Jen McComb

The systemic fungicide phosphite is increasingly being used to treat oomycete plant diseases in horticultural crops and native ecosystems around the world. The effects of phosphite application on a plant are variable. Resistance depends on the species, the time of application and the method of application. Very little is known about how phosphite is transported and stored within plants, or how these processes are affected by the physiological status of the plant. This is mainly due to the unavailability of a simple, efficient method for measuring the concentration of phosphite. We have developed an enzymatic assay that enables phosphite to be quantified as an alternative to high-performance liquid chromatography or gas chromatography. Phosphite is oxidised to phosphate by a modified phosphite dehydrogenase. NADH formed in the reaction reduces p-iodonitrotetrazolium violet (INT) via an intermediate electron carrier to produce a stable red formazan that can be measured spectrophotometrically at 490 nm. This method is being further developed to quantify phosphite in plant-tissue samples. Dried plant material is powdered and resuspended in water. Phosphite is separated from the plant extract solution by dialysis and determined with the phosphite dehydrogenase assay. The method is rapid and inexpensive and could potentially be developed to measure the phosphite concentration within plant samples in the field.

The potential fungitoxic qualities of the root exudates of A. pulchella var. goadbyi on P. cinnamomi were tested. Techniques were developed to ascertain the mycelial inhibition and chlamydospore suppression of P. cinnamomi to a range of concentrations of exudates which were collected from in vitro grown A. pulchella plants. The controlled conditions under which the experiments were conducted prevented other environmental factors interfering with the effects of root exudates on P. cinnamomi. This was an important goal and the system enabled continuous collection of root exudates for laboratory bioassays. Bioassay procedures consisted of incubating mycelial disks of P. cinnamomi in a range of concentrations of A. pulchella exudates incorporated into a basal liquid medium. Significant inhibition of, P. cinnamomi mycelial growth was recorded in the solutions with A. pulchella var. goadbyi root exudates compared to the control solutions. The root exudates were fractioned with hexane and the bioassays conducted with the chlamydospore suspensions showed collapse and low germination of chlamydospores in the A. pulchella exudates compared to the controls.

Resistance to Phytophthora cinnamomi in jarrah (Eucalyptus marginata) is under strong genetic control. It has high heritability, is probably polygenic, and is durable in field trials. Seedlings from healthy mother trees, either growing on long-tenn dieback sites or from a provenance collection made across the range of the species, were grown in the glasshouse and resistant seedlings selected from inoculation trials. These were micropropagated by tissue culture. The resulting clonal lines were planted in field validation trials on dieback-affected sites and soil at the base of the plants was also inoculated with P.'cinnamomi to test survival and growth of resistant plants. In spite of some drought deaths, survival of most resistant lines has been high. Some 45 unrelated resistant lines have been selected. However, due to high costs of production and establishment problems in forest sites, it is not feasible to use the clonal jarrah directly in large-scale operational plantings. Clonal seed orchards are now being planted to supply seed of resistant jarrah for use in the rehabilitation of dieback-affected forest and plantings on cleared land.

The fungicide phosphite was found to reduce pollen fertility in Australian and exotic species. In the perennial Dryandra sessilis the reduction was evident for a year after treatment with 2.5-10 gL-1 phosphite sprayed to run off. Pterochaeta paniculata, an annual, showed reduced pollen fertility in flowers that opened 16-30 days after spraying. The horticultural species Pelunia hybrida and Tradescantia virginiana also displayed reduced pollen fertility after phosphite treatment. Pollen mother cells of Tradescantia had a significant percentage of abnormal first and second divisions and micronuclei in the microspores for up to one month after spraying. There was evidence that phosphite induced premature tapetum breakdown in Pelunia but not Tradescantia. The percentage of abnormal meiotic cells and the frequency of premature tapetum breakdown appeared insufficient to account for the high levels of pollen infertility observed after phosphite treatment.

Soil moisture and the type of organic matter colonised by Phylophlhora cinnamomi significantly affected long-term survival of the pathogen. Banksia grandis stem pieces, and root tips of Eucalyptus marginata (jarrah) colonised with P. cinnamomi were placed into pots filled with soil from the jarrah forest or an adjacent rehabilitated bauxite mine site in the south west of Western Australia. The soil was either maintained at container capacity, or allowed to dry-out slowly from container capacity. Samples were harvested over a 210-day period and assessed for P. cinnamomi survival. P. cinnamomi was recovered after 210 days from banksia stems (98% colonisation) and eucalypt root tips (45% colonisation) from both soil types when the soil was maintained at container capacity. However when the soils were allowed to dry, the pathogen was not recovered after 112 days from either banksia stems or eucalypt roots. Soil origin did not influence P. cinnamomi survival for either inoculum type. These findings indicate that under moist conditions the pathogen can survive in small pieces of organic matter for extended periods of time.

Techniques were evaluated to develop an effective controlled-pollination technique for jarrah (Eucalyptus marginata). Pollination success was scored by counting pollen tubes in squashed styles. The most effective method to isolate the flowers after emasculation was to wrap each in a twist of aluminium foil. Pollen application was best when several freshly-picked, newly-opened flowers were wiped across each receptive stigma. Stigmas were receptive from 3-6 days after anthesis and most pollen tubes were observed pollination in the middle of the flowering season. Very few mature fruits resulted from any controlled-pollution technique. Bud, flower and fruit-loss rates under natural conditions were recorded (in five clones and five wild trees) and 0-13% of buds remained on the trees to develop into fruits. The percentage of pollen germination from different trees was very variable. Pollen was most fertile in the middle of the flowering season, environmental conditions had little effect. Fresh pollen in anthers was more fertile than pollen separated using a fine sieve.

To extend the range of plants that can be grown on saline land and give a direct financial return, hybrids were produced between Eucalyptus camaldulensis lines selected for salt tolerance, and E. globulus selected for high pulp quality. There was a wide range of compatibility between the parents in terms of numbers of seeds per capsule, percentage germination and abnormal plants. Morphological traits inherited from the male E. globulus parent made it easy to confirm the hybrid status of the seedlings. Variability in leaf morphology made it impossible to distinguish the hybrids from the parents on the basis of leaf shape and size in the field, but the flowers and fruits of the hybrids were intermediate between the parents in shape and size. Hybrids were reproductively fertile and backcrossing with E. globulus was possible.

The eucalypt species Eucalyptus marginata which is harvested for high quality timber comprises three subspecies recognized by morphological characters; E. marginata ssp. marginata, ssp. thalassica, and ssp. elegantella. Genetic diversity and phylogenetic relationships between the subspecies were examined using anonymous nuclear RFLP loci, with Eucalyptus staerei included as an outgroup in the phylogenetic analysis. The level of diversity within the nuclear genome was lower than that found in comparative studies with other eucalypts (A = 2. 7, H4 = 0.345). Most of the variation occurred with ill the populations (96. 9%, H4 = 0.334) The two populations sampled for each of ssp. thalassica and ssp. elegantella clustered together in the UPGMA analysis, however there was little differentiation between the three subspecies overall (D = 0.029). Eucalyptus marginata was clearly distinct from its closest relative E. staerei (0 = 0.16). There is little genetic support for the separation of the subspecies.

In woody plants the periderm provides a formidable barrier to mechanical damage, and only a few fungal pathogens have been shown to directly penetrate suberised and/or lignified phellem cells. Recent research has shown that when the non-wounded periderm of Eucalyptus marginata (jarrah) seedlings are exposed to motile zoospores of P. cinnamomi, infection occurs and may result in seedling death. The current histological study was undertaken to determine how zoospores of P. cinnamomi circumvent the periderm.