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Emerging infectious diseases pose a major threat to human, animal, and plant health. The risk of species-extinctions increases when pathogens can survive in the absence of the host, for example in environmental reservoirs. However, identifying such reservoirs and modes of infection is often highly challenging. In this study, we investigated the presence and nature of an environmental reservoir for the ascomycete fungus Pseudogymnoascus destructans, the causative agent of white-nose disease. We also characterised the modes and timing of transmission of the pathogen; key elements to better understand the disease dynamics. Using 18 microsatellite markers, we determined the genotypic and genic (based on allele frequencies) differentiation between 1,497 P. destructans isolates collected from nine closely situated hibernacula in North-Eastern Germany. One hibernaculum was the focus of intensive sampling in which both the bats and walls of the site were sampled at regular intervals over five consecutive winter seasons (1,062 isolates). We found significant genic differentiation between sites and few multi-locus genotypes shared across hibernacula (genotypic differentiation). This demonstrates that each hibernaculum has an essentially unique population of the fungus. This would be expected if bats purge viable P. destructans over the summer, preventing the mixing and exchange of the pathogen in maternity colonies, where bats from all of the studied hibernacula meet. Results from the intensively sampled site show higher measures of genotypic richness on walls compared to bats, the absence of genic differentiation between bats and walls, and stable relative abundance of multi-locus genotypes over multiple winter seasons. This clearly implicates hibernacula walls as the main environmental reservoir of the pathogen, from which bats become re-infected annually.

Emerging infectious diseases rank among the most important threats to human and wildlife health. A comprehensive understanding of the mode of infection and presence of potential reservoirs is critical for the development of effective counter strategies. Fungal pathogens can remain viable in environmental reservoirs for extended periods of time before infecting susceptible individuals. This may be the case for Pseudogymnoascus destructans ( Pd ), the causative agent of bat white-nose disease. Owing to its cold-loving nature, this fungal pathogen only grows on bats during hibernation, when their body temperature is reduced. Bats only spend part of their life cycle in hibernation and do not typically show signs of infection in summer, raising the question of whether Pd remains viable in hibernacula during this period (roughly six months). If so, this could facilitate the re-infection of bats when they return to the sites the following winter. In a laboratory experiment, we determined the germination rate of Pd spores kept under constant conditions on a wall-like substrate, over the course of two years. Results showed that the seasonal pattern in Pd germination mirrored the life cycle of the bats, with an increased germination rate at times when hibernating bats would naturally be present and lower germination rates during their absence. We suggest that Pd is dependent on the presence of hibernating bats and has therefore coupled its germination rate to host availability. Furthermore, we demonstrate that Pd spores survive extended periods of host absence and can remain viable for at least two years. There is, however, a strong decrease in spore viability between the first and second years (98%). Pd viability for at least two years on a solid mineral-based substrate establishes the potential for environmental reservoirs in hibernacula walls and has strong implications for the efficacy of certain management strategies (e.g. bat culling).

Emerging infectious diseases are a severe conservation threat for a variety of plants and animals. In North America, several bat species are threatened by white-nose disease, which has caused an unprecedented mass mortality of > 6 million bats since 2006. The fungus Pseudogymnoascus (Geomyces) destructans is the causative agent of the disease. Though asexual reproduction is the norm, sexual reproduction is possible as two mating types exist. Sexual reproduction has been implicated in the emergence of virulent strains of fungi but to date no rapid means of mating type characterisation was available for P. destructans. In this study, three mating type-specific primer sets were designed and tested on 80 isolates. The primers were multiplexed with microsatellite loci allowing for rapid and concurrent genotyping and mating type assignment. These markers will have great utility in better understanding and predicting the population dynamics and evolutionary potential of this fungus, including the emergence of virulent strains.