The Role of Secondary Metabolites in the Fungal Community of Black Apples

Fungi inhabit most parts of our planet, and they are crucial for most ecosystems due to their lifestyle as nutrient recyclers. Besides being prolific producers of secreted enzymes for degrading substrate, filamentous fungi also encode and produce a remarkably vast amount of structurally diverse and complex, small molecules. These metabolites are not directly involved with the primary metabolic pathways essential for growth and are thus often referred to as secondary metabolites (SMs). Fungal SMs have been exploited by humans for decades as pharmaceuticals like penicillin and lovastatin. On the contrary, filamentous fungi and their SMs have major negative impact as causal agents of food and crops spoilage and of deadly infections in immunocompromised patients. For these reasons, fungal SMs have been studied immensely in the laboratory, however, the true ecological roles of SMs in the natural habitat of the producer is not clear. Many SMs display antibiotic activity in high concentrations in vitro, which has led to the conventional notions of SMs mainly being agents of chemical warfare against competing species. However, more nuanced roles might be revealed, when studying them within the context of systems that better resemble the natural environment of the producer.

Microbial communities are inherently structurally and chemically complex and dynamic systems, which makes them difficult to study. This PhD thesis aims at generating insight into the ecological roles of fungal SMs by studying the fungal community of windfall, mouldy black apples as a model ecosystem.

With a multidisciplinary approach, using longread whole genome sequencing combined with untargeted mass spectrometry, the phylogenomic diversity and biosynthetic potential of black apples were examined, revealing a rich diversity of SMs in the system, and pointing to specific important SMproducing species. The two key SMproducing fungi of the system were found to be Penicillium expansum and Monilinia fructigena. To examine the effect of specific SMs, markerfree CRISPRCas9 genetic engineering tools were established in P. expansum, enabling the deletion of core biosynthetic genes. A library of SMdeficient mutant strains were grown in coculture with M. fructigena on apple puree agar and the effect of the mutations on the competitiveness of P. expansum was evaluated. Specifically, the P. expansum mutant strain deficient in patulin production was unable to antagonise M. fructigena. Even so, M. fructigena was shown to biotransform patulin into the degradation product, ascladiol, in coculture with the wild type P. expansum. Patulin has previously been shown to play a role in the virulence of P. expansum in axenic cultures on apples and as an unspecific toxin, but the results presented in this thesis suggest a broader, multifaceted role of patulin in the interaction with M. fructigena, which is an ecological relevant species. It further highlights the importance of studying the fate of SMs in microbial communities, as the success of a species not only depends on the SMs produced, but also on its ability to resist toxic SMs from other species inhabiting the same ecosystem.

Collectively, this thesis explores the complex world of fungal secondary metabolites and their ecological relevance in microbial communities within the context of mouldy, black apples. It challenges conventional perceptions of these metabolites as chemical weapons and highlights the importance of studying them in their natural environments. As emerging resistance to antibiotics and pesticides becomes a global concern, this research contributes to a deeper understanding of the chemical ecology of fungi, which is essential for managing harmful species, addressing antibiotic resistance and harnessing their diverse capabilities for new drug development.