Development of new, molecular-based, diagnostic tools for high priority exotic viruses of winter cereals (2014)

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1 Development of new, molecular-based, diagnostic tools for high priority exotic viruses of winter cereals (2014) L. Zheng 1,2, A. Freeman 1,2 and B. Rodoni 1,2 1 Department of Economic Development, Jobs, Transport and Resources, AgriBio, 5 Ring Road, La Trobe University, Bundoora, Victoria 3083, Australia, 2 Plant Biosecurity Cooperative Research Centre, LPO Box 5012, Bruce, Australian Capital Territory 2617, Australia A number of seedborne and seed-carried viruses have serious impact on cereal production in Europe and many other production zones. The increase in global movement of germplasm increases the risk of the accidental importation of these exotic viruses into Australia, however winter cereals are not subject to post-entry quarantine virus-testing. The main aim of this project is to develop rapid and cost-effective technologies for the detection of high priority exotic viruses of cereals which may enter Australia in imported germplasm and then develop protocols suitable for high-throughput post-entry quarantine virus-testing and surveillance. The diagnostic tools developed in this project include three genus-specific molecular tests for the Hordeivirus, Furovirus and Pecluvirus genera which have been validated in field surveys. A rapid, field-based, molecular surveillance method is being developed to enable rapid response to exotic virus incursions. The test utilises the loop-mediated isothermal amplification (LAMP) technology platform, which enables the generation of test results within an hour of sampling. The method has been used for field surveys of wheat/barley crops in Victoria using Barley yellow dwarf virus as a model. An alternate pathway for importation of these viruses (and potentially other unknown viruses) has been identified as the importation of clonal ornamental grasses. A massive parallel sequencing pipeline for one-step detection and diagnosis of known and previously unknown plant pathogens was used to screen clonal grasses for the presence of plant viruses and its potential for use in post-entry quarantine is being assessed. Presenting author s biography Linda Zheng obtained her doctorate in Ecology, Evolution and Systematics from The Australian National University, Canberra. During her candidature, Linda investigated the implications of plant virus discovery in the genus Potyvirus on their detection and developed a novel method of designing group-specific universal primers for virus detection. Her PhD studies also introduced her to the field of applied bioinformatics. Upon the completion of her PhD studies, Linda worked on various projects in the field of biosecurity where she continued to explore the world of plant virus diagnostics and epidemiology. Linda is currently involved in a PBCRC project aiming to develop new molecular tests for high priority viruses of winter cereals. Her research interests include applied bioinformatics, virus detection and diagnostics, virus evolution and epidemiology. Linda Zheng (03) linda.zheng@ecodev.vic.gov.au Science Exchange 2015 Conference Handbook 35

2 Session Four: Faster, cheaper, more accurate identification of plant pests Presentation title: Development of new, molecularbased, diagnostic tools for high priority exotic viruses of winter cereals Presenter: Linda Zheng

3 PBCRC 2014 New tools for field grains surveillance and diagnostics for high priority grains pests Linda Zheng Plant Biosecurity Cooperative Research Centre

4 Problem being addressed A major gap in diagnostic capability for exotic cereal viruses Inadequate cereal post-entry quarantine requirements - Seed-borne and/or seed-carried viruses post a threat to the grains industry The identification of an alternate entry pathway for cereal viruses into Australia in clonal, ornamental grasses A lack of field-based molecular diagnostic tools for identifying exotic cereal viruses in the case of an incursion.

5 Problem being addressed by: developing a list of high priority exotic cereal viruses with PHA developing new molecular diagnostic tests for high priority pests of grains examining the potential of new full length sequencing technologies for identifying sequence of known viruses and potential new viruses investigating and developing new molecular tools suitable for field-based detection and diagnosis of viruses

6 Results so far new tests for high priority pests Furovirus Hordeivirus Rymovirus Published and used in surveys to screen for exotic viruses in cereal crops in Victoria (2080 plants) (2000 plants) - All samples tested negative Protocols and surveys are first of their kind in Australia Hordeivirus-specific protocol drafted for submission to SPHD for endorsement Barley stripe mosaic virus Protocol developed, validated and written up for submission to SPHD for endorsement Used in surveys in 2013 and 2014 Used in fee-for-service diagnostics Pecluvirus Protocol validated SOP generated First time these exotic viruses were screened for in Victorian cereal crops. Negative results provided evidence of freedom for these virus species.

7 Results so far field-based virus surveillance tools LAMP (Loop-mediated isothermal amplification) - Validated test for rapid detection of Barley yellow dwarf virus (BYDV) in the field Optimising crude RNA extraction methods for use in LAMP - Testing on 100 samples collected in cereal virus survey in the Wimmera valley in min LAMP assay for on-site BYDV detection was successful. LAMP assays can now be developed for the rapid detection of other high priority viruses. 40 min

8 Results so far NGS for plant virus diagnostics Explore the use of high throughput sequencing for simultaneous detection and identification of multiple known and unknown pathogens in plants - Case study using clonal grasses intercepted in post-entry quarantine suspected of virus infection based on symptoms - Clonal grass sample A (Pennisetum spp.) Widespread genus in the grass family ( species) Widely used in landscaping Tested using several genus-specific test with no positive results

9 Results so far NGS for plant virus diagnostics dsrna extracted from clonal grass sample A and used for NGS A total of 32,867 reads mapped to the reference genome of Maize chlorotic dwarf virus (MCDV) Consensus sequence of 11,772 nucleotides in length Genome coverage is 99.5% Nucleotide identity is 98.7% MCDV-severe strain found in clonal grass sample A AY MCDV M1 U67839 MCDV Tennessee AY MCDV Severe 100 ClonalA_MCDV MCDV - genus: Waikavirus Exotic to Australia Overwinters in Johnson grass Infects corn, sorghum, wheat and sudan grass Identification of MCDV in this case confirms the importation of clonal grass as an alternative pathway for cereal viruses to enter Australia including those not seed-borne, therefore not in the seed importation pathway

10 Who will use research? At the border - Improved protocols for PEQ of cereals (cereal virus diagnostics) - Next generation sequencing to identify unknown viruses in PEQ Post border - Biosecurity scientists and incursion managers (LAMP and cereal virus diagnostics) - Diagnosticians (cereal virus diagnostics, next generation sequencing)

11 Who will directly benefit from the research? Industry protecting industry from incursions - Australia free of the exotic viruses described - Breeding for resistance has not been very successful - Infected spores of the fungus-transmitted viruses can remain viable for 20 years preventing cereal production in these areas. Biosecurity scientists - Provide tools which will facilitate the development of improved PEQ protocols for cereals - Provide a field surveillance tool (LAMP) to facilitate incursion management - Provide tools for diagnosticians and scientists to enable testing for these viruses.

12 Challenges and issues arising from the research The development of new diagnostic tools has been successful- the challenge is to deliver them to end users Molecular diagnostics for exotic cereal viruses Discussions with Department of Agriculture, PHA, etc to develop improved PEQ protocols for cereals LAMP field surveillance technology Next generation sequencing Developing diagnostic tests for high priority viruses Training scientists to use and adopt the technology How will these complex technologies fit into the Australian Biosecurity landscape (ie. In PEQ, as a diagnostic test, costs and availability, etc.)

13 Impact delivery plan Publication of newly developed protocols in peerreviewed journals Submission of protocols to SPHD for endorsement Training of diagnosticians at PEQ stations in the molecular diagnostics Training of grains virologists in the use of LAMP Engaging with the Department of Agriculture and biosecurity community to improve the PEQ protocols for cereals Consultation with industry for uptake of technology

14 Impact delivery plan wisdom knowledge Information Data data improvement

15 The End-User Advocate s perspective The strategy was to identify and mitigate biosecurity risks to the grains industry Lack of information and research on cereal quarantine The project brings together a team with varied skills to develop the technical tools to deliver the biosecurity outcomes New model for diagnostics needed?

16 Thank you For more information, please Acknowledgement - Angela Freeman (VIC DEDJTR) - Brendan Rodoni (VIC DEDJTR) - Jenny Davison (SARDI) - Shivraj Braich - Hugh Mason