THE ROLE OF STILBENES IN RESISTANCE OF SITKA SPRUCE (PICEA-SITCHENSIS (BONG) CARR) TO ENTRY OF FUNGAL PATHOGENS

The major constitutive antifungal compounds present in the bark of Sitka spruce (Picea sitchensis) were the stilbene glucosides astringin (5,3'',4'',-trihydroxystilbene-3-.beta.-D-glucoside) and rhaponticin (5,3''-dihydroxy-4''-methoxystilbene-3-.beta.-D-glucoside). In healthy bark, levels of astringin and rhapoticin of around 10-15 mg g-1 tissue (fresh weight) were found. At concentrations above 1 mg ml-1 these compounds inhibited the growth in vitro of two weakly pathogenic decay-causing fungi Phaeolus schweinitzii and Sparassis crispa. Other, more pathogenic fungi responded in a different way. Heterobasidion annosum and Rhizinia undulata were less severely inhibited, whereas Armillaria ostoyae and Stereum sanguinolentum were stimulated. In excised bark discs, maintained in vitro, levels of the glucosides fell markedly following fungal challenge. Concomitant with this loss, their aglycones, astringenin (5,3,3'',4''-tetrahydroxystilbene) and isorhapontigenin (5,3,3,''-trihydroxy-4''-methoxystilbene) accumulated. Based on estimates of ED50 values, the antifungal activity in vitro of isorhapontigenin against P. schweinitzii was six times greater than that of rhaponticin. Loss of stilbene glucosides from challenged discs appeared to be associated with the production of .beta.-glucosidase enzymes by the invading fungi. Sensitivity to rhaponticin of the six test fungi appeared to be correlated broadly with their ability to metabolize this compound in vitro. After incubation with rhaponticin, culture filtrates of the two weak pathogens contained substantial quantities of both the glucoside and the aglycone isorhapontigenin, whereas only small amounts of these compounds remained in the medium after incubation with the other more pathogenic species tested. Colonization of bark discs by P. schweinitzii, measured by a chitin assay, was considered in relation to levels of stilbene aglycones produced following challenge. During the period from 3 to 10 days after challenge when stilbene aglycone levels were increasing, chitin levels remained static. Quantities of chitin increased 12 days after challenge concomitantly with a fall in the level of stilbene aglycones. Penetration of excised bask discs by the six fungi showed only a doubtful relationship with pathogenicity, with H. amnosum showing the most mycelial growth and S. sanguinolentum the greatest depth of penetration. The necrotic response of the host tissues was similar with all rapidly growing species. Stilbene aglycones also accumulated around wounds made into root bark of standing trees, although because of fluctuating environmental conditions, results were much more variable than with the excised bark disc system. The excised bark disc system proved to be of considerable value in the study of the chemical responses of Sitka spruce roots to fungal challenge, overcoming the physical and environmental problems associated with such experiments conducted on trees in the field. The significance of these chemical mechanisms in the defence of Sitka spruce root bark against the ingress of potential pathogens and decay-causing fungi is discussed.