Abstract
In this case study, the mycelium growth of Sclerospora graminicola in the infected tissues of pearl millet and the process of sporulation and liberation of sporangia and zoospores were observed using four different microscopic techniques. The cotton blue-stained samples observed under light microscope revealed the formation of zoospores with germ tubes, appressoria and initiation of haustorium into the host cells, while the environmental scanning electron microscopy showed the rapid emergence of sporangiophores with dispersed sporangia around the stomata. For fluorescence microscopy, the infected leaf samples were stained with Fluorescent Brightener 28 and Calcofluor White, which react with β-glucans present in the mycelial walls, sporangiophores and sporangia. Calcoflour White was found to be the most suitable for studying the structural morphology of the pathogen. Therefore, samples observed by confocal laser scanning microscopy (CLSM) were pre-treated with Calcofluor White, as well as with Syto-13 that can stain the cell nuclei. Among the four microscopic techniques, CLSM is ideal for observing live host-pathogen interaction and studying the developmental processes of the pathogen in the host tissues. The use of different microscopic bioimaging techniques to study pathogenesis will enhance our understanding of the morphological features and development of the infectious propagules in the host.
Highlights
S. graminicola oospore viability assay by light microscopy, and symptoms of S. graminicola-infected pearl millet
Prior to the seed coating, the viability of oospores used in the coating was tested by staining with 2,3,5-triphenyltetrazolium chloride (TTC) and examined under light microscope
The downy mildew pathogen S. graminicola of pearl millet was examined by using light microscopy, fluorescence laser microscopy, confocal laser scanning microscopy (CLSM), and Environmental scanning electron microscopy (ESEM)
Summary
Phyllody is the reason why the disease is referred to as ‘witch’s broom’, ‘crazy top’ or ‘green ear disease’[14] These symptoms are common among the many host crop species, including pearl millet, infected by downy mildew pathogens belonging to the genera Peronosclerospora, Sclerospora and Sclerophthora[13,15]. Mycelial structures and asexual reproduction of S. graminicola was examined for the first time in infected leaves using light, fluorescence, confocal, and environmental scanning electron microscopic techniques in combination with various stains. We aimed to test the suitability and simplicity of different microscopic techniques in identifying the developmental stages of the asexual structures of S. graminicola so that scientists can select the most appropriate approach(es) to obtain information on the host resistance and susceptibility mechanisms, such as papillae and callose formation, and molecular understanding of the host-pathogen interactions
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