Early and accurate detection of bacterial pathogens

biosecurity built on science

Early and accurate detection of bacterial pathogens

Rachel MannResearch Scientist DEDJTR

Plant Biosecurity Cooperative Research Centre


Project TeamPBCRC 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

http://www.pbcrc.com.au/about/organisations/kansas-state-university

http://www.pbcrc.com.au/about/organisations/kansas-state-university

http://www.pbcrc.com.au/about/organisations/new-south-wales-department-primary-industries

http://www.pbcrc.com.au/about/organisations/new-south-wales-department-primary-industries


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

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During the 1997 fire blight incursion a false positive result from samples in the Adelaide

Botanical Gardens caused the shut down of trade


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


What are we doing about it?

Using genome-informed diagnostic design to improve detection of exotic phytopathogenicbacteria that pose a significant threat to Australian Agriculture.

Relevant pathogens:

FireblightErwinia amylovora

Zebra ChipCandidatus Liberibactersolanacearum

Citrus CankerXanthomonas citri pv. citir Bacterial canker of kiwifruit

Pseudomonas syringae pv. actinidiae


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



Key outputs– knowledge, strategy, tools and capacityTools: 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 BlightErwinia amylovora

Zebra ChipCandidatus Liberibactersolanacearum

Citrus CankerXanthomonas citri pv. citir

Bacterial canker of kiwifruitPseudomonas 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



Key outputs – knowledge, strategy, tools and capacityCapacity: 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)


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


How will this research be delivered?

In-field validation


How will this research be delivered?

Biosecurity Staff – “Training the Trainer”


How will this research be delivered?Biosecurity Staff –

“Hands-on”


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


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


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


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


Thank you

For more information, please email [email protected]

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PBCRC is established and supported under the Australian Government Cooperative Research Centres Programme

Early and accurate detection of bacterial pathogens

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