Tomato (Solanum lycopersicum) is one of the most important crops in Mexico, and its production is affected by Meloidogyne enterolobii. Traditional management in Mexico relies mainly on synthetic pesticides. However, efforts to reduce their use have led to the evaluation of crop rotation and biological control strategies (Lamberti, 1997; Tapia-Vázquez et al., 2022). For example, arbuscular mycorrhizal fungi such as Acaulospora longulata (Campos et al., 2013; de Sá and Campos, 2020) and filamentous fungi such as Pochonia chlamydosporia (Silva et al., 2017), Paraboeremia taiwanensis (Liang et al., 2020), Trichoderma harzianum (Jindapunnapat et al., 2013), and Purpureocillium lilacinum stand out for their nematicidal capacity. Likewise, bacteria from the genera Pseudomonas, Paenibacillus (Sohrabi et al., 2018), and Bacillus spp. (Basyony and Abo-Zaid, 2018; Díaz-Manzano et al., 2023; Vieira de Carvalho Júnior et al., 2022) have shown potential.

In Mexico, several commercial microbial-based products exist (Tapia-Vázquez et al., 2022); however, farmers show resistance to their use due to the need for frequent applications and slower action compared to synthetic nematicides. Therefore, management strategies that increase the efficacy of bioproducts are required. The use of microbial combinations may represent a valuable alternative. For instance, Nesha and Siddiqui (2017) evaluated Paecilomyces lilacinus and Aspergillus niger individually and combined in carrot (Daucus carota), showing significant reductions in gall formation by M. javanica. Additionally, Bacillus species are known plant growth promoters and have been reported to contribute to pest control. Suppression of Meloidogyne populations by B. subtilis has been documented in chili (Capsicum annuum) (Mokbel et al., 2022; Soto Deza et al., 2012) and tomato (Basyony and Abo-Zaid, 2018). Strains BMH e INV, related to B. velezensis, reduced more than 90% of galls and eggs of M. incognita in tomato (Cruz-Magalhães et al., 2022). Likewise, B. velezensis Bv-25 reduced galling by 73.8% in cucumber roots (Cucumis sativus) through root colonization and volatile compound production (Tian et al., 2022). Moreover, B. velezensis VB7 inhibited egg viability and caused mortality of M. incognita juveniles in vitro and induced innate immune responses in tomato (Kamalanathan et al., 2023).

The strain B. velezensis 83 is commercialized in Mexico as a foliar biofungicide (Fungifree AB®), used for biological control of phytopathogenic fungi in various crops (Galindo et al., 2013). Additionally, Balderas-Ruíz et al. (2021) demonstrated its capacity to promote tomato growth and control Botrytis cinerea, improving yield and quality.

This study aimed to evaluate individually and in combination B. velezensis 83 and the environmental strain P. lilacinum MTL01 against M. enterolobii infection in tomato, as well as their efficiency in increasing yield.

Nematode isolation. A Meloidogyne enterolobii population was established in the laboratory for biological control studies. M. enterolobii nematodes (GenBank OM721664) were obtained from infected tomato roots intended for nematode propagation, originating from a chard crop (Beta vulgaris var. cicla) and collections from Veracruz (Salinas-Castro et al., 2022). Egg masses were extracted from infected roots, and second-stage juveniles (J2) were recovered by a modification of the Baermann funnel method (Tintori et al., 2022).

These juveniles were used to establish laboratory populations on tomato cv. Calafia F1 (Harris Moran), due to its susceptibility to this nematode.

Fungal isolation and identification. During independent propagation experiments of Meloidogyne paranaensis in a tomato microculture system, the survival of one of five seedlings inoculated with egg masses was observed. In the surviving plant, J2s failed to hatch because of a fungal infection. An axenic fungal culture was obtained on potato dextrose agar (PDA) supplemented with ampicillin (100 µg mL⁻¹) and kanamycin (50 µg mL⁻¹). The isolate was characterized morphologically and identified molecularly by amplification and sequencing of intergenic ITS region of ribosomal DNA gene with the primers ITS1 (5′-TCCGTAGGTGAACCTGCG-3′) and ITS4 (5′-

TCCTCCGCTTATTGATATG-3′) (White et al., 1990), followed by phylogenetic comparison with related strains of the same genus and species. Phylogenetic reconstruction was performed using the TN93 model (Tamura and Nei, 1993), with branch support values calculated using the aLRT (SH-like) algorithm and visualized with the SeaView software.

Interaction between M. enterolobii and P. lilacinum MTL01. To evaluate the interaction and possible parasitism of P. lilacinum MTL01 on M. enterolobii, in vitro assays and observations of organisms recovered from infected tomato plants were carried out. First, a confrontation assay was performed on PDA medium supplemented with kanamycin (50 µg mL⁻¹) and ampicillin (100 µg mL⁻¹), in which M. enterolobii egg masses were placed alongside a P. lilacinum colony, approximately 5 cm apart. After 5 days, egg parasitism was observed. In addition, at the end of the greenhouse experiments, females from galls collected from tomato roots treated with P. lilacinum were recovered in water, and fungal mycelium emerged after 48 h. Egg masses obtained from the same treatment were disinfected with 3% NaOCl and washed four times with sterile water. The recovered eggs were incubated on antibiotic-supplemented PDA to evaluate fungal colonization.

Evaluation of the combination of P. lilacinum MTL01 and B. velezensis 83 as control agents against M. enterolobii in tomato plants. We aimed to determine whether there was a synergistic effect on M. enterolobii control when P. lilacinum MTL01 and B. velezensis 83 (commercial product Fungifree AB®, LMG S-30921) are inoculated together in tomato plants, compared with the effects of each microorganism applied separately. The experimental design was intended to favor early colonization of the MTL01 fungus in tomato seedlings during germination, and to introduce B. velezensis 83 treatments two weeks after germination, followed by two additional applications every 20 days. For the biocontrol assays, tomato seeds were surface-disinfected with 10% NaOCl and 70% ethanol, followed by washes with sterile distilled water. Then seeds were incubated for 24 h in a suspension of 1x10⁶ spores mL⁻¹ of MTL01 in 10% sterile glycerol before sowing. After germination and transplanting, B. velezensis 83 was applied to the substrate as a suspension containing 1x10⁸ CFU per pot, with two additional applications every 20 days. Three weeks after transplanting, plants were inoculated with a combination of J2s and eggs of M. enterolobii to apply between 1,200 and 3,400 nematodes per plant, depending on each experiment.

Experiments were repeated three times over time in a greenhouse under a completely randomized design, at different seasons of the year: Experiment 1, July to October 2022; Experiment 2, February to June 2023; and Experiment 3, September 2024 to January 2025. In each experiment, four treatments were evaluated (with and without M. enterolobii): 1) no microorganisms; 2) P. lilacinum MTL01 only; 3) B. velezensis 83 only; and 4) the combination of P. lilacinum MTL01 + B. velezensis 83. Six plants were included per treatment. Plants were monitored for eight weeks after nematode inoculation, and the following variables were quantified: total length, aboveground biomass (fresh and dry weight), number of flowers, root fresh weight, number of galls, and galling index (Taylor and Sasser, 1978). Data were analysed by one-way ANOVA, and means were compared using Tukey’s test (p ≤ 0.05) in GraphPad Prism.

Characterization of P. lilacinum MTL01 and interaction with M. enterolobii. Based on morphological and molecular characteristics of the fungal isolate obtained from M. paranaensis eggs (Figure 1A), Purpureocillium lilacinum identification was confirmed, and the isolate was designated as strain MTL01, with 100% identity and 100% coverage for the ITS region (GenBank OR470716). On PDA medium, the colony was initially white-beige, with a spongy-cottony texture, and gradually became lilac to violet as sporulation progressed (Figure 1C and 1D). Microscopic observation showed branched conidiophores, elongated phialides, and oval conidia (Figure 1B) (Nguyen Thi H et al., 2025).

Figure 1

Meloidogyne enterolobii successfully established and completed its life cycle in the roots of Calafia F1 tomato, causing the formation of characteristic galls associated with root-knot nematode infection (Figure 2A), which led to overall plant decline. These structures contained adult females and egg masses associated with the nematode reproductive cycle (Tapia-Vázquez et al., 2022). A close-up of a root knot showed an egg mass protruding from the tissue (Figure 2B), while observations under a stereomicroscope revealed adult females with their characteristic pear-shaped morphology (Tapia-Vázquez et al., 2022), accompanied by egg masses (Figure 2C). Recently hatched J2s were motile and survived in water for up to two weeks (Figure 2D).

Figure 2

In the in vitro confrontation assays, M. enterolobii eggs placed in the presence of P. lilacinum MTL01 were progressively colonized by the fungus, preventing juvenile hatching (Figures 1E and 1F). Meanwhile, females recovered from galls from roots treated with MTL01 showed visible mycelial growth after incubation in sterile distilled water (Figures 1G and 1H). Likewise, eggs obtained from those galls produced P. lilacinum colonies when incubated on PDA supplemented with kanamycin and ampicillin, confirming the nematophagous potential of this strain.

In the three greenhouse experiments, variable responses were observed in vegetative growth parameters (Tables 1, 2, and 3), possibly due to the weather conditions of the season of the year. In Experiment 1, the combined treatment of P. lilacinum MTL01 and B. velezensis 83 increased shoot length in the absence of nematodes; however, this effect was not observed in plants inoculated with M. enterolobii. Differences in root weight were not significant; nevertheless, nematode-infected roots were heavier, possibly due to gall formation (Table 1). In Experiment 2, no consistent effects on plant growth associated with any treatment were detected (Table 2). In Experiment 3, individual inoculation with P. lilacinum MTL01 reduced root fresh weight (27 g) in nematode-infested plants, while the other parameters remained comparable among treatments (Table 3). This suggests that the growth-promoting responses induced by Purpureocillium lilacinum MTL01 and Bacillus velezensis 83 were influenced by the specific greenhouse conditions, as has been reported for tomato plants (Jeong et al., 2026).

Table 1

Treatment	Height (cm)		Shoot fresh weight (g)		Shoot dry weight (g)		Root fresh weight (g)
	Control	+ M. enterolobii	Control	+ M. enterolobii	Control	+ M. enterolobii	Control	+ M. enterolobii
Control	122.3±12.2a	118.3±12.7a	38.7±5.0a	27.9±7.8a	5.0±0.6ab	3.7±1.0b	17.6±5.4abc	22.8±8.4ab
MTL01	116.3±9.9a	104.8±8.5a	32.3±6.1a	30.6±7.8a	4.34±0.7ab	4.0±0.9b	12.5±2.2c	24.5±6.8a
B.v. 83	109.8±8.4a	116.8±8.0a	27.4±8.1a	36.3±5.8a	4.0±1.2b	5.3±0.8ab	15.6±4.7abc	19.7±3.7abc
MTL01 + B.v. 83	121.7±12.0a	108.5±3.9a	39.1±12.3a	39.0±5.8a	6.1±1.6a	5.4±0.8ab	14.2±2.2bc	23.0±3.6ab

*Different letters indicate statistically significant differences (one-way ANOVA, Tukey’s test; 𝑝 < 0.05).

Table 2

Treatment	Height (cm)		Shoot fresh weight (g)		Shoot dry weight (g)		Root fresh weight (g)
	Control	+ M. enterolobii	Control	+ M. enterolobii	Control	+ M. enterolobii	Control	+ M. enterolobii
Control	104.9±7.5ab	106.7±4.4ab	36.6±2.4a	36.6±2.4a	7.2±1.0a	5.9±1.3abc	18.5±1.1ab	18.6±4.6ab
MTL01	108.8±4.4a	107.0±8.0a	31.1±3.0ab	31.1±3.0ab	5.0±0.9bc	5.4±0.4abc	16.2±4.2bc	13.6±1.2bc
B.v. 83	108.4±3.8a	95.2±6.2b	32.9±4.0ab	32.9±4.0ab	6.0±1.5ab	5.3±0.4bc	22.8±4.6a	13.6±1.5bc
MTL01 + B.v. 83	104.5±7.9ab	104.1±6.0ab	27.4±3.9b	27.4±3.9b	4.3±0.9bc	4.2±0.7c	16.2±2.3bc	11.6±2.1c

* Different letters indicate statistically significant differences (one-way ANOVA, Tukey’s test; 𝑝 < 0.05).

Table 3

Treatment	Height (cm)		Shoot fresh weight (g)		Shoot dry weight (g)		Root fresh weight (g)
	Control	+ M. enterolobii	Control	+ M. enterolobii	Control	+ M. enterolobii	Control	+ M. enterolobii
Control	120.4±18.9ab	121.3±10.1ab	45.5±11.1a	48.1±3.8a	8.2±2.3a	8.4±0.8a	31.6±17.3ab	35.8±3.3a
MTL01	126.7±6.8a	94.7±14.4b	39.0±4.9ab	29.8±7.7b	6.9±0.9a	5.5±1.9a	11.9±0.5d	27.0±3.9abc
B.v. 83	107.8±13.9ab	104.1±12.7ab	40.9±7.8ab	41.6±4.0ab	8.2±1.8a	7.9±0.9a	20.4±7.4bcd	32.43±4.1ab
MTL01 + B.v. 83	115.5±18.9ab	106.3±21.9ab	40.3±10.1ab	37.0±9.7ab	7.6±2.0a	6.4±1.9a	16.4±2.8cd	30.1±7.2ab

*Different letters indicate statistically significant differences (one-way ANOVA, Tukey’s test; 𝑝 < 0.05).

In contrast to the variability observed in vegetative growth parameters, some effects showed more reproducible trends across experiments. In particular, two key parameters stand out: a) number of flowers and b) number of galls. Although the flowering response was also variable among experiments, combined inoculation with P. lilacinum MTL01 and B. velezensis 83 showed a consistent tendency toward higher flowering levels in all cases (Figures 3A, 3C, and 3E). In Experiment 1, the combination of both microorganisms resulted in an increase of 1.3-fold more flowers than the uninoculated control plants without nematodes, and 2.3-fold more flowers in plants infected with M. enterolobii (Figure 3A).

Figure 3

In Experiment 2, differences among treatments were less pronounced in the absence of nematodes (1.3-fold increase); however, the combination of both microorganisms produced almost twice as many flowers (1.94-fold increase) as the control in the presence of M. enterolobii (Figure 3C). Experiment 3 showed the largest differences under the combined microbial treatment, with a 4.3-fold increase in flower number in the absence of nematodes and a 1.6-fold increase in plants with nematodes (Figure 3E).

Galling index decreased significantly in each experiment. In Experiment 1, P. lilacinum MTL01 and B. velezensis 83, individually, reduced gall formation by approximately 60%, while the combined treatment reduced it by 66%, based on one gram of peripheral roots (Figure 3B). In Experiment 2, reductions were approximately 71% (MTL01), 43% (B. velezensis 83), and 78% for the combination (Figure 3D). Finally, in Experiment 3, galling was reduced by approximately 56% (MTL01), 67% (B. velezensis 83), and 81% (combination), confirming the reproducibility of the effect across assays (Figure 3F and Table 4). These results emphasize that both P. lilacinum MTL01 and B. velezensis 83 contribute to improved biocontrol of M. enterolobii, with their combined application being the most effective strategy, in agreement with other reports (Díaz-Manzano et al., 2023; de Paula et al., 2024; Huang et al., 2016).

Table 4

Treatment	Experiment 1		Experiment 2		Experiment 3
	No. of galls	Reduction (%)	No. of galls	Reduction (%)	No. of galls	Reduction (%)
Control	117.0±76.0a	-	13.9±4.5a	-	85.2±25.4a	-
P. lilacinum MTL01	45.5±22.3b	61 %	4.1±2.6b	71 %	37.7±9.1b	56 %
B. velezensis 83	45.7±12.4b	61 %	7.9±2.1bc	43 %	28.0±10.4b	67 %
MTL01+ 83	39.5±10.0b	66 %	3.1±1.3c	78 %	15.8±3.3b	81%

* Different letters indicate statistically significant differences (one-way ANOVA, Tukey’s test; 𝑝 < 0.05).

The positive impact of the combined treatment on flowering and gall reduction across the experiments demonstrated a more stable and reproducible biological response. These results indicate that although growth promotion may depend on environmental conditions, the protective and reproductive effects conferred by the microbial combination are more consistent.

The combination of P. lilacinum MTL01 and B. velezensis 83 proved to be an effective strategy for the biocontrol of Meloidogyne enterolobii in tomato plants under greenhouse conditions. Combined inoculation of both microorganisms reduced galling by more than 60% compared with plants infected with the nematode and consistently promoted an increase in flowering ranging from 1.3- to 4.3-fold relative to the control, both in the presence and absence of the parasite. These results provide a foundation for further studies to evaluate their effectiveness under field conditions and to optimize the formulation and dosage of this treatment for sustainable agricultural applications.

Limitations. The main limitation of this study was the small sample size used in the experiments. Nevertheless, the use of certified seeds helped maintain low variability among the technical replicates of each experiment, making it possible to perform reliable statistical comparisons of the results obtained.

Conflict of interest. The authors declare that they have no conflicts of interest regarding the research, authorship, and/or publication of this note.

Funding. This work was funded by DGAPA-PAPIIT project (IN203622) to C. Martínez-Anaya, and by the Programa de Becas de Estancias Posdoctorales Fellowship Program for Mexico of the National Council for Science and Technology (CONACYT) awarded to I. Tapia-Vázquez.

Acknowledgments. The authors thank Verónica Rojo and Chris Wood, from the Laboratorio Nacional de Microscopía Avanzada (LNMA), Instituto de Biotecnología, UNAM, for their technical support. The authors also thank Leobardo Serrano and Enrique Galindo for their critical review of this work.

Author contributions. Conceptualization, ITV and CMA; Methodology, ELG, ASC, and GLC; Formal analysis, ELG, ITV, and CMA; Investigation, ITV and CMA; Resources, CMA, ASC, and GLC; Data curation, ITV and ELG; Writing - original draft, CMA; Writing - review and editing, ELG, ASC, GLC, CMA, and ITV; Funding, CMA. All authors have read and agreed to the final version of the manuscript.