Combinatorial effect of mutagenesis and medium component optimization on Bacillus amyloliquefaciens antifungal activity and efficacy in eradicating Botrytis cinerea

Abstract

This work is directed towards Bacillus amyloliquefaciens strain BLB371 metabolite production for biocontrol of fungal phytopathogens. In order to maximise antifungal metabolite production by this strain, two approaches were combined: random mutagenesis and medium component optimization. After three rounds of mutagenesis, a hyper active mutant, named M3-7, was obtained. It produces 7 fold more antifungal metabolites (1800AU/mL) than the wild strain in MC medium. A hybrid design was applied to optimise a new medium to enhance antifungal metabolite production by M3-7. The new optimized medium (35g/L of peptone, 32.5g/L of sucrose, 10.5g/L of yeast extract, 2.4g/L of KH₂PO₄, 1.3g/L of MgSO₄ and 23mg/L of MnSO₄) achieved 1.62 fold enhancement in antifungal compound production (3000AU/mL) by this mutant, compared to that achieved in MC medium. Therefore, combinatory effect of these two approaches (mutagenesis and medium component optimization) allowed 12 fold improvement in antifungal activity (from 250UA/mL to 3000UA/mL). This improvement was confirmed against several phytopathogenic fungi with an increase of MIC and MFC over than 50%. More interestingly, a total eradication of gray mold was obtained on tomato fruits infected by Botrytis cinerea and treated by M3-7, compared to those treated by BLB371. From the practical point of view, combining random mutagenesis and medium optimization could be considered as an excellent tool for obtaining promising biological products useful against phytopathogenic fungi.

Keywords: Antifungal metabolites; Bacillus amyloliquefaciens; Biological control; Hybrid design; Medium optimization; Mutagenesis.