An optimized, rhamnolipid-containing cell-free filtrate from Pseudomonas aeruginosa 8–7 exhibits broad-spectrum antifungal activity and exceptional environmental stability

Introduction The biocontrol strain Pseudomonas aeruginosa 8-7 (PA8-7) produces antimicrobial metabolites, yet its application is limited by low yield and insufficiently characterized environmental stability. Methods Fermentation was optimized through medium screening, single-factor experiments, and orthogonal array design. The resulting cell‑free filtrate (CFF) was evaluated for antifungal activity against 19 tropical crop pathogens, environmental stability (thermal, pH, UV, reductants, metal ions), rhamnolipid composition (TLC, HPLC‑MS, ¹H NMR), field efficacy against rubber tree powdery mildew ( Oidium heveae ), and biosafety on soybean seedlings. Results The optimal medium (15 g/L acid‑hydrolyzed casein, 25 mL/L glycerol, 1 g/L MgSO₄·7H₂O, 1.5 g/L K₂HPO₄, pH 6.5) and culture conditions (50 mL/500 mL flask, 220 rpm, 10% inoculum, 36 °C, 5 days) increased antifungal activity against Colletotrichum alienum by 54% to 89.81%. The CFF exhibited broad‑spectrum activity against all 19 pathogens, with the highest inhibition against Phytophthora litchii (94.56 ± 1.38%) and Phytophthora melonis (93.87 ± 1.69%). It retained >87% activity after autoclaving (121 °C, 30 min), remained stable across pH 1–12, and tolerated UV, reductants and most metal ions. Eighteen rhamnolipid congeners (9 mono‑, 9 di‑) were identified. Field trials showed that a 10‑fold diluted CFF reduced the disease index of rubber tree powdery mildew from 23.55 to 21.11 within 7 days (73.23% control efficacy), with no phytotoxicity on soybean seedlings. Conclusion The optimized CFF combines broad‑spectrum activity, exceptional stability, and field efficacy with biosafety, making it a promising low‑cost biocontrol candidate for sustainable tropical agriculture.