The south-west of Western Australia has a Mediterranean climate and flora endemic to this area, including the keystone species, jarrah (Eucalyptus marginata), have adapted to the droughted summer conditions. The introduction of an exotic soil borne pathogen, Phytophthora cinnamomi, has challenged the survival of this and many other species. The expectation might be that plants stressed by drought are more susceptible to disease and this study examined the development of disease caused by P. cinnamomi in E. marginata and the significance of water status to that development. Seedlings of E. marginata, clonal plants resistant to P. cinnamomi and clonal plants susceptible to P. cinnamomi, were subjected to different watering regimes in a number of field and glasshouse experiments. To determine the level of drought stress that could be imposed on container-grown E. marginata seedlings without killing them, a preliminary experiment progressively lowered the moisture levels of the substrate in their containers, until the plants reached wilting point, at which time moisture was restored to a predetermined droughted level and the process repeated. With each subsequent droughting the wilting point was lower until it was found that the seedlings could survive when only 5% of the moisture lost from container capacity to wilting point was restored. No deaths had occurred after seedlings had been maintained at this low level for 14 days (Chapter 2). Based on these findings, the level of droughting maintained in all experiments conducted under controlled glasshouse conditions was 10% restoration. After testing the appropriateness of underbark inoculation, and a zoospore inoculation method for which no wounding was necessary, a new, non-invasive stem inoculation technique was developed. Stems were moistened in a pre-treatment, then agar plugs colonized with P. cinnamomi mycelium were held against the stem with wads of wet cotton wool and bound in place with tape. This technique resulted in a high proportion of infection in E. marginata (Chapter 4) without the need for underbark inoculation or the use of zoospores (Chapter 3). It was successfully used in a large field trial in a rehabilitated bauxite mine site with 2-year-old E. marginata clonal plants, resistant to P. cinnamomi (Chapter 5). Inoculation was in late spring after the winter and spring rainfall. This timing was to allow comparison of disease development in stressed plants under normal droughted summer conditions compared with itsdevelopment in non-stressed, irrigated plants. However, two months after inoculation, the area was deluged with unseasonal and abnormally heavy summer rainfall, negating any difference in the treatments and causing an outbreak of P. cinnamomi in the soil from an adjacent infested site. This resulted in the infection and death of some noninoculated control clones. Monitoring of the site continued for twelve months and the advance of P. cinnamomi at the site was mapped. To test the effect of drought on the expression of P. cinnamomi under more controlled conditions, a series of glasshouse experiments was set up that simulated two possible summer conditions; drought or drought followed by abnormally high summer rainfall. These experiments utilised E. marginata seedlings and clonal plants, some resistant and some susceptible to P. cinnamomi. Plants were inoculated with P. cinnamomi prior to or after droughting. Results were compared to those of control plants that had not experienced water deficit. In both seedlings and clonal plants, the greatest extent of colonization was found in plants which had experienced no water deficit. These results indicated that drought stress played a role in inhibiting the in planta development of P. cinnamomi in all genotypes (Chapter 8). This finding was consistent for both clones, susceptible and resistant to P. cinnamomi. Most recoveries were made from non-stressed clonal plants, resistant to P. cinnamomi (Chapter 6) and more colonization was found in non-stressed clonal plants, susceptible to P. cinnamomi (Chapter 7), than was recorded for droughted plants. The results of the field trial showed that P. cinnamomi was not recovered from some inoculated stems, which had obvious lesions, when segments were plated onto selective agar. This led to an intensive in vitro investigation into improved methods of recovery. Dark brown exudates from some segments of inoculated stems stained the surrounding agar onto which they were plated, suggesting the presence of phenolic compounds. Recovery of the pathogen from stems increased by about 10% when segments were first soaked in distilled water to leach out the phenolic compounds, then replated onto agar. Other recovery methods were also tested, including (1) baiting with Pimelea ferruginea leaves floated on the surface of water or soil filtrate, in which the infected stem segments were immersed and (2) the application of different light and temperature regimes. It was clearly shown that exudates from infected stems of field grown E. marginata inhibited the outgrowth of P. cinnamomi onto the agar. To counter the possible toxic effect that oxidized phenolics had on the growth of the P. cinnamomi, an antioxidant was added to the agar. P. cinnamomi was grown on media whichincorporated exudates from infected stems and different concentrations of ascorbic acid, with and without adjusted pH levels. There was a pronounced pH effect, with less growth on media with lower pH and no significant increase in growth of the mycelium with increased ascorbic acid concentration on pH adjusted agar (Chapter 9). The inhibitory effect of the exudates from the stem segments led to an investigation of the possibility that, if seedlings to be planted in the rehabilitation process could be pre-treated with phenolic compounds to render them more resistant, they may have an advantage when establishing in areas where there was a potential threat of P. cinnamomi. E. marginata seeds were germinated and the seedlings grown hydroponically in a constant temperature growth room. Different concentrations of synthetic catechol, a phenolic compound naturally occurring in E. marginata, were added to the nutrient solution. Roots remained immersed in the catechol solutions for three days, before being inoculated at the root tip with zoospores of P. cinnamomi. Roots in higher concentrations of catechol were less colonized than those in lower concentrations, indicating an increased resistance to the pathogen (Chapter 10). Further work is required to determine if seedlings treated before being planted in areas threatened by an outbreak of P. cinnamomi have a greater capacity for survival, and for how long the protection persists. The improved recovery of P. cinnamomi from infected plants is important for accurate assessment of the spread of the disease in an area and for the subsequent implementation of management strategies of containment and control. An outbreak of P. cinnamomi can impact on the revegetation of rehabilitated mine sites and the aetiology of the pathogen in mine sites needs to be more fully understood. The interaction of plant defences with the invasive pathogen has been examined in a range of environments in the field, the glasshouse, in a hydroponics system and in vitro. The results indicate that summer droughting increases the resistance of E. marginata to P. cinnamomi. However, more work is required to understand the mechanisms involved. The study also indicates that clones of E. marginata, selected as resistant to P. cinnamomi, are not resistant under all conditions and that environmental interactions should be further investigated. Lastly, for effective management strategies to be implemented it is critical that the pathogen can be confidently isolated from plants. It was shown that exudates from infected hosts inhibit the recovery of P. cinnamomi. Recovery methods that can overcome these inhibitory compounds are required. The findings invite further research into the complexity of host-pathogen relationships.

Phosphite is currently used for the management of Phytophthora cinnamomi in native plant communities. A greater understanding of how phosphite affects the host-pathogen interaction is required in order to determine the most effective treatment. This thesis aimed to investigate the effects of applied phosphite concentration on phytotoxicity, in planta concentration of phosphite, disease development and anatomical responses of Eucalyptus marginata. Spraying the foliage to run-off with 7.5 and 10 g phosphite/L led to the development of severe leaf necrosis within 7 days, with greater than 60% of the leaf area damaged. Moderate phytotoxicity was observed after treatment with 5 g phosphite/L. In planta concentration of phosphite in stems, lignotubers and roots did not differ significantly between applied concentrations of phosphite. Stem tissue contained the largest concentration of phosphite at one week after spraying, with approximately 210 and 420 µg phosphite/g dry weight detected after treatment with 5 and 10 g phosphite/L, respectively. In a subsequent field trial, the applied concentration of phosphite was found to affect the duration of effectiveness of phosphite in protecting E. marginata seedlings from stem colonisation by P. cinnamomi. Plants were wound-inoculated with P. cinnamomi at 6-monthly intervals after spraying with phosphite. The 2.5 and 5 g phosphite/L treatments were effective against colonisation by P. cinnamomi when inoculated 0 and 6 months after spraying, but only the 5 g phosphite/L treatment inhibited P. cinnamomi within 12 months of spraying. Phosphite had no effect on colonisation by P. cinnamomi when plants were inoculated at 17 months after spraying. The in planta concentration of phosphite detected in the leaves, stems and roots of plants treated with 5 g phosphite/L did not differ significantly between the time of harvest or tissue type at 0.2 and 6 months after spraying. P. cinnamomi remained viable in plants treated with phosphite.Treatment with 2.5 and 5 g phosphite/L when P. cinnamomi was well established in the stems was ineffective at preventing the death of E. marginata. Between 45 and 89% of plants were girdled on the day of spraying. Spraying plants with 2.5 and 5 g phosphite/L when conditions were less favourable for the pathogen reduced the mortality of E. marginata for up to 10 months. E. marginata seedlings responded to damage by P. cinnamomi with the production of kino veins and woundwood. Bark lesions were in the process of being sloughed off by 7 months after inoculation in plants that remained alive. In plants of a resistant (RR) clonal line and susceptible (SS) clonal line, phosphite treatment inhibited lesion extension in stems, but lesions did not indicate the amount of stem colonised by P. cinnamomi. The pathogen was isolated from up to 17 cm beyond the lesion front in the RR clonal line. Treatments that reduced the mortality of E. marginata were 5 g phosphite/L in the RR clonal line (RR/5) and 10 g phosphite/L in the SS clonal line (SS/10). Uninoculated plants were wounded with liquid nitrogen to determine the microscopic responses to injury in the absence of the pathogen. Wound closure was achieved within 21 days of wounding, with callus formation and vascular cambium regeneration. A wound periderm separated wounded tissue from healthy tissue, adjacent to a lignified boundary zone. Two types of phellem were observed – thin-walled phellem (TnP) and thick-walled phellem (TkP). The first-formed TnP layers contained variable-shaped cells, while subsequent layers were more cubical in shape. Multiple TnP layers developed up to 42 days after wounding, with TkP cells sandwiched between the TnP layers. Genotype and phosphite treatment did not affect the wound responses. Inoculated plants with a restricted lesion extension also formed a wound periderm to separate damaged tissue from healthy tissue. Phosphite treatment stimulated the responses to P. cinnamomi in both clonal lines. Early development of the wound periderm was visible by 6 days after phosphite treatment. It waspreceded by the formation of a ligno-suberised boundary zone in the cambial zone and in phloem parenchyma cells existing prior to injury. Suberin was not detected in the SS/0 treatment. TnP layers completely surrounded lesioned tissue in plants still alive by 24 days after phosphite treatment. Extensive callus production was evident in the SS/10, RR/5 and RR/10 treatments. Temperature affected the post-inoculation efficacy of phosphite and anatomical responses of E. marginata. At 20°C, lesion extension was restricted in both clonal lines of E. marginata, irrespective of phosphite treatment. Greater than 70% of inoculated plants in all treatments produced a ligno-suberised boundary zone at 20°C and between 30 and 70% formed a wound periderm. At 28°C, lesion extension was reduced in phosphite-treated plants at 7 days after treatment. However, lesions continued to extend up to 5 mm per day in the SS clonal line and very few SS plants formed a wound periderm at the lesion front. This contrasted with the strong responses to abiotic wounding observed in uninoculated SS plants at 28°C. The most extensive responses to P. cinnamomi were detected in the RR/5 treatment at 28°C, with a ligno-suberised boundary zone and differentiated TnP of a wound periderm observed in greater than 70% of plants. This treatment resulted in significantly less girdled plants than all other treatments at 28°C, including the RR/0 treatment. At 23 and 24°C, there was no significant difference in acropetal lesion extension or circumferential lesion spread between clonal lines. The inoculation technique and environmental conditions may have resulted in too high a disease pressure for a full expression of resistance in the RR clonal line. This thesis demonstrates that phosphite has the potential to enhance the resistance of young E. marginata and enable them to survive infection by P. cinnamomi. However, its effectiveness is dependent upon a number of factors, including host resistance, environmental conditions, the applied phosphite concentration and the timing of application.