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Abstract
The plant root growth niche is a hotspot for microbial life and interactions of different character between species and kingdoms, ranging from mutualistic and synergistic to antagonistic. The multispecies microbial communities that inhabit the rhizosphere greatly impact plant health. Among the microbial inhabitants, plant growth-promoting rhizobacteria (PGPR) offer beneficial effects to the plant through various mechanisms. Members of the Bacillus genus are regularly isolated from the rhizosphere, rhizoplane or phylloplane and possess plant beneficial properties including modulation of nutrient availability, phosphate solubilization, enhancement of abiotic stress tolerance, and plant growth promotion. In addition, Bacillus spp. have the potential to produce a vast array of bioactive compounds potentially inhibitory to plant pathogens, positioning these bacteria as promising candidates for biological control strategies in agriculture, particularly for combating fungal phytopathogens.
Alike Bacillus spp., the black mold fungus Aspergillus niger is frequently identified in soil samples, however, little is known about the interaction between these species. Using bacterial-fungal co-cultures, we investigated the hyphal colonization by Bacillus subtilis biofilm mutants and demonstrated that extracellular matrix components are essential for robust biofilm formation on the mycelium. Furthermore, we showed that matrix components are shared, as defective biofilm formation can be rescued by addition of a producing strain. The interaction between the species was further explored by adaptive laboratory evolution of B. subtilis in the presence of A. niger. The repeated bacterial- fungal co-cultivation promoted enhanced B. subtilis niche colonization facilitated by increased colony expansion. The spreading behavior was attributed to increased biosynthesis of the lipopeptide surfactin, which caused fungal cell wall stress and reduced acidification of the medium by the fungus. This phenotype was correlated to genetic changes of the regulatory system DegS-DegU.
The Bacilli subjected to iterative planktonic growth cycles, in the presence or absence of the grey mold fungus Botrytis cinerea, accumulated flagellum-related mutations, conferring loss of motility to the evolved strains. Repetitive growth on solid medium, in the presence or absence of the filamentous fungus Fusarium culmorum, fostered adaptation of the quorum sensing system ComP-ComA associated with enhanced niche colonization in confrontation with the fungus, reduced production of antifungal lipopeptides, and increased volatilome effect on fungal growth. In both experimental evolution campaigns, further adaptative changes were detected in transcriptional regulators and cell differentiation pathways, which were associated with multiple phenotypic changes such as altered biofilm morphology, modulated biosynthesis of specialized metabolites and fungal growth inhibition potency. Several adaptation mechanisms focused on energy preservation effects by compromising carbon- and energy-demanding metabolic pathways, demonstrating an evolutionary selection for growth.
In summary, this Ph.D. project contributed to the knowledge of bacterial-fungal interactions and understanding of the Bacillus adapted response to fungal co-cultivation. The evaluation of Bacillus antifungal potency highlighted the importance of specialized metabolites and additional alternative fungal inhibition mechanisms. Furthermore, the project investigated experimental evolution as a tool for strain improvement for biocontrol purposes.
Alike Bacillus spp., the black mold fungus Aspergillus niger is frequently identified in soil samples, however, little is known about the interaction between these species. Using bacterial-fungal co-cultures, we investigated the hyphal colonization by Bacillus subtilis biofilm mutants and demonstrated that extracellular matrix components are essential for robust biofilm formation on the mycelium. Furthermore, we showed that matrix components are shared, as defective biofilm formation can be rescued by addition of a producing strain. The interaction between the species was further explored by adaptive laboratory evolution of B. subtilis in the presence of A. niger. The repeated bacterial- fungal co-cultivation promoted enhanced B. subtilis niche colonization facilitated by increased colony expansion. The spreading behavior was attributed to increased biosynthesis of the lipopeptide surfactin, which caused fungal cell wall stress and reduced acidification of the medium by the fungus. This phenotype was correlated to genetic changes of the regulatory system DegS-DegU.
Inspired by these results, we explored experimental evolution of natural Bacillus isolates in co-culture with phytopathogenic fungi as a strategy to improve the antifungal properties of the bacteria for biocontrol applications. To assess to inhibitory potency of evolved strains, we developed two high throughput screening methods, allowing quantification of antifungal properties and facilitating easy comparison to the respective ancestors. The methods provided insights of the antifungal potency of culture supernatant components or accounted for all the antagonistic mechanisms by co-inoculation of the species.
Original language | English |
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Place of Publication | Kgs. Lyngby, Denmark |
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Publisher | DTU Bioengineering |
Number of pages | 332 |
Publication status | Published - 2023 |
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Improving antifungal properties of bacillus spp
Stancheva, B. K. (PhD Student), Kuipers, O. (Examiner), Kovács, Á. T. (Main Supervisor), Dominguez-Cuevas, P. (Supervisor), da Fonseca, C. S. (Supervisor) & Andersen, M. Ø. (Examiner)
01/12/2018 → 16/11/2023
Project: PhD