Serratia entomophila, the causative agent of amber disease, is an endemic bacterium used for the biocontrol of New Zealand grass grub larvae. Although the available biopesticide is effective, its use is limited to areas where sub-surface application is feasible, and is also impacted by soil conditions such as moisture levels and osmolarity. The aim of this study was to elucidate the responses of S. entomophila to osmotic and desiccation stresses in relation to challenges encountered during production, storage and soil application, with the goal of developing a more robust and versatile biocontrol agent. RpoS is a key factor in the stress response of many enteric bacteria. In order to dissociate the effect of RpoS from subsequent cellular stress studies, an rpoS mutant was constructed by site-directed mutagenesis. Assessment of the rpoS mutant showed that RpoS was not implicated in NaC1 or desiccation tolerance of S. entomophila. The rpoS mutant was instead found to have enhanced salt tolerance and could be distinguished from the wild-type by the ability to ferment arabinose, a phenotype that was confirmed through complementation. Complete abolition of the amber disease process was observed using an rpoS strain also missing the Sep virulence genes, suggesting that RpoS is a regulator of the S. entomophila anti-feeding prophage (Afp). These findings indicate a subtle interplay between NaC1 tolerance, virulence and RpoS-mediated regulation of amber disease in S. entomophila. A transposon mutagenesis screen was carried out to identify genes associated with NaC1 tolerance in S. entomophila. Fourteen mutants displaying NaC1 sensitivity were identified, two of which had mutations in genes with potential implications for the formulation of the bacterium as a biocontrol agent. The gene leuO that encodes a LysR-family transcriptional regulator was found to be essential for S. entomophila NaC1 tolerance. The toxicity of increased cellular LeuO from an over-expression vector led to the investigation of the effects of leuO mutation on the proteome. Multiple protein changes observed by two-dimensional gel analysis suggested that LeuO may be a global regulator in S. entomophila, as has been hypothesised for Salmonella species. A second NaC1-sensitive mutant contained an insertion in afp15, the product of which is thought to be involved in assembly of the Afp. As well as being sensitive to NaC1, the afp15 mutant was unable to induce the anti-feeding component of amber disease, again highlighting the link between stress tolerance and virulence in S. entomophila. This study also determined that pre-exposure to NaC1 in conjunction with the provision of exogenous glycine betaine significantly enhanced the survival of S. entomophila either in a desiccated state or after application to soil, regardless of the soil moisture content. The implication of this finding on the future formulation of S. entomophila led to investigation of the underlying genetic mechanisms involved in glycine betaine synthesis and NaC1 tolerance. The genes involved in glycine betaine biosynthesis from choline were identified through genomic comparison, degenerate PCR and primer walking. A 6.5 kb region was sequenced and found to contain four genes with homology and similar chromosomal arrangement to the E. coli bet genes (betTIBA). The S. entomophila betIBA genes comprised an operon, flanked by the divergently-transcribed betT gene whose product is responsible for choline transport. To ascertain the relative transcription levels of components of the bet operon, quantitative RT-PCR was performed. Results of qRT-PCR showed that choline in conjunction with NaC1 induced the greatest levels of bet gene transcription, and that levels of the betA transcript were significantly lower than those of the other bet genes. Examination of the betA 5� non-coding region identified a previously undetected hairpin region, possibly accounting for the observed decrease in betA transcript levels. The findings of this study have significantly advanced our understanding of how S. entomophiia responds to stress, and will contribute to the development of formulation strategies for the production of a robust product capable of application to pasture by a range of teclmiques. In addition, there is significant potential to utilise these findings in the development of other bacterial inocula for a range of biotechnological applications.

Errata inserted at leaf 162. / Bibliography: leaves 136-161. / xiii, 161 leaves, [8] 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, 1995?

Errata inserted at leaf 162. Bibliography: leaves 136-161.

Bibliography: leaves 207-220. Pseudomonas corrigata strain 2140 (Pc2140), isolated from wheat field soil in Australia, antagonises the take-all fungus, Gaeumannomyces graminis var. tritici (Ggt) in vitro and significantly reduces take-all symptoms on wheat in pot trials. This study investigates the mechanisms by which the biocontrol agent reduces the disease symptoms. Biochemical analysis of metabolites of P. corrugata 2140 reveal a number of compounds potentially antagonistic to Ggt and which may play a role in disease control. These include water-soluble antibiotics, siderophores, proteases, peptides and volatiles including hydrogen cyanide.

Copy of author's previously published work inserted. Bibliography: leaves 114-136. The study concentrates on: investigation of the biological properties and role of agrocin 434 and other factors in the bicontrol process of crown gall disease; isolation of the complete range of K84 derivatives carrying different combinations of plasmids pAgK84, pAgK434 and pAtK84b in the same host background; development of a rapid plant bioassay to assess the efficacy of biocontrol strains by leaf disc tumorigenesis assay; and characterization of agrocin 434 synthesis and immunity genes.

Bibliography: leaves 175-191. Trissolcus basalis (Wollaston) is a solitary endoparasitoid of the eggs of many pentatomid bugs. It is a quasi-gregarious scelenoid and aggressively defends host patches. In the Adelaide (S. Aust.) region its most abundant host is the native horehound bug, Agonoscelis rutila (Fabricius). This study of patch defence draws together important areas of research in parasitoid behavioural ecology, including the theories of adaptive superparasitism, sex ratio decisions, foraging behaviour and contest resolution.

Specialist and generalist phytoseiid mites are widely used for biological control of spider mites. Understanding dispersal attributes of these mites is important to implementing more stable, cost-efficient integrated pest management. In this regard, we studied dispersal of phytoseiid mites from a local ambulatory phase of movement within a prey patch to a longer-range phase that includes emigration (aerial take-off and dispersal distance) and immigration to a new plant. Specialist phytoseiids showed higher ambulatory and aerial dispersal than generalist species. Somewhat different dispersal strategies were seen between Neoseiulus fallacis (more specialized predator) and N. calfornicus (more generalized predator): The most important difference was earlier and continuous dispersal of N. californicus from a prey patch. Cues from spider mite infestation suppressed the dispersal rates for specialists, but either increased or did not change the dispersal rates for generalist phytoseiids. Aerodynamic calculations support the hypothesis that Phytoseiulus persimilis may not require a standing take-off behavior to become airborne. However, with less vertical profile, a mite may become airborne more by standing erect (N. fallacis), than a species that does not show standing take-off (N. calfornicus). Jumping behavior by P. persimilis was observed for the first time among Phytoseiidae. Falling speed ranged from 0.4 to 0.73 m/s for 13 phytoseiid species and 0.79 to 0.81 for two-spotted spider mite. These values were quite similar to theoretical estimates for specialist phytoseiids, but less for generalists. From falling speed estimates and other morphological data, it was possible to predict aerial dispersal distance of phytoseiids using analytical models. After landing on bare soil following aerial dispersal, high mortality of N. fallacis was observed in the field during summer. Distance from the landing point to target plants showed negative log linear relationships. Soil surfaces and management actions influenced survival and recovery. Environmental conditions greatly affected survival of the predator. We speculated that phytoseiids that fell on ground were moving to the target plants via both ambulatory and aerial means. / Graduation date: 2001

Yeast biocontrol agents that were applied to 'Bose' and 'Anjou' pears in the field up to three weeks prior to harvest were found to survive on the fruit at high population levels on both pear varieties. Cryptococcus infirmo-miniatus, Cryptococcus laurentii, and Rhodotorula glutinis maintained populations averaging 5 x 10��� cfu/fruit for three weeks. Candida oleophila had high initial populations, but the population size quickly declined to levels similar to the total yeast populations on untreated fruit. After a storage period of 2-4 months, fruit that were treated with C. infirmo-miniatus three weeks before harvest showed significantly lower incidence of decay at wounds than did untreated fruit. Combinations of biocontrol agents with reduced rates of the postharvest fungicides captan and thiabendazole were effective in reducing incidence and severity of blue mold decay caused by Penicillium expansum on 'Bosc' pears. Calcium chloride was also effective in combination with some biocontrol agents. Chitosan caused reductions in decay when used alone, but not when combined with most biocontrol agents. The compounds L-asparagine, L-proline, and 2-deoxy-D-glucose were not consistently effective either alone or combined with biocontrol agents. Storage of 'Bosc' and 'Anjou' pears in atmospheres with carbon dioxide concentrations of 12% or 20% for up to six weeks significantly reduced incidence and severity of gray mold decay caused by Botrytis cinerea, but decay was not reduced when the atmospheres was only 3% CO���. In contrast, the 12% or 20% CO��� atmospheres did not have significant effects on decay caused by P. expansum. Use of the biocontrol agents C. infirmo-miniatus, C. laurentii, or R. glutinis led to reductions in decay in all atmospheres, with C. infirmo-miniatus being the most consistently effective. The biocontrol products BioSave-110 (EcoScience Corp.) and Aspire (Ecogen Corp.) were less effective than the yeasts C. laurentii, R. glutinis, and C. infirmo-miniatus which were grown in the lab. / Graduation date: 1999

In the first part of this study we monitored the development of biological control of purple loosestrife Lythrum salicaria over a six-year period at Morgan Lake in western Oregon. In 1992, two beetles, Galerucella pusilla and G. calmariensis (Coleoptera: Chrysomelidae), were released to control the wetland weed at this test site. Our purpose was to estimate quantitative performance parameters that might be generally applied in monitoring biological weed control. Our six performance measures were: 1) biological control agent establishment, 2) the rate of increase of the agents, 3) the rate of spread of the agents, 4) the effect of the agents on individual target plants, 5) the effect of the agents on the population of the target plants, and 6) the indirect impact of the biological control agents on the local plant community. The beetles established viable populations that increased during the study with an intrinsic rate of increase (r), based on the growth rate in damage, estimated at 2.24/year. Within six years after introduction, the beetles spread to saturate the entire purple loosestrife habitat (4100 m��) around the lake. The rate of spread, estimated by calculating a diffusion coefficient (D), was 57.5 m��/year. Adult beetles made seasonal, exploratory movements up to 30 m away from the host plant stand into surrounding crop fields, which suggests a disturbance-free buffer should be established in the habitat surrounding the loosestrife stand. By 1997, both flowering success and median stem density (per 0.125 m�� plot) of purple loosestrife declined to zero. Mean above-ground biomass decreased to 8.4% of its 1994 level. Biomass of native plant species increased by only 3% between 1996 and 1997. Overall, G. pusilla and G. calmariensis reduced the abundance of the target plant at our site. Our monitoring methods were effective at quantitatively measuring the establishment, increase, spread, and damage of the biological control agent, the subsequent decline of the target plant, and the impact on the local plant community. The second part of our study used field and greenhouse experiments to assess non-target effects of two introduced biological control organisms (Galerucella pusilla Duftschmid and G. calmariensis L.: Chrysomelidae) on the economically important ornamental plant, crape myrtle (Lagerstroemia indica L: Lythraceae). Prior host specificity tests performed in the laboratory found that beetles fed, but were unable to complete their life cycle, on this non-target plant. However, there was concern over damage that might occur when the two plant species existed together. This study extended prior tests into a field environment in order to compare the physiological host range revealed in greenhouse tests with the ecological host range revealed in the field. We assumed, based on prior evidence, that the control agents would not complete development on the non-target plant, and therefore, when the non-target organism was isolated from populations of the target organism the direct effects of the biological control agents would be negligible. When the target and non-target organisms existed together, the magnitude of indirect effect of the target organism on the non-target organism via the control agent was expected to increase with: 1) decreasing distance between the target and non-target organisms, and 2) increasing dispersal capability of the control agents. As expected from prior studies beetle feeding and oviposition occurred on crape myrtle but the beetles could not complete development on this non-target plant in our greenhouse and field tests. Leaf damage inflicted by the beetle was lower on crape myrtle than on purple loosestrife plants used as controls and extensive defoliation to the non-target plant was limited to within 30 m from the edge of the purple loosestrife stand. Biomass of crape myrtle was significantly reduced near the stand compared with plants that remained relatively untouched at greater distances. Purple loosestrife biomass exhibited a greater reduction with decreasing distance from the source of beetle colonization. In this thesis we construct and implement strategies for quantitatively assessing success of biological control programs and risk of introduced biological agents to non-target organisms. Through these observations and experiments we hope to increase the predictability and safety of biological control programs. / Graduation date: 1998

The feasibility of managing codling moth, Cydia pomonella (L.), with mixtures of Bacillus thuringiensis-based microbial and selected chemical insecticides was studied under laboratory and field conditions. Joint actions (synergism, additivity and antagonism) of these mixtures were determined for both egg and larval stages. Higher rates of microbial control agents (MCAs), Dipel�� 2X and MVP��, combined with low to medium rates of chemical insecticides gave additive or synergistic egg and/or larval mortality. Esfenvalerate and permethrin synergised the effect of these MCAs, whereas carbaryl, azinphosmethyl, phosmet and diflubenzuron had an additive effects. For all chemical insecticides, often the interactions were better with Dipel than MVP. Generally, egg exposure to low rates of the chemical insecticides alone or in combination with MCAs, caused the larvae hatching from these eggs to be more susceptible to insecticides-MCA mixtures. Most mixtures produced better results when treated at the blackhead stage of egg development. Adult and larval exposure to sublethal rates of azinphosmethyl, phosmet, carbaryl, esfenvalerate and permethrin manifested short- and long-term effects. Adult exposure caused deleterious effects on the survival and oviposition, and the larval exposures affected survival and pupal formation. Carbaryl, esfenvalerate and permethrin affected the pupal survival and subsequent eclosion of adults. Only esfenvalerate and permethrin significantly extended the larval period and reduced pupal weight and subsequent egg deposition. The deleterious sublethal effects of pyrethroids observed in the present study may provide added control of codling moth in the field. Diflubenzuron and MCAs had neither adulticidal activity nor long-term effects on codling moth biology. Results of field efficacy tests indicate that utilization of mixtures of Dipel with low rates of pyrethroids (0.1-0.2X) or diflubenzuron (0.25-0.50X) in the codling moth management is possible and indeed might be beneficial. Dipel-diflubenzuron mixtures were only suitable when applied 3 times/generation. These mixtures were environmentally safe and did not cause any outbreak of phytophagous mites. Standard application rate (2 times/generation) and timing were found to be appropriate for Dipel-pyrethroid (esfenvalerate and permethrin) mixtures. These pyrethroids at all test rates (0.1-1.0X), applied alone or mixed with MCAs, were toxic to predatory phytoseiids, thus caused an outbreak of European red mite and yellow spider mites particularly at higher rates. However, mixtures of Dipel and 0.1X of esfenvalerate/permethrin were less disruptive than corresponding 0.2-1.0X standard rate. These mixtures were selective to most predatory insects, spiders and Zetzellia malt. Hence, use of these mixtures instead of full rate of pyrethroids may improve the prospects of biological control of insect and mite pests in apple orchards. / Graduation date: 1996