P-167 - A first step toward in vitro cultivation of Plasmodiophora brassicae.

Abstract Text: Plasmodiophora brassicae, a soil-borne Chromist pathogen, causes clubroot in brassica crops. While resistant cultivars offer the most effective management strategy, their effectiveness is often short-lived because of the rapid increase of virulent pathotypes. As an obligate pathogen, P. brassicae relies on a host to complete its lifecycle, which restricts research into its biology, virulence mechanisms, and host-pathogen interactions. The pathogen is unable to utilize inorganic compounds in culture media because of the absence of essential metabolic genes, which means that the pathogen cannot currently be grown in vitro. This study aims to develop an in vitro culture method by meeting the specialized nutritional requirements of P. brassicae to support cell multiplication. Resting spores were subjected to enzymatic treatments to create protoplasts. The protoplasts were cultured in various liquid media formulations and cell multiplication was assessed. Growth was quantified at days 1, 7, and 12 using quantitative PCR. Initial observations revealed a decline in cell numbers at day 3, followed by an increase above the initial number at day 6, and a subsequent decline at day 12, likely due to nutrient depletion in the absence of media replenishment. These first signs of cell multiplication of P. brassicae in culture represent a promising step toward establishing a sustainable in vitro culture system. This method has the potential to benefit other non-culturable pathogens, providing a foundation for future studies on their biology and management.