Combined application of plant growth-promoting bacteria and iron oxide nanoparticles ameliorates the toxic effects of arsenic in Ajwain (Trachyspermum ammi L.)

Abstract

Soil contamination with toxic heavy metals [such as arsenic (As)] is becoming a serious global problem because of the rapid development of the social economy. Although plant growth-promoting bacteria (PGPB) and nanoparticles (NPs) are the major protectants to alleviate metal toxicity, the study of these chemicals in combination to ameliorate the toxic effects of As is limited. Therefore, the present study was conducted to investigate the combined effects of different levels of Providencia vermicola (5 ppm and 10 ppm) and iron oxide nanoparticles (FeO-NPs) (50 mg/l^-1 and 100 mg/l^-1) on plant growth and biomass, photosynthetic pigments, gas exchange attributes, oxidative stress and response of antioxidant compounds (enzymatic and non-enzymatic), and their specific gene expression, sugars, nutritional status of the plant, organic acid exudation pattern As accumulation from the different parts of the plants, and electron microscopy under the soil, which was spiked with different levels of As [0 μM (i.e., no As), 50 μM, and 100 μM] in Ajwain (Trachyspermum ammi L.) seedlings. Results from the present study showed that the increasing levels of As in the soil significantly (p< 0.05) decreased plant growth and biomass, photosynthetic pigments, gas exchange attributes, sugars, and nutritional contents from the roots and shoots of the plants, and destroyed the ultra-structure of membrane-bound organelles. In contrast, increasing levels of As in the soil significantly (p< 0.05) increased oxidative stress indicators in term of malondialdehyde, hydrogen peroxide, and electrolyte leakage, and also increased organic acid exudation patter in the roots of T. ammi seedlings. The negative impact of As toxicity can overcome the application of PGPB (P. vermicola) and FeO-NPs, which ultimately increased plant growth and biomass by capturing the reactive oxygen species, and decreased oxidative stress in T. ammi seedlings by decreasing the As contents in the roots and shoots of the plants. Our results also showed that the FeO-NPs were more sever and showed better results when we compared with PGPB (P. vermicola) under the same treatment of As in the soil. Research findings, therefore, suggest that the combined application of P. vermicola and FeO-NPs can ameliorate As toxicity in T. ammi seedlings, resulting in improved plant growth and composition under metal stress, as depicted by balanced exudation of organic acids.

Keywords: Providencia vermicola; electron microanalysis; heavy metal; herbaceous crop; nano-technology.

Figure 1

Effect of combined application of various levels of iron oxide nanoparticles (FeO-NPs) (i.e., 50 mg/l⁻¹ and 100 mg/l⁻¹) and plant growth-promoting bacteria (Providencia vermicola) (i.e., 5 ppm and 10 ppm) on root length (A), shoot length (B), root fresh weight (C), shoot fresh weight (D), root dry weight (E), and shoot dry weight (F) of Ajwain (Trachyspermum ammi seedlings) grown under various stress levels of arsenic (i.e., 0 μM, 50 μM, and 100 μM). Values are demonstrated as means of four replicates, along with standard deviation (SD; n = 4). Two-way ANOVA was performed and means differences were tested by Tukey’s highly significant difference post-hoc test (p< 0.05). Different lowercase letters on the error bars indicate significant difference between the treatments.

Figure 2

Effect of combined application of various levels of iron oxide nanoparticles (FeO-NPs) (i.e., 50 mg/l⁻¹ and 100 mg/l⁻¹) and plant growth-promoting bacteria (Providencia vermicola) (i.e., 5 ppm and 10 ppm) on chlorophyll-a content (A), chlorophyll-b content (B), total chlorophyll content (C), carotenoid content (D), net photosynthesis, (E) stomatal conductance (F), transpiration rate (G), and intercellular CO₂ (H) of Ajwain (Trachyspermum ammi seedlings) grown under various stress levels of arsenic (i.e., 0 μM, 50 μM, and 100 μM). Values are demonstrated as means of four replicates along with standard deviation (SD; n = 4). Two-way ANOVA was performed and means differences were tested by Tukey’s highly significant difference post-hoc test (p< 0.05). Different lowercase letters on the error bars indicate significant difference between the treatments.

Figure 3

Effect of combined application of various levels of iron oxide nanoparticles (FeO-NPs) (i.e., 50 mg/l⁻¹ and 100 mg/l⁻¹) and plant growth-promoting bacteria (Providencia vermicola) (i.e., 5 ppm and 10 ppm) on malondialdehyde (MDA) contents in the roots (A), MDA contents in the leaves (B), hydrogen peroxide (H₂O₂) contents in the roots (C), H₂O₂ contents in the leaves (D), EL percentage in the roots (E), and EL percentage in the leaves (F) of Ajwain (Trachyspermum ammi seedlings) grown under various stress levels of arsenic (i.e., 0 μM, 50 μM, and 100 μM). Values are demonstrated as means of four replicates along with standard deviation (SD; n = 4). Two-way ANOVA was performed and means differences were tested by Tukey’s highly significant difference post-hoc test (p< 0.05). Different lowercase letters on the error bars indicate significant difference between the treatments.

Figure 4

Effect of combined application of various levels of iron oxide nanoparticle (FeO-NPs) (i.e., 50 mg/l⁻¹ and 100 mg/l⁻¹) and plant growth-promoting bacteria (Providencia vermicola) (i.e., 5 ppm and 10 ppm) on superoxide dismutase (SOD) activity in the roots (A), SOD activity in the shoots (B), peroxidase (POD) activity in the roots (C), POD activity in the shoots (D) catalase (CAT) activity in the roots (E), CAT activity in the shoots (F), ascorbate peroxidase (APX) activity in the roots, (G) and APX activity in the shoots (H) of Ajwain (Trachyspermum ammi seedlings) grown under various stress levels of arsenic (i.e., 0 μM, 50 μM, and 100 μM). Values are demonstrated as means of four replicates along with standard deviation (SD; n = 4). Two-way ANOVA was performed and means differences were tested by Tukey’s highly significant difference post-hoc test (p< 0.05). Different lowercase letters on the error bars indicate significant difference between the treatments.

Figure 5

Effect of combined application of various levels of iron oxide nanoparticles (FeO-NPs) (i.e., 50 mg/l⁻¹ and 100 mg/l⁻¹) and plant growth-promoting bacteria (Providencia vermicola) (i.e., 5 ppm and 10 ppm) on iron superoxidase dismutase (Fe-SOD) (A), peroxidase (POD) (B), catalase (CAT) (C), and ascorbate peroxidase (APX) (D) of Ajwain (Trachyspermum ammi seedlings) grown under various stress levels of arsenic (i.e., 0 μM, 50 μM, and 100 μM). Values are demonstrated as means of four replicates along with standard deviation (SD; n = 4). Two-way ANOVA was performed and means differences were tested by Tukey’s highly significant difference post-hoc test (p< 0.05). Different lowercase letters on the error bars indicate significant difference between the treatments.

Figure 6

Effect of combined application of various levels of iron oxide nanoparticles (FeO-NPs) (i.e., 50 mg/l⁻¹ and 100 mg/l⁻¹) and plant growth-promoting bacteria (Providencia vermicola) (i.e., 5 ppm and 10 ppm) on phenolic contents (A), flavonoid contents (B), ascorbic acid contents (C), anthocyanin contents (D), soluble sugar contents (E), reducing sugar contents (F), non-reducing sugar contents (G), and proline contents (H) of Ajwain (Trachyspermum ammi seedlings) grown under various stress levels of arsenic (i.e., 0 μM, 50 μM, and 100 μM). Values are demonstrated as means of four replicates along with standard deviation (SD; n = 4). Two-way ANOVA was performed and means differences were tested by Tukey’s highly significant difference post-hoc test (p< 0.05). Different lowercase letters on the error bars indicate significant difference between the treatments.

Figure 7

Effect of combined application of various levels of iron oxide nanoparticles (FeO-NPs) (i.e., 50 mg/l⁻¹ and 100 mg/l⁻¹) and plant growth-promoting bacteria (Providencia vermicola) (i.e., 5 ppm and 10 ppm) on iron contents in the roots (A), iron contents in the shoots (B), magnesium contents in the roots (C), magnesium contents in the shoots (D), calcium contents in the roots (E), calcium contents in the shoots (F), phosphorus contents in the roots (G), and phosphorus contents in the shoots (H) of Ajwain (Trachyspermum ammi seedlings) grown under various stress levels of arsenic (0 i.e., 0 μM, 50 μM, and 100 μM). Values are demonstrated as means of four replicates along with standard deviation (SD; n = 4). Two-way ANOVA was performed and means differences were tested by Tukey’s highly significant difference post-hoc test (p< 0.05). Different lowercase letters on the error bars indicate significant difference between the treatments.

Figure 8

Effect of combined application of various levels of iron oxide nanoparticles (FeO-NPs) (i.e., 50 mg/l⁻¹ and 100 mg/l⁻¹) and plant growth-promoting bacteria (Providencia vermicola) (i.e., 5 ppm and 10 ppm) on oxalic acid contents (A), malic acid contents (B), citric acid contents (C), acetic acid contents (D), formic acid contents (E), fumaric acid contents (F), in the roots and As contents in the roots (G), and As contents in the shoots (H) of Ajwain (Trachyspermum ammi seedlings) grown under various stress levels of arsenic (i.e., 0 μM, 50 μM, and 100 μM). Values are demonstrated as means of four replicates along with standard deviation (SD; n = 4). Two-way ANOVA was performed and means differences were tested by Tukey’s highly significant difference post-hoc test (p< 0.05). Different lowercase letters on the error bars indicate significant difference between the treatments.

Figure 9

Transmission electron microscopy (TEM) photos of Ajwain (Trachyspermum ammi seedlings) grown under various stress levels of As (i.e., 0 μM, 50 μM, and 100 μM). Different lowercase abbreviations in the TEM photos are presented as: C, chloroplast; CW, cell wall; M, mitochondria; FV, food vacuole; SG, starch grain; Po, peroxisome; and Pl, plastoglobuli. Different uppercase abbreviations are used for the various treatments of iron oxide nanoparticles (FeO-NPs) (i.e., 50 mg/l⁻¹ and 100 mg/l⁻¹) and plant growth-promoting bacteria [Providencia vermicola (P. vermicola)] (i.e., 5 ppm and 10 ppm) grown under various stress levels of As (i.e., 0 μM, 50 μM, and 100 μM). For example: (A) FeO-NPs 0 mg/l⁻¹, P. vermicola 0 ppm, and As concentration 0 μM; (B) FeO-NPs 50 mg/l⁻¹, P. vermicola 0 ppm, and As concentration 0 μM; (C) FeO-NPs 100 mg/l⁻¹, P. vermicola 0 ppm, and As concentration 0 μM; (D) FeO-NPs 0 mg/l⁻¹, P. vermicola 5 ppm, and As concentration 0 μM; (E) FeO-NPs 0 mg/l⁻¹, P. vermicola 10 ppm, and As concentration 0 μM; (F) FeO-NPs 0 mg/l⁻¹, P. vermicola 0 ppm, and As concentration 50 μM; (G) FeO-NPs 50 mg/l⁻¹, P. vermicola 0 ppm, and As concentration 50 μM; (H) FeO-NPs 100 mg/l⁻¹, P. vermicola 0 ppm, and As concentration 50 μM; (I) FeO-NPs 0 mg/l⁻¹, P. vermicola 5 ppm, and As concentration 50 μM; (J) FeO-NPs 0 mg/l⁻¹, P. vermicola 10 ppm, and As concentration 50 μM; (K) FeO-NPs 0 mg/l⁻¹, P. vermicola 0 ppm, and As concentration 100 μM; (L) FeO-NPs 50 mg/l⁻¹, P. vermicola 0 ppm, and As concentration 100 μM; (M) FeO-NPs 100 mg/l⁻¹, P. vermicola 0 ppm, and As concentration 100 μM; (N) FeO-NPs 0 mg/l⁻¹, P. vermicola 5 ppm, and As concentration 100 μM; and (O) FeO-NPs 0 mg/l⁻¹, P. vermicola 10 ppm, and As concentration 100 μM.

Figure 10

Schematic presentation of the findings from this study under the application of Providencia vermicola (P. vermicola) and iron oxide nanoparticles (FeO-NPs) in arsenic (As)-stressed Ajwain [Trachyspermum ammi (T. ammi)] seedlings grown under different levels of As stress (i.e., 50 μM and 100 μM) in sandy loam soil. The figure shows As sources in the natural soil and its toxic effects on the plants. The figure also shows that As toxicity can be overcome by the application of P. vermicola and FeO-NPs, which decreased oxidative stress in membrane-bound organelles by decreasing As content in various parts of the plants. Overall, this scheme presents the complete description of this experiment and the important findings that we have evaluated from the application of P. vermicola and FeO-NPs in As-stressed Ajwain (T. ammi) seedlings.