Genomic and Chemical Diversity of Bacillus subtilis Secondary Metabolites against Plant Pathogenic Fungi

Abstract

Bacillus subtilis produces a wide range of secondary metabolites providing diverse plant growth-promoting and biocontrol abilities. These secondary metabolites include nonribosomal peptides with strong antimicrobial properties, causing either cell lysis, pore formation in fungal membranes, inhibition of certain enzymes, or bacterial protein synthesis. However, the natural products of B. subtilis are mostly studied either in laboratory strains or in individual isolates, and therefore, a comparative overview of secondary metabolites from various environmental B. subtilis strains is missing. In this study, we isolated 23 B. subtilis strains from 11 sampling sites, compared the fungal inhibition profiles of wild types and their nonribosomal peptide mutants, followed the production of targeted lipopeptides, and determined the complete genomes of 13 soil isolates. We discovered that nonribosomal peptide production varied among B. subtilis strains coisolated from the same soil samples. In vitro antagonism assays revealed that biocontrol properties depend on the targeted plant pathogenic fungus and the tested B. subtilis isolate. While plipastatin alone is sufficient to inhibit Fusarium spp., a combination of plipastatin and surfactin is required to hinder growth of Botrytis cinerea. Detailed genomic analysis revealed that altered nonribosomal peptide production profiles in specific isolates are due to missing core genes, nonsense mutation, or potentially altered gene regulation. Our study combines microbiological antagonism assays with chemical nonribosomal peptide detection and biosynthetic gene cluster predictions in diverse B. subtilis soil isolates to provide a broader overview of the secondary metabolite chemodiversity of B. subtilis. IMPORTANCE Secondary or specialized metabolites with antimicrobial activities define the biocontrol properties of microorganisms. Members of the Bacillus genus produce a plethora of secondary metabolites, of which nonribosomally produced lipopeptides in particular display strong antifungal activity. To facilitate the prediction of the biocontrol potential of new Bacillus subtilis isolates, we have explored the in vitro antifungal inhibitory profiles of recent B. subtilis isolates, combined with analytical natural product chemistry, mutational analysis, and detailed genome analysis of biosynthetic gene clusters. Such a comparative analysis helped to explain why selected B. subtilis isolates lack the production of certain secondary metabolites.

Development of quantitative high-throughput screening methods for identification of antifungal biocontrol strains

“…The so-called dual-culture assay is among the most common screening methods to identify potent fungal inhibitors from microbial collections [17,21,30–32]. Typically, the assay is performed by inoculating potential biocontrol agents at a fixed distance from the pathogenic fungal inoculum on a petri dish as illustrated in Fig.…”

Metabolic Profiling of Rhizobacteria Serratia plymuthica and Bacillus subtilis Revealed Intra- and Interspecific Differences and Elicitation of Plipastatins and Short Peptides Due to Co-cultivation

“…One class of metabolic features, which differed in later stages of interaction between S. plymuthica 4Rx13 and B. subtilis B2g belonged to compounds of the plipastatin family. Plipastatins are bioactive lipopeptides produced by isolates of Bacillus subtilis isolates (Nishikiori et al, 1986;Ongena et al, 2007;Hussein, 2019), mainly known for their striking antifungal activity (Kaspar et al, 2019;Kiesewalter et al, 2021). They occur as various isomers characterized by different structures (A1, A2, B1, and B2; Kaspar et al, 2019) of whom only specific isomers were increasingly produced by B. subtilis B2g in the cocultivation with S. plymuthica 4Rx13.…”

Deletion of Rap‐Phr systems in Bacillus subtilis influences in vitro biofilm formation and plant root colonization

“…Furthermore, studying biofilms is of special interest due to their detrimental impact in clinical and industrial settings (Di Pippo et al, 2018; Stewart, 2002) as well as their promising potential within the biotechnology industry (Blake et al, 2021; Singh et al, 2006). Regarding the latter, the gram‐positive bacterium Bacillus subtilis has in the last two decades gained interest due to its promising potential as a biocontrol agent within agriculture (Kiesewalter et al, 2021; Ongena & Jacques, 2008). In its natural habitat, the soil‐dwelling bacterium colonizes plants by forming a biofilm on the root (Bais et al, 2004; Beauregard et al, 2013; Chen et al, 2012).…”

“…Regarding the latter, the gram-positive bacterium Bacillus subtilis has in the last two decades gained interest due to its promising potential as a biocontrol agent within agriculture (Kiesewalter et al, 2021;Ongena & Jacques, 2008). In its natural habitat, the soildwelling bacterium colonizes plants by forming a biofilm on the root (Bais et al, 2004;Beauregard et al, 2013;Chen et al, 2012).…”