1.9.36
PEROXIDASE ACTIVITY IN BETULA PLATYPHYLLA VAR. JAPONICA PLANTLETS INFECTED BY A WHITE-ROT FUNGUS CORIOLUS HIRSUTUS

S YOKOTA1, M YAGISAWA1, H SASAMOTO2 and N YOSHIZAWA1

1Department of Forest Science, Utsunomiya University, 350 Mine-machi, Utsunomiya, Tochigi 321-8505, Japan; 2Forestry and Forest Products Research Institute, Ministry of Agriculture, Forestry and Fisheries, PO Box 16, Tsukiba, lbaraid 305, Japan

Background and objectives
It is well known that rapid production of active oxygen species, the oxidative burst, occurs at the early stage of systemically induced resistance after plants have been challenged by pathogens [1]. H2O2 production and peroxidase reactions were observed during the event [2]. Although the oxidative burst has been reported in many herbaceous plants, it has not been examined extensively in woody plants. In the present study, peroxidase activity and its isozymes were investigated in birch plantlets infected by a white-rot fungus.

Materials and methods
An aseptic Betula platyphylla var. japonica plantlet No. 8 was used as a host plant. It was cultured in the solid Murashige & Skoog medium containing 2.5 M indole-3-butyric acid and 0.1 M 1-naphthaleneacetic acid. It was subcultured every 2 months to the same fresh medium. A white-rot fungus, Coriolus hirsutus CRH7038, was employed as a pathogen. The fungus was inoculated on the medium near the basal part of the stem of a 2-month-old plantlet. Un-inoculated plantlets were used as controls. At intervals after incubation, both infected and control plantlets were deep-frozen with liquid nitrogen, powdered with a mortar and pestle, and extracted with buffer to prepare crude enzyme solutions. Protein content was determined according to the method of Bradford using chicken ovalbumin as a standard. The crude enzyme preparations obtained, containing 0.51 g protein, were applied to polyacrylamide gels containing 5% carrier ampholyte (pH 3.5-9.5), followed by isoelectric focusing (IEF). After IEF, the gel was stained for peroxidase with using 3-amino-ethylcarbazole as a substrate. Peroxidase activity was also assayed spectrophotometrically using guaiacol as a substrate.

Results and conclusions
After 2 weeks of culture, two specific bands near pI 5.0 appeared on the IEF gel of the sample from the infected plantlet. These isozymes are considered to have been induced in the plantlets by the fungal infection, and to be involved in consumption of H2O2 produced by the oxidative burst. However, the specific roles of these peroxidases, such as biosynthesis of barrier lignin against fungal invasion, have not been confirmed.

References
1. Doke N, 1983. Physiological Plant Pathology 23, 345-357.
2. Apostol I, Heinstein PF, Low PS, 1989. Plant Physiology 90, 109-116.