Wound responses of Eucalyptus globulus and E. nitens : anatomy and chemistry

The anatomical and chemical host responses of two economically important Eucalyptus species grown in southern Australia (E globulus and E. nitens) to different wounding treatments (mechanical, chemical and biological) were investigated. Initially, the project primarily focused on the role of kino veins as barrier zones in eucalypt tree defence but then later also included the characterization of the new tissue formed subsequent to wounding, an area of study previously never been studied in any eucalypt species. The developmental anatomy and structure of kino veins was examined in three juvenile Eucalyptus species (E. nitens, E. globulus and E. obliqua) to treatment with an ethylenereleasing compound, 2-chloroethyl phosphonic acid (CEPA). Unlike E. globulus and E. obliqua, E. nitens failed to produce kino veins in response to the hormone treatment, confirming anecdotal evidence that this species does not readily form kino veins. During this study, histochemical stains including p-dimethylaminocinnamaldehyde (DMACA) and Coomassie brilliant blue G-250 (BBG-250) were refined to allow the localization of condensed tannins and hydolyzable tannins, respectively in wood sections. The production of dark extractives (non-structural components of wood and bark) was regularly observed as part of the wound response in both the phloem and/or xylem tissue and regardless of the cause of injury. The various wound treatments examined included drill wounding with fungal inoculations, dry ice wounding, natural infection to stem canker (Cytonaema sp.) and prune wounding. Analysis of dark extractives extracted from the new tissue formed after wounding using gas chromatography combined with mass spectrometry (GC-MS), high performance liquid chromatography (HPLC-UV) coupled to negative ion electrospray mass spectrometry (ESI-MS) revealed that a complex range of secondary metabolites was present, including hydrolyzable tannins, proanthocyanidins, flavonone glycoside, stilbene glycosides, formylated phloroglucinol compounds (FPCs) and volatile terpenes. These compounds were either undetectable in healthy tissue or else present at significantly lower concentrations than in wound-associated tissue. In particular, the detection of FPCs in the wound-associated wood was a novel finding, hitherto detected in the leaves of various eucalypt species. We suggest that the diverse range of secondary metabolites detected in the wound-associated tissue may have a multi-functional role in relation to tree wound repair and defence. Crude wound wood extracts were shown to possess in vitro antimicrobial activity against decay fungi and gram-positive bacteria as well as in vitro antioxidant activity. We detail the first reported case of traumatic oil glands induced by wounding in eucalypts. Histological examinations revealed the new phloem tissue formed in the two years following green pruning in 5-year-old E. globulus to be largely composed of secretory cavities similar in appearance to oil glands. Subsequent analysis of extracts by GC-MS confirmed the presence of volatile terpenes and phenols. The total oil content determined for wound associated phloem extracts was significantly higher (>50 times) than for healthy stem phloem extracts. Based on these chemical and morphological findings, we propose a reassessment of the importance of wound wood in tree compartmentalization response for E. globulus and E. nitens, particularly in comparison with other woody tree species.