Taxonomy and Ecology of Novel Pythium sensu stricto Clade B Species Isolated from Cucurbits

Pythium species are globally distributed oomycetes of major ecological and phytopathological importance, yet species boundaries and character evolution within Pythium sensu stricto remain incompletely understood. Using an integrative taxonomic approach, we investigated oomycete diversity associated with greenhouse-grown cucurbits by combining morphology, multilocus phylogenetics, ecological analyses, and pathogenicity assays. A total of 351 oomycete isolates were recovered from root tissues, rhizosphere soil, and irrigation water, of which 196 belonged to Pythium sensu stricto. Multilocus analyses based on nuclear (βtub) and mitochondrial (cox1, cox2) loci resolved four strongly supported and genetically distinct lineages within Clade B, designated P. macrosporangium sp. nov., P. melonis sp. nov., P. tardioosporum sp. nov., and P. tumidoantherum sp. nov. Each lineage exhibited stable phylogenetic placement and diagnostic morphological traits. Notably, P. macrosporangium produced globose to subglobose sporangia, a feature exceptional within Pythium sensu stricto, in which sporangia are characteristically filamentous. Ecological analyses revealed pronounced substrate-dependent structuring, with irrigation water harboring minimal diversity and root tissues supporting the highest alpha diversity. Pythium macrosporangium dominated in all hosts, locations, and ecological substrates. Pathogenicity assays demonstrated that all lineages were pathogenic to cucurbit hosts, causing severe root rot and damping-off. These results reveal previously unrecognized diversity within Pythium Clade B and reveal a morphologically and ecologically distinctive lineage that challenges traditional diagnostic concepts of Pythium sensu stricto.