New approaches to winter cereal post-entry quarantine

1. SESSION 1: STRENGTHENING BORDER BIOSECURITY
New approaches to winter cereal post-entry quarantine
Angela Freeman

2. biosecurity built on science
New approaches to winter cereal post-entry quarantine
Angela Freeman
Senior Research Scientist, Microbial
Science, Pests and Diseases, DEDJTR, Vic
Plant Biosecurity Cooperative Research Centre

3. biosecurity built on science
Introduction - Australia’s grains industry
Barley for grain
15%
Lupins for grain
1%
Canola
4%
Cotton
2%
Nurseries
9%
Sugar cane for crushing
6%
Crops for hay
20%
Grain sorghum
6%
Oats for grain
3%
Wheat for grain
34%
 34 million tonnes of grain each year from
an area of about 20 million hectares
 Annual gross value of more than $9
billion at farm gate
 New germplasm imported each year
 Post-entry quarantine of imported cereal
seeds
- considered low risk in the past
- currently based on plant grow outs with
visual inspection
- no virus testing
winter cereals are the largest grains
industry in Australia

4. biosecurity built on science
 The Australian grains industry depends on importation of seed as
germplasm for crop breeding and bulk grain for processing and animal
feed.
 Rapid global movement of seed, soil contaminations, equipment and
people is causing rapid spread of grains pathogens.
 Lack of post-entry quarantine testing for seed- and soil- borne viruses.
 Soil-borne cereal viruses vectored by the soil-borne plasmodiophorid
protist, Polymyxa graminis, whose tiny spores can be carried on cereal
seed.
 P. graminis has been identified in Queensland and Western Australia.
Dormant spores can survive in the soil for 15 years rendering areas
unsuitable for cereal production.
A changing world necessitates new approaches to cereal PEQ

5. biosecurity built on science
Soil-borne (P.graminis-transmitted) and seed-borne viruses of winter cereals
 Viruses vectored by P.graminis cause economically important soil-borne diseases
- increasingly widespread worldwide
- reported in all continents except Australia
 Yield losses may be as high as 70%
 “Soil” potential risk and import of other semi-processed material from “positive” regions
 Development of resistant varieties only means of preventing severe losses

6. biosecurity built on science
Virus transmission Acronym Genus Natural hosts Distribution
Soil-borne, P. graminis
Barley mild mosaic virus BaMMV Bymovirus Barley Europe, Japan, China, Korea
Barley yellow mosaic virus BaYMV Bymovirus Barley Europe, Japan, China, Korea
Oat mosaic virus OMV Bymovirus Oats Oats Europe, USA
Wheat spindle streak mosaic virus WSSMV Bymovirus Wheat, rye, triticale North America, Europe
Wheat yellow mosaic virus WYMV Bymovirus Wheat Japan, China
Chinese wheat mosaic virus CWMV Furovirus Wheat China
Oat golden stripe virus OGSV Furovirus Oats Europe, USA
Soil-borne cereal mosaic virus SBCMV Furovirus Wheat, rye, tricale Europe
Soil-borne wheat mosaic virus SBWMV Furovirus Wheat, barley, rye, triticale North America
Sorghum chlorotic spot virus SrCSV Furovirus Sorghum, wheat, barley USA
Peanut clump virus /
Indian peanut clump virus
PCV Pecluvirus Peanut, sorghum, wheat India, West Africa
Aubian wheat mosaic virus ?? AWMV Unassigned Wheat France
Seedborne
Barley stripe mosaic virus BSMV Hordeivirus Barley, wheat, oats, maize,
rye
Worldwide
Brome mosaic virus BMV Bromovirus Barley, wheat, oats, rye USA, Europe, South Africa
Soil-borne (P.graminis-transmitted) and seed-borne viruses of winter cereals
12 viruses
in 3
genera
14 viruses
in total
5 out of 14
viruses
discussed
are PHA
Priority EPPs

7. biosecurity built on science
Import pathways for high priority, exotic cereal viruses
 Poaceae family one of the largest plant families (~700 genera and
10,000 species)
 Wheat, rice and maize belong to this family
 Almost 20% of known plant viruses infect Poaceae
 Many members of the Poaceae imported for a range of industries via
import pathways such seed, tissue culture plantlets and clonal
grasses, and
 These pathways represent a potential risk of entry of cereal viruses
 The key import pathways are:
- Winter Cereals
- Coarse grains
- Sugarcane
- Clonal grasses
- Stockfeed

8. biosecurity built on science
Import risk pathways and quarantinable viruses of cereals-levels of risk
High risk:
• P. Graminis-transmitted, soil-
borne viruses, all pathways
• Seed-borne viruses in cereal
seed
• Unknown number of viruses
in clonal grass import
pathway

9. biosecurity built on science
 Clonal grasses- Clonal grass sample A (Pennisetum sp.) showing virus symptoms in PEQ
• tested using several genus-specific test with no positive results
• dsRNA extracted from clonal grass “sample A” and used for NGS
MCDV-severe strain found in clonal grass “sample A”
• MCDV - genus: Waikavirus, widespread genus in the grass family (80-140 species), exotic to Australia
• overwinters in Johnson grass, infects corn, sorghum, wheat and many grasses, including Pennisetum
spp.
• Pennisetum spp. widely used in landscaping, high degree of weediness, many species establishing in
agricultural and bushland areas
Recently identified risks in the import pathways
 P. graminis virus vector identified in Australia
• identified in Queensland (2009) and WA (2012)
• likely widespread
 Recent incursion and establishment of Russian wheat aphid (RWA)
• Vector of Brome mosaic virus
AY829112|MCDVM1
U67839|MCDVTennessee
AY362551|MCDVSevere
ClonalA_MCDV
100
100
0.05

10. biosecurity built on science
Imported seed
 Development and validation of genus-specific virus diagnostic tests (RT-PCR) for the four genera of
P. graminis- transmitted, soil-borne viruses- bromoviruses, bymoviruses, furoviruses, and
pecluviruses, and for hordeiviruses
 Specific RT-PCR tests for:
- the two seed-borne HPPs of cereals- Barley stripe mosaic virus, Brome mosaic virus
- viruses listed on Plant Health Australia (PHA) Threat Summary Table for Grains as HPPs (European and
American viruses, not Asian viruses of limited distribution)
 Sequencing of PCR products
 Full length sequencing (NGS) of samples testing positive
 Multi-faced approach for screening “usual samples”
- Imported antisera for ELISA and TBIA for European P. graminis-transmitted viruses
Clonal grasses
 Full length sequencing (NGS) of samples
 Confirmation with a second test
Diagnostics developed for high risk, exotic viruses (HPPs) of cereals and other Poaceae

11. biosecurity built on science
 Summary
- Current winter cereal PEQ regulations are inadequate and represent an unacceptable
risk to the cereal industry
- High risk exotic viruses (HPPs) have been identified based on the PHA Exotic Threats
Table for Grains
- All P. graminis-transmitted viruses of cereals have been identified and included in the
proposed list of targeted quarantinable viruses
- Diagnostic tests and protocols have been developed for the 14 quarantinable viruses of
winter cereals identified
 Recommendations
- Proposed improved PEQ protocols for winter cereals and associated risk pathways
discussed with stakeholders and implemented
Summary and recommendations

12. biosecurity built on science
Diagnostics developed for high risk, exotic viruses (HPPs) of cereals and other Poaceae
Cereals PEQ manual that contains SOPs
for viruses/pathogens to screen
germplasm at the Australian border
Plant sample
Examine symptoms
sampling
RNA extraction
Housekeeping RT-PCR
(NAD and/or MDH gene)
- +
RT-PCR
(BSMVF-R)
-+
If negative:
Stop. BSMV not
detected
Repeat RNA
extraction and all
PCR tests
If positive:
Sequence PCR product for
verification of infection
Worlds best practice for
cereal PEQ
Enhances the biosecurity
at the Australian grains
industry
Adoption of SOPs by other
biosecurity agencies

13. biosecurity built on science
 The project:
- CRC2014 New tools for field grains surveillance and diagnostics of high priority pests
 The team:
- Angela Freeman, Linda Zheng, Brendan Rodoni
Agribio, Biosciences Research, DEDJTR, Victoria
 For more information, please email angela.freeman@ecodev.vic.gov.au
Thank you