Heterogeneity in spore germination dynamics in Aspergillus niger

Abstract

The genus Aspergillus is ubiquitous and impacts human health, food safety, and industrial biotechnology through its resilient asexual spores, or conidia. Successful colonization and the subsequent beneficial or detrimental effects of Aspergillus species depend on spore germination. This thesis investigates the phenotypic heterogeneity in spore germination dynamics within and between Aspergillus species to inform targeted antifungal strategies and optimize industrial applications. The research used high-throughput oCelloScope imaging and an asymmetric kinetic model to quantify swelling and germ tube formation at single-spore resolution across controlled nutritional and population settings. It systematically tested the impacts of signaling molecules, nutrient availability, spore density, population heterogeneity, and spore maturity on germination dynamics. Finally, it introduces a refined, size-independent analysis to robustly compare mixed or variably sized populations. In Chapter 2, the minimal nutrient requirements for A. niger conidia germination are defined. Glucose combined with at least one inorganic component (phosphate, nitrate, or magnesium sulfate) was essential, whereas water or glucose alone yielded <1% germination. Specific amino acids were strong germination triggers; 10 mM proline and alanine were more effective than 50 mM glucose, while most other amino acids were weak inducers. Chapter 3 revealed significant inter- and intra-species heterogeneity in germination dynamics. When comparing A. niger to four other species (A. oryzae, A. clavatus, A. nidulans, and A. terreus), differential germination responses were observed across various nutrient conditions. Some species (e.g., A. nidulans, A. clavatus) germinated even in water, whereas A. niger and A. terreus required nutrients; proline and alanine robustly induced germination in four species but not in A. terreus. Within species, sub-populations grouped by initial size/contrast exhibited distinct germination dynamics, with larger conidia often reaching higher maximal probabilities of swelling or germ tube formation. This heterogeneity suggests a "bet-hedging" mechanism, where heterogeneous spore populations optimize survival under fluctuating environmental conditions. Chapter 4 showed density-dependent germination dynamics in A. niger. Increasing A. niger spore density reduced germination in proline and alanine, and co-culturing with spores from other Aspergillus species further inhibited A. niger germination. Constant amino acid concentrations during assays indicate inhibition mechanisms other than nutrient depletion, potentially through physical signaling or microbial competition. In Chapter 5, the impact of spore maturity was explained using an improved analytical methodology based on tracking the percentage change in spore area and circularity over time, reducing bias associated with fixed thresholds for heterogeneous populations. Mature (8-day-old) A. niger conidia consistently displayed enhanced germination compared to immature (38-hour-old) spores when exposed to strong triggers like alanine, proline, and glucose, whereas immature spores germinated better in arginine. A compatible-solute-deficient mutant (ΔtpsABC, ΔmpdA) germinated similarly to mature reference spores in amino acids but not in glucose, suggesting compatible solutes are nonessential for amino acid-induced germination initiation. Overall, this research highlights that Aspergillus spore germination is a highly regulated and heterogeneous process, influenced by a complex interplay of environmental signals and intrinsic spore properties. The insights gained are essential for advancing food preservation strategies and enhancing industrial fermentation yields.