Molecular test strategies target exotic cereal viruses

Taking ‘rapid threat assessment’ from the laboratory to the field is the aim of new viral detection tests under development

Photo of Dr Linda Zheng

Dr Linda Zheng is using LAMP technology to develop a new, rapid, in-field test that confirmed the presence of barley yellow dwarf virus in crops in Horsham last year.

PHOTO: Plant Biosecurity CRC

Ornamental clonal grasses have become one of Australia’s landscaping darlings, with widespread plantings in both public and private settings. However, new research has revealed that imported grasses, like these, are a disease risk for Australia’s cereal crops.

Based at the Victorian Government and La Trobe University’s Centre for Agribioscience (AgriBio) in Melbourne, Dr Linda Zheng says genetic testing has identified a virulent strain of maize chlorotic dwarf virus (MCDV) in a Pennisetum species of clonal grass intercepted during quarantine checks in 2013.

This strain of MCDV was first identified in the US in 2004, where MCDV is one of the most economically significant diseases affecting maize. It also affects wheat and sorghum.

Dr Zheng says she was provided with samples of a plant that had been identified as suffering from a viral infection. However, tests for known pathogens all produced negative results. Plant ribonucleic acid (RNA) was extracted from plant samples and the whole genome was sequenced, which simultaneously sequenced the pathogen responsible for the infection. A search through GenBank, an international database for sequence depository, including that of known plant viruses, found a 98.7 per cent match with a virulent strain of MCDV. Other known hosts for the MCDV include Johnson grass (Sorghum halepense), designated as a noxious weed in NSW, and Sudan grass (Sorghum sudanese).

The testing was undertaken as part of Dr Zheng’s work for the Plant Biosecurity Cooperative Research Centre (PBCRC). The GRDC contributes to this work through funding for PBCRC.

She says next-generation genetic sequencing is the most effective option to identify “unknown” plant pathogens and is becoming more cost effective as technology improves.

Seed-borne viruses

Dr Zheng is also developing broad-spectrum molecular tests for three high-priority groups of pathogens already identified as threats: Furovirus, Hordeivirus and Rymovirus.

These can all be imported with cereal seeds, either in the seed coating or within the seed itself. Imported seed of winter cereals is not virus-tested during post-entry quarantine grow-out.

Wheat streak mosaic virus is thought to have arrived in Australia on imported seed. It was first detected in 2003 and caused yield losses of 50 to 80 per cent in South Australia and NSW in 2005.

Specific viruses Dr Zheng is targeting include barley stripe mosaic virus, soil-borne cereal mosaic virus and soil-borne wheat mosaic virus.

These are all transmitted by the soil-borne protist (fungus-like) Polymyxa graminis and would be a major economic threat to Australian cereal crops if introduced. They are carried in the tiny resting spores of P. graminis, and can survive through stresses such as drought for more than 20 years.

Dr Zheng says while none of these viruses has yet been detected in Australia, there have been two confirmed reports of P. graminis in the past four years – one in Queensland and one in Western Australia.

As part of the PBCRC project broad-spectrum molecular diagnostic tests for high-risk exotic viruses of cereals have been developed. The aim is to incorporate molecular testing into the post-entry quarantine program for cereals to reduce the risk of virus entry on infected seed.

In-field testing

Dr Zheng is also working on in-field test kits for a range of viruses. The test kits can produce results quickly and eliminate the need to move infected plant material to a laboratory.

In a trial in 2015 she used barley yellow dwarf virus (BYDV) as a model for a rapid testing procedure, applying loop-mediated isothermal amplification (LAMP) technology. LAMP uses a species-specific DNA amplification process, with comparable accuracy to the standard laboratory-based polymerase chain reaction tests.

LAMP technology has been widely used in Europe as part of a surveillance and control program for the fungal infection that causes ash dieback but is relatively new to Australia.

The testing technique takes a plant sample in the field, crushes it and air-dries it on specialised filter paper. A small piece of the filter paper is then washed with processing agents and added to a portable LAMP machine.

Dr Zheng’s field trial in Horsham last year took less than an hour to confirm BYDV infection in a wheat crop.

She says once the specific tests for other viruses have been developed, LAMP technology will offer real potential to improve surveillance and diagnosis for both existing and exotic viral outbreaks. The portability of the equipment and simplified process means testing could be undertaken by extension officers, agronomists or growers themselves.

More information:

Dr Linda Zheng,
03 9032 7352,
linda.zheng@ecodev.vic.gov.au

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