Dihydromaitophilin

Background: Anthracnose caused by Colletotrichum fructicola is one of the most important diseases in pear fruit, resulting in huge economic losses. Public awareness of protecting the environment and food safety, together with pathogen resistance to many key fungicides have led to an urgent need to develop alternative strategies for controlling fruit diseases. Here, the antifungal activity of a natural product, dihydromaltophilin [heat-stable antifungal factor (HSAF)], against C. fructicola in vitro and in vivo was investigated to determine its efficacy for anthracnose management. Results: HSAF exhibited pronounced antifungal activity against in vitro mycelial growth of C. fructicola, with a half-inhibition concentration of 0.43 mg L-1 . Hyphae treated with HSAF showed defects such as hyperbranching, swelling and depolarized growth. Conidia germination in the pathogen was inhibited by HSAF in a dose-dependent manner. In the presence of 4 mg L-1 HSAF, conidia germination was significantly delayed, and germ tube growth was inhibited. HSAF at 8 mg L-1 completely blocked conidia germination in C. fructicola. In addition, HSAF disrupted coordination of cytokinesis with growth and nuclear division, induced reactive oxygen species production in conidia, and damaged the integrity of the conidia cell wall. Moreover, an in vivo test confirmed that 50 mg L-1 HSAF significantly reduced the development of anthracnose decay in pear fruit caused by C. fructicola. Conclusion: HSAF was highly effective in reducing pear anthracnose caused by C. fructicola and has great potential to become a new type of fruit preservative.

Antifungal HSAF (heat-stable antifungal factor, dihydromaltophilin) is a polycyclic tetramate macrolactam from the biocontrol agent Lysobacter enzymogenes. Its biosynthetic gene cluster contains only a single-module polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS), although two separate hexaketide chains are required to assemble the skeleton. To address the unusual biosynthetic mechanism, we expressed the biosynthetic genes in two "clean" strains of Streptomyces and showed the production of HSAF analogues and a polyene tetramate intermediate. We then expressed the PKS module in Escherichia coli and purified the enzyme. Upon incubation of the enzyme with acyl-coenzyme A and reduced nicotinamide adenine dinucleotide phosphate (NADPH), a polyene was detected in the tryptic acyl carrier protein (ACP). Finally, we incubated the polyene-PKS with the NRPS module in the presence of ornithine and adenosine triphosphate (ATP), and we detected the same polyene tetramate as that in Streptomyces transformed with the PKS-NRPS alone. Together, our results provide evidence for an unusual iterative biosynthetic mechanism for bacterial polyketide-peptide natural products.

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.