Rhizobacteria modify soil biological indices and induce tolerance to osmotic stress in tomato depending on the salinity level and bacteria species

Abstract

Salinity is a major abiotic stress that impacts crop productivity globally. Plant growth-promoting rhizobacteria (PGPRs) exploit several mechanisms to not only decrease soil salinity but also improve the systemic tolerance of plants to osmotic stress. In this work, the effect of five PGPR strains was investigated on the growth and physiological responses of tomato plants, including stomatal closure, proline, and K⁺ and Na⁺ content under a range of salt stress, 0, 2.5, 5, 7.5, and 10 dS m⁻¹. The effect of PGPR strains and salinity levels on the soil biological characteristics was also investigated. Salt stress affected the plant growth and physiological factors and soil biological factors in a dose-dependent manner. The highest saline stress, 10 dS m⁻¹, reduced shoot and root dry weight and root volume up to 51.3, 41.5, and 51.8%, respectively. It also increased stomatal resistance and proline content 2.01- and 3.66-folds and decreased K⁺/Na⁺ ratio 4.16-folds, respectively. It also reduced basal respiration, substrate-induced respiration, and microbial biomass carbon up to 2.25-, 4.83-, and 6.7-folds and increased qCO2 3.18-folds, respectively. PGPR strains were able to modulate salt tolerance mechanisms, improve plant growth factors, and improve soil biological indicators. Bacillus megaterium P2 was the best strain in the balancing K⁺/Na⁺ uptake at least at 10 dS m⁻¹. However, the efficiency of strains was dependent on the magnitude of salt stress. Therefore, it is possible to introduce PGPR strains based on soil salt level or exploit rhizobacteria consortia to manage salt stress in different conditions.

Introduction

Soil salinization is the main threat to crop production in many agricultural lands and affects almost 1 billion ha worldwide. Many of the widely used crops are susceptible to salt stress higher than 4 dS m⁻¹. Natural salinity, inappropriate irrigation/drainage practices, and climate change may increase saline landscapes [1, 2]. A large quantity of Na⁺ and Cl⁻ ions in saline soils not only are phytotoxic but also can lead to a decrease in accessibility and uptake of other elements such as N, P, K, and Mg [1, 3–5]. Soil salinization also induces the biosynthesis of ethylene that inhibits root growth and the biosynthesis of ABA that elicits stomatal closure [6–8]. These effects maintain turgor pressure but may cause a reduction in water uptake and affect the balance in nutrient uptake by the seedling [1, 9]. Salinity also elicits reactive oxygen species (ROS) accumulation and osmotic stress, which decreases photosynthesis, chlorophyll content, and stomatal conductance, and increases membrane peroxidation [3–5, 9]. Altogether, salinity affects all steps in crop production, from seed germination to grain production and even grain quality [3, 10, 11].

Soil Salinity not only affects soil physical and chemical factors, but also changes soil microbiome and microbial activities [12–15]. Although microbes represent less than 0.5% of soil weight, they play a critical role in soil productivity [16]. It is reported that soil salinity decreases soil microbial features and increases soil metabolic quotient (qCO₂) which decrease nutrient mineralization and soil fertility [17–19]. Thus, soil salinity can indirectly affect plant growth via alteration of microbial abundance and their activities.

Microbial inoculants are promising technology for improving plant health in saline conditions. These microbes not only can ameliorate salt stress, but also promote plant growth and control plant pests and diseases [1, 7, 8, 20]. These beneficial microbes are known as plant growth-promoting rhizobacteria or fungi (PGPR or PGPF) or plant probiotics [7, 21]. PGPR exploits several mechanisms to alleviate soil salinity or induce systemic tolerance (IST) to osmotic stress in plants [2, 5, 22]. PGPRs reduce the soil Na⁺ level by producing extracellular polysaccharides (EPS) or by reducing soil pH. Pseudomonas aeruginosa increased sunflower tolerance to salt, while its EPS mutant failed to ameliorate salt stress [23]. Cyanobacteria also mitigate soil salinity by reducing pH from 8.8 to 5 and reducing Na⁺ from 0.78 to 0.60 ppm [24]. However, PGPRs in the most of cases modulate plant systemic tolerance to osmotic stress [15]. Bacillus subtilis GB03 modulates nitric oxide, a central signal molecule, which regulates the expression of sodium transporters such as HKT3;5 and HKT1 [4, 25]. These transporters promote basipetal translocation and extruding Na⁺ from root cells, respectively. Rhizobacteria also can protect plants from the adverse effects of osmotic stress by inducing the accumulation of osmoprotectants such as proline, glycine betaine, and soluble sugars or by reducing stress hormones ethylene and ABA [1, 7, 8]. Gluconacetobacter diazotrophicus increased the trehalose and α-tocopherol content in rice roots (Oryza sativa). This bacterium also improved root system architecture by inducing some genes related to auxin and cytokinin signaling [26, 27]. 1-Aminocyclopropane-1-carboxylic acid (ACC) deaminase produced by a wide range of rhizobacteria converts ACC, ethylene precursor, to a-ketobutyrate [7, 28]. ACC conversion not only improves root proliferation, but also promotes plant tolerance to abiotic stresses [7].

In this study, the effect of salinization stress levels was investigated on the growth and some physiological parameters of tomato plants and soil biological characteristics under greenhouse condition. In addition, the ability of plant probiotic bacteria from different taxonomic positions was checked on the amelioration of osmotic stress either in physiologic and phonotypic levels.

Materials and methods

A factorial experiment was conducted in the greenhouse situation with two factors, different bacterial strains and a range of salinity levels. The effects of exploited factors and their interactions were investigated on the plant growth factors, stomatal resistance, proline content, Na⁺ and K⁺ ratio, and soil biological indices.

Physical and chemical characteristics of the soil

The soil samples were collected from an agricultural field in the college of agriculture, Razi University, and transported to soil analysis labs. Soil samples were subjected to analysis for some soil physical and chemical properties based on standard procedures. Soil samples were air-dried and passed through a 2-mm sieve before measuring their properties. The analysis methods were as follows: soil texture was analyzed by Bouyoucos hydrometer method [29] and total N by the microkejeldal technique [30], and organic carbon was measured by Walkly [31] procedure. The soil pH was determined based on 1:2 soil water suspension by a glass electrode [32]. The electrical conductivity (EC) was checked in the saturated extract [33]. The calcium carbonate equivalent was determined by acid neutralization method [34]. Cation exchange capacity (CEC) was measured using sodium acetate procedure at pH 8.2 [35]. Available phosphorus was determined by Bray No. 1 method [36]. Potassium was determined by means of the flame photometry method [37].

Rhizobacteria strains

Five bacterial strains (Pseudomonas geniculata B11, Achromobacter sp. B124, Bacillus megaterium P2, Lysinibacillus sphaericus B19, and Bacillus cereus B40) were obtained from the culture collection of probiotic bacteria, Plant Protection Department, Razi University, Kermanshah, Iran. According to tomato growth promotion, these strains had been screened from more than 250 isolates derived from tomato rhizosphere and endorhizosphere [38]. A commercial bacterial strain B. megaterium P2 was kindly provided by Hezar-dane Arman Co, Kermanshah, Iran. The bacterial inoculum was prepared in 250-mL flask containing 150 mL nutrient broth and incubated for 48 h at 30 °C on a 120 rpm rotary shaker. After centrifugation, the pellet was re-suspended in sterile distilled water containing 1% carboxymethyl cellulose. The concentration of bacterial suspension was adjusted to 1 × 10⁹ CFU/mL.

Pot experiment

Tomato seeds (cultivar Karoon) were sown in transplant trays filled with sterilized coco peat and kept in greenhouse conditions at 25 ± 4° C for 30 days. For bacteria inoculation, the roots of 30 days old tomato seedlings were dipped in each bacteria suspension (1 × 10⁹ CFU/mL) containing 1% carboxymethyl cellulose for 20 min. Solution of 1% carboxymethyl cellulose without bacteria was used as control. Plastic pots with 18 cm diameter and 16 cm height were filled by sterilized a 2:1 mixture of field soil and sand. For the first 2 weeks, the pots were irrigated with tap water every other day. Salt levels, 0, 2.5, 5, 7.5, and 10 dS m⁻¹ were applied using equal ratio of sodium chloride and calcium chloride at cultivation time [14, 39]. All the plants were irrigated enough to ensure drainage. The pots were kept in a greenhouse (25 ± 4° C and 50–80% humidity) and irrigated every 2 days. Sixty days after planting, the plants were harvested, and their growth factors including root and shoot dry weight and root volume were measured.

Plant physiological factors

Stomatal resistance was calculated for each salt stress period using a porometer (Decagon Devices Inc. version 1.06). Proline content was determined as described by the modified Bates et al. [40], at 30 days after the onset of the experiment. Leaf tissues (0.25 g) were placed in a tube with 10 mL of 3% (w/v) aqueous sulfosalicylic acid in water. Proline was extracted from 1 mL sample using 1 mL ninhydrin reagent and heated for 1 h at 100 °C. Tubes were cooled, and 2 mL of toluene was added to each tube and vortexed for 2 min. Toluene phase was decanted, and the absorbance of toluene phase was read at 520 nm. Proline concentration was measured using a standard curve of L-proline. To determine leaf sodium and potassium content, dried leaves were ground and passed through the 40 mesh and analyzed by flame photometer [41]. Next, the concentration of Na⁺ and K⁺ was calculated using the calibration curve based on dry tissue.

Soil biological indices

At harvesting time, 50 g of soil was passed through a 4-mm sieve to determine soil microbial biomass. The soil samples were pre-incubated for 7 days and fumigated with chloroform for 24 h. Soil microbial biomass carbon (MBC) was measured using the fumigation–incubation method [42, 43]. Basal soil respiration (BR) was determined in an incubation experiment for 10 days and expressed as mg C kg¹ soil day¹. Substrate-induced respiration (SIR) was determined same as BR in the presence of 1 mL of 1% glucose as carbon substrate [42, 43]. Soil microbial metabolic quotient (qCO2) was calculated as the ratio of the MBC in the total organic C per unit time [19, 42].

Data analysis

Factorial experiments were carried out based on a completely randomized design with four replications. Analysis of variance was performed using SAS V9.1 statistical software. The mean values were compared using the least significant difference (LSD) test at the 5% level.

Results and discussion

Soil analysis

Soil analysis showed that the tested soil had a silty clay loam texture. EC was 0.42 dS m⁻¹, and sodium adsorption ratio and available potassium content were 0.14 meq Lit⁻¹ and 330 mg kg⁻¹, respectively (Table 1).

Table 1.

Physical and chemical characteristics of the soil

Characteristics	Unit	Value
Soil texture	-	Silty clay loam
OC	%	0.84
pH	-	7.12
CEC	meq 100gr−1	24.5
EC	dS m−1	0.8
Pava	mg kg−1	15
CaCO3	%	26.7
Kava	mg kg−1	330
SAR		0.14

The effect of bacterial strains on the plant growth

Plant growth was investigated in this soil in the presence of 0, 2.5, 5, 7.5, and 10 dS m⁻¹ salt levels. Analysis of variance showed that bacterial strains and salt stress levels did not have significant interaction on shoot and root dry weight and root volume (Table 2). So, the simple effect of each factor was interpreted in detail. Results showed that salt levels negatively affected the measured plant growth factors in a dose-dependent manner (Fig. 1a–c). Even the low level of salt stress decreased plant growth significantly. However, the highest level of salt stress, 10 dS m⁻¹, decreased shoot and root dry weight and root volume up to 51.3, 41.5, and 51.8% compared to the control, respectively. Root inoculation by rhizobacteria alleviated salt stress and improved the plant growth in the presence of different salt levels. Bacillus cereus B40 had the lowest effect on the alleviation of salt stress. However, this strain increased shoot dry weight up to 12.5% compared to the control (Fig. 1b). Pseudomonas geniculata B11, Achromobacter sp. B124, and Bacillus megaterium P2 had the highest effect on improving shoot dry weight. Pseudomonas geniculata B11 increased root dry weight and root volume 62 and 35%, respectively. Achromobacter sp. B124 also increased root dry weight and root volume 32 and 48%, respectively.

Table 2.

Analysis of variance for the effect of salt levels and bacterial strains on the plant growth factors

Mean of squares
Sources of variation	df	Shoot dry weight	Root dry weight	Root volume
Rep	3	26.80*	0.63*	53.37*
Bacteria	5	28.80*	1.62*	73.50*
Salt	4	145.64*	2.43*	288.94*
Bacteria × salt	20	2.52 ns	0.02 ns	1.22 ns
Error	90	13.94	0.02	3.53
C.V.(%)		13.48	8.78	15.10

Fig. 1.

The simple effect of different salt levels (a, b, and c) and five rhizobacteria strains (d, e, and f) on the tomato growth factors. Tomato seedlings were sown in pots adjusted to saline conditions 0, 2.5, 5, 7.5, and 10 dS m⁻.¹. Rhizobacteria were applied by seedling root inoculation method. Data were collected 60 days post sowing time. Different letters indicate significant differences between treatments according to Fisher’s LSD test at P < 0.05

The effect of bacterial strains on the stress tolerance mechanisms

The effect of salt levels and bacterial strains were investigated on the plant’s physiological and nutritional features (Table 3). Increased salt concentration modulates defensive mechanisms, including stomatal resistance, proline content, and Na⁺/K⁺ ratio in plant tissues. In comparison to the control, the highest salt level, 10 dS m⁻¹, increased stomatal resistance from 55.2 to 111 mM m⁻² s⁻¹, proline content from 1.89 to 6.92 µM g⁻¹ DW⁻¹, sodium content from 38.89 to 121.97 mg/kg, and decreased potassium content from 7.29 to 5.48 g/kg (Table 3) and K⁺/Na⁺ ratio 4.16-folds (data not shown).

Table 3.

The effect of different salt levels and rhizobacteria strains on tomato physiological and nutritional characteristics

Factors	Salt levels			Bacteria strains
		Lysinibacillus sphaericus B19	Pseudomonas geniculate B11	Achromobacter sp. B124	Bacillus megaterium P2	Bacillus cereus 40	Control
Stomatal resistance (mM m−2 s−1)	0	37.28 ± 3.35 no	49.40 ± 4.65 m	52.12 ± 5.64 klm	51.25 ± 2.86 lm	31.62 ± 4.65 o	55.25 ± 3.78 j-m
	2.5	39.28 ± 1.05 n	51.40 ± 2.57 klm	54.17 ± 3.39 jklm	53.25 ± 0.31 klm	33.62 ± 1.32 no	57.52 ± 0.93 ijkl
	5	57.40 ± 4.86 ijkl	58.80 ± 6.07 ijk	61.22 ± 3.67 ij	63.18 ± 2.01 hi	51.58 ± 2.16 klm	63.91 ± 2.46 hi
	7.5	77.80 ± 2.71 ef	77.51 ± 5.75 f	69.62 ± 9.25 gh	75.83 ± 4.59 fg	81.80 ± 2.91 ef	84.52 ± 1.03 e
	10	103.65 ± 4.64 bc	98.53 ± 14.04 bcd	104.37 ± 4.43 b	97.47 ± 3.38 cd	93.28 ± 8.28 cd	111.1 ± 2.13 a
Proline (µM g−1 DW−1)	0	2.32 ± 0.34 h	2.22 ± 0.29 h	2.14 ± 0.25 h	2.29 ± 0.38 h	1.95 ± 0.34 h	1.89 ± 0.26 h
	2.5	2.40 ± 0.27 h	2.31 ± 0.14 h	2.23 ± 0.18 h	2.38 ± 0.67 h	2.36 ± 0.48 h	1.98 ± 0.34 h
	5	3.69 ± 0.16 f	3.45 ± 0.19 fg	3.06 ± 0.16 g	3.54 ± 0.16 fg	3.45 ± 0.46 fg	2.41 ± 0.32 h
	7.5	5.73 ± 0.10 c	5.37 ± 0.33 cd	5.27 ± 0.26 cd	5.26 ± 0.48 cd	5.11 ± 0.22 d	4.35 ± 0.24 e
	10	7.21 ± 0.33 b	8.38 ± 0.25 a	7.49 ± 0.85 b	8.31 ± 0.36 a	8.35 ± 0.25 a	6.92 ± 0.27 b
Leaf sodium (g/kg)	0	27.49 ± 0.31 p	33.32 ± 0.36 n	30.43 ± 0.29 o	32.69 ± 0.61 n	30.28 ± 0.33 o	38.89 ± 0.86 l
	2.5	36.68 ± 0.41 m	37.04 ± 0.57 lm	33.64 ± 0.52 n	37.58 ± 0.70 lm	37.77 ± 0.85 lm	59.45 ± 1.09 k
	5	61.36 ± 1.10 j	63.77 ± 0.96 i	65.71 ± 0.97 h	61.85 ± 2.41 j	65.22 ± 1.12hi	81.28 ± 1.18 g
	7.5	79.73 ± 1.02 g	84.93 ± 1.05 ef	86.53 ± 2.44 e	83.36 ± 1.20 f	80.77 ± 1.97 g	99.02 ± 1.11 d
	10	102.97 ± 1.31 c	108.14 ± 1.16 b	103.43 ± 1.85 c	100.15 ± 2.14 d	104.76 ± 1.95 c	121.97 ± 1.43 a
Leaf Potassium (g/kg)	0	7.62 ± 0.14 a	7.49 ± 0.26 ab	7.62 ± 0.15 a	7.44 ± 0.09 ab	7.31 ± 0.37 a-d	7.29 ± 0.17 bcd
	2.5	7.41 ± 0.26 abc	7.41 ± 0.16 abc	7.41 ± 0.18 abc	7.24 ± 0.16 bcd	7.11 ± 0.16 cde	7.08 ± 0.24 def
	5	6.83 ± 0.29 e–h	6.70 ± 0.32 ghij	6.80 ± 0.34 e-i	6.78 ± 0.33 f-j	6.80 ± 0.10 e-i	6.62 ± 0.21 g-j
	7.5	6.47 ± 0.31 jk	6.90 ± 0.19 efg	6.50 ± 0.28 ijk	6.57 ± 0.21 h–k	6.29 ± 0.17 k	6.27 ± 0.13 k
	10	5.48 ± 0.12 n	5.60 ± 0.36 mn	5.58 ± 0.31 mn	5.91 ± 0.24 l	5.86 ± 0.11 ml	5.48 ± 0.17 n

Different letters indicate significant differences between treatments according to Fisher’s LSD test at P < 0.05

Rhizobacteria inoculation promotes physiological and nutritional mechanisms to induce plant systemic tolerance to osmotic stress, thereby improving plant growth. All bacterial strains reduced stomatal resistance. B. cereus B40 in non-salinized condition decreased stomatal resistance up to 42.77%. Compared to bacterial inoculation control, this strain also reduced stomatal resistance up to 56, 44, and 20% in co-treatment with 2.5, 5, and 10 dS m⁻¹, respectively. However, Achromobacter sp. B124 was the best strain only in 7.5 dS m⁻¹ of salt stress and decreased stomatal resistance up to 17.63% compared to non-inoculated control.

The proline content increased linearly depending on the increase in salinization level. In the absence of rhizobacteria, the highest proline level was 6.92 µM g⁻¹ DW⁻¹. P. geniculata B11, B. megaterium P2, and Achromobacter sp. B124 increased the proline content in the same salinization level up to 21.10, 20.8, and 8.24%, respectively (Table 3).

Bacterial strains also decrease plant sodium accumulation either in non-salinized condition or in the treatment with different salt levels. In non-salinized control, all bacterial strains decrease sodium content noticeably (Table 3). L. sphaericus B19 exhibited the highest impact and reduced sodium content up to 30% compared to control. However, in the highest salt concentration, B. megaterium P2 as the best strain reduced sodium assimilation up to 17.88% compared to the non-inoculated control. In this salt level, P. geniculata B11 showed the lowest impact and decreased the sodium content only 12.78%.

In contrast to sodium, bacterial strains increase potassium uptake significantly. L. sphaericus B19 and Achromobacter sp. B124 increased potassium accumulation in non-salinize control. But in 2.5 dS m⁻¹ salt level, all rhizobacteria strains improved the potassium content, noticeably. However, most bacterial strains could not increase the potassium content in 10 dS m⁻¹ of salinization. The highest effect in this salt concentration was recorded in the case of B. megaterium P2, which increased the potassium accumulation just 7.84% compared to the non-inoculated control.

Soil biological indices

Soil salinity modified soil biological features, noticeably (Table 4). At 10 dS m⁻¹ of salinization, BR, SIR, and MBC reduced up to 2.25-, 4.83-, 6.70-folds, respectively. In contrast, qCO2 increased 3.46 times in this salinity level. In non-salinized soil, bacterial strains Achromobacter sp. B124, P. geniculata B11, and P. geniculata B11 increased BR, SIR, and MBC up to 47.14%, 39.70%, and 35.05%, respectively. However, bacteria strains did not significantly affect qCO₂ in non-salinized soil. At 2.5, 5, 7.5, and 10 dS m⁻¹ soil salinity, bacteria strains P. geniculata B11, Achromobacter sp. B124, P. geniculata B11, and B. megaterium P2 had the highest effect on increasing BR, and bacterial strains P. geniculata B11, Achromobacter sp. B124, P. geniculata B11, and P. geniculata B11 had the highest impact on increasing BR, and bacterial strains P. geniculata B11, Achromobacter sp. B124, B. megaterium P2, and L. sphaericus B19 had the highest effect on increasing MBC, respectively. Bacteria strains did not significantly affect qCO₂ at 0, 2.5, and 5 dS m⁻¹ soil salinity, but strains L. sphaericus B19 and P. geniculata B11 were the best strains in reducing qCO₂ up to 43.82% and 42.58% at 7.5 and 10 dS m⁻¹ soil salinity, respectively.

Table 4.

Mean comparison of interaction between salt levels and bacterial strains on soil biological characteristics

Bacterial strains
Biological indices	Salt Levels dS m−1	Bacillus cereus B40	Bacillus megaterium P2	Achromobacter sp. B124	Pseudomonas geniculate B11		Lysinibacillus sphaericus B19	شاهد Control
Microbial biomass carbon (mg kg−1 soil)	0	423.23 ± 5.57 f	514.30 ± 4.04 c	611.60 ± 7.02 a	621.67 ± 18.01 a		501.30 ± 10.60 cd	404.00 ± 3.511 g
	2.5	362.67 ± 7.00 i	387.33 ± 3.06 gh	496.08 ± 5.13 cd	580.20 ± 9.00 b		488.30 ± 8.00 de	370.33 ± 9.50 hi
	5	342.00 ± 6.00 j	268.67 ± 3.51 m	325.13 ± 7.00 jk	425.67 ± 8.02 f		475.67 ± 8.02 e	290.33 ± 12.50 l
	7.5	157.33 ± 7.02 qr	195.30 ± 2.52 lm	201.20 ± 8.02 o	318.67 ± 2.51 k		300.34 ± 13.50 l	184.30 ± 4.50 op
	10	88.00 ± 6.56 u	171.30 ± 4.50 pq	134.33 ± 5.69 s	222.33 ± 6.01 n		140.03 ± 5.00 rs	111.30 ± 7.20 t
Basal respiration (mg C kg−1 soil day−1)	0	19.80 ± 1.05 f	22.75 ± 0.70 de	28.47 ± 0.50 a	27.20 ± 0.60 b		23.20 ± 0.65 cd	16.76 ± 0.75 hij
	2.5	15.70 ± 0.70 jkl	17.03 ± 0.85 hi	21.83 ± 0.80 e	24.00 ± 0.90 c		19.30 ± 0.60 f	15.13 ± 0.8 klm
	5	16.37 ± 0.65 ijk	14.76 ± 0.76 lmn	19.23 ± 0.25 f	17.83 ± 0.55 gh		18.76 ± 0.61 fg	14.23 ± 0.55 mno
	7.5	11.80 ± 0.80 rs	12.76 ± 0.25 pqr	12.80 ± 0.80 pqr	14.20 ± 0.95 mno		13.36 ± 0.75 opq	12.56 ± 0.60 p-s
	10	11.56 ± 0.75 rs	13.50 ± 0.86 nop	12.10 ± 1.10 qrs	12.93 ± 0.91 o-r		12.13 ± 0.61 qrs	11.23 ± 0.70 s
Substrate-induced respiration (mg C kg−1 soil day−1)	0	20.03 ± 0.55 hi	29.50 ± 0.50 c	31.83 ± 0.80 b	33.33 ± 0.35 a		24.56 ± 0.60 e	20.10 ± 0.75 hi
	2.5	19.83 ± 0.32 hi	24.26 ± 0.56 e	26.43 ± 0.95 d	28.36 ± 0.95 c		20.73 ± 1.28 gh	17.76 ± 0.79 k
	5	17.73 ± 0.65 k	19.06 ± 0.75 ij	22.20 ± 0.82 f	21.50 ± 0.50 fg		19.93 ± 1.13 hi	15.36 ± 0.80 l
	7.5	12.90 ± 0.60 mn	18.13 ± 0.85 jk	14.06 ± 0.79 m	20.50 ± 0.80 gh		13.73 ± 0.75 m	13.03 ± 0.90 mn
	10	13.13 ± 0.45 mn	12.73 ± 0.22 mn	11.80 ± 0.80 n	16.26 ± 0.66 l		12.73 ± 0.70 mn	12.20 ± 0.70 n
Metabolic quotient (mg C gMBC−1 day−1)	0	0.047 ± 0.0031 jk	0.044 ± 0.0011 k	0.047 ± 0.0014 jk	0.69 ± 0.0003 k		0.045 ± 0.0004 k	0.041 ± 0.0022 k
	2.5	0.76 ± 0.0028 jk	0.044 ± 0.0026 k	0.043 ± 0.0012 k	0.69 ± 0.0022 k		0.040 ± 0.0006 k	0.040 ± 0.0010 k
	5	0.048 ± 0.0011 jk	0.055 ± 0.0021 ij	0.059 ± 0.0021 hi	0.042 ± 0.0021 k	0.040 ± 0.0006 k		0.049 ± 0.0003 jk
	7.5	0.075 ± 0.0018 ef	0.065 ± 0.0017 gh	0.064 ± 0.0061 gh	0.045 ± 0.0034 k	0.045 ± 0.0005 k		0.068 ± 0.0049 fg
	10	0.132 ± 0.0186 a	0.079 ± 0.0030 de	0.090 ± 0.0121 c	0.058 ± 0.0026 hi	0.087 ± 0.0014 cd		0.101 ± 0.0002 b

Different letters indicate significant differences between treatments according to Fisher’s LSD test at P < 0.05

Discussion

Salinity and drought stress are two interconnected stresses causing water deficiency and osmotic stress in plants [3, 6]. In both stresses, the plant suffers from the toxicity of some elements such as Na⁺ and Cl⁻ and reactive oxygen species (ROS) [1, 3, 6]. ROS accumulation increases lipid membrane peroxidase and has a negative effect on the photosynthesis and respiration pathways. That’s why, salinized soil above 2 dS m⁻¹ has a negative effect on tomato growth and yield [10, 11]. Our results also showed that salinization reduced tomato shoot and root growth concentration-dependent (Fig. 1a–c). However, PGPR strains were able to rescue plant growth under high salinity conditions (Fig. 1d–f).

PGPRs employ several direct and indirect mechanisms to support plant growth and productivity under salinity stress. PGPRs alleviate saline stress by regulating sodium and potassium uptake and their homeostasis in plant tissues [3, 4, 25]. They also improve lateral and root hair proliferation, increasing plant water use efficiency [26, 44]. Our results also showed that although salinization decreases root proliferation noticeably, rhizobacteria such as Achromobacter sp. B124 improve root volume in the presence of different salinity levels (Fig. 1c and f). Rhizobacteria improve root volume by mechanisms such as basipetal movement of auxin [44] and inhibition of ethylene biosynthesis in root tissue [7, 28]. Moreover, rhizobacteria also can reduce the hydraulic conductivity of roots, which can be sensed by plants as slight stress to increase root growth. Thus, even in the absence of salt stress, some rhizobacteria induce plant root growth as well as an increase in stomatal resistance [45, 46]. Plants close their stomata and increase osmoprotectants such as proline to protect cells from osmotic stress without inhibiting enzymatic activities [47, 48]. Osmoprotectants also improve membrane stabilization in the presence of radial oxygens [9, 48]. In the current study, rhizobacteria increased proline content in all salt levels, significantly. They also decreased stomatal closure to improve water uptake and respiration. Rhizobacteria also decreased sodium accumulation and increased the K⁺/Na⁺ ratio to avoid sodium toxicity. So, rhizobacteria regulate sodium/potassium homeostasis by regulating sodium transporter such as SOS1 and HKT1 in root cells in a sophisticated manner to extrude Na⁺ from root cells and improve plant K⁺/Na⁺ balance [4, 25].

PGPRs can also modulate soil microbial characteristics by modifying root exudates or directly change microbiome when it introduced in high density. Several studies report the effect of PGPR inoculation on improving the soil biological indices [12–14]. For instance, introducing microbes can degrade soil organic matter and release labile C sources such as methanol and acetone, which changes the microbial community [49]. Bacteria can also release metabolites as growth stimulators, growth inhibitors, or quorum sensingrelated signals to shape soil microbial communities and their activities [49, 50]. They also can promote the synthesis and release of such compounds by host plants [51, 52].

Conclusion

Briefly, rhizobacteria used in the current study, except Bacillus cereus B40, improved the root proliferation and increased shoot growth. Bacillus cereus B40 did not significantly affect on root volume and root dry weight, but reduced stomatal resistance even in non-salt stress condition. It indicates that the bacterium constitutively decreases stomatal resistance in tomato which interfere in fine-tuning this phenomenon in the host plant. However, other strains primed the stomatal opening only in stress condition. Rhizobacteria also reduced the Na⁺ content and decreased its toxicity in plant tissues and increased proline to protect cell physiological function and alleviate osmotic stress. However, rhizobacteria were able to reduce Na⁺ content in non-stress condition. They may adsorb Na⁺ by negative charge EPS or induce Na⁺ extrusion from the plant root.

The efficiency of PGPR strains was dependent to salinity levels and type of plant growth and physiological factors. Indeed, rhizobacteria exploit several plant growth promotion and stress tolerance induction mechanisms in strains specific manner [3, 8, 53]. In our study, each strain relies more on a specific mechanism. B. megaterium P2 was the best strain in the balancing sodium/potassium uptake at least in 10 dS m⁻¹. Pseudomonas geniculate B11 was the best strain in improving soil indices. However, strains had different abilities according to salinity levels. Therefore, preparing and application of rhizobacteria consortia are a promising method to protect plants under different salinity conditions.

Author contribution

Sheida Naseri, conceptualization, data collection, and methodology. Ali Beheshti Ale Agha, conceptualization, supervision, investigation, and data collection. Rouhallah Sharifi, conceptualization, supervision, methodology, investigation, and writing — review and editing. Sohbat Bahraminedjad, supervision, methodology, and validation.

Funding

This study was funded by Razi University, Kermanshah, Iran (grant number 26.4.97).

Declarations

Conflict of interest

The authors declare no competing interests.