New approaches for diagnosing bacterial pathogens of horticulture

1. biosecurity built on science
Early and accurate detection of
bacterial pathogens
Rachel Mann
Research Scientist DEDJTR
Plant Biosecurity Cooperative Research Centre

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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

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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

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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

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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

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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

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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

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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)

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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

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How will this research be delivered?
In-field validation

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How will this research be delivered?
Biosecurity Staff – “Training the Trainer”

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How will this research be delivered?
Biosecurity Staff –
“Hands-on”

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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

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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

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