Persistence under simulated rainfall
We carried out rainfall experiments in a controlled laboratory
environment using a pressure sprayer filled with deionized water and an
adjustable nozzle to alter droplet size from a direct stream to a fine
mist. To simulate vegetation, we used six tomatoes and six peaches to
provide a comparison between smooth and textured surfaces. Tomatoes and
peaches were first washed and dried to remove any external
contamination, before 3µl of slurry were deposited onto five of the six
pieces of fruit, leaving one as a negative control. Once the slurry had
dried, we placed four tomatoes and four peaches onto an
~35 x 25 cm metallic mesh grate, which was first cleaned
with 10% bleach solution and rinsed. The last tomato and peach were
kept as positive controls. Fruit were then sprayed to simulate rainfall
using a handheld pressure sprayer from one meter away with a maximum
working pressure of 36 PSI (2.48 Bar) and maximum water exit speed of
approximately 10 meters per second; which would result in comparable
speed and kinetic energy of heavy rainfall (van Boxel, 1997; Tilg,
Vejen, Hasager, & Nielsen, 2020). We began by using a large nozzle
opening to represent large droplets during heavy rain and sprayed 220 ml
of water over the fruit. Using nitrile gloves, all pieces of fruit were
then placed alone into two-liter buckets (cleaned with 1% bleach and
double rinsed) filled with a liter of deionized water to remove any eDNA
remaining on their surface (Valentin et al., 2018). Fruits were then
removed from the buckets and the resultant solution filtered through a
10 µm PCTE filter to capture intracellular eDNA. We then rewashed the
tomatoes and peaches in a 1% bleach solution then double rinsed in
deionized water and randomly assigned one of each as a negative each
time to control for decontamination errors. We repeated the above
process with an intermediate nozzle opening to represent light rainfall
and sprayed 220 ml of water, then once again after changing the nozzle
to simulate a misty rain event and spraying 220 ml of water. We repeated
the mist process an additional three times, halving the volume of mist
applied each time (i.e. 110 ml, 55 ml, 27 ml). We then extracted the DNA
from the filters using the HotSHOT method and tested for target
intracellular eDNA via qPCR in an Applied Biosystems 7500 real-time PCR
machine in duplicate following the above protocol.