Myxomycetes (plasmodial slime molds or myxogastrids) have long attracted scientific attention for their complex life cycles and unique behaviors, yet they remain underexplored in applied research contexts such as plant pathology. This study established reproducible, stepwise protocols for the cultivation, maintenance, and induction of sclerotia and sporocarps under defined polyxenic
conditions of four species of myxomycetes, Didymium squamulosum, D. bahiense, Physarum cinereum, and P. compressum originally collected from maize. Starting from spore-based cultures, we optimized feeding regimes using bacterial and yeast mixtures, introduced a purification method combining antibiotic agar and low-heat-killed yeast, and demonstrated long-term culture maintenance at low temperature. Growth dynamics were modeled via the area of microbial feed
consumed, revealing strong species- and temperature-dependent differences. Controlled sclerotization yielded sclerotia with quantifiable macrocyst activity, assessed by phase-contrast microscopy of cytoplasmic movement as a reproducible proxy for macrocyst functionality. Sporulation was successfully induced for all four species, with interspecific variation in sporocarp formation and spore germination rates. Together, these methods enable full life cycle completion in the laboratory and provide standardized inoculum sources in small scale for downstream applications.