Chili pepper (Capsicum annuum) is the third most cultivated and consumed vegetable worldwide and holds significant economic importance in producer countries such as Mexico (Lozana et al., 2022; Bobadilla-Larios et al., 2017). In 2023, chili production in Mexico reached 3,681,061.47 t, with a market value of 45,997.65 million pesos (SIAP, 2023). However, crop yield is threatened by various biotic factors, with fungal diseases being among the most significant.

Species of the Fusarium genus pose one of the main phytosanitary threats to chili cultivation, causing diseases such as wilt and root rot, which can lead to severe yield losses (Sam-on et al., 2024; Gabrekiristos and Demiyo, 2020). Among these, wilt caused by Fusarium oxysporum is one of the most destructive, capable of reducing crop yield by 68 to 71% (Sam-on et al., 2024; Adina et al., 2021; Gabrekiristos and Demiyo, 2020). This is because the fungus colonizes the vascular system, blocking the transport of water and nutrients, which leads to the progressive weakening and, in many cases, death of the affected plants (Bashir et al., 2018).

The persistence of Fusarium in the soil and its ability to form resistant structures complicate its control, making it a recurring issue in chili-producing areas (Campbell, 1990). Control strategies have relied mainly on chemical fungicides, whose indiscriminate use has caused environmental issues, pathogen resistance, and risks to human health (Shaheen et al., 2021; Sela-Burlage et al., 2001).

As a sustainable alternative, the use of beneficial microorganisms with biocontrol and plant growth-promoting capabilities has been proposed. In this context, the Bacillus genus has proven to be a viable option due to its ability to produce antimicrobial compounds, induce plant resistance against pathogens, and enhance plant development (Tiwari et al., 2019; Shafi et al., 2017).

Various Bacillus species have shown promising effects in controlling Fusarium spp. and promoting plant growth. For example, Bacillus subtilis QST713 has been shown to inhibit the growth of F. oxysporum through the production of lipopeptide antibiotics such as iturins and fengycins (Kulkova et al., 2023; Zhou et al., 2021). Similarly, B. amyloliquefaciens FZB42 induces systemic resistance in tomato and chili plants by producing surfactins that inhibit the growth of Fusarium sp. (Chowdhury et al., 2015). In addition, B. velezensis S3-1 suppresses Fusarium spp. infections through the production of bacillomycins and the activation of plant defense mechanisms (Antil et al., 2022; Kumar et al., 2020).

B. cereus has also been reported as an effective biocontrol agent against Fusarium spp., promoting plant growth through phosphorus solubilization and the production of indoleacetic acid (Malik et al., 2022; Yanti et al., 2017). Strains such as B. cereus SLBE.1AP have shown antifungal activity and promoted chili seedling growth (Yanti et al., 2021), while B. cereus EC9 has induced systemic defense responses in tomato plants (Pazarlar et al., 2022). Likewise, B. cereus NK91 was characterized and found to produce chitinases that help control F. oxysporum in vitro (Thakur et al., 2022). B. thuringiensis has been evaluated not only for its insecticidal action but also as a growth promoter in horticultural crops, inducing increases in biomass and plant height due to the production of phytohormones and siderophores (Hyder et al., 2020; Jo et al., 2020; Praça et al., 2012). Strains such as B. thuringiensis CHGP12 have shown effectiveness in controlling F. oxysporum through lipase production (Fatima et al., 2023), while B. thuringiensis IBRC-M11096 induces defense responses in tomato (Zibanezhadian et al., 2020).

However, the use of B. cereus and B. thuringiensis as biocontrol agents and plant growth promoters (PGPs) in jalapeño chili remains insufficiently studied. This is especially relevant considering that the strains used in this study are native to the soil of the chili-producing region, specifically the south-central area of the state of Chihuahua. This characteristic could provide them with a greater capacity for adaptation and effectiveness against local pathogens such as F. oxysporum. Moreover, identifying microorganisms with biocontrol and PGP properties native to the same production area may offer a more efficient and sustainable strategy for managing Fusarium oxysporum wilt in jalapeño chili. Based on this premise, it is hypothesized that the application of B. cereus and B. thuringiensis will reduce the severity of Fusarium oxysporum wilt and promote jalapeño chili growth under greenhouse conditions. Therefore, the objective of this study was to evaluate the biocontrol and PGP potential of B. cereus and B. thuringiensis, as well as their effects on morphological variables of jalapeño chili and the reduction of wilt severity. The findings of this study will provide evidence of the potential of these microorganisms as ecological alternatives to chemical control, contributing to sustainable crop management strategies.

The strains Bacillus cereus Bc28-2 (Bc28-2), B. cereus Bc28-5 (Bc28-5), and B. thuringiensis Bt24 (Bt24) were used as potential biocontrol agents and plant growth promoters (PGPs). These strains were isolated from soil in the jalapeño chili-producing region of Lázaro Cárdenas, Municipality of Meoqui, Chihuahua, and belong to the Applied Microbiology, Phytopathology, and Postharvest Physiology Laboratory (MAFFP) of the Faculty of Agrotechnological Sciences at the Autonomous University of Chihuahua (UACH) (Hernández-Huerta et al., 2023). The commercial strain Bacillus subtilis QST713 (PC), from the product Serenade®, was used as a positive control.

The Fusarium oxysporum strains FM1, FM2, FL1, FL5, FL6, and FL2, belonging to the MAFFP laboratory, were used in this study. These strains were isolated from jalapeño chili plants exhibiting characteristic wilt symptoms in the chili-producing agricultural area of Irrigation District 005 in Delicias, Chihuahua. For the assays, jalapeño chili seeds of cultivar M (Southern Star Seeds S. de R.L. de C.V., Mexico) were used. This cultivar is a common commercial variety in the chili-producing region where the microorganisms were obtained.

Preparation of the bacterial inoculum. Bacillus strains were cultured in nutrient broth (NB; BD Difco Laboratories, Sparks, Maryland, MD, USA) for 72 h at 28 °C. The bacterial suspensions were then centrifuged at 7,000 rpm for 10 min at 4 °C, and the supernatant was discarded. The resulting pellet was resuspended in 20 mL of sterile 0.85% saline solution and adjusted to a concentration of 1 × 10⁸ CFU mL⁻¹, corresponding to an optical density of 0.4 at 600 nm (Chandrasekaran et al., 2017).

Preparation of Fusarium spore suspensions. Spore suspensions were obtained by culturing the fungi on potato dextrose agar (PDA; BD Difco Laboratories, Sparks, Maryland, MD, USA) for 7 days at 28 °C. Spores were collected by scraping and filtering with sterile fiberglass. The spore concentration was then adjusted to 1 × 10⁶ spores mL⁻¹ using a Neubauer chamber (Weber Scientific International Ltd., Teddington, UK).

Pathogenicity and virulence tests of Fusarium strains. This test was conducted during the germination stage of jalapeño chili seeds under in vitro conditions. Seeds were surface-disinfested with 4% (v/v) NaClO under agitation for 5 min, followed by three rinses with sterile distilled water, each lasting 5 min. The sterile seeds were then immersed in fungal spore suspensions and agitated at 120 rpm for 20 min. After inoculation, the seeds were dried and placed in Petri dishes containing moist sterile paper, with five seeds per dish. The dishes were sealed and placed in a growth chamber under a photoperiod of 16 h light at 28 °C and 8 h darkness at 18 °C.

Disease incidence was evaluated as the percentage of infected seeds per dish, and the severity index (SI) was determined 10 days after inoculation using the scale by Robles- Hernández et al. (2015): 0 = asymptomatic germinated seed; 1 = seed with mycelial growth on the seed coat; 2 = isolated necrotic spots and/or necrotic lesions of 1-5 mm; 3

= necrotic lesions of 6-10 mm; and 4 = germinated seeds with total necrosis, necrotic lesions over 11 mm, or seedlings with necrotic hypocotyls. The SI was calculated using the formula proposed by Galanihe et al. (2004).

I S = ∑ P Q M N x 100

where IS = severity index, P = severity category, Q = number of plants in the same category, M = total number of plants observed, and N = maximum value on the rating scale.

To assess disease progression caused by each fungal strain over time, the area under the disease progress curve (AUDPC) was calculated using the formula proposed by Saner and Finney (1997) and the method of Campbell and Madden (1990).

The assay was conducted under a completely randomized design (CRD) with four replicates per treatment (Petri dishes with inoculated seeds). Treatments consisted of fungal spore suspensions, and sterile distilled water was used as the control.

In vitro dual culture antagonism assay between Bacillus and Fusarium. The antagonism assay was conducted in sterile 96-well microplates (Biologix Steril 07-6096, Biologix Research) under in vitro conditions. Each well was filled with 150 μL of bacterial suspensions (1 × 10⁸ CFU mL⁻¹). Then, 150 μL of fungal spore suspensions (1

× 10⁶ spores mL⁻¹) prepared in potato dextrose broth (BD Difco Laboratories, Sparks, Maryland, MD, USA) were added. The inoculated microplates were sealed and incubated at 28 °C for 48 h in darkness. To assess fungal spore degradation, 10 μL were taken from each well, and the spores present were counted directly using a Neubauer chamber.

The assay was conducted using a 4×6 completely randomized factorial design, evaluating two factors: bacterial strains (Bc28-2, Bc28-5, Bt24, and PC) and fungal isolates (FM1, FM2, FL1, FL5, FL6, and FL2), resulting in 24 unique treatments corresponding to each bacteria-fungus combination. Fungal spore suspensions alone were used as controls. All treatments were performed in triplicate.

Growth promotion in chili seedlings by Bacillus spp. To evaluate the effect of bacteria on the development of jalapeño chili seedlings, sterile seeds were sown in 20-cavity polystyrene trays filled with sterile horticultural perlite (1 h at 120 °C, 15 lb/in²). The trays were irrigated with a nutrient solution (Nutrient Solution for Vegetables®; pH 5.5 and electrical conductivity of 1.5 mS). The seedlings were grown under greenhouse conditions at 27 °C ± 2 °C and 75% relative humidity (RH).

Seedlings were inoculated at the stem base with 5 mL of bacterial suspensions of Bc28-5, Bc28-2, Bt24, and the PC (1 × 10⁸ CFU mL⁻¹) at 10 days after sowing. Inoculation was repeated every six days for a total of five applications. Thirty days after the first inoculation, morphological variables were evaluated: seedling height, root length, stem diameter, number of leaves, leaf area (Canopeo App; https://canopeoapp.com), and fresh and dry biomass of leaves, stem, and root. Photosynthetic pigment content (chlorophyll a, chlorophyll b, and carotenoids) was also assessed following the method of Lichtenthaler and Wellburn (1983).

The assay was established under a completely randomized design (CRD) with four replicates per treatment. Treatments consisted of bacterial solutions applied to the seedling trays, and uninoculated seedlings were used as the control.

Biocontrol of Fusarium by Bacillus spp. under greenhouse conditions. This assay used 40-day-old jalapeño chili seedlings that had been previously grown under the described conditions and inoculated with strains Bc28-2, Bc28-5, Bt24, and PC. The seedlings were transplanted into 10 cm diameter pots filled with sterile peat moss previously inoculated with spores of Fusarium oxysporum FL2-a strain characterized by a high severity index, the largest area under the disease progress curve, and resistance to spore degradation.

Before transplanting, the sterile peat moss (1 h at 120 °C, 15 lb/in²) was inoculated with a fungal spore suspension (1 × 10⁶ spores mL⁻¹) and incubated at 28 °C for five days. The inoculated seedlings were then placed under greenhouse conditions at 27 °C ± 2 °C and 75% relative humidity. Plants were irrigated every third day with nutrient solution.

Thirty days after transplanting, the disease severity index (SI) was evaluated using the following scale: 0 = healthy root system; 1 = 1-25% root damage; 2 = 25-50% root damage; 3 = 50-75% root damage; and 4 = 75-100% root damage. The SI was calculated using the formula proposed by Galanihe et al. (2004).

Morphological variables were also assessed, including seedling height, root length, stem diameter, number of leaves, leaf area (Canopeo App), root volume, and fresh and dry biomass of leaves, stem, and root. Photosynthetic pigment content was determined following the methodology of Lichtenthaler and Wellburn (1983). The experiment was conducted under a completely randomized design (CRD) with four replicates per treatment, as described in Table 1.

Table 1

Treatments	Treatment code
Fusarium oxysporum FL2	FL2
Bacillus subtilis QST713 + F. oxysporum FL2	CP/FL2
B. cereus Bc28-2 + F. oxysporum FL2	Bc28-2/FL2
B. cereus Bc28-5 + F. oxysporum FL2	Bc28-5/FL2
B. thuringiensis Bt24 + F. oxysporum FL2	Bt24/FL2
Plants without microbial treatment	Control

Statistical analysis. The data from the assays were subjected to Shapiro-Wilk and Levene’s tests to assess normality and homogeneity of variance prior to statistical analysis.

Data from the in vitro pathogenicity and virulence tests were analyzed using the non- parametric Kruskal-Wallis test followed by the Conover-Iman test (p < 0.05). Results from the in vitro antagonism assay evaluating fungal spore degradation were analyzed using a factorial analysis to assess the main effects (bacteria and fungi) and their interactions on spore degradation. Multiple comparisons were then performed using the Scott-Knott test (p < 0.05).

Data from the assay on seedling growth promotion by Bacillus spp. and from the greenhouse assay evaluating growth promotion in plants interacting with F. oxysporum were analyzed by analysis of variance (ANOVA), followed by Tukey’s post hoc test for mean separation (p < 0.05).

The severity index data from the greenhouse interaction between bacterial strains and F. oxysporum FL2 were analyzed using the Kruskal-Wallis test and the Conover-Iman test (p < 0.05).

A principal component analysis (PCA) was conducted to assess the influence of bacterial treatments on biocontrol and plant growth promotion in interaction with F. oxysporum FL2. Prior to PCA, Bartlett’s test of sphericity (p < 0.01) was performed and the Kaiser-Meyer-Olkin (KMO) index was calculated, with KMO values above 0.60 considered acceptable (Tabachnick and Fidell, 2007).

All statistical analyses were performed using InfoStat software (InfoStat 2021v, Grupo InfoStat, Argentina) and JAMOVI version 2.5.2.0 (The Jamovi Project, 2024).

Pathogenicity and virulence tests of Fusarium strains. The in vitro pathogenicity and virulence tests conducted with Fusarium isolates during jalapeño chili seed germination showed that all fungal strains caused disease symptoms 10 days after inoculation, with no significant differences among strains. Observed symptoms included necrotic spots on the radicle and hypocotyl, complete necrosis of the radicle, mycelial growth on the seed coat (either alone or accompanied by exudate production), and ungerminated seeds with internal necrosis. All treatments showed 100% disease incidence and an average severity of 89.9%.

Disease progression, measured as the area under the disease progress curve (AUDPC), had an average value of 26.7, indicating rapid and severe disease development (Table 2). In contrast, the control group showed healthy seed germination and seedling development. After the assay, fungi were re-isolated from infected plants, and their morphological characteristics matched those of the original inoculum, thus fulfilling Koch’s postulates.

Table 2

Isolated	Incidence (%)	Severity (%)	ABCDEx
F. oxysporum FM1	100	95.8a	30.2a
F. oxysporum FM2	100	89.6a	27.3a
F. oxysporum FL1	100	89.6a	27.4a
F. oxysporum FL5	100	81.3a	21.0a
F. oxysporum FL6	100	87.5a	23.7a
F. oxysporum FL2	100	95.8a	30.5a
Control	0	0b	0

^x

AUDPC = area under the disease progress curve. Values in each column followed by the same letter are not significantly different according to the Kruskal-Wallis and Conover-Iman tests (p ≤ 0.05).

In vitro antagonism of Bacillus vs. Fusarium. The analysis of Fusarium spore degradation by Bacillus spp. showed that the commercial strain PC exhibited the highest spore degradation percentages compared to the native Bacillus strains evaluated (Figure 1). In interaction with PC, no significant statistical differences were observed among the fungal isolates, with an average degradation rate of 86.7%.

In contrast, when interacting with strain Bc28-5, the fungus FM2 showed greater resistance to degradation, with an average of 41.4%, while the remaining fungi were degraded at an average rate of 84.2%. In the case of strain Bc28-2, FL6 showed the highest resistance to spore degradation at 18%, followed by FL2 and FL1 with 42.9%. The other fungal isolates were degraded at an average of 84.9%.

Bt24 was the least effective strain in degrading spores. It only succeeded in degrading two fungi (FL1 and FM1) at a rate of 92.85%, followed by FL6 and FL2 at 73.15%. The most resistant fungi were FL5 and FM2, with spore degradation percentages of 32.8% and 52.2%, respectively.

Figure 1

Growth promotion in chili seedlings. Bacterial inoculation significantly improved chili seedling growth under controlled conditions at 30 days (Table 3).

Table 3

Parameters	Control	Bacillus subtilis PC	B. cereus Bc28-2	B. cereus Bc28-5	B. thuringiensis Bt24	LSD
SH (cm)	8.2±0.5bc	6.6±0.7d	8.9±1.1ab	7.7±0.5cd	9.8±0.2a	1.1
RL (cm)	8.9±0.8a	8.9±1.5a	9.9±0.8a	9.5±1.4a	9.4±1.4a	2.0
SD (mm)	2.2±0.1c	2.0±0.3c	2.8±0.3ab	2.3±0.5bc	2.8±0.3a	0.5
NL	8.0±0.6b	10.0±2.8ab	12.0±1.3a	9.3±1.5ab	12.0±0.9a	2.7
LA (cm2 plant−1)	51.1±7.1c	48.9±2.1c	115.1±6.7a	76.3±8.5b	103.6±10.7a	20.5
LFW (g)	0.6±0.1c	0.6±0.3c	1.6±0.1a	1.1±0.1b	1.5±0.2a	0.3
SFW (g)	0.3±0.03c	0.3±0.1c	0.7±0.2a	0.5±0.1b	0.7±0.1a	0.2
RFW (g)	0.2±0.1c	0.2±0.09c	0.6±0.1a	0.4±0.1b	0.7±0.1a	0.2
LDW (g)	0.1±0.01c	0.1±0.03c	0.3±0.1a	0.2±0.03b	0.3±0.1a	0.1
SDW (g)	0.03±0.01b	0.02±0.01b	0.1±0.01a	0.03±0.01b	0.1±0.01ª	0.01
RDW (g)	0.02±0.01cd	0.02±0.003d	0.1±0.01b	0.03±0.004c	0.1±0.01a	0.01
Chl a (mg/g FW-1)	1.5±0.1b	1.7±0.1ab	2.1±0.4a	2.2±0.3a	1.9±0.2ab	0.6
Chl b (mg/g FW-1)	0.9±0.3a	0.9±0.1a	1.2±0.4a	1.2±0.2a	1.3±0.1a	0.5
Carotenoids (mg/g FW-1)	0.2±0.1a	0.2±0.03a	0.2±0.2a	0.2±0.1a	0.1±0.1a	0.2

SH= seedling height; RL = root length; SD = stem diameter; NL = number of leaves; LA = leaf area; LFW = leaf fresh weight; SFW = stem fresh weight; RFW = root fresh weight; LDW = leaf dry weight; SDW = stem dry weight; RDW = root dry weight; Chl a = chlorophyll a; Chl b = chlorophyll b; LSD = least significant difference. PC = B. subtilis QST713; Bc28-2 and Bc28-5 = B. cereus; Bt24 = B. thuringiensis. Data represent the means ± standard deviation of four replicates. Values in rows followed by different letters indicate significant differences (p ≤ 0.05) according to ANOVA and Tukey’s test.

Seedlings inoculated with Bt24 exhibited the greatest height, reaching an average of 9.8 cm, which represents a 19.5% increase compared to the control (8.2 cm). In contrast, the PC treatment resulted in the shortest height (6.6 cm), reflecting a 19.5% reduction compared to the control. Regarding root length, treatments showed no significant differences, with an average of 9.3 cm. Stem diameter was significantly greater in the Bc28-2 and Bt24 treatments (2.8 mm), representing a 27.3% increase compared to the control (2.2 mm).

The number of leaves was significantly higher in seedlings treated with Bt24 (12) and Bc28-2 (12), representing a 50% increase compared to the control (8 leaves). Similarly, leaf area was greater in seedlings inoculated with Bc28-2 and Bt24 (109.4 cm² plant⁻¹), representing a 114.0% increase compared to the control (51.1 cm² plant⁻¹).

Regarding fresh and dry biomass, leaf fresh weight was significantly higher in the Bt24 and Bc28-2 treatments (1.6 g), which corresponds to a 166.7% increase compared to the control (0.6 g). Stem fresh weight also increased with Bt24 and Bc28-2 (0.7 g), representing a 133.3% increase compared to the control (0.3 g). Root fresh weight was significantly higher as well in the Bt24 and Bc28-2 treatments (0.7 g), a 250% increase over the control (0.2 g).

Leaf dry weight showed a significant increase with Bt24 and Bc28-2 (0.3 g), representing a 200% increase over the control (0.1 g). Stem dry weight also increased significantly with Bt24 and Bc28-2 (0.1 g), reflecting a 233.3% increase compared to the control (0.03 g). For root dry weight, the Bt24 treatment (0.1 g) showed the highest increase, with a 400% gain compared to the control (0.02 g).

In terms of photosynthetic pigment content, chlorophyll a was significantly higher in the Bc28-5 and Bc28-2 treatments (2.2 mg/g FW⁻¹), a 46.7% increase over the control (1.5 mg/g FW⁻¹). In contrast, chlorophyll b content showed no significant differences among treatments, with an average of 1.1 mg/g FW⁻¹. Carotenoid content also showed no significant differences, with an average of 0.2 mg/g FW⁻¹.

Growth of chili plants in interaction with Fusarium. Bacterial inoculation significantly improved chili plant growth in interaction with the fungus (Table 4).

Table 4

Parameters	Control	FL2	PC/FL2	Bc28-2/FL2	Bc28-5/FL2	Bt24/FL2	LSD
SH (cm)	22.1±1.7b	14.4±1.9cd	26.4±1.0a	23.1±1.3ab	17.8±1.3c	13.0±2.3d	3.7
RL (cm)	19.4±1.7a	11.5±0.8b	18.4±1.4a	17.9±1.5a	18.1±1.4a	11.6±0.6b	2.9
SD (mm)	4.5±0.5bc	5.1±0.7abc	5.9±0.6ab	4.7±0.5abc	6.1±1.0a	3.7±0.7c	1.6
NL	35.5±3.3a	20.3±2.8b	35.0±5.7a	25.0±2.2b	37.3±3.3a	19.0±2.6b	7.9
LA (cm2 plant−1)	516.3±88.7ab	196.1±21.0c	564.0±77.0a	405.4±58.7b	583.8±79.5a	252.2±19.2c	143.2
LFW (g)	10.5±0.9a	3.0±1.0d	9.7±0.9ab	6.5±0.8c	7.5±1.7bc	3.6±0.4d	2.3
SFW (g)	6.9±1.2abc	3.2±1.0cd	10.0±2.8a	5.6±1.2bcd	9.4±1.5ab	3.0±0.9d	3.8
RFW (g)	4.0±0.5ab	1.6±0.4c	4.8±0.7a	3.1±0.7b	4.3±0.5ab	1.1±0.4c	1.2
LDW (g)	1.9±0.2a	0.6±0.3c	1.6±0.1ab	0.8±0.1c	1.4±0.2b	0.5±0.1c	0.4
SDW (g)	0.8±0.3abc	0.3±0.2c	1.0±0.3ab	0.5±0.1abc	1.0±0.3a	0.5±0.2bc	0.5
RDW (g)	0.5±0.1ab	0.2±0.03c	0.5±0.1ab	0.4±0.1b	0.5±0.04a	0.1±0.04c	0.1
RV (cm3)	5.4±0.6a	1.5±0.6c	4.8±0.7ab	3.9±0.5b	4.1±0.6ab	1.1±0.6c	1.4
Chl a (mg/g FW-1)	2.0±0.5a	1.8±0.4a	2.0±0.4a	1.8±0.4a	2.0±0.4a	1.8±0.3a	0.9
Chl b (mg/g FW-1)	0.9±0.3a	1.0±0.3a	1.2±0.3a	1.0±0.1a	1.2±0.3a	1.0±0.4a	0.7
Carotenoids (mg/g FW-1)	0.3±0.1a	0.1±0.1a	0.2±0.1a	0.2±0.1a	0.1±0.03a	0.3±0.1a	0.2

SH = seedling height; RL = root length; SD = stem diameter; NL = number of leaves; LA = leaf area; LFW = leaf fresh weight; SFW = stem fresh weight; RFW = root fresh weight; LDW = leaf dry weight; SDW = stem dry weight; RDW = root dry weight; RV = root volume; Chl a = chlorophyll a; Chl b = chlorophyll b; FL2 = F. oxysporum; PC = B. subtilis QST713; Bc28-2 and Bc28-5 = B. cereus; Bt24 = B. thuringiensis; LSD = least significant difference. Data represent the means ± standard deviation of four replicates. Values in rows followed by different letters indicate significant differences according to ANOVA and Tukey’s test (P < 0.05).

Plants inoculated with PC/FL2 showed the greatest height, averaging 26.4 cm, which represents a 20% increase compared to the control (22.1 cm). In contrast, the treatments with FL2 alone and the Bt24/FL2 interaction recorded the lowest heights (14.4 cm and 13.0 cm, respectively), reflecting a reduction of 34.8% and 41.2%, respectively, compared to the control.

Regarding root length, most bacteria-pathogen interaction treatments maintained a length similar to the control (18.6 cm), except for Bt24/FL2 and FL2 alone, which showed a 40.5% reduction (11.6 cm) compared to the control. Stem diameter was significantly greater in the Bc28-5/FL2 treatment (6.1 mm), representing a 35.6% increase compared to the control (4.5 mm). On the other hand, the Bt24/FL2 interaction resulted in the smallest stem diameter (3.7 mm), a 17.8% decrease relative to the control.

The number of leaves did not differ significantly in the PC/FL2 and Bc28-5/FL2 treatments compared to the control, with an average of 35.9 leaves. However, the Bc28- 2/FL2 and Bt24/FL2 interactions, as well as the FL2 pathogen alone, showed a significant reduction, averaging 21.4 leaves, which represents a 39.6% decrease compared to the control (35.5 leaves).

Leaf area was significantly greater in the Bc28-5/FL2 and PC/FL2 treatments (573.9 cm³). In contrast, the Bt24/FL2 and FL2 treatments showed the lowest leaf area (224.2 cm³), reflecting a 43.4% reduction compared to the control (516.3 cm³).

Regarding fresh biomass, only the PC/FL2 treatment showed a leaf fresh weight similar to the control (9.7 g). The Bt24/FL2 interaction and FL2 treatment alone showed the lowest leaf fresh weight (3.3 g), corresponding to a 68.6% decrease compared to the control (10.5 g). For stem and root fresh weight, only the PC/FL2 and Bc28-5/FL2 treatments maintained values comparable to the control (9.7 g and 4.55 g, respectively), while Bt24/FL2 and FL2 recorded the lowest values: 3.1 g for stem and 1.35 g for root, representing reductions of 44.9% and 66.3%, respectively.

As for dry biomass, leaf dry weight did not show significant increases in any treatment, except for PC/FL2, which matched the control (1.6 g). The lowest leaf dry weight was observed in Bc28-5/FL2, Bt24/FL2, and FL2 (0.6 g), a 66.7% decrease relative to the control (1.9 g). For stem dry weight, most treatments showed no significant differences (0.8 g), except for Bt24/FL2 and FL2, which resulted in a 50% reduction. In the case of root dry weight, only PC/FL2 and Bc28-5/FL2 maintained values similar to the control (0.5 g), while Bt24/FL2 and FL2 showed the lowest values (0.15 g), representing a 70% reduction.

Root volume did not show significant increases, remaining comparable to the control in the PC/FL2 and Bc28-5/FL2 treatments (4.5 cm³). The lowest root volume was observed in the Bt24/FL2 and FL2 treatments (1.3 cm³), representing a 75.9% reduction compared to the control (5.4 cm³). Regarding photosynthetic pigment content, no significant differences were observed among treatments for chlorophyll a (2.28 mg/g FW⁻¹), chlorophyll b (1.26 mg/g FW⁻¹), or carotenoids (0.24 mg/g FW⁻¹).

The severity index results showed significant differences among treatments (Figure 2a). Plants treated with FL2 alone exhibited a severity index (SI) of 87.5%, significantly higher than all other treatments. In contrast, the bacteria-pathogen interaction treatments PC/FL2, Bc28-2/FL2, and Bc28-5/FL2 reduced the SI by 60.4% compared to the pathogen (FL2) alone. Conversely, the Bt24/FL2 treatment did not show a significant reduction in SI, with a value of 81.3%, which was statistically equivalent to the pathogen treatment.

Plant development in interaction with the pathogen also varied depending on the treatment applied (Figure 2b). Plants that did not receive microbial treatment developed healthily and vigorously. In contrast, plants treated with the pathogen alone exhibited severe disease symptoms, particularly in the root system, resulting in stunted growth. Plants treated with the bacteria-pathogen combinations PC/FL2, Bc28-2/FL2, and Bc28- 5/FL2 showed development similar to the control treatment, characterized by healthy aerial growth and reduced disease symptoms in the root system. In contrast, plants treated with Bt24/FL2 displayed severe symptoms comparable to those observed in plants treated with the pathogen alone.

Figure 2

Principal component analysis allowed visualization of the variability in the developmental response of jalapeño chili plants to the different Fusarium biocontrol treatments (Figure 3). The first principal component (Dim 1) explained 75.4% of the variability, while the second component (Dim 2) accounted for 11.3%, together capturing 86.7% of the total variability.

Figure 3

The pathogen and the Bt24/FL2 treatment exhibited similar behavior patterns, showing a negative relationship with most vegetative parameters and a positive relationship with the severity index (SI), indicating that Bt24 did not contribute to disease control. In contrast, a strong positive interrelationship was observed between the bacterial treatments PC/FL2, Bc28-2/FL2, and Bc28-5/FL2, as well as the control, across vegetative parameters, along with a negative relationship with the SI. This indicates effective disease control and plant growth promotion by the bacterial treatments.

The results of this study show that inoculation with native strains of Bacillus cereus (Bc28-5 and Bc28-2) and B. thuringiensis (Bt24) in jalapeño chili plants under controlled conditions has a significant impact on vegetative growth and on reducing the severity of wilt caused by Fusarium oxysporum. Additionally, some Bacillus strains demonstrated the ability to effectively degrade Fusarium spores in in vitro assays. However, the effectiveness varied depending on the bacterial and fungal strain involved.

The pathogenicity and virulence tests confirmed that all Fusarium strains were pathogenic, causing 100% disease incidence and an average severity of 89.9%. This confirms the virulence of the evaluated strains on jalapeño chili seedlings under in vitro conditions. Observed symptoms included necrosis in the radicle and hypocotyl, which is consistent with previous studies on the ability of Fusarium oxysporum to affect the root system of crops such as jalapeño chili (Robles-Hernández et al., 2015).

The antagonistic capacity of Bacillus strains in degrading Fusarium spores varied significantly. The commercial strain PC showed the highest spore degradation percentage (86.7%) compared to the native strains. Antagonistic activity was also evident with Bc28- 5, whereas Bt24 demonstrated lower effectiveness, particularly against the more resistant Fusarium strains (FL5 and FM2).

The degradation mechanism may be associated with the production of antifungal lipopeptides, as previous studies have shown that B. cereus and B. thuringiensis can control F. oxysporum f. sp. lycopersici through the production of antimicrobial peptides (Sarangi et al., 2017). Ajesh et al. (2013) reported that soil-derived B. cereus produces the lipopeptide kurstakin, which has antifungal activity. In addition, Bacillus spp. can produce lytic enzymes such as chitinases, glucanases, and proteases that hydrolyze fungal cell walls (Chenniappan et al., 2019; Ku et al., 2018). In the case of B. thuringiensis, recent studies have reported its ability to control phytopathogenic fungi through the production of bacteriocins, lactonases, chitinases, antibiotics, and hydrogen cyanide (Gupta et al., 2024; Azizoglu et al., 2023; Yoshida et al., 2019).

In terms of growth promotion, Bt24 showed the greatest increase in seedling height and biomass. Strains Bc28-2 and Bc28-5 also significantly promoted growth by increasing leaf area and both fresh and dry plant biomass. This effect may be linked to plant growth-promoting mechanisms previously reported for B. cereus. Kumar et al. (2020) noted that B. cereus can produce indole-3-acetic acid (IAA) and gibberellins as mechanisms for promoting plant growth. Similarly, Ku et al. (2018) reported that this bacterium solubilizes both organic and inorganic phosphorus, which supports plant development. Yanti and Nasution (2017) indicated that this group of bacteria rapidly colonizes the plant rhizosphere due to exopolysaccharide production, and B. cereus has been shown to form biofilms that facilitate root colonization (Gao et al., 2019; Wijman et al., 2007).

On the other hand, the growth-promoting effect of B. thuringiensis may be associated with its ability to produce IAA and 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which act as plant growth regulators (Cherif-Silini et al., 2016). It has also been reported to produce siderophores that enhance nutrient uptake by the plant (Delfim, 2021), and to solubilize soil phosphorus through the production of organic acids, pH modification, and the secretion of acid and alkaline phosphatases (Cherif-Silini et al., 2016; Delfim, 2020).

In addition, the biocontrol of Fusarium in jalapeño chili plants under greenhouse conditions showed that treatments with B. cereus (Bc28-2 and Bc28-5) significantly reduced disease severity caused by Fusarium FL2, compared to the treatment with the pathogen alone. These results may be attributed to the ability of B. cereus to reduce fungal disease severity through the production of antimicrobial compounds, as mentioned earlier, and by inducing systemic resistance (ISR) in plants (Boulahouat, 2023).

Several studies have shown that B. cereus induces ISR against fungi such as Botrytis cinerea in tomato through the production of oxalic acid (Yu et al., 2022; Nie et al., 2017). Likewise, B. cereus MH778713 has been reported to help control F. oxysporum in tomato via ISR (Nie et al., 2017), and B. cereus EC9 has been identified as an inducer of resistance against F. oxysporum in kalanchoe plants (Madriz-Ordeñana et al., 2022). Furthermore, B. cereus C1L has been reported to induce ISR through the production of volatile compounds such as dimethyl disulfide (Piechulla et al., 2017).

The growth observed during the interaction between Fusarium spp. FL2 and B. cereus may be explained by findings from Leibman-Markus et al. (2023), who suggest that growth promotion and induced resistance can be codependent processes. In some cases, inoculation with bacteria such as Bacillus can enhance developmental processes and improve plant performance in biocontrol interactions (Gupta et al., 2022).

However, the Bt24/FL2 treatment showed an unexpected pattern, with results similar to the pathogen-only treatment in terms of both disease severity and growth parameters. This may be due to the limited antagonistic capacity of Bt24 against certain Fusarium strains, highlighting the importance of evaluating the specificity of bacteria-pathogen interactions.

The results suggest that the use of specific Bacillus strains can contribute to the integrated management of Fusarium in agricultural systems by enhancing plant growth while reducing disease severity. This opens the possibility of developing targeted formulations, particularly with strains such as Bc28-2 and Bc28-5, for application under both greenhouse and field conditions.

This study demonstrated that all Fusarium oxysporum strains isolated from soil are pathogenic to jalapeño chili seedlings, causing 100% disease incidence and an average severity of 89.9%. Disease progression showed a rapid increase under controlled conditions.

The in vitro biocontrol assay revealed that B. subtilis (PC) achieved the highest spore degradation of F. oxysporum (86.7%), followed by B. cereus (84.2%), while B. thuringiensis was less effective. This suggests that antagonistic effects depend on the specific antagonist and pathogen strains evaluated.

Greenhouse biocontrol with Bacillus spp. showed variability in effectiveness depending on the strain used. B. cereus Bc28-2 and Bc28-5 reduced disease severity by 60.4% and promoted chili plant growth. In contrast, Bt24 did not reduce disease severity, showing results similar to the pathogen-only treatment. However, both Bt24 and Bc28-2 promoted vegetative growth in chili seedlings.

These findings highlight the potential of Bacillus cereus as a bioinoculant for jalapeño chili. Nonetheless, further validation under field conditions is necessary, as well as exploration of its integration into sustainable agricultural management strategies.