Molecular diagnostic tools can meet the needs of biosecurity agencies Brendan Rodoni Senior research scientist Vic DPI Cooperative Research Centre for National Plant Biosecurity
Whattam challenges: “Its expensive to develop diagnostic protocols for every EPP that is a threat to Australia – can we develop one test that detects multiple targets?”
“We have a capability to detect known EPPs – what about the unknown threats?” “Escalating costs of managing EPP outbreaks with decreasing budgets and resources likely to lead to establishment of more pest incursions” Cooperative Research Centre for National Plant Biosecurity
Whattam Biosecurity R&D Needs Surveillance tools •Proactive surveillance at high risk border sites •Up-to-date records of plant pests and pathogens in Australia in APPD/ALA Detection systems that are rapid, reliable, accurate, cheap, sensitive and user-friendly so they can be delivered at the border in a timely fashion Implementation from research lab to quarantine frontline Improve disconnect between researchers and regulatory authorities (e.g. importing EPPs for diagnostic/contingency planning)
Cooperative Research Centre for National Plant Biosecurity
CRC60037 - The application of a genomics approach to develop a reliable diagnostic tool for the detection of Erwinia amylovora. Rachel Powney PhD (submission date June 2012)
- 5 referred papers -Accurate multi-locus PCR assay for fire blight pathogen -Strategy developed formed the basis of PBCRC Exemplar “Bacterial Pathovars”
E. amylovora pan-genome “Fast, Accurate,
Reliable, Easy and Affordable Diagnostic tests”
Cooperative Research Centre for National Plant Biosecurity
PBCRC Program 2 Exemplar: “New approaches for diagnosing bacterial pathovars� 1. Define scope of diagnostic tool Generate voucher specimen/s Review existing diagnostic protocols 2. Define pan-genome - Sequence and annotate genome/s
Research Strategy (per diagnostic target):
3. Diagnostic design – consider multiple platforms (lab and field) 4. Validation of protocols - regional surveillance (Aus and USA) 5. Produce protocols, train end users Cooperative Research Centre for National Plant Biosecurity
Detection of New and Emerging Pathogens
Rumsfold, D and Bush G (2001): Proceedings of the USDA APHIS Post Entry Quarantine Congress. Whitehouse, Washington D.C. USA
Cooperative Research Centre for National Plant Biosecurity
CRC40035 - The development of diagnostic platforms for Post Entry Quarantine (PEQ) and market access Strategy: •Develop PCR tests that detect groups of pathogens -Plant Virus Genera •Use group-specific primers as a “diagnostic tool” and not as a research tool – big difference •Winter Cereals (Furovirus, Rymovirus, Hordeivirus)
Outputs: -3 diagnostic tools developed -Diagnostic capability transferred to Horsham labs (Vic DPI) -Two years survey data in NW Victoria -Used to screen clonal grasses in AQIS PEQ - Will result in routine screening of winter cereals in PEQ -(PBCRC project – Freeman et al) Known unknowns Cooperative Research Centre for National Plant Biosecurity Known unknowns
CRC40180 - The submission of a validated protocol for the detection of potyviruses (virus genus) to SPHDS that can detect over 40 potyvirus species
Research Output
Product Potyvirus Diagnostic Manual Linda Zheng and Brendan Rodoni Department of Primary Industries
Peer reviewed paper on science supporting the diagnostic test
Diagnostic manual validated by NZ MAFF and ratified by SPHDS for use in diagnostic labs From research lab to quarantine frontline
Cooperative Research Centre for National Plant Biosecurity
Vic DPI – Crop Health Services (CHS) Fee-for-Service Plant Diagnostic laboratory
Test
2007 2008 2009 2010 2011
Poty 104
80
32
42
78
All
179
107
272
362
150
•Currently use group-specific PCR to detect 17 genera/families of plant viruses, phytoplasmas and Liberibacter species •Detected over 100 virus and phytoplasma species in our hands •Detected 5 “new” viruses, 3 new phytoplasmas From research lab to quarantine frontline
Potyvirus Furovirus Hordeivirus Rymovirus Begomovirus Carlavirus Betaflexiviridae Potexvirus Tobamovirus Tosopovirus Closteroviridae Ampelovirus Crinivirus Comoviridae Secoviridae Nepovirus (A&B) Ilavirus Liberibacter Phytoplasma
Cooperative Research Centre for National Plant Biosecurity
CRC100164 - Potato spindle tuber viroid (PSTVd): Phylogeny, Epidemiology and Pathogenicity of isolates associated with recent incursions in Australia • Alison
Mackie PhD at UWA (Martin Barbetti, Roger Jones and Simon McKirdy)
•Improved understanding of PSTVd epidemiology within and around incursion zones •Tomato isolates are highly pathogenic on potatoes
This is science supporting DAFF policy!!! Implications of research findings: • Scientific evidence to support DAFF policy on the importation of
Tomato seed •Commercial tomato seed is a pathway for PSTVd •PSTVd isolates associated with tomato seed are highly pathogenic to potatoes Disconnect between researchers and regulatory authorities Cooperative Research Centre for National Plant Biosecurity
CRC 20093: Plant Biosecurity: Technological research and training for improved pest diagnostics in Thailand and Australia (Gary Kong – Project Leader) - Victorian perspective: Facilitate the export of Australian seed potatoes
Project activities: •Training of Thai PEQ scientists – Increase biosecurity in region •First record of a tospovirus in potatoes in Northern Thailand Partnerships between Thai and Australian government agencies: •Transparent certification protocols in Australia and PEQ protocols at the Thai border Facilitate trade of Victorian seed potatoes: •Re-opened trade of seed potatoes ($3 million) in 2003 •Annual trade of Victorian seed potatoes has increased to approx $6 million (0.5% of trade attributed to Scientific partnerships = $30,000/yr for the last 10 years)
From research lab to quarantine frontline Cooperative Research Centre for National Plant Biosecurity
-“Point-of-Care” Molecular Diagnostics - Diagnostics to be completed in the field or at remote locations - More affordable surveillance programs - Faster more effective response - Ongoing passive surveillance Is not the ultimate diagnosis but… confident we can identify a negative
Applications of LAMP on Xanthomonas arboricola pv. Pruni, Potato virus Y and Potato spindle tuber viroid - A. Bühlmann, B Duffy, L Zheng, B Rodoni
LFDs that detect nucleic acid
qPCR
Cooperative Research Centre for National Plant Biosecurity
Isothermal multiplex AmpliDet RNA
Helicase-dependent isothermal amplification
Plant Biosecurity requirements for easy, affordable surveillance tools
Next Generation Sequencing (NGS) A powerful tool for Plant Microbial Discovery An unbiased approach to detect unknown viruses - Gayfeather mild mottle virus (Adams et al., 2008) Ecogenomics – discovery of hundreds of new viruses in plants - The majority of viruses are non-pathogenic - Area de Conservacion Guanacaste of Costa Rica (Roossinck et al. 2010) - Tall grass Prairie Preserve (TPP) (Oklahoma, USA) Wren et al., 2006)
Bacteria: Metagenomic profiles of “environmental samples”
Unknown unknowns Cooperative Research Centre for National Plant Biosecurity
Challenges I know what’s challenging!!!
Bioinformatics: - skills - tools
Microbial discovery - What do we do with all these names?
Maintain Biology Capability - Molecular based milestones are short and easy to fund…But… - pathogenicity and other biological parameters are equally important Cooperative Research Centre for National Plant Biosecurity
Thank you
Linda Zheng Rachel Powney Jo luck Jane Moran Angela Freeman Merrin spackman Shane King Aftab Plant micro team
Gerard Clover Jo Tang
Kim Plummer Alison Mackie (DAWA/UWA Roger Jones (DAWA/UWA Martin Barbetti (UWA) Gary Kong (QDPI/CRCNPB) Simon McKirdy
Brion Duffy
Steve Beer
GRDC
Cooperative Research Centre for National Plant Biosecurity