Thesis (Ph. D.)--University of Missouri-Columbia, 2003. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.

No description available.

No description available.

No description available.

Diseases caused by micro-organisms are still a major threat to the agro-industry worldwide. Diseases not only have negative effects on crop yields, but also they can affect the quality of crops post-harvest. Genetic engineering is one of several strategies that have been developed to control plant diseases and to enhance plant disease resistance to pathogens. Although some genetic strategies have provided plants with enhanced disease resistance, some pathogens can easily overcome this resistance by rapid evolution resulting in a lack of durability in the field. The oomycete Phytophthora infestans, the causal pathogen of late blight disease of potato, is an example of a crop pathogen that causes a major problem in one of the most important crops worldwide. Many efforts have been made trying to control this pathogen including chemical controls and genetic engineering, but unfortunately it remains a severe problem and the control measures are rarely very successful. Due to the complexity of this pathogen, and to limit the need for chemical control, breeding programmes to incorporate durable forms of genetic resistance are crucially needed. Although, this type of resistance is believed to be effective against all known races of P. infestans and provide in additional some level of general resistance, until now the genetic bases of this type of resistance is still unknown and the molecular mechanisms poorly understood. This project set out to isolate and identify gene sequences that are induced during the compatible interaction between cultured potato plants and P. infestans, specifically those leading to the establishment of durable resistance. It was demonstrated that the potato variety Stirling is capable of developing this type of resistance as judged by the development of resistant shoots during the interaction with Phytophthora. These shoots showed very strong resistance not only to Phytophthora but also to other potato pathogens (R. solani and F. sulphureum) even after two generations of culturing the plants in the absence of the pathogen. The fast production of ROS and the tight deposition of callose surrounding the hypersensitive cells, which deprive the pathogen of nutrients and limit pathogen growth to a small region of the plant, may be important factors in the success of the durable plants in defending themselves against the pathogen attack.

In order to identify potential avirulence factors of <i>Phytophthora infestans</i>, secreted protein profiles of six strains (IPO-0, CBS431.90, IPO-655-2A, IPO-428-2, 88069 and 90128) with different avirulence phenotypes were analysed by proteomics.  The proteins from culture filtrates were visualised on 2D gels with a total number between 350 and 1000.  PiAVR X was found only in avirulent strains and considered therefore as a possible avirulence determinant.  Its expression increased in all strains tested when grown in Modified Henniger medium.  Single nucleotide polymorphisms (SNPs) were detected within a 735 nt fragment of <i>PiAvrX</i> but none was linked to avirulence characteristics.  The consensus EST database facilitated identification of approximately 50% of 45 most abundant proteins excised from strain 88069.  Beta-glucosidase, Pi-NIP2 (<i>Phytophthora infestans</i> necrosis inducing protein-like protein), enoyl CoA hydratase, glutathione S transferase, peptidylprolyl isomerase, acidic chitinase and Pi-PR1 (<i>Phytophthora infestans </i>homologue of plant PR-1 protein) were among the identified proteins with a recognisable signal sequence.  Enolase, quinine-oxido reductase, nucleoside di-phosphate kinase, actin depolymerisation factor, thioredoxin, ubiquitin and 14-3-3 protein were also identified but without a signal peptide.  The occurrence and expression of <i>Pi-nip2, Pi-chi1 </i>and <i>Pi-pr1</i> were confirmed in the strains tested.  Transformants were obtained from <i>P. infestans</i> strain T30-4 via a biolistic particle delivery approach using a single plasmid vector containing <i>Pi-pr1</i>.  Detailed analysis of these transformants did not demonstrate induction of homology-dependent gene silencing.  It was found that transformation rates were different among the tested <i>P. infestans</i> strains.

Thesis (Ph. D.)--University of Hawaii at Manoa, 1995. / Includes bibliographical references (leaves 109-111). / Microfiche. / ix, 113 leaves, bound ill. 29 cm

Phytophthora cinnamomi Rands is a common and devastating pathogen of cultivated proteas worldwide. Webb (1997) described a Sudden Death plant disease of proteas in Western Australia (WA) protea plantations. Proteas that suffer the syndrome display symptoms such as stunted growth, wilting, chlorosis and often death. In the current study, a number of protea plantations in the southwest of WA were visited to quantify the extent that P. cinnamomi was attributing to deaths of cultivated proteas. The survey indicated that P. cinnamomi is the major cause of Sudden Death in proteas. A range of other fungi (Fusarium, Botryosphaeria, Pestalotiopsis, Alternaria) and pests (nematodes, mealy bug, scale insects) were also identified to be contributing to protea death and decline in WA plantations. In many cases the factors contributing to protea disease appeared complex, with a range of physical factors or nutritional imbalances commonly associated with these pathogens and pests. As P. cinnamomi was the major cause of death of cultivated proteas the remainder of the experiments described in this dissertation investigated its control in horticultural plantings. Biofumigation has the potential to become an important technique in an overall integrated management approach to P. cinnamomi. In this thesis, biofumigation refers to the suppression of pathogens and pests by the incorporation of Brassica plants into the soil. Two biofumigants (Brassica juncea (L.) Czern., B. napus L.) were screened for their effect on the in vitro growth of five common Phytophthora species (P. cinnamomi, P. cactorum (Lebert & Colin) Schroeter., P. citricola Sawada, P. cryptogea Pethyb. & Laff. and P. megasperma Drechsler). Growth was determined by the measuring dry weight and radial growth of vegetative hyphae. B. juncea was found to be superior in its suppressive effect compared to B. napus. There was also significant variation in the sensitivity of the Phytophthora species to the suppressive effects of the biofumigants. P. cinnamomi was the most sensitive of the five species investigated. Where the rates of the biofumigant were sufficient to suppress growth of Phytophthora, the suppressive effect was mostly fungicidal. To determine how B. juncea and B. napus affect the infective ability and survival of P. cinnamomi, their effects on sporangia and chlamydospores production in soil was investigated in vitro. P. cinnamomi colonised Miracloth discs were added to soil amended with the two Brassica species, before being removed every two days over an eight day period for the determination of sporangia production, chlamydospore production and infective ability. Only the soils amended with B. juncea significantly reduced sporangia production in P. cinnamomi. Both Brassica species increased the percentage of aborted or immature sporangia and reduced the infective ability of the pathogen. Neither Brassica species had any effect on zoospore release or chlamydospore production in P. cinnamomi. Soil cores and soil leachate were collected from biofumigant-amended field soils to determine the inoculum potential and infective ability of the pathogen under glasshouse conditions. Amending the soil with both Brassica species had an immediate suppressive effect on the inoculum potential and infective ability of the P. cinnamomi. However, after this initial suppression there was a gradual increase in the recovery of the pathogen over the monitoring period of four weeks. To determine if the suppression would result in decreased disease incidence in a susceptible host, Lupinus angustifolius L. seeds were planted in the biofumigant amended soil. B. juncea amended soils reduced the disease incidence of P. cinnamomi by 25%. B. napus had no effect on disease incidence in L. angustifolius. Although the current study had demonstrated that biofumigants could suppress the growth, sporulation and infection of P. cinnamomi, it was unclear if this would equate to a reduction in disease incidence when applied in the field. A field trial was conducted on a protea plantation in the southwest of Western Australia that compared biofumigation with B. juncea to chemical fumigation (metham sodium) and soil solarisation. The three soil treatments were used in an integrated management approach to control P. cinnamomi that included the use of a hardwood compost, mulch and water sterilisation. All treatments were monitored during their application to ensure the treatments were conducted successfully. The three soil treatments significantly reduced the recovery of the pathogen and the infective ability of the pathogen to a soil depth of 20 cm. Metham sodium was the most suppressive soil treatment and soil solarisation was the least suppressive treatment. Only the metham sodium treatment resulted in a significant reduction in the incidence of root rot in Leucadendron salignum P.J. Bergius x laureolum (Lam.) Fourc (c.v. Safari Sunset) over the monitoring period of three years. Another field trial was conducted on the same protea plantation to compare the effectiveness of B. juncea and B. napus, without the use of other control strategies, to reduce the incidence of P. cinnamomi infection of Leucadendron Safari Sunset. The concentration of isothiocyanates was monitored for seven days after the incorporation of the biofumigants. Although both Brassica species reduced the recovery and infective ability of the pathogen, neither biofumigant reduced the incidence of root rot in Leucadendron Safari Sunset. In conclusion, P. cinnamomi is the most common and devastating pathogen in WA protea plantations. The current study demonstrated that P. cinnamomi is sensitive to the suppressive nature of biofumigants. Biofumigants can suppress the in vitro growth, sporulation, infective ability of P. cinnamomi and reduce the incidence of the disease caused by the pathogen in the glasshouse. Of the two Brassica species investigated, B. juncea was superior in its ability to control P. cinnamomi compared to B. napus. When applied in the field, biofumigation using B. juncea was found to be more suppressive that soil solarisation, but not as effective as metham sodium.

Phytophthora diseases have caused worldwide economic, social and environmental impacts for decades. Once their presence is confirmed, they are difficult to eradicate. To reduce and manage the damage inflicted by the pathogen, fast and reliable disease management protocols are required. Tests that enable the rapid and reliable identification of the pathogen assist greatly in disease management. Phytophthora species are traditionally not only detected by baiting but also by plating of symptomatic tissue on selective media. Species can be identified by the characteristics of the mycelium growing out of the bait. However, the method is low throughput, labour intensive, and prone to false negatives. An alternative approach would be to detect the pathogen by the presence of its DNA. This involves amplification of the pathogen DNA using Polymerase Chain Reaction (PCR) and detection of the amplification product. Detection is usually by agarose gel electrophoresis. However, this is also a labour intensive process involving pouring, loading, running, and staining of the gels. The aim of this thesis is to explore the use of Matrix Assisted Laser Desorption/ Ionisation Time-of-Flight (MALDI-TOF) mass spectrometry for detection of PCR products. This procedure enables the analysis of large numbers of samples within a very short time-frame as the average time for analysis of each sample is in the order of milliseconds. The assay involves annealing an extension (genotyping) primer to the PCR product and its extension by a single nucleotide. The nature of the nucleotide added differentiates species as does the site to which the primer anneals. Multiple extension (genotyping) primers can be used together in a single reaction for detection of multiple species. In this project four genotyping primers (GPs) were designed from the ITS regions of Phytophthora palmivora, Phytophthora cinnamomi, Phytophthora citricola, and Phytophthora cambivora. The extension primers were tested for their specificity on the DNA of the target species. The four primers designed were specific for their intended targets except for GPpalm3 which in addition to being extended by ddT when tested with DNA from P. palmivora, was also extended by ddC when tested with DNA from other species of Phytophthora or Pythium. These primers were also tested for their ability to detect multiple Phytophthora species in a single reaction (multiplexing). Mixtures of primers were added to mixed DNA templates and the primer extension reaction carried out. The primers were designed so that their masses were sufficiently different for them to be identified from a mixture. Six replicates were analysed for each reaction. In general only about 1-3 of the six replicates gave a positive reaction. This indicates that there may be some interference between primers, or that the presence of all four nucleotides interfered with the primer extension reaction. Increasing either the amount of enzyme, the amount of nucleotides or both did not improve the results. The sensitivity of detection was tested by the addition of different amounts of mycelium to soil. The detection sensitivity depended on the primer pair used for PCR amplification. The ITS1/2 primer pair was more sensitive than the ITS1/4 pair. The limit of detection was 1 ìg mycelium g soil-1. However using nested PCR, levels of sensitivity comparable to those obtained using the ITS1/2 primer pair could be achieved. Primers to other regions of the genome such as the beta cinnamomin elicitin gene gave very low levels of sensitivity compared to the ITS primers. In comparison with DNA detection we found that the limit of detection using baiting was 4 ìg mycelium g soil-1. Results below this limit were unreliable. The method suffered from the additional disadvantage that it took a long time in comparison to DNA detection. DNA detection methods do not distinguish between living and dead organisms in the soil. However it can be hypothesised that DNA is unlikely to persist for any significant length of time in soil. To test this, we added plasmid DNA to soil and tested the persistence of this DNA using a variety of methods such as precipitation of labelled DNA, southern blotting and PCR amplification. It was found that in general, in soils from different ecosystems, the bulk of the DNA was undetectable after 24 hours. The rate of DNA breakdown differed with the soil type. In some soils, the added DNA was not detected even after 2 hours, whereas in others it could be observed after 10 hours. The detection depended on the method. Southern blotting showed that although DNA could be observed at 10 hours, by 24 hours it was completely degraded. In contrast a PCR product could be obtained from the soil extracts up to 24 hours. In a separate experiment, plasmid DNA was detectable over a 24 hour incubation period in 5 soil samples from 5 different sites. The results suggest that DNA is degraded rapidly in soil and is unlikely to persist longer than 24 hours. The results in this thesis demonstrate that MALDI-TOF MS is a suitable alternative to agarose gel electrophoresis for analysis of PCR products. The technique is rapid, differentiates species from mixtures, is high-throughput and amenable to automation. Implementation will require further research to automate the primer extension assay to reduce the sensitivity to impurities in the DNA and to design parameters for sampling asymptomatic material.