Synergistic application of Bacillus subtilis IAGS174 and thiamine to mitigate salinity and lead stress in Helianthus annuus

Inoculum preparation and soil inoculation

To prepare inoculation, a loopful of rhizobacteria (B. subtilis IAGS174) was inoculated into Luria Bertani (LB) broth media, which was then incubated in a mechanical shaker for 2 days at 27 °C. The optical densities were then measured to establish a uniform population of 108–109 CFU mL⁻¹. Approximately 8–10 mL of bacterial culture was applied to the soil with the help of a sterile syringe

Pot experiment

A pot experiment was performed to determine the positive effect of rhizobacteria and thiamine on Pb and salt stress in sunflower plants. The plastic pots were filled with soil (1.5 kg) obtained from the agricultural fields of the University of the Punjab, Pakistan. Salt and heavy metal stress were applied by amending sodium chloride (NaCl) (500 mg/kg soil) and Pb (NO₃)₂ (150 mg/kg) in the soil. The pots were moistened up to field capacity and then kept for 14 days to blend up the pollutants in the soil. The sunflower seeds were procured from the Roshan Seed shop in Lahore, Pakistan. For surface sterilization, seeds were immersed in 70% ethanol for 30 s and 1% sodium hypochlorite for an additional 60 s, following multiple washes with distilled water. The healthy seeds (eight to ten) were planted in each pot, and thinning was performed after germination to keep five plants in each pot. After three weeks of planting, growth regulator (PGPR, thiamine) treatments were applied. The bacterial inoculum (10 mL) was drenched into the soil and thiamine (100 ppm) was supplemented by foliar application to the sunflower plants. The experiment was done in a wirehouse. The environmental conditions in the wirehouse were a temperature of 28–35 °C, a night/day period of 11/13 h, and humidity of 75 ± 5%. The plants were watered at regular intervals to keep optimum moisture in the soil. Each treatment was replicated thrice by a completely randomized design (CRD). A total of 48 pots for 16 treatments were planted with sunflower seeds, and these plants were uprooted after 120 days. Hence, total of 16 treatments was planned by using either single or combined treatments of both growth promoters (microbe, thiamine) and single and co-application of pollutants (Na, Pb) such as C; control, B; Bacteria, T; thiamine, B+T; Bacteria+ thiamine, S; Salinity, S+B; Salinity+ Bacteria, S+T; Salinity and Thiamine, S+B+T; Salinity and Bacteria +Thiamine, Pb; lead, Pb+B; Lead+Bacteria, Pb+T; Lead+Thiamine, Pb+B+T; Lead+Bacteria+Thiamine, Pb+S; Lead+ Salinity, Pb+S+B; Lead+Salinity+Bacteria, Pb+S+T; Lead+Salinity+Thiamine, and Pb+S+B+T; Lead+Salinity+Bacteria+Thiamine.

Assessment of photosynthetic pigments

The fully mature and widened fresh leaves from the center of each plant were selected and removed. To estimate chlorophyll (Chl.) pigments, 0.25 g of fresh leaf tissue was blended with 80% acetone to make a plant extract. After extraction, the mixtures were centrifuged at 12,000× g for 10 min. The amount of Chl. a and Chl. b was measured by monitoring the absorbance of the supernatant at wavelengths of 663 nm and 645 nm, respectively, with the help of a spectrophotometer (UV/VIS, Cecil Aquarius CE 7200). The relative absorbance of the supernatant was obtained at 480 nm for examining carotenoid content (Liu et al., 2024). The formulas used for calculating the chlorophyll content are:

Chl. ‘a’ (mg g⁻¹ FW) = $\frac{\left(12.7\times \mathrm{A}663\right)-\left(2.69\times \mathrm{A}645\right)\times \text{Sample volume}}{1000\times \mathrm{W}}$

Chl.‘b’(mg g⁻¹ FW) = $\frac{\left(12.7\times \mathrm{A}645\right)-\left(4.68\times \mathrm{A}663\right)\times \text{Sample volume}}{1000\times \mathrm{W}}$

Total chlorophyll (mg g⁻¹ FW) = $\frac{20\left(\mathrm{A}645\right)+8.02\left(\mathrm{A}663\right)\times \text{Sample volume}}{1000\times \mathrm{W}}$

Carotenoids (mg g⁻¹ FW) = OD480+0.114 (OD663)−0.638(OD645).

Antioxidant enzymes analysis

Enzyme extract was obtained by adding one g of the plant’s fresh leaves to a mortar and crushing with a pestle while pouring liquid N₂. Afterwards, the plant biomass was homogenized in 2.0 mL of an ice-cold 50 mM sodium phosphate buffer. The homogenous mixture was centrifuged at 10,000× g for 20 min at 4 °C. The resulting supernatant was employed to assess different antioxidative enzymes with the help of a spectrophotometer (Rao & Sresty, 2000). Each analysis was replicated thrice.

Superoxide dismutase (SOD, EC: 1.5.1.1 ) content was observed by pouring 0.1 mL of enzyme extract into 1.5 mL 50 mM sodium phosphate (pH 7.8), 0.300 mL 750 µM nitro blue tetrazolium (NBT), 0.300 mL 20 µM riboflavin, 0.3 mL 130 µM methionine, 0.3 mL 100 µM EDTA-N Spectrophotometer (UV/VIS, Cecil Aquarius CE 7200) was used to note reading at 560 nm after the reaction mixture was illuminated under light of 4,000 flux for 20 min (Bin et al., 2010). Peroxidase (POD, EC: 1.11.1.7) activity was analyzed using 50 µL enzyme extract and 1.0 mL 0.3% H₂O₂, 1.0 mL 50 mM sodium phosphate (pH 5.5), and 0.95 mL 0.2% guaiacol. This was recorded at 470 nm using a spectrophotometer (UV/VIS, Cecil Aquarius CE 7200). A reaction mixture containing 500 µL 0.1 M H₂O₂, 3.0 mL 50 mM sodium phosphate (pH 7.8), 1.0 mL deionized water, and 200 µL enzyme extract was prepared for catalase (CAT, EC: 1.11.1.6) activity. A spectrophotometer (UV/VIS, Cecil Aquarius CE 7200) was used to note the absorbance at 240 nm (Rhaman et al., 2024).

Quantification of hydrogen peroxide and malondialdehyde

For malondialdehyde (MDA) determination, 0.2 g of plant leaf sample was crushed in five mL of 0.1% TCA and centrifuged for 5 min at 10,000 g. The reaction mixture was made by adding one mL of the supernatant aliquot to four mL of 20% TCA with 0.5% thiobarbituric acid (TBA). The absorbance of the reaction mixture was estimated at 532 nm by a spectrophotometer (UV/VIS, Cecil Aquarius CE 7200), and the reading for non-specific absorption at 450 nm and 600 nm was subtracted (Zhang et al., 2013. The MDA level (mol g⁻¹ FW) was recorded using (= 155 mM⁻¹ cm⁻¹). The hydrogen peroxide (H₂O₂) content was estimated by crushing 0.5 g of fresh plant leaf with five mL of trichloroacetic acid (TCA 0.1%) and then centrifuging at 12,000 g for 20 min at 4 °C. Afterward, 0.5 mL of supernatant liquor was homogenized with 0.5 mL of 10 mM KPO₄ buffer. The reading of absorbance was noted at 390 nm by a spectrophotometer (UV/VIS, Cecil Aquarius CE 7200) (Velikova, Yordanov & Edreva, 2000).

Estimation of flavonoid and phenolic content

A volumetric flask was filled with one mL of plant extracts and four mL of distilled water to determine the flavonoid concentration. The solution was mixed with 0.3 mL of 5% NaNO₂ after 5 min. 0.3 mL of 10% AlCl₃ was added to it. After 6 min, two mL of 1 M NaOH was added to the solution. Distilled water was added to make the volume 10 mL. The absorbance of the solution was measured at 510 by a spectrophotometer (UV/VIS, Cecil Aquarius CE 7200) (Zhishen, Mengcheng & Jianming, 1999). Phenol contents were determined by immersing two g of plant leaf in 10 mL of 80% methanol for 15 min at 65 ^∘C. Afterwards, one mL of plant extract was mixed with 250 µL of Folin-Ciocalteau reagent (1N) and five mL of distilled water. The reaction mixture was stored at 30 degrees Celsius. To identify the exact number of phenols, the absorbance at 725 nm was measured by a spectrophotometer (UV/VIS, Cecil Aquarius CE 7200) and compared to the gallic acid curve (Zieslin & Ben-Zaken, 1993).

Proline estimation

A sample of dried and prewashed leaf (100 mg) was placed into a flask containing 10 mL of sulfosalicylic acid (3%). The mixture was vortexed and then filtered through Whatman’s no-filter paper. Then, two mL of the filtrate was transferred to a glass tube containing as much glacial acetic acid as there was acid ninhydrin. It was placed in a water bath at 100 °C for 0.5 h. Then, four mL of toluene was added, and the chromophore was aspirated. This reaction mixture was incubated at 25 °C for 0.5 h, and its colorimetric value was recorded at 520 nm by a spectrophotometer (UV/VIS, Cecil Aquarius CE 7200) and compared to the standard curve (Bates, Waldren & Teare, 1973).

Plant growth and biomass production assessment

After 120 days, the plants were removed from the pots and properly rinsed with distilled water. Plant parts, including roots, shoots, and leaves, were separated. The lengths of the roots and shoots were measured. The values of fresh biomass production from root and shoot were also measured using an electrical balance. The root and shoot samples were placed in an oven at 700 ^∘C for one day. The root and shoot dry weights were also calculated.

Heavy metal analysis

The dried plant samples were ground into a fine powder, and 1 g was mixed with 10 mL of HNO₃ and five mL of HCLO₄. The mixture was put in a flask and transferred to the hot plate, which was then placed in the hot plate at 1,500 °C until the solution turned transparent and reduced to five mL. After the digestion, 45 mL of distilled water was mixed into this solution, making a solution of 50 mL. The solution was filtered twice using the Whatman 42 filter paper. The filtrate was shifted to sample bottles, and distilled water was poured to make the sample up to 100 mL. The Pb quantity was estimated using an atomic absorption spectrophotometer (AAS; PerkinElmer AAnalyst 800; PerkinElmer, Waltham, MA, USA), while Na was analyzed by a flame photometer (Model PFP7, 90–125 V).

The metal tolerance index, translocation factor (TF) and bio-concentration factor (BCF) were calculated by the methodology proposed by Wu et al. (2018). The metal tolerance index was analyzed for the evaluation of the ability of the plant to grow in the presence of metal concentration by a formula:

Metal tolerance index = $\frac{\text{Mass of treated plant}}{\text{Mass of control plant}}\times 100$

The translocation factor was determined as;

TF = $\frac{\text{or Na conc}.\text{in shoot}}{\text{Pbor Na conc}.\text{in root}}$

The bio-concentration factor (BCF) was determined by:

BC = $\frac{\text{Pb or Na conc}.\text{in shoot}}{\text{or Na conc}.\text{in soil}}$.

Statistical analysis

The experiment was performed with three replicates of each treatment in a completely randomized design (CRD). The obtained results were demonstrated as mean ±standard deviation. For statistical analysis, a one-way analysis of variance (ANOVA) was performed with the help of DSAASTAT software. Significance among multiple treatments was checked by performing Duncan’s multiple range test (DMRT) at P ≤ 0.05 significance level using DSAASTAT software.

Effect B. subtilis IAGS174 and thiamine on growth parameters of H. annuus subjected to salinity and Pb stress

The effects of different treatments were significant on shoot length (SL), root length (RL), shoot fresh weight (SFW), shoot dry weight (SDW), root fresh weight (RFW), and root dry weight (RDW) of sunflower. Both salinity and Pb stress and their combination significantly hampered shoot and root length, dry weight, and fresh weight of the shoot and root of sunflower compared to the control treatment. The Pb, S and Pb+S treatments declined the shoot length of sunflower by 38%, 25% and 49% respectively as compared to untreated control. However, the individual application of PGPR and thiamine and their combined treatment enhanced the growth of sunflower plants under normal and stress conditions. The efficacy of B. subtilis IAGS174 and thiamine on sunflower was proved better under un-stressed conditions. The combined B+T treatment under Pb and salt stress was found to be very effective and meaningfully improved shoot length, root length, shoot fresh weight, shoot dry weight, root fresh weight, and root dry weight by 28.12%, 60%, 6.84%, 9.3%, 16.3% and 32.25% as compared to Pb+S treatment (Table 1).

Influence B. subtilis IAGS174 and thiamine on photosynthetic attributes of H. annuus subjected to salinity and Pb stress

Both Pb and salinity stress either single or combined significantly reduced the Chl. a, Chl. b, total chlorophyll and carotenoid content in sunflower plants. Combined Pb+S stress significantly (DMRT at p ≤ 0.05) reduced Chl. a by 44.5%, Chl. b by 54.81%, total Chl. by 47.33%, and carotenoid by 45.21% as compared to the untreated control. However, the combined application of bacteria and thiamine (B+T) treatment on sunflower under Pb and salt stress was the most effective in improving the Chl. a, Chl. b, total chlorophyll and carotenoid content by 16.33%, 16.39%, 15.66%, and 14.28%, respectively, as compared to the Pb+S treatment (Table 2).

Impact of B. subtilis IAGS174 and thiamine on antioxidant enzymes of H. annuus subjected to salinity and Pb stress

The antioxidant enzyme parameters like SOD, POD, and CAT showed a significant change in sunflower under all the treatments. The amount of SOD, POD, and CAT showed a significant (DMRT at p ≤ 0.05) increase in sunflower under salt (220.5%, 226.58%, and 220%, respectively) and Pb stress (412.8%, 358.38%, and 97.14%) as compared to the control. While combined Pb+ S treatment significantly (DMRT at p ≤ 0.05) increased the antioxidant machinery of the sunflower plant by 6.62-fold in SOD, 5-fold on POD, and 3.2-fold in CAT as compared to the control. The application of PGPR to sunflower exposed to Pb and salinity stressed soil further increased the antioxidative enzymes., The application of thiamine also augmented the antioxidant enzyme levels in sunflower plants under stressed regimes compared to the relevant control, but less than the bacterial and B+T treatments. The B+T treatment significantly (DMRT at p ≤ 0.05) improved the activity of SOD, POD, and CAT in sunflower plants growing in Pb and salt-stressed soil by 67.74%, 65.08%, and 34.82%, respectively, as compared to Pb+S treatment (Fig. 1).

Effect of B. subtilis IAGS174 and thiamine on phenol, flavonoid and proline contents of H. annuus exposed to salinity and Pb stress

The sunflower plants exhibited an increasing trend in phenol, flavonoid, and proline contents under single or combined Pb and salinity stress regimes. However, PGPR and thiamine application alone or in combination further enhanced phenol, flavonoid, and proline content of plants exposed to Pb and Na stress as compared to the control plants. The combined Pb+S treatment significantly (DMRT at p ≤ 0.05) increased phenol content by 84.13%, flavonoid content by 2.14-fold, and proline content by 3.31-fold. Combined application of B+T under stress (Pb+S) conditions also resulted in a substantial (DMRT at p ≤ 0.05) increase in phenol, flavonoid, and proline content by 38%, 45% and 49% as compared to the respective control (Table 2).

Influence of B. subtilis IAGS174 and thiamine on malondialdehyde and hydrogen peroxide levels of H. annuus exposed to salinity and Pb stress

Single or combined Pb and salinity stress attenuated the levels of MDA and H₂O₂ in sunflower compared with the untreated control. Lead and salt stress significantly (DMRT at p ≤ 0.05) increased the MDA and H₂O₂ levels in sunflower plants by 2.28-fold and 2.08-fold in the Pb+S treatment compared to the relevant control. The B. subtilis IAGS174 and thiamine-assisted H. annuus plants exhibited a decline in the MDA and H₂O₂ contents during toxic regimes. The treatment, including PGPR and thiamine in combination, significantly (DMRT at p ≤ 0.05) reduced the MDA and H₂O₂ levels in sunflower plants raised under Pb and salt stress by 26.57% and 39.70%, respectively, as compared to the Pb+ S treatment (Fig. 2).

Effect of B. subtilis IAGS174 and thiamine on lead and sodium uptake in H. annuus

Under salt and Pb stress conditions, sodium and Pb uptake was observed in root and shoot tissues of sunflower. However, the PGPR inoculation enhanced the Na uptake, while thiamine application reduced the Na uptake. The amount of salt was much higher when treated with PGPR than with thiamine, as PGPR acts as a phyto-extractant, which tends to absorb salt from the soil. Thiamine does phyto-stabilization by keeping the metal-stabilized near roots, preventing them from entering the plants (Table 3).

A similar trend was observed with Pb concentration in the root and shoot of the sunflower. Under thiamine influence, Pb uptake was reduced by 28% in the shoot and 18.6% in the root of the sunflower. After PGPR inoculation, significantly (DMRT at p ≤ 0.05) enhanced Pb uptake was observed in the root and shoot of sunflower by 30.7% and 36.94%, respectively, as compared to the Pb treatment. The combined treatment (B+T) showed high translocation values but less than the PGPR treatment. Similarly, increased translocation of Pb and Na wereobserved in B. subtilis IAGS174 assisted sunflower exposed to Pb+S treatment, while reduced translocation was noted applying thiamine. Table 3 shows the TF, BCF, and MTI values for thiamine and bacteria/PGPR strain (Table 3).

Principal component analysis

The biplot (loading and score) resulted from the principal component analysis (PCA) to evaluate the efficacy of PGPR strain and thiamine-induced ameliorative effects on the biochemical and physiological attributes of sunflower (H. annuus). PCA of plants exposed to Pb and salt stress are given in Fig. 3. The first two components of PCA, i.e., PC1 and PC2, showed maximum contribution and accounted for 86.5% of the total variance in the given database. The PC1 accounted for a 75.8% variance, while PC2 accounted for a 10.7% variance in the given dataset, correspondingly. All 16 treatments were dispersed successfully by the first two principal components. The distribution pattern provided a clear indication that bacterial strain and thiamine supplementation under combined (Pb and salt) stress had a prominent beneficial effect on various growth characteristics of sunflower than the control. The variables aligned with PC1 were correlated positively, such as root L, shoot L, root FW, root DW, shoot FW, shoot DW, total carotenoids, total Chl., Chl. a and Chl. b. However, a highly negative relationship between the variables of PC1 and PC2 was observed: shoot Pb; root Pb; POD; SOD; CAT; total flavonoid content; total phenolic content; proline; MDA; H₂O₂; shoot Na and root Na (Fig. 3).

Treatments under no stress exhibit scores clustered within a relatively small range, having values such as C (4.03, −0.04), B (6.16, 0.57), T (5.55, 0.41), and B+T (7.19, 0.72). However, treatments under salinity stress (S) exhibit scores clustered within a small range with positive PC1 scores and negative PC2 scores, such as S (0.04, −2.38), S+B (0.90, −2.40), S+T (0.83, −1.33) and S+B+T (1.34, −1.92). Treatment under lead (Pb) stress showed negative PC1 scores and positive PC2 scores, such as Pb (−2.09, 1.23), Pb+B (−1.50, 2.52), Pb+T (−1.33, 1.36) and Pb+B+T (−1.15, 2.50). Meanwhile, treatment with combined Pb and salinity stress (Pb+S) showed negative scores for PC1 and PC2. Pb+S (−4.93, −1.01), Pb+S+B (−5.33, −0.29), Pb+S+T (−4.72, −0.08) and Pb+S+B+T (−5.004, 0.14). All the treatments showed great variability in the PCA biplot.

Correlation analysis

A Pearson correlation matrix was performed among the studied parameters of sunflower (Fig. 4) indicated by the color scale. Blue, showing a highly positive correlation, light yellow indicating no correlation between the parameters, and dark red showing a highly negative correlation between the parameters. Shoot length (shoot L) was negatively correlated with total phenolics content; total flavonoid content; proline content; SOD; CAT; POD; MDA; H₂O₂; root Pb; shoot Pb; shoot Na, and root Na. There was a slightly negative correlation between root Na, shoot Na, and root Pb, shoot Pb. On the other hand, shoot length (shoot L) was positively correlated with root L, shoot FW, shoot DW, root FW, root DW, and Chl. a; Chl. b; total Chl. and total carotenoids. All of these were significantly and positively correlated with each other as well. The treatments demonstrate (*) within rows that depict significance (p ≤ 0.05) level.