Teliosporogenesis of the Peanut Smut Fungus Thecaphora frezzii in Arachis hypogaea: A Correlative Multiscale Microscopy Study

The smut fungus <i>Thecaphora frezzii</i> causes severe yield losses in peanuts (<i>Arachis hypogaea</i>) in Argentina. Previous work established its fully intracellular biotrophic progression through subterranean organs and its exclusive sporulation within the seed coat, yet the ontogeny of teliospore formation in planta remained unresolved. Here, we applied a pragmatic correlative multiscale microscopy approach based on serial paraffin sections examined by stereomicroscopy, light microscopy, confocal laser scanning microscopy, and scanning electron microscopy, enabling spatial correlation of fungal structures within their tissue context. Using this integrative framework, we characterized the organization and progression of sporogenic structures associated with teliosporogenesis. Teliosporogenesis proved to be tightly synchronized with host tissue context and seed developmental stage, and was consistently preceded by a marked reorganization of sporogenous hyphae into three-dimensional coiled hyphal aggregates embedded in a mucilaginous matrix. These precursors undergo hyphal fragmentation followed by central-peripheral differentiation, whereby a small number of central units enlarge and individualize into teliospore initials while peripheral elements collapse, yielding stable teliospore balls as the final sporogenic product. This developmental sequence defines a distinct ontogenetic pattern not captured by current schemes of sporogenesis, here designated the Teliospore-ball type. Our results clarify the developmental pathways of <i>T. frezzii</i> sporulation in planta and demonstrate how accessible multiscale microscopy can be used to integrate structural information across spatial scales in complex plant-fungus interactions.