T YAMADA and T NAKASHIMA

Forest Biology Division, Forestry and Forest Products Research Institute, Tsukuba, Ibaraki 305-8687, Japan

Background and objectives
Infection of sapwood of living trees with pathogenic fungi induces extended wood discoloration. Active responses such as phytoalexin accumulation play an important defensive role in sapwood of the Japanese cedar, Cryptomeria japonica [1, 2]. However, the time course of defensive responses and fungal spread in the infected sapwood has not previously been clarified. It is also necessary to consider the relationship between host defences and pathogen virulence. This paper describes the chemical defence responses of the Japanese cedar sapwood in relation to fungal spread following inoculation.

Materials and methods

Japanese cedar trees were inoculated with a virulent isolate, MA18, or an avirulent isolate, MA19, of the Guignardia dieback fungus Guignardia cryptomeriae (Botryosphaeria sp.). Spread of wood discoloration and fungal hyphae in the inoculated tree were determined. The boundary of wood discoloration was observed with a light microscope, and methanol extracts of the discolored region were assayed and analysed by thin-layer chromatography and gas chromatography.

Results and conclusions
Wood discoloration was recognized 1 week after inoculation. The spread of wood discoloration preceded the spread of fungal hyphae. Wood discoloration and fungal hyphae of the virulent isolate spread quickly during the first 2 weeks following inoculation. Then, the spread of both virulent and avirulent isolates was delayed, and was completely prevented between 2 and 4 weeks after inoculation. The extent of wood discoloration varied depending on the virulence of inoculated fungal isolates.

Within 1 week after inoculation, the moisture content of the discoloured region decreased, and an increase in NAD diaphorase activity, and secretion of oil droplet-like deposits containing norlignans and terpenes, began in the same zone. An antifungal norlignan, hinokiresinol [1], accumulated quickly, and the concentration of hinokiresinol reached ca 20 and ca 50% of the maximum concentration 1 and 2 weeks after inoculation, respectively. Maximum concentration of hinokiresinol was observed 2-3 months after inoculation. Agatharesinol and sequirin-C, norlignans which have little antifungal activity, and terpenes, accumulated slowly in the reaction zone barrier. These results strongly suggest that accumulation of antimicrobial substances contributes to the restriction of fungal colonization.

Concentrations of norlignans and terpenes in the reaction zone barrier were higher in fungus-inoculated trees than in wounded trees. This fact suggests that the induction of defence responses is enhanced by the presence of pathogenic fungi.

References
1. Yamada T, Tamura H, Mineo K, 1988. Physiological and Molecular Plant Pathology 33, 429-442.
2. Yamada T, 1994. 5th International Mycological Congress, Abstract 248.