Extreme environments are known to be distributed over large portions of the Earth. These environments are characterized by different physicochemical, chemical, and biological conditions, such as extremely high or low temperatures, high concentrations of heavy metals, very pronounced pH ranges, etc. [1]. This environment includes hydrothermal and volcanic sites, which have been found on the Earth’s surface and the deep sea [2].

Various microorganisms capable of adapting to geothermal ecosystems have been identified. These include bacteria [3], fungi [4], algae [5], and cyanobacteria [6]. These capabilities are related to survival under these hostile conditions. Classification of these microorganisms is based on the physicochemical conditions in which they can adapt and develop extensively [7]. For example, those that live in high temperature conditions are considered thermophiles (45°C to 60°C) and hyperthermophiles (>80°C), whereas those developing and reproducing at low temperatures are referred to as psychrophiles (<10°C). Microorganisms that tolerate high salt concentrations are referred to as halophiles. Finally, those that tolerate various pH ranges are divided into three groups: acidophiles (living in a pH range of 0 to 5), alkalines (tolerating a pH range of 5 to 8), and alkalophiles (those surviving at a pH above 8) [8,9].

The genus Paenibacillus predominates in this category [10]. These Gram-positive, spore-forming bacteria exhibit facultative anaerobic metabolism and belong to the phylum Firmicutes [11]. Bacteria belonging to this genus have been identified in different extreme ecosystems, one of them is the active volcano El Chichón, which is located northwest of the State of Chiapas, Mexico, with a crater lake formed after its eruption in 1982. This complex is considered a highly hydrodynamic system due to the geochemical composition, variations in temperature and pH of the lake, and geothermal activity due to the presence of minerals, heavy metals and sulfur, which gives the lake acid-thermal characteristics [12]. Due to the hostile qualities of the crater lake, microbial life is limited; however, there are reports where different microbial strains have been identified that can produce phytohormones, solubilize phosphates, fix nitrogen, and synthesize siderophores [13]. In summary, Paenibacillus bacteria play an essential role in soil ecology and agriculture, and their continued study may have beneficial applications in sustainable agriculture and biotechnology.

Biosurfactants are surface metabolites produced by microbes [14]. According to reports, the genera Acinetobacter, Arthrobacter, Pseudomonas, Halomonas, Bacillus, Rhodococcus, and Enterobacter, as well as some Paenibacillus species such as P. polymyxa, P. macerans, P. dendritiformis, etc., can produce these metabolites [15,16]. Most biosurfactants are produced by metabolic activities, where the metabolic substrates are lipids, alkanes, and sugars. These compounds are classified according to their molecular structure, where categories such as glycolipids, phospholipids, neutral lipids, and lipopeptides can be found [17]. Lipopeptides are defined as low molecular weight compounds with a peptide chain variation between 6 and 13 amino acids and several variations along the fatty acid chain [18]. They can form pores in phytopathogens while possessing siderophore activity, biofilm inhibition, and detachment activity. Lastly, they can prevent colonization by phytopathogens [19].

The use of microorganisms and their secreted metabolites for the suppression of phytopathogenic fungi and promotion of plant growth has numerous advantages [20]. The degradation of these metabolites is easy and environmentally friendly, and they can even become part of the cycles of natural elements, which is in stark contrast with the chemical products used nowadays. However, a critical scientific problem lies in the understanding of the genetic and metabolic mechanisms involved in the biosynthesis of antifungal compounds in thermophilic bacteria. This knowledge gap hinders the development of new biotechnological strategies for the control of phytopathogens and the promotion of plant growth. Based on this, we hypothesize that Paenibacillus strain VCA1 presents unique genomic and metabolic characteristics as a biocontrol agent and plant growth promoter. Therefore, this work aimed to evaluate the antifungal and genomic potential, along with the biosurfactant activity of the Paenibacillus sp. strain VCA1 isolated from the crater lake of El Chichón volcano, thus attempting to provide scientific evidence for its application as a next-generation microbial biocontrol agent in sustainable agriculture.

Bioinformatics techniques based on whole genome information and used for taxonomic assignment of microorganisms help us broadly delimit the comparison between related species [31]. According to data obtained for strain P1XP2, it had higher TETRA, ANIb, and ANIm values than those shown for strains Y412MC10 and YC0573, as seen in Table 2. Although there are insufficient reports for the identification of Paenibacillus species by use of TETRA, it has proved to be effective in identifying several species in a genus, such as Thioalkalivibrio [29], Paraburkholderia [32] and Salinimomas [33], as different authors have reported that a Z value closer to a value of 1.0 indicates similarity between them at the species level. On the other hand, it has been stated that the cutoff level between the compared genomes must be above the 95%–96% similarity range to assert that they are the same species, according to the ANIb and ANIm analysis [34]. However, although data for percentage similarity and TETRA analysis generated species-level matches with the P1XP2 strain, ANIn, and ANIb analysis determined that it is a new species, according to the recommended cutoff level. However, [35] demonstrated that if ANI values are below 95% but within an 80%–90% range, it is still the same species and is a new subspecies instead. These results were demonstrated via DNA-DNA hybridization analysis (Supplementary Material Table S3) with the GGDC method, which helped determine the genomic relationship between microorganisms. This technique is based on the combination of two single DNA strands, which give way to a double-stranded molecule by base pairing. When the percentage of this pairing is above 70%, it is concluded that it is a new subspecies. The percentage of hybridization similarity was 84.5% with Paenibacillus sp. P1XP2, meaning there is high similarity between strain VCA1 and P1XP2. However, it could be a new subspecies. The identification of new thermophilic isolates is desirable for research expansion in the biotechnology and agriculture fields. Also, this information is highly useful for visualizing the genomic similarities and differences between different species and subspecies within the same bacterial genus [36]. The ecological niche of strain VCA1 is markedly distinct from that of mesophilic P. polymyxa strains commonly isolated from soil environments (Table 1). Strain VCA1, isolated from the geothermal environment of El Chichón volcano, occupies a thermophilic niche that contrasts sharply with mesophilic soil-derived P. polymyxa strains, which typically grow optimally around 30°C–32°C (soil isolates; Optima ~30°C–32°C) (Grady et al., 2016). The adaptation of VCA1 to high-temperature environments is supported by the presence of thermotolerance-related genes, such as Hsp20 and Hsp70, which are well-documented in thermophilic bacteria as key factors for protein stabilization under thermal stress [30].

On the other hand, according to the genomic functionality data from strain VCA1, a diversity of subsystems in the genome was found. One of the most important functions for living things is the ability to metabolize carbohydrates, as this allows them to produce the energy needed to live and reproduce [37]. Although this function is essential among bacteria, small differences can be observed between the metabolic pathways of existing genera, which then provide information on how each microorganism adapts to the surrounding environment [38]. Other notable functions in the genome of strain VCA1 include the ability to metabolize iron, sulfur metabolism, amino acid and fatty acid metabolism, motility, and chemotaxis. It is worth mentioning that sulfur metabolism is of the utmost importance, as strain VCA1 was isolated from an ecosystem in which several sulfur-containing species are present [39].

Likewise, when analyzing the subsystems found in the complete genome of strain VCA1, it was found that several functions are involved in biocontrol and plant growth promotion. One of these functions is the synthesis of isoprenoids, whose type of metabolite is classified under secondary metabolites, meaning they are involved in the defense against phytopathogens that cause damage and subsequent death to the plant [38]. Likewise, it was observed that it has flagellar motility capabilities, whereby a certain number of genes are involved in the motility that benefits bacteria, as they can move to colonize plant roots and carry out their promoter and biocontrol functions [40]. On the other hand, the function involved in bacterial chemotaxis, which allows microorganisms to have the ability to move according to the different chemical gradients that exist in their environment, also allows them to adapt and is in addition to flagellar mobility [41]. This mechanism belongs to the signal translation system, which controls the movement of bacteria. This implies that it oversees converting an extracellular stimulus into an intracellular signal, creating a response that will establish guidelines to approach or move away from the existing chemical gradient [42]. These extracellular stimuli can come from the environment or a neighboring cell. Another function observed was responsible for the production of N-acetylglucosaminidase. This enzyme is involved in the degradation of chitin, which can weaken cell structure and decrease the ability to infect [43]. The degradation of this polymer is of utmost importance for biological control because, as the fungal cell wall degrades, fungal cells become exposed and are more vulnerable to various molecules produced by bacteria, which are responsible for inhibiting phytopathogenic functions [44]. Finally, the role of the production of siderophores is iron-chelating compounds secreted by microorganisms [45]. It has been reported that iron tends to limit agricultural productivity in various environments. Therefore, chelating compounds are of extreme importance for iron assimilation and utilization in plants, as they can create a mechanism that promotes plant growth [46].

All the above can be compared with the study by [38], where different genes and functions were reported for the Paenibacillus pasadensis R16 strain, which was isolated from the rhizosphere in agricultural soils. This strain was capable of controlling phytopathogens such as Botrytis cinerea, Fusarium graminearum, and Aspergillus niger. Likewise, it has been reported that the Paenibacillus genus could biocontrol other genera, such as Alternaria and Chrysosporum [47], and serves as a plant growth promoter. Belonging to the same bacterial genus, strain VCA1 presents this functional similarity.

Regarding the analysis used to determine biosynthetic gene clusters (BGC), genes involved in cyclic lipopeptide biosynthesis are assembled as large gene groups that encode multifunctional enzyme complexes (NRPS), which have a structure with multiple domains that affect activation and recognition for amino acid ordering, this system functions as an enzymatic assembly line where each domain incorporates an amino acid to the peptide of interest. For the synthesis of iturin, 4 domains are necessary: (1) Domain A (Adelination), which recognizes and activates the amino acids necessary for the formation of lipopeptides, where each free amino acid is activated with ATP to form an aminoacyl-AMP; (2) Domain T (Thioester), which oversees transporting the activated amino acids. This domain contains a phosphopantetheine group that binds to the activated amino acid using a thioester bond; (3) C domain (condensation), this domain forms the peptide bonds between the new amino acid and the previous one, in this way the reaction is catalyzed between the amino group and the carboxyl group of the previous amino acid giving way to the binding of a new residue to the peptide in formation; (4) TE domain (Thioesterase), this domain is in charge of releasing the peptide complex [48].

Upon further analysis, it was found that the production of the 4-phosphopantetheinyl transferase enzyme is occurring within the analyzed region. It has been reported that this enzyme can perform post-translational modification for the active functionality of NRPS [49]. This enzyme is encoded by the sfp gene, which plays an essential role in priming some synthases and NRPS, such as cyclic lipopeptides and, more specifically, surfactins, iturins, and fengycins [21].

All these results indicate that Paenibacillus sp. strain VCA1 can inhibit disease-causing phytopathogens in various plants of agricultural and commercial interest, which could contribute to reducing crop losses and improving productivity. It is essential to mention that the VCA1 strain isolated from the extreme environment of El Chichón Volcano has some advantages for agricultural applications, as it can withstand temperatures ranging from 28°C to 65°C and pH ranges from 3 to 5.

In this study, the antagonistic activity of Paenibacillus sp. VCA1 against the fungal pathogen Fusarium oxysporum 45ta had an inhibition of 49.4%, a result comparable to that reported by [50], where Paenibacillus polymyxa SK1 obtained an inhibition of 55.5% against the fungus Fusarium oxysporum. Similarly, [51] reported 59.2% inhibition with Paenibacillus peoriae CFCC and 46.9% inhibition with Paenibacillus enshidis CCTCCAB when they tested the antifungal activity against the plant pathogen Fusarium graminearum. A similar result to that obtained with VCA1 was reported by [52], where the Paenibacillus kribbensis T-9 strain showed broad antifungal activity against several phytopathogens such as Phytophthora capsici, Botrytis cinerea and Colletrotrichum acutatum, which are mainly transmitted through soil. Although the inhibition rate of VCA1 is slightly lower than some of the reported values, this variation can be attributed to several factors, including differences in experimental conditions (e.g., medium composition, temperature, pH, and incubation time), the specificity of interactions between the bacterial strain and the phytopathogen tested, and the quantity and diversity of antifungal metabolites produced. Furthermore, the extreme environment from which VCA1 was isolated (the crater lake of El Chichón volcano) may have shaped unique metabolic adaptations that limit the optimal production of bioactive compounds under conventional laboratory conditions. These findings highlight the importance of optimizing culture parameters to enhance the synthesis of antifungal secondary metabolites (Ngo et al., 2025) and exploring alternative strategies, such as co-cultivation or metabolic engineering, to improve its efficacy as a biocontrol agent.

Thus, it is worth mentioning that not only hydrolytic enzymes could function as antifungals, but some biosurfactants (lipopeptides) are associated with this activity and have been reported to inhibit the growth of phytopathogenic fungi [53]. These biotensoactive compounds not only reduce the surface tension of water but also play a direct role in biocontrol mechanisms against phytopathogenic diseases. The mechanism by which they reduce the surface tension of the fungal membrane is through the degradation of phospholipids [54]. Finally, they also offer advantages for ecological purposes, as they have low toxicity, are easily degradable, have good environmental compatibility, and are highly selective in extreme environments [55].

According to the genomic analyses performed, DNA-DNA digital hybridization (dDDH) showed 84.5% similarity with the compared genomes, suggesting that Paenibacillus sp. VCA1 may represent a new subspecies of this genus. Functional genome annotation revealed the presence of genes associated with plant growth promotion, biocontrol mechanisms, and biosynthesis of antifungal and antimicrobial lipopeptides applicable to agriculture. Since the bacterium produces compounds that have biocontrol activity, the VCA1 strain could inhibit the growth of phytopathogenic fungi isolated from tomato plants. This antagonistic effect is likely mediated by the production of bioactive compounds capable of degrading fungal cell walls and membranes. Therefore, in this work, the use of Paenibacillus sp. VCA1 strain isolated from the lake crater of El Chichón Volcano was elucidated for the first time against a phytopathogenic fungus that causes significant losses of crops. Consequently, it’s important to carry out field experiments to understand the behavior of the bacterial strain in the presence of crops, specifically tomato crops with symptoms of diseases caused by phytopathogens and its possible action as a plant growth promoter, in addition to the implications of working with a thermophilic bacterium, since there are challenges such as the viability and stability of crops, the formulation and production at scale of these microorganisms, which are little explored, is of interest in the scientific and technological field for the development of new bioinoculants.