Tomato (Solanum lycopersicum) is cultivated across all states of the Mexican Republic on approximately 49 000 ha, with an average yield exceeding 70 t ha⁻¹. Mexico ranks first among tomato-exporting countries, holding a market share of more than 19% in the global market (Montaño et al., 2021). Of the total area under tomato cultivation, Chiapas contributed 3.3% (1 638 ha) and 2.4% (82 729 t) of national production, ranking eighth and fourteenth in these categories, respectively (SIAP, 2023). Conventional management of tomato pests and diseases relies on synthetic chemical formulations (McGovern, 2015).

Bacteria such as Bacillus subtilis and fungi such as Trichoderma spp. are considered biological control agents of plant diseases with broad potential in modern agriculture (Prabhakaran et al., 2015). Both can be applied as live or dormant cells to soil, foliage, or seeds (Minchev et al., 2021). Trichoderma spp. is ubiquitous in the environment, easy to isolate and culture, capable of growing on diverse substrates, and able to control a wide range of phytopathogenic microorganisms. In tomato plants, Trichoderma spp. has been reported to reduce the incidence of Fusarium wilt caused by Fusarium oxysporum f. sp. lycopersici by 85-90% (Alshammari et al., 2022). Similarly, Bacillus sp. reduces wilting caused by Fusarium oxysporum f. sp. lycopersici race 2 by 75% (Qi et al., 2016). In light of the above, the objective of the present study was to determine the level of Fusarium wilt (Fusarium oxysporum) in tomato (Solanum lycopersicum) using Trichoderma asperellum Jc01 and Bacillus subtilis ANT01, two microorganisms with biocontrol potential.

Study site. The study was conducted at the "Ayol" Agroecological Station, Municipality of Tapachula, Chiapas, Mexico (14°49'46" N; 92°17'42" W; mean annual rainfall 2,600 mm; relative humidity 80%; mean annual temperature 26 °C).

Culturing of T. asperellum Jc01 and B. subtilis ANT01. The strains were obtained from the culture collection of the Institute of Biosciences of the Autonomous University of Chiapas. T. asperellum Jc01 was grown to sporulation on Potato Dextrose Agar (PDA) at pH 5.6 ± 0.2 and 25-28 °C in Petri dishes. For mass spore production, a disk of actively growing mycelium (1 cm diameter) was suspended in 10 mL of isotonic solution, and 150 µL of the suspension was spread onto PDA in 500 mL Roux bottles. The microorganism was grown under the conditions described above until sporulation. Spore harvesting was performed using 10 mL of isotonic solution containing 0.5% Tween 80. Spore concentration was determined by direct counting with a Neubauer hemocytometer.

B. subtilis ANT01 was cultured in Potato Dextrose Broth (PDB). A single colony with the characteristic morphology of the strain was transferred with a bacteriological loop into 5 mL of PDB in a 16 × 100 mm test tube and incubated with rotary shaking (150 rpm) at 25 °C for 24 h. The culture was then transferred to 100 mL of PDB and incubated under the same conditions. Bacterial concentration was determined by serial dilution using the Most Probable Number (MPN) method. Following incubation under the conditions described above, microbial growth was assessed spectrophotometrically at 560 nm against uninoculated PDB.

Plant material. S. lycopersicum seedlings were raised in a compost-vermicompost mixture (4:1) in 70-cell trays. Seeds were surface-sterilized with sodium hypochlorite (1.5%) for 10 min, rinsed three times with sterile water, and allowed to dry on absorbent paper. Three seeds per cell were sown at a depth of 1 cm. Germinated seedlings were selected and maintained for 30 days under constant moisture through regular irrigation until reaching a height of 10-12 cm.

Field crop establishment. For open-field cultivation, four planting beds were prepared, each 12 m long, 45 cm wide, and 30 cm high, with 200 cm between beds. Healthy seedlings (30 per treatment) with well-developed green leaves, an erect habit, and a well-developed white root system were transplanted at 30 cm between plants.

Treatment design. A complete 2² factorial treatment design was used (Table 1), with

B. subtilis ANT01 (1×10⁶ CFU g⁻¹ soil) and T. asperellum Jc01 (1×10⁴ spores g⁻¹ soil) as factors, each at two levels: absence (−1) and presence (+1). Treatments were applied weekly within a 30 cm radius around each plant to a depth of 30 cm, from the day of transplanting through fruit harvest (15 applications in total).

Crop management. Soil fertilization was carried out with 2 L of biol every 7 days throughout the crop cycle, and 1 kg of compost plus 2 kg of vermicompost every 56 days. Biofertilizers were applied at the drip line of each plant. Weed control was performed manually, and pest control was achieved by spraying a 10% aqueous extract of neem (Azadirachta indica) leaves every 7 days. Irrigation to field capacity was applied as needed, and stakes were placed to support plant growth.

Response variables. Plants in all treatments were inspected weekly for the characteristic visual symptoms of Fusarium wilt (yellowing and wilting of lower leaves, vascular discoloration of the stem) resulting from natural infection. Disease incidence (I) was calculated for each treatment using the following formula: I = (number of plants showing Fusarium wilt symptoms) × (total plants)⁻¹. In addition, the total number of flowers and fruits produced per plant was recorded for each treatment.

Data analysis. Incidence, flower, and fruit production data were subjected to analysis of the effects of B. subtilis ANT01 and/or T. asperellum Jc01, and the statistical significance of treatment effects on Fusarium wilt incidence was determined in accordance with the procedures established for a complete factorial design.

Six weeks after transplanting (WAT) into the field, the first plants displaying characteristic symptoms of Fusarium wilt were observed. In plants treated with B. subtilis ANT01, the first symptomatic plants were not observed until 9 WAT, whereas in plants treated with T. asperellum Jc01 or with both microorganisms, they were recorded at 6 and 7 WAT, respectively. As the crop cycle progressed, the number of symptomatic plants continued to increase; plants in the untreated control reached the highest incidence (0.50), while plants in the B. subtilis ANT01, T. asperellum Jc01, and dual-inoculation treatments showed 60, 16, and 28% lower incidence at 15 WAT, respectively, compared to the control (Figure 1).

Figure 1

Analysis of the effects of B. subtilis ANT01 and T. asperellum Jc01 on Fusarium wilt incidence showed that B. subtilis ANT01 was the only factor with negative values, whereas the values for T. asperellum Jc01 and the B. subtilis ANT01 × T. asperellum Jc01 interaction were positive (Table 1).

The B. subtilis ANT01 treatment yielded an average of 70 flowers plant⁻¹, while the

B. subtilis ANT01 + T. asperellum Jc01, T. asperellum Jc01, and control treatments produced 42.1, 30.7, and 30.0 flowers plant⁻¹, respectively. Fruit production in the B. subtilis ANT01 treatment was 47.9 fruits plant⁻¹, whereas the control, T. asperellum Jc01, and B. subtilis ANT01 × T. asperellum Jc01 treatments produced 7.6, 18.0, and 27.3 fruits plant⁻¹, respectively.

Table 1

Effect	Incidence	Flowers plant-1	Fruits plant-1
Total	0.266	40.8	25.2
B. subtilis ANT01	-0.064	10.5	12.4
T. asperellum Jc01	0.025	-9.2	-2.6
B. subtilis ANT01+T. asperellum Jc01	0.051	-9.5	-7.7

Analysis of the effects of B. subtilis ANT01 and T. asperellum Jc01 and their interaction on flower and fruit production in tomato plants showed that B. subtilis ANT01 was the only factor that improved production of both variables (Table 1). The Fusarium wilt incidence observed in the present study (Figure 1) compared favorably to that reported for squash (Cucurbita maxima) using B. subtilis SQR 9 as a biocontrol agent, which achieved only a 55% reduction (Cao et al., 2011). The biocontrol activity of B. subtilis ANT01 against Fusarium wilt may result from efficient colonization of tomato roots, as suggested by Cao et al. (2011) for B. subtilis strain SQR 9 and by Zhao et al. (2013) for B. subtilis strain Y-IVI; from induction of systemic resistance in the plant, as reported for B. subtilis strains CBMT51, B2, and IAGS174 (Mejía-Bautista et al., 2016; Corrales et al., 2012; Akram et al., 2014); from the production of antibiotic compounds by B. subtilis strain B2 (Corrales et al., 2012); or from nutrient competition or production of lytic enzymes by

B. subtilis strain TV-125 (Wang et al., 2018; Senol et al., 2014).

On the other hand, the failure of T. asperellum Jc01 to control Fusarium wilt (Figure 1) merely indicates that this strain likely does not produce metabolites or enzymes capable of inhibiting F. oxysporum growth, and exhibits neither competitive ability nor capacity to induce systemic resistance in the plant. Nevertheless, T. asperellum Jc01 appears to exert a negative effect on the activity of B. subtilis ANT01; although the interaction reduced Fusarium wilt incidence, the reduction was not statistically significant (Table 1). Finally, the fruit yield (fruits plant⁻¹) obtained in this study falls within the range reported for this crop, which varies depending on cultivar, planting density, irrigation, and whether production takes place under greenhouse or open-field conditions, among other factors (Márquez-Hernández et al., 2013). Further studies are needed to characterize the fungus associated with Fusarium wilt through morphological and molecular identification, and to determine whether it exhibits resistance to the biocontrol organisms evaluated in this study.

Bacillus subtilis ANT01 reduced Fusarium wilt incidence associated with Fusarium oxysporum in tomato (Solanum lycopersicum) plants grown under field conditions by 60%, while Trichoderma asperellum Jc01 and the combination of both microorganisms reduced incidence by 16 and 28%, respectively. The lower Fusarium wilt incidence and severity observed in tomato plants treated with Bacillus subtilis ANT01 translated into greater flower and fruit production per plant, with 70 flowers and 48 fruits recorded, respectively.

Limitations. The present report demonstrates the potential of strain ANT01 as a biocontrol agent against the pathogen; however, further field and laboratory trials are needed to corroborate its efficacy. Additionally, the molecular identification of the antagonistic strains has not been published and has only been reported at the thesis level. Furthermore, the molecular taxonomic identification of the pathogen is still lacking, as it has been characterized solely on the basis of morphology and plant symptomatology. Finally, although all plants in the field experiment were assumed to be exposed to the same inoculum density in the soil and uniform sowing conditions, these variables should be more rigorously controlled in future studies.

Conflict of interest. The authors declare no conflict of interest.

Funding. Not applicable.

Acknowledgements. The authors thank the support staff of the Ayol Agroecological Station for all the facilities provided for the conduct of this research.

Author contributions. All authors contributed actively to the development of the present work.

Supplementary material. Not applicable.