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• W31662457 abstract "Sugarcane is an agronomically important crop worldwide and in Australia it is worth up to AU$2 billion per annum in export. Sugarcane grown in continuous monoculture is prone to numerous diseases including disease caused by pathogens potentially involved in the syndrome of yield decline. Yield decline is defined as the diminishing ability of caneland to produce sugar per harvested hectare. The soilborne pathogens that possibly contribute to this syndrome include species belonging to the oomycete genera Pythium and Pachymetra. The overall aim of the research described in this thesis is to elucidate the relative importance of soilborne pathogens of sugarcane in yield decline through the development of diagnostic tests. The pathogens targeted in this thesis are Pachymetra chaunorhiza, Pythium arrhenomanes and Pythium graminicola. The first step towards developing diagnostic tests for the abovementioned species was to assess the genetic diversity within and among the different pathogens. To this end, the ribosomal RNA genes including the internal transcribed spacer (ITS) and 5.8S regions were selected to provide variable and conserved DNA sequence information for analysis. Representative isolates of Pythium arrhenomanes and Pythium graminicola were sequenced in conjunction with isolates representing twelve other Pythium species (including other possible sugarcane pathogens) to determine phylogenetic relationships within this genus. In addition, DNA sequence analysis of the same genomic region was undertaken for Pachymetra chaunorhiza. This pathogen is unique to Australia and very little is known about it, especially at the molecular level. Due to its devastating effect on sugarcane it is an important target for the development of diagnostic tests. A comparison of these three oomycete pathogens was made with database sequences of Phytophthora (another oomycete genus). Phylogenies based on these sequences showed that Pythium species associated with root rot of sugarcane (P arrhenomanes, P. catenulatum, P dissotocum, P. graminicola, P. myriotylum) were phylogenetically diverse and readily distinguished from each other and from other Pythium species. In particular, it was possible to use ribosomal sequence information to distinguish the morphologically similar P. arrhenomanes and P. graminicola. It was also apparent that the genus Pythium displays greater genetic diversity than Phytophthora and that some Pythium species (Pythium vexans and Pythium ostracodes) represent groups that are phylogenetically closer to Phytophthora than Pythium as currently defined. These findings highlight the necessity for aligning relationships based on traditional taxonomic criteria with relationships based on DNA sequence analysis. Analysis of ITS ribosomal sequences indicated very high similarity (g95%) amongst isolates within each of the three target species: Pachymetra chaunorhiza, Pythium arrhenomanes and Pythium graminicola. It was possible to identify regions of sequence conservation and variability that enabled the design of species-specific PCR primers that captured genetically diverse members within a species but excluded other closely related species. Pythium arrhenomanes and Pythium graminicola display a high level of morphological similarity and a multiplex polymerase chain reaction (PCR) assay based on ribosomal DNA differences allowed differentiation of these two species. Three real time PCR tests, highly specific and sensitive for each pathogen, were successfully developed. These tests were based on TaqMant real time PCR and involved the design of species-specific primers and fluorescent probes. Subsequent work involved optimisation and laboratory validation of these tests on cloned target and genomic DNA templates. Development of a multiplex real time PCR assay for the simultaneous detection of Pythium graminicola and Pachymetra chaunorhiza was explored, as well as the development of a general oomycete assay using a universal TaqMan probe that could detect all three pathogens simultaneously but without differentiating them. Such an assay could provide a diagnostic tool towards managing yield decline in sugarcane where all three target pathogens are involved. Finally, extension of the real time PCR assay to root and soil samples was explored. Pachymetra chaunorhiza was selected for detection in real samples as it is currently considered to be the most destructive soilborne pathogen of sugarcane and there is a real need for quantitative soil assays to determine infestation levels of Pachymetra oospores. Root samples of known resistance/susceptibility ratings were collected from a glasshouse trial for quantification of the level of Pachymetra chaunorhiza within them. Soil samples were collected from 15 field sites representing diverse locations and soil types, and manual oospore counts of these samples were correlated with the specific TaqMan assay for Pachymetra chaunorhiza. Initially, diseased root samples were targeted for Pachymetra chaunorhiza detection and P. chaunorhiza DNA could be detected by the specific real time PCR assay with amounts varying from 0.78 - 60.9 ng per mg of original root tissue (dry weight). No correlation was observed between resistance/susceptibility rating of all varieties tested and the amount of P chaunorhiza detected but P chaunorhiza could be reliably detected in roots. Analysis of 15 field soil samples representing various soil types also displayed no correlation between oospore counts and the amount of P chaunorhiza detected. However, correlation of these two factors was apparent if results from only one soil type were considered. As a result, a soil-based real time PCR assay could provide an extremely useful diagnostic tool especially if the effect of different soil types on extraction of DNA and PCR inhibition is understood. Another issue that needs consideration is the age and condition of oospores and their resulting viability. While these issues were not studied in this thesis an analysis of oospore infestation levels in a soil dilution series was undertaken. This experiment removed the variables of soil type and varying oospore age within a sample. The soil extraction procedure and real time PCR assay yielded a linear relationship between oospore count and amount of P chaunorhiza DNA detected for this soil dilution series. The significantly high correlation coefficient found in this experiment validates the quantitative assay for a given soil type and oospore quality. Further experiments will be needed to explore the effect of variation in soil type and oospore viability on quantitative PCR data. This thesis describes the successful development, from phylogenetic analysis to PCR design and optimisation, of diagnostic assays for the abovementioned oomycete species. These assays are of considerable potential benefit to the Australian sugarcane industry in assisting research and management of the syndrome of root disease and yield decline.n" @default.
• W31662457 created "2016-06-24" @default.
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• W31662457 date "2002-01-01" @default.
• W31662457 modified "2023-09-27" @default.
• W31662457 title "Development and application of DNA-based technologies for identification and analysis of soilborne oomycetes associated with yield decline in sugarcane" @default.
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