Life Cycle of Plasmodiophora brassicae Koji Kageyama �� Takahiro Asano Received: 4 March 2009 / Accepted: 10 March 2009 / Published online: 29 April 2009 �� Springer Science+Business Media, LLC 2009 Abstract Plasmodiphora brassicae is a soil-borne obli- gate parasite. The pathogen has three stages in its life cycle: survival in soil, root hair infection, and cortical infection. Resting spores of P. brassicae have a great ability to survive in soil. These resting spores release pri- mary zoospores. When a zoospore reaches the surface of a root hair, it penetrates through the cell wall. This stage is termed the root hair infection stage. Inside root hairs the pathogen forms primary plasmodia. A number of nuclear divisions occur synchronously in the plasmodia, followed by cleavage into zoosporangia. Later, 4���16 secondary zoospores are formed in each zoosporangium and released into the soil. Secondary zoospores penetrate the cortical tissues of the main roots, a process called cortical infection. Inside invaded roots cells, the pathogen develops into secondary plasmodia which are associated with cellular hypertrophy, followed by gall formation in the tissues. The plasmodia finally develop into a new generation of resting spores, followed by their release back into soil as survival structures. In vitro dual cultures of P. brassicae with hairy root culture and suspension cultures have been developed to provide a way to nondestructively observe the growth of this pathogen within host cells. The development of P. brassicae in the hairy roots was similar to that found in intact plants. The observations of the cortical infection stage suggest that swelling of P. brassicae-infected cells and abnormal cell division of P. brassicae-infected and adjacent cells will induce hypertrophy and that movement of plasmodia by cytoplasmic streaming increases the number of P. brassicae-infected cells during cell division. Keywords Life cycle Plasmodiophora brassicae Resting spore Plasmodium Zoosporangium Root hair infection Clubroot Dual culture in vitro Life Cycle In Vivo Plasmodiphora brassicae is a soil-borne obligate parasite. The pathogen has three stages in its life cycle: survival in soil, root hair infection, and cortical infection (Fig. 1) (Ayers 1944 Ingram and Tommerup 1972 Naiki 1987). Primary inoculum is composed of resting spores dispersed from rotten host tissue into the surrounding soil. The resting spore is about 3 lm in size and subspherical to spherical (Figs. 1a, 2a) (Buczacki and Cadd 1976). The surface of each resting spore is covered with spines (Fig. 2b) (Williams and McNabola 1967 Ikegami and others 1978). A primary zoospore is released from each resting spore, spindle-shaped or pyriform, 2.8���5.9 lm long, and biflagellate (Fig. 1b) (Ayers 1944). The flagellae have two shapes: a shorter flagellum with a blunt end and a longer flagellum with a whiplash or tail piece. When the zoospore reaches the surface of a root hair, it penetrates the cell wall. This stage is termed the root hair infection stage or primary infection stage. In root hairs the pathogen forms primary plasmodia (Fig. 1c). A number of nuclear divi- sions occur synchronously in the plasmodia, followed by cleaving into zoosporangia. The zoosporangia form clus- ters in the root hair (Fig. 1d) and sometimes in epidermal cells. Later, 4���16 secondary zoospores are formed in each K. Kageyama (&) T. Asano River Basin Research Centre, Gifu University, Gifu 501-1193, Japan e-mail: kageyama@green.gifu-u.ac.jp T. Asano Geological Isolation Research and Development Directorate, Japan Atomic Energy Agency, 4-33 Muramatsu, Tokai-Mura, Naka-Gun, Ibaraki 319-1194, Japan 123 J Plant Growth Regul (2009) 28:203���211 DOI 10.1007/s00344-009-9101-z

zoosporangium. After releasing these zoospores, the empty zoosporangia remain in the root hairs (Fig. 1e). The secondary zoospores cannot be visually differenti- ated from the primary zoospores. Binucleate zoospores are sometimes found and interpreted as having formed by the fusion of two distinct zoospores, not from division within nuclei (Tommerup and Ingram 1971 Ingram and Tomm- erup 1972). The secondary zoospores penetrate the cortical tissues, a process called cortical infection or secondary infection stage. Inside infected cells the pathogen develops into secondary plasmodia which proliferate and are asso- ciated with cellular hypertrophy (Fig. 1f, g), followed by gall formation in root tissues. After a number of nuclear divisions, the secondary plasmodia contain two nuclei in the early stages of growth and then develop into multinu- clear plasmodia (Garber and Aist 1979). In plasmodia with haploid nuclei, the nuclei may fuse forming diploid nuclei. Futhermore, meiotic cleavage may occur in the diploid plasmodia, indicating that the plasmodia return to the haploid state again (Buczacki 1983). This hypothesis is not universally accepted. The plasmodia finally develop into resting spores (Fig. 1h, i) (Ikegayami and others 1982), followed by their release into soil as survival structures. During these complex cleavages, the pathogen produces resting spores and increases its genetic diversity. Resting-Spore Germination Resting spores of Plasmodiophora brassicae have a great ability to survive in soil. Gibbs (1931) reported that resting spores may survive without host plants for 5 years. Wal- lenhammar (1996) found that the half-life of an inoculum was 3���6 years in heavily infested fields and that the level of infestation declined to below a detectable level after a period of 17.3 years. The resting spores remained active when held at 40��C for 24 h but were inactivated by treat- ment at 30��C for 14 days (White and Buczacki 1979). Fig. 1 Life cycle of Plasmodiophora brassicae. a Resting spore. b Primary zoospore. c Primary plasmodium in root hair. d Zoosporangial cluster in root hair. e Empty zoosporangium. f, g Secondary plasmodia in cortical cells. h, i Resting spores in cortical cells Fig. 2 Scanning electron micrographs of immature and mature resting spores in clubroot of turnip. a Immature resting spores. b Mature resting spores. Scale bar = 1 lm 204 J Plant Growth Regul (2009) 28:203���211 123