Growth, occurrence and development of septa in Plasmopara viticola and other members of the Peronosporaceae using light- and epifluorescence-microscopy

Abstract

Although Plasmopara viticola causes grape downy mildew in most, if not all, wine producing countries, many basic biological or chemical aspects are still unknown and thus the histopathological changes during development of this oomycete pathogen were studied. The fluorochromes Aniline blue and Uvitex 2B were successfully used in whole leaf stainings to visualise intercellular hyphae and to investigate septal-development in sporangiophores. The occurrence and transfer of cytoplasm in sporangiophores was studied with the aid of Chlorazole Black E and Phloxin-B. Application of Chlorazole Black E was the most reliable method to differentiate between cytoplasm and septa in sporangiophores due to a high contrast between the dark cytoplasm and the pale septa. In P. viticola, septa were found in the stem and branches of the sporangiophores, but not in the intercellular hyphae, which was in contrast to other oomycetes, such as P. tabacina, Pseudoperonospora cubensis and P. humuli, where septa were frequently found in the mycelium. In order to verify chemical composition of septa, sporangiophores were digested using chitinase and beta-1,3-glucanase. After staining with Aniline blue and Uvitex 2B, the intense fluorescence of septa was conserved after the application of chitinase but not after beta-1,3-glucanase, indicating that septa are mainly composed of beta-1,3-glucans. Pretreatments of infected vine leaves with 2-deoxy-D-glucose (2-DOG) at low concentrations (1-5 mM) led to a disorganisation of the sporangiophore structure of P. viticola, whereas the production of septa was unaffected. Application of 2-DOG at higher concentrations resulted in a reduced length of intercellular hyphae (10 mM) or total inhibition (50 mM) of the intercellular mycelium. Thus, 2 DOG or its analogues interferes with morphogenesis in Plasmopara viticola and may have a deleterious effect on the spread of this downy mildew between plants.