Studies on amoebae and cysts associated with the isolation of Spongospora subterranea f.sp subterranea in vitro

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 18 degreesC 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.