Histochemical demonstrations of dehydrogenases in the rice plant tissues affected by Cochliobolus miyabeanus and in the causal fungus

Abstract

Dehydrogenase activity in the rice plant tissues inoculated with Cochliobolus miyabeanus (Helminthosporium leaf-spot fungus) and in the causal fungus was investigated using blue tetrazolium (BT). This reaction was positive in the sections of healthy leaf tissues after 2 hours' incubation at 30∼32°C and the rate of the staining became constant after 8 hours' incubation. The reaction was negative when the sections were pretreated with formalin (10%, 1hr.) or treated at 50∼60°C, 10min. In leaf tissues, the parenchymatous cells were most strongly stained and the sites of the enzyme activity seemed to lie on grana-like region in chloroplasts.The leaf spot caused by the present fungus became visible 20 hours after inoculation. The dehydrogenase activity in this early stage of infection was demonstrated in cells near browned epidermis. With the enlargement of spotted area, the tissue around the lesion stained with BT; but the reaction became constant 48 hours after inoculation, although the enlargement of the lesion was still proceeding. In the typical leaf spot produced 5∼7 days after inoculation, localization and intensity of formazan deposition could not be distinguished from those in healthy leaf tissue.The reaction was weak in leaf-sheath cells even when the pathogen penetrated them. On the other hand, the penetrating hyphae, especially the appressoria and the germ tubes, showed strong, reaction. When the spores germinated on slide-glass, the reaction was detected on the terminal cells of the spores and on the germ tubes as well as on the appressoria. This positive reaction on spores of the present fungus seemed to localize on mitochondria of the fungus.