New DNA tool helps researchers screen for plant viruses

Together a 'probe' and 'hairpins' are helping to detect plant viruses

Diseases
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Virus detection tool has promise for plant breeding and mass screening.

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Queensland Department of Agriculture and Fisheries researcher, Dr Fiona Filardo, is part of research efforts into screening for groups of viruses and identifying specific viruses. PHOTO Ngaire Naran

Queensland Department of Agriculture and Fisheries researcher, Dr Fiona Filardo, is part of research efforts into screening for groups of viruses and identifying specific viruses. PHOTO Ngaire Naran

Using a DNA 'probe' and 'hairpins', researchers have developed a diagnostic tool that can screen plants en masse for viruses.

The tool - called the Tissue Blot-Hybridisation Chain Reaction (TB-HCR) - can be used as a general screen for a group of viruses, or to identify a specific virus.

It has already been shown to work with the Turnip yellows virus/Beet western yellows virus in pulse crops and canola, and is now being tested with other viruses.

The work, led by Dr Paul Campbell and Dr Fiona Filardo, at the Queensland Department of Agriculture and Fisheries (DAF), is important for diagnosing plant viruses and has particular relevance to breeding and for mass screenings.

"Having a cost-effective tool will help breeders to screen new cultivars for resistance, which would mean resistant varieties are released more quickly to the industry," Dr Filardo says.

"But the tool will also be useful in the field, where viral infections can be difficult to distinguish from other disorders - such as nutrition problems."

It could also prove useful in detecting new and emerging viruses. This is because the TB-HCR tool is different to other diagnostic tools, which require antibodies to aid in virus detection.

Having a cost-effective tool will help breeders to screen new cultivars for resistance, which would mean resistant varieties are released more quickly to the industry. - Queensland Department of Agriculture and Fisheries researcher Dr Fiona Filardo

Current testing method

Plants are currently tested for viruses using a method called tissue blot immunoassay (TBIA), which includes the use of antibodies. Antibodies are specific to certain viruses or their families and bind to the virus, helping researchers to 'see' them.

Although this system is fairly fast and cheap, it is limited by antibody availability.

"You cannot undertake this process unless you have the antibody - and these are difficult, expensive and time-consuming to produce," Dr Filardo says.

"As researchers, we are often limited by a lack of antibodies - as in the physical quantities needed and the specifics.

"You may be limited to testing for a family of viruses and then, if positive, you need to do further, more-specific testing.

"We wanted to develop an alternative that could be easily modified to target any virus and potentially other pathogens too."

New method

The TB-HCR method works by using DNA probes and hairpins.

The DNA probe works in a similar way to Velcro and is made specifically to bind to either a family, group or specific virus.

Dr Filardo says the probe is created to complement the targeted virus's DNA.

"We look at the DNA sequence of the virus and design a complementary DNA probe to match it. If the probe encounters the virus it sticks to it," she says.

To alert researchers to a virus, two specially designed DNA hairpins then come into play. These hairpins act as initial messengers.

One attaches to the probe and the other attaches to the first hairpin. Hairpin number two contains a biotin label - akin to a 'sticky note' which, via a chain reaction, alerts researchers to plant tissue containing a virus.

"It causes a cascading reaction and amplifies the signal of a virus being there," Dr Filardo says.

Without the need for antibodies, researchers can undertake other virus screenings - for example, where new and emerging viruses are suspected and antibodies for them do not exist.

The tool could also be used to screen large amounts of plants coming into Australia, or researchers could take the tool overseas and test for viruses.

Dr Filardo also sees that, in the future, more probes could be added to the tool to test for multiple viruses at once.

The method being developed to screen plants en masse for viruses works by using a probe and DNA hairpins. The DNA probe is designed to bind to either a family, group or specific virus. To alert researchers to a virus, two specially designed DNA hairpins then act as initial messengers. SOURCE: Dr Paul Campbell

The method being developed to screen plants en masse for viruses works by using a probe and DNA hairpins. The DNA probe is designed to bind to either a family, group or specific virus. To alert researchers to a virus, two specially designed DNA hairpins then act as initial messengers. SOURCE: Dr Paul Campbell

"We could increase the number of viruses being identified across a range of crops," Dr Filardo says.

"This would help in the screening and breeding work already being done.

"For example, despite the drought, last year we surveyed about 230 plants for more than eight viruses using the older technology of TBIA.

"And in breeding terms, plant pathologists often screen up to 30,000 plants for more than seven viruses annually."

Turnip Yellows Virus

The tool has already proved successful in diagnosing Turnip yellows virus (TuYV).

Early infection of canola with TuYV up to the rosette stage can result in high yield losses. Canola can still become infected at later stages of crop development, but it is unlikely to cause significant yield and quality losses at later growth stages.

Although the TuYV infection causes plant stunting, reddening, yellowing and stiffening of lower leaves, symptoms are not a definitive diagnostic as they are similar to nutrient disorders or physiological stress.

The TB-HCR is being developed as part of a collaboration between GRDC and Queensland DAF.

See also:

GRDC Research Code DAN00202

More information: Dr Fiona Filardo, fiona.filardo@daf.qld.gov.au

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