Epifluorescence and Scanning Electron Microscopy of Host-Pathogen Interactions between Pythium porphyrae (Peronosporales, Oomycota) and Porphyra yezoensis (Bangiales, Rhodophyta)

Abstract

The initial steps of host-pathogen interactions leading to successful colonization of Porphyra yezoensis thalli by zoospores of Pythium porphyrae were studied using epifluorescence and scanning electron microscopy. Biflagellated zoospores act as the infective agents for the red rot causing microbial pathogen Pythium porphyrae. These biflagellate zoospores were similar in morphology to primary and secondary zoospores of diplanetic Oomycetes. The flagella lie in a longitudinal groove on the ventral zoospore surface, with long, fine mastigonemes occurring on the anterior tinsel flagellum. There was no evidence of zoospore chemotaxis. The zoospores came into contact, attached and encysted onto the host thallus. The membrane-bound zoospores did not stain with calcofluor, which is specific for chitin and glucans, while the cell walled cysts and mycelia specifically stained with calcofluor when viewed by epifluorescence microscopy, allowing in situ identification of the encysted zoospores, the first stage of colonization on the host thallus. Cysts attached to the host thallus developed a cell wall, germinated, formed appressoria (an essential prelude of penetration), and penetrated the host surface near the site of cyst attachment. The cysts appeared to be adhering to the host thallus by forming a cementing matrix around encysted zoospores when observed with a scanning electron microscope. Absence of inward deformations at penetration sites suggested that enzymatic action rather than mechanical pressure was responsible for initial penetration of the host cell wall. The specific staining of spore walls with calcofluor fluorescent brightner is an inexpensive, easy and rapid technique for early identification of parasitized Porphyra thalli.