session 1: strengthening border biosecurity improving - post entry quarantine: researchers and...

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


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)


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)


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.


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)


Viruses and viroids can be identified rapidly and reliably

Future vision: High risk plants spend less than 12 months in PEQ!


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


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


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


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


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


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


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)


6. Questions (All)

Two or three questions from audience


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 $$)?