A simple protocol for whole leaf preparation to investigate the interaction between Puccinia psidii and Eucalyptus grandis

During the infection of a susceptible Eucalyptus tree by the biotrophic fungus Puccinia psidii a series of processes occur, starting with germination of the fungal spore, through hyphal penetration, colonization and finally sporulation. Not all of these processes occur when the fungus tries to infect a resistant Eucalyptus tree. Such interactions need to be studied to allow future molecular analyses to be correlated with the physical/biological state of the infection in both resistant and susceptible hosts. In this manuscript we describe a simple protocol that allowed us to follow the infection cycle, both on the surface and within the leaf tissue, of Puccinia psidii on its host Eucalyptus grandis. The procedure consists of fixing the tissue with ethanol and acetic acid, clearing with hot KOH and staining with calcofluor before fluorescence detection. All of the previously mentioned infection steps, including pustule rupture and spore release, were observed. Germ tubes, appressoria, haustorial mother cells and urediniospores were all clearly seen. This protocol also proved useful in tissue preparation for scanning electron microscopy and confocal microscopy.