2.9.1
SYMBIOTIC ASSOCIATION BETWEEN CHINESE CABBAGE AND BIOCONTROL ENDOPHYTIC FUNGUS HETEROCONIUM CHARTOSPIRA

K NARISAWA1 and T HASHIBA2

1Plant Biotechnology Institute Ibaraki Agricultural Center, Ago, Iwama, Nishi-Ibaraki 319-02 Japan; 2Laboratory of Plant Pathology, Faculty of Agriculture,Tohoku University, Sendai 981, Japan

Background and objectives
Clubroot disease of Chinese cabbage is difficult to control by chemical methods and therefore the development of resistant varieties was an important breeding objective. Clubroot resistant varieties have been developed but most were not widely accepted by growers because of a low quality. In our previous pot trials, sixteen of 322 isolates of root-colonizing fungi reduced the incidence of clubroot in Chinese cabbage caused by P. ;brassicae in sterile soil [1]. Among them, two isolates of Heteroconium chaetospira suppressed clubroot in nonsterile soil [1]. We report successful biocontrol of clubroot disease under field conditions and anatomical observation of root endophytic fungus, H. ;chaetospira.

Materials and methods
Pieces of colonies were inoculated into autoclaved peat pellets containing medium. One month later, surface-disinfected seeds of Chinese cabbage were placed on the peat pellets on which fungal colonies were growing. The seedlings growing on the peat pellets were incubated in a growth chamber. Two experimental fields were located at Iwama, Ibaraki, Japan, on land cropped to Chinese cabbage and infested with P. ;brassicae. One month after planting, Chinese cabbage seedlings were transplanted to the fields. Disease symptoms and the number of diseased plants were assessed 3 ;months after transplanting. The disease index was calculated. Endophytic fungus were reisolated from the roots of Chinese cabbage using the procedure reported by Tokumasu [2] with slight modifications. Small pieces of roots were cut and stained with 0.005% cotton blue in 50% acetic acid. Roots were observed under a microscope.

Results and conclusions
The endophytic fungus, H. ;chaetospira was effective in reducing the incidence of clubroot disease in fields infested with resting spores of P. brassicae. After fields trials, the frequencies of recovery of the fungus were 40% and hyphae of the fungus colonized the cortical tissues of Chinese cabbage. Conidia or root fragments of H. ;chaetospira germinated and produced small amounts of hyphae on the root surfaces. The extensive hyphae contacted with roots and formed appressoria. The fungus penetrated into outer epidermal cells to form an infection peg. Following the penetration of the epidermis and exodermal cells, hyphae passed into inner cortex of the roots. Intracellular hyphae developed in cortical cells, but no hyphal branch was formed. Hyphae proliferated intracellulary along root tips. Morphologically, Chinese cabbage treated with H. ;chaetospira appeared healthy and did not show any external symptoms. These results suggest that development of the symbiotic association between the plant and fungus is an important for the biocontrol of clubroot disease in the fields.

References
1. Narisawa K, Hashiba T, 1998. Plant Pathology, in press.
2. Tokumasu S, 1978. Transactions of the Mycological Society of Japan 19, 383-390.