Development of a molecular based diagnostic capability for the detection of cereal viruses

Development of a molecular based diagnostic capability for the detection of cereal viruses Linda Zheng Research Scientist Merrin Spackman, Angela Freeman, Brendan Rodoni biosecurity built on science Cooperative Research Centre for National Plant Biosecurity

Important cereal viruses Wheat - Triticum

Barley yellow dwarf virus, Wheat dwarf virus, Soil-borne wheat mosaic virus, Wheat spindle streak mosaic virus, Soil-borne cereal mosaic virus, Wheat yellow mosaic virus, Aubian wheat mosaic virus, Barley yellow striate mosaic virus, Indian peanut clump virus, Wheat rosette stunt virus, Wheat American striate virus

Barley - Hordeum

Barley yellow dwarf virus, Wheat dwarf virus, Barley mild mosaic virus, Barley yellow mosaic virus, Barley stripe mosaic virus, Northern cereal mosaic virus, Barley yellow streak mosaic virus, Arabis mosaic virus, Tobacco rattle virus

Rye - Secale

Barley yellow dwarf virus, Wheat dwarf virus, Soil-borne cereal mosaic virus, Wheat spindle streak mosaic

Triticale Triticosecale

Barley yellow dwarf virus, Wheat dwarf virus, Soil-borne cereal mosaic virus, Wheat spindle streak mosaic virus

Oat - Avena

Barley yellow dwarf virus, Oat sterile dwarf virus, Oat golden stripe virus, Oat chlorotic stunt virus, Oat mosaic virus, Wheat dwarf virus

Corn - Zea

Maize dwarf mosaic virus, Johnsongrass mosaic virus, Sugarcane mosaic virus, Maize rough dwarf virus, Maize chlorotic mottle virus, Maize chlorotic dwarf virus, Maize bushy stunt virus, Cereal chlorotic mottle virus, Barley yellow dwarf virus,Sorghum chlorotic spot virus, High Plains virus, Wheat streak mosaic virus

Rice - Oryza

Rice tungro virus, Rice dwarf virus, Rice gall dwarf virus, Rice grassy stunt virus, Rice hoja blanca virus, Rice necrosis mosaic virus, Rice ragged stunt virus, Rice stripe necrosis virus, Rice yellow mottle virus, Barley yellow dwarf virus

Sorghum / Millet Sorghum/Pennisetu m

Maize dwarf mosaic virus, Sorghum yellow banding virus, Sorghum chlorotic spot virus

Serological and molecular tests available for some of these viruses i.e. WSMV, HPV, BYDV – but not all biosecurity built on science

Threats: Seed-borne/carried viruses that have not been recorded in Australian Winter Cereals Seed-borne viruses: - Barley stripe mosaic virus (Hordeivirus) -No known vector -Interceptions in barley seed imports -Soil-borne cereal mosaic virus (Furovirus) (Budge et al., 2008) -Soil-borne wheat mosaic virus (Furovirus) - detected in NZ (Lebas et al., 2009) - suspected seed transmission (Lebas et al., 2009)

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Recent threat: viruses transmitted by Polymyxa graminis

Kanyuka (2003) Molecular Plant Pathology 4(5) 393-406

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Records of P. graminis - Murrumburra (NSW) 1959 confirmed?

- Wondai (Qld) 2009 Serious implications for incursions of these viruses if P. graminis becomes widespread biosecurity built on science

Post Entry Quarantine  No active testing for any viral disease in imported grains materials  No specific tests for exotic viruses of cereals that could be carried inside/outside of seed  All diagnosis in quarantine facilities are by visual inspection (Rodoni 2009) Concern about seed borne/carried viruses coming in at low levels and not being detected Concern about fungus borne viruses if P. graminis becomes widespread Rodoni, B. and Zheng, L. (2009) A review of the legal importation of grains material into Australia and New Zealand: Identification of critical control points to better manage biosecurity risks biosecurity built on science

Project Objectives  Development of broad-spectrum molecular diagnostic tests to support Post entry quarantine (PEQ) for cereal crops - Cheaper, faster and more reliable molecular tests - PCR assays with the use of universal primers designed to detect a group of closely-related species biosecurity built on science

Broad-spectrum molecular tests  Based on the development model for potyvirus detection (Zheng et al, 2010) - Validated in Australian and NZ labs - Optimised primer design and assay conditions - Submitted as SPHDS manual and routinely used in a diagnostic setting – with a list of virus isolates detected - 87 tests have been done using the assay since it’s development in 2008

Zheng et al (2010) Plant Pathology 59, 211-220

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Broad-spectrum molecular tests  Assays developed for furovirus, hordeivirus and rymoviruses - Validated in Australia and NZ labs - Evaluated by USDA - Currently being evaluated in diagnostic labs in Australia (mini surveys in Victoria) - Used on clonal grass samples intercepted by Eastern Creek NSW - Paper in preparation

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Cereal virus detection assays Detection assays

Furovirus

Viruses targeted 1.Soil-borne wheat mosaic virus 2.Soil-borne cereal mosaic virus 3.Chinese wheat mosaic virus 4.Oat golden stripe virus 5.Sorghum chlorotic spot virus

Hordeivirus

1.Barley stripe mosaic virus 2.Poa semilatent virus 3.Lychnis ringspot virus 4.Anthoxanthum latent blanching virus

Rymovirus

1.Ryegrass mosaic virus 2.Agropyrum mosaic virus 3.Hordeum mosaic virus

Tritimovirus

1.Wheat streak mosaic virus 2.Brome streak mosaic virus 3.Oat necrotic mottle virus biosecurity built on science

When – October 2010 Where – radiating out from Horsham How – Random sampling What – 12 wheat crops 11 barley crops

1

- Horsham

2 – Goroke to Nhill 3 - Balmoral

1

4 – Lake Bolac

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Survey results Lanes 1-16 – survey samples 1

2

9 10

3

4

5

11 12 13

6

7

8

14 15 16

Lanes 1-20 – survey samples M 1

2

3

4

5

6

7

8

00bp

NTC + H

-

M 9 10

11 12 13

M 17 18 19 20

-

14 15 16

+

H

Lanes 1-24 – survey samples M 1

2

M 9 10

3

4

5

11 12 13

M 17 18 19 20 21

6

7

8

14 15 16

22 23

24

Good news! All negative -

+

H

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When – October 2011 Where – Radiating out from Horsham How – Random sampling What – 170 wheat crops 150 barley crops All were negative All samples were tested using the 3 generic assays

1 - Horsham 2 - Antwerp 3 - Brim

4 2

3 1

4 - Beyal 5 - Minnera 6 - Hamilton 6

5

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What now?  Continue surveys - Provide evidence of absence for phytosanitary status required for market access - Screen for more virus groups and unknowns  Scope for new technology platform development

- Screen for polymyxa graminis (vector) as well as the viruses that it transmits

 Develop protocols for use in PEQ - Develop and validate virus specific protocols - PEQ manual for cereal virus diagnostics

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Acknowledgement  CRC NPB  GRDC  Horsham virology team

- Shane King, Mohammad Aftab, Virginia McQueen

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AQIS (Doris Mercado-Escueta) AWCC (Australian Winter Cereals Collection) NZ MAFF (Gerard Clover) USDA-APHIS (Laurene Levy)

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