Diagnostics and genomic diversity of Erwinia amylovora
thesis
posted on 2023-01-18, 16:56 authored by Rachel MannSubmission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the Department of Botany, School of Life Sciences, Faculty of Science, Technology and Engineering, La Trobe University, Bundoora.
Fire blight is a destructive disease of plants in the family Rosaceae which has significant economic impacts on pear and apple production in countries where the disease is present. The causal agent, Erwinia amylovora, is not found in Australia and is a major biosecurity threat to the Australian pome fruit industry. Molecular diagnostics routinely used for detection of E. amylovora were unsuitable for use in a quarantine situation as they did not detect all strains of the species or gave false positive results for bacteria that were not E. amylovora. This project has taken a genomics approach to evaluation and design of tests for the detection of the pathogen, taking intra-species and inter-species genetic diversity into consideration when identifying molecular diagnostic targets. Intra-species genetic diversity of E. amylovora was determined using the genomes of 12 diverse strains to identify the pan-genome. The accessory genome, genes not shared by all strains, was analysed to identify potential host specificity factors, including variation in type III effector proteins, carbon utilisation pathways and a secondary metabolite pathway, that differed between the two host-specific groupings of E. amylovora, Spiraeoideae-infecting and Rubus-infecting strains. A genomics-based diagnostic target identification and development pipeline which utilised the core genome of E. amylovora (genes shared by all strains of the species), genomes of closely related species, and sequences housed in international databases, was developed to identify species-specific targets for E. amylovora. Putative targets were further analysed for their suitability for use in diagnostics and used to develop multiplex PCR diagnostic tests for E. amylovora and for the two host-specific groupings of the pathogen. This pipeline, and the holistic principles outlined in this project to develop and evaluate diagnostic tests, are applicable to diagnostic development for a wide variety of microbes.
Fire blight is a destructive disease of plants in the family Rosaceae which has significant economic impacts on pear and apple production in countries where the disease is present. The causal agent, Erwinia amylovora, is not found in Australia and is a major biosecurity threat to the Australian pome fruit industry. Molecular diagnostics routinely used for detection of E. amylovora were unsuitable for use in a quarantine situation as they did not detect all strains of the species or gave false positive results for bacteria that were not E. amylovora. This project has taken a genomics approach to evaluation and design of tests for the detection of the pathogen, taking intra-species and inter-species genetic diversity into consideration when identifying molecular diagnostic targets. Intra-species genetic diversity of E. amylovora was determined using the genomes of 12 diverse strains to identify the pan-genome. The accessory genome, genes not shared by all strains, was analysed to identify potential host specificity factors, including variation in type III effector proteins, carbon utilisation pathways and a secondary metabolite pathway, that differed between the two host-specific groupings of E. amylovora, Spiraeoideae-infecting and Rubus-infecting strains. A genomics-based diagnostic target identification and development pipeline which utilised the core genome of E. amylovora (genes shared by all strains of the species), genomes of closely related species, and sequences housed in international databases, was developed to identify species-specific targets for E. amylovora. Putative targets were further analysed for their suitability for use in diagnostics and used to develop multiplex PCR diagnostic tests for E. amylovora and for the two host-specific groupings of the pathogen. This pipeline, and the holistic principles outlined in this project to develop and evaluate diagnostic tests, are applicable to diagnostic development for a wide variety of microbes.
History
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Faculty of Science, Technology and Engineering. School of Life Sciences. Department of Botany.Thesis type
- Ph. D.
Awarding institution
La Trobe UniversityYear Awarded
2013Rights Statement
This thesis contains third party copyright material which has been reproduced here with permission. Any further use requires permission of the copyright owner. It also contained third party copyright material which has been removed pending the receipt of written permission(s). The thesis author has been contacted with a request to seek the appropriate permission(s) to allow the material to be reproduced. The thesis author retains all proprietary rights (such as copyright and patent rights) over all other content of this thesis, and has granted La Trobe University permission to reproduce and communicate this version of the thesis. The author has declared that any third party copyright material contained within the thesis made available here is reproduced and communicated with permission. If you believe that any material has been made available without permission of the copyright owner please contact us with the details.Data source
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