Effects of Bacillus halotolerans as a Plant Growth-Promoting Rhizobacterium and Root Phytopathogen Biocontrol Agent in Solanum lycopersicum Under Field Conditions

Abstract

Tomato is the most widely consumed vegetable worldwide and serves as an important source of vitamins and minerals. Using the Bacillus species as biocontrol agents and plant growth promoters is a sustainable approach to optimize production and mitigate the effects of root-infecting phytopathogenic fungi, thereby reducing reliance on chemical inputs. This study evaluated the effectiveness of a Bacillus sp.-based bioinoculant, produced in a 7 L bioreactor, for controlling root phytopathogens and enhancing tomato yields under field conditions. The trial was conducted at an experimental field of the Universidad Nacional Agraria La Molina (Lima, Peru) using a randomized complete block design with four blocks. Treatment means were compared using Tukey’s multiple range test (α = 0.05) to evaluate treatment effects. The treatments included three concentrations of the bioinoculant (10%, 20%, and 30%) derived from an initial concentration of 1 × 10⁸ CFU/mL of a Bacillus halotolerans IcBac2.1 strain sourced from the LEMyB laboratory strain collection, a commercial biological product (1 × 10⁹ CFU/g), and uninoculated control. Applications were made for the following four key stages of crop development: 10 days after germination, when transplanting through root dipping, 7 days after transplanting, and at the onset of flowering. In all treated groups, applications were directed to the plant crown, whereas the control group received no treatment. The evaluated variables included plant height (cm), stem diameter (mm), root disease incidence (%), chlorophyll index (SPAD), °Brix, pH, vitamin C (mg/100 g), total protein (mg/100 g) and crop yield (t/ha). The greatest plant growth-promoting effects were observed in plants inoculated with the 20% bioinoculant and in the commercial product treatment, as evidenced by increased plant height, greater fruit diameter, caliber, and length, as well as lower root disease incidence (2.86% and 1.43%, respectively). In addition, yields were highest in these treatments (29.9 and 25.2 t ha⁻¹, respectively) compared with 14.5 t ha⁻¹ in the control. These results indicate that a 20% B. halotolerans-based bioformulation, similar to the commercial formulation, promotes plant growth, improves agronomic performance, and reduces root disease incidence in tomato crops.

1. Introduction

After potatoes, tomatoes are the second most widely cultivated vegetable worldwide [1]. In addition to serving as a key source of income for farm households [2] tomatoes contribute to poverty reduction. Consumption of tomatoes, whether raw or cooked, preserves their nutritional value, providing essential nutrients such as lycopene, potassium, iron, folate, vitamin C, and antioxidants including beta-carotene [3,4,5]. Tomatoes are among the most economically valuable vegetables worldwide, with an approximate production of 192 million t in 2023, growing on around 5.4 million hectares. China is the leading producer, followed by India and Turkey, according to the Food and Agriculture Organization of the United Nations [6]. In Peru, during the first quarter of 2024, 4037 t of tomatoes were exported, showing a 5.7% increase in value compared to the previous year [7].

In addition, tomatoes are among the most economically valuable vegetables, with a global production of approximately 186 million t from 4.9 million ha, according to the Food and Agriculture Organization of the United Nations [6]. In 2022, tomato production in Peru reached 211,339 t across 5135 ha, with an average yield of 41.16 t/ha [6]

Tomato production for both fresh and processed markets faces significant constraints from over 200 diseases caused by various pathogens worldwide [1,8], resulting in substantial economic losses [9]. The key soilborne pathogens include Fusarium oxysporum and Phytophthora sp., which can reduce yields by 45–55%, with effects amplified under conditions favorable for their growth [9]. The most commonly employed control method is the application of chemical fungicides. However, their prolonged use can lead to significant adverse effects, including environmental contamination [10], residual toxicity, and the development of pathogen resistance [11].

Considering these limitations, the use of beneficial microorganisms has become increasingly important for biological control [12]. Several microorganisms are currently used in agriculture as biocontrol agents, whereas others show considerable potential for future application [13]. Inoculation with beneficial microorganisms enhances plant resistance to infection and improves crop yield [14]. Species of the genus Bacillus are among the most extensively studied PGPR, as they simultaneously exhibit both direct growth-promoting traits and indirect biocontrol activities. Currently, numerous commercial products based on microorganisms or their derivatives, including cellular formulations, enzyme products, and secondary metabolites, are available [15,16].

Currently, numerous commercial products based on microorganisms or their derivatives, including cellular formulations, enzyme products, and secondary metabolites, are available [15,16].

Several species of the genus Bacillus have shown promise as biocontrol agents because of their ability to produce broad-spectrum secondary metabolites, such as lipopeptides [17]. In addition, these microorganisms can activate natural plant defenses and stimulate plant growth [18]. Plant growth stimulation is associated with the production of volatile compounds and lytic enzymes, as well as with the activation of metabolic pathways related to induced systemic resistance, which is regulated by multiple complex molecular mechanisms involving hormones such as salicylic acid, jasmonate, and ethylene [19,20] reported that native strains of Bacillus subtilis and Bacillus amyloliquefaciens, isolated from high Andean regions, inhibited Rhizoctonia solani infection in seedlings of two potato varieties (Ccompis and Andina). Analyses of secondary metabolites from Bacillus spp. have revealed a broad range of compounds with herbicidal, insecticidal, and antimicrobial activities, including subtilosin A, bacillibactin, bacillaene, and bacilysin, which contribute to biocontrol of pathogens and agricultural sustainability [21].

The use of biological agents represents a sustainable strategy to enhance soil fertility and control diseases across various crops, playing a key role in the development of bioinoculants and biopesticides [22]. In this context, the present study aimed to evaluate, under field conditions, the effects of Bacillus halotolerans IcBac2.1 applied at different concentrations as a plant growth-promoting rhizobacterium and as a biocontrol agent against root wilt-associated phytopathogens in tomato (Solanum lycopersicum), using a liquid bioinoculant produced in a 7 L bioreactor. The application of this strain was expected to reduce disease incidence while enhancing plant growth, physiological performance, fruit quality, and yield.

2. Materials and Methods

2.1. Location of the Experimental Field

The experimental field was situated in the Horticulture Department of the Universidad Nacional Agraria La Molina, district La Molina, Province and Department of Lima, Peru (12°05′06″ S, 76°57′07″ W; 243.7 m a.s.l.). The study was conducted during the summer season. Data from the weather station at Universidad Nacional Agraria La Molina orchard indicated that precipitation was low, ranging from 0 to 1.2 mm. Maximum temperatures ranged from 30.2 °C to 31.6 °C, whereas minimum temperatures ranged from 15.6 °C to 17.7 °C. Relative humidity varied between 64.4% and 86.9%.

2.2. Preparation of the Plant Material

The plant material used was tomato (Solanum lycopersicum) var. Ktya, a hybrid developed by Hazera. This variety exhibits semi-erect determinate growth and poro-type fruits and can be cultivated year-round. Seeds were germinated in 198-cell trays (one seed per cell) filled with imported sterile Sphagnum peat substrate. The seeds were then moistened and placed in a germination chamber for 3 days at a constant temperature of 25 °C. At the onset of germination and emergence of the first cotyledons, seedlings were transferred into the greenhouse for 28 days before being transplanted to the final field.

2.3. Soil Sampling for Physicochemical Analysis

Several soil subsamples were collected across the field and combined to obtain a total of 1 kg of soil. Analysis of a representative soil sample from the experimental area indicated that the soil was slightly alkaline (pH 7.6) and exhibited high organic matter content, high to very high phosphorus and potassium levels, and a sandy loam texture. For field preparation, soil was loosened using a polydisk implement and leveled with a disk plow. Furrows were spaced 1.2 m between rows and 0.6 m between plants. Crop management followed a gravity irrigation system, with irrigation performed 1 day before transplanting (DBT).

2.4. Bacillus halotolerans IcBac2.1 Strain and Bacterial Growth Conditions

The bacterium used in this experiment was obtained from the Strain Bank (IcBac2.1) of the Microbial Ecology and Biotechnology Laboratory of Universidad Nacional Agraria La Molina. The bacterial strain was isolated from the arid zones of the Ica region of Peru, based on previous studies by [23], and was identified as Bacillus halotolerans IcBac2.1 based on whole-genome sequencing, showing 90.2% dDDH and 98.52% ANIb with B. halotolerans ATCC 25096ᵀ strains [24]. The strain was selected for its ability to produce various volatile and non-volatile compounds exhibiting antagonistic activity against various soilborne pathogens [24]. The bioinoculant was produced in a 7 L bioreactor for three days, yielding a final cell density of 1 × 10⁸ CFU/mL.

2.5. Production of the Bacillus halotolerans IcBac2.1 Bioinoculant

The bioinoculant was produced in a 7 L Applikon^® bioreactor over a 72 h fermentation period using a culture medium composed of starch (15 g/L), yeast extract (8 g/L), NaCl (0.1 g/L), FeSO3 (0.1 g/L), KH₂PO₄ (1 g/L), and MgSO₄ (0.5 g/L). Fermentation was conducted under controlled conditions with an aeration rate of 0.5 VVM, an agitation speed of 300 rpm, and a temperature of 28 °C, resulting in an optimized fermented broth with a final cell density of 1 × 10⁸ CFU/mL and a pH ranging from 8.0 to 8.5. The components used for bioinoculant optimization were selected based on previously reported studies [23].

2.6. Final Field Establishment

Seedlings were transplanted 28 days after sowing, when they had reached approximately 15 cm in height. Seedlings were placed on one side of the furrow at 0.5 m spacing, corresponding to a planting density of 20,000 plants/ha, under a gravity-fed irrigation system. Crop management was carried out under an organic system; that is, no chemical inputs were applied for pest control, and nutrients were supplied via foliar application of an organic biofertilizer (biol). The experiment included five treatments with five replicates each, resulting in a total of 25 experimental units arranged in five blocks. Each experimental unit consisted of three furrows with 14 plants each, resulting in 42 plants per experimental unit. The treatments consisted of inoculating plants with the bacterial bioinoculant (1 × 10⁸ CFU/mL) produced in a 7 L bioreactor at three concentrations (10% [T2], 20% [T3], and 30% [T4]), a commercial B. subtilis formulation (T5), and an uninoculated control (T1) (

Table 1. Description of treatments applied in the experimental field.

Treatment	Key	Formulation Concentration (%)	Dose Applied (L/ha)	Number of Applications
Control	C-T1	0	0	0
Dose 1	BIO1 T2	10	6	4
Dose 2	BIO2 T3	20	6	4
Dose 3	BIO3 T4	30	6	4
Commercial P. (B. subtilis)	BC T5	7	6	4

).

2.7. Timing of Bioinoculant Application

The bioinoculant of each biological formulation was first applied 8 days after seedling germination, at a rate of 1 mL per seedling or per germination cell. The trays containing the seedlings were labeled according to the treatment used for field establishment. The second application was conducted before transplanting by immersing seedlings in 3 L of inoculum for each treatment concentration for 1 min. The third inoculation was performed 7 days after transplanting (DAT) to the final field, whereas the fourth was applied at the onset of flowering, 30 days after the third inoculation.

The third and fourth applications were performed as soil drenches, with the nozzle fully open and directed at the plant collar. Before application, the equipment was calibrated with water to deliver the required volume for 42 plants per experimental unit. The uninoculated control received water instead of the inoculant in all four applications.

2.8. Experimental Design

A randomized complete block design with five treatments and five replicates was used. The results were compared using analysis of variance. Differences between treatments were determined using Tukey’s multiple comparison test (α = 0.05). The data were processed using the statistical software InfoStat 2020 [25].

2.9. Effectiveness of a Bacillus halotolerans IcBac2.1 Bioinoculant in the Biocontrol of Root Diseases

• Incidence of root pathogens in plants

The number of diseased plants showing secondary symptoms of basal leaf yellowing, decline, and wilting caused by root pathogens (e.g., Fusarium oxysporum and Phytophthora spp.) was evaluated at 10, 20, 35, and 50 days after transplanting (DAT). Plant incidence was calculated using the following equation:

$$\% \mathrm{Incidence} = {\displaystyle \frac{\mathrm{N}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{p}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{t}\mathrm{s} \mathrm{a}\mathrm{f}\mathrm{f}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{e}\mathrm{d}}{\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{p}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{t}\mathrm{s} \mathrm{o}\mathrm{b}\mathrm{s}\mathrm{e}\mathrm{r}\mathrm{v}\mathrm{e}\mathrm{d}}} \times 100$$

• Area under the disease progress curve

Cumulative incidence (%) of root diseases (e.g., Phytophthora, R. solani, and Fusarium sp.) was assessed at 7 DAT and every 15 days thereafter. The area under the disease progress curve (AUDPC) was calculated following the formula described by [26], as commonly applied in modern studies of plant disease epidemiology [27,28]:

$$\mathrm{A}\mathrm{U}\mathrm{D}\mathrm{P}\mathrm{C}=\sum _{\mathrm{i}=1}^{\mathrm{n}-1}\left({\displaystyle \frac{{\mathrm{Y}}_{\mathrm{i}}+{\mathrm{Y}}_{\mathrm{i}+1}}{2}}\right)\left({\mathrm{t}}_{\mathrm{i}+1}-{\mathrm{t}}_{\mathrm{i}}\right)$$

where n is the number of evaluations, y is the incidence, and t is the number of days post-transplanting at which the evaluation was performed. The point (t, y) = (0, 0) was included as the initial evaluation.

• Identification of the causal agent of plant wilt

Through targeted sampling of plants exhibiting wilt symptoms, molecular identification at the genus level was performed for fungi isolated from roots and stems at the diagnostic clinic of the Department of Phytopathology of Universidad Nacional Agraria La Molina.

• PGPR Activity of the Bacillus spp. Bioinoculant in Tomato Plants
• Plant height (cm)

Plant height was measured from the plant collar to the apex of the highest leaf using a tape measure. Measurements were taken at 60 DAT thereafter until full flowering.

• Stem diameter (mm)

Plants selected for aerial growth measurements were considered as samples, and a digital caliper was used. Evaluations were conducted at 45 DAT thereafter until full flowering.

• Chlorophyll index (SPAD)

Chlorophyll content was indirectly evaluated using a SPAD-502 Plus chlorophyll meter [29]; verifier reading 69.0 ± 3.0, expressed in SPAD units). Two measurements were performed: the first when flowering was below 50% and the second during the fruiting stage. In each evaluation, three fully expanded leaves from the middle third of each plant were randomly selected within each experimental unit (42 plants), using five representative plants per unit. For each leaf, three readings were taken and averaged to obtain the final value per plant.

The SPAD-502 Plus chlorophyll meter is a portable, non-destructive device widely used to measure the chlorophyll content of leaves, providing rapid and immediate readings. It operates by measuring light absorbance at two wavelengths, 650 nm and 940 nm, corresponding to chlorophyll absorption. The device calculates a SPAD value proportional to the chlorophyll concentration in the leaf. Its small measuring area (2 mm × 3 mm), ease of use via a clip-on sensor, and waterproof design make it ideal for assessing plant health and optimizing fertilizer application in various crops such as wheat, rice, corn, and cotton. The meter displays results within 2 s and can store up to 30 measurements in a trend graph for monitoring changes over time [29].

• Morphological and physicochemical characteristics of the fruits

The length and diameter of the sampled fruits (mm) were measured using a Vernier caliper. A representative sample of 10 fruits per experimental unit was selected to ensure statistical validity and uniformity in maturity characteristics. Using the same fruits measured for length and diameter, physicochemical variables including °Brix, pH, vitamin C, and total protein content were determined from 1 kg composite samples per experimental unit.

• Yield (t/ha)

Fruits were harvested once a week for 8 weeks, selecting only those with pigmentation. The fruits were weighed, and data was recorded in a field notebook. After each harvest, the fruits of each experimental unit were weighed separately. The weights obtained in each harvest were then summed to calculate the total yield (t/ha).

3. Results

3.1. Effect of Biocontrol Inoculation Under Field Conditions

3.1.1. Disease Incidence

The incidence (%) of tomato plants with wilt symptoms caused by the genera Phytophthora, Rhizoctonia, and Fusarium varied significantly among treatments during the 55 days after transplanting (DDt) (Figure 1). In the untreated control (T1), disease incidence increased steadily from 1.4% at 10 DDt to 14.3% at 55 DDt, representing the highest severity levels. Accordingly, the treatments with Bacillus sp. showed a reduction in incidence. The dose of the 10% formulation (T2) exhibited a gradual increase, reaching 5.7% at 55 DDt., While the 20% concentration(T3) showed a low and stable incidence, with values of 2.9% maintained from 35 DDt until the end of the evaluation period. The higher concentration of the 30% formulation (T4) showed an increase up to 4.3% at 35 DDt, remaining constant at the same value on the last evaluation day, whereas the commercial Bacillus formulation (T5) presented the lowest incidence, recording only 1.4%. Overall, the incidence values demonstrated a clear differentiation among treatments, with the untreated control exhibiting the highest levels, while Bacillus-based treatments significantly limited disease progression under field conditions.

3.1.2. Area Under the Disease Progress Curve (AUDPC)

Disease incidence (%) and the area under the disease progress curve (AUDPC) showed significant differences between the treatments and the control (

Table 2. Disease incidence and AUDPC across treatment groups (α = 0.05).

Treatments	Incidence (%)	Sig.	Percent Effectiveness	AUDPC	Sig.
T1: Control	14.29	a	0.00	414.29	a
T2: 10% concentration	5.71	abc	60.00	171.43	bc
T3: 20% concentration	2.86	bc	80.00	85.71	c
T4: 30% concentration	4.29	abc	70.00	128.57	c
T5: Commercial P. (B. subtilis)	1.43	c	90.00	14.29	c

Note. Different letters indicate statistically significant differences between treatments, as determined by Tukey’s test (p ≤ 0.05).

). The control (T1) exhibited the highest incidence (14.29%) and the highest AUDPC value (414.29), reflecting high susceptibility to the pathogen. Among the bioinoculant treatments, the 20% concentration (T3) achieved the best performance, with an AUDPC of 85.71, followed by the 30% concentration (T4) with 128.57 and the 10% concentration (T2) with 171.43. Although no significant differences were detected among the bioinoculant formulations, all of them significantly reduced the disease compared to the control.

3.2. PGPR Activity of Bacillus spp. Bioinoculant in Tomato Plants

3.2.1. Morphometric Characteristics

At 60 DAT, field evaluation revealed that tomato plants inoculated with Bacillus spp. had significantly greater height and stem diameter than control plants. Aerial growth was highest in T3 (73.96 cm), whereas stem diameter was significantly greater in T3 (17.12 mm) and T5 (17.03 mm) than in T1 (11.96 mm);

Table 3. Stem height and diameter across treatment groups at 60 DAT (α = 0.05).

Treatments	Height (cm)	Sig.	Diameter (mm)	Sig.
T1: Control	65.76	b	11.96	b
T2: 10% concentration	72.86	ab	15.82	a
T3: 20% concentration	73.96	ab	17.03	a
T4: 30% concentration	71.90	a	16.42	a
T5: Commercial P. (B. subtilis)	73.08	a	17.12	a

Different letters indicate statistically significant differences between the groups according to Tukey’s test (p ≤ 0.05).

.

Chlorophyll content showed significant differences among treatments at 55 and 70 days after transplanting (DAT) (

Table 4. Chlorophyll content at 55 and 70 DAT.

Treatments	Chlorophyll 1 55 DAT	Sig.	Chlorophyll 2 70 DAT	Sig.
T1: Control	46.36	c	49.37	b
T2: 10% concentration	56.01	ab	54.91	ab
T3: 20% concentration	57.01	ab	56.44	a
T4: 30% concentration	56.25	ab	53.60	ab
T5: Commercial P. (B. subtilis)	59.65	a	56.71	a

Different letters indicate statistically significant differences between the groups according to Tukey’s test (p ≤ 0.05).

). At 55 DAT, values ranged from 46.36 to 59.65 SPAD units. The commercial product (B. subtilis) recorded the highest chlorophyll content (59.65 SPAD), differing significantly from the control (46.36 SPAD), while the 10%, 20%, and 30% bioinoculant treatments showed intermediate values (56.01–57.01 SPAD).

At 70 DAT, the 10% and 20% bioinoculant treatments, as well as the commercial product, maintained the highest chlorophyll values (54.91–56.71 SPAD), which were statistically superior to the control (49.37 SPAD). Overall, these findings demonstrate that both the 10% and 20% bioinoculant concentrations and the commercial product enhanced chlorophyll accumulation compared to the control, highlighting their positive effect on plant physiology.

Overall, these results indicate that the 10% and 20% bioinoculant concentrations, as well as the commercial product, promoted greater chlorophyll accumulation compared to the control, suggesting a positive effect on the photosynthetic physiology of tomato plants.

3.2.2. Biochemical Characteristics

The analysis of harvested fruits revealed significant differences in soluble solid contents (°Brix) and pH among treatments (

Table 5. °Brix, pH, vitamin C content, and total protein content across treatment groups (α = 0.05).

Treatments	°Brix		pH		Vitamin C (mg/100 g)		Total Protein (g/100 g)
T1	5.15	a	4.27	a	11.14	A	1.57	a
T2	5.73	b	4.07	bc	17.04	B	1.87	b
T3	6.5	c	4.01	c	16.86	B	2.16	c
T4	5.8	b	4.11	bc	16.56	B	1.85	b
T5	5.9	b	4.13	b	*	*	*	*

Different letters indicate statistically significant differences between the groups according to Tukey’s test (p ≤ 0.05). * Not analyzed.

). The control (T1) showed the lowest °Brix value (5.15) and the highest pH (4.70). In contrast, T3 (20% bioinoculant) recorded the highest °Brix (6.50) and a significantly lower pH (4.01) compared with the commercial product (T5).

The vitamin C content in the treatments with the Bacillus halotolerans formulation T2 (10%), T3 (20%), and T4 (30%) showed statistically significantly higher levels compared to the control (T1), while no significant differences were detected among these three treatments (Table 5). Total protein content (g/100 g) showed statistically significant differences among the treatments. The highest protein content was observed in T3, which differed significantly from the control (T1), T2, and T4, while T2 and T4 also showed significant differences compared with the uninoculated control. The commercial Bacillus subtilis-based product (T5) was not analyzed for vitamin C or total protein and therefore was excluded from the statistical comparison of these variables.

At harvest, fruit yield and growth parameters showed significant differences among the treatments (

Table 6. Length, diameter, and yield across treatment groups (α = 0.05).

Treatments	Length (cm)	Sig.	Diameter (cm)	Sig.	Weight (g)	Sig.	Yield (t/ha)	Sig.
T1	5.95	d	4.80	b	71.48	C	14.50	c
T2	6.32	bc	5.42	a	89.46	Ab	22.02	ab
T3	6.71	a	5.43	a	102.83	A	29.92	a
T4	6.27	c	5.10	ab	95.28	Ab	21.54	ab
T5	6.53	ab	5.23	ab	92.88	Ab	25.24	a

Different letters indicate statistically significant differences according to Tukey’s test (p ≤ 0.05).

). The highest yield was observed in T3 (29.92 t/ha), which showed a tendency to be higher than the other treatments, while being significantly greater than the uninoculated control (T1, 14.50 t/ha). These results showed a positive effect of the bioinoculant on overall crop productivity.

Growth parameters followed a similar trend: T3 produced the longest fruits (6.71 cm) and the heaviest individual fruits (102.83 g), both significantly greater than those of T1. Fruit diameter was significantly greater in T2 and T3 (5.42 and 5.43 cm, respectively) compared to T1, while T4 and T5 showed intermediate values. Overall, treatments with the Bacillus formulations (T2–T5) improved fruit size, weight, and yield compared to the control, likely due to the ability of Bacillus strains to enhance plant growth, improve nutrient uptake, and provide protection against phytopathogens [19]. The bromatological composition of the tomato fruits observed in this study falls within the ranges previously reported for Solanum lycopersicum in different cultivation systems, coinciding with the values described [30].

4. Discussion

This study evaluated the capacity of a Bacillus halotolerans IcBac2.1 strain, applied at different concentrations under field conditions, to enhance yield and control root diseases caused by various phytopathogens. The results indicated that the inoculant exerted a biocontrol effect against the pathogens responsible for wilting in tomato plants at all three concentrations tested. A previous study [23] highlighted the ability of Bacillus halotolerans IcBac1.2 to produce various volatile and non-volatile compounds with biocontrol activity against Fusarium oxysporum, Sclerotinia sclerotiorum, and Rhizoctonia solani, as well as its potential to induce plant growth. Whole-genome sequencing of this strain confirmed its assignment to the species Bacillus halotolerans [24]. It is possible that volatile and non-volatile compounds produced by Bacillus control plant pathogens by inducing pore formation in microbial cell walls and membranes, thereby inhibiting their growth and indirectly stimulating plant defense responses [12].

It is important to note that the control treatment consisted of repeated applications of water without microbial inoculation, a standard practice in field and greenhouse studies with PGPR [9]. Repeated irrigation of the soil can dilute or transiently redistribute soluble nutrients and root exudates in the rhizosphere, particularly in sandy or sandy-loam soils [19]. However, these effects tend to be transient, and in the present study, all treatments were subjected to uniform application volumes and frequencies. Field experiments designed to evaluate the effects of PGPR generally include uninoculated control treatments subjected to identical irrigation regimes, allowing for adequate differentiation of effects attributable to microbial inoculation from those associated solely with irrigation [31]. Therefore, any possible water-induced change in the root zone environment would have occurred uniformly across all treatments and is unlikely to explain the consistent differences observed between inoculated and non-inoculated plants.

Similarly, ref. [32] identified a novel strain, B. halotolerans Cal.l.30, with high plant colonization capacity and the ability to produce secondary metabolites associated with antifungal activity, antibiosis, and the induction of systemic resistance in plants. Likewise, species of the genus Bacillus have demonstrated key roles of their antimicrobial compounds in the biological control of diseases under in vivo conditions [33].

The results demonstrated that the Bacillus halotolerans strain was able to modulate morphometric traits, such as plant height and stem diameter, in a manner comparable to that of the commercial product. This effect may be related to the strain’s capacity to produce phytohormones, such as auxins and gibberellins, which promote seedling growth and stem thickening, as reported by [34]. Consequently, the interpretations of this study are strictly limited to the observed effects of the Bacillus halotolerans strain treatments and the commercial product evaluated. Previous studies [35] have reported that different Bacillus species, such as B. megaterium and B. licheniformis, can increase stem diameter and other growth parameters in tomato plants when applied at sowing or through periodic foliar applications. Although these species were not used in the present study, the growth-promoting potential of the genus Bacillus is highlighted.

According to [36], auxin is the most abundant hormone secreted by plant growth-promoting bacteria and is the main factor driving increased growth in plants treated with these microorganisms. Similarly, ref. [37] reported that certain bacteria can activate multiple growth-promoting mechanisms. For example, B. subtilis produces auxins that enhance tomato growth and induce systemic resistance against F. oxysporum. However, the expression of genes involved in plant growth and development varies according to environmental conditions, which influence gene regulation in these microorganisms [38].

Ref. [39] investigated two bacterial strains with antimicrobial activity, B. subtilis and Pseudomonas fluorescens, and compared them with commercial antimicrobial agents. The results showed an increase in rhizosphere soil biodiversity and an increase in plant growth and disease control through mechanisms such as ACC deaminase activity, chitinase production, and phosphorus solubilization.

One of the most significant results was that the intermediate concentration of the B. halotolerans formulation (20%, T3) showed a superior effect compared to the highest concentration treatment (30%, T4) across multiple agronomic indicators, chlorophyll content, and yield. This indicates that the use of a higher amount of bioinoculant does not necessarily generate greater benefits in Solanum lycopersicum. On the contrary, the results suggest that there is an appropriate or optimal concentration at which the plant responds more favorably, whereas higher concentrations do not improve the response and may even reduce its effect.

These findings are consistent with those reported by [40], which indicated that excessive application of B. amyloliquefaciens at 80% reduced plant growth. Therefore, it is unlikely that inoculants applied at high concentrations positively influence plant development, as they may generate neutral responses or inhibit growth by altering the hormonal balance. The effect observed at the highest concentration (30%) used in this study may be related to an increase in indolic compounds and other metabolites derived from the fermented broth of Bacillus halotolerans, which could modify endogenous levels in the plant beyond the optimal threshold, resulting in the induction or inhibition of plant growth [40]. In addition, high bacterial density in the rhizosphere may induce stress responses or cause a reallocation of metabolic resources from growth toward defense mechanisms [41].

The determination of chlorophyll content in plant tissues is a widely used method to assess crop nutritional status due to the close relationship between chlorophyll and nitrogen levels [42]. In addition, key physiological processes such as leaf transpiration and photosynthesis are essential mechanisms for improving plant growth following exposure to PGPR. In this study, chlorophyll content was significantly higher in plants treated with bioinoculants than in control plants. This suggests that the bacteria promoted chlorophyll accumulation, which may be associated with enhanced photosynthetic efficiency. This effect is believed to result from the activation of reactive oxygen species (ROS)-scavenging enzymes, which play a key role in mitigating oxidative stress caused by adverse environmental factors [43,44].

In this context, stress signals may be associated with both biotic agents, such as root pathogens, and abiotic factors to which plants were exposed during the trial. This was evidenced by lower chlorophyll index values in the control group, suggesting greater susceptibility to stress in the absence of PGPR. These findings are consistent with those of [45], who reported that the application of B. mycoides-based biostimulants improved water status and photosynthetic capacity in bean plants under saline stress, as indicated by increased chlorophyll content. Similarly, in the present study, increased chlorophyll content in plants inoculated with B. halotolerans and B. subtilis may exert a protective effect against pathogen-induced stress and adverse environmental conditions.

Previous studies have indicated that PGPR inoculation can improve crop quality and commercial value. For example, ref. [46] reported that the B. subtilis strain CBR05 significantly improved fruit quality. In addition, these bacteria are known to increase the availability of essential nutrients such as phosphorus and iron, thereby optimizing plant nutritional status in the rhizosphere [47]. Collectively, these results support the importance of PGPR in stress mitigation and optimization of plant development, demonstrating their potential utility in sustainable agricultural strategies to improve crop productivity and quality.

Biochemical analysis of fruits harvested in this study revealed significant differences in sugar levels, vitamin C, and total protein content between inoculated and non-inoculated treatments. According to [47], plant–bacteria interactions can activate multiple metabolic pathways related to sucrose—the main non-reducing sugar—which, together with glucose and fructose, plays a key role in stimulating plant growth. Several studies have reported that PGPR inoculation improves the nutritional content and organoleptic characteristics of tomatoes, contributing to increased total protein levels. In this study, fruit acidity decreased following inoculation with the B. halotolerans bioformulation at a concentration of 20%. This effect may be attributed to increased metabolic activity during ripening, which promotes the release of enzymes that degrade polysaccharides, proteins, and lipids into simpler sugars.

Overall, several authors have suggested that increased levels of total free proteins may be associated with resistant induction processes linked to the activation of specific enzymes, as cited by [48]. In addition, ref. [49] reported that multiple factors, such as crop type and cultivation practices, can influence the bromatological composition of tomatoes, particularly mineral (ash), protein, and fat contents.

Study [50] reported that increased fruit sugar levels may be related to enhanced metabolic activity, which stimulates the production of compounds associated with disease resistance. Consequently, the results suggest that fruits with higher sugar content were produced by plants with lower disease incidence, whereas fruits with lower sugar content were associated with higher wilting rates and greater disease incidence.

When fruit yield was evaluated, plants inoculated with bacteria showed significantly higher yields than the control group. This positive effect is related to other evaluated parameters, which collectively contribute to increased productivity in the presence of beneficial microorganisms. In this study, the highest yields were obtained from plants inoculated with B. halotolerans and B. subtilis. One of the tested concentrations resulted in yield increases comparable to those achieved with the commercial product. Specifically, the B. halotolerans bioinoculant applied at a 20% concentration was associated with the highest values for key agronomic traits, including plant height, stem diameter, fruit size, and chlorophyll index. These results are consistent with those reported by [51], who evaluated Bacillus sp. and Bradyrhizobium strains under field conditions and observed increased chlorophyll content, enhanced plant growth, and significant improvements in parameters such as fresh and dry weight of plants and pods in tarwi.

Similarly, ref. [52] demonstrated that tomato plants treated with Bacillus strains exhibited significant increases in stem diameter, chlorophyll content, and yield compared to plants inoculated only with phytopathogens. In their study, B. amyloliquefaciens and B. subtilis strains promoted a 30% increase in plant height, root length, and yield, while the phytopathogen F. oxysporum reduced yield by the same percentage. Therefore, the results of the present study are consistent with previous reports, supporting the ability of the genus Bacillus to promote plant growth and exert antagonistic effects against pathogens. In addition, the potential of B. halotolerans as a bioformulant was highlighted, as its efficacy can be maintained when an appropriate application concentration is established, resulting in significant improvements in key agronomic variables that directly influence tomato crop yield.

The results confirm the PGPR capacity of Bacillus, as evidenced by enhanced metabolic activity and antagonistic effects against phytopathogens. The agronomic performance of B. halotolerans IcBac2.1 is likely associated with its ecological origin, as this strain was isolated from rhizosphere soils of the coastal desert of Ica, Peru, characterized by arid conditions, alkaline pH, low organic matter, limited water retention, and high salinity. These selective environments may favor microorganisms with high stress tolerance and metabolic versatility, making this strain particularly interesting for effective application under field conditions [24]. Furthermore, incorporating B. halotolerans into a bioformulation can provide agronomic benefits, provided that an appropriate application concentration is used. The positive relationship between bacterial inoculation and yield parameters underscores its potential as a biotechnological strategy to optimize tomato production.

5. Conclusions

The results indicated that a Bacillus halotolerans bioinoculant tested at different concentrations and a commercial Bacillus subtilis product were effective in promoting vegetative development of tomato cv. Ktya. Notably, the 20% B. halotolerans-based bioinoculant achieved a higher chlorophyll index, °Brix, pH (lower acidity), vitamin C content, and fruit yield than the uninoculated control.z

These results are relevant for enhancing the yield and organoleptic and nutritional quality of the fruit. Therefore, applying a 20% B. halotolerans-based bioinoculant to tomato plants can be considered a sustainable strategy for improving crop nutrition and health.