifugation for 15 minutes at a speed of 10,000 rpm. The resulting supernatant was then collected and combined with an equal volume of 2% AlCl₃ solution, which had been diluted in 95% ethanol. After a 20 minutes incubation at room temperature, the absorbance was measured spectrophotometrically at a wavelength of 390 nm. Determination of glutathione and total polyphenol contents The sample was ground in liquid nitrogen, and 1 mL 10% TCA was added and then gently vortexed. The resultant solution was centrifuged at 1,000 rpm, 4 ◦ C for 15 minutes. The amount of 350 µL extract was transferred to a fresh 1.5 tube and then added with 150 µL Ellman regent and 1ml phosphate buffer (pH 7.0 and 150 mM) and mixed thoroughly using vortex mixer. The 96-well microplate was triple-pipetted with 150 µL of the solution, and the absorbance was measured at 420 nm using a spectrophotometer (Al Kharusi et al. 2019 ). Total polyphenolic content was assessed by utilizing the Folin–Ciocalteu reagent, and samples were analyzed for absorbance at a wavelength of 750 nm as described by previous research (Haghighi and Saharkhiz 2021 ). Confirmation of bacterial isolates in inoculated plants In order to confirm bacterial isolates, we removed soil from the roots of both control and inoculated plants. Then, the roots were ground into a fine powder using mortar and pestle in the presence of liquid nitrogen and the total DNA was extracted from the ground root samples using the CTAB method (Asif et al. 2023 ). The extracted DNA was then used as a template for PCR amplification of the 16S rRNA gene using the 27 F (5′-AGAGTTTGATC(AC)TGGCTCAG-3′) and 1492R (5′-CGG(CT)TACCTTGTTACGACTT-3′) primers which are complementary to the 5′ and 3′ ends of the bacterial 16 S rRNA. PCR products were sequenced, and the obtained sequences were compared to existing sequences in the NCBI database using BLAST search software. EzTaxon-e was used for further taxonomic identification based on the 16S rRNA gene sequences. Statistically analysis All experimental data were carried out in five biological replicates, with data from each replicate combined uncombined from using analysis of variance and Duncan’s multiple range test. The means values of different various treatments were compared using a completely randomized design. Results Assessment of IAA production, phosphate solubilization, and siderophore production The SPSB2 strain was found to produce 97.4 ± 0.12 µg/ml of IAA by applying colorimetric assay using Salkowski reagents. To determine the phosphate solubilizing (PSB) activity the SPSB2 strain was cultivated on the NBRIP plate. SPSB2 strain colonies developed distinct halazones. The first qualitative sign of PSB is the halo that forms around the colonies Conversely, CAS reagent color shift from blur to orange in CAS agar plates was used to screen siderophore-positive isolates. The presence of orange colonies on incubation plates, after the SPSB2 bacterial strain exposure indicates the strain's capacity for siderophore production. Additionally, the strain showed appreciable phosphate-solubilizing capabilities. This strain was chosen for further study because of its potential to promote plant growth via phosphate solubilization, siderophore, and IAA synthesis. Bacterial identification and phylogenetic analysis The bacterial strain SPSB2 (OQ380690) was identified, and its phylogenetic position was inferred by using BLAST search analysis ( http://www.ncbi.nlm.nih.gov/ ) of the 16S ribosomal RNA sequences of the isolates against the sequences in the NCBI database. A greater degree of 16S sequence identity was found between SPSB2 and Nitratireductor aquimarinus . Parameters for gap opening penalty and gap extension penalty in both pairwise and multiple sequence alignment were set at 15.00 and 6.66, respectively. After sequence alignment with Clustal W (version 7.222) the phylogenetic tree of the aligned 16S rRNA sequences was constructed by the neighbor-joining method of the MEGA 11 software, with 1,000 times bootstrap replicates (BS) ( Figure S1 ). Effect of SPSB2 and mannitol treatment on plant biomasses of tomato plant under NaCl and As stress Our results revealed that the bacterial inoculation significantly induced tomato plants' shoot and root length by 17% and 53%, respectively, compared to control plants under normal conditions (Figs. 1 and 2 ). The results indicate that the length of shoot and root decreased significantly under NaCl stress compared to control plants. Our finding showed that the SPSB2 inoculation has a significant positive effect on tomato plant growth and mitigates the negative impact of NaCl stress, resulting in a considerable enhancement of both shoot and root length by 84.8% and 152.5%, respectively, compared to their non-inoculated counterparts. Mannitol treatment also significantly promoted shoot length by 13.8%, and surprisingly, the same mannitol treatment led to a significant decrease in root length, reducing it by about 45% under NaCl stress conditions. The combined treated plants with SPSB2 and mannitol exhibited significantly increased root length (94.1%) but unexpectedly reduced shoot length by 8.3% compared to plants treated with NaCl. Similarly, SPSB2 inoculated plants showed a considerable increase in fresh weight of shoot and root, i.e. 82.1% and 105.7%, respectively, compared to the plants in the control condition (Fig. 2 C and D) . Similar trends were observed in the plants under NaCl stress conditions. Combined treated plants with SPSB2 and mannitol showed enhanced fresh root weight (31.6%) when compared to control plants. Conversely, there was a significant decrease in fresh shoot weight, with a reduction of 14% compared to untreated plants. Similarly, under As stress condition, inoculation with plants with SPSB2 bacteria led to a significant increase in the shoot length by 104.8% and in the root length by 36.1%. The results showed that the combined treatment of SPSB2 and mannitol had a positive effect on the shoot and root length of tomato plants compared to untreated plants, as the combined treatment resulted in a notable increase of 66.4% in shoot length and 5.3% in root length when compared to the control and mannitol-treated plants. The results revealed that under As stress conditions, a significant increase occurred in the fresh weight of the tomato plant's shoot and root by 63.1% and 45.5%, respectively, through inoculation with SPSB2. Additionally, the combined application of SPSB2 and mannitol also influenced the fresh weight of both shoot and root compared to the control. Effect of SPSB2 and mannitol treatment on chlorophyll and relative water contents of tomato plant under NaCl and As stress The results in Figs. 3 illustrated that SPSB2-treated plants significantly increased the photosynthetic contents in the control conditions compared to untreated plants. The combined treatment of SPSB2 and mannitol has a consistent positive influence on both chlorophyll-a and carotenoid and significantly increased their contents under both NaCl (76.3% and 123.6%) and As (43.3%, and 50%) stress conditions respectively (Fig. 3 A and 3 C). Moreover, the combined treatment of SPSB2 and mannitol resulted in a significant increase (78%) in chlorophyll-b content in plants. Interestingly, plants treated with As dramatically increased Chlorophyll-b content, while SPSB2 inoculation significantly decreased (Fig. 3 B). Similarly, SPSB2-treated plants significantly decreased their carotenoid content by 35% compared to NaCl stress plants. The combined treated plants with SPSB2 and mannitol exhibited increased carotenoid concentration in NaCl and As stress conditions. The RWC of the tomato leaves was significantly affected by SPSB2 inoculation under control conditions. Similarly, all treatments, including SPSB2, mannitol, and a combination of both, led to a significant increase in RWC in plants under NaCl stress, i.e., 71.1%, 56.5% and 59.9%, respectively. Furthermore, under As stress, the application of mannitol significantly improved (36.1%) RWC, while SPSB2 and combined treatments showed a 19.4% and 37.1% decrease, respectively, when compared to As stressed plants (Fig. 3 D). Effect of SPSB2 and mannitol treatment on catalase and protein contents of tomato plant under NaCl and As stress Our results in Fig. 4 A showed that the catalase contents of tomato plants increased in SPSB2-treated plants, while with mannitol treatment, a reduction of 3% occurred in the control plants. The catalase content in plants treated with SPSB2 and mannitol was similar to that of plants treated with 400 mM NaCl alone. In comparison, mannitol-treated plants showed a slight increase of 2.5% in catalase content compared to SPSB2-treated plants. Catalase contents were analyzed in inoculated bacteria and mannitol under the As stress condition. The results revealed that SPSB2 treatments significantly increased the catalase content by 3.4% in tomato plants. Similar catalase contents were observed in tomato plants treated with the combination of SPSB2 and mannitol. In Fig. 4 B, a reduction in protein content was recorded in plants treated with mannitol. Meanwhile, in control conditions, a significant increase of 14.5% in protein content was observed in plants inoculated with SPSB2. A decrease in protein content was observed when the tomato plants were treated with NaCl and As stress conditions. However, SPSB2-and mannitol-treated plants showed increased protein content by 45.1% and 18.3% under 400 mM stress conditions, respectively. The protein content in plants treated with SPSB2, mannitol individually, and a combination of both was higher compared to As-stressed plants. Mannitol treatment notably induced a significant increase in protein content, with a boost of 54.3% compared to As stressed plants. Effect of SPSB2 and mannitol treatment on sugar, starch and flavonols contents of tomato plant under NaCl and As stress In Figs. 5 , results indicated that mannitol treatments significantly increased sugar and starch contents by 90.3% and 68.9%, respectively, under control conditions. Under the NaCl stress condition, sugar and starch levels rose 36.6% and 32.1%, respectively. Under the As stress, the corresponding increases were 39.8% for sugar and 37.1% for starch. The sugar contents significantly increased in plants treated with mannitol compared to the NaCl stress plants. However, they were lower than SPSB2-treated plants under NaCl and As stress conditions. At the same time, the sugar and starch content of combined treatment plants showed similar results to those treated with NaCl stress, but their concentration significantly decreased under As stress conditions. The results presented in Fig. 5 C indicate a significant effect of the inoculated SPSB2 on the flavonols contents compared to the control condition. Furthermore, inoculated SPSB2 and mannitol treatments led to a considerable 44.9% and 68.4% increase in the flavonol contents of the plants under 400 mM NaCl. However, we observed that combined treatment of SPSB2 and mannitol significantly inhibited flavonol contents, e.g., a 5.9% decrease compared to 400 mM NaCl stress condition. Similarly, in the case of As stress, the SPSB2 and mannitol significantly increase the flavonol contents. Effect of SPSB2 and mannitol treatment on reduced glutathione and total polyphenol contents of tomato plant under NaCl and As stress Reduced glutathione contents were observed to be decreased by 5.6% and 9.4% with SPSB2 and mannitol treatments, respectively, compared to non-treated plants under the control conditions. Furthermore, NaCl-treated plants exhibited significantly enhanced glutathione contents and reduced 15.7%, 33.1%, and 25.4% in glutathione contents by applying inoculated SPSB2, mannitol and combined treatments. The results of glutathione contents in As stress were opposite in plants treated with NaCl stress (Fig. 6 A). The combined stress of NaCl and As significantly increased the glutathione content by 25.2%, and an almost 12.4% increase was observed in SPSB2-inoculated plants compared to As-stressed plants. The PPO contents decreased by 18.1% in plants receiving the mannitol treatment and increased by 19% in plants treated with bacteria under control conditions (Fig. 6 B). The PPO contents in tomato plants dramatically decreased under salinity stress; however, the application of inoculated bacteria improved the PPO contents by 85.0%. Interestingly, both the individual and combined treatment of mannitol and SPSB2 exhibited almost similar effects on PPO contents as the individual treatment with mannitol resulted in a 35.9% increase, while the combined treatment showed a slightly lower increase of 25.9% under NaCl stress condition. The results revealed that PPO contents were almost similar in NaCl and As-treated plants; however, the mannitol and SPSB2 treatment significantly increased the content by 54.7% and 76.1% under As stress conditions. PCA and correlation trait of tomato plants under NaCl and As stress Principal Component Analysis (PCA) results explored the variability in morpho-physiological, biochemical, and antioxidative traits of tomato plants under control, SPSB2 inoculated, and stress conditions of NaCl and As stress (Fig. 7 ). The data was collected from untreated control, SPSB2 inoculated, and mannitol-treated plants under control and stress conditions of NaCl and As. SPSB2 bacterial strain alleviated the salinity and As stress conditions in tomato plants. Our results showed that PC1 has greater values than PC2 in all three plots (Fig. 7 A, B, and C ). The eigenvalue increased and decreased one by one, but the cumulative value was induced in stress conditions. In control, 400 mM NaCl and 0.5 mM As stress conditions were recognized in biplots as 73.12%, 54.75%, and 55.27% cumulative values (Table 1 ). The PCA analysis showed that plants treated with SPSB2 were prominently represented in the PC1, and the SPSB2 treatment significantly impacted the plants' physiological, biochemical, and antioxidant contents under control conditions. Catalase was negatively correlated with plant morphological traits with combined treatments. Moreover, Pearson correlation analysis was conducted to determine the extent of the relationship among the characteristics. The results significantly correlated the morphological, biochemical, and antioxidant traits under control and stress conditions of NaCl and As in both inoculated and non-inoculated plants. Surprisingly, shoot, root length, FWS, FWR, and protein contents were positively correlated with other traits. On the other hand, RWC has a significantly negative correlation with other features shown in (Fig. 7 D). Overall, the results showed insight correlation interaction among different growth parameters in tomato plants under salt and arsenic stress and highlighted the potential of using SPSB2-treated plants to improve growth and stress tolerance. Table 1 Eigenvalue, variance, and cumulative of Control, NaCl, and As stress. Eigenvalue Percentage of Variance Cumulative Control 10.96779 73.12% 73.12% 4.03221 26.88% 100.00% 0 0.00% 100.00% 8.21311 54.75% 54.75% NaCl 400 mM 4.51075 30.07% 84.83% 2.27615 15.17% 100.00% 0 0.00% 100.00% 8.29107 55.27% 55.27% As 0.5 mM 4.94274 32.95% 88.23% 1.76619 11.77% 100.00% 0 0.00% 100.00% Effect of SPSB2 and mannitol treatment on stomata of tomato plant under NaCl and As stress Generally, abiotic stresses elevate the ROS levels and induce osmotic stress in plant cells. Abscisic acids (ABA) play a vital role in closing stomatal cells, thereby preventing the dehydration of transpiration pathways. Stomatal morphology was screened in the microscope to check the effect of inoculated SPSB2 bacterial strain and mannitol under salt and heavy metal stress conditions. As shown in Fig. 8 , under NaCl stress, a higher stomatal density accompanied by a decrease in size was observed compared to SPSB2-inoculated plants. The stomatal density of the plants treated with NaCl alone and those treated with NaCl + B was observed, and the number of stomata increased in the NaCl + B group. Similar trends were observed in the NaCl + M and SPSB2 + mannitol-treated groups, indicating that bacterial (B) and mannitol treatments reduced the number of stomata. Moreover, plants treated with As stress exhibited smaller and open stomata compared to control and SPSB2-treated plants. On the other hand, in SPSB2 inoculated plants, larger and open stomata were observed, and mannitol treatment increased the size and openness of stomata under As stress. Discussion Tomato is an essential vegetable crop grown globally (Kimura and Sinha 2008 ; Rothan et al. 2019 ). It serves as a model plant to study the impacts of abiotic stresses on various aspects of plant life, including growth, development, physiology, and molecular and biochemical processes (Shrivastava and Kumar 2015 ). Excessive salinity may negatively impact plants' growth, metabolism, and general physiology (Zhu 2002 ). Plants may experience water stress (Gupta et al. 2022 ) and dehydration due to excess salts (Zhao et al. 2021 ). Reduced water intake, blocked nutritional absorption, and stunted development are all possible outcomes (Zhao et al. 2020 ). High sodium and chloride ions concentrations build up in plant tissues during salt stress, disrupting vital metabolic processes (Zaheer et al. 2022 ). Similarly, the stress induced by heavy metals, including arsenic, cadmium, mercury, and lead, is prevalent in agricultural lands and poses significant harm to plants globally (Finnegan and Chen 2012 ). High concentrations of heavy metals can inhibit plant functions, photosynthesis, and enzyme processes and produce reactive oxygen species ROS, which cause oxidative stress and are harmful to cells within the plants (Imran et al. 2021 ). Modern technology, such as molecular breeding techniques (Ashraf and Akram 2009 ), CRISPR and transcriptomic analysis (Gonzalez Guzman et al. 2022 ) have modified the crop under abiotic stress (Khan et al. 2021 ). Furthermore, these methods might not always be feasible and can seriously affect the ecosystem (Aizaz et al. 2023 ). Extensive research reveals the beneficial effects of halotolerant and halophilic microorganisms on plant growth (Kadyan et al. 2013 ; Essghaier et al. 2014 ). Bacterial strains exhibiting tolerance to grow in saline and heavy metal-contaminated soil produce several biological metabolites, which could be considered promising keys to sustaining plant growth and protection under different stressful conditions (Wang et al. 2022 ). In this context, in the current study, we tried to isolate bacterial strains endowed with multiple biological activities to promote plant growth under salt stress and in the presence of phytotoxic concentrations of heavy metals. Moreover, the exogenous application of mannitol has been observed to mitigate the adverse effects of salinity and heavy metal stress. Studies indicate that mannitol treatment increases the tolerance of plants to salt and heavy metal stress by ion homeostasis, improving antioxidants, and decreasing ROS (Rathor et al. 2020 ; Habiba et al. 2019 ; Adrees et al. 2015 ). Furthermore, mannitol can enhance plant biomasses, water, and nutrient uptake under abiotic conditions (Slama et al. 2007 ; Aizaz et al. 2023 ). The primary objective of the current study is to investigate the beneficial role of halotolerant bacteria strain and mannitol in mitigating the impacts the impacts of NaCl and As stress conditions on tomato plants. Our finding illustrated that the bacterial strain used in this study produces IAA, a plant growth-stimulating hormone and exhibits both phosphate-solubilizing and siderophore activities. According to previous work, plant growth promoting bacteria can enhance plant growth directly or indirectly by increasing available P, fixing nitrogen, sequestering iron by siderophores, and producing antibiotics and plant hormones (Glick et al. 1998 ; Mantelin and Touraine 2004 ). In current work, tomato plants treated with SPSB2 halotolerant bacterial strain and mannitol showed enhanced growth parameters such as shoot, root, length, and fresh weight compared to control plants by diminishing the adverse effects of NaCl and Arsenic stress. Consistent with our results, a more recent study has revealed that inoculation of two halophilic bacteria ( V. marismortui and T. halophilus ) to tomato seeds improves stem growth compared to the uninoculated control plants(Essghaier et al. 2014 ). Previous studies also investigated that halotolerant and halophilic bacterial isolates have PGP traits that can alleviate salt stress (Orhan 2021 ). The results are also consistent with previous results (Dias et al. 2009 ; Desale et al. 2014 ). The production of IAA by halotolerant and halophilic bacteria is essential for plant development because this phytohormone plays an important role in root initiation, cell expansion, and cell division. The increased root length caused by IAA-producing bacteria can positively affect nutrient absorption from the soil by plants (Boiero et al. 2007 ). Similarly, according to previous research, PGPB can maintain nutritional status and modify phytohormonal balance by producing plant growth regulators, improving plant tolerance to heavy metals and other climatic challenges and stimulating plant growth in metal-contaminated soil (Afzal et al. 2019 ; Hewage et al. 2020 ). Our results agree with previous studies that the subjective of endophyte bacteria and mannitol have been shown to influence the photosynthesis pigment of plants [54,66] positively. The results revealed that SPSB2 bacteria and mannitol induced the chlorophyll and carotenoid contents in plants, decreasing ion toxicity (Almuhayawi et al. 2021 ) and inducing chloroplast metabolism (Das and Roychoudhury 2014 ). Various environmental stress leads to the excessive generation of ROS. It causes cell death (Auten and Davis 2009 ; Schieber and Chandel 2014 ) and damage to nucleic acid, protein, lipid, and other membrane organelles (Redza-Dutordoir and Averill-Bates 2016 ; QAMER et al. 2021 ). Excessive ROS has a harmful effect on plants. Thus, plants utilize enzymatic and non-enzymatic mechanisms to prevent deleterious ROS production (Ahmad et al. 2009; Sarker and Oba 2018; Aizaz et al. 2023 a). Our results agree with those of (Rabiei et al. 2020 ), who found that catalase content shows a significant increase with inoculation of bacteria under non-stressed conditions, while mannitol treatment showed no significant change in catalase contents compared to control plants. However, when tomato plants were inoculated with the SPSB2-bacteria ( N. aquimarinus) , the catalase content increased significantly compared to mannitol treatment under the same non-stressed condition. Our results agreed with previous studies that mannitol showed a positive effect on catalase content under NaCl stress conditions when compared to their respective control plants. In contrast, the bacterial inoculation individually and combined with mannitol showed a slightly positive effect on catalase content (Khare et al. 2010 ; Ajmal et al. 2022 ). We found that inoculating A. esculentus with B. megaterium UPMR2 and Enterobacter sp. UPMR18 increased the expression of stress-related genes such SOD, APX, CAT, GR, and DHAR in response to NaCl stress, which is consistent with previous research (Habib et al. 2016 ). In As stress condition, catalase contents were observed to be significantly increased by bacteria inoculation and mannitol treatment. However, catalase contents were slightly decreased in control and mannitol-treated plants compared to SPSB2 inoculated plants. The inoculated bacteria increased the protein contents, while a decrease was observed in mannitol-treated plants under control conditions. Furthermore, in combined treated plants with SPSB2 and mannitol, protein contents were significantly decreased in NaCl stress, identical to previous studies (Vı́tová et al. 2002 ). Sugar and starch content were significantly higher in mannitol treated plants than control and bacterial inoculated plants under control conditions. Our results are consistent with previous reports that bacterial inoculation significantly increased the sugar and starch contents in salt and heavy metals stress conditions (Hajiabadi et al. 2021 ; Jan et al. 2019 ). Furthermore, inoculated bacteria enhanced the flavanol content compared to the control condition and under NaCl stress, flavanol content was significantly raised in plants with mannitol treatments compared to other treatments, contrasting with previous studies (Ahmed et al. 2016 ). However, inoculated bacteria had significantly higher content than mannitol and combined treatment under As stress. Stomatal pores are the major routes for gaseous exchange across the impermeable cuticle of leaves. Stomatal density and size influence gaseous exchange (Blatt 2016 ). In the current study, the number of stomata studied in control and NaCl-treated plants was fewer but large as compared to SPSB2 treated plants. Our results are in agreement with previous reports that plants sustain their physiological and metabolic processes, improve their resistance to heavy metals by multiplying their stomata, which expands their surface area and facilitates better uptake of CO 2 and water (Wahid et al. 2008 ). Conclusions As chemical pesticides and fertilizers have detrimental effects on human and environmental health, farmers are urged to employ more ecologically friendly substitutes. Using halotolerant plant growth-promoting bacteria is a sustainable solution that helps crops adapt to increasing salinity levels. In recent years, PGPR has had a more positive influence on agriculture, leading to the harvesting of commercially significant crops. The bacterial strains known as halotolerant plant growth promoters (HT-PGP) increase crop yield in salty conditions via several physiological and molecular processes. Using biopreparations based on non-pathogenic living microorganisms can offer several benefits for sustainable and environmentally friendly agricultural practices. Our findings supported the application of halotolerant bacteria as a potential tool to mitigate the effect of salt and heavy metal stress in tomato plants. Continued research is essential to focus on uncovering the underlying mechanisms of plant growth promotion under salinity and heavy metal stress and the practical aspects of integrating these strategies into mainstream agricultural practices to contribute to the resilience and productivity of agriculture globally. Declarations CRediT authorship contribution statement Lubna, Mohammed Aizaz, Shima Ahmed Ali Alrumaidhi, Rawan Ahmed Mohammed Alhinai, and Reem Saif Mohammed AL Kalbani performed experimental and analysis. Ibrahim Khan extracted DNA and microbes identification. Saqib Bilal performed phytohormone and antioxidant analysis, and Sajjad Asaf wrote the draft manuscript and statistical analysis. Ahmed Al-Harrasi, supervision and arranging resources. Funding: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. 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The innovation 1 (1) Zhao S, Zhang Q, Liu M, Zhou H, Ma C, Wang P (2021) Regulation of plant responses to salt stress. International Journal of Molecular Sciences 22 (9):4609 Zhu J-K (2002) Salt and drought stress signal transduction in plants. Annual review of plant biology 53 (1):247-273 Additional Declarations Competing interest reported. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests Supplementary Files FigureS1.tif Supplementary figures Figure S1.Molecular phylogenetic analysis of the bacterial strain (SPSB2) used in this study from the 16S region using the neighbour joining (NJ) method. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4798297","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":333555267,"identity":"30317ff2-e535-42bb-a151-0fc3990c9e6b","order_by":0,"name":"Lubna #","email":"","orcid":"","institution":"University of Nizwa","correspondingAuthor":false,"prefix":"","firstName":"Lubna","middleName":"","lastName":"#","suffix":""},{"id":333555272,"identity":"712ef9cc-855c-48cb-b37f-2d60095c8363","order_by":1,"name":"Muhammad Aizaz","email":"","orcid":"","institution":"University of Nizwa","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"","lastName":"Aizaz","suffix":""},{"id":333555275,"identity":"6b416b08-60aa-4e3e-ac68-313902844790","order_by":2,"name":"Shima Ahmed Ali Alrumaidhi","email":"","orcid":"","institution":"University of Nizwa","correspondingAuthor":false,"prefix":"","firstName":"Shima","middleName":"Ahmed Ali","lastName":"Alrumaidhi","suffix":""},{"id":333555277,"identity":"ada422c8-376c-47f7-bf68-2e98a3622bcd","order_by":3,"name":"Rawan Ahmed Mohammed Alhinai","email":"","orcid":"","institution":"University of Nizwa","correspondingAuthor":false,"prefix":"","firstName":"Rawan","middleName":"Ahmed Mohammed","lastName":"Alhinai","suffix":""},{"id":333555278,"identity":"c40c44c9-50ab-44ed-af98-8a549e611d84","order_by":4,"name":"Reem Saif Mohammed AL Kalbani","email":"","orcid":"","institution":"University of Nizwa","correspondingAuthor":false,"prefix":"","firstName":"Reem","middleName":"Saif Mohammed AL","lastName":"Kalbani","suffix":""},{"id":333555279,"identity":"53132760-4c41-4b30-a0e9-9c14a5ad252e","order_by":5,"name":"Ibrahim Khan","email":"","orcid":"","institution":"University of Nizwa","correspondingAuthor":false,"prefix":"","firstName":"Ibrahim","middleName":"","lastName":"Khan","suffix":""},{"id":333555281,"identity":"63f0dc3c-b086-4a94-9964-db6d094a88c3","order_by":6,"name":"Saqib Bilal","email":"","orcid":"","institution":"University of Nizwa","correspondingAuthor":false,"prefix":"","firstName":"Saqib","middleName":"","lastName":"Bilal","suffix":""},{"id":333555285,"identity":"b88cdafa-130a-44eb-bc25-39d5db88dc3b","order_by":7,"name":"Sajjad Asaf","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBklEQVRIie3RMWvCQBTA8XccxOVJ55JCvoJdhE5+kC4HgUzeniEe1yVZpK4tWPsV2sX5JJAuB10DXeLu4CSCIL50skMSx4L3HwKP3A/ycgAu1/9MAAJgL3vSRsQ0c64vI2jzVbW1NWGdBGoCWEbh/Wtajx0kyMx6u0nUHcJ46PcXk8ebjMg+XjaSgRXh7bzIEcESWX7Jl5xpNrU/zQSE8NEzOGLTmhRSE+EsbSbBrAoPeFSIHInMC/neRaAUkd9POaLn0fo6kR9dZFBW0cPbM+2CnH5yYeQnkVXbLsFsHJabnRphsKarTJRcfNMF7eOWDwMU51P++zQt56nen/eq/bDL5XJdZSfLZV6Wvo9zqAAAAABJRU5ErkJggg==","orcid":"","institution":"University of Nizwa","correspondingAuthor":true,"prefix":"","firstName":"Sajjad","middleName":"","lastName":"Asaf","suffix":""},{"id":333555287,"identity":"f553fb01-520d-4682-b8a8-5388c6216ab6","order_by":8,"name":"Ahmed AL-Harrasi","email":"","orcid":"","institution":"University of Nizwa","correspondingAuthor":false,"prefix":"","firstName":"Ahmed","middleName":"","lastName":"AL-Harrasi","suffix":""}],"badges":[],"createdAt":"2024-07-25 01:46:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4798297/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4798297/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":63251343,"identity":"45766847-acee-4fef-83d8-9c850df5b26d","added_by":"auto","created_at":"2024-08-26 07:11:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2632251,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of inoculation of SPSB2 and mannitol on tomato plant growth under control, 400 mM NaCl, and 0.5 mM As stress conditions.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4798297/v1/8d1462d9ea0a6458d1894fa7.png"},{"id":63251342,"identity":"7d26e5b0-5102-4e1e-bc36-b02903d0f9c2","added_by":"auto","created_at":"2024-08-26 07:11:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":708062,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Shoot length, \u003cstrong\u003e(B)\u003c/strong\u003eRoot length, \u003cstrong\u003e(C)\u003c/strong\u003e Fresh shoot weight, \u003cstrong\u003e(D)\u003c/strong\u003e Fresh root weight. Error bars are represented by standard bars with significant differences among control, SPSB2 inoculation, mannitol, and combined treatment of (SPSB2+mannitol) evaluated by DMRT analysis. The given treatments are indicated as C=control, B=bacteria, and M=mannitol.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4798297/v1/76342be12ab3a609cdaf80d3.png"},{"id":63251346,"identity":"333784d0-3d7d-40f2-9c14-85d80b3d50f8","added_by":"auto","created_at":"2024-08-26 07:11:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":744564,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of inoculation of SPSB2 and mannitol on the biomass of tomato plant under control, 400 mM NaCl, and 0.5 mM As stress condition. \u003cstrong\u003e(A)\u003c/strong\u003e Chlorophyll a, \u003cstrong\u003e(B)\u003c/strong\u003e Chlorophyll b, \u003cstrong\u003e(C)\u003c/strong\u003eCarotenoid, \u003cstrong\u003e(D)\u003c/strong\u003e RWC. Error bars are represented by standard bars with significant differences among control, SPSB2 inoculation, mannitol, and combined treatment of (SPSB2+mannitol) evaluated by DMRT analysis. The given treatments are indicated as C=control, B=bacteria, and M=mannitol.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4798297/v1/e36d7cd3b9cba57e2a15713d.png"},{"id":63251345,"identity":"e987e437-850d-4708-9387-ab38e549cb01","added_by":"auto","created_at":"2024-08-26 07:11:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":349329,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of inoculation of SPSB2 and mannitol on the biomass of tomato plant under control, 400 mM NaCl, and 0.5 mM As stress condition. \u003cstrong\u003e(A)\u003c/strong\u003e Catalase \u003cstrong\u003e(B)\u003c/strong\u003e Protein. Error bars are represented by standard bars with significant differences among control, SPSB2 inoculation, mannitol, and combined treatment of (SPSB2+mannitol) evaluated by DMRT analysis. The given treatments are indicated as C=control, B=bacteria and M=mannitol.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4798297/v1/5abe1f7c86230fa9b9c0c4c4.png"},{"id":63251349,"identity":"663795c3-6732-482c-89ac-671b4669fd40","added_by":"auto","created_at":"2024-08-26 07:11:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":648649,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of inoculation of SPSB2 and mannitol on the biomass of tomato plant under control, 400 mM NaCl, and 0.5 mM As stress condition. \u003cstrong\u003e(A)\u003c/strong\u003e Sugar\u003cstrong\u003e (B)\u003c/strong\u003e Starch \u003cstrong\u003e(C)\u003c/strong\u003e flavanol. Error bars are represented by standard bars with significant differences among control, SPSB2 inoculation, mannitol, and combined treatment of (SPSB2+mannitol) evaluated by DMRT analysis. The given treatments are indicated as C=control, B=bacteria, and M=mannitol.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4798297/v1/76c913cd2075a877b7f036c7.png"},{"id":63251348,"identity":"0a2ebdf0-02a1-48a5-b99c-b03bdc5cd959","added_by":"auto","created_at":"2024-08-26 07:11:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":343129,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of inoculation of SPSB2 and mannitol on the biomass of tomato plant under control, 400 mM NaCl, and 0.5 mM stress condition. \u003cstrong\u003e(A)\u003c/strong\u003e Glutathione \u003cstrong\u003e(B)\u003c/strong\u003e Total polyphenol Contents. Error bars are represented by standard bars with significant differences among control, SPSB2 inoculation, mannitol, and combined treatment of (SPSB2+mannitol) evaluated by DMRT analysis. The given treatments are indicated as C=control, B=bacteria, and M=mannitol.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4798297/v1/baefcb54c26da7084ef9079a.png"},{"id":63251946,"identity":"16736c7e-f9e4-47db-95e1-9c746dc56d60","added_by":"auto","created_at":"2024-08-26 07:19:39","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2676961,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal component analysis (PCA) biplot of tomato plant \u003cstrong\u003e(A) \u003c/strong\u003eControl, \u003cstrong\u003e(B) \u003c/strong\u003eNaCl stress, \u003cstrong\u003e(C) \u003c/strong\u003eArsenic stress, and \u003cstrong\u003e(D) \u003c/strong\u003ePearson correlation analysis between plant physiological, biochemical, and antioxidant with mannitol and SPSB2 inoculation in different stress condition. Correlation is displayed in yellow light positive and blue light negative.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4798297/v1/00ac2104e99125215475b9bd.png"},{"id":63251947,"identity":"896c2d15-801b-45c3-aa34-2da656ba8bc1","added_by":"auto","created_at":"2024-08-26 07:19:39","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":18812517,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of inoculation of SPSB2 and mannitol on stomata under control, 400 mM NaCl, and 0.5 mM stress condition. (A) 400 mM NaCl (B) 0.5 mM As.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-4798297/v1/b9e06e6a36f2f8b5b07ac31b.png"},{"id":63251340,"identity":"43f47019-56a4-41c7-a786-a53532a32abc","added_by":"auto","created_at":"2024-08-26 07:11:38","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2743513,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary figures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S1.\u003c/strong\u003eMolecular phylogenetic analysis of the bacterial strain (SPSB2) used in this study from the 16S region using the neighbour joining (NJ) method.\u003c/p\u003e","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4798297/v1/a617b1c5ffd19d2dbd383eee.tif"}],"financialInterests":"Competing interest reported. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests","formattedTitle":"Exploring the Interactive Mechanisms of Halophilic Bacterium SPSB2 and Mannitol in Mitigating Sodium Chloride and Arsenic Stress in Tomato Plants","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThreats to agricultural sustainability in the 21st century arise from the expanding human population and decreasing agricultural land. Agriculture's problems are worsened by the worldwide water shortage, environmental contamination, and the increasing soil and water salinity (Shahbaz and Ashraf \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Salinity stress negatively impacts seed germination, plant vigor, and agricultural productivity (Chakma et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). There are two ways in which soil salts might prevent plants from flourishing. First, salt in soil solutions reduces plants' ability to absorb water, hindering their growth and development. Second, suppose too much salt enters the plant via the transpiration stream. In that case, it may cause damage to the cells and the transpiring leaves, leading to a further slowing of development. This effect is described as the ion-excess or salt-specific impact of salinity (Greenway and Munns \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1980\u003c/span\u003e; Patil et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In addition to salt stress, heavy metals like As, Ni, Cd, Fe, Mn, Cu, Co, Zn, and Hg have been accumulating in soils as a result of the disposal of industrial waste. However, many metals are essential micronutrients needed in trace amounts for numerous biological processes. Still, an excess of these metals adversely affects senescence, metabolism, physiology, and plant growth and development (Ghori et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTomatoes are the most popular and economically significant fruits and vegetables worldwide. The demand for tomatoes has increased significantly worldwide in recent years due to their wide range of uses in raw, cooked, and processed foods (Chaudhary et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Being a temperate crop, tomatoes are grown in various climate zones, making their production more difficult, and abiotic stressors, including drought, salt, and heat, significantly negatively impact agricultural productivity and output (Rao et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Many unconventional tomato-producing sites have embraced greenhouse-based agriculture to guarantee a consistent year-round supply. Therefore, improving tomato cultivars' stress tolerance is more financially viable and sustainable. The principal problems limiting tomato production include extreme water and temperature regimes, nutritional imbalances, various abiotic stresses, elemental toxicity, and high salt in the soil substrate (Conti et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Abiotic stresses become more complex in agricultural settings due to the frequent coexistence of many types of stresses. To feed the world's population, we need to breed crops that can withstand and recover from a wide range of abiotic stresses while still producing high yields (Patil et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePopulation growth is outpacing the increase in demand for agricultural products and food security (Pawlak and Kołodziejczak \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Farming methods have changed in recent decades to consider this and respond to this global problem. Applying chemical fertilizers and agrochemicals can improve food production by boosting crop yield, enhancing quality, and prolonging shelf life. However, these actions are costly in terms of ecosystem destruction, global warming, soil erosion, and biodiversity loss (Fasusi et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Mitter et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). For decades, soil health has steadily declined, primarily due to the careless use of agrochemicals (El-Ghamry et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Guha et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Microbes, with their eco-friendly, cost-effective, and protective attributes against various biotic and abiotic pressures, may offer a viable alternative approach to overcoming constraints on agricultural output (Zaidi et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHalotolerant plant growth-promoting rhizobacteria (HT-PGPR) are a varied group of saltwater soil microorganisms that help plants survive in salt habitats and boost their development and other soil-related properties (Hidri et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The role of HT-PGPR in mitigating the adverse effects of salt stress on crops has become prominent in recent years. Through the production of exopolysaccharides, siderophores, volatile organic compounds (VOCs), suitable osmolytes, and phytohormones, HT-PGPR boosts the productivity of the saline-agroecosystem (Bhat et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) directly by preventing the impacts of the phytopathogens, or indirectly by controlling the expression of stress-related genes (Prasad et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). \u003cem\u003eRhizobium\u003c/em\u003e, \u003cem\u003eArthrobacter, Flavobacterium, Alcaligenes, Pseudomonas\u003c/em\u003e, and \u003cem\u003eAzospirillum\u003c/em\u003e are only a few halotolerant bacteria reported to increase crop salinity tolerance (Saghafi et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhen assessing halobacteria for their potential to promote plant development and shield certain crops, toxicity to heavy metals has also been considered. In fact, (Masmoudi et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) found that \u003cem\u003eB. velenzensis\u003c/em\u003e and \u003cem\u003eB. subtilis\u003c/em\u003e were effective in boosting the development of tomato plants (\u003cem\u003eS. lycopersicum\u003c/em\u003e) subjected to salt stress and heavy metal contamination (Co, Ni, Cu, and Cr). Endophytic and rhizospheric bacteria may decrease metal toxicity in plants via their metal resistance mechanism and boost plant development under metal stress; hence, phytoremediation with bacterial strains has recently been highly recommended for cleaning up metal-polluted soils(Asaf et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Because they are affordable, eco-friendly, and capable of protecting plants from biotic pressures, microorganisms offer a viable alternative approach to overcoming the constraints on the agricultural output caused by abiotic stress and for improved plant health and protection (Zaidi et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Moreover, the most effective method for removing hazardous metals. This approach is favored due to its natural, eco-friendly nature, cost-effectiveness, and widespread acceptance as a technology for metal removal. Utilizing bacteria for the bioremediation of heavy metal-contaminated soil to encourage plant growth is one such method (Adesemoye et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Chamkhi et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Shaffique et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) .\u003c/p\u003e \u003cp\u003eIn the current study, we proposed that the SPSB2 halophilic bacterial strain has the potential to serve as an effective mitigator of salt and heavy metal stress in tomato plants. The purpose of the present studyis that bacteria strains may positively influence plant growth, modulate the antioxidative system, and stimulate the production of phytohormones. This contribution could advance the development of an eco-friendly phytoremediation strategy for efficiently addressing salt and heavy metal stress while promoting the growth of tomato plants. We investigated the in vitro and in vivo plant growth-promoting abilities of the \u003cem\u003eNitratrieducator aquimarinus\u003c/em\u003e SPSB2 strain in a greenhouse experiment, subjecting tomato plants to both control and high NaCl and As stress conditions.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIsolation of halophilic bacteria\u003c/h2\u003e \u003cp\u003eHalophilic bacteria were isolated from Muscat, Oman, as described in recent studies. Sediment weighing 10 gm was mixed into 90 mL of 5% or 15% NaCl (w/v) sterile brine and incubated at 37\u0026deg;C for an hour while being shaken at 200 revolutions per minute. To isolate bacteria, aliquots of the resultant slurry (0.1 mL) were distributed onto Petri plates and serially diluted with sterilized NaCl brine (5% or 15%, w/v). Colony counts were triple checked by plating three copies of each medium on separate plates. NaCl (5 or 15% w/v) and nystatin (50 mg/L) were added to all agar plates to prevent the development of non-bacterial fungi. One to six days were spent in a 37\u0026deg;C incubator with the Petri plates. Size and color were used to select colonies for further purification on inorganic salt-starch agar or TSA with 5% or 15% (w/v) NaCl. Long-term preservation of the purified colonies was accomplished by suspending them in glycerol (20% v/v) and stored at -20\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eScreening bacterial strains for IAA production\u003c/h2\u003e \u003cp\u003eThe examination of bacterial strains for their IAA synthesis using the colorimetric method (Asif et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The bacterial strains were cultured in nutrient broth at 30\u0026deg;C of bacterial suspension. After seven days, the samples were filtered, and the IAA concentrations were determined by adding 1 mL of Salkowski reagent to 2 mL of each culture filtrate, followed by 30 minutes of incubation in the dark. The optical density was determined at 530 nm using a UV spectrophotometer. IAA production was calculated using a standard curve based on indole acetic acid (Gordon and Weber \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1951\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePhosphate solubilizations activity\u003c/h2\u003e \u003cp\u003eThe quantitative assessment of phosphate (P) solubilizations was performed using the vanado-molybdat phosphoric method (Murphy and Riley \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1962\u003c/span\u003e). we autoclaved a 50 mL volume of NBRIP broth medium in a 250 mL flask and pH-adjusted to 7.0 and after that, a new 200 \u0026micro;L inoculum was added, and the flask was shaken for seven days at 28\u0026deg;C and 120 rpm. An autoclaved and uninoculated NBRIP broth medium was used as a control. After the incubation period ended, 2 mL of the culture was centrifuged at 10,000 rpm for 15 minutes, and the molybdenum blue colorimetric technique was used to determine the amount of soluble Phosphate present. A UV-VIS spectrophotometer was used to determine the optical density at 430 nm. Triplicates of all conditions were carried out throughout the experiment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eSiderophore production\u003c/h2\u003e \u003cp\u003eThe Chrome-Azurol S (CAS) medium and the Universal Chemical Assay were used to evaluate the siderophore synthesis of all of the bacterial strains (Senthilkumar et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Four independent tests were performed to ensure reliable findings. In a nutshell, bacterial strains were cultured into CAS plates for four days at 28\u0026deg;C. An orange ring around the colonies indicated were production of siderophore.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eBacterial identification and phylogenetic analysis\u003c/h2\u003e \u003cp\u003eThe isolated bacterial culture was hydrolyzed using enzymes to isolate their genomic DNA as prescribed by (Saito and Miura \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e1963\u003c/span\u003e). To amplify the whole 1.4\u0026ndash;1.5 kb 16S rRNA gene, universal bacterial primers 27 F (5\u0026prime;-AGA GTT TGA TCC TGG CTC AG-3\u0026prime;) and 1541R (5\u0026prime;-AAG GAG GTG ATC CAG CCG CA-3\u0026prime;) were used in a PCR reaction. To amplify the 16S rRNA region, a DNA engine gradient thermal cycler was used (BIO-RAD, USA). The 50 \u0026micro;L PCR reaction mixture comprised 4 \u0026micro;L of 2.5 U/\u0026micro;LTaq DNA polymerase (Tiangen, Beijing), 5 \u0026micro;L of 10\u0026times; buffer (Tiangen), 1 \u0026micro;L of 20 mM dNTPs (Tiangen), 37 \u0026micro;L of SDW, 1 \u0026micro;L of 50 \u0026micro;M per primer, and 1 \u0026micro;L of the template was prepared. Initial denaturation was performed at 94\u0026deg;C for 4 minutes, followed by 30 amplification cycles at 94\u0026deg;C for 1 minute, 56\u0026deg;C for 1 minute, and 72\u0026deg;C for 2 minutes, and finally elongation was performed at 72\u0026deg;C for 10 minutes. The amplified region was sequenced by Macrojen company, South Korea. The GenBank databases (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/genbank/\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/genbank/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were searched for matching data to compare the sequences. The MEGA-11 software (Tamura et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) with Clustal-W alignment and the neighbor-joining (NJ) technique was used to construct the phylogenetic tree of the SPSB2 bacterial strain with other downloaded matching bacterial sequences. Each node in the phylogenetic trees was statistically supported using 1,000 bootstrap replications.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSeed sterilization and germination\u003c/h2\u003e \u003cp\u003eThe experiments were conducted within the confines of the Biotechnology and OMICs laboratory's greenhouse at the University of Nizwa Oman. High yielding of tomato seeds cultivar (\u003cem\u003eS. lycopersicum cv.Yegwang\u003c/em\u003e) was acquired from Kyungpook National University, South Korea and assessed for viability before use. HgCl\u003csub\u003e2\u003c/sub\u003e solution was used for surface seed sterilization for one minute, followed by a double wash with 70% ethanol and two additional washes in ddH\u003csub\u003e2\u003c/sub\u003eO. The seeds were transferred to autoclaved filter papers and placed in Petri dishes, and 3 mL of distilled water was added. After five days, germinated seeds were carefully transferred to 10 \u0026times; 9-cm plastic pots filled with horticulture substrate containing coco peat moss (10\u0026ndash;15%), coco peat (45\u0026ndash;50%), perlite (35\u0026ndash;40), NH+ (ca.0.09 mg/g), KO (c.0.1 mg/g), zeolite (6\u0026ndash;8%) with NO3 (ca. 0.205 mg/g), and PO (ca.0.35 mg/g) autoclaved soil. The pots were placed in a growth chamber and maintained environmental conditions with day and nighttime temperatures range and relative humidity between 60% and 70%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eExperimental design\u003c/h2\u003e \u003cp\u003eIn this experiment, we used different stress conditions on tomato plants: (a) control group, treated with dH\u003csub\u003e2\u003c/sub\u003e0, (b) Inoculated plants with SPSB2 bacterial strain, (c) Plant treated with 100 mM mannitol, (d) Plant treated with 400 mM NaCl, (e) Plant treated with 0.5 mM As, (f) Plant treated with NaCl\u0026thinsp;+\u0026thinsp;SPSB2, (g) Plant treated with NaCl\u0026thinsp;+\u0026thinsp;mannitol, (h) Plant treated with NaCl\u0026thinsp;+\u0026thinsp;bacteria\u0026thinsp;+\u0026thinsp;mannitol, (i) As treated plants, (j) Plant treated with As +\u0026thinsp;SPSB2, (k) Plant treated with As +\u0026thinsp;mannitol, (l) Plant treated with As +\u0026thinsp;SPSB2\u0026thinsp;+\u0026thinsp;mannitol. As a result, eleven distinct treatments were administered, each with five replicates, requiring a total of 35 pots for implementation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of plant biomasses and relative water contents (RWC)\u003c/h2\u003e \u003cp\u003ePlants were harvested after 60 days and various growth parameters such as root and shoot length, and fresh weight of root and shoot were measured. The collected samples were quickly placed into liquid nitrogen and preserved at -80\u0026deg;C for future analysis. RWC of the leaves was assessed using a previously established method (Silveira et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Fresh leaves from the plants were immersed in distilled water for 4 hours to note their turgid weight. Subsequently, the leaf samples were subjected to a 24-hour drying process in an oven maintained at a temperature of 80\u0026deg;C to acquire their dry mass. The following formula was used to calculate the RWC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eRWC (%) =(FW\u0026thinsp;\u0026minus;\u0026thinsp;DW)/(TW\u0026thinsp;\u0026minus;\u0026thinsp;DW) \u0026times; 100\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003eDetermination of stomatal density and size\u003c/h2\u003e \u003cp\u003eWith slight modifications, we used the nail polish and stick tape method as described by (Silveira et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), to determine the stomatal density. Three mature fresh tomato leaves from each experimental plant were carefully selected and removed from the stem. A thin layer of nail polish was spread on the lower leaf surface. When the nail polish was dried, a strip of clear stick tape was placed over the leaf area, and the stick tape was immediately pressed down to contact it. Carefully removed, the tape was fixed to the microscopic slide and then the stomatal density and structure were examined under a compound microscope.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of chlorophyll contents\u003c/h2\u003e \u003cp\u003eFresh tomato plant leaves were ground in liquid nitrogen, and a 200 mg sample was mixed with 1 mL of 80% acetone in a 1.5 mL test tube. After a brief vertexing, the tube was centrifuged at 1,000 rpm for 5 minutes. Subsequently, 150 \u0026micro;L of the supernatant was transferred into a 96-well microplate. Data was recorded using a spectrometer for chlorophyll a, b, and carotenoids at 663 nm, 645 nm, and 470 nm, respectively. Photosynthetic pigment concentrations were calculated using the extension coefficients and equation given in Barnes\u0026rsquo;s method (Barnes et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1992\u003c/span\u003e) with slight modifications.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of protein and catalase contents\u003c/h2\u003e \u003cp\u003eThe method described by (Bradford, M. M., 1976) was used for protein content estimation. Fresh leaves (200mg) were crushed into fine powder in liquid nitrogen and added with 1mL of extracted buffer containing (50 mM Tris HCl (pH 7.0), 10%, glycerol, 3mM, MgCl\u003csub\u003e2\u003c/sub\u003e, 1mM EDTA, and 1% pvp) and mixed gently. The homogenate samples were centrifuged at 4,000 rpm for 10 minutes at 2\u0026deg;C. Transfer 240 \u0026micro;L supernatant in a new tube and mixed with an equal volume of phosphate buffer of 0.1 mM (pH 7.0) and 120 \u0026micro;L of 0.2 M H\u003csub\u003e2\u003c/sub\u003e0\u003csub\u003e2\u003c/sub\u003e, and the optical density (OD) was measured at 240 nm using spectrophotometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of sugar, starch, and flavonols\u003c/h2\u003e \u003cp\u003eSugar and starch contents were analyzed using the ethanol method. A 0.5 g grained tomato plant sample was taken, added with 1mL 80% ethanol, and kept at room temperature for 24 hours. The ethanolic solution was centrifuged at 3,000 rpm for 10 minutes. After centrifuging the solution, sugar and starch content were measured at 490 nm and 630 nm by spectrophotometer. The method described by the previous research (Levon and Klymenko \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e)was used to extract and quantify total flavonol contents. Fresh leaf samples were meticulously ground using a mortar and pestle with liquid nitrogen. Subsequently, 0.5 g of the resulting powder was added with 1 mL of 80% methanol, and this mixture was left at room temperature for 24 hours. Following this incubation period, the mixture underwent centrifugation for 15 minutes at a speed of 10,000 rpm. The resulting supernatant was then collected and combined with an equal volume of 2% AlCl₃ solution, which had been diluted in 95% ethanol. After a 20 minutes incubation at room temperature, the absorbance was measured spectrophotometrically at a wavelength of 390 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of glutathione and total polyphenol contents\u003c/h2\u003e \u003cp\u003eThe sample was ground in liquid nitrogen, and 1 mL 10% TCA was added and then gently vortexed. The resultant solution was centrifuged at 1,000 rpm, 4 \u003csup\u003e◦\u003c/sup\u003eC for 15 minutes. The amount of 350 \u0026micro;L extract was transferred to a fresh 1.5 tube and then added with 150 \u0026micro;L Ellman regent and 1ml phosphate buffer (pH 7.0 and 150 mM) and mixed thoroughly using vortex mixer. The 96-well microplate was triple-pipetted with 150 \u0026micro;L of the solution, and the absorbance was measured at 420 nm using a spectrophotometer (Al Kharusi et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Total polyphenolic content was assessed by utilizing the Folin\u0026ndash;Ciocalteu reagent, and samples were analyzed for absorbance at a wavelength of 750 nm as described by previous research (Haghighi and Saharkhiz \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eConfirmation of bacterial isolates in inoculated plants\u003c/h2\u003e \u003cp\u003eIn order to confirm bacterial isolates, we removed soil from the roots of both control and inoculated plants. Then, the roots were ground into a fine powder using mortar and pestle in the presence of liquid nitrogen and the total DNA was extracted from the ground root samples using the CTAB method (Asif et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The extracted DNA was then used as a template for PCR amplification of the 16S rRNA gene using the 27 F (5\u0026prime;-AGAGTTTGATC(AC)TGGCTCAG-3\u0026prime;) and 1492R (5\u0026prime;-CGG(CT)TACCTTGTTACGACTT-3\u0026prime;) primers which are complementary to the 5\u0026prime; and 3\u0026prime; ends of the bacterial 16 S rRNA. PCR products were sequenced, and the obtained sequences were compared to existing sequences in the NCBI database using BLAST search software. EzTaxon-e was used for further taxonomic identification based on the 16S rRNA gene sequences.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eStatistically analysis\u003c/h2\u003e \u003cp\u003eAll experimental data were carried out in five biological replicates, with data from each replicate combined uncombined from using analysis of variance and Duncan\u0026rsquo;s multiple range test. The means values of different various treatments were compared using a completely randomized design.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of IAA production, phosphate solubilization, and siderophore production\u003c/h2\u003e \u003cp\u003eThe SPSB2 strain was found to produce 97.4\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.12 \u0026micro;g/ml of IAA by applying colorimetric assay using Salkowski reagents. To determine the phosphate solubilizing (PSB) activity the SPSB2 strain was cultivated on the NBRIP plate. SPSB2 strain colonies developed distinct halazones. The first qualitative sign of PSB is the halo that forms around the colonies Conversely, CAS reagent color shift from blur to orange in CAS agar plates was used to screen siderophore-positive isolates. The presence of orange colonies on incubation plates, after the SPSB2 bacterial strain exposure indicates the strain's capacity for siderophore production. Additionally, the strain showed appreciable phosphate-solubilizing capabilities. This strain was chosen for further study because of its potential to promote plant growth via phosphate solubilization, siderophore, and IAA synthesis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eBacterial identification and phylogenetic analysis\u003c/h2\u003e \u003cp\u003eThe bacterial strain SPSB2 (OQ380690) was identified, and its phylogenetic position was inferred by using BLAST search analysis (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) of the 16S ribosomal RNA sequences of the isolates against the sequences in the NCBI database. A greater degree of 16S sequence identity was found between SPSB2 and \u003cem\u003eNitratireductor aquimarinus\u003c/em\u003e. Parameters for gap opening penalty and gap extension penalty in both pairwise and multiple sequence alignment were set at 15.00 and 6.66, respectively. After sequence alignment with Clustal W (version 7.222) the phylogenetic tree of the aligned 16S rRNA sequences was constructed by the neighbor-joining method of the MEGA 11 software, with 1,000 times bootstrap replicates (BS) (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of SPSB2 and mannitol treatment on plant biomasses of tomato plant under NaCl and As stress\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOur results revealed that the bacterial inoculation significantly induced tomato plants' shoot and root length by 17% and 53%, respectively, compared to control plants under normal conditions (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The results indicate that the length of shoot and root decreased significantly under NaCl stress compared to control plants. Our finding showed that the SPSB2 inoculation has a significant positive effect on tomato plant growth and mitigates the negative impact of NaCl stress, resulting in a considerable enhancement of both shoot and root length by 84.8% and 152.5%, respectively, compared to their non-inoculated counterparts. Mannitol treatment also significantly promoted shoot length by 13.8%, and surprisingly, the same mannitol treatment led to a significant decrease in root length, reducing it by about 45% under NaCl stress conditions. The combined treated plants with SPSB2 and mannitol exhibited significantly increased root length (94.1%) but unexpectedly reduced shoot length by 8.3% compared to plants treated with NaCl. Similarly, SPSB2 inoculated plants showed a considerable increase in fresh weight of shoot and root, i.e. 82.1% and 105.7%, respectively, compared to the plants in the control condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cb\u003eD)\u003c/b\u003e. Similar trends were observed in the plants under NaCl stress conditions. Combined treated plants with SPSB2 and mannitol showed enhanced fresh root weight (31.6%) when compared to control plants. Conversely, there was a significant decrease in fresh shoot weight, with a reduction of 14% compared to untreated plants. Similarly, under As stress condition, inoculation with plants with SPSB2 bacteria led to a significant increase in the shoot length by 104.8% and in the root length by 36.1%. The results showed that the combined treatment of SPSB2 and mannitol had a positive effect on the shoot and root length of tomato plants compared to untreated plants, as the combined treatment resulted in a notable increase of 66.4% in shoot length and 5.3% in root length when compared to the control and mannitol-treated plants. The results revealed that under As stress conditions, a significant increase occurred in the fresh weight of the tomato plant's shoot and root by 63.1% and 45.5%, respectively, through inoculation with SPSB2. Additionally, the combined application of SPSB2 and mannitol also influenced the fresh weight of both shoot and root compared to the control.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of SPSB2 and mannitol treatment on chlorophyll and relative water contents of tomato plant under NaCl and As stress\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe results in Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e illustrated that SPSB2-treated plants significantly increased the photosynthetic contents in the control conditions compared to untreated plants. The combined treatment of SPSB2 and mannitol has a consistent positive influence on both chlorophyll-a and carotenoid and significantly increased their contents under both NaCl (76.3% and 123.6%) and As (43.3%, and 50%) stress conditions respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Moreover, the combined treatment of SPSB2 and mannitol resulted in a significant increase (78%) in chlorophyll-b content in plants. Interestingly, plants treated with As dramatically increased Chlorophyll-b content, while SPSB2 inoculation significantly decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Similarly, SPSB2-treated plants significantly decreased their carotenoid content by 35% compared to NaCl stress plants. The combined treated plants with SPSB2 and mannitol exhibited increased carotenoid concentration in NaCl and As stress conditions. The RWC of the tomato leaves was significantly affected by SPSB2 inoculation under control conditions. Similarly, all treatments, including SPSB2, mannitol, and a combination of both, led to a significant increase in RWC in plants under NaCl stress, i.e., 71.1%, 56.5% and 59.9%, respectively. Furthermore, under As stress, the application of mannitol significantly improved (36.1%) RWC, while SPSB2 and combined treatments showed a 19.4% and 37.1% decrease, respectively, when compared to As stressed plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of SPSB2 and mannitol treatment on catalase and protein contents of tomato plant under NaCl and As stress\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOur results in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA showed that the catalase contents of tomato plants increased in SPSB2-treated plants, while with mannitol treatment, a reduction of 3% occurred in the control plants. The catalase content in plants treated with SPSB2 and mannitol was similar to that of plants treated with 400 mM NaCl alone. In comparison, mannitol-treated plants showed a slight increase of 2.5% in catalase content compared to SPSB2-treated plants. Catalase contents were analyzed in inoculated bacteria and mannitol under the As stress condition. The results revealed that SPSB2 treatments significantly increased the catalase content by 3.4% in tomato plants. Similar catalase contents were observed in tomato plants treated with the combination of SPSB2 and mannitol. In Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, a reduction in protein content was recorded in plants treated with mannitol. Meanwhile, in control conditions, a significant increase of 14.5% in protein content was observed in plants inoculated with SPSB2. A decrease in protein content was observed when the tomato plants were treated with NaCl and As stress conditions. However, SPSB2-and mannitol-treated plants showed increased protein content by 45.1% and 18.3% under 400 mM stress conditions, respectively. The protein content in plants treated with SPSB2, mannitol individually, and a combination of both was higher compared to As-stressed plants. Mannitol treatment notably induced a significant increase in protein content, with a boost of 54.3% compared to As stressed plants.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of SPSB2 and mannitol treatment on sugar, starch and flavonols contents of tomato plant under NaCl and As stress\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e, results indicated that mannitol treatments significantly increased sugar and starch contents by 90.3% and 68.9%, respectively, under control conditions. Under the NaCl stress condition, sugar and starch levels rose 36.6% and 32.1%, respectively. Under the As stress, the corresponding increases were 39.8% for sugar and 37.1% for starch. The sugar contents significantly increased in plants treated with mannitol compared to the NaCl stress plants. However, they were lower than SPSB2-treated plants under NaCl and As stress conditions. At the same time, the sugar and starch content of combined treatment plants showed similar results to those treated with NaCl stress, but their concentration significantly decreased under As stress conditions. The results presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eC indicate a significant effect of the inoculated SPSB2 on the flavonols contents compared to the control condition. Furthermore, inoculated SPSB2 and mannitol treatments led to a considerable 44.9% and 68.4% increase in the flavonol contents of the plants under 400 mM NaCl. However, we observed that combined treatment of SPSB2 and mannitol significantly inhibited flavonol contents, e.g., a 5.9% decrease compared to 400 mM NaCl stress condition. Similarly, in the case of As stress, the SPSB2 and mannitol significantly increase the flavonol contents.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of SPSB2 and mannitol treatment on reduced glutathione and total polyphenol contents of tomato plant under NaCl and As stress\u003c/b\u003e \u003c/p\u003e \u003cp\u003eReduced glutathione contents were observed to be decreased by 5.6% and 9.4% with SPSB2 and mannitol treatments, respectively, compared to non-treated plants under the control conditions. Furthermore, NaCl-treated plants exhibited significantly enhanced glutathione contents and reduced 15.7%, 33.1%, and 25.4% in glutathione contents by applying inoculated SPSB2, mannitol and combined treatments. The results of glutathione contents in As stress were opposite in plants treated with NaCl stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The combined stress of NaCl and As significantly increased the glutathione content by 25.2%, and an almost 12.4% increase was observed in SPSB2-inoculated plants compared to As-stressed plants. The PPO contents decreased by 18.1% in plants receiving the mannitol treatment and increased by 19% in plants treated with bacteria under control conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). The PPO contents in tomato plants dramatically decreased under salinity stress; however, the application of inoculated bacteria improved the PPO contents by 85.0%. Interestingly, both the individual and combined treatment of mannitol and SPSB2 exhibited almost similar effects on PPO contents as the individual treatment with mannitol resulted in a 35.9% increase, while the combined treatment showed a slightly lower increase of 25.9% under NaCl stress condition. The results revealed that PPO contents were almost similar in NaCl and As-treated plants; however, the mannitol and SPSB2 treatment significantly increased the content by 54.7% and 76.1% under As stress conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003ePCA and correlation trait of tomato plants under NaCl and As stress\u003c/h2\u003e \u003cp\u003ePrincipal Component Analysis (PCA) results explored the variability in morpho-physiological, biochemical, and antioxidative traits of tomato plants under control, SPSB2 inoculated, and stress conditions of NaCl and As stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The data was collected from untreated control, SPSB2 inoculated, and mannitol-treated plants under control and stress conditions of NaCl and As. SPSB2 bacterial strain alleviated the salinity and As stress conditions in tomato plants. Our results showed that PC1 has greater values than PC2 in all three plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B, \u003cb\u003eand C\u003c/b\u003e). The eigenvalue increased and decreased one by one, but the cumulative value was induced in stress conditions. In control, 400 mM NaCl and 0.5 mM As stress conditions were recognized in biplots as 73.12%, 54.75%, and 55.27% cumulative values (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The PCA analysis showed that plants treated with SPSB2 were prominently represented in the PC1, and the SPSB2 treatment significantly impacted the plants' physiological, biochemical, and antioxidant contents under control conditions. Catalase was negatively correlated with plant morphological traits with combined treatments. Moreover, Pearson correlation analysis was conducted to determine the extent of the relationship among the characteristics. The results significantly correlated the morphological, biochemical, and antioxidant traits under control and stress conditions of NaCl and As in both inoculated and non-inoculated plants. Surprisingly, shoot, root length, FWS, FWR, and protein contents were positively correlated with other traits. On the other hand, RWC has a significantly negative correlation with other features shown in (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Overall, the results showed insight correlation interaction among different growth parameters in tomato plants under salt and arsenic stress and highlighted the potential of using SPSB2-treated plants to improve growth and stress tolerance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEigenvalue, variance, and cumulative of Control, NaCl, and As stress.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEigenvalue\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePercentage of Variance\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCumulative\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.96779\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e73.12%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e73.12%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.03221\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e26.88%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100.00%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.00%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100.00%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.21311\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e54.75%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e54.75%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eNaCl 400 mM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.51075\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e30.07%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e84.83%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.27615\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15.17%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100.00%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.00%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100.00%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.29107\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e55.27%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e55.27%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eAs 0.5 mM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.94274\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32.95%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e88.23%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.76619\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11.77%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100.00%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.00%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100.00%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of SPSB2 and mannitol treatment on stomata of tomato plant under NaCl and As stress\u003c/b\u003e \u003c/p\u003e \u003cp\u003eGenerally, abiotic stresses elevate the ROS levels and induce osmotic stress in plant cells. Abscisic acids (ABA) play a vital role in closing stomatal cells, thereby preventing the dehydration of transpiration pathways. Stomatal morphology was screened in the microscope to check the effect of inoculated SPSB2 bacterial strain and mannitol under salt and heavy metal stress conditions. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003e, under NaCl stress, a higher stomatal density accompanied by a decrease in size was observed compared to SPSB2-inoculated plants. The stomatal density of the plants treated with NaCl alone and those treated with NaCl\u0026thinsp;+\u0026thinsp;B was observed, and the number of stomata increased in the NaCl\u0026thinsp;+\u0026thinsp;B group. Similar trends were observed in the NaCl\u0026thinsp;+\u0026thinsp;M and SPSB2\u0026thinsp;+\u0026thinsp;mannitol-treated groups, indicating that bacterial (B) and mannitol treatments reduced the number of stomata. Moreover, plants treated with As stress exhibited smaller and open stomata compared to control and SPSB2-treated plants. On the other hand, in SPSB2 inoculated plants, larger and open stomata were observed, and mannitol treatment increased the size and openness of stomata under As stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eTomato is an essential vegetable crop grown globally (Kimura and Sinha \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Rothan et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). It serves as a model plant to study the impacts of abiotic stresses on various aspects of plant life, including growth, development, physiology, and molecular and biochemical processes (Shrivastava and Kumar \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Excessive salinity may negatively impact plants' growth, metabolism, and general physiology (Zhu \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Plants may experience water stress (Gupta et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and dehydration due to excess salts (Zhao et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Reduced water intake, blocked nutritional absorption, and stunted development are all possible outcomes (Zhao et al. \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). High sodium and chloride ions concentrations build up in plant tissues during salt stress, disrupting vital metabolic processes (Zaheer et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Similarly, the stress induced by heavy metals, including arsenic, cadmium, mercury, and lead, is prevalent in agricultural lands and poses significant harm to plants globally (Finnegan and Chen \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). High concentrations of heavy metals can inhibit plant functions, photosynthesis, and enzyme processes and produce reactive oxygen species ROS, which cause oxidative stress and are harmful to cells within the plants (Imran et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Modern technology, such as molecular breeding techniques (Ashraf and Akram \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), CRISPR and transcriptomic analysis (Gonzalez Guzman et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) have modified the crop under abiotic stress (Khan et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Furthermore, these methods might not always be feasible and can seriously affect the ecosystem (Aizaz et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Extensive research reveals the beneficial effects of halotolerant and halophilic microorganisms on plant growth (Kadyan et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Essghaier et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Bacterial strains exhibiting tolerance to grow in saline and heavy metal-contaminated soil produce several biological metabolites, which could be considered promising keys to sustaining plant growth and protection under different stressful conditions (Wang et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In this context, in the current study, we tried to isolate bacterial strains endowed with multiple biological activities to promote plant growth under salt stress and in the presence of phytotoxic concentrations of heavy metals. Moreover, the exogenous application of mannitol has been observed to mitigate the adverse effects of salinity and heavy metal stress. Studies indicate that mannitol treatment increases the tolerance of plants to salt and heavy metal stress by ion homeostasis, improving antioxidants, and decreasing ROS (Rathor et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Habiba et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Adrees et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Furthermore, mannitol can enhance plant biomasses, water, and nutrient uptake under abiotic conditions (Slama et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Aizaz et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The primary objective of the current study is to investigate the beneficial role of halotolerant bacteria strain and mannitol in mitigating the impacts the impacts of NaCl and As stress conditions on tomato plants. Our finding illustrated that the bacterial strain used in this study produces IAA, a plant growth-stimulating hormone and exhibits both phosphate-solubilizing and siderophore activities. According to previous work, plant growth promoting bacteria can enhance plant growth directly or indirectly by increasing available P, fixing nitrogen, sequestering iron by siderophores, and producing antibiotics and plant hormones (Glick et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Mantelin and Touraine \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). In current work, tomato plants treated with SPSB2 halotolerant bacterial strain and mannitol showed enhanced growth parameters such as shoot, root, length, and fresh weight compared to control plants by diminishing the adverse effects of NaCl and Arsenic stress. Consistent with our results, a more recent study has revealed that inoculation of two halophilic bacteria (\u003cem\u003eV. marismortui\u003c/em\u003e and \u003cem\u003eT. halophilus\u003c/em\u003e) to tomato seeds improves stem growth compared to the uninoculated control plants(Essghaier et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Previous studies also investigated that halotolerant and halophilic bacterial isolates have PGP traits that can alleviate salt stress (Orhan \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The results are also consistent with previous results (Dias et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Desale et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The production of IAA by halotolerant and halophilic bacteria is essential for plant development because this phytohormone plays an important role in root initiation, cell expansion, and cell division. The increased root length caused by IAA-producing bacteria can positively affect nutrient absorption from the soil by plants (Boiero et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Similarly, according to previous research, PGPB can maintain nutritional status and modify phytohormonal balance by producing plant growth regulators, improving plant tolerance to heavy metals and other climatic challenges and stimulating plant growth in metal-contaminated soil (Afzal et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Hewage et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Our results agree with previous studies that the subjective of endophyte bacteria and mannitol have been shown to influence the photosynthesis pigment of plants [54,66] positively. The results revealed that SPSB2 bacteria and mannitol induced the chlorophyll and carotenoid contents in plants, decreasing ion toxicity (Almuhayawi et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and inducing chloroplast metabolism (Das and Roychoudhury \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Various environmental stress leads to the excessive generation of ROS. It causes cell death (Auten and Davis \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Schieber and Chandel \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and damage to nucleic acid, protein, lipid, and other membrane organelles (Redza-Dutordoir and Averill-Bates \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; QAMER et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Excessive ROS has a harmful effect on plants. Thus, plants utilize enzymatic and non-enzymatic mechanisms to prevent deleterious ROS production (Ahmad et al. 2009; Sarker and Oba 2018; Aizaz et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003ea). Our results agree with those of (Rabiei et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), who found that catalase content shows a significant increase with inoculation of bacteria under non-stressed conditions, while mannitol treatment showed no significant change in catalase contents compared to control plants. However, when tomato plants were inoculated with the SPSB2-bacteria (\u003cem\u003eN. aquimarinus)\u003c/em\u003e, the catalase content increased significantly compared to mannitol treatment under the same non-stressed condition. Our results agreed with previous studies that mannitol showed a positive effect on catalase content under NaCl stress conditions when compared to their respective control plants. In contrast, the bacterial inoculation individually and combined with mannitol showed a slightly positive effect on catalase content (Khare et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Ajmal et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). We found that inoculating \u003cem\u003eA. esculentus\u003c/em\u003e with \u003cem\u003eB. megaterium\u003c/em\u003e UPMR2 and \u003cem\u003eEnterobacter sp.\u003c/em\u003e UPMR18 increased the expression of stress-related genes such SOD, APX, CAT, GR, and DHAR in response to NaCl stress, which is consistent with previous research (Habib et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In As stress condition, catalase contents were observed to be significantly increased by bacteria inoculation and mannitol treatment. However, catalase contents were slightly decreased in control and mannitol-treated plants compared to SPSB2 inoculated plants. The inoculated bacteria increased the protein contents, while a decrease was observed in mannitol-treated plants under control conditions. Furthermore, in combined treated plants with SPSB2 and mannitol, protein contents were significantly decreased in NaCl stress, identical to previous studies (Vı́tov\u0026aacute; et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Sugar and starch content were significantly higher in mannitol treated plants than control and bacterial inoculated plants under control conditions. Our results are consistent with previous reports that bacterial inoculation significantly increased the sugar and starch contents in salt and heavy metals stress conditions (Hajiabadi et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Jan et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Furthermore, inoculated bacteria enhanced the flavanol content compared to the control condition and under NaCl stress, flavanol content was significantly raised in plants with mannitol treatments compared to other treatments, contrasting with previous studies (Ahmed et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). However, inoculated bacteria had significantly higher content than mannitol and combined treatment under As stress. Stomatal pores are the major routes for gaseous exchange across the impermeable cuticle of leaves. Stomatal density and size influence gaseous exchange (Blatt \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In the current study, the number of stomata studied in control and NaCl-treated plants was fewer but large as compared to SPSB2 treated plants. Our results are in agreement with previous reports that plants sustain their physiological and metabolic processes, improve their resistance to heavy metals by multiplying their stomata, which expands their surface area and facilitates better uptake of CO\u003csub\u003e2\u003c/sub\u003e and water (Wahid et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eAs chemical pesticides and fertilizers have detrimental effects on human and environmental health, farmers are urged to employ more ecologically friendly substitutes. Using halotolerant plant growth-promoting bacteria is a sustainable solution that helps crops adapt to increasing salinity levels. In recent years, PGPR has had a more positive influence on agriculture, leading to the harvesting of commercially significant crops. The bacterial strains known as halotolerant plant growth promoters (HT-PGP) increase crop yield in salty conditions via several physiological and molecular processes. Using biopreparations based on non-pathogenic living microorganisms can offer several benefits for sustainable and environmentally friendly agricultural practices. Our findings supported the application of halotolerant bacteria as a potential tool to mitigate the effect of salt and heavy metal stress in tomato plants. Continued research is essential to focus on uncovering the underlying mechanisms of plant growth promotion under salinity and heavy metal stress and the practical aspects of integrating these strategies into mainstream agricultural practices to contribute to the resilience and productivity of agriculture globally.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Lubna, Mohammed Aizaz, Shima Ahmed Ali Alrumaidhi, Rawan Ahmed Mohammed Alhinai, and Reem Saif Mohammed AL Kalbani performed experimental and analysis. Ibrahim Khan extracted DNA and microbes identification. Saqib Bilal performed phytohormone and antioxidant analysis, and Sajjad Asaf wrote the draft manuscript and statistical analysis. Ahmed Al-Harrasi, supervision and arranging resources.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe authors have no competing interests to declare that are relevant to the content of this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAdesemoye A, Torbert H, Kloepper J (2009) Plant growth-promoting rhizobacteria allow reduced application rates of chemical fertilizers. 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Annual review of plant biology 53 (1):247-273\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":false,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"halotolerant bacteria, tomato, salt and heavy metal stress, mannitol","lastPublishedDoi":"10.21203/rs.3.rs-4798297/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4798297/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAgricultural productivity is adversely affected by soil salinization and contamination with heavy metals, emphasizing the necessity for environmentally friendly technologies. This study investigates the impact of sodium chloride (NaCl) and arsenic (As) stress on tomato seedlings and explores the stress-alleviating effects of mannitol and a halophilic bacterium, \u003cem\u003eNitratrieducator aquimarinus\u003c/em\u003e SPSB2. Our results revealed that bacteria strainSPSB2 establishes a symbiotic relationship with tomato plants, which modulates the secondary metabolites and antioxidant system in tomato plants exposed to both NaCl and As stress. Under the NaCl and As stress tomato seedling growth was significantly reduced, although this reduction was mitigated by bacteria strain SPSB2 and mannitol treatment. When exposed to NaCl stress, the bacterial strain enhances shoot and root length by 84.8% and 152.5%, respectively. Similarly, under the As stress conditions, bacteria strain SPSB2 inoculation increased the shoot and root weights by 63.1% and 45.5%, respectively. Bacteria strain SPSB2 inoculation also significantly enhanced the chlorophyll a, b, and carotenoid contents by 76.3%, 78%, and 50%, respectively, compared to their non-inoculated counterparts under As stress conditions. Furthermore, during NaCl and As stress conditions, treatments with SPSB2 and mannitol increase the levels of enzymatic components (catalase, polyphenol oxidases) and non-enzymatic components (flavonol protein, sugar, starch), indicating a stress-alleviating effect of bacteria strain SPSB2 and mannitol. In the current study, the bacteria strain SPSB2 was more effective than mannitol in improving tomato plants' salinity and heavy metal tolerance regarding growth and physiological attributes. The symbiotic relationship between SPSB2 and tomato plants positively impacted various parameters, including plant growth, chlorophyll content, and antioxidant system activity. Moreover, the study suggests that SPSB2 is more effective than mannitol in improving tomato plants' salinity and heavy metal tolerance. These findings contribute to the understanding of environmentally friendly strategies for managing soil salinization and heavy metal contamination in agriculture, and the potential use of SPSB2 in microbial-assisted phytoremediation of polluted saline soils.\u003c/p\u003e","manuscriptTitle":"Exploring the Interactive Mechanisms of Halophilic Bacterium SPSB2 and Mannitol in Mitigating Sodium Chloride and Arsenic Stress in Tomato Plants","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-26 07:11:33","doi":"10.21203/rs.3.rs-4798297/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"12fe1600-5da4-4e9c-b0e4-3fb31333a1d7","owner":[],"postedDate":"August 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-07T13:09:28+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-26 07:11:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4798297","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4798297","identity":"rs-4798297","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}