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Investigating the impacts of continuous artificial culture systems on Phytophthora infestans virulence

Eric Larson: University of Wisconsin-Madison

<div>It is widely accepted, and documented for <em>Phythophthora infestans</em>, that continued sub-culturing on artificial media results in virulence reduction. Typically, passage of the cultured pathogens back through host plant tissue brings about a return in virulence, a response that suggests that changes are not due to genetic mutation. Rather, changes may be the result of a more sophisticated and fluid method of regulation. Elucidating this mechanism could shed light on <em>P. infestans’</em> ability to rapidly overcome host resistance. We investigated the virulence of 5 <em>P. infestans</em> isolates (Gu68, US-1, US-8, US-23, and US-24) after sub-culturing on artificial media, and we analyzed changes in effector expression within the pathogen at key stages. Each isolate was sub-cultured and maintained at 12°C on Rye-A medium for at least 8 times over 6 months. The oldest, most recent, and median sub-cultures of each isolate were tested by plug inoculation on both susceptible (‘Katahdin’ potato) and resistant (‘Katahdin’ potato transformed with RB) hosts in a conducive 100% humidity environment. Disease severity was determined by measuring lesion size and a significant interaction (p=0.054) was found between the host type and age of culture. In preparation for expression analysis, a subset of 6 <em>P. infestans</em> effectors were verified by PCR in each of the isolates. RNA was extracted from infected plants and RT-qPCR analyses were conducted to assess changes in effector expression.</div>