**Background:** Plant diseases caused by *Fusarium oxysporum* and *Rhizoctonia solani* are major constraints to amaranth production, causing root-rot, stem decay, and damping-off with estimated crop losses of ~50–60%. Chemical fungicides pose risks to human, animal, and environmental health, driving the need for biological alternatives. *Bacillus subtilis* is known to produce a wide array of antimicrobial secondary metabolites, including polyketides such as macrolactin. This study aimed to evaluate the biocontrol potential of *B. subtilis* BS-58 against these two pathogens and to identify the antifungal metabolite responsible.
**Methods:** *B. subtilis* BS-58 was previously isolated from non-rhizospheric soil in Salamkhet, Uttarakhand, India. In vitro antagonism was assessed using dual culture plate assays. Scanning electron microscopy (SEM) was used to examine hyphal deformities at the interaction zone. Cell-free supernatant (CFS) was tested for antifungal activity by agar well diffusion, and its stability was evaluated after heat treatment (70°C and 100°C for 20 min) and proteinase K treatment (100 μg/ml, 37°C for 30 min). The antifungal metabolite was purified via TLC-guided column chromatography (silica gel, mobile phase ethyl acetate:methanol 60:40). Identification was performed using LC–MS (ESI positive mode, scan range 150–1000 m/z) and FT-IR (400–4000 cm⁻¹). The *mln* gene was detected by PCR with gene-specific primers and sequenced (accession MT726941). A 30-day pot trial compared seed treatment with BS-58 (talc formulation, 10 g/kg seed) against negative controls (pathogen-infested soil, no treatment) and positive controls (carbendazim 2.0 g/kg seed) in sterilized potting mix (sand:soil:farmyard manure 1:1:1).
**Key Results:** In dual culture, BS-58 inhibited *F. oxysporum* by 68.25% and *R. solani* by 64.50%. SEM revealed hyphal lysis, swelling, perforation, shrinkage, and mycelial shredding in both pathogens. CFS showed 65.57% inhibition of *F. oxysporum* and 61.66% inhibition of *R. solani*. Heat treatment reduced activity modestly (62.76% and 58.8% for *F. oxysporum* at 70°C and 100°C; 59.33% and 57.6% for *R. solani*), while proteinase K substantially reduced inhibition to 28.96% and 22.24%, respectively. LC–MS of the most active column elute (F7) showed a major peak at m/z 403 [M+H]⁺ and a sodium adduct at m/z 425, corresponding to macrolactin A (402 Da). FT-IR identified alcohol (3421.31 cm⁻¹), alkane (2932.57, 2962.76, 2875.92, 1410.34 cm⁻¹), and carbonyl (1728.01 cm⁻¹) functional groups consistent with macrolactin A. The *mln* gene was amplified (554 bp fragment) and showed 98.1% homology with known macrolactin genes. In pot trials, BS-58 seed treatment reduced seedling mortality by approximately 54% in *F. oxysporum*-infested soil (17.85% mortality vs. 38.89% in negative control) and by 43.76% in *R. solani*-infested soil (17.85% vs. 31.74%). Disease suppression was nearly equivalent to carbendazim. BS-58 also increased shoot length, root length, fresh weight, and dry weight under pathogen stress. SEM of recovered seedling roots confirmed hyphal disintegration.
**Clinical Implications:** This study provides strong evidence that *B. subtilis* BS-58, through production of macrolactin A, is an effective biocontrol agent against *F. oxysporum* and *R. solani* in amaranth. The heat stability and extracellular nature of the metabolite are advantageous for industrial formulation. The strain's performance was comparable to the chemical fungicide carbendazim, suggesting it could serve as a safe, eco-friendly alternative for managing soilborne diseases in amaranth and potentially other crops. Further field trials and formulation development are warranted to translate these findings into agricultural practice.