Extraction, Purification and Characterization of Antifungal Compounds Produced by Bacillus velezensis CMRP4489

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Agricultural production is constantly affected by fungal phytopathogens that threaten sustainable food production. The use of biological control by microorganisms is an alternative of increasing interest to reduce the losses caused by these phytopathogens. The strain Bacillus velezensis CMRP4489 stands out for having great biotechnological potential in the control of fungal phytopathogens, and, according to in silico analyses, twelve important gene clusters have been identified in the codification of biosynthesis pathways of secondary metabolites, eight some are responsible for the production of antimicrobial metabolites. However, the isolated expression and activity of these metabolites have not yet been fully elucidated. The aim of the present study was to produce, extract, purify, identify, and evaluate antifungal metabolites produced by B. velezensis CMRP4489 with activity against S. sclerotiorum, a fungus responsible for white mold. For the production of antifungal metabolites, B. velezensis CMRP4489 was grown in a medium formulated with different macro and micronutrients (Patent BR 10 2020 013481 7), at 25°C for 72 h at 200 rpm and then the culture was centrifuged. The cell-free supernatant was lyophilized and fractionated by means of Vacuum liquid chromatography (VLC), followed by flash liquid chromatography (FLC) and a final purification step by preparative high-performance liquid chromatography (prep HPLC). All fractions were tested using disk diffusion tests and Spot-on-the-lawn assays, all of which were conducted using the phytopathogenic fungus S. sclerotiorum. The compound with the highest activity was identified by Nuclear Magnetic Resonance 1D (1H NMR and 13C NMR) and 2D (COSY, HSQC, HMBC and NOESY) spectroscopy. The most active fraction of VLC, found by disk-diffusion tests, was F2 (16 ± 0.8 mm). From it, 9 fractions were obtained in FLC, of which F6F was the one with the highest activity against S. sclerotiorum (26.25 ± 1.8 mm). F6F was purified by prep. HPLC, resulting in four compounds, of which F6F.1 was the one with the highest activity (27.5 ± 2.0 mm). Through the NMRs it was possible to verify that F6F.1 is probably a Bacillopeptin, a molecule of the Iturin family. However, it will be necessary others experiments for proper confirmation. If confirmed, the present study will be the first to describe the antifungal activity of bacillopeptin against S. sclerotiorum.

  • 1 Departamento de Microbiologia / Centro de Ciências Biológicas / Universidade Estadual de Londrina
  • 2 Universidade Estadual do Norte do Paraná
  • Innovation and Biotechnology
Sclerotinia sclerotiorum
Biological control