RKNs (Meloidogyne spp.) are one of the most important groups of nematodes due to their broad host range and the level of damage caused after their establishment in the root tissues of their hosts (Jones et al., 2013), causing agricultural losses of around $157 billion per year (Mokrini et al., 2018). They establish a sophisticated relationship within the host plants where they induce a local swelling called gall with the aid of specific effectors. Within the galls, they also trigger the formation of specialized nourishing cells called giant cells (Escobar et al., 2015). The intensive cropping with susceptible varieties, lack of awareness of farmers, and global warming have a significant impact on nematode disease aggravation and their emergence in new areas. Yet, nematode control is difficult due to its broad host range, soil-hidden nature, lack of resistant varieties, and the ban of many chemical nematicides because of their high environmental toxicity. Hence, there is an increasing demand for sustainable and efficient nematode-management strategies. These methods should be combined in the integrated pest management (IPM) system (Riyaz et al., 2022), which consists of combining different tactics, including host resistance (when available), rotation with nonhosts, sanitation, destruction of residual crop roots, biological control, among others.

Increasing the knowledge of IPM systems, including the development of new biotechnological tools against PPNs, is nowadays on focus (Alemu, 2020). In this respect, biological control using the soil rhizobacteria of the genus Bacillus is an alternative. They have shown the ability to produce antibacterial and antifungal secondary metabolites and to promote plant growth (Oliveira et al., 2014; Cao et al., 2019). Therefore, they drawn significant attention in recent years because of their safety for the environment and ability to suppress nematode populations in the soil (Engelbrecht et al., 2018; Topalović et al., 2020). Yet, the ability of different strains of the genus Bacillus spp. for PPNs control, reside in diverse effects. Some can be opportunistic, and feed from the nematodes, producing also proteases that affects and finally kill the nematode, they can increase plant tolerance due to its plant growth promoting effect (reviewed in Ahmad et al., 2021), or secrete toxic proteins as Cry5 that kill juveniles of M. incognita (Geng et al., 2017).

Rhizobacteria displays different methods of action to quell PPNs (Migunova and Sasanelli, 2021). The mechanism of nematode suppression can be categorized mainly in two major ways: a direct effect on the pre-infective juveniles, and an indirect effect on the post-infective juveniles and adults’ phase by triggering an immune response in the plant, delaying nematode development and reproduction. The direct effect involves production of enzymes (Lian et al., 2007; Corrales-Ramírez et al., 2017), antibiotics (Nadeem et al., 2021), volatile organic compounds and soluble secondary metabolites that are delivered around the rhizosphere (Oliveira et al., 2014). Some species of Bacillus spp. protects plants against nematodes through their high potential to form a biofilm on the roots which is a physical barrier to the nematode’s penetration. They can also form multi-species biofilms increasing their efficacy by combining the different strain-specific functions and diversity in the soil (Ansari and Ahmad, 2019). On the other hand, indirect mechanisms happen through interference with nematode behavior, by altering the root diffusates and affecting its recognition by the nematodes or altering the nematode feeding site development or sex female/male ratio inside the root tissue, or even by promoting plant growth (Mhatre et al., 2019). So far, some secondary metabolites can also trigger an immune reaction in the host plants, which leads to systemic resistance (Choudhary and Johri, 2009; Adam et al., 2014), rendering the plant less susceptible to pathogen infection. Some examples also point to enhanced beneficial effects combining both the plant growth attributes and their effect on nematodes control after co-inoculation of different rhizobacteria species (Ansari and Ahmad, 2019; El-Nagdi and Abd-El-Khair, 2019; Topalović et al., 2020).

Plant growth promoting rhizobacteria (PGPR; Lim and Kim, 2009) facilitate plant development by producing phytohormones (auxins, cytokines, ethylene, abscisic acid, etc.; Poveda and González-Andrés, 2021) that promote plant growth, helping to decrease disease severity, as the plants show more tolerance to the nematodes (Backer et al., 2018; Mhatre et al., 2019). Moreover, the phytohormones produced by rhizobacteria promote cell division and cell elongation (Khan et al., 2009; Arora et al., 2013), particularly, microbial auxins hormones increase the lateral and adventitious rooting leading to improved mineral and nutrient uptake (Arora et al., 2013), increasing nitrogen fixing and phosphate solubilization in the soil (Saeid et al., 2018; Jain et al., 2021). In this way, several studies demonstrated that inoculation of auxins producing PGPR in the field by seed application resulted in enhanced plant growth and nematicidal activity (Ruanpanun et al., 2010; Khan et al., 2012; Gamalero and Glick, 2020).

In this context, we use and evaluated a commercial blend of the dual-strain combination of B. paralicheniformis FMCH001 (previously identified as B. licheniformis) and B. subtilis FMCH002 on reducing nematode infection in plants. This blend is currently available as bionematicides in the market for managing different nematode species (Machado, 2022). We show that this specific dual-combination has nematicidal properties against Meloidogyne spp., interfering with egg hatching, juvenile penetration, female development, and reproduction during the nematode - Solanum lycopersicum interaction. The interference in the different stages of the nematode cycle is a result of the multiple modes of action of this Bacillus dual-strain bacteria combination that could effectively control RKNs in tomato.

There is an increasing need to search for nematode control strategies compatible with IPM systems. In this respect, rhizobacteria within the Bacillus genus show multiple modes of action against PPNs that can act alone or in combination (reviewed in Miljaković et al., 2020; Ahmad et al., 2021). Thus, we evaluated the dual-strain combination of B. paralicheniformis FMCH001 and B. subtilis FMCH002 on PPNs control, with particular emphasis on RKNs. Firstly, we proved a direct effect on the pre-infective J2, resulting in high mortality rates, as well as reduction on egg hatching after the treatment with the dual-strain bacteria combination ( Figure 1 ). Moreover, bacterial supernatant of each of the two Bacillus strains (FMCH001and FMCH002) caused also direct mortality after 48 hours incubation of Meloidogyne spp. J2 ( Figure 2 ). These findings suggest that the secondary metabolites produced by each of the strains during vegetative cells growth, could be, at least partially, responsible for the direct nematicidal effect observed. It has been described that direct nematicidal activity can be due to secretion of secondary metabolites by Bacillus spp. (reviewed in Ahmad et al., 2021), one example is that diverse volatile compounds (ketones, acids, alkyls, sulfide, etc.) present in B. subtilis strain Bs-1, interfere with hatching and produce J2 mortality on M. incognita (Cao et al., 2019), or Bacillus mycoides strain R2 that produce the volatile styrene, very effective as a repellent and nematicidal against M. incognita (Luo et al., 2018).

It was also observed a significant reduction on juvenile attraction and penetration during the early stages of the tomato-M. javanica interaction in plants from bacteria treated roots as compared to untreated ( Figure 3 ). Similar effects were also described with Bacillus subtilis strain Bs-1, which showed repellence of M. incognita, as well as effects on mortality and egg hatching (Cao et al., 2019). GC-MS assays detected that the major gas of the volatile compounds (VOCs) produced by this BS-1 strain was CO₂ and other substances such as acetic acid, in this respect, the alterations of Meloidogyne spp. attraction by high concentrations of CO₂ and variations on pH are well described (Pline and Dusenbery, 1987; Wang et al., 2009). Other examples are the treatment with bacterium B. cereus in soil that inhibited the penetration of M. javanica J2s (Oka et al., 1993) and caused repellence and B. cereus strain BCM2 with M. incognita, in tomato (Li et al., 2019). Therefore, it might be possible that the dual-strain combination of B. paralicheniformis FMCH001 and B. subtilis FMCH002 here studied, when colonizing the roots, can interfere with the recognition of plant exudates by the nematodes. Accordingly, it is known that rhizobacteria consume the exudates thereby truncating the nematode’s identification of root penetration points (reviewed in Lamovsek et al., 2013).

However, the dual-strain combination used, also remarkably reduced the number of galls per plant in vitro ( Figure 4 ), as well as the reproduction parameters in soil, and this last effect was independent of the bacterial application mode, either before or after inoculation with the nematodes ( Figure 7 ), hence, the versatility of those treatments seems a positive characteristic for IPM uses. Several studies observed a similar reduction in egg masses or root galling after Meloidogyne spp. infection in tomato, such as those treated with Bacillus spp. strains isolated from Iranian tomato fields (Ramezani-Moghaddam et al., 2014) or the B. cereus strain Jdm1 that decreased the root galling severity of M. incognita (43%) and promoted tomato growth performance (Xiao et al., 2018). Interestingly, the size of the galls, as well as GCs size was also reduced by more than 50% in the in vitro bacteria treated plants ( Figures 4 , 5 ). These results indicate that the development of the feeding sites is somehow affected by the dual-strain combination of B. paralicheniformis FMCH001 and B. subtilis FMCH002 treatment. Similarly, M. incognita also produced few small galls in the tomato roots treated with B. mycoides R2 broth as compared to the untreated controls (Luo et al., 2018) and the treatment of tomato plants in greenhouses with B. subtilis showed fewer and smaller galls than those on water control plants (Cao et al., 2019). However, to our knowledge this is the first time that specifically, GCs size alterations have been reported after Bacillus treatments during Meloidogyne spp. infection. Additionally, we confirmed by confocal microscopy that the bacteria are present in the plant tissues and therefore can colonize the plant roots, including the galls, after the seeds coating treatment ( Figure 6 , S4 ). The effects observed in reducing the galls, but also the GCs growth could be directly due to altered plant responses to the presence of the bacteria, and/or to a direct or indirect effect of the bacteria on the nematodes at the J2, J3, J4 living stages, that could interfere with the proper GCs development. This last, could be due for example to alterations of the plant-nematode molecular crosstalk necessary for GCs formation and development. In this respect, bacterial metabolites are also able to produce a plant defense response that controls Meloidogyne spp. on tomato like hydrogen cyanide (Siddiqui et al., 2006). However, functional mechanisms for these described effects have not yet been proposed. Further research will elucidate the putative transduction pathways involved in those altered morphogenetic responses in galls and GCs after the bacterial treatment. Besides, the bacterial combination used was also effective in soybean, that showed a considerable reduction of the reproduction parameters after the dual-strain combination treatment, which was enhanced after two infection cycles ( Figure 8 ). Moreover, the infection of nematodes of the genus Pratylenchus spp. was also considerably affected after the dual-strain treatment in soybean. This opens the possibility, that the dual-strain combination of B. paralicheniformis FMCH001 and B. subtilis FMCH002 could be effective against other groups of PPNs and in different crops.

In conclusion, our analysis confirmed the effectiveness of a combination of bacteria as a biological control method for Meloidogyne spp. in tomato in controlled conditions. We have evidence that direct mechanisms may act on the J2 pre-infective stage and on the eggs of M. javanica. Additionally, direct and/or indirect mechanisms promoted a decrease in the attraction and penetration of the J2 to the tomato roots after bacteria treatment, which indicates that the dual-strain combination could also interfere with root recognition. Furthermore, different mechanisms, still not well known, also interfered with galling and reproduction that were severely reduced. These altered reproduction parameters could be a consequence of the reduction in the GCs size, either through effects on the nematode (J3 and J4 life-stages), or on the plant development processes that lead to GCs formation (Escobar et al., 2015). Our findings are aligned with other studies showing different nematode control mechanisms by bacteria within the Bacillus genus. Some examples are B. velezensis strain Bv-25, that reduced nematode infection in laboratory assessments exhibiting also direct nematicidal against M. incognita in vitro (Tian et al., 2022). Likewise, inoculation of B. megaterium stimulated plant biomass, root length, shoot length, and nutrient uptake in tomato plants with declined RKNs population (El-Hadad et al., 2011). M. incognita were also controlled using an eco-friendly formulation from B. subtilis under laboratory and greenhouse conditions (Silva et al., 2017; Basyony and Abo-Zaid, 2018). Consequently, this study identified a potential bionematicide based on a dual-strain combination of B. paralicheniformis FMCH001 and B. subtilis FMCH002 that could be effective for better management of RKNs within an IPM scheme and described different modes of action of the dual-strain, including effects on GCs development. Considering the existing environmental challenges, it is essential to restrict the chemical nematicides available on the market as the regulations of all the states are demanding, yet many of them are already banned by many countries (Gad, 2014).

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

CE, LM, DA, and FED-M conceived and designed the experiments. CE and LM provided resources and funding. CE coordinated the experiments. FED-M and AG-M performed most of the experiments. CE, FED-M, and DA wrote the manuscript. All authors contributed to the article and approved the submitted version.