Evidence for the growth of Plasmodiophora brassicae in vitro

Plasmodiophora brassicae is a root-invading pathogen that causes club root disease of brassica crops. Although generally regarded as an obligate biotroph, a number of reports over the last century have suggested that the organism may have a free living saprotrophic amoebal stage. Here we present new evidence to support the contention that P. brassicae is capable of at least a short-lived period of saprotrophic amoebal growth. Spores extracted from surface sterilized blocks of club root tissue were incubated at 25 degrees C on 1/4 strength Ringer's agar spread previously with a suspension of Escherichia coli. After a period of 5-10 d, outgrowth of amoebae commonly appeared around the inoculum. Amoebae were subcultured on to fresh Ringer's-E. coli plates, where they continued to grow and eventually encysted. An amoebal cyst suspension was prepared from these plates and used to inoculate Chinese cabbage (Brassica campestris var. chinensis) and Savoy cabbage (B. oleracea) seedlings. Of 27 amoebal isolates tested, nine were found which induced typical club root disease symptoms. However, when these putative positive P. brassicae amoebal isolates were subcultured, the subsequent generation of amoebal cysts failed to induce disease symptoms. A random genomic library was constructed in pUC18 using BamH 1-digested DNA extracted from P. brassicae resting spores. The inserts from nine individual clones were then used to probe DNA extracted from 12 amoeba cultures; three showed positive hybridization to the P. brassicae probes but this hybridization signal was lost after subculture of the amoebae. These results are discussed in relation to the ability to culture P. brassicae in vitro.