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. Environmental reservoirs can facilitate this. 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. Using 18 microsatellite markers, we determined the genotypic differentiation between 1,497 P. destructans isolates collected from nine closely situated underground sites where bats hibernate (i.e., hibernacula) in Northeastern Germany. This approach was unique in that it ensured that every isolate and resulting multi-locus genotype was not only present, but also viable and therefore theoretically capable of infecting a bat. The distinct distribution of multi-locus genotypes across hibernacula demonstrates that each hibernaculum has an essentially unique fungal population. This would be expected if bats become infected in their hibernaculum (i.e., the site they spend winter in to hibernate) rather than in other sites visited before they start hibernating. In one hibernaculum where both the walls and the hibernating bats were sampled at regular intervals over five consecutive winter seasons (1,062 isolates), higher genotypic richness was found on walls compared to bats and multi-locus genotypes showed a stable frequency over multiple winters. This clearly implicates hibernacula walls as the main environmental reservoir of the pathogen, from which bats become re-infected annually during hibernation.

To genotype isolates of P. destructans, 18 microsatellite markers (Drees et al., 2017b) were used in 4 PCR multiplexes as described in Dool et al. (2020). Genotyping was carried out using an ABI 3130 Genetic Analyser (Applied Biosystems). GeneMapper® Software v.5 (Applied Biosystems) was used for fragment analysis.

From Dool et al. (2020): "Eighteen microsatellite loci and the three mating type markers (apn2_Short, sla2_Short with two forward primers and sla2_Long) were optimised into four multiplex reactions (Table 2). Reactions were carried out in 6 μL volumes containing 1 μL of DNA extract, 1 × Type-it Microsatellite PCR Master Mix (QIAGEN) and primer concentrations as reported in Table 2. Amplification conditions were: 5 min at 95 °C; 30 cycles of 95 °C for 30 s, 58 °C for 90 s, 72 °C for 30sec; 30 min at 60 °C. PCR products were run on an ABI PRISM 3130XL Genetic Analyser (Applied Biosystems) and sized with the internal lane standard 600-LIZ using GeneMapper v.5.0 (Applied Biosystems)".

Dool SE, Altewischer A, Fischer NM, et al. (2020) Mating type determination within a microsatellite multiplex for the fungal pathogen Pseudogymnoascus destructans, the causative agent of white-nose disease in bats. Conservation Genetics Resources 12, 45-48.

Drees KP, Parise KL, Rivas SM, et al. (2017) Characterization of microsatellites in Pseudogymnoascus destructans for White-nose Syndrome genetic analysis. Journal of Wildlife Diseases 53, 869-874.

Information for the file 'Data_Reservoir_v1.csv' containing all the data used in the manuscript.

culture: name of the culture

country: country where the sample was collected

province: province where the sample was collected

site: city where the sample was collected

date: date when the sample was collected

labNo: name to identify all cultures from the same sample (e.g. 'Gd00298': three cultures [Gd_00298-aa, Gd_00298-ab, Gd_00298-ac] with the 'Gd00298' identifier have been made from sample '20150302_B_ELD_21')

sample: sample (swab) name

species: bat species from which the sample was collected (if the sample was collected from a bat)

substrate: substrate from which the sample was collected, here 'bat' or 'wall'

mtype: mating type inferred from the 'Sla2_Long' and/or 'Sla2_Short' markers

sampling_event: combined information on year of sampling, season, and substrate (used in Table 3 for example)

winter: winter season (used in Figure 4 for example)

Pd1- Pd22: microsatellite allele ('NA': missing value)

Sla2_Long: mating-type marker (519: MAT1-1; 547: MAT1-2; 'NA': missing value)

Sla2_Short: mating-type marker (240: MAT1-1; 208: MAT1-2; 'NA': missing value)

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The file 'Rscript_V8.R' containins the R script used to analyse the data contained in the file 'Data_Reservoir_v1.csv'.

The script is annotated.