The Potential of Bacillus subtilis and Phosphorus in Improving the Growth of Wheat under Chromium Stress.

doi:10.21203/rs.3.rs-983650/v1

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The Potential of Bacillus subtilis and Phosphorus in Improving the Growth of Wheat under Chromium Stress.

https://doi.org/10.21203/rs.3.rs-983650/v1

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The high magnitude of heavy metals contamination in daily food attracts the attention of researchers for finding efficient and eco-friendly approaches that can help resolve this problem. Hexavalent Chromium (Cr⁺⁶) is one of the most toxic heavy metals that have deteriorating effects on the growth and quality of the end product of wheat. In this research, the role of Bacillus subtilis and phosphorus fertilizer on wheat development in Cr⁺⁶ stress was evaluated. The statistical analysis of the data showed that the co-application of Bacillus subtilis and phosphorus gave considerably significant (p<0.05) results compared to their separate application. The increase in shoot length, root length, shoot fresh weight, and root fresh weight was 38.17%, 29.31%, 47.89%, and 45.85%, respectively, compared to untreated stress-facing plants. The use of Bacillus subtilis and phosphorus improved the process of osmolytes (proline 39.98%, and sugar 41.30%) production in plants which results in high water uptake and structural stability of proteins (86.65%) and improved cell membranes (66.66%) as well. They also augmented the production of antioxidants by 67.71% (SOD), 95.39% (APX), and 60.88% (CAT), respectively. The Cr⁺⁶ accumulations were reduced by 54.24%, 59.19%, and 90.26% in the shoot, root, and wheat grains, respectively. The combined application of Bacillus subtilis and phosphorus can reduce the heavy metals toxicity in crops.

Renewable Resources

Environmental Engineering

Bacillus subtilis

heavy metal

chromium

wheat

Heavy metals have been causing serious challenges to crops for the last many decades. They enter the environment from natural and anthropogenic sources, including industrial waste agrochemicals, mining, and vehicle exhausts. Once heavy metals enter the environment, they get accumulated. These elements stay there forever and enter into the food chain and, by bio-magnification, become more toxic and cause serious health issues in animals and humans [1]. Chromium (Cr) has been considered the seventh most abundant element present on the earth. Fossil fuel burning, electroplating, pigment oxidants, tanneries, pulp, paper production, fertilizers, sewage sludge, and steel production are the major sources of Cr release into the surroundings. Chromium is severely noxious to plants, and it drastically reduces plant development and growth. Hexavalent Cr is more mutagenic and highly carcinogenic. Cr disturbs normal growth processes of plants, including chlorophyll content, germination, shoot, and root length, net photosynthesis, and stomatal conductance. Its accumulation in plants causes chlorosis, inhibition of respiration and photosynthesis, and delays the process of germination. Furthermore, wheat is highly susceptible to chromium stress [2]. The uptake of contaminated food results in serious health problems, including kidney and lung diseases, different types of cancers, skin and respiratory diseases, gastrointestinal problems, and anemia[3].

Phosphorous is one of the most essential fertilizers for improving crop yield. Insufficient availability of phosphorus fertilizer decreases crop yields, so it is continually added to the fields for better crop yield. The application of phosphorus reduces the uptake and accumulation of heavy metals in plants. They improve the growth of plants, which improves soil fertility, maintains soil structure, reduces the chances of soil erosion, and stabilizes contaminated sites [4]. They improve the nutritional status of plants and make them resistant to heavy metal stress. The application of phosphorus restored the diversity of plants in the heavy metal contaminated areas. [5]. Applying P and Zn fertilizer increase essential oil content in Matricaria recutita in 50% field capacity. They also improve other traits of plants and results in better growth and yield [6]. The use of P increases nutrients uptake, proline, sugar, amino acids, chlorophyll content, and stress tolerance in Annona squamosal. They also increase potassium content in the shoot, which improves pigments and enhances the efficiency of PSII, resulting in better growth even in unfavorable conditions [7].

Besides their uses, the runoff of excess phosphorus from agricultural fields contributes to eutrophication, the consequent increase in algal biomass in lakes, rivers, and coastal oceans that often lead to “dead zones” in which oxygen has been depleted by microbial decomposition of decaying blooms. Meeting these dual issues is incredibly challenging, given the essential role of phosphorus in sustaining biological production. Economic damages due to eutrophication of freshwaters worldwide have been estimated to be 2.2 billion annually. [8, 9].

Wheat demands a high amount of phosphorus, an essential constituent for plant growth, and is applied in the form of organic or/and inorganic phosphorus in the soil because of its importance for the continual crop system. It is also effective for reducing heavy metal stress in wheat. It has been observed that the phytase-producing bacteria from Himalayan soils possess the ability to solubilize inorganic phosphate, can produce phytase, IAA, and ammonia, and increase the availability of IAA, P, and ammonia, leading to enhance plant growth. The combined application of conventional fertilizer with phosphate solubilizing bacteria (PSB). Hence, a combination of plant growth-promoting rhizobacteria (PGPR) with phosphorus is more efficient when compared with separate applications. PGPR can control the release of phosphorus and other nutrients in the rhizosphere and make a conducive environment for plant growth even under unfavorable conditions [10].

PGPR live in a symbiotic relationship with plants, increase plants' competitiveness for space and nutrients, improve plant growth, and increase plant resistance to withstand external stresses. These microbes get their nutritional needs from root exudates and, in return, impart many benefits to plants, including nitrogen fixation, plant growth promotion, growth hormone production, lytic enzymes production, antagonism against pathogenicity [11]. Bacillus subtilis is one of the most important bacterial strains that can detoxify heavy metals and are helpful in the management of contaminated soil [12]. The use of Bacillus subtilis and Pseudomonas pseudoalcaligenes increases stress tolerance in Glycine max by activating different defense systems and improving its growth [13]. They are also efficient in immobilizing heavy metals in the soil and reducing their uptake in plants. They also reduce oxidative stress and convert the more toxic form of heavy metal ions into less toxic form by oxidation-reduction reactions [14]. The mechanisms include the formation of polyphosphate, thiophosphate, glutathione, and biofilm, which protects roots from the damaging effects of heavy metals [15]

The scale of toxicities of heavy metals and their repercussions in flora and fauna warrant quick steps to device cost-effective and sustainable ways for remediating these noxious compounds. Many physical and chemical processes have been used to remediate metal-contaminated environments, each with its own merits and demerits. Bioremediation techniques involving the plant-microbe ecosystem have attracted the attention of many groups of researchers because of their cost-effectiveness and sustainability and have yielded better and long-lasting results. The plant-microbe combinations help to improve the metal remediation techniques. [16] .

This study was designed to isolate the efficient heavy metal tolerant strains of PGPR and find the effect of separate and combined application of phosphate and PGPR on wheat growth under chromium stress.

Soil sampling

Soil samples were collected from the discharge point of Kohinoor Textile Mills Rawalpindi, Pakistan. The soil sample was properly cleaned and placed in a sterile labeled zipper plastic bag and was then stored in the refrigerator at 4°C. Soil pH was measured by mixing an equal amount of water and soil in a beaker, shaken vigorously, and let it sit. The pH was measured by a pH meter. To measure soil electrical conductivity, the sample was mixed with 200 mL distilled water, and after shaking, filter the solution and measure EC using an EC meter [17]. The texture, organic matter, and chromium content were also analyzed. The soil sample (2 g) was mixed with 5 mL HNO₃ and 5 mL distilled water and heated in a digestion block for 30 minutes. The solution was reheated after adding HNO₃ until the oxidation process was complete. Then the sample was heated again to reduce the digestion liquid to 5 mL, and further H₂O₂ and H₂O were added to the sample and heated and evaporated to 5 mL. The sample was kept at room temperature, and after cooling, HCl was added to the sample and placed at 95°C for 15 min. The solution was filtered and diluted with water to make the final volume 100 mL, and chromium analysis was done by atomic absorption spectrophotometer [18].

Isolation of bacterial strains and their analysis

Bacterial strains were isolated by serial dilution. 0.1 g of the soil sample was mixed with 9 mL of 1% saline distilled water and was vortexed. Nutrient agar media was used, and bacterial colonies were examined after 24 and 48 hours. The purification of bacterial colonies was done by streaking. All of the selected isolates were analyzed for their tolerance to heavy metals by the agar dilution. In this process, freshly growing cultures were streaked on agar containing Petri plates amended with K₂Cr₂O₇ at different concentrations (10, 15, and 25 mg/L). Heavy metal tolerance was determined by the appearance of bacterial growth after 3-4 days of incubation [19]. To analyze siderophore production, bacterial strains were grown on Chrome Azurol S agar at 28°C for 48 h and observed for the appearance of an orange zone as an indication of siderophore activity [20]. Tryptic soy broth media was used to grow bacterial strains for 24 hours at 200 rpm. Bacterial strains were inoculated in Dworkin and Foster media having ACC and kept at 30°C for 48 hours (200 rpm). The production of α-ketobutyrate was calculated using the standard curve of α-ketobutyrate [21]. The phosphate solubilization ability of various isolates was estimated by observing their activity on agar plates having tri-calcium phosphate. The agar media was poured into Petri plates, and then after solidification, the isolates were spot inoculated on the center of agar plates. Then the plates were incubated for 5 days at 28°C. The phosphate solubilization index was measured as the ratio of total diameter (colony + halo zone) to the diameter of control (colony) [22]. The most tolerant bacterial strain A3 was sequenced and submitted to NCBI.

Experimental layout

The soil was collected from an undisturbed field area near the glasshouse of the Department of Botany, PMAS Arid Agriculture University Rawalpindi, Pakistan. The soil sample was analyzed for pH, texture, Ec, organic matter, nutrients, and Cr content by following the methods mentioned in the soil sampling section (Table. 1). The soil sample (5 g) was mixed with 10 mM CaCl₂ (50 mL) having dilution of KCl₂ (0.2, 0.4, 1, 2, 4 mmol/L). The sample was shaken properly and allowed to sit for 24 hours. After filtration, the analysis of potassium was done by a flame photometer [23]. A 5 g soil was mixed with 100 mL NaHCO₃ reagent to measure phosphorus and placed on a reciprocating shaker at 20°C for 30 minutes. The sample was filtered, and 5 mL soil extract solution was treated with 1.5 M H₂SO₄ (5 mL). After that, ammonium molybdate was added to the sample, and absorbance was taken at 880 nm. Blank was also prepared. The standard solution of phosphorus was used to make the curve. To measure nitrogen content, 0.5 g soil was treated with 5 mL H₂SO₄, selenium catalyst (0.4%), and 0.1 g salicylic acid mixed with agitation. After 30 min. 0.1 g of zinc dust was added to the solution and heated at 310°C for 4.5 hours. The sample was cooled, and H₂SO₄ was added to make the volume 5 mL. Then 5 mL distilled water was added, and auto-analysis was done as given by [24]. The bacterial strain was grown in Luria Broth medium, and it was inoculated in the soil at the rate of 10⁻⁹ CFU/mL. Ammonium Phosphate was used at the rate of 2%. The soil was incubated for 30 days, and plastics pots having a diameter of 7 cm (height 8 cm) were filled with that soil (8 kg/pot). Two wheat varieties were selected, i.e., V1-NARC 2011 and V2-NARC 2009. Seeds of wheat varieties were surface sterilized using 0.5% sodium hypochlorite and washed with sterile distilled water. The sowing was done in October when average temperatures were 21±5°C. Treatments included; T0 = control, T1 = Cr⁺⁶ stress, T2 = Phosphate + Cr⁺⁶ stress, T3 = Bacillus subtilis + Cr⁺⁶ stress, T4 = Phosphate + Bacillus subtilis + Cr⁺⁶ stress. When the plants reached the stem elongation stage, chromium stress was applied using 25 mg/L K₂Cr₂O₇ solutions (1000 mL solution per pot). Leaf samples were collected after 20 days of imposing stress and analyzed for morphological, physiological, and biochemical parameters.

Analysis of plant length and biomass

Plants were uprooted, and the length of shoot and roots were measured by using a meter rod. The plants were washed with distilled water, and their fresh weights were taken after separating their roots and shoots. They were placed in an oven at 65°C for 1 week, and dry weight was measured [25].

Leaf Chlorophyll contents

To measure leaf chlorophyll content, 0.2 g flag leaves were homogenized with 80% acetone then their absorbance was checked by spectrophotometer. The absorbance was taken at 663 nm and 645 nm. Calculations were done by using the formula as given below [26].

Chlorophyll a = 12.7 (A633) – 2.7 (A645)

Chlorophyll b = 22.9 (A645) – 4.79 (A633)

Determination of Proline content

For the determination of proline content, the protocol of Bates et al. [27] was used. Plant material (0.5 g) was homogenized in 4 mL sulfosalicylic acid, and after filtration, the supernatant was placed in separate test tubes, having ninhydrin (2 mL) and acetic acid (2 mL). The whole mixture was shaken. The samples were boiled in the water bath for one hour, then 4 mL toluene was added to the solution, and after mixing, the upper layer was used to measure absorbance at 520 nm. The standard curve of proline was used for the estimation of proline content in the sample.

Determination of Soluble sugar and protein

It was determined by the method as given by Dubois et al. [28]. The plant material (0.5 g) was mixed in 80% of 10 mL ethanol, and the mixture was heated at 80°C for 60 minutes. The extract (0.5 mL) was mixed with 18% of 1 mL phenol and incubated for 60 minutes. After that 2.5 mL, sulphuric acid was added to the mixture, and the absorbance was taken at 490 nm. The standard curve of glucose was used for calculation. To find soluble protein, 0.2 g plant material was taken and ground in 4 mL sodium phosphate buffer; the extract was collected after centrifugation. In 0.5 mL extract, 3 mL of coomassie bio red dye and 0.5 mL of distilled water were added. The absorbance was calculated at 595 nm [29].

Membrane Stability Index (M.S.I) and Relative water content

The membrane stability index (MSI) was calculated by Bhusan Bal et al. [30] method. The leaf discs (100mg) were rinsed with tap water and chopped into pieces. Then these pieces were placed in a water bath at 40°C for 30 minutes. The electrical conductivity (C1) was recorded by an EC meter. After 10 minutes same samples were placed in a water bath at 100°C, and EC was calculated (C2). The formula used for calculation is given below.

MSI = [1-(C1/C2)] ×100.

The fresh weight of the flag leaf was recorded to calculate relative water content [30]. The leaves were dipped in water for 24 hours, and their turgid weight was measured. Leaves were dried for 48 hours at 70°C, and their dry weight was measured. The following formula was used for calculation;

RWC = [(Fresh weight – dry weight) / (turgid weight – dry weight)

Analysis of antioxidant enzymes

The Giannopolitis and Ries [31] method was used for superoxide dismutase (SOD) estimation. The fresh plant material (0.5 g) was homogenized in 10 mL phosphate buffer (pH 7). The samples were centrifuged at 15000 rpm for 15 min, and the supernatant (0.1 mL) was added to two sets of test tubes (A and B). Then 0.1 mL riboflavin stock, and 3 mL buffer (EDTA + NTB + methionine) was added and mixed. Set A test tubes were kept in fluorescent light (40 W), set B was kept in the dark for 8 minutes until conversion of yellow color to dark. The absorbance was taken at 560 nm for both sets. To measure ascorbate, peroxidase, and catalase activity, enzyme extract was prepared. The sample (1 g) was treated with 10 mL extraction buffer (1% polyvinyl polypyrrolidone in 50 mM potassium phosphate buffer). The homogenized mixture was centrifuged at 15,000 g for 30 minutes, and the supernatant was collected. The ascorbate peroxidase reaction mixture was prepared using 100 mM phosphate buffer, 100 µL enzyme extract, 0.1 mM EDTA, 0.062 mM H₂O_2, and 0.3 mM ascorbic acid. The activity of ascorbate peroxidase was calculated by measuring the oxidation rate of ascorbate at 290 nm [32]. The reaction mixture for catalase was prepared by using 200 µL of 50 mM K-phosphate buffer, 250 µL enzyme extract, 100 µL of H₂O₂ (60 ηM), and 450 µL DH₂O. To initiate the reaction, H₂O₂ was added to the mixture. The decrease in absorbance was taken at 470 nm [33].

Analysis of heavy metals in wheat

The plant samples were collected at the milking stage to find the content of heavy metals in roots, shoots, and wheat seeds. The uptake of heavy metals was measured by using the method of Ouzounidou et al. [34]. The dried sample (0.6 g) was digested with HNO₃ (5 mL), and after 12 h, H₂O₂ (0.5 mL) was added to the sample and placed in a muffle furnace (at 155°C) for 4.5 hrs. The remaining product was dissolved in distilled water to make the final volume 30 mL. Atomic absorption spectrophotometry was used to measure heavy metals in every sample individually.

Statistical analysis

To analyze data, Statistix 8.1 software was used for statistical analysis. ANOVA was applied, the design was completely randomized, Tukey honest significant test was used, and post-hoc was also done to measure the difference among treatments. The varieties were also compared, and it was observed that V2 was better than V1.

Heavy metal tolerance and plant growth-promoting characteristics of bacterial strains

In this research-heavy metal contaminated soil samples were collected from the effluent point of Kohinoor Textile Mills Rawalpindi, Pakistan. Bacterial strains were isolated and tested for Cr tolerance in the medium (Table 2). It was observed that all strains showed tolerance to 10 mg/L Cr stress while only 3 bacterial strains grew in the presence of 15 mg/L Cr concentration. Two strains named A2 and A3 tolerated 25 mg/L Cr stress. The plant growth-promoting characteristics like siderophore, ACC deaminase production, and phosphate solubilization were also tested for all bacterial strains. The strains named A3 performed better in all assessments, so it was selected for inoculation in wheat after its sequencing by 16S rRNA. It was identified as Bacillus subtilis with accession No. MZ 461936.

Table 1. Analysis of different parameters of soil used in pot experiment.

Parameters
pH	7.1
Texture	Clay sandy loam
Ec	1.92 ds/m
Organic matter	0.56%
P (extractable)	3.6 ppm
N (extractable)	23 ppm
K (extractable)	145 ppm
Cr (extractable)	0.00 mg/kg

Table 2. Heavy metal tolerance and plant growth promoting characteristics of bacterial strains.

Bacterial strains	Cr-10 mg/L)	Cr-15 mg/L)	Cr-25 mg/L)	Siderophore production	ACC deaminase	P solubilization
A1	+	-	-	+	+	+
A2	+	+	+	-	-	+
A3	+	+	+	+	+	+
A4	+	-	-	+	+	-
A5	+	+	-	+	+	+

Morphological parameters

Chromium treatment considerably decreased shoot length, root length, and biomass of wheat (Fig. 1). The application of phosphorus and Bacillus subtilis has a positive interaction and supports plants' development in unfavorable conditions. The plants treated with phosphorus showed an increase of 12.64% (V1), 7.55% (V1), 22.62% (V2), and 12.60% (V2) in shoot and root length, respectively, as compared to untreated stress exposed plants. The increase in these parameters by the inoculation of Bacillus subtilis was 25.50%, 17.73% in V1, and 33.73% and 22.46% in V2, respectively. The co-application of phosphorus and Bacillus subtilis further augmented the growth of plants. They increased shoot length and root length by 30.02% and 28.19% in V1 and 38.71% and 29.31% in V2, respectively. These treatments also enhanced the biomass of plants significantly at p<0.05. The application of phosphorus increased shoot fresh weight, shoot dry weight, root fresh weight, and root dry weight by 9.48%, 11.06%, 29.41, and 58.49 in V1 and 15.71%, 14.00%, 31.21%, and 62.29% in V2, respectively as compared to untreated stress facing plants. Bacillus subtilis also increased wheat biomass to 24.13%, 16.60%, 38.12%, 69.81% in V1 and 30.25%, 23.22%, 42.92%, and 73.77% in V2, respectively as compared to stress exposed untreated plants. The mixed phosphorus and Bacillus subtilis enhanced the biomass by 45.68%, 28.65%, 41.98%, 75.47% in V1, and 47.89%, 30.67%, 45.85%, and 83.60% in V2, respectively. It was observed that the increase was phosphate < Bacillus subtilis < Phosphate + Bacillus subtilis.

Physiological parameters

The increase in plant biomass is linked with the rate of photosynthesis and chlorophyll content. In this study, the amount of chlorophyll a and b content was decreased by 51.60% and 43.15% in V1 and 50.99%, and 41.81% in V2 compared to control (Fig. 2a and b). The supplementation of phosphorus increased the amount of chlorophyll a and b content by 16.89% and 12.96% in V1 and 43.70%, and 18.96% in V2 compared to their stress-exposed controls. The inoculation of Bacillus subtilis was better as compared to phosphorus. It enhanced the rate of chlorophyll synthesis by 31.75% (chlorophyll a, V1), 25.92% (chlorophyll b, V1), 62.91% (chlorophyll a, V2), and 31.01% (chlorophyll b, V2). The co-application of phosphorus and Bacillus subtilis gave significant results as compared to their separate application. They enhanced the amount of chlorophyll content by 52.70% (chlorophyll a, V1), 35.18% (chlorophyll b. V1), 70.19% (chlorophyll a, V2), and 43.10% (chlorophyll b. V2) as compared to untreated stress-facing plants. The trend was phosphate < Bacillus subtilis < Phosphate + Bacillus subtilis in both varieties of wheat.

The production of osmolytes was also increased in plants under stress conditions (Fig. 2c and d). The separate application of phosphorus increased the proline and sugar content by 8.92% and 19.56% in V1 and 13.24% and 22.15% in V2 compared to the untreated one. The presence of Bacillus subtilis in plants rhizosphere enhanced the synthesis of proline and sugar to 15.02% (V1), 31.52% (V1), 18.26% (V2), and 33.86% (V2), respectively. The maximum amount of proline and sugar was reported in plants receiving the co-application of phosphorus and Bacillus subtilis, i.e., the increase was 17.83%, 39.98%, 19.63%, and 41.30% as compared to their respective control.

The amount of protein was decreased in plants facing chromium stress (Fig. 2e). The decrease was 64.01% and 62.26% in V1 and V2, respectively. It was observed that phosphorus and Bacillus subtilis enhanced protein synthesis by 32.47%, 71.67% in V1 and 40.93%, and 75.45% in V2, respectively. In contrast, the maximum amount of protein was found in plants receiving the co-application of phosphorus and Bacillus subtilis 76.84% and 86.65% compared to un-inoculated ones. Chromium stress also decreased membrane stability (56.25%) and relative water content (34.24%) in plants (Fig. 2f and g). Phosphorus treatment improved the membrane stability and water content in stress-exposed plants by 40.00% (V1), 12.5% (V1), 42.85% (V2), and 15.68% (V2), respectively. Bacillus subtilis inoculation also remarkably increased the membrane stability (59.52%) and water content (23.52%). However, co-inoculation of phosphorous and Bacillus subtilis gave efficient results. They enhanced the membrane stability and relative water content to 62.85% (V1), 29.16% (V1), 66.66% (V2), and 31.37% (V2).

Biochemical parameters

The rate of antioxidants production, including superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT), was also measured in both wheat varieties (Fig. 3). The data analysis showed that the alone and combined application of phosphorus and Bacillus subtilis significantly (p<0.05) improved the activity of antioxidants in plants facing chromium stress. Phosphorus application enhanced the amount of SOD by 25.75% and 27.68% in V1 and V2 compared to untreated stress (Cr⁺⁶) facing plants. Bacillus subtilis also increased the rate of SOD synthesis by 41.71% (V1) and 54.58% (V2). The combined inoculation of phosphorus and Bacillus subtilis considerably improved the synthesis of SOD in plants, i.e., 60.41% and 67.71% increase was noted in V1 and V2 respectively compared to untreated stress (Cr⁺⁶) facing plants.

The activity of APX and CAT was also increased by the treatment of phosphorous to 61.04%, 21.47%, 64.28%, and 24.91% in V1 and V2, respectively, compared to untreated stress (Cr⁺⁶) facing plants. The improvement in APX and CAT by the inoculation of Bacillus subtilis was 73.19%, 34.03%, 79.15%, and 50.19% in V1 and V2, respectively. The Co-application of phosphorus and Bacillus subtilis gave more pronounced results, i.e., the increase was 91.05%, 55.21%, 95.39%, and 60.88% in V1 and V2, respectively, as compared to untreated stress (Cr⁺⁶) facing plants.

Heavy metal content

The amount of Cr⁺⁶ was analyzed in the shoot, root, and grains of wheat to evaluate the effect of phosphorus and Bacillus subtilis in reducing the toxicity and uptake of heavy metals in plants (Fig. 4). Phosphorus amendments reduce the amount of Cr⁺⁶ in shoot and roots by 31.99%, 17.28%, 33.70%, and 19.07% compared to untreated stress (Cr⁺⁶) facing plants. While the use of Bacillus subtilis further reduced the amount of Cr⁺⁶ in shoot and roots of wheat, i.e., 40.40%, 24.32%, 42.61%, and 25.67% decrease was observed in both wheat varieties. However, the co-application of phosphorus and Bacillus subtilis strongly reduced the uptake and accumulation of Cr⁺⁶ in plants. They decreased the amount of Cr⁺⁶ by 54.24%, 28.71%, 59.19%, and 30.53%. The content of Cr⁺⁶ was also significantly reduced in the grains of wheat. The decrease was 73.71% and 74.16% by using phosphorus, while Bacillus subtilis decreases the amount of Cr⁺⁶ by 80.93% and 83.57% compared to untreated stress (Cr⁺⁶) facing plants. The co-application of phosphorus and Bacillus subtilis considerably decreased the amount of Cr⁺⁶ in wheat grains (86.45% and 90.26% decrease was noted), and it gave the best results in both wheat varieties.

Heavy metals pollution is a severe threat to the environment, including agricultural soils. Heavy metals are toxic to soil structure and a severe issue to vegetation, including food crops and other plants. Heavy metal toxicity affects several cellular and biochemical processes in plants. Crops grown in heavy metal contaminated soil are hazardous (contained excess amount of metals) for living organisms when consumed [35]. Chromium is considered one of the most toxic heavy metals for plants and animals. It affects the soil's biogeochemical activities. It has been reported that chromium was most abundant among toxic heavy metals, and its concentration mostly exceeded the threshold limits [36–38]. Most often, the discharged chromium is gathered at the thin topsoil layer. Its contamination in the agricultural lands mainly results in low fertility, lack of proper structure, decreased aeration, and low yield because of disturbed plant growth. Chromium disturbs normal processes of plants, including germination, shoot, and root length, net photosynthesis, and stomatal conductance. Its accumulation in the plant causes chlorosis, decreases respiration and photosynthesis, and delays germination.

Wheat is susceptible to chromium stress [39]. The present study was designed to find the amelioration ability of phosphate fertilizer and plant growth-promoting rhizobacteria for wheat crops under chromium stress. Plant growth-promoting rhizobacteria have the strong ability to tolerate heavy metal stress in culture medium and contaminated soil. Due to their beneficial PGP characteristics, they enable the plants to tolerate metal stress and promote the growth of crops, including wheat. Heavy metal contaminated soil was used to isolate the PGPR, and Bacillus subtilis was selected based on its high heavy metal tolerance ability. It was further evaluated for the analysis of plant growth-promoting characteristics. This study elaborates on the positive impact of the co-application of phosphorus and Bacillus subtilis on wheat.

Chromium is severely noxious to plants, and it drastically reduces plant development and growth. Hexavalent Cr is more mutagenic and highly carcinogenic for many crops. Perveen et al. [40] found that vegetables irrigated with wastewater accumulated more concentrations of Cr ranging from 3.74-3.95 mg kg⁻¹ compared to control having 0.004 mg kg⁻¹ concentrations. All these noxious metals have been proposed to produce serious health problems, including kidney and lung diseases, different types of cancers, skin and respiratory diseases, gastrointestinal problems and, anemia. [41]. In this study, Cr⁺⁶ stress also reduced the growth of wheat while the application of phosphorus and Bacillus subtilis significantly improved the size and biomass of plants. The co-application of phosphorus and Bacillus subtilis acts in synergism to reduce the toxic effect of stress. Phosphorus is an essential macronutrient, and the inoculation of phosphorus solubilizing bacterial strains enhanced P availability and uptake in plants. The use of phosphorus fertilizer increased the biomass of plants under unfavorable conditions [42]. The application of phosphorus increased the growth of Corchorus capsularis by improving the rate of photosynthesis and reducing oxidative stress under unfavorable conditions [43]. It was also observed that PGPR having ACC deaminase activity increased the growth of Cajanus cajan and Triticum aestivum, and it also reduced the toxic effects of Cd stress on plants [44]. Moustaine et al. [45] reported the ability of different strains of Bacillus sp. to boost both roots and shoot lengths of T. aestivum compared to plants without PGPR inoculation. It was noted that Pantoeagglomerans bacterial strains gave satisfactory results in terms of fresh weight and chlorophyll content of wheat. Swarnalakshmi et al. [46] reported combined inoculation of mixtures and bio inoculants (i.e., Azotobacter chroococcum + Anabaena torulosa + Pseudomonas striata and/or Anabaena torulosa) were best over single inoculation and chemical fertilizer in terms of nutrient uptake and plant growth. Baris et al. [47] revealed that the plants receiving mixed inoculation of PGPR (Bacillus megaterium, Bacillus subtilis, Azospirillum brasilense) have a high uptake of plant nutrients as compared to the untreated ones. The application of phosphorus solubilizing bacteria and 50% recommended phosphate fertilizer considerably increase shoot length (21%), shoot dry weight (97%), P uptake (122%), and yield (50%) of O. sativa [48]. The co-application and Se and Bradyrhizobium japonicum improved plant biomass, osmolytes content, and antioxidant activity. These treatments improve the availability and uptake of nutrients in plants which improves the growth of plants in unfavorable conditions [49].

It was observed that there was a negative correlation between plant growth and Cr stress. They downregulate the physiological attributes, including the reduced rate of photosynthesis, transpiration, and decreased water using efficiency. Chlorophyll pigment production and protein contents were also reduced due to the toxicity of heavy metals in plants. The decrease in chlorophyll content was 25.83% and 22.77% in Nicotiana tobaccum facing heavy metal stress. [50]. Saleem et al. [51] reported that phosphorus improved biomass, photosynthetic pigments, gaseous exchange attributes, and yield and quality of end product in crops. Phosphorous fertilizer enhanced the content of chlorophyll pigments and decreased the bioavailability of heavy metals in the rhizosphere, making them less available to the roots directly, which improved plant growth. Chromium toxicity disturbs the metabolic processes in plants. Mushtaq et al. [52] reported that the cumulative use of press mud and PGPR increase photosynthetic rate, root length and, shoot length by 31%, 37%, and 19% in Abelmoschus esculents. They convert the Cr (VI) to Cr (III), which is less toxic, and the negative effect of Cr toxicity was reduced. The application of ACC deaminase producing PGPR and Fe-fortification enhances the amount of chlorophyll a and b content by 51.1% and 55.5%, respectively, in the presence of Cr⁺⁶ stress. This increase was due to the improved uptake of nutrients from the soil. The amount of K, P and N in leaves and roots was increased by 3.40 fold, 183.3%, 64.7%, 97.3%, 122.2%, and 25.6% compared to control [52]. Hamid et al. [53] reported that salinity stress decreases the amount of chlorophyll content and the rate of photosynthesis of Helianthus annuus by 43-53% and 39-53%, respectively. While the combined application of Bacillus subtilis and biogas slurry improved the chlorophyll content, photosynthetic rate, and transpiration rate by 78%, 84%, and 59%, respectively, compared to untreated control. This increase was due to reduced ROS, improved P uptake, and the production of growth hormones. The co-application of Bacillus subtilis and biogas slurry maintains water content in plants, and this study also corroborated our findings.

Proline and sugar are essential osmoprotectants accumulated in plant cells in response to different stresses. In this study, the amount of proline and sugar increased significantly by applying phosphorus and Bacillus subtilis in Cr⁺⁶ exposed plants. These treatments also maintained the structure of proteins, enhanced membrane stability, and relative water content of both wheat varieties. These results were supported by the study of Khanna et al. [54]. They reported that PGPR supplementation increased proline, reducing sugars, carbohydrates, and free amino acids 54.8%, 64.5%, 94%, and 63%, respectively. The increase in phenol, flavonoids, polyphenols, and anthocyanin was also significant as compared to control. The cumulative application of nutrients with biofertilizer improved chlorophyll content, osmolyte production, and antioxidant activities in plants. This improvement was linked with the activation of genes responsible for the synthesis of these metabolites. They reduce the toxicity of heavy metals by bio-sorption and cation exchange capacity. The production of osmolytes like proline and sugar reduces the toxic effects of free radicals and maintains membrane stability. The increase in water content was also associated with the mechanism of osmotic adjustment performed by these osmolytes in plants [55, 56].

Plants produce different antioxidants to cope with stress conditions, but when exposed to the high concentration of heavy metals, they cannot maintain homeostasis inside their cells. The decrease in antioxidants activities was may be due to the denaturation of the protein and distortion of the structure of enzymes which causes the reduction in their activity. In this study, the application of phosphorus and Bacillus subtilis increased the production of SOD, APX, and CAT in plants. The literature also reported that inoculation of PGPR (Pseudomonas aeruginosa) releases plant growth regulators like HCN, indole acetic acid, siderophore, and various other metabolites that improve plant growth in heavy metal (Ni and Cr) stress. They also increased the synthesis of SOD, GR, CAT, and APX in plants and decreased the heavy metal uptake by roots and their accumulation in plant tissues [57]. The inoculation of Rhizobium also increased SOD, POD, and CAT in leaves and roots of Vicia faba facing Cu stress [58]. When the plants face stress, their immune response becomes active, and the free radicals are detected by associated microbes, activating molecular patterns for reducing the damaging effects of stress. It was observed that the use of phosphate solubilizing bacterial strains and phosphate fertilizer increase catalase and peroxidase activity in Mentha piperita by 16% and 52%, respectively. [59].

The application of phosphorus increases the fertility of the soil, maintains soil pH, and increases the diversity of vegetation. They reduce the toxicity of heavy metals, increase plant resistance, and helps in the recovery of heavy metals polluted ecosystems [5]. It was noted that many studies showed that microorganisms resist, absorb and endure heavy metals by mechanisms like changing metabolic pathways, producing secondary metabolites, and their valance state [60]. In this study, the co-application of phosphorus and Bacillus subtilis considerably decreased the accumulation of Cr⁺⁶ in shoot, root, and wheat grains. Such findings were also documented by Wani and Khan [61], who has stated that the inoculation of Bacillus sp. decreases the uptake of chromium in the root, shoot, and grains of Cicer aretianum. Pseudomonas putida decreased the accumulation of Cr, Pb, Ni, and Cd in leaves and grains of Zea mays. They control the movement of heavy metals and bind them to make them unavailable. They also control the leaching of heavy metals. They modulate the movements of ions through the membrane transporters; all these factors contribute to the low uptake and accumulation of heavy metals in plants [62].

Bacillus subtilis has powerful potential to decrease heavy metal stress in contaminated soils and improves plant growth under heavy metal stress through different mechanisms. It was observed in this research that the sole and combined application of phosphorus and Bacillus subtilis increased the morphological, physiological, and biochemical attributes of plants. The co-application showed considerable improvement in plants facing chromium stress. The uptake and accumulation of Cr⁺⁶ were also significantly (p<0.05) decreased under these treatments. The direct and indirect mechanisms used by phosphorus and Bacillus subtilis were responsible for the better growth of plants in stress conditions, and they showed strong potential to be used to ameliorate heavy metals stress in food plants.

Availability of data and materials

All data mentioned in this paper has been provided in tables and Figures

Competing interests

Authors declare no conflict of interests

Funding

This research did not receive any specific funding

Authors' contributions

NI: Investigation; Methodology; original draft; Writing, NA: Conceptualization; Supervision; AN: Methodology, Data curation; Formal analysis, RQ: Project administration; AMS: Resources, RZS: Writing; Reviewing-editing.

Acknowledgements

The authors deeply thank to Chairperson of Botany Department, PMAS Arid Agriculture University Rawalpindi for providing resources.

Authors' information

¹Department of Botany, PMAS-Arid Agriculture University, 46300, Rawalpindi, Pakistan

²Rice Research Program, Crop Sciences Institute (CSI), National Agricultural Research Center (NARC), Islamabad, Pakistan

³Department of Microbiology, P.S.G.V.P. Mandal’s, Arts, Science, and Commerce College, Shahada 425409, India

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The Potential of Bacillus subtilis and Phosphorus in Improving the Growth of Wheat under Chromium Stress.

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Abstract

Figures

Introduction

Materials And Methods

Soil sampling

Isolation of bacterial strains and their analysis

Experimental layout

Analysis of plant length and biomass

Leaf Chlorophyll contents

Determination of Proline content

Determination of Soluble sugar and protein

Membrane Stability Index (M.S.I) and Relative water content

Analysis of antioxidant enzymes

Analysis of heavy metals in wheat

Statistical analysis

Results

Heavy metal tolerance and plant growth-promoting characteristics of bacterial strains

Morphological parameters

Physiological parameters

Biochemical parameters

Heavy metal content

Discussion

Conclusion

Declarations

References

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