session 1: strengthening border biosecurity improving - post entry quarantine: researchers and...
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SESSION 1: STRENGTHENING BORDER BIOSECURITY
Improving post entry quarantine: researchers and end-users working together
Mark Whattam, Frank Greenhalgh and Roberto Barrero
biosecurity built on science
Strengthening border biosecurityImproving Post Entry Quarantine: Researchers and End-Users
Working Together
End User Advocate: Mark Whattam, OSS Director
Science Researcher: Roberto Barrero, PBCRC Project Leader
Industry Beneficiary: Frank Greenhalgh, Chair VSICA
Plant Biosecurity Cooperative Research Centre
biosecurity built on science
Presentation Outline
1. Challenges with current PEQ virus/viroid diagnostic platforms (Mark)
2. Using NGS to find viruses and viroids in imported plants (Roberto)
3. Results from PBCRC project (Roberto)
4. Potential impact from a strawberry industry perspective (Frank)
5. Where to from here? (Mark)
6. Questions
7. ……….. a challenge (Mark)
biosecurity built on science
1. Current PEQ virus diagnostic platforms (Mark)
Approx. 500 high risk plants imported annually mainly stone and pome fruit, citrus, potato, strawberry, grapevine, blueberry and raspberry
Tests include: Visual and biological indicators (herbaceous & woody indicators) Transmission electron microscope (TEM) Serological (ELISA) Molecular (PCR)
biosecurity built on science
Challenges with current PEQ diagnostic platforms
Classical diagnostic protocols can be slow (biological indicators 2+ years) => extends PEQ period
Declining availability of expertise to conduct biological indexing and results can be ambiguous (false +ve’s/-ve’s)
Typically target known pathogens (e.g. recent Vitis Biosecurity Import Risk Analysis recommended x16 PCR tests)
Expensive in terms of GH resources and wide range of tests required => e.g. fruit tree $4,000/cv.
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2. Using NGS to find viruses in imported plants? (Roberto)
As part of the ‘immune response’ to virus infection, plants produce ‘dicer’enzymes that cut up the infecting RNA from the virus (Andika et al., Plant J., 2015)
RNA can be sequenced (millions of them) and the virus genome reconstructed
Supercomputers and specialist software used to ID virus - no prior knowledge required
Nucleus
Invading virus
Plant cells produce DiceEnzymes to ‘attack’ the virus
Cutting into small RNAs
Small RNAs are extracted andSent to sequence provider
Which produce massiveAmounts of raw sequence data
Super computers and web basedBioinformatics analyse aad re-Assembles the data to work out
the identity of the virus
Virus puzzle
small RNA NGS (sRNA NGS)
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Viruses and viroids can be identified rapidly and reliably
Future vision: High risk plants spend less than 12 months in PEQ!
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3. Results of current PBCRC project (Roberto)
25 samples 9 samples 5 samples
Virus 24 (96%) 2 (22.3%) 3 (60%) 3 (100%)
Viroid 24 (96%) 0 0 1 (33.3%
Both 23 (92%) 0 0 1 (33.3%)
Vitaceae Solanaceae Rosaceae
Rutaceae
3 samples
NGS tests align well with known virus infections; more viruses were found in grapevine and raspberry samples using NGS c.f. current PEQ protocols
Overall 42 (68%) out of 62 samples were positive for viruses and/or viroids with remaining sample all being negative
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3. Results of current PBCRC project (Cont.)
Demonstrated use of sRNA NGS to reliably detect ssRNA(+), dsDNA, ssDNA viruses & viroids in 42 known +ve controls
Developed a fully automated, user friendly web-based bioinformatics toolkit (Yabi) and training manual for the diagnosis of viral pathogens
End-user training (Melbourne and Auckland workshops 2015 & 2016)
Presented findings to NZ/AUS policy regulators and industry groups (May & June 16)
Adopted sRNA NGS as the new PEQ test for screening imported clonal grasses
Engagement with the Agence Nationale de Sécurité Sanitaire (France) in trialling NGS
Auckland 23rd April 2015
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Benefits for plant industries Cheaper PEQ screening
- Reduce diagnostic costs from $4,000+ to only ~$700 (for virus/viroid testing only)
Rapid access and improved profitability- Reduces PEQ time to 6-12 months thereby providing faster access to new genetics and
market opportunities
Protect business investment- Reduce the change of viral outbreaks
Facilitates surveillance strategies- Faster diagnostics will support quicker responses to incursions
Identify new and emerging threats- Facilitates evaluation of potential threats before they establish
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4. Potential impacts for the strawberry industry (Frank)
Introduction of new and improved cvs. and production of healthy runners of these cvs. are critical for the productivity and sustainability of the Australian strawberry industry (GVP - more than $400m p.a.)
Technology to reduce the time in PEQ is important for the industry, therefore the R&D on NGS is strongly supported
The following diagram provides context for PEQ as one of the stakeholders in the production of healthy planting stock of improved cvs. for strawberry fruit growers in Australia
biosecurity built on science
Australian program
Nucleus Cvs. maintained in G/H & pathogen-tested annually
Daughter Nucleus stock
Foundation stock Produced in soil- less mix in a screenhouse
Mother stock Produced in fumigated soil by Toolangi runner growers
Certified runners Produced in fumigated soil by Toolangi runner growers
Strawberry breeding
Australian strawberry fruit crops
Overseas’ programs
PEQ Quarantine
Approved source
Plant breeders
Quarantine plant pathologists
AgriBio plant pathologists
Victorian Strawberry Industry Certification Authority
Runner growers
Fruit growers
Licensees of cvs.
O/S diagnosticians
Victorian Strawberry Runner Certification Scheme & Stakeholders
Produced in soil-less mix in a G/H
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Issues from a strawberry industry perspective Speed and sensitivity of detection of viruses/viroids are not the only considerationsIndustry needs confidence in NGS tests to maintain the integrity of pathogen-tested schemes and prevent the dissemination of pathogens around the country (possibly resulting in litigation) Factors affecting industry’s confidence in NGS include:
- Comparisons between NGS, PCR and biological methods- Evaluation of NGS methodology developed in Australia against different isolates of the most significant
exotic viruses/viroids- Studies on the likelihood of false positives, and detection of non-significant or unknown organisms- Publication of the work in a refereed journal(s)
NGS doesn’t detect fungal, bacterial and phytoplasma pathogens Licensees, VSICA & the runner industry are keen to develop partnerships with PEQ/PBCRC to further evaluate NGS and combinations with other diagnostic tests
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5. Where to from here (Mark)
System is essentially running - bioinformatics software is at a point where you click two buttons, walk away and come back after lunch
PBCRC project recently extended for additional 12 months to:Collect further evidence from O/S NGS studies and conduct additional ‘side by side’ trials comparing current PEQ diagnostic platforms to NGS (Oct-Mar 17)Prepare online training material and an end-user program including ongoing end user access to open source bioinformatics (Mar 17)Run additional workshops with industry and policy regulators in Aust. and NZ as part of policy development (Aug 16 & April 17)Prepare an issues paper on ‘challenges/solutions using NGS in a PEQ setting’ (April 17)Submit papers to peer reviewed journals and write up final PBCRC report (June 17)Offer industry (at their expense) option to conduct NGS along with mandatory PEQ testing to provide additional confidence (July 17)
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6. Questions (All)
Two or three questions from audience
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7. Challenges for everyone to consider over next few days (Mark)
PBCRC talk about leaving a ‘legacy’ - apart from peer reviewed papers, what legacy are you going to leave with your project?
How are the outputs from your research project going to be adopted and implemented by end users post life of the project?
Does your industry/agency have very clear R&D priorities and support for projects to implement R&D outputs (inc $$)?