Heavy Metal-Resistant Plant Growth-Promoting Citrobacter werkmanii Strain WWN1 and Enterobacter cloacae Strain JWM6 Enhance Wheat (Triticum aestivum L.) Growth by Modulating Physiological Attributes and Some Key Antioxidants Under Multi-Metal Stress

Abstract

Due to wastewater irrigation, heavy metal (HM) exposure of agricultural soils is a major limiting factor for crop productivity. Plant growth-promoting bacteria (PGPB) may lower the risk of HM toxicity and increase crop yield. In this context, we evaluated two HM-resistant PGPB strains, i.e., Citrobacter werkmanii strain WWN1 and Enterobacter cloacae strain JWM6 isolated from wastewater-irrigated agricultural soils, for their efficacy to mitigate HM (Cd, Ni, and Pb) stress in a pot experiment. Increasing concentrations (0, 50, 100, and 200 ppm) of each HM were used to challenge wheat plants. Heavy metal stress negatively affected wheat growth, biomass, and physiology. The plants under elevated HM concentration accumulated significantly higher amounts of heavy metals (HMs) in shoots and roots, resulting in increased oxidative stress, which was evident from increased malondialdehyde (MDA) content in roots and shoots. Moreover, alterations in antioxidants like superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), and catalase (CAT) were observed in plants under HM stress. The severity of damage was more pronounced with rising HM concentration. However, inoculating wheat with Citrobacter werkmanii strain WWN1 and Enterobacter cloacae strain JWM6 (10⁷ CFU ml^-1) improved plant shoot length (11-42%), root length (19-125%), fresh weight (41-143%), dry weight (65-179%), and chlorophyll a (14%-24%) and chlorophyll b content (2-24%) under HM stress. Citrobacter werkmanii strain WWN1 and Enterobacter cloacae strain JWM6 either alone or in co-inoculation enhanced the antioxidant enzyme activity, which may lower oxidative stress in plants. However, seeds treated with the bacterial consortium showed an overall better outcome in altering oxidative stress and decreasing HM accumulation in wheat shoot and root tissues. Fourier transform infrared spectroscopy indicated the changes induced by HMs in functional groups on the biomass surface that display effective removal of HMs from aqueous medium using PGPB. Thus, the studied bacterial strains may have adequate fertilization and remediation potential for wheat cultivated in wastewater-irrigated soils. However, molecular investigation of mechanisms adopted by these bacteria to alleviate HM stress in wheat is required to be conducted.

Keywords: Citrobacter werkmanii and Enterobacter cloacae; bioremediation and biofertilization; cadmium (Cd); heavy metals contamination; lead (Pb); nickle (Ni); plant growth promoting bacteria; wastewater irrigated agricultural soils.

FIGURE 1

Bacterial colonization [CFU per gram rhizospheric soil (×10⁶)] in the rhizosphere of wheat under control and heavy metal (Cd, Ni, Pb) stress conditions. All the values are the mean of three replicates. Bars represent the standard error of means. Different letters above the bars indicate statistically significant difference between treatments at P ≤ 0.05. A, Citrobacter werkmanii strain WWN1; B, Enterobacter cloacae strain JWM6; A + B, Citrobacter + Enterobacter consortium; 50 ppm, 50 ppm of Cd, Ni, and Pb; 100 ppm, 100 ppm of Cd, Ni, and Pb; 200 ppm, 200 ppm of Cd, Ni, and Pb.

FIGURE 2

(A) Effect of heavy metal–resistant plant growth–promoting bacteria on the length of shoots and roots of wheat (Triticum aestivum) under control and heavy metal (Cd, Ni, Pb) stress conditions. Values are mean of three replicates. Bars represent the standard error of means. Different letters above the bars indicate statistically significant difference between treatments at P ≤ 0.05. Details of treatments are the same as those in Figure 1. (B) Effect of heavy metal (a) 0 ppm, (b) 50 ppm, (c) 100 ppm, and (d) 200 ppm resistant plant growth–promoting bacteria on the growth of wheat (Triticum aestivum) under control and heavy metal (Cd, Ni, Pb) stress conditions. Details of treatments are the same as those in Figure 1.

FIGURE 3

(A) Effect of heavy metal–resistant plant growth–promoting bacteria on the fresh and dry weights of the shoots of wheat (Triticum aestivum) under control and heavy metal (Cd, Ni, Pb) stress conditions. Values are the mean of three replicates. Bars represent the standard error of means. Different letters above the bars indicate statistically significant difference between treatments at P ≤ 0.05. Details of treatments are the same as those in Figure 1. (B) Effect of heavy metal–resistant plant growth–promoting bacteria on the fresh and dry weights of the roots of wheat (Triticum aestivum) under control and heavy metal (Cd, Ni, Pb) stress conditions. Values are the mean of three replicates. Bars represent the standard error of means. Different letters above the bars indicate statistically significant difference between treatments at P ≤ 0.05. Details of treatments are the same as those in Figure 1.

FIGURE 4

Effect of heavy metal–resistant plant growth–promoting bacteria on chlorophyll a and chlorophyll b of wheat (Triticum aestivum) under control and heavy metal (Cd, Ni, Pb) stress conditions. Values are the mean of three replicates. Bars represent the standard error of means. Different letters above the bars indicate statistically significant difference between treatments at P ≤ 0.05. Details of treatments are the same as those in Figure 1.

FIGURE 5

Effect of heavy metal–resistant plant growth–promoting bacteria on (A) MDA and (B) proline activity of wheat (Triticum aestivum L.) in plants under control and heavy metal (Cd, Ni, Pb) stress conditions. Values are the mean of three replicates. Bars represent the standard error of means. Different letters above the bars indicate statistically significant difference between treatments at P ≤ 0.05. Details of treatments are the same as those in Figure 1.

FIGURE 6

Effect of heavy metal–resistant plant growth–promoting bacteria on (A) SOD, (B) POD, (C) CAT, and (D) APX activity of wheat (Triticum aestivum L.) in plants under control and heavy metal (Cd, Ni, Pb) stress conditions. Values are the mean of three replicates. Bars represent the standard error of means. Different letters above bars the indicate statistically significant difference between treatments at P ≤ 0.05. Details of treatments are the same as those in Figure 1.

FIGURE 7

Heavy metal (A) cadmium, (B) lead, and (C) nickel uptake by shoots and roots of wheat (Triticum aestivum L.) grown under different concentrations of multi-HMs. Values are the mean of three replicates. Bars represent the standard error of means. Different letters above the bars indicate statistically significant difference between treatments at P ≤ 0.05. Details of treatments are the same as those in Figure 1.

FIGURE 8

Infrared spectra of Citrobacter werkmanii strain WWN1 and Enterobacter cloacae strain JWM6 biomass in the presence of heavy metals (HMs) (Ni, Cd, Ni).

FIGURE 9

Pearson’s correlation coefficient (r) of growth attributes and antioxidant enzymes of wheat root (A) and shoot (B) with and without the inoculation of heavy metal–resistant plant growth–promoting bacteria under Cd, Ni, and Pb stress. RL (root length), RFW (root fresh weight), and RDW (shoot dry weight). SL, shoot length; SFW, shoot fresh weight; SDW, shoot dry weight; Chl a, chlorophyll a; Chl b, chlorophyll b. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

FIGURE 10

Principal coordinate analysis (PCA) of the effect of bacterial inoculation on RL, root length; RFW, root fresh weight; and RDW, root dry weight and antioxidants in wheat roots. Details of treatments are the same as those in Figure 1.