Session 1: New approaches to winter cereal post-entry quarantine

SESSION 1: STRENGTHENING BORDER BIOSECURITY

New approaches to winter cereal post-entry quarantine

Angela Freeman

biosecurity built on science

New approaches to winter cereal post-entry quarantineAngela Freeman

Senior Research Scientist, Microbial Science, Pests and Diseases, DEDJTR, Vic

Plant Biosecurity Cooperative Research Centre


Introduction - Australia’s grains industry

Barley for grain15%

Lupins for grain1%

Canola4%

Cotton2%

Nurseries9%

Sugar cane for crushing6%

Crops for hay20%

Grain sorghum6%

Oats for grain3%

Wheat for grain34%

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


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


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


Virus transmission Acronym Genus Natural hosts DistributionSoil-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, USAWheat spindle streak mosaic virus WSSMV Bymovirus Wheat, rye, triticale North America, EuropeWheat yellow mosaic virus WYMV Bymovirus Wheat Japan, ChinaChinese wheat mosaic virus CWMV Furovirus Wheat ChinaOat golden stripe virus OGSV Furovirus Oats Europe, USASoil-borne cereal mosaic virus SBCMV Furovirus Wheat, rye, tricale EuropeSoil-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 FranceSeedborne Barley stripe mosaic virus BSMV Hordeivirus Barley, wheat, oats, maize,

ryeWorldwide

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


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

https://www.google.com.au/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwijzYnDve7PAhVCtJQKHfecAIAQjRwIBw&url=https://mulching.wordpress.com/&bvm=bv.136593572,d.cGc&psig=AFQjCNEkzwKSAX4EbUB54tktGNCldyBJsg&ust=1477228031330577


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


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|MCDV M1 U67839|MCDV Tennessee

AY362551|MCDV Severe ClonalA_MCDV

100

100

0.05


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


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


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


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 [email protected]

Thank you