Three-dimensional surface reconstruction and in situ site-specific cutting of the teliospores of Puccinia miscanthi causing leaf rust of the biomass plant Miscanthus sinensis

The rust fungus Puccinia miscanthi on the naturally infected leaves of eulalia grass were investigated by light and electron microscopy. Many black telia were present on both the adaxial and abaxial leaf surfaces. The telia were often confluent, protruding distinctly from the leaf surface, and had numerous teliospores. Light microscopy revealed that the teliospores were club-like in shape (clavate), chestnut brown, and produced pedicels from one end. They were measured to be approximately 45 μm × 20 μm in size, and slightly constricted at the septa. Using stereo imaging, it was possible to generate images with more three-dimensionality than conventional scanning electron micrographs. Without the need for the mechanical sectioning of telia, the height of telia from the leaf epidermis could be measured at approximately 230 μm using three-dimensional surface reconstruction. Urediniospores were also found on the leaf surface, and possessed spines on the surface (echinulate). The internal features of the nonembedded teliospores could be unveiled using a focused ion beam system with a field emission scanning electron microscope, allowing for the in situ site-specific cutting of the non-embedded teliospores. These results show that three-dimensional surface reconstruction and targeted cutting are a potential alternative to mechanical sectioning of resin-embedded blocks, and have the potential to eliminate the uncertainty involved in selecting the region of interest.