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Phytophthora medicaginis is an important soil-borne oomycete pathogen of lucerne (Medicago sativa) and chickpea (Cicer arietinum) within Australia and overseas. To understand the host/pathogen interaction, a pathosystem was developed using the model legume Medicago truncatula. Using the resources developed for genetics and molecular characterisation in this model plant, the aim of this research was to understand the interaction between M. truncatula and P. medicaginis, with a view to improving resistance to this important pathogen in related legumes. To observe and characterise the interaction between M. truncatula and P. medicaginis, a pathosystem was developed by first screening a germplasm collection of 99 M. truncatula accessions. This revealed a continuous distribution in disease phenotypes with variable extremes in natural resistance to P. medicaginis culture UQ5750, isolated originally from M. sativa. P. medicaginis zoospore inoculation of 1-2 week-old seedlings in glasshouse experiments proved to be a robust and repeatable method to consistently confirm the responses observed for six key M. truncatula accessions; SA8618 and SA8623 exhibit high natural resistance to this pathogen, accession A17 is moderately resistant, A20 is moderately susceptible and accessions Borung and SA30199 are susceptible. To characterise the genetic basis of resistance to P. medicaginis, two reciprocal F2 populations from cross pollinations between A17 and Borung and SA8618 and SA30199 were produced and then phenotyped for disease symptoms. Genetic segregation patterns indicated the involvement of a gene with a major effect in both reciprocal populations. In particular, a 3:1 segregation ratio for resistance in the F2 populations from cross pollinations between A17 and Borung indicated the possibility of a single dominant gene for moderate resistance. Further phenotyping of F3 families is required to verify this. A M. truncatula linkage map was constructed using 50 F2 individuals of the A17 X Borung population and 49 F2 individuals from the Borung X A17 population. The map, covering 519.3 cM, is comprised of 84 SSR markers with an average distance between markers of 8.7 cM. These are evenly spaced over 7 linkage groups, including a super linkage group conferred by a translocation event between LG4 and LG8 of accession A17. Quantitative trait locus (QTL) analysis confirmed there was a QTL with a major effect in the A17/Borung reciprocal populations. A significant QTL was determined by quantifying two symptoms of P. medicaginis infection - proportion of dead/chlorotic leaves and root fresh weight. The trait loci for both symptoms were located on the same linkage group within the same region, supporting the putative position of the QTL and the authenticity of its involvement in resistance to P. medicaginis. This QTL was located on LG6 and accounted for 69.5% of the observed variation in proportion of dead/chlorotic leaves or 38.1% of the variation in root fresh weight within the inoculated populations. The effect of this QTL on resistance to P. medicaginis translated into 27.5% less dead/chlorotic leaves or 0.86 g more root fresh weight. Other QTLs with minor effects that are potentially involved in the interaction are located elsewhere on LG6 and LG2. However, the marker density of the linkage map and the population size need to be increased to verify this. In parallel to this, an F7 recombinant inbred line (RIL) population of chickpea (BG212 X Jimbour), developed by breeders at the New South Wales Department of Primary Industries (NSW DPI), was also assessed for the genetic basis of resistance to P. medicaginis. Variance component analysis of phenotype scores for this intraspecific RIL population indicated that 57.15% of the differences in between-family and withinfamily variance could be attributed to a genetic component. However, gene-based markers developed in M. truncatula and established simple sequence repeat (SSR) markers of chickpea were not sufficiently polymorphic in size to produce a linkage map for further QTL analysis. An interspecific cross between C. arietinum and C. echinospermum (Howzat X ILWC246) was also performed by breeders at the NSW DPI to develop RILs. In the duration of this research these interspecific RILs were bred to generation F3 and phenotyping assessment had not been performed. However, marker screening of the parents revealed 122 size polymorphic chickpea SSR markers. A sufficient linkage map could be produced for QTL analysis once field assessment of this population is performed. Initial screening of the M. truncatula gene-based markers on the parents of this interspecific cross also revealed that 50% show a sequence-identified base pair difference. A chickpea linkage map incorporating these markers could be comparatively mapped with M. truncatula. Molecular investigations of the M. truncatula/P. medicaginis pathosystem were performed to elucidate the possible underlying defence mechanisms involved in the observed resistance. To determine the function of ethylene in the resistant response, the characterisation of defence associated mutants of M. truncatula and Agrobacterium rhizogenes-mediated ‘hairy root’ transformations were employed. Comparison of response to inoculation of an ethylene insensitive mutant of M. truncatula (sickle) with the moderately resistant background genotype A17 showed that sickle was hypersensitive to P. medicaginis. This indicated that ethylene insensitivity was not the source of resistance to this pathogen and importantly that ethylene is a key defence signalling molecule in the moderate resistance of A17 to P. medicaginis. Agrobacterium-mediated ‘hairy root’ transformations of M. truncatula with 4GCC::Luc constructs, revealed that the production of ethylene and consequently ethylene response factors (ERFs) after inoculation by P. medicaginis was a general defence reaction by all accessions. The two susceptible M. truncatula accessions exhibited a much stronger and earlier response to inoculation than the highly resistant and moderately resistant accessions. This indicated that the resistant response may be directed by a transcriptional component governed by the host genotype, downstream of ethylene production. The M. truncatula/P. medicaginis ‘hairy root’ transformation assay has scope to be a powerful functional genomics tool for this pathogen interaction. Reverse transcriptase quantitative polymerase chain reaction (RTqPCR) was employed to determine the general patterns of gene expression and function underlying the response to P. medicaginis infection. Relative changes in gene expression of key enzymes in each of the salicylic acid, jasmonic acid, ethylene and isoflavonoid defence pathways and in genes encoding downstream target proteins revealed potential genes involved in the resistance to P. medicaginis. There was a distinct molecular difference in the response between the high and moderately resistant M. truncatula phenotypes to this pathogen. Moderate resistance to P. medicaginis in M. truncatula is possibly mediated by ethylene and involves the considerable induction of pathogenesis related protein 5 (PR5), which was not the same defence response that conferred the high resistance to P. medicaginis. Early and consistent expression of genes encoding key enzymes of the isoflavonoid pathway by the highly resistant accession indicated that phytoalexin response could be associated with the high resistance. Confirmation of the involvement of isoflavonoid phytoalexins in the high resistance response to P. medicaginis merits further investigation.

Bibliography: leaves 212-227.

Fruit-tree borer, Maroga melanostigma (Wallengren), is a native Australian pest in many species of trees. It is of particular economic importance in prune (Prunus domestica) trees because the presence of this wood boring insect can reduce productivity by an average of 5% per tree. Large areas of orchards can be affected. There are currently no chemicals registered for control of this pest. Young, New South Wales is the second largest prune-growing district in Australia and the area most seriously affected by M. melanostigma. Prune growers in the district utilise integrated pest management and were supportive of a project to investigate biological control options for this economically damaging pest. The two main objectives of the project were 1) to understand the life cycle of M. melanostigma, so biological controls could be timed appropriately; and 2) to investigate biological control options for this pest. Life cycle studies were commenced in the first season (2003/04) using field cages and light trapping. These investigations continued throughout the project. In Young, moths were found to emerge from wood over a two month period (December and January). Oviposition was assumed to be during this period however, even after extensive searches of trees, no eggs were observed. Historical data were collated to determine locations and timings of moth emergence elsewhere in Australia. The data showed that M. melanostigma has been found in every state and territory Australia, with moths observed from October through to March. The biological control options reviewed were egg parasitoids (Trichogramma species only), entomopathogenic nematodes and entomopathogenic fungi. Trichogramma were favoured because of previous research undertaken against the same pest in pecans in Moree, NSW. Entomopathogenic nematodes were also investigated due to research indicating their effectiveness in cryptic situations, such as borer tunnels in trees. Fungi were considered but dismissed due to lack of literature supporting their effectiveness in reducing lepidopteran pest damage in trees. A major field trial was designed with the assistance of a biometrician and the trial blocks laid out based on this advice. There were three trial sites, each containing four blocks of approximately 200 trees (~800 trees/site). Two blocks were designated as release blocks and two as non-release blocks to correspond with the trial’s two treatments. In the first season (2003/04) an initial visual assessment of borer damage was undertaken after leaf fall on each of the trees in the trial. This information was used as baseline data, to compare against damage levels following biological control releases in the second and third years of the project. Natural parasitism in the field was assessed using cultured eggs of Helicoverpa armigera (Hübner) before and between Trichogramma carverae (Oatman and Pinto) releases. Temperature and relative humidity were recorded in each of the trial sites, for the duration of the trial, using commercially available data loggers. In the second season (2004/05), Trichogramma releases were made during the period of moth activity and H. armigera eggs were used to monitor parasitism in the trial orchards. Parasitised eggs were reared through and all parasitoids were identified as T. carverae. Damage assessments were again carried out after leaf fall to compare release versus non-release blocks, as well as to determine if there was any change in borer activity. Early instar larvae were collected from non-trial blocks and exposed to the entomopathogenic nematode Steinernema carpocapsae in a laboratory investigation. Results from this limited bioassay were inconclusive. In the third season (2005/06), Trichogramma releases were again made during the period of moth activity and H. armigera eggs were used to monitor parasitism in the trial orchards. Parasitised eggs were reared through to emergence. The emerged parasites were identified as T. carverae, T. pretiosum and T. nr brassicae. Damage assessments were again made of all the trees in the trial. Results were statistically analysed to detect any differences between treatments. There was no statistically significant evidence that the releases of T. carverae reduced damage from M. melanostigma over the duration of the trial. Although damage increased across both release and non-release treatments in most blocks during the trial investigations, the increase was slightly lower in trees in which Trichogramma had been released. It should be noted that the experiments were affected by serious drought conditions which prevailed during the three seasons of the trial. / Master of Science (Hons)

Ready-to-eat vegetable salads containing lettuce as a main ingredient have become popular food items in recent years. Microorganisms associated with these products determine their shelf-life, sensory appeal and safety. This thesis investigates the bacterial ecology of lettuce, aspects of their pre-harvest contamination with microorganisms, and the presence of antimicrobial constituents in such produce. Commercial pesticides (insecticides, herbicides, fungicides), used during lettuce cultivation were examined as potential sources of microbial contaminants. None of the pesticide concentrates contained viable microorganisms. After reconstitution in water, two of the pesticides supported growth of inoculated species of Pseudomonas, Salmonella and Escherichia coli. Pesticides reconstituted in agricultural waters (bore, dam and river) supported the growth of microorganisms (e.g. Pseudomonas, Acinetobacter, Aeromonas spp. and coliforms) naturally present in these waters. Unless properly managed, pesticide application could contribute microbial contaminants to vegetable produce, thereby affecting their quality. Bacterial species associated with retail samples of lettuce were examined by plate culture on Tryptone Soy Agar and PCR-DGGE analysis. Macerates and rinses of lettuce sub-samples with and without addition of Tween 80 were examined to maximize bacterial recoveries. Predominant bacteria isolated by agar culture included species of Pseudomonas, Agrobacterium, Curtobacterium and Burkholderia, at populations of 103-106 cfu/g. PCR-DGGE was unable to recover the same incidence of species as agar culture and failed to detect bacteria in many samples. In some samples, PCR-DGGE detected species of Bacillus, Pseudomonas, Serratia and Acinetobacter, not found by culture. Failure of the PCR-DGGE analyses was attributed to interference by plant chloroplast DNA. Preparative agarose gel electrophoresis of lettuce macerates was necessary to remove chloroplast DNA before application of PCR-DGGE analysis. Thirty percent of lettuce samples contained Acinetobacter species at 101-104 cfu/g when examined after culture on minimal salts agar or enrichment in Baumann enrichment medium. Other Acinetobacter media failed to give reliable isolation of these species from lettuce and salad vegetables. Lettuce could be an environmental source of Acinetobacter nosocomial infections. Juices, solvent extracts and supercritical fluid carbon dioxide extracts of lettuce and capsicum samples did not exhibit antimicrobial action against a range of food spoilage and pathogenic bacteria.

Bibliography : leaves [70]-79. / ix, 80 leaves, [6] leaves of plates : ill. (some col) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Crop Protection, 1994

Bibliography: leaves 212-227. / viii, 227 leaves, [19] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Applied and Molecular Ecology, 2001

iv, 114 leaves : ill. ; 25 cm / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Pathology, Waite Agricultural Research Institute, 1972

Bibliography: leaves 119-143 / v, 148 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, 2000

Bibliography : leaves [70]-79.