A sand–solution culture technique used to observe the effect of calcium and pH on root hair and cortical stages of infection by Plasmodiophora brassicae

A technique that enables observation of primary and secondary stages of the life cycle of Plasmodiophora brassicae in brassica roots is described. Brassica seeds were sown directly into 5 mL pippette tips containing an acid-washed, coarse, mixed-grade sand. These were suspended in a buffered nutrient solution (pH 5.5, 6.5, 7.5 or 8.0) containing 2.5, 5, 10 or 20 m m calcium that moved up the tube by capillary action. Seedlings were inoculated when the cotyledons were fully expanded. Ten days after inoculation, primary (root hair) stages of infection were observed in up to 84% of root hairs. The effect of calcium on the development of P. brassicae in the root hairs was dependent on pH. At low pH (5.5), a significant delay in the developmental progress of the pathogen was observed only at the highest level of added calcium (20 m m ). At intermediate pH (6.5), all calcium amendments caused a significant delay in the developmental progress of the pathogen. As pH increased to pH 8.0, the number of infected roots and the effect of calcium amendment decreased. Several secondary plasmodia were evident in the cortex at the lowest pH (5.5) and root galls were subsequently observed, mainly on plants grown at pH 5.5 and 6.5. This technique should prove useful for studying the life history of P. brassicae and mechanisms of control and resistance.