Introduction

Nigella sativa L. is a plant that comes from dry and semi-arid areas. In the Ranunculaceae family, Nigella sativa L. is an annual herb that is native to several countries in southern Europe and Asia, including Turkey, Syria, Saudi Arabia, India, and Pakistan1. Nigella sativa L. has been recommended in various conventional medical practices, including Unani, Ayurveda, and Tibb. The antioxidant, anti-inflammatory, anticancer, neuroprotective, cardioprotective, and hepatoprotective properties of Nigella sativa L. make it a medicinally important plant2,3. The essential oil of Nigella sativa L. contains medicinally important and pharmacologically important compounds such as thymoquinone (TQ), carvacrol, nigellidine, nigellicine, and hederin4. It is also vital that nutraceuticals, as their components, support the immune system5.

However, cultivation of this vital crop is affected by soil salinity6. When excessive fertilizer is applied to the soil, crop production is lost, and the soil becomes highly salinized7,8. To withstand excess salt ions, plants undergo biochemical and physiological changes9,10. Moreover, the enhanced activity of antioxidant enzymes reduces the damage caused by ROS due to salinity stress11. The regulation of these physiological and biochemical processes is based on the activities of the crop microbiome. Salinity may negatively affect the growth, yield, and physiological processes of Nigella sativaL12,13,14,15..

Additionally, it has been observed that farming methods significantly impact crop growth in saline soils15. Ploughing down or tilling more deeply increases evaporation of soil water, leaving behind excess salts. Additionally, salts in irrigation water can increase soil salinity, thereby reducing productivity16. Traditional physical and chemical methods of salinity mitigation have certain limitations17. To ensure sustainable agricultural production on saline soils, sustainable measures should be employed alongside salt-tolerant plant varieties and chemical neutralization techniques18. Recent studies have demonstrated that plant growth-promoting rhizobacteria (PGPR) can mitigate salinity while boosting soil fertility and crop yield19,20. PGPR works by fixing nitrogen, producing phytohormones, enzymes, and solubilizing minerals to enhance plant growth under biotic and abiotic stresses21,22,23,24,25. Thus, PGPR can convert unfertile soils to fertile soils, thereby enhancing plant adaptation to various biotic and abiotic stresses26,27,28. Overall, PGPR as biofertilizers offers an affordable and environmentally acceptable method to improve crop growth and yield under salinity stress, making them a practical and necessary tool to support sustainable agriculture. This study aimed to evaluate the impact of PGPR on the growth and physiological properties of Nigella sativa L. in saline soil. We hypothesize that inoculating salt-tolerant PGPR could improve the growth, morphological traits, and physiological properties of Nigella sativa L. in saline soil.

Methods

Soil and plant growth-promoting bacteria

Saline soil collected from the Surkhandaryo Region (Kumkurgan District), Uzbekistan (37.8208° N, 67.6007° E), was used in the present study. Bacillus subtilis IGPEB 1, Bacillus altitudinis IGPEB 8, Bacillus endophyticus IGPEB 33, and Pseudomonas koreensis IGPEB 17, obtained from the Institute of Genetics and Plant Experimental Biology in Tashkent, were employed as PGPR. These cultures were grown in a nutrient broth containing 3 g beef extract, 5 g peptone, and 1000 mL of water. Black cumin (Nigella sativa L.) seeds were obtained from Tashkent Botanical Garden, named after the Academy. F.N. Rusanov of the Institute of Botany of the Academy of Sciences of the Republic of Uzbekistan. N. sativa L. seeds were bacterized with a fresh culture of PGPR (5 × 107 cells/mL), followed by air-drying and sowing in pots. All treatments were replicated five times. Forty days after sowing (DOS), plant height, fresh weight of shoot, fresh weight of root, and photosynthetic pigments were measured.

Measurement of root morphological traits of Nigella sativa L

The water-washed root system was analyzed using a scanning system (Expression 4990, Epson, CA), and the digital images were analyzed to measure the total root length, the root surface area, the root volume, the projected area, and the root diameter using Win RHIZO software (Régent Instruments, Québec, Canada).

Physiological parameters measurement

The total chlorophyll, chlorophyll a, chlorophyll b, and carotenoid contents in Nigella sativa L. were measured according to the methods of Havaux and Kloppstech30 using the following equations –

$$\:\text{C}\text{h}\text{l}\:a\:(\text{m}\text{g}/\text{g})=2.7\left(\text{A}663\right)-2.69\left(\text{A}645\right)x\frac{\text{V}}{\text{W}}$$
$$\:\text{C}\text{h}\text{l}\:b\:(\text{m}\text{g}/\text{g})=22.9\left(\text{A}663\right)-2.69\left(\text{A}645\right)x\frac{\text{V}}{\text{W}}$$
$$\:\text{T}\text{o}\text{t}\text{a}\text{l}\:\text{C}\text{h}\text{l}\:\:(\text{m}\text{g}/\text{g})=20.2\left(\text{A}645\right)-8.02\left(\text{A}663\right)x\frac{\text{V}}{\text{W}}$$
$$\:\text{T}\text{o}\text{t}\text{a}\text{l}\:\text{c}\text{a}\text{r}\text{o}\text{t}\text{e}\text{n}\text{o}\text{i}\text{d}\text{s}\:\:(\text{m}\text{g}/\text{g})=\left[\right(1000\:\text{x}\:\text{A}470)\:\--\:(3.27\:\text{x}\:\text{C}\text{h}\text{l}\:a\:+\:104\:\text{x}\:\text{C}\text{h}\text{l}\:b\left)\right]\:x\frac{\text{V}}{\text{W}}$$

.

Measurement of relative water content

The relative water content of the leaves was measured using the Barrs and Weatherly method31. 30 to 100 mg of fully expanded was incubated in double-distilled water for four h at 28 °C, and the fresh weight was. The leaf was removed, blotted dry, and its turgid weight (TW) was recorded. After that, the samples were oven-dried at 70 °C overnight, and the dry weight (DW) was recorded. RWC was calculated as follows-

$$\:\text{R}\text{W}\text{C}\:\left(\text{\%}\right)=\frac{\text{F}\text{W}-\text{D}\text{W}}{\left(\text{T}\text{W}-\text{D}\text{W}\right)}\text{x}100$$

Measurement of total antioxidant capacity (TAC)

Total antioxidant capacity was measured according to the method of Prieto et al.32. A 0.1 mL mixture of 0.6 M sulphuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate was incubated at 95 °C for 90 min, cooled, and then the absorbance was measured at 695 nm against a blank containing ascorbic acid. The TAC was calculated from the calibration curve of ascorbic acid using the following linear equation and expressed as µg of ascorbic acid equivalent per mL of extract (µg AAE/mL)

$$\:\text{Y}\:=\:0.0011\text{X}++\:0.0121\:(\text{R}2\:=\:0.9963)$$

Where,

Y is the absorbance at 695 nm.

X is the concentration of ascorbic acid (µg/mL).

Measurement of antioxidant enzymes

For screening of antioxidant enzymes, namely, superoxide dismutase (SOD), catalase (CAT), glutathione oxidase (GSH), and peroxidase (PO), and stress-relieving enzyme ACC deaminase (ACCD), each isolate was separately grown in each minimal medium (MM) at 30◦C for 24 h at 120 rpm. Following 24 h of incubation, each broth was centrifuged at 1,000 rpm for 10 min to obtain a cell homogenate.ACCD activity was assayed according to the Honma and Smmomura method33. The number of mM of α-ketobutyrate produced by this reaction is determined by comparing the absorbance at 540 nm of a sample to a standard curve of α-ketobutyrate (0.1mM-1.0 mM).

For SOD activity, a 100 µL cell homogenate was mixed with 100 µL pyrogallol in EDTA buffer (pH 7.0), and the absorbance was measured at 420 nm34. One unit of SOD is defined as the amount of SOD required for preventing 50% auto-oxidation of pyrogallol.

For measuring CAT activity, 100 µL of cell homogenate was mixed with 100 µL of H2O2 in phosphate buffer (pH 7.0), and the absorbance was measured at 240 nm35. One unit of CAT is defined as the amount of mM of H2O2 decomposed per minute.

For measuring GSH activity, 100 µL of cell homogenate was mixed with 100 µL of GSH in phosphate buffer (pH 7.0), and the absorbance was measured at 240 nm36. GSH activity was measured as the reduction in µM of GSH per min.

For measuring POD activity, 100 µL of cell homogenate was mixed with 4 µL 4-methylcatechol in sodium phosphate buffer (pH 7.0). POD activity was taken as an increase in the absorbance at 420 nm resulting from the oxidation of 4-methyl catechol by H2O2. Under assay conditions, one unit of enzyme activity is defined as a 0.001 change in absorbance per min37.

Statistical analysis

The data were statistically analyzed using Analysis of Variance (ANOVA) with IBM SPSS Statistics version 25. A one-way ANOVA with Duncan’s multiple range test (DMRT) was used to determine the level of significance (p < 0.05). The values were compared to assess differences in the effects of various treatments on the morphological and physiological properties of Nigella sativa L. The heat map was created using MetaboAnalyst 6.0.

Results

Plant growth parameters of Nigella sativa L

The effects of PGPR on some plant growth parameters under saline soil were investigated. B. endophyticus 33 treatment enhances the shoot length (cm) by 9.67 ± 0.40, the fresh weight of the shoot (cm) by 0.68 ± 0.03, and the fresh weight (cm) of the root by 0.39 ± 0.01 compared to B. subtilis IGPEB 1, P. koreensis IGPEB 17 treatments, and control, as shown in Table 1. Treatment of B. altitudinis IGPEB 8 shoot length 9.23 ± 0.35, 0.65 ± 0.02 fresh weight of shoot, and 0.37 ± 0.02 fresh weight of root compared to B. subtilis IGPEB 1, P. koreensis IGPEB 17, and control. Treatment of B. subtilis IGPEB 1 enhances the shoot lengths 8.30 ± 0.10, fresh weight of shoot 0.51 ± 0.03, and fresh weight of root 0.29 ± 0.02 compared to the control.

Table 1 Impact of PGPR on the growth of Nigella sativa L. under saline soils.
Full size table

B. endophyticus 33 treatment maximizes Nigella sativa L. root morphological traits in saline soil compared to B. altitudinis IGPEB 8, B. subtilis IGPEB, P. koreensis IGPEB 17, and control. Root diameter (mm) 0.45 ± 0.05, root volume (cm3) 0.35 ± 0.05, root surface area (cm2) 9.65 ± 0.21, projected area (cm2) 7.90 ± 0.29, and total root length (cm) 76.03 ± 1.61 when treated with B. endophyticus 33. Table 2 represents the root morphological traits of Nigella sativa L. under saline soils. Treated with B. altitudinis IGPEB 8, the root diameter (mm) was 0.45 ± 0.05, root volume (cm3) was 0.35 ± 0.05, root surface area (cm2) was 8.44 ± 0.41, projected area (cm2) was 7.78 ± 0.95, and total root length (cm) was 64.64 ± 4.29 compared to the control. B. subtilis IGPEB 1treatment the root diameter (mm) 0.41 ± 0.03, root volume (cm3) 0.32 ± 0.05, root surface area (cm2) 7.97 ± 0.28, projected area (cm2) 6.42 ± 0.32, and total root length (cm) 54.94 ± 6.86.

Table 2 Impact of PGPR on root morphological traits of Nigella sativa L. under saline soils.
Full size table

Physiological properties of Nigella sativa L

The chlorophyll a content (mg/g) increased when treated with B. endophyticus 33 and B. altitudinis IGPEB 8 compared to B. subtilis IGPEB 1, P. koreensis IGPEB 17, and control. No significant (chlorophyll a content) difference between B. endophyticus 33 and B. altitudinis IGPEB 8. Treatment with B. subtilis IGPEB 1 chlorophyll a content (mg/g) increased compared to the P. koreensis IGPEB 17 and control. P. koreensis IGPEB 17 treatment increased the chlorophyll a content compared to the control. Chlorophyll b content is maximum with B. altitudinis IGPEB 8 treatment compared to P. koreensis IGPEB 17, B. endophyticus 33, B. subtilis IGPEB 1 treatments, and control. P. koreensis IGPEB 17 treatment showed a significant difference in chlorophyll b content compared to the B. endophyticus 33, B. subtilis IGPEB 1 treatment, and control. B. endophyticus 33 showed a significant increase in chlorophyll b content compared to the B. subtilis IGPEB 1 treatment and control (Fig. 1).

Fig. 1
figure 1

Impact of PGPR on the (a) Chlorophyll a, (b) Chlorophyll b, total chlorophyll, and d) total carotenoid contents.

Full size image

T1- Control, T2- B. subtilis IGPEB 1, T3- B. altitudinis IGPEB 8, T4- B. endophyticus 33, T5- P. koreensis IGPEB 17.

Values are the average of triplicates (n = 3). + = standard deviation. Different letters indicate significant values at p < 0.05. The values were compared to assess differences in the effects of various treatments on photosynthetic pigments in N. sativa L.

The maximum content of total chlorophyll by B. altitudinis IGPEB8 and B. endophyticus 33 treatments compared to P. koreensis IGPEB17 and B. subtilis IGPEB1, concerning the control. P. koreensis IGPEB17 and B. subtilis IGPEB1 treatments showed no significant difference in total chlorophyll content, but a significant difference compared to B. altitudinis IGPEB8, B. endophyticus 33, and the control. The total carotenoid content (mg/g) increased by the B. endophyticus 33 treatment compared to the B. altitudinis IGPEB 8, B. subtilis IGPEB 1, P. koreensis IGPEB 17 treatments, and control (Fig. 1). B. altitudinis IGPEB 8 treatment produces more carotenoid content than the B. subtilis IGPEB 1, P. koreensis IGPEB 17 treatments, and the control.

The relative water content of the leaf increased when treated with B. endophyticus 33, B. altitudinis IGPEB 8, B. subtilis IGPEB 1, and P. koreensis IGPEB 17 compared to the control, as shown in Fig. 2a. No significant differences in RWC were recorded between B. endophyticus 33, B. altitudinis IGPEB 8, B. subtilis IGPEB 1, and P. koreensis IGPEB 17.

Fig. 2
figure 2

Impact of PGPR on the (a) relative water content and leaf water storage (b) of the leaf.

Full size image

T1- Control, T2- B. subtilis IGPEB 1, T3- B. altitudinis IGPEB 8, T4- B. endophyticus 33, T5- P. koreensis IGPEB 17. Values are the average of triplicates (n = 3). + = standard deviation. Different letters indicate significant values at p < 0.05. The values were compared to assess differences in the effects of various treatments on the relative water content of N. sativa L.

The leaf water storage increased when treated with B. altitudinis IGPEB 8, B. endophyticus 33, and P. koreensis IGPEB 17 compared to the B. subtilis IGPEB 1 and control treatments (Fig. 2b). B. subtilis IGPEB 1 treatment significantly differed from the control in leaf water storage.

The effects of plant growth-promoting bacteria on growth parameters, root morphological traits, and physiological properties of Nigella sativa L. under saline soil were analyzed using a heat map and clustering (Fig. 3). Figure 3 indicates the differences between the morphological and physiological traits of inoculated and uninoculated plants. From the dendrogram, it was analyzed that the treatments were categorized into three classes: untreated control, with the least effect; plants treated with B. subtilis IGPEB 1 and P. koreensis IGPEB 17, which showed a better impact on growth promotion; and B. altitudinis IGPEB 8 and B. endophyticus 33-treated plants, which showed the most noticeable results. T3 and T4 were the most effective treatments, showing the most significant growth promotion and increases in physiological and biochemical traits. All treatments were significantly different from one another, indicating that each bacterium had a distinct effect on Nigella sativa L. growth.

Fig. 3
figure 3

The heat map, based on Pearson and Ward clustering to determine distances, shows the effect of PGPR on growth parameters and the physiological properties of N. sativa L. under salinity stress.

Full size image

T1 − Control (uninoculated), T2 − B. subtilis IGPEB 1, T3 − B. altitudinis IGPEB 8, T4 − B. endophyticus 33, T5 − P. koreensis IGPEB 17.

Total antioxidant capacity (TAC) of Nigella sativa L

All the treatments (except the control) improved the levels of antioxidant enzymes in plant leaves. However, treatments T3 and T4 yielded maximum TAC (13.60 ± 0.18 and 12.92 ± 0.11 AAE/mL) compared to the control (7 ± 0.11 AAE/mL) (Table 3).

Antioxidant enzymes of N. sativa L

The highest ACCD activity was observed in the T3 and T4 treatments (0.59 ± 0.03 and 0.62 ± 0.02 nM α-ketobutyrate mg protein/h, respectively) compared with other treatments (Table 3). ACCD activities of these treatments were 18% and 22%, respectively, higher than in the control.

All the treatments (except the control) improved the levels of antioxidant enzymes in plant leaves. However, Treatment T3 and T4 significantly enhanced the activities of SOD, CAT, GSH, and POD. Treatment T3 and T4 revealed SDO activity of 21.22 ± 0.03 and 22.01 ± 0.03 IU/mg, CAT activity of 2.011 ± 0.02 and 1.99 ± 0.02 mM of H2O2 decomposed/min, GSH activity of 31.97 ± 0.01 and 32.03 ± 0.01µM GSH reduced/min, and POD activity of 11.97 ± 0.01 and 12.03 ± 0.01 µg/mL (Table 3). SOD, CAT, SGH, and POD activities were significantly higher by 24%, 29%, 27%, and 31%, respectively, over the control.

Table 3 TAC and activities of antioxidant enzymes following the various treatments.
Full size table

Discussion

Plant salt uptake indirectly affects plant growth, as it first influences turgor state and photosynthesis, and simultaneously impacts enzyme activity, as well as morphological, physiological, and biochemical processes38,39. The accumulation of salt ions in old leaves accelerates cell death and hinders the transport of carbohydrates and growth hormones to growth tissues. Excessive accumulation of salt ions hinders plant growth by reducing photosynthetic rate and generating growth-inhibiting metabolites. Earlier reports have shown the negative impacts of salinity on plant growth15,40,41.

The growth and health of crop plants rely on adequate amounts of essential nutrients. The crop microbiome plays a pivotal role in supplying these nutrients to its host plants. Crop microbiomes secrete a diverse array of plant-beneficial traits to enhance plant growth42. A broad range of PGPR, including Bacillus spp., has been identified for its role in promoting plant growth under both normal and saline stress conditions. Using PGPR under saline circumstances is a promising method of increasing crop productivity. PGPR interacts with plant roots and has both direct and indirect favorable effects on plant growth and the reduction of biotic and abiotic stressors43.

Khan and Kunuc13 reported the adverse effects of soil salinity and salinity levels on plant height, vegetative dry weight, and oil content of black cumin. They further found that soil salinity and dits levels reduce the number of capsules and seed weight. Douka et al.44 found that endophytic PGPR exert beneficial effects on the health of black cumin. They isolated endophytic Bacillus halotolerans from N. sativa leaves, which demonstrated multiple plant growth-promoting traits that support plant growth, colonization, and tolerance to abiotic stress. Darekh et al.45 reported a substantial increase in chlorophyll a content in N. sativa following inoculation with Pseudomonas fluorescens.

Chlorophyll content in leaves is an indicator of salt tolerance and also plays a role in responding to increased salinity46. In the present study, chlorophyll a, chlorophyll b, total chlorophyll, and total carotenoid contents of the leaf increased following the inoculation with PGPR compared to the control. Accordingly, several studies have shown that inoculation with PGPR increases plant growth and chlorophyll content under salt stress conditions47,48,49. The PGPR can enhance photosynthetic pigments by increasing photosynthetic potential and facilitating the absorption of water and ions50. On the other hand, the inoculated salt-stressed coriander plants showed higher chlorophyll content and dark-green leaves due to the presence of ACC deaminase-producing PGPR51. ACCD activity helps plants withstand salinity and other stress conditions while promoting growth and development41. PGPR significantly improves the photosynthetic pigments, such as chlorophyll and carotenoid content, of plants under various saline soil conditions52,53,54,55,56,57.

Kusale et al.58,59 observed a negative impact of excess salt on wheat growth in saline soil, reporting substantial reductions in root length, shoot height, and chlorophyll content in wheat seedlings after 40 days of salinity stress. However, they noted that the inoculation of halophilic Klebsiella sp. mitigated this adverse effect and significantly improved plant growth parameters, such as root length, shoot length, and chlorophyll content, in wheat60.

Abiotic stress negatively affects plant growth, root morphological traits, and physiological properties of different plants61,62,63,64. The PGPR treatment increased the relative water content and leaf water storage compared to the control. Inoculation with PGPR improved growth yield and plant-water relations, consequently enhancing quinoa yield. Salinity reduces the plant’s water content and causes the buildup of excess ions, thereby lowering the osmotic potential66.

Soil salinity affects plant growth and development, reducing crop yield. Plants often adapt to salt stress through the metabolic activities of their PGPR. The interplay of metabolites from PGPR induces biochemical and physiological changes in plants, enabling them to withstand salinity stress67. There are various mechanisms, both direct and indirect, by which PGPR improve plant growth, nutrient content, and antioxidant capacities under salt-stressed conditions58,59,68.

Many PGPR improve total antioxidant capacity by producing a wide range of antioxidant enzymes69, which protect plants from oxidation70. Kapadia et al.67 isolated halophilic Bacillus halotolerans from the saline soil of Dandi, India, and reported the production of various antioxidant enzymes under salt stress, which improved tomato growth under salinity.

ACC deaminase-producing PGPR is vital in withstanding plant stress conditions and helps plants grow in the presence of excess salt71. Consequently, the production of ACCD reduced ethylene concentration, which exhibits ROS activity. The enzyme ACCD reduces ACC levels in root exudates, thereby preventing excessive ethylene accumulation in plant roots and promoting better root growth and length. Plants with longer root lengths absorb more nutrients67,72. The effect of Pseudomonas entomophila PE3 on growth parameters and physiological traits, and on salinity stress in sunflowers under saline conditions was studied by Fatima and Arora73. They reported that Pseudomonas entomophila PE3 enhances plant growth, physiological characteristics, and salinity-stress tolerance. Moradzadeh et al.74 and Devkot et al.75 observed higher oil and macronutrient contents in Nigella sativa L. and other crops following inoculation with rhizobacteria and the application of biochemical fertilizers76,77,78. Plant growth-promoting bacteria enhance the growth and physiological characteristics of plants under saline conditions79,80,81,82.

The widespread use of agrochemicals to increase crop yields has harmed water supplies, agricultural soils, beneficial organisms, human health, and the ecosystem. PGPR, which encompasses numerous bacterial species, can effectively enhance growth characteristics and yield under various conditions by mitigating multiple abiotic factors76,81,83. Plants benefit from PGPR in various physiological activities, including the solubilization of mineral nutrients and the production of phytohormones83. The inoculation of Nigella sativa L. plants with B. endophyticus IGPEB33 and B. altitudinis IGPEB8 had essential impacts on the tolerance of Nigella sativa L. to salinity stress. Our results suggest that salt-tolerant plant growth-promoting bacterial strains B. endophyticus IGPEB 33 and B. altitudinis IGPEB 8 can be applied to mitigate the effects of salinity stress while improving the growth and yield of Nigella sativa L.