Genome informed diagnostics

biosecurity built on science

Genome informed Diagnostics

Plant Biosecurity Cooperative Research Centre

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


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


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


• 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


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


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


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


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)


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)


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)”

http://plantbiosecuritydiagnostics.net.au/


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


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


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!!


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


Thank you

For more information, please email [email protected]
