Feature: Biodefender

With the wettest spring on record, 2010 saw an old nemesis return in force to ravage eastern Australia’s vineyards. Downy mildew (Plasmopora viticola) had lain quiescent in vineyards for almost two decades since the last major epidemic in 1992-1993 seriously damaged grape crops.

Many growers were unprepared, especially irrigated wine-grape producers in the Murray Valley. Late October rains exacerbated the epidemic at the worst possible time; vines were flowering, which ensured damage to developing bunches would be severe.

Demand rapidly exhausted supplies of the few systemic fungicides capable of eliminating downy mildew infections, and the recent economic turbulence that hit the wine industry left many grape growers unable to afford the fungicides they needed.

Many organic vineyards lost almost everything, because organic production protocols limit growers to naturally occurring copper and sulfur compounds that arrest – but don’t eliminate – downy mildew infections.

What would Australian grape growers pay to be rid of downy mildew and other fungus diseases, like powdery mildew (Erysiphe necator) and botrytis (Botrytis cinerea) also rife in eastern Australian vineyards this year?

Researchers at the CSIRO have the technology, and now the genes, to potentially make all grapevines completely resistant to downy and powdery mildew. The question is whether Australia’s grape industries – the wine industry in particular – and risk-averse consumers are ready for the change.

CSIRO Plant Industry vine disease expert, Dr Ian Dry, acknowledges these obstacles to solving the problem of disease in domesticated Vitis vinifera grapevines.

Dry is growing a number of genetically modified grapevine clones in a biosecure glasshouse at Glen Osmond, near Adelaide. He is confident that some will prove fully resistant to downy mildew or powdery mildew, although not yet both.

Each GM clone contains one of seven candidate mildew-resistance genes cloned from a cluster of resistance genes from the North American native Concord grape, Muscadinia rotundifolia. In the 1960s French wine grape breeder, Alain Bouquet, of the National Agricultural Research Institute (INRA) produced a vinifera x muscadinia hybrid, as the two are interfertile.

However, the hybrids inherited the unpleasant foxy flavour of their Muscadine parents, so Bouquet back-crossed them to traditional V. vinifera varieties to segregate the resistance and flavour traits.

Inevitably, hybridisation and back-crossing broke up the elite constellations of genes that gives varieties like Shiraz, Cabernet Sauvignon, Sauvignon Blanc and Chardonnay their unique varietal characters. While breeders might eventually get back to a Shiraz-like wine through multiple back-crosses to Shiraz, “you couldn’t call it Shiraz,” Dry said.

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“Consumers have been familiar with the names and the styles of these traditional varieties for at least 150 years, and there has been a lot of selection for particular clones of varieties like Shiraz. You’d be lucky to find something that would appeal to the connoisseur or the general public.”

The only way to preserve the familiar is to transfer the Muscadinia resistance genes into an otherwise-intact Shiraz or Chardonnay genome with recombinant DNA technology. However, the mere idea of genetically modified (GM) vines causes the deeply conservative winemakers of France, Germany and Italy to pop their corks. Consumers there oppose all things GM, almost reflexively.

But under pressure from risk-averse consumers, EU governments are moving to a zero-tolerance policy on fungicide residues in wine. That could only be realised by the industry – and consumers – embracing GM vines. CSIRO and INRA have spent more than a decade trying to locate and clone the mildew-resistance genes in Alain Bouquet’s vinifera x muscadinia hybrid.

Natural defences

So far, the project led by Dr Dry has been a success. “We’re fairly confident we have generated a number of transgenic vines with individual candidate genes from the resistance locus.”

This resistance locus houses a complex of seven individual genes. Their similarities at DNA-sequence level indicate they arose through serial duplications from a single, ancestral gene. Several may be non-functional, obsolescent casualties of “breakthrough” mildew strains.

But Dry is optimistic that one of the genes confers complete resistance to powdery mildew because it has made a formerly highly susceptible variety – Gordo Blanco or muscatel – impervious to infection.

Remarkably, a neighbouring gene also seems to confer highly specific resistance to downy mildew. Despite their sequence similarity, the powdery mildew fungus, Uncinula necator, is not related to the downy mildew fungus. However, they do infect vines in similar ways.

Dry said powdery mildew infects the leaf’s epidermal layer, while downy mildew enters via the stomata on the underside of the leaves – the tiny pores through which the plant “inhales” carbon dioxide and expires water vapour. Both fungi then insert tiny feeding structures, called haustoria, into the interior of the leaf, to obtain nutrients.

He said the resistance genes behave like alarm bells: they detect a specific protein secreted by the fungus during infection, and activate a network of “suicide” genes that rapidly kill off the cells around the point of infection, quarantining the fungus within a patch of dead tissue.

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In contrast, Dry said, the “noble rot” fungus, Botrytis cinerea, feeds only on dead tissue by secreting an enzyme that liberates nutrients from dead cells. Mildew outbreaks commonly precede Botrytis infections.

“There has been a revolution in the past 20 years in our understanding of how fungus diseases infect plants,” he said. “We now know that plants have a type of basic immune response that protects against most fungus strains. You can expose a vine to 150 strains of powdery mildew, and it will be resistant to 149 of them, but the 150th strain somehow breaks through the basal resistance.

So plants have evolved a second line of defence in the form of resistance genes like the ones we have found in Muscadine grapes. These resistance genes evolved to plug holes that the fungi have found in the plant’s defences. There is a lot of research interest in how some fungus strains manage to break through this basic immune response.”

As well as having the means to make vines resistant to downy mildew and powdery mildew, CSIRO researchers also pioneered an RNA interference technology that uses man-made genes to detect and destroy the genetic material of invading plant viruses before they can replicate in plant cells.

With a single synthesised gene, it is now theoretically possible to protect any crop or ornamental plant against all of its major virus diseases. As very long-lived plants, grapevines eventually accumulate a vigour-sapping collection of plant viruses. The only way to protect crops against virus diseases is to spray pesticides to kill the aphids and other sap-sucking insects that transmit them.

While overhauling traditional grape varieties to make them resistant fungal and viral diseases, why not implant a “designer” gene to switch off the polyphenol oxidase (PPO) gene, which codes for the oxidizing enzyme that darkens dried fruit that has been exposed to rain.

That would allow Australian growers to produce golden-coloured fruit even in very wet years, like 2009 and 2010. And for good measure, the anti-PPO transgene would also spare winemakers of the cost of removing brown colouration from white grape juice.

Research by the CSIRO, the Australian Wine Research Institute and other agencies led to a cultural and technological revolution in Australia’s wine industry in the 1980s, and played a major role in the industry’s phenomenal export success. Australia’s rivals have now caught up, and many believe it’s time to go to the next level and use gene technology to make grape growing cleaner, cheaper and to improve wine quality.

But Ian Dry says there’s another obstacle: the cost of transforming the scores of different clones of each grape variety and subjecting them individually to the enormously expensive process that the US Food and Drug Administration demands of all new GM crops to confirm they are safe for the environment and human consumption.

While this safety regime is intended to reassure consumers, if anything, it has only reinforced perceptions that rigorous testing is needed to stave off potential hazards.