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biosecurity built on science
Genome informed Diagnostics
Plant Biosecurity Cooperative Research Centre
biosecurity built on science
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
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
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22)
End
emic
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1
End
emic
sp.
2
End
emic
sp.
3
During the 1997 fire blight incursion a false positive result from samples in the Adelaide Botanical Gardens caused the
shut down of trade
biosecurity built on science
• 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
biosecurity built on science
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
biosecurity built on science
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
biosecurity built on science
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
biosecurity built on science
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)
biosecurity built on science
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)
biosecurity built on science
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)”
biosecurity built on science
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
biosecurity built on science
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
biosecurity built on science
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!!
biosecurity built on science
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