| Summary: | Endophytic microorganisms (EMs) have recently attracted interest for applications in plant protection, mainly due to their bioactive compound-producing capacity. Therefore, we underwent the task of isolating olive tree EMs and investigating their bioactivity against the devastating pathogen <i>Colletotrichum acutatum</i>. Several EMs were isolated; however, the <i>Bacillus</i> sp. PTA13 isolate exhibited the highest toxicity to the phytopathogen. Bacteria of the genus <i>Bacillus</i> exhibit superior bioactive metabolite-producing capacity, with the lipopeptides (LPs) of surfactin, iturin, and fengycin groups being the most studied. A total LP extract and several fractions were obtained, and their bioactivity was assessed against <i>C. acutatum</i> strains. LPs of the major surfactin, iturin, and fengycin groups and the minor gageotetrin and bacilotetrin groups were annotated. The results confirmed the bioactivity of the major LPs, with fengycins being the most fungitoxic. Interestingly, the minor LP fraction exhibited selective toxicity to the fungicide-resistant <i>C. acutatum</i> isolate, an observation that highlights the significance of our approach to comprehensively mine the total LP extract. This work represents a proof of concept of the exploitation of EMs in customized olive tree plant protection and aligns well with strategies that focus on the sustainability and safety of food production via the development of next-generation plant protection products.
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