In Vitro Screening of Microbial Extracts Against the Oomycetes <em>Phytophthora capsici</em> and <em>Pythium ultimum</em>

Oomycetes are a predominantly plant-pathogenic group of organisms often considered and managed as fungi; however, due to their evolutionary divergence from true fungi, many conventional fungicides are ineffective against them. Their unique physiological characteristics make them challenging to work with, highlighting the need for a standardized and reproducible procedure for anti-oomycete assays. Previous studies describe methods to obtain sporulation forms in the laboratory, but there remains a disconnect between spore production and the subsequent screening process for potential biological pesticides based on microbial organic extracts. This protocol bridges that gap by providing a complete and reliable workflow from spore production to screening. In this study, we present an efficient in vitro protocol to identify microbial extracts with activity against <i>Phytophthora capsici</i> and <i>Pythium ultimum.</i> The protocol includes a method for obtaining zoospores of <i>P. capsici</i> and oospores of <i>P. ultimum</i>, followed by a simple and rapid screening assay to detect microbial extracts that inhibit the growth of these pathogens. The extracts are dispensed onto plates in two concentrations and allowed to dry. This facilitates pauses in the protocol and allows for storage of the plates until the biological material is ready for the assay. The protocol's effectiveness has been validated with these two oomycetes, resulting in the identification of active extracts in both cases. Moreover, it can be adapted to other pathogens. Key features • This protocol is designed for in vitro susceptibility testing of oomycetes, using <i>Phytophthora</i> zoospores and <i>Pythium</i> oospores as inoculum sources. • This protocol can be easily used for the identification of natural compounds with anti-oomycete activity.