Fusarium wilt caused by Fusarium oxysporum f. sp. pisi (Fop) poses significant threats to pea cultivation worldwide. Controlling this disease is mainly achieved through the integration of various disease management procedures, among which biological control has proven to be a safe and effective approach. This study aims to extract and identify antifungal secondary metabolites from Streptomyces alboflavus KRO3 strain and assess their effectiveness in inhibiting the in vitro growth of Fop. This bacterial strain exerts in vitro antagonistic activity against Fop, achieving highly significant inhibition over one week. The ethyl acetate extract, obtained from its ISP2 agar medium culture, also exhibited strong antifungal activity, maintaining an inhibition rate of approximately 90% at concentrations up to 250 µg/plug compared to the control. Thus, the organic extract has been fractionated using chromatographic techniques and its bioguided purification allowed us to isolate the main bioactive compound. This latter was identified as metacycloprodigiosin using nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and specific optical rotation data. Metacycloprodigiosin demonstrates dose-dependent inhibitory activity against the phytopathogen with an effective concentration of 125 µg/plug. The other secondary metabolites present in the ethyl acetate extract were also identified by gas chromatography–mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR). This study highlighted the potential of S. alboflavus KRO3 strain and its antimicrobial compounds for the management of the pea pathogen Fusarium oxysporum f. sp. pisi.
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