Michael Calver

The area of Australian native vegetation in temperate and tropical Australia affected by Phytophthora cinnamomi exceeds many hundreds of thousands of hectares, and continues to increase. In Western Australia alone, greater than 6000 km2 are now infested and 41% of the approximately 6000 plant species in the South West Botanical Province are susceptible. P. cinnamomi and the disease caused by it is a ‘key threatening process to Australia’s Biodiversity’. While the pathogen is widespread and large areas are now infested, many areas of high conservation value remain free of the pathogen. Pathogen free areas could remain so, given effective hygiene and quarantine measures are applied, and if effective methods can be developed to eliminate incursions of the pathogen. To date, there are no robust methods available to eradicate P. cinnamomi from spot infestations or to contain the spread of the pathogen along an active disease front. The need to eradicate or contain the pathogen is now paramount to ensure threatened flora or threatened ecological communities are protected for the long-term. The aim of this study was to develop protocols that can be used to contain and eradicate spot infestations of P. cinnamomi that, if untreated, are likely to threaten extensive areas of native vegetation or areas of high conservation value. Treatment regimes were guided by two assumptions: 1) within the selected sites transmission of the pathogen is by root-to-root contact; and 2) the pathogen is a weakly competitive saprotroph. In Cape Riche, Western Australia, treatment and control plots were set up along an active disease front within scrub-heath vegetation dominated by Banksia spp. Treatments applied sequentially and in combination, included: 1) destruction of the largest plants within disease free vegetation forward of the disease front; 2) destruction of all plants to create a fallow or ‘dead zone’; 3) installation of physical root barriers and subsurface irrigation for the application of fungicide/s; 4) surface applications of fungicides selective against Oomycetes (triadiazole and Metalaxyl-M) and; 5) surface injection and deep (± 1 m) treatments with the soil fumigant methamsodium. In a separate experiment in Narawntapu National Park (NP), Tasmania, two treatment regimes were applied to experimental heath plots with active disease centres within a Eucalyptus-Banksia woodland. Treatments were: 1) a combined treatment including vegetation removal, Metalaxyl-M and metham-sodium and root barriers and; 2) with Metalaxyl-M and root barriers alone. Standard baiting techniques were used to recover P. cinnamomi from combined soil and root samples, down to 1.5 m deep at Cape Riche, and to 1 m at Narawntapu NP. Research into natural and induced resistance in Australian native vegetation of Phytophthora cinnamomi and innovative methods to contain and/or eradicate within localised incursions in areas of high biodiversity in Australia.

To test the efficacy of treatments against a pathogen it is necessary to have a standard pathosystem. We assessed the suitability of intact Arabidopsis plants from 20 ecotypes, root inoculated with 29 P. cinnamomi isolates, as Arabidopsis offers so many advantages for physiological and molecular work and allows fast throughput of trials. It was shown that intact Arabidopsis plants, grown hydroponically and root inoculated with P. cinnamomi zoospores are not a suitable pathosystem, as although root mass is reduced, shoots are qualitatively and quantitatively unaffected and plants remain healthy. An ideal pathosystem is one where susceptible Arabidopsis ecotypes die and resistant Arabidopsis ecotypes survive. Detached leaves of 4-week old Arabidopsis (ecotype Landsberg erecta) plants inoculated with zoospores or mycelium of P. cinnamomi provided a good model system for some of the analysis of the effects of phosphite as infection could be assessed through lesion size and abundance of callose papillae. Leaves treated with phosphite showed reduced lesion size and increased numbers of callose papillae. Using qPCR an increase in the level of expression of the defense gene PRI was quantified. Although not an ideal pathosystem, Arabidopsis can be used to examine very early defense responses (in the first few days following inoculation) after treatment with phosphite. A rapid assay was developed to compare the effect of phosphite and metabolic inhibitors on pathogenicity of P. cinnamomi. Filter paper discs overgrown with P. cinnamomi were treated with 20 μL drops of phosphite or inhibitors, then tested for pathogenicity (ability to colonise lupin roots), or growth on NARPH plates. It was shown that c-AMP is likely to be involved in the reduction by phosphite of P. cinnamomi pathogenicity. The technique provides a means of screening compounds that might enhance phosphite efficacy, and to explain the mode of action of phosphite. Quantification of phosphite uptake and movement in the plant is hampered by the lack of a quick, cheap method of measuring accurately the concentration of phosphite in different plant tissues. Costs of the existing HPLC method (~$30 per sample) prevent such studies, and analyses in the region of $1 - $5 per sample are required. We examined two methods (a silver nitrate assay, and a phosphite dehydrogenase assay) as potential methods of accurately and cost effectively measuring phosphite in plant tissues. Both methods proved promising and represent exciting advances in phosphite analysis. Some additional fine tuning is required to ensure that the methods are reliable across a range of plant species from different families. A cheap, accurate and robust analytical method will allow many important questions about phosphite uptake and movement to be investigated.

Phosphite is of major importance in controlling root disease caused by Phytophthora cinnamomi. It acts both directly and indirectly on the pathogen. In order to maximise the efficacy of phosphite we need to understand how the physiological status of the plant at the time of phosphite application affects control. The physiological status of plants is not constant but varies over time depending on developmental gene expression (e.g. leaf phenology, flowering/fruiting and senescence) and interactions with the environment (e.g. temperature, moisture, light, fire, nutrients and other biota). In Mediterranean environments in particular, plants experience stresses due to extremes in water availability and the incidence of wild fire is high. Furthermore, individuals and species of plants are not in synchrony due to differences in recruitment, ontogeny, longevity and rest periods. Therefore, from a management perspective when considering all of these stresses native plant communities are subjected to, it is critical to know when to apply phosphite to ensure optimal disease control. We examined each of the key environmental stresses (water excess, water deficit, fire and flowering) independently, on the efficacy of phosphite to control disease.