Abstract
Determining the mode of action of microbial biocontrol agents plays a key role in their development and registration as commercial biopesticides. The biocontrol rhizobacterium Lysobacter capsici AZ78 (AZ78) is able to inhibit a vast array of plant pathogenic oomycetes and Gram-positive bacteria due to the release of antimicrobial secondary metabolites. A combination of MALDI-qTOF-MSI and UHPLC-HRMS/M was applied to finely dissect the AZ78 metabolome and identify the main secondary metabolites involved in the inhibition of plant pathogenic microorganisms. Under nutritionally limited conditions, MALDI-qTOF-MSI revealed that AZ78 is able to release a relevant number of antimicrobial secondary metabolites belonging to the families of 2,5-diketopiperazines, cyclic lipodepsipeptides, macrolactones and macrolides. In vitro tests confirmed the presence of secondary metabolites toxic against Pythium ultimum and Rhodococcus fascians in AZ78 cell-free extracts. Subsequently, UHPLC-HRMS/MS was used to confirm the results achieved with MALDI-qTOF-MSI and investigate for further putative antimicrobial secondary metabolites known to be produced by Lysobacter spp. This technique confirmed the presence of several 2,5-diketopiperazines in AZ78 cell-free extracts and provided the first evidence of the production of the cyclic depsipeptide WAP-8294A2 in a member of L. capsici species. Moreover, UHPLC-HRMS/MS confirmed the presence of dihydromaltophilin/Heat Stable Antifungal Factor (HSAF) in AZ78 cell-free extracts. Due to the production of HSAF by AZ78, cell-free supernatants were effective in controlling Plasmopara viticola on grapevine leaf disks after exposure to high temperatures. Overall, our work determined the main secondary metabolites involved in the biocontrol activity of AZ78 against plant pathogenic oomycetes and Gram-positive bacteria. These results might be useful for the future development of this bacterial strain as the active ingredient of a microbial biopesticide that might contribute to a reduction in the chemical input in agriculture.