Suppression of clubroot disease under neutral pH caused by inhibition of spore germination of Plasmodiophora brassicae in the rhizosphere

Abstract

To elucidate the mechanism of clubroot suppression under neutral soil pH, a highly reproducible germination assay system under soil culture conditions was designed based on the hypothesis that germinated spores of Plasmodiophora brassicae could be identified by the absence of a nucleus (i.e. having released a zoospore to infect a root hair of the host plant). Brassica rapa var. perviridis seedlings were inoculated with a spore suspension of P. brassicae at a rate of 2.0 × 106 spores g-1 soil and grown in a growth chamber for 7 days. The spores were recovered from rhizosphere and non-rhizosphere soils and stained with both Fluorescent Brightener 28 (cell-wall-specific) and SYTO 82 orange fluorescent nucleic-acid stain (nucleus-specific stain). Total numbers of spores were counted under UV-excitation, and spores with a nucleus that fluoresced orange under G-excitation were counted. The significant increase in the percentage of spores without a nucleus (germinated spores) in the rhizosphere after 7 days' cultivation and the correlation with root-hair infections validated the assay system. Applications of calcium-rich compost or calcium carbonate to neutralize the soil significantly reduced the percentage of germinated spores in the rhizosphere, as well as the number of root-hair infections. The present study provides direct evidence that the inhibition of spore germination is the primary cause of disease suppression under neutral soil pH. © 2008 The Authors.