Potato is one of the most important crops grown in Canada and all over the world. Late blight caused by P. infestans is one of the major diseases of potato and is mainly managed by fungicides application. The extensive use of fungicides not only causes adverse effects on the environment but also accelerates the development of resistance in this pathogen. Horizontal resistance is considered as the best choice to control P. infestans as it is durable over years. Breeding for durable resistance requires evaluation of hundreds of breeding lines in greenhouses and in the field. This is usually done by testing several epidemiological parameters such as infection efficiency, lesion size, latent period, and area under disease progress curve (AUDPC). These methods are time-consuming and expensive. The present study reports standardization of metabolic profiling protocols and exploration of metabolic profiling based on GC/MS as an additional tool to discriminate resistance in potato against late blight. Potato cultivars varying in horizontal resistance against late blight have been inoculated with water or the pathogen and more than 100 metabolites have been tentatively identified by GC/MS. Univariate analysis has been used to identify several pathogenesis related (PR) and defense related (DR) metabolites that have potential for application as resistance biomarker metabolites. Multivariate analysis of the abundances of metabolites (the mass spectral (MS) ion trap detector outputs were obtained using Saturn Lab Software Version 5.52 and these abundances are positively proportional to the concentration of mass ions of metabolites) in cultivars were mainly used to identify pathogenesis and resistance functions. Following pathogen inoculation, several metabolites such as amino acids, organic acids, fatty acids and sugars, were significantly increased in abundances, especially in the resistant cultivar. Other metabolites such as phenylalanine, tyrosine, shikimic acid and malonic acid detected here are well known for their direct participation in the shikimic acid, the phenylpropanoid, and the malonic acid metabolic pathways. These pathways lead to the production of several defense metabolites including antimicrobial compounds including phenolics, flavonoids and phytoalexins. The metabolic profiling technology developed here has the potential application for screening of potato breeding lines for horizontal resistance against late blight.

Thesis (M.S.)--University of Hawaii at Manoa, 2006. / Includes bibliographical references (leaves 103-108). / 108 leaves, bound col. ill. 29 cm

Phytophthora cinnamomi has been recognised as a key threatening process to Australia’s biodiversity by the Commonwealth’s Environment Protection and Biodiversity Conservation Act 1999. Despite over 80 years of extensive research, its exact mode of survival is still poorly understood. It is widely accepted that thin- and thick-walled chlamydospores are the main survival propagules while oospores are assumed to play no role in the survival of the pathogen in the Australian environment, yet evidence is limited. The saprophytic ability of the pathogen is still unresolved despite the important role this could play in the ability of the pathogen to survive in the absence of susceptible hosts. This thesis aimed to investigate chlamydospores, oospores and the saprophytic ability of P. cinnamomi to determine their contribution to survival. Phytophthora cinnamomi did not show saprophytic ability in non-sterile soils. The production of thick-walled chlamydospores and selfed oospores of P. cinnamomi in vitro was documented. Thick-walled chlamydospores were sporadically formed under sterile and non-sterile conditions in vitro but exact conditions for stimulating their formation could not be determined. The formation of thick-walled chlamydospores emerging from mycelium of similar wall thickness was observed, challenging the current knowledge of chlamydospore formation. Selfed oospores were abundant in vitro on modified Ribeiro’s minimal medium in one isolate. Three other isolates tested also produced oospores but not in large numbers. Although the selfed oospores did not germinate on a range of media, at least 16 % were found to be viable using Thiozolyl Blue Tetrazolium Bromide staining and staining of the nuclei with 4´, 6-diamidino-2-phenylindole.2HCl (DAPI). This indicated the potential of selfed oospores as survival structures and their ability to exist dormantly. The ability of phosphite to kill chlamydospores and selfed oospores was studied in vitro. Results challenged the efficacy of this chemical and revealed the necessity for further study of its effect on survival propagules of P. cinnamomi in the natural environment. Phosphite was shown to induce dormancy in thin-walled chlamydospores if present during their formation in vitro. Interestingly, dormancy was only induced by phosphite in isolates previously reported as sensitive to phosphite and not those reported as tolerant. Chlamydospores were produced uniformly across the radius of the colony on control modified Ribeiro’s minimal medium but on medium containing phosphite (40 or 100 µg ml-1), chlamydospore production was initially inhibited before being stimulated during the log phase of growth. This corresponded to a point in the colony morphology where mycelial density changed from tightly packed mycelium to sparse on medium containing phosphite. This change in morphology did not occur when the pathogen was grown on liquid media refreshed every four days, and chlamydospores were evenly distributed across the radius of these colonies. This trend was not observed in selfed oospores produced in the presence of phosphite. Selfed oospore production was found to be inhibited by phosphite at the same concentrations that stimulated chlamydospore production. Isolates of P. cinnamomi were transformed using a protoplast/ polyethylene glycol method to contain the Green Fluorescent Protein and geneticin resistance genes to aid in future studies on survival properties of the organism. Although time constraints meant the stability of the transgene could not be determined, it was effective in differentiating propagules of the transformed P. cinnamomi from spores of other microrganisms in a non-sterile environment. Two different sized chlamydospores (approximately 30 µm diameter and < 20 µm diameter) were observed in preliminary trials of transformed P. cinnamomi inoculated lupin roots floated in non-sterile soil extracts and these were easily distinguished from microbial propagules of other species. The growth and pathogenicity was reduced in two putative transformants and their ability to fluoresce declined over ten subcultures but they still remained resistant to geneticin. This study has improved our knowledge on the survival abilities of P. cinnamomi in vitro and has provided a useful tool for studying these abilities under more natural glasshouse conditions. Important implications of phosphite as a control have been raised.

This project assesses two aspects of DNA detection of Phytophthora species from soil samples. Firstly, a nested PCR protocol was established with both primary and nested PCR specific for P. cinnamomi detection. PCR amplification of P. cinnamomi DNA isolated from soil was optimised with the addition of bovine serum albumin and formamide. This was found to improve both the specificity and sensitivity of PCR amplification of DNA in the presence of inhibitors co-extracted along with the target DNA from soil samples. The application of diagnostic nested PCR with the addition of BSA and formamide was verified by comparison with routine culture based detection methods. In all cases, nested PCR detection incorporating BSA and formamide was found to be considerably more sensitive than the culture based detection methods. The second component of this thesis investigates the simultaneous detection of multiple species of Phytophthora using microarray analysis. Microarray based detection has been previously limited by variable and inconsistent hybridisation intensities across the diversity of probes used in each array. In this study a novel concept for the differentiation of detection targets using duplex melting kinetics is introduced. A microarray assay was developed on a PamChip „¥ microarray enabling the differentiation of target Phytophthora species using the melting kinetics of probe-target duplexes. In the majority of cases the hybridization kinetics of target and non-target duplexes differed significantly. Analysis of the melting kinetics of duplexes formed by probes with target and non-target DNA was found to be an effective method for determining specific hybridization and was independent of fluctuations in hybridization signal intensity. This form of analysis was more robust than the traditional approach based on hybridisation intensity, and allowed the detection of individual Phytophthora species and mixtures there of.

Thesis (Ph.D)--Murdoch University, 2003. / Thesis submitted to the Division of Science and Engineering. Bibliography: leaves 219-235.

Thesis (Ph.D.)--Murdoch University, 2005. / Thesis submitted to the Division of Science and Engineering. Bibliography: leaves 160-177.

Thesis (M.S.)--Oregon State University, 2009. / Printout. Includes bibliographical references (leaves 58-61). Also available on the World Wide Web.

Thesis (M.S.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 69-72). Also available on the World Wide Web.

Thesis (Ph. D.)--Ohio State University, 2005. / Document formatted into pages; contains 215 p. Includes bibliographical references. Abstract available online via OhioLINK's ETD Center; full text release delayed at author's request until 2006 March 3.

Univ., Diss., 2008--Kassel.