Early and accurate detection of bacterial pathogens
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This document summarizes a research project that aims to develop accurate and rapid diagnostic tools for detecting exotic bacterial plant pathogens through genome-informed design. The project is developing PCR and LAMP-based diagnostics for key pathogens like Erwinia amylovora and Xanthomonas citri. Field-deployable tools like LAMP and RPA are also being validated for "smart surveillance" directly in the field. The diagnostics and training provided by this research will benefit biosecurity agencies, diagnostic laboratories, and the horticultural industry by facilitating early detection and a rapid response to incursions.
Early and accurate detection of bacterial pathogens
- 1. biosecurity built on science Early and accurate detection of bacterial pathogens Rachel Mann Research Scientist DEDJTR Plant Biosecurity Cooperative Research Centre
- 2. biosecurity built on science Project Team PBCRC 2156 - Deployment of Validated Genome-Informed Bacterial Diagnostics KSU: James Stack, Grethel Busot, Mohammed Arif PFR-NZ: Grant Smith, Sarah Thompson, Rebekah Frampton, Kerry Sullivan DEDJTR VIC - AgriBio: Brendan Rodoni, Rachel Mann, Jason Shiller DPI NSW: Toni Chapman PHD students: Jacqui Morris, Rebecca Roach
- 3. biosecurity built on science What is the problem? For most plant pathogenic bacteria, accurate, rapid, low cost tools are not currently available (Palacio‐Bielsa et al. 2009) Accurate, rapid, low cost tools for detecting exotic plant pests are the foundation for: - secure border protection - rapid response to incursions - large‐scale active surveillance programs Correct identification is critical - Identification failures result in inappropriate responses - False negative, false positive Marker H20control E.amylovora(Ea322) Endemicsp.1 Endemicsp.2 Endemicsp.3 During the 1997 fire blight incursion a false positive result from samples in the Adelaide Botanical Gardens caused the shut down of trade
- 4. biosecurity built on science What is the problem? In particular, we are designing detection tools to differentiate at sub‐specific levels. For example: - Xanthomonas citri pv. citri, Citrus Canker (not in Australia) from X. citri pv. malvacearum, bacterial wilt of cotton (in Australia) - Pseudomonas syringae pv. actinidiae (Psa) high virulence strains (not in Australia) from low virulence strains (in Australia) - Candidatus liberibacter solanacearum haplotypes The best way to identify new diagnostic targets is by comparing the genomes of these populations of bacteria and identifying DNA targets specific to each group Genome-informed diagnostic design
- 5. biosecurity built on science What are we doing about it? Using genome-informed diagnostic design to improve detection of exotic phytopathogenic bacteria that pose a significant threat to Australian Agriculture. Relevant pathogens: Fireblight Erwinia amylovora Zebra Chip Candidatus Liberibacter solanacearum Citrus Canker Xanthomonas citri pv. citir Bacterial canker of kiwifruit Pseudomonas syringae pv. actinidiae
- 6. biosecurity built on science What are we doing about it and how will it be delivered? Key outputs – knowledge, strategy, tools and capacity Knowledge: A more fundamental understanding of key plant pathogenic bacteria and the closely associated species that can confuse phytosanitary procedures. Strategy: A generalised genomics-based strategy to develop diagnostic tools for plant pathogenic bacteria. Delivery Reports and scientific publications
- 7. biosecurity built on science What are we doing about it and how will it be delivered? Key outputs– knowledge, strategy, tools and capacity Tools: Detection and diagnosis tools to support national diagnostic networks and regulatory phytosanitary programs such as Post Entry Quarantine (PEQ) facilities. Delivery National diagnostic protocols (NDPs) validated in Australia for the Subcommittee on Plant Health Diagnostics (SPHDs) Field-deployable molecular tools for smart surveillance Scientific publications Fire Blight Erwinia amylovora Zebra Chip Candidatus Liberibacter solanacearum Citrus Canker Xanthomonas citri pv. citir Bacterial canker of kiwifruit Pseudomonas syringae pv. actinidiae - PCR diagnostics designed and validated - LAMP test validated - NDP protocol submitted to SPHDS - PCR diagnostics designed & validated - Field diagnostics developed & validated - PCR diagnostics designed - PCR diagnostics designed and validated - Field diagnostics developed & validated
- 8. biosecurity built on science What are we doing about it and how will it be delivered? Key outputs – knowledge, strategy, tools and capacity Capacity: Training in the disciplines and technologies that underpin plant biosecurity. Delivery Training of people across disease surveillance and diagnostic networks both domestically and internationally (e.g. training/ workshops with biosecurity officers & plant diagnosticians)
- 9. biosecurity built on science Field-deployable diagnostic tools for “Smart Surveillance” What are the field-deployable tools? - LAMP (Loop-mediated isothermal amplification) - RPA (Recombinase Polymerase Amplification) Both isothermal (stable temperature) DNA amplification Varying ways to visualise result
- 10. biosecurity built on science How will this research be delivered? In-field validation
- 11. biosecurity built on science How will this research be delivered? Biosecurity Staff – “Training the Trainer”
- 12. biosecurity built on science How will this research be delivered? Biosecurity Staff – “Hands-on”
- 13. biosecurity built on science Who will benefit from the research? PHC/SPHD/NPBDN USDA-APHIS-PPQ; USA NPDN Federal and State biosecurity agencies State and federal diagnostic laboratories Diagnostic laboratories of trade partners NAQS PEQ Biosecurity field officers /Surveillance teams Researchers INDUSTRY
- 14. biosecurity built on science Benefit for our horticultural industries Short Term Accurate detection tools with multiple stable targets Fast results with the ability to detect pathogens in-field Rapid and accurate diagnostics facilitate early pathogen detection and rapid response times. This minimises: - economic loss - environmental impact - social impact on farming communities Longer Term Building a bank of reliable diagnostics for use in Horticulture Establishing capability which will accelerate delivery of diagnostics for newly evolved pathogens
- 15. biosecurity built on science End-User Advocate’s Perspective “Currently many early detection surveillance activities are conducted through visual inspection, with suspect samples sent to the diagnostic laboratory. A test that could be run in field would reduce the number of suspect samples and this would in turn reduce the surveillance turnaround time.” - Dr. Louise Rossiter, Biosecurity NSW Leader of Plant Pest Surveillance
- 16. biosecurity built on science Future Complete NDPs for key pathogens Finalise field-deployable “smart surveillance” tools and protocols for key bacteria Continue to improve diagnostics for other important plant pathogenic bacteria The Ralstonia solanacearum species complex Rathayibacter toxicus
- 17. biosecurity built on science Thank you For more information, please email rachel.mann@ecodev.vic.gov.au This image cannot currently be displayed. PBCRC is established and supported under the Australian Government Cooperative Research Centres Programme