Rhizobacteria Mitigate the Negative Effect of Aluminum on Pea Growth by Immobilizing the Toxicant and Modulating Root Exudation

Abstract

High soil acidity is one of the main unfavorable soil factors that inhibit the growth and mineral nutrition of plants. This is largely due to the toxicity of aluminum (Al), the mobility of which increases significantly in acidic soils. Symbiotic microorganisms have a wide range of beneficial properties for plants, protecting them against abiotic stress factors. This report describes the mechanisms of positive effects of plant growth-promoting rhizobacteria Pseudomonas fluorescens SPB2137 on four pea (Pisum sativum L.) genotypes grown in hydroponics and treated with 80 µM AlCl3. In batch culture, the bacteria produced auxins, possessed 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, alkalized the medium and immobilized Al, forming biofilm-like structures and insoluble phosphates. Inoculation with Ps. fluorescens SPB2137 increased root and/or shoot biomass of Al-treated plants. The bacteria alkalized the nutrient solution and transferred Al from the solution to the residue, which contained phosphorus that was exuded by roots. As a result, the Al concentration in roots decreased, while the amount of precipitated Al correlated negatively with its concentration in the solution, positively with the solution pH and negatively with Al concentration in roots and shoots. Treatment with Al induced root exudation of organic acids, amino acids and sugars. The bacteria modulated root exudation via utilization and/or stimulation processes. The effects of Al and bacteria on plants varied depending on pea genotype, but all the effects had a positive direction and the variability was mostly quantitative. Thus, Ps. fluorescens SPB2137 improved the Al tolerance of pea due to immobilization and exclusion of toxicants from the root zone.

Keywords: PGPR; aluminum; immobilization; pea; phosphorus; rhizosphere; root exudation; soil acidity; symbiosis.

Figure 1

The amount of aluminum in solution and residue (a), pH of the solution (b), the amount of P (c) and the number of bacteria (d) in batch culture of Ps. fluorescens SPB2137 supplemented with 80 or 160 µM AlCl₃. Treatments: −Pf—sterile control, +Pf—inoculated with Pseudomonas fluorescens SPB2137. Vertical bars show standard errors. Different letters show significant differences between treatments (least significant difference test, p < 0.05, n = 3). CFU stands for colony-forming units.

Figure 2

Confocal laser scanning images of Pseudomonas fluorescens SPB2137gfp batch culture in the absence of Al (a,b) and in the presence of 80 (c,d) or 160 (e,f) µM AlCl₃. The bacteria are colored green. Images are single optical sections (a,c,e) and 10 merged optical sections (b,d,f). Scale bar (20 µm) shown in e is the same for all images.

Figure 3

The number (a) and size (b,c) of clumps formed by Pseudomonas fluorescens SPB2137gfp in batch culture supplemented with 80 or 160 µm AlCl₃. Vertical bars show standard errors. Different letters show significant differences between treatments (Student’s t-test, p < 0.05, n varied from 5 to 18 depending on the parameter and treatment). nd stands for not detected.

Figure 4

Root (a), shoot (b) and whole plant (c) biomass of pea genotypes VIR1903, VIR8473, VIR7307 and VIR8353 inoculated with Pseudomonas fluorescens SPB2137 and treated with 80 µm AlCl₃. Treatments: −Al−Pf—Al-untreated and uninoculated control plants, −Al+Pf—Al-untreated and inoculated plants, +Al−Pf—Al-treated and uninoculated plants, +Al+Pf—Al-treated and inoculated plants. Vertical bars show standard errors. Different lowercase letters show significant differences between treatments (least significant difference test, p < 0.05, n varied from 16 to 24 depending on pea genotype and treatment). FW stands for fresh weight.

Figure 5

The number of Pseudomonas fluorescens SPB2137 in the nutrient solution (a) and on roots (b) of pea genotypes VIR1903, VIR8473, VIR7307 and VIR8353 untreated and treated with 80 µM AlCl₃. Treatments: −Al+Pf—Al-untreated and inoculated plants, +Al+Pf—Al-treated and inoculated plants. Vertical bars show standard errors. Different lowercase letters show significant differences between treatments (least significant difference test, p < 0.05, n varied from 4 to 12 depending on pea genotype and treatment). FW stands for fresh weight. CFU stands for colony-forming units.

Figure 6

Confocal laser scanning images showing localization examples of Pseudomonas fluorescens SPB2137gfp attached to the root surface (a) and root hairs (b) of pea genotype VIR1903 treated with 80 µM AlCl₃. The bacteria are colored in green. Images are 10 merged optical sections.

Figure 7

Effect of Pseudomonas fluorescens SPB2137 on Al concentration in the nutrient solution (a), aluminum amount in the residue in growth media (b) and aluminum concentration in roots (c) or shoots (d) of pea genotypes VIR1903, VIR8473, VIR7307 and VIR8353 untreated and treated with 80 µm AlCl₃ in the end of experiments. Treatments: +Al−Pf—Al-treated and uninoculated plants, +Al+Pf—Al-treated and inoculated plants. Vertical bars show standard errors. Different lowercase letters show significant differences between treatments (least significant difference test, p < 0.05, n = 3). DW stands for dry weight.

Figure 8

Effect of Pseudomonas fluorescens SPB2137 on solution pH (a) and amount of P in the residue in growth media (b) where pea genotypes VIR1903, VIR8473, VIR7307 and VIR8353 untreated and treated with 80 µM AlCl₃ were cultivated. Treatments: +Al−Pf—Al-treated and uninoculated plants, +Al+Pf—Al-treated and inoculated plants. Vertical bars show standard errors. Different lowercase letters show significant differences between treatments (least significant difference test, p < 0.05, n = 3).

Figure 9

Effect of Pseudomonas fluorescens SPB2137 on concentration of P in roots (a) and shoots (b) where pea genotypes VIR1903, VIR8473, VIR7307 and VIR8353 untreated and treated with 80 µm AlCl₃. Treatments: −Al−Pf—Al-untreated and uninoculated control plants, −Al+Pf—Al-untreated and inoculated plants, +Al−Pf—Al-treated and uninoculated plants, +Al+Pf—Al-treated and inoculated plants. Vertical bars show standard errors. Different lowercase letters show significant differences between treatments (least significant difference test, p < 0.05, n = 3). DW stands for dry weight.

Figure 10

Effect of Pseudomonas fluorescens SPB2137 on total root exudation of organic acids (a), amino acids (b) and sugars (c) by pea genotypes VIR1903, VIR8473, VIR7307 and VIR8353 untreated and treated with 80 µM AlCl₃. Treatments: −Al−Pf—Al-untreated and uninoculated control plants, −Al+Pf—Al-untreated and inoculated plants, +Al−Pf—Al-treated and uninoculated plants, +Al+Pf—Al-treated and inoculated plants. The data present the amount of exuded compounds of each category in sum for the period of plant cultivation divided by the root biomass. Vertical bars show standard errors. Different lowercase letters show significant differences between treatments (least significant difference test, p < 0.05, n = 3). DW stands for dry weight.