Potential for Growth and Spread of the Salamander-Devouring Fungus, Batrachochytrium salamandrivorans, in Eastern North America.

McGrath-Blaser, Sarah

Field Methods - Collect soil from locations along the A.T. in Great Smoky Mountains National Park to perform the subsequent experiments. Experimental Methods – In order to implement efficient conservation strategies to counteract disease spread, it is pivotal that we first understand the pathogen’s life history and transmission limitations. In this case, we have the rare opportunity to experimentally test these constraints for a given area before the pathogen is actually present, allowing us to develop preemptive instead of reactive mitigation strategies. Therefore, the goals of this study are to provide an understanding of 1) Bsal’s growth potential in North American soils and 2) potential routes of Bsal dispersal by human vectors. We hypothesize that soil composition, microbiome structure and environmental conditions will determine the persistence of Bsal. To test their relative importance, we will conduct biological growth assays using 5–400g soil samples collected from salamander habitats along the Appalachian Trail in Great Smoky Mountains National Park, a location at the zenith of salamander biodiversity as well as a passageway to thousands of humans annually. Soil will be partitioned into 12–well culturing plates (2g/well) for inoculation at two different concentrations of Bsal (Fig. 1A). Half of the soil from each site will be autoclaved to help delimit potential drivers of Bsal growth as naturally occurring microbial organisms may interact by inhibiting or promoting growth. This will help us understand if external environmental conditions or soil communities are more important contributors to fungal proliferation. Media (broth) plates will be included as a positive control to show normal Bsal growth under optimal nutrient conditions. Heat killed Bsal inoculations will serve as a negative control to demonstrate that live Bsal zoospores are required for growth (Fig. 1A). Treatments within each plate will be plated in triplicate and plates will be made for each of 3 different time points to extract DNA at day 0, day 7, and day 14. Extraction and quantitative Polymerase Chain Reaction (qPCR) will be completed following the forest soil sampling protocol of Stegen et al. 2017 to quantify Bsal growth. Preliminary testing of these methods on soil samples has been conducted to demonstrate feasibility. Results from this preliminary test are in Fig. 2. After quantifying the thresholds for Bsal growth, we will test realistic scenarios of disease spread. Human vector transmission delimitation will consist of experiments wherein human subjects move through trays of soil (both wet and dry) inoculated with Bsal into trays of pathogen free soil (and vice versa) (Fig 1, B). Trays can accommodate two regular-gait strides and transmission rates can be extrapolated for distance. This will help us determine to what extent Bsal zoospores can attach to the matrix of human clothing and equipment and then be transported across substrates. Following subject movement through an experimental array, soil, clothing, and equipment will be collected/swabbed and processed similarly to the biological growth assays for presence/absence of the pathogen. Because Bsal has both a motile, swimming zoospore stage as well as an encysted one that can remain at virulent levels in soils, samples from soil and boots/clothing, etc. will be placed on a slide to determine which zoospore type is transmitted.