Characterisation of the Antibiotic Profile of Lysobacter capsici AZ78, an Effective Biological Control Agent of Plant Pathogenic Microorganisms

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.

Plasmopara viticola the Causal Agent of Downy Mildew of Grapevine: From Its Taxonomy to Disease Management

“…Copper-resistant Lysobacter capsici AZ78, isolated from tobacco rhizosphere, showed efficiency against grapevine downy mildew under controlled conditions, where disease severity on leaves was reduced similarly to application of copper fungicide (Puopolo et al, 2014b ; Brescia et al, 2021 ). Direct inhibition of the pathogen was reached by production of the secondary metabolite 2,5-diketopiperazine that was found to be toxic against sporangia of some plant pathogenic oomycetes, including P. viticola (Puopolo et al, 2014a ; Cimmino et al, 2020 ).…”

Advances and Perspectives in the Use of Biocontrol Agents against Fungal Plant Diseases

“…The growth of the BCA Papiliotrema terrestris LS28 in apple wounds is affected by ROS and makes necessary the expression of genes involved in resistance to ROS-generated oxidative stress. This was first suggested by Castoria et al [176], and recently corroborated with a functional genetic approach by knocking out the gene encoding the oxidative stress-responsive transcription factor YAP1. The deleted mutants displayed a significant reduction of biocontrol activity [199].…”

“…Other bacterial genera, such as Streptomyces, Burkholderia, Serratia, Pantoea, Lysobacter and Enterobacter are known as producers of antibiotics with antibacterial and antifungal properties (e.g., iturin lipopeptides produced by Bacillus spp.) and have been tested against postharvest fungal pathogens [114,[173][174][175][176][177][178]. Among filamentous fungi, different Trichoderma species are known to produce antibiotic compounds active against a wide range of microorganisms [179,180].…”

Biological Control of Plant Diseases: An Evolutionary and Eco-Economic Consideration

“…Plants can defend themselves by producing compounds to kill pathogens or promote the growth of beneficial microbes [54]. These compounds can be extracted from plants and used in combination with antimicrobials or metabolism produced by beneficial microbes such as BCAs [55,56]. For example, many bacterial and fungal endophytes produce myriad secondary metabolites that have antagonistic, inhibitory, and deterrent properties that defer plant pathogens [57,58].…”

“…There is also an urgent need to develop a new generation of BCAs to meet increasing societal demand for more production of high-quality and diverse foods with "green" effects on ecosystems [81,103]. Many BCAs used currently act either as an antagonism against pathogens or as a promotor to increase host immunity [13,26,36,50,55]. The first category may be very effective at the beginning, but its effectiveness can be lost quickly due to the evolution of pathogens.…”