The Role of Different Rhizobacteria in Mitigating Aluminum Stress in Rice (Oriza sativa L.)

Abstract

Aluminum toxicity in acidic soils threatens rice (Oryza sativa L.) cultivation, hindering agricultural productivity. This study explores the potential of plant growth-promoting rhizobacteria (PGPR) as a novel and sustainable approach to mitigate aluminum stress in rice. Two rice varieties, INIAP-4M and SUPREMA I-1480, were selected for controlled laboratory experiments. Seedlings were exposed to varying aluminum concentrations (0, 2, 4, 8, and 16 mM) in the presence of four PGPR strains: Serratia marcescens (MO4), Enterobacter asburiae (MO5), Pseudomonas veronii (R4), and Pseudomonas protegens (CHAO). The INIAP-4M variety exhibited greater tolerance to aluminum than SUPREMA I-1480, maintaining 100% germination up to 4 mM and higher vigor index values. The study revealed that rhizobacteria exhibited different responses to aluminum concentrations. P. protegens and S. marcescens showed the highest viability at 0 mM (2.65 × 10¹⁰ and 1.71 × 10¹⁰ CFU mL⁻¹, respectively). However, P. veronii and S. marcescens exhibited the highest viability at aluminum concentrations of 2 and 4 mM, indicating their superior tolerance and adaptability under moderate aluminum stress. At 16 mM, all strains experienced a decrease, with P. protegens and E. asburiae being the most sensitive. The application of a microbial consortium significantly enhanced plant growth, increasing plant height to 73.75 cm, root fresh weight to 2.50 g, and leaf fresh weight to 6 g compared to the control (42.75 cm, 0.88 g, and 3.63 g, respectively). These findings suggest that PGPR offer a promising and sustainable strategy to bolster rice resilience against aluminum stress and potentially improve crop productivity in heavy metal-contaminated soils.

1. Introduction

Rice is essential for global food security and is the primary food source for over half of the world’s population [1,2]. According to the Food and Agriculture Organization of the United Nations (FAO), this cereal is the food base of more than 3.5 billion people and provides approximately 27% of dietary calories per capita worldwide [3,4]. Demand for rice is projected to increase by 30% by 2050, a challenge amplified by population growth and rising incomes in Asia, Africa, and Latin America [3]. However, rice production faces significant threats, one of which is aluminum toxicity, exacerbated by intensive agricultural practices that increase the presence of this metal in soils [5,6,7].

Aluminum inhibits root growth and restricts the absorption of essential nutrients, resulting in poor crop development and reduced grain production [8,9]. Approximately 30–40% of the world’s arable soils are classified as acidic, with toxic aluminum affecting up to 50% of major food crops, including rice [10,11,12]. In these soils, aluminum toxicity can reduce crop yields.

The growing awareness of environmental challenges necessitates continuous advancements in remediation techniques. Furthermore, traditional phytoremediation methods often rely on hyperaccumulators with restricted target contaminant ranges or slow remediation cycles, which can take several months or even years to achieve significant results [13,14,15]. This poses a particular challenge for crops like rice, where rapid remediation is crucial for maintaining food security. Therefore, traditional methods often prove impractical or unsustainable in the long run.

Using rhizobacteria to increase aluminum tolerance in rice crops innovatively addresses one of the most critical challenges in acid soil agriculture [16,17]. The high presence of aluminum (Al³⁺) in these soils, due to a pH lower than 5, constitutes a severe limitation for plant growth and productivity [11,18]. Aluminum fundamentally affects root development and the ability of plants to absorb water and nutrients [9,19]. Al³⁺ can interfere with physiological and biochemical processes such as the inhibition of cell elongation and division in root tips, the alteration of the integrity of the plasma membrane, and the absorption and transport of essential nutrients such as calcium, magnesium, and phosphorus [8,20].

Recent research has demonstrated the relevance of adjusting soil pH to reduce the availability of aluminum and its phytotoxic effects [18,20]. Simultaneously, the topic of how plants are able to counteract these abiotic stresses through their biological mechanisms, such as the secretion of organic acids, has been explored [21,22]. In this framework, plant growth-promoting rhizobacteria (PGPR) emerge as a promising strategy to strengthen the resilience of rice crops against hostile environments, taking advantage of plants’ natural mechanisms to neutralize the effects of aluminum and promote optimal development under conditions of acid soil [23,24,25].

PGPR can mitigate aluminum toxicity by different mechanisms, such as the production of organic acids that chelate toxic Al³⁺ ions, making them less available to plants [26]. Furthermore, PGPR enhance the secretion of exopolysaccharides, which form protective biofilms around root surfaces, reducing Al³⁺ uptake [27]. They also produce phytohormones, such as indole-3-acetic acid (IAA), that promote root growth and reduce damage caused by aluminum stress [28].

In agriculture, PGPR have been used for their ability to increase crop yields in a sustainable manner [25,29,30]. In recent years, PGPR have demonstrated great potential to increase rice productivity, mainly through nitrogen fixation, phosphate solubilization, the synthesis of growth regulators, and the biological control of pests and diseases [31]. Bacteria are able to improve plant mineral nutrition through associated nitrogen fixation [32,33], phosphate mobilization in the soil [34], siderophore production [35], the stimulation of the development of mycorrhizal symbiosis, and the modulation of root architecture [36]. PGPR can also activate phytopathogens resistance [37] and reduce abiotic stress caused by factors such as drought [38], salinity [39], and pollution by heavy metals [40,41]. PGPR enhance the bioremediation and biocontrol of aluminum contamination in soils. Various studies report significant increases in parameters such as plant height, number of tillers, grain weight, and final yield in rice inoculated with PGPR compared to non-inoculated controls [42,43,44].

This study adopts an integrated and novel approach to mitigating aluminum toxicity in rice, focusing on the evaluation of aluminum-tolerant PGPR strains. While previous research has explored the use of PGPR in promoting plant growth and stress tolerance, few have specifically investigated their potential to address aluminum toxicity in Oryza sativa L. Although this study will not cover all the complexities of aluminum’s impact on acidic soils, it provides a solid foundation for developing targeted and sustainable strategies for rice cultivation in such soils.

2. Materials and Methods

2.1. Genetic Material

For the development of this study, four strains of rhizobacteria were selected, which were conserved in the strain bank of the Molecular Biology laboratory of the Universidad Técnica Estatal de Quevedo (UTEQ) in coastal Ecuador. These previously characterized and reported strains, maintained at a temperature of −40 °C [45], included Serratia marcescens and Enterobacter asburiae, isolated from the rhizosphere of banana plants in Valencia, Ecuador; Pseudomonas veronii, isolated from grape cultivation under controlled conditions in Valparaíso, Chile; and Pseudomonas protegens, isolated from tobacco seeds in Morens, Fribourg Canton, Switzerland. The bacteria were cultivated under controlled conditions using King B medium, specifically formulated to favor the growth of Pseudomonas species. The medium was prepared with glycerol (10 g L⁻¹), peptone (20 g L⁻¹), magnesium sulfate heptahydrate (MgSO₄·7H₂O, 1.5 g L⁻¹), and monopotassium phosphate (KH₂PO₄, 1.5 g L⁻¹), with the pH adjusted to 7.2 before sterilization at 121 °C for 15 min. These strains were used in bacterial inoculation trials to evaluate their effect on two rice varieties, specifically INIAP-4M and SUPREMA I-1480, under aluminum stress conditions (

Table 1. Characterization of rhizobacteria by their antagonistic and PGP properties.

Rhizobacteria	Strain	PR	HCN	Prn	DAPG	Siderophores	Indole Acetic Acid
Serratia marcescens	M04	−	−	−	−	+	+
Enterobacter asburiae	M05	−	−	−	−	+	+
Pseudomonas veronii	R4	+	−	+	−	+	+
Pseudomonas protegens	CHA0	+	+	+	+	+	+

+: positive, −: negative, PR: protease, HCN: hydrogen cyanide, Prn: pyrrolnitrin, DAPG: 2,4-diacetylphloroglucinol.

). The selection of these strains was based on their ability to produce a variety of metabolites with potential antagonistic and plant growth-promoting (PGP) properties [46,47], which could give rice plants greater tolerance to abiotic stress generated by the presence of aluminum in the soil (Table 1).

2.2. Germination Trial for Sensitivity of Oryza sativa L. to Aluminum

Two genotypes of Oryza sativa L. (INIAP-4M and SUPREMA I-1480), obtained from the germplasm bank of the Instituto Nacional de Investigaciones Agropecuarias (INIAP), Pichilingue station, located in Quevedo, Ecuador, were used to determine the degree of sensitivity towards aluminum. These genotypes were selected based on their contrasting characteristics reported in previous studies and regional agronomic evaluations. One hundred rice seeds washed superficially with alcohol (70%) were sterilized for 30 s and NaOCl (1% V/V) for 60 s, and then rinsed three times with sterile distilled water. Ten seeds were placed in sterile Petri dishes containing 5% water–agar culture medium supplemented with different concentrations of aluminum sulfate Al₂(SO₄)₃ (0, 2, 8, and 16 mM) [48]. The Petri dishes were sealed and incubated at room temperature (25 °C) under a 12 h light–12 h dark photoperiod for 96 h [5].

Variables to Evaluate in Germination

All variables described below were measured on nine plants per treatment.

Hypocotyl Length (HL): The length of the hypocotyls (the stem-like structure located below the seed leaves but above the root) was measured using a digital Vernier caliper (cm).

Root Length (RL): The root length was measured from the base of the hypocotyl to the tip of the primary root (cm). This was achieved using a digital Vernier caliper after ten days to examine the development of the main root.

Vigor Index (VI): The vigor index was calculated to reflect the capacity for germination and development of essential structures. The equation to calculate the vigor index is as follows [49]:

$$\mathrm{V}\mathrm{I}=(\mathrm{R}\mathrm{L}+\mathrm{H}\mathrm{L})\times {\displaystyle \frac{\left(\mathrm{G}\mathrm{P}\right)}{100}}$$

(1)

where RL: average root length; HL: average hypocotyl length; and GP: germination percentage.

Germination Percentage (GP): The germination percentage was calculated by determining the ratio of seeds that successfully germinated to the total number of seeds sown. This value was expressed as a percentage (%), based on the number of seeds that exhibited visible radicle growth during the observation period.

Relative Growth Index (RGI): This was used to compare root growth under treatment versus control. This was evaluated using the following formula [50]:

RGI = RLS/RLC

(2)

In this equation, RLS represents the root length of nine plants from each treatment with the study substance or condition, while RLC is the root length in the control.

Germination Index (GI): This provides a comprehensive measure that combines seed germination and root growth. It was calculated using the following equation [51]:

$$\mathrm{GI}={\displaystyle \frac{\mathrm{RLS} \times \mathrm{GSS} \times 100}{\mathrm{R}\mathrm{L}\mathrm{C}\times \mathrm{G}\mathrm{S}\mathrm{C}}}$$

(3)

In this equation, RLS represents the root length in the treatment, GSS is the number of germinated seeds in the treatment, RLC is the root length in the control, and GSC is the number of germinated seeds in the control.

2.3. Degree of Protection of Rhizobacteria in the Presence of Aluminum

To determine the resistance of rhizobacteria to aluminum, a protocol was implemented using bacterial strains from the UTEQ germplasm bank. Each strain was inoculated individually in 50 mL Erlenmeyer flasks containing liquid King B culture medium (KBL), composed of peptone (10 g L⁻¹), magnesium sulfate (1.5 g L⁻¹), dipotassium phosphate (1.5 g L⁻¹), and glycerin (15 mL L⁻¹). This medium was enriched with different concentrations of aluminum sulfate Al₂(SO₄)₃, specifically 0, 2, 4, 8, and 16 mM, following the method described by Farh et al. [48]. The rhizobacteria used in this study included Serratia marcescens M04, Enterobacter asburiae M05, Pseudomonas veronii R4, and Pseudomonas protegens CHA0.

Samples were incubated at 28 °C with constant shaking at 150 rpm for seven days. Subsequently, pH was evaluated using a pH meter, the concentration of colony-forming units per milliliter (CFU mL⁻¹) was determined by the plate counting method by surface plating, and the absorbance was measured at 600 nm. Each of these measurements was performed in triplicate to ensure the precision and reproducibility of the results. These procedures allowed us to evaluate the ability of rhizobacteria to survive and potentially protect themselves in the presence of variable concentrations of aluminum, being a critical factor in agricultural environments affected by soil acidification.

Variables Evaluated

Hydrogen Potential (pH): The pH of the samples was measured using a calibrated pH meter (OHAUS Starter 5000 Bench Type, manufactured by OHAUS Corporation, Parsippany, NJ, USA) to ensure accuracy. This measurement is critical for understanding how aluminum affects bacterial activity, as pH influences the solubility of aluminum. Measurements were performed in triplicate for reproducibility [52].

Colony-Forming Units (CFU mL⁻¹): CFU mL⁻¹ was determined using the plate count method, calculating the number of viable cells through serial dilutions and applying the following standard formula [53]:

$$\mathrm{CFU} {\mathrm{mL}}^{-1}={\displaystyle \frac{\mathrm{Number} \mathrm{of} \mathrm{colonies} \times \mathrm{Plate} \mathrm{volumen}}{\begin{array}{c}\mathrm{Dilution} \mathrm{factor}\end{array}}}$$

(4)

Measurements were performed in triplicate.

Optical Density (OD600): Optical density at 600 nm was measured using a spectrophotometer (HACH DR3900, manufactured by Hach Company, headquartered in Loveland, CO, USA), providing an indirect estimate of bacterial biomass. OD600 measurements, also performed in triplicate, are a fast and effective method for monitoring cell growth.

The combination of CFU mL⁻¹ and OD600 provides a comprehensive evaluation of bacterial response to varying aluminum concentrations [54].

2.4. Effect of Application of Aluminum-Tolerant Rhizobacteria in Acidic Soils with the Presence of Aluminum

For rice sowing, the seeds were pre-germinated by being submerged in distilled water for 24 h. Then, they were placed on filter paper moistened with sterile distilled water, and then, the seeds were covered with filter paper. They were left covered with black plastic for 72 h until the appearance of radicles was observed. Ten pregerminated seeds were sown per treatment in transparent plastic cups, which contained 100 g of acidic soil with the presence of Al. Then, 2 mL was applied at a concentration of 1 × 10⁸ CFU mL⁻¹ of bacteria solution separately and in consortium (M04, M05, R4, and CHA0).

Variables Evaluated from the Third Experiment

The length of the plant and root was measured with a ruler from the base to the apex, and from the root collar to its tip, 25 days after the treatment. Fresh weight was determined by carefully extracting the plants, cleaning the roots and leaves, and immediately weighing them on a precision scale to assess biomass. Subsequently, to determine the seedlings’ root and leaf dry weight, the samples were subjected to a drying process in an oven at 65 °C for 48 h.

Determination of CFU g⁻¹: To calculate the concentration of colony-forming units per gram (CFU g⁻¹) in the soil and thus evaluate the abundance of bacteria, the viable bacteria counting methodology described by Bhuyan et al. [55] was used. This procedure involved performing serial dilutions followed by plate counting, facilitating accurate quantification of the presence and bacterial activity in the soil samples inoculated with the rhizobacteria.

2.5. Design and Statistical Analysis

For the tolerance assessment, protective capacity, and biostimulant effect experiments, a completely randomized design (CRD) was used. In the first experiment, the CRD was implemented to determine the degree of tolerance of two rice genetic materials under different aluminum concentrations (0, 2, 4, 8, and 16 mM). In the second experiment, the same design was applied to investigate the protective capacity of rhizobacteria strains M04, M05, R4, and CHA0 against aluminum stress. These experiments used five treatments, each with three repetitions and three experimental units per repetition.

For the biostimulant effect experiment, six treatments (M04, M05, R4, CHA0, a bacterial consortium, and a control) were evaluated with three repetitions and three experimental units per repetition.

Statistical analysis of the data was performed using Stat Graphics 18.1.08 software. An ANOVA was performed to detect significant differences among treatments and Tukey’s multiple range test was applied for comparisons of means with a significance level of p ≤ 0.05. Before performing the ANOVA, the assumptions of normality and homogeneity of variances were verified using the Shapiro–Wilk test and Bartlett test, respectively.

3. Results

3.1. Degree of Aluminum Tolerance in the Germination Process of Two Rice Varieties

In the SUPREMA I-1480 variety, the germination percentage exhibited significant differences (p ≤ 0.05) among the evaluated concentrations. At a concentration of 0 mM, germination reached 100%, which was significantly higher than at 2 and 4 mM, where it remained at 80% (p ≤ 0.05). At 8 mM, germination significantly decreased to 50%, demonstrating the sensitivity of this variety to aluminum at higher concentrations. Regarding hypocotyl length, 0 mM exhibited the highest value (52.33 cm), which was significantly superior (p ≤ 0.05) to the values obtained at 2, 4, and 8 mM, which ranged from 20.67 to 26.33 cm. Root length was significantly greater (p ≤ 0.05) at 0 mM (39.33 cm) compared to 2 mM (11.50 cm), while no measurable values were recorded at 4 and 8 mM. In terms of vigor index, the highest value was observed at 0 mM (655), significantly decreasing to 149 at 2 mM and reaching 52 at 8 mM. The relative growth index (RGI) was 1 at 0 mM, significantly decreasing to 0.15 at 2 mM (p ≤ 0.05). Similarly, the germination index (GI) also demonstrated significant differences (p ≤ 0.05), with a value of 1 at 0 mM, reducing to 0.23 at 2 mM (

Table 2. Aluminum tolerance of two rice varieties.

Rice Variety	Al2(SO4)3	GP (%)	HL (cm)	RL (cm)	VI	RGI	GI
SUPREMA I-1480	0 mM	100 ± 0.00 a	52.33 ± 0.44 a	39.33 ± 0.93 a	655 ± 5.00 a	1 ± 0.00 a	1 ± 0.00 a
SUPREMA I-1480	2 mM	80 ± 10.00 b	25.67 ± 1.17 b	11.50 ± 0.25 b	149 ± 7.86 b	0.28 ± 0.02 b	0.23 ± 0.05 b
SUPREMA I-1480	4 mM	80 ± 7.07 b	26.33 ± 0.93 b	-	105 ± 7.42 c	-	-
SUPREMA I-1480	8 mM	50 ± 8.66 c	20.67 ± 0.17 b	-	52 ± 0.83 d	-	-
SUPREMA I-1480	16 mM	-	-	-	-	-	-
INIAP-4M	0 mM	100 ± 0.00 a	47.00 ± 2.65 a	55.67 ± 2.89 a	767 ± 4.41 a	1 ± 0.00 a	1 ± 0.00 a
INIAP-4M	2 mM	100 ± 0.00 a	31.67 ± 0.93 b	3.90 ± 1.53 b	178 ± 2.24 b	0.07 ± 0.04 b	0.07 ± 0.02 b
INIAP-4M	4 mM	100 ± 0.00 a	25.50 ± 0.14 b	0.60 ± 0.05 c	129 ± 0.73 c	0.01 ± 0.06 b	0.01 ± 0.02 b
INIAP-4M	8 mM	70 ± 7.07 b	23.83 ± 0.46 bc	-	83 ± 3.25 d	-	-
INIAP-4M	16 mM	60 ± 7.07 c	15.67 ± 0.33 c	-	47 ± 2.00 e	-	-

GP: germination percentage, HL: hypocotyl length, RL: root length, VI: vigor index, RGI: relative growth index, GI: germination index. The bars indicate the individual SD (standard deviation) for treatment (±). Means with equal letters in the column do not differ significantly, according to Tukey’s test (p ≤ 0.05).

).

In the INIAP-4M variety, the germination percentage did not show significant differences (p > 0.05) among 0, 2, and 4 mM, remaining at 100%. However, at 8 mM, germination significantly decreased to 70% (p ≤ 0.05) and further declined to 60% at 16 mM (p ≤ 0.05). Hypocotyl length was significantly higher (p ≤ 0.05) at 0 mM (47.00 cm) compared to concentrations of 2 and 4 mM. Root length, which was significantly greater at 0 mM (55.67 cm, p ≤ 0.05), decreased to 3.90 cm and 0.60 cm at 2 and 4 mM, respectively (p ≤ 0.05). No measurable values were recorded at higher concentrations. For the vigor index, a significant decrease was observed from 767 to 129 between 0 and 4 mM (p ≤ 0.05). The relative growth index (RGI) significantly decreased from 1 ± 0.00 to 0.03 between 0 and 2 mM (p ≤ 0.05), reaching 0.01 at 4 mM. Finally, the germination index (GI) showed values of 1 ± 0.00 at 0 mM, significantly decreasing to 7.55 at 2 mM and 0.54 at 4 mM (p ≤ 0.05) (Table 2).

Figure 1 illustrates the effects of aluminum on the roots of the SUPREMA I-1480 and INIAP-4M varieties. In SUPREMA I-1480, a significant reduction in root development was observed at concentrations exceeding 4 mM, with severe necrosis at 8 and 16 mM (indicated by red arrows). In contrast, INIAP-4M exhibited better root development at 2 and 4 mM, although necrosis was also present at 8 and 16 mM, indicating greater relative tolerance at lower concentrations. Both varieties were severely affected at 8 and 16 mM, confirming that these concentrations surpass their tolerance capacity.

3.2. Degree of Protection of Rhizobacteria to Aluminum

3.2.1. Viability of Rhizobacteria to Aluminum

The viability of aluminum-tolerant rhizobacteria was evaluated at different concentrations of aluminum sulfate (Al₂(SO₄)₃), measured as colony-forming units per milliliter (CFU mL⁻¹) (Figure 2 and Figure 3). At 0 mM aluminum, P. protegens CHA0 exhibited the highest CFU count (2.65 × 10¹⁰ CFU mL⁻¹), followed by S. marcescens M04 (1.71 × 10¹⁰ CFU mL⁻¹), with no statistically significant difference between these strains (p > 0.05). P. veronii R4 and E. asburiae M05 displayed slightly lower viabilities (1.33 × 10¹⁰ and 1.30 × 10¹⁰ CFU mL⁻¹, respectively), and these were not significantly different from the other strains (p > 0.05).

At 2 mM Al, all strains showed a decrease in viability. S. marcescens M04 maintained the highest viability (2.51 × 10¹⁰ CFU mL⁻¹), significantly higher than P. protegens CHA0 (2.50 × 10⁹ CFU mL⁻¹), E. asburiae M05 (2.27 × 10⁹ CFU mL⁻¹), and P. veronii R4 (1.44 × 10¹⁰ CFU mL⁻¹) (p ≤ 0.05). However, P. veronii R4 showed significantly higher viability compared to both P. protegens CHA0 and E. asburiae M05 (p ≤ 0.05) (Figure 2 and Figure 3).

At 4 mM Al, P. veronii R4 exhibited the highest CFU count (2.55 × 10¹⁰ CFU mL⁻¹), which was significantly higher than that of S. marcescens M04 (1.33 × 10¹⁰ CFU mL⁻¹), P. protegens CHA0 (1.28 × 10⁸ CFU mL⁻¹), and E. asburiae M05 (8.82 × 10⁹ CFU mL⁻¹). At this concentration, the susceptibility of P. protegens CHA0 became more pronounced, with a significant reduction compared to lower concentrations (p ≤ 0.05) (Figure 2 and Figure 3).

At 8 mM aluminum, all strains experienced a marked reduction in viability. S. marcescens M04 and P. veronii R4 demonstrated relatively greater resistance, with CFU counts of 1.50 × 10⁹ and 2.37 × 10⁹ CFU mL⁻¹, respectively, both significantly higher than P. protegens CHA0 (1.57 × 10⁸ CFU mL⁻¹) and E. asburiae M05 (1.60 × 10⁸ CFU mL⁻¹) (p ≤ 0.05) (Figure 2 and Figure 3).

At the maximum concentration of 16 mM, P. veronii R4 retained viability (7 × 10⁸ CFU mL⁻¹), significantly higher than S. marcescens M04 (1.59 × 10⁵ CFU mL⁻¹), which demonstrated extreme sensitivity. Both P. protegens CHA0 and E. asburiae M05 showed complete inhibition, with CFU counts below detectable limits, indicating their inability to tolerate aluminum at this concentration (Figure 2 and Figure 3).

Figure 2 and Figure 3 illustrate the variability in aluminum tolerance among the evaluated strains. While Figure 2 presents the quantitative results (CFU mL⁻¹) and statistical differences, Figure 3 provides visual confirmation of these trends, showcasing the density of colonies across the aluminum concentration gradient.

3.2.2. Optical Density Analysis of Tolerant Rhizobacteria

Optical density at 600 nm was measured to assess the tolerance of four rhizobacterial strains to different concentrations of aluminum sulfate (Al₂(SO₄)₃) (Figure 4). At 0 mM aluminum, P. protegens CHA0 showed the highest optical density (1.230); however, no statistically significant difference was observed between the other strains (p > 0.05).

At 2 mM aluminum, P. veronii R4 presented the highest optical density (0.590), showing significant differences with P. protegens CHA0, S. marcescens M04, and E. asburiae M05 (p ≤ 0.05). At 4 mM aluminum, optical density values for all strains were reduced compared to 0 mM. S. marcescens M04 (0.550), which did not show significant differences with P. veronii R4 (0.500); however, both strains showed significant differences with E. asburiae M05 (0.430) and P. protegens CHA0 (0.400) (p ≤ 0.05).

At higher concentrations of aluminum (8 and 16 mM), all strains experienced a decrease in optical density, suggesting an adverse effect of aluminum on bacterial growth. P. veronii R4 and S. marcescens M04 maintained the highest optical densities under these conditions, showing significant differences with P. protegens and E. asburiae M05 (p ≤ 0.05). At 16 mM, E. asburiae M05 produced the lowest optical density, reflecting the lowest tolerance among the strains studied (Figure 4).

3.2.3. Determination of pH at Aluminum Concentrations Inoculated with Rhizobacteria

After seven days of culture, significant increases in pH were noted in the media inoculated with the bacteria compared to the control, which presented a decrease dependent on the aluminum concentration. As a result, it ranged from an initial pH of 7.50 in the absence of aluminum to a pH of 3.50 at the maximum concentration tested. Specifically, P. veronii R4 demonstrated a notable ability to alter the medium’s acidity, raising the pH to 8.18, 7.83, and 5.80 at aluminum concentrations of 2, 4, and 8 mM, respectively (Figure 5).

At 0 mM aluminum, P. veronii R4 demonstrated the highest pH value (8.27), which was statistically different (p ≤ 0.05) from P. protegens CHA0 (7.84). No significant differences were detected among P. veronii R4, S. marcescens M04 (8.09), and E. asburiae M05 (8.09), indicating a similar ability to increase pH in the absence of aluminum stress. The control presented a lower pH (7.52), showing that all strains enhanced the medium’s alkalinity.

At 2, 4, and 8 mM aluminum, a consistent pattern emerged. P. veronii R4 maintained the highest pH values (8.18, 7.83, and 5.80, respectively), significantly higher than S. marcescens M04 (8.10, 7.81, and 5.50) and E. asburiae M05 (8.07, 7.60, and 5.61) (p ≤ 0.05). However, no significant differences were observed between S. marcescens M04 and E. asburiae M05 at these concentrations (p > 0.05). P. protegens CHA0 maintained lower pH values (7.39, 7.81, and 5.62), but higher than the control at each concentration. The control exhibited a steady decline in pH, dropping from 7.24 at 2 mM to 5.11 at 8 mM.

At 16 mM Al, all strains experienced a marked reduction in pH. P. veronii R4 (4.00) showed the highest pH, with no significant difference (p > 0.05) from the other strains. However, all strains maintained significantly higher pH levels than the control (3.53) (p ≤ 0.05) (Figure 5).

3.3. Application of Rhizobacteria in Soil Contaminated with Aluminum on the Vegetative Development of Oryza sativa L.

3.3.1. Plant Height and Root Length

The microbial consortium resulted in the highest plant height (73.75 cm) and root length (23.25 cm), significantly outperforming S. marcescens M04, P. protegens CHA0, E. asburiae M05, and the control (p ≤ 0.05). However, no statistically significant differences were observed between the consortium and P. veronii R4 (p > 0.05) (Figure 6).

The treatments with E. asburiae M05 and the control resulted in the lowest plant height (43.50 cm and 42.75 cm, respectively), with no significant differences between them (p > 0.05). However, in root length, all strains were significantly superior to the control treatment (p ≤ 0.05) (Figure 6).

3.3.2. Fresh and Dry Root Weight

The microbial consortium exhibited the highest values for root fresh weight (2.50 g) and dry weight (2.33 g), significantly surpassing all other treatments (p ≤ 0.05). These results highlight the synergistic effect of the consortium in promoting root biomass under aluminum stress. The single-strain treatments did not present significant differences among themselves (p > 0.05). However, in terms of fresh weight, S. marcescens M04, P. veronii R4, and E. asburiae M05 were superior to the control treatment (p > 0.05). P. protegens CHA0 exhibited lower root fresh weight (1.25 g) and dry weight (0.90 g) (Figure 7).

3.3.3. Fresh Weight and Dry Leaf

The microbial consortium demonstrated the highest leaf fresh weight (6.00 g) and dry weight (2.70 g), significantly outperforming all other treatments (p ≤ 0.05). For fresh leaf weight, no significant differences (p > 0.05) were observed among S. marcescens M04 (5.00 g), P. veronii R4 (4.75 g), P. protegens CHA0 (4.38 g), and E. asburiae M05 (4.38 g). However, S. marcescens M04 and P. veronii R4 were statistically superior to the control treatment (p ≤ 0.05). Regarding dry leaf weight, P. veronii R4 (2.15 g) and S. marcescens M04 (2.08 g) did not show significant differences (p > 0.05) but were significantly higher than E. asburiae M05 (1.63 g) (p ≤ 0.05). The control exhibited the lowest dry weight (1.45 g) (Figure 8).

3.3.4. Content of Colony-Forming Units in Soil (CFU g⁻¹)

The microbial consortium demonstrated the highest bacterial population in the soil, with a mean value of 1.28 × 10⁹ CFU g⁻¹, significantly surpassing all other treatments (p ≤ 0.05). Among the single-strain treatments, S. marcescens M04 achieved a bacterial population of 8.10 × 10⁸ CFU g⁻¹, showing significant differences (p ≤ 0.05) from the other strains. The bacterial populations of P. veronii R4 (9.08 × 10⁷ CFU g⁻¹), P. protegens CHA0 (4.53 × 10⁷ CFU g⁻¹), and E. asburiae M05 (3.63 × 10⁷ CFU g⁻¹) did not show significant differences among themselves (p > 0.05). The control treatment exhibited no measurable bacterial population, highlighting the absence of introduced microorganisms (Figure 9).

4. Discussion

4.1. Aluminum Tolerance in the Germination Process of Two Varieties of Oryza sativa L.

Aluminum in acidic soils is highly toxic to plants, inhibiting root growth [56]. This toxicity reduces water and nutrient absorption, affecting plant development [57]. In Oryza sativa, aluminum alters root structure by reducing metaxylem vessels and increasing sclerenchyma cells [56]. Additionally, aluminum induces oxidative stress, increasing reactive oxygen species (ROS) that damage cell membranes [57].

This study allowed the evaluation of tolerance to aluminum in two varieties of Oryza sativa L., INIAP-4M and SUPREMA I-1480, by exposing them to various aluminum concentrations. The varieties exhibited negative effects on root growth as aluminum concentrations increased, confirming the toxic impact of aluminum on rice development. INIAP-4M demonstrated a higher relative tolerance to aluminum compared to SUPREMA I-1480, particularly in terms of germination percentage, which remained at 100% even at concentrations of 4 mM, while SUPREMA I-1480 decreased to 80% at 2 mM. However, root growth in INIAP-4M was drastically reduced, reaching only 3.90 cm at 2 mM and 0.60 cm at 4 mM, indicating that while seeds germinated successfully, root elongation was severely inhibited.

In SUPREMA I-1480, germination and root growth were even more sensitive to aluminum. Germination was significantly decreased at 2 mM (80%), and root elongation ceased completely at 4 mM, highlighting its lower relative tolerance. Severe root necrosis was observed in both varieties at 8 and 16 mM, confirming that these concentrations exceed the tolerance capacity of both genotypes. These results suggest that INIAP-4M exhibits partial tolerance to aluminum at low to moderate concentrations (up to 4 mM), as demonstrated by its ability to maintain germination and some root development.

These findings align with previous studies by Bhadra and Roy [58], who analyzed 14 rice genotypes with different degrees of tolerance to aluminum, determining that germination varied significantly between them, ranging from 72 to 99.50%. Most genotypes indicated a high germination rate, suggesting a general trend toward aluminum tolerance in these varieties.

Azura et al. [59] found that root length decreases with increasing aluminum concentration, highlighting that the rice variety MR 219 is less tolerant to aluminum than other varieties. This finding is particularly relevant in acidic soils, which, as Zhao et al. [60] point out, are characterized by high concentrations of ammonium and aluminum, which increases the potential for toxicity.

Furthermore, the studies by Awasthi et al. [5] on 24 wild Indian rice varieties exposed to 100 μM of Al indicated significant differences in growth parameters, including relative growth rate and root tolerance index. These results indicate that aluminum toxicity variably affects different rice varieties. Similarly, Chang et al. [61] observed that certain Japanese rice varieties yielded greater tolerance to aluminum than Indian varieties, which was corroborated by the relative elongation of the root.

4.2. Degree of Protection of Rhizobacteria with Aluminum Concentrations

Plant growth-promoting rhizobacteria (PGPR) mitigate aluminum toxicity through various mechanisms. PGPR immobilize aluminum in the rhizosphere and reduce its availability to plants [17]. Certain species promote the exudation of organic acids that chelate aluminum and protect the roots [62]. Biofilms formed around the roots create a barrier that prevents aluminum from entering plant tissues [63]. PGPR release various low-molecular-weight organic acids into the rhizosphere, such as acetic, lactic, malic, succinic, tartaric, gluconic, 2-ketogluconic, oxalic, and citric acids [64,65]. These compounds play a crucial role in phosphate solubilization and metal chelation, including aluminum, forming stable complexes that reduce their availability and toxicity to plants. Other mechanisms include the production of antioxidants that neutralize oxidative stress caused by aluminum [66].

In addition, PGPR synthesize siderophores, low-molecular-weight compounds with a high affinity for metals such as iron and aluminum. These siderophores chelate Al³⁺, forming stable complexes that limit its availability to plants and, therefore, reduce its toxicity [67].

This study investigated the tolerance of rhizobacteria P. veronii (R4) and S. marcensces (M04) to aluminum concentrations. While both strains displayed viability at high aluminum levels, their growth significantly decreased at 16 mM. This was reflected in lower CFU mL⁻¹ and OD, indicating a reduced ability to thrive under high aluminum stress. This suggests a potential limitation in the production of secondary metabolites essential for aluminum sequestration at such high concentrations.

However, at intermediate aluminum concentrations (2–8 mM), both strains demonstrated higher viability and optical density, suggesting the presence of effective mitigation mechanisms within these ranges. These results indicate significant differences in the capacity of aluminum stress tolerance between the strains, highlighting their potential in moderately acidic soils while limiting their applicability in environments with high aluminum toxicity.

Pseudomonas species have been shown to reduce aluminum toxicity through metabolic adaptations, including the overexpression of enzymes such as NADP-dependent isocitrate dehydrogenase and glucose-6-phosphate dehydrogenase. These enzymes promote a reducing environment within the cell, allowing the bacteria to counteract the toxic effects of aluminum and maintain their viability under conditions of high aluminum concentrations [68]. In addition, Pseudomonas species produce auxins, possess 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, and alkalinize the medium, contributing to the immobilization of aluminum. This process includes the formation of biofilm-like structures and insoluble phosphates, which reduces the toxicity of aluminum to plants and improves their growth in acidic soils [69].

Our findings align with the research of Jiang et al. [70], who isolated 16 endophytic bacteria from roots and found that Leifsonia shinshuensis had strong aluminum resistance. Similarly, Bisht and Kumar [71] reported aluminum tolerance in Cedecea davisae M1. These studies highlight the diverse aluminum tolerance capabilities among various bacterial strains.

Furthermore, the study of Gyaneshwar et al. [72] provides additional context, demonstrating how different conditions and concentrations of substances in the medium can influence bacterial viability and growth. In particular, Gyaneshwar et al. [72] observed that the number of bacteria varies significantly depending on the plant’s location, while Feld et al. [73] indicated how Cypermethrin concentrations affect cell density and generation time.

The influence of environmental factors on bacterial viability is well documented. Soil pH, in particular, plays a crucial role. As noted by Wang et al. [74] strongly acidic environments can significantly alter microbial metabolisms. Zhang et al. [75] delve into this topic, highlighting how resistance to oxidative stress is crucial for microbial tolerance in acidic aluminum-rich soils. Further investigation is needed to determine the specific mechanisms by which P. veronii (R4) and S. marcescens (M04) tolerate aluminum and how these mechanisms might be affected by environmental factors like soil pH.

4.3. Determination of the Morphological Characteristics of the Oryza sativa L. Plant by the Effect of Rhizobacteria and Aluminum

Plant growth-promoting rhizobacteria (PGPR) play a crucial role in mitigating stress caused by minerals such as aluminum. PGPR like Pseudomonas and Glutamicibacter modulate the exudation of organic compounds in the rhizosphere, reducing aluminum absorption and improving root growth and plant biomass [17]. Halotolerant rhizobacteria, such as Bacillus licheniformis and Enterobacter asburiae, increase phosphate solubilization and enhance nutrient uptake in plants under saline stress [76]. Similarly, bacterial consortia including Bacillus megaterium reduce aluminum absorption and improve the growth of Vigna radiata through the production of antioxidants and osmoprotectants under drought and aluminum stress conditions [66].

Plant growth-promoting rhizobacteria (PGPR) can mitigate aluminum stress in plants by immobilizing the metal in the rhizosphere, reducing its absorption, and improving root growth [17]. They also stimulate the exudation of organic acids that protect roots from aluminum toxicity [12]. Under saline conditions, these bacteria enhance nutrient uptake and promote plant growth [77]. Additionally, in acidic soils, strains like Bacillus megaterium reduce aluminum absorption and improve plant growth through antioxidant production [66].

The use of PGPR has proven to be essential in improving the morphology of the rice plant Oryza sativa L., particularly under stress conditions caused by the presence of aluminum in acidic soils. The application of the PGPR consortium can significantly enhance plant biomass, which positively affects both plant height and the weight of leaf and root tissues, leading to increased plant height and weight of both leaf and root tissues (fresh and dry weight). This positive effect is likely due to the ability of PGPR to improve the microbial community in acidic soils.

These outcomes can be attributed to the synergistic effects of the consortia, which likely enhanced the production of plant growth-promoting metabolites such as indole-3-acetic acid (IAA) and siderophores, facilitating nutrient availability and reducing aluminum toxicity through chelation [78,79]. Additionally, the joint production of exopolysaccharides (EPSs) may have formed protective biofilms around the roots, stabilizing the rhizosphere and minimizing aluminum uptake [80]. This highlights the potential of consortia to improve plant development under stress conditions.

Several studies support the use of PGPR to mitigate aluminum stress in rice. Mozumder et al. [81] and Zhang et al. [75] demonstrated that inoculation with specific PGPR strains, such as A7, improved root length and biomass production in chickpeas under aluminum toxicity. These studies also revealed the positive modulation of plant responses to aluminum stress, potentially involving the production of antibiotics by specific bacterial genera like Bacillus spp. and Pseudomonas spp., among others, in extremely acidic soils.

The research by Bhuyan et al. [55] and Mitra et al. [82] emphasizes that specific inoculation with strain S2 in rice not only improves morphological and biochemical characteristics under cadmium (Cd) stress but also suggests a prolonged time for adaptation and elimination of heavy metals. This paves the way for utilizing PGPR as biofertilizers and bioremediation tools in sustainable agriculture.

5. Conclusions

This study demonstrated that INIAP-4M exhibits greater relative tolerance to aluminum compared to SUPREMA I-1480, maintaining 100% germination and measurable root growth up to 4 mM. However, both varieties showed severe necrosis and growth inhibition at 8 and 16 mM, highlighting the need for further research to enhance aluminum tolerance in rice.

Secondly, the study evaluated the aluminum tolerance of rhizobacteria P. veronii R4 and S. marcescens M04. While these strains displayed viability at various aluminum concentrations, their growth significantly decreased at the highest concentration (16 mM). This suggests a limitation in their tolerance at very high aluminum levels.

Finally, applying a PGPR consortium improved rice plant morphology, particularly root growth and biomass, under aluminum stress. This finding suggests that PGPR can protect rice plants from aluminum toxicity and promote healthier growth under adverse conditions.