Cystosori of Spongospora subterranea (Wallr.) Lagerh., with cysts at different stages in development, were found on the surface of a potato powdery scab lesion in situ at harvest time and are described using light and transmission electron microscopy. The resting cyst cytoplasm has a five-layered envelope separating it from periplasmic space and is contained within a wall consisting of three layers (W1, W2, and W3). Protuberances on the outer surface of the cysts are outgrowths of the outermost (W1) wall layer. The differentiation of a primary zoospore within each cyst is marked by a reorganisation of the cytoplasmic envelope, the appearance of multivesicular bodies in the cytoplasm, and a germ pore with a thickened W3 wall rim. The ultrastructure of the cysts is compared with the resting spore and encysted primary zoospore of Plasmodiophora brassicae.
New evidence is presented to support the contention that the amoeba/cyst colonies isolated from surface-sterilized Spongospora subterranea f.sp. subterranea-infected potato tubers and spore balls have a saprophytic phase but are contaminants and not S. subterranea. Amoebae isolated from infected tissues and spore balls formed colonies associated with bacteria on 1% water agar at 188C and encysted after 5±7 days. These cysts were morphologically distinct from the resting spores of S. subterranea and were formed singly or in a layer, unlike the spore ball (cystosorus) of S. subterranea. Amoebae, cysts and mixtures of amoebae and cysts in primary, secondary and tertiary subcultures failed to infect tomato roots. PCR amplification of DNA from amoebae, cysts and spore balls using the S. subterranea-specific primer pair SsF/R generated a 434-bp product from S. subterranea spore balls only and not from amoebae or cysts. When an amoeba/cyst-specific primer pair AmF/R was designed and used for PCR amplification, a single 411-bp product was generated from DNA of amoebae and cysts, but not from DNA of S. subterranea spore balls. These results are discussed in relation to earlier reports claiming the successful isolation of S. subterranea and other plasmodiophorids in vitro.
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