Alleviation of Cadmium and Nickel Toxicity and Phyto-Stimulation of Tomato Plant L. by Endophytic Micrococcus luteus and Enterobacter cloacae

Abstract

Cadmium (Cd) and nickel (Ni) are two of the most toxic metals, wreaking havoc on human health and agricultural output. Furthermore, high levels of Cd and Ni in the soil environment, particularly in the root zone, may slow plant development, resulting in lower plant biomass. On the other hand, endophytic bacteria offer great promise for reducing Cd and Ni. Moreover, they boost plants' resistance to heavy metal stress. Different bacterium strains were isolated from tomato roots. These isolates were identified as Micrococcus luteus and Enterobacter cloacae using 16SrDNA and were utilized to investigate their involvement in mitigating the detrimental effects of heavy metal stress. The two bacterial strains can solubilize phosphorus and create phytohormones as well as siderophores. Therefore, the objective of this study was to see how endophytic bacteria (Micrococcus luteus and Enterobactercloacae) affected the mitigation of stress from Cd and Ni in tomato plants grown in 50 μM Cd or Ni-contaminated soil. According to the findings, Cd and Ni considerably lowered growth, biomass, chlorophyll (Chl) content, and photosynthetic properties. Furthermore, the content of proline, phenol, malondialdehyde (MDA), H₂O₂, OH, O₂, the antioxidant defense system, and heavy metal (HM) contents were significantly raised under HM-stress conditions. However, endophytic bacteria greatly improved the resistance of tomato plants to HM stress by boosting enzymatic antioxidant defenses (i.e., catalase, peroxidase, superoxide dismutase, glutathione reductase, ascorbate peroxidase, lipoxygenase activity, and nitrate reductase), antioxidant, non-enzymatic defenses, and osmolyte substances such as proline, mineral content, and specific regulatory defense genes. Moreover, the plants treated had a higher value for bioconcentration factor (BCF) and translocation factor (TF) due to more extensive loss of Cd and Ni content from the soil. To summarize, the promotion of endophytic bacterium-induced HM resistance in tomato plants is essentially dependent on the influence of endophytic bacteria on antioxidant capacity and osmoregulation.

Keywords: antioxidant defenses; bioconcentration factor; heavy metals; specific regulatory defense genes; translocation factor.

Figure 1

NA plates of two heavy metal tolerant strains, Micrococcus luteus, and Enterobacter cloacae.

Figure 2

Based on 1000 replicates, the neighbor-joining tree of the 16S rDNA gene of the isolate Enterobacter cloacae (Accession no. OM519328) and 23 Enterobacter cloacae sequences (a). Micrococcus luteus (Accession number OM519327) and 13 Micrococcus luteus sequences (b).

Figure 3

The influence of endophytic bacteria on (a) shoot length, (b) length of the root, (c) fresh weight of shoot, (d) dry weight of shoots, (e) fresh weight of the root, (f) dry weight of the root in tomato plants growing under HM stress. Fisher’s test at p < 0.05 reveals significant variations in means (±standard error), which are different letters (a–g) on the same bars. *, ** and *** imply significance levels of 0.01 and 0.05, respectively. T1: (Control with tap water), T2: Micrococcus luteus, T3: Enterobacter cloacae, T4: 50 μM Cd, T5: Micrococcus luteus + 50 µM Cd, T6: Enterobacter cloacae + 50 µM Cd T7: 50 µM Ni, T8: Micrococcus luteus + 50 µM Ni, T9: Enterobacter cloacae + 50 µM Ni.

Figure 4

The influence of two different endophytic bacteria on (a) SPAD chlorophyll values, (b) net photosynthetic rate (PN), (c) stomatal conductance (gs), (d) intercellular CO₂ concentration (Ci), and (e) transpiration rate (E) in tomato plants growing under HM stress. Fisher’s test at p < 0.05 reveals significant variations in means (±standard error), which are different letters (a–i) on the same bars. Thus, *, ** and *** imply significance levels of 0.01 and 0.05, respectively. T1: (Control with tap water), T2: Micrococcus luteus, T3: Enterobacter cloacae, T4: 50 μM Cd, T5: Micrococcus luteus + 50 µM Cd, T6: Enterobacter cloacae + 50 µM Cd T7: 50 µM Ni, T8: Micrococcus luteus + 50 µM Ni, T9: Enterobacter cloacae + 50 µM Ni.

Figure 5

The influence of two different endophytic bacteria on (a) proline, (b) phenol, (c) lipid peroxidation (MDA), (d) hydrogen peroxide (H₂O₂), (e) hydroxyl radicals (OH), and (f) superoxide anion (O₂) in tomato plants growing under HM stress. Fisher’s test at p < 0.05 reveals significant variations in means (±standard error) of different letters (a–h) on the same bars. Thus, ** and *** imply significance levels of 0.01 and 0.05, respectively. T1: (Control with tap water), T2: Micrococcus luteus, T3: Enterobacter cloacae, T4: 50 μM Cd, T5: Micrococcus luteus + 50 µM Cd, T6: Enterobacter cloacae + 50 µM Cd T7: 50 µM Ni, T8: Micrococcus luteus + 50 µM Ni, T9: Enterobacter cloacae + 50 µM Ni.

Figure 6

The influence of two endophytic bacteria on (a) catalase, (b) superoxide dismutase, (c) peroxidase, (d) ascorbate peroxidase, (e) glutathione reductase, (f) lipoxygenase, (g) carbonic anhydrase and (h) nitrate reductase activity in tomato plants growing under HM stress. Fisher’s test at p < 0.05 reveals significant variations in means (±standard error) of different letters (a–i) on the same bars. Thus, ** and *** imply significance levels of 0.01 and 0.05, respectively. T1: (Control with tap water), T2: Micrococcus luteus, T3: Enterobacter cloacae, T4: 50 μM Cd, T5: Micrococcus luteus + 50 µM Cd, T6: Enterobacter cloacae + 50 µM Cd T7: 50 µM Ni, T8: Micrococcus luteus + 50 µM Ni, T9: Enterobacter cloacae + 50 µM Ni.

Figure 7

The influence of two endophytic bacteria on (a) α-Tocopherol, (b) lignin, (c) ethylene, (d) nitrogen (N), (e) phosphor (P), and (f) potassium (K) content in tomato plants growing under HM stress. Fishers test at p < 0.05 reveals significant variations in means (±standard error) of different letters (a–i) on the same bars Thus, ** and *** imply significance levels of 0.01 and 0.05, respectively. T1: (Control with tap water), T2: Micrococcus luteus, T3: Enterobacter cloacae, T4: 50 μM Cd, T5: Micrococcus luteus + 50 µM Cd, T6: Enterobacter cloacae + 50 µM Cd T7: 50 µM Ni, T8: Micrococcus luteus + 50 µM Ni, T9: Enterobacter cloacae + 50 µM Ni.

Figure 8

The influence of two endophytic bacteria on Cd, and Ni concentration in (a) leaves, (b) roots and BCF of Cd and Ni in (c) leaves, and (d) roots of tomato plants growing under HM stress. Fisher’s test at p < 0.05 reveals significant variations in means (±standard error) of different letters (a–f) on the same bars. T4: 50 μM Cd, T5: Micrococcus luteus + 50 µM Cd, T6: Enterobacter cloacae + 50 µM Cd T7: 50 µM Ni, T8: Micrococcus luteus + 50 µM Ni, T9: Enterobacter cloacae + 50 µM Ni.

Figure 9

The influence of two endophytic bacteria on antioxidant enzyme gene expression ((a) PAL gene, (b) PPO gene, (c) GPOX gene, and (d) GST gene) in tomato plants growing under HM stress. Fisher’s test at p < 0.05 reveals significant variations in means (±standard error) of different letters (a–e) on the same bars. T4: 50 μM Cd, T5: Micrococcus luteus + 50 µM Cd, T6: Enterobacter cloacae + 50 µM Cd T7: 50 µM Ni, T8: Micrococcus luteus + 50 µM Ni, T9: Enterobacter cloacae + 50 µM Ni.

Figure 10

(a) The heat map confirms the association between quantitative statistical parameters based on the mean values of different parameters reported in this research. (b) Using principal component analysis (PCA) to analyze the correlations between treatment variables in tomato plants. Shoot length (SL), root length (RL), F W. of the shoot (FS), D W. of the shoot (DS), F W. of root (FR), D W. of the root (DR), SPAD chlorophyll value (SPAD), stomatal conductance (gs), net photosynthetic rate (PN), transpiration rate (E), internal CO₂ concentration (Ci), proline (Pro), phenol, MDA, H₂O₂, OH, O₂, CAT, POX, SOD, GR, APX, LOX, carbonic anhydrase (CA), nitrate reductase activity (NR), α-Tocopherol (α-Toc), lignin, ethylene, metal concentration in leaves (MCL), metal concentration in roots (MCR), BFC leaves (BFCL), BFC roots (BFCR), PAL gene (PAL gn), PPO gene (PPO gn), GPOX gene (GPOX gn), and GST gene (GST gn).

Figure 11

Mechanism of endophytic bacteria to promote plant growth and metabolites.