An improved culture solution technique for Plasmodiophora brassicae infection and the dynamic infection in the root hair

Abstract

We developed an improved solution culture technique that enabled clear and comprehensive observation of dynamic root hair infection by Plasmodiophora brassicae. Brassica oleracea seedlings were sown directly into 10 mL centrifuge tubes containing half-strength Hoagland nutrient solution. Seedlings were fastened in 1 cm thick foam in the tube top and tubes were wrapped with black tape to block light and prevent algal growth. Maximal root hair infection was observed under conditions of pH 5.5, temperature of 20 to 25 °C, and an inoculation concentration of 1 × 107 spores/mL. Infection efficiency was obviously higher in fresh nutrient. Under optimum infection conditions, plasmodia were found in the root hair 4 days after inoculation (DAI), with some undergoing cleavage and developing into round zoosporangia 3 days later. At ten DAI, the number of root hairs containing zoosporangia per root reached 208, and the cytoplasm within the zoosporangia began to cleave and form incipient zoospores. Most zoosporangia released the zoospores and became diaphanous at ten DAI. Root hairs became swollen during the experimental trial and small galls appeared clearly on the root at 20 DAI. In addition, sectioned specimens showed that some cortical cells contained resting spores. We sequentially tested the germination of the resting spores in tubes from 1 to 11 DAI and the temporal dynamics of germination corresponding to the infection. Zoosporangia were found in epidermal cells at the same time as in the root hair.