Draft Genome Sequence of Bacillus sp. Strain 11B20, a Promising Plant-Growth Promoting Bacterium Associated with Maize (Zea mays L.) in the Yaqui Valley, Mexico

Abstract

Strain 11B20 was isolated from a commercial field of maize (Zea mays L.) located in the Yaqui Valley, Mexico. The draft genome sequence revealed a genomic size of 3,759,824 bp, 41.6% G + C content, 973,288 bp N50, 2 L50, and 29 contigs. According to the 16S rRNA gene, strain 11B20 belongs to the genus Bacillus. Genome annotation revealed 3952 coding DNA sequences (CDSs) grouped into 319 subsystems. Among these, several CDSs were associated with traits related to plant growth promotion, including (i) virulence, disease, and defense (33 CDSs); (ii) iron acquisition and metabolism (28 CDSs); and (iii) secondary metabolism (6 CDSs), among others. In vitro, metabolic analysis (IAA, siderophore biosynthesis; phosphorus solubilization; and tolerance to thermal, hydric, and saline stress) confirmed the genomic background of this strain. Finally, in planta assays showed that the inoculation of Bacillus sp. 11B20 significantly (p ≤ 0.05) increased the root length (48.2%) and root dry weight (35.4%) versus non-inoculated maize plants. Thus, this is the first report of Bacillus sp. 11B20 as a promising beneficial strain for sustainable corn production, and further research is needed to ensure the success of the application of this strain in agriculture.

1. Introduction

As the world population and the threatening effects of climate change continue to increase, the global demand for crop production is also expected to grow, it is estimated that by 2050, food production is required to escalate by 70–100%, and cereals will keep playing an important role in food security since they ensure an adequate calorie and protein intake in diets [1,2]. Whole grains such as corn offer various dietary elements that can help address the triple challenge of undernutrition, micronutrient deficiencies, and overnutrition (overweight, obesity, and non-communicable diseases) [3]. Global corn production averages 1127 million tons per year; this crop thrives across a wide range of agroecological conditions, including varying temperatures, altitudes, latitudes, and soil types; however, yields per hectare can differ significantly [1]. In 2024, total corn grain production in Mexico exceeded 24.3 million metric tons, representing a 11.7% decrease compared to the previous year, which could be driven by climate variability, water scarcity, and stress conditions [4,5,6,7]. Corn, like multiple crops in the face of the effects of climate change, suffers biotic (i.e., Fusarium verticillioides) and abiotic (i.e., hydric, saline, and temperature) stress, affecting plant physiology and thereby causing significant losses of approximately 50–82% in crop productivity [8]. Furthermore, high-intensity farming methods have been employed to boost crop yields, leading to increased environmental problems, such as soil degradation, water scarcity, eutrophication, and deforestation, as well as negative human health effects [9].

The Yaqui Valley, located in the State of Sonora, Mexico, is recognized as the birthplace of the Green Revolution. As one of the most intensively farmed regions globally, it utilizes irrigation systems, fertilizers, improved cultivars, and other agricultural inputs to produce wheat, soybeans, corn, safflower, and sorghum [10,11]. In 2024, the State of Sonora, Mexico, contributed to national production with an average yield of 171,694 tons of corn. However, the soil used for corn production has resulted in low organic matter content (<1.5%), severe wind erosion due to arid climate (200 ton ha⁻¹ year⁻¹), saline or alkaline conditions, and reduced fertilizer-use efficiency, due to the excessive use of agrochemicals and intensive agronomic practices [11]. Studies in the Yaqui Valley have shown that agricultural management affects the structure and diversity of cultivable microbes. Moreover, conventional tillage and soil amendments alter bacterial community composition compared to more sustainable practices, highlighting the interplay between management and microbial ecology [12,13].

Thus, there is a growing need for the development of innovative agricultural practices to meet the sustainability challenges of global food demand. Beneficial microorganisms, particularly plant growth-promoting microorganisms (PGPMs), are crucial for maintaining functional and healthy agroecosystems [14]. Nowadays, PGPMs are widely employed as microbial inoculants for biofertilization; they act through direct mechanisms like nitrogen fixation, solubilization of both organic and inorganic phosphates, and the production of siderophores. They also promote plant growth by synthesizing phytohormones (indole acetic acid, gibberellins, and cytokinins), as well as ACC deaminase, which reduces stress caused by ethylene. In addition to these direct effects, PGPMs help control phytopathogens through direct mechanisms, like enhancing systemic resistance, competing for nutrients and space, and producing siderophores to capture iron. Furthermore, they produce various bioactive compounds, including antibiotics, lytic enzymes, hydrogen cyanide, and exopolysaccharides, which aid in defending plants against harmful pathogens [14,15]. As a result, the bioprospection of beneficial microorganisms has gained importance, driven by the need to boost crop production while reducing the reliance on agrochemicals and enhancing soil fertility [16,17]. The application of microbial inoculants, formulated from plant growth-promoting bacteria (PGPB), including genera like Azospirillum, Rhizobium, Azotobacter, Pseudomonas, and Bacillus, has emerged as a key strategy in sustainable agriculture over recent decades [18]. Among the abovementioned genera, Bacillus has gained significant attention due to its ability to produce heat-resistant endospores, a trait first described by Cohn in 1872; this genus, belonging to the phylum Firmicutes, class Bacilli, order Bacillales, and family Bacillaceae, is characterized by aerobic or facultative anaerobic growth, a Gram-positive bacillary morphology, flagellar motility, and a variable size (0.5 to 10 µm) [19]. Most Bacillus species thrive at neutral pH and are mesophilic, with optimal growth temperatures between 30 and 45 °C [20]. Genomic analyses of Bacillus species widely used in agriculture, such as B. subtilis, B. amyloliquefaciens, B. safensis, and B. pumilus, have revealed genetic determinants associated with plant growth promotion, nutrient acquisition, and stress tolerance, supporting the use of genome-based approaches to identify beneficial traits in novel isolates [21]. Currently, Bacillus strains make up the active ingredient in over 85% of bacterial inoculants sold globally, due to their notable and diverse metabolic capacities and multifaceted mechanisms of action [14,20]. Several species within this genus are being investigated for their potential to enhance crop production and yields through different mechanisms, such as lipopeptide production, nitrogen fixation, siderophore production, phosphate solubilization, lytic enzymes, toxins, and the induction of systemic resistance in plants [22]. Strain 11B20 was originally isolated during a broader study aimed at characterizing the cultivable soil microbiota associated with maize in the Yaqui Valley, Mexico. It is one of the most abundant isolates, motivating further genomic and functional characterization.

In this sense, this study presents a comprehensive genomic analysis and mining of strain 11B20, isolated from a commercial maize field (Zea mays L.) in the Yaqui Valley, Mexico. This process enabled the identification of genes associated with plant–bacteria interactions related to growth promotion, which was further validated through metabolic and in planta tests.

2. Materials and Methods

2.1. Bacterial Isolation and Culture Conditions

Strain 11B20 was isolated from a monoculture commercial field of maize (Zea mays L., Hipopótamo variaty—Asgrow) located in the Yaqui Valley, Mexico (27°25′38.3″ N 110°06′27.8″ W), a region characterized by semi-arid conditions, low annual precipitation (mean 384.5 mm), extreme temperatures (from −2 °C to 44 °C), and high relative humidity (annual mean 69%), as well as by low organic matter content (<1%), high salinity levels (EC > 4 dS/m), and alkaline soils (pH: 8.9) [13,23]. For this, 1 ha was subdivided into evenly distributed grid (100 m²) points, and soil subsamples were collected across the field, ensuring coverage of spatial heterogeneity within the plot. These subsamples were homogenized to obtain a composite (n = 10) soil sample of 10 kg. Then, 10 g was homogenized with 90 mL sterile distilled water, applying the serial dilution technique, up to 10⁻⁴; and 100 µL of 10⁻³ dilution was spread onto nutrient agar (NA) contained in Petri dishes, in triplicate, and incubated at 28 °C for two days. Strain 11B20 was characterized based on its morphological traits, such as cell and colony shape, color, elevation, and opacity. The Schaeffer–Fulton method was assessed to observe endospores [24]. The strain was cryopreserved at −80 °C in nutrient broth (NB) with glycerol and stored in the Native Endophytes and Soil Microorganisms Collection (COLMENA) (itson.mx/colmena, accessed on 16 August 2024) [9]. In addition, Fusarium verticillioides, a phytopathogenic fungus that reduces the maize yield in the field, was reactivated from a cryopreserved glycerol Eppendorf tube preserved in the microbial culture collection of Universidad Autónoma de Occidente (UAdeO), Unidad Regional Los Mochis, using a Petri dish containing potato dextrose agar (PDA), and incubated for seven days at 28 °C.

2.2. Whole Genome Sequence and Genomic Analysis

To explore the genomic background of strain 11B20 in relation to beneficial traits for crops, high-quality genomic DNA was extracted from a fresh culture, obtaining 1 × 10⁶ CFU/mL. A 40 µL aliquot of the cell suspension was lysed using 120 µL of TE buffer containing lysozyme (final concentration 0.1 mg/mL) and RNase A (ITW Reagents, Barcelona, Spain) (final concentration 0.1 mg/mL), followed by incubation at 37 °C for 25 min. Proteinase K (VWR Chemicals, Solon, OH, USA) at 0.1 mg/mL and SDS (Sigma-Aldrich, St. Louis, MO, USA) at 0.5% (v/v) were added, and the mixture was incubated for 5 min at 65 °C. The genomic DNA was then purified using an equal volume of SPRI beads and resuspended in EB buffer (10 mM Tris-HCl, pH 8.0). The extracted DNA (DNA ≥ 1 µg, concentration ≥ 20 ng/µL) was quantified with the Quant-iT dsDNA HS kit (ThermoFisher Scientific, Waltham, MA, USA) assay in an Eppendorf AF2200 plate reader (Eppendorf UK Ltd., Stevenage, Hertfordshire, UK) and diluted as appropriate [25].

To sequence that high-quality genomic DNA, the Illumina NovaSeq platform (2 × 250 bp) was used, and genomic DNA libraries were prepared using the Nextera XT Library Prep Kit (Illumina, San Diego, CA, USA), according to the manufacturer’s protocol, but with the following modifications: used DNA was doubled, and PCR elongation time was increased to 45 s. DNA quantification and library preparation were performed on a Hamilton Microlab STAR automated liquid handling system (Hamilton Bonaduz AG, Bonaduz, Switzerland) [26]. Libraries were then sequenced on an Illumina NovaSeq 6000 (Illumina, San Diego, CA, USA) using a 250 bp paired-end protocol, and Trimmomatic version 0.30 was used to remove adapter sequences and low-quality bases, with a sliding window-quality cutoff of 15. Reads shorter than 200 bp were discarded. Furthermore, a de novo assembly was generated using SPAdes version 3.15.4, under the “careful” parameter for error correction in reads [27]. The assembled contigs were ordered using Mauve Contig Mover version 2.4.0 according to the reference genome of Bacillus safensis subsp. safensis FO-36b^T (GenBank accession number ASJD01000027) based on the similarity (100%) and completeness (100%) based on the 16S rRNA gene, which was submitted to the EzBioCloud database to identify close phylogenetic relatives (species delimitation threshold of >98.7% similarity) [28,29]. Also, plasmid detection was carried out using PlasmidFinder 2.0 [30]. The quality of the assembly was evaluated using Quast version 4.4 and CheckM version 1.0.18 [31,32]. In addition, a phylogenetic tree based on the 16S rRNA gene was constructed using CLC Sequence Viewer version 8.0 (CLC bio A/S, Qiagen, Aarhus, Denmark), with the neighbor-joining method, and Bacillus vallismortis DV1-F-3^T (GenBank accession number JH600237) was designated as the outgroup. The 16S rRNA sequence of 11B20 was deposited in the NCBI GenBank under the accession number PV037057. To compare the genome of strain 11B20 with that of closely related Bacillus safensis strains, a protein clustering analysis using the OrthoVenn3 platform was performed [33,34,35,36,37,38]. Seven genomes obtained from NCBI (Bacillus safensis FB03, Bacillus safensis P1.5S, Bacillus safensis RGM 2450, Bacillus safensis VK, Bacillus safensis 1TAz, Bacillus safensis 32PB, and Bacillus safensis 8Taz) and strain 11B20’s genome were submitted for comparison. The protein sequences used for the analysis were obtained from annotated genomes, and clustering was based on sequence similarity and orthology criteria.

2.3. Genome Annotation

The genome annotation of strain 11B20 was performed by the Rapid Annotation Using Subsystem Technology (RAST) server version 2.0 (https://rast.nmpdr.org/), using the RASTtk pipeline based on the PathoSystems Resource Integration Center (PATRIC) (accessed on 2 November 2024) [39]. Additionally, Proksee version 1.2.0 (accessed on 5 November 2024) [40] was also used, a platform that incorporates the Rapid Prokaryotic Genome Annotation (Prokka) [41], to generate a circular chromosome map of strain 11B20, displaying CDSs, tRNAs, rRNAs, and guanine–cytosine (GC) skew content.

2.4. Genome Mining

The prediction of plant growth-promoting traits was done using the web resource PLaBAse (version 1.02) (https://plabase.cs.uni-tuebingen.de/pb/plabase.php, accessed on 12 November 2024) with the tool PGPT-Pred (accessed on 12 November 2024) [42]. For this, the protein FASTA file (.faa), generated through annotation in RAST, was uploaded to PLaBAse with the PGPT-Pred function to identify genomic features of strain 11B20 associated with plant growth promotion. Also, based on the OrthoVenn3 analysis, a protein FASTA file (.faa) of unique genetic features of strain 11B20 was submitted to such a platform to identify exclusive genomic traits. To identify biosynthetic gene clusters (BGCs) associated with biocontrol, genome mining of strain 11B20 was performed on the Antibiotics & Secondary Metabolites Analysis Shell (antiSMASH) version 7.0 web server (https://antismash.secondarymetabolites.org, accessed on 29 November 2024) under the “relaxed” parameter. Additional antiSMASH modules enabled included KnownClusterBlast, SubClusterBlast, ActiveSiteFinder, RREFinder, and TFBS analysis. The pipeline was configured to identify different types of biosynthetic pathways involved in the production of secondary metabolites, for example, gene clusters encoding non-ribosomal peptide synthetases (NRPSs); type I and type II polyketide synthases (PKSs); and the ribosomally synthesized and post-translationally modified peptide (RiPP) classes of lanthipeptides, lasso peptides, sactipeptides, and thiopeptides [43].

2.5. Metabolic Characterization

Strain 11B20 was metabolically characterized in terms of key biochemical activities most commonly associated with plant growth promotion, identified in genome mining. Production of indole acetic acid (IAA) [44] was assessed by inoculating, in triplicate, 1 × 10⁴ colony-forming units (CFU) in 10 mL of NB supplemented with 100 ppm of L-tryptophan. The bacterial culture was incubated at 30 ± 2 °C for 5 days under continuous agitation at 120 rpm in a rotary shaker. Then, 1 mL of the culture was centrifuged at 13,000 rpm for 10 min. The production of IAA was determined by adding 200 µL of the Salkowski reagent to 100 µL of supernatant, incubating at room temperature for 30 min in the dark, and then the optical density at 530 nm (OD₅₃₀) was measured by using a BioTek ELx800 Absorbance^TM Microplate Reader (BioTek Instruments, Winooski, VT, USA), and correlated to an IAA standard curve. The capacity to solubilize phosphate was analyzed using Pikovskaya (PVK) agar [45], inoculating 1 × 10⁴ CFU of this strain, in triplicate, in a Petri dish and incubating at 28 °C for 7 days; the presence of a transparent halo around the inoculated colony was noted as a positive result, and to determine the efficiency of solubilization of phosphate (ES), the formula ES = $\left({\displaystyle \frac{\mathrm{H}\mathrm{D}-\mathrm{C}\mathrm{D}}{\mathrm{C}\mathrm{D}}}\right)\times 100$ was used, where HD means the halo diameter, and CD is the colony diameter. The ability to produce siderophores was determined using Chrome Azurol S (CAS) agar [46]. The medium was inoculated with 1 × 10⁴ CFU of strain 11B20, in triplicate, and incubated for 7 days at 28 °C. A positive result was indicated by the appearance of a yellow-orange halo around the inoculated colony, and to determine the efficiency of production of siderophores (EP), the formula EP = $\left({\displaystyle \frac{\mathrm{H}\mathrm{D}-\mathrm{C}\mathrm{D}}{\mathrm{C}\mathrm{D}}}\right)\times 100$ was used, where HD means the halo diameter, and CD is the colony diameter.

In addition, the tolerance to abiotic stress was analyzed by inoculating 1 × 10⁴ CFU strain 11B20 on Falcon tubes with NB containing distinct supplements such as (i) Sodium Chloride (5%, 68 dS/m) to determine saline stress, and (ii) Polyethylene Glycol 6000 (10%, −0.84 mPa) to simulate water deficit conditions; both assays had an incubation of 28 °C and 120 rpm for three days [47]. For thermal stress, strain 11B20 (1 × 10⁴ CFU) was inoculated on Falcon tubes containing NB and incubated at 43.5 °C and 120 rpm for three days, and a control treatment consisting of strain 11B20 was inoculated on a Falcon tube containing NB, incubated at 28 °C and 120 rpm for three days. Each assay was carried out in triplicate. Stress tolerance (ST) of strain 11B20 was calculated with the formula %ST = $(100-\left({\displaystyle \frac{\mathrm{B}\mathrm{G}\mathrm{s}-\mathrm{B}\mathrm{G}}{\mathrm{B}\mathrm{G}}}\right))\times 100$, where BGs refers to the colony-forming units obtained under stress conditions, and BG refers to the colony-forming units of the control treatment (without abiotic stress) [48].

2.6. Biological Control Assay

Dual confrontation assays of strain 11B20 against maize phytopathogens Fusarium verticillioides, Curvularia sp. H3-5, and Fusarium sp. H2-3 were conducted. Fusarium sp. H2-3 and Curvularia sp. H3-5 were previously isolated from symptomatic maize plants collected from a commercial field in the Yaqui Valley, Mexico. For each phytopathogen, dual culture assays were performed on potato dextrose agar (PDA) plates. A 0.5 mycelial plug from a grown culture was placed at the center of the plate, while two of the bacterial biomasses were positioned equidistantly, 1 cm from the edge of the plate. Plates were incubated at 30 °C for 5 days [49]. The experiment was conducted with three independent replicates, comparing control treatments only inoculated with the studied phytopathogens. Radial fungal growth was measured five days post-inoculation.

2.7. Maize Growth Promotion Assay

To study the maize plant and strain 11B20 interaction, a greenhouse assay was carried out, where twenty-four maize seeds (DK-4050) were sown in a forest tray (cavity diameter of 4.7 cm) that contained commercial substrate GTX PRO-MIX (PRO-MIX, Québec, QC, Canada). Strain 11B20 was grown on an Erlenmeyer flask containing 250 mL of NB and incubated for 2 days at 30 °C, with constant shaking at 120 rpm. After incubation, the bacterial suspension was centrifuged at 3600 rpm for 10 min, and the resulting pellet was washed three times before being resuspended in 100 mL of sterile water. Subsequently, optical density was measured (630 nm) and adjusted to an OD of 0.5 (8.3 × 10⁷ CFU/mL) using sterile water. Thus, 3 mL of strain 11B20 was inoculated into the substrate immediately after sowing each seed in the forest tray; the control treatment consisted of non-inoculated plants, replacing the cell suspension with 3 mL of distilled water. After three weeks from inoculation, plant biometric parameters were evaluated, such as stem height, root length, and root and plant fresh and dry weight [50].

2.8. Statistical Analysis

All statistical analyses were performed using Student’s t-test (p ≤ 0.05) in STATGRAPHICS Plus version 5.1 (Statgraphics Technologies, Inc., Virginia, VA, USA).

3. Results

3.1. Morphological Characterization

Strain 11B20, isolated as one of the most abundant bacterial strains associated with maize in this study, presented morphological traits of white cream, round, irregular, flat, entire to slightly undulate margins, and opaque colonies and Gram-positive rod-shaped cells, with a size of 1.21 × 0.47 µm (Figure 1). Spore staining confirmed that strain 11B20 produces endospores, which appeared green within pink/red vegetative cells. Microscopic analysis revealed that the endospores were predominantly centrally located, indicating typical Bacillus-like sporulation under nutrient-limited conditions (Figure 1).

3.2. Whole-Genome Sequence, Genomic Analysis, and Phylogenetic Analysis

After sequencing the genomic DNA of strain 11B20, a total of 937,698 paired-end reads (2 × 250 bp) were obtained. The assembled genome presented 19 contigs (≥200 bp), resulting in 3,759,824 bp, 41.6% G + C content, an N50 value of 973,288 bp, and an L50 of 2. No plasmids were detected. This Whole-Genome Shotgun project was deposited in DDBJ/ENA/GenBank under the accession number JBLZWZ000000000. The version described in this paper is version JBLZWZ010000000. The length of the 16S rRNA gene sequence of strain 11B20 was 1139 bp. Based on the 16S rRNA gene, strain 11B20 showed 100% similarity to Bacillus safensis subsp. safensis FO-36b^T, as well as to B. safensis subsp. osmophilus BC09^T and B. zhangzhouensis DW5-4^T (

Table 1. 16S rRNA-based similarity of strain 11B20.

Taxon Name	Strain	GenBank Accession Number	Similarity (%)
Bacillus safensis subsp. safensis	FO-36bT	ASJD01000027	100
Bacillus safensis subsp. osmophilus	BC09T	KY990920	100
Bacillus zhangzhouensis	DW5-4T	JOTP01000061	100
Bacillus pumilus	ATCC 7061T	ABRX01000007	99.91
Bacillus australimaris	NH7I_1T	JX680098	99.91
Bacillus altitudinis	41KF2bT	ASJC01000029	99.64
Bacillus xiamenensis	HYC-10T	AMSH01000114	99.55

). According to the 16S rRNA-based phylogenetic tree and morphological traits, strain 11B20 was taxonomically affiliated to the genus Bacillus (Figure 2).

To identify shared and unique genomic features, the genome of Bacillus sp. 11B20 was compared with seven publicly available genomes of Bacillus safensis using OrthoVenn3. The analysis revealed a core set of 3367 orthologous gene clusters shared among all strains analyzed (Figure 3). Due to the number of genomes analyzed (n = 8), an UpSet plot was generated instead of a Venn diagram; this approach enabled a more efficient and interpretable analysis of conserved and unique gene families. Notably, strain 11B20 also possesses 172 singleton clusters, i.e., gene clusters not present in any of the other seven genomes included in the comparison (

Table 2. Proteins, clusters, and singletons of Bacillus sp. 11B20, Bacillus safensis RGM 2450, Bacillus safensis VK, Bacillus safensis 8TAz, Bacillus safensis 1TAz, Bacillus safensis P1.5S, Bacillus safensis 32PB, and Bacillus safensis FB03, according to OrthoVenn3.

Species	Proteins	Clusters	Singletons
Bacillus sp. 11B20	3952	3767	172
Bacillus safensis RGM 2450	3978	3790	171
Bacillus safensis VK	3950	3677	260
Bacillus safensis 8TAz	3847	3819	20
Bacillus safensis 1TAz	3839	3819	13
Bacillus safensis P1.5S	3830	3692	128
Bacillus safensis 32PB	3826	3815	6
Bacillus safensis FB03	3761	3678	72

).

3.3. Genome Annotation

RAST annotation identified a total of 80 RNAs and 3952 coding DNA sequences (CDSs) in the genome of Bacillus sp. 11B20, distributed across 319 subsystems (Figure S1). Genes related to biocontrol were detected, including those associated with virulence, disease, and defense (33 CDSs), such as antibiotics and toxic compound resistance (14 CDSs); invasion and intracellular resistance (12 CDSs); and bacteriocins, ribosomally synthesized antibacterial peptides (7 CDSs). Other CDSs involved in plant growth promotion were found, including (i) iron acquisition and metabolism (28 CDSs), specifically siderophores (16 CDSs), which function as iron chelators, limiting iron availability to phytopathogens; (ii) secondary metabolism (6 CDSs), including plant hormones (4 CDSs); and (iii) phosphorus metabolism (10 CDSs). Also, subsystems contributing to bacterial resilience for potential agricultural applications were identified, including stress response (43 CDSs), such as osmotic stress (14 CDSs) and oxidative stress 13 (CDSs). Moreover, in support of these findings, the circular chromosome map generated via Prokka identified a total of 3812 CDSs, 74 tRNAs, and 1 tmRNA (Figure 4).

3.4. Genome Mining

Based on mining the genome of Bacillus sp. 11B20, 27% of its genome is related to colonizing plant systems, 21% to competitive exclusion, 21% to stress control and biocontrol, 12% to biofertilization, 10% to phytohormones and plant signal production, 7% to bioremediation, and 2% to plant immune response stimulation (Figure 5 and Table S1). Additionally, the PGPT Predictor identified 23 genes associated with iron acquisition via siderophore production: achromobactin (1), bacillibactin (5), bisucaberin (1), coprogen (1), desferrioxamine (1), enterobactin/enterochelin (6), ferrichrome/ferrioxamine (1), mycobactin (1), petrobactin (1), rhizobactin (2), and staphyloferrin b (2) (Table S1). A total of 112 genes involved in phosphate solubilization were found (Table S1), comprehending genes involved in biosynthesis and transport of acetic acid (8), aconitic (3), butyric (6), citric (5), formic (2), fumaric (5), galactonic (1), gluconic (4), and glycolic acid (1). Also, 382 genes related to abiotic stress tolerance were detected, such as high-temperature (21) and low-temperature (7) tolerance, osmotic stress (54), salt stress (176), osmotic stress (54), and oxidative stress (92). Following biotic stress, 43 genes involved in the metabolism of bactericidal compounds were identified. In addition, PLaBAse analysis revealed that the unique protein-coding genes of strain 11B20 were related to colonizing the plant system, competitive exclusion, phytohormones and plant signal production, and stress control (Figure 6 and Table S2).

On the other hand, the antiSMASH version 7.0 identified three regions, detecting three biosynthetic gene clusters (BGCs) in regions 5, 7, and 9: lichenysin, a lipopeptide produced non-ribosomally; bacilysin, a dipeptide antibiotic; and bacillibactin/bacillibactin E/bacillibactin F, chelating agents (

Table 3. BGCs found in Bacillus sp. 11B20, identified by antiSMASH version 7.0.

Region	Most Similar Known Cluster	Similarity (%)	Type	From	To
5.1	Lichenysin	92	NRPS	177,309	261,032
9.1	Bacilysin	85	Other	679,296	720,717
9.2	Bacillibactin/bacillibactin E/bacillibactin F	80	NRP-metallophore and NRPS	953,627	1,005,355

and Figure 4).

3.5. Metabolic Characterization and Biological Control Assay

Bacillus sp. 11B20 exhibited functional traits linked to plant growth promotion, including the production of IAA, phosphorus solubilization, and siderophore production, as well as tolerance to abiotic stress (

Table 4. Metabolic traits of Bacillus sp. 11B20.

Production of IAA (µg/mL)	Phosphate Solubilization (%)	Siderophore Production (%)	Tolerance to Thermal Stress (%)	Tolerance to Saline Stress (%)	Tolerance to Hydric Stress (%)
3.7 ± 0.1	39.4 ± 1.3	7.4 ± 1.3	99.94	99.5	98.4

). Strain 11B20 did not show biological control against the tested phytopathogens (Fusarium verticillioides, Fusarium sp. H2-3, and Curvularia sp. H3-5; Figure S2).

3.6. Maize Growth Promotion Assays

The inoculation of Bacillus sp. 11B20 in maize plants showed significant (p ≤ 0.05) increments compared to non-inoculated treatments in root length (48.2%) and root dry weight (35.4%), while plant dry weight presented a decrease (−67.7%), and stem height did not show significant differences (

Table 5. Maize plants’ growth promotion by the inoculation of Bacillus sp. 11B20.

	Non-Inoculated Maize (Control)	Inoculated Maize (Bacillus sp. 11B20)	Increment vs. Control (%)
Stem height (cm)	8.81 ± 2.26	8.8 ± 1.96	0.11%
Root length (cm)	5.68 ± 1.21	8.42 ± 1.48 *	48.24%
Stem dry weight (mg)	172.93 ± 40	55.8 ± 0.01 *	−67.73%
Root dry weight (mg)	91.89 ± 13	124.43 ± 14 *	35.41%

Asterisks (*) indicate statistically significant differences among treatments. Means (n = 24).

).

4. Discussion

The genus Bacillus is a highly adaptable and effective beneficial agent for crops, due to its genomic and metabolic background [22]. These attributes underscore the importance of this genus in advancing sustainable practices in agriculture. In this sense, strain 11B20 was isolated from a commercial field of maize, which presented white cream, round, irregular, flat, entire to slightly undulate margins, and opaque colonies, typical characteristics of Bacillus strains [10,48]. At the microscopic level, Gram-positive rod-shaped cells are observed, and this observation agrees with previous descriptions of this genus [20,51]. After its genome sequencing, the 16S rRNA gene was obtained and used for its taxonomic affiliation due to its high conservation and variable regions that allow for differentiation between bacterial genera [52,53]. The analysis of the 16S rRNA gene (Table 1; Figure 2) confirmed strongly that strain 11B20 belongs to the genus Bacillus. In addition, comparative genomic analysis supported the taxonomic placement of Bacillus sp. 11B20 within the B. safensis clade and revealed 3367 orthologous clusters shared with other strains of the species (Figure 3), supporting a strong genomic core. Further genomic exploration of the 172 unique orthologous clusters in Bacillus sp. 11B20 revealed genes potentially associated with adaptive functions relevant to rhizosphere fitness and plant-growth promotion (Figure 6 and Table S2). For instance, puuE, involved in polyamine metabolism, may contribute to stress tolerance in the host plant via regulation of putrescine pathways, a known plant stress signal [54]. Genes such as fadD and ABC_CD_A suggest enhanced capacities for lipid metabolism and transport, important under drought or osmotic stress [55]. The presence of lantibiotic-associated genes (nisF, spaF, cprA, and epiF) also points to uncharacterized antimicrobial potential, even though no fungal antagonism was observed [56]. Additionally, stress-response regulators such as hipB and DNA repair genes (dut, xerD, and nrdB) could support bacterial survival in competitive and fluctuating soil environments [57,58]. These findings underscore the value of comparative genomic approaches in identifying hidden genomic traits with biotechnological relevance [59,60]. These genes should be regarded as candidates for future functional characterization rather than direct evidence of specific ecological traits. Similar patterns have been reported in other Bacillus genomes, where core plant-beneficial functions are conserved, but strain-specific genes are commonly involved in regulation and metabolism related to niche adaptation [61,62]. The genome of Bacillus sp. 11B20 harbored a wide array of genes associated with sporulation and germination that likely underpin its environmental resilience. The presence of spore coat protein genes such as cotA, cotD, cotE, cotH, cotN, cotV, cotX, cotY, and cotZ suggests a robust coat architecture capable of resisting heat, desiccation, and chemical stress, consistent with well-characterized assembly frameworks in B. subtilis (cotE and cotY form essential coat and crust layers) [63,64]. This genomic potential was phenotypically supported by malachite green staining, which revealed green-stained endospores within pink/red vegetative cells, indicating that Bacillus sp. 11B20 can form endospores under nutrient-limited conditions. Additionally, the detection of germination-related genes, including those in the gerAA, gerAB, gerAC, gerBA, gerBB, gerBC, gerD, gerK, gerKC, gerPA, gerPB, gerPC, gerPE, gerPF, and gerPQ operons, indicates that strain 11B20 contains the molecular machinery needed for rapid exit from dormancy under favorable conditions, as seen in B. subtilis models [65,66,67]. Moreover, regulatory elements, such as spo0A, spo0B, spo0E, spo0F, spo0M, and components of the sporulation-specific sigma factor cascade—spoIIIA through spoIIID, spoIIAA, spoIIAB, spoIIB, and spoIVFB—are present and suggestively functional [68,69,70,71,72,73]. These genes orchestrate the initiation and progression of sporulation. Taken together, the conservation of these genes underscores the ecological robustness of Bacillus sp. 11B20 and supports its potential use in microbial formulations where spore stability, longevity, and predictable germination are essential for field development.

To explore the beneficial role of Bacillus sp. 11B20 in corn production, its genome was mined and correlated with metabolic and functional traits. Regarding biological control activity, genes related to this bioactivity were detected in the genome of strain 11B20 (Table 3), such as lchAA, lchAB, lchAC, and lchAD, detected during PGPT-Pred analysis, which belong to the lycA-TE gene cluster and encode lichenysin. Nevertheless, Prokka annotation identified genes srfAA, srfAB, and srfAC (Figure 4), which are key components of the surfactin biosynthetic pathway; this apparent discrepancy may reflect the high genetic and structural similarity between surfactin and lichenysin, two closely related lipopeptides that share homologous non-ribosomal peptide synthetase modules but differ slightly in their amino acid composition and regulatory elements [74,75,76]. Such overlap often leads to inconsistencies among annotation and mining pipelines, emphasizing the importance of combining multiple genome-mining tools to improve metabolite prediction reliability. In addition, genes involved in converting prephenate to bacilysin, causing bacterial transcriptomic and phenotypical changes, and bacilysin extracellular expression, were detected, i.e., bacA, bacB, bacC, bacD, bacF, bacG, degU, and scoC [77]. Also, dhbA, dhbB, dhbC, and dhbE genes were detected, which are the core biosynthetic genes for bacillibactin, providing function during iron deficiency in soil and associated with the biocontrol of pathogens [78]. Thus, the biological control activity of strain 11B20 was tested against Fusarium verticillioides, Fusarium sp. H2-3, and Curvularia sp. H3-5; however, fungal growth inhibition was not observed (Figure S2). This finding could be explained: (i) lichenysin, a surface-active lipopeptide that seems to be synthesized during growth under aerobic and anaerobic conditions, has been reported to have antibiotic properties against important bacterial in humans (Pseudomonas aeruginosa, Salmonella typhi, Shigella flexneri, and Escherichia coli) [79,80]; (ii) bacilysin is an antibiotic compound that causes cell lysis in pathogenic bacteria (Microcystis aeruginosa, Aphanizomenon flos-aquae, Nostoc sp., Anabaena sp., E. coli, and Staphylococcus aureus) [77]; and (iii) bacillibactin/bacillibactin E/bacillibactin/F is involved in chelating Fe and acting as an antibacterial agent against P. syringae, P. marginalis, S. aureus, E. faecallis, and Klebsiella pneumoniae [81,82,83]. Although the BGCs are found in the genome of Bacillus sp. 11B20 have previously been reported in the biosynthesis of metabolites with antibacterial activity, Bacillus sp. 11B20 does not possess metabolites involved in the biocontrol of fungal phytopathogens Fusarium verticillioides, Curvularia sp. H3-5, or Fusarium sp. H2-3; these findings suggest that its antifungal potential may be limited or mediated by mechanisms not detected under the conditions tested, thereby warranting further investigation [84]. Additionally, it is important to consider the biosafety implications of these findings. Lichenysin, although reported to exhibit antibacterial properties, has also been described as a cytotoxic lipopeptide with potential food safety concerns when produced at high levels, as shown in Bacillus licheniformis strains associated with foodborne illness [76,85]. Therefore, future studies should assess whether lichenysin is actively produced by strain 11B20 to evaluate any potential risks associated with its use in agricultural applications.

On the other hand, according to the RAST annotation, in the genome of Bacillus sp. 11B20, four CDSs related to tryptophan biosynthesis were detected: APRT, PRAI, TSa, and TSb. Other genes of interest are trpA, trpB, trpC, trpD, trpF, and trpS, which contribute to the production of IAA, as well as ethylene and ammonia [78]. Also, the gene aldH was detected, since aldehyde dehydrogenase genes are key to the indole-3-pyruvic acid pathway (IPA), one of the most common pathways for IAA biosynthesis in microorganisms. In this pathway, tryptophan is converted to indole-3-pyruvic acid (IPA) and then to indole-3-acetaldehyde (IAAld); finally, IAAld is converted to IAA via the action of aldehyde dehydrogenases [86]. This metabolic ability of Bacillus sp. 11B20 was confirmed by using the Salkowski method, showing a production of 3.7 ± 0.1 µg/mL of IAA. This is a desirable metabolism in beneficial microorganisms since it is involved in the regulation of plant growth and development, such as cell division, elongation, fruit development, and senescence [86,87].

Additionally, plant-associated microbiota can enhance nutrient availability by mobilizing otherwise inaccessible resources, such as inorganic phosphate and iron, through processes like solubilization, mineralization, or the secretion of iron-chelating siderophores [88]. This strain showed genes related to phosphate solubilization, which results from the combination of metabolites and the functional genes that regulate them; among these genes, pqq and GDH are involved in the conversion of glucose to gluconic acid, the principal organic acid produced for the dissolution of phosphorus [89,90]. In addition, the putative genes phoA and phn (as well as other related genes) are involved in the release of inorganic phosphorus by promoting the synthesis of enzymes to hydrolyze organic phosphorus in soil [89,91]. Phosphate solubilization by bacteria is primarily driven by the secretion of low-molecular-weight organic acids, which chelate metal cations (e.g., Ca²⁺, Fe³⁺, and Al³⁺) and lower the pH in the rhizosphere, increasing the solubility of otherwise insoluble phosphates. Organic acids such as gluconic, citric, acetic, and formic acid have been reported in a variety of phosphate-solubilizing bacteria and are associated with enhanced phosphate release from mineral sources [92,93,94]. Bacillus sp. 11B20 showed the capacity to solubilize phosphates in 39.4 ± 1.3%, confirming the functionality of the detected genes related to phosphorus solubilization. This metabolic trait is a desired trait in beneficial bacteria since phosphorus is an essential nutrient for plant growth and development, contributing significantly to metabolic processes and various physiological functions; in arid and semi-arid soils, phosphorus is often the primary limiting factor for crop yield [95].

On the other hand, the genome of Bacillus sp. 11B20 revealed 16 CDSs related to siderophore production, such as Sdab, Sdad, SbsM, PchA, PchB, PchC, PchD, and PchEPchF, among others. Genes associated with iron acquisition and metabolism were detected, such as (i) feuA, feuB, feuC, and fecD (iron transportation) [78]; and (ii) dhbA, dhbB, dhbC, dhbE, dhbF, and YusV (siderophore synthesis) [96]. Under iron-deficient conditions, the dhbACEF gene cluster drives the synthesis of bacillibactin, which is then secreted into the environment to chelate iron. The Fe–siderophore complex is subsequently imported into the cell via the feuABC-YusV uptake system [96]. This genomic background was confirmed by the ability of Bacillus sp. 11B20 to produce siderophores in CAS medium, with a 7.4 ± 1.3%. Siderophores play a key role in various ecological processes, including iron biogeochemical cycling in soils, pathogen competition, plant growth promotion, and cross-kingdom signaling; the widespread presence of siderophore gene clusters across diverse biosynthetic pathways suggests that traits enhancing competition for scarce resources confer a selective advantage to microorganisms, aiding in their successful colonization of plant roots [88].

Shifting the focus to abiotic stress, Bacillus sp. 11B20 showed 99.5% tolerance to saline, 98.4% to hydric, and 99.95% to thermal stress, respectively [84]. These metabolic traits could be associated with the 14 CDSs related to osmotic stress that were detected in the genome of the studied strain. Some of them were BetA, betB, BetC, betI, BetT, OpuAA, OpuAB, and OpuAC, which are involved in bacterial osmoprotectant by facilitating the uptake and metabolism of choline into the osmoprotectant glycine betaine, which helps microorganisms survive high salt or drought conditions, as is important under arid or semi-arid environmental conditions [97,98] such as those observed in the Yaqui Vally, Mexico, where Bacillus sp. 11B20 was isolated.

Thus, based on genome mining and metabolic tests, Bacillus sp. 11B20 is a promising strain to enhance the growth of plants, and its promise was confirmed since its inoculation significantly (p ≤ 0.05) increased root length by 48.2% and root dry weight by 35.4% versus non-inoculated plants (Table 5). The development of root surface area is directly related to the plant’s efficiency in acquiring nutrients [99,100]. Similarly, this root development enhances the interaction with the surrounding soil by releasing carbon compounds that stimulate microbial activity, influencing both short- and long-term soil nutrient pools [101]. For example, for low-mobility nutrients like phosphorus, fine roots play a crucial role in absorption, contributing to increased biomass and enhanced stress tolerance in later growth stages [64,66,67]. Also, auxin biosynthesis may contribute to root elongation and lateral root development in bacterized plants; however, this relationship was not directly evaluated in this study. Interestingly, the stem dry weight decreased by 67.7% in inoculated plants. Such a shift may reflect an early-stage resource allocation trade-off, where the plant diverts assimilates from aboveground to belowground growth to prioritize root establishment. Similar patterns—increased root: shoot ratios under early stress or inoculation—have been reported in plants exposed to drought or nutrient limitation, where roots are preferentially supported to enhance survival and resource acquisition. It is plausible that, given additional time and under field-like conditions, initial investment in root development could benefit subsequent shoot and total biomass accumulation. Future studies should thus include longer growth durations and more realistic soil systems to evaluate long-term implications of this physiological allocation strategy [102,103,104,105]. Integrative approaches such as time-course evaluation, biomass allocation patterns, quantification of bacterial colonization, and metabolite profiling to detect plant hormones (e.g., auxins, cytokinins, and ethylene) or lipopeptides that might modulate growth responses have been recommended to disentangle plant–microbiome interactions that cause shifts in biomass allocation [104,105]. The present study contributes significantly to the biotechnological exploration of native microbial resources from the Yaqui Valley, a region facing increasing abiotic stress due to climate change. The performance of plant growth-promoting bacteria is known to be influenced by environmental factors. Strain Bacillus sp. 11B20 was subjected to an integrative analysis that combined morphological, biochemical, genomic, and in planta evaluations, reinforcing its potential as a dual-function strain with both plant growth-promoting and antagonistic properties. Further studies should evaluate strain 11B20 across multiple locations, soil types, and maize varieties to assess its robustness and practical applicability. Importantly, this work underscores the value of coupling phenotypic validation with bioinformatics-driven genome mining to identify robust candidates for bioinoculant development, particularly for sustainable maize production in semi-arid agroecosystems.

5. Conclusions

The bioprospection of PGPB through genomic and metabolic approaches offers a suitable alternative for identifying promising active ingredients for microbial inoculants used in sustainable agriculture. Strain 11B20, taxonomically affiliated with the genus Bacillus, revealed diverse gene clusters related to plant growth promotion that were validated through metabolic and in planta assays. The integration of genomic approaches with in vitro and in planta studies is crucial for advancing microbial-based solutions for sustainable agriculture. Thus, this is the first report of Bacillus sp. 11B20 as a promising beneficial strain for sustainable corn production; in this sense, further research (i.e., additional action modes, bioformulation, and in-field studies) is needed to ensure the success of the application of Bacillus sp. 11B20 in agriculture.