Investigation of chemistry of filamentous fungi in relation to beneficial crop outcomes

Fungi inhabit nearly all environments on Earth and play essential ecological roles as decomposers and symbionts. Beyond being enzyme producers, they can produce a vast array of small, complex molecules known as secondary metabolites (SMs). These metabolites are not directly associated with growth or reproduction but rather confer specific ecological or physiological benefits to improve their fitness within the environment. Historically, drug discovery efforts have shaped the perception of SMs primarily as chemical weapons used to compete against microbial competitors. However, this perspective only captures a fraction of their true ecological significance. Microbe-centered research has revealed that SMs function as key chemical mediators, driving interactions between microbes, as well as between microbes and their hosts, both vital for overall ecosystem functioning. Despite their importance, the ecological roles of these molecules remain poorly understood.

This thesis explores the diversity of filamentous fungi associated with wheat roots and investigates the role of SMs in shaping cross-kingdom interactions between inhabitants of the wheat rhizosphere. Through a culture-dependent approach combined with polyphasic taxonomic method, we curated a library of 385 fungal strains obtained from the bulk soil, rhizosphere and rhizoplane of both greenhouse-grown and field-grown wheat. Taxonomic analysis revealed a strong dominance of the Penicillium species. In addition, we demonstrated a decrease in fungal diversity along the bulk soil–rhizosphere–rhizoplane continuum, with SM-rich Penicillium species enriched in the rhizosphere and rhizoplane. In contrast, Mucor and Rhizopus species, known to be poor producers of SMs, were completely absent from the rhizoplane. Our survey of fungal biodiversity led to the discovery of Penicillium croceum, a new species within the Penicillium section Ramosum, closely related to Penicillium virgatum. A comparative untargeted metabolomics approach, combined with macromorphological analysis, confirmed the distinction between P. croceum and P. virgatum, with P. croceum consistently producing cycloaspeptides and cyclopiazonic acid.

To explore the role of SMs, we employed untargeted metabolomics, mass spectrometry imagining (MSI) and molecular approaches, to decipher the molecular mechanism that governs the interaction between P. hordei and Bacillus subtilis, a consistent bacterial rhizosphere member. We demonstrated that fungal-derived organic acid, terrestric acid, trigger the precipitation of bacterial lipopeptides (LPs) to confer a protective mechanism for the fungus. We further revealed that the absence of LP production allowed P. hordei to invade and overgrow the B. subtilis colony. Finally, our findings indicated that LP precipitation is a conserved mechanism among Penicillium species that produce terrestric acid, indicating the widespread distribution of this defense strategy.

Surfactin is perhaps the most extensively studied SM of the Bacillus genus. Using bioreporter strains, molecular techniques and MSI, we demonstrated that subtilosin A production is increased in B. subtilis mutants deficient in surfactin. We further confirmed the roles of Rok and AbrB as repressors, and ResD as an activator, of the subtilosin A biosynthetic gene cluster (BGC) Our results suggest that an unknown regulatory mechanism mediates the reduction of subtilosin A production in the presence of surfactin.

Untargeted metabolomics is a powerful tool in microbial ecology, enabling the exploration of the chemical landscape of microbial SM networks. However, this approach often generates complex datasets with thousands of unique features, many of which remain unannotated, making data interpretation a significant challenge. Utilizing co-culture, taxonomic, and ecological datasets, we demonstrated that Metadata-Based Molecular Networks (MBMN), generated with the newly developed tool MolNetInvert, improved the visual clarity and interpretability of molecular networks produced by the Global Natural Products Social Molecular Networking (GNPS) platform.

Penicillium species are indispensable workhorses in the discovery and production of novel antibiotics and bioactive compounds with applications in agriculture and biotechnology. Overall, the work presented in this thesis provides a curated collection of Penicillium species, offering a valuable resource for future research focused on understanding the functional roles of these fungi in promoting plant growth and health. Furthermore, it provides insight into the ecological roles of fungal and B. subtilis SMs in mediating cross-kingdom interactions. Uncovering the molecular mechanisms and ecological consequences of these SM-mediated interactions will deepen our understanding of microbial community dynamics and their contributions to broader ecosystem functioning.