Effects of B. subtilis on plant morphological traits

B. subtilis showed a significant effect on morphological traits in Brassica. Pb treatment significantly inhibited plant height compared to the control. Interestingly, after supplementation of the B. subtilis with Pb treatment, plant height increased (Figure 2A). Brassica plant weight (biomass) was significantly inhibited in response to Pb, while it was significantly increased in response to B. subtilis (Figure 2B). The Pb stress significantly inhibited the development of root length and weight. However, Pb combined with B. subtilis significantly improved root length in Brassica (Figure 2C), and the plant showed a similar fashion in root weight (Figure 2D).

Analysis of plant morphology. A) plant height (cm), B) plant weight (gm), C) Root length (cm), and D) Root weight (gm). Values are expressed as Mean ± SEM. Different letters among the error bars indicate a significant difference (p < 0.05) among the group means.

Effect of B. subtilis supplementation on Brassica alba leaf traits

Pb-treatment in Brassica negatively influenced leaf-related traits (leaf number, length, width, weight, and branching). The number of Brassica leaves significantly declined in response to Pb treatment, while Pb combined with B. subtilis significantly increased the leaf number in Brassica (Figure 3A). The increased potential of leaf number was almost similar to that of the control, and plant response to single B. subtilis treatment showed the highest leaf number. In terms of root length, there were no significant differences among control, Pb, and Pb with B. subtilis treatments (Figure 3B). The leaf width trait no significant difference between Pb and Pb combined with B. subtilis (Figure 2C). The leaf weight showed almost a similar response between the Pb and Pb combined with B. subtilis (Figure 3D). Interestingly, the number of Brassica branching significantly declined, but it increased in Pb combined with B. subtilis treatment (Figure 3E).

Analysis of plant leaf morphology. A) Leaf number, B) Leaf length (cm), C) Leaf width (cm), D) Leaf weight (gm), and E) Branching number. Values are expressed as Mean ± SEM. Different letters among the error bars indicate a significant difference (p < 0.05) among the group means.

Regulation of Brassica leaf photosynthetic pigments in response to B. subtilis

Photosynthetic pigments, including chlorophyll a, b, total chlorophyll, and carotenoid accumulation, considerably declined in response to Pb treatment. However, chlorophyll slightly increased in response to B. subtilis (Figure 4A). The chlorophyll b significantly declined under Pb but reverted in response to Pb combined with B. subtilis treatment (Figure 4B). In the case of total chlorophyll calculation, no substantial variation was observed (Figure 4C). Another key pigment, carotenoid content, significantly declined under Pb stress, while it increased significantly after supplementation of B. subtilis with that Pb treatment (Figure 4D).

Biochemical Appearance A) Chlorophyll a (mg/L), B) Chlorophyll b (mg/L), C) Total Chlorophyll (mg/L), and D) Carotenoid. Values are expressed as Mean ± SEM. Different letters among the error bars indicate a significant difference (p < 0.05) among the group means.

Alteration of soluble protein content in response to B. subtilis in Brassica

Protein content declined significantly under Pb stress compared to the control. However, it increased slightly in response to Pb combined with B. subtilis (Figure 5). The soluble protein content improved in response to B. subtilis (single treatment as positive control).

Analysis of Protein concentration (µg/L). Values are expressed as Mean ± SEM. Different letters among the error bars indicate a significant difference (p < 0.05) among the group means.