Molecular characterization and functional evaluation of a halotolerant endophytic Priestia megaterium with dual plant growth–promoting and antifungal activities under salinity stress

Caracterização molecular e avaliação funcional de Priestia megaterium endofítica halotolerante com dupla atividade de promoção do crescimento vegetal e atividade antifúngica sob estresse salino

Abstract

Soil salinity is a major constraint on global agricultural productivity, particularly in arid and coastal ecosystems. In this study, we isolated and characterized a halotolerant endophytic bacterium from the leaf tissue of mangrove plants collected from the Sehat mangrove ecosystem in Saudi Arabia. Molecular identification based on 16S rRNA gene sequencing confirmed the isolate as Priestia megaterium, showing over 98% sequence similarity; the sequence was deposited in GenBank under accession number PV018403. P. megaterium exhibited strong antifungal activity against Fusarium solani, achieving up to 96% inhibition of mycelial growth after 48 h of incubation with cell-free culture supernatant. Microscopic analysis revealed severe hyphal deformation, fragmentation, and structural disruption, indicating a fungicidal mode of action. This activity was associated with the production of cyclic lipopeptides and hydrogen cyanide, independent of siderophore production. Additionally, the strain demonstrated multiple plant growth–promoting traits and tolerated salinity up to 10% NaCl, significantly enhancing tomato germination, root development, and biomass under salt stress. Co-application with ACC further improved plant performance. These findings highlight P. megaterium as a promising bioinoculant for sustainable agriculture in salt-affected environments.

Keywords:
endophytic bacteria; Priestia megaterium; salinity stress; halotolerance; plant growth–promoting bacteria

Resumo

A salinidade do solo constitui uma das principais limitações à produtividade agrícola global, especialmente em regiões áridas e costeiras. Neste estudo, foi isolada e caracterizada uma bactéria endofítica halotolerante de tecidos foliares de plantas de mangue coletadas no ecossistema de manguezal de Sehat, na Arábia Saudita. A identificação molecular baseada no sequenciamento do gene 16S rRNA confirmou o isolado como Priestia megaterium, com mais de 98% de similaridade de sequência; a sequência foi depositada no GenBank sob o número de acesso PV018403. Priestia megaterium apresentou forte atividade antifúngica contra Fusarium solani, alcançando até 96% de inibição do crescimento micelial após 48 h de incubação com sobrenadante livre de células. A análise microscópica revelou deformações severas das hifas, fragmentação e perda de integridade estrutural, indicando um modo de ação fungicida. Essa atividade esteve associada à produção de lipopeptídeos cíclicos e cianeto de hidrogênio, independentemente da produção de sideróforos. Além disso, a estirpe demonstrou múltiplas características promotoras de crescimento vegetal e tolerou salinidade de até 10% de NaCl, melhorando significativamente a germinação, o desenvolvimento radicular e a biomassa de tomate sob estresse salino. A coaplicação com ACC potencializou ainda mais o desempenho das plantas. Esses resultados destacam P. megaterium como um bioinoculante promissor para a agricultura sustentável em ambientes afetados pela salinidade.

Palavras-chave:
bactérias endofíticas; Priestia megaterium; estresse salino; halotolerância; promoção do crescimento vegetal

1. Introduction

Soil salinity represents one of the most severe abiotic constraints limiting agricultural productivity on a global scale, especially in arid and semi-arid regions where evapotranspiration exceeds precipitation, leading to progressive salt accumulation in the rhizosphere Mwesige, 2025. Elevated salt concentrations impose osmotic and ionic stress on plants, disrupting water balance, impairing selective nutrient uptake, inhibiting photosynthetic efficiency, and altering enzymatic and metabolic activities. These physiological and biochemical perturbations culminate in growth retardation, reduced biomass accumulation, and substantial yield losses (Atta et al., 2023). The intensification of irrigated agriculture, coupled with inadequate drainage systems and the accelerating impacts of climate change, has further exacerbated the expansion of salt-affected soils worldwide. In response to this escalating challenge, considerable attention has been directed toward the development of sustainable and environmentally compatible mitigation strategies (Waheed et al., 2024). Among these, the utilization of beneficial plant-associated microorganisms particularly endophytic bacteria has emerged as a highly promising approach due to their capacity to enhance plant stress tolerance, modulate phytohormonal balance, and improve nutrient acquisition under saline conditions (Aizaz et al., 2023). Endophytic bacteria are non-pathogenic microorganisms that reside within plant tissues without causing disease, establishing intricate and often mutualistic interactions with their hosts. These microorganisms have garnered significant scientific interest owing to their diverse functional attributes, including the promotion of plant growth, enhancement of tolerance to abiotic stresses, and suppression of phytopathogenic organisms through various direct and indirect mechanisms (Ali et al., 2023).

Mangrove ecosystems, which thrive under extreme environmental pressures such as elevated salinity, periodic waterlogging, and fluctuating oxygen availability, harbor highly specialized and diverse microbial assemblages (Kesawat et al., 2023). The persistent exposure to such harsh conditions has selected for endophytic communities possessing remarkable halotolerance and adaptive resilience (Kamińska et al., 2022). Consequently, mangrove-associated endophytes represent a valuable reservoir of stress-adapted microorganisms with considerable potential for application in saline and other stress-prone agroecosystems (Soldan et al., 2019; Lacava et al., 2022; Yuan et al., 2023).

Recent advances in microbial ecology and plant–microbe interactions have underscored the biotechnological relevance of mangrove-derived endophytic bacteria as strategic resources for sustainable agriculture in salt-affected environments. Halotolerant members of genera such as Bacillus, Pseudomonas, Serratia, and Halomonas have been reported to exhibit a wide array of plant growth-promoting (PGP) functions under saline stress (Szymańska et al., 2022). These functional attributes encompass the biosynthesis of phytohormones particularly indole-3-acetic acid (IAA) mineral phosphate solubilization, siderophore-mediated iron acquisition, atmospheric nitrogen fixation, and modulation of plant stress-responsive pathways. Collectively, these mechanisms contribute to improved nutrient use efficiency, enhanced osmotic adjustment, and stabilization of plant physiological performance under high salinity (Wu et al., 2021).

In addition to their growth-promoting capacities, many mangrove-associated endophytes produce structurally diverse antifungal metabolites and extracellular hydrolytic enzymes, including chitinases and β-1,3-glucanases, which target key structural components of fungal cell walls (Mohamad et al., 2020). Such bioactive compounds have demonstrated inhibitory activity against economically important phytopathogens, including Rhizoctonia, Botrytis cinerea, and Aspergillus spp., thereby conferring an integrated benefit that combines stress mitigation with biological disease control (Chaudhary et al., 2022).

The present study was designed to isolate and perform a comprehensive phenotypic and functional characterization of halotolerant endophytic bacteria obtained from mangrove plant tissues collected in the Sehat coastal ecosystem, located in the Eastern Province. Specifically, we aimed to (i) evaluate their plant growth-promoting traits under saline conditions, (ii) assess their antifungal potential against major soil-borne pathogens, and (iii) elucidate key physiological and biochemical mechanisms underpinning salt stress tolerance. By identifying robust, stress-adapted microbial candidate with multifunctional properties, this work seeks to contribute to the development of next-generation bioinoculants capable of enhancing crop productivity, ecological resilience, and sustainability in arid and salinity-prone agroecosystems facing intensifying climate-driven pressures.

2. Material and Methods

2.1. Isolation of mangrove-derived endophytic bacteria

Endophytic bacterial strains were recovered from symptom-free mangrove leaves collected under sterile conditions from the intertidal mangrove habitats of Sehat (26°30'13.8"N, 50°02'36.9"E), located in the Eastern Province. Immediately after collection, plant materials were transported to the laboratory in sterile containers for microbiological processing.

To eliminate epiphytic microorganisms, leaf samples were subjected to a rigorous surface-sterilization procedure consisting of sequential immersion in 70% (v/v) ethanol for 1 min, followed by treatment with 10% (v/v) sodium hypochlorite for 5 min. The tissues were then rinsed multiple times with sterile distilled water to ensure complete removal of residual sterilizing agents and surface contaminants, in accordance with protocols adapted from Pallavi et al., 2023. The effectiveness of sterilization was verified by plating the final rinse water onto nutrient agar to confirm the absence of external microbial growth.

Surface-sterilized tissues were aseptically excised into small segments and placed onto nutrient agar (NA) plates, which were incubated at 28°C for 48 h. Bacterial colonies emerging from within the plant tissues were considered putative endophytes. Morphologically distinct colonies were selected, repeatedly sub-cultured to obtain pure isolates, and subsequently preserved under appropriate storage conditions for downstream phenotypic and functional characterization.

2.2. Assessment of halotolerance

The salt tolerance capacity of the isolated bacterial strains was evaluated by monitoring their growth on NA plates supplemented with increasing concentrations of sodium chloride (NaCl). Briefly, isolates were spot-inoculated onto NA plates amended with 0%, 5%, 10%, 15%, and 20% (w/v) NaCl. The inoculated plates were incubated at 28°C for 48–72 h under aerobic conditions. Bacterial growth was assessed qualitatively based on the presence of visible colonies at each salinity level. The degree of halotolerance was defined as the highest NaCl concentration supporting detectable growth for each isolate, following criteria adapted from Akimbekov et al. (2025).

2.3. Molecular identification of the halotolerant endophytic bacterium

The halotolerant endophytic bacterium was identified by 16S rRNA gene sequencing. Genomic DNA was extracted using the Zymo DNA Extraction Kit (Zymo Research, USA), and the nearly full-length 16S rRNA gene (~1400 bp) was amplified using universal primers 27F and 1492R. PCR reactions (25 µL) contained MyTaq Red Mix (Bioline, UK), primers (10 µM each), genomic DNA (~100 ng), and nuclease-free water. Amplification was performed under standard cycling conditions with 35 cycles and an annealing temperature of 52 °C. PCR products were verified on 1% agarose gel, purified using a spin column purification kit (Zymo Research), and subjected to Sanger sequencing (1st BASE Asia, Malaysia). The resulting sequences were edited and assembled using Chromas Pro and DNA MAN software, and taxonomic identity was determined by BLASTn comparison against reference sequences in the NCBI GenBank database (Hammami et al., 2009; Hammami et al., 2013).

2.4. Evaluation of Plant Growth-Promoting (PGP) traits

2.4.1. Nutrient acquisition potential

The ability of the isolates to solubilize inorganic phosphate was determined using Pikovskaya’s agar medium. Bacterial strains were spot-inoculated onto the medium and incubated at 28°C for 5–7 days. The formation of clear halo zones surrounding the colonies indicated phosphate solubilization. The solubilization index (SI) was calculated by dividing the total diameter by the colony diameter, as described by Sanchez-Gonzalez et al. (2023).

The nitrogen-fixing potential of the isolates was evaluated using nitrogen-free semi-solid Burk’s medium. Strains were inoculated into the medium and incubated at 28°C for up to 5 days. The development of a subsurface pellicle indicated bacterial growth under nitrogen-limited conditions, suggesting diazotrophic capability. In addition, the appearance of pink coloration in the medium was considered indicative of nitrogenase activity, following the methodology reported by Keleshyan et al. (2022).

2.4.2. Phytohormone production

Indole-3-AceticAcid (IAA) Production was quantified colorimetrically. Bacterial isolates were cultured in tryptic soy broth (TSB) supplemented with 500 µg/mL L-tryptophan and incubated at 28°C for 48 h under shaking conditions. After centrifugation, the supernatant was mixed with Salkowski’s reagent and incubated in the dark for color development. The intensity of the pink coloration, corresponding to IAA concentration, was measured at 530 nm using a UV–Vis spectrophotometer. IAA levels were determined based on a standard calibration curve, following the protocol of Feng et al. (2023).

2.4.3. Evaluation of antifungal activity

The antifungal potential of the selected bacterial isolates was assessed against the phytopathogenic fungus Fusarium solani (NCBI accession: EU326473.1). The fungal strain was cultured on potato dextrose agar (PDA; pH 5.6 ± 0.2) and incubated at 27 ± 2°C for 5 days to obtain actively growing mycelia. Stock cultures were maintained on PDA slants at 4°C for short-term preservation (Hammami et al., 2011).

Antagonistic activity was initially evaluated using a dual culture confrontation assay, following the method described by Aljameel et al. (2026). A mycelial plug (5 mm diameter) excised from the actively growing margin of a 5-day-old fungal culture was placed at the center of a PDA plate, while the bacterial isolate was streaked at a defined distance from the fungal plug. Plates were incubated at 28 ± 2°C under strictly controlled and standardized physicochemical conditions, including temperature (28 ± 2°C), medium pH (5.6 ± 0.2), and moisture content, maintained consistently across all treatments, and the extent of fungal growth inhibition was recorded after 48–72 h. Control plates without bacterial inoculation were included to assess normal fungal growth.

2.4.4. Quantitative assessment of mycelial growth inhibition

The antifungal activity of bacterial cell-free supernatants (CFS) was quantitatively evaluated using a poisoned medium assay to assess their inhibitory effect on the radial growth of phytopathogenic fungi, following Aljameel et al. (2026). Briefly, bacterial cultures were centrifuged at 8,000 × g for 10 min to remove cellular biomass, and the resulting supernatants were sterilized by sequential filtration through 0.45 µm and 0.22 µm membrane filters (Millipore, USA) to obtain cell-free extracellular metabolites.

Sterile CFS was incorporated into molten potato dextrose agar (PDA; pH 5.6 ± 0.2) at final concentrations of 15%, 25%, and 50% (v/v), and the amended media were aseptically dispensed into sterile Petri dishes. Control plates without CFS were included as negative controls, while plates supplemented with the commercial fungicide Hymexazol (100 µg/mL) were used as positive controls. To account for potential dilution and nutrient variation effects associated with CFS addition, additional control plates supplemented with an equivalent volume of sterile uninoculated broth were also included.

After solidification, each plate was inoculated with a 5-mm diameter mycelial plug excised from the actively growing margin of a 7-day-old fungal culture and incubated at 28 ± 2°C for 7 days under strictly controlled and standardized physicochemical conditions, including temperature (28 ± 2°C), medium pH (5.6 ± 0.2), and moisture conditions maintained by uniform agar volume and incubation environment across all treatments. All treatments were performed in triplicate (n = 3) to ensure reproducibility.

The radial growth of fungal colonies was measured using a digital caliper, and antifungal activity was expressed as the percentage of mycelial growth inhibition (PI), calculated using the following Formula 1:

$\text{PI (\%)}=\frac{R_1-R_2}{R_1}\times 100$ (1)

where $\text{R}1$and $\text{R}2$represent the mean colony diameter of the control and treated plates, respectively. Data were subjected to analysis of variance (ANOVA), and significant differences among treatments were determined using Tukey’s multiple comparison test at a significance level of p ≤ 0.05.

2.4.5. Mode of action of endophytic P. megaterium CFS on fungal growth

To investigate the mode of action of the cell-free supernatant (CFS) and to distinguish between fungicidal and fungistatic effects, agar plugs from fungal colonies exhibiting complete growth inhibition were aseptically transferred onto fresh potato dextrose agar (PDA; pH 5.6 ± 0.2) plates without CFS supplementation. The plates were incubated at 28 ± 2°C for 5 days under strictly controlled and standardized physicochemical conditions, including temperature (28 ± 2°C), medium pH (5.6 ± 0.2), and moisture maintained by identical agar volume and uniform incubation conditions across all treatments, and monitored for mycelial regrowth. The absence of fungal recovery after transfer was interpreted as indicative of a fungicidal effect, whereas resumed growth was considered consistent with a fungistatic response. All treatments were performed in triplicate (n = 3) to ensure reproducibility (Hammami et al., 2011).

In addition, morphological alterations in F. solani hyphae following CFS treatment were examined using light microscopy (Olympus BH-2, Tokyo, Japan) at 40× magnification. Structural changes, including hyphal fragmentation, deformation, swelling, vacuolization, and apparent cell lysis, were documented and compared with untreated controls.

2.4.6. Characterization of antagonistic and enzymatic properties

Multiple functional assays were conducted to evaluate the antagonistic potential of the endophytic isolate P. megaterium and to elucidate the underlying mechanisms involved in phytopathogen suppression. Hydrogen cyanide (HCN) production was assessed by inoculating the isolate onto nutrient agar supplemented with glycine and incubating at 28 °C for 4 days. Cyanide production was detected using sterile filter paper impregnated with picric acid–sodium carbonate solution placed in the lid of the Petri dish; a color change from yellow to orange–brown was considered indicative of HCN emission. (Uzair et al., 2018).

Siderophore production was evaluated using chrome azurol S (CAS) agar medium, and the nature of the siderophore compounds was characterized by determining their reactivity to chemical tests specific for catecholates and hydroxamates. The contribution of siderophores to antifungal activity was further examined by supplementing dual culture plates with an excess of iron (FeCl₃) to assess a reduction in inhibition zones. Cyclic lipopeptide (CLP) production was qualitatively tested using a drop-collapse assay on parafilm and by emulsification index measurement, both indicators of biosurfactant production (Uzair et al., 2018).

The production of cyclic lipopeptides (CLPs) and related biosurfactants was qualitatively assessed using the drop-collapse assay on parafilm and by determining the emulsification index (E24), both of which serve as indirect indicators of surface-active metabolite production. These amphiphilic biosurfactants are known to interact with fungal plasma membranes and may contribute to membrane destabilization, pore formation, and leakage of intracellular components. Additionally, they are likely associated with the induction of oxidative stress via reactive oxygen species (ROS) accumulation, which could ultimately lead to membrane disruption and fungal cell death (Uzair et al., 2018).

Extracellular enzymatic activities associated with antifungal activity and rhizosphere competence were evaluated using substrate-specific plate assays (Kaleh et al., 2022)

Protease activity was assessed on nutrient agar supplemented with 3% (w/v) skim milk, where the formation of clear hydrolysis zones indicated casein degradation mediated by extracellular proteases. Cellulase activity was screened on carboxymethyl cellulose (CMC) agar plates, followed by Congo red staining and destaining with 1 M NaCl to visualize zones of cellulose hydrolysis. Amylase activity was evaluated on starch agar plates, and starch degradation was confirmed by iodine staining, with the appearance of clear halos indicating enzymatic activity.

Chitinolytic activity was assessed using colloidal chitin agar containing 1% (w/v) chitin as the sole carbon source, following Hammami et al. (2013) with minor modifications. The isolate was spot-inoculated onto the medium and incubated at 28 °C for 5 days. The formation of distinct transparent zones surrounding bacterial colonies indicated extracellular chitinase activity resulting from the enzymatic degradation of chitin, a major structural component of fungal cell walls.

All assays were performed in triplicate to ensure experimental reproducibility and reliability.

2.4.7. Assessment of salt stress tolerance mechanisms

The 1-aminocyclopropane-1-carboxylate deaminase (ACCD) activity of endophytic isolate was evaluated based on its ability to utilize ACC as the sole nitrogen source, following the method of Simarmata, et al. (2023). Briefly, the endophytic strain was cultured in nitrogen-free minimal medium (MM) supplemented with 3 mM ACC and incubated at 28 °C for 48 h under continuous shaking at 180 rpm. Bacterial growth under these conditions was considered indicative of ACC utilization and potential ACCD activity. Quantitative determination of ACCD activity was performed by measuring the production of α-ketobutyrate, the enzymatic cleavage product of ACC, using a colorimetric assay adapted from Simarmata, et al. (2023). Cell-free extracts were incubated with ACC substrate, and the resulting α-ketobutyrate was derivatized with 2,4-dinitrophenylhydrazine (DNPH). The absorbance of the resulting complex was measured at 540 nm using a UV–Vis spectrophotometer. Enzyme activity was expressed as µmol of α-ketobutyrate produced per mg of protein per hour. Total protein concentration in the cell extracts was determined using the bicinchoninic acid (BCA) assay, based on the formation of a stable purple complex between bicinchoninic acid and reduced copper ions (Cu⁺), with absorbance measured at 562 nm. Bovine serum albumin (BSA) was used as the standard for protein quantification, following the protocol of Cortés-Ríos et al. (2020).

To further evaluate the oxidative stress mitigation potential endophytic isolate, the activities of key antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR), were determined using spectrophotometric methods. SOD activity was measured based on the inhibition of nitroblue tetrazolium (NBT) photoreduction, as described by Wood and Sorensen (2001). The reduction of NBT was monitored at 560 nm, and one unit of SOD activity was defined as the amount of enzyme required to inhibit 50% of NBT photoreduction under the assay conditions. Catalase (CAT) activity was determined by monitoring the decomposition of hydrogen peroxide (H₂O₂) at 240 nm, following the method of Hadwan et al. (2024). One unit of CAT activity was defined as the amount of enzyme required to decompose 1 µmol of H₂O₂ per minute under standard assay conditions. Glutathione reductase (GR) activity was evaluated based on the NADPH-dependent reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH), according to Yannarelli et al. (2007). The rate of NADPH oxidation was monitored spectrophotometrically at 340 nm, and one unit of GR activity was defined as the amount of enzyme catalyzing the oxidation of 1 µmol of NADPH per minute.

2.4.8. Assessment of salt tolerance

The halotolerance of the endophytic bacterium P. megaterium was evaluated using both optical density measurements and viable cell counts to assess its growth response under increasing salinity conditions following the method of Peng et al. (2024). Overnight cultures were initially grown in nutrient broth (NB) at 30 ± 2 °C under standard conditions. The bacterial suspension was then adjusted to an initial density of approximately 1 × 10⁷ CFU/mL and inoculated into NB supplemented with increasing concentrations of sodium chloride (NaCl) at 0%, 1%, 3%, 5%, 10%, and 15% (w/v). The cultures were incubated at 30 ± 2 °C with continuous agitation at 150 rpm for 48 h to ensure homogeneous growth conditions.

Bacterial growth was monitored spectrophotometrically by measuring the optical density at 600 nm (OD₆₀₀) at 24-h intervals, providing a quantitative estimate of biomass accumulation. In parallel, viable cell counts were determined using the standard serial dilution and plate count method. Briefly, aliquots from each culture were serially diluted in sterile saline solution and plated onto nutrient agar (NA), followed by incubation at 30 ± 2 °C to determine colony-forming units per milliliter (CFU/mL).

Salt tolerance was defined by the ability of the isolate to maintain measurable growth (OD₆₀₀ ≥ 0.1) and to produce viable colonies under saline conditions. The maximum tolerance concentration (MTC) was defined as the highest NaCl concentration supporting sustained bacterial growth and viability. All experiments were conducted in triplicate, and the results were expressed as mean values ± standard deviation, ensuring statistical reliability and reproducibility.

2.4.9. Effect of P. megaterium inoculation on tomato growth under salinity stress

To evaluate the effect of P. megaterium on tomato seed germination and early seedling development under salinity stress, a controlled in vitro assay was performed following Patani et al. (2023). Tomato seeds (Solanum lycopersicum var. cerasiforme) were surface-sterilized by sequential immersion in 70% (v/v) ethanol for 1 min and 2% (w/v) sodium hypochlorite for 5 min, followed by thorough rinsing with sterile distilled water.Salinity stress was imposed using NaCl at 0.5%, 1.5%, and 3.0% (w/v), corresponding to low, moderate, and high stress levels, respectively. To assess the involvement of ACC deaminase activity in stress alleviation, ACC (1-aminocyclopropane-1-carboxylic acid) was applied at 3 mM in designated treatments.

P. megaterium was cultured in nutrient broth at 30°C for 72 h and adjusted to approximately 10⁸ CFU mL⁻¹ prior to inoculation. Seeds assigned to inoculation treatments were soaked in the bacterial suspension for 2 h, whereas control seeds were treated with sterile broth under identical conditions. The experiment was conducted using a completely randomized design. Each treatment comprised three independent replicates (n = 3), with 10 seeds per replicate, placed on sterile germination paper in Petri dishes. Plates were incubated at 25 ± 1°C under dark conditions during the germination phase; following radicle emergence, seedlings were maintained under a 16 h light / 8 h dark photoperiod, with a photosynthetic photon flux density of ~150 µmol m⁻² s⁻¹ and relative humidity maintained at 60–70%, and irrigated daily with the corresponding NaCl or NaCl + ACC solutions.

A total of twelve treatment groups were established (Table 1), integrating salinity levels, bacterial inoculation, and ACC application. Germination rate and seedling growth parameters, including root length, shoot length, and shoot dry biomass, were recorded at 10 and 30 days post-incubation.

Table 1
Description of the treatments.

Code	Treatment Description
S0.5-C	Surface-sterilized seeds, no P. megaterium, no ACC, irrigated with 0.5% NaCl solution
S0.5-PM	Surface-sterilized seeds + P. megaterium suspension, irrigated with 0.5% NaCl solution
S1.5-C	Surface-sterilized seeds, no P. megaterium, no ACC, irrigated with 1.5% NaCl solution
S1.5-PM	Surface-sterilized seeds + P. megaterium suspension, irrigated with 1.5% NaCl solution
S3-C	Surface-sterilized seeds, no P. megaterium, no ACC, irrigated with 3.0% NaCl solution
S3-PM	Surface-sterilized seeds + P. megaterium suspension, irrigated with 3.0% NaCl solution
S0.5-ACC	Surface-sterilized seeds + ACC (3 mM), no P. megaterium, irrigated with 0.5% NaCl + ACC solution
S0.5-PM+ACC	Surface-sterilized seeds + P. megaterium + ACC (3 mM), irrigated with 0.5% NaCl + ACC solution
S1.5-ACC	Surface-sterilized seeds + ACC (3 mM), no P. megaterium, irrigated with 1.5% NaCl + ACC solution
S1.5-PM+ACC	Surface-sterilized seeds + P. megaterium + ACC (3 mM), irrigated with 1.5% NaCl + ACC solution
S3-ACC	Surface-sterilized seeds + ACC (3 mM), no P. megaterium, irrigated with 3.0% NaCl + ACC solution
S3-PM+ACC	Surface-sterilized seeds + P. megaterium+ ACC (3 mM), irrigated with 3.0% NaCl + ACC solution

All experiments were conducted in triplicate, and data were expressed as mean values for subsequent statistical analysis.

2.4.10. Statistical analysis

Data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s HSD post hoc test for multiple comparisons. Results are expressed as mean ± standard deviation (SD). Exact p-values were calculated and reported where applicable, and 95% confidence intervals (CI) were determined for key measurements. In addition, effect sizes (η²) were computed to quantify the magnitude of treatment effects. Statistical significance was set at p < 0.05, and all analyses were performed using IBM SPSS Statistics (Version 26).

3. Result and Discussion

An endophytic bacterium was isolated from mangrove leaf tissue collected from the Sehat mangrove ecosystem, Saudi Arabia, following a rigorous surface sterilization protocol. The isolate was identified as Priestia megaterium based on 16S rRNA gene sequencing, showing over 98% sequence similarity, and the sequence was deposited in GenBank under accession number PV018404. The results of this study clearly demonstrate the strong antifungal potential of P. megaterium against F. solani. As shown in Table 2, the antifungal activity of the cell-free supernatant increased progressively with incubation time. Fungal radial growth inhibition reached 40 ± 0.7% at 12 h, increased to 56.2 ± 1.0% at 24 h, and further rose to 80.0 ± 1.0% at 36 h, before reaching a maximum of 96 ± 0.5% at 48 h. A significant decline to 60 ± 0.5% was observed at 60 h, indicating reduced antifungal efficacy at prolonged incubation. These differences were statistically significant (one-way ANOVA, p < 0.001, η² = 0.91), with post hoc analysis confirming significant differences between time points (Tukey’s test, p < 0.05).The pronounced antifungal activity observed at 36–48 h likely reflects the peak accumulation of bioactive secondary metabolites, which may act synergistically to disrupt fungal cell membranes, compromise cell wall integrity, and inhibit hyphal expansion. Comparable metabolite-mediated antifungal mechanisms have been reported in other Prestia species (Raaijmakers et al., 2010), further supporting the strong biocontrol potential of P. megaterium against F. solani.

Table 2
Antifungal, enzymatic, and plant growth-promoting activities of endophytic bacteria P. megaterium.

Test Category	Test	Result
Antifungal Activity	% fungal radial growth inhibition	(± SD)
	12 h Cell-free supernatant	40 ± 0.7ᵃ **
	24 h Cell-free supernatant	56.2 ± 1.0ᵇ **
	36 h Cell-free supernatant	80.00 ± 1.0ᶜ **
	48 h Cell-free supernatant	96± 0.5 d**
	60h Cell-free supernatant	60± 0.5 e **
Antagonistic Properties	Hydrogen cyanide production1	+
	Cyclic lipopeptide surfactant production2	+
	Siderophore production3	–
Enzymatic Properties	Protease	+
	Cellulase	+
	Amylase	+
	Chitinase	+
Plant Growth-Promoting Traits	Phosphate solubilization2	+
	Nitrogenase activity	+
	Indole-3-acetic acid (IAA) production	+

• ᵃ^,ᵇ^,ᶜ^,d,e: Values within a column followed by different superscript letters are significantly different (p ≤ 0.01) based on one-way ANOVA and Tukey’s post hoc test.
• ¹
  Detected by colorimetric assay using glycine-supplemented nutrient agar.
• ²
  Detected using oil displacement and Pikovskaya’s agar, respectively.
• ³
  Evaluated using CAS blue agar.
• ⁺
  indicates a positive result;
• −indicates a negative result, and
• ^**
  indicates a statistically significant difference at p < 0.01.

Microscopic analysis of fungal mycelia treated with 48 h cell-free supernatant (CFS) revealed pronounced morphological alterations compared with the untreated control (Figure 1), consistent with strong antifungal activity. In the control, hyphae appeared smooth, intact, and well organized, forming continuous, thin filaments characteristic of normal growth. In contrast, CFS-treated samples exhibited marked structural irregularities, including hyphal thickening, swelling, distortion, and fragmentation. Similar morphological alterations have been reported in fungi exposed to antimicrobial metabolites produced by Bacillus and related genera, particularly cyclic lipopeptides, which disrupt membrane integrity and induce hyphal deformation (Raaijmakers et al., 2010). Notably, Ling et al. (2022) demonstrated that the antifungal activity of Bacillus licheniformis is associated with the production of extracellular proteases capable of degrading key structural components of fungal cells, resulting in comparable hyphal deformation and growth inhibition. Similarly, Mohamad et al. (2020) reported pronounced hyphal damage in Fusarium spp. following treatment with endophytic bacteria.

Figure 1
Antifungal activity of P. megaterium cell-free supernatant (CFS) at different concentrations against F. solani, assessed by the radial mycelial growth inhibition assay over 72 h.

The antifungal potential of P. megaterium cell-free supernatant (CFS) was evaluated using a radial mycelial growth inhibition assay against F. solani at increasing concentrations (15–50%). A clear concentration-dependent suppression of mycelial growth was observed, with inhibition ranging from 15% ± 0.5 at 15% CFS to complete inhibition (100% ± 0.4) at 50% CFS (Figure 1, Table 2). These differences were statistically significant (one-way ANOVA, p < 0.001, η² = 0.95), and post hoc analysis confirmed significant differences among concentrations (Tukey’s test, p < 0.05). These results demonstrate the strong, dose-dependent antifungal activity of P. megaterium CFS and support its potential as an effective biocontrol agent against phytopathogenic fungi.

The fungistatic or fungicidal effect of the cell-free supernatant (CFS) was determined by transferring the inhibited mycelial discs obtained from plates treated with 50% CFS onto fresh, untreated potato dextrose agar (PDA) and evaluating their capacity for regrowth. No mycelial recovery was observed after 72 h of incubation, demonstrating that the inhibitory effect was irreversible and confirming the fungicidal nature of the CFS. Microscopic analysis revealed clear morphological differences between untreated and treated samples of F. solani. In the untreated control, the fungus exhibited normal morphological features, including dense, hyaline, septate, and regularly branched hyphae forming a well-organized and structurally intact mycelial network. Numerous well-developed, multicellular macroconidia with typical elongated, fusiform morphology were observed, indicating active conidiogenesis and normal reproductive capacity. In contrast, exposure to 50% CFS resulted in severe morphological alterations, including a substantial reduction in macroconidia production, suggesting inhibition of sporulation. Additionally, the hyphae displayed pronounced structural abnormalities, such as irregular diameter, distortion, fragmentation, and reduced density, accompanied by disorganization and partial collapse of the mycelial architecture. These findings suggest that the CFS may induce irreversible structural damage to fungal cells, thereby impairing both vegetative growth and reproductive development. Similar antifungal effects have been reported by Imran et al. (2026), who demonstrated that Bacillus-derived culture filtrates, particularly in combination with plant-derived compounds, suppress Fusarium infection through disruption of hyphal integrity and inhibition of fungal development. The agreement between these observations and the present results supports the hypothesis that extracellular metabolites produced by P. megaterium may contribute to antifungal activity.

The antifungal activity observed in this study is strongly supported by the production of hydrogen cyanide (HCN) and cyclic lipopeptides (CLPs), two well-characterized secondary metabolites with potent antifungal properties. HCN, a volatile compound, inhibits cytochrome c oxidase within the mitochondrial electron transport chain, thereby disrupting oxidative phosphorylation, suppressing ATP synthesis, and ultimately inducing fungal cell death (Huang et al., 2026). In parallel, CLPs including iturins, fengycins, and surfactins contribute to antifungal efficacy through complementary and synergistic mechanisms. These amphiphilic biosurfactants insert into fungal plasma membranes, destabilize lipid bilayers, induce pore formation, promote leakage of intracellular components, and trigger oxidative stress via reactive oxygen species (ROS) accumulation, ultimately leading to membrane collapse and programmed cell death (Markelova and Chumak, 2025).

The co-production of volatile (HCN) and diffusible (CLPs) metabolites by P. megaterium reflects a multi-layered antagonistic strategy that enhances overall biocontrol performance. The combination of respiratory inhibition and membrane disruption may underlie the high levels of fungal growth suppression observed in vitro. Comparable metabolite-driven synergistic antifungal mechanisms have been reported in other Priestia spp. (Abiala et al., 2023).

Notably, P. megaterium displayed pronounced antifungal activity despite the absence of siderophore production, a trait traditionally associated with microbial antagonism through iron sequestration. This suggests that fungal suppression in this strain may operate via siderophore-independent pathways, potentially mediated by bioactive secondary metabolites. Similar observations were reported by Zelaya-Molina et al. (2023) who demonstrated that siderophore-deficient P. megaterium strains retained strong antifungal capacity through the secretion of CLPs and volatile organic compounds. These results highlight the metabolic plasticity of P. megaterium and support the concept that diffusible and volatile metabolites can play a dominant role in fungal growth inhibition independent of iron competition.

Beyond metabolite-mediated antagonism, P. megaterium exhibited a broad extracellular enzymatic repertoire, including protease, cellulase, amylase, and chitinase activities (Table 1), which likely contribute to fungal cell wall degradation. Chitinase is particularly critical, as it hydrolyzes chitin polymers within fungal cell walls, leading to structural destabilization, cytoplasmic leakage, and impaired hyphal development (Dutilloy et al., 2024). Proteases may further attenuate fungal virulence by degrading extracellular enzymes and pathogenicity-associated proteins. Meanwhile, cellulases and amylases facilitate controlled plant tissue colonization and organic matter turnover without inducing host damage.

Collectively, the integration of volatile inhibition (HCN), membrane-targeting CLPs, and lytic enzyme production establishes a robust, multifactorial antagonistic system. This combination not only ensures effective suppression of phytopathogenic fungi but also contributes to nutrient cycling and rhizosphere fertility through organic matter mineralization (Dobrzyński et al., 2023). Such metabolic versatility underscores the potential of P. megaterium as a sustainable and efficient biocontrol agent for agricultural applications.

Furthermore, the multifactorial antagonistic mechanisms exhibited by P. megaterium highlight its remarkable metabolic versatility and reinforce its potential as a robust biocontrol agent in sustainable agricultural systems. The ability of this strain to produce both volatile and diffusible antifungal metabolites, in combination with cell wall–degrading enzymes, ensures effective inhibition of fungal pathogens through simultaneous disruption of essential cellular functions and structural integrity. This integrated mode of action not only enhances antifungal efficacy but also reduces the likelihood of resistance development, as multiple cellular targets are affected concurrently. In addition, the enzymatic activities observed in this study, including chitinase, protease, cellulase, and amylase, may facilitate rhizosphere colonization and competitive establishment, thereby strengthening the persistence and ecological fitness of P. megaterium in plant-associated environments. These traits are particularly advantageous for long-term biocontrol applications, as they support both direct antagonism against phytopathogens and indirect promotion of plant health through improved nutrient cycling and microbial balance. Overall, the strong fungicidal activity of the CFS, supported by microscopic evidence of irreversible structural damage in F. solani, underscores the significant potential of P. megaterium as an environmentally friendly alternative to chemical fungicides for the management of fungal diseases in agricultural crops.

The multifunctionality of P. megaterium exemplifies its dual ecological role as a potent biocontrol agent and a plant growth-promoting endophyte (PGPE). Its capacity to inhabit internal plant tissues provides a strategic advantage in delivering localized antifungal metabolites and enzymes, while simultaneously modulating plant defense signaling pathways.

Beyond its antagonistic activities, P. megaterium displayed several plant growth-promoting (PGP) traits, such as phosphate solubilization, indole-3-acetic acid (IAA) production, and nitrogenase activity (Table 1). These functions are crucial for improving nutrient availability and stimulating plant development under stress conditions (Egamberdieva et al., 2017). Furthermore, the strain tolerated high levels of salinity, maintaining growth up to 10% NaCl and showing reduced but detectable viability at 15% (Figure 2). This halotolerance is consistent with the adaptation of endophytes from mangrove environments and highlights the potential of P. megaterium for application in saline soils (Vurukonda et al., 2016). This biochemical trait not only alleviates phosphorus deficiency but also promotes root proliferation and nutrient uptake, synergizing with the auxin-mediated effects of IAA, a phytohormone pivotal for root system architecture modulation and improved absorptive capacity (Uzair et al., 2018).

Figure 2
Microscopic analysis (40× magnification) of hyphal morphology in Fusarium solani under control conditions and following treatment with 50% P. megaterium cell-free supernatant (CFS). (A) Control (F. solani without treatment); (B) F. solani Treated with 50% cell-free supernatant (CFS).

Crucially, P. megaterium nitrogenase activity confirms its capacity for biological nitrogen fixation, an energetically demanding process that converts atmospheric nitrogen into ammonia, thus supplementing soil nitrogen pools and enhancing plant nutrition in nitrogen-poor environments (Ntabo et al., 2018). This integration of phosphate solubilization, phytohormone production, and nitrogen fixation positions P. megaterium as a multifunctional PGP endophyte capable of mitigating nutrient deficiencies while enhancing plant growth.

When compared to other well-characterized endophytic genera, such as Azospirillum and Pseudomonas, P. megaterium exhibits distinct advantages linked to its robust stress tolerance and environmental persistence. Azospirillum spp., while highly efficient in nitrogen fixation and IAA synthesis, often exhibit limited resilience under abiotic stresses like salinity and drought, which constrains their field application under marginal conditions (Sun et al., 2024). In contrast, the spore-forming nature of P. megaterium confers enhanced survival and activity in harsh soil environments, ensuring sustained colonization and PGP efficacy (Tsotetsi et al., 2022).

In summary, the convergent PGP traits and intrinsic resilience of P. megaterium underline its promise as a next-generation bioinoculant tailored for sustainable agriculture, particularly under nutrient-limited and stress-prone conditions. Its multifunctionality, combining nutrient mobilization, hormone production, nitrogen fixation, and stress tolerance, represents an integrative strategy to enhance crop productivity while reducing reliance on synthetic agrochemicals.

The halotolerance of P. megaterium was systematically evaluated under increasing NaCl concentrations using optical density (OD₆₀₀) measurements (Table 3). The strain exhibited robust growth at low salinity levels (0–3% NaCl), with OD₆₀₀ values increasing from 1.38 ± 0.05 to 1.64 ± 0.05 at 0% NaCl, from 1.26 ± 0.05 to 1.50 ± 0.05 at 1% NaCl, and from 1.15 ± 0.05 to 1.38 ± 0.05 at 3% NaCl after 48 h, corresponding to strong growth. At 5% NaCl, growth was moderately reduced, with OD₆₀₀ values increasing from 1.00 ± 0.03 at 24 h to 1.20 ± 0.05 at 48 h, indicating moderate osmotic stress but sustained viability. A pronounced reduction in bacterial growth was observed at 10% NaCl, with OD₆₀₀ values of 0.44 ± 0.03 at 24 h and 0.63 ± 0.05 at 48 h, reflecting limited cellular proliferation under elevated salinity. No detectable growth occurred at 15% NaCl confirming complete inhibition and absence of viable cells. Based on these findings, 10% NaCl was identified as the maximum tolerance concentration (MTC) for P. megaterium. This strong halotolerant phenotype highlights the strain’s ability to adapt to saline environments and supports its potential application in saline agroecosystems and coastal soils.

Table 3
Growth of P. megaterium at different NaCl concentrations, measured as optical density at 600 nm (OD₆₀₀).

NaCl concentration (%)	0% NaCl	1% NaCl	3% NaCl	5% NaCl	10% NaCl	15% NaCl
Growth after 24 h (OD600 ± SD)	1.38 ± 0.05	1.26 ± 0.05	1.15 ± 0.05	1.00 ± 0.03	0.44 ± 0.03	0.00
Growth after 48 h (OD600 ± SD)	1.64 ± 0.05	1.50 ± 0.05	1.38 ± 0.05	1.20 ± 0.05	0.63 ± 0.05	0.00
Growth level+	+++	+++	+++	++	+	(no viability)

• Values are presented as mean ± SD (n = 3).
• ⁺
  ++indicates strong growth;
• ⁺
  +indicates moderate growth;
• ⁺
  indicates weak growth.

This tolerance to elevated salinity suggests that P. megaterium possesses efficient osmoadaptive mechanisms that enable maintenance of cellular homeostasis under hyperosmotic conditions. Halotolerant bacteria typically respond to salt stress through the accumulation of compatible solutes such as proline, glycine betaine, and trehalose, which balance intracellular osmotic pressure without disrupting metabolic processes. Additionally, adaptive changes in membrane lipid composition and enhanced activity of ion transport systems contribute to maintaining membrane integrity and cellular functionality under saline stress. The gradual decline in OD₆₀₀ values with increasing NaCl concentrations reflects the physiological burden associated with osmotic adaptation, while the complete growth inhibition at 15% NaCl indicates that this concentration exceeds the strain’s osmophysiological tolerance threshold. Importantly, halotolerance represents a critical ecological and functional trait for plant-associated beneficial bacteria, as it enables survival, colonization, and sustained metabolic activity in saline soils. This characteristic is particularly relevant for biocontrol applications, as it ensures that P. megaterium can maintain its antagonistic activity and persistence in salt-affected agricultural environments, thereby enhancing its effectiveness as a resilient and sustainable biocontrol agent.

Many halotolerant Prestia species respond to osmotic stress through the accumulation of compatible solutes such as trehalose, proline, and glycine betaine, which play a crucial role in maintaining intracellular osmotic balance and stabilizing proteins, membranes, and other macromolecules without disrupting essential metabolic functions (Akimbekov et al., 2025). In addition, P. megaterium likely produces exopolysaccharides (EPS), which contribute to biofilm formation and provide a protective barrier against osmotic stress by retaining water and reducing cellular dehydration. EPS-mediated biofilm formation also enhances bacterial adhesion to plant root surfaces and promotes stable colonization, a key trait widely reported in endophytic and rhizospheric Bacillus species (Ntabo et al., 2018). Furthermore, the ability of P. megaterium to form highly resistant endospores confers an additional survival advantage under extreme salinity and fluctuating environmental conditions, allowing the strain to persist during unfavorable periods and rapidly resume growth when conditions improve (Tsotetsi et al., 2022).

Salinity stress markedly impaired tomato seed germination and early seedling development, as evidenced by progressive reductions in root elongation, shoot growth, and biomass accumulation with increasing NaCl concentrations (Table 4). In uninoculated controls, severe salinity (3% NaCl) significantly reduced germination to 48% and restricted root elongation to only 3 cm after 30 days, highlighting the inhibitory effects of osmotic stress and ion toxicity on early plant establishment. Even under moderate salinity (1.5% NaCl), substantial growth limitations were observed, indicating that salt stress disrupts cellular expansion processes and alters biomass allocation during seedling development.

Table 4
Effect of P. megaterium inoculation and/or ACC application on tomato seed germination and seedling growth under different salinity levels.

Treatment	Germination (%)	Root Length (cm) 10 days	Root Length (cm) 30 days	Shoot Length (cm) 10 days	Shoot Length (cm) 30 days	Shoot Dry Weight (g plant−1) 30 days
S0.5-C	55 ± 1ᵇ	2 ± 0.4ᵈ	4.5 ± 0.3ᵉ	9.7 ± 0.3ᵈ	12.5 ± 0.3ᵉ	8.7 ± 0.5ᵈ
S0.5-PM	63 ± 1ᵃ	4 ± 0.6ᵃ	8.5 ± 0.3ᶜ	10.5 ± 0.5ᵇ	14.5 ± 0.5ᵇ	12.0 ± 1.0ᵇ
S1.5-C	68 ± 1ᵇ	5 ± 0.4ᵈ	5.5 ± 0.3ᵉ	14.4 ± 0.3ᵈ	16 ± 0.2ᶠ	15.2 ± 0.5ᵉ
S1.5-PM	79 ± 2ᵇ	8± 0.5ᶜ	11 ± 0.5ᵇ	16 ± 0.5ᶜ	19 ± 0.3ᵈ	18 ± 0.8ᵃᵇ
S3-C	48± 1ᶜ	1.5 ± 0.2ᵈ	3 ± 0.2ᶠ	5 ± 0.2ᵉ	9.8 ± 0.2ᶠ	11.5 ± 0.4ᵉ
S3-PM	51 ± 1ᵇ	1.8 ± 0.4ᶜ	7± 0.3ᶜ	8 ± 0.4ᵇ	11 ± 0.3ᵈ	12 ± 0.8ᶜ
S0.5-ACC	51 ± 2ᵇ	3 ± 0.3ᶜᵈ	6 ± 0.3ᶜ	10 ± 0.3ᶜ	14 ± 0.3ᵉ	10.0 ± 0.3ᵇ
S0.5-PM+ACC	77 ± 1ᵃ	5 ± 0.4ᵃ	8 ± 0.5ᵃ	14 ± 0.5ᵃ	16 ± 0.5ᵃ	11 ± 0.4ᵃ
S1.5-ACC	63 ± 3ᵇ	4 ± 0.3ᵈ	7.5 ± 0.4ᵇ	16 ± 0.5ᵇ	18 ± 0.2ᶠ	14 ± 0.5ᵇ
S1.5-PM+ACC	85 ± 2ᵃ	7 ± 0.3ᵇ	13 ± 0.5ᵃ	18 ± 0.5ᵃ	25 ± 0.5ᵃ	18 ± 0.2ᵃ
S3-ACC	48 ± 2ᶜ	2 ± 0.3ᵈ	5.5 ± 0.3ᵇ	12 ± 0.3ᶜ	19 ± 0.2ᵉ	12± 0.4ᵇ
S3-PM+ACC	77 ± 2ᵃ	4 ± 0.3ᶜ	10 ± 0.5ᵃ	12± 0.5ᵃ	21 ± 0.4ᵃ	13.5± 0.5ᵃ

• Values are presented as mean ± SD (n = 3). Different superscript letters within each column indicate significant differences among treatments (p ≤ 0.05; Tukey’s HSD test).

Inoculation with P. megaterium significantly mitigated the detrimental effects of salinity across all tested NaCl concentrations (p ≤ 0.05). Under mild salinity (0.5% NaCl), bacterial treatment nearly doubled root elongation after 30 days (8.5 cm compared with 4.5 cm in the uninoculated control) and increased shoot dry biomass by approximately 38%, suggesting improved root system architecture and enhanced nutrient and water acquisition. Under moderate salinity (1.5% NaCl), P. megaterium further enhanced germination (79% versus 68%) and promoted substantial root elongation (11 cm versus 5.5 cm), demonstrating an improved capacity of inoculated plants to withstand osmotic stress. Remarkably, even under severe salinity (3% NaCl), bacterial inoculation partially restored root growth (7 cm compared with 3 cm in the control) and improved shoot biomass, confirming the strain’s ability to alleviate salt-induced growth inhibition.

ACC supplementation alone provided moderate protective effects; however, the combined treatment (P. megaterium + ACC) produced the most pronounced improvements across all measured parameters. The S1.5-PM+ACC treatment exhibited the highest overall performance, achieving maximal germination (85%), enhanced root elongation (13 cm at 30 days), increased shoot length (25 cm), and the greatest biomass accumulation (18 g plant⁻¹), significantly outperforming all individual treatments (p ≤ 0.05). Under severe salinity (3% NaCl), the synergistic treatment (S3-PM+ACC) dramatically improved germination (77%) and increased root length more than threefold relative to the saline control, indicating a substantial mitigation of salt-induced growth suppression.

Statistical analysis further confirms that bacterial inoculation, particularly in combination with ACC, significantly enhances plant performance under saline conditions. These improvements in germination, root development, and biomass production strongly suggest that P. megaterium contributes to salinity tolerance through integrated physiological mechanisms. A key component of this response likely involves the modulation of ethylene homeostasis via bacterial ACC deaminase activity. Under salt stress, elevated ethylene levels typically inhibit root elongation and accelerate senescence. By degrading ACC, the bacterium may reduce stress-induced ethylene accumulation, thereby contributing to alleviation of growth inhibition and promotion of root system expansion. The pronounced enhancement observed in PM+ACC treatments strongly supports the hypothesis that bacterial regulation of ethylene signaling plays a central role in plant adaptation to saline environments.

Beyond ethylene modulation, the marked stimulation of root development suggests improved water and nutrient acquisition, contributing to enhanced osmotic adjustment and physiological stability under salt stress. The greater magnitude of growth promotion under moderate salinity (1.5% NaCl) further indicates that P. megaterium optimizes plant stress adaptation within a responsive physiological range rather than merely compensating for extreme damage. Such regulation likely involves coordinated hormonal balance, improved stress signaling, and metabolic stabilization.

Collectively, these adaptive mechanisms, combined with the strain’s demonstrated fungicidal activity against F solani, extracellular hydrolytic enzyme production, and tolerance to elevated salinity, position P. megaterium as a multifunctional and resilient bio-inoculant. Its capacity to simultaneously enhance stress tolerance and suppress phytopathogens underscores its strong potential for application in saline agroecosystems, where integrated stress resilience and biocontrol performance are essential for sustainable crop productivity.

4. Conclusion

This study identifies P. megaterium as a highly promising, stress-resilient bio-inoculant for saline agriculture. The strain demonstrated strong antifungal activity, significant halotolerance, and multiple plant growth–promoting traits, including phytohormone production, nutrient mobilization, and extracellular enzymatic activity. It effectively enhanced tomato germination, root development, and biomass under salt stress, highlighting its capacity to improve plant resilience and productivity in saline environments. These combined traits emphasize the functional versatility of P. megaterium and support its potential as a sustainable solution for crop production in salt-affected agroecosystems.

Future research should focus on validating its performance under greenhouse and field conditions to confirm its consistency in real agricultural settings. Additionally, the development of stable bio-inoculant formulations and scalable application strategies will be crucial for its practical implementation.

Acknowledgements

The author gratefully acknowledges the financial support provided by Imam Abdulrahman Bin Faisal University. This study was approved by the Institutional Review Board (IRB) of Imam Abdulrahman Bin Faisal University (IRB No. IRB-PGS-2024-10-470).

Data Availability Statement

The entire data set that supports the results of this study was published in the article itself.

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Edited by

• Editor:
  Takako Matsumura Tundisi

Publication Dates

• Publication in this collection
  03 Aug 2026
• Date of issue
  2026

History

• Received
  26 Mar 2026
• Accepted
  01 June 2026