Phytopathogenic nematodes (PPNs) are one of the greatest threats to agricultural, ornamental and forestry activities (Archidona-Yuste et al., 2019). According to the report of the American Society of Phytopathology (APS), losses in agricultural production caused by PPNs are approximately 14%, which translates into almost 125 billion dollars annually (Mesa-Valle et al., 2020). A common feature of PPNs is the stylet located in the stoma or in the mouth that allows them to cross the host cell wall and inject enzymes that partially digest the cellular content before the nematode sucks it into its digestive system (Kimenju et al., 2009). According to their life cycle and feeding behavior, PPNs are classified into three groups: endoparasites, ectoparasites and semiendoparasites (Gullino et al., 2020). Endoparasites penetrate inside the plant, where all or part of their life cycle occurs; they are mainly root-knot nematodes (e.g., Meloidogyne spp.), encysted nematodes (e.g., Heterodera spp., Globodera spp.), and root-injuring nematodes (e.g., Pratylenchus spp., Hirschmanniella spp., Radopholus spp.), among others (Pires et al., 2022). In contrast, the ectoparasite life cycle occurs entirely outside the plant, piercing the epidermis or the superficial layers of the root cortex of the host plant with the stylet (e.g., Xiphinema spp., Trichodorus spp., Paratrichodorus spp.); therefore, they can also be found in seeds, stems and the aerial part of the plant (leaves and flowers; Pires et al., 2022). Finally, semiendoparasites can partially penetrate the plant, leaving part of the nematode inside the plant and another part outside, laying eggs toward the outside (e.g., Sphaeronema spp., Hoplolaimus spp., and Helycotylenchus spp.; Mesa-Valle et al., 2020).

Any agricultural crop can be affected by the action of some PPNs, and their effect is often underestimated as they produce nonspecific symptoms that are often confused with situations of water stress, nutritional disorders, soil fertility problems, or other secondary fungal or bacterial infections (Pires et al., 2022). Additionally, PPNs have fast generation times, high reproductive rates, and cryptic behaviors, contributing to the difficulties experienced in effectively controlling PPNs infections (Naz and Khan, 2013; Horak et al., 2019). Several strategies have been reported for the control of PPNs infections, such as nonhost crop rotation, soil solarization and the use of chemical nematicides, which include halogenated aliphatic hydrocarbons (e.g., 1,3-dichloropropene), methyl isothiocyanate, oxamyl, thionazine, and carbofuran (Engelbrecht et al., 2018; Naz et al., 2021). Although the use of chemical nematicides is the most successful strategy for the control of PPNs, their long-term repeated use leads to increased nematode resistance, exacerbating the difficulty of nematode management (Chen et al., 2020). In addition, there is evidence of their negative impact on the environment, for example, the groundwater contamination, disturbance of soil fertility, and the accumulation of chemical residues within the trophic levels of soil ecosystems (Engelbrecht et al., 2018). As eco-friendly alternatives, the use of microorganisms and/or their metabolites as biological control agents has attracted scientific interest due to their minimal environmental side effects (He et al., 2020).

Previous studies have shown that certain plant-associated rhizosphere microorganisms belonging to the Pasteuria, Pseudomonas, Streptomyces, and Bacillus genera control and antagonize PPNs through their ability to colonize the rhizosphere and secrete enzymes and/or secondary metabolites that are toxic to PPNs and/or elicit an induced systemic response (ISR) in plants (Berini et al., 2018; Zhao et al., 2018; Horak et al., 2019). However, in addition to their nematode control capabilities, this kind of microorganism must thrive under adverse conditions such as droughts, high temperatures and/or soil salinity promoted by climate change, which has been dramatically accelerated in recent years (Bokhari et al., 2020). Indeed, recent research has focused on the bioprospecting of plant-associated beneficial microorganisms capable of adapting to adverse conditions (Engelbrecht et al., 2018; Bokhari et al., 2020).

Desert soils are characterized by extreme conditions, such as high temperatures, water scarcity, high soil salinity, low nutrient levels and high UV radiation (Alsharif et al., 2020; Franklin et al., 2021). In addition, the survival of plants in desert areas is intrinsically related to their microbial communities (Sayed et al., 2019; Bokhari et al., 2020). Alsharif et al. (2020), Bokhari et al. (2020), and Franklin et al. (2021) showed that desert microorganisms not only relieve water stress in plants and promote growth but also protect the plant against pathogens, such as PPNs. Moreover, Viljoen and Labuschagne (2019) and Abd-Elgawad and Mahfouz (2020) found that Streptomyces and Bacillus strains isolated from the Egyptian desert are capable of effectively controlling PPNs and surviving in extreme conditions of drought and water stress. In this way, the plant-associated microbiota from deserts is a perfect ecological niche to isolate new microorganisms that are well adapted to extreme conditions and have potential for the biological control of phytopathogens, including PPNs.

The Sonoran Desert is one of the warmest regions in North America and in the world, where temperatures above 50°C have been recorded and cover a territory of 260,000 km², including southern Arizona and southeastern California, EUA; northeast, north and south-central Baja California; and north-central and western Sonora, Mexico (Zolotokrylin et al., 2020; Flower et al., 2021). However, unlike other deserts, the Sonoran Desert is the most biodiverse; this is the case for flora, with approximately 2,500 species identified (Andrew et al., 2012; del Conagua, 2020; Franklin et al., 2021). This is relevant under the premise that the microbial communities are important for the survival of these plants, constituting a diverse and untapped pool of microorganisms with the potential to control phytopathogenic agents, such as PPNs, and to support extreme biotic and abiotic conditions or to synthesize thermostable or operatively stable metabolites. For example, Galaviz et al. (2018) and Bashan et al. (2012) found that the Sonoran Desert is home to plant growth-promoting rhizobacteria (PGPRs), such as Azospirillum brasilense and Bacillus pumilus. However, research on the potential of microorganisms isolated from this ecological niche for biological control of PPNs is scarce. Hence, the objectives of this research were to isolate, characterize and apply bacteria from the rhizosphere of plants growing in the Sonoran Desert with the potential for PPNs control.

Harsh abiotic conditions existing in desert environments (e.g., extreme temperatures and radiation, water scarcity, erosion, low organic matter content, and sedimentation, among others) negatively impact living organism development especially in plants and microorganisms (Alsharif et al., 2020). However, several species have developed adaptations to remain metabolically active (Schultz and Soares, 2020) and to be able to proliferate under these adverse conditions. Such is the case for the Sonoran Desert, which possesses the greatest biodiversity of flora and fauna among deserts worldwide, although the temperatures in summer can exceed 45–50°C (Zolotokrylin et al., 2020). Desert soils worldwide possess a huge diversity of microorganisms dominated by Actinobacteria (36.8%), Proteobacteria (23.8%), Firmicutes (8.6%), and Acidobacteria (5.5%) (Dean et al., 2020). According to this microbial diversity distribution and the well-known potential of Actinobacteria as control agents (Bhatti et al., 2017; Cruz Silva et al., 2022); ISP2 medium was employed in the first screening, looking for isolates belonging to this phylum or to Firmicutes (Tian et al., 2022). Since, although ISP2 is considered as a selective medium for actinomycetes, it allows the growth of some other bacterial genera, such as Bacillus, too (Paz et al., 2016; Zhou et al., 2019).

Specifically, regarding the Sonoran Desert, there is scarce information about rhizospheric and soil microbiota composition and the biotechnological potential from their microorganisms. Sutto-Ortiz et al. (2017) and Bacame-Valenzuela et al. (2015) reported the isolation of thermotolerant actinomycetes and fungal strains capable of synthesizing enzymes of industrial interest, such as phospholipase A and feruloyl esterases, respectively. Additionally, Camacho-Ruiz et al. (2020) reported the isolation from Sonoran Desert soils of Streptomyces, Bacillus, Aspergillus, and Aureobasidium strains able to produce gastrointestinal lipase inhibitors. Moreover, Baqueiro et al. (2019) reported the biotechnological potential of Bacillus strains capable of producing stable proteases. Regardless of agricultural applications, some plant growth-promoting rhizobacteria (PGPRs), such as Azospirilum brasilense and Bacillus pumilus, have been isolated (Bashan et al., 2012; Galaviz et al., 2018), but there are no reports focusing on PPNs control. Indeed, this is the first work to explore and demonstrate the potential of microorganisms from the Sonoran Desert for the control of PPNs.

In the current work, from 100 thermotolerant bacteria isolated from rhizospheric soils collected in the Sonoran Desert, the secretomes from 27 of them showed some nematicidal activity in vitro (3–96%) against M. incognita. Of these isolates, 10 produced high mortality in M. incognita larvae (70–90%), but the secretome from the TB197 strain produced the highest (>95%) (Figure 1). Consequently, it was selected for identification and evaluation in greenhouse and open-field tests. Sequence analysis of the 16S rRNA gene had the most significant impact on the identification and taxonomic classification of the TB197 strain. Even so, analyses of the housekeeping gene sequences gyrA and groEL were also included because previous studies mention difficulties in the differentiation and phylogenetic relationships of certain groups of Bacillus based on 16S rRNA gene sequences due to their similarities (Table 2). Additionally, the sequencing of housekeeping genes has been shown to be more appropriate than 16S rRNA sequencing for discriminating certain species of Bacillus (Ozdemir and Arslan, 2022). Therefore, the use of these housekeeping genes provides a high resolution between species closely related to groups of Bacillus, as they are generally more conserved between bacterial species than the 16S rRNA gene (Liu et al., 2013; Franco-sierra et al., 2020). This information allowed the robust identification of the TB197 strain as Bacillus paralicheniformis.

In agriculture, many strains from the Bacillus genus have been employed in biofungicides and biofertilizer formulations due to their biological activities, including antagonism against phytopathogenic agents, such as bacteria, fungi and nematodes; induction of plant systemic resistance against phytopathogens; and promotion of plant growth and development (Villarreal-delgado et al., 2018; Valenzuela-Ruiz et al., 2019). Furthermore, Bacillus is one of the three most important genera for PPNs control by several mechanisms: direct parasitism, antibiosis, reduction in plant root penetration and competition for essential nutrients (Engelbrecht et al., 2018). Antibiosis has been identified as the main control mechanism of PPNs through the production of antibiotics and other secondary metabolites (lipopeptides, siderophores, and endotoxins, among others) and lytic enzymes secreted during growth and the stationary phase (Xiong et al., 2015; Gao et al., 2016; Engelbrecht et al., 2018; Liu et al., 2020a). Nevertheless, information on secondary metabolites produced by B. paralicheniformis with nematicidal activity is almost nonexistent. Valenzuela-Ruiz et al. (2019) reported that the draft genome of B. paralicheniformis TRQ65, isolated from wheat fields in the Yaqui Valley, Mexico, revealed the presence of putative genes related to osmotic and oxidative stress responses and auxin, lipopeptides, siderophores and antibiotic biosynthesis.

For preliminary chemical characterization, extracts obtained by lixiviation of freeze-dried supernatants with methanol were employed. TLC plates from samples obtained by methanolic extraction are shown in Figure 3, revealing the presence of several fractions with different Rf values. The spots from TLC plates containing the methanolic extract can be visualized by different methods (UV and stains). Almost all of them were reactive to ninhydrin and iodine vapors (Figures 3C,D), but only some of them were active to UVA and/or UVC (Figures 3A,B) with slight activity for UV light but high activity for iodine vapors and ninhydrin (Figure 3). The reactivity to ninhydrin suggests the presence of peptide-natural compounds (Dlamini et al., 2020) and probably molecules that contain unsaturated fatty acids due to reaction with iodine vapors (Meyers and Meyers, 2008). In the case of some UV-active spots, the molecules could include aromatic compounds, such as aromatic amino acids.

The information provided by TLC analyses fits with the description of siderophores and cyclic lipopeptides usually produced by Bacillus species and the presence of putative genes to produce them, according to the draft genome of B. paralicheniformis TRQ65 (Horak et al., 2019; Valenzuela-Ruiz et al., 2019). In general, lipopeptides contain a hydrophobic fatty acid chain bonded to a hydrophilic cyclic peptide. According to the nature of the cyclic peptide, lipopeptides produced by Bacillus species, are generally classified into three families: surfactin, fengycin, and iturin (Falardeau et al., 2013; Yang et al., 2015). Members from the fengycin and iturin families usually contain aromatic amino acids in their peptide ring, such as tyrosine (Zhao et al., 2017), which would explain the UV activity of some compounds (Figure 3). Molecules with Rf values between 0 and 0.3 or 0.2 and 0.4 could belong to the fengycin and iturin families, respectively, as mentioned by Geissler et al. (2016). On the other hand, low-molecular-weight metal-chelating compounds of the hydroxamate and catecholate types, which are involved in the uptake of iron (siderophores) by microorganisms, have been reported as metabolites produced by several Bacillus species (Sansinenea and Ortiz, 2011). The results of the TLC analysis strongly suggest that secondary metabolites synthesized by the B. paralicheniformis TB197 strain could be siderophores and/or cyclic lipopeptides. However, an exhaustive chemical characterization of its secretome is currently ongoing to verify this hypothesis.

This study demonstrated the potential of the secondary metabolites contained in the secretome from B. paralicheniformis TB197 to effectively control infections caused by M. enterolobii in open-field assays. The GI and the infection severity percentage observed in tomato roots were very low and similar to the conventional management performed by the farmer. In this sense, there are few studies evaluating the effectiveness of secretomes of Bacillus species for the control of PPNs in agricultural fields. For example, Liu et al. (2020b) showed that the application of 16 mL/m² of various secretomes of Bacillus strains in tomato roots resulted in a GI of 1, while the GI reported in the present work by employing the secretome of the TB197 strain was 0.2. Therefore, the secretome of B. paralicheniformis TB197 appears to be more effective as a lower concentration (0.8 mL/m²) was applied and obtained better GI results.

However, additional work over the application of secretomes regarding biosafety, doses, application times, optimization of culture medium, culture conditions and the exhaustive characterization of their bioactive metabolites is required. Although the chemical nematicide fluopyram (pyridinyl-ethyl-benzamide) was highly effective in suppressing infection by Meloidogyne species, there are reports that this chemical shows a reversible inhibition phenomenon that could lead to possible crop infestation even after application (Watson, 2022). In addition, fluopyram is reported to be a recalcitrant compound that modifies the general soil structure and the activity and function of beneficial soil microorganisms (Zhang et al., 2014). Consequently, as an advancement toward sustainable agriculture, the secondary metabolites contained in the secretome of the TB197 strain appear to be a promising alternative to chemical nematicides.

Although bacterial metabolites look promising for PPNs control, the use of cells or endospores from Bacillus species is the most common way for formulating commercial products (Zhou et al., 2016; Engelbrecht et al., 2018). The use of live cells and/or spores offers several additional advantages over the use of metabolites, such as the ability to combine multiple mechanisms, like interference in nematode-host recognition, competition for nutrients, promotion of plant growth, and induction of systemic resistance (Chinheya et al., 2017; Engelbrecht et al., 2018; Ramírez-Cariño et al., 2020). Additionally, endospores are preferred to prepare commercial formulations because they are highly resistant to adverse environmental conditions, making them suitable for long-term storage and convenient for formulating powdered products for agricultural application (Zhou et al., 2019). In this study, the capacity of B. paralicheniformis TB197 to control PPNs was evaluated for the first time. In the greenhouse assay, both the TB197 strain and fluopyram demonstrated a control efficacy of M. incognita greater than 98% based on the reduction in galling severity (Figure 5A).

On the other hand, several studies mention that success of controlling PPNs at the greenhouse level does not guarantee bacterial performance at the field level (Tian et al., 2007; Abdel-Salam et al., 2018; Zhao et al., 2020; Wang et al., 2021), usually because strains are affected by unfavorable factors, such as climatic changes, physical and chemical properties of soil and the competition of native microorganisms (Wang et al., 2021). However, our results show the consistent ability of B. paralicheniformis TB197 to control PPNs even at the field level. One assay was performed against root-knot PPN comparing the bacterial effectiveness with a probed commercial biopesticide LilaPlus^® (P. lilacinum). Both treatments were highly effective and showed consistent results for the control and reduction of the severity of M. incognita infection (~84% compared with the control, Figure 5B). Several studies and patents report Bacillus cells or endospores as biological control agents, particularly against root-knot nematodes from the Meloidogyne genus; however, none of them surpassed the effectiveness reported here for B. paralicheniformis TB197. For example, the percent reduction in GI in field assays through the use of Bacillus altitudinis AMCC1040 (Wang et al., 2021), B. subtilis Bs-1, B. cereus Bc-cm103 (Cao et al., 2019), B. aryabhattai KMT-4 (Yin et al., 2021), B. velezensis Bv-25 (Antil et al., 2021), B. marisflavi CRB2 (Tian et al., 2022), B. subtilis CRB7 and B. methylotrophicus (Gowda et al., 2022) was <70%.

A second assay was performed against the burrowing nematode R. similis, a migratory, polyphagous endoparasite with a wide global distribution mainly found in tropical and subtropical regions that infects banana crops. The control effectiveness of the TB197 strain was similar to that of Nemacem^®, an effective commercial botanical extract from Tagetes erecta, employed as a positive control. In both cases, the percentage of root necrosis and the nematode population density were reduced by 81.0 and 71.5% (Figure 5C), respectively, compared to the control (water). The main active metabolite from the botanical extract (Nemacem^®) is alpha terthienyl, a well-known molecule acting on the central and peripheral nervous systems of nematodes, causing their immobilization and subsequent death (Plantoria, 2022). Although this extract is considered an environmentally friendly alternative, its half-life is very short (̴ 4 h, according to the National Institute of Ecology and Climate Change of Mexico²), which could be a disadvantage compared with bacterial-based nematicides, that in the case of molecules synthetized by the Bacillus genus, are stable at extreme soil pH, temperature and salinity conditions for more than 25 days (Raaijmakers et al., 2010; Engelbrecht et al., 2018). Our results demonstrate that both the bioactive metabolites contained in the secretome of the B. paralicheniformis TB197 strain and their endospores can effectively control PPNs infections. They provide an ecological alternative to chemically synthesized nematicides, and can compete successfully with commercial botanical and microbiological bionematicides.

Despite the fact that the TB197 strain was isolated from the desert rhizosphere and showed an optimum growth temperature range between 40 and 45°C (data not shown), its ability to growth in different environmental conditions is highlighted. For example, the greenhouse trials were developed under controlled conditions of temperature and humidity (25°C and 60%, respectively), while the field trials on tomato plants were performed in Aguascalientes, Mexico, where the temperature ranged from 13 to 30°C with a relative humidity of 6.7%. By the contrary, the trials on banana crops were carried out in Colima, Mexico, where the temperature varies from 23 to 31°C with a relative humidity of 81%, higher than greenhouse and field trials conditions. These facts suggest that the TB197 strain has the ability to colonize different crop roots under distinct environmental conditions, possibly due to a unique adaptation mechanism inherent to its niche of isolation. However, further studies are required to establish more precisely the colonization capacity of the TB197 strain under extreme conditions (ongoing studies).

Plant-associated bacteria from the Sonoran Desert show potential for controlling PPNs. In particular, the new Bacillus paralicheniformis TB197 strain exhibited a high capacity for PPNs control in vitro, greenhouse and agricultural field tests. Moreover, the TB197 strain successfully proliferated in various rhizospheric soils with different climate, soil conditions and crops, indicating its high adaptability to colonize environments different from its niche of isolation. The effective reduction of root infection by PPNs and the resulting damage, achieved through the application of the secretome or the endospores of TB197 strain, are comparable to those obtained with commercial chemically synthesized and natural nematicides. These results support B. paralicheniformis TB197 as a potential control agent for the development of commercial bionematicidal formulations.

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

EC-Q, VC-J, AC-F, FD, and AA-T contributed to the study conception and design. AC-F performed the secretome field and the greenhouse assays. VC-J obtained the secretomes for the in vitro nematicidal assays. FD performed the molecular characterization. EC-Q performed the chemical characterization of the secretome, analyzed the data, and wrote the original manuscript. AA-T and VC-J supervised the research and reviewed the manuscript. All authors contributed to the article and approved the submitted version.

This work was supported by Grant N° 20624 Innovak Global-CIAD A.C.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.