Stress-Tolerant Bacillus Strains for Enhancing Tomato Growth and Biocontrol of Fusarium oxysporum under Saline Conditions: Functional and Genomic Characterization

Stress-Tolerant Bacillus Strains for Enhancing Tomato Growth and Biocontrol of Fusarium oxysporum under Saline Conditions: Functional and Genomic Characterization | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Stress-Tolerant Bacillus Strains for Enhancing Tomato Growth and Biocontrol of Fusarium oxysporum under Saline Conditions: Functional and Genomic Characterization María F. Valencia-Marin, Salvador Chávez-Avila, Edgardo Sepúlveda, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5671788/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Mar, 2025 Read the published version in World Journal of Microbiology and Biotechnology → Version 1 posted 11 You are reading this latest preprint version Abstract Soil salinity is a major limiting factor for agricultural crops, which increases their susceptibility to pathogenic attacks. This is particularly relevant for tomato ( Solanum lycopersicum ), a salt-sensitive crop. Fusarium wilt, caused by Fusarium oxysporum f. sp. lycopersici , is a significant threat to tomato production in both greenhouse and field environments. This study evaluated the salinity tolerance, biocontrol, and plant growth-promoting properties of Bacillus velezensis AF12 and Bacillus halotolerans AF23, isolated from soil affected by underground fires and selected for their resistance to saline conditions (up to 1000 mM NaCl). In vitro assays confirmed that both strains produced siderophores, indole-3-acetic acid (IAA), and proteases, and exhibited phosphate solubilization under saline stress (100–200 mM NaCl). AF23 exhibited synergistic interactions with AF12, and inoculation with either strain individually or in combination significantly improved the growth of the Bonny Best tomato cultivar under 200 mM saline stress, leading to increased shoot and root weight, enhanced chlorophyll content, and higher total biomass. The biocontrol potential of AF12 and AF23 was evaluated in tomato plants infected with F. oxysporum . Both strains, individually or combined, increased shoot and root weight, chlorophyll content, and total biomass under non-saline conditions, promoting growth and reducing infection rates under saline stress (100 mM NaCl). Genomic analysis revealed that both strains harbored genes related to salt stress tolerance, biocontrol, and plant growth promotion. In conclusion, Bacillus AF23 and AF12 demonstrated strong potential as bioinoculants for enhancing tomato growth and providing protection against F. oxysporum in saline-affected soils. Bacilli biocontrol Fusarium wilt disease genome halotolerant Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Tomato ( Solanum lycopersicum L.) is one of the most widely cultivated vegetables worldwide. It is an important aspect of the human diet and contains vitamins A and C and minerals(Adedayo et al. 2022a ; Olowe et al. 2022 ). Tomatoes also contain antioxidants such as lycopene, which has been found to be effective in preventing cancer. As a result of the global consumption of this vegetable(Emmanuel and Babalola 2020 ), it recorded over 186.82 million tonnes of production in 2022, which accounted for 36.97t/ha production. However, despite the importance of this vegetable, it continues to face a continual threat of both biotic and abiotic stressors, which cause a decline in tomato yield and quality(Fadiji et al. 2022 ). Among these stress factors, excessive accumulation of soluble soil salinity has been found to severely impact plant growth and reduce crop yields, leading to economic losses and land degradation. High concentrations of soluble salts such as sodium chloride (NaCl), calcium chloride (CaCl2), and magnesium chloride (MgCl2) contribute to the high electrical conductivity of saline soils. Among these, NaCl accounts for most of the soluble salts in soils with salinity problems(Gupta et al. 2022 ). Soil was classified as saline when its electrical conductivity in the root zone (from 0 to 60 cm depth) exceeded 4 dS m − 1 (approximately 40 mM NaCl)(Bonarota et al. 2022 ). Soil salinity is a significant challenge for agriculture worldwide, particularly in arid and semi-arid regions, and can arise from both natural and human-induced processes(Huang et al. 2017 ). Naturally occurring salinity often develops in arid and semi-arid regions, where high evaporation rates lead to high salt concentrations at the soil surface. In coastal areas, seawater intrusion can bring salt into freshwater aquifers, thereby contaminating agricultural soils(Stavi et al. 2021 ). Climate change is a primary driver of increasing soil salinity because global temperature increases will lead to increased evaporation rates and a rise in sea levels. Additionally, altered precipitation patterns can result in more frequent and intense droughts, further worsening the soil salinity(Eswar et al. 2021 ; Singh 2022 ). Human activities also contribute to soil salinization. Overirrigation, particularly in arid regions, can lead to waterlogging, which prevents salts from leaching out of the soil profile. Simultaneously, increased groundwater pumping for irrigation depletes freshwater resources, forcing farmers to rely on deeper aquifers, which often contain higher salt content. Moreover, deforestation and land mismanagement can disrupt the natural balance of water and salt in the soil, exacerbating this problem(Stavi et al. 2021 ; Maertens et al. 2022 ). Fusarium oxysporum , which causes wilt diseases, is a major pathogen of tomatoes that accounts for a significant loss in yield and crop quality(Olowe et al. 2022 ). Moreover, the interaction of Fusarium oxysporum , which causes wilt diseases, with salt stress has been found to vary depending on the formae speciales (f. sp.) and host plants involved(Akanmu et al. 2024 ). Moreover, increased disease incidence following irrigation with high-salinity water has been reported in earlier investigations. However, the global population is constantly increasing, making one of the main challenges for agriculture to meet the growing demand for food. This indicates the urgent need to boost agricultural production in the coming years(Jiménez-Mejía et al. 2022 ). However, the yield of economically important crops is affected by both biotic and abiotic stressors(Kumar et al. 2020 ). Given the challenges posed by salinity and pathogenic Fusarium oxysporum in tomatoes, several strategies have been developed to mitigate its negative effects on plant growth(Adedayo et al. 2022b ). One approach involves the generation of salt-resistant plants through genetic modifications. However, owing to the time and costs involved, this is often not a viable solution for large-scale agriculture. As an alternative, the use of halotolerant microorganisms such as plant growth-promoting bacteria (PGPB) has emerged as a promising method for enhancing crop growth under saline stress conditions(Sunita et al. 2020 ). PGPB are bacterial species that significantly affect plant growth, yield, and disease resistance via various mechanisms(Khatoon et al. 2020 ; Morales-Cedeño et al. 2021 ). Bacteria can either directly alleviate the harmful effects of salinity or indirectly enhance plant tolerance to salinity. One direct method is to take up excess ions from the soil to prevent their accumulation in the root zone. Additionally, bacteria can produce compatible solutes that help plants maintain their osmotic balance and prevent water loss. They also produce antioxidants that protect plants from the oxidative stress caused by salinity. Indirectly, bacteria promote plant growth and development, making them more resilient to stress(Liu et al. 2022 ), and contributing to the improvement of soil structure and nutrient availability by producing organic matter and enhancing soil aggregation, which facilitates water infiltration and nutrient uptake(Kumar Arora et al. 2020 ). The beneficial effects of bacteria on plant salinity tolerance and disease management are well documented(Adedayo and Babalola 2023 ). Several studies have shown that the inoculation of plants with salt-tolerant bacteria can improve their growth and yield in saline soils, and specific bacterial genes and proteins that contribute to enhancing plant salt tolerance have been identified(Shilev 2020 ). In this context, the use of halotolerant PGPB offers a promising alternative for reducing biotic and abiotic stress in plants. This study aimed to analyze the plant growth-promoting action of Bacillus halotolerans AF23 and Bacillus velezensis AF12, isolated from soils affected by underground fires under saline stress conditions, both in vitro and in tomato plants. Additionally, we sought to identify potential genes involved in salinity tolerance and plant growth promotion. Materials and Methods Biological Material Bacillus velezensis AF12 and B. halotolerans AF23 were isolated from soils affected by underground fires in the community of Pueblo Viejo, in the municipality of Venustiano Carranza, Michoacán de Ocampo, Mexico (20°23′27″–17°53′50″N and 100°03′32″–103°44′49″W). The strains were cultured at 30°C for 18-24 hours on nutrient agar (NA) medium and preserved at 4°C for routine use in the laboratory. Additionally, for long-term storage, the strains were stored in a 30% (v/v) glycerol solution at -20°C. Tomato seeds ( S. lycopersicum ) of the Bonny Best cultivar were provided by the Microbiology Laboratory at the Center for Scientific Research and Higher Education at Ensenada (CICESE). F. oxysporum f. sp. lycopersici (also referred to in later sections as "Fol") was provided by the Microbiology Laboratory at CICESE. The fungus was cultured on potato dextrose agar (PDA), incubated at 28°C for 4 days, then stored at 4°C for routine use. Phylogenetic tree with halotolerant strains A phylogenetic tree was constructed with 48 Bacillus strains isolated from soils affected by underground fires, most of which were identified previously. The 16S ribosomal RNA gene sequence was used with Escherichia coli ATCC 1177 as an outgroup. The tree was built using MEGA software version 7.9, employing the maximum likelihood method, with a bootstrap support of 1000 repetitions (Kumar et al. 2016). To determine halotolerance, 48 Bacillus strains were cultured on nutrient agar (NA) supplemented with NaCl at 1.2% and 2.9% w/v (200 mM and 490 mM, respectively), concentrations corresponding to the growth range of halotolerant microorganisms(Merino et al. 2019a). The highest concentration was used as the criterion for classifying the strains as halotolerant or non-halotolerant. In each Petri dish (100 × 15 mm), four bacteria were streaked, incubated at 30°C for 16 h, and subsequently, the growth of the strains was reported. Evaluation of salt tolerance in liquid medium The salt tolerance of B. velezensis AF12 and B. halotolerans was measured after 12, 24, 36, and 48 h of growth in LB medium supplemented with different concentrations of NaCl (25, 50, 100, 200, 500, and 1000 mM). bacterial pre-inocula were initially incubated with shaking at 150 rpm and 30°C for 12 h in LB medium without NaCl. Once the culture reached an optical density of 0.1 (A590nm), 500 µL was inoculated into the culture medium (supplemented with NaCl) in a final volume of 5 mL and incubated at 30°C with shaking at 150 rpm. Each treatment was performed in triplicate, and uninoculated LB medium served as a blank. The absorbance was measured at 590 nm at each time interval(Orozco-Mosqueda et al. 2019). Plant growth promoting traits in vitro under saline conditions Siderophores The production of siderophores was assessed on Chrome Azurol S (CAS) agar plates (Schwyn and Neilands 1987)with NaCl concentrations of 25, 50, 100, and 200 mM. The Bacteria were inoculated at the center of the Petri dish and incubated at 30°C for 48 h. A color change from blue to orange in the medium indicated a positive result. Indole acetic acid To determine IAA production of indole acetic acid, cells were cultured in nutrient broth supplemented with 1% tryptophan and NaCl (0, 25, 50, 200, and 200 mM) for 24 h at 30°C with shaking at 150 rpm until reaching an optical density of 1 (A590nm). The cells were centrifuged at 3000 rpm for 30 min, and 350 µL of the supernatant was mixed with 700 µL of Salkowski reagent. After incubating for 30 min in the dark, the absorbance was measured at 530 nm. Measurements were compared with a calibration curve constructed using dilutions of a standard indole solution (Fluka, Switzerland), and the uninoculated medium with Salkowski reagent served as a control. (Rojas-Solis et al. 2020) Phosphate solubilization The phosphate solubilization assay was carried out by inoculating the bacterial strains on Pikovskaya agar plates(Djuuna et al. 2022), supplemented with different concentrations of NaCl, and using bromocresol purple as a dye in the medium. The plates were incubated at 30°C for 48 h. A color change from purple to yellow in the medium indicated a positive result. Proteases Skim milk agar medium was prepared to evaluate protease production (Abbasi et al. 2019) and the aforementioned concentrations of NaCl were added. The Petri dish was inoculated in the center and incubated at 30°C for 48 h. A clear zone around the bacteria resulted in a positive reaction, indicating the production of protease. Biofilm Biofilm production was evaluated following the protocol of Wei and Zhang (2006) with slight modifications. Cells were cultured in nutrient broth (supplemented with NaCl) for 24 h at 30°C with shaking at 150 rpm until reaching an optical density of 1 (A570nm). Subsequently, a 1:100 dilution of the cultured cells was prepared with sterile distilled water. 500 mL of the dilution was transferred to an Eppendorf tube and incubated at 30°C without shaking for 24, 48, and 72 h. The biofilm was quantified at each time interval. Subsequently, 500 mL of 0.1% (w/v) crystal violet was added and incubated for 15 min at room temperature. The dye was decanted and the cells were washed with sterile distilled water to remove residual dye and non-adherent cells. Ethanol (95%) was used to solubilize the dye from biofilm cells. The absorbance of the solubilized dye was measured (A570nm), and 95% ethanol was used as a blank. For siderophores, protease production, and phosphate solubilization, the diameter of the halo of positive reactions was reported at the end of the incubation period. Inoculation tests of tomato plants under salt stress (200 mM NaCl) Preparation of bacterial inoculum To prepare the bacterial inoculum, an isolated colony of each of the strain ( B. velezensis AF12 and B. halotolerans AF23 separately) was placed in 25 mL of LB culture medium and incubated at 30°C at 110 rpm for 18 h. When the cultures reached an optical density of 0.5 (A590nm), they were used to inoculate the LB culture medium at a concentration of ~2 × 10 8 CFU/mL in a final volume of 50 mL. In-vitro planting and inoculation of tomato seedlings Experiments on tomato plants were performed following the methodology described inCorral-Federico et al. (2024). Tomato seeds of the Bonny Best cultivar were sterilized by immersion in solutions of 70% ethanol (v/v) for 3 min and 2% NaOCl (v/v) for 2 min, after which they were rinsed three times with water. sterile. The seeds were placed on plates with 1.5% agar, once germinated, they were transferred to germination trays with cosmopeat substrate, and kept in a growth chamber at 26°C with light and dark periods of 16 and 8 h, respectively, for two weeks. Finally, seedlings with an average height of 5 cm in the presence of true leaves were selected. Seedlings were transplanted into 6-inch pots and inoculated near the stem with 5 mL of bacterial solution at a concentration of approximately 2 × 10 8 CFU/mL. For the AF12 and AF23 consortium, 2.5 mL of each bacterium was inoculated. Control plants (not inoculated) received 5 mL sterile LB medium. Eight days after the first inoculation of the seedlings, a booster inoculation was performed with 5 mL of bacterial solution at a concentration of ~2 × 10 8 CFU/mL applied near the stems of the plants. At 24 hours after inoculation, the plants were irrigated with saline water (NaCl at 200 mM), which was initially carried out every two days and was subsequently irrigated on demand until the end of the experiment. Eight treatments were carried out as follows: 1) plants without bacteria and without NaCl, 2) plants inoculated with AF12, 3) plants inoculated with AF23, 4) plants inoculated with the AF12 and AF23 consortium, 5) NaCl plants, 6) plants inoculated with AF12 and NaCl, 7) plants inoculated with AF23 and NaCl, and 8) plants inoculated with the AF12 and AF23 consortium and NaCl. Eight replicates were prepared for each treatment, and the pots were arranged in the greenhouse of the Microbiology Department of the Center for Scientific Research and Higher Education at Ensenada in a completely randomized design at an average temperature of 24°C on the day and 20 °C at night. Fifty days after transplanting into a pot, the plants were harvested, and different physiological parameters were measured, such as length, fresh and dry weight of the aerial part and root, and chlorophyll content in the leaves. Biocontrol assays of AF12 and AF23 towards F. oxysporum in tomato plants under greenhouse conditions The methodology described previously (Delgado-Ramírez et al. 2021) was followed with some modifications. The seeds were disinfected and germinated as described above. To evaluate the biocontrol of strains AF12 and AF23, on day one after transplanting into a pot, the plants were inoculated near the stem with 5 mL of bacterial solution at a concentration of approximately 2 × 10 8 CFU/mL for the AF12 consortium. and AF23 were inoculated with 2.5 mL of each bacterium. Control plants (not inoculated) received 5 mL sterile LB medium. Eight days later, a booster inoculation was applied, and 15 days later, F. oxysporum f. sp. lycopersici race 1 was inoculated into the stem at a concentration of approximately 2 × 10 6 total spores/mL per plant. Initially, the plants were watered every third day, and subsequently, irrigation was performed on demand. Five treatments were carried out: 1) plants without bacteria and without Fusarium , 2) plants inoculated with Fusarium; 3) plants inoculated with Fusarium and AF12, 4) plants inoculated with Fusarium and AF23, and 5) plants inoculated with Fusarium and the AF12 and AF23 consortium. Eight replicates were prepared for each treatment and maintained in a greenhouse with a completely randomized design, as previously described. Sixty days after transplanting into a pot, the plants were harvested and different physiological parameters were measured, such as length, fresh and dry weights of the aerial part and the root, and the chlorophyll content in the leaves. Isolation and detection of Fusarium in inoculated plants The stems were examined using vertical cuts to observe the presence of the pathogen. Three centimeters of the basal part of the stem of the plants was taken and superficially sterilized with a flame. Subsequently, a longitudinal cut was made in the stems, which were placed in Petri dishes containing PDA medium and incubated for 5 days at 28°C. Biocontrol assays of AF12 and AF23 towards F. oxysporum in tomato plants under salinity conditions (100 mM NaCl) The seeds were sterilized and germinated as previously described(Delgado-Ramírez et al. 2021). Seedlings with a height of 5 cm and the presence of a true leaf were transplanted into 4-inch pots and inoculated at the base of the stem with 5 mL of a bacterial suspension at a concentration of 2.5 x 10 6 CFU/ml. For the AF12 and AF23 consortium, 2.5 mL of each bacterium was inoculated. Next, the pots were randomly placed in a growth chamber at 26°C with light and dark periods of 16 and 8 hours. Booster inoculation was performed 8 days later. After 72 h, the plants were placed under greenhouse conditions in a completely randomized design and infected with F. oxysporum f. sp. lycopersici race 1. Before transplanting into pots, the seedlings were watered with purified water and a spray bottle was used to keep the substrate moist. After transplanting into a pot, the plants were watered every third day with 50 mL of water for four weeks, and then the volume was increased to 100 mL. Irrigation was alternated with unsalted water and saline water at a concentration of 100 mM (NaCl). Eight treatments were carried out: 1) plants without bacteria, without Fusarium and without NaCl; 2) plants inoculated with AF12 and NaCl; 3) plants inoculated with AF23 and NaCl; 4) plants inoculated with AF12, AF23 consortium, and NaCl ; 5) plants inoculated with Fusarium and NaCl; 6) plants inoculated with Fusarium , AF12, and NaCl; 7) plants inoculated with Fusarium , AF23, and NaCl; and 8) plants inoculated with Fusarium, AF12, AF23 consortium, and NaCl. Ten replicates were prepared for each treatment. 45 days after infection, the plants were harvested, and different physiological parameters were measured. The stems were examined using vertical cuts to observe the presence of the pathogen. The fungal inoculum was prepared as follows: three mycelial discs from a culture of F. oxysporum f. sp. lycopersici race 1 were placed in a glass jar with 50 g of sterile rice. The cultures were incubated for 14 d at 28°C. For the infection of the plants, the substrate was removed from the surface, then 0.5 g of the fungal inoculum (~2.5 x 10 4 total spores per plant) was placed and mixed with the substrate. In all plant trials, eight replicates per treatment were used. Statistical analyzes The experiments were performed at least three times, and the results were analyzed with Statistica 8.0, using one-way ANOVA and comparison of means through the Duncan test (p ≤ 0.05). Genome assembly, OGRI and antiSMASH analysis Strains AF12 and AF23 were initially cultured on nutrient agar. A single colony from each strain was transferred to 5 mL of nutrient broth (NB) and incubated overnight at 28°C with shaking at 150 rpm. Genomic DNA was extracted using the SDS/proteinase K method followed by polysaccharide precipitation in a high-salt environment(Mahuku 2004). The quality and concentration of the extracted DNA were evaluated using 1% agarose gel electrophoresis and a NanoDrop 1000 spectrophotometer (Thermo Scientific). High-quality DNA was sent to Mr. DNA (Shallowater, Texas, USA) for Illumina sequencing. The genome assembly was performed as previously described Chávez-Ávila et al. (2024). Briefly, quality control of sequencing reads was performed using FastQC version 0.11.5(Andrews 2010). Adapter sequences and low-quality bases were trimmed using Trimmomatic version 0.32(Bolger et al. 2014). De novo assembly was conducted using SPAdes version 3.10.1(Bankevich et al. 2012), employing the default parameters for error correction. The assembled contigs were aligned with Mauve Contig Mover (MCM) (Rissman et al. 2009)using the reference genomes of Bacillus velezensis CBMB205 T and Bacillus halotolerans FJAT-2398 T . Genome annotation was performed using the BV-BRC software(Olson et al. 2023), and functional genomic analysis was performed using the RAST server(Aziz et al. 2008). The genomes of AF12 and AF23 are available in NCBI GenBank under accession numbers NZ_JAJHVX000000000.1 and NZ_JAJHVY000000000.1, respectively, with associated Bioproject numbers PRJNA715922 and PRJNA715770. Additionally, the functions of the putative genes linked to the biological control of phytopathogens within the genomes of strains AF12 and AF23 were predicted using antiSMASH(Blin et al. 2023). Detection of stress, biocontrol and plant growth-promoting genes Genes involved in stress resistance and plant growth promotion in B. velezensis AF12 and B. halotolerans AF23 were identified according to previously reported bacterial genes(Nascimento et al. 2020a; Yin et al. 2022). The search for genes was performed using the annotation generated by the National Center for Biotechnology Information (NCBI) Prokaryotic Genome Annotation Pipeline (PGAP) database of genomes AF12 and AF23. Results Phylogenetic and genomic analysis In an initial assessment, a search for halotolerant bacteria was conducted from a collection of strains isolated from Pueblo Viejo, where underground fires occur recurrently. Of the 48 strains evaluated, 6 showed no growth in 2.9% NaCl: AF24, AF30, AF38, AF58, AF64, and AF65 (Suppl. Table 1); therefore, they were classified as non-halotolerant. The remaining strains exhibited optimal growth at both AF23 and AF12 concentrations. In the phylogenetic tree, no grouping of the six non-halotolerant bacteria was observed; instead, they were distributed across the different branches of the tree. Interestingly, the non-halotolerant strains AF64 and AF65, identified only at the genus level, were positioned closer to the strain used as an outgroup. In the phylogenetic tree, a grouping of strains was observed according to species. Strain AF23 had a closer phylogenetic relationship with B. halotolerans AF29, and curiously, these two bacteria were the only ones not grouped with the other strains of the same species (Figure 1). [Place Figure 1 here] Once strains AF12 and AF23 were selected, their draft genomes were sequenced with complete coverage. The genome sizes were 3,995,228 bp (base pairs) for AF12 and 4,256,474 bp AF23 strain. The other genomic features are shown in Table 1. Similarly, Table 2 displays the Overall Genome Relatedness Index (OGRI) values for each strain, where AF12 showed high similarity to the genome of Bacillus velezensis CBMB205T and AF23 showed the greatest similarity to Bacillus halotolerans FJAT-2398T (Table 3). Based on these values, which were also greater than ≥98.7% in the 16S ribosomal RNA gene sequence, it was determined that strain AF12 belonged to the species Bacillus velezensis and AF23 to Bacillus halotolerans . [Place Tables 1, 2 and 3 here] Evaluation of Salinity Tolerance in Liquid Medium The salt tolerance of B. velezensis AF12 and B. halotolerans AF23 was assessed by adding 0, 25, 50, 100, 200, 500, and 1000 mM NaCl to the LB liquid culture medium. The growth of AF12 was faster and reached higher levels under low salinity conditions (0 mM). As the salt concentration increased, growth significantly decreased, especially at higher salinity concentrations (500 mM and 1000 mM), until 36 h. At 48 h, a statistically significant increase in growth was observed at 500 mM compared to that at 0 mM, which may be due to a delay in the adaptation phase (Figure 2). [Place Figure 2 here] In AF23, the results showed that after 12 h of incubation, there was a significant increase in growth at 50 mM compared to the control, whereas at 12 and 36 h, greater growth was observed at the 100 mM concentration. Finally, at 48 h, at 500 mM concentration, AF23 showed greater growth in the culture medium, reaching an optical density of almost 1. At the highest NaCl concentration evaluated (1000 mM), a statistically significant decrease was recorded throughout the incubation period with AF23, indicating that AF23 can grow at higher NaCl concentrations than AF12 (Figure 2). Additionally, AF23 adaptation time to the different salinity concentrations evaluated was shorter than that of AF12. Mechanisms of In Vitro Plant Growth Promotion Figure 3 shows the plant growth-promotion mechanisms evaluated in vitro. The production of siderophores by B. velezensis and B. halotolerans AF23 was evaluated by the generation of a halo, which reflects the production of these molecules in solid culture media. Although both bacteria produced siderophores at all evaluated concentrations, AF23 generated brighter halos. In AF12, phosphorus solubilization was higher starting from 25 mM NaCl concentration compared to the control (0 mM). The AF23 activity was slightly to moderate. Protease production in AF12 decreased at 100 and 200 mM NaCl compared to that at the other concentrations. AF23 showed consistent protease production, except at 200 mM NaCl. Indole acetic acid production was subtle in both the strains. No biofilm formation was observed in the experiments conducted in the presence or absence of NaCl. [Place Figure 3 here] Inoculation Trials on Tomato Plants under Saline Stress (200 mM NaCl) The results from the in vitro experiments suggest that B. halotolerans AF23 can act as a plant growth-promoting bacterium. Therefore, greenhouse inoculation experiments were conducted on tomato plants grown under saline conditions. To induce salt stress, the plants were irrigated with 200 mM NaCl. Additionally, tomato plants were inoculated with B. velezensis AF12 to evaluate the interaction of plant growth with this strain. Under non-saline conditions, AF12 increased the chlorophyll content by 64% compared with that of the control plants. Inoculation with AF23 and the bacterial consortium increased this parameter by 39% and 40%, respectively. NaCl led to a decrease in chlorophyll in the plants; however, this inhibitory effect was counteracted when AF12 was inoculated (Figure 4a). Shoot length decreased in salt-stressed plants compared to non-salt plants; however, in NaCl-treated plants, there was a statistically significant increase of 17% when the AF12 and AF23 consortium was inoculated compared to non-salt plants (Figure 4b). Shoot fresh weight remained similar in non-salt plants. On the other hand, in salt-stressed plants, a significant increase of 48% was observed with AF12, whereas with AF23 and both strains, it increased by 34% and 24%, respectively, compared to the control (Figure 4c). This was reflected in the shoot dry weight, where AF12 significantly increased this parameter by 69%, whereas AF23 and the consortium improved the dry weight by 57% and 51%, respectively, compared to the control (Figure 4d). Root length remained unchanged between treatments, except in non-salt plants, where AF12 increased root length by 19%, and a 3% improvement was observed when the consortium was inoculated (Figure 4e). The fresh weight of the roots remained similar in plants without NaCl. However, it decreased significantly in salt-stressed plants; however, AF12 and the bacterial consortium increased the dry weight of the root by 35% and 34%, respectively, compared to the control (Figure 4f). The dry weight of the roots increased by 41% with AF12 in non-salt plants. As expected, it decreased in plants treated with NaCl, and interestingly, when the strains were inoculated together, the dry weight increased by 17% (Figure 4g). The total biomass remained similar in the non-salt plants. In contrast, AF12 increased biomass by 53% in salt-stressed plants compared to the control, and when AF23 and both strains were inoculated together, there was also an increase in plant biomass of 38% and 41%, respectively (Figure 4h). These results suggest that AF12 and AF23 differentially promote tomato growth under both normal and saline stress conditions. Figure 4 (lower panel) shows representative images of each treatment. [Place Figure 4 here] Biocontrol Trials of AF12 and AF23 against F. oxysporum in Tomato Plants under Greenhouse Conditions Chlorophyll content decreased in plants infected with F . oxysporum , and there were no statistically significant differences between plants inoculated with AF12 and the bacterial consortium compared to control plants (Figure 5a). Shoot length did not differ significantly between the treatments (Figure 5b). AF12 increased the fresh weight of the shoots by 36% and AF23 by 27% (Figure 5c). Similarly, AF12 increased the dry weight of the shoot by 40% compared to the control and AF23 by 37%, while the consortium increased the dry weight by 30% (Figure 5d). There were no differences in the root lengths of the plants (Figure 5e). AF12 increased the fresh weight of the roots by 39% compared to that of the control plants (Figure 5f). Dry weight remained without statistically significant differences between the treatments (Figure 5g). Figure 5h shows the effect of bacterial strain inoculation on the total biomass of plants infected with F. oxysporum. AF12, AF23, and the bacterial consortium increased the total biomass by 34%, 28%, and 24%, respectively, compared with plants without the fungal pathogen. Figure 5 (lower panel) shows representative images of each treatment. [Place Figure 5 here] Isolation and Detection of Fusarium in Inoculated Plants In the stems of plants infected with F. oxysporum f. sp. lycopersici, a dark brown line was observed, which corresponded to the initial symptoms of vascular wilting. This was not observed in the control plants (Figure 6, upper panel). The pathogen was re-isolated from the stems of the infected tomato plants once they were placed on the PDA medium (Figure 6, lower panel). Macroscopic growth of the fungus coincided with the routine growth of Fol race 1. With this, the presence of the fungus was detected in the stems of the infected plants. [Place Figure 6 here] Biocontrol Trials of AF12 and AF23 against F. oxysporum in Tomato Plants under Salinity Conditions (100 mM NaCl) In the trials conducted to evaluate the biocontrol of the AF12 and AF23 strains against F. oxysporum f. sp. lycopersici under saline stress conditions, all plants were irrigated with saline water at a concentration of 100 mM NaCl. Shoot length in plants without the pathogen decreased with the co-inoculation of both strains compared to the control; the other treatments remained similar to each other (Figure 7a). The fresh weight of the shoots increased with individual inoculation of AF12 and AF23, and in the consortium in non-Fusarium-infected plants (17%, 16%, and 12%, respectively), and in the presence of the pathogen, the bacterial consortium inoculation increased by 14% compared to the control (Figure 7b). Interestingly, AF23 increased the dry weight of the shoot (39%), and bacterial consortium inoculation increased this parameter by 31% in non-infected plants compared with the control. There were no statistically significant differences between the treatments when the pathogen was present (Figure 7c). An increase in total biomass was observed with the inoculation of AF23 and the bacterial consortium by 32% and 28%, respectively, in the non -F. oxysporum- infected plants (Figure 7d). The root length significantly increased by 28% with the bacterial consortium without the pathogen, compared to the control plants; treatments of infected plants remained unchanged (Figure 7e). Strain AF12 increased the fresh weight of the roots (44%), and there was a 23% increase in the co-inoculation of both strains in non- Fusarium -infected plants compared to control plants. When AF12 was inoculated into plants infected with the pathogen, a recovery in the fresh weight of the root was observed, similar to that observed in non-infected plants (Figure 7f). The dry weight of the roots significantly increased by 27% in plants inoculated with AF12 without Fusarium, compared to the control plants (Figure 7g). It is important to mention that when examining the stems of plants infected with Fol, not all plants showed lesions caused by the pathogen; that is, the inoculation of B. velezensis AF12 and B. halotolerans AF23 significantly reduced infection in the plants. With AF12, only 40% of the plants were infected, whereas with AF23, it was 80%; however, with the co-inoculation of both strains, the infection percentage was 20% (Figure 7h). These results suggest that AF12 and AF23 are biocontrol agents for plants subjected to saline stress. Figure 7 (lower panel) shows representative images of each treatment. [Place Figure 7 here] Gene Identification Genes with predicted functions in adaptation to high salinity were identified in the genomes of B. velezensis AF12 and B. halotolerans AF23 (NCBI GenBank under accession numbers NZ_JAJHVX000000000.1 and NZ_JAJHVY000000000.1; Figure 8). For example, genes related to the synthesis of osmoprotective compounds such as proline ( proA , proB , and proC ) and the synthesis and transport of glycine betaine/proline ( betB , opuA, opuAB, opuE ). Genes encoding Na+/H+ antiporters ( nhaC ), Na+ transporters ( natA ), and K+ transporters ( ktrD and ktrC ) were also present. [Place Figure 8 here] Given that this strain was isolated from soils affected by underground fires and is thermotolerant, AF12 and AF23 contain various genes involved in temperature-stress resistance. For example, dnaJ, dnaK , and groES encode chaperone-like proteins and three genes related to cold shock ( cspB , cspC , and cspD ) were also found. Genes involved in protection against oxidative and nitrosative stress are present, including the sodA gene, the product of which is the enzyme superoxide dismutase SodA, which participates in the degradation of superoxide anion radicals. Other genes involved in the breakdown of hydrogen peroxide ( ahpC , efeB , etc.) and a transcriptional repressor sensitive to nitric oxide ( nsrR ) have also been identified. Other genes found in the AF strains are involved in nitrogen, sulfur, and phosphorus metabolism. The nirB gene, necessary for nitrite reduction; the alkaline phosphatase PhoA encoded by the phoA gene, which is important in the solubilization of organic phosphate; and genes involved in sulfate transport ( cysK ), to name a few. In addition, both bacilli strains possess genes such as besA , whose product is a ferri-bacillibactin esterase BesA, and dhbC , which encodes an isochorismate synthase DhbC that is involved in siderophore production, an important mechanism in plant growth promotion. An important aspect of plants is the production and modulation of hormones by prostaglandin B (PGPB). The genomes of both strains contained genes involved in IAA and cytokinin synthesis. trpB encodes the beta subunit of tryptophan synthase, which is crucial for the synthesis of L-tryptophan from indole and L-serine (the main precursor of IAA). The dhaS gene is also present and catalyzes the conversion of indole-3-acetaldehyde to IAA. Genes related to production and degradation of VOCs in both strains. The alsS gene is involved in acetoin synthesis and the bdhA gene is related to butanediol degradation. Genes related to chemotaxis ( cheA, cheB, cheD, cheR, cheV, cheY, cheW, motA ), flagellar biosynthesis ( flgB, flgC, flgK, flhA ), and biofilm formation ( tasA and Efp ) were also detected in the bacterial genomes. Genes involved in the transport and resistance to heavy metals, such as zinc, copper, and arsenate, and genes related to sporulation ( yabP, yjcZ, yjcD, ytfJ, yunB, gerD, yutH, cotJC ) were also identified. The presence of these genes indicates that both strains possess genetic machinery to cope with different types of stress, including salinity, and promote tomato growth through various mechanisms (Figure 9). [Place Figure 9 here] According to antiSMASH analysis, gene clusters involved in the synthesis of metabolites with potential antimicrobial activity, such as surfactin and fengycin, were also identified (Table 4). Both lipopeptides are widely characterized as antifungal agents that play a significant role in the biocontrol of crop diseases. The similarity percentages were greater than 70%, suggesting a high likelihood of functional activity; however, this requires further experimental confirmation. [Place Table 4 here] Discussion In the present study, the biocontrol and plant growth-promoting traits of strains AF12 and AF23 isolated from soils affected by underground fires were evaluated under normal and salinity-stress conditions. There are earlier reports of bacteria, particularly Bacilli species, which successfully promote plant growth under similar environmental stress conditions(Jamali et al. 2019 ; Bokhari et al. 2019 ; Pranaw et al. 2020 ) Besides, the potential PGP Bacillus spp. AF12 and AF23 salinity tolerance of were evaluated both in vitro and in vivo by prescreening several Bacillus strains isolated from soils affected by underground fires(Flores-Piña et al. 2024 ). This location has been a wonderful site for the detection of potential PGP and biocontrol bacteria with thermotolerance and halotolerance capacities, in addition to exhibiting beneficial effects on plants of agronomic importance(Chávez-Avila et al. 2023 ). Strain AF23 was identified as Bacillus halotolerans through OGRI, including average nucleotide identity (ANI) analyses, genome-to-genome distance calculator (GGDC), and phylogeny based on the 16S rRNA gene sequence, obtaining 98.9%, 97.92%, and 100%, respectively. The AF23 genome has a size of 4.2 Mb. AF12 was identified as a Bacillus velezensis species according to the ANI, GGDC, and 16S rRNA with values ​​of 98.15, 98.54%, and 99.68%, respectively, and a genome size of 3.9 Mb. Recently, Wu and colleagues ( 2022 ) a strain of Bacillus halotolerans KKD1 with biocontrol and plant growth-promoting capabilities was identified in wheat. This strain induced the production of phytohormones such as 6-benzylaminopurine and gibberellic acid under salt stress conditions. Additionally, genome mining of KKD1 has revealed interesting genes involved in biocontrol and plant growth promotion. For Bacillus velezensis , there is also evidence of its beneficial effects under saline conditions. For instance, Bai and coauthors (2023) the salt-tolerant bacterium Bacillus velezensis strain JB0319 acts as a plant growth promoter. The JB0319 strain successfully stimulated the growth of lettuce plants under saline conditions by enhancing antioxidant activities (superoxide dismutase and peroxidase) and reducing MDA accumulation. Other studies have highlighted the great potential of these two Bacillus species as halotolerant and beneficial microorganisms include works byChen et al. ( 2025 ), Masmoudi et al. ( 2021 ), and Çam et al. ( 2023 ). Some strains, such as Bacillus velezensis GB03, have been commercially available for some time(Jang et al. 2023 ). Thus, strains AF12 and AF23 represent excellent candidates as potential bioinoculants for saline soils and regions with high temperatures, as they can grow at 50°C (Chávez-Ávila et al., 2025, unpublished results). With bacterial identification at the species level, the initial assumption was that AF23 was a salt-tolerant bacterium. Therefore, growth was evaluated at different NaCl concentrations (25, 50, 100, 200, 500, and 1000 mM) at various time intervals. The results showed that this strain could grow under high-salinity conditions. When comparing the growth of AF12 and AF23 with that of B. subtilis 168 in halotolerance experiments, the growth of B. subtilis 168 was minimal (data not shown), as previously reported (under NaCl stress conditions)(Rath et al. 2020 ). In contrast, the growth of AF12 and AF23 was in the range of NaCl designated for microorganisms termed halotolerants(Merino et al. 2019b ; Reang et al. 2022 ). It is important to mention that although both bacteria can grow under saline conditions, in the assays evaluated, AF23 showed greater growth in the medium supplemented with NaCl. In the present study, the mechanisms of direct and indirect plant growth promotion (pathogen antagonism) were determined. One of the mechanisms of plant growth promotion is nutrient facilitation such as iron acquisition through siderophore production (Khan et al., 2019). AF23 produced siderophores under saline conditions, with 200 mM NaCl being the highest concentration tested. Similar siderophore production has been reported in Bacillus strains, such as the salt-tolerant B. aryabhattai MS3 at 100 and 200 mM NaCl(Sultana et al. 2021 ). Genomic analysis revealed that AF23 harbors gene clusters with 100% similarity to those encoding the siderophore bacillibactin, including besA and dhbC , the latter of which codes for isochorismate synthase. These genes have been identified in other species such as B. megaterium STB1(Nascimento et al. 2020). Siderophore production, a dual mechanism of plant growth promotion, not only enhances iron uptake, but also limits plant pathogen growth(Di Francesco and Baraldi 2021 ). Bacillibactin production by AF23 may explain its antifungal activity, as this siderophore is linked to its biocontrol properties(Dimopoulou et al. 2021 ). In vitro assays showed that AF23 produced IAA at all tested concentrations (25, 50, 100, and 200 mM), with a slight reduction observed at 200 mM. IAA synthesis is a key trait for stress mitigation by PGPB. Genes involved in IAA synthesis have been identified in B. halotolerans AF23. IAA regulates key plant growth processes such as cell division, elongation, and root hair formation, which improve water and nutrient uptake(Luo et al. 2022 ). AF23 also produced proteases under saline conditions, although its activity decreased at 200 mM NaCl compared to that under non-saline conditions. Similar activity has been reported for B. velezensis DMB06 under saline conditions(Na et al. 2022 ). PGPB mitigates salinity stress by supplying nutrients such as phosphorus(Gao et al. 2022 ). AF23 solubilized phosphate, with larger halos observed at 25–100 mM NaCl than at 0 mM NaCl. Genomic analysis revealed genes involved in phosphate solubilization. This differs from B. pumilus JPVS11, in which higher salinity reduces phosphate solubilization, although it improves rice growth under saline stress(Kumar et al. 2021 ). The positive in vitro results prompted the evaluation of AF23 in tomato plants under different conditions: 1) 200 mM saline stress, 2) infection with F. oxysporum f. sp. lycopersici (Fol), and 3) 100 mM saline stress combined with Fol infection. A 200 mM NaCl concentration was selected based on AF23's optimal growth and previous reports(Abdelshafy Mohamad et al. 2020 ; Sultana et al. 2021 ). The 100 mM NaCl solution was chosen because of the optimal growth of Fol at this concentration. Tomato experiments evaluated B. velezensis AF12 and B. halotolerans AF23 individually and in consortia under biotic and abiotic stresses, leveraging prior findings and literature on their plant growth promotion and antifungal activities(Dong et al. 2023 ). Salinity negatively affected the chlorophyll content. AF12 (data not shown) and AF23 siderophore production may explain the increased chlorophyll content in tomatoes, because Fe is essential for chlorophyll biosynthesis(Sultana et al. 2021 ). AF12 and AF23 inoculation, alone or in combination, significantly improved shoot dry weight under 200 mM NaCl by 50–69%, reflected in a 38–53% increase in total biomass. In the absence of NaCl, root dry weight increased by 41% after AF12 inoculation compared with uninoculated plants. Saleem et al. ( 2021 ) observed that 200 mM NaCl severely reduced cotton growth parameters, but inoculation with Bacillus spp. partially restored root and shoot length, chlorophyll content, and reduced Na + uptake. Notably, the greatest effects of AF12 and AF23 inoculation occurred under saline conditions, likely linked to the absence of genomic traits under non-saline conditions. Tomato wilt, caused by Fol, remains a major challenge. B. halotolerans Cal.l.30 significantly reduced disease incidence in grape and cherry tomatoes inoculated with Botrytis cinerea , as well as in vitro antagonism towards B. cinerea , Rhizoctonia solani , and Fol(Tsalgatidou et al. 2023 ). Similarly, B. velezensis AP-3 reduced the severity of Fusarium wilt in tomatoes by 50%(Medeiros and Bettiol 2021 ). Greenhouse experiments confirmed that AF12 and AF23 improved tomato biomass during Fol infection. Shoot dry weight increased by 40% with AF12 and 37% with AF23. Total biomass increased by 34% and 28%, respectively. Under combined saline and Fusarium stress (100 mM NaCl), inoculation with AF12, AF23, or their combination reduced disease incidence by 60–80%. Comparable findings include halotolerant Bacillus spp. that promote tomato growth and reduce disease severity under saline conditions(Abdelshafy Mohamad et al. 2020 ). The impact of the inoculation of strains AF12 and AF23 on the rhizosphere microbiome of tomato plants remains to be investigated, as some bioinoculant agents have been observed to impact the structure and modulation of functions of other microorganisms associated with the same host plant. For example, a study Ji et al. ( 2022 ) revealed that the microbial agents Bacillus subtilis HG-15 and Bacillus velezensis JC-K3, when applied to wheat growth under low salt stress, affected fungal communities, including pathogenic and beneficial arbuscular mycorrhizal fungi (AMF). The results showed that inoculation with bacterial agents increased wheat yield, chlorophyll content, photosynthesis, and water use efficiency, while reducing disease incidence by 79.80%. Analysis of fungal communities revealed that inoculation decreased the abundance of harmful fungi such as Gibberella and Fusarium while promoting beneficial AMF species such as Glomus . These findings suggest that Bacillus -based PGPR can improve plant growth, disease resistance, and soil microbiome dynamics, thereby providing insights into sustainable disease control. In conclusion, this study highlights the functional and genomic basis of salinity tolerance and plant growth-promoting traits of AF, particularly in synergy. Genes linked to osmotic and oxidative stress responses, volatile organic compound (VOC) synthesis, and metal resistance were identified (Fig. 9 ). These findings demonstrate the potential of AF strains to mitigate combined biotic and abiotic stresses in economically important crops. Declarations Funding G.S. thanks CIC-UMSNH (2024–2025), and ICTI-Michoacán (ICTI-PICIR23-000) for their support with the research projects. Author Contribution FVM, SCA, ES, CSDR, JJMC, and RHM contributed to the implementation of the research, analysis of sequence data, and writing of the manuscript. FVM created the figures. MCOM, SDLV, and OBB contributed to the data analysis and editing of the manuscript. GS conceptualized, designed the experiments, and wrote the draft and final manuscript. All authors read and approved the final manuscript. Acknowledgement F.V.M. and S.C.A. received a Master's in Science scholarship from CONAHCyT-Mexico. 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Saudi J Biol Sci 28:. https://doi.org/10.1016/j.sjbs.2021.05.056 Schwyn B, Neilands BB (1987) Universal Chemical Assay for the Detection and Determination of Siderophores’. Analytical Biochemestry 160:47–56 Shilev S (2020) Plant-growth-promoting bacteria mitigating soil salinity stress in plants. Applied Sciences (Switzerland) 10 Singh A (2022) Soil salinity: A global threat to sustainable development. Soil Use Manag 38 Stavi I, Thevs N, Priori S (2021) Soil Salinity and Sodicity in Drylands: A Review of Causes, Effects, Monitoring, and Restoration Measures. Front Environ Sci 9 Sultana S, Alam S, Karim MM (2021) Screening of siderophore-producing salt-tolerant rhizobacteria suitable for supporting plant growth in saline soils with iron limitation. J Agric Food Res 4:. https://doi.org/10.1016/j.jafr.2021.100150 Sunita K, Mishra I, Mishra J, et al (2020) Secondary Metabolites From Halotolerant Plant Growth Promoting Rhizobacteria for Ameliorating Salinity Stress in Plants. Front Microbiol 11 Tsalgatidou PC, Thomloudi EE, Delis C, et al (2023) Compatible Consortium of Endophytic Bacillus halotolerans Strains Cal.l.30 and Cal.f.4 Promotes Plant Growth and Induces Systemic Resistance against Botrytis cinerea. Biology (Basel) 12:. https://doi.org/10.3390/biology12060779 Wei HL, Zhang LQ (2006) Quorum-sensing system influences root colonization and biological control ability in Pseudomonas fluorescens 2P24. Antonie Van Leeuwenhoek 89:267–280. https://doi.org/10.1007/s10482-005-9028-8 Wu X, Fan Y, Wang R, et al (2022) Bacillus halotolerans KKD1 induces physiological, metabolic and molecular reprogramming in wheat under saline condition. Front Plant Sci 13:. https://doi.org/10.3389/fpls.2022.978066 Yin Z, Wang X, Hu Y, et al (2022) Metabacillus dongyingensis sp. nov. Is Represented by the Plant Growth-Promoting Bacterium BY2G20 Isolated from Saline-Alkaline Soil and Enhances the Growth of Zea mays L. under Salt Stress . mSystems 7:1–16. https://doi.org/10.1128/msystems.01426-21 Tables Tables 1 to 4 are not available with this version. Additional Declarations No competing interests reported. Supplementary Files Tables.docx Suppl.docx Cite Share Download PDF Status: Published Journal Publication published 07 Mar, 2025 Read the published version in World Journal of Microbiology and Biotechnology → Version 1 posted Editorial decision: Revision requested 29 Jan, 2025 Reviews received at journal 17 Jan, 2025 Reviewers agreed at journal 13 Jan, 2025 Reviews received at journal 06 Jan, 2025 Reviewers agreed at journal 28 Dec, 2024 Reviewers agreed at journal 25 Dec, 2024 Reviewers agreed at journal 25 Dec, 2024 Reviewers invited by journal 25 Dec, 2024 Editor assigned by journal 20 Dec, 2024 Submission checks completed at journal 20 Dec, 2024 First submitted to journal 18 Dec, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5671788","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":392954235,"identity":"9d3068cc-44bb-4478-9069-40f54979af58","order_by":0,"name":"María F. Valencia-Marin","email":"","orcid":"","institution":"Universidad Michoacana de San Nicolás de Hidalgo","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"F.","lastName":"Valencia-Marin","suffix":""},{"id":392954236,"identity":"5ba557b8-92de-4db8-9867-01fea4754999","order_by":1,"name":"Salvador Chávez-Avila","email":"","orcid":"","institution":"Universidad Michoacana de San Nicolás de Hidalgo","correspondingAuthor":false,"prefix":"","firstName":"Salvador","middleName":"","lastName":"Chávez-Avila","suffix":""},{"id":392954237,"identity":"8cc7cde4-9ccf-4b02-9363-a5ed721fa3f1","order_by":2,"name":"Edgardo Sepúlveda","email":"","orcid":"","institution":"CICESE","correspondingAuthor":false,"prefix":"","firstName":"Edgardo","middleName":"","lastName":"Sepúlveda","suffix":""},{"id":392954238,"identity":"91fcb0b7-8ab2-4ef9-997c-a166a70cb519","order_by":3,"name":"Carmen S. Delgado-Ramírez","email":"","orcid":"","institution":"CICESE","correspondingAuthor":false,"prefix":"","firstName":"Carmen","middleName":"S.","lastName":"Delgado-Ramírez","suffix":""},{"id":392954239,"identity":"76b84c89-ef41-4620-a27e-efdf16c2b872","order_by":4,"name":"Jenny J. Meza-Contreras","email":"","orcid":"","institution":"CICESE","correspondingAuthor":false,"prefix":"","firstName":"Jenny","middleName":"J.","lastName":"Meza-Contreras","suffix":""},{"id":392954240,"identity":"0a39b4fe-06a8-4d4b-af5d-70bcf2abe1d0","order_by":5,"name":"Ma del Carmen Orozco-Mosqueda","email":"","orcid":"","institution":"Tecnológico Nacional de México en Celaya","correspondingAuthor":false,"prefix":"","firstName":"Ma","middleName":"del Carmen","lastName":"Orozco-Mosqueda","suffix":""},{"id":392954241,"identity":"001354ce-f999-4d66-84e2-6e0830a1cd5b","order_by":6,"name":"Sergio Santos-Villalobos","email":"","orcid":"","institution":"Instituto Tecnológico de Sonora","correspondingAuthor":false,"prefix":"","firstName":"Sergio","middleName":"","lastName":"Santos-Villalobos","suffix":""},{"id":392954242,"identity":"90cc7f5c-db22-4c7e-a2d3-8aea44a3c7e1","order_by":7,"name":"Olubukola Oluranti Babalola","email":"","orcid":"","institution":"North-West University","correspondingAuthor":false,"prefix":"","firstName":"Olubukola","middleName":"Oluranti","lastName":"Babalola","suffix":""},{"id":392954243,"identity":"bac4c14a-64de-4800-848c-478a7427bf9e","order_by":8,"name":"Rufina Hernández-Martinez","email":"","orcid":"","institution":"CICESE","correspondingAuthor":false,"prefix":"","firstName":"Rufina","middleName":"","lastName":"Hernández-Martinez","suffix":""},{"id":392954244,"identity":"a24f628b-3e5c-40d1-9100-9b2fc555efd8","order_by":9,"name":"Gustavo Santoyo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIiWNgGAWjYCgAOQYGxgZmAooYG5AZxqRrSQQReLXIR6Q/f/CzzYZBvv3s8Qcf22zSN1w73Pi5gGFbYgMOLYY3cgwbe9vSGAzO5CU2zmxLy91wO7FZegbDbWNcthjOyGFs4DlzmMGAIcewmbftMEhLGzMPw2053FrSHzb+AWqR738D1pJuANXCg9MvEgmGzTwVhxkYbkBsSTAgZIsBzxvD2TIVaTwGN94YzpxxLs1wJsgvPAa4/SLfnv7g4xsDGzn5/hyDDx/KbOT5bqc//MxTcRtniBkcgNAQlzOywcVx2QG0BdWsP7hVjoJRMApGwcgFAB/QWsPo1HAMAAAAAElFTkSuQmCC","orcid":"","institution":"Universidad Michoacana de San Nicolás de Hidalgo","correspondingAuthor":true,"prefix":"","firstName":"Gustavo","middleName":"","lastName":"Santoyo","suffix":""}],"badges":[],"createdAt":"2024-12-18 19:23:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5671788/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5671788/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11274-025-04308-8","type":"published","date":"2025-03-07T15:58:19+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":72212005,"identity":"cb5c53fe-80d9-4e69-85de-1f9ba1cd3b2a","added_by":"auto","created_at":"2024-12-23 18:03:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":710121,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree of 49 \u003cem\u003eBacillus\u003c/em\u003estrains based on the 16S rRNA gene, using \u003cem\u003eE. coli\u003c/em\u003e ATCC 11775 as the outgroup. The tree was constructed using the Maximum Likelihood method with bootstrap support of 1000 replicates. \u003cem\u003eB. halotolerans\u003c/em\u003e AF23 is highlighted in a red box, gray circles represent halotolerant bacteria, and blue circles represent non-halotolerant bacteria.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5671788/v1/2b223c772f85b2d6efab16b4.png"},{"id":72211151,"identity":"ddf8f4e7-48f5-4323-93e6-3152e382e8d8","added_by":"auto","created_at":"2024-12-23 17:47:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":626970,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth of strains AF12 and AF23 at different NaCl concentrations, reported at 12, 24, 36, and 48 hours. The data correspond to absorbance values (O.D. 590 nm) of the cultures at the indicated time points and are presented as the mean ± standard error. Asterisks indicate significant differences compared to the 0 mM concentration at each evaluated time interval, according to Duncan's test (\u003cem\u003ep\u003c/em\u003e ≤ 0.05).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5671788/v1/8c5f53543e5bdf6199eddb6f.png"},{"id":72211161,"identity":"eb91e53a-5b40-4770-8ea2-87a4060b00ec","added_by":"auto","created_at":"2024-12-23 17:47:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2221179,"visible":true,"origin":"","legend":"\u003cp\u003eBiocontrol and plant growth promotion mechanisms evaluated in strains AF12 and AF23. The production of siderophores, phosphate solubilization, protease production, and indole acetic acid production were assessed under different salinity conditions. A \"+\" indicates low activity, \"++\" medium activity, and \"+++\" high activity.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5671788/v1/855fba61a708b1a7b6519813.png"},{"id":72211154,"identity":"1d93ff21-a51d-4eff-ba25-4a07db311d79","added_by":"auto","created_at":"2024-12-23 17:47:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1358533,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of inoculation with \u003cem\u003eB. velezensis\u003c/em\u003e AF12 and \u003cem\u003eB. halotolerans\u003c/em\u003e AF23 on tomato plants under saline stress conditions was evaluated. a) Chlorophyll content; b) Shoot length; c) Shoot fresh weight; d) Shoot dry weight; e) Root length; f) Root fresh weight; g) Root dry weight; h) Total biomass. Bars represent the mean ± standard error of the plants for each treatment. The bottom panel shows representative images of tomato plants (\u003cem\u003eS. lycopersicum\u003c/em\u003e) inoculated with strains AF12 and AF23 under saline stress conditions at day 50 after transplanting to pots. Control plants were neither inoculated nor irrigated with NaCl.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5671788/v1/ef17360918080d73c6044920.png"},{"id":72212006,"identity":"85635d7b-ec63-4650-87b1-298f28aab5e1","added_by":"auto","created_at":"2024-12-23 18:03:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1299799,"visible":true,"origin":"","legend":"\u003cp\u003eThe biocontrol effect of strains AF12 and AF23 on tomato plants inoculated with \u003cem\u003eF. oxysporum\u003c/em\u003e was evaluated through various parameters: a) chlorophyll content; b) shoot length; c) shoot fresh weight; d) shoot dry weight; e) root length; f) root fresh weight; g) root dry weight; h) total biomass. Bars represent the mean ± standard error of the plants for each treatment. Letters above the bars indicate significant differences, according to Duncan's test (\u003cem\u003ep\u003c/em\u003e ≤ 0.05). The bottom panel shows representative images of tomato plants (\u003cem\u003eS. lycopersicum\u003c/em\u003e) inoculated with AF12, AF23, and \u003cem\u003eF. oxysporum\u003c/em\u003e at day 60 after transplanting to pots. Control plants were not inoculated.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5671788/v1/09fbeade31fb0b07454d6e0f.png"},{"id":72212664,"identity":"518a16ad-2de2-4b0c-b79c-2205b5fc3103","added_by":"auto","created_at":"2024-12-23 18:11:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3035090,"visible":true,"origin":"","legend":"\u003cp\u003eInitial symptoms of wilt caused by \u003cem\u003eF. oxysporum\u003c/em\u003e in tomato plant stems after 60 days. The control was not infected with the pathogen, white arrows indicate the inoculation site, and red arrows point to the dark brown lines, which are the initial symptoms of vascular wilt. Bottom panel: Re-isolation of \u003cem\u003eF. oxysporum\u003c/em\u003e from tomato stems.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5671788/v1/1a4c1f35acebbe26b16f65a3.png"},{"id":72211797,"identity":"2c53b4b4-4ec5-49de-a30e-ce9264e0aeae","added_by":"auto","created_at":"2024-12-23 17:55:39","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1447125,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of biocontrol by AF12 and AF23 on tomato plants inoculated with \u003cem\u003eF. oxysporum\u003c/em\u003e under saline stress conditions. a) Shoot length; b) Shoot fresh weight; c) Shoot dry weight; d) Total biomass; e) Root length; f) Root fresh weight; g) Root dry weight; h) Percentage of infected plants. Bars represent the mean ± standard error of the plants for each treatment. Letters above the bars indicate significant differences, according to Duncan's test (\u003cem\u003ep\u003c/em\u003e ≤ 0.05). Bottom panel: Tomato plants (\u003cem\u003eS. lycopersicum\u003c/em\u003e) inoculated with AF12, AF23, and \u003cem\u003eF. oxysporum\u003c/em\u003eunder saline stress conditions. Representative images of the plants for each treatment at day 45 after transplanting to pots. Control plants were neither inoculated nor irrigated with salt.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-5671788/v1/9ba901d0c505499103fbad05.png"},{"id":72211794,"identity":"1194ec86-636e-4a1d-b91b-09b4604436b5","added_by":"auto","created_at":"2024-12-23 17:55:39","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1265622,"visible":true,"origin":"","legend":"\u003cp\u003eGraphs of the draft genomes of \u003cem\u003eB. velezensis\u003c/em\u003e AF12 and \u003cem\u003eB. halotolerans\u003c/em\u003e AF23.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-5671788/v1/863680cca4516259a57ca16c.png"},{"id":72212009,"identity":"bde9917e-6476-4045-b69a-b393a7c61fbd","added_by":"auto","created_at":"2024-12-23 18:03:39","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":2098337,"visible":true,"origin":"","legend":"\u003cp\u003eIllustration of the shared stress-gene capacities of both genomes \u003cem\u003eB. velezensis\u003c/em\u003e AF12 and \u003cem\u003eB. halotolerans\u003c/em\u003e AF23. The figure shows the production of protective osmolytes such as proline and trehalose; heat-shock proteins; as well as osmoprotective transporters for Na+, K+, Ca+; antioxidant functions, among others.\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-5671788/v1/eb42957adb6daa8f37d4cb98.png"},{"id":78190425,"identity":"328bef76-b5e0-40e4-96bd-eadd0035193f","added_by":"auto","created_at":"2025-03-10 19:49:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":19558101,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5671788/v1/d35673c7-ad21-4534-8c67-7a9ae5b12d97.pdf"},{"id":72211149,"identity":"6e7125e2-68ba-4792-b36b-19b310aee187","added_by":"auto","created_at":"2024-12-23 17:47:38","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":574779,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-5671788/v1/0c0d3cde64d0089533d54be5.docx"},{"id":72211148,"identity":"e7d5ef13-b341-4f15-8185-07d7c5915978","added_by":"auto","created_at":"2024-12-23 17:47:38","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":18806,"visible":true,"origin":"","legend":"","description":"","filename":"Suppl.docx","url":"https://assets-eu.researchsquare.com/files/rs-5671788/v1/6e5337c4e155070e306b53aa.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Stress-Tolerant Bacillus Strains for Enhancing Tomato Growth and Biocontrol of Fusarium oxysporum under Saline Conditions: Functional and Genomic Characterization","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e L.) is one of the most widely cultivated vegetables worldwide. It is an important aspect of the human diet and contains vitamins A and C and minerals(Adedayo et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e; Olowe et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Tomatoes also contain antioxidants such as lycopene, which has been found to be effective in preventing cancer. As a result of the global consumption of this vegetable(Emmanuel and Babalola \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), it recorded over 186.82\u0026nbsp;million tonnes of production in 2022, which accounted for 36.97t/ha production. However, despite the importance of this vegetable, it continues to face a continual threat of both biotic and abiotic stressors, which cause a decline in tomato yield and quality(Fadiji et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Among these stress factors, excessive accumulation of soluble soil salinity has been found to severely impact plant growth and reduce crop yields, leading to economic losses and land degradation. High concentrations of soluble salts such as sodium chloride (NaCl), calcium chloride (CaCl2), and magnesium chloride (MgCl2) contribute to the high electrical conductivity of saline soils. Among these, NaCl accounts for most of the soluble salts in soils with salinity problems(Gupta et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Soil was classified as saline when its electrical conductivity in the root zone (from 0 to 60 cm depth) exceeded 4 dS m\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (approximately 40 mM NaCl)(Bonarota et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSoil salinity is a significant challenge for agriculture worldwide, particularly in arid and semi-arid regions, and can arise from both natural and human-induced processes(Huang et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Naturally occurring salinity often develops in arid and semi-arid regions, where high evaporation rates lead to high salt concentrations at the soil surface. In coastal areas, seawater intrusion can bring salt into freshwater aquifers, thereby contaminating agricultural soils(Stavi et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Climate change is a primary driver of increasing soil salinity because global temperature increases will lead to increased evaporation rates and a rise in sea levels. Additionally, altered precipitation patterns can result in more frequent and intense droughts, further worsening the soil salinity(Eswar et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Singh \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Human activities also contribute to soil salinization. Overirrigation, particularly in arid regions, can lead to waterlogging, which prevents salts from leaching out of the soil profile. Simultaneously, increased groundwater pumping for irrigation depletes freshwater resources, forcing farmers to rely on deeper aquifers, which often contain higher salt content. Moreover, deforestation and land mismanagement can disrupt the natural balance of water and salt in the soil, exacerbating this problem(Stavi et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Maertens et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eFusarium oxysporum\u003c/em\u003e, which causes wilt diseases, is a major pathogen of tomatoes that accounts for a significant loss in yield and crop quality(Olowe et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Moreover, the interaction of \u003cem\u003eFusarium oxysporum\u003c/em\u003e, which causes wilt diseases, with salt stress has been found to vary depending on the formae speciales (f. sp.) and host plants involved(Akanmu et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Moreover, increased disease incidence following irrigation with high-salinity water has been reported in earlier investigations. However, the global population is constantly increasing, making one of the main challenges for agriculture to meet the growing demand for food. This indicates the urgent need to boost agricultural production in the coming years(Jim\u0026eacute;nez-Mej\u0026iacute;a et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, the yield of economically important crops is affected by both biotic and abiotic stressors(Kumar et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGiven the challenges posed by salinity and pathogenic \u003cem\u003eFusarium oxysporum\u003c/em\u003e in tomatoes, several strategies have been developed to mitigate its negative effects on plant growth(Adedayo et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e). One approach involves the generation of salt-resistant plants through genetic modifications. However, owing to the time and costs involved, this is often not a viable solution for large-scale agriculture. As an alternative, the use of halotolerant microorganisms such as plant growth-promoting bacteria (PGPB) has emerged as a promising method for enhancing crop growth under saline stress conditions(Sunita et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). PGPB are bacterial species that significantly affect plant growth, yield, and disease resistance via various mechanisms(Khatoon et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Morales-Cede\u0026ntilde;o et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Bacteria can either directly alleviate the harmful effects of salinity or indirectly enhance plant tolerance to salinity. One direct method is to take up excess ions from the soil to prevent their accumulation in the root zone. Additionally, bacteria can produce compatible solutes that help plants maintain their osmotic balance and prevent water loss. They also produce antioxidants that protect plants from the oxidative stress caused by salinity. Indirectly, bacteria promote plant growth and development, making them more resilient to stress(Liu et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and contributing to the improvement of soil structure and nutrient availability by producing organic matter and enhancing soil aggregation, which facilitates water infiltration and nutrient uptake(Kumar Arora et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe beneficial effects of bacteria on plant salinity tolerance and disease management are well documented(Adedayo and Babalola \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Several studies have shown that the inoculation of plants with salt-tolerant bacteria can improve their growth and yield in saline soils, and specific bacterial genes and proteins that contribute to enhancing plant salt tolerance have been identified(Shilev \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In this context, the use of halotolerant PGPB offers a promising alternative for reducing biotic and abiotic stress in plants. This study aimed to analyze the plant growth-promoting action of \u003cem\u003eBacillus halotolerans\u003c/em\u003e AF23 and \u003cem\u003eBacillus velezensis\u003c/em\u003e AF12, isolated from soils affected by underground fires under saline stress conditions, both in vitro and in tomato plants. Additionally, we sought to identify potential genes involved in salinity tolerance and plant growth promotion.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eBiological Material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eBacillus velezensis\u003c/em\u003e AF12 and \u003cem\u003eB. halotolerans\u003c/em\u003e AF23 were isolated from soils affected by underground fires in the community of Pueblo Viejo, in the municipality of Venustiano Carranza, Michoacán de Ocampo, Mexico (20°23′27″–17°53′50″N and 100°03′32″–103°44′49″W). The strains were cultured at 30°C for 18-24 hours on nutrient agar (NA) medium and preserved at 4°C for routine use in the laboratory. Additionally, for long-term storage, the strains were stored in a 30% (v/v) glycerol solution at -20°C. Tomato seeds (\u003cem\u003eS. lycopersicum\u003c/em\u003e) of the Bonny Best cultivar were provided by the Microbiology Laboratory at the Center for Scientific Research and Higher Education at Ensenada (CICESE). \u003cem\u003eF. oxysporum\u0026nbsp;\u003c/em\u003ef. sp. \u003cem\u003elycopersici\u0026nbsp;\u003c/em\u003e(also referred to in later sections as \"Fol\") was provided by the Microbiology Laboratory at CICESE. The fungus was cultured on potato dextrose agar (PDA), incubated at 28°C for 4 days, then stored at 4°C for routine use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic tree with halotolerant strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA phylogenetic tree was constructed with 48 Bacillus strains isolated from soils affected by underground fires, most of which were identified previously. The 16S ribosomal RNA gene sequence was used with \u003cem\u003eEscherichia coli\u0026nbsp;\u003c/em\u003eATCC 1177 as an outgroup. The tree was built using MEGA software version 7.9, employing the maximum likelihood method, with a bootstrap support of 1000 repetitions\u0026nbsp;(Kumar et al. 2016).\u003c/p\u003e\n\u003cp\u003eTo determine halotolerance, 48 \u003cem\u003eBacillus\u003c/em\u003e strains were cultured on nutrient agar (NA) supplemented with NaCl at 1.2% and 2.9% w/v (200 mM and 490 mM, respectively), concentrations corresponding to the growth range of halotolerant microorganisms(Merino et al. 2019a).\u0026nbsp;The highest concentration was used as the criterion for classifying the strains as halotolerant or non-halotolerant. In each Petri dish (100 × 15 mm), four bacteria were streaked, incubated at 30°C for 16 h, and subsequently, the growth of the strains was reported.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of salt tolerance in liquid medium\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe salt tolerance of \u003cem\u003eB. velezensis\u0026nbsp;\u003c/em\u003eAF12 and \u003cem\u003eB. halotolerans\u003c/em\u003e was measured after 12, 24, 36, and 48 h of growth in LB medium supplemented with different concentrations of NaCl (25, 50, 100, 200, 500, and 1000 mM). bacterial pre-inocula were initially incubated with shaking at 150 rpm and 30°C for 12 h in LB medium without NaCl. Once the culture reached an optical density of 0.1 (A590nm), 500 µL was inoculated into the culture medium (supplemented with NaCl) in a final volume of 5 mL and incubated at 30°C with shaking at 150 rpm. Each treatment was performed in triplicate, and uninoculated LB medium served as a blank. The absorbance was measured at 590 nm at each time interval(Orozco-Mosqueda et al. 2019).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlant growth promoting traits in vitro under saline conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSiderophores\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe production of siderophores was assessed on Chrome Azurol S (CAS) agar plates (Schwyn and Neilands 1987)with NaCl concentrations of 25, 50, 100, and 200 mM. The Bacteria were inoculated at the center of the Petri dish and incubated at 30°C for 48 h. A color change from blue to orange in the medium indicated a positive result.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIndole acetic acid\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine IAA production of indole acetic acid, cells were cultured in nutrient broth supplemented with 1% tryptophan and NaCl (0, 25, 50, 200, and 200 mM) for 24 h at 30°C with shaking at 150 rpm until reaching an optical density of 1 (A590nm). The cells were centrifuged at 3000 rpm for 30 min, and 350 µL of the supernatant was mixed with 700 µL of Salkowski reagent. After incubating for 30 min in the dark, the absorbance was measured at 530 nm. Measurements were compared with a calibration curve constructed using dilutions of a standard indole solution (Fluka, Switzerland), and the uninoculated medium with Salkowski reagent served as a control. (Rojas-Solis et al. 2020)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhosphate solubilization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe phosphate solubilization assay was carried out by inoculating the bacterial strains on Pikovskaya agar plates(Djuuna et al. 2022),\u0026nbsp;supplemented with different concentrations of NaCl, and using bromocresol purple as a dye in the medium. The plates were incubated at 30°C for 48 h. A color change from purple to yellow in the medium indicated a positive result.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProteases\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSkim milk agar medium was prepared to evaluate protease production (Abbasi et al. 2019) and the aforementioned concentrations of NaCl were added. The Petri dish was inoculated in the center and incubated at 30°C for 48 h. A clear zone around the bacteria resulted in a positive reaction, indicating the production of protease.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiofilm\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBiofilm production was evaluated following the protocol of Wei and Zhang (2006)\u0026nbsp;with slight modifications. Cells were cultured in nutrient broth (supplemented with NaCl) for 24 h at 30°C with shaking at 150 rpm until reaching an optical density of 1 (A570nm). Subsequently, a 1:100 dilution of the cultured cells was prepared with sterile distilled water. 500 mL of the dilution was transferred to an Eppendorf tube and incubated at 30°C without shaking for 24, 48, and 72 h. The biofilm was quantified at each time interval. Subsequently, 500 mL of 0.1% (w/v) crystal violet was added and incubated for 15 min at room temperature. The dye was decanted and the cells were washed with sterile distilled water to remove residual dye and non-adherent cells. Ethanol (95%) was used to solubilize the dye from biofilm cells. The absorbance of the solubilized dye was measured (A570nm), and 95% ethanol was used as a blank. For siderophores, protease production, and phosphate solubilization, the diameter of the halo of positive reactions was reported at the end of the incubation period.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInoculation tests of tomato plants under salt stress (200 mM NaCl)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePreparation of bacterial inoculum\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo prepare the bacterial inoculum, an isolated colony of each of the strain (\u003cem\u003eB. velezensis\u0026nbsp;\u003c/em\u003eAF12 and \u003cem\u003eB. halotolerans\u003c/em\u003e AF23 separately) was placed in 25 mL of LB culture medium and incubated at 30°C at 110 rpm for 18 h. When the cultures reached an optical density of 0.5 (A590nm), they were used to inoculate the LB culture medium at a concentration of ~2 × 10\u003csup\u003e8\u0026nbsp;\u003c/sup\u003eCFU/mL in a final volume of 50 mL.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn-vitro\u003c/em\u003e \u003cem\u003eplanting and inoculation of tomato seedlings\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eExperiments on tomato plants were performed following the methodology described inCorral-Federico et al. (2024). Tomato seeds of the Bonny Best cultivar were sterilized by immersion in solutions of 70% ethanol (v/v) for 3 min and 2% NaOCl (v/v) for 2 min, after which they were rinsed three times with water. sterile. The seeds were placed on plates with 1.5% agar, once germinated, they were transferred to germination trays with cosmopeat substrate, and kept in a growth chamber at 26°C with light and dark periods of 16 and 8 h, respectively, for two weeks. Finally, seedlings with an average height of 5 cm in the presence of true leaves were selected. Seedlings were transplanted into 6-inch pots and inoculated near the stem with 5 mL of bacterial solution at a concentration of approximately 2 × 10\u003csup\u003e8\u0026nbsp;\u003c/sup\u003eCFU/mL. For the AF12 and AF23 consortium, 2.5 mL of each bacterium was inoculated. Control plants (not inoculated) received 5 mL sterile LB medium. Eight days after the first inoculation of the seedlings, a booster inoculation was performed with 5 mL of bacterial solution at a concentration of ~2 × 10\u003csup\u003e8\u0026nbsp;\u003c/sup\u003eCFU/mL applied near the stems of the plants. At 24 hours after inoculation, the plants were irrigated with saline water (NaCl at 200 mM), which was initially carried out every two days and was subsequently irrigated on demand until the end of the experiment.\u003c/p\u003e\n\u003cp\u003eEight treatments were carried out as follows: 1) plants without bacteria and without NaCl, 2) plants inoculated with AF12, 3) plants inoculated with AF23, 4) plants inoculated with the AF12 and AF23 consortium, 5) NaCl plants, 6) plants inoculated with AF12 and NaCl, 7) plants inoculated with AF23 and NaCl, and 8) plants inoculated with the AF12 and AF23 consortium and NaCl. Eight replicates were prepared for each treatment, and the pots were arranged in the greenhouse of the Microbiology Department of the Center for Scientific Research and Higher Education at Ensenada in a completely randomized design at an average temperature of 24°C on the day and 20 °C at night. Fifty days after transplanting into a pot, the plants were harvested, and different physiological parameters were measured, such as length, fresh and dry weight of the aerial part and root, and chlorophyll content in the leaves.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiocontrol assays of AF12 and AF23 towards \u003cem\u003eF. oxysporum\u003c/em\u003e in tomato plants under greenhouse conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe methodology described previously (Delgado-Ramírez et al. 2021)\u0026nbsp;was followed with some modifications. The seeds were disinfected and germinated as described above. To evaluate the biocontrol of strains AF12 and AF23, on day one after transplanting into a pot, the plants were inoculated near the stem with 5 mL of bacterial solution at a concentration of approximately 2 × 10\u003csup\u003e8\u003c/sup\u003e CFU/mL for the AF12 consortium. and AF23 were inoculated with 2.5 mL of each bacterium. Control plants (not inoculated) received 5 mL sterile LB medium. Eight days later, a booster inoculation was applied, and 15 days later, \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e race 1 was inoculated into the stem at a concentration of approximately 2 × 10\u003csup\u003e6\u003c/sup\u003e total spores/mL per plant.\u003c/p\u003e\n\u003cp\u003eInitially, the plants were watered every third day, and subsequently, irrigation was performed on demand. Five treatments were carried out: 1) plants without bacteria and without \u003cem\u003eFusarium\u003c/em\u003e, 2) plants inoculated with \u003cem\u003eFusarium;\u003c/em\u003e 3) plants inoculated with \u003cem\u003eFusarium\u003c/em\u003e and AF12, 4) plants inoculated with \u003cem\u003eFusarium\u003c/em\u003e and AF23, and 5) plants inoculated with \u003cem\u003eFusarium\u003c/em\u003e and the AF12 and AF23 consortium. Eight replicates were prepared for each treatment and maintained in a greenhouse with a completely randomized design, as previously described. Sixty days after transplanting into a pot, the plants were harvested and different physiological parameters were measured, such as length, fresh and dry weights of the aerial part and the root, and the chlorophyll content in the leaves.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation and detection of \u003cem\u003eFusarium\u003c/em\u003e in inoculated plants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe stems were examined using vertical cuts to observe the presence of the pathogen. Three centimeters of the basal part of the stem of the plants was taken and superficially sterilized with a flame. Subsequently, a longitudinal cut was made in the stems, which were placed in Petri dishes containing PDA medium and incubated for 5 days at 28°C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiocontrol assays of AF12 and AF23 towards \u003cem\u003eF. oxysporum\u003c/em\u003e in tomato plants under salinity conditions (100 mM NaCl)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe seeds were sterilized and germinated as previously described(Delgado-Ramírez et al. 2021).\u0026nbsp;Seedlings with a height of 5 cm and the presence of a true leaf were transplanted into 4-inch pots and inoculated at the base of the stem with 5 mL of a bacterial suspension at a concentration of 2.5 x 10\u003csup\u003e6\u003c/sup\u003e CFU/ml. For the AF12 and AF23 consortium, 2.5 mL of each bacterium was inoculated. Next, the pots were randomly placed in a growth chamber at 26°C with light and dark periods of 16 and 8 hours. Booster inoculation was performed 8 days later. After 72 h, the plants were placed under greenhouse conditions in a completely randomized design and infected with \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u0026nbsp;\u003c/em\u003erace 1.\u003c/p\u003e\n\u003cp\u003eBefore transplanting into pots, the seedlings were watered with purified water and a spray bottle was used to keep the substrate moist. After transplanting into a pot, the plants were watered every third day with 50 mL of water for four weeks, and then the volume was increased to 100 mL. Irrigation was alternated with unsalted water and saline water at a concentration of 100 mM (NaCl). Eight treatments were carried out: 1) plants without bacteria, without \u003cem\u003eFusarium\u0026nbsp;\u003c/em\u003eand without NaCl; 2) plants inoculated with AF12 and NaCl; 3) plants inoculated with AF23 and NaCl; 4) plants inoculated with AF12, AF23 consortium, and NaCl ; 5) plants inoculated with \u003cem\u003eFusarium\u003c/em\u003e and NaCl; 6) plants inoculated with \u003cem\u003eFusarium\u003c/em\u003e, AF12, and NaCl; 7) plants inoculated with\u0026nbsp;\u003cem\u003eFusarium\u003c/em\u003e, AF23, and NaCl; and 8) plants inoculated with\u0026nbsp;\u003cem\u003eFusarium,\u003c/em\u003e AF12, AF23 consortium, and\u0026nbsp;NaCl. Ten replicates were prepared for each treatment. 45 days after infection, the plants were harvested, and different physiological parameters were measured. The stems were examined using vertical cuts to observe the presence of the pathogen.\u003c/p\u003e\n\u003cp\u003eThe fungal inoculum was prepared as follows: three mycelial discs from a culture of \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u0026nbsp;\u003c/em\u003erace 1 were placed in a glass jar with 50 g of sterile rice. The cultures were incubated for 14 d at 28°C. For the infection of the plants, the substrate was removed from the surface, then 0.5 g of the fungal inoculum (~2.5 x 10\u003csup\u003e4\u003c/sup\u003e total spores per plant) was placed and mixed with the substrate. In all plant trials, eight replicates per treatment were used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analyzes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experiments were performed at least three times, and the results were analyzed with Statistica 8.0, using one-way ANOVA and comparison of means through the Duncan test (p ≤ 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenome assembly,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eOGRI and antiSMASH\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStrains AF12 and AF23 were initially cultured on nutrient agar. A single colony from each strain was transferred to 5 mL of nutrient broth (NB) and incubated overnight at 28°C with shaking at 150 rpm. Genomic DNA was extracted using the SDS/proteinase K method followed by polysaccharide precipitation in a high-salt environment(Mahuku 2004). The quality and concentration of the extracted DNA were evaluated using 1% agarose gel electrophoresis and a NanoDrop 1000 spectrophotometer (Thermo Scientific). High-quality DNA was sent to Mr. DNA (Shallowater, Texas, USA) for Illumina sequencing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe genome assembly was performed as previously described Chávez-Ávila et al. (2024). Briefly, quality control of sequencing reads was performed using FastQC version 0.11.5(Andrews 2010). Adapter sequences and low-quality bases were trimmed using Trimmomatic version 0.32(Bolger et al. 2014). De novo assembly was conducted using SPAdes version 3.10.1(Bankevich et al. 2012), employing the default parameters for error correction. The assembled contigs were aligned with Mauve Contig Mover (MCM) (Rissman et al. 2009)using the reference genomes of \u003cem\u003eBacillus velezensis\u003c/em\u003e CBMB205\u003csup\u003eT\u003c/sup\u003e and \u003cem\u003eBacillus halotolerans\u003c/em\u003e FJAT-2398\u003csup\u003eT\u003c/sup\u003e. Genome annotation was performed using the BV-BRC software(Olson et al. 2023), and functional genomic analysis was performed using the RAST server(Aziz et al. 2008).\u003c/p\u003e\n\u003cp\u003eThe genomes of AF12 and AF23 are available in NCBI GenBank under accession numbers NZ_JAJHVX000000000.1 and NZ_JAJHVY000000000.1, respectively, with associated Bioproject numbers PRJNA715922 and PRJNA715770. Additionally, the functions of the putative genes linked to the biological control of phytopathogens within the genomes of strains AF12 and AF23 were predicted using antiSMASH(Blin et al. 2023).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of stress, biocontrol and plant growth-promoting genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenes involved in stress resistance and plant growth promotion in \u003cem\u003eB. velezensis\u003c/em\u003e AF12 and \u003cem\u003eB. halotolerans\u003c/em\u003e AF23 were identified according to previously reported bacterial genes(Nascimento et al. 2020a; Yin et al. 2022). The search for genes was performed using the annotation generated by the National Center for Biotechnology Information (NCBI) Prokaryotic Genome Annotation Pipeline (PGAP) database of genomes AF12 and AF23.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003ePhylogenetic and genomic analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn an initial assessment, a search for halotolerant bacteria was conducted from a collection of strains isolated from Pueblo Viejo, where underground fires occur recurrently. Of the 48 strains evaluated, 6 showed no growth in 2.9% NaCl: AF24, AF30, AF38, AF58, AF64, and AF65 (Suppl. Table 1); therefore, they were classified as non-halotolerant. The remaining strains exhibited optimal growth at both AF23 and AF12 concentrations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the phylogenetic tree, no grouping of the six non-halotolerant bacteria was observed; instead, they were distributed across the different branches of the tree. Interestingly, the non-halotolerant strains AF64 and AF65, identified only at the genus level, were positioned closer to the strain used as an outgroup. In the phylogenetic tree, a grouping of strains was observed according to species. Strain AF23 had a closer phylogenetic relationship with \u003cem\u003eB. halotolerans\u003c/em\u003e AF29, and curiously, these two bacteria were the only ones not grouped with the other strains of the same species (Figure 1). \u003cstrong\u003e[Place Figure 1 here]\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOnce strains AF12 and AF23 were selected, their draft genomes were sequenced with complete coverage. The genome sizes were 3,995,228 bp (base pairs) for AF12 and 4,256,474 bp AF23 strain. The other genomic features are shown in Table 1. Similarly, Table 2 displays the Overall Genome Relatedness Index (OGRI) values for each strain, where AF12 showed high similarity to the genome of \u003cem\u003eBacillus velezensis\u003c/em\u003e CBMB205T and AF23 showed the greatest similarity to \u003cem\u003eBacillus halotolerans\u003c/em\u003e FJAT-2398T (Table 3). Based on these values, which were also greater than ≥98.7% in the 16S ribosomal RNA gene sequence, it was determined that strain AF12 belonged to the species \u003cem\u003eBacillus velezensis\u003c/em\u003e and AF23 to \u003cem\u003eBacillus halotolerans\u003c/em\u003e. \u003cstrong\u003e[Place Tables 1, 2 and 3 here]\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of Salinity Tolerance in Liquid Medium\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe salt tolerance of \u003cem\u003eB. velezensis\u003c/em\u003e AF12 and \u003cem\u003eB. halotolerans\u0026nbsp;\u003c/em\u003eAF23 was assessed by adding 0, 25, 50, 100, 200, 500, and 1000 mM NaCl to the LB liquid culture medium. The growth of AF12 was faster and reached higher levels under low salinity conditions (0 mM). As the salt concentration increased, growth significantly decreased, especially at higher salinity concentrations (500 mM and 1000 mM), until 36 h. At 48 h, a statistically significant increase in growth was observed at 500 mM compared to that at 0 mM, which may be due to a delay in the adaptation phase (Figure 2).\u0026nbsp;\u003cstrong\u003e[Place Figure 2 here]\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn AF23, the results showed that after 12 h of incubation, there was a significant increase in growth at 50 mM compared to the control, whereas at 12 and 36 h, greater growth was observed at the 100 mM concentration. Finally, at 48 h, at 500 mM concentration, AF23 showed greater growth in the culture medium, reaching an optical density of almost 1. At the highest NaCl concentration evaluated (1000 mM), a statistically significant decrease was recorded throughout the incubation period with AF23, indicating that AF23 can grow at higher NaCl concentrations than AF12 (Figure 2). Additionally, AF23 adaptation time to the different salinity concentrations evaluated was shorter than that of AF12.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMechanisms of In Vitro Plant Growth Promotion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 3 shows the plant growth-promotion mechanisms evaluated \u003cem\u003ein vitro.\u003c/em\u003eThe production of siderophores by \u003cem\u003eB. velezensis\u003c/em\u003e and \u003cem\u003eB. halotolerans\u003c/em\u003e AF23 was evaluated by the generation of a halo,\u0026nbsp;which reflects the production of these molecules in solid culture media. Although both bacteria produced\u0026nbsp;siderophores at all evaluated concentrations, AF23 generated brighter halos. In AF12, phosphorus solubilization was higher starting from 25 mM NaCl concentration compared to the control (0 mM).\u0026nbsp;The AF23 activity was slightly to moderate.\u0026nbsp;Protease production in AF12 decreased at 100 and 200 mM NaCl compared to that at the other concentrations.\u0026nbsp;AF23 showed consistent protease production, except at 200 mM NaCl.\u0026nbsp;Indole acetic acid production was subtle in both the strains.\u0026nbsp;No biofilm formation was observed in the experiments conducted in the presence or absence of NaCl. \u003cstrong\u003e[Place Figure 3 here]\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInoculation Trials on Tomato Plants under Saline Stress (200 mM NaCl)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results from the in vitro experiments suggest that\u0026nbsp;\u003cem\u003eB. halotolerans\u003c/em\u003e AF23 can act as a plant growth-promoting bacterium. Therefore, greenhouse inoculation experiments were conducted on tomato plants\u0026nbsp;grown under saline conditions. To induce salt stress, the plants were irrigated with\u0026nbsp;200 mM NaCl. Additionally,\u0026nbsp;tomato plants were inoculated with \u003cem\u003eB. velezensis\u003c/em\u003e AF12 to evaluate\u0026nbsp;the interaction of plant growth\u0026nbsp;with this strain.\u003c/p\u003e\n\u003cp\u003eUnder non-saline conditions, AF12 increased the chlorophyll content by 64% compared with that of the control plants. Inoculation with AF23 and the bacterial consortium increased this parameter by 39% and 40%, respectively. NaCl led to a decrease in chlorophyll in the plants; however, this inhibitory effect was counteracted when AF12 was inoculated (Figure 4a). Shoot length decreased in salt-stressed plants compared to non-salt plants; however, in NaCl-treated plants, there was a statistically significant increase of 17% when the AF12 and AF23 consortium was inoculated compared to non-salt plants (Figure 4b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eShoot fresh weight remained similar in non-salt plants. On the other hand, in salt-stressed plants, a significant increase of 48% was observed with AF12, whereas with AF23 and both strains, it increased by 34% and 24%, respectively, compared to the control (Figure 4c). This was reflected in the shoot dry weight, where AF12 significantly increased this parameter by 69%, whereas AF23 and the consortium improved the dry weight by 57% and 51%, respectively, compared to the control (Figure 4d). Root length remained unchanged between treatments, except in non-salt plants, where AF12 increased root length by 19%, and a 3% improvement was observed when the consortium was inoculated (Figure 4e). The fresh weight of the roots remained similar in plants without NaCl. However, it decreased significantly in salt-stressed plants; however, AF12 and the bacterial consortium increased the dry weight of the root by 35% and 34%, respectively, compared to the control (Figure 4f). The dry weight of the roots increased by 41% with AF12 in non-salt plants. As expected, it decreased in plants treated with NaCl, and interestingly, when the strains were inoculated together, the dry weight increased by 17% (Figure 4g). The total biomass remained similar in the non-salt plants. In contrast, AF12 increased biomass by 53% in salt-stressed plants compared to the control, and when AF23 and both strains were inoculated together, there was also an increase in plant biomass of 38% and 41%, respectively (Figure 4h). These results suggest that AF12 and AF23 differentially promote tomato growth under both normal and saline stress conditions.\u0026nbsp;Figure 4 (lower panel) shows representative images of each treatment.\u0026nbsp;\u003cstrong\u003e[Place Figure 4 here]\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiocontrol Trials of AF12 and AF23 against F. \u003cem\u003eoxysporum\u003c/em\u003e in Tomato Plants under Greenhouse Conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChlorophyll content decreased in plants infected with \u003cem\u003eF\u003c/em\u003e. \u003cem\u003eoxysporum\u003c/em\u003e, and there were no statistically significant differences between plants inoculated with AF12 and the bacterial consortium compared to control plants (Figure 5a). Shoot length did not differ significantly between the treatments (Figure 5b). AF12 increased the fresh weight of the shoots by 36% and AF23 by 27% (Figure 5c). Similarly, AF12 increased the dry weight of the shoot by 40% compared to the control and AF23 by 37%, while the consortium increased the dry weight by 30% (Figure 5d). There were no differences in the root lengths of the plants (Figure 5e). AF12 increased the fresh weight of the roots by 39% compared to that of the control plants (Figure 5f). Dry weight remained without statistically significant differences between the treatments (Figure 5g). Figure 5h shows the effect of bacterial strain inoculation on the total biomass of plants infected with \u003cem\u003eF. oxysporum.\u0026nbsp;\u003c/em\u003eAF12, AF23, and the bacterial consortium increased the total biomass by 34%, 28%, and 24%, respectively, compared with plants without the fungal pathogen. Figure 5 (lower panel) shows representative images of each treatment. \u003cstrong\u003e[Place Figure 5 here]\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation and Detection of \u003cem\u003eFusarium\u003c/em\u003e in Inoculated Plants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the stems of plants infected with \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici,\u0026nbsp;\u003c/em\u003ea dark brown line was observed, which corresponded to the initial symptoms of vascular wilting. This was not observed in the control plants (Figure 6, upper panel). The pathogen was re-isolated from the stems of the infected tomato plants once they were placed on the PDA medium (Figure 6, lower panel). Macroscopic growth of the fungus coincided with the routine growth of Fol race 1. With this, the presence of the fungus was detected in the stems of the infected plants. \u003cstrong\u003e[Place Figure 6 here]\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiocontrol Trials of AF12 and AF23 against \u003cem\u003eF. oxysporum\u0026nbsp;\u003c/em\u003ein Tomato Plants under Salinity Conditions (100 mM NaCl)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the trials conducted to evaluate the biocontrol of the AF12 and AF23 strains against\u0026nbsp;\u003cem\u003eF. oxysporum\u0026nbsp;\u003c/em\u003ef. sp. \u003cem\u003elycopersici\u003c/em\u003e under saline stress conditions, all plants were irrigated with saline water at a concentration of 100 mM NaCl. Shoot length in plants without the pathogen decreased with the co-inoculation of both strains compared to the control; the other treatments remained similar to each other (Figure 7a). The fresh weight of the shoots\u0026nbsp;increased with individual inoculation of AF12 and AF23, and in\u0026nbsp;the consortium in non-Fusarium-infected plants (17%, 16%, and 12%, respectively),\u0026nbsp;and in the presence of the pathogen, the bacterial consortium inoculation increased by 14% compared to the control (Figure\u0026nbsp;7b). Interestingly, AF23 increased the dry weight of the shoot (39%), and bacterial consortium inoculation increased this parameter by 31% in non-infected plants compared with the control. There were no statistically significant differences between\u0026nbsp;the treatments when the pathogen was present (Figure\u0026nbsp;7c). An increase in total biomass was observed with the inoculation of AF23 and the bacterial consortium by 32% and 28%, respectively, in\u0026nbsp;the non\u003cem\u003e-F. oxysporum-\u003c/em\u003einfected plants (Figure 7d). The root length significantly increased by 28% with the bacterial consortium without the pathogen, compared to\u0026nbsp;the control plants; treatments of infected plants remained unchanged (Figure\u0026nbsp;7e). Strain AF12 increased the fresh weight of the roots (44%), and there was a 23% increase in the co-inoculation of both strains in non-\u003cem\u003eFusarium\u003c/em\u003e-infected plants compared to control plants. When AF12 was inoculated into plants infected with the pathogen, a recovery in the fresh weight of the root was observed,\u0026nbsp;similar to\u0026nbsp;that observed in non-infected plants (Figure\u0026nbsp;7f). The dry weight of the roots significantly increased by 27% in plants inoculated with AF12 without Fusarium, compared to the control plants (Figure\u0026nbsp;7g).\u003c/p\u003e\n\u003cp\u003eIt is important to mention that when examining the stems of plants infected with Fol, not all plants showed lesions caused by the pathogen; that is, the inoculation of\u003cem\u003e\u0026nbsp;B. velezensis\u003c/em\u003e AF12 and \u003cem\u003eB. halotolerans\u003c/em\u003e AF23 significantly reduced infection in the plants. With AF12, only 40% of the plants were infected, whereas with AF23, it was 80%; however, with the co-inoculation of both strains, the infection percentage was 20% (Figure 7h). These results suggest that AF12 and AF23 are biocontrol agents for plants subjected to saline stress. Figure 7 (lower panel) shows representative images of each treatment. \u003cstrong\u003e[Place Figure 7 here]\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene Identification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenes with predicted functions in adaptation to high salinity were identified in the genomes of \u003cem\u003eB. velezensis\u003c/em\u003e AF12 and \u003cem\u003eB. halotolerans\u003c/em\u003e AF23 (NCBI GenBank under accession numbers NZ_JAJHVX000000000.1 and NZ_JAJHVY000000000.1; Figure 8). For example, genes related to the synthesis of osmoprotective compounds such as proline (\u003cem\u003eproA\u003c/em\u003e, \u003cem\u003eproB\u003c/em\u003e, and \u003cem\u003eproC\u003c/em\u003e) and the synthesis and transport of glycine betaine/proline (\u003cem\u003ebetB\u003c/em\u003e, \u003cem\u003eopuA, opuAB, opuE\u003c/em\u003e). Genes encoding Na+/H+ antiporters (\u003cem\u003enhaC\u003c/em\u003e), Na+ transporters (\u003cem\u003enatA\u003c/em\u003e), and K+ transporters (\u003cem\u003ektrD\u003c/em\u003e and \u003cem\u003ektrC\u003c/em\u003e) were also present. \u003cstrong\u003e[Place Figure 8 here]\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven that this strain was isolated from soils affected by underground fires and is thermotolerant, AF12 and AF23 contain various genes involved in temperature-stress resistance. For example, \u003cem\u003ednaJ, dnaK\u003c/em\u003e, and \u003cem\u003egroES\u003c/em\u003e encode chaperone-like proteins and three genes related to cold shock (\u003cem\u003ecspB\u003c/em\u003e, \u003cem\u003ecspC\u003c/em\u003e, and \u003cem\u003ecspD\u003c/em\u003e) were also found. Genes involved in protection against oxidative and nitrosative stress are present, including the \u003cem\u003esodA\u003c/em\u003e gene, the product of which is the enzyme superoxide dismutase SodA, which participates in the degradation of superoxide anion radicals. Other genes involved in the breakdown of hydrogen peroxide (\u003cem\u003eahpC\u003c/em\u003e, \u003cem\u003eefeB\u003c/em\u003e, etc.) and a transcriptional repressor sensitive to nitric oxide (\u003cem\u003ensrR\u003c/em\u003e) have also been identified.\u003c/p\u003e\n\u003cp\u003eOther genes found in the AF strains are involved in nitrogen, sulfur, and phosphorus metabolism. The \u003cem\u003enirB\u003c/em\u003e gene, necessary for nitrite reduction; the alkaline phosphatase PhoA encoded by the \u003cem\u003ephoA\u003c/em\u003e gene, which is important in the solubilization of organic phosphate; and genes involved in sulfate transport (\u003cem\u003ecysK\u003c/em\u003e), to name a few. In addition, both bacilli strains possess genes such as \u003cem\u003ebesA\u003c/em\u003e, whose product is a ferri-bacillibactin esterase BesA, and \u003cem\u003edhbC\u003c/em\u003e, which encodes an isochorismate synthase DhbC that is involved in siderophore production, an important mechanism in plant growth promotion. An important aspect of plants is the production and modulation of hormones by prostaglandin B (PGPB). The genomes of both strains contained genes involved in IAA and cytokinin synthesis.\u0026nbsp;\u003cem\u003etrpB\u003c/em\u003e encodes the beta subunit of tryptophan synthase,\u0026nbsp;which is crucial for the synthesis of L-tryptophan from indole and L-serine (the main precursor of IAA). The\u0026nbsp;\u003cem\u003edhaS\u003c/em\u003e gene is also present and catalyzes the conversion of indole-3-acetaldehyde to IAA.\u003c/p\u003e\n\u003cp\u003eGenes related to production and degradation of VOCs in both strains. The \u003cem\u003ealsS\u003c/em\u003e gene is involved in acetoin synthesis and the \u003cem\u003ebdhA\u003c/em\u003e gene is related to butanediol degradation. Genes related to chemotaxis (\u003cem\u003echeA, cheB, cheD,\u003c/em\u003e \u003cem\u003echeR, cheV, cheY, cheW, motA\u003c/em\u003e), flagellar biosynthesis (\u003cem\u003eflgB, flgC, flgK, flhA\u003c/em\u003e), and biofilm formation (\u003cem\u003etasA\u003c/em\u003e and \u003cem\u003eEfp\u003c/em\u003e) were also detected in the bacterial genomes. Genes involved in the transport and resistance to heavy metals, such as zinc, copper, and arsenate, and genes related to sporulation (\u003cem\u003eyabP, yjcZ, yjcD, ytfJ, yunB, gerD, yutH, cotJC\u003c/em\u003e) were also identified. The presence of these genes indicates that both strains possess genetic machinery to cope with different types of stress, including salinity, and promote tomato growth through various mechanisms (Figure 9).\u0026nbsp;\u003cstrong\u003e[Place Figure 9 here]\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to antiSMASH analysis, gene clusters involved in the synthesis of metabolites with potential antimicrobial activity, such as surfactin and fengycin, were also identified (Table 4). Both lipopeptides are widely characterized as antifungal agents that play a significant role in the biocontrol of crop diseases. The similarity percentages were greater than 70%, suggesting a high likelihood of functional activity; however, this requires further experimental confirmation.\u0026nbsp;\u003cstrong\u003e[Place Table 4 here]\u003c/strong\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, the biocontrol and plant growth-promoting traits of strains AF12 and AF23 isolated from soils affected by underground fires were evaluated under normal and salinity-stress conditions. There are earlier reports of bacteria, particularly Bacilli species, which successfully promote plant growth under similar environmental stress conditions(Jamali et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Bokhari et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Pranaw et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) Besides, the potential PGP \u003cem\u003eBacillus\u003c/em\u003e spp. AF12 and AF23 salinity tolerance of were evaluated both in vitro and in vivo by prescreening several Bacillus strains isolated from soils affected by underground fires(Flores-Pi\u0026ntilde;a et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This location has been a wonderful site for the detection of potential PGP and biocontrol bacteria with thermotolerance and halotolerance capacities, in addition to exhibiting beneficial effects on plants of agronomic importance(Ch\u0026aacute;vez-Avila et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eStrain AF23 was identified as \u003cem\u003eBacillus halotolerans\u003c/em\u003e through OGRI, including average nucleotide identity (ANI) analyses, genome-to-genome distance calculator (GGDC), and phylogeny based on the 16S rRNA gene sequence, obtaining 98.9%, 97.92%, and 100%, respectively. The AF23 genome has a size of 4.2 Mb. AF12 was identified as a \u003cem\u003eBacillus velezensis\u003c/em\u003e species according to the ANI, GGDC, and 16S rRNA with values ​​of 98.15, 98.54%, and 99.68%, respectively, and a genome size of 3.9 Mb. Recently, Wu and colleagues (\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) a strain of \u003cem\u003eBacillus halotolerans\u003c/em\u003e KKD1 with biocontrol and plant growth-promoting capabilities was identified in wheat. This strain induced the production of phytohormones such as 6-benzylaminopurine and gibberellic acid under salt stress conditions. Additionally, genome mining of KKD1 has revealed interesting genes involved in biocontrol and plant growth promotion. For \u003cem\u003eBacillus velezensis\u003c/em\u003e, there is also evidence of its beneficial effects under saline conditions. For instance, Bai and coauthors (2023) the salt-tolerant bacterium \u003cem\u003eBacillus velezensis\u003c/em\u003e strain JB0319 acts as a plant growth promoter. The JB0319 strain successfully stimulated the growth of lettuce plants under saline conditions by enhancing antioxidant activities (superoxide dismutase and peroxidase) and reducing MDA accumulation. Other studies have highlighted the great potential of these two \u003cem\u003eBacillus\u003c/em\u003e species as halotolerant and beneficial microorganisms include works byChen et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), Masmoudi et al. (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and \u0026Ccedil;am et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Some strains, such as \u003cem\u003eBacillus velezensis\u003c/em\u003e GB03, have been commercially available for some time(Jang et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Thus, strains AF12 and AF23 represent excellent candidates as potential bioinoculants for saline soils and regions with high temperatures, as they can grow at 50\u0026deg;C (Ch\u0026aacute;vez-\u0026Aacute;vila et al., 2025, unpublished results).\u003c/p\u003e \u003cp\u003eWith bacterial identification at the species level, the initial assumption was that AF23 was a salt-tolerant bacterium. Therefore, growth was evaluated at different NaCl concentrations (25, 50, 100, 200, 500, and 1000 mM) at various time intervals. The results showed that this strain could grow under high-salinity conditions. When comparing the growth of AF12 and AF23 with that of \u003cem\u003eB. subtilis\u003c/em\u003e 168 in halotolerance experiments, the growth of \u003cem\u003eB. subtilis\u003c/em\u003e 168 was minimal (data not shown), as previously reported (under NaCl stress conditions)(Rath et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In contrast, the growth of AF12 and AF23 was in the range of NaCl designated for microorganisms termed halotolerants(Merino et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e; Reang et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). It is important to mention that although both bacteria can grow under saline conditions, in the assays evaluated, AF23 showed greater growth in the medium supplemented with NaCl.\u003c/p\u003e \u003cp\u003eIn the present study, the mechanisms of direct and indirect plant growth promotion (pathogen antagonism) were determined. One of the mechanisms of plant growth promotion is nutrient facilitation such as iron acquisition through siderophore production (Khan et al., 2019). AF23 produced siderophores under saline conditions, with 200 mM NaCl being the highest concentration tested. Similar siderophore production has been reported in \u003cem\u003eBacillus\u003c/em\u003e strains, such as the salt-tolerant \u003cem\u003eB. aryabhattai\u003c/em\u003e MS3 at 100 and 200 mM NaCl(Sultana et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Genomic analysis revealed that AF23 harbors gene clusters with 100% similarity to those encoding the siderophore bacillibactin, including \u003cem\u003ebesA\u003c/em\u003e and \u003cem\u003edhbC\u003c/em\u003e, the latter of which codes for isochorismate synthase. These genes have been identified in other species such as \u003cem\u003eB. megaterium\u003c/em\u003e STB1(Nascimento et al. 2020). Siderophore production, a dual mechanism of plant growth promotion, not only enhances iron uptake, but also limits plant pathogen growth(Di Francesco and Baraldi \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Bacillibactin production by AF23 may explain its antifungal activity, as this siderophore is linked to its biocontrol properties(Dimopoulou et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn vitro assays showed that AF23 produced IAA at all tested concentrations (25, 50, 100, and 200 mM), with a slight reduction observed at 200 mM. IAA synthesis is a key trait for stress mitigation by PGPB. Genes involved in IAA synthesis have been identified in \u003cem\u003eB. halotolerans\u003c/em\u003e AF23. IAA regulates key plant growth processes such as cell division, elongation, and root hair formation, which improve water and nutrient uptake(Luo et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). AF23 also produced proteases under saline conditions, although its activity decreased at 200 mM NaCl compared to that under non-saline conditions. Similar activity has been reported for \u003cem\u003eB. velezensis\u003c/em\u003e DMB06 under saline conditions(Na et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePGPB mitigates salinity stress by supplying nutrients such as phosphorus(Gao et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). AF23 solubilized phosphate, with larger halos observed at 25\u0026ndash;100 mM NaCl than at 0 mM NaCl. Genomic analysis revealed genes involved in phosphate solubilization. This differs from \u003cem\u003eB. pumilus\u003c/em\u003e JPVS11, in which higher salinity reduces phosphate solubilization, although it improves rice growth under saline stress(Kumar et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The positive in vitro results prompted the evaluation of AF23 in tomato plants under different conditions: 1) 200 mM saline stress, 2) infection with \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e (Fol), and 3) 100 mM saline stress combined with Fol infection. A 200 mM NaCl concentration was selected based on AF23's optimal growth and previous reports(Abdelshafy Mohamad et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Sultana et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The 100 mM NaCl solution was chosen because of the optimal growth of Fol at this concentration. Tomato experiments evaluated \u003cem\u003eB. velezensis\u003c/em\u003e AF12 and \u003cem\u003eB. halotolerans\u003c/em\u003e AF23 individually and in consortia under biotic and abiotic stresses, leveraging prior findings and literature on their plant growth promotion and antifungal activities(Dong et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Salinity negatively affected the chlorophyll content. AF12 (data not shown) and AF23 siderophore production may explain the increased chlorophyll content in tomatoes, because Fe is essential for chlorophyll biosynthesis(Sultana et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAF12 and AF23 inoculation, alone or in combination, significantly improved shoot dry weight under 200 mM NaCl by 50\u0026ndash;69%, reflected in a 38\u0026ndash;53% increase in total biomass. In the absence of NaCl, root dry weight increased by 41% after AF12 inoculation compared with uninoculated plants. Saleem et al. (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) observed that 200 mM NaCl severely reduced cotton growth parameters, but inoculation with \u003cem\u003eBacillus\u003c/em\u003e spp. partially restored root and shoot length, chlorophyll content, and reduced Na\u0026thinsp;+\u0026thinsp;uptake. Notably, the greatest effects of AF12 and AF23 inoculation occurred under saline conditions, likely linked to the absence of genomic traits under non-saline conditions.\u003c/p\u003e \u003cp\u003eTomato wilt, caused by Fol, remains a major challenge. \u003cem\u003eB. halotolerans\u003c/em\u003e Cal.l.30 significantly reduced disease incidence in grape and cherry tomatoes inoculated with \u003cem\u003eBotrytis cinerea\u003c/em\u003e, as well as \u003cem\u003ein vitro\u003c/em\u003e antagonism towards \u003cem\u003eB. cinerea\u003c/em\u003e, \u003cem\u003eRhizoctonia solani\u003c/em\u003e, and Fol(Tsalgatidou et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Similarly, \u003cem\u003eB. velezensis\u003c/em\u003e AP-3 reduced the severity of Fusarium wilt in tomatoes by 50%(Medeiros and Bettiol \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Greenhouse experiments confirmed that AF12 and AF23 improved tomato biomass during Fol infection. Shoot dry weight increased by 40% with AF12 and 37% with AF23. Total biomass increased by 34% and 28%, respectively. Under combined saline and Fusarium stress (100 mM NaCl), inoculation with AF12, AF23, or their combination reduced disease incidence by 60\u0026ndash;80%. Comparable findings include halotolerant \u003cem\u003eBacillus\u003c/em\u003e spp. that promote tomato growth and reduce disease severity under saline conditions(Abdelshafy Mohamad et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe impact of the inoculation of strains AF12 and AF23 on the rhizosphere microbiome of tomato plants remains to be investigated, as some bioinoculant agents have been observed to impact the structure and modulation of functions of other microorganisms associated with the same host plant. For example, a study Ji et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) revealed that the microbial agents \u003cem\u003eBacillus subtilis\u003c/em\u003e HG-15 and \u003cem\u003eBacillus velezensis\u003c/em\u003e JC-K3, when applied to wheat growth under low salt stress, affected fungal communities, including pathogenic and beneficial arbuscular mycorrhizal fungi (AMF). The results showed that inoculation with bacterial agents increased wheat yield, chlorophyll content, photosynthesis, and water use efficiency, while reducing disease incidence by 79.80%. Analysis of fungal communities revealed that inoculation decreased the abundance of harmful fungi such as \u003cem\u003eGibberella\u003c/em\u003e and \u003cem\u003eFusarium\u003c/em\u003e while promoting beneficial AMF species such as \u003cem\u003eGlomus\u003c/em\u003e. These findings suggest that \u003cem\u003eBacillus\u003c/em\u003e-based PGPR can improve plant growth, disease resistance, and soil microbiome dynamics, thereby providing insights into sustainable disease control.\u003c/p\u003e \u003cp\u003eIn conclusion, this study highlights the functional and genomic basis of salinity tolerance and plant growth-promoting traits of AF, particularly in synergy. Genes linked to osmotic and oxidative stress responses, volatile organic compound (VOC) synthesis, and metal resistance were identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). These findings demonstrate the potential of AF strains to mitigate combined biotic and abiotic stresses in economically important crops.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eG.S. thanks CIC-UMSNH (2024\u0026ndash;2025), and ICTI-Michoac\u0026aacute;n (ICTI-PICIR23-000) for their support with the research projects.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eFVM, SCA, ES, CSDR, JJMC, and RHM contributed to the implementation of the research, analysis of sequence data, and writing of the manuscript. FVM created the figures. MCOM, SDLV, and OBB contributed to the data analysis and editing of the manuscript. GS conceptualized, designed the experiments, and wrote the draft and final manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eF.V.M. and S.C.A. received a Master's in Science scholarship from CONAHCyT-Mexico.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe genomes of AF12 and AF23 are available in NCBI GenBank under accession numbers NZ_JAJHVX000000000.1 and NZ_JAJHVY000000000.1, respectively, with associated Bioproject numbers PRJNA715922 and PRJNA715770.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbbasi S, Safaie N, Sadeghi A, Shamsbakhsh M (2019) Streptomyces Strains Induce Resistance to Fusarium oxysporum f. Sp. Lycopersici Race 3 in Tomato through Different Molecular Mechanisms. 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Is Represented by the Plant Growth-Promoting Bacterium BY2G20 Isolated from Saline-Alkaline Soil and Enhances the Growth of Zea mays L. under Salt Stress . mSystems 7:1\u0026ndash;16. https://doi.org/10.1128/msystems.01426-21\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 4 are not available with this version.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-microbiology-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wibi","sideBox":"Learn more about [World Journal of Microbiology and Biotechnology](https://www.springer.com/journal/11274)","snPcode":"11274","submissionUrl":"https://submission.nature.com/new-submission/11274/3","title":"World Journal of Microbiology and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Bacilli, biocontrol, Fusarium wilt disease, genome, halotolerant","lastPublishedDoi":"10.21203/rs.3.rs-5671788/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5671788/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSoil salinity is a major limiting factor for agricultural crops, which increases their susceptibility to pathogenic attacks. This is particularly relevant for tomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e), a salt-sensitive crop. Fusarium wilt, caused by \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e, is a significant threat to tomato production in both greenhouse and field environments. This study evaluated the salinity tolerance, biocontrol, and plant growth-promoting properties of \u003cem\u003eBacillus velezensis\u003c/em\u003e AF12 and \u003cem\u003eBacillus halotolerans\u003c/em\u003e AF23, isolated from soil affected by underground fires and selected for their resistance to saline conditions (up to 1000 mM NaCl). In vitro assays confirmed that both strains produced siderophores, indole-3-acetic acid (IAA), and proteases, and exhibited phosphate solubilization under saline stress (100\u0026ndash;200 mM NaCl). AF23 exhibited synergistic interactions with AF12, and inoculation with either strain individually or in combination significantly improved the growth of the Bonny Best tomato cultivar under 200 mM saline stress, leading to increased shoot and root weight, enhanced chlorophyll content, and higher total biomass. The biocontrol potential of AF12 and AF23 was evaluated in tomato plants infected with \u003cem\u003eF. oxysporum\u003c/em\u003e. Both strains, individually or combined, increased shoot and root weight, chlorophyll content, and total biomass under non-saline conditions, promoting growth and reducing infection rates under saline stress (100 mM NaCl). Genomic analysis revealed that both strains harbored genes related to salt stress tolerance, biocontrol, and plant growth promotion. In conclusion, \u003cem\u003eBacillus\u003c/em\u003e AF23 and AF12 demonstrated strong potential as bioinoculants for enhancing tomato growth and providing protection against \u003cem\u003eF. oxysporum\u003c/em\u003e in saline-affected soils.\u003c/p\u003e","manuscriptTitle":"Stress-Tolerant Bacillus Strains for Enhancing Tomato Growth and Biocontrol of Fusarium oxysporum under Saline Conditions: Functional and Genomic Characterization","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-23 17:47:33","doi":"10.21203/rs.3.rs-5671788/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-01-29T14:17:20+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-17T11:13:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"331959950632405224558222740691567789895","date":"2025-01-13T06:50:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-06T09:46:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"43479236004503300274007772020305884103","date":"2024-12-28T09:19:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"209954262423687674475413168690073389254","date":"2024-12-26T04:10:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"70995863517538067122689379973302139372","date":"2024-12-25T23:26:34+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-12-25T19:45:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-12-20T16:39:56+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-12-20T15:03:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"World Journal of Microbiology and Biotechnology","date":"2024-12-18T19:14:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-microbiology-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wibi","sideBox":"Learn more about [World Journal of Microbiology and Biotechnology](https://www.springer.com/journal/11274)","snPcode":"11274","submissionUrl":"https://submission.nature.com/new-submission/11274/3","title":"World Journal of Microbiology and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5762f0f4-2b92-48e2-904f-e1f902c9b08b","owner":[],"postedDate":"December 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-03-10T19:45:50+00:00","versionOfRecord":{"articleIdentity":"rs-5671788","link":"https://doi.org/10.1007/s11274-025-04308-8","journal":{"identity":"world-journal-of-microbiology-and-biotechnology","isVorOnly":false,"title":"World Journal of Microbiology and Biotechnology"},"publishedOn":"2025-03-07 15:58:19","publishedOnDateReadable":"March 7th, 2025"},"versionCreatedAt":"2024-12-23 17:47:33","video":"","vorDoi":"10.1007/s11274-025-04308-8","vorDoiUrl":"https://doi.org/10.1007/s11274-025-04308-8","workflowStages":[]},"version":"v1","identity":"rs-5671788","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5671788","identity":"rs-5671788","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}