An indole dimer antifungal metabolite from a rice seed endophyte inhibits ergosterol biosynthesis in fungal pathogens

ABSTRACT

The increasing prevalence of fungal phytopathogens and the widespread emergence of fungicide resistance necessitate the development of alternative antifungal strategies with reduced environmental impact. Here, we report the isolation and characterization of a novel antifungal metabolite, SM06, produced by the rice seed-associated endophytic bacterium Phytobacter sp. RSE02. SM06 exhibited broad-spectrum antifungal activity against plant and human pathogenic fungi, including Curvularia lunata, Fusarium oxysporum, and Candida albicans. In vitro assays and micromorphological analyses revealed that SM06, an indole dimer, disrupts fungal cell membrane integrity, while in planta experiments demonstrated significant suppression of brown leaf spot disease in tomato and rice. Molecular docking suggested that SM06 binds to lanosterol 14α-demethylase (ERG11), a key enzyme in fungal sterol biosynthesis. Consistent with this prediction, LC-MS–based analyses confirmed a significant reduction in ergosterol content in SM06-treated fungal cells. Together, these findings identify SM06 as a biologically active antifungal metabolite produced by a plant-associated bacterium, highlighting its potential application in sustainable fungal disease management.

IMPORTANCE

Fungal diseases cause major losses in crop production and contribute to the growing challenge of antifungal resistance, underscoring the need for sustainable alternatives to chemical fungicides. This study identifies SM06, a novel indole dimer produced by the rice seed endophyte Phytobacter sp. RSE02, with strong antifungal activity against economically important plant pathogens and clinically relevant fungi. Through integrated chemical, cellular, and in planta analyses, we demonstrate that SM06 disrupts fungal membrane integrity by inhibiting ergosterol biosynthesis. The compound is biocompatible, stable, and effective in plant disease suppression, highlighting its translational potential for crop protection. These findings reveal seed endophytes as an important yet underexplored source of antifungal metabolites and provide a mechanistic foundation for developing eco-friendly biocontrol strategies with implications beyond agriculture.

INTRODUCTION

Ensuring sustainable agricultural productivity has become a global priority in the face of rising food demand and increasing environmental pressures associated with conventional farming practices (1). Among the major constraints to crop production, fungal phytopathogens represent a persistent and widespread threat, contributing to substantial pre- and post-harvest losses, contamination of food commodities with mycotoxins, and reduced market value. It is estimated that fungal diseases alone account for yield losses exceeding 20% across several economically important crops worldwide (2, 3). Synthetic fungicides, such as carbendazim and thiophanate-methyl, have been extensively employed to manage these infections (4); however, their long-term use has resulted in significant drawbacks, including environmental persistence, toxicity to non-target organisms, and increasing regulatory restrictions. Moreover, repeated exposure has accelerated the emergence of fungicide-resistant pathogen populations, diminishing their effectiveness over time (5, 6). Resistance has been widely reported in genera such as Fusarium, Botrytis, Alternaria, and Penicillium, often arising through point mutations in target enzymes, overexpression of efflux transporters, alterations in target site architecture, or enhanced metabolic detoxification pathways (7).

In response to these limitations, biological control strategies have gained increasing attention as environmentally compatible alternatives for fungal disease management (8). The plant-associated bacteria are particularly attractive as they synthesize a wide array of secondary metabolites with antifungal properties (9). Endophytic bacteria, which reside asymptomatically within plant tissues, contribute to host fitness by promoting growth and enhancing tolerance to biotic and abiotic stresses. Members of the family Enterobacteriaceae, including species of the genus Phytobacter, have been reported to exhibit plant growth-promoting traits and antagonistic activity against phytopathogens (10). The application of such beneficial endophytes aligns with sustainable agriculture goals by reducing reliance on synthetic fungicides while supporting crop productivity and soil health (11). Consequently, exploring underexplored microbial taxa such as Phytobacter for antifungal metabolite production represents a promising avenue for the development of next-generation biocontrol agents (12).

A key attribute of endophytic microorganisms is their capacity to produce structurally diverse bioactive secondary metabolites, including alkaloids, polyketides, terpenoids, peptides, phenolics, and lipopeptides, many of which possess strong antimicrobial activities (13, 14). Endophytic fungi have historically yielded several clinically and agriculturally relevant antifungal compounds, such as griseofulvin and trichothecenes. Similarly, bacterial endophytes produce antifungal metabolites, including pyrrolnitrin, phenazine-1-carboxylic acid, and various biosurfactants, some of which also elicit induced systemic resistance in plants (15). These observations highlight endophytes as prolific reservoirs of chemically and functionally diverse antifungal agents. Such agents also have very specific targets present among respective target organisms. The uniqueness of these targets makes an antifungal agent specific toward a group without having toxicity against other organisms. At the molecular level, ergosterol biosynthesis remains one of the most effective targets for antifungal intervention. The cytochrome P450 enzyme lanosterol 14α-demethylase (ERG11) catalyzes a critical step in this pathway by removing the 14α-methyl group from lanosterol and is the primary target of azole-class antifungals such as fluconazole and itraconazole (16). However, resistance to azoles frequently arises through mutations in ERG11 that reduce inhibitor binding efficiency (17). Heterocyclic scaffolds, particularly indole-based compounds, are of interest for antifungal drug design due to their ability to engage in π–π stacking interactions with the heme group and aromatic residues within the ERG11 active site (18). In silico docking approaches provide valuable insights into ligand orientation, residue-level interactions, and binding affinities, thereby guiding experimental validation and aiding in the identification of compounds with improved efficacy and resistance resilience. These considerations emphasize the need for environmentally benign antifungal agents that operate through well-defined molecular mechanisms (19).

Here, we investigated Phytobacter sp. RSE02, a rice seed-associated endophytic bacterium previously identified as a plant growth-promoting strain (20), was studied for its antifungal metabolite production. Preliminary assays revealed that culture supernatants of RSE02 exhibited broad-spectrum antifungal activity against multiple fungal pathogens (20). Here, we report the purification and structural characterization of an indole dimer antifungal compound, designated SM06, produced by RSE02. We further examine its antifungal efficacy, effects on fungal cell integrity, and potential mechanism of action, including interaction with ERG11. The identification of SM06 highlights the untapped metabolic potential of seed-derived bacterial endophytes and underscores their relevance as sources of biologically active antifungal metabolites for sustainable disease management.

MATERIALS AND METHODS

Preliminary screening for antimicrobial property

In this study, we have investigated the bioactive compounds produced by the endophytic bacterium Phytobacter sp. RSE02 (GenBank accession no. OM403299), which was isolated from viable rice seeds, as previously described by (20, 20). Using Mueller-Hinton (MH) agar media and the conventional agar-diffusion method, the antimicrobial activity of the strain RSE02 was evaluated (21). The test organisms used in this method were Staphylococcus aureus (MTCC 96; gram-positive), Escherichia coli (MTCC 1687; gram-negative), plant fungal pathogen Curvularia lunata (MTCC 8018), Fusarium oxysporum (MTCC 284), human fungal pathogen Candida albicans (MTCC 183), and Rhizopus stolonifer (MTCC 4886). In total, 2- to 3-day-old RSE02 culture supernatant was used to check the antibiosis against the aforesaid strains. The plates were incubated at 37°C for 3–4 days.

Optimization of SM06 production

Optimization of SM06 production was performed by evaluating different culture media and incubation times. During these experiments, inoculum size (1%), shaking speed (180 rpm), and temperature (30°C) were maintained constant. The optimal production was observed at 16 h of incubation under these conditions.

Production, extraction, and purification of SM06

Overnight grown culture of RSE02 (1%, vol/vol) was inoculated in 2 L of sterile Luria-Bertani medium and incubated at 30°C for 16 h with 180 rpm of shaking. The culture medium was centrifuged at 13,000 rpm for 15 min to collect the supernatant. An equal volume of ethyl acetate was added to the supernatant, and the mixture was shaken properly to ensure thorough extraction of the active compound (22). The active compound was collected after drying the organic phase using a rotary evaporator. After resuspension with 200 μL ethyl acetate, the fraction was run on a thin-layer chromatography plate (silica gel 60 F254) using PET:ethyl acetate (9:1, vol/vol; Rf = 0.40). To obtain the required product, silica gel column chromatography (60–120 mesh size) was applied to the crude product with the same solvent eluent ratio. The solvent was removed from the eluted fractions using a rotary evaporator, and the pure product was obtained as an ash-gray solid. The 2 L extract solutions yielded approximately 42 mg of pure product. The product was then further employed for its chemical and functional characterization.

Antimycotic activity of RSE02 crude extract and SM06 against fungal pathogens

To evaluate the antifungal activity of RSE02 crude extract and the SM06 compound against C. lunata, F. oxysporum, C. albicans, and R. stolonifer, we followed the methodology outlined in a previous study by Ruhil et al. (23). Briefly, each fungal suspension (100 μL) was uniformly spread onto Mueller-Hinton agar plates. A volume of 4 μL containing RSE02 crude, SM06, Nystatin, Cycloheximide, or biogenic silver nanoparticles (AgNPs) was individually applied to the inoculated plates. After incubation at 37°C for 3 days, the diameters of inhibition zones were measured.

To assess the concentration-dependent inhibition of C. lunata biomass by SM06, sterile 2 mL culture tubes were each filled with 900 µL of the sterilized culture medium. Fresh mycelial inocula (100 µL) were added to all tubes. Subsequently, SM06 was added at increasing volumes of 5, 10, and 20 µL from a 15 µg/mL stock solution. Cycloheximide and nystatin were included as positive controls. Following 7 days of incubation at 37°C, the mycelial growth inhibition was quantified by comparing biomass between treated and control samples, expressed as the percentage inhibition of mycelial growth (24). The thermal stability of the compound was evaluated by incubating it at temperatures ranging from 30°C to 90°C for 30 min, followed by testing its activity against the indicated pathogens. The antibiosis against both gram-positive and gram-negative bacteria was also tested, taking S. aureus and E. coli, respectively, as standard strains (20, 21).

Structural elucidation of SM06

The solvents were distilled and dried following the standard procedures (25). High-resolution mass spectrometry (HRMS) was carried out using a Waters XEVO G2-XS QTOF instrument by using acetonitrile (ACN) as the solvent. Bruker Avance 300 (300 MHz) and Bruker Avance Neo HD 400 (400 MHz) were used to record the ¹H and ¹³C NMR. For NMR spectra, CDCl₃ was used as a solvent, and TMS as an internal standard. Chemical shifts are expressed in δ ppm units and ¹H–¹H, ¹H–¹³C coupling constants in Hz. The following abbreviations describe spin multiplicities in ¹H NMR spectra: s = singlet; d = doublet; t = triplet; and m = multiplet. UV spectra were recorded on an Ava Spec-ULS2048L-USB2-UARS spectrometer with a temperature-controlled cuvette holder. Fourier transform infrared (FTIR) spectroscopy data were collected using an FTIR-7600S (Lambda-Scientific) spectrometer.

Assessment of antifungal activity

Sporicidal and mycelium disruption efficacy of SM06 against C. lunata

To evaluate the inhibitory effect of SM06 on spores and mycelia of C. lunata, assays were conducted in sterile 2 mL culture tubes, each containing 900 µL of sterilized culture medium (26). A working concentration of SM06 (15 µg/mL) was prepared; 100 µL of C. lunata spore or mycelium suspension was introduced into each tube. Tubes containing only the spores or mycelia suspension and devoid of SM06 served as negative controls. Following inoculation, all tubes were incubated at 37°C for 16 h. After incubation, spore germination and hyphal (mycelial) morphology were assessed microscopically using an Olympus cellSens imaging system and scanning electron microscope (SEM) system developed by Carl Zeiss Microscopy GmbH. We also performed time-kill kinetics; after certain time intervals (0, 1.5, and 2.5 h), fungal hyphae from treated and control samples were examined under a light microscope (Olympus cellSens) to observe morphological alteration (21).

We assessed the membrane integrity of the test fungal strain in both compound-treated and untreated hyphae by staining with propidium iodide (10 µg/mL), a membrane-impermeant fluorescent dye, and visualized them using fluorescence microscopy (27). The fluorescence intensity of individual hyphal segments was then quantified using ImageJ software (version 1.54g).

Membrane integrity assessment via extracellular protein leakage

By monitoring the leakage of cell constituents, such as proteins, into the cell suspension, the integrity of the cell membrane was assessed (28). Centrifugation at 6,000 rpm for 5 min was used to collect the actively growing fungal mycelial pellet. This pellet was washed with phosphate-buffered saline (PBS) (pH 7.0) three times. To assess membrane integrity, the pellet was treated with the minimum inhibitory concentration (MIC) of SM06 (15 µg/mL) and incubated over multiple time intervals (0, 1, 4, 8, 12, 16, 20, and 24 h). At each time point, 1 mL of the suspension was collected and centrifuged at 6,000 rpm for 10 min. The total proteins present in the supernatant were measured through the Bradford assay procedure (29). Each treatment and measurement was performed in triplicate to ensure reproducibility and statistical reliability.

Antifungal efficacy of SM06 in combination with nystatin

To determine the combinatorial effect of SM06 with frequently used antifungal antibiotics like nystatin, a checkerboard assay was performed following the standard protocol as mentioned in a previous study by Sardana et al. (30). Antibiotic concentrations ranged from the MIC to 1/6 of the MIC equivalent. The final antibiotic concentrations were as follows: nystatin: 0.08, 0.16, 0.32, 0.64, 1.28, 2.56, and 5.12 μg/mL; and SM06: 0.8, 1.6, 3.2, 6.4, 12.8, 25, and 50 μg/mL. Experiments were conducted in a 96-well plate with a final volume of 100 µL per well, consisting of 40 µL Mueller-Hinton (MH) broth, 50 µL of freshly prepared fungal suspension (1 × 10⁶ spores/mL final inoculum), 5 µL of SM06, and 5 µL of the respective antibiotic. Each well, therefore, contained the test pathogen (C. lunata) together with the individual antibiotic treatment, with or without SM06 as indicated. The cells were incubated for 48 h at 30°C. The fractional inhibitory concentration index (FICI was used to quantify in vitro interaction, and the formula for FICI calculation is (MIC of drug A in combination/MIC of drug A alone) + (MIC of drug B in combination/MIC of drug B alone). FICIs were interpreted as follows: <0.5, synergy; 0.5–0.75, partial synergy; 0.76–1.0, additive effect; 1.0–4.0, indifference; and >4.0, antagonism. The varying levels of synergy between two given compounds were determined (30). The experiment was performed in triplicate.

In vitro toxicity testing and localization profiling of the SM06 compound in zebrafish embryos and mammalian cells

Zebrafish maintenance

Adult zebrafish of the AB strain, with ages between 3 and 6 months, were used in this study for spawning. Fish were maintained in customized aquarium tanks at a density of about five fish per liter at 28°C water temperature and fed three times daily. Collection of zebrafish embryos was performed after natural mating and maintained in E3 medium at a temperature of 28°C up to 4 days post-fertilization (dpf).

Generation of germ-free zebrafish embryos and treatment with SM06

The generation of germ-free embryos and evaluation of the effect of SM06 on zebrafish embryos were done as previously described with minor modifications (31). Briefly, after 24 hpf (hours post-fertilization), the embryos were dechorionated and divided into two groups: conventionally raised (CV) and germ-free (GF). The CV larvae were maintained at room temperature, while the GF embryos underwent sterilization by immersion in sterilized gentamicin (100 µg/mL) for 1 h, followed by treatment in 0.003% hypochlorite solution and subsequently washed in sterile E3 medium under a laminar hood to ensure sterility. The non-lethal dose was determined by exposing 24 hpf zebrafish embryos to three metabolite concentrations according to the in vitro dose (50, 100, and 250 µg/mL).

A total of 10 zebrafish embryos at 24 hpf were placed in a Petri dish containing 3 mL of E3 medium supplemented with SM06 (50, 100, and 250 µg/mL) and exposed for 3 days, with the medium renewed daily. Embryos exposed to 0.1% DMSO in E3 medium served as the control group.

Fluorescence microscopic analysis of embryonic gut lumen

The 96 hpf zebrafish embryos after exposure were anesthetized with tricaine and mounted on the grooved slide using 0.1% Methyl cellulose (M7027, Sigma, St. Louis, MO, USA) as described by Mukherjee et al. (32) and photographed by using an Olympus BX53F2 fluorescent microscope (Olympus, Tokyo, Japan) under 335 nm wavelength filter to observe the fluorescent intensity of the bacteria metabolite in the gut lumen of the embryo.

Mammalian cell maintenance

HeLa cell lines were prepared from a continuous culture in Dulbecco’s modified Eagle’s medium (Sigma Chemical Co., St. Louis, MO) supplemented with 10% fetal bovine serum (Invitrogen), penicillin (100 μg/mL), and streptomycin (100 μg/mL). After the cells reached the logarithmic phase, the cell density was adjusted to 1.0 × 10⁵ per culture dish in culture media and then used to inoculate with 1.0 mL (1.0 × 10⁴ cells) of cell suspension in each glass-bottom dish.

We have performed an MTT assay to evaluate the cytotoxicity of compound SM06 on the HeLa cell line by a standard protocol (33). A 96-well polystyrene-coated plate with 100 μL of 7,500 cells was incubated for 24 h at 37°C in an incubator having 5% CO₂ and 95% air. Each SM06 dilution was mixed (1×, 2×, 3×, and 4× MIC μg/mL) (MIC is 15 μg/mL) in the medium taken in wells of a 96-well plate. Each dilution point was taken in triplicate; 20 μL MTT reagent was added to each well and incubated for an additional 3 h. After the formation of the purple formazan crystals, 150 μL of the MTT solvent was added to each well of the microplate and scanned using a microplate reader Synergy H1 (Agilent-Biotek) at 590 nm. The reading from the wells containing medium, SM06, and MTT reagent but no cells was considered a blank.

In silico interactions between indole dimer and ERG11 complex

The in silico molecular docking study was performed between the indole dimer and ERG11 complex with the CB-Dock tool (34). CB-Dock is an online docking tool, while the docking algorithm is programmed by Auto Dock Vina (34). The crystal structure of the ERG11 protein was retrieved from the Protein Data Bank (PDB: 5EQB). However, the established structure of the ligand molecules (such as normal indole, fluconazole [positive control], and ergosterol [substrate of ERG11]) was retrieved from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/). The 3D structure of the stacked indole dimer was designed using Avogadro software (35). The energy-minimized structure of the ligand molecules was used for in silico docking analysis. The energy minimization of the ligands was performed with Avogadro software using the MMFF94 force field (36). The docking result was analyzed using PyMOL (Schrodinger, v 2.5.8) (37) and Discovery Studio Client software (v 24.1.0.23298).

In silico ADMET prediction of the indole and indole dimer

The ADMET and physicochemical properties of the indole and indole dimer were collected from the ADMET-AI prediction tool server (https://greenstonebio.com/ai/). It is a web-based platform to find out the ADMET property of the unknown compound (38).

Estimation of ergosterol from C. lunata upon SM06 treatment

To evaluate the time-dependent effects of SM06 on ergosterol biosynthesis in C. lunata mycelia, ergosterol was extracted using a chloroform:methanol solvent system following the protocols described in previous studies (39, 39). The resulting lipid extracts were analyzed by LC-MS to quantify ergosterol abundance across treatment time points using a Waters Xevo-XS Q-TOF (ESI)–UPLC system, with separation on a reverse-phase C18 column and identification in positive ion mode based on characteristic m/z values from extracted ion chromatograms.

In planta antagonistic activity of SM06 against Curvularia in tomato and rice plants

Detection of indole dimer synthesis by RSE02 inside tomato plants

We investigated whether Phytobacter sp. RSE02, when present inside tomato plants, can synthesize SM06. We first monitored the localization of the RSE02 (labeled with red-fluorescent protein) within tomato plants following the laboratory standard described in a study by Jana et al. (40). Briefly, a bacterial suspension of Phytobacter sp. RSE02 (1 × 10⁸ CFU/mL) was prepared and applied directly to the root zone of tomato plants as a soil drench (10 mL per plant). Following inoculation with the fungal pathogen C. lunata, RSE02 treatments were administered at 3-day intervals over a total period of 10 days. Subsequent analysis confirmed that RSE02 was able to enter and colonize the internal tissues of tomato plants infected with C. lunata. We further aimed to perform the LC-MS analysis to detect SM06 from tissues of healthy plants and C. lunata-infected plants with RSE02 treatment. Harvested tissues were extracted with ethyl acetate. Following extraction, a rotary evaporator was used to collect and dry the active organic phase and then run through LC-QTOF-MS (Agilent), with SM06 spiked in set as our standard control for indole dimer detection. Retention time, mass/charge (m/z) values, and peak shapes for SM06 were established under these conditions (41).

Pathogenic challenge of tomato and rice with Curvularia species

C. lunata was cultivated on potato dextrose broth for 10 days at 30°C in the dark to yield spores. Thirty-day-old tomato and rice plants were infected with the pathogen using the punch inoculation method, as described by Wang et al. (42). Spores were used at a concentration of 5 × 10⁵/ mL for punch inoculation. After lightly pounding each plant leaf with a puncher, 5 µL of spore suspension was added. After that, the spore suspension was kept in place by covering both sides with opaque adhesive tape. The inoculated leaves were photographed 12 days following the inoculation.

Pre-infection and post-infection treatments with SM06 in C. lunata-infected tomato and rice plants

After the successful infection of the tomato and rice plants with C. lunata, SM06 was applied. SM06 (60 µg/mL) was applied as foliar sprays or root drenches on both plants (43, 44). There was a total of four experimental sets: set I consisted of plants without pathogen infection; set II consisted of infected plants without SM06 treatment (positive control); set III consisted of infected plants with SM06 treatment; and set IV consisted of SM06 pre-treated (from 10 days after seedling germination) plants before pathogen infection. For these studies, all plants were maintained in a growth chamber at 28°C, relative humidity (60%–65%) under a 16 h light/8 h dark photoperiod with a light intensity of approximately 200 µmol m⁻² s⁻¹. Depending on the severity of the infection, we applied the SM06 compound solutions at 7-day intervals for 20 days prior to pathogen challenge.

Microscopic evaluation of antibiosis by SM06

For the microscopic visualization of the in planta antifungal activity of the SM06 against the fungal pathogen, transverse sections of 30-day-old growing roots of rice plants (pathogen colonization was found to be better in roots than the leaves possibly for having thicker, more structurally robust leaf tissues with a waxy cuticle, which can limit efficient pathogen entry) and leaves of tomato plants (better pathogen establishment in tomato leaves) were prepared and placed on grease-free slides. The section was stained with lactophenol-cotton blue and observed under a microscope. Images were captured with Olympus cellSens standard software under 10× magnification (45).

Measurement of necrotic lesions

H₂O₂ accumulation from the necrotic tissues of the tomato leaf lesions was detected using 3,3′-diaminobenzidine (DAB) staining following the methods described in a previous study by Roschzttardtz et al. (46). Leaf samples were subsequently washed with water and submerged in DAB (100 mg DAB, 25 µL Tween 20, and 2.5 mL of 200 mM NaHPO₄ for 100 mL solution) solution. Then, the samples were vacuum-infiltrated for 15 min and incubated in the dark for 8 h. The leaves were kept in a water bath at 70°C for 10 min, and bleaching solution (ethanol:acetic acid:glycerol) was added until the chlorophyll was removed and the leaves turned pale or transparent. The samples were washed with water and then imaged.

Investigation of genetic clusters present in the RSE02 genome

The gene clusters associated with the synthesis of secondary metabolites of RSE02 were analyzed using Proksee (https://proksee.ca) and the antibiotics and Secondary Metabolite Analysis Shell (antiSMASH) version 8.0.1, a comprehensive bioinformatics tool designed for the identification and annotation of biosynthetic gene clusters (BGCs) in bacterial genomes (47). The analysis was conducted with relaxed and loose strictness settings to allow for broader detection of putative biosynthetic gene clusters, including borderline or less-conserved clusters that might be missed under stricter parameters. This approach maximizes sensitivity and captures a more comprehensive secondary metabolite profile.

To enhance cluster identification and comparative analysis, the following similarity search modules were activated. KnownClusterBlast was used to compare the predicted gene clusters to experimentally characterized clusters in the MIBiG database to identify known metabolites with similar biosynthetic origins, whereas ClusterBlast performs genome-wide comparisons of predicted clusters against a large database of microbial genomes to find related gene clusters in other organisms. SubClusterBlast helps identify conserved sub-clusters or gene modules shared between biosynthetic clusters, providing insights into cluster modularity and evolution.

Statistical interpretation

All experiments were performed with a minimum of three replicates, and the results were expressed as the mean ± standard deviation. For the statistical analysis, one‐way and two‐way analysis of variance (ANOVA), followed by Dunnett’s multiple comparisons test, and mean values in each column do not differ significantly at P < 0.05.

Survival data were analyzed using the Kaplan–Meier method. For the zebrafish embryo survival assays, experiments were performed in triplicate with a total of 10 embryos per condition.

All graphs and data were subjected to an analysis of variance test by using the GraphPad Prism 8.0.1 version software and OriginPro 8.0 version software (20).

RESULTS

SM06 produced by RSE02 effectively inhibits plant and human pathogenic fungi

SM06 displayed potent antifungal activity against C. lunata, F. oxysporum, and C. albicans, as evidenced by pronounced inhibition zones around wells treated with RSE02 culture supernatant, purified SM06, and reference antifungals (Fig. 1AI through IV). However, it failed to inhibit the growth of R. stolonifer. SM06 also remained ineffective against tested gram-positive and gram-negative bacteria (see Fig. S1A and B at https://doi.org/10.5281/zenodo.19136837). Spore germination assays further revealed extensive germ tube elongation in the untreated controls (Fig. 1BI), whereas SM06 treatment markedly reduced both the spore number and germ tube length (Fig. 1BII). Biomass quantification of C. lunata revealed a clear dose-dependent suppression in growth by SM06 (Fig. 1C), comparable to the inhibitory effects of nystatin and cycloheximide. Consistent with these findings, disc-diffusion assays on a solid plate also confirmed the substantial antifungal potency of purified SM06 in comparison to the DMSO controls (Fig. 1D). Furthermore, SM06 retained full functional activity across 30°C–90°C for 30 min, demonstrating high thermal stability (Fig. 1E).

Fig 1.

Broad-spectrum antifungal performance and thermal resilience of SM06. (A) Antifungal efficacy of SM06 against (I) C. lunata, (II) F. oxysporum, (III) C. albicans, and (IV) R. stolonifer (where 1 is cycloheximide; 2, nystatin; 3, SM06; 4, crude SM06; and 5, AgNPs). (B) Sporicidal effect of SM06 against C. lunata spores. (C) Concentration-dependent biomass inhibition of C. lunata by SM06. (D) Comparison study of indole dimer with the known indole molecule against C. lunata. (E) Temperature stability of SM06 compound. Mean values in each column do not differ significantly at P < 0.05. Comparison between control and treatment mean values was made by Dunnett’s multiple comparisons test, where **** = P < 0.0001, ** = P < 0.01, and ns = data not significant. Data were subjected to a one-way ANOVA test by using the GraphPad Prism 8.0.1 version software.

SM06 is a novel biomolecule consisting of π–π–stacked indole units

SM06 was obtained as a water-insoluble ash-gray solid and purified by column chromatography using EtOAc:PET (1:9). HRMS analysis showed a dominant [M]⁺ ion at m/z 235.1235 with a secondary peak at m/z 236.1246 (Fig. 2A). The combined ¹H and ¹³C NMR data (Table 1) established the molecular formula as C₁₆H₁₄N₂. UV–visible and fluorescence measurements revealed absorption and emission maxima at 280 and 335 nm, respectively (Table 1; Fig. S1A through D), which are consistent with an indole chromophore. The IR bands at 1,483 and 1,597 cm⁻¹ correspond to aromatic C=C stretching, while shifts in the N–H stretching region near 3,400 cm⁻¹ indicate hydrogen bonding associated with π–π–stacked indole interactions. A broad absorption at 1,974 cm⁻¹ further reflects strong hydrogen bonding between paired N–H groups in the stacked structure (Fig. 2B). Detailed NMR analyzes, including COSY, HMBC, DEPT-135, and 1H/13C assignments (see Figs. S3 to S7 at https://doi.org/10.5281/zenodo.19136837), supported an indole-1H–derived scaffold (Fig. 2C). The mass spectrometry data confirmed the presence of an indole dimer (molecular weight 235.1235). Several possible spatial configurations of the dimer were proposed (see Fig. S8 at https://doi.org/10.5281/zenodo.19136837) and evaluated using DFT calculations (6-31G**; CPCM-H₂O), which identified pattern four as the most energetically favorable structure (see Fig. S9 at https://doi.org/10.5281/zenodo.19136837).

Fig 2.

Characterization of compound SM06. (A) HRMS spectra of SM06. (B) FTIR spectra of SM06 compound. (C) Structural elucidation of compound SM06.

TABLE 1.

Characteristics of the compound SM06

Characteristic	Details
Appearance	Ash-gray solid
Molecular formula	C16H14N2
Molecular weight	235.1235
HRMS m/z	(M+H)+
Found	235.1235 and 236.1246
Absorbance	280 nm
Emission	335 nm
IR (KBr) cm−1	1,483, 1,597, 1,974, 2,166, and 3,029
1H NMR (400 MHz, CDCl3)	δppm 8.2 (s, 1H), 7.68 (d, 1H, J = 8.0 Hz), 7.41 (d, 1H, J = 8.0 Hz), 7.22 (d, 1H, J = 16 Hz), 7.14 (t, 2H, J = 12 Hz), 6.59 (broad singlet, 1H)
13C NMR (100 MHz, CDCl3)	δppm 135.8, 127.87, 124.16, 122.01, 120.76, 119.84, 111.05, and 102.64
Chemical name identified	1H-indole

SM06 leads to the damage of cellular compartments and causes cellular leakage

Scanning electron microscopic (SEM) analysis revealed marked morphological alterations in SM06-treated fungi. Untreated hyphae appeared smooth and structurally intact (Fig. 3AI), whereas SM06 treatment caused swelling, septal distortion, and loss of cell wall integrity (Fig. 3AII, also see Fig. S10 at https://doi.org/10.5281/zenodo.19136837). Similarly, control spores retained normal surface morphology (Fig. 3AIII), while treated spores showed pronounced surface disruption and fragmentation (Fig. 3AIV). Propidium iodide (PI) staining further confirmed membrane damage where the untreated hyphae exhibited minimal fluorescence (Fig. 3BI and II) as a result of very limited or no entry of PI, but the SM06-treated hyphae showed strong PI uptake at damaged regions (Fig. 3BIII and IV), with significantly elevated fluorescence intensity (Fig. 3C). Protein leakage assays demonstrated a time-dependent increase in extracellular protein over 24 h following treatment at the MIC (15 µg/mL) level of SM06, consistent with progressive membrane permeabilization (Fig. 3D). These results suggest that SM06 disrupts membrane permeability, leading to cytoplasmic leakage and cellular lysis. Antifungal susceptibility testing showed an MIC of 15 µg/mL for SM06 and 1.28 µg/mL for nystatin against C. lunata. Combination assays yielded MICs of 1.6 µg/mL (SM06 + nystatin) and 0.64 µg/mL (nystatin + SM06), corresponding to a FICI of 0.60 (Table 2; also see Fig. S11AI through III at https://doi.org/10.5281/zenodo.19136837), indicating partial synergistic interaction.

Fig 3.

Sporicidal and mycelial disruption by SM06. (A) Untreated (I and III) and SM06-treated (II and IV) (15 µg/mL) (for 16 h) C. lunata mycelium and spore under SEM (orange arrow indicates the disruption of both fungal mycelium and spore). (B) Untreated (I and III) and SM06-treated (II and IV) (15 µg/mL) C. lunata mycelium treated (for 30 min) with PI. (C) Relative fluorescence intensity of SM06-treated fungal cell membrane stained with PI. Images from five distinct microscopic fields were analyzed using ImageJ v1.54g software to quantify relative fluorescence intensity. Statistical analysis was performed using GraphPad Prism (version 8.0). Data were expressed as mean ± standard error of the mean (SEM). (D) Total protein leakage of C. lunata strain treated with SM06 for different time periods.

TABLE 2.

Synergic effect of compound SM06 along with nystatin against C. lunata

Test organism	Antibiotics	MIC (μg/mL)	FICIa	Interpretation
Curvularia sp.	SM06	15	0.60	Partial synergy
	Nystatin	1.28
	SM06 + nystatin	1.6
	Nystatin + SM06	0.64

^a FICIs were interpreted as follows: ≤0.5, synergy; 0.5–0.75, partial synergy; 0.76–1.0, additive effect; 1.0–4.0, indifference; and >4.0, antagonism.

SM06 is biocompatible and nontoxic for zebrafish embryos and mammalian cell lines

SM06 exhibited no detectable toxicity. Zebrafish embryos exposed to concentrations up to 250 µg/mL from 24–96 hpf showed no significant mortality, and fluorescence imaging indicated the preferential localization of the compound within the intestinal lumen (Fig. 4A through C). Consistently, HeLa cells treated with SM06 at 4× MIC (15 µg/mL) displayed no measurable cytotoxic effects (Fig. 4D). These findings demonstrate that SM06 is biocompatible under the tested conditions and does not elicit adverse effects in zebrafish embryos or mammalian cell lines.

Fig 4.

Toxicity and localization profiling of SM06 in zebrafish embryos and mammalian cells. (A) For 24 hpf, zebrafish embryos (N = 10) were grown in germ-free conditions and treated with SM06 (50, 100, and 250 µg/mL), followed by observation after 72 h of treatment at 96 hpf stage. (B) Representative image of zebrafish embryo at 96 hpf stage showing localization of SM06 after treating with either DMSO as a control (upper panel) or SM06 (lower panel) for 72 h. Visualization of SM06 localized in the embryonic premature gut (marked by arrow) observed at 335 nm excitation under fluorescent microscope (magnification 4×; scale bar 100 μm). (C) Kaplan–Meier survival curve of germ-free zebrafish embryos (N = 10) after exposure to SM06 (control, 50 µg/mL, 100 µg/mL, and 250 µg/mL; each treatment set contains equal amount of DMSO as solvent). (D) Effect of SM06 on HeLa cells. Data are expressed as mean ± SE (n = 3). Statistical analysis was performed using two-way ANOVA followed by Dunnett’s multiple comparisons test (GraphPad Prism 8.0.1). Significance levels: **** = P < 0.0001, ** = P < 0.01, and ns = data not significant.

SM06 binds with fungal ERG11 (lanosterol 14-α-demethylase) and inhibits ergosterol biosynthesis

The active site of ERG11 contains a heme cofactor targeted by azole antifungals (e.g., fluconazole) and is positioned within the core catalytic cavity (Fig. 5A through C). Docking analysis showed that lanosterol (substrate for the enzyme ERG11), fluconazole (inhibitor of the ERG11), and indole dimer (SM06) occupied the same binding pocket of ERG11, with a cavity volume of 5,267 ų (Fig. 5D through H). SM06 exhibited a docking score of –10.5 kcal/mol, which was comparable to that of lanosterol (–10.3 kcal/mol) and stronger than that of fluconazole (–7.0 kcal/mol). Lanosterol formed 20 interactions at the active site (Fig. 5D), whereas fluconazole engaged in 11 interactions (Fig. 5E), which is consistent with their reported binding modes (Fig. 5F; also see Table S1 at https://doi.org/10.5281/zenodo.19136837). SM06 established four key interactions: three hydrophobic contacts with Leu95, Phe241, and Phe384, and a hydrogen bond with Ala125 (Fig. 5G; see Table S1 at https://doi.org/10.5281/zenodo.19136837). Although the amino acids involved did not fully overlap with the lanosterol interactome, SM06 was bound in the same catalytic pocket as lanosterol and fluconazole (Fig. 5H and I). ERG11 contains a distinct tunnel that ends at its catalytic site and possibly functions as an entry point for lanosterol. The indole dimer SM06 binds to the residues present in the tunnel, impairing the entrance of lanosterol into the catalytic site. Additional cavity morphology comparisons between SM06 and indole are shown in Fig. S12 at https://doi.org/10.5281/zenodo.19136837.

Fig 5.

Interaction between indole dimer and ERG11. The docking results were obtained using PyMOL (Schrodinger, v 2.5.8). (A and B) The figures depict the crystal structure of EGR11 (PDB: 5EQB), where A is a surface view of the space-field model, and B, the ribbon view. Both show the active site (heme group) of the protein, along with the iron atom in brick-red color and the porphyrin group in green. (C) The figure depicts the zoom-in active site. (D–I) The figures show ERG11 docked with lanosterol (green color), fluconazole (red color), lanosterol and fluconazole, indole dimer (blue color), lanosterol and indole dimer, and lanosterol, fluconazole, and indole dimer, respectively. The binding residues are highlighted in light blue.

Pharmacokinetic and ADMET profiling reveal enhanced oral bioavailability and stability of SM06

In silico physicochemical and ADMET analyses demonstrated the favorable pharmacokinetic properties of SM06 (see Table S2 at https://doi.org/10.5281/zenodo.19136837). Compared to indole, the indole dimer structure showed increased lipophilicity (~1.34-fold) and higher predicted plasma protein binding (~1.61-fold), along with reduced aqueous solubility (20.86% vs. 64.33%, respectively). SM06 also exhibited an elevated TPSA (16.05% vs. 7.95%), indicating greater hydrophilicity. Notably, oral bioavailability was improved (57.58% vs. 52.19%), and the predicted excretion half-life was longer (53.59% vs. 48.89%), suggesting an enhanced metabolic stability. Collectively, these pharmacokinetic features support the superior drug-likeness and biological efficiency of SM06 relative to the parent indole molecule.

SM06 treatment leads to a significant reduction in ergosterol levels

Ergosterol extraction and LC-MS analysis revealed that treatment with SM06 led to a marked, time-dependent reduction in ergosterol in C. lunata (Fig. 6A through C). As shown in the extracted ion chromatograms (m/z 413.2588), the untreated culture displayed a strong ergosterol peak at day 3, whereas equal biomass of SM06-treated samples showed a substantial decrease in peak intensity after 1.5 days and an almost complete depletion after 3 days of incubation (Fig. 6B). Quantitative analysis further corroborated this trend; ergosterol levels were significantly reduced at 1.5 days compared to the untreated control, and by 3 days, ergosterol was nearly undetectable (Fig. 6C). Collectively, these results demonstrate that SM06 effectively inhibits ergosterol biosynthesis in a time-dependent manner, indicating strong interference with the sterol production pathway.

Fig 6.

Time-dependent reduction of ergosterol upon SM06 treatment. (A) Workflow of ergosterol extraction and LC-MS analysis from control and treated cultures harvested at 1.5 and 3 days. (B) LC-MS spectra showing the ergosterol ion (m/z 413.2588) in untreated and treated samples, indicating a marked decrease after treatment, especially at 3 days. (C) Quantified ergosterol abundance demonstrating significant, time-dependent depletion of ergosterol in SM06-treated cells compared to control. *** = P < 0.001, **** = P < 0.0001.

SM06 cures the plant from Curvularia pathogenesis and is produced by RSE02 inside the host plant

Phytobacter sp. RSE02 enters and colonizes inside tomato plants infected with the fungal pathogen C. lunata. Confocal microscopic analysis showed that the RFP-labeled RSE02 was primarily found along the perivascular and epidermal layers (see Fig. S13 at https://doi.org/10.5281/zenodo.19136837). Active in-plant proliferation is indicated by these persistent fluorescence aggregates. In planta transmission and colonization were also established following time-dependent counts of the labeled RSE02 from various plant tissues (20). Untreated plants showed significant wilting and chlorosis as the disease progressed (see Fig. S14AII at https://doi.org/10.5281/zenodo.19136837), while healthy controls showed no changes (see Fig. S14AI at https://doi.org/10.5281/zenodo.19136837). Remarkably, RSE02-treated pathogen-infected plants showed significant recovery, with recovered turgor, greenness, and nearly normal morphology (see Fig. S14AIII at https://doi.org/10.5281/zenodo.19136837).

The indole dimer metabolite at m/z 235.1252 [M+H]⁺ was identified by chemical profiling of the tissue extracts from RSE02-treated, infected plants (see Fig. S14B at https://doi.org/10.5281/zenodo.19136837). This characteristic matched the SM06 reference standard in terms of retention time, precise mass, and fragmentation pattern (see Fig. S14C at https://doi.org/10.5281/zenodo.19136837). The lack of endogenous synthesis was indicated by the absence of such a signal from the tissue extract of a plant not infected with RSE02. These findings indicate that RSE02 colonization in plants ensures endogenous accumulation of SM06 indole dimer metabolite, which could essentially help to reduce the disease symptoms caused by fungal pathogens.

SM06 controls brown leaf spot of tomato and rice

This study was designed to assess the protective efficacy of SM06 against C. lunata-induced brown leaf spot disease in tomato plants and fruits. The preventive and curative activities of SM06 were evaluated by applying foliar sprays of the compound at its MIC to tomato leaves and fruits exhibiting freshly induced lesions (Fig. 7). Pathogen inoculation resulted in severe foliar wilting and chlorosis (Fig. 7AII), whereas both preventive and post-infection SM06 treatments markedly reduced symptom severity and restored plant vigor (Fig. 7AIII and IV). Microscopic observations revealed an intact tissue architecture in the control plants (Fig. 7BI) and dense hyphal colonization in the infected samples (Fig. 7BII). Curative application of SM06 substantially restricted hyphal spread (Fig. 7BIII), and preventive treatment completely inhibited pathogen ingress (Fig. 7BIV).

Fig 7.

Antifungal activity of SM06 in C. lunata-infected tomato plant. (A) Images showing growth and infection parameters of the experimental set of control (I), C. lunata-infected plant (II), SM06-treated C. lunata-infected plant (III), and SM06-treated plant before C. lunata infection (IV). (B) Microscopic visualization of the tomato leaf surface of the experimental sets A I–IV, respectively. The arrows (orange) indicate the colonization of C. lunata mycelium in the intercellular spaces on the leaf (AII and III). (C) Images of the leaves of the plants from experimental sets of AI–IV, and DAB staining of tomato leaves to show the necrotic tissues. (D) Effect of SM06 on control (I), C. lunata-infected tomato (II), SM06-treated C. lunata-infected tomato (III), and SM06-treated tomato before C. lunata infection (IV). All microscopic images are taken at 10×, and the bar shown in the images (BI–IV) is 100 μm.

Histochemical staining further supported these findings, as pathogen-infected leaves exhibited strong necrotic coloration (Fig. 7CI and II), whereas SM06-treated leaves showed minimal cell death (Fig. 7CIII and IV). Infection assays in fruits also confirmed reduced necrosis following SM06 application (Fig. 7DI through IV). Consistent with the results in tomato, SM06 also provides robust protection against brown leaf spot in rice, significantly limiting lesion formation and chlorosis (see Fig. S15 at https://doi.org/10.5281/zenodo.19136837). Together, these results establish SM06 as a broad-spectrum antifungal agent with strong preventive and curative activities in multiple host species.

RSE02 contains biosynthetic gene clusters for indole-like and other secondary metabolites

Genome mining revealed that Phytobacter sp. RSE02 encodes a diverse array of BGCs linked to secondary metabolite formation (Fig. 8). Indole-related genes (trpA, trpB, and tnaA) were identified (Fig. 8A), along with multiple non-ribosomal peptide synthetase (NRPS) and PKS clusters predicted to synthesize aromatic polyketides and complex peptide metabolites.

Fig 8.

Biosynthetic gene clusters from the RSE02 genome. (A) Indole biosynthesis gene cluster (trpA, trpB and tnaA) in the RSE02 genome. (B) The figures show a physical map of the biosynthetic gene clusters for azole-containing-RiPP (I), terpene-precursor (II), RiPP-like (III), arylpolyene (IV), and NRPS (V). (C) The figures show highly similar antimicrobial gene clusters of RSE02 (I to III) compared with known clusters in the antiSMASH database. In the right panels, the secondary metabolites are predicted to be synthesized from the respective gene clusters.

AntiSMASH analysis showed the presence of five BGCs in the genome of Phytobacter sp. strain RSE02. Clusters are found to represent various secondary metabolites producing genes (Fig. 8BI through V) such as azole-containing-RiPP, terpene-precursor, RiPP-like, arylpolyene, and NRPS. However, the direct relation between such clusters and the production of the compound SM06 has not been established.

The genome of Phytobacter sp. strain RSE02 harbors BGC (Fig. 8CI), which showed 100% and 94% similarities with gene clusters producing aryl polyene and APE Ec (aryl polyene from E. coli), respectively. Additionally, the genome encodes a siderophore biosynthetic gene cluster (Fig. 8CII and III), exhibiting 100% sequence identity to the NRPS-dependent cluster responsible for trichrysobactin, cyclic trichrysobactin, chrysobactin, and dichrysobactin biosynthesis, whereas 90% similarity to the enterobactin gene cluster and 61% similarity to the viobactin gene cluster. This metabolic architecture indicates that RSE02 possesses a strong biosynthetic capacity for producing indole derivatives and additional bioactive compounds, with the diversity of its biosynthetic gene clusters suggesting a significant potential to synthesize a broad spectrum of metabolites consistent with its observed antifungal and plant beneficial phenotypes.

DISCUSSION

The present study identified Phytobacter sp. RSE02 is a seed-associated “plant probiotic” endophyte that produces a previously uncharacterized indole dimer antifungal metabolite, SM06, and establishes its broad-spectrum efficacy against diverse plant and clinical fungal pathogens. In contrast to our findings, a previous study demonstrated that di-indole metabolites derived from commensal microbiota modulate host xenobiotic and metabolic pathways (48). Our mechanistic, structural, and ecological analyses collectively indicate that SM06 acts primarily through competitive inhibition of fungal lanosterol 14-α-demethylase (ERG11), which causes perturbation of ergosterol balance and ultimately leads to impaired membrane integrity and fungal cell death. While this study highlights the bioactive potential of Phytobacter sp. RSE02, future investigations involving a broader collection of endophytic isolates will be valuable to assess the prevalence, diversity, and reproducibility of similar antifungal traits across related strains.

Mechanistic basis of SM06 antifungal action

Multiple lines of evidence indicate that SM06 targets the fungal cell membrane as its primary site of action. Enhanced PI uptake following SM06 treatment indicated the loss of membrane integrity, consistent with necrotic cell death and in line with the established behavior of membrane-disruptive antifungals (49). Scanning electron microscopy further revealed severe morphological aberrations, including hyphal swelling, septal distortion, and conidial surface collapse, consistent with catastrophic membrane and cell wall destabilization (50). The thermal stability of SM06 across a temperature range (30°C–90°C) suggests that its antifungal activity is not easily abrogated, making it a promising candidate for various applications. Additionally, its broad-spectrum activity against diverse fungal pathogens underscores its potential as a versatile antifungal agent. Protein leakage assays revealed a time-dependent increase in extracellular protein content following SM06 treatment, indicating progressive disruption of the fungal cell membrane. The time-dependent leakage of intracellular proteins reinforces the conclusion that SM06 progressively compromises membrane permeability, a hallmark of lytic antifungal mechanisms (51). From a detailed LC-MS analysis, it has also been found that there is a significant reduction in fungal ergosterol levels (m/z 413.2588) upon SM06 treatment, which strongly supports the interference with sterol biosynthesis (52). Ergosterol depletion is functionally congruent with the observed morphological abnormalities, given the central role of sterols in maintaining fungal membrane fluidity and structural order (53, 54). Molecular docking further supports this mechanism, revealing that SM06 exhibits competitive binding within the catalytic pocket of ERG11, partially overlapping with the binding surfaces of both lanosterol and fluconazole. This mode of inhibition parallels the action of azoles but involves distinct structural interactions that may circumvent conventional azole resistance pathways, including ERG11 mutations and efflux activation (55, 56). The proposed inhibition of ERG11 is supported by both molecular docking analysis and experimental evidence demonstrating reduced ergosterol content in treated fungal cells, indicating that the mechanism is not inferred solely from in silico predictions but is corroborated by biochemical validation. Although ERG11 is conserved across fungi, intrinsic resistance to ERG11-targeting compounds has been widely reported in Mucorales, including Rhizopus species. Structural differences in lanosterol 14α-demethylase, altered sterol composition, or reduced drug-binding affinity may contribute to decreased susceptibility. Such mechanisms may explain the limited response of Rhizopus sp. observed. The partial synergism observed in combination assays with nystatin (FICI = 0.60) aligns with their complementary biochemical activities, SM06 disrupting sterol biosynthesis and nystatin binding to ergosterol to further destabilize the membrane structure (57). Collectively, these results establish that SM06 disrupts fungal cell integrity through dual mechanisms: direct membrane permeabilization and impairment of ergosterol biosynthesis.

Structure-function comparisons with known antifungal indoles

The superior performance of SM06 in comparison to monomeric indoles can be rationalized by structural considerations. Prior studies have highlighted that dimeric or conjugated indole architectures exhibit enhanced biological potency due to increased hydrophobicity and improved target engagement (58, 59). Similar patterns are reflected in synthetic 1,3,4-thiadiazole-indoles with strong activity against plant pathogens (60) and in dimeric indole-diterpenoids derived from Penicillium species that display strong receptor interactions (61). In contrast, monomeric indoles exhibit moderate antifungal effects (62), suggesting that dimerization confers superior bioactivity and metabolic stability to the resulting dimers. Molecular docking suggested that within the fungal ERG11 active site, the two indole rings preferentially adopt a π–π–stacked configuration, further stabilized by hydrogen bonding interactions between the N–H group of the dimer and key amino acid residues. This aligns with our ADMET profiling, which indicated improved lipophilicity, plasma protein binding, and extended half-life of SM06 relative to that of normal indole. The enhanced affinity of SM06 for ERG11 compared to fluconazole further underscores the functional advantage imparted by this dimeric architecture (63, 64).

Ecological relevance and implications for plant protection

Endophytic bacteria that synthesize antifungal metabolites represent an important ecological strategy for suppressing plant diseases (65, 66). Consistent with this paradigm, Phytobacter sp. RSE02 not only inhibited fungal pathogens in vitro but also conferred robust protection to tomato and rice plants against C. lunata infections. Preventive application of SM06 completely blocked fungal colonization, whereas curative application significantly limited lesion expansion and reduced the spread of hyphae. These findings parallel previous reports demonstrating the antifungal activities of small molecules against Curvularia sp. (67, 68) and expand the repertoire of endophyte-derived metabolites with agricultural potential.

The ability of RSE02 to colonize in plant tissues, as confirmed by confocal microscopy, further suggests that the endogenous production of SM06 within host tissues contributes to disease mitigation. The detection of SM06-like signatures (m/z 235.1252) in infected plants treated with RSE02 supports in planta biosynthesis or accumulation of this compound. This phenomenon is consistent with the established ecological roles of endophytes, which routinely synthesize protective metabolites under pathogen-challenge conditions (69, 70). Beyond disease suppression, SM06 exhibited preservative benefits in tomato fruits, reducing infection-induced necrosis and oxidative damage. These effects align with the known natural preservative compounds in tomatoes (e.g., carotenoids and phenolic acids) (71) and support the utility of SM06 as a post-harvest protective agent (72). In a nutshell, multiple independent lines of evidence, such as purified SM06 inhibits C. lunata, exogenous application of pure SM06 suppresses disease symptoms in planta, and LC-MS analysis confirms the presence of SM06 in plant tissues colonized by RSE02 but not in untreated controls, indicate the effect of SM06 against C. lunata. Although a definitive confirmation of its causal role in disease suppression will require validation through targeted gene disruption or knockout of the SM06 biosynthetic pathway in RSE02 and its impact on disease prevention, which will be pursued in future studies.

Genomic context and metabolic potential of RSE02

Genome mining of RSE02 revealed multiple BGCs, including tryptophan-derived indole pathways (trpA, trpB, and tnaA) and NRPS/PKS clusters (Fig. 8). This metabolic repertoire is characteristic of plant-associated Enterobacterales and is often linked to antifungal activity, competition, and beneficial host traits (73, 74). The presence of indole biosynthetic genes supports the endogenous synthesis of indole dimer metabolites, such as SM06, while NRPS/PKS clusters suggest additional cryptic metabolites that may synergize with SM06 or exert independent protective effects.

Conclusion

Overall, our results position Phytobacter sp. RSE02 and its indole dimer metabolite, SM06, are potent fungal antagonists with mechanistic, structural, and ecological features well-suited for agricultural and biotechnological applications (Fig. 9). SM06 couples membrane-disruptive activity with targeted inhibition of ergosterol biosynthesis, conferring broad antifungal efficacy, including strong activity against Curvularia pathogens in multiple crop systems. The capacity of RSE02 to colonize host tissues and synthesize SM06 in planta highlights a promising route for eco-friendly, endophyte-based disease management, which aligns with global efforts to reduce dependence on synthetic fungicides.

Fig 9.

Schematic overview of the discovery of antifungal metabolite SM06 from a rice seed endophyte. The image depicts broad-spectrum antifungal activity, structural elucidation, and the application of SM06.

ETHICS APPROVAL

All zebrafish husbandry and experiments were conducted in accordance with institutional and national animal ethics guidelines and were approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Department of Biochemistry, University of Calcutta, India (approval no. IAEC-04/BIOCHEM/CU/02-2022/03-SH)

DATA AVAILABILITY

All relevant data are within the paper.