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The overall aim of the research project was to evaluate the potential of the fungal pathogen <I>Ascochyta caulina</I> as a biological control agent against <I>Chenopodium album</I>, a major weed in arable crops. A number of isolates of <I>Ascochyta caulina</I> were evaluated but isolate W 90-1 from Holland proved to be the most promising candidate because of its high virulence. It was therefore selected for more detailed greenhouse and field studies into the environmental parameters required for infection and disease development. Results of these studies showed that in order to achieve the maximum infection, a temperature of between 20-30°C, a relative humidity of >95% for 24 h and a spore density of approximately of 1-2 x 10⁶ spores per ml spore suspension were required. Mortality and plant necrosis levels after application of <I>A. caulina</I> decreased with plant age and treatment of <I>C. album</I> shortly after emergence or to juvenile plants (before 4-leaf growth stage) was most effective. The requirement for long periods of high relative humidity and the inability of <I>A. caulina </I>to cause satisfactory disease after the 4 leaf growth stage are the most important limiting factors for the development of <I>A. caulina</I> as a bioherbicide for <I>C. album. </I>A range of spore formulations was studied with the aim of reducing the requirement for long periods of high relative humidity for disease development. Studies indicated that disease development could be increased by incorporation of surfactants (Tween 80 or Sylgard) and nutrients (Czapek-Dox Broth and Yeast Extract) into inoculum suspension. Results of field trials indicated that if application were properly timed and optimum environmental conditions can be achieved the pathogen can give satisfactory control of the weed.

Successful bacterial pathogens manipulate crucial intracellular host processes as a virulence strategy. One particular potent mechanism utilized by bacterial phytopathogens is to inject virulence factors (effectors) directly into the host cell. While many effectors have been identified and shown to suppress plant immune responses, very few have well-characterized enzymatic activities or host targets. To overcome the challenges of functional analysis of effectors, I designed two heterologous screens to characterize effector proteins of the bacterial phytopathogen Pseudomonas syringae. Specifically, my objective was to identify those P. syringae effectors that target evolutionarily conserved host proteins or processes and to subsequently elucidate the molecular mechanisms of these effectors. The first heterologous screen that I performed was to utilize tandem-affinity-purification (TAP)-tagged effectors in human cells to identify potential interacting host proteins. The second heterologous screen iii utilized a high-throughput genomics approach in yeast, known as the pathogenic genetic array (PGA), to characterize P. syringae effectors. Using the first heterologous approach, I have identified HopZ1a as the first bacterial phytopathogen effector that binds tubulin. I have shown that HopZ1a is an acetyltransferase activated by the eukaryotic co-factor, phytic acid. In vitro, activated HopZ1a acetylates itself and tubulin. In Arabidopsis thaliana, activated HopZ1a causes microtubule destruction, disrupts the secretory pathway and suppresses cell wall-mediated defense. The acetyltransferase activity of HopZ1a is dependent on the conserved catalytic cysteine residue (C216) and a conserved lysine residue (K289). Using the second heterologous screen in yeast, I have shown that HopZ1a may target the mitogen-activated protein kinase (MAPK) signaling cascades. Together, my work has identified novel eukaryotic targets and elucidated the virulence functions of HopZ1a.

Successful bacterial pathogens manipulate crucial intracellular host processes as a virulence strategy. One particular potent mechanism utilized by bacterial phytopathogens is to inject virulence factors (effectors) directly into the host cell. While many effectors have been identified and shown to suppress plant immune responses, very few have well-characterized enzymatic activities or host targets. To overcome the challenges of functional analysis of effectors, I designed two heterologous screens to characterize effector proteins of the bacterial phytopathogen Pseudomonas syringae. Specifically, my objective was to identify those P. syringae effectors that target evolutionarily conserved host proteins or processes and to subsequently elucidate the molecular mechanisms of these effectors. The first heterologous screen that I performed was to utilize tandem-affinity-purification (TAP)-tagged effectors in human cells to identify potential interacting host proteins. The second heterologous screen iii utilized a high-throughput genomics approach in yeast, known as the pathogenic genetic array (PGA), to characterize P. syringae effectors. Using the first heterologous approach, I have identified HopZ1a as the first bacterial phytopathogen effector that binds tubulin. I have shown that HopZ1a is an acetyltransferase activated by the eukaryotic co-factor, phytic acid. In vitro, activated HopZ1a acetylates itself and tubulin. In Arabidopsis thaliana, activated HopZ1a causes microtubule destruction, disrupts the secretory pathway and suppresses cell wall-mediated defense. The acetyltransferase activity of HopZ1a is dependent on the conserved catalytic cysteine residue (C216) and a conserved lysine residue (K289). Using the second heterologous screen in yeast, I have shown that HopZ1a may target the mitogen-activated protein kinase (MAPK) signaling cascades. Together, my work has identified novel eukaryotic targets and elucidated the virulence functions of HopZ1a.

The enteric pathogen Escherichia albertii represents an emerging food safety challenge. It has been mistakenly identified as Hafnia alvei, Shigella, or as a member of the Enteropathogenic E. coli (EPEC). Isolates of certain strains of the organism are known to possess genes encoding pathogenesis factors capable of inducing attaching/effacing (A/E) lesions, cytolethal distending toxin and a variant Shiga toxin. The pathogen has been isolated from infants and adults and has been identified as a causative agent from an outbreak of foodborne disease occurring in an industrialized nation. Recent reports have detailed the ability of this pathogen to survive on ground beef and to resist several classes of therapeutic antibiotics. The objectives of this study were to: (i) determine the efficacy of E. albertii isolates to survive lactic acid exposure as a function of solution pH, and (ii) verify its inactivation in ground beef according to the USDA recommendations for in-home preparation. Rifampicin resistant (RifR) isolates of E. albertii (ATCC 9194, 19982, 10457) were obtained according to published methods. Thermal resistance of parent and RifR isolates were compared in vitro at 55 °C to confirm no significant differences in tolerance to heat as a result of antibiotic resistance capacity. Tolerance to food grade lactic acid (Purac, Olathe, KS) (3.0% w/v) at differing pH levels (3.0, 4.0, 5.0, 7.0) was examined in physiological saline at 35 °C. Finally, ground beef patties (80% lean) inoculated with individual RifR isolates were cooked to internal temperatures of 62, 71, or 76 °C to determine resistance of different internal temperatures. Experiments comparing the in vitro tolerances of parent and RifR E. albertii isolates indicated no differences between parent and mutant with regards to heat and lactic acid challenge. E. albertii inactivation in lactic acid increased as the pH of the solution was decreased; maximum reduction at pH 3.0 was at 30 min and maximum reduction for pH 4.0 at 2.5 hours. Changes in populations of E. albertii at pH 5.0 were not different than that at pH 7.0. Cooking of beef to 62 °C internal temperature produced reductions of all RifR isolates to non-detectable levels.

To determine the impact of stress response on enumeration, cell association status and the viability of Escherichia coli DH5, Staphylococcus aureus ATCC 13565 and Listeria monocytogenes CDC 7762 were evaluated using fluorescence microscopy and were compared with the outcomes of traditional plate count and optical density measurements. Fluorescence microscopy revealed that organic acid stress (acetic and lactic, pH 2.7-3.3) induced cell clumping with little loss of viability in Escherichia coli DH5. Significantly lower values for cell enumeration were found for plate counts and OD600 measurement, likely due to cell clumping in response to organic acid stress. Gram-negative bacteria Escherichia coli DH5 showed higher levels of clumping and subsequent resistance against organic acid stress. Increased cell surface hydrophobicity was found in cells that exhibited more evident clumping. However, inorganic acid stress (hydrochloric and sulfuric, pH 3.0-3.3) induced only very low level of clumping in stationary-phase Escherichia coli DH5 and almost no clumping in other cultures. Osmotic stress, heat and cold shock were not found to induce cell clumping. It has been determined that traditional enumeration methods have significantly underestimated the number of viable bacterial cells when organic acid stress is involved. Plate counts and OD600 measurement therefore need to be reassessed as tools for accurate evaluation of pathogens in food industry. / Food Science and Technology

Polymerase Chain Reaction (PCR) detection paves the way to reliable and rapid diagnosis of diseases and has been used extensively since its introduction. Many miniaturized PCR systems were presented by microfluidics and lab-on-a-chip community. However, most of the developed systems did not employ real-time detection and thus required post-PCR processes to obtain results. Among the few real-time PCR systems, almost all of them aimed for medical applications and those for plant pathogen diagnosis systems are almost non-existent in the literature. In this work, we are presenting a portable system that employs microfluidics PCR system with integrated optical systems to accomplish real-time quantitative PCR for plant pathogen diagnosis. The system is comprised of a PCR chip that has a chamber for PCR sample with integrated metal heaters fabricated by standard microfabrication procedures, an optical system that includes lenses, filters, a dichroic mirror and a photomultiplier tube (PMT) to achieve sensitive fluorescence measurement capability and a computer control system for Proportional Integral Derivative (PID) control and data acquisition. The optical detection system employs portable components and has a size of 3.9 x 5.9 x 11.9 cm which makes it possible to be used in field settings. On the device side, two different designs are used. The first design includes a single chamber in a 25.4 x 25.4 mm device and the capacity of the chamber is 9 micro-liters which is sufficient to do gel electrophoresis verification. The second design has three 2.2 micro-liter chambers squeezed in the same size device while having smaller volume to increase high throughput of the system. The operation of the system was demonstrated using Fusarium oxysporum spf. lycopersici which is a fungal plant pathogen that affects crops in the USA. In the presence of the plant pathogen, noticeable increases in the photomultiplier tube output were observed which means successful amplifications and detections occurred. The results were confirmed using gel electrophoresis which is a conventional post-PCR process to determine the existence and length of the amplified DNA. Clear bands located in the expected position were observed following the gel electrophoresis. Overall, we have presented a portable PCR system that has the capability of detecting plant pathogens.

This thesis examines a broad spectrum of physiological and silvicultural features of the highly valued woody angiosperm hemi-parasite Santalurn album L. (Indian sandalwood) in relation to its culture in plantations in northern Western Australia. Topics covered include allometry of host and Santalum when grown as single plant pairings in both field and pot culture, nutritional interactions between Santalum and beneficial and non-beneficial hosts, deleterious influences of parasitism on plantation productivity and heartwood induction in young trees. In Western Australia sandalwood is grown in the nursery for 8 months before establishment in the field and during this time a pot host is introduced. Survival of Santalurn after field establishment and its subsequent growth were significantly affected by the time of introduction of the pot host, Alternanthera nana. Increasing the period of the Santalum : Alternanthera association in the nursery to 109 days prior to field establishment markedly increased early growth of Salztalum plantations. Introduction at 134 days prior to field establishment was detrimental to the parasite as the Alternanthera was too vigorous for the small Santalum seedlings. Santalurn plants had a lower root : shoot ratio lower when cultured with Alternanthera in the nursery prior to field establishment compared with seedlings grown without Alternanthera. Alterrzantlzera survival in the field was high when it had been grown with Santalum for 12 weeks or more in the nursery prior to field establishment. After 1 1 weeks in the field a strong negative linear relationship was shown between Santalunz root : shoot ratio and Alternarzthera dry weight, and a positive linear relationship between Salztalum DW and Alternanthera DW. In Western Australia Santalu~n is established in the field with an intermediate host which nourishes the parasite for 3-5 years before Santalum becomes dependent on its long-term host and the intermediate host dies. The relationship between Santalum and several species tested as intermediate hosts was examined by pairing Santalum seedlings with intermediate host seedlings in 25 litre pots over a 10 month period. Growth of Santalum in pot culture with three N2-fixing woody intermediate hosts (Sesbania forrnosa, Acacia traclzycarpa and A. ampliceps), the woody non N2-fixing Eucalyptus camaldulensis or without a host varied considerably between host treatments. Santalum growth was greater and root : shoot ratio lower for seedlings grown with N2-fixing hosts compared with seedlings grown with E. carnaldulensis or with no host. The root : shoot ratio of unattached Santalum increased exponentially over time, whereas for all other treatments it remained relatively constant. An assessment of the value of the hosts, termed host use efficiency, was computed as Santalum shoot DW / host shoot DW. The host use efficiency of A. trachycalpa was greater than that of the other hosts. The xylem sap of hosts and Sarztalum, and ethanolic extracts of endophytic tissue of haustoria of Santalzkm were analysed for amino acids, organic acids and sugars to determine which solutes were available in the host and which were extracted by the Santalum haustoria from different hosts. There were similarities between Santalum and legume hosts in concentration and composition of xylem sap amino acids, and in the amino acid spectra of the corresponding Santalum endophytic tissue, whereas there were low N levels in xylem sap of E. camaldulensis and dissimilarities between its amino acid composition and that of Santalum. This indicated substantial direct intake of xylem N by Santalum from legume hosts but little N from the xylem sap of E. canzaldulensis. There were high concentrations of asparagine, glutamate, aspartate and y-amino glutamate in the xylem sap of the legume hosts, while in the non-legume the most common amino acids were glutamate, aspartate, glutamine and arginine. Proline, the predominant amino acid in the xylem sap of Santalum acurninatum growing in natural vegetation (Tennakoon et al. 1997) was not detected or present in very low concentrations in Santalurn album under these conditions. in the non-legume. Xylem sap of hosts contained variable amounts of sugars (sucrose, glucose and fructose) and organic acids (fumaric, citric and malic acid), whereas that of the parasitic Santalum was dominated by fructose and malic acid. Dissimilarities in the proportional amounts of xylem-borne sugars and organic acids were particularly evident for the E. camaldulensis : Santalum partnership. Diurnal profiles of photosynthesis and transpiration of Santalum were closely similar to those for corresponding hosts, whereas the midday leaf water potential of Santalum was consistently more negative than that of corresponding hosts. Net photosynthesis and water use efficiency was lower, but transpiration rates were similar to that of corresponding hosts. Nitrogen concentrations of foliage of Santalum were higher than their hosts, and higher when on legume hosts than on E. camaldulensis, or without a host. Nitrogen concentrations of Santalum foliage was strongly correlated with net photosynthesis and water use efficiency of Santalum. 813C values of shoot dry matter of Santalum were poorly correlated with instantaneous water use efficiency of Santalum. Tissue water relations of Santalum were similar to that of water-stress tolerant species. S. formosa proved the best host followed by Acacia ampliceps and A. traclzycarpa based on dry matter gains of Santalum. Estimates of heterotrophic gain of C of Santalum when grown in association with the legume hosts over a nine week period indicate 57.9% of C was derived from A. ampliceps, 45.5% from A. trachycarpa and 34.6% fiom S. fomosa. Abundance of haustorial attachments on roots of hosts was poorly correlated to Santalum shoot DW. Root nodules of legume hosts were parasitised by a small proportion of Santalum haustoria. Sodium and phosphorus concentrations of foliage of Santalum were generally higher than that of corresponding hosts. Net gains of calcium, potassium, phosphorus and sodium in Santalum was greatest when grown in association with hosts richest in the corresponding element. Net losses or only small gains of calcium, potassium, phosphorus and sodium were recorded when Santalum was grown with E. camaldulensis or without a host suggesting that Santalum has limited ability for uptake of those minerals through its own root system. To understand the effect of hosts on the productivity of a Santalum plantation a young plantation of Santalum with three host species Cathormion umbellatum, Sesbania formosa and Acacia anuera was selected to study the relationship between host quality and distance of hosts from Santalunz on Santalum health. The selected plantation showed marked decline in health and vigour of both Santalum and hosts between years 3 and 5. Parameters of the host plants were assessed to select the best predictor of Santalunz crown health. The height and diameter growth increment of Santalum between years 3 and 5 was strongly correlated to Santalum crown health. Santaluin crown health and growth increased as host quality increased, and the distance of host fiom Santalum decreased. An index, which combined host quality and the distance of the host from that of Santalum, was a better predictor of Santalum crown health than host distance or quality alone. The age at which heartwood is initiated in Santalum album under plantation conditions in Western Australia in unknown, but in natural stands in India it occurs between 10-13 years of age (Rai 1990). A field experiment was conducted to determine the efficacy of stem injections of paraquat andlor ethrel in initiating heartwood formation in five year old Santalum trees in a plantation. Trees injected with paraquat alone had a significantly greater extension of induced heartwood, both radially and vertically, than those trees injected with ethrel alone or distilled water. Eight months after treatment with paraquat or ethrel or a combination of these chemicals induced heartwood was formed, which had high lipid, and low starch and polysaccharide concentrations compared to the sapwood. Induced heartwood from both chemical treatments and their combinations contained total volatile oil and santalol oil (alpha and beta santalol) concentrations that were equal to or greater than that of naturally formed heartwood and greater than that of sapwood. Moisture content, and concentrations of K and Mg, and in some treatments Ca of induced heartwood were significantly lower than that of sapwood. The thesis concludes with a synthesis of the findings and suggestions for future research, with special reference to mid-rotation aspects of Santaltrm plantation silviculture.

Thesis (Ph. D.)--University of Melbourne, 1994. / Typescript (photocopy). Includes bibliographical references (leaves 235-246).

Thesis (Ph. D.)--Ohio State University, 2004. / Title from first page of PDF file. Document formatted into pages; contains xvii, 232 p.; also includes graphics (some col.). Includes bibliographical references (p. 206-232).