Antimicrobial defence in the sapwood of Eucalyptus nitens: studies of the reaction zone

Decay arising from pruning wounds in plantation-grown Eucalyptus spp. is a concern for the Australian solid wood products industry, as the potential for decay spread within the tree is unknown at present. Mechanisms of antimicrobial defence in the xylem of Eucalyptus nitens were investigated. Key factors investigated within the host-fungal interaction included phenolics, moisture content, mineral content and anatomical alterations (tyloses and suberization). A purple reaction zone was regularly observed at the sapwood interface of decay lesions developing from naturally-infected pruning wounds in plantation-grown E. nitens. Total phenols levels were increased substantially and vessel tyloses were abundant in the reaction zone. A log incubation experiment showed that after 9 months the reaction zone remained intact while sapwood became decayed. The reaction zone was significantly drier than healthy sapwood, with lower levels of potassium, and a lower pH than both sapwood and heartwood. These trends are more similar to conifer reaction zones than deciduous angiosperms. The mode of reaction zone defence appears to be a dynamic process (also similar to conifers) rather than a static process typical of deciduous angiosperms. The initial stages of antimicrobial defence formation were also studied. Xylem defence responses occurring in young pot-grown E. nitens saplings were analysed within the first few weeks following stem wounding and inoculation with the decay fungus Ganoderma adspersum (which displayed relatively slow colonization of the wounded xylem). Events occurring most quickly after wounding and inoculation were tylose formation and phenol accumulation, which indicates that they are key defences. NMR imaging revealed that changes in xylem moisture content were not rapid after wounding. Reaction zone extracts were inhibitory to test-fungi in thin-layer chromatography bioassays. Analysis of extracts by liquid chromatography — electrospray ionization mass spectrometry revealed that a diverse range of hydrolyzable tannins were present in E. nitens wood, including over thirty gallotannins, ellagitannins and phenols. A number of ellagitannins (particularly pedunculagin) were considerably more abundant in the reaction zone than the healthy sapwood and may contribute to the effectiveness of the reaction zone as an antimicrobial barrier. The process of reaction zone formation may be an accentuation of processes that occur when heartwood forms. Heartwood extracts were qualitatively similar to reaction zone extracts but actual increases in ellagitannins were not as great. Conditions such as season of wounding and the type of fungi which become established in wounds, appear to effect the degree of defence response. This was indicated by phenol levels. More extensive decay lesions were generally associated with more phenolic production. Sterile inoculations and weakly-aggressive fungi were associated with no or little reaction zone formation, while aggressive fungi elicited more discolouration and phenolic accumulation in advance of infection. This suggests that non-aggressive fungi are less tolerant of the inherent xylem microenvironment and active defence responses are not "required" or elicited. Xylem defence responses appear to be relatively successful in E. nitens. This may be facilitated (at least in part) by a fast and considerable accumulation of phenolic compounds. Qualitative studies of phenolic compounds may be a useful indicator of defensive capacity.