This document summarizes a project that aims to develop diagnostic tools for detecting exotic plant pathogens using a genome-informed approach. The project generates knowledge of plant pathogenic bacteria and strategies for diagnostic development. Key outputs include scientific publications, diagnostic protocols validated in Australia, and tools to facilitate early detection and response. The diagnostics produced will help secure border protection and support trade by enabling accurate identification of pathogens.

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Genome informed Diagnostics
Plant Biosecurity Cooperative Research Centre

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PBCRC 2002/2156 – Project goal
 Use a genome-informed approach to develop
diagnostic tools for the detection of exotic
phytopathogenic bacteria that pose a significant
threat to Australian Agriculture.
 Relevant pathogens:
Fire blight
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 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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• 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
Research Strategy
 We are designing detection tools to differentiate at species
and 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

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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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Benefit for industry
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 Agriculture
 Establishing capability which will accelerate delivery of
diagnostics for newly evolved pathogens

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Research Impact – an end user’s perspective:
• Science Capability
• 9 scientists (post docs and PhD aligned with PBCRC2156)
• Re-established Plant Bacteriology capability in Australia
• Peer reviewed science
• The diagnostic pipeline
• Diagnostic tools are/will be published
• In country validation (surveys)
• Smart Surveillance Tools
• LAMP vs RPA vs ????
• SNPHS

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End User Perspective - Diagnostician
PBCRC 2002/2156 Outputs:
 National diagnostic protocols (NDPs) validated in Australia for
the Subcommittee on Plant Health Diagnostics (SPHD)
- 4 (plus!!!) National Diagnostic Protocols
Why are NDPs important?
- Provide a minimum standard to detect a pest/pathogen
- Accurate, reliable diagnostics are needed to support
quarantine responses and trade related decisions
- Provide a baseline diagnostic assay to facilitate comparison of
test results between diagnostic labs
- NDPs are endorsed by Plant Health Committee (PHC)

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Intergovernmental
Agreement on
Biosecurity
National Plant Biosecurity
Strategy
National Plant
Biosecurity
Surveillance
Strategy
National Plant
Biosecurity
Diagnostics
Strategy
National Plant Biosecurity Strategy
Subcommittee for
National Plant
Health Surveillance
(SNPHS)
Subcommittee for
Plant Health
Diagnostics
(SPHD)
Plant Health Committee (PHC)

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Subcommittee for Plant Health Diagnostics
(SPHD)
• Facilitate the development of a diagnostic capability and capacity for all High
Priority Pests
• Develop and recommend national standard processes relating to plant pest
diagnostics
• Promote and facilitate the development of National Diagnostic Protocols
(NDPs) for EPPs and endemic pests of national significance
• The National Plant Biosecurity Network (NBPDN)
(http://plantbiosecuritydiagnostics.net.au/)
- SPHD Reference Standard No. 2:
“Development of Diagnostic Protocols Instructions to Authors”
- Based on the IPPC ISPM No 27 “Diagnostic protocols for
Regulated Pests (IPPC 2006)”

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There are four outcomes when you conduct a
diagnostic assay for a “Target Species” on a
field sample:
Positive Negative False
Negative
False
Positive
Assay detects all
known subspecies,
pathovars, strains,
haplotypes within
the target species
- Assay detects
closely related
species/
organisms
- Lab
contamination
Target species was
not present within
the detectable
limits of the assay
- Assay fails to
detect a
pathovar/strain
of the target
species
- Lab error
- Inhibition of test
assay
NATA, Proficiency testing

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Positive Negative False
Negative
False
Positive
The Bacterial Pathovars project (PBCRC 2002/2156) and
associated PhDs are using a genomics approach to design and
validate molecular assays that
• detect all known subspecies, pathovars, strains, haplotypes
within the target species
• do not detect closely related species/organisms
The risk of false negative or false positive results can be reduced
by well designed diagnostic tools using a genomics approach and
appropriate test validation
4 NDPs under development

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Candidatus Liberibacter solanacearum
 CRC2002 Established proof of concept: bioinformatic selection of
differential diagnostic loci in a fastidious bacterial species
- Tripled the number of available useful CLso genomes in Genbank
- Established international CLso genomics/ diagnostics research network
- Thompson et al (2015). Genomes of ‘Candidatus Liberibacter solanacearum’
haplotype A from New Zealand and the United States suggest significant
genome plasticity in the species. Phytopathology 105: 863-871.
• CRC2156 SPHD NDP for new CLso diagnostic being drafted
• Validation of NDP under Australian conditions
• Adapt diagnostic for enduser use in field/ in situ
• CRC62116 Microflora analyses of the Australian eggplant psyllid
(Jacqueline Morris – PhD candidate)
• Candidatus Liberibacter brunswickensis
• HLB diagnostics detect Lbr False
positive!!

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Challenges and issues arising from the research
Challenges:
 Complexity of microbial ecology
 Bioinformatics
 Hypothetical proteins
 Field deployable molecular diagnostics
Issues:
 A systems approach is required to align genomic data
with pathogen biology

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Thank you
 For more information, please email
brendan.rodoni@ecodev.vic.gov.au

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