Evaluation of Phosphate-Solubilizing Bacteria (PSB) on Phosphorus Availability in Agricultural Soils and the Growth of Wheat (Triticum aestivum L.)

Abstract

The objective of this research was to determine the effect of phosphate-solubilizing bacteria (PSB) on phosphorus availability in agricultural soils and the growth of wheat (Triticum aestivum L.). This applied research considered PSB and phosphorus availability in the soil as variables. An experimental design was employed, comprising four groups of pots containing 1 kg of wheat-cultivated soil (no inoculum, 5% inoculum, 10% inoculum, and 15% inoculum), with three replicates each, using a bacterial suspension of 3 × 10⁸ CFU/mL. Wheat seedling development parameters were evaluated on days 29 and 45, and soil phosphorus availability was assessed on day 45. The 10% inoculum treatment yielded superior results in seedling development: plant height, aerial dry biomass, and root dry biomass showed highly significant differences (p < 0.0001). A 10% PSB dose improved soil phosphorus availability from 72.77 ± 0.13 ppm to 96.68 ± 0.58 ppm compared to the control. These findings highlight PSB as a sustainable alternative for enhancing agricultural productivity, thereby reducing dependence on chemical fertilizers.

1. Introduction

Population growth drives a constant demand for food, compelling agriculture to heavily rely on chemical fertilizers to enhance crop yields. These fertilizers, primarily composed of nitrogen (N), phosphorus (P), and potassium (K), are essential for plant development. However, their excessive use has led to significant soil degradation, alterations in microbiota, and contamination of water resources [1,2]. In particular, phosphorus, despite being the second most required macronutrient for plants, exhibits low availability for absorption due to its fixation in insoluble forms under acidic pH conditions and interactions with aluminum, iron, and calcium ions [3].

Soil degradation, exacerbated by the indiscriminate use of chemical fertilizers, impacts not only agricultural productivity but also the health of soil microbiota, reducing communities of bacteria, fungi, and protozoa [4,5]. This degradation constitutes a significant disruption of soil ecosystems, as it interferes with nutrient cycling functions and alters symbiotic relationships with plants [6], leading to negative effects on crop productivity [7].

In Latin America, countries such as Mexico apply significant amounts of fertilizers, ranging from 3 to 5.5 t/ha per crop cycle, to meet the growing food demand [8,9]. On average, these fertilizers contain 23% nitrogen (N), 13% phosphorus (P), and 26% potassium (K) [10]. However, the tendency toward overapplication has caused negative impacts on soil microbiota and its ecological balance, affecting both soil quality and agricultural sustainability [11]. Similarly, in Peru, the intensive use of inorganic fertilizers such as urea, diammonium phosphate, ammonium nitrate, ammonium sulfate, and potassium sulfate has resulted in the accumulation of residues in the soil and leachates that contaminate groundwater [12,13]. This accumulation, along with toxic elements such as selenium (Se) and arsenic (As), has led to severe erosion, loss of microbiota, nutrient imbalances, and contamination of surface and underground water [14,15].

Wheat (Triticum aestivum L.) is one of the most important cereals worldwide, achieving a global production of 789 million tons during the 2021/2022 season [16]. In Peru, it holds particular relevance in the Andes, with an annual production of approximately 200,000 tons [17]. However, its cultivation heavily depends on inorganic fertilizers (NPK), which can alter soil conditions and reduce populations of beneficial microorganisms, thereby negatively affecting nutrient cycling efficiency and the long-term productivity of these crops [18]. Furthermore, phosphorus availability, essential for the growth of these plants, is often limited or rendered insoluble [19].

This scenario has driven the search for sustainable alternatives to reduce reliance on chemical inputs and promote soil fertility regeneration. Among these alternatives, phosphate-solubilizing bacteria (PSB) have demonstrated significant potential as biological agents capable of converting insoluble phosphorus into forms accessible to plants [20]. These bacteria function by releasing organic acids, such as citric and lactic acids, which lower soil pH and solubilize insoluble phosphate compounds, thereby making them available to plants [21]. Additionally, they can produce phytohormones such as auxins, which promote root development, and siderophores, which enhance iron availability [22]. The use of PSB in agriculture not only reduces dependency on chemical fertilizers but also enhances soil fertility and fosters a more sustainable ecological balance. Integrating PSB into agricultural practices represents a key ecological strategy to achieve greater sustainability in crop production while simultaneously protecting soil microbiota [23,24].

The general research question is as follows: What is the effect of PSB on phosphorus availability in agricultural soils and the growth of wheat crops (Triticum aestivum L.)? Specific research questions include the following: (1) Which PSB strains can be biochemically isolated and identified? (2) What is the effect of PSB application on the growth of wheat crops? (3) What is the available phosphorus content in the soil under different PSB application doses? (4) What is the effect of PSB on the fertility of agricultural soil?

This research is justified for its potential scientific, environmental, and social contributions. Scientifically, it provides insights into the use of PSB as biological agents to improve phosphorus availability in agricultural soils, particularly those impacted by excessive inorganic fertilizer use. This approach offers a promising alternative to increase soil fertility and enhance crop growth [25]. Environmentally, PSB applications can reduce dependence on chemical fertilizers, thereby decreasing soil and groundwater contamination associated with their use. This directly promotes more sustainable and eco-friendly agricultural practices. Socially, incorporating PSB into agricultural practices benefits farmers by improving soil microbiological quality, potentially leading to higher productivity and better economic returns. Additionally, strengthening soil microbiota fosters more resilient and healthier agricultural systems over the long term.

The overarching objective of this study is to evaluate the effect of PSB on agricultural soil fertility and the growth of wheat crops (Triticum aestivum L.) as an alternative to reduce reliance on chemical fertilizers. The specific objectives are to (1) isolate and biochemically identify PSB strains from wheat cultivation soils, (2) assess the effect of PSB application on wheat seedling growth, (3) determine the available phosphorus content in the soil under varying levels of PSB application, and (4) evaluate the impact of PSB on the fertility of agricultural soils.

2. Materials and Methods

2.1. Agricultural Soil Sampling

Soil samples were collected from a wheat (Triticum aestivum L.) cultivation area measuring 3771.2 m² located in Otuzco, La Libertad, Peru (see Figure 1). The sampling followed the guidelines outlined in the “Guide for Soil Sampling 2014–MINAM”, employing a composite sampling method with a two-dimensional grid to a depth of 30 cm. Ten subsamples of 0.5 kg each were collected and homogenized to create a composite sample weighing 5 kg. The samples were labeled and stored at 4 °C until further analysis at the Scientific and Technological Research Institute Laboratory of Universidad César Vallejo, Trujillo.

2.2. Characterization of Agricultural Soil for Wheat Cultivation

The results of the characterization are presented in

Table 1. Characterization of the agricultural soil sample.

Parameters	Unit	Values
pH	-	6.52 ± 0.01
Electrical Conductivity (EC)	(mS/cm)	0.464 ± 0.01
Available Phosphorus (P)	(ppm)	33.67 ± 0.10
Organic Matter (OM)	(%)	1.05 ± 0.01
Potassium Concentration (K)	(ppm)	442.17 ± 0.01
Saturations	(%)	34.00 ± 1
Calcium Carbonate Content (CaCO3)	(%)	0.00 ± 0.00

. The characterization of the agricultural soil was performed based on the following parameters: pH and electrical conductivity (EC, mS/cm), measured directly using a calibrated HI98194 multiparameter meter with a 1:2.5 suspension of soil and distilled water; available phosphorus (P, ppm), quantified using UV-Vis spectrophotometry at 880 nm following the Olsen method; organic matter (OM, %), determined through the Walkley–Black method based on chemical oxidation; potassium concentration (K, ppm), assessed via flame emission photometry using an ammonium acetate extracting solution; saturation (%), calculated based on cation exchange capacity (CEC); and calcium carbonate content (CaCO₃, %), determined through chemical titration with hydrochloric acid (HCl).

2.3. Isolation and Selection of Phosphate-Solubilizing Bacteria (PSB)

Soil samples were mixed and sieved to remove debris that could interfere with the results. From the sieved sample, 10 g was taken to prepare serial dilutions up to 10⁵ using sterile distilled water. From the last two dilutions, duplicate surface plating was performed on nutrient agar with the following composition: peptone (5 g/L), meat extract (3 g/L), sodium chloride (NaCl, 8 g/L), and agar (15 g/L). The plates were then incubated at 35 °C for 24 to 48 h.

For the selection of PSB, Pikovskaya agar was used with the following composition: yeast extract (0.5 g/L), dextrose (C₆H₁₂O₆, 10 g/L), calcium phosphate (Ca₃(PO₄)₂, 5 g/L), ammonium sulfate ((NH₄)₂SO₄, 0.5 g/L), potassium chloride (KCl, 0.2 g/L), magnesium sulfate (MgSO₄, 0.1 g/L), and manganese sulfate (MnSO₄, 0.0001 g/L). Bacterial growth was observed, and macroscopic and microscopic characteristics were recorded using Gram staining. Pure cultures of each isolate were prepared on inclined nutrient agar, and streak plating was performed on Pikovskaya agar, followed by incubation at 30 °C for five days. After incubation, bacterial growth on Pikovskaya agar was observed. Strains capable of growing on this medium were selected and preserved on inclined nutrient agar for subsequent phosphate solubilization testing and biochemical identification.

2.4. Phosphate Solubilization Test In Vitro

Two isolated strains, coded as MT-13 and MT-8, were cultivated using the medium described by Osorio and Habte (2001), which consists of several components, namely sodium chloride (NaCl, 1 g/L), calcium chloride dihydrate (CaCl₂·2H₂O, 0.2 g/L), magnesium sulfate pentahydrate (MgSO₄·7H₂O, 0.4 g/L), ammonium nitrate (NH₄NO₃, 1 g/L), glucose (C₆H₁₂O₆, 10 g/L), agar (7 g/L), and phosphate rock (3.5 g/L), in a liquid medium using test tubes [26]. Following inoculation, the phosphate solubilization capacity of the strains was evaluated by recording pH and color changes after 72 h of incubation at 25 °C.

2.5. Biochemical Identification of Strains MT-13 and MT-8

The biochemical identification of strains MT-13 and MT-8 was carried out using the VITEK-2 Compact Microbiological Autoanalyzer (BioMerieux, Lyon, France). For this, suspensions of the strains were prepared in two test tubes with 3 mL of sterile saline solution. The concentration of the microorganisms was then adjusted to 1 on the McFarland scale, equivalent to 3 × 10⁸ CFU/mL in both cases, and the solution was homogenized. The optical density of the suspensions was measured using the VITEK-2 Densichek Plus (BioMerieux, Lyon, France), applying the McFarland method, until a range between 0.50 and 0.63 was achieved. Next, the identification cards and test tubes with the suspensions were placed in the VITEK-2 Compact cassette. The density was configured according to the values established by the manufacturer for the Gram-negative bacterial identification card. The analysis time for the identification of the strains was approximately 4.83 and 5.82 h. Finally, complementary microscopic observations were made using Gram staining to evaluate the morphological and microscopic characteristics of the microorganisms.

2.6. Preparation of Pots and Inoculation of Wheat Seeds with PSB

Ten pots, each containing 1 kg of sifted agricultural soil, were used. Wheat seeds, previously disinfected and germinated, were sown, with six seeds per pot. The treatments were divided into four groups, namely the control group (CG), an experimental group inoculated at 5% (EG-5), an experimental group inoculated at 10% (EG-10), and an experimental group inoculated at 15% (EG-15), with three repetitions for each group (see Figure 2).

The strains were cultivated on Petri plates with nutrient agar to obtain pure biomass, which was then transferred to a liquid medium of tryptic soy broth (TSB) and incubated at 30 °C with constant agitation at 80 rpm for 24 h. Subsequently, PSB suspensions were prepared and adjusted to 3 × 10⁸ CFU/mL by measuring absorbance at 660 nm and inoculated onto the wheat seeds at concentrations of 5%, 10%, and 15%. The inoculum was applied initially during sowing and at two additional intervals (days 14 and 30). A control group without inoculation was established for comparison.

The inoculation rates were expressed as percentages of the original inoculum volume, previously adjusted to a 1.0 McFarland standard. This approach allowed for the standardization of application, ensuring uniformity between treatments and facilitating the reproducibility of the study.

2.7. Evaluation of Plant Indicators

The first evaluation was conducted on day 29, and a final evaluation was performed on day 45. The following plant indicators were assessed: plant height (cm), leaf area (cm²), fresh aerial biomass (g), dry aerial biomass (g), fresh root biomass (g), dry root biomass (g), and stem diameter (mm). Plant height and stem diameter were measured using a calibrated digital caliper (Truper CALDI-6MP, Jilotepec, Mexico). Fresh and dry aerial biomass, as well as fresh and dry root biomass, were measured using an analytical balance (Boeco BAS 31 PLUS, Hamburg, Germany). The leaf area was assessed using ImageJ (version 1.54d) software.

Finally, chemical parameters were measured to evaluate the effect of the PSB on soil fertility on day 45. These included available phosphorus (ppm), pH, organic matter (%), electrical conductivity (mS/cm), potassium (K) (ppm), saturation (%), and calcium carbonate (CaCO₃) (%). The methods used for these measurements were previously described in Section 2.2. The rate of change was calculated using the following equation:

$$\mathrm{Rate} \mathrm{of} \mathrm{change} \left(\%\right)={\displaystyle \frac{\mathrm{Final} \mathrm{value}-\mathrm{Initial} \mathrm{value}}{\mathrm{Initial} \mathrm{value}}}\times 100$$

(1)

2.8. Data Analysis Method

The Fisher’s LSD test was used to identify the optimal PSB inoculation dose for wheat (Triticum aestivum L.) growth and to evaluate its effect on phosphorus (P) availability in the soil. Additionally, Pearson’s correlation was applied to analyze the relationship between available P and the growth parameters of wheat seedlings inoculated with the optimal PSB dose. Furthermore, the Shapiro–Wilk normality test was performed to check if the data followed a normal distribution. If the data did not meet this assumption, Spearman’s correlation test was used to assess the relationship between PSB inoculation and P availability in the soil.

3. Results and Discussion

3.1. Isolation and Identification of Phosphate-Solubilizing Bacteria (PSB)

3.1.1. Isolation of PSB and In Vitro Phosphorus Solubilization Test

From the soil samples, the MT-8 and MT-13 strains were isolated on Pikovskaya medium. Although no halos were observed in the solid medium, both strains demonstrated the ability to solubilize phosphorus in liquid medium (Osorio and Habte, 2001) [26], as evidenced by color changes to magenta and pH reductions after 72 h of incubation (Figure 3). The MT-8 strain reduced the pH from 6.8 to 5.0, while MT-13 reached a pH of 3.8, showing greater efficiency in phosphorus solubilization. The control group maintained the initial pH value.

In the study conducted by Solanki, Kundu, and Nehra (2018), a total of 49 microorganisms were isolated from wheat cultivation, of which 8 demonstrated the ability to solubilize phosphate using Pikovskaya medium [27]. On the other hand, Mohamed et al. (2019) isolated 40 microorganisms from the root zone of wheat plants using NBRIP medium [28]. These researchers observed the formation of a halo around the colonies. However, in the present study, no such halo, which is an indicator of phosphate solubilization, was observed. Similarly, Vargas-Barrante and Castro-Barquero (2019) initially isolated 54 microorganisms. After a selection process, the sample was reduced to 19 microorganisms, including 8 fungi, 6 yeasts, and 5 bacteria. During the experiment, a liquid medium was produced, showing a color change and a significant decrease in the pH of the medium inoculated with bacteria [29]. These findings are consistent with the results obtained in this study. The reduction in pH in the medium is explained by the fact that PSB produce organic acids, which leads to a decrease in pH during the phosphate solubilization mechanism [30].

3.1.2. Biochemical Identification of MT-8 and MT-13 Strains

The biochemical identification using the VITEK 2 Compact System revealed that the MT-8 strain corresponds to the genus Pantoea spp., with a 97% probability and excellent confidence level. Meanwhile, the MT-13 strain was identified as Lelliottia amnigena 2, with a 99% probability and excellent confidence level. In both cases, the strains were Gram-negative bacteria (

Table 2. Biochemical identification information by VITEK 2.

Biochemical Identification Information	Isolation Code
	MT-8	MT-13
Organism Identified Card Analysis Time Probability Confidence Level Bionumber	Pantoea spp. Gram-negative 5.82 h 97% Excellent 4625730551130010	Lelliottia amnigena 2 Gram-negative 4.83 h 99% Excellent 4627734543572010

).

In

Table 3. Gram staining test for strain MT-8.

Characteristics	Description	Microscopic Observation
Isolated Strain Incubation Time Color Morphology Quantity Magnification Bacteria Type	Code MT-8 48 h Red Bacillus Abundant 100× Gram-negative
Macroscopic Characteristics	Elongated and cylindrical

and

Table 4. Gram staining test for strain MT-13.

Characteristics	Description	Microscopic Observation
Isolated Strain Incubation Time Color Morphology Quantity Magnification Bacteria Type	Code MT-13 48 h Pink Bacillus Abundant 100× Gram-negative
Macroscopic Characteristics	Elongated and cylindrical

, the results of the Gram staining tests are shown, confirming that the MT-8 and MT-13 strains are Gram-negative bacilli, with similar microscopic and macroscopic characteristics. Both strains appeared as elongated, cylindrical bacteria, cultured on Pikovskaya agar.

These results are similar to those obtained by Osei et al. (2022) and Li et al. (2021), who used molecular identification through the 16 rRNA gene to identify bacterial strains coded as PC3, ZJ62, and ZJ3-12. PC3 was identified as Lelliottia amnigena with a 99.44% similarity, and ZJ3-12 were identified as Pantoea dispersa and Pantoea ananatis, respectively, with a 99.93% similarity in both cases [31,32]. Further, phosphate-solubilizing bacteria identified as Pantoea spp. and Lelliottia amnigena 2 belong to the Enterobacter family. Scattareggia (2016) mentions that the Enterobacter family is one of the most commonly used PSB groups in the agricultural field [33].

3.2. Effect of PSB on Wheat (Triticum aestivum L.) Growth

The wheat seedlings were evaluated on days 29 and 45, where Fisher’s LSD test was used to compare the means and determine if the application of different doses resulted in a statistically significant difference. Based on this, the following hypotheses were formulated:

H₀: 

The growth of the seedlings is not different from the application of PSB doses.

H₁: 

The growth of the seedlings is different from the application of PSB doses.

In

Table 5. Fisher’s LSD test for the evaluation of growth and development of wheat seedlings on Day 29 by different PSB inoculum doses applied.

PSB Inoculum Dose	Height (cm)	Leaf Area (cm2)	Fresh Aerial Biomass (g)	Dry Aerial Biomass (g)	Fresh Radical Biomass (g)	Fresh Radical Biomass (g)	Stem Diameter (mm)
No inoculum	19.20 ± 0.62 B	4.47 ± 2.3 A	0.05 ± 0.05 A	0.30 ± 0.13 A	0.01 ± 0.02 A	0.01 ± 0.01 A	0.10 ± 0.06 A
5% inoculum	28.17 ± 0.62 D	13.87 ± 2.3 B	0.34 ± 0.05 B	0.38 ± 0.13 A	0.04 ± 0.02 A	0.02 ± 0.01 A	0.50 ± 0.06 B
10% inoculum	23.77 ± 0.62 C	14.43 ± 2.3 B	0.49 ± 0.05 C	0.12 ± 0.13 A	0.12 ± 0.02 B	0.05 ± 0.01 B	1.17 ± 0.06 C
15% inoculum	12.13 ± 0.62 A	3.70 ± 2.3 A	0.12 ± 0.05 A	0.03 ± 0.13 A	0.03 ± 0.02 A	0.02 ± 0.01 A	0.37 ± 0.06 B

Means with the same letter are not significantly different (p > 0.05). The values are expressed as mean ± standard error (S.E.).

, the results of Fisher’s LSD test show the evaluation of wheat seedling growth and development on day 29 with different PSB inoculum doses applied. The plant height results showed that the 5% inoculation had the most significant positive effect on wheat seedling growth on day 29, reaching an average height of 28.17 cm, with a highly significant difference (p < 0.0001), followed by the 10% inoculation with an average height of 23.77 cm. In contrast, the 15% inoculation had a negative effect on seedling growth, with an average height of 12.1 cm, which was lower than the control (19.2 cm) and other inoculation groups. Regarding the leaf area, the results showed that the 5% and 10% inoculations had a significant difference (p < 0.0161) and a positive impact on the leaf area of the wheat seedlings on day 29, with average values of 13.87 cm² and 14.43 cm², respectively. In contrast, the 15% inoculation resulted in an average leaf area of 3.70 cm², which was significantly lower than the other PSB inoculated groups and the control (4.47 cm²). These results support that lower doses, such as 5% and 10%, promote a larger leaf area in wheat seedlings, while higher doses, such as 15%, may limit leaf development. Fresh aerial biomass data revealed that the 10% inoculation was highly significant (p < 0.0004), showing the highest efficiency in increasing fresh aerial biomass with an average weight of 0.49 g. The 5% inoculum also showed a positive impact with an average of 0.34 g, whereas the 15% inoculum produced a value lower than the other inoculated groups, with a mean of 0.12 g, but was still higher than the control (0.05 g). In dry aerial biomass, no significant differences (p > 0.2821) were found among the treatment doses, showing a slight increase with the 5% inoculation, with an average value of 0.38 g compared to the control (0.3 g). The 10% and 15% doses showed slightly lower values than the control, with average values of 0.12 g and 0.03 g, respectively. Therefore, no significant effect on biomass was observed with the different PSB doses. Fresh root biomass results showed that with the 10% inoculation, a significant difference (p < 0.0120) was observed compared to the other treatments, showing a substantial increase in fresh root biomass with an average of 0.12 g, followed by the 5% inoculation with an average of 0.04 g. In contrast, the 15% inoculation produced lower results compared to the other PSB doses, with an average of 0.03 g. Dry root biomass results showed that the wheat seedlings inoculated with 10% PSB had slightly higher results with an average weight of 0.05 g, showing a statistically significant difference (p < 0.0255). Seedlings treated with 5% inoculation had an average value of 0.02 g, while the 15% treatment presented a value of 0.02 g, similar to the 5% inoculated group. Regarding the stem diameter, PSB application had a positive impact on stem diameter, particularly with the 10% inoculation, which showed a highly significant difference (p < 0.0001), reaching an average value of 1.17 mm. The 5% inoculation also showed a positive increase, as seedlings with this dose reached an average of 0.5 mm. In contrast, the 15% dose led to a smaller stem diameter compared to other applied doses, with a mean of 0.37 mm.

In Figure 4, the growth of wheat seedlings at 29 days after inoculation with different doses of Pantone spp. and Lelliottia amnigena 2, as well as the control treatment, is shown. In Figure 4a, corresponding to the 5% dose, the seedlings exhibited vigorous growth, with thick stems and long roots. However, signs of stress were also detected, such as pale or yellowish leaves, which could be related to the low concentration of the inoculum [34]. In Figure 4b, representing the 10% dose, more robust growth is observed compared to the 5% treatment. The roots and stems show greater extension, suggesting that the higher inoculation concentration may have favored better seedling development at this stage of the experiment. Finally, in Figure 4c, corresponding to the 15% dose, slowed growth is evident in both the aerial and root parts of the seedlings, with some differences in vigor, suggesting a less favorable response to the inoculation at this concentration. In all three treatments, an improvement in plant growth was observed compared to the control, indicating the beneficial effects of PSB application.

Table 6. Fisher’s LSD test for the evaluation of growth and development of wheat seedlings on day 45 by different PSB inoculum doses applied.

PSB Inoculum Dose	Height (cm)	Leaf Area (cm2)	Fresh Aerial Biomass (g)	Dry Aerial Biomass (g)	Fresh Radical Biomass (g)	Fresh Radical Biomass (g)	Stem Diameter (mm)
No inoculum	27.8 ± 1.29 B	12.14 ± 1.34 A	0.10 ± 0.11 A	0.06 ± 0.01 A	0.01 ± 0.02 A	0.01 ± 0.01 A	0.58 ± 0.07 A
5% inoculum	36.27 ± 1.29 C	21.08 ± 1.34 B	0.40 ± 0.11 A	0.18 ± 0.01 B	0.06 ± 0.02 A	0.03 ± 0.01 B	1.02 ± 0.07 B
10% inoculum	39.00 ± 1.29 C	17.51 ± 1.34 B	1.01 ± 0.11 B	0.54 ± 0.01 C	0.13 ± 0.02 B	0.11 ± 0.01 C	1.57 ± 0.07 C
15% inoculum	18.15 ± 1.29 A	7.97 ± 1.34 A	0.17 ± 0.11 A	0.08 ± 0.01 A	0.05 ± 0.02 A	0.05 ± 0.01 B	0.43 ± 0.07 A

Means with the same letter are not significantly different (p > 0.05). The values are expressed as mean ± standard error (S.E.).

shows the results of Fisher’s LSD test for the evaluation of the growth and development of wheat seedlings on day 45 after applying different doses of PSB inoculum. Regarding plant height, it was noted that the 5% and 10% doses had a greater effect on wheat seedling height, with average values of 36.27 cm and 39 cm, respectively. These values also showed a highly significant difference (p < 0.0001). On the other hand, the 15% inoculation was unfavorable for seedling growth, as its mean was 18.15 cm, which was lower than the non-inoculated group (27.8 cm). In terms of leaf area, the 5% and 10% inoculation doses were the most effective in terms of leaf area, with average values of 21.08 cm² and 17.51 cm², respectively, showing a highly significant difference (p < 0.0006). In contrast, the 15% dose had an average of 7.97 cm², which was below the control group (12.14 cm²), with no statistically significant differences between these two latter treatments. The results for fresh aerial biomass showed that with a 10% inoculum application, there was a considerable increase in fresh aerial biomass of the seedlings, with an average weight of 1.01 g. The 10% inoculation showed a significant difference (p < 0.0016) compared to the treatments without inoculum, 5% inoculum, and 15%, which had average values of 0.1 g, 0.4 g, and 0.17 g, respectively. The dry aerial biomass of the wheat seedlings showed better development with a 10% dose, yielding an average weight of 0.54 g, which was highly significant (p < 0.0001). Second, the 5% inoculation also showed a positive effect with an average weight of 0.18 g, compared to the 15% inoculation, which had the lowest value of the applied PSB doses, with an average weight of 0.08 g. In terms of fresh root biomass, it was observed that with a 10% PSB application, the highest average of fresh root biomass was obtained, with a value of 0.13 g and a significant difference (p < 0.0051) compared to the treatments without inoculum, 5% inoculum, and 15%, which had average results of 0.01 g, 0.05 g, and 0.06 g, respectively. The dry root biomass showed that the 10% PSB application had the most positive effect on dry root biomass, with an average weight of 0.11 g, which was highly significant (p < 0.0001) compared to the treatments without inoculum, 5% inoculum, and 15%, which had average weights of 0.01 g, 0.03 g, and 0.05 g, respectively. As for the stem diameter, it was observed that the stem diameter in wheat seedlings was greater at a 10% dosage, with a value of 1.57 mm, showing a highly significant difference (p < 0.0001). The second-best treatment was with a 5% inoculation, yielding a value of 1.02 mm. In contrast, the 15% dose had the lowest result among the inoculated groups, with 0.43 mm, which was also lower than the non-inoculated treatment (0.58 mm).

Therefore, the 10% inoculation emerged as the most effective dose for the growth and development of wheat seedlings, excelling in most of the analyzed parameters, including height, both fresh and dry aerial and root biomass, and stem diameter. This treatment demonstrated superior performance, positioning it as the optimal choice for promoting overall development. On the other hand, the 5% inoculum also had positive effects, particularly on the leaf area, although its performance was lower compared to the 10%. In contrast, the highest dose (15%) proved detrimental, registering the lowest values in almost all indicators, indicating that it should be avoided.

Figure 5 shows the growth of wheat seedlings 45 days after inoculation with different doses of Pantone spp. and Lelliottia amnigena 2. In Figure 5a, corresponding to the 5% dose, notable growth is observed, with seedlings having developed long leaves and stems, along with a considerable root system. In Figure 5b, associated with the 10% dose, outstanding growth is observed, with long, vigorous leaves, thick stems, and well-formed roots. Additionally, good development of wheat spikes is seen, indicating an advanced stage in the growth cycle. On the other hand, in Figure 5c, representing the 15% dose, growth continues to be slowed, with shorter stems and less elongated leaves, some of which show slight yellowing. Overall, with the 5% and 10% doses, significant improvements were achieved compared to the control, while the 15% dose did not show the same benefits, exhibiting more limited growth.

The results are consistent with those obtained by Da Costa et al. (2020), who observed an improvement in the height of rice seedlings at 30 days, reaching 63 cm, a result higher than the control (51 cm) [35]. Javeed et al. (2019) applied the bacterium Enterobacter sakazakii J129 and observed an increase in the height of maize seedlings to 136.14 cm, compared to the uninoculated control, which was 124.78 cm, at 95 days [36]. The increase in seedling height is attributed to the fact that PSB are plant growth promoters [37]. Additionally, Mahdi et al. (2020) applied the bacterial strain Bacillus licheniformis and observed, at 45 days, an increase in dry aerial biomass to 0.24 g/plant, compared to the control (0.12 g/plant), while with the strain Enterobacter asburiae, they achieved a 0.25 g increase in root dry weight at day 45, compared to the control (0.14 g) [38]. Furthermore, Javeed et al. (2019) showed an improvement in dry biomass, reaching 341.66 g at 95 days after inoculating the bacterium Enterobacter sakazakii J129, a result superior to the control (302.44 g) [36]. Also, Mohamed et al. (2019) applied a PGPB consortium (Enterobacter aerogenes, Pantoea sp., and Enterobacter sp.) to wheat cultivation and showed an increase in root dry weight to 0.157 g, surpassing the control (0.090 g) at 60 days after inoculation [28].

On day 29, the results indicated that the 15% inoculation resulted in a lower plant height and leaf area compared to the control but exhibited a higher weight in fresh aerial biomass. This could be attributed to an osmotic stress effect or a physiological response of the plants to the high concentration of PSB, which might have favored the accumulation of reserves in the aerial biomass at the expense of height and leaf area growth [39,40]. Furthermore, it is possible that the excess inoculum disrupted the soil microbiota, affecting nutrient availability and altering the growth pattern of the seedlings. To clarify these discrepancies, additional studies are needed to analyze the effects of the 15% inoculum in terms of plant metabolism and resource distribution. In the meantime, the data support the idea that lower doses, such as 10% and 5%, are more consistent in promoting balanced development in the seedlings.

The observed inconsistency between fresh and dry biomass results suggests that PSB treatments influence the plants’ ability to absorb and retain water [41]. In particular, the higher doses (15%) appear to generate an adverse effect, possibly related to osmotic stress or imbalances in nutrient availability. This effect could cause the plants to accumulate less dry biomass and a higher proportion of water in their tissues, leading to noticeable differences between fresh and dry weights [42]. In contrast, the lower doses (5% and 10%) seem to promote a more appropriate balance between water content and dry biomass accumulation, explaining their more consistent performance in both parameters. These doses may enhance optimal microbial activity in the soil, improving nutrient and water availability for the plants.

Table 7. Rate of change in the growth parameters of wheat treated with PSB at 10% and non-inoculated on day 45.

Parameters	10% Inoculum	No Inoculum	Rate of Change (%)
Height (cm)	39.00 ± 1.29	27.8 ± 1.29	40.29
Leaf Area (cm2)	17.51 ± 1.34	12.14 ± 1.34	44.23
Fresh Aerial Biomass (g)	1.01 ± 0.11	0.10 ± 0.11	910
Dry Aerial Biomass (g)	0.54 ± 0.01	0.06 ± 0.01	800
Fresh Radical Biomass (g)	0.13 ± 0.02	0.01 ± 0.02	1200
Fresh Radical Biomass (g)	0.11 ± 0.01	0.01 ± 0.01	1000
Stem Diameter (mm)	1.57 ± 0.07	0.58 ± 0.07	170.69

presents a comparative analysis of wheat growth parameters under two conditions: the application of a 10% PSB dose and the control without inoculation. The results show a significant improvement in all evaluated indicators for PSB-treated plants, with substantial increases reflected in the percentage change rate. In terms of height, PSB-treated plants exhibited a 40.29% increase compared to the control. This result suggests that PSB not only enhances phosphorus availability in the soil—a nutrient essential for cell elongation and vertical plant development—but also optimizes physiological processes such as photosynthesis and energy metabolism, promoting overall crop growth [43]. Regarding the leaf area, the treated plants achieved a 44.23% increase over the control. This improvement reflects an enhanced photosynthetic capacity, as PSB contribute to greater phosphorus availability, which is necessary for chlorophyll formation and efficient conversion of sunlight into chemical energy [44]. Fresh aerial biomass displayed an extraordinary increase, reaching a 910% change rate in treated plants compared to the control. This result relates to the role of PSB in stimulating plant growth by improving soluble phosphorus availability, which is fundamental for the formation of structural compounds and water storage in tissues [45]. For dry aerial biomass, the treated plants recorded an 800% increase over the control. This rise in dry biomass indicates a greater accumulation of structural organic matter, consistent with the role of phosphorus in synthesizing carbohydrates and lipids essential for strengthening and stabilizing plant structures [46]. Fresh root biomass in PSB-treated plants showed a 1200% increase compared to the control. This remarkable effect can be attributed to PSB’s ability to release organic and inorganic phosphorus in the soil, promoting greater root system development, which in turn enhances water and essential nutrient absorption [47]. Dry root biomass followed a similar pattern, with a 1000% increase. This outcome confirms that PSB not only increase root volume but also strengthen root structures, enabling them to withstand adverse soil conditions and store nutrients more efficiently [48]. Finally, the stem diameter increased by 170.69% over the control. This parameter directly reflects the robustness of treated plants, which could be linked to the greater availability of phosphorus and structural compounds that reinforce stem tissues [49]. These findings highlight the potential of PSB as an effective biotechnological tool for sustainably optimizing crop yields.

Table 8. Pearson correlation with 10% PSB inoculum.

	P (ppm)	Height (cm)	Leaf Area (cm2)	Fresh Aerial Biomass (g)	Dry Aerial Biomass (g)	Fresh Radical Biomass (g)	Fresh Radical Biomass (g)	Stem Diameter (mm)
P (ppm)	1	0.80	0.15	0.29	0.77	0.05	0.77	0.57
Height (cm)	−0.31	1	0.94	0.50	0.02	0.75	0.03	0.63
Leaf Area (cm2)	−0.97	0.09	1	0.44	0.92	0.2	0.92	0.43
Fresh Aerial Biomass (g)	−0.89	0.7	0.77	1	0.48	0.25	0.47	0.87
Dry Aerial Biomass (g)	−0.35	1	0.13	0.73	1	0.72	4.40 × 10−3	0.65
Fresh Radical Biomass (g)	−1	0.38	0.95	0.93	0.42	1	0.72	0.62
Fresh Radical Biomass (g)	0.36	−1	−0.13	−0.74	−1	−0.43	1	0.66
Stem Diameter (mm)	−0.62	−0.55	0.78	0.2	−0.52	0.56	0.51	1

La correlación es significativa en el nivel 0.05.

presents the Pearson correlation coefficients for the relationship between available phosphorus (P) and the parameters of wheat seedlings, showing statistically significant correlations (p < 0.05) and positive relationships, with very high and high correlation types. Plant height exhibited a very high correlation, with an R-value of 0.80. Following this, both dry aerial biomass and dry root biomass showed high correlations, with R-values of 0.77 in both cases. Therefore, when evaluating the growth parameters of wheat seedlings with available P under 10% inoculation, significant correlations were found between available P and plant height, dry aerial biomass, and dry root biomass.

The application of PSB at 10% proved to be the most effective dose for the growth of wheat seedlings, demonstrating superior development compared to other concentrations. Seedlings treated with this dose displayed more robust stems, longer and more vigorous leaves, and a better-developed root system, indicating greater nutrient absorption efficiency [50]. These findings position 10% PSB inoculation as a viable alternative for organic fertilization, offering multiple benefits such as improved phosphate availability, reduced reliance on chemical fertilizers, and the promotion of sustainable agricultural practices that enhance soil and ecosystem health [51].

3.3. Effect of PSB on Soil P Availability

To determine the effect of PSB on soil P availability, Fisher’s LSD test was used to identify whether significant differences exist among the doses applied in the treatments by comparing the means of available phosphorus. The following hypotheses were proposed:

H₀: 

Soil P availability is the same for each dose of PSB applied.

H₁: 

Soil P availability differs for each dose of PSB applied.

The tables below present the results of the analysis of variance (ANOVA) and Fisher’s LSD test employed to determine the optimal PSB dose for enhancing soil P availability.

In

Table 9. Analysis of variance for soil P availability across PSB doses.

F.V.	SC	gl	CM	F	p-Value
Model	1976.84	3	658.95	8.67	<0.0068
PSB dose	1976.84	3	658.95	8.67	<0.0068
Error	607.83	8	75.98
Total	2584.67	11

, it is observed that the p-value < 0.05, supporting the acceptance of the alternative hypothesis: soil P availability differs for each dose of PSB applied. Additionally, a significant difference (p < 0.0068) was found among the doses.

As shown in

Table 10. Fisher’s LSD test for evaluating soil P availability at different PSB inoculum doses.

PSB Dose	Mean de P (ppm)	n	E.E.
No inoculum	72.77	3	5.03	A
10% inoculum	96.68	3	5.03	B
5% inoculum	99.94	3	5.03	B
15% inoculum	106.88	3	5.03	B

Means with the same letter are not significantly different (p > 0.05).

, the application of phosphate-solubilizing bacteria (PSB) at different levels (5%, 10%, and 15%) had a positive effect on soil P availability, with mean values of 96.68 ppm, 99.94 ppm, and 106.88 ppm, respectively. A significant difference (p < 0.0068) was observed when compared to the control treatment, which yielded 72.77 ppm. Similarly, Kumar et al. (2021) isolated and identified the JPVS11 strain corresponding to Bacillus pumilus, suspended at 1 × 10⁸ CFU/mL, and demonstrated an improvement in soil P availability, reaching 38.5 kg h⁻¹, which was superior to the control (27.7 kg h⁻¹) [52]. Likewise, Hipólito-Romero et al. (2017) reported that the application of PSB strains Chromobacterium violaceum and Acinetobacter calcoaceticus in consortium at a suspension of 1.7 × 10⁹ CFU/mL improved soil P availability, reaching 33.5 mg/kg compared to the control, which showed 11 mg/kg [53].

To analyze the relationship between PSB doses and available soil phosphorus, and to determine whether there is a positive or negative trend, the Shapiro–Wilks normality test was applied, followed by Spearman’s correlation coefficient. The following hypotheses were proposed:

H₀: 

The data follow a normal distribution.

H₁: 

The data do not follow a normal distribution.

In

Table 11. Shapiro–Wilk normality test for the evaluation of phosphorus availability in soil by different PSB treatments.

Variable	n	Mean	S.D.	W*	P (One-Tailed D)
P (ppm)	12	94.07	15.33	0.84	0.0499
Treatments	12	2.50	1.17	0.83	0.0340

, the Shapiro–Wilks normality test was performed, as the sample sizes were less than 30. For the variable P (ppm), the p-value was (p < 0.0499), and for the variable treatments, the p-value was (p < 0.0340). These results indicate that the variables of available P (ppm) and treatments do not follow a normal distribution (p < 0.05). Therefore, the alternative hypothesis was accepted, and Spearman’s correlation test was applied.

Table 12. Spearman correlation coefficients for evaluating soil P availability across PSB treatments.

Variable	P (ppm)	Treatments
P (ppm)	1.00	0.02
Treatments	0.65	1.00

also shows the Spearman correlation analysis, which indicates a moderate positive relationship (r = 0.65, p = 0.02) between the PSB dose applied and the availability of phosphorus in the soil. This result suggests that increasing PSB doses is associated with higher levels of available phosphorus, likely due to the microbial activity promoted by PSB. Moreover, the average p-values across the different treatments show a progressive increase, with significant differences identified using the post hoc Tukey’s HSD test, further reinforcing the role of PSB in enhancing soil fertility. Several researchers have reported that PSB influence soil P availability by releasing phosphorus bound to metallic ions such as calcium, magnesium, aluminum, and iron [54]. This process occurs through the production of organic acids by PSB, which convert insoluble phosphorus into a soluble form, making it accessible to plants. This effect was evident in the improved development of plants observed in this study [55].

3.4. Effect of PSB on Agricultural Soil Fertility

The table below presents measurements taken on day 45 of certain physicochemical soil parameters for each treatment with varying PSB doses, aiming to evaluate their effect on soil fertility.

In

Table 13. Effect of PSB doses on soil physicochemical parameters after 45 days of treatment.

PSB Doses	pH	O.M. (%)	P (ppm)	K (ppm)	Satur. (%)	C.E. (mS/cm)	CaCO3 (%)
No inoculum	6.25 ± 0.02	1.62 ± 0.04	72.77 ± 0.13	709.85 ± 0.20	27.00 ± 0.09	3.942 ± 0.15	2.10 ± 0.07
5% inoculum	6.47 ± 0.09	1.61 ± 0.02	99.94 ± 0.82	720.91 ± 0.60	27.67 ± 0.28	7.98 ± 0.11	2.35 ± 0.05
10% inoculum	6.52 ± 0.07	1.70 ± 0.16	96.68 ± 0.58	616.94 ± 0.52	28.00 ± 0.10	9.83 ± 0.57	2.40 ± 0.10
15% inoculum	6.61 ± 0.01	1.59 ± 0.03	106.88 ± 0.13	715.38 ± 0.56	29.00 ± 0.10	12.16 ± 0.08	2.80 ± 0.20

, the results of the effect of PSB on soil fertility are presented, evaluating various physicochemical parameters. A gradual increase in pH was observed, from an initial value of 6.52 ± 0.01, slightly acidic, to 6.61 ± 0.01 with the 15% PSB treatment, indicating a moderate neutralization of soil acidity, possibly due to the release of alkaline compounds by PSB [56]. Electrical conductivity (EC) showed a significant increase, from 0.464 ± 0.01 mS/cm initially to 12.16 ± 0.08 mS/cm with the 15% PSB treatment, reflecting a higher concentration of soluble salts derived from PSB, which enhances the availability of essential nutrients [57]. Regarding available phosphorus (P), there was a notable increase, rising from an initial value of 33.67 ± 0.1 ppm to a range between 72.77 ± 0.13 ppm (no inoculation) and 106.88 ± 0.13 ppm (15% PSB). This remarkable increase, nearly tripling the initial values, highlights the ability of PSB to release phosphorus, a critical element for plant development, thus improving soil fertility [58]. As for organic matter, the initial values (1.05 ± 0.01%) remained relatively stable after the treatments, varying slightly between 1.59 ± 0.03% and 1.70 ± 0.16%. These results suggest that while PSB did not significantly alter the organic matter content, this parameter maintained stability consistent with its structural and functional role in the soil [59]. Potassium concentration showed a significant increase, rising from an initial value of 442.17 ± 0.01 ppm to a range between 709.85 ± 0.20 ppm and 720.91 ± 0.60 ppm. This substantial increase demonstrates the contribution of PSB as a source of potassium, an essential nutrient for plant physiology, promoting metabolic processes in plants [60]. In terms of saturation, a slight increase was observed compared to the initial value of 34.00 ± 1%. After 45 days, this parameter varied between 27.67 ± 0.28% and 29 ± 0.10%, from the lowest to the highest dose. Although the changes were not drastic, they reflect an improvement in the soil’s capacity to retain and mobilize nutrients. Finally, calcium carbonate content, initially nonexistent, showed a progressive increase, reaching 2.80 ± 0.20% in the treatment with 15% PSB. This result suggests an accumulation of calcium compounds possibly associated with mineralization processes promoted by the treatment [61]. These results indicate that the use of PSB significantly impacts key parameters such as electrical conductivity, available phosphorus, and potassium, while pH and saturation showed moderate changes that were consistent with the increasing doses. This analysis highlights the effectiveness of PSB in improving soil quality, offering potential benefits for its use in agricultural systems.

4. Conclusions

This research demonstrated that phosphate-solubilizing bacteria (PSB) are an effective and sustainable strategy to enhance soil fertility and wheat growth (Triticum aestivum L.). Indigenous strains (Pantoea spp. and Lelliottia amnigena 2) were successfully isolated and identified, showcasing high efficiency in solubilizing phosphorus under controlled conditions. Inoculation with 10% PSB significantly enhanced wheat growth compared to non-inoculated treatments, with notable increases in plant metrics: height (40.29%), leaf area (44.23%), stem diameter (170.69%), and dry shoot and root biomass (800–1200%). Additionally, PSB applications increased phosphorus availability in the soil from 72.77 ± 0.13 ppm to 96.68 ± 0.58 ppm, reducing the need for chemical fertilizers. These findings highlight the potential of PSB as a key biotechnological tool for achieving sustainable agriculture by reducing chemical fertilizer dependency, improving crop productivity, and preserving environmental health.

For future research, it is recommended to employ the Osorio and Habte method to detect microorganisms capable of producing organic acids, as these are pivotal in phosphate solubilization. Furthermore, conducting molecular analyses of the Pantoea spp. and Lelliottia amnigena 2 strains is essential for more precise genetic-level identification of the PSB. Finally, it is advised to compare the effects of PSB with conventional chemical fertilizers to assess their economic viability and environmental impact. Such evaluations could solidify PSB as a sustainable alternative in modern agriculture.