Rhizosphere bacteria enhance selenium accumulation and volatilization by indian mustard

Abstract

Indian mustard (Brassica juncea L.) accumulates high tissue Se concentrations and volatilizes Se in relatively nontoxic forms, such as dimethylselenide. This study showed that the presence of bacteria in the rhizosphere of Indian mustard was necessary to achieve the best rates of plant Se accumulation and volatilization of selenate. Experiments with the antibiotic ampicillin showed that bacteria facilitated 35% of plant Se volatilization and 70% of plant tissue accumulation. These results were confirmed by inoculating axenic plants with rhizosphere bacteria. Compared with axenic controls, plants inoculated with rhizosphere bacteria had 5-fold higher Se concentrations in roots (the site of volatilization) and 4-fold higher rates of Se volatilization. Plants with bacteria contained a heat-labile compound in their root exudate; when this compound was added to the rhizosphere of axenic plants, Se accumulation in plant tissues increased. Plants with bacteria had an increased root surface area compared with axenic plants; the increased area was unlikely to have caused their increased tissue Se accumulation because they did not accumulate more Se when supplied with selenite or selenomethionine. Rhizosphere bacteria also possibly increased plant Se volatilization because they enabled plants to overcome a rate-limiting step in the Se volatilization pathway, i.e. Se accumulation in plant tissues.

Figure 1

Role of bacteria in Se volatilization (A) and accumulation (B) from plants supplied with 20 μm selenate. Ampicillin at 100 mg L⁻¹ was used as an antibiotic to inhibit rhizosphere bacteria. The mean and sd values of three replicates are shown. Differences in Se accumulation and volatilization between ampicillin-treated and untreated plants were significant (P < 0.05). DW, Dry weight.

Figure 2

Inoculation of bacterial isolates into the rhizosphere of axenic plants leads to increased Se accumulation in shoots (A) and roots (B) of plants supplied with 20 μm selenate. The mean and sd values of three replicates are shown. All bacterial strains were isolated from the rhizosphere of Indian mustard plants except for strains BM3 and BA5, which were isolated from the rhizosphere of salt-marsh bulrush plants. The differences in tissue Se accumulation for axenic plants compared with plants inoculated with bacteria were significant in most cases (P < 0.05). The following bacteria-treated plants showed nonsignificant differences from axenic tissue Se accumulation: shoot Se accumulation for strains BJ13, BJ6, BJ11, and BJ15, and both root and shoot Se accumulation for strain BM3. DW, Dry weight.

Figure 3

Inoculation of bacterial strains BJ2 and BJ15 into the rhizosphere of axenic plants leads to increased Se volatilization (A). Inoculation of these bacteria into the rhizosphere of axenic plants also increases the protein content of the root exudate (B) and Se accumulation in plant tissues (C). Bacterial strains BJ2 and BJ15 were identified as superior strains for plant tissue Se accumulation (Fig. 2). The mean and sd values of three replicates are shown. Differences in Se accumulation and volatilization between bacteria-treated and untreated plants were significant (P < 0.05). The amount of Se volatilized during a 24-h period by strains BJ2 and BJ15 alone (0.08 and 0.37 μg d⁻¹) was subtracted from the amount volatilized by the plant-bacteria combination during the same period. DW, Dry weight.

Figure 4

Axenic plants infected with bacterial strain BJ2 (A and C) had increased root-hair production compared with axenic plants (B and D) supplied with 0 and 20 μm selenate in agar medium. Seedlings were germinated from surface-sterilized seeds coated with or without bacteria in a methylcellulose paste, stained with acridine orange, and observed at 5× magnification. Similar results were observed when strain BJ15 was used and when the root tips were observed at 10× magnification by confocal microscopy. Bars = 100 μm.

Figure 5

Effect of bacteria and different Se concentrations on growth and Se accumulation by axenic plants grown in agar. A, The length of the longest root; B, fresh weight of individual seedlings; and C, Se concentration in seedling tissue (root plus shoot). All differences between bacteria-treated and untreated axenic plants were statistically significant (P < 0.05) except for root lengths at 250 μm Se and fresh weights at 0, 20, and 100 μm Se. DW, Dry weight.

Figure 6

The amino acid and protein contents of 0.22-μm filtered root exudate from plants inoculated with strain BJ2 were higher than those from axenic plants; all were supplied with 20 μm selenate. The amino acid concentration in root exudate from bacteria-supplied plants (0.5–23.6 nmol mL⁻¹) was divided by that of axenic plants (0.2–9.2 nmol mL⁻¹). The protein content shown in the inset was calculated from the amino acid content.

Figure 7

The bacteria-plant combination produced a heat-labile compound in diluted root exudate that, when supplied to axenic plants, enhanced the Se concentration in roots 5-fold. The mean and sd values of three replicates are shown. Differences in root Se concentrations of axenic plants treated with growth medium (diluted root exudate) supplied with bacteria and axenic plants treated with root exudate or boiled exudate were significant (P < 0.05). The root exudate from axenic plants or plants grown with strain BJ2 was filtered through a 0.22-μm filter to remove bacteria, and then added to a fresh batch of axenic plants with 20 μm selenate.