The impact of inter- and intra-species spore density on germination of the food spoilage fungus Aspergillus niger.

Aspergilli can be used to produce food but can spoil it as well. Both food production and spoilage are initiated by germination of the conidia of these fungi that have been introduced by inoculation and contamination, respectively. Germination of these spores includes activation, swelling, establishment of cell polarity, and formation of a germ tube. So far, only quantitative single-species germination studies of fungal spores have been performed. Here, spore germination of the food spoilage fungus Aspergillus niger was studied quantitatively in mono-culture or when mixed with other food-relevant aspergilli (Aspergillus nidulans, Aspergillus terreus, Aspergillus clavatus, and Aspergillus oryzae). In the presence of the germination inducing amino acids proline or alanine, but not in the case of the lowly inducing amino acid arginine, the incidence of swelling and germ tube formation was reduced when 35,000 extra conidia of Aspergillus niger were added to wells containing 5000 of these spores. Adding 35,000 spores of one of the other aspergilli also did not have an effect on germination in the presence of arginine, but the germination inhibition was stronger when compared to the extra A. niger spores in the case of alanine. A similar effect was obtained with proline. Together, results show that the germination of A. niger conidia is impacted by the density of its own spores and that of other aspergilli under favorable nutritional conditions. These results increase our understanding of food spoilage by fungi and can be used to optimize food production with fungi.

Compatible solutes determine the heat resistance of conidia.

BACKGROUND: Asexually developed fungal spores (conidia) are key for the massive proliferation and dispersal of filamentous fungi. Germination of conidia and subsequent formation of a mycelium network give rise to many societal problems related to human and animal fungal diseases, post-harvest food spoilage, loss of harvest caused by plant-pathogenic fungi and moulding of buildings. Conidia are highly stress resistant compared to the vegetative mycelium and therefore even more difficult to tackle. RESULTS: In this study, complementary approaches are used to show that accumulation of mannitol and trehalose as the main compatible solutes during spore maturation is a key factor for heat resistance of conidia. Compatible solute concentrations increase during conidia maturation, correlating with increased heat resistance of mature conidia. This maturation only occurs when conidia are attached to the conidiophore. Moreover, conidia of a mutant Aspergillus niger strain, constructed by deleting genes involved in mannitol and trehalose synthesis and consequently containing low concentrations of these compatible solutes, exhibit a sixteen orders of magnitude more sensitive heat shock phenotype compared to wild-type conidia. Cultivation at elevated temperature results in adaptation of conidia with increased heat resistance. Transcriptomic and proteomic analyses revealed two putative heat shock proteins to be upregulated under these conditions. However, conidia of knock-out strains lacking these putative heat shock proteins did not show a reduced heat resistance. CONCLUSIONS: Heat stress resistance of fungal conidia is mainly determined by the compatible solute composition established during conidia maturation. To prevent heat resistant fungal spore contaminants, food processing protocols should consider environmental conditions stimulating compatible solute accumulation and potentially use compatible solute biosynthesis as a novel food preservation target.

Inter- and intra-species heterogeneity in germination of Aspergillus conidia.

Aspergilli are among the most abundant fungi worldwide. They degrade organic material and can be pathogens of plants and animals. Aspergilli spread by forming high numbers of conidia. Germination of these stress resistant asexual spores is characterized by a swelling and a germ tube stage. Here, we show that conidia of Aspergillus niger, Aspergillus oryzae, Aspergillus clavatus, Aspergillus nidulans and Aspergillus terreus show different swelling and germ tube formation dynamics in pure water or in water supplemented with (in)organic nutrients. Apart from inter-species heterogeneity, intra-species heterogeneity was observed within spore populations of the aspergilli except for A. terreus. Sub-populations of conidia differing in size and/or contrast showed different swelling and germ tube formation dynamics. Together, data imply that aspergilli differ in their competitive potential depending on the substrate. Moreover, results suggest that intra-species heterogeneity provides a bet hedging mechanism to optimize survival of aspergilli.

Minimal nutrient requirements for induction of germination of Aspergillus niger conidia.

Aspergillus niger reproduces asexually by forming conidia. Here, the minimal nutrient requirements were studied that activate germination of A. niger conidia. To this end, germination was monitored in time using an oCelloScope imager. Data was used as input in an asymmetric model to describe the process of swelling and germ tube formation. The maximum number of spores (Pmax) that were activated to swell and to form germ tubes was 32.54% and 20.51%, respectively, in minimal medium with 50 mM glucose. In contrast, Pmax of swelling and germ tube formation was <1% in water or 50 mM glucose. Combining 50 mM glucose with either NaNO3, KH2PO4, or MgSO4 increased Pmax of swelling and germination up to 15.25% and 5.4%, respectively, while combining glucose with two of these inorganic components further increased these Pmax values up to 25.85% and 10.99%. Next, 10 mM amino acid was combined with a phosphate buffer and MgSO4. High (e.g. proline), intermediate and low (e.g. cysteine) inducing amino acids were distinguished. Together, a combination of an inducing carbon source with either inorganic phosphate, inorganic nitrogen or magnesium sulphate is the minimum requirement for A. niger conidia to germinate.